Measles virus minigenomes encoding two autofluorescent proteins reveal cell-to-cell variation in reporter expression dependent on viral sequences between the transcription units.

Transcription from morbillivirus genomes commences at a single promoter in the 3' non-coding terminus, with the six genes being transcribed sequentially. The 3' and 5' untranslated regions (UTRs) of the genes (mRNA sense), together with the intergenic trinucleotide spacer, comprise the non-coding sequences (NCS) of the virus and contain the conserved gene end and gene start signals, respectively. Bicistronic minigenomes containing transcription units (TUs) encoding autofluorescent reporter proteins separated by measles virus (MV) NCS were used to give a direct estimation of gene expression in single, living cells by assessing the relative amounts of each fluorescent protein in each cell. Initially, five minigenomes containing each of the MV NCS were generated. Assays were developed to determine the amount of each fluorescent protein in cells at both cell population and single-cell levels. This revealed significant variations in gene expression between cells expressing the same NCS-containing minigenome. The minigenome containing the M/F NCS produced significantly lower amounts of fluorescent protein from the second TU (TU2), compared with the other minigenomes. A minigenome with a truncated F 5' UTR had increased expression from TU2. This UTR is 524 nt longer than the other MV 5' UTRs. Insertions into the 5' UTR of the enhanced green fluorescent protein gene in the minigenome containing the N/P NCS showed that specific sequences, rather than just the additional length of F 5' UTR, govern this decreased expression from TU2.

Identifying strategies to maximise recruitment and retention of practices and patients in a multicentre randomised controlled trial of an intervention to optimise secondary prevention for coronary heart disease in primary care.

Background Recruitment and retention of patients and healthcare providers in randomised controlled trials (RCTs) is important in order to determine the effectiveness of interventions. However, failure to achieve recruitment targets is common and reasons why a particular recruitment strategy works for one study and not another remain unclear. We sought to describe a strategy used in a multicentre RCT in primary care, to report researchers’ and participants’ experiences of its implementation and to inform future strategies to maximise recruitment and retention. Methods In total 48 general practices and 903 patients were recruited from three different areas of Ireland to a RCT of an intervention designed to optimise secondary prevention of coronary heart disease. The recruitment process involved telephoning practices, posting information, visiting practices, identifying potential participants, posting invitations and obtaining consent. Retention involved patients attending reviews and responding to questionnaires and practices facilitating data collection. Results We achieved high retention rates for practices (100%) and for patients (85%) over an 18-month intervention period. Pilot work, knowledge of the setting, awareness of change in staff and organisation amongst participant sites, rapid responses to queries and acknowledgement of practitioners’ contributions were identified as being important. Minor variations in protocol and research support helped to meet varied, complex and changing individual needs of practitioners and patients and encouraged retention in the trial. A collaborative relationship between researcher and practice staff which required time to develop was perceived as vital for both recruitment and retention. Conclusions Recruiting and retaining the numbers of practices and patients estimated as required to provide findings with adequate power contributes to increased confidence in the validity and generalisability of RCT results. A continuous dynamic process of monitoring progress within trials and tailoring strategies to particular circumstances, whilst not compromising trial protocols, should allow maximal recruitment and retention.

The design and performance of a 2.5-GHz telecommand link for wireless biomedical monitoring

This paper details the implementation and operational performance of a minimum-power 2.45-GHz pulse receiver and a companion on-off keyed transmitter for use in a semi-active duplex RF biomedical transponder. A 50-Ohm microstrip stub-matched zero-bias diode detector forms the heart of a body-worn receiver that has a CMOS baseband amplifier consuming 20 microamps from +3 V and achieves a tangential sensitivity of -53 dBm. The base transmitter generates 0.5 W of peak RF output power into 50 Ohms. Both linear and right-hand circularly polarized Tx-Rx antenna sets were employed in system reliability trials carried out in a hospital Coronary Care Unit, For transmitting antenna heights between 0.3 and 2.2 m above floor level, transponder interrogations were 95% reliable within the 67-m-sq area of the ward, falling to an average of 46 % in the surrounding rooms and corridors. Overall, the circular antenna set gave the higher reliability and lower propagation power decay index.

A Ligand predication tool based on modeling and reasoning with imprecise probabilistic knowledge

Ligand prediction has been driven by a fundamental desire to understand more about how biomolecules recognize their ligands and by the commercial imperative to develop new drugs. Most of the current available software systems are very complex and time-consuming to use. Therefore, developing simple and efficient tools to perform initial screening of interesting compounds is an appealing idea. In this paper, we introduce our tool for very rapid screening for likely ligands (either substrates or inhibitors) based on reasoning with imprecise probabilistic knowledge elicited from past experiments. Probabilistic knowledge is input to the system via a user-friendly interface showing a base compound structure. A prediction of whether a particular compound is a substrate is queried against the acquired probabilistic knowledge base and a probability is returned as an indication of the prediction. This tool will be particularly useful in situations where a number of similar compounds have been screened experimentally, but information is not available for all possible members of that group of compounds. We use two case studies to demonstrate how to use the tool.