The Application and Development of Metabolomics Methodologies for the Profiling of Food and Cellular Toxicity

Metabolomics is a rapidly growing field of study. Its growth reflects advancements in technology and an improved understanding of the impact of the environment on metabolism. As a result, metabolomics is now commonly employed to investigate and characterize human and plant metabolism. The first chapter of this thesis provides an introduction to metabolomics and an overview of the protocols for sample preparation, data collection and statistical analysis. The second thesis chapter describes in explicit detail the step-by-step process of extracting and analyzing metabolites collected from mammalian cells, specifically brain tissue with a focus on Parkinson’s disease. The chapter highlights important factors to consider including experiment design, sample collection, and data processing. Chapters 3 and 4 include the application of metabolomics to evaluate how the metabolome responds to the environment. Chapter 3 focuses on the neuronal response to the xenobiotic arsenic. It demonstrates how astrocytes increase glutathione production through an up regulation of the citric acid cycle and glycolytic processes. Arsenic was also observed to decreases related metabolites including citrate and lactate. These metabolites are important intermediates to ATP production and illustrate the interconnection of metabolomic processes. Chapter 4 shows how metabolite profiles can be used to evaluate the impact of environmental conditions on wines. Metabolite profiles of Pinot Noir derived from the same scion clone (Pinot noir 667) and grown in different regions along the Pacific coast were compared. NMR and a differential sensing array were used to profile the chemical composition of the samples. We observed how environmental conditions resulted in different metabolite profiles in the various wine samples. This thesis aims to highlight the application of metabolomic to various biological studies in order to evaluate the impact of external stimuli. Advisor: Robert Power

Carbon Oxidation State in Microbial Polar Lipids Suggests Adaptation to Hot Spring Temperature and Redox Gradients

The influence of oxidation-reduction (redox) potential on the expression of biomolecules is a topic of ongoing exploration in geobiology. In this study, we investigate the novel possibility that structures and compositions of lipids produced by microbial communities are sensitive to environmental redox conditions. We extracted lipids from microbial biomass collected along the thermal and redox gradients of four alkaline hot springs in Yellowstone National Park (YNP) and investigated patterns in the average oxidation state of carbon (ZC), a metric calculated from the chemical formulae of lipid structures. Carbon in intact polar lipids (IPLs) and their alkyl chains becomes more oxidized (higher ZC) with increasing distance from each of the four hot spring sources. This coincides with decreased water temperature and increased concentrations of oxidized inorganic solutes, such as dissolved oxygen, sulfate, and nitrate. Carbon in IPLs is most reduced (lowest ZC) in the hot, reduced conditions upstream, with abundance-weighted ZC values between −1.68 and −1.56. These values increase gradually downstream to around −1.36 to −1.33 in microbial communities living between 29.0 and 38.1◦C. This near-linear increase in ZC can be attributed to a shift from ether-linked to ester-linked alkyl chains, a decrease in average aliphatic carbons per chain (nC), an increase in average degree of unsaturation per chain (nUnsat), and increased cyclization in tetraether lipids. The ZC of lipid headgroups and backbones did not change significantly downstream. Expression of lipids with relatively reduced carbon under reduced conditions and oxidized lipids under oxidized conditions may indicate microbial adaptation across environmental gradients in temperature and electron donor/acceptor supply

Physical and Anthropometric Characteristics Do Not Differ According to Birth Year Quartile in High-Level Junior Australian Football Players

The aim of this study was to explore differences in the physical fitness and anthropometric profiles between birth year quartiles of players attending the Australian Football League (AFL) National Draft Combine. Date of birth, anthropometric, 20 m sprint, vertical and running vertical jump, AFL planned agility, and 20 m Multi-Stage Fitness Test (MSFT) data were obtained for players selected to attend the Combine between 1999 and 2019 (n = 1549; Mage = 18.1; SDage = 0.3). The underlying density distributions of the data were visually explored using violin plots overlaid with box and whisker plots. A multivariate analysis of variance (MANOVA) was then used to model the main effect of birth quartile (four levels) on the physical and anthropometric scores. Results showed that physical and anthropometric test scores did not significantly differ according to birth quartile (V = 0.008, F = 0.880, p = 0.631). We conclude that the physical and anthropometric profiles of high-level junior Australian Football players were similar according to birth year quartile across the modeled period. Therefore, how players utilize their physical and anthropometric attributes during game-play via contextualized, representative assessments, such as small-sided games, should be considered when examining potential causes of a RAE

Development of a sports technology quality framework

Identifying tools and processes to effectively and efficiently evaluate technologies is an area of need for many sport stakeholders. This study aimed to develop a standardised, evidence-based framework to guide the evaluation of sports technologies. In developing the framework, a review of standards, guidelines and research into sports technology was conducted. Following this, 55 experts across the sports industry were presented with a draft framework for feedback. Following a two-round Delphi survey, the final framework consisted of 25 measurable features grouped under five quality pillars. These were 1) Quality Assurance & Measurement (Accuracy, Repeatability, Reproducibility, Specifications), 2) Established Benefit (Construct Validity, Concurrent Validity, Predictive Validity, Functionality), 3) Ethics & Security (Compliance, Privacy, Ownership, Safety, Transparency, Environmental Sustainability), 4) User Experience (Usability, Robustness, Data Representation, Customer Support & Training, Accessibility) & 5) Data Management (Data Standardisation, Interoperability, Maintainability, Scalability). The framework can be used to help design and refine sports technology in order to optimise quality and maintain industry standards, as well as guide purchasing decisions by organisations. It may also serve to create a common language for organisations, manufacturers, investors, and consumers to improve the efficiency of their decision-making relating to sports technology

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p&lt;0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p&lt;0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p&lt;0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP &gt;5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

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

The Application and Development of Metabolomics Methodologies for the Profiling of Food and Cellular Toxicity

Metabolomics is a rapidly growing field of study. Its growth reflects advancements in technology and an improved understanding of the impact of the environment on metabolism. As a result, metabolomics is now commonly employed to investigate and characterize human and plant metabolism. The first chapter of this thesis provides an introduction to metabolomics and an overview of the protocols for sample preparation, data collection and statistical analysis. The second thesis chapter describes in explicit detail the step-by-step process of extracting and analyzing metabolites collected from mammalian cells, specifically brain tissue with a focus on Parkinson’s disease. The chapter highlights important factors to consider including experiment design, sample collection, and data processing. Chapters 3 and 4 include the application of metabolomics to evaluate how the metabolome responds to the environment. Chapter 3 focuses on the neuronal response to the xenobiotic arsenic. It demonstrates how astrocytes increase glutathione production through an up regulation of the citric acid cycle and glycolytic processes. Arsenic was also observed to decreases related metabolites including citrate and lactate. These metabolites are important intermediates to ATP production and illustrate the interconnection of metabolomic processes. Chapter 4 shows how metabolite profiles can be used to evaluate the impact of environmental conditions on wines. Metabolite profiles of Pinot Noir derived from the same scion clone (Pinot noir 667) and grown in different regions along the Pacific coast were compared. NMR and a differential sensing array were used to profile the chemical composition of the samples. We observed how environmental conditions resulted in different metabolite profiles in the various wine samples. This thesis aims to highlight the application of metabolomic to various biological studies in order to evaluate the impact of external stimuli. Advisor: Robert Power

Carbon Oxidation State in Microbial Polar Lipids Suggests Adaptation to Hot Spring Temperature and Redox Gradients

The influence of oxidation-reduction (redox) potential on the expression of biomolecules is a topic of ongoing exploration in geobiology. In this study, we investigate the novel possibility that structures and compositions of lipids produced by microbial communities are sensitive to environmental redox conditions. We extracted lipids from microbial biomass collected along the thermal and redox gradients of four alkaline hot springs in Yellowstone National Park (YNP) and investigated patterns in the average oxidation state of carbon (ZC), a metric calculated from the chemical formulae of lipid structures. Carbon in intact polar lipids (IPLs) and their alkyl chains becomes more oxidized (higher ZC) with increasing distance from each of the four hot spring sources. This coincides with decreased water temperature and increased concentrations of oxidized inorganic solutes, such as dissolved oxygen, sulfate, and nitrate. Carbon in IPLs is most reduced (lowest ZC) in the hot, reduced conditions upstream, with abundance-weighted ZC values between −1.68 and −1.56. These values increase gradually downstream to around −1.36 to −1.33 in microbial communities living between 29.0 and 38.1◦C. This near-linear increase in ZC can be attributed to a shift from ether-linked to ester-linked alkyl chains, a decrease in average aliphatic carbons per chain (nC), an increase in average degree of unsaturation per chain (nUnsat), and increased cyclization in tetraether lipids. The ZC of lipid headgroups and backbones did not change significantly downstream. Expression of lipids with relatively reduced carbon under reduced conditions and oxidized lipids under oxidized conditions may indicate microbial adaptation across environmental gradients in temperature and electron donor/acceptor supply