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In protein design, the ultimate test of success is that the designs function as desired. Here, we discuss the utility of cell free protein synthesis (CFPS) as a rapid, convenient and versatile method to screen for activity. We champion the use of CFPS in screening potential designs. Compared to in vivo protein screening, a wider range of different activities can be evaluated using CFPS, and the scale on which it can easily be used-screening tens to hundreds of designed proteins-is ideally suited to current needs. Protein design using physics-based strategies tended to have a relatively low success rate, compared with current machine-learning based methods. Screening steps (such as yeast display) were often used to identify proteins that displayed the desired activity from many designs that were highly ranked computationally. We also describe how CFPS is well-suited to identify the reasons designs fail, which may include problems with transcription, translation, and solubility, in addition to not achieving the desired structure and function., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

We present a new method for the surface capture of proteins in cell-free protein synthesis (CFPS). We demonstrate the spontaneous self-assembly of the protein BslA into functionalizable surfaces on the surface of a CFPS reaction chamber. We show that proteins can be covalently captured by such surfaces, using "Catcher/Tag" technology. Importantly, proteins of interest can be captured either when synthesised in situ by CFPS above the BslA surfaces, or when added as pure protein. The simplicity and cost efficiency of this method suggest that it will find many applications in cell-free-based methods., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Thornton, Paterson, Gidden, Horrocks, Laohakunakorn and Regan.)

A monomer that contained the RGD ligand motif was synthesized and copolymerized with 2-hydroxyethyl methacrylate using polymerization-induced phase separation methods to form poly(2-hydroxyethyl methacrylate)-based hydrogel sponges. The sponges had morphologies of aggregated polymer droplets and interconnected pores, the pores having dimensions in the order of 10 μm typical of PHEMA sponges. RGD-containing moieties appeared to be evenly distributed through the polymer droplets. Compared to PHEMA sponges that were not functionalized with RGD, the new sponges containing RGD allowed greater invasion by human corneal epithelial cells, by advancing the attachment of cells to the surface of the polymer droplets., (© 2013.)

Methylphenidate (MPH) is a drug that is licensed for treatment of ADHD and also narcolepsy. Monitoring of the parent drug and its major metabolite ritalinic acid (RA) in urine is considered necessary to ensure compliance with treatment programmes. A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the determination of MPH and its metabolite RA in human urine. After urine was diluted with water, methylphenidate, the major metabolite ritalinic acid, and d₆-amphetamine as the internal standard were resolved on a PFP propyl column using gradient elution of 0.02% ammonium formate and acetonitrile. The total analysis time was 13.5 min. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 5-5000 μg/L (r>0.997), bias was ≤ ±20%, intra- and inter-day coefficients of variation (imprecision) were <8% and the limit of detection was 5 μg/L. The limit of quantitation was set at 100 μg/L. Matrix effects were up to 140% but these were accounted for by the internal standard. The assay is being used successfully in clinical practice to enhance the safe and effective use of methylphenidate., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objectives: To compare the early effects of local corticosteroid injection, naproxen, and placebo as treatments for tennis elbow in primary care. Specifically, to find out whether the extra pain reduction experienced by patients who are given the steroid injection in the short-term would be realized within the first 5 days of treatment and to attempt to assess how much extra pain may be associated with the injection initially., Methods: A randomized controlled trial carried out in 23 family practices in the United Kingdom. A total of 164 patients aged 18 to 70 years presenting with a new episode of tennis elbow were recruited and invited to keep a daily record of their pain intensity and medication use over the first 5 days of randomized treatment using a "diary.", Results: On day 1, pain scores were higher in the injection group compared with the naproxen group and placebo group, and the injection group was also taking more painkillers. By day 4, the converse was true, pain scores were significantly lower in the injection group than the other 2 groups, and patients given an injection were less likely to be taking painkillers than those in the placebo group., Discussion: Steroid injection was associated with an increase in reported pain for the first 24 hours of treatment, but the therapeutic benefits compared with naproxen and placebo were evident 3 to 4 days after the start of treatment.

Background: In randomised clinical trials (RCTs), outcome may be influenced by the opinions of the participants about the efficacy of treatments., Aim: To examine how initial treatment preferences of participants in a shoulder pain trial affected functional outcome and future treatment preferences., Design of Study: Observational cohort study nested within a multicentre, pragmatic RCT of steroid injection versus physiotherapy for unilateral shoulder pain., Setting: Nine general practices in north Staffordshire., Method: Two hundred and seven adults were randomised in the trial. Disability scores and preferences of the participants for the trial treatments were elicited at two points: prior to randomisation and 6 months post-randomisation. A good functional outcome was defined as at least a halving in the disability score at the 6 months follow-up point., Results: Pre-randomisation preferences were: 40% for injection and 20% for physiotherapy, and 40% gave no preference. A good outcome was achieved in a higher percentage of participants who gave a pre-randomisation treatment preference compared with those who did not (62% compared with 48% percentage difference = 14%; 95% confidence interval [CI] = -1 to 27%) with similar percentages in each preferred treatment group. However, receiving the preferred treatment did not confer any additional benefit in those who expressed a preference (receiving preferred treatment = 56%; not receiving preferred treatment = 69%). At 6 months post-randomisation, participants with a good, as opposed to poor, outcome were more likely to report as their preferred treatment the one to which they had been randomised, irrespective of pre-randomisation preference and whether the preferred treatment was received., Conclusion: This analysis suggests that preferences prior to treatment can affect outcome, but that treatment outcome is a stronger influence on post-treatment preferences. We present some empirical evidence to support the statement that treatment preferences can have important effects on the results of RCTs.

Objectives: This study investigates the influence of manual work on the persistence of lateral epicondylitis during a 12-month follow-up period after consultation in general practice., Methods: A cohort study was made of 164 adult consulters with tennis elbow, who had initially been recruited to a randomized controlled trial from 23 general practices in North Staffordshire and South Cheshire. As part of the recruitment process prior to treatment randomization, self-reported questionnaires on symptoms and sociodemographic characteristics were completed. Four weeks, six months, and twelve months later, the schedules were repeated. The main outcomes were measures of elbow symptoms (pain and function) based on a ten-point Likert scale. Total pain and function for the whole 12-month period were assessed using an area-under-the-curve (AUC) analysis. The pain and function scores were analyzed according to employment status (manual versus nonmanual work) and severe versus mild upper-limb stress in manual occupations., Results: The AUC scores for elbow pain and function were significantly higher for the manual workers, and manual work was associated with increased elbow pain and decreased function in the 4-week and 6-month follow-ups. The greater the severity of upper-limb stresses, the worse the outcome., Conclusions: Previous epidemiologic studies have shown that manual occupations are a risk factor for developing lateral epicondylitis. It is feasible that resuming manual work after treatment may hinder recovery or increase the risk of relapse. The study lends support to this hypothesis.