Bio21 - Research Publications
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ItemVariability of allergens in commercial fish extracts for skin prick testing(WILEY, 2019-07)BACKGROUND: Commercial allergen extracts for allergy skin prick testing (SPT) are widely used for diagnosing fish allergy. However, there is currently no regulatory requirement for standardization of protein and allergen content, potentially impacting the diagnostic reliability of SPTs. We therefore sought to analyse commercial fish extracts for the presence and concentration of fish proteins and in vitro IgE reactivity using serum from fish-allergic patients. METHODS: Twenty-six commercial fish extracts from five different manufacturers were examined. The protein concentrations were determined, protein compositions analysed by mass spectrometry, followed by SDS-PAGE and subsequent immunoblotting with antibodies detecting 4 fish allergens (parvalbumin, tropomyosin, aldolase and collagen). IgE-reactive proteins were identified using serum from 16 children with confirmed IgE-mediated fish allergy, with focus on cod, tuna and salmon extracts. RESULTS: The total protein, allergen concentration and IgE reactivity of the commercial extracts varied over 10-fold between different manufacturers and fish species. The major fish allergen parvalbumin was not detected by immunoblotting in 6/26 extracts. In 7/12 extracts, five known fish allergens were detected by mass spectrometry. For cod and tuna, almost 70% of patients demonstrated the strongest IgE reactivity to collagen, tropomyosin, aldolase A or β-enolase but not parvalbumin. CONCLUSIONS: Commercial fish extracts often contain insufficient amounts of important allergens including parvalbumin and collagen, resulting in low IgE reactivity. A comprehensive proteomic approach for the evaluation of SPT extracts for their utility in allergy diagnostics is presented. There is an urgent need for standardized allergen extracts, which will improve the diagnosis and management of fish allergy.
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ItemOperational Experience of an Open-Access, Subscription-Based Mass Spectrometry and Proteomics Facility(AMER CHEMICAL SOC, 2018-03)This paper discusses the successful adoption of a subscription-based, open-access model of service delivery for a mass spectrometry and proteomics facility. In 2009, the Mass Spectrometry and Proteomics Facility at the University of Melbourne (Australia) moved away from the standard fee for service model of service provision. Instead, the facility adopted a subscription- or membership-based, open-access model of service delivery. For a low fixed yearly cost, users could directly operate the instrumentation but, more importantly, there were no limits on usage other than the necessity to share available instrument time with all other users. All necessary training from platform staff and many of the base reagents were also provided as part of the membership cost. These changes proved to be very successful in terms of financial outcomes for the facility, instrument access and usage, and overall research output. This article describes the systems put in place as well as the overall successes and challenges associated with the operation of a mass spectrometry/proteomics core in this manner. Graphical abstract ᅟ.
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ItemNo Preview AvailableA type III effector antagonizes death receptor signalling during bacterial gut infection(NATURE PUBLISHING GROUP, 2013-09-12)Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonize the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) Escherichia coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonization and interfere with antimicrobial host responses. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death-domain-containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death-inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death-receptor-induced apoptosis. This inhibition depended on the N-acetylglucosamine transferase activity of NleB1, which specifically modified Arg 117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing pathogens antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response.
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ItemNo Preview AvailableA Modular BAM Complex in the Outer Membrane of the α-Proteobacterium Caulobacter crescentus(PUBLIC LIBRARY SCIENCE, 2010-01-08)Mitochondria are organelles derived from an intracellular alpha-proteobacterium. The biogenesis of mitochondria relies on the assembly of beta-barrel proteins into the mitochondrial outer membrane, a process inherited from the bacterial ancestor. Caulobacter crescentus is an alpha-proteobacterium, and the BAM (beta-barrel assembly machinery) complex was purified and characterized from this model organism. Like the mitochondrial sorting and assembly machinery complex, we find the BAM complex to be modular in nature. A approximately 150 kDa core BAM complex containing BamA, BamB, BamD, and BamE associates with additional modules in the outer membrane. One of these modules, Pal, is a lipoprotein that provides a means for anchorage to the peptidoglycan layer of the cell wall. We suggest the modular design of the BAM complex facilitates access to substrates from the protein translocase in the inner membrane.
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ItemStable isotope shifted matrices enable the use of low mass ion precursor scanning for targeted metabolite identification(BMC, 2011-01-17)We describe a method to identify metabolites of proteins that eliminates endogenous background by using stable isotope labeled matrices. This technique allows selective screening of the intact therapeutic molecule and all metabolites using a modified precursor ion scan that monitors low molecular weight fragment ions produced during MS/MS. This distinct set of low mass ions differs between isotopically labeled and natural isotope containing species allowing excellent discrimination between endogenous compounds and target analytes. All compounds containing amino acids that consist of naturally abundant isotopes can be selected using this scanning technique for further analysis, including metabolites of the parent molecule. The sensitivity and selectivity of this technique is discussed with specific examples of insulin metabolites identified within a complex matrix using a range of different validated low mass target ions.
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ItemA rigorous method to enrich for exosomes from brain tissue(TAYLOR & FRANCIS LTD, 2017-07-26)Extracellular vesicles, including exosomes, are released by all cells, including those of the nervous system. Capable of delivering lipid, protein and nucleic acids to both nearby and distal cells, exosomes have been hypothesized to play a role in progression of many diseases of the nervous system. To date, most analyses on the role of these vesicles in the healthy and diseased state have relied on studying vesicles from in vitro sources, such as conditioned cell culture media, or body fluids. Here we have taken a critical approach to the enrichment and characterization of exosomes from human frontal cortex. This method maintains the integrity of the vesicles and their cargo, and comprehensive proteomic and genomic characterization confirms the legitimacy of the resulting extracellular vesicles as endosome-derived exosomes. This method will enable neuroscientists to acquire more detailed information about exosomes in the brain and explore the role(s) this form of intercellular communication and unique source of lipid, protein and RNA has in healthy brain function and pathogenic conditions. Furthermore, this method may have important utility in the isolation of exosomes from other tissues.
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ItemIntersectin-1 interacts with the golgin GCC88 to couple the actin network and Golgi architecture(AMER SOC CELL BIOLOGY, 2019-02-01)The maintenance of the Golgi ribbon relies on a dynamic balance between the actin and microtubule networks; however, the pathways controlling actin networks remain poorly defined. Previously, we showed that the trans-Golgi network (TGN) membrane tether/golgin, GCC88, modulates the Golgi ribbon architecture. Here, we show that dispersal of the Golgi ribbon by GCC88 is dependent on actin and the involvement of nonmuscle myosin IIA. We have identified the long isoform of intersectin-1 (ITSN-1), a guanine nucleotide exchange factor for Cdc42, as a novel Golgi component and an interaction partner of GCC88 responsible for mediating the actin-dependent dispersal of the Golgi ribbon. We show that perturbation of Golgi morphology by changes in membrane flux, mediated by silencing the retromer subunit Vps26, or in a model of neurodegeneration, induced by Tau overexpression, are also dependent on the ITSN-1-GCC88 interaction. Overall, our study reveals a role for a TGN golgin and ITSN-1 in linking to the actin cytoskeleton and regulating the balance between a compact Golgi ribbon and a dispersed Golgi, a pathway with relevance to pathophysiological conditions.
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ItemDafachronic acid promotes larval development in Haemonchus contortus by modulating dauer signalling and lipid metabolism(PUBLIC LIBRARY SCIENCE, 2019-07)Here, we discovered an endogenous dafachronic acid (DA) in the socioeconomically important parasitic nematode Haemonchus contortus. We demonstrate that DA promotes larval exsheathment and development in this nematode via a relatively conserved nuclear hormone receptor (DAF-12). This stimulatory effect is dose- and time-dependent, and relates to a modulation of dauer-like signalling, and glycerolipid and glycerophospholipid metabolism, likely via a negative feedback loop. Specific chemical inhibition of DAF-9 (cytochrome P450) was shown to significantly reduce the amount of endogenous DA in H. contortus; compromise both larval exsheathment and development in vitro; and modulate lipid metabolism. Taken together, this evidence shows that DA plays a key functional role in the developmental transition from the free-living to the parasitic stage of H. contortus by modulating the dauer-like signalling pathway and lipid metabolism. Understanding the intricacies of the DA-DAF-12 system and associated networks in H. contortus and related parasitic nematodes could pave the way to new, nematode-specific treatments.
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ItemA proteomic characterization shows differences in the milk fat globule membrane of buffalo and bovine milk(Elsevier, 2017-09-01)The proteins of the milk fat globule membrane (MFGM) have a number of functions, such as the regulation of milk fat secretion and metabolism, the uptake and transportation of fatty acids in the intestine, and potential protection from bacterial or viral infection. While the proteome of the MFGM in bovine milk has been extensively characterized, knowledge of these proteins in buffalo milk is limited. In this study, a proteomic approach was used to characterize the proteome of the buffalo MFGM. Multiple extraction techniques were used to increase the number of proteins identified, while label free relative quantitative liquid chromatography tandem mass spectrometry was used for comparison between the buffalo and bovine MFGM proteomes. A total of 220 buffalo MFGM proteins and 234 bovine MFGM proteins were identified after being filtered from the initial dataset of 757 and 680 proteins, respectively. A sixfold higher concentration of xanthine oxidoreductase was identified per mass of buffalo MFGM protein extracted, together with significantly greater concentrations of platelet glycoprotein 4, heat shock cognate and calcineurin B homologous protein. The expression of xanthine oxidoreductase in the MFGM of buffalo milk, which can affect milk shelf-life and flavor, was confirmed by Western blot analysis and a heterogeneous distribution of this protein observed in situ on the surface of the MFGM. The high concentration of fat in buffalo milk, together with the differences in the MFGM proteome provide insights into the differences in nutritional profile, biological function and properties of these two milk products.
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ItemQuantitative proteomic analyses of dynamic signalling events in cortical neurons undergoing excitotoxic cell death(NATURE PUBLISHING GROUP, 2019-03-01)Excitotoxicity, caused by overstimulation or dysregulation of ionotropic glutamate receptors (iGluRs), is a pathological process directing neuronal death in many neurological disorders. The aberrantly stimulated iGluRs direct massive influx of calcium ions into the affected neurons, leading to changes in expression and phosphorylation of specific proteins to modulate their functions and direct their participation in the signalling pathways that induce excitotoxic neuronal death. To define these pathways, we used quantitative proteomic approaches to identify these neuronal proteins (referred to as the changed proteins) and determine how their expression and/or phosphorylation dynamically changed in association with excitotoxic cell death. Our data, available in ProteomeXchange with identifier PXD008353, identified over 100 changed proteins exhibiting significant alterations in abundance and/or phosphorylation levels at different time points (5-240 min) in neurons after glutamate overstimulation. Bioinformatic analyses predicted that many of them are components of signalling networks directing defective neuronal morphology and functions. Among them, the well-known neuronal survival regulators including mitogen-activated protein kinases Erk1/2, glycogen synthase kinase 3 (GSK3) and microtubule-associated protein (Tau), were selected for validation by biochemical approaches, which confirmed the findings of the proteomic analysis. Bioinformatic analysis predicted Protein Kinase B (Akt), c-Jun kinase (JNK), cyclin-dependent protein kinase 5 (Cdk5), MAP kinase kinase (MEK), Casein kinase 2 (CK2), Rho-activated protein kinase (Rock) and Serum/glucocorticoid-regulated kinase 1 (SGK1) as the potential upstream kinases phosphorylating some of the changed proteins. Further biochemical investigation confirmed the predictions of sustained changes of the activation states of neuronal Akt and CK2 in excitotoxicity. Thus, future investigation to define the signalling pathways directing the dynamic alterations in abundance and phosphorylation of the identified changed neuronal proteins will help elucidate the molecular mechanism of neuronal death in excitotoxicity.