Luke Whiley

Parkinson's disease (PD) is a progressive neurodegenerative disorder defined by motor impairments. However, people with PD (PwPD) experience a defined spectrum of non-motor symptoms, with gastrointestinal dysfunction the most common and earliest-presenting. Evidence suggests that PD pathology may originate in the gut, where microbial dysbiosis and immune dysregulation contribute to neuroinflammation, although mechanisms underlying this are unclear. PwPD (n = 31) and healthy controls (n = 28) were evaluated for clinical and gastrointestinal symptoms, faecal and plasma sample metabolomics, and comprehensive blood immunophenotyping. In PwPD, faecal samples exhibited reduced glutamate, succinate, and uracil concentrations, while plasma showed decreased 3-hydroxybutyrate and elevated creatine, succinate, and alanine levels. Immunophenotyping revealed a reduction in T cells, with evidence of altered effector capacity and functionality in CD4, CD8, MAIT and Vδ2 compartments. NK cells were expanded, while B cells were decreased in frequency with an enrichment of memory-like cells. Immune perturbations were correlated with levels of immunomodulatory metabolite succinate. Finally, clustering of blood parameters identified two PD endophenotypes distinguishable by gastrointestinal symptoms and T cell phenotypes associated with gut- and brain-tropism. These findings contribute to the growing understanding of metabolite-associated immune dysregulation in PD and highlight potential targets for early intervention in individuals presenting with gastrointestinal dysfunction.

Background
In Alzheimer’s disease (AD), astrocytes undergo reactive changes that can exert both protective and detrimental effects on neurons, thereby influencing neuronal survival and contributing to disease progression. Increasing attention has been directed toward elucidating the mechanisms underlying astrocyte dysfunction and neuroinflammation to identify novel therapeutic targets. Butyrate—a short‐chain fatty acid produced by gut microbiota—has demonstrated anti‐inflammatory and neuroprotective properties. Similarly, lauric acid (LA), a medium‐chain fatty acid, has shown potential in attenuating amyloid‐beta (Aβ)‐induced neurotoxicity, enhancing mitochondrial function, and modulating neuronal excitability. Despite these findings, the mechanisms by which they modulate astrocyte and neuronal function remain poorly understood. This study investigates the effects of butyrate and LA on oxidative stress, mitochondrial dysfunction, and lipidomic alterations in human‐induced pluripotent stem cells (iPSCs)‐derived astrocytes, neurons, and spontaneous cultures.

Method
Astrocytes, neurons, and spontaneous co‐cultures were differentiated from iPSCs derived from a healthy donor. Cultures were treated with synthetic Aβ (20 µM), sodium butyrate (NaB) (100 µM), or LA (100 µM), alone and in combination. Oxidative stress, mitochondrial dysfunction and the lipidomic changes of these cells was measured using Quantitative PCR, Catalase activity, Mitochondrial ToxGlo™ Assay and Liquid Chromatography‐Mass Spectrometry (LC‐MS) respectively.

Result
Aβ exposure significantly suppressed catalase expression in astrocytes, indicating elevated oxidative stress. Treatment with NaB and LA exhibit partial restoration of catalase expression, with the most effects observed in mixed cultures, suggesting enhanced cellular resilience. Furthermore, expression of Mitofusin‐1, a key regulator of mitochondrial fusion, was downregulated following Aβ treatment. Functional assays revealed butyrate and LA enhance antioxidant enzyme activity and improved mitochondrial integrity over time, with astrocytes and mixed cultures showing the most pronounced protective effect. The lipid profiles indicate that Aβ induces greater lipid variation in neurons compared to astrocytes and spontaneous cultures.

Conclusion
This study highlights the modulatory roles of butyrate and lauric acid on oxidative stress pathways, mitochondrial dynamics, and lipidomic profiles in iPSC‐derived brain cell cultures subjected to amyloid‐beta toxicity. These results support further exploration of butyrate and LA as therapeutic agents targeting neuroinflammation and metabolic dysregulation in AD.

Background
Central adiposity is a modifiable risk factor for age-related cognitive decline and has been linked to lipid dysregulation. However, the mechanisms underlying this relationship, particularly the role of plasma lipids at the species level, remain poorly understood. This study investigates whether lipids mediate the relationship between central adiposity and cognition in cognitively unimpaired older adults.

Methods
Ninety-four cognitively normal older adults (n = 94, mean age 69.0 ± 5.0 years, 54% female) were included in this study. Cognitive composite scores were derived from z-standardised neuropsychological assessments, and central adiposity was measured using the waist–hip ratio (WHR). Lipidomic profiling identified 918 lipid species, which were clustered into modules of highly correlated lipids using a Weighted Gene Co-Expression Network Analysis (WGCNA). Modules associated with the WHR and cognition were identified via partial Spearman’s correlation analysis, followed by a mediation analysis.

Results
Of the 39 lipid modules identified, 1 enriched with phosphatidylglycerol (PG) lipids containing an arachidic acid (20:0) sidechain was positively correlated with cognition (ρ = 0.32, FDR p < 0.05) and negatively correlated with the WHR (ρ = −0.43, FDR p < 0.001). Mediation analysis revealed that this arachidic acid-carrying PG lipid-enriched module mediated the WHR–cognition relationship, with individual species PG (20:0_16:1), PG (20:0_18:1), and PG (20:0_18:2) also contributing individually. Conclusions: Arachidic acid-carrying PG lipids statistically mediate the WHR–cognition relationship in cognitively unimpaired older adults. These findings suggest that adiposity-related lipid pathways are detectable in cognitively unimpaired older adults and may represent targets for early intervention to preserve cognitive health.

Pooled quality control (PQC) samples are the gold standard for data quality monitoring in metabolic phenotyping studies. Typically composed of equal parts from all study samples, PQCs can be challenging to generate in large cohorts or when sample volumes are low. As an alternative, externally sourced matrix-matched surrogate QCs (sQC) have been proposed. This study evaluates the performance of sQCs against PQCs for assessing analytical variation, data pre-processing, and downstream data analysis in a targeted lipidomics workflow.
Plasma samples (n = 701) from the Microbiome Understanding in Maternity Study, along with PQC (n = 80) and sQC (n = 80) samples, were analyzed using a lipidomics assay targeting 1162 lipids. QC samples were injected throughout acquisition, and data pre-processing was performed using each strategy. For simplicity, a subset (n = 381) of the study samples was used to assess differences in downstream statistical analyses.
Both QC approaches demonstrated high analytical repeatability. While PQC and sQC compositions differed, use of PQCs retained less than 4 % more lipid species during pre-processing. Univariate analysis identified more statistically significant lipids with PQC-based pre-processing, but multivariate model performance was similar between datasets.
This study provides a comprehensive comparison of QC strategies and emphasizes the importance of careful QC workflow selection. While PQCs offer advantages, sQCs serve as a suitable alternative for quality assessment and pre-processing. Their commercial availability also supports use as intra- and inter-laboratory long-term references, aiding data harmonization across studies and laboratories.
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•Comparison of two quality control workflows; pooled study and surrogate QC samples.•In-depth assessment of lipid composition, precision, and filtering.•OPLS-DA model predictive power maintained with both QC pre-processing strategies.•Surrogate QC samples are a robust alternative to a pooled QC in targeted lipidomics.

SARS-CoV-2 infections in children lead to symptoms from mild respiratory illness to severe postacute sequelae of COVID-19, including multisystem inflammatory syndrome in Children (MIS-C). We conducted a metabolic profiling of 147 children's serum samples, including acute COVID-19 patients, MIS-C patients, and healthy controls. Using nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry, we measured 1101 metabolites. The results revealed distinct metabolic profiles in acute COVID-19 and MIS-C patients, with significant alterations in lipid classes. Both conditions exhibited an elevated Apo-B100/Apo-A1 ratio and increased serum inflammatory markers. MIS-C patients showed unique disruptions, including increased triglycerides and altered lipoprotein composition. Despite milder clinical respiratory symptoms, children's metabolic disturbances mirrored those seen in severe adult COVID-19 patients, indicating a shared inflammatory response to SARS-CoV-2. This suggests potential long-term health impacts, underscoring the need for continued research into the metabolic consequences of COVID-19 in children.

Purpose
Approximately 20% of road fatalities can be attributed to driver fatigue; however, there are relatively few options available for police to specifically address this danger. Salivary biomarkers are a promising solution as saliva collection is non-invasive, quick, and easy to perform in roadside settings. Metabolomics is emerging as a useful tool for biomarker detection because it allows for the comprehensive profiling of small molecules, providing insights into subtle biochemical changes that may be 8associated with fatigue. This pilot study aims to explore the potential of metabolomic approaches in discovering fatigue biomarkers in saliva.

Methods
Saliva samples were collected from participants (n = 12) at baseline (well-rested) and following sleep deprivation. Participants also provided subjective ratings of perceived fatigue and cognitive inhibition was assessed via the three-minute psychomotor vigilance task (PVT). Saliva samples were analysed using proton nuclear magnetic resonance (1H NMR) spectroscopy. 1H NMR data was interrogated using multivariate (O-PLS) and univariate (Kruskal-Wallis, Spearman’s Correlation) analyses to identify metabolites associated with fatigue.

Results
O-PLS identified seven metabolites as potential biomarkers of fatigue, but only scyllo-inositol reached statistical significance when interrogated univariately. No significant correlation was observed between PVT scores and self-reported fatigue, raising questions about the validity of specifically the three-minute PVT, compared to either the five or ten-minute variety, as a measure of cognitive inhibition.

Conclusion
This pilot study highlights scyllo-inositol as a potential salivary biomarker for fatigue, but further validation in larger cohorts is necessary. Additionally, recommendations are made for improving similar research.

Dried blood spot (DBS) sample collections can offer a minimally invasive, cost-effective alternative to traditional venepuncture for remote sampling and high-frequency metabolic profiling. We present an optimised protocol for DBS-based extraction and comprehensive untargeted 4D lipid profiling using ultrahigh-performance liquid chromatography coupled with high-resolution mass spectrometry, designed to support large-scale applications in population-wide lipidomics research. Inclusion of stable isotopically labelled internal standards allowed for semi-quantitative subclass-level correction for 10 μL DBS samples, enhancing the number of reproducible lipids within our curated target list (focussed on 432 unique rule-based lipid annotations out of 6845 features) across positive and negative heated electrospray ionisation modes. The reproducibility of unique lipid features detected in replicate DBS (n = 6) was assessed on both peak areas (351 lipids < 25 % CV) and calculated concentrations relative to internal standards (432 lipids < 25 % CV), underscoring the benefit of internal standard addition. Storage conditions for DBS were also evaluated to determine short-term lipid stability at different temperatures (-20 ˚C, 4 ˚C, room temperature, and 45 ˚C). The majority of lipid subclasses, excluding a minority of glycerophospholipids and oxylipins, were stable up to 1 week at -20 ˚C and 4 ˚C (log2-fold change < 30 % difference), which supports the short-term storage capacity for DBS in field and clinical settings. Similar stability was observed within a week at room temperature, excluding phosphatidylethanolamines and phosphatidylglycerols (log2-fold change > 30 % difference). Application of the optimised workflow to a microsampling device (n = 6) identified 432 lipid features (CV < 25 %) with three repeated samplings over an hour showing minimal impact on lipid profiles by principal component analysis, showing promise for high-frequency, longitudinal DBS monitoring in population health. This work represents a significant advance, highlighting the potential for reliable lipid analysis from DBS samples with short-term stability under various storage conditions, an important logistical benefit for remote or resource-limited settings.
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•Dried blood spots enable minimally invasive, cost-effective sampling in lipidomics•The developed untargeted 4D-lipidomic method annotates 432 lipids in 10 μL DBS•Majority of lipid subclasses are stable on DBS up to 1 week, ideal at -20°C and 4°C•Commercial microsampling devices suit remote, high-frequency lipid profiling

We investigated plasma and serum blood derivatives from capillary blood microsamples (500 μL, MiniCollect tubes) and corresponding venous blood (10 mL vacutainers). Samples from 20 healthy participants were analyzed by 1 H NMR, and 112 lipoprotein subfraction parameters; 3 supramolecular phospholipid composite (SPC) parameters from SPC 1 , SPC 2 , and SPC 3 subfractions; 2 N-acetyl signals from α-1-acid glycoprotein (Glyc), GlycA, and GlycB; and 3 calculated parameters, SPC (total), SPC 3 /SPC 2 , and Glyc (total) were assessed. Using linear regression between capillary and venous collection sites, we explained that agreement (Adj. R 2 ≥ 0.8, p < 0.001) was witnessed for 86% of plasma parameters (103/120) and 88% of serum parameters (106/120), indicating that capillary lipoprotein, SPC, and Glyc concentrations follow changes in venous concentrations. These results indicate that capillary blood microsamples are suitable for sampling in remote areas and for high-frequency longitudinal sampling of the majority of lipoproteins, SPCs, and Glycs.

To ensure biological validity in metabolic phenotyping, findings must be replicated in independent sample sets. Targeted workflows have long been heralded as ideal platforms for such validation due to their robust quantitative capability. We evaluated the capability of liquid chromatography-mass spectrometry (LC-MS) assays targeting organic acids and bile acids to validate metabolic phenotypes of SARS-CoV-2 infection. Two independent sample sets were collected: (1) Australia: plasma, SARS-CoV-2 positive (
= 20), noninfected healthy controls (
= 22) and COVID-19 disease-like symptoms but negative for SARS-CoV-2 infection (
= 22). (2) Spain: serum, SARS-CoV-2 positive (
= 33) and noninfected healthy controls (
= 39). Multivariate modeling using orthogonal projections to latent structures discriminant analyses (OPLS-DA) classified healthy controls from SARS-CoV-2 positive (Australia;
= 0.17, ROC-AUC = 1; Spain
= 0.20, ROC-AUC = 1). Univariate analyses revealed 23 significantly different (
< 0.05) metabolites between healthy controls and SARS-CoV-2 positive individuals across both cohorts. Significant metabolites revealed consistent perturbations in cellular energy metabolism (pyruvic acid, and 2-oxoglutaric acid), oxidative stress (lactic acid, 2-hydroxybutyric acid), hypoxia (2-hydroxyglutaric acid, 5-aminolevulinic acid), liver activity (primary bile acids), and host-gut microbial cometabolism (hippuric acid, phenylpropionic acid, indole-3-propionic acid). These data support targeted LC-MS metabolic phenotyping workflows for biological validation in independent sample sets.

Whilst wound repair in severe burns has received substantial research attention, non-severe burns (<20% total body surface area) remain relatively understudied, despite causing considerable physiological impact and constituting most of the hospital admissions for burns. Early prediction of healing outcomes would decrease financial and patient burden, and aid in preventing long-term complications from poor wound healing. Lipids have been implicated in inflammation and tissue repair and may play essential roles in burn wound healing. In this study, plasma samples were collected from 20 non-severe burn patients over 6 weeks from admission, including surgery, and analysed by liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance spectroscopy to detect 850 lipids and 112 lipoproteins. Orthogonal projections to latent structures-discriminant analysis was performed to identify changes associated with re-epithelialisation and delayed re-epithelisation.
We demonstrated that the lipid and lipoprotein profiles at admission could predict re-epithelisation outcomes at 2 weeks post-surgery, and that these discriminatory profiles were maintained up to 6 weeks post-burn. Inflammatory markers GlycB and C-reactive protein indicated divergent systemic responses to the burn injury at admission. Triacylglycerols, diacylglycerols and low-density lipoprotein subfractions were associated with delayed wound closure (p-value <0.02, Cliff’s delta >0.7), whilst high-density lipoprotein subfractions, phosphatidylinositols, phosphatidylcholines, and phosphatidylserines were associated with re-epithelisation at 2 weeks post-surgery (p-value <0.01, Cliff’s delta <-0.7). Further model validation will potentially lead to personalised intervention strategies to reduce the risk of chronic complications post-burn injury.
Abbreviations

AUROCArea under receiver operating characteristicsCABINRandomised placebo-controlled trial of Celecoxib for Acute Burn Inflammation and FeverCRPC-reactive proteinCVCross-validatedDIREDiffusion and relaxation editingDGDiacylglycerolDREDelayed wound re-epithelisationFAFatty acylHDLHigh-density lipoproteinIDLIntermediate-density lipoproteinIPAIsopropyl alcoholIQRInter-quartile rangeLC-QQQ-MSLiquid chromatography-tandem mass spectrometryLDCHLow-density lipoprotein cholesterolLDHDLow-density lipoprotein / high-density lipoprotein ratioLDLLow-density lipoproteinLDPLLow-density lipoprotein phospholipidsLPILysophosphatidylinositolNMRNuclear magnetic resonanceOPLS-DAOrthogonal projections to latent structures-discriminant analysisPCPhosphotidylcholinePCAPrincipal component analysisPGPhosphatidylglycerolPIPhosphatidylinositolPLSPartial least squaresPOSASPatient and observer scar assessment scalePSPhosphotidylserineQCQuality controlQTRAPQuadrupole ion trapREWound re-epithelisationSDStandard deviationTBSATotal body surface areaTGTriacylglyerolVLDLVery low-density lipoprotein