Bio21 Molecular Science and Biotechnology Institute

Parkville, Australia

Bio21 Molecular Science and Biotechnology Institute

Parkville, Australia
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Akinyi S.,Emory Vaccine Center | Hanssen E.,Bio21 Molecular Science and Biotechnology Institute | Hanssen E.,University of Melbourne | Meyer E.V.S.,Emory Vaccine Center | And 9 more authors.
Molecular Microbiology | Year: 2012

Plasmodium vivax and P. cynomolgi produce numerous caveola-vesicle complex (CVC) structures within the surface of the infected erythrocyte membrane. These contrast with the electron-dense knob protrusions expressed at the surface of Plasmodium falciparum-infected erythrocytes. Here we investigate the three-dimensional (3-D) structure of the CVCs and the identity of a predominantly expressed 95kDa CVC protein. Liquid chromatography - tandem mass spectrometry analysis of immunoprecipitates by monoclonal antibodies from P. cynomolgi extracts identified this protein as a member of the Plasmodium helical interspersed subtelomeric (PHIST) superfamily with a calculated mass of 81kDa. We named the orthologous proteins PvPHIST/CVC-81 95 and PcyPHIST/CVC-81 95, analysed their structural features, including a PEXEL motif, repeated sequences and a C-terminal PHIST domain, and show that PHIST/CVC-81 95 is most highly expressed in trophozoites. We generated images of CVCs in 3-D using electron tomography (ET), and used immuno-ET to show PHIST/CVC-81 95 localizes to the cytoplasmic side of the CVC tubular extensions. Targeted gene disruptions were attempted in vivo. The pcyphist/cvc-81 95 gene was not disrupted, but parasites containing episomes with the tgdhfr selection cassette were retrieved by selection with pyrimethamine. This suggests that PHIST/CVC-81 95 is essential for survival of these malaria parasites. © 2012 Blackwell Publishing Ltd.

Oehme D.P.,IBM | Downton M.T.,IBM | Wagner J.,IBM | Gidley M.J.,University of Melbourne | And 2 more authors.
Plant Physiology | Year: 2015

The question of how many chains an elementary cellulose microfibril contains is critical to understanding the molecular mechanism(s) of cellulose biosynthesis and regulation. Given the hexagonal nature of the cellulose synthase rosette, it is assumed that the number of chains must be a multiple of six. We present molecular dynamics simulations on three different models of Ib cellulose microfibrils, 18, 24, and 36 chains, to investigate their structure and dynamics in a hydrated environment. The 36-chain model stays in a conformational space that is very similar to the initial crystalline phase, while the 18- and 24-chain models sample a conformational space different from the crystalline structure yet similar to conformations observed in recent high-temperature molecular dynamics simulations. Major differences in the conformations sampled between the different models result from changes to the tilt of chains in different layers, specifically a second stage of tilt, increased rotation about the O2-C2 dihedral, and a greater sampling of non-TG exocyclic conformations, particularly the GG conformation in center layers and GT conformation in solvent-exposed exocyclic groups. With a reinterpretation of nuclear magnetic resonance data, specifically for contributions made to the C6 peak, data from the simulations suggest that the 18- and 24-chain structures are more viable models for an elementary cellulose microfibril, which also correlates with recent scattering and diffraction experimental data. These data inform biochemical and molecular studies that must explain how a six-particle cellulose synthase complex rosette synthesizes microfibrils likely comprised of either 18 or 24 chains. © 2014 American Society of Plant Biologists. All Rights Reserved.

Swarbrick C.M.D.,Charles Sturt University | Perugini M.A.,La Trobe University | Perugini M.A.,Bio21 Molecular Science and Biotechnology Institute | Cowieson N.,Australian Synchrotron | Forwood J.K.,Charles Sturt University
Acta Crystallographica Section D: Biological Crystallography | Year: 2015

Acyl-CoA thioesterases catalyse the hydrolysis of the thioester bonds present within a wide range of acyl-CoA substrates, releasing free CoASH and the corresponding fatty-acyl conjugate. The TesB-type thioesterases are members of the TE4 thioesterase family, one of 25 thioesterase enzyme families characterized to date, and contain two fused hotdog domains in both prokaryote and eukaryote homologues. Only two structures have been elucidated within this enzyme family, and much of the current understanding of the TesB thioesterases has been based on the Escherichia coli structure. Yersinia pestis, a highly virulent bacterium, encodes only one TesB-type thioesterase in its genome; here, the structural and functional characterization of this enzyme are reported, revealing unique elements both within the protomer and quaternary arrangements of the hotdog domains which have not been reported previously in any thioesterase family. The quaternary structure, confirmed using a range of structural and biophysical techniques including crystallography, small-angle X-ray scattering, analytical ultracentrifugation and size-exclusion chromatography, exhibits a unique octameric arrangement of hotdog domains. Interestingly, the same biological unit appears to be present in both TesB structures solved to date, and is likely to be a conserved and distinguishing feature of TesB-type thioesterases. Analysis of the Y. pestis TesB thioesterase activity revealed a strong preference for octanoyl-CoA and this is supported by structural analysis of the active site. Overall, the results provide novel insights into the structure of TesB thioesterases which are likely to be conserved and distinguishing features of the TE4 thioesterase family.

Shi Z.,University of Melbourne | Shi Z.,Bio21 Molecular Science and Biotechnology Institute | Wedd A.G.,University of Melbourne | Wedd A.G.,Bio21 Molecular Science and Biotechnology Institute | And 2 more authors.
PLoS ONE | Year: 2013

The development of synthetic biology requires rapid batch construction of large gene networks from combinations of smaller units. Despite the availability of computational predictions for well-characterized enzymes, the optimization of most synthetic biology projects requires combinational constructions and tests. A new building-brick-style parallel DNA assembly framework for simple and flexible batch construction is presented here. It is based on robust recombination steps and allows a variety of DNA assembly techniques to be organized for complex constructions (with or without scars). The assembly of five DNA fragments into a host genome was performed as an experimental demonstration. © 2013 Shi et al.

MacRae J.I.,Bio21 Molecular Science and Biotechnology Institute | Dixon M.W.A.,Bio21 Molecular Science and Biotechnology Institute | Dixon M.W.A.,University of Melbourne | Dearnley M.K.,Bio21 Molecular Science and Biotechnology Institute | And 10 more authors.
BMC Biology | Year: 2013

Background: The carbon metabolism of the blood stages of Plasmodium falciparum, comprising rapidly dividing asexual stages and non-dividing gametocytes, is thought to be highly streamlined, with glycolysis providing most of the cellular ATP. However, these parasitic stages express all the enzymes needed for a canonical mitochondrial tricarboxylic acid (TCA) cycle, and it was recently proposed that they may catabolize glutamine via an atypical branched TCA cycle. Whether these stages catabolize glucose in the TCA cycle and what is the functional significance of mitochondrial metabolism remains unresolved.Results: We reassessed the central carbon metabolism of P. falciparum asexual and sexual blood stages, by metabolically labeling each stage with 13C-glucose and 13C-glutamine, and analyzing isotopic enrichment in key pathways using mass spectrometry. In contrast to previous findings, we found that carbon skeletons derived from both glucose and glutamine are catabolized in a canonical oxidative TCA cycle in both the asexual and sexual blood stages. Flux of glucose carbon skeletons into the TCA cycle is low in the asexual blood stages, with glutamine providing most of the carbon skeletons, but increases dramatically in the gametocyte stages. Increased glucose catabolism in the gametocyte TCA cycle was associated with increased glucose uptake, suggesting that the energy requirements of this stage are high. Significantly, whereas chemical inhibition of the TCA cycle had little effect on the growth or viability of asexual stages, inhibition of the gametocyte TCA cycle led to arrested development and death.Conclusions: Our metabolomics approach has allowed us to revise current models of P. falciparum carbon metabolism. In particular, we found that both asexual and sexual blood stages utilize a conventional TCA cycle to catabolize glucose and glutamine. Gametocyte differentiation is associated with a programmed remodeling of central carbon metabolism that may be required for parasite survival either before or after uptake by the mosquito vector. The increased sensitivity of gametocyte stages to TCA-cycle inhibitors provides a potential target for transmission-blocking drugs. © 2013 MacRae et al.; licensee BioMed Central Ltd.

Riglar D.T.,Walter and Eliza Hall Institute of Medical Research | Riglar D.T.,University of Melbourne | Rogers K.L.,Walter and Eliza Hall Institute of Medical Research | Rogers K.L.,University of Melbourne | And 17 more authors.
Nature Communications | Year: 2013

Export of proteins into the infected erythrocyte is critical for malaria parasite survival. The majority of effector proteins are thought to export via a proteinaceous translocon, resident in the parasitophorous vacuole membrane surrounding the parasite. Identification of the Plasmodium translocon of exported proteins and its biochemical association with exported proteins suggests it performs this role. Direct evidence for this, however, is lacking. Here using viable purified Plasmodium falciparum merozoites and three-dimensional structured illumination microscopy, we investigate remodelling events immediately following parasite invasion.We show that multiple complexes of the Plasmodium translocon of exported proteins localize together in foci that dynamically change in clustering behaviour. Furthermore, we provide conclusive evidence of spatial association between exported proteins and exported protein 2, a core component of the Plasmodium translocon of exported proteins, during native conditions and upon generation of translocation intermediates. These data provide the most direct cellular evidence to date that protein export occurs at regions of the parasitophorous vacuole membrane housing the Plasmodium translocon of exported proteins complex. © 2013 Macmillan Publishers Limited.

Paterson B.M.,University of Melbourne | Paterson B.M.,Bio21 Molecular Science and Biotechnology Institute | Donnelly P.S.,University of Melbourne | Donnelly P.S.,Bio21 Molecular Science and Biotechnology Institute
Chemical Society Reviews | Year: 2011

The molecules known as bis(thiosemicarbazones) derived from 1,2-diones can act as tetradentate ligands for Cu(ii), forming stable, neutral complexes. As a family, these complexes possess fascinating biological activity. This critical review presents an historical perspective of their progression from potential chemotherapeutics through to more recent applications in nuclear medicine. Methods of synthesis are presented followed by studies focusing on their potential application as anti-cancer agents and more recent investigations into their potential as therapeutics for Alzheimer's disease. The Cu(ii) complexes are of sufficient stability to be used to coordinate copper radioisotopes for application in diagnostic and therapeutic radiopharmaceuticals. Detailed understanding of the coordination chemistry has allowed careful manipulation of the metal based properties to engineer specific biological activities. Perhaps the most promising complex radiolabelled with copper radioisotopes to date is Cu II(atsm), which has progressed to clinical trials in humans (162 references). © 2011 The Royal Society of Chemistry.

Hliscs M.,Bio21 Molecular Science and Biotechnology Institute | Hliscs M.,University of Melbourne | Millet C.,Bio21 Molecular Science and Biotechnology Institute | Millet C.,University of Melbourne | And 7 more authors.
Cellular Microbiology | Year: 2015

In preparation for transmission to its mosquito vector, Plasmodium falciparum, the most virulent of the human malaria parasites, adopts an unusual elongated shape. Here we describe a previously unrecognized actin-based cytoskeleton that is assembled in maturing P.falciparum gametocytes. Differential extraction reveals the presence of a highly stabilized population of F-actin at all stages of development. Super-resolution microscopy reveals an F-actin cytoskeleton that is concentrated at the ends of the elongating gametocyte but extends inward along the microtubule cytoskeleton. Formin-1 is also concentrated at the gametocyte ends suggesting a role in actin stabilization. Immunoelectron microscopy confirms that the actin cytoskeleton is located under the inner membrane complex rather than in the sub-alveolar space. In stage V gametocytes, the actin and microtubule cytoskeletons are reorganized in a coordinated fashion. The actin-depolymerizing agent, cytochalasin D, depletes actin from the end of the gametocytes, whereas the actin-stabilizing compound, jasplakinolide, induces formation of large bundles and prevents late-stage disassembly of the actin cytoskeleton. Long-term treatment with these compounds is associated with disruption of the normal mitochondrial organization and decreased gametocyte viability. © 2014 John Wiley & Sons Ltd.

Armstrong C.W.,University of Melbourne | McGregor N.R.,University of Melbourne | Butt H.L.,Bio21 Molecular Science and Biotechnology Institute | Gooley P.R.,University of Melbourne
Advances in Clinical Chemistry | Year: 2014

Chronic fatigue syndrome (CFS) is a poorly understood condition that presents as long-term physical and mental fatigue with associated symptoms of pain and sensitivity across a broad range of systems in the body. The poor understanding of the disorder comes from the varying clinical diagnostic definitions as well as the broad array of body systems from which its symptoms present. Studies on metabolism and CFS suggest irregularities in energy metabolism, amino acid metabolism, nucleotide metabolism, nitrogen metabolism, hormone metabolism, and oxidative stress metabolism. The overwhelming body of evidence suggests an oxidative environment with the minimal utilization of mitochondria for efficient energy production. This is coupled with a reduced excretion of amino acids and nitrogen in general. Metabolomics is a developing field that studies metabolism within a living system under varying conditions of stimuli. Through its development, there has been the optimisation of techniques to do large-scale hypothesis-generating untargeted studies as well as hypothesis-testing targeted studies. These techniques are introduced and show an important future direction for research into complex illnesses such as CFS. © 2014 Elsevier Inc.

Watt A.D.,University of Melbourne | Watt A.D.,Bio21 Molecular Science and Biotechnology Institute | Perez K.A.,University of Melbourne | Perez K.A.,Bio21 Molecular Science and Biotechnology Institute | And 19 more authors.
Acta Neuropathologica | Year: 2013

The formation of low-order oligomers of β-amyloid (Aβ) within the brain is widely believed to be a central component of Alzheimer's disease (AD) pathogenesis. However, despite advances in high-throughput and high-resolution techniques such as xMAP and mass spectrometry (MS), investigations into these oligomeric species have remained reliant on low-resolution Western blots and enzyme-linked immunosorbent assays. The current investigation compared Aβ profiles within human cortical tissue using sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE), xMAP and surface enhanced laser desorption/ionization time-of-flight MS and found that whilst there was significant correlation across the techniques regarding levels of monomeric Aβ, only SDS-PAGE was capable of detecting dimeric isoforms of Aβ. The addition of synthetic di-tyrosine cross-linked Aβ1-40Met 35(O) to the AD tissue demonstrated that the MS methodology was capable of observing dimeric Aβ at femto-molar concentrations, with no noticeable effect on monomeric Aβ levels. Focus turned to the association between SDS-PAGE and levels of observable dimeric Aβ within the AD brain tissue. These investigations revealed that increased levels of dimeric Aβ were observed with increasing concentrations of SDS in the sample buffer. This finding was subsequently confirmed using synthetic Aβ1-42 and suggests that SDS was inducing the formation of dimeric Aβ. The findings that SDS promotes Aβ dimerization have significant implications for the putative role of low-order oligomers in AD pathogenesis and draw into question the utility of oligomeric Aβ as a therapeutic target. © 2013 Springer-Verlag Berlin Heidelberg.

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