ANSTO :: Browsing by Author "Le Brun, AP"

Browsing by Author "Le Brun, AP"

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90° magnetic coupling in a NiFe/FeMn/biased NiFe multilayer spin valve component investigated by polarized neutron reflectometry
(American Institute of Physics, 2014-07-17) Callori, SJ; Bertinshaw, J; Cortie, DL; Cai, JW; Le Brun, AP; Zhu, T; Klose, F
We have observed 90° magnetic coupling in a NiFe/FeMn/biased NiFe multilayer system using polarized neutron reflectometry. Magnetometry results show magnetic switching for both the biased and free NiFe layers, the latter of which reverses at low applied fields. As these measurements are only capable of providing information about the total magnetization within a sample, polarized neutron reflectometry was used to investigate the reversal behavior of the NiFe layers individually. Both the non-spin-flip and spin-flip neutron reflectometry signals were tracked around the free NiFe layer hysteresis loop and were used to detail the evolution of the magnetization during reversal. At low magnetic fields near the free NiFe coercive field, a large spin-flip signal was observed, indicating magnetization aligned perpendicular to both the applied field and pinned layer. © 2020 AIP Publishing LLC.
Annexin V-containing cubosomes for targeted early detection of apoptosis in degenerative retinal tissue
(Royal Society of Chemistry, 2018-10-26) Ding, Y; Chow, SH; Liu, GS; Wang, B; Lin, TW; Hsu, HY; Duff, AP; Le Brun, AP; Shen, HH
New drug delivery materials targeting damaged ocular tissues are of particular interest. In this work, we have formulated annexin/phosphatidylserine/phytantriol and annexin/phosphatidylserine/monoolein cubosomes based on incorporation of 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (PS) lipid and annexin V (ANX) protein with phytantriol (Phy) and monoolein (MO) respectively. The incorporation of ANX is important because it can be used as a diagnostic tool for in vivo apoptosis detection due to its high affinity to phosphatidylserine in the presence of Ca2+. We have also prepared PS–Phy and PS–MO cubosomes without ANX as a comparison, and characterized them using dynamic light scattering, cryo-TEM images and small-angle X-ray scattering, showing that PS–Phy cubosomes have greater chemical stability, and that ANX–PS–Phy cubosomes have the potential for in vivo drug delivery. In addition, we have reconstituted an apoptotic biomimetic membrane on a surface to gain insights into cubosome–bilayer interactions using a quartz-crystal microbalance and neutron reflectometry. The neutron reflectivity data reveal that there is exchange of materials between the biomimetic apoptotic bilayer and ANX–PS–Phy cubosomes, with an accumulation of ANX between the membrane and cubosomes possibly being the reason for the reduced cytotoxicity of ANX–PS–Phy cubosomes. A rat model of laser-induced choroidal neovascularization showed that ANX–PS–Phy cubosomes specifically targeted apoptotic cells in vivo. We propose that ANX–PS–Phy cubosomes are a potential candidate for ocular drug delivery for eye diseases. © The Royal Society of Chemistry 2018
The antimicrobial peptide aurein 1.2 disrupts model membranes via the carpet mechanism
(Royal Society of Chemistry, 2012-01-01) Fernandez, DI; Le Brun, AP; Whitwell, TC; Sani, MA; James, M; Separovic, F
The membrane interactions of the antimicrobial peptide aurein 1.2 were studied using a range of biophysical techniques to determine the location and the mechanism of action in DMPC (dimyristoylphosphatidylcholine) and DMPC/DMPG (dimyristoylphosphatidylglycerol) model membranes that mimic characteristics of eukaryotic and prokaryotic membranes, respectively. Neutron reflectometry and solid-state NMR revealed subtle changes in membrane structure caused by the peptide. Quartz crystal microbalance with dissipation, vesicle dye leakage and atomic force microscopy measurements were used to investigate the global mode of peptide interaction. Aurein 1.2 displayed an enhanced interaction with the anionic DMPC/DMPG membrane while exhibiting primarily a surface interaction with both types of model membranes, which led to bilayer disruption and membrane lysis. The antimicrobial peptide interaction is consistent with the carpet mechanism for aurein 1.2 with discrete structural changes depending on the type of phospholipid membrane. © 2012, Royal Society of Chemistry
Asymmetric phospholipid: lipopolysaccharide bilayers; a Gram-negative bacterial outer membrane mimic
(The Royal Society, 2013-10-16) Clifton, LA; Skoda, MWA; Daulton, E; Hughes, AV; Le Brun, AP; Lakey, JH; Holt, SA
The Gram-negative bacterial outer membrane (OM) is a complex and highly asymmetric biological barrier but the small size of bacteria has hindered advances in in vivo examination of membrane dynamics. Thus, model OMs, amenable to physical study, are important sources of data. Here, we present data from asymmetric bilayers which emulate the OM and are formed by a simple two-step approach. The bilayers were deposited on an SiO2 surface by Langmuir–Blodgett deposition of phosphatidylcholine as the inner leaflet and, via Langmuir–Schaefer deposition, an outer leaflet of either Lipid A or Escherichia coli rough lipopolysaccharides (LPS). The membranes were examined using neutron reflectometry (NR) to examine the coverage and mixing of lipids between the bilayer leaflets. NR data showed that in all cases, the initial deposition asymmetry was mostly maintained for more than 16 h. This stability enabled the sizes of the headgroups and bilayer roughness of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and Lipid A, Rc-LPS and Ra-LPS to be clearly resolved. The results show that rough LPS can be manipulated like phospholipids and used to fabricate advanced asymmetric bacterial membrane models using well-known bilayer deposition techniques. Such models will enable OM dynamics and interactions to be studied under in vivo-like conditions. © 2013, The Royal Society.
Developments on the platypus neutron reflectometer
(Australian Institute of Nuclear Science and Engineering (AINSE), 2020-11-11) Nelson, A; Le Brun, AP; Huang, TY; Paul, O; Holt, SA
PLATYPUS is the initial neutron reflectometer at the Australian Centre for Neutron Scattering with the capability to study surface and interface systems ranging from biomolecules, soft matter through to magnetic thin films [1-3]. There have been a number of significant improvements to both the instrument and data reduction and treatment software [4] over the last two years. On the hardware front the original detector has been replaced yielding higher count-rate capabilities, greater detection efficiency at shorter wavelengths and significantly lower background. The slits which define the neutron beam have been replaced with upgraded positioning mechanisms enabling greater flexibility in experimental setup. These changes have significantly enhanced the instrument performance with improved reproducibility. This presentation will highlight the enhancements and recent publications.
Hot commissioning and first user experiments on the Spatz neutron reflectometer
(Australian Nuclear Science and Technology Organisation, 2021-11-26) Le Brun, AP; Huang, TY; Pullen, SA; Nelson, A; Holt, SA
The Spatz neutron beam instrument is the latest to be installed and commissioned in the Neutron Guide Hall at the 20 MW OPAL Research Reactor. Spatz is a time-of-flight neutron reflectometer used for studying nanoscale structures at surfaces and interfaces and utilises a vertical sample geometry / horizontal scattering geometry. The instrument is situated at the end position of the CG2B neutron guide and views the cold neutron source (CNS). The disc chopper cascade that pulses the neutron beam to produce the time-of-flight is very configurable to provide a wavelength resolution between 1 to 12 %. The detector is a helium-3 two dimensional detector that is capable of measuring both specular and off-specular reflectivity. The sample stage can support a range of different sample environments including multiple solid-liquid cells, an atmospheric chamber with temperature control, the ATR-FT-IR spectrometer for simultaneous infra-red spectroscopy and neutron reflectometry measurements, electrochemical cells, etc. The geometry of the instrument and the sample environment available means that Spatz is well suited to studying phenomena at the gas-solid interface and solid-liquid interface. The Spatz instrument has been fully commissioned with neutrons and the results of the commissioning are presented. This includes measurements using the ‘Bragg mirror’ consisting of 25 bilayers of nickel and titanium, different solid substrates of silicon, quartz and sapphire, spin-coated polymer samples, and films under liquid. Reflectivity down to 10-7 can be achieved within 1 hour measuring time with good counting statistics in most cases. Early user experiments cover a range of science including investigating the thermal stability of organic solar cell materials and proteins interacting with biomimetic phospholipid cell membranes. © 2021 The Authors
The impact of pH on packing in tethered lipid bilayers
(Australian Institute of Nuclear Science and Engineering, 2016-11-29) Cranfield, CG; Berry, T; Holt, SA; Le Brun, AP; Valenzuela, SM; Coster, HGL; Cornell, BA
We report that increasing the H3O+ concentration when lowering the pH reduces the intrinsic ionic conduction through phospholipid bilayers (Fig 1A), which is counter to what might be expected from increasing the H3O+ concentration. We attribute the conduction decrease to a reduction of the molecular area per lipid (ao)[1]. These effects are seen at H3O+ concentrations in the range nM to µM despite these being very low concentrations compared to that of a typical bathing electrolyte solution of 135mM ionic concentration. We present a model, in which the pH dependent reduction in ao favours an increase in lipid packing. To support this model, we provide evidence of the effects of the hydronium ion on lipid geometry using neutron reflectometry (Fig 1C). Further examples will be given of the impact of the H3O ion concentration on the hydrogen bonding within the polar groups of lipid.
In situ study of the impact of acidic and neutral deposition pH on alkane phosphate film formation and stability on TiO2
(Royal Society Chemistry, 2013-02-28) Holt, SA; Le Brun, AP; Nelson, A; Lakey, JH
TiO2 has been used as a model system for the surface of medical implant devices based upon titanium alloys. Self Assembled Monolayers (SAM) can be applied via a specific phosphate interaction to modify the surface properties of the TiO2 substrate. Here it is demonstrated via in situ Quartz Crystal Microbalance and Neutron Reflectometry experiments that the deposition of dodecyl phosphate onto TiO2 proceeds more rapidly at pH 4.5 than at pH 7.0. Conversely, the film stability was enhanced for films deposited at pH 7.0, demonstrating that while the initial association with the surface is driven by electrostatics it does not determine the SAM density. While the adsorbed amount appeared to be relatively constant after a few minutes incubation time it was found that washing with buffer removed about 50% of the adsorbed material after these short incubation times. With incubation time of the order of hours the proportion of the film washed off the surface decreased demonstrating that the specific phosphate-TiO2 interaction was a slow process. The slower initial surface interaction at pH 7.0 therefore allowed greater re-arrangement of the dodecyl phosphate resulting in more complete and robust monolayers than at pH 4.5. This was demonstrated by washing the film with buffer of increasing pH of up to 9.5. For SAM longevity on titanium alloys it is clear that slow deposition at pH 7 produces more robust films than rapid deposition at lower pH values.© 2013, Royal Society of Chemistry.
An in vitro model to investigate the interactions between antimicrobial peptides and the outer membrane of gram-negative pathogens
(Australian Institute of Nuclear Science and Engineering, 2016-11-29) Han, ML; Shen, HH; Zhu, Y; Le Brun, AP; Holt, SA; Roberts, K; Song, JN; Cooper, MA; Moskowitz, SM; Velkov, T; Li, J
Increasing antibiotic resistance in Gram-negative bacteria led to polymyxins as the last therapy. Polymyxins present their antimicrobial activity through an initial electronical interaction with lipid A in the outer membrane (OM) of GNB, and the most common mechanism of polymyxin resistance is through modifications of lipid A with positively charged groups, such as 4-amino-L-arabinose (L-Aar4N) or phosphoethanolamine (pEtN). However, it is notable that Gram-negative bacteria employ a combination of charge-charge repulsion mechanism and the modification to fatty acyl chains of lipid A to obtain high-level polymyxin resistance. Hence, we designed hydrophobic polymyxin-related lipopeptides in order to overcome modified lipid A to insert into the outer membrane of Gram-negative bacteria. In this study, we employed neutron reflectometry (NR) study to investigate the interactions between lipid A and polymyxins. Lipid A was extracted from polymyxin-susceptible and -resistant pseudomonas aeruginosa strains, and analysed using ESI-MS in the negative ion mode. The asymmetric lipid A: deuterated DPPC bilayers were deposited on SiO2 surfaces by combined Langmuir-Blodgett and Langmuir-Schaefer disposition methods, and characterised by neutron reflectometer. Our results showed L-Ara4N modified lipid A was observed in polymyxin-resistant PAKpmrB6 strain, but not in the wild-type PAK strain. The NR data obtained from unmodified lipid A: DPPC bilayer was fitted into a five-layer model. Whereas, a six-layer model containing an extra outer headgroup was established for L-Ara4N modified lipid A: d-DPPC bilayer. Our results showed a dense of PMB (volume fraction of >20%) bound to the surface of both unmodified and modified lipid A: DPPC bilayers. While it is notable that the significant changes in NR profiles obtained from H2O contrast indicated about 15.8% and 6.1% of PMB penetrated into the wild-type lipid A headgroup and fatty acyl chains, respectively, but without penetration into L-Ara4N-lipid A: d-DPPC bilayer. However, the employment of octpeptin A3 induced higher hydrophobic interactions with L-Ara4N-lipid A: d-DPPC bilayer. Our study provides an in vitro model to investigate the interactions of polymyxins with OM bilayers in GNB, and confirmed that lipid A modification with L-Ara4N was certainly to reduce the penetration of PMB into bacterial membranes. Remarkably, the higher binding affinity between octapeptin A3 and L-Ara4N modified lipid A indicated its potential to be the new generation antibiotics for the therapy of infections caused by multi-drug resistant Gram negative bacteria.
Investigating the interactions of the 18 kDa translocator protein and its ligand PK11195 in planar lipid bilayers
(Elsevier, 2014-03) Hatty, CR; Le Brun, AP; Lake, V; Clifton, LA; Liu, GJ; James, M; Banati, RB
The functional effects of a drug ligand may be due not only to an interaction with its membrane protein target, but also with the surrounding lipid membrane. We have investigated the interaction of a drug ligand, PK11195, with its primary protein target, the integral membrane 18 kDa translocator protein (TSPO), and model membranes using Langmuir monolayers, quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). We found that PK11195 is incorporated into lipid monolayers and lipid bilayers, causing a decrease in lipid area/molecule and an increase in lipid bilayer rigidity. NR revealed that PK11195 is incorporated into the lipid chain region at a volume fraction of ~ 10%. We reconstituted isolated mouse TSPO into a lipid bilayer and studied its interaction with PK11195 using QCM-D, which revealed a larger than expected frequency response and indicated a possible conformational change of the protein. NR measurements revealed a TSPO surface coverage of 23% when immobilised to a modified surface via its polyhistidine tag, and a thickness of 51 Å for the TSPO layer. These techniques allowed us to probe both the interaction of TSPO with PK11195, and PK11195 with model membranes. It is possible that previously reported TSPO-independent effects of PK11195 are due to incorporation into the lipid bilayer and alteration of its physical properties. There are also implications for the variable binding profiles observed for TSPO ligands, as drug–membrane interactions may contribute to the apparent affinity of TSPO ligands. © 2013, Elsevier B.V.
The membrane activity of the antimicrobial peptide caerin 1.1 is pH dependent
(Science Direct, 2023-03-21) Sani, MA; Le Brun, AP; Rajput, S; Attard, T; Separovic, F
Antimicrobial peptides are an important class of membrane-active peptides that can provide alternatives or complements to classic antibiotics. Among the many classes of AMPs, the histidine-rich family is of particular interest since they may induce pH-sensitive interactions with cell membranes. The AMP caerin 1.1 (Cae-1), from Australian tree frogs, has three histidine residues, and thus we studied the pH dependence of its interactions with model cell membranes. Using NMR spectroscopy and molecular dynamics simulations, we showed that Cae-1 induced greater perturbation of the lipid dynamics and water penetrations within the membrane interior in an acidic environment compared with physiological conditions. Using 31P solid-state NMR, the packing, chemical environment, and dynamics of the lipid headgroup were monitored. 2H solid-state NMR showed that Cae-1 ordered the acyl chains of the hydrophobic core of the bilayer. These results supported the molecular dynamics data, which showed that Cae-1 was mainly inserted within the lipid bilayer for both neutral and negatively charged membranes, with the charged residues pulling the water and phosphate groups inward. This could be an early step in the mechanism of membrane disruption by histidine-rich antimicrobial peptides and indicated that Cae-1 acts via a transmembrane mechanism in bilayers of neutral and anionic phospholipid membranes, especially in acidic conditions. © 2023 Elsevier B.V.
Neutron reflectometry studies define prion protein N-terminal peptide membrane binding
(Elsevier, 2014-11-18) Le Brun, AP; Haigh, CL; Drew, SC; James, M; Boland, MP; Collins, SJ
The prion protein (PrP), widely recognized to misfold into the causative agent of the transmissible spongiform encephalopathies, has previously been shown to bind to lipid membranes with binding influenced by both membrane composition and pH. Aside from the misfolding events associated with prion pathogenesis, PrP can undergo various posttranslational modifications, including internal cleavage events. Alpha- and beta-cleavage of PrP produces two N-terminal fragments, N1 and N2, respectively, which interact specifically with negatively charged phospholipids at low pH. Our previous work probing N1 and N2 interactions with supported bilayers raised the possibility that the peptides could insert deeply with minimal disruption. In the current study we aimed to refine the binding parameters of these peptides with lipid bilayers. To this end, we used neutron reflectometry to define the structural details of this interaction in combination with quartz crystal microbalance interrogation. Neutron reflectometry confirmed that peptides equivalent to N1 and N2 insert into the interstitial space between the phospholipid headgroups but do not penetrate into the acyl tail region. In accord with our previous studies, interaction was stronger for the N1 fragment than for the N2, with more peptide bound per lipid. Neutron reflectometry analysis also detected lengthening of the lipid acyl tails, with a concurrent decrease in lipid area. This was most evident for the N1 peptide and suggests an induction of increased lipid order in the absence of phase transition. These observations stand in clear contrast to the findings of analogous studies of Ab and α-synuclein and thereby support the possibility of a functional role for such N-terminal fragment-membrane interactions. © 2014,Biophysical Society.
Polarization "down under": the polarized time-of-flight neutron reflectometer PLATYPUS
(American Institute of Physics, 2012-08-01) Saerbeck, T; Klose, F; Le Brun, AP; Füzi, J; Brûlé, A; Nelson, A; Holt, SA; James, M
This review presents the implementation and full characterization of the polarization equipment of the time-of-flightneutron reflectometer PLATYPUS at the Australian Nuclear Science and Technology Organisation (ANSTO). The functionality and efficiency of individual components are evaluated and found to maintain a high neutron beam polarization with a maximum of 99.3% through polarizing Fe/Si supermirrors. Neutron spin-flippers with efficiencies of 99.7% give full control over the incident and scattered neutron spin direction over the whole wavelength spectrum available in the instrument. The first scientific experiments illustrate data correction mechanisms for finite polarizations and reveal an extraordinarily high reproducibility for measuring magnetic thin film samples. The setup is now fully commissioned and available for users through the neutron beam proposal system of the Bragg Institute at ANSTO. © 2012, American Institute of Physics.
Polymyxin-induced lipid A deacylation in pseudomonas aeruginosa perturbs polymyxin penetration and confers high-level resistance
(ACS Publications, 2017-11-28) Han, ML; Velkov, T; Zhu, Y; Roberts, KD; Le Brun, AP; Chow, SH; Gutu, AD; Moskowitz, SM; Shen, HH; Li, J
Polymyxins are last-line antibiotics against life-threatening multidrug-resistant Gram-negative bacteria. Unfortunately, polymyxin resistance is increasingly reported, leaving a total lack of therapies. Using lipidomics and transcriptomics, we discovered that polymyxin B induced lipid A deacylation viapagL in both polymyxin-resistant and -susceptible Pseudomonas aeruginosa. Our results demonstrated that the deacylation of lipid A is an “innate immunity” response to polymyxins and a key compensatory mechanism to the aminoarabinose modification to confer high-level polymyxin resistance in P. aeruginosa. Furthermore, cutting-edge neutron reflectometry studies revealed that an assembled outer membrane (OM) with the less hydrophobic penta-acylated lipid A decreased polymyxin B penetration, compared to the hexa-acylated form. Polymyxin analogues with enhanced hydrophobicity displayed superior penetration into the tail regions of the penta-acylated lipid A OM. Our findings reveal a previously undiscovered mechanism of polymyxin resistance, wherein polymyxin-induced lipid A remodeling affects the OM packing and hydrophobicity, perturbs polymyxin penetration, and thereby confers high-level resistance. © 2017 American Chemical Society
A polytherapy based approach to combat antimicrobial resistance using cubosomes
(Springer Nature, 2022-01-17) Lai, XF; Han, ML; Ding, Y; Chow, SH; Le Brun, AP; Wu, CM; Bergen, PJ; Jiang, JH; Hsu, HY; Muir, BW; White, J; Song, JN; Shen, HH
A depleted antimicrobial drug pipeline combined with an increasing prevalence of Gram-negative ‘superbugs’ has increased interest in nano therapies to treat antibiotic resistance. As cubosomes and polymyxins disrupt the outer membrane of Gram-negative bacteria via different mechanisms, we herein examine the antimicrobial activity of polymyxin-loaded cubosomes and explore an alternative strategy via the polytherapy treatment of pathogens with cubosomes in combination with polymyxin. The polytherapy treatment substantially increases antimicrobial activity compared to polymyxin B-loaded cubosomes or polymyxin and cubosomes alone. Confocal microscopy and neutron reflectometry suggest the superior polytherapy activity is achieved via a two-step process. Firstly, electrostatic interactions between polymyxin and lipid A initially destabilize the outer membrane. Subsequently, an influx of cubosomes results in further membrane disruption via a lipid exchange process. These findings demonstrate that nanoparticle-based polytherapy treatments may potentially serve as improved alternatives to the conventional use of drug-loaded lipid nanoparticles for the treatment of “superbugs”. Open Access: This article is licensed under a Creative Commons Attribution 4.0 International Licence.
Progress with hot commissioning and first user experiments on the SPATZ neutron reflectometer
(Australian Institute of Nuclear Science and Engineering (AINSE), 2020-11-11) Le Brun, AP; Huang, TY; Nelson, A; Holt, SA
The SPATZ neutron reflectometer was originally the V18 BioRef reflectometer at the BER-II Research Reactor (Berlin, Germany) [1, 2], and was transferred from HZB to ANSTO ownership during October 2016 to February 2017. The reflectometer was re-installed at ANSTO’s 20 MW OPAL Research Reactor in the Neutron Guide Hall on the end position of the newly installed CG2B cold-neutron guide. The hot commissioning licence was issued in October 2018 with first neutrons in November 2018. Radiation surveys and shielding improvements were completed in May 2019. Since this time, hot commissioning has continued and has involved evaluating the performance of the instrument. SPATZ is a time-of-flight instrument, and the ability to translate chopper 2 makes the instrument highly configurable with the ability to select a time resolution from 1 to 12 %. A number of different sample environments have been tested including: solid-liquid cells with a HPLC pump for solvent contrast exchange, circulating water baths for temperature control, and the infrared spectrometer for simultaneous neutron reflectometry and ATR-FT-IR spectroscopy. The operating license was issued in December 2019, and first experiments are scheduled for the last quarter of 2020. SPATZ is now available for user proposals in the ACNS portal [3]. This presentation will highlight the instrument performance and achievements during hot commissioning and give an overview of the user program to date and first data collected.
Self-assembly of protein monolayers engineered for improved monoclonal immunoglobulin G binding
(MDPI Publishing, 2011-08-01) Le Brun, AP; Shah, DSH; Athey, D; Holt, SA; Lakey, JH
Bacterial outer membrane proteins, along with a filling lipid molecule can be modified to form stable self-assembled monolayers on gold. The transmembrane domain of Escherichia coli outer membrane protein A has been engineered to create a scaffold protein to which functional motifs can be fused. In earlier work we described the assembly and structure of an antibody-binding array where the Z domain of Staphylococcus aureus protein A was fused to the scaffold protein. Whilst the binding of rabbit polyclonal immunoglobulin G (IgG) to the array is very strong, mouse monoclonal IgG dissociates from the array easily. This is a problem since many immunodiagnostic tests rely upon the use of mouse monoclonal antibodies. Here we describe a strategy to develop an antibody-binding array that will bind mouse monoclonal IgG with lowered dissociation from the array. A novel protein consisting of the scaffold protein fused to two pairs of Z domains separated by a long flexible linker was manufactured. Using surface plasmon resonance the self-assembly of the new protein on gold and the improved binding of mouse monoclonal IgG were demonstrated. © The Authors - This is an open access article distributed under the Creative Commons Attribution License
SPATZ: The second time-of-flight neutron reflectometer at the OPAL Research Reactor
(International Conference on Neutron Scattering, 2017-07-12) Le Brun, AP; Pullen, SA; Constantine, P; Spedding, J; Roach, D; McGregor, A; Affleck, J; Christoforidis, J
In September 2015, an agreement was signed between HZB and ANSTO to transfer the V18 ‘BioRef’ time-of-flight neutron reflectometer [1, 2], which was situated at the BER-II Research Reactor, to the OPAL Research Reactor. In September 2016 a joint team of ANSTO and HZB personnel spent four weeks carefully disassembling BioRef and packing it into shipping containers for transport to ANSTO. The instrument safely arrived in Sydney in February 2017 [3], and will be known as SPATZ (German for Sparrow). SPATZ will be the 15th neutron-scattering instrument at OPAL. SPATZ has a vertical sample geometry, which complements the current reflectometer, PLATYPUS, which has a horizontal sample geometry. The vertical sample geometry will allow for use of sample environments which cannot be currently used on PLATYPUS due to geometry constraints and allows for wide-angle diffraction from multilayers and lamellarstacks. SPATZ will continue to be equipped for simultaneous infra-red spectroscopy and reflectometry experiments, and will come with equipment for upgrades for polarisation and spin-echo techniques. The instrument will view the OPAL cold neutron source (CNS) by taking the end position of the CG2B guide. Currently, the CG2B guide is installed between the primary and secondary shutters and part of the project scope is to complete the installation of the CG2B guide beyond the secondary shutter into the Neutron Guide Hall. The CG2B guide will accommodate SPATZ and an additional upstream instrument to be determined in the future. This presentation will provide an overview of the project, its current status, and future direction.
SPATZ: The second time-of-flight neutron reflectometer at the OPAL Research Reactor
(Australian Institute of Nuclear Science and Engineering, 2016-11-29) Le Brun, AP; Pullen, SA; Constantine, P; Spedding, J; Roach, D; McGregor, A; Affleck, J; Christoforidis, J; Trapp, M; Steitz, R
In September 2015, an agreement was signed between HZB and ANSTO to transfer the V18 ‘BioRef’ time-of-flight neutron reflectometer [1], currently situated at the 10 MW BER-II Research Reactor, to the OPAL Research Reactor. During 2016 preparations have been made to carry out the transfer of a neutron-scattering instrument halfway around the globe. This has involved a joint team of ANSTO and HZB personnel spending four weeks carefully disassembling BioRef and packing it into shipping containers for transport to ANSTO. Once the instrument arrives it will be known as SPATZ (German for Sparrow) and will be the 15th neutron-scattering instrument at OPAL. SPATZ has a vertical sample geometry, which complements the current reflectometer, PLATYPUS, which has a horizontal sample geometry. The vertical sample geometry will allow for use of sample environments which cannot be currently used on PLATYPUS due to geometry constraints and allows for wide-angle diffraction from multilayers and lamellar stacks. SPATZ will also be equipped for simultaneous infra-red spectroscopy and reflectometry experiments, and will come with equipment for upgrades for polarisation and spin-echo techniques. The instrument will view the OPAL cold neutron source (CNS) by taking the end position of the CG2B guide. Currently, the CG2B guide is installed between the primary and secondary shutters and part of the project scope is to complete the installation of the CG2B guide beyond the secondary shutter into the Neutron Guide Hall. The CG2B guide will accommodate SPATZ and an additional upstream instrument to be determined in the future. This presentation will provide an overview of the project, its current status, and future direction. Feedback from the neutron scattering community is encouraged.
Structural characterization of a model gram-negative bacterial surface using lipopolysaccharides from rough strains of escherichia coli
(American Chemical Society, 2013-06-01) Le Brun, AP; Clifton, LA; Halbert, CE; Lin, B; Meron, M; Holden, PJ; Lakey, JH; Holt, SA
Lipopolysaccharides (LPS) make up approximately 75% of the Gram-negative bacterial outer membrane (OM) surface, but because of the complexity of the molecule, there are very few model OMs that include LPS. The LPS molecule consists of lipid A, which anchors the LPS within the OM, a core polysaccharide region, and a variable O-antigen polysaccharide chain. In this work we used RcLPS (consisting of lipid A plus the first seven sugars of the core polysaccharide) from a rough strain of Escherichia coli to form stable monolayers of LPS at the air?liquid interface. The vertical structure RcLPS monolayers were characterized using neutron and X-ray reflectometry, while the lateral structure was investigated using grazing incidence X-ray diffraction and Brewster angle microscopy. It was found that RcLPS monolayers at surface pressures of 20 mN m(-1) and above are resolved as hydrocarbon tails, an inner headgroup, and an outer headgroup of polysaccharide with increasing solvation from tails to outer headgroups. The lateral organization of the hydrocarbon lipid chains displays an oblique hexagonal unit cell at all surface pressures, with only the chain tilt angle changing with surface pressure. This is in contrast to lipid A, which displays hexagonal or, above 20 mN m(-1), distorted hexagonal packing. This work provides the first complete structural analysis of a realistic E. coli OM surface model. © 2013, American Chemical Society.

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