School of BioSciences - Research Publications
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ItemEpidermal bladder cells confer salinity stress tolerance in the halophyte quinoa and Atriplex species(WILEY, 2017-09)Epidermal bladder cells (EBCs) have been postulated to assist halophytes in coping with saline environments. However, little direct supporting evidence is available. Here, Chenopodium quinoa plants were grown under saline conditions for 5 weeks. One day prior to salinity treatment, EBCs from all leaves and petioles were gently removed by using a soft cosmetic brush and physiological, ionic and metabolic changes in brushed and non-brushed leaves were compared. Gentle removal of EBC neither initiated wound metabolism nor affected the physiology and biochemistry of control-grown plants but did have a pronounced effect on salt-grown plants, resulting in a salt-sensitive phenotype. Of 91 detected metabolites, more than half were significantly affected by salinity. Removal of EBC dramatically modified these metabolic changes, with the biggest differences reported for gamma-aminobutyric acid (GABA), proline, sucrose and inositol, affecting ion transport across cellular membranes (as shown in electrophysiological experiments). This work provides the first direct evidence for a role of EBC in salt tolerance in halophytes and attributes this to (1) a key role of EBC as a salt dump for external sequestration of sodium; (2) improved K+ retention in leaf mesophyll and (3) EBC as a storage space for several metabolites known to modulate plant ionic relations.
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ItemLipidomics reveal the protective effects of a vegetable-derived isothiocyanate against retinal degeneration.(F1000 Research Ltd, 2019)Background: Age-related macular degeneration (AMD) is a leading cause of blindness in the ageing population. Without effective treatment strategies that can prevent disease progression, there is an urgent need for novel therapeutic interventions to reduce the burden of vision loss and improve patients' quality of life. Dysfunctional innate immune responses to oxidative stress observed in AMD can be caused by the formation of oxidised lipids, whilst polyunsaturated fatty acids have shown to increase the risk of AMD and disease progression in affected individuals. Previously, our laboratory has shown that the vegetable-derived isothiocyanate, L-sulforaphane (LSF), can protect human adult pigment epithelial cells from oxidative damage by upregulating gene expression of the oxidative stress enzyme Glutathione-S-Transferase µ1. This study aims to validate the protective effects of LSF on human retinal cells under oxidative stress conditions and to reveal the key players in fatty acid and lipid metabolism that may facilitate this protection. Methods: The in vitro oxidative stress model of AMD was based on the exposure of an adult retinal pigment epithelium-19 cell line to 200µM hydrogen peroxide. Percentage cell proliferation following LSF treatment was measured using tetrazolium salt-based assays. Untargeted fatty acid profiling was performed by gas chromatography-mass spectrometry. Untargeted lipid profiling was performed by liquid chromatography-mass spectrometry. Results: Under hydrogen peroxide-induced oxidative stress conditions, LSF treatment induced dose-dependent cell proliferation. The key fatty acids that were increased by LSF treatment of the retinal cells include oleic acid and eicosatrienoic acid. LSF treatment also increased levels of the lipid classes phosphatidylcholine, cholesteryl ester and oxo-phytodienoic acid but decreased levels of phosphatidylethanolamine lipids. Conclusions: We propose that retinal cells at risk of oxidative damage and apoptosis can be pre-conditioned with LSF to regulate levels of selected fatty acids and lipids known to be implicated in the pathogenesis and progression of AMD.
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ItemAndrogen receptor antagonism accelerates disease onset in the SOD1G93A mouse model of amyotrophic lateral sclerosis(WILEY, 2019-07)BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease typically more common in males, implicating androgens in progression of both patients and mouse models. Androgen effects are mediated by androgen receptor which is highly expressed in spinal motor neurons and skeletal muscles. To clarify the role of androgen receptors in ALS, we therefore examined the effect of androgen receptor antagonism in the SOD1G93A mouse model. EXPERIMENTAL APPROACH: The androgen receptor antagonist, flutamide, was administered to presymptomatic SOD1G93A mice as a slow-release subcutaneous implant (5 mg·day-1 ). Testosterone, flutamide, and metabolite levels were measured in blood and spinal cord tissue by LC-MS-MS. Effects on disease onset and progression were assessed using motor function tests, survival, muscle, and neuropathological analyses. KEY RESULTS: Flutamide was metabolised to 2-hydroxyflutamide achieving steady-state plasma levels across the study duration and reached the spinal cord at pharmacologically active concentrations. Flutamide treatment accelerated disease onset and locomotor dysfunction in male SOD1G93A mice, but not female mice, without affecting survival. Analysis of hindlimb muscles revealed exacerbation of myofibre atrophy in male SOD1G93A mice treated with flutamide, although motor neuron pathology was not affected. CONCLUSION AND IMPLICATIONS: The androgen receptor antagonist accelerated disease onset in male SOD1G93A mice, leading to exacerbated muscle pathology, consistent with a role of androgens in modulating disease severity, sexual dimorphism, and peripheral pathology in ALS. These results also demonstrate a key contribution of skeletal muscle pathology to disease onset, but not outcome, in this mouse model of ALS.
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ItemSingle cell-type analysis of cellular lipid remodelling in response to salinity in the epidermal bladder cells of the model halophyte Mesembryanthemum crystallinum(WILEY, 2018-10)Salt stress causes dramatic changes in the organization and dynamic properties of membranes, however, little is known about the underlying mechanisms involved. Modified trichomes, known as epidermal bladder cells (EBC), on the leaves and stems of the halophyte Mesembryanthemum crystallinum can be successfully exploited as a single-cell-type system to investigate salt-induced changes to cellular lipid composition. In this study, alterations in key molecular species from different lipid classes highlighted an increase in phospholipid species, particularly those from phosphatidylcholine and phosphatidic acid (PA), where the latter is central to the synthesis of membrane lipids. Triacylglycerol (TG) species decreased during salinity, while there was little change in plastidic galactolipids. EBC transcriptomic and proteomic data mining revealed changes in genes and proteins involved in lipid metabolism and the upregulation of transcripts for PIPKIB, PI5PII, PIPKIII, and phospholipase D delta suggested the induction of signalling processes mediated by phosphoinositides and PA. TEM and flow cytometry showed the dynamic nature of lipid droplets in these cells under salt stress. Altogether, this work indicates that the metabolism of TG might play an important role in EBC response to salinity as either an energy reserve for sodium accumulation and/or driving membrane biosynthesis for EBC expansion.
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ItemTwo strings to the systems biology bow: Co-extracting the metabolome and proteome of yeast(Springer Science and Business Media LLC, 2013-02-01)
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ItemIdentification of Plant-like Galactolipids in Chromera velia, a Photosynthetic Relative of Malaria Parasites(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2011-08-26)Apicomplexa are protist parasites that include Plasmodium spp., the causative agents of malaria, and Toxoplasma gondii, responsible for toxoplasmosis. Most Apicomplexa possess a relict plastid, the apicoplast, which was acquired by secondary endosymbiosis of a red alga. Despite being nonphotosynthetic, the apicoplast is otherwise metabolically similar to algal and plant plastids and is essential for parasite survival. Previous studies of Toxoplasma gondii identified membrane lipids with some structural features of plastid galactolipids, the major plastid lipid class. However, direct evidence for the plant-like enzymes responsible for galactolipid synthesis in Apicomplexan parasites has not been obtained. Chromera velia is an Apicomplexan relative recently discovered in Australian corals. C. velia retains a photosynthetic plastid, providing a unique model to study the evolution of the apicoplast. Here, we report the unambiguous presence of plant-like monogalactosyldiacylglycerol and digalactosyldiacylglycerol in C. velia and localize digalactosyldiacylglycerol to the plastid. We also provide evidence for a plant-like biosynthesis pathway and identify candidate galactosyltranferases responsible for galactolipid synthesis. Our study provides new insights in the evolution of these important enzymes in plastid-containing eukaryotes and will help reconstruct the evolution of glycerolipid metabolism in important parasites such as Plasmodium and Toxoplasma.
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ItemMuscle p70S6K phosphorylation in response to soy and dairy rich meals in middle aged men with metabolic syndrome: a randomised crossover trial(BIOMED CENTRAL LTD, 2014-09-30)BACKGROUND: The mammalian target of rapamycin (mTOR) pathway is the primary regulator of muscle protein synthesis. Metabolic syndrome (MetS) is characterized by central obesity and insulin resistance; little is known about how MetS affects the sensitivity of the mTOR pathway to feeding. METHODS: The responsiveness of mTOR pathway targets such as p706Sk to a high protein meal containing either dairy or soy foods was investigated in healthy insulin sensitive middle-aged men and those presenting with metabolic syndrome (MetS). Twenty male subjects (10 healthy controls, 10 MetS) participated in a single-blinded randomized cross-over study. In a random sequence, subjects ingested energy-matched breakfasts composed predominately of either dairy-protein or soy-protein foods. Skeletal muscle biopsies were collected in the fasted state and at 2 and 4 h post-meal ingestion for the analysis of mTOR- and insulin-signalling kinase activation. RESULTS: Phosphorylated Akt and Insulin receptor substrate 1 (IRS1) increased during the postabsorptive period with no difference between groups. mTOR (Ser448) and ribosomal protein S6 phosphorylation increased 2 h following dairy meal consumption only. p70S6K (Thr389) phosphorylation was increased after feeding only in the control subjects and not in the MetS group. CONCLUSIONS: These data demonstrate that the consumption of a dairy-protein rich but not a soy-protein rich breakfast activates the phosphorylation of mTOR and ribosomal protein S6, required for protein synthesis in human skeletal muscle. Unlike healthy controls, subjects with MetS did not increase muscle p70S6K(Thr389) phosphorylation in response to a mixed meal. TRIAL REGISTRATION: This trial was registered with the Australian New Zealand Clinical Trials Registry (ANZCTR) as ACTRN12610000562077.
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ItemLipidomic Profiling of Adipose Tissue Reveals an Inflammatory Signature in Cancer-Related and Primary Lymphedema(PUBLIC LIBRARY SCIENCE, 2016-05-16)Cancer-related and primary lymphedema (LE) are associated with the production of adipose tissue (AT). Nothing is known, however, about the lipid-based molecules that comprise LE AT. We therefore analyzed lipid molecules in lipoaspirates and serum obtained from LE patients, and compared them to lipoaspirates from cosmetic surgery patients and healthy control cohort serum. LE patient serum analysis demonstrated that triglycerides, HDL- and LDL-cholesterol and lipid transport molecules remained within the normal range, with no alterations in individual fatty acids. The lipidomic analysis also identified 275 lipid-based molecules, including triacylglycerides, diacylglycerides, fatty acids and phospholipids in AT oil and fat. Although the majority of lipid molecules were present in a similar abundance in LE and non-LE samples, there were several small changes: increased C20:5-containing triacylglycerides, reduced C10:0 caprinic and C24:1 nervonic acids. LE AT oil also contained a signature of increased cyclopropane-type fatty acids and inflammatory mediators arachidonic acid and ceramides. Interestingly C20:5 and C22:6 omega-3-type lipids are increased in LE AT, correlating with LE years. Hence, LE AT has a normal lipid profile containing a signature of inflammation and omega-3-lipids. It remains unclear, however, whether these differences reflect a small-scale global metabolic disturbance or effects within localised inflammatory foci.
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ItemPhospholipase A2 activity during the replication cycle of the flavivirus West Nile virus(PUBLIC LIBRARY SCIENCE, 2018-04)Positive-sense RNA virus intracellular replication is intimately associated with membrane platforms that are derived from host organelles and comprised of distinct lipid composition. For flaviviruses, such as West Nile virus strain Kunjin virus (WNVKUN) we have observed that these membrane platforms are derived from the endoplasmic reticulum and are rich in (at least) cholesterol. To extend these studies and identify the cellular lipids critical for WNVKUN replication we utilized a whole cell lipidomics approach and revealed an elevation in phospholipase A2 (PLA2) activity to produce lyso-phosphatidylcholine (lyso-PChol). We observed that the PLA2 enzyme family is activated in WNVKUN-infected cells and the generated lyso-PChol lipid moieties are sequestered to the subcellular sites of viral replication. The requirement for lyso-PChol was confirmed using chemical inhibition of PLA2, where WNVKUN replication and production of infectious virus was duly affected in the presence of the inhibitors. Importantly, we could rescue chemical-induced inhibition with the exogenous addition of lyso-PChol species. Additionally, electron microscopy results indicate that lyso-PChol appears to contribute to the formation of the WNVKUN membranous replication complex (RC); particularly affecting the morphology and membrane curvature of vesicles comprising the RC. These results extend our current understanding of how flaviviruses manipulate lipid homeostasis to favour their own intracellular replication.