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Context: Symptom management is essential in the end-of-life care of long-term care facility residents., Objectives: To study discrepancies and possible associated factors in staff and family carers' symptom assessment scores for residents in the last week of life., Methods: A postmortem survey in Belgium, The Netherlands, and Finland: staff and family carers completed the End-of-Life in Dementia-Comfort Assessment in Dying scale, rating 14 symptoms on a one-point to three-point scale. Higher scores reflect better comfort. We calculated mean paired differences in symptom, subscale, and total scores at a group level and inter-rater agreement and percentage of perfect agreement at a resident level., Results: Mean staff scores significantly reflected better comfort than those of family carers for the total End-of-Life in Dementia-Comfort Assessment in Dying (31.61 vs. 29.81; P < 0.001) and the physical distress (8.64 vs. 7.62; P < 0.001) and dying symptoms (8.95 vs. 8.25; P < 0.001) subscales. No significant differences were found for emotional distress and well-being. The largest discrepancies were found for gurgling, discomfort, restlessness, and choking for which staff answered not at all, whereas the family carer answered a lot, in respectively, 9.5%, 7.3%, 6.7%, and 6.1% of cases. Inter-rater agreement κ ranged from 0.106 to 0.204, the extent of perfect agreement from 40.8 for lack of serenity to 68.7% for crying., Conclusion: There is a need for improved communication between staff and family and discussion about symptom burden in the dying phase in long-term care facilities., (Copyright © 2020. Published by Elsevier Inc.)

Background/objectives: Opioids relieve symptoms in terminal care. We studied opioid underuse in long-term care facilities, defined as residents without opioid prescription despite pain and/or dyspnoea, 3 days prior to death., Design and Setting: In a proportionally stratified randomly selected sample of long-term care facilities in six European Union countries, nurses and long-term care facility management completed structured after-death questionnaires within 3 months of residents' death., Measurements: Nurses assessed pain/dyspnoea with Comfort Assessment in Dying with Dementia scale and checked opioid prescription by chart review. We estimated opioid underuse per country and per symptom and calculated associations of opioid underuse by multilevel, multivariable analysis., Results: Nurses' response rate was 81.6%, 95.7% for managers. Of 901 deceased residents with pain/dyspnoea reported in the last week, 10.6% had dyspnoea, 34.4% had pain and 55.0% had both symptoms. Opioid underuse per country was 19.2% (95% confidence interval: 12.9-27.2) in the Netherlands, 25.2% (18.3-33.6) in Belgium, 29.3% (16.9-45.8) in England, 33.7% (26.2-42.2) in Finland, 64.6% (52.0-75.4) in Italy and 79.1% (71.2-85.3) in Poland ( p  < 0.001). Opioid underuse was 57.2% (33.0-78.4) for dyspnoea, 41.2% (95% confidence interval: 21.9-63.8) for pain and 37.4% (19.4-59.6) for both symptoms ( p  = 0.013). Odds of opioid underuse were lower (odds ratio: 0.33; 95% confidence interval: 0.20-0.54) when pain was assessed., Conclusion: Opioid underuse differs between countries. Pain and dyspnoea should be formally assessed at the end-of-life and taken into account in physicians orders.

Background: The PACE 'Steps to Success' programme is a complex educational and development intervention for staff to improve palliative care in long-term care facilities (LTCFs). In a cluster randomized controlled trial, this programme has been implemented in 37 LTCFs in 7 European countries. Alongside an effectiveness study, a process evaluation study was conducted. This paper reports on the results of this process evaluation, of which the aim was to provide a more detailed understanding of the implementation of the PACE Programme across and within countries., Methods: The process evaluation followed the Reach, Effectiveness, Adoption, Implementation, Maintenance (RE-AIM) framework and involved various measures and tools, including diaries for country trainers, evaluation questionnaires for care staff, attendance lists and interviews (online and face-to-face, individual and in groups) with country trainers, managers, PACE coordinators and other staff members. Based on key elements of the PACE Programme, a priori criteria for a high, medium and low level of the RE-AIM components Reach, Adoption, Implementation and intention to Maintenance were defined. Qualitative data on factors affecting each RE-AIM component gathered in the online discussion groups and interviews were analysed according to the principles of thematic analysis., Results: The performance of the PACE Programme on the RE-AIM components was highly variable within and across countries, with a high or medium score for in total 28 (out of 37) LTCFs on Reach, for 26 LTCFs on Adoption, for 35 LTCFs on Implementation and for 34 LTCFs on intention to Maintenance. The factors affecting performance on the different RE-AIM components could be classified into three major categories: (1) the PACE Programme itself and its way of delivery, (2) people working with the PACE Programme and (3) contextual factors. Several country-specific challenges in implementing the PACE Programme were identified., Conclusions: The implementation of the PACE Programme was feasible but leaves room for improvement. Our analysis helps to better understand the optimal levels of training and facilitation and provides recommendations to improve implementation in the LTC setting. The results of the process evaluation will be used to further adapt and improve the PACE Programme prior to its further dissemination., Trial Registration: The PACE study was registered at www.isrctn.com-ISRCTN14741671 (FP7-HEALTH-2013-INNOVATION-1 603111) July 30, 2015.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave many of nature's most recalcitrant polysaccharides by acting on the C1- and/or C4-carbon of the glycosidic bond. Here, the results of an extensive mutagenesis study on three LPMO representatives, Phanerochaete chrysosporium LPMO9D (C1-oxidizer), Neurospora crassa LPMO9C (C4), and Hypocrea jecorina LPMO9A (C1/C4), are reported. Using a previously published indicator diagram, the authors demonstrate that several structural determinants of LPMOs play an important role in their oxidative regioselectivity. N-glycan removal and alterations of the aromatic residues on the substrate-binding surface are shown to alter C1/C4-oxidation ratios. Removing the carbohydrate binding module (CBM) is found not to alter the regioselectivity of HjLPMO9A, although the effect of mutational changes is shown to increase in a CBM-free context. The accessibility to the solvent-exposed axial position of the copper-site reveales not to be a major regioselectivity indicator, at least not in PcLPMO9D. Interestingly, a HjLPMO9A variant lacking two surface exposed aromatic residues combines decreased binding capacity with a 22% increase in synergetic efficiency. Similarly to recent LPMO10 findings, our results suggest a complex matrix of surface-interactions that enables LPMO9s not only to bind their substrate, but also to accurately direct their oxidative force., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Opioids, antipsychotics and hypnotics are recommended for comfort care in dying. We studied their prescription during the last 3 days in residents deceased in the long-term care facility (LTCF)., Methods: In a retrospective, cross-sectional survey in Belgium, England, Finland, Italy, the Netherlands and Poland, LTCFs, selected by proportional stratified random sampling, reported all deaths over the previous 3 months. The nurse most involved in the residents' care reviewed the chart for opioid, antipsychotic and hypnotic prescription, cause of death and comorbidities. Multivariable logistic regression was performed to adjust for resident characteristics., Results: Response rate was 81.6%. We included 1079 deceased residents in 322 LCTFs. Opioid prescription ranged from 18.5% (95% CI: 13.0-25.8) of residents in Poland to 77.9% (95% CI: 69.5-84.5) in the Netherlands, antipsychotic prescription from 4.8% (95% CI: 2.4-9.1) in Finland to 22.4% (95% CI: 14.7-32.4) in Italy, hypnotic prescription from 7.8% (95% CI: 4.6-12.8) in Finland to 47.9% (95% CI: 38.5-57.3) in the Netherlands. Differences in opioid, antipsychotic and hypnotic prescription between countries remained significant (P < 0.001) when controlling for age, gender, length of stay, cognitive status, cause of death in multilevel, multivariable analyses. Dying from cancer showed higher odds for receiving opioids (OR 3.51; P < 0.001) and hypnotics (OR 2.10; P = 0.010)., Conclusions: Opioid, antipsychotic and hypnotic prescription in the dying phase differed significantly between six European countries. Further research should determine the appropriateness of their prescription and refine guidelines especially for LTCF residents dying of non-cancer diseases.

Background: Several studies have highlighted the need for improvement in palliative care delivered to older people long-term care facilities. However, the available evidence on how to improve palliative care in these settings is weak, especially in Europe. We describe the protocol of the PACE trial aimed to 1) evaluate the effectiveness and cost-effectiveness of the 'PACE Steps to Success' palliative care intervention for older people in long-term care facilities, and 2) assess the implementation process and identify facilitators and barriers for implementation in different countries., Methods: We will conduct a multi-facility cluster randomised controlled trial in Belgium, Finland, Italy, the Netherlands, Poland, Switzerland and England. In total, 72 facilities will be randomized to receive the 'Pace Steps to Success intervention' or to 'care as usual'. Primary outcome at resident level: quality of dying (CAD-EOLD); and at staff level: staff knowledge of palliative care (Palliative Care Survey)., Secondary Outcomes: resident's quality of end-of-life care, staff self-efficacy, self-perceived educational needs, and opinions on palliative care. Economic outcomes: direct costs and quality-adjusted life years (QALYs). Measurements are performed at baseline and after the intervention. For the resident-level outcomes, facilities report all deaths of residents in and outside the facilities over a previous four-month period and structured questionnaires are sent to (1) the administrator, (2) staff member most involved in care (3) treating general practitioner, and (4) a relative. For the staff-level outcomes, all staff who are working in the facilities are asked to complete a structured questionnaire. A process evaluation will run alongside the effectiveness evaluation in the intervention group using the RE-AIM framework., Discussion: The lack of high quality trials in palliative care has been recognized throughout the field of palliative care research. This cross-national cluster RCT designed to evaluate the impact of the palliative care intervention for long-term care facilities 'PACE Steps to Success' in seven countries, will provide important evidence concerning the effectiveness as well as the preconditions for optimal implementation of palliative care in nursing homes, and this within different health care systems., Trial Registration: The study is registered at www.isrctn.com - ISRCTN14741671 (FP7-HEALTH-2013-INNOVATION-1 603111) Registration date: July 30, 2015.

Lytic polysaccharide monooxygenases (LPMOs) have changed our understanding of lignocellulosic degradation dramatically over the last years. These metalloproteins catalyze oxidative cleavage of recalcitrant polysaccharides and can act on the C1 and/or C4 position of glycosidic bonds. Structural data have led to several hypotheses, but we are still a long way from reaching complete understanding of the factors that determine their divergent regioselectivity. Site-directed mutagenesis enables the investigation of structure-function relationship in enzymes and will be of major importance in unraveling this intriguing matter. In this context, it is crucial to have an enzyme assay or screening approach with a direct correlation with the desired functionality. LPMOs render this search extra challenging due to their insoluble substrates, complex pattern of reaction products and lack of synthetic standards of most oxidized products. Here, we describe a regioselectivity indicator diagram based on the time-course of only 2 HPAEC-PAD signals. The diagram was successfully used to confirm the hypothesis that aromatic surface residues influence the C1/C4 oxidation ratio in Hypocrea jecorina LPMO9A. Consequently, the diagram should become a valuable tool in the search towards better understanding and engineering of regioselectivity in LPMOs.

Lytic polysaccharide monooxygenases (LPMOs) are crucial components of cellulase mixtures but their stability has not yet been studied in detail, let alone been engineered for industrial applications. In this work, we have evaluated the importance of disulfide bridges for the thermodynamic stability of Streptomyces coelicolor LPMO10C. Interestingly, this enzyme was found to retain 34% of its activity after 2-h incubation at 80°C while its apparent melting temperature (Tm) is only 51°C. When its three disulfide bridges were broken, however, irreversible unfolding occurred and no residual activity could be detected after a similar heat treatment. Based on these findings, additional disulfide bridges were introduced, as predicted by computational tools (MOdelling of DIsulfide bridges in Proteins (MODiP) and Disulfide by Design (DbD)) and using the most flexible positions in the structure as target sites. Four out of 16 variants displayed an improvement in Tm, ranging from 2 to 9°C. Combining the positive mutations yielded additional improvements (up to 19°C) but aberrant unfolding patterns became apparent in some cases, resulting in a diminished capacity for heat resistance. Nonetheless, the best variant, a combination of A143C-P183C and S73C-A115C, displayed a 12°C increase in Tm and was able to retain and was able to retain no less than 60% of its activity after heat treatment., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The auxiliary activity family 9 (AA9, formerly GH61) harbors a recently discovered group of oxidative enzymes that boost cellulose degradation. Indeed, these lytic polysaccharide monooxygenases (LPMOs) are able to disrupt the crystalline structure of cellulose, thereby facilitating the work of hydrolytic enzymes involved in biomass degradation. Since these enzymes require an N-terminal histidine residue for activity, their recombinant production as secreted protein is not straightforward. We here report the expression optimization of Trichoderma reesei Cel61A (TrCel61A) in the host Pichia pastoris. The use of the native TrCel61A secretion signal instead of the alpha-mating factor from Saccharomyces cerevisiae was found to be crucial, not only to obtain high protein yields (>400 mg/L during fermentation) but also to enable the correct processing of the N-terminus. Furthermore, the LPMO activity of the enzyme is demonstrated here for the first time, based on its degradation profile of a cellulosic substrate.

First trimester screening for Down syndrome is yet to become the first intention strategy in France. This screening program at 11-14 weeks of gestation using maternal age, fetal nuchal translucency, maternal serum free beta-human chorionic gonadotropin and pregnancy-associated plasma protein-A has already been validated for a long time by many international studies. It seems to improve detection rate and decrease false positive rates. We report here five years prospective experience., (Copyright 2010 Elsevier Masson SAS. All rights reserved.)

We report and analyze four new cases of spontaneous twin anemia-polycythemia sequence (TAPS) and discuss antenatal management by fetoscopic laser coagulation for this uncommon form of chronic intertwin transfusion. The clinical course and placental characteristics of four pairs of monochorionic-diamniotic (MCDA) twins with spontaneous TAPS, of which one was treated with fetoscopic laser surgery, are described. For the three cases that did not undergo intrauterine intervention, serial Doppler measurement revealed a gradual increase in the middle cerebral artery peak systolic velocity (MCA-PSV) in the donor and a concomitant decrease in the recipient. These twins were born at between 32 and 34 weeks' gestation by Cesarean section. At birth, the donor twins were severely anemic and the recipient twins were polycythemic. Placental injection studies revealed a few small arteriovenous (AV) and venoarterial anastomoses. In the fourth case, because of the high suspicion of TAPS on MCA-PSV data at 24 weeks' gestation, fetoscopic laser coagulation of three small AV anastomoses was successfully performed. No hematological abnormalities were detected at birth. TAPS is a newly described form of chronic twin-to-twin transfusion associated with chronic anemia in the donor and polycythemia in the recipient, without twin oligo-polyhydramnios sequence (TOPS). The monitoring of MCDA pregnancies should include measurement of MCA-PSV in both fetuses even in the absence of intertwin discordance in amniotic fluid volume. Early detection of TAPS could indicate fetoscopic laser coagulation of AV anastomoses., (Copyright 2009 ISUOG. Published by John Wiley & Sons, Ltd.)

Objective: Many studies have shown that an increased nuchal translucency (NT) may be a good marker of fetal heart malformation, but the extent to which NT is suitable for identifying the population at risk remains unclear. We aimed to determine the value of NT measurement and of the presence of cystic hygroma colli in the screening of euploid fetuses for congenital heart defects (CHD)., Methods: We carried out a retrospective analysis of 12 910 euploid pregnancies examined between January 1995 and August 2007 at our institution. The screening performance of NT measurements in identifying fetuses with CHD was assessed, with comparison between the use of cut-offs defined as absolute values, multiples of the median (MoM) and percentiles. The presence of cystic hygroma colli was also assessed in the prediction of CHD., Results: The incidence of major CHD was 3.4 per thousand (44/12 910). The sensitivity of NT measurement in screening for major CHD was 54.5% if the threshold was set at the 95(th) percentile, 45.4% if it was set at 3 mm, 27.3% for 3.5 mm, 50.0% for 1.5 MoM and 45.5% for 1.75 MoM. The false-positive rates for these thresholds were 8.4, 6.6, 1.7, 8.9 and 6.3%, respectively. The incidence of major CHD was 1.2% (10/813) in cases of thick NT (> 95(th) centile) and 4.3% (13/304) in cases of hygroma colli., Conclusions: NT measurement during the first trimester is potentially useful for screening for fetal major CHD. Screening performance is consistent whether NT values are expressed as MoMs, percentiles or absolute values. The incidence of major CHD seems to be higher in cases of cystic hygroma colli., ((c) 2010 ISUOG. Published by John Wiley & Sons, Ltd.)

Objective: Fetal biometric data are a major part of prenatal ultrasound screening in the general population. The aim of this study was to analyze the effect of choice of reference curve on the quality of screening for growth abnormalities, using a statistical tool based on Z-scores., Methods: The biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC) and femur length (FL) were measured in 9699 ultrasound scans during the second trimester (20-24 weeks of gestation) and 8100 scans during the third trimester (30-34 weeks of gestation). These biometric data were all transformed retrospectively into Z-scores, calculated using five reference curves: those published by Snijders and Nicolaides (1994), Chitty et al. (1994), Kurmanavicius et al. (1999) and Salomon et al. (2006), and curves used at our ultrasound unit generated from a sample of the local population. The Z-score distribution was compared with the expected normal distribution by calculation of the mean and SD, and using the Kolmogorov-Smirnov test. The sensitivity and specificity of each reference curve were calculated to determine the capacity of these curves to identify fetuses with measurements < 5(th) percentile or > 95(th) percentile for each parameter., Results: Most of the distribution curves determined from the Z-scores of the measurements taken differed significantly from a non-skewed standard normal curve (mean of 0 and SD of 1). In our population, the Chitty reference curves gave the best results for identifying fetuses with abnormal (< 5(th) percentile or > 95(th) percentile) BPD (sensitivity, 100%; specificity, 97.24%), HC (sensitivity, 96.07%; specificity, 98.89%) and FL (sensitivity, 96.46%; specificity, 98.80%). The best reference for AC was the Salomon curve (sensitivity, 72.25%; specificity, 99.64%)., Conclusions: Checking for good concordance between the study population and chosen reference data is a key initial step in quality control. Z-scores are a simple tool for evaluating the performance of each reference curve for a given population in order to optimize the sensitivity and specificity of screening for fetal growth abnormalities.

Objective: To determine whether, in a selected high-risk population, Doppler velocimetry of the ductus venosus can improve the predictive capacity of increased nuchal translucency in the detection of major congenital heart defects in chromosomally normal fetuses at 11-14 weeks of gestation., Methods: Ductus venosus Doppler ultrasound blood velocity waveforms were obtained prospectively at 11-14 weeks of gestation in 1040 consecutive singleton pregnancies. Waveforms were classified either as normal in the presence of a positive A-wave, or as abnormal if the A-wave was absent or negative. All cases were screened for chromosomal defects by a combination of maternal age and fetal nuchal translucency thickness. In 484 cases karyotyping was performed. Those fetuses found to be chromosomally normal by prenatal cytogenetic analysis, and which had abnormally increased nuchal translucency and/or abnormal ductus venosus Doppler velocimetry, underwent fetal echocardiography at 14-16 weeks of gestation. Ultrasound examination was repeated at 22-24 weeks of gestation in all women. The sensitivity, specificity and positive and negative predictive values for the detection of major cardiac defects of increased nuchal translucency thickness alone, ductus venosus Doppler alone and increased nuchal translucency thickness in association with abnormal ductus venosus Doppler were determined., Results: In 29 of 998 fetuses presumed to be chromosomally normal, reversed or absent flow during atrial contraction was associated with increased (> 95(th) centile for crown-rump length) nuchal translucency. Major cardiac defects were observed in 9 of these 29 fetuses. No other major cardiac abnormalities were found in chromosomally normal fetuses in spite of the presence of either increased nuchal translucency alone or abnormal ductus venosus velocimetry. A total of 25 cardiac malformations were observed in the population. Fifteen were associated with aneuploidy and 10 fetuses had a normal karyotype. Nine of the 10 had major cardiac anomalies and one had a ventricular septal defect. The nine cases with normal karyotype and major cardiac anomalies had both increased nuchal translucency and abnormal ductus venosus flow velocity waveforms., Conclusion: In chromosomally normal fetuses with increased nuchal translucency, assessment of ductus venosus blood flow velocimetry could improve the predictive capacity for an underlying major cardiac defect., (Copyright 2003 ISUOG. Published by John Wiley & Sons, Ltd.)