The Australian Synchrotron is a 3 GeV synchrotron radiation facility built in Melbourne, Victoria and opened on 31 July 2007.The circular building was designed by Architectus in conjunction with Thiess, while the lattice design was performed substantially by Professor John Boldeman. The Synchrotron building is located in Clayton near the Monash University Clayton Campus.The Australian Synchrotron is a light source facility . It uses particle accelerators to produce a beam of high energy electrons which are placed within a storage ring that circulates the electrons to create synchrotron light. The light is directed down separate beamlines at the end of which may be placed a variety of experimental equipment contained within the endstations. Wikipedia.
News Article | May 8, 2017
Their experiment was the first to use a newly installed x-ray detector, called Maia, mounted at NSLS-II's Submicron Resolution X-Ray Spectroscopy (SRX) beamline. Scientists from around the world come to SRX to create high-definition images of mineral deposits, aerosols, algae—just about anything they need to examine with millionth-of-a-meter resolution. Maia, developed by a collaboration between NSLS-II, Brookhaven's Instrumentation Division and Australia's Commonwealth Scientific and Industrial Research Organization (CSIRO), can scan centimeter-scale sample areas at micron scale resolution in just a few hours—a process that used to take weeks. "The Maia detector is a game-changer," said Juergen Thieme, lead scientist at the SRX beamline. "Milliseconds per image pixel instead of seconds is a huge difference." SRX beamline users now have time to gather detailed data about larger areas, rather than choosing a few zones to focus on. This greatly enhances the chance to capture rare "needle in a haystack" clues to ore forming processes, for example. "This is important when you are trying to publish a paper," said Thieme. "Editors want to make sure that your claim is based on many examples and not one random event." "We've already gathered enough data for one, if not two papers," said Margaux Le Vaillant, one of the visiting users from CSIRO and principal investigator for this experiment. Collaborator Giada Iacono Marziano of the French National Center for Scientific Research added, "Because we can now look at a larger image in detail, we might see things—like certain elemental associations—that we didn't predict." These kinds of surprises pose unexpected questions to scientists, pushing their research in new directions. Siddons and his collaborators at Brookhaven Lab and CSIRO have provided Maia detectors to synchrotron light sources around the world—CHESS at Cornell University in New York, PETRA-III at the DESY laboratory in Hamburg, Germany, and the Australian Synchrotron in Melbourne. The detector at SRX offers the advantage of using beams from NSLS-II, the brightest light source of its kind in the world. When scientists shine the x-ray beams at samples, they excite the material's atoms. As the atoms relax back to their original state they fluoresce, emitting x-ray light that the detector picks up. Different chemical elements will emit different characteristic wavelengths of light, so this x-ray fluorescence mapping is a kind of chemical fingerprinting, allowing the detector to create images of the sample's chemical makeup. The Maia detector has several features that help it map samples at high speeds and in fine detail. "Maia doesn't 'stop and measure' like other detectors," said physicist Pete Siddons, who led Brookhaven's half of the project. Most detectors work in steps, analyzing each spot on a sample one at a time, he explained, but the Maia detector scans continuously. Siddons' team has programmed Maia with a process called dynamic analysis to pick apart the x-ray spectral data collected and resolve where different elements are present. Maia's analysis systems also make it possible for scientists to watch images of their samples appear on the computer screen in real-time as Maia scans. If samples are very similar, Maia will recycle the dynamic analysis algorithms it used to create multi-element images from the first sample's fluorescence signals to build the subsequent sample's images in real-time, without computational lag. Part of Maia's speed is also attributable to the 384 tiny photon-sensing detector elements that make up the large detector. This large grid of sensors can pick up more re-emitted x-rays than standard detectors, which typically use less than 10 elements. Siddons' instrumentation team designed special readout chips to deal with the large number of sensors and allow for efficient detection. The 20-by-20 grid of detectors has a hole in the middle, but that's intentional, Siddons explained. "The hole lets us put the detector much closer to the sample," Siddons said. Rather than placing the sample in front of the x-ray beam and the detector off to the side, SRX beamline scientists have aligned the beam, sample, and detector so that the x-ray beam shines through the hole to reach the sample. With this arrangement, the detector covers a wide angle and captures a large fraction of fluoresced x-rays. That sensitivity allows researchers to scan faster, which can be used either to save time or to cut back on the intensity of x-rays striking the sample, reducing any damage the rays might cause. Siddons noted that the team is currently developing new readout chips for the detector, and incorporating a new type of sensor, called a silicon drift detector array. Together these will heighten the detector's ability to distinguish between photons of similar energy, unfolding detail in complex spectra and making for even more accurate chemical maps. Explore further: Multilaboratory collaboration brings new X-ray detector to light
News Article | May 15, 2017
Everything we see with the unaided eye in a painting – from the Australian outback images of Albert Namatjira or Russell Drysdale, to the vibrant works of Pro Hart – is thanks to the mix of colours that form part of the visible spectrum. But if we look at the painting in a different way, at a part of the spectrum that is invisible to our eyes, then we can see something very different. As our recently published research shows, it could even help us detect art fraud. The electromagnetic spectrum ranges from very high-frequency gamma rays down to the extremely low-frequency radiation of just a few hertz. Hertz is the unit of measurement for frequency. The frequency of colours in the visible spectrum range from blue, at about 800 terahertz (THz), through to red at about 400THz (1 THz = 1012 or 1,000,000,000,000 hertz). If we drop to frequencies below the visible spectrum we find the near-infrared at about 300THz and then the mid-infrared at about 30THz. Then comes the far-infrared and at last we meet the frequencies around 1THz. Continuing even further brings us to microwaves and radio waves where frequencies range from the gigahertz down to kilohertz. Thus the terahertz part of the electromagnetic spectrum lies between the radio and the visible parts – in other words, between electronics and photonics. Things can look very different when viewed with "eyes" that can see in the terahertz range. Some things that are transparent to visible light, such as water, are opaque to terahertz light. Conversely, some things that visible light won't penetrate, such as black plastic, readily transmit terahertz radiation. Intriguingly, two objects that have the same colour when viewed by the unassisted eye may transmit terahertz radiation differently. So their terahertz signal can be used to tell them apart. This points to the potential use of terahertz radiation in differentiating paints and pigments. Terahertz spectroscopy can distinguish different pigments with similar colours. We recently used terahertz spectroscopy to distinguish between three related pigments. All come from a family of chemical compounds called quinacridones. These are used widely in producing stable, reproducible pigments that range in colour from red to violet. Measurements at the University of Wollongong provided the experimental data in the range of 1THz to 10THz. Numerical modelling at Syracuse University (New York) reproduced the experimental data, and gave physical insight into the origin of the features observed. The combined experimental and theoretical work, published last month in the Journal of Physical Chemistry, unequivocally demonstrates that terahertz spectroscopy is able to distinguish three different quinacridones. This brings us to the subject of art authentication – or more importantly, detecting cases of art fraud. Museums, galleries and collectors are typically very protective of their art collections, but terahertz spectroscopy is well suited to examining their works. While terahertz spectrometers are often located in laboratories, there are also portable models. Unlike an analysis that requires removing and consuming some material (by reacting it with chemicals, or burning it), there is no contact made with the material, and thus no harm done to the artwork. The terahertz radiation simply shines on the painting, and the transmitted radiation is measured. The low energy and low density of terahertz radiation means that the painting is not damaged in any way. This all makes it suitable for examining art in a way that does not damage it and can be performed where it is located – in a gallery, or home, or almost anywhere. So how can terahertz spectroscopy assist in detecting art fraud in practice? Here's an example. Let's say terahertz spectroscopy picks up a quinacridone pigment in a painting. Quinacridone is an artificial material that was first synthesised in 1935, so the painting must date from 1935 or later. Any claim that the painting is a work by Leonardo da Vinci (who died in 1519), Vincent van Gogh (died 1890) or Claude Monet (died 1926) could therefore be dismissed. Any claim the the work was by an artist who worked after 1935 could not be so easily disproved on this basis. Of course, other physical methods than terahertz spectroscopy may be applied to analyse paintings. One direct way to analyse art work is by sophisticated, quantitative measurements of the visible spectrum. Artworks may also be interrogated by other species of light that lie above the blue end visible spectrum. Here the ultraviolet (uv) photons are higher in energy than visible photons. That means they can put energy into a material that is re-radiated as visible photons. This is the phenomenon of fluorescence, and uv-fluorescence is an established tool in art conservation. Moving further above the ultraviolet, X-rays may be used to examine works of art. For example, X-ray fluorescence at the Australian Synchrotron has been used to find hidden layers in works by Degas and Streeton. There are many aspects to authenticating an artwork, the physical examination being but one of them. Nonetheless, technical analysis of the materials used – the paints, the canvas, the frames – plays a fundamental role, and that is where terahertz spectroscopy contributes. But other approaches also play a role. For example, documentation such as records of sales may provide key evidence, as may the more subtle appraisal of style by art historians. The perceptions of people who assess and buy art is itself an important factor. The word of the artist might be thought to be definitive, but even this has been overruled by expert opinion, as in the case of Lucian Freud. Finally, the legal dimension is critical, as has been reported recently in the quashing of the art fraud convictions of Peter Gant and Mohamed Siddique. These related to the paintings Blue Lavender Bay, Orange Lavender Bay, and Through the Window. At issue was whether the paintings were the work of Brett Whiteley. Of course, art fraud is just one application of terahertz spectroscopy. There are many more. Able to penetrate paper and cardboard, terahertz radiation can be used to look inside envelopes for contraband, or inside packaged food for contamination. Terahertz methods have been used to assess burns and to monitor the hydration of plants. As better terahertz sources, detectors and components are developed, the range of applications will further expand. Explore further: Researchers nearly double the continuous output power of a type of terahertz laser
News Article | May 18, 2017
In research published in Nature Scientific Reports, a team of investigators led by ANSTO biologist Nicholas Howell and Prof Richard Banati provided evidence of previously unseen spatial patterns in the distribution of metals that do not appear to be linked to physical characteristics in the feathers. Because the patterns are not linked to pigmentation, thickness or other structural characteristics in the feathers, the authors suggest another unidentified mechanism may be at work. "Our collaboration has produced some remarkable depictions of the feathers that let us see into complex and pattern-forming, biochemical processes in cells," said Prof Banati. High resolution images collected using the X-ray fluorescence microprobe and Maia spectroscopic detector at the Australian Synchrotron, revealed independent distribution of zinc, calcium, bromine, copper and iron. In this investigation, the technique was applied to the whole feather, and required no subsampling or extraction procedures in order to accurately identify elements. "Using this powerful instrument and Maia detector, David Paterson and Daryl Howard were able to scan samples that were several centimetres in length at micron resolution," said Howell. X-ray fluorescence microscopy allows you to view hard biological structures in their natural state. The detector system speeds up the scanning of the sample in real time and delivers data at unprecedented resolution. The images, which have previously unachieved sensitivity and resolution, provide a distribution map of a range of chemical elements in the feather. Understanding the development of bird feathers is important for understanding the evolution of birds, formation of organs, tissue regeneration and the health status of individual animals. The findings also have significant potential application more broadly in developmental biology. "The same basic biochemical mechanisms that allow feathers to develop in birds are at work in other animals and humans, "said Howell. For example, the identification of a distinct, repetitious pattern in the concentration of zinc in all samples was of particular interest. Zinc is an essential element in birds for growth, the formation of enzymes, the development of the skeleton and a range of physiological functions. These zinc bands resembled but were not related to distinct growth bands. The exact mechanism that leads to the regular deposits of zinc is unknown but the scientists noticed that the number of zinc bands appears to be the same as the number of days the feather grows, e.g. the duration of the moulting period. "We do not have entirely accurate data on the rate of feather growth in a migratory seabird, which needs to be observed under conditions of the animal's natural life-cycle," said Howell. "Nonetheless, such highly regular, biological patterns hold important information, because similar to tree rings , they are a natural time stamp that records events during the growth of these patterns." said Howell. Therefore, the patterns in the feathers may be useful in assessing the bird's health and nutritional status retrospectively, in the way that tree rings indicate past environmental events, such as droughts and floods. The feathers came from three species of migratory shearwaters, birds that are known to travel over 60,000 kilometres per year on their migration to breeding areas. Mr Howell said none of the work would have been possible without the painstaking field work in remote locations. Single breast and wing feathers from the fleshfooted, streaked and short-tailed shearwater were collected on Lord Howe Island, several Japanese islands and Bundeena Beach (NSW) under the direction of co-author Dr Jennifer Lavers of the Institute of Marine and Antarctic Studies at the University of Tasmania. "It is very difficult to image and measure metals in biological samples, but it is something we can do with a variety of techniques at ANSTO using X-rays, neutrons and isotopes," said Howell. Last year, a similar approach was used to detect and measure strontium in the vertebrae of sharks. Explore further: Feathers hold key to proof of bird health More information: Nicholas R. Howell et al. The Topobiology of Chemical Elements in Seabird Feathers, Scientific Reports (2017). DOI: 10.1038/s41598-017-01878-y
News Article | May 10, 2017
Patients who were included in the study all had Goodpasture disease and fulfilled the following key diagnostic criteria: (1) serum anti-α3(IV)NC1 IgG by enzyme-linked immunosorbent assay (ELISA), (2) linear IgG staining of the GBM and (3) necrotizing and crescentic glomerulonephritis. HLA-DR15 typing of patients was done by monoclonal antibody staining (BIH0596, One Lambda) and flow cytometry. Blood from HLA-typed healthy humans was collected via the Australian Bone Marrow Donor Registry. HLA-DR15, HLA-DR1 and HLA-DR15/DR1 donors were molecularly typed and were excluded if they expressed DQB1*03:02, which is potentially weakly associated with susceptibility to anti-GBM disease2. Studies were approved by the Australian Bone Marrow Donor Registry and Monash Health Research Ethics Committees, and informed consent was obtained from each individual. Mouse MHCII deficient, DR15 transgenic mice and mouse MHCII deficient, DR1 transgenic mice were derived from existing HLA transgenic colonies and intercrossed so that they were on the same background as previously described4. The background was as follows: 50% C57BL/10, 43.8% C57BL/6, 6.2% DBA/2; or with an Fcgr2b−/− background: 72% C57BL/6, 25% C57BL/10 and 3% DBA/2. To generate mice transgenic for both HLA-DR15 and HLA-DR1, mice transgenic for either HLA-DR15 or HLA-DR1 were intercrossed. FcγRIIb intact HLA transgenic mice and cells were used for all experiments, except those in experimental Goodpasture disease, where Fcgr2b−/− HLA transgenic strains were used. While DR15+ mice readily break tolerance to α3(IV)NC1 when immunized with human α3 or mouse α3 , renal disease is mild4. As genetic changes in fragment crystallizable (Fc) receptors have been implicated in the development of nephritis in rodents and in humans18, Fcgr2b−/− HLA transgenic strains were used when end organ injury was an important endpoint. For in vitro experiments, cells from either male or female mice were used. For in vivo experiments both male and female mice were used, for immunization aged 8–12 weeks and for the induction of experimental Goodpasture disease aged 8–10 weeks. Experiments were approved by the Monash University Animal Ethics Committee (MMCB2011/05 and MMCB2013/21). HLA-DR15-α3 and HLA-DR1-α3 were produced in High Five insect cells (Trichoplusia ni BTI-Tn-5B1-4 cells, Invitrogen) using the baculovirus expression system essentially as described previously for HLA-DQ2/DQ8 proteins19, 20. Briefly, synthetic DNA (Integrated DNA Technologies, Iowa, USA) encoding the α- and β-chain extracellular domains of HLA-DR15 (HLA-DR1A*0101, HLA-DRB1*15:01), HLA-DR1 (HLA-DR1A*0101, HLA-DRB1*01:01) and the α3 peptide were cloned into the pZIP3 baculovirus vector19, 20. To promote correct pairing, the carboxy (C) termini of the HLA-DR15 and HLA-DR1 α- and β-chain encoded enterokinase cleavable Fos and Jun leucine zippers, respectively. The β-chains also encoded a C-terminal BirA ligase recognition sequence for biotinylation and a poly-histidine tag for purification. HLA-DR15-α3 and HLA-DR1-α3 were purified from baculovirus-infected High Five insect cell supernatants through successive steps of immobilized metal ion affinity (Ni Sepharose 6 Fast-Flow, GE Healthcare), size exclusion (S200 Superdex 16/600, GE Healthcare) and anion exchange (HiTrap Q HP, GE Healthcare) chromatography. For crystallization, the leucine zipper and associated tags were removed by enterokinase digestion (Genscript, New Jersey, USA) further purified by anion exchange chromatography, buffer exchanged into 10 mM Tris, pH 8.0, 150 mM NaCl and concentrated to 7 mg ml−1. Purified HLA-DR15-α3 and HLA-DR1-α3 proteins were buffer exchanged into 10 mM Tris pH 8.0, biotinylated using BirA ligase and tetramers assembled by addition of Streptavidin-PE (BD Biosciences) as previously described19. In mice, 107 splenocytes or cells from kidneys were digested with 5 mg ml−1 collagenase D (Roche Diagnostics, Indianapolis, Indiana, USA) and 100 mg ml−1 DNase I (Roche Diagnostics) in HBBS (Sigma-Aldrich) for 30 min at 37 °C, then filtered, erythrocytes lysed and the CD45+ leukocyte population isolated by MACS using mouse CD45 microbeads (Miltenyi Biotec); they were then surface stained with Pacific Blue-labelled anti-mouse CD4 (BD), antigen-presenting cell (APC)-Cy7-labelled anti-mouse CD8 (BioLegend) and 10 nM PE-labelled tetramer. Cells were then incubated with a Live/Dead fixable Near IR Dead Cell Stain (Thermo Scientific), permeabilized using a Foxp3 Fix/Perm Buffer Set (BioLegend) and stained with Alexa Fluor 647-labelled anti-mouse Foxp3 antibody (FJK16 s). To determine Vα2 and Vβ6 usage, cells were stained with PerCP/Cy5.5 anti-mouse Vα2 (B20.1, Biolegend) and antigen-presenting cell labelled anti-mouse Vβ6 (RR4-7, Biolegend). For each mouse a minimum of 100 cells were analysed. The tetramer+ gate was set on the basis of the CD8+ population. In humans, 3 × 107 white blood cells were surface stained with BV510-labelled anti-human CD3 (BioLegend), Pacific Blue-labelled anti-human CD4 (BioLegend), PE-Cy7-labelled anti-human CD127 (BioLegend), FITC-labelled anti-human CD25 (BioLegend) and 10 nM PE-labelled tetramer. Then, cells were incubated with a Live/Dead fixable Near IR Dead Cell Stain (Life Technologies), permeabilized using a Foxp3 Fix/Perm Buffer Set (BioLegend) and stained with Alexa Fluor 647-labelled anti-human Foxp3 antibody (150D). The tetramer+ gate was set on the basis of the CD3+CD4− population. As validation controls, we found that HLA-DR1-α3 tetramer+ cells did not bind to HLA-DR1-CLIP tetramers (data not shown). The human α3 peptide (GWISLWKGFSF), the mouse α3 peptide (DWVSLWKGFSF) and control OVA peptide (ISQAVHAAHAEINEAGR) were synthesized at >95% purity, confirmed by high-performance liquid chromatography (Mimotopes). Recombinant murine α3(IV)NC1 was generated using a baculovirus system21 and recombinant human α3(IV)NC1 expressed in HEK 293 cells22. The murine α3(IV)NC1 peptide library, which consists of 28 20-amino-acid long peptides overlapping by 12 amino acids, was synthesized as a PepSet (Mimotopes). To measure peptide specific recall responses, IFN-γ and IL-17A ELISPOTs and [3H]thymidine proliferation assays were used (Mabtech for human ELISPOTs and BD Biosciences for mouse ELISPOTs). To measure pro-inflammatory responses of HLA-DR15-α3 tetramer+ CD4+ T cells in patients with Goodpasture disease, HLA-DR15-α3 tetramer+ CD4+ T cells were enumerated then isolated from peripheral blood mononuclear cells of patients with Goodpasture disease (frozen at the time of presentation) by magnetic bead separation (Miltenyi Biotec) then co-cultured at a frequency of 400 HLA-DR15-α3 tetramer+ CD4+ T cells per well with 2 × 106 HLA-DR15-α3 tetramer-depleted mitomycin C-treated white blood cells and stimulated with either no antigens, α3 (10 μg ml−1) or whole recombinant human α3(IV)NC1 (10 μg ml−1) in supplemented RPMI media (10% male AB serum, 2 mM l-glutamine, 50 μM 2-ME, 100 U ml−1 penicillin and 0.1 mg ml−1 streptomycin) (Sigma-Aldrich). Cells were cultured for 18 h at 37 °C, 5% CO and the data expressed as numbers of IFN-γ or IL-17A spots per well. To measure pro-inflammatory responses of HLA-DR15-α3 tetramer+ CD4+ T cells in DR15+ transgenic mice, HLA-DR15-α3 tetramer+ CD4+ T cells were enumerated then isolated from pooled spleen and lymph node cells of DR15+ transgenic mice, immunized with mouse α3 10 days previously by magnetic bead separation. They were then co-cultured at a frequency of 400 HLA-DR15-α3 tetramer+ CD4+ T cells per well with 106 HLA-DR15-α3 tetramer-depleted mitomycin C-treated white blood cells and stimulated with either no antigens, mouse α3 (10 μg ml−1), human α3 (10 μg ml−1), whole recombinant mα3(IV)NC1 (10 μg ml−1) or whole recombinant hα3(IV)NC1 (10 μg ml−1) in supplemented RPMI media (10% FCS, 2 mM l-glutamine, 50 μM 2-ME, 100 U ml−1 penicillin and 0.1 mg ml−1 streptomycin). Cells were cultured for 18 h at 37 °C, 5% CO and the data expressed as numbers of IFN-γ or IL-17A spots per well. To determine the immunogenic portions of α3(IV)NC1, mice were immunized subcutaneously with peptide pools (containing α3 amino acids 1–92, 81–164, or 153–233; 10 μg per peptide per mouse), the individual peptide or in some experiments mα3 at 10 μg per mouse in Freund’s complete adjuvant (Sigma-Aldrich). Draining lymph node cells were harvested 10 days after immunization and stimulated in vitro (5 × 105 cells per well) with no antigen, peptide (10 μg ml−1) or whole α3(IV)NC1 (10 μg ml−1) in supplemented RPMI media (10% FCS, 2 mM l-glutamine, 50 μM 2-ME, 100 U ml−1 penicillin and 0.1 mg ml streptomycin). For [3H]thymidine proliferation assays, cells were cultured in triplicate for 72 h with [3H]thymidine added to culture for the last 16 h. To measure human α3 - or mouse α3 -specific responses in CD4+ T cells from naive transgenic mice or blood of healthy humans, we used a modification of a previously published protocol23. One million CD4+ T cells were cultured with 106 mitomycin-treated CD4-depleted splenocytes for 8 days in 96-well plates with or without 100 μg ml−1 of human α3 or mouse α3 . T cells were depleted from mouse cultures by sorting out CD4+CD25+ and in humans by sorting out CD4+CD25hiCD127lo cells using antibodies and a cell sorter. Cytokine secretion was detected in the cultured supernatants by cytometric bead array (BD Biosciences) or ELISA (R&D Systems). To determine proliferation, magnetically separated CD4+ T cells were labelled with CellTrace Violet (CTV; Thermo Scientific) before culture. To measure the expansion of T cells, mice were immunized with 100 μg of α3 emulsified in Freund’s complete adjuvant, then boosted 7 days later in Freund’s incomplete adjuvant. Draining lymph node cells were stained with the HLA-DR15-α3 tetramer, CD3, CD4, CXCR5, PD-1, CD8 and Live/Dead Viability dye. To determine the potency of HLA-DR1-α3 tetramer+ T cells, 106 cells per well of CD4+CD25− T effectors isolated by CD4+ magnetic beads and CD25− cell sorting from naive DR15+DR1+ mice were co-cultured with CD4+CD25+ T cells with or without depletion of HLA-DR1-α3 tetramer+ T cells from DR1+ mice at different concentrations: 0, 12.5 × 103, 25 × 103, 50 × 103 and 100 × 103 cells per well in the presence of 106 CD4-depleted mitomycin C-treated spleen and lymph node cells from DR15+DR1+mice in supplemented RPMI media (10% FCS, 2 mM l-glutamine, 50 μM 2-ME, 100 U ml−1 penicillin and 0.1 mg ml−1 streptomycin) containing 100 μg ml−1 of mouse α3 . To determine proliferation, the CD4+CD25− T effector cells were labelled with CTV before culture. Cells were cultured in triplicate for 8 days in 96-well plates. HLA transgenic mice, on an Fcgr2b−/− background, were immunized with 100 μg of α3 or mα3 subcutaneously on days 0, 7 and 14, first in Freund’s complete, and then in Freund’s incomplete, adjuvant. Mice were killed on day 42. Albuminuria was assessed in urine collected during the last 24 h by ELISA (Bethyl Laboratories) and expressed as milligrams per micromole of urine creatinine. Blood urea nitrogen and urine creatinine were measured using an autoanalyser at Monash Health. Glomerular necrosis and crescent formation were assessed on periodic acid-Schiff (PAS)-stained sections; fibrin deposition using anti-murine fibrinogen antibody (R-4025) and DAB (Sigma); CD4+ T cells, macrophages and neutrophils were detected using anti-CD4 (GK1.5), anti-CD68 (FA/11) and anti-Gr-1 (RB6-8C5) antibodies. The investigators were not blinded to allocation during experiments and outcome assessment, except in histological and immunohistochemical assessment of kidney sections. To deplete regulatory T cells, mice were injected intraperitoneally with 1 mg of an anti-CD25 monoclonal antibody (clone PC61) or rat IgG (control) 2 days before induction of disease. In these experiments, mice were randomly assigned to receive control or anti-CD25 antibodies. Individual DR15-α3 -specific CD4+ T cells were sorted into wells of a 96-well plate. Multiplex single-cell reverse transcription and PCR amplification of TCR CDR3α and CDR3β regions were performed using a panel of TRBV- and TRAV-specific oligonucleotides, as described24, 25. Briefly, mRNA was reverse transcribed in 2.5 μl using a Superscript III VILO cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) (containing 1× Vilo reaction mix, 1× superscript RT, 0.1% Triton X-100), and incubated at 25 °C for 10 min, 42 °C for 120 min and 85 °C for 5 min. The entire volume was then used in a 25 μl first-round PCR reaction with 1.5 U Taq DNA polymerase, 1× PCR buffer, 1.5 mM MgCl , 0.25 mM dNTPs and a mix of 25 mouse TRAV or 40 human TRAV external sense primers and a TRAC external antisense primer, along with 19 mouse TRBV or 28 human TRBV external sense primers and a TRBC external antisense primer (each at 5 pmol μl−1), using standard PCR conditions. For the second-round nested PCR, a 2.5 μl aliquot of the first-round PCR product was used in separate TRBV- and TRAV-specific PCRs, using the same reaction mix described above; however, a set of 25 mouse TRAV or 40 human TRAV internal sense primers and a TRAC internal antisense primer, or a set of 19 mouse TRBV or 28 human TRBV internal sense primers and a TRBV internal antisense primer, were used. Second-round PCR products were visualized on a gel and positive reactions were purified with ExoSAP-IT reagent. Purified products were used as template in sequencing reactions with internal TRAC or TRBC antisense primers, as described. TCR gene segments were assigned using the IMGT (International ImMunoGeneTics) database26. In mouse experiments, three mice were pooled per HLA and the number of sequences obtained were as follows. For TRAV: DR15, n = 81; DR1 n = 84; for TRBV: DR15, n = 100; DR1 n = 87; for TRAJ: DR15, n = 81; DR1 n = 84; and for TCR beta joining (TRBJ): DR15, n = 100; DR1 n = 87. Red-blood-cell-lysed splenocytes from DR1+ and DRB15+DR1+ mice were sorted on the basis of surface expression of CD4 and CD25 and being either DR1-α3 tetramer positive or negative into three groups: (1) CD4+CD25−HLA-DR1-α3 tetramer− T cells; (2) CD4+CD25+HLA-DR1-α3 tetramer− T cells; and (3) CD4+CD25+HLA-DR1-α3 tetramer+ T cells. A minimum of 1,000 cells were sorted. Immediately after sorting, the RNA was isolated and complementary DNA (cDNA) generated using a Cells to Ct Kit (Ambion) followed by a preamplification reaction using Taqman Pre Amp Master Mix (Applied Biosystems), which preamplified the following cDNAs: Il2ra, Foxp3, Ctla4, Tnfrsf18, Il7r, Sell, Pdcd1, Entpd1, Cd44, Tgfb3, Itgae, Ccr6, Lag3, Lgals1, Ikzf2, Tnfrsf25, Nrp1, Il10. The preamplified cDNA was used for RT–PCR reactions in duplicate using Taqman probes for the aforementioned genes. Each gene was expressed relative to 18S, logarithmically transformed and presented as a heat map. The Epstein-Barr-virus-transformed human B lymphoblastoid cell lines IHW09013 (SCHU, DR15-DR51-DQ6) and IHW09004 (JESTHOM, DR1-DQ5) were maintained in RPMI (Invitrogen) supplemented with 10% FCS, 50 IU ml−1 penicillin and 50 μg ml−1 streptomycin. Confirmatory tissue typing of these cells was performed by the Victorian Transplantation and Immunogenetics Service. The B-cell hybridoma LB3.1 (anti-DR) was grown in RPMI-1640 with 5% FCS at 37 °C and secreted antibody purified using protein A sepharose (BioRad). HLA-DR-presented peptides were isolated from naive DR15+Fcgr2b+/+ or DR1+Fcgr2b+/+ mice. Spleens and lymph nodes (pooled from five mice in each group) or frozen pellets of human B lymphoblastoid cell lines (triplicate samples of 109 cells) were cryogenically milled and solubilized as previously described12, 27, cleared by ultracentrifugation and MHC peptide complexes purified using LB3.1 coupled to protein A (GE Healthcare). Bound HLA complexes were eluted from each column by acidification with 10% acetic acid. The eluted mixture of peptides and HLA heavy chains was fractionated by reversed-phase high-performance liquid chromatography as previously described10. Peptide-containing fractions were analysed by nano-liquid chromatography–tandem mass spectrometry (nano-LC–MS/MS) using a ThermoFisher Q-Exactive Plus mass spectrometer (ThermoFisher Scientific, Bremen, Germany) operated as described previously10. LC–MS/MS data were searched against mouse or human proteomes (Uniprot/Swissprot v2016_11) using ProteinPilot software (SCIEX) and resulting peptide identities subjected to strict bioinformatic criteria including the use of a decoy database to calculate the false discovery rate28. A 5% false discovery rate cut-off was applied, and the filtered data set was further analysed manually to exclude redundant peptides and known contaminants as previously described29. The mass spectrometry data have been deposited in the ProteomeXchange Consortium via the PRIDE30 partner repository with the data set identifier PXD005935. Minimal core sequences found within nested sets of peptides with either N- or C-terminal extensions were extracted and aligned using MEME (http://meme.nbcr.net/meme/), where motif width was set to 9–15 and motif distribution to ‘one per sequence’31. Graphical representation of the motif was generated using IceLogo32. Crystal trials were set up at 20 °C using the hanging drop vapour diffusion method. Crystals of HLA-DR15-α3 were grown in 25% PEG 3350, 0.2 M KNO and 0.1 M Bis-Tris-propane (pH 7.5), and crystals of HLA-DR1-α3 were grown in 23% PEG 3350, 0.1 M KNO , and 0.1 M Bis-Tris-propane (pH 7.0). Crystals were washed with mother liquor supplemented with 20% ethylene glycol and flash frozen in liquid nitrogen before data collection. Data were collected using the MX1 (ref. 33) and MX2 beamlines at the Australian Synchrotron, and processed with iMosflm and Scala from the CCP4 program suite34. The structures were solved by molecular replacement in PHASER35 and refined by iterative rounds of model building using COOT36 and restrained refinement using Phenix37 (see Extended Data Table 2 for data collection and refinement statistics). No statistical methods were used to predetermine sample size. For normally distributed data, an unpaired two-tailed t-test (when comparing two groups). For non-normally distributed data, non-parametric tests (Mann–Whitney U-test for two groups or a Kruskal–Wallis test with Dunn’s multiple comparison) were used. Statistical analyses, except for TCR usage, was by GraphPad Prism (GraphPad Software). For each TCR type/region (TRAV, TRBV, TRAJ, TRBJ), we compared the TCR distribution (frequencies of different TCRs) between DR15 and DR1 using Fisher’s exact test. This was applied both to mice and to human samples. The P values associated with those TCR distributions are indicated above the pie-charts. To correct for multiple testing for individual TCRs, we used Holm’s method. *P < 0.05, **P < 0.01, ***P < 0.001. The data that support the findings of this study are available from the corresponding authors upon request. Self-peptide repertoires have been deposited in the Proteomics Identifications Database archive with the accession code PXD005935. Structural information has been deposited in the Protein Data Bank under accession numbers 5V4M and 5V4N.
News Article | May 11, 2017
Achieving an enhanced rate of ion flow through channels and porous membranes is important for a range of applications, such as energy storage and water desalination, but it is challenging. The collaboration of researchers from Deakin University and ANSTO in Australia, the Sorbonne in France and Drexel University in the US, has just published the study in The Journal of the American Chemical Society. Boron nitride nanosheets are usually hydrophilic and the team used an understanding of the nanosheet interactions in solution during a filtration process to allowing nanosheets to self-assemble into the special structure in aqueous solution. ANSTO instrument scientist Chris Garvey and Guang Wang an AINSE Post Graduate Research Award recipient from Deakin University, used small angle X-ray scattering (SAXS) at the Australian Synchrotron as a structural tool to probe the material and characterise the nanofluidic channels in a dry and fully hydrated boron nitride membrane. "The interaction of the nanoparticles in solution allowed the nanosheets to self-assemble into material with an interesting structure as a thin film with enhanced conductivity," explained Garvey. "As you remove the water during the manufacturing/filtration process, the particles come closer together and the interactions between the particles become important in the self-assembly process and the final structure," said Garvey. The boron nitride nanosheets stacked up in a well-aligned manner and formed a lamellar membrane structure. Thousands of parallel slit shaped ionic channels formed in a particular orientation on the membrane that acted as a nanofluidic conduit. "By contrast to an electron microscope, with SAXS you can look inside a material and see how it is assembled, we can see what happens when you put water and salt in a nanosized compartment," said Garvey. Measurements at the Australian Synchrotron at the SAXS beamline allowed them to determine the average spacing between the layers. "The X-ray beam, which is about 200-300 microns in diameter, is well suited for analysing a many nanolayers, giving a statistical perspective on structure," said Garvey. SAXS measurements perpendicular to the beam indicated a lack of structural order along the lateral direction of the membrane, which had also been reported for nanosheets of graphene oxide. The overall structural perspective suggested the ions were being excluded from the inner spaces of the channels in the membrane. Measurement parallel to the boron nitride membrane allowed them to determine that water molecules and ions remained in the intra-layer channels. The way ions pass through the nanoscale fluidic channels is significantly different from the way ions pass through the bulk. The authors concluded that a negative surface charge at the interface between the channel wall and the electrolyte was found to play an important role in ion transport. Garvey said that the physics of filtration processes was not well understood, with further understanding having relevance for many applications, such as assembly of these materials but also including the way clay soils behave. Boron nitride membranes could be an attractive and promising replacement for current 2D nanomaterials subject to harsh conditions. Explore further: New nontoxic process promises larger ultrathin sheets of 2-D nanomaterials More information: Si Qin et al. High and Stable Ionic Conductivity in 2D Nanofluidic Ion Channels between Boron Nitride Layers, Journal of the American Chemical Society (2017). DOI: 10.1021/jacs.6b11100
Schuettfort T.,University of Cambridge |
Thomsen L.,Australian Synchrotron |
McNeill C.R.,Monash University
Journal of the American Chemical Society | Year: 2013
The molecular orientation and microstructure of films of the high-mobility semiconducting polymer poly(N,N-bis-2-octyldodecylnaphthalene-1,4,5,8-bis- dicarboximide-2,6-diyl-alt-5,5-2,2-bithiophene) (P(NDI2OD-T2)) are probed using a combination of grazing-incidence wide-angle X-ray scattering (GIWAXS) and near-edge X-ray absorption fine-structure (NEXAFS) spectroscopy. In particular a novel approach is used whereby the bulk molecular orientation and surface molecular orientation are simultaneously measured on the same sample using NEXAFS spectroscopy in an angle-resolved transmission experiment. Furthermore, the acquisition of bulk-sensitive NEXAFS data enables a direct comparison of the information provided by GIWAXS and NEXAFS. By comparison of the bulk-sensitive and surface-sensitive NEXAFS data, a distinctly different molecular orientation is observed at the surface of the film compared to the bulk. While a more "face-on" orientation of the conjugated backbone is observed in the bulk of the film, consistent with the lamella orientation observed by GIWAXS, a more "edge-on" orientation is observed at the surface of the film with surface-sensitive NEXAFS spectroscopy. This distinct edge-on surface orientation explains the high in-plane mobility that is achieved in top-gate P(NDI2OD-T2) field-effect transistors (FETs), while the bulk face-on texture explains the high out-of-plane mobilities that are observed in time-of-flight and diode measurements. These results also stress that GIWAXS lacks the surface sensitivity required to probe the microstructure of the accumulation layer that supports charge transport in organic FETs and hence may not necessarily be appropriate for correlating film microstructure and FET charge transport. © 2012 American Chemical Society.
News Article | September 12, 2016
New research has found that venom extracted from a species of marine cone snail could hold the key to developing 'ultra-fast-acting' insulins, leading to more efficient therapies for diabetes management. Researchers from Australia and the US have successfully determined the three-dimensional structure of a cone snail venom insulin, revealing how these highly efficient natural proteins called Con-Ins G1 can operate faster than human insulin. The teams also discovered that Con-Ins G1 was able bind to human insulin receptors, signifying the potential for its translation into a human therapeutic. Associate Professor Mike Lawrence from Melbourne's Walter and Eliza Hall Institute of Medical Research led a collaborative study between the University of Utah, the Monash Institute of Pharmaceutical Sciences, La Trobe University and Flinders University in Australia. Associate Professor Lawrence, a specialist in the structure of insulins and their receptors, said the teams utilised the Australian Synchrotron to create and analyse the three-dimensional structure of this cone snail venom insulin protein with exciting results. "We found that cone snail venom insulins work faster than human insulins by avoiding the structural changes that human insulins undergo in order to function -- they are essentially primed and ready to bind to their receptors, " Associate Professor Lawrence said. Associate Professor Lawrence said human insulins could be considered 'clunky' by comparison. "The structure of human insulins contain an extra 'hinge' component that has to open before any 'molecular handshake' or connection between insulin and receptor can take place. "By studying the three-dimensional structure of this snail venom insulin we've found how to dispense with this 'hinge' entirely, which may accelerate the cell signalling process and thus the speed with which the insulin takes effect." Associate Professor Lawrence said. Published today in Nature Structural and Molecular Biology, the team's findings build on earlier studies from 2015, when the University of Utah reported that the marine cone snail Conus geographus used an insulin-based venom to trap its prey. Unsuspecting fish prey would swim into the invisible trap and immediately become immobilised in a state of hyperglycaemic shock induced by the venom. Dr Helena Safavi-Hemami from the University of Utah said it was fascinating to uncover how the cone snail insulin was able to have such a rapid effect on its prey and, furthermore, that the peptide had therapeutic potential in humans. "We were thrilled to find that the principles of cone snail venom insulins could be applied to a human setting," Dr Safavi-Hemami said. "Our Flinders University colleagues have shown that the cone snail insulin can 'switch on' human insulin cell signalling pathways, meaning the cone snail insulin is able to successfully bind to human receptors," Dr Safavi-Hemami said. "The next step in our research, which is already underway, is to apply these findings to the design of new and better treatments for diabetes, giving patients access to faster-acting insulins," she said.
News Article | February 27, 2017
New light on a key factor involved in diseases such as Parkinson's disease, gastric cancer and melanoma has been cast through latest University of Otago, New Zealand, research carried out in collaboration with Australian scientists. In new findings published in leading international journal PNAS, the team of researchers, led by Otago Department of Biochemistry's Dr Peter Mace, studied a protein called Apoptosis signal-regulating kinase 1 (ASK1). Along with other kinases, ASK1 acts as a signalling protein that controls many aspects of cellular behaviour. Kinases put tags onto other proteins that can turn them on, off, which in turn can make a cell divide, die, move or any number of other responses. Dr Mace says ASK1 plays an important role in controlling how a cell responds to cell damage, and can push the cell towards a process of programmed cell death for the good of the body, if damage to a cell is too great. This key role is reflected in ASK1's name - apoptosis is an Ancient Greek word meaning "falling off" - and is used to describe the process of programmed dying of cells, rather than their loss by injury. The research team determined ASK1's molecular structure through crystallography studies and also performed biochemical experiments to better understand the protein. They found that ASK1 has unexpected parts to its structure that help control how the protein is turned on, and that an entire family of ASK kinases share these features. "We now know a lot more about how ASK1 gets turned on and off - this is important because in diseases such as Parkinson's, stomach cancer and melanoma, there can be either too much or too little ASK1 activity". Dr Mace says that the new findings add to our understanding of how cells can trigger specific responses to different threats or damage encountered. Such threats can include oxidants, which damage the body's tissues by causing inflammation. He adds that kinases are excellent targets for developing new drugs because they have a "pocket" in their structure that such compounds can bind to, but to develop better drugs we need to understand far more about how they are controlled. This is the goal of several projects in his lab, he says. The study is a collaboration between Otago researchers and scientists at the Walter and Eliza Hall Institute (WEHI) in Melbourne, and at the Australian Synchrotron. Otago alumnus Tom Caradoc-Davies, who works at the MX Beamline, collected data that was critical to the project. Synchrotron access was enabled by the New Zealand Synchrotron Group, which is coordinated by the Royal Society of New Zealand and supported by all New Zealand universities in partnership with the Government. The synchrotron is crucial to many other research projects from Otago and throughout New Zealand.
News Article | February 27, 2017
In new findings published in leading international journal PNAS, the team of researchers, led by Otago Department of Biochemistry's Dr Peter Mace, studied a protein called Apoptosis signal-regulating kinase 1 (ASK1). Along with other kinases, ASK1 acts as a signalling protein that controls many aspects of cellular behaviour. Kinases put tags onto other proteins that can turn them on, off, which in turn can make a cell divide, die, move or any number of other responses. Dr Mace says ASK1 plays an important role in controlling how a cell responds to cell damage, and can push the cell towards a process of programmed cell death for the good of the body, if damage to a cell is too great. This key role is reflected in ASK1's name - apoptosis is an Ancient Greek word meaning "falling off" - and is used to describe the process of programmed dying of cells, rather than their loss by injury. The research team determined ASK1's molecular structure through crystallography studies and also performed biochemical experiments to better understand the protein. They found that ASK1 has unexpected parts to its structure that help control how the protein is turned on, and that an entire family of ASK kinases share these features. "We now know a lot more about how ASK1 gets turned on and off - this is important because in diseases such as Parkinson's, stomach cancer and melanoma, there can be either too much or too little ASK1 activity". Dr Mace says that the new findings add to our understanding of how cells can trigger specific responses to different threats or damage encountered. Such threats can include oxidants, which damage the body's tissues by causing inflammation. He adds that kinases are excellent targets for developing new drugs because they have a "pocket" in their structure that such compounds can bind to, but to develop better drugs we need to understand far more about how they are controlled. This is the goal of several projects in his lab, he says. The study is a collaboration between Otago researchers and scientists at the Walter and Eliza Hall Institute (WEHI) in Melbourne, and at the Australian Synchrotron. Otago alumnus Tom Caradoc-Davies, who works at the MX Beamline, collected data that was critical to the project. Synchrotron access was enabled by the New Zealand Synchrotron Group, which is coordinated by the Royal Society of New Zealand and supported by all New Zealand universities in partnership with the Government. The synchrotron is crucial to many other research projects from Otago and throughout New Zealand. More information: Structural basis of autoregulatory scaffolding by apoptosis signal-regulating kinase 1, PNAS, www.pnas.org/cgi/doi/10.1073/pnas.1620813114
De Jonge M.D.,Australian Synchrotron |
Vogt S.,Argonne National Laboratory
Current Opinion in Structural Biology | Year: 2010
Hard X-ray fluorescence microscopy is well-suited to in-situ investigations of trace metal distributions within whole, unstained, biological tissue, with sub-parts-per-million detection achievable in whole cells. The high penetration of X-rays indicates the use of X-ray fluorescence tomography for structural visualization, and recent measurements have realised sub-500-nm tomography on a 10-μm cell. Limitations of present approaches impact the duration of an experiment and imaging fidelity. Developments in X-ray resolution, detector speed, cryogenic environments, and the incorporation of auxiliary signals are being pursued within the synchrotron community. Several complementary approaches to X-ray fluorescence tomography will be routinely available to the biologist in the near future. We discuss these approaches and review applications of biological relevance. © 2010 Elsevier Ltd.