Studies on the AROM Pentafunctional Enzyme in Saccharomyces cerevisiae and Aspergillus nidulans

Aromatic amino acids are synthesised via the shikimate pathway. AROM is a pentafunctional enzyme which catalyses the five central steps of the shikimate pathway and is found in fungi and yeast. This thesis describes the purification and characterisation of AROM from overexpressing strains of Saccharomyces cerevisiae and Aspergillus nidulans. S. cerevisiae AROM was purified 30-fold with a 10% yield from a yeast overexpression strain which exploited the ubiquitin-fusion system of yeast. The purified protein was found to possess all five enzyme activities in a similar ratio to that observed in crude extract and had a subunit molecular weight of 175kDa. The main S. cerevisiae protein was shown to have several minor, lower molecular weight contaminants following SDS PAGE and three of these were found to cross-react with anti-AROM antibodies raised against Neurospora crassa AROM. The peak AROM fraction eluted from gel filtration chromatography was found to be composed of two proteins which were separable by native PAGE and both of which were shown to have shikimate DH activity. The poor recovery and multiple protein bands suggested that during the AROM preparation limited proteolysis was occurring despite a number of anti-proteinase measures. No means of eliminating limited proteolysis during S. cerevisiae AROM isolation were found and purification studies were carried out on the AROM of A. nidulans in the hope that proteolysis might not be as problematic in this species. A rapid procedure for the purification of A. nidulans AROM from the overexpresion strain A. nidulans 1314 has been developed which results in 13-fold purification and a 9% yield. The subunit molecular weight was estimated at 175kDa and the native molecular weight suggests that the protein is a dimer. The N-terminal DHQ synthase activity in the AROM protein was found to be severly deficient in both crude extract and the purified protein from A. nidulans 1314. This was independently attributed to the introduction of a missense muta tion in this region of the polypeptide. A preliminary limited proteolysis study was carried out on AROM purified from A. nidulans 1314 which suggested that the proteolysis pattern is complicated and which show that the DHQase and shikimate DH enzyme activities are least susceptible to limited proteolysis

Development of a measure to assess the quality of proxy decisions about research participation on behalf of adults lacking capacity to consent : the Combined Scale for Proxy Informed Consent Decisions (CONCORD scale)

Acknowledgements We would like to thank the participants who generously volunteered their time to participate in the interviews and the lay advisory group who provide invaluable insight and support for this research programme. Funding This study was conducted as part of a National Institute of Health Research Advanced Fellowship (CONSULT) held by VS and funded by the Welsh Government through Health and Care Research Wales (NIHR-FS(A)-2021). The funding body did not participate in the study design, data collection, analysis, or interpretation in writing this manuscript. Primary and Emergency Care (PRIME) Research Centre Wales) is funded by the Welsh Government through Health and Care Research Wales and the Centre for Trials Research is funded by Health and Care Research Wales and Cancer Research UK.Peer reviewedPublisher PD

Feasibility, effectiveness and costs of a decision support intervention for consultees and legal representatives of adults lacking capacity to consent (CONSULT) : protocol for a randomised Study Within a Trial

Acknowledgements We would like to thank the lay advisory group who provide invaluable insight and support for this research programme. Funding This research was funded by Health and Care Research Wales as part of an NIHR Advanced Fellowship (NIHR-FS(A)-2021). For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising. Primary and Emergency Care (PRIME) Research Centre Wales) is funded by the Welsh Government through Health and Care Research Wales and the Centre for Trials Research is funded by Health and Care Research Wales and Cancer Research UK. © 2022. The Author(s).Peer reviewedPublisher PD

Recruitment interventions for trials involving adults lacking capacity to consent: methodological and ethical considerations for designing Studies Within a Trial (SWATs)

Background: The number of interventions to improve recruitment and retention of participants in trials is rising, with a corresponding growth in randomised Studies Within Trials (SWATs) to evaluate their (cost-)efectiveness. Despite recognised challenges in conducting trials involving adults who lack capacity to consent, until now, no individual-level recruitment interventions have focused on this population. Following the development of a decision aid for family members making non-emergency trial participation decisions on behalf of people with impaired capacity, we have designed a SWAT to evaluate the decision aid in a number of host trials (CONSULT). Unlike in recruitment SWATs to date, the CONSULT intervention is aimed at a ‘proxy’ decision-maker (a family member) who is not a participant in the host trial and does not receive the trial intervention. This commentary explores the methodological and ethical considerations encountered when designing such SWATs, using the CONSULT SWAT as a case example. Potential solutions to address these issues are also presented. Discussion: We encountered practical issues around informed consent, data collection, and follow-up which involves linking the intervention receiver (the proxy) with recruitment and retention data from the host trial, as well as issues around randomisation level, resource use, and maintaining the integrity of the host trial. Unless addressed, methodological uncertainty about diferential recruitment and heterogeneity between trial populations could potentially limit the scope for drawing robust inferences and harmonising data from diferent SWAT host trials. Proxy consent is itself ethically complex, and so when conducting a SWAT which aims to disrupt and enhance proxy consent decisions, there are additional ethical issues to be considered. Conclusions: Designing a SWAT to evaluate a recruitment intervention for non-emergency trials with adults lacking capacity to consent has raised a number of methodological and ethical considerations. Explicating these challenges, and some potential ways to address them, creates a starting point for discussions about conducting these potentially more challenging SWATs. Increasing the evidence base for the conduct of trials involving adults lacking capacity to consent is intended to improve both the ability to conduct these trials and their quality, and so help build research capacity for this under-served populatio

The effectiveness and cost-effectiveness of resuscitative endovascular balloon occlusion of the aorta (REBOA) for trauma patients with uncontrolled torso haemorrhage : study protocol for a randomised clinical trial (the UK-REBOA trial)

Acknowledgements The UK-REBOA trial grantholders include Jan O. Jansen, University of Aberdeen, UK, and University of Alabama at Birmingham, USA; Marion K. Campbell, University of Aberdeen, UK; Chris Moran, Nottingham University Hospital Trust, UK; Karim Brohi, Queen Mary University of London, UK; Fiona Lecky, University of Sheffield, UK; Robbie Lendrum, Bart’s Health NHS Trust, UK; Graeme MacLennan, University of Aberdeen, UK; Jonathan J. Morrison, University of Maryland, USA; Nigel Tai, Academic Department of Military Surgery and Trauma, Royal Centre for Defence Medicine, UK; Tim Harris, Bart’s Health NHS Trust, UK; John Norrie, University of Edinburgh, UK; Dwayne Boyers, University of Aberdeen, UK; Alan Paterson, University of Strathclyde, UK; and Nick Welch. Funding {4} This study/project is funded by the National Institute for Health Research (NIHR) HTA Programme (reference 14/199/09). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. The funder has/had no role in the design of the study; the collection, analysis, and interpretation of data; or writing the manuscript. The Health Services Research Unit, Institute of Applied Health Sciences (University of Aberdeen), is core-funded by the Chief Scientist Office of the Scottish Government Health and Social Care Directorates.Peer reviewedPublisher PD

Cerebrospinal fluid cortisol levels are higher in patients with delirium versus controls

<p>Abstract</p> <p>Background</p> <p>High plasma cortisol levels can cause acute cognitive and neuropsychiatric dysfunction, and have been linked with delirium. CSF cortisol levels more closely reflect brain exposure to cortisol, but there are no studies of CSF cortisol levels in delirium. In this pilot study we acquired CSF specimens at the onset of spinal anaesthesia in patients undergoing hip fracture surgery, and compared CSF and plasma cortisol levels in delirium cases versus controls.</p> <p>Findings</p> <p>Delirium assessments were performed the evening before or on the morning of operation with a standard battery comprising cognitive tests, mental status assessments and the Confusion Assessment Method. CSF and plasma samples were obtained at the onset of the operation and cortisol levels measured. Twenty patients (15 female, 5 male) aged 62 - 93 years were studied. Seven patients were diagnosed with delirium. The mean ages of cases (81.4 (SD 7.2)) and controls (80.5 (SD 8.7)) were not significantly different (p = 0.88). The median (interquartile range) CSF cortisol levels were significantly higher in cases (63.9 (40.4-102.1) nmol/L) than controls (31.4 (21.7-43.3) nmol/L; Mann-Whitney U, p = 0.029). The median (interquartile range) of plasma cortisol was also significantly higher in cases (968.8 (886.2-1394.4) nmol/L, than controls (809.4 (544.0-986.4) nmol/L; Mann Whitney U, p = 0.036).</p> <p>Conclusions</p> <p>These findings support an association between higher CSF cortisol levels and delirium. This extends previous findings linking higher plasma cortisol and delirium, and suggests that more definitive studies of the relationship between cortisol levels and delirium are now required.</p

Overcoming barriers to engaging socio-economically disadvantaged populations in CHD primary prevention: a qualitative study

&lt;p&gt;&lt;b&gt;Background:&lt;/b&gt; Preventative medicine has become increasingly important in efforts to reduce the burden of chronic disease in industrialised countries. However, interventions that fail to recruit socio-economically representative samples may widen existing health inequalities. This paper explores the barriers and facilitators to engaging a socio-economically disadvantaged (SED) population in primary prevention for coronary heart disease (CHD).&lt;/p&gt; &lt;p&gt;&lt;b&gt;Methods:&lt;/b&gt; The primary prevention element of Have a Heart Paisley (HaHP) offered risk screening to all eligible individuals. The programme employed two approaches to engaging with the community: a) a social marketing campaign and b) a community development project adopting primarily face-to-face canvassing. Individuals living in areas of SED were under-recruited via the social marketing approach, but successfully recruited via face-to-face canvassing. This paper reports on focus group discussions with participants, exploring their perceptions about and experiences of both approaches.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; Various reasons were identified for low uptake of risk screening amongst individuals living in areas of high SED in response to the social marketing campaign and a number of ways in which the face-to-face canvassing approach overcame these barriers were identified. These have been categorised into four main themes: (1) processes of engagement; (2) issues of understanding; (3) design of the screening service and (4) the priority accorded to screening. The most immediate barriers to recruitment were the invitation letter, which often failed to reach its target, and the general distrust of postal correspondence. In contrast, participants were positive about the face-to-face canvassing approach. Participants expressed a lack of knowledge and understanding about CHD and their risk of developing it and felt there was a lack of clarity in the information provided in the mailing in terms of the process and value of screening. In contrast, direct face-to-face contact meant that outreach workers could explain what to expect. Participants felt that the procedure for uptake of screening was demanding and inflexible, but that the drop-in sessions employed by the community development project had a major impact on recruitment and retention.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Conclusion:&lt;/b&gt; Socio-economically disadvantaged individuals can be hard-to-reach; engagement requires strategies tailored to the needs of the target population rather than a population-wide approach.&lt;/p&gt

PYY plays a key role in the resolution of diabetes following bariatric surgery in humans.

BACKGROUND: Bariatric surgery leads to early and long-lasting remission of type 2 diabetes (T2D). However, the mechanisms behind this phenomenon remain unclear. Among several factors, gut hormones are thought to be crucial mediators of this effect. Unlike GLP-1, the role of the hormone peptide tyrosine tyrosine (PYY) in bariatric surgery in humans has been limited to appetite regulation and its impact on pancreatic islet secretory function and glucose metabolism remains under-studied. METHODS: Changes in PYY concentrations were examined in obese patients after bariatric surgery and compared to healthy controls. Human pancreatic islet function was tested upon treatment with sera from patients before and after the surgery, in presence or absence of PYY. Alterations in intra-islet PYY release and insulin secretion were analysed after stimulation with short chain fatty acids (SCFAs), bile acids and the cytokine IL-22. FINDINGS: We demonstrate that PYY is a key effector of the early recovery of impaired glucose-mediated insulin and glucagon secretion in bariatric surgery. We establish that the short chain fatty acid propionate and bile acids, which are elevated after surgery, can trigger PYY release not only from enteroendocrine cells but also from human pancreatic islets. In addition, we identify IL-22 as a new factor which is modulated by bariatric surgery in humans and which directly regulates PYY expression and release. INTERPRETATION: This study shows that some major metabolic benefits of bariatric surgery can be emulated ex vivo. Our findings are expected to have a direct impact on the development of new non-surgical therapy for T2D correction

Job satisfaction and its modeling among township health center employees: a quantitative study in poor rural China

<p>Abstract</p> <p>Background</p> <p>Job satisfaction is important to staff management of township health centers (THCs), as it is associated with organizational performance, quality of care and employee retention. The purpose of this study was to measure job satisfaction level of THC employees in poor rural China and to identify relevant features in order to provide policy advice on human resource development of health service institutions in poor regions.</p> <p>Methods</p> <p>A self-completion questionnaire was used to assess the job satisfaction and relevant features (response rate: 90.5%) among 172 employees (i.e., clinic doctors, medico-technical workers and public health workers) of 17 THCs in Anhui and Xinjiang provinces of China. The study covered a time period of two months in 2007.</p> <p>Results</p> <p>The mean staff job satisfaction scored 83.3, which was in the category of "somewhat satisfied" on a scale ranging from 0 (extremely dissatisfied) to 100 (extremely satisfied) by employing Likert's transformation formula. Exploratory factor analysis (EFA) revealed eight domains involved in modeling of job satisfaction, among which, the caregivers were more satisfied with job significance (88.2), job competency (87.9) and teamwork (87.7), as compared with work reward (72.9) and working conditions (79.7). Mean job satisfaction in Xinjiang (89.7) was higher than that in Anhui (75.5).</p> <p>Conclusions</p> <p>Employees of THCs have moderate job satisfactions in poor areas, which need to be raised further by improving their working conditions and reward.</p

Effect of a Perioperative, Cardiac Output-Guided Hemodynamic Therapy Algorithm on Outcomes Following Major Gastrointestinal Surgery A Randomized Clinical Trial and Systematic Review

Importance: small trials suggest that postoperative outcomes may be improved by the use of cardiac output monitoring to guide administration of intravenous fluid and inotropic drugs as part of a hemodynamic therapy algorithm.Objective: to evaluate the clinical effectiveness of a perioperative, cardiac output–guided hemodynamic therapy algorithm.Design, setting, and participants: OPTIMISE was a pragmatic, multicenter, randomized, observer-blinded trial of 734 high-risk patients aged 50 years or older undergoing major gastrointestinal surgery at 17 acute care hospitals in the United Kingdom. An updated systematic review and meta-analysis were also conducted including randomized trials published from 1966 to February 2014.Interventions: patients were randomly assigned to a cardiac output–guided hemodynamic therapy algorithm for intravenous fluid and inotrope (dopexamine) infusion during and 6 hours following surgery (n=368) or to usual care (n=366).Main outcomes and measures: the primary outcome was a composite of predefined 30-day moderate or major complications and mortality. Secondary outcomes were morbidity on day 7; infection, critical care–free days, and all-cause mortality at 30 days; all-cause mortality at 180 days; and length of hospital stay.Results: baseline patient characteristics, clinical care, and volumes of intravenous fluid were similar between groups. Care was nonadherent to the allocated treatment for less than 10% of patients in each group. The primary outcome occurred in 36.6% of intervention and 43.4% of usual care participants (relative risk [RR], 0.84 [95% CI, 0.71-1.01]; absolute risk reduction, 6.8% [95% CI, ?0.3% to 13.9%]; P?=?.07). There was no significant difference between groups for any secondary outcomes. Five intervention patients (1.4%) experienced cardiovascular serious adverse events within 24 hours compared with none in the usual care group. Findings of the meta-analysis of 38 trials, including data from this study, suggest that the intervention is associated with fewer complications (intervention, 488/1548 [31.5%] vs control, 614/1476 [41.6%]; RR, 0.77 [95% CI, 0.71-0.83]) and a nonsignificant reduction in hospital, 28-day, or 30-day mortality (intervention, 159/3215 deaths [4.9%] vs control, 206/3160 deaths [6.5%]; RR, 0.82 [95% CI, 0.67-1.01]) and mortality at longest follow-up (intervention, 267/3215 deaths [8.3%] vs control, 327/3160 deaths [10.3%]; RR, 0.86 [95% CI, 0.74-1.00]).Conclusions and relevance: in a randomized trial of high-risk patients undergoing major gastrointestinal surgery, use of a cardiac output–guided hemodynamic therapy algorithm compared with usual care did not reduce a composite outcome of complications and 30-day mortality. However, inclusion of these data in an updated meta-analysis indicates that the intervention was associated with a reduction in complication rate