Developing Methods to Assess Enzyme Stability in Liquid Laundry Detergents

This thesis presents work focused on developing established protein analysis methods for use in studying enzyme inactivation in laundry detergent systems. In a multi-billion dollar per year industry, basic, labour intensive procedures still dominate commercial stability studies, with extensive storage tests and activity assays remaining the industry standard. These methods are both inefficient and provide little insight into inactivation processes, leading to a 'trial and error' approach to product development. This slows the introduction new formulations and enzyme variants to the market. Furthermore, a valuable opportunity is being missed, harnessing available resources in the detergent industry to advance both protein analysis technologies and understanding of protein denaturation processes. Transfer from these basic, low throughput methods to those favoured by other protein-focused industries has been hindered by sample complexity and the presence of high concentrations of the surfactant, LAS. In this work, two novel approaches to enzyme analysis in LAS-rich media will be presented. The first employing an analogous surfactant, SDS, which yields similar effects on protein stability but does not affect UV detection, and the second, exploiting the irreversible nature of detergent enzyme unfolding to enable manipulation of formulations to within instrument specifications. These approaches will allow for incorporation of ultra-high throughput screening methods, such as DSF, as well as techniques which provide further insight into protein unfolding processes, such as CD, to the available suite of analytical techniques. Thermal data arising from this work were compared with rates of degradation obtained through conventional storage tests. Empirical fittings suggest a linear relationship between Tm values and long-term storage stability, enabling the use of thermal analysis as a tool for prediction of degradation rates. Further work is required to refine these models, however, before expanding to more complex systems

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570