FRET characterisation for cross-bridge dynamics in single-skinned rigor muscle fibres

In this work we demonstrate for the first time the use of Förster resonance energy transfer (FRET) as an assay to monitor the dynamics of cross-bridge conformational changes directly in single muscle fibres. The advantage of FRET imaging is its ability to measure distances in the nanometre range, relevant for structural changes in actomyosin cross-bridges. To reach this goal we have used several FRET couples to investigate different locations in the actomyosin complex. We exchanged the native essential light chain of myosin with a recombinant essential light chain labelled with various thiol-reactive chromophores. The second fluorophore of the FRET couple was introduced by three approaches: labelling actin, labelling SH1 cysteine and binding an adenosine triphosphate (ATP) analogue. We characterise FRET in rigor cross-bridges: in this condition muscle fibres are well described by a single FRET population model which allows us to evaluate the true FRET efficiency for a single couple and the consequent donor–acceptor distance. The results obtained are in good agreement with the distances expected from crystallographic data. The FRET characterisation presented herein is essential before moving onto dynamic measurements, as the FRET efficiency differences to be detected in an active muscle fibre are on the order of 10–15% of the FRET efficiencies evaluated here. This means that, to obtain reliable results to monitor the dynamics of cross-bridge conformational changes, we had to fully characterise the system in a steady-state condition, demonstrating firstly the possibility to detect FRET and secondly the viability of the present approach to distinguish small FRET variations

Lamina propria macrophage phenotypes in relation to Escherichia coli in Crohn's disease

Background: Abnormal handling of E. coli by lamina propria (LP) macrophages may contribute to Crohn’s disease (CD) pathogenesis. We aimed to determine LP macrophage phenotypes in CD, ulcerative colitis (UC) and healthy controls (HC), and in CD, to compare macrophage phenotypes according to E. coli carriage. Methods: Mucosal biopsies were taken from 35 patients with CD, 9 with UC and 18 HCs. Laser capture microdissection was used to isolate E. coli-laden and unladen LP macrophages from ileal or colonic biopsies. From these macrophages, mRNA was extracted and cytokine and activation marker expression measured using RT-qPCR. Results: E. coli-laden LP macrophages were identified commonly in mucosal biopsies from CD patients (25/35, 71 %), rarely in UC (1/9, 11 %) and not at all in healthy controls (0/18). LP macrophage cytokine mRNA expression was greater in CD and UC than healthy controls. In CD, E. coli-laden macrophages expressed high IL-10 & CD163 and lower TNFα, IL-23 & iNOS irrespective of macroscopic inflammation. In inflamed tissue, E. coli-unladen macrophages expressed high TNFα, IL-23 & iNOS and lower IL-10 & CD163. In uninflamed tissue, unladen macrophages had low cytokine mRNA expression, closer to that of healthy controls. Conclusion: In CD, intra-macrophage E. coli are commonly found and LP macrophages express characteristic cytokine mRNA profiles according to E. coli carriage. Persistence of E. coli within LP macrophages may provide a stimulus for chronic inflammation

A comparison of methods for temporal analysis of aoristic crime

Objectives: To test the accuracy of various methods previously proposed (and one new method) to estimate offence times where the actual time of the event is not known. Methods: For 303 thefts of pedal cycles from railway stations, the actual offence time was determined from closed-circuit television and the resulting temporal distribution compared against commonly-used estimated distributions using circular statistics and analysis of residuals. Results: Aoristic analysis and allocation of a random time to each offence allow accurate estimation of peak offence times. Commonly-used deterministic methods were found to be inaccurate and to produce misleading results. Conclusions: It is important that analysts use the most accurate methods for temporal distribution approximation to ensure any resource decisions made on the basis of peak times are reliable

A core outcome set for pre‐eclampsia research: an international consensus development study

Objective To develop a core outcome set for pre‐eclampsia. Design Consensus development study. Setting International. Population Two hundred and eight‐one healthcare professionals, 41 researchers and 110 patients, representing 56 countries, participated. Methods Modified Delphi method and Modified Nominal Group Technique. Results A long‐list of 116 potential core outcomes was developed by combining the outcomes reported in 79 pre‐eclampsia trials with those derived from thematic analysis of 30 in‐depth interviews of women with lived experience of pre‐eclampsia. Forty‐seven consensus outcomes were identified from the Delphi process following which 14 maternal and eight offspring core outcomes were agreed at the consensus development meeting. Maternal core outcomes: death, eclampsia, stroke, cortical blindness, retinal detachment, pulmonary oedema, acute kidney injury, liver haematoma or rupture, abruption, postpartum haemorrhage, raised liver enzymes, low platelets, admission to intensive care required, and intubation and ventilation. Offspring core outcomes: stillbirth, gestational age at delivery, birthweight, small‐for‐gestational‐age, neonatal mortality, seizures, admission to neonatal unit required and respiratory support. Conclusions The core outcome set for pre‐eclampsia should underpin future randomised trials and systematic reviews. Such implementation should ensure that future research holds the necessary reach and relevance to inform clinical practice, enhance women's care and improve the outcomes of pregnant women and their babies

Long-term safety and efficacy of elexacaftor/tezacaftor/ivacaftor in children aged ⩾6 years with cystic fibrosis and at least one F508del allele: a phase 3, open-label clinical trial.

Rationale: A 24-week, phase 3, open-label study showed elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) was safe and efficacious in children aged 6-11 years with cystic fibrosis (CF) and one or more F508del-CFTR alleles. Objectives: To assess long-term safety and efficacy of ELX/TEZ/IVA in children who completed the pivotal 24-week phase 3 trial. Methods: In this phase 3, two-part (part A and part B), open-label extension study, children aged ⩾6 years with CF heterozygous for F508del and a minimal function CFTR mutation (F/MF genotypes) or homozygous for F508del (F/F genotype) who completed the 24-week parent study received ELX/TEZ/IVA based on weight. Children weighing <30 kg received ELX 100 mg once daily/TEZ 50 mg once daily/IVA 75 mg every 12 hours, whereas children weighing ⩾30 kg received ELX 200 mg once daily/TEZ 100 mg once daily/IVA 150 mg every 12 hours (adult dose). The 96-week analysis of part A of this extension study is reported here. Measurements and Main Results: Sixty-four children (F/MF genotypes, n = 36; F/F genotype, n = 28) were enrolled and received one or more doses of ELX/TEZ/IVA. Mean (SD) period of exposure to ELX/TEZ/IVA was 93.9 (11.1) weeks. The primary endpoint was safety and tolerability. Adverse events and serious adverse events were consistent with common manifestations of CF disease. Overall, exposure-adjusted rates of adverse events and serious adverse events (407.74 and 4.72 events per 100 patient-years) were lower than in the parent study (987.04 and 8.68 events per 100 patient-years). One child (1.6%) had an adverse event of aggression that was moderate in severity and resolved after study drug discontinuation. From parent study baseline at Week 96 of this extension study, the mean percent predicted FEV1 increased (11.2 [95% confidence interval (CI), 8.3 to 14.2] percentage points), sweat chloride concentration decreased (-62.3 [95% CI, -65.9 to -58.8] mmol/L), Cystic Fibrosis Questionnaire-Revised respiratory domain score increased (13.3 [95% CI, 11.4 to 15.1] points), and lung clearance index 2.5 decreased (-2.00 [95% CI, -2.45 to -1.55] units). Increases in growth parameters were also observed. The estimated pulmonary exacerbation rate per 48 weeks was 0.04. The annualized rate of change in percent predicted FEV1 was 0.51 (95% CI, -0.73 to 1.75) percentage points per year. Conclusions: ELX/TEZ/IVA continued to be generally safe and well tolerated in children aged ⩾6 years through an additional 96 weeks of treatment. Improvements in lung function, respiratory symptoms, and CFTR function observed in the parent study were maintained. These results demonstrate the favorable long-term safety profile and durable clinical benefits of ELX/TEZ/IVA in this pediatric population. Clinical trial registered with www.clinicaltrials.gov (NCT04183790)