Site selective labelling of proteins via native chemical ligation

A novel protein labelling strategy has been developed based on a chemical reaction known as Native Chemical Ligation (NCL). NCL is a highly specific and facile reaction, ligating a thioester specifically to an N-terminal cysteine residue and can be used to tag proteins in a selective and quantitative manner. This has advantages over traditional labelling methods such as use of green fluorescent protein: labelling with a small molecule is less likely to interfere with the protein’s native biological activity, and its physicochemical properties can be fine-tuned by synthesis. The key challenge is generating the protein bearing an N-terminal cysteine. The strategy developed in this thesis utilises the Foot-and-Mouth Disease Virus 3C protease (3Cpro), a highly selective protease that cleaves at PAKQ X with no apparent specificity for the P1’ position. To demonstrate the application of 3Cpro for NCL-mediated labelling, fluorescein was specifically ligated to the N-terminus of the PH domain of Protein Kinase B (PKB), which when defective is a key protagonist in a number of cancers. An expression–purification strategy was developed to express the protein as a fusion with a GST affinity tag, linked by the sequence PAKQC. The GST-PAKQC-PH protein was cleaved using 3Cpro and purified, yielding the N-terminal cysteine protein, C-PH, which was subsequently coupled to a fluorescein thioester via NCL. The labelled PH domain was successfully used as a probe of membrane bound phosphoinositol lipids in a fixed cell imaging assay. Suitable thioester tags are generated by converting an activated succinimide ester to a mercaptoethanesulfonate thioester using mercaptoethanesulfonate. This reaction was used to build up a labelling library of labels ranging from fluorescent dyes for imaging to biotin for pull-down studies, all of which were successfully ligated on the C-PH domain. This labelling strategy was then taken one step further to allow the specific labelling of the target from within a protein mixture. NCL is a generic method, requiring only a thioester and a cysteine N-terminus, therefore the versatility of this system can be exploited to create an easy to use labelling ‘toolbox’. The constructs and chemical tools produced here should enable facile production of a wide range of N-terminally labelled recombinant proteins for a vast array of studies

Weight management assessment and advice in primary care

Background: Although primary care settings provide a large-scale and high-reach opportunity for weight management and obesity prevention, the proportion of adults in the United Kingdom (UK) who report receiving weight management advice is limited. This study examines the self-reported frequency of assessing weight and providing weight management advice by General Practitioners (GPs) and Practice Nurses (PNs) working in primary care in the UK, and differences by practitioner characteristics. Methods: Cross-sectional survey with GPs and PNs in the UK (n = 2020), conducted January–March 2017. A mock consultation exercise assessed what factors led to calculating a patient’s Body Mass Index (BMI) and whether weight management advice was given after determining the patient had an obese BMI. For all patients, practitioners were asked how often they calculated BMI, how often they gave weight management advice to patients with an obese BMI, and how often they utilised different advice or referral options (each: Always/Often vs. Less often/Never). Binary logistic regressions examined whether frequency of assessing weight and providing advice was associated with practitioner characteristics. Results: In the mock consultation, physical cues (40%) were most likely to prompt calculation of BMI, and half of practitioners (56%) provided weight management advice after determining the patient had an obese BMI, with GPs less likely to do so than PNs (Odds Ratio [OR] = 0.59, 95% CI: 0.47–0.75). Half of practitioners (58%) said they calculated the BMI of all patients Always/Often, with GPs less likely to do so than PNs (OR = 0.27, 95% CI: 0.21–0.34). Three quarters (78%) said they provided weight management advice to patients with an obese BMI Always/Often, with GPs less likely to do so than PNs (OR = 0.63, 95% CI: 0.47–0.85). Weight management advice was provided more frequently than referrals, particularly suggesting increased physical activity (93%) and diet modification (89%). Conclusions: Consistent with previous research, the findings suggest that opportunities to provide weight management advice in primary care, including to patients with an obese BMI, are potentially missed. Future research should test alternative mechanisms to increase weight assessment and advice provision, examine the effectiveness of advice frequently given, and seek solutions to reported barriers for providing weight management advice

Youth engagement with alcohol brands in the UK

Report from Cancer Research UK, University of the West of England and University of Stirling which explores how young people engage with alcohol brand

Area deprivation, screen time and consumption of food and drink high in fat salt and sugar (HFSS) in young people: Results from a cross sectional study in the UK

ObjectivesTo investigate associations between deprivation in young people and consumption of foods high in fat, salt and sugar (HFSS), screen time exposure and health knowledge.DesignAn online cross-sectional survey with people aged 11–19 years in the UK, where participants reported consumption behaviours across 13 HFSS and two non-HFSS groups; screen time for commercial television and streaming services; and knowledge of health conditions and their links to obesity.SettingUKParticipantsA total of 3348 young people aged 11–19 years across the UK.Main outcome measuresThe study assessed the consumption behaviours, commercial screen time exposure and the health knowledge of 3348 people aged 11–19 years. Multivariate binary regression analysis, controlling for age and gender, was performed.ResultsDeprivation level was associated with increases in consumption of six of the HFSS products including energy drinks (OR: 2.943, p<0.001) and sugary drinks (OR: 1.938, p<0.001) and a reduction in consumption in the two non-HFSS products included in the study, fruit (OR: 0.668, p=0.004) and vegetables (OR: 0.306, p<0.001). Deprivation was associated with high weekly screen time of both television (OR: 2.477, p<0.001) and streaming (OR: 1.679, p=0.001). Health knowledge was also associated with deprivation. There was lower awareness of the association of obesity and cancer (OR: 0.697, p=0.003), type 2 diabetes (OR: 0.64, p=0.004) and heart disease (OR: 0.519, p<0.001) in the most deprived.ConclusionsYoung people from the more deprived areas of the UK were more likely to consume a range of HFSS products, report increased exposure to HFSS advertising and have a poorer awareness of health conditions associated with overweight and obesity. The findings suggest that population-level measures addressing childhood obesity should account for consumption patterns among different groups of children and young people and the factors that may influence these

New national alcohol guidelines in the UK: public awareness, understanding and behavioural intentions

Background: Alcohol consumption places a significant burden on the NHS and is an important risk factor for cancer, associated with 12 800 UK cases/year. New alcohol guidelines were published in 2016, taking into account the increasing evidence of the health harms of alcohol. Methods: A survey of the UK drinker population (n = 972) was conducted 1 week before and 1 month after the release of the guidelines to capture drinking habits, guideline awareness and intended behaviour change. Results: Overall, 71% were aware of the new alcohol guidelines, however, just 8% knew what the recommended limits were. Higher socioeconomic groups were more likely to know these limits (ABC1 = 9% versus C2DE = 4%, P = 0.009). Participants who recognized the message that alcohol causes cancer were more likely to correctly identify the new guidelines (message recognition = 12% versus no recognition = 6%, P = 0.004); and were more likely to self-report an intention to reduce their alcohol consumption (message recognition = 10% versus no recognition = 6%, P = 0.01). Conclusion: The majority of the population knew the guidelines had been updated, however, communication of the new limits needs to be improved. Raising awareness of the links between alcohol and cancer may improve understanding of alcohol guidelines and could prompt behaviour change for those motivated to reduce their alcohol consumption

Public awareness and healthcare professional advice for obesity as a risk factor for cancer in the UK:a cross-sectional survey

BACKGROUND: Overweight and obesity is the second biggest preventable cause of cancer after smoking, causing ~3.4 million deaths worldwide. This study provides current UK data on awareness of the link between obesity and cancer by socio-demographic factors, including BMI, and explores to what degree healthcare professionals provide weight management advice to patients. METHODS: Cross-sectional survey of 3293 adults completed an online survey in February/March 2016, weighted to be representative of the UK population aged 18+. RESULTS: Public awareness of the link between obesity and cancer is low (25.4% unprompted and 57.5% prompted). Higher levels of awareness existed for least deprived groups (P < 0.001), compared to more deprived groups. Most respondents had seen a healthcare practitioner in the past 12 months (91.6%) and 17.4% had received advice about their weight, although 48.4% of the sample were overweight/obese. CONCLUSION: Cancer is not at the forefront of people’s minds when considering health conditions associated with overweight or obesity. Socio-economic disparities exist in health knowledge across the UK population, with adults from more affluent groups being most aware. Healthcare professionals are uniquely positioned to provide advice about weight, but opportunities for intervention are currently under-utilized in healthcare settings

Cost-Effectiveness of Strategies to Improve HIV Testing and Receipt of Results: Economic Analysis of a Randomized Controlled Trial

The CDC recommends routine voluntary HIV testing of all patients 13-64 years of age. Despite this recommendation, HIV testing rates are low even among those at identifiable risk, and many patients do not return to receive their results. To examine the costs and benefits of strategies to improve HIV testing and receipt of results. Cost-effectiveness analysis based on a Markov model. Acceptance of testing, return rates, and related costs were derived from a randomized trial of 251 patients; long-term costs and health outcomes were derived from the literature. Primary-care patients with unknown HIV status. Comparison of three intervention models for HIV counseling and testing: Model A = traditional HIV counseling and testing; Model B = nurse-initiated routine screening with traditional HIV testing and counseling; Model C = nurse-initiated routine screening with rapid HIV testing and streamlined counseling. Life-years, quality-adjusted life-years (QALYs), costs and incremental cost-effectiveness. Without consideration of the benefit from reduced HIV transmission, Model A resulted in per-patient lifetime discounted costs of $48,650 and benefits of 16.271 QALYs. Model B increased lifetime costs by$53 and benefits by 0.0013 QALYs (corresponding to 0.48 quality-adjusted life days). Model C cost $66 more than Model A with an increase of 0.0018 QALYs (0.66 quality-adjusted life days) and an incremental cost-effectiveness of$36,390/QALY. When we included the benefit from reduced HIV transmission, Model C cost $10,660/QALY relative to Model A. The cost-effectiveness of Model C was robust in sensitivity analyses. In a primary-care population, nurse-initiated routine screening with rapid HIV testing and streamlined counseling increased rates of testing and receipt of test results and was cost-effective compared with traditional HIV testing strategies

Bone Marrow Cells in Murine Colitis: Multi-Signal Analysis Confirms Pericryptal Myofibroblast Engraftment without Epithelial Involvement

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credite

Comics, graphic narratives, and lesbian lives

Lesbian comics and graphic narratives have gained unprecedented cultural presence in the twenty-first century. Yet despite the surge in interest in the work of artists such as Alison Bechdel, and despite the existence of a substantial online archive about lesbian comics created by artists, readers, and collectors, relatively little critical attention has been directed to this work. The chapter begins to fill this gap. Taking the Bechdel’s work as its start-and-end point, it provides an overview of major developments in lesbian comics and contextualises them including in relation to the gendered conditions of possibility that define comics culture