Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time

Fluorescence lifetime imaging (FLIM) combined with optical projection tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3-D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis. Although apoptosis is a naturally occurring process during development, it can also be triggered in a variety of ways, including through gamma irradiation. FLIM OPT is shown here to enable apoptosis to be monitored over time, in live zebrafish larvae via changes in Caspase 3 activation following gamma irradiation at 24 hours post fertilisation. Significant apoptosis was observed at 3.5 hours post irradiation, predominantly in the head region

Hearing Characteristics of Stroke Patients: Prevalence and Characteristics of Hearing Impairment and Auditory Processing Disorders in Stroke Patients

BACKGROUND: Stroke survivors may suffer from a range of hearing impairments that may restrict their participation in postacute rehabilitation programs. Hearing impairment may have a significant impact on listening, linguistic skills, and overall communication of the affected stroke patient. However, no studies sought to systematically characterize auditory function of stroke patients in detail, to establish the different types of hearing impairments in this cohort of patients. Such information would be clinically useful in understanding and addressing the hearing needs of stroke survivors. PURPOSE: The present study aimed to characterize and classify the hearing impairments, using a detailed audiological assessment test battery, in order to determine the level of clinical need and inform appropriate rehabilitation for this patient population. RESEARCH DESIGN: A case‐control study. STUDY SAMPLE: Forty-two recruited stroke patients who were discharged from a stroke unit and 40 control participants matched for age. DATA COLLECTION AND ANALYSIS: All participants underwent pure-tone audiometry and immittance measurements including acoustic reflex threshold, transient-evoked otoacoustic emissions, auditory-evoked brainstem response, and a central auditory processing assessment battery, performed in a single session. Hearing impairments were classified as peripheral hearing loss (cochlear and neural type), central auditory processing disorder (CAPD), and as a combination of CAPD and peripheral hearing loss. RESULTS: Overall mean hearing thresholds were not significantly different between the control and stroke groups. The most common type of hearing impairment in stroke patients was the combination type, “peripheral and CAPD,” in the 61- to 80-yr-old subgroup (in 55%), and auditory processing deficits in 18- to 60-yr-olds (in 40%), which were both significantly higher than in controls. CONCLUSIONS: This is the first study to examine hearing function in detail in stroke patients. Given the importance of hearing for the efficiency of communication, it is essential to identify hearing impairments and differentiate peripheral and central deficits to define an appropriate intervention plan

Galaxy mass profiles from strong lensing II: The elliptical power-law model

We present a systematic analysis of the constraints σγ on the mass profile slope γ obtainable when fitting a singular power-law ellipsoid model to a typical strong lensing observation of an extended source. These results extend our previous analysis of circular systems, Paper I. We draw our results from 676 mock observations covering a range of image configurations, each created with a fixed signal to noise ratio S = 100 in the images. We analyse the results using a combination of theory and a simplified model which identifies the contribution to the constraints of the individual fluxes and positions in each of the lensed images. The main results are: 1. For any lens ellipticity, the constraints σγ for two image systems are well described by the results of Paper I, transformed to elliptical coordinates; 2. We derive an analytical expression for σγ for systems with the source aligned with the axis of the lens; 3. For both two-image systems and aligned systems, σγ is limited by the flux uncertainties; 4. The constraints for off-axis four-image systems are a factor of two to eight better, depending on source size, than for two-image systems, and improve with increasing lens ellipticity. We show that the constraints on γ in these systems derive from the complementary positional information of the images alone, without using flux. The complementarity improves as the offset of the source from the axis increases, such that the best constraints σγ < 0.01, for S = 100, occur when the source approaches the caustic

Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100's to 1000's of FOV enables unbiased quantitative analysis with high statistical power. Plotting average donor lifetime vs. acceptor/donor intensity ratio clearly identifies protein interactions and fitting to double exponential donor decay models provides estimates of interacting population fractions that, with calibrated donor and acceptor fluorescence intensities, can yield dissociation constants. We demonstrate the application to identify binding partners of MST1 kinase and estimate interaction strength among the members of the RASSF protein family, which have important roles in apoptosis via the Hippo signalling pathway. KD values broadly agree with published biochemical measurements

An automated multiwell plate reading film microscope for live cell autofluorescence lifetime assays

Fluorescence lifetime imaging (FLIM) is increasingly used to read out cellular autofluorescence originating from the coenzyme NADH in the context of investigating cell metabolic state. We present here an automated multiwell plate reading FLIM microscope optimized for UV illumination with the goal of extending high content fluorescence lifetime assays to readouts of metabolism. We demonstrate its application to automated cellular autofluorescence lifetime imaging and discuss the key practical issues associated with its implementation. In particular, we illustrate its capability to read out the NADH-lifetime response of cells to metabolic modulators, thereby illustrating the potential of the instrument for cytotoxicity studies, assays for drug discovery and stratified medicine

A multi-layer extension of the stochastic heat equation

Motivated by recent developments on solvable directed polymer models, we define a 'multi-layer' extension of the stochastic heat equation involving non-intersecting Brownian motions.Comment: v4: substantially extended and revised versio

Об одном из возможных путей создания свободновихревых насосов типа "TURO" малой быстроходности

BACKGROUND: Interventions promoting physical activity by General Practitioners (GPs) lack a strong evidence base. Recruiting participants to trials in primary care is challenging. We investigated the feasibility of (i) delivering three interventions to promote physical activity in inactive participants and (ii) different methods of participant recruitment and randomised allocation. METHODS: We recruited general practices from Devon, Bristol and Coventry. We used a 2-by-2 factorial design for participant recruitment and randomisation. Recruitment strategies were either opportunistic (approaching patients attending their GP surgery) or systematic (selecting patients from practice lists and approaching them by letter). Randomisation strategies were either individual or by practice cluster. Feasibility outcomes included time taken to recruit the target number of participants within each practice. Participants were randomly allocated to one of three interventions: (i) written advice (control); (ii) brief GP advice (written advice plus GP advice on physical activity), and (iii) brief GP advice plus a pedometer to self-monitor physical activity during the trial. Participants allocated to written advice or brief advice each received a sealed pedometer to record their physical activity, and were instructed not to unseal the pedometer before the scheduled day of data collection. Participant level outcomes were reported descriptively and included the mean number of pedometer steps over a 7-day period, and European Quality of Life (EuroQoL)-5 dimensions (EQ-5D) scores, recorded at 12 weeks' follow-up. RESULTS: We recruited 24 practices (12 using each recruitment method; 18 randomising by cluster, 6 randomising by individual participant), encompassing 131 participants. Opportunistic recruitment was associated with less time to target recruitment compared with systematic (mean difference (days) -54.9, 95% confidence interval (CI) -103.6; -6.2) but with greater loss to follow up (28.8% versus. 6.9%; mean difference 21.9% (95% CI 9.6%; 34.1%)). There were differences in the socio-demographic characteristics of participants according to recruitment method. There was no clear pattern of change in participant level outcomes from baseline to 12 weeks across the three arms. CONCLUSIONS: Delivering and trialling GP-led interventions to promote physical activity is feasible, but trial design influences time to participant recruitment, participant withdrawal, and possibly, the socio-demographic characteristics of participants. TRIAL REGISTRATION NUMBER: ISRCTN73725618

Evaluation of different recruitment and randomisation methods in a trial of general practitioner-led interventions to increase physical activity: a randomised controlled feasibility study with factorial design

Comparative StudyJournal ArticleMulticenter StudyRandomized Controlled TrialThis is a freely-available open access publication. Please cite the published version which is available via the DOI link in this record.There is another ORE record for this publication: http://hdl.handle.net/10871/39760BACKGROUND: Interventions promoting physical activity by General Practitioners (GPs) lack a strong evidence base. Recruiting participants to trials in primary care is challenging. We investigated the feasibility of (i) delivering three interventions to promote physical activity in inactive participants and (ii) different methods of participant recruitment and randomised allocation. METHODS: We recruited general practices from Devon, Bristol and Coventry. We used a 2-by-2 factorial design for participant recruitment and randomisation. Recruitment strategies were either opportunistic (approaching patients attending their GP surgery) or systematic (selecting patients from practice lists and approaching them by letter). Randomisation strategies were either individual or by practice cluster. Feasibility outcomes included time taken to recruit the target number of participants within each practice. Participants were randomly allocated to one of three interventions: (i) written advice (control); (ii) brief GP advice (written advice plus GP advice on physical activity), and (iii) brief GP advice plus a pedometer to self-monitor physical activity during the trial. Participants allocated to written advice or brief advice each received a sealed pedometer to record their physical activity, and were instructed not to unseal the pedometer before the scheduled day of data collection. Participant level outcomes were reported descriptively and included the mean number of pedometer steps over a 7-day period, and European Quality of Life (EuroQoL)-5 dimensions (EQ-5D) scores, recorded at 12 weeks' follow-up. RESULTS: We recruited 24 practices (12 using each recruitment method; 18 randomising by cluster, 6 randomising by individual participant), encompassing 131 participants. Opportunistic recruitment was associated with less time to target recruitment compared with systematic (mean difference (days) -54.9, 95% confidence interval (CI) -103.6; -6.2) but with greater loss to follow up (28.8% versus. 6.9%; mean difference 21.9% (95% CI 9.6%; 34.1%)). There were differences in the socio-demographic characteristics of participants according to recruitment method. There was no clear pattern of change in participant level outcomes from baseline to 12 weeks across the three arms. CONCLUSIONS: Delivering and trialling GP-led interventions to promote physical activity is feasible, but trial design influences time to participant recruitment, participant withdrawal, and possibly, the socio-demographic characteristics of participants. TRIAL REGISTRATION NUMBER: ISRCTN73725618.Medical Research Counci

Successful ACL reconstruction with a variant of the pes anserinus

An anatomical variant of the pes anserinus encountered during anterior cruciate ligament reconstructive surgery which has not been previously described is discussed. During routine harvesting, the sartorius fascia was incised and the semitendinosus and gracilis tendons were identified. At the distal portion, it was noted that each tendon gave off an additional tendinous slip. The slip from the semitendinosus tendon had attached to the gracilis tendon and vice versa, thereby creating a double pes anserinus. This variant was used to construct the graft, and at 1 year review the patient had returned to full sporting activities with no complications encountered