School of Biomedical Sciences - Research Publications

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    Insect Antennal Morphology: The Evolution of Diverse Solutions to Odorant Perception
    Elgar, MA ; Zhang, D ; Wang, Q ; Wittwer, B ; Hieu, TP ; Johnson, TL ; Freelance, CB ; Coquilleau, M (Yale University, 2018-12-01)
    Chemical communication involves the production, transmission, and perception of odors. Most adult insects rely on chemical signals and cues to locate food resources, oviposition sites or reproductive partners and, consequently, numerous odors provide a vital source of information. Insects detect these odors with receptors mostly located on the antennae, and the diverse shapes and sizes of these antennae (and sensilla) are both astonishing and puzzling: what selective pressures are responsible for these different solutions to the same problem - to perceive signals and cues? This review describes the selection pressures derived from chemical communication that are responsible for shaping the diversity of insect antennal morphology. In particular, we highlight new technologies and techniques that offer exciting opportunities for addressing this surprisingly neglected and yet crucial component of chemical communication.
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    The potentially beneficial central nervous system activity profile of ivacaftor and its metabolites (vol 4, 00127, 2018)
    Schneider, EK ; McQuade, RM ; Carbone, VC ; Reyes-Ortega, F ; Wilson, JW ; Button, B ; Saito, A ; Poole, DP ; Hoyer, D ; Li, J ; Velkov, T (EUROPEAN RESPIRATORY SOC JOURNALS LTD, 2018-10-01)
    [This corrects the article DOI: 10.1183/23120541.00127-2017.].
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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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    Hypoxic preconditioning of myoblasts implanted in a tissue engineering chamber significantly increases local angiogenesis via upregulation of myoblast vascular endothelial growth factor-A expression and downregulation of miRNA-1, miRNA-206 and angiopoietin-1
    Taylor, CJ ; Church, JE ; Williams, MD ; Gerrand, Y-W ; Keramidaris, E ; Palmer, JA ; Galea, LA ; Penington, AJ ; Morrison, WA ; Mitchell, GM (WILEY, 2018-01)
    Vascularization is a major hurdle for growing three-dimensional tissue engineered constructs. This study investigated the mechanisms involved in hypoxic preconditioning of primary rat myoblasts in vitro and their influence on local angiogenesis postimplantation. Primary rat myoblast cultures were exposed to 90 min hypoxia at <1% oxygen followed by normoxia for 24 h. Real time (RT) polymerase chain reaction evaluation indicated that 90 min hypoxia resulted in significant downregulation of miR-1 and miR-206 (p < 0.05) and angiopoietin-1 (p < 0.05) with upregulation of vascular endothelial growth factor-A (VEGF-A; p < 0.05). The miR-1 and angiopoietin-1 responses remained significantly downregulated after a 24 h rest phase. In addition, direct inhibition of miR-206 in L6 myoblasts caused a significant increase in VEGF-A expression (p < 0.05), further establishing that changes in VEGF-A expression are influenced by miR-206. Of the myogenic genes examined, MyoD was significantly upregulated, only after 24 h rest (p < 0.05). Preconditioned or control myoblasts were implanted with Matrigel™ into isolated bilateral tissue engineering chambers incorporating a flow-through epigastric vascular pedicle in severe combined immunodeficiency mice and the chamber tissue harvested 14 days later. Chambers implanted with preconditioned myoblasts had a significantly increased percentage volume of blood vessels (p = 0.0325) compared with chambers implanted with control myoblasts. Hypoxic preconditioned myoblasts promote vascularization of constructs via VEGF upregulation and downregulation of angiopoietin-1, miR-1 and miR-206. The relatively simple strategy of hypoxic preconditioning of implanted cells - including non-stem cell types - has broad, future applications in tissue engineering of skeletal muscle and other tissues, as a technique to significantly increase implant site angiogenesis.
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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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    Effect of NADPH oxidase 1 and 4 blockade in activated human retinal endothelial cells
    Appukuttan, B ; Ma, Y ; Stempel, A ; Ashander, LM ; Deliyanti, D ; Wilkinson-Berka, JL ; Smith, JR (WILEY, 2018-08)
    BACKGROUND: Over-production of reactive oxygen species (ROS) and resulting oxidative stress contribute to retinal damage in vascular diseases that include diabetic retinopathy, retinopathy of prematurity and major retinal vessel occlusions. NADPH oxidase (Nox) proteins are professional ROS-generating enzymes, and therapeutic targeting in these diseases has strong appeal. Pharmacological inhibition of Nox4 reduces the severity of experimental retinal vasculopathy. We investigated the potential application of this drug approach in humans. METHODS: Differential Nox enzyme expression was studied by real-time-quantitative polymerase chain reaction in primary human retinal endothelial cell isolates and a characterized human retinal endothelial cell line. Oxidative stress was triggered chemically in endothelial cells, by treatment with dimethyloxalylglycine (DMOG; 100 μM); Nox4 and vascular endothelial growth factor (VEGFA) transcript were measured; and production of ROS was detected by 2',7'-dichlorofluorescein. DMOG-stimulated endothelial cells were treated with two Nox1/Nox4 inhibitors, GKT136901 and GKT137831; cell growth was monitored by DNA quantification, in addition to VEGFA transcript and ROS production. RESULTS: Nox4 (isoform Nox4A) was the predominant Nox enzyme expressed by human retinal endothelial cells. Treatment with DMOG significantly increased endothelial cell expression of Nox4 over 72 h, accompanied by ROS production and increased VEGFA expression. Treatment with GKT136901 or GKT137831 significantly reduced DMOG-induced ROS production and VEGFA expression by endothelial cells, and the inhibitory effect of DMOG on cell growth. CONCLUSIONS: Our findings in experiments on activated human retinal endothelial cells provide translational corroboration of studies in experimental models of retinal vasculopathy and support the therapeutic application of Nox4 inhibition by GKT136901 and GKT137831 in patients with retinal vascular diseases.
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    Antennal asymmetry is not associated with social behaviour in Australian Hymenoptera
    Freelance, CB ; Majoe, M ; Tierney, SM ; Elgar, MA (WILEY, 2019-08)
    Abstract Lateralisation of biological form and function are well known for vertebrates and are being increasingly documented among invertebrates in recent years. Behavioural lateralisation in insects, together with asymmetrical distributions of antennal sensilla, has been linked to the communication challenges faced by social, but not solitary, insects. Recent evidence on patterns of asymmetry in insects outside of the Hymenoptera suggests that this explanation for antennal sensilla asymmetry may not be phylogenetically constrained. We explore this possibility by examining the distribution of antennal sensilla in three species of ants (Formicidae), the meat ant Iridomyrmex purpureus (Dolichoderinae), the green tree ant Oecophylla smaragdina (Formicinae) and the shield ant Meranoplus sp. (Myrmicinae) in which colony organisation is eusocial, and two species of nomiine bees, Mellitidia tomentifera and Reepenia bituberculata (Halictidae: Nomiinae), where colony organisation is not eusocial. Our results demonstrate that while there are differences in the left–right asymmetry of antennal sensilla basiconica in workers of the formicine ant I. purpureus, there is no consistent sensilla asymmetry across the five species. We find a negative correlation between antennal sensilla density and body size in R. bituberculata, which was not apparent in the other species. Our results contradict the suggestion that asymmetrical distribution of antennal sensilla is associated with the evolution of eusocial behaviour.
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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.