School of Biomedical Sciences - Research Publications

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    Charge Has a Marked Influence on Hyperbranched Polymer Nanoparticle Association in Whole Human Blood
    Glass, JJ ; Chen, L ; Alcantara, S ; Crampin, EJ ; Thurecht, KJ ; De Rose, R ; Kent, SJ (AMER CHEMICAL SOC, 2017-06)
    In this study, we synthesize charge-varied hyperbranched polymers (HBPs) and demonstrate surface charge as a key parameter directing their association with specific human blood cell types. Using fresh human blood, we investigate the association of 5 nm HBPs with six white blood cell populations in their natural milieu by flow cytometry. While most cell types associate with cationic HBPs at 4 °C, at 37 °C phagocytic cells display similar (monocyte, dendritic cell) or greater (granulocyte) association with anionic HBPs compared to cationic HBPs. Neutral HBPs display remarkable stealth properties. Notably, these charge-association patterns are not solely defined by the plasma protein corona and are material and/or size dependent. As HBPs progress toward clinical use as imaging and drug delivery agents, the ability to engineer HBPs with defined biological properties is increasingly important. This knowledge can be used in the rational design of HBPs for more effective delivery to desired cell targets.
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    Observational study of alternative therapies among paediatric emergency department patients
    Ding, J-L ; Taylor, DM ; Lee, M ; Johnson, OG ; Ashok, A ; Griffiths, M ; Simma, L ; Craig, SS ; Cheek, JA ; Babl, FE (WILEY, 2017-04)
    OBJECTIVE: While complementary medicine use among ED paediatric patients is common, the use of alternative therapies (ATs; physical or spiritual therapies) is unknown. We aimed to determine the 12 month period prevalence and nature of AT use among paediatric patients and parent perceptions of AT use. METHODS: We undertook a cross-sectional survey of a convenience sample of parents of paediatric patients in three EDs in metropolitan Melbourne, Australia (January-June, 2015). Parents were invited to complete a validated, anonymous, self-administered questionnaire. The main outcomes were AT use by the patient and parent perceptions of ATs. RESULTS: A total of 806 parents were enrolled. In the previous 12 months, 393 (48.8%) patients had received at least one AT. There were no gender or ethnicity differences between AT users and non-users. AT use was more common among older patients (P < 0.05). Patients with chronic illness tended to use more ATs (P = 0.12). A total of 1091 courses of 43 different ATs had been provided. The most common were massage (16% of patients), chiropractic therapy (9.8%), relaxation (7.2%), meditation (6.2%) and aromatherapy (6.1%). ATs were generally used for musculoskeletal problems, health maintenance, stress and anxiety. Parents who arranged the ATs were significantly more likely to report that ATs are safe, prevent and treat illness, assist prescription medicines and offer a more holistic approach to healthcare (P < 0.001). CONCLUSION: AT use is common among paediatric ED patients. Parents who arrange AT have differing perceptions of AT usefulness and safety from those who do not.
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    Cell cycle-regulated PLEIADE/AtMAP65-3 links membrane and microtubule dynamics during plant cytokinesis
    Steiner, A ; Rybak, K ; Altmann, M ; McFarlane, HE ; Klaeger, S ; Ngoc, N ; Facher, E ; Ivakov, A ; Wanner, G ; Kuster, B ; Persson, S ; Braun, P ; Hauser, M-T ; Assaad, FF (WILEY-BLACKWELL, 2016-11)
    Cytokinesis, the partitioning of the cytoplasm following nuclear division, requires extensive coordination between cell cycle cues, membrane trafficking and microtubule dynamics. Plant cytokinesis occurs within a transient membrane compartment known as the cell plate, to which vesicles are delivered by a plant-specific microtubule array, the phragmoplast. While membrane proteins required for cytokinesis are known, how these are coordinated with microtubule dynamics and regulated by cell cycle cues remains unclear. Here, we document physical and genetic interactions between Transport Protein Particle II (TRAPPII) tethering factors and microtubule-associated proteins of the PLEIADE/AtMAP65 family. These interactions do not specifically affect the recruitment of either TRAPPII or MAP65 proteins to the cell plate or midzone. Rather, and based on single versus double mutant phenotypes, it appears that they are required to coordinate cytokinesis with the nuclear division cycle. As MAP65 family members are known to be targets of cell cycle-regulated kinases, our results provide a conceptual framework for how membrane and microtubule dynamics may be coordinated with each other and with the nuclear cycle during plant cytokinesis.
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    Structural, kinetic and computational investigation of Vitis vinifera DHDPS reveals new insight into the mechanism of lysine-mediated allosteric inhibition
    Atkinson, SC ; Dogovski, C ; Downton, MT ; Czabotar, PE ; Dobson, RCJ ; Gerrard, JA ; Wagner, J ; Perugini, MA (SPRINGER, 2013-03)
    Lysine is one of the most limiting amino acids in plants and its biosynthesis is carefully regulated through inhibition of the first committed step in the pathway catalyzed by dihydrodipicolinate synthase (DHDPS). This is mediated via a feedback mechanism involving the binding of lysine to the allosteric cleft of DHDPS. However, the precise allosteric mechanism is yet to be defined. We present a thorough enzyme kinetic and thermodynamic analysis of lysine inhibition of DHDPS from the common grapevine, Vitis vinifera (Vv). Our studies demonstrate that lysine binding is both tight (relative to bacterial DHDPS orthologs) and cooperative. The crystal structure of the enzyme bound to lysine (2.4 Å) identifies the allosteric binding site and clearly shows a conformational change of several residues within the allosteric and active sites. Molecular dynamics simulations comparing the lysine-bound (PDB ID 4HNN) and lysine free (PDB ID 3TUU) structures show that Tyr132, a key catalytic site residue, undergoes significant rotational motion upon lysine binding. This suggests proton relay through the catalytic triad is attenuated in the presence of lysine. Our study reveals for the first time the structural mechanism for allosteric inhibition of DHDPS from the common grapevine.
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    From Knock-Out Phenotype to Three-Dimensional Structure of a Promising Antibiotic Target from Streptococcus pneumoniae
    Dogovski, C ; Gorman, MA ; Ketaren, NE ; Praszkier, J ; Zammit, LM ; Mertens, HD ; Bryant, G ; Yang, J ; Griffin, MDW ; Pearce, FG ; Gerrard, JA ; Jameson, GB ; Parker, MW ; Robins-Browne, RM ; Perugini, MA ; Taylor, P (PUBLIC LIBRARY SCIENCE, 2013-12-13)
    Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S. pneumoniae (sp) lacking the DHDPS gene is unable to grow unless supplemented with lysine-rich media. We subsequently set out to characterize the structure, function and stability of the enzyme drug target. Our studies show that sp-DHDPS is folded and active with a k(cat) = 22 s(-1), K(M)(PYR) = 2.55 ± 0.05 mM and K(M)(ASA) = 0.044 ± 0.003 mM. Thermal denaturation experiments demonstrate sp-DHDPS exhibits an apparent melting temperature (T(M)(app)) of 72 °C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (T(M)(app) = 59 °C). Sedimentation studies show that sp-DHDPS exists in a dimer-tetramer equilibrium with a K(D)(4→2) = 1.7 nM, which is considerably tighter than its E. coli ortholog (K(D)(4→2) = 76 nM). To further characterize the structure of the enzyme and probe its enhanced stability, we solved the high resolution (1.9 Å) crystal structure of sp-DHDPS (PDB ID 3VFL). The enzyme is tetrameric in the crystal state, consistent with biophysical measurements in solution. Although the sp-DHDPS and Ec-DHDPS active sites are almost identical, the tetramerization interface of the s. pneumoniae enzyme is significantly different in composition and has greater buried surface area (800 Å(2)) compared to its E. coli counterpart (500 Å(2)). This larger interface area is consistent with our solution studies demonstrating that sp-DHDPS is considerably more thermally and thermodynamically stable than Ec-DHDPS. Our study describe for the first time the knock-out phenotype, solution properties, stability and crystal structure of DHDPS from S. pneumoniae, a promising antimicrobial target.
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    A method for quantifying pulmonary Legionella pneumophila infection in mouse lungs by flow cytometry.
    Ang, DKY ; Ong, SY ; Brown, AS ; Hartland, EL ; van Driel, IR (Springer Science and Business Media LLC, 2012-08-20)
    BACKGROUND: Pulmonary load of Legionella pneumophila in mice is normally determined by counting serial dilutions of bacterial colony forming units (CFU) on agar plates. This process is often tedious and time consuming. We describe a novel, rapid and versatile flow cytometric method that detects bacteria phagocytosed by neutrophils. FINDINGS: Mice were infected with L. pneumophila via intratracheal or intranasal administration. At various times after bacteria inoculation, mouse lungs were harvested and analysed concurrently for bacterial load by colony counting and flow cytometry analysis. The number of L. pneumophila-containing neutrophils correlated strongly with CFU obtained by bacteriological culture. CONCLUSIONS: This technique can be utilised to determine pulmonary bacterial load and may be used in conjunction with other flow cytometric based analyses of the resulting immune response.
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    Crystal, Solution and In silico Structural Studies of Dihydrodipicolinate Synthase from the Common Grapevine
    Atkinson, SC ; Dogovski, C ; Downton, MT ; Pearce, FG ; Reboul, CF ; Buckle, AM ; Gerrard, JA ; Dobson, RCJ ; Wagner, J ; Perugini, MA ; Kursula, P (PUBLIC LIBRARY SCIENCE, 2012-06-25)
    Dihydrodipicolinate synthase (DHDPS) catalyzes the rate limiting step in lysine biosynthesis in bacteria and plants. The structure of DHDPS has been determined from several bacterial species and shown in most cases to form a homotetramer or dimer of dimers. However, only one plant DHDPS structure has been determined to date from the wild tobacco species, Nicotiana sylvestris (Blickling et al. (1997) J. Mol. Biol. 274, 608-621). Whilst N. sylvestris DHDPS also forms a homotetramer, the plant enzyme adopts a 'back-to-back' dimer of dimers compared to the 'head-to-head' architecture observed for bacterial DHDPS tetramers. This raises the question of whether the alternative quaternary architecture observed for N. sylvestris DHDPS is common to all plant DHDPS enzymes. Here, we describe the structure of DHDPS from the grapevine plant, Vitis vinifera, and show using analytical ultracentrifugation, small-angle X-ray scattering and X-ray crystallography that V. vinifera DHDPS forms a 'back-to-back' homotetramer, consistent with N. sylvestris DHDPS. This study is the first to demonstrate using both crystal and solution state measurements that DHDPS from the grapevine plant adopts an alternative tetrameric architecture to the bacterial form, which is important for optimizing protein dynamics as suggested by molecular dynamics simulations reported in this study.
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    A Convenient Model of Severe, High Incidence Autoimmune Gastritis Caused by Polyclonal Effector T Cells and without Perturbation of Regulatory T Cells
    Tu, E ; Ang, DKY ; Hogan, TV ; Read, S ; Chia, CPZ ; Gleeson, PA ; van Driel, IR ; Piccirillo, CA (PUBLIC LIBRARY SCIENCE, 2011-11-09)
    Autoimmune gastritis results from the breakdown of T cell tolerance to the gastric H(+)/K(+) ATPase. The gastric H(+)/K(+) ATPase is responsible for the acidification of gastric juice and consists of an α subunit (H/Kα) and a β subunit (H/Kβ). Here we show that CD4(+) T cells from H/Kα-deficient mice (H/Kα(-/-)) are highly pathogenic and autoimmune gastritis can be induced in sublethally irradiated wildtype mice by adoptive transfer of unfractionated CD4(+) T cells from H/Kα(-/-) mice. All recipient mice consistently developed the most severe form of autoimmune gastritis 8 weeks after the transfer, featuring hypertrophy of the gastric mucosa, complete depletion of the parietal and zymogenic cells, and presence of autoantibodies to H(+)/K(+) ATPase in the serum. Furthermore, we demonstrated that the disease significantly affected stomach weight and stomach pH of recipient mice. Depletion of parietal cells in this disease model required the presence of both H/Kα and H/Kβ since transfer of H/Kα(-/-) CD4(+) T cells did not result in depletion of parietal cells in H/Kα(-/-) or H/Kβ(-/-) recipient mice. The consistency of disease severity, the use of polyclonal T cells and a specific T cell response to the gastric autoantigen make this an ideal disease model for the study of many aspects of organ-specific autoimmunity including prevention and treatment of the disease.
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    The Golgi apparatus in the endomembrane-rich gastric parietal cells exist as functional stable mini-stacks dispersed throughout the cytoplasm
    Gunn, PA ; Gliddon, BL ; Londrigan, SL ; Lew, AM ; van Driel, IR ; Gleeson, PA (PORTLAND PRESS LTD, 2011-12)
    BACKGROUND INFORMATION: Acid-secreting gastric parietal cells are polarized epithelial cells that harbour highly abundant and specialized, H+,K+ ATPase-containing, tubulovesicular membranes in the apical cytoplasm. The Golgi apparatus has been implicated in the biogenesis of the tubulovesicular membranes; however, an unanswered question is how a typical Golgi organization could regulate normal membrane transport within the membrane-dense cytoplasm of parietal cells. RESULTS: Here, we demonstrate that the Golgi apparatus of parietal cells is not the typical juxta-nuclear ribbon of stacks, but rather individual Golgi units are scattered throughout the cytoplasm. The Golgi membrane structures labelled with markers of both cis- and trans-Golgi membrane, indicating the presence of intact Golgi stacks. The parietal cell Golgi stacks were closely aligned with the microtubule network and were shown to participate in both anterograde and retrograde transport pathways. Dispersed Golgi stacks were also observed in parietal cells from H+,K+ ATPase-deficient mice that lack tubulovesicular membranes. CONCLUSIONS: These results indicate that the unusual organization of individual Golgi stacks dispersed throughout the cytoplasm of these terminally differentiated cells is likely to be a developmentally regulated event.
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    Inhibition of Breast Cancer Resistance Protein (ABCG2) in Human Myeloid Dendritic Cells Induces Potent Tolerogenic Functions during LPS Stimulation
    Jin, J-O ; Zhang, W ; Wong, K-W ; Kwak, M ; van Driel, IR ; Yu, Q ; Wu, Y (PUBLIC LIBRARY SCIENCE, 2014-08-11)
    Breast cancer resistance protein (ABCG2), a member of the ATP-binding cassette transporters has been identified as a major determinant of multidrug resistance (MDR) in cancer cells, but ABC transporter inhibition has limited therapeutic value in vivo. In this research, we demonstrated that inhibition of efflux transporters ABCG2 induced the generation of tolerogenic DCs from human peripheral blood myeloid DCs (mDCs). ABCG2 expression was present in mDCs and was further increased by LPS stimulation. Treatment of CD1c+ mDCs with an ABCG2 inhibitor, Ko143, during LPS stimulation caused increased production of IL-10 and decreased production of pro-inflammatory cytokines and decreased expression of CD83 and CD86. Moreover, inhibition of ABCG2 in monocyte-derived DCs (MDDCs) abrogated the up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines in these cells in response to LPS. Furthermore, CD1c+ mDCs stimulated with LPS plus Ko143 inhibited the proliferation of allogeneic and superantigen-specific syngenic CD4+ T cells and promoted expansion of CD25+FOXP3+ regulatory T (Treg) cells in an IL-10-dependent fashion. These tolerogenic effects of ABCG2 inhibition could be abolished by ERK inhibition. Thus, we demonstrated that inhibition of ABCG2 in LPS-stimulated mDCs can potently induce tolerogenic potentials in these cells, providing crucial new information that could lead to development of better strategies to combat MDR cancer.