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Our understanding of Alzheimer’s disease (AD) pathogenesis has developed with several hypotheses over the last 40 years, including the Amyloid and Tau hypotheses, respectively. More recently, the p53 protein, well-known as ‘the guardian of the genome,’ has gained attention for its role in the early evolution of AD. This is due to p53’s central role in the control of oxidative stress and potential involvement in both Amyloid and Tau pathways. p53 is commonly regulated by post-translational modifications (PTMs), which affect its conformation, increasing its capacity to adopt multiple structural and functional states, including those that can influence several processes in AD. The following review will explore the impact of p53 post-translational modifications (PTMs) on its function and consequential involvement in AD pathogenesis.

Introduction This study examines the cost-effectiveness of PredictSURE in guiding the early use of biologic therapy in newly diagnosed CD patients, at high-risk of requiring early and frequent treatment escalations in the UK. PredictSURE IBD™ is a 17-gene, whole blood-based qPCR-based classifier that predicts long-term outcome in IBD, enabling early personalised treatment strategies through the early use of biologics in high-risk patients. Methods A decision tree leading into a Markov state-transition model was constructed in MS Excel to compare two treatment approaches: 1) standard of care therapy following established UK clinical guidelines, consisting of sequences of immunomodulator followed by biologic upon relapse (‘step-up’ treatment), 2) targeted therapy guided by PredictSURE, whereby patients identified as high-risk receive sequences of anti-TNF biologic treatment followed by other biologic classes upon relapse (‘top-down’ treatment), figure 1. Parameters were informed by patient data from PredictSURE clinical studies and the literature. Results Top-down treatment guided by PredictSURE resulted in an incremental cost-effectiveness ratio (ICER) of £7,179 per quality-adjusted life year (QALY), with £1,852 incremental costs and 0.258 incremental QALYs vs. standard of care generated over a 15-year time horizon. Additional costs relating to earlier biologic use were offset by reductions in the costs of flares, hospitalisations and surgery. Incremental QALYs were driven by increased time spent in remission and improved quality of life from reduced flares and surgery. The model was most sensitive to the time horizon, rates of mucosal healing on top-down vs. step-up therapy, the costs of hospitalisation and the costs and quality of life in the severe disease health state. Conclusion Modelling shows that upfront use of biologic guided by PredictSURE could substantially improve clinical outcomes for high-risk patients by increasing remission rates and reducing flares, surgery and treatment escalations. The ICER for PredictSURE was well below the £20-£30 k/QALY threshold used by the UK National Institute for Health and Care Excellence (NICE). Top-down treatment guided by PredictSURE would not only represent a treatment paradigm shift for CD patients but would also be a highly cost-effective use of resources in the UK National Health Service.

Background PredictSURE IBD™ is a CE-marked whole blood-based biomarker test that predicts long-term clinical outcomes in inflammatory bowel disease (Crohn’s disease, CD and ulcerative colitis, UC). PredictSURE IBD™ uses a 17-gene qPCR-based classifier to stratify patients into two prognostic subgroups, high and low risk. High-risk patients experience significantly more aggressive disease than low-risk patients, with the need for earlier and more frequent treatment escalation over time. Early stratification could enable personalised treatment strategies, such as ‘top-down’ use of biologics in high-risk patients. Our objective was to examine the cost-effectiveness of PredictSURE IBD™ in guiding the use of early biologic therapy in newly diagnosed CD patients in the UK. Methods A decision tree leading into a Markov state-transition model was constructed in MS Excel to compare two treatment approaches: (1) standard of care therapy following established UK clinical guidelines, consisting of sequences of immunomodulator followed by biologic upon relapse (‘step-up’ treatment), (2) targeted therapy guided by PredictSURE IBD™, whereby patients identified as high-risk receive sequences of anti-TNF biologic treatment followed by other biologic classes upon relapse (‘top-down’ treatment), Figure 1. Parameters were informed by patient data from PredictSURE IBD™ clinical studies and the literature. Results Top-down treatment guided by PredictSURE IBD™ resulted in an incremental cost-effectiveness ratio (ICER) of £7,179 per quality-adjusted life-year (QALY), with £1,852 incremental costs and 0.258 incremental QALYs vs. standard of care generated over a 15-year time horizon. Additional costs relating to earlier biologic use were offset by reductions in the costs of flares, hospitalisations and surgery. Incremental QALYs were driven by increased time spent in remission and improved quality of life from reduced flares and surgery. The model was most sensitive to the time horizon, rates of mucosal healing on top-down vs. step-up therapy, the costs of hospitalisation and the costs and quality of life in the severe disease health state. Conclusion Modelling shows that upfront use of biologic guided by PredictSURE IBD™ could substantially improve clinical outcomes for high-risk patients by increasing remission rates and reducing flares, surgery and treatment escalations. The ICER for PredictSURE IBD™ was well below the £20–£30k/QALY threshold used by the UK National Institute for Health and Care Excellence (NICE). Top-down treatment guided by PredictSURE IBD™ would not only represent a treatment paradigm shift for CD patients but would also be a highly cost-effective use of resources in the UK National Health Service.

The extraction and amplification of DNA from biological samples is laborious and time-consuming, requiring numerous instruments and sample handling steps. An integrated, single-use, poly(methyl methacrylate) (PMMA) microdevice for DNA extraction and amplification would benefit clinical and forensic communities, providing a completely closed system with rapid sample-in-PCR-product-out capability. Here, we show the design and simple flow control required for enzyme-based DNA preparation and PCR from buccal swabs or liquid whole blood samples with an ~5-fold reduction in time. A swab containing cells or DNA could be loaded into a novel receptacle together with the DNA liberation reagents, heated using an infrared heating system, mixed with PCR reagents for one of three different target sets under syringe-driven flow, and thermally-cycled in less than 45 min, an ~6-fold reduction in analysis time as compared to conventional methods. The 4 : 1 PCR reagents : DNA ratio required to provide the correct final concentration of all PCR components for effective amplification was verified using image analysis of colored dyes in the PCR chamber. Novel single-actuation, 'normally-open' adhesive valves were shown to effectively seal the PCR chamber during thermal cycling, preventing air bubble expansion. The effectiveness of the device was demonstrated using three target sets: the sex-typing gene Amelogenin, co-amplification of the β-globin and gelsolin genes, and the amplification of 15 short tandem repeat (STR) loci plus Amelogenin. The use of the integrated microdevice was expanded to the analysis of liquid blood samples which, when incubated with the DNA liberation reagents, form a brown precipitate that inhibits PCR. A simple centrifugation of the integrated microchips (on a custom centrifuge), mobilized the precipitate away from the microchannel entrance, improving amplification of the β-globin and gelsolin gene fragments by ~6-fold. This plastic integrated microdevice represents a microfluidic platform with potential for evolution into point-of-care prototypes for application to both clinical and forensic analyses, providing a 5-fold reduction from conventional analysis time.

Extraction of DNA from forensic samples typically uses either an organic extraction protocol or solid phase extraction (SPE) and these methods generally involve numerous sample transfer, wash and centrifugation steps. Although SPE has been successfully adapted to the microdevice, it can be problematic because of lengthy load times and uneven packing of the solid phase. A closed-tube enzyme-based DNA preparation method has recently been developed which uses a neutral proteinase to lyse cells and degrade proteins and nucleases [14]. Following a 20min incubation of the buccal or whole blood sample with this proteinase, DNA is polymerase chain reaction (PCR)-ready. This paper describes the optimization and quantitation of DNA yield using this method, and application to forensic biological samples, including UV- and heat-degraded whole blood samples on cotton or blue denim substrates. Results demonstrate that DNA yield can be increased from 1.42 (±0.21)ng/μL to 7.78 (±1.40)ng/μL by increasing the quantity of enzyme per reaction by 3-fold. Additionally, there is a linear relationship between the amount of starting cellular material added and the concentration of DNA in the solution, thereby allowing DNA yield estimations to be made. In addition, short tandem repeat (STR) profile results obtained using DNA prepared with the enzyme method were comparable to those obtained with a conventional SPE method, resulting in full STR profiles (16 of 16 loci) from liquid samples (buccal swab eluate and whole blood), dried buccal swabs and bloodstains and partial profiles from UV or heat-degraded bloodstains on cotton or blue denim substrates. Finally, the DNA preparation method is shown to be adaptable to glass or poly(methyl methacrylate) (PMMA) microdevices with little impact on STR peak height but providing a 20-fold reduction in incubation time (as little as 60s), leading to a ≥1h reduction in DNA preparation time.