Use of horseradish peroxidase for gene-directed enzyme prodrug therapy with paracetamol

Gene therapy is a potential method of treating cancer with a greater degree of targeting than conventional therapies. In addition, therapy can be directed towards cells within the tumour population that are traditionally resistant to current treatment schedules. Horseradish peroxidase (HRP) can oxidise paracetamol to N-acetyl-p-benzoquinoneimine via a one-electron pathway. Incubation of human cells expressing HRP with 0.5–10 mm paracetamol reduced clonogenic survival, but had little effect on control cells. A small increase in apoptosis was seen and a decrease in the number of cells undergoing mitosis, consistent with reports in hepatocytes using higher paracetamol concentrations. The cytotoxicity was also seen under conditions of severe hypoxia (catalyst induced anoxia), indicating that the HRP/paracetamol combination may be suitable for hypoxia-targeted gene therapy

Comparison of the benefits of cochlear implantation versus contra-lateral routing of signal hearing aids in adult patients with single-sided deafness: study protocol for a prospective within-subject longitudinal trial

Background Individuals with a unilateral severe-to-profound hearing loss, or single-sided deafness, report difficulty with listening in many everyday situations despite having access to well-preserved acoustic hearing in one ear. The standard of care for single-sided deafness available on the UK National Health Service is a contra-lateral routing of signals hearing aid which transfers sounds from the impaired ear to the non-impaired ear. This hearing aid has been found to improve speech understanding in noise when the signal-to-noise ratio is more favourable at the impaired ear than the non-impaired ear. However, the indiscriminate routing of signals to a single ear can have detrimental effects when interfering sounds are located on the side of the impaired ear. Recent published evidence has suggested that cochlear implantation in individuals with a single-sided deafness can restore access to the binaural cues which underpin the ability to localise sounds and segregate speech from other interfering sounds. Methods/Design The current trial was designed to assess the efficacy of cochlear implantation compared to a contra-lateral routing of signals hearing aid in restoring binaural hearing in adults with acquired single-sided deafness. Patients are assessed at baseline and after receiving a contra-lateral routing of signals hearing aid. A cochlear implant is then provided to those patients who do not receive sufficient benefit from the hearing aid. This within-subject longitudinal design reflects the expected care pathway should cochlear implantation be provided for single-sided deafness on the UK National Health Service. The primary endpoints are measures of binaural hearing at baseline, after provision of a contra-lateral routing of signals hearing aid, and after cochlear implantation. Binaural hearing is assessed in terms of the accuracy with which sounds are localised and speech is perceived in background noise. The trial is also designed to measure the impact of the interventions on hearing- and health-related quality of life. Discussion This multi-centre trial was designed to provide evidence for the efficacy of cochlear implantation compared to the contra-lateral routing of signals. A purpose-built sound presentation system and established measurement techniques will provide reliable and precise measures of binaural hearing. Trial registration Current Controlled Trials http://www.controlled-trials.com/ISRCTN33301739 (05/JUL/2013

Gene to Diagnostic: Self immobilizing protein for silica microparticle biosensor, modelled with sarcosine oxidase.

A rational design approach is proposed for a multifunctional enzyme reagent for point-of-care diagnostics. The biomaterial reduces downstream isolation steps and eliminates immobilization coupling chemicals for integration in a diagnostic platform. Fusion con-structs combined the central functional assay protein (e.g. monomeric sarcosine oxidase, mSOx, horseradish peroxidase, HRP), a visualizing protein (e.g. mCherry) and an in-built immobilization peptide (e.g. R5). Monitoring protein expression in E.coli was facilitated by following the increase in mCherry fluorescence, which could be matched to a color card, indicating when good protein expression has occurred. The R5 peptide (SSKKSGSYSGSKGSKRRIL) provided inbuilt affinity for silica and an immobilization capability for a silica based diagnostic, without requiring additional chemical coupling reagents. Silica particles extracted from beach sand were used to collect protein from crude protein extract with 85-95% selective uptake. The silica immobilized R5 pro-teins were stable for more than 2 months at room temperature. The Km for the silica-R52-mCh-mSOx-R5-6H was 16.5±0.9mM (com-pared with 16.5±0.4 mM, 16.3±0.3 mM, and 16.1±0.4 mM for R52-mCh-mSOx-R5-6H, mSOx-R5-6H and mSOx-6H respectively in solution). The use of the “silica-enzymes” in sarcosine and peroxide assays was shown, and a design using particle sedimentation through the sample was examined. Using shadowgraphy and particle image velocimetry the particle trajectory through the sample was mapped and an hourglass design with a narrow waist shown to give good control of particle position. The hourglass biosensor was demonstrated for sarcosine assay in the clinically useful range of 2.5 to 10 µM in both a dynamic and end point measurement regime

Research data supporting "Gene to Diagnostic: Self immobilizing protein for silica microparticle biosensor, modelled with sarcosine oxidase"

Analytical data supporting manuscript, including fluorescent data, laser scattering, protein sequence

Developments in integrating nucleic acid isothermal amplification and detection systems for point-of-care diagnostics.

Early disease detection through point-of-care (POC) testing is vital for quickly treating patients and preventing the spread of harmful pathogens. Disease diagnosis is generally accomplished using quantitative polymerase chain reaction (qPCR) to amplify nucleic acids in patient samples, permitting detection even at low target concentrations. However, qPCR requires expensive equipment, trained personnel, and significant time. These resources are not available in POC settings, driving researchers to instead utilize isothermal amplification, conducted at a single temperature, as an alternative. Common isothermal amplification methods include loop-mediated isothermal amplification, recombinase polymerase amplification, rolling circle amplification, nucleic acid sequence-based amplification, and helicase-dependent amplification. There has been a growing interest in combining such amplification methods with POC detection methods to enable the development of diagnostic tests that are well suited for resource-limited settings as well as developed countries performing mass screenings. Exciting developments have been made in the integration of these two research areas due to the significant impact that such approaches can have on healthcare. This review will primarily focus on advances made by North American research groups between 2015 and June 2020, and will emphasize integrated approaches that reduce user steps, reliance on expensive equipment, and the system's time-to-result