Chaikovska I.,University Paris - Sud |
Bruni C.,University Paris - Sud |
Chance S.,University Paris - Sud |
Gamelin A.,University Paris - Sud |
And 2 more authors.
IPAC 2016 - Proceedings of the 7th International Particle Accelerator Conference | Year: 2016
ThomX is a compact Compton based X-ray source under construction at LAL in Orsay (France). The ThomX facility is composed by a 50-70 MeV linac, a transfer line and a 18 meters long storage ring. Compton scattering between the 50 MeV electron bunch of 1 nC and the 30 mJ laser pulses stacked in the Fabry-Perot cavity results in the production of photons with energies (up to 90 keV) with a maximum flux of 1013 photons per second. The ThomX construction will start shortly aiming to be completed in the middle of 2017. The preparation to the storage ring commissioning as far as a control system and beam physics applications are concerned is progressing gradually in order to prepare and test all the tools well ahead the start of the machine. The storage ring commissioning will face with many challenges providing the low energy, compactness, the nonlinear beam dynamics, the limited beam storage and need for the precision and stabilization in the Interaction Region. Several techniques used at the Synchrotron Light Sources should be modified/adapted to meet all the specificity of the ThomX. This is a report on preparation of the ThomX storage ring commissioning, its status, planning, main challenges and expectations. Copyright © 2016 CC-BY-3.0 and by the respective authors.
Chwiej J.,AGH University of Science and Technology |
Dulinska J.,AGH University of Science and Technology |
Janeczko K.,Jagiellonian University |
Dumas P.,SOLEIL |
And 3 more authors.
Journal of Chemical Neuroanatomy | Year: 2010
In the present work, synchrotron radiation Fourier transform infrared (SRFTIR) micro-spectroscopy and imaging were used for topographic and semi-quantitative biochemical analysis of rat brain tissue in cases of pilocarpine-induced epilepsy. The tissue samples were analyzed with a beam defined by small apertures and spatial resolution steps of 10μm which allowed us to probe the selected cellular layers of hippocampal formation. Raster scanning of the samples has generated 2D chemical cartographies revealing the distribution of proteins, lipids and nucleic acids. Spectral analysis has shown changes in the saturation level of phospholipids and relative secondary structure of proteins. Special interest was put in the analysis of two areas of the hippocampal formation (sector 3 of the Ammon's horn, CA3 and dentate gyrus, DG) in which elemental abnormalities were observed during our previous studies. Statistically significant increase in the saturation level of phospholipids (increased ratio of the absorption intensities at around 2921 and 2958cm-1) as well as conformational changes of proteins (β-type structure discrepancies as shown by the increased ratio of the absorbance intensities at around 1631 and 1657cm-1 as well as the ratio of the absorbance at 1548 and 1657cm-1) were detected in pyramidal cells of CA3 area as well as in the multiform and molecular layers of DG. The findings presented here suggest that abnormalities in the protein secondary structure and increases in the level of phospholipid saturation could be involved in mechanisms of neurodegenerative changes following the oxidative stress evoked in brain areas affected by pilocarpine-induced seizures. © 2010 Elsevier B.V.
Rankovic M.L.,University of Belgrade |
Giuliani A.,SOLEIL |
Giuliani A.,French National Institute for Agricultural Research |
Milosavljevic A.R.,University of Belgrade |
Milosavljevic A.R.,University of Notre Dame
Applied Physics Letters | Year: 2016
We have performed inner-shell electron impact action spectroscopy of mass and charge selected macromolecular ions. For this purpose, we have coupled a focusing electron gun with a linear quadrupole ion trap mass spectrometer. This experiment represents a proof of principle that an energy-tunable electron beam can be used in combination with radio frequency traps as an activation method in tandem mass spectrometry (MS2) and allows performing action spectroscopy. Electron impact MS2 spectra of multiply protonated ubiquitin protein ion have been recorded at incident electron energies around the carbon 1 s excitation. Both MS2 and single ionization energy dependence spectra are compared with literature data obtained using the soft X-ray activation conditions. © 2016 AIP Publishing LLC.
News Article | March 4, 2016
No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. The strains, plasmids and oligonucleotides used in this study are listed in the Supplementary Table. The enteroaggregative E. coli EAEC strain 17-2 and its ∆tssA, ∆tssBC, ∆tssE, ∆tssF, ∆tssG, ∆tssK, ∆tssL, ∆tssM, ∆hcp, ∆vgrG and tssB-mCherry isogenic derivatives were used for this study11, 15, 25. The E. coli K-12 DH5α, W3110, BTH101 and T7 Iq pLys strains were used for cloning steps, co-immunoprecipitation, bacterial two-hybrid and protein purification, respectively. Strains were routinely grown in LB rich medium (or Terrific broth medium for protein purification) or in Sci-1 inducing medium (SIM; M9 minimal medium, glycerol 0.2%, vitamin B1 1 μg ml−1, casaminoacids 100 μg ml−1, LB 10%, supplemented or not with bactoagar 1.5%) with shaking at 37 °C31. Plasmids were maintained by the addition of ampicillin (100 μg ml−1 for E. coli K-12, 200 μg ml−1 for EAEC), kanamycin (50 μg ml−1) or chloramphenicol (30 μg ml−1). Expression of genes from pBAD, pETG20A/pRSF or pASK-IBA vectors was induced at A ≈ 0.6 with 0.02% of l-arabinose (Sigma-Aldrich) for 45 min, 0.5–1 mM of isopropyl-β-d-thio-galactopyrannoside (IPTG, Eurobio) for 14 h or 0.02 μg ml−1 of anhydrotetracyclin (AHT, IBA Technologies) for 45 min, respectively. For BACTH experiments, plates were supplemented with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal, Eurobio, 40 μg ml−1). The tssA gene was deleted into the enteroaggregative E. coli 17-2 strain using a modified one-step inactivation procedure32 as previously described11 using plasmid pKOBEG33. In brief, a kanamycin cassette was amplified from plasmid pKD46 using oligonucleotides carrying 50-nucleotide extensions homologous to regions adjacent to tssA. After electroporation of 600 ng of column-purified PCR product, kanamycin-resistant clones were selected and verified by colony-PCR. The kanamycin cassette was then excised using plasmid pCP20 (ref. 32). The deletion of tssA was confirmed by colony-PCR. The same procedure was used to introduce the mCherry-coding sequence upstream the stop codon of the tssB gene (vector pmCh-KD4 as template for PCR amplification) or the sfGFP-coding sequence downstream the start codon (vector pKD4-sfGFP as template) or upstream the stop codon (vector psfGFP-KD4 as template) of the tssA gene to yield strains producing TssB–mCherry, sfGFP–TssA or TssA–sfGFP from their original chromosomal loci. All bacterial two-hybrid plasmids and the plasmid producing the TssJLM membrane core complex (pRSF-TssJST-FLTssL-6HisTssM, pRSF-TssJLM) have been described previously13, 25. PCR was performed using a Biometra thermocycler using the Q5 (New England Biolabs) or Pfu Turbo (Agilent Technologies) DNA polymerases. Restriction enzymes were purchased from New England Biolabs and used according to the manufacturer’s instructions. Custom oligonucleotides were synthesized by Sigma Aldrich and are listed in the Supplementary Table. Enteroaggregative E. coli 17-2 chromosomal DNA was used as a template for all PCR. E. coli strain DH5α was used for cloning procedures. All the plasmids (except for pETG20A and pDEST17 derivatives) have been constructed by restriction-free cloning34 as previously described25. In brief, the gene of interest was amplified using oligonucleotides introducing extensions annealing to the target vector. The double-stranded product of the first PCR has then been used as oligonucleotides for a second PCR using the target vector as template. PCR products were then treated with DpnI to eliminate template plasmids and transformed into DH5α-competent cells. For protein purification, the sequences encoding the full-length TssA (residues 1–542), the TssA (residues 1–392), the TssA (residues 221–377) and TssA (residues 393–542) domains, the N-terminal domain of VgrG (residues 1– 490), the full-length TssE or both TssB and TssC were cloned into the pETG-20A (TssA, TssA , VgrG , TssE) or pDEST17 (TssA , TssBC) expression vector (gifts from A. Geerlof, EMBL, Hamburg) according to standard Gateway protocols. Proteins produced from pETG20A derivatives are fused to an N-terminal 6 × His-tagged thioredoxin (TRX) followed by a cleavage site for the Tobacco etch virus (TEV) protease whereas proteins produced from pDEST17 are fused to an N-terminal 6 × His tag followed by a TEV protease cleavage site. All constructs have been verified by restriction analyses and DNA sequencing (Eurofins or MWG). The adenylate cyclase-based bacterial two-hybrid technique35 was used as previously published36. In brief, the proteins to be tested were fused to the isolated T18 and T25 catalytic domains of the Bordetella adenylate cyclase. After introduction of the two plasmids producing the fusion proteins into the reporter BTH101 strain, plates were incubated at 30 °C for 48 h. Three independent colonies for each transformation were inoculated into 600 μl of LB medium supplemented with ampicillin, kanamycin and IPTG (0.5 mM). After overnight growth at 30 °C, 10 μl of each culture were dropped onto LB plates supplemented with ampicillin, kanamycin, IPTG and X-Gal and incubated for 16 h at 30 °C. The experiments were done at least in triplicate and a representative result is shown. Fluorescence microscopy experiments have been performed essentially as described13, 15, 21, 25. In brief, cells were grown overnight in LB medium and diluted to an A ≈ 0.04 into Sci-1 inducing medium (SIM). Exponentially growing cells (A ≈ 0.8–1) were harvested, washed in phosphate buffered saline buffer (PBS), resuspended in PBS to an A ≈ 50, spotted on a thin pad of 1.5% agarose in PBS, covered with a cover slip and incubated for one hour at 37 °C before microscopy acquisition. For each experiment, ten independent fields were manually defined with a motorized stage (Prior Scientific) and stored (x, y, z, PFS-offset) in our custom automation system designed for time-lapse experiments. Fluorescence and phase contrast micrographs were captured every 30 s, using an automated and inverted epifluorescence microscope TE2000-E-PFS (Nikon, France) equipped with a perfect focus system (PFS). PFS automatically maintains focus so that the point of interest within a specimen is always kept in sharp focus at all times despite mechanical or thermal perturbations. Images were recorded with a CoolSNAP HQ 2 (Roper Scientific, Roper Scientific SARL, France) and a 100× /1.4 DLL objective. The excitation light was emitted by a 120 W metal halide light. All fluorescence images were acquired with a minimal exposure time to minimize bleaching and phototoxicity effects. The sfGFP images were recorded by using the ET-GFP filter set (Chroma 49002) using an exposure time of 200–400 ms. The mCherry images were recorded by using the ET-mCherry filter set (Chroma 49008) using an exposure time of 100–200 ms. Slight movements of the whole field during the time of the experiment were corrected by registering individual frames using StackReg and Image Stabilizer plugins for ImageJ. sfGFP and mCherry fluorescence channels were adjusted and merged using ImageJ (http://rsb.info.nih.gov/ij/). For statistical analyses, fluorescent foci were automatically detected. First, noise and background were reduced using the ‘Subtract Background’ (20 pixels Rolling Ball) plugin from Fiji37. The sfGFP foci were automatically detected by a simple image processing: (1) create a mask of cell surface and dilate; (2) detect the individual cells using the ‘Analyse particle’ plugin of Fiji; (3) sfGFP foci were identified by the ‘Find Maxima’ process in Fiji. To avoid false positives, each event was manually controlled in the original data. Box-and-whisker representations of the number of foci per cell were made with R software. t-tests were performed in R to statistically compare each population. Kymographs were obtained after background fluorescence substraction and sectioning using the Kymoreslicewide plug-in under Fiji37. Fluorescent foci were detected using a local and sub-pixel resolution maxima detection algorithm and tracked over time with a specifically developed plug-in for ImageJ. The x and y coordinates were obtained for each fluorescent focus on each frame. The mean square displacement (MSD) was calculated as the distance of the foci from its location at t = 0 at each time using R software and plotted over time. For each strain tested, the MSD of at least 10 individual focus trajectories was calculated. For statistical analyses of mobile trajectories, kymograph analyses were performed and the percentage of fixed, mobile with random dynamics and mobile with unidirectional trajectory foci are reported. FLIM experiments were carried on the same microscope device used for the time-lapse microscopy experiments except with a laser of 488 nm. For each cell a region of interest that corresponds to the size of the laser beam was focused away from TssB–mCherry sheath-labelled sfGFP–TssA for a time of 3 s at a maximum intensity of 100%. The extinction of the complete sfGFP–TssA pool was checked by (i) the absence of recovery of bleached sfGFP–TssA-membrane clusters and (ii) by the overall drop and lack of recovery in intracellular intensity. E. coli T7 Iq pLysS cells bearing pETG20A or pDEST17 derivatives were grown at 37 °C in Terrific Broth to an A ≈ 0.9 and gene expression was inducted with 0.5 mM IPTG for 16 h at 17 °C. Cells were harvested, resuspended in Tris-HCl 20 mM pH 8.0, NaCl 150 mM and lysozyme (0.25 mg ml−1) and broken by sonication. Soluble proteins were separated from inclusion bodies and cell debris by centrifugation 30 min at 20,000g. The His-tagged fusions were purified using ion metal Ni2+ affinity chromatography (IMAC) using a 5-ml HisTrap column (GE Healthcare) and eluted with a step gradient of imidazole. The fusion proteins were further digested overnight at 4 °C by a hexahistidine-tagged TEV protease using a 1:10 (w/w) protease:protein ratio. The TEV protease and contaminants were retained by a second IMAC and the purified proteins were collected in the flow through. Proteins were further separated on preparative Superdex 200 or Superose 6 gel filtration column (GE Healthcare) equilibrated in Tris-HCl 20 mM pH 8.0, NaCl 150 mM. The fractions containing the purified protein were pooled and concentrated by ultrafiltration using the Amicon technology (Millipore, California, USA). The seleno-methionine (SeMet) derivatives of the N- and C-terminal domains of TssA were produced in minimal medium supplemented with 100 mg l−1 of lysine, phenylalanine and threonine, 50 mg l−1 of isoleucine, leucine, valine and seleno-methionine. Gene induction and protein purification were performed as described above. The full-length TssA protein was subjected to Proteinase K limited proteolysis (1:10 protease:protein ratio). The reaction was quenched at different time points by the addition of 1 mM PMSF and further boiling for 10 min at 96 °C. Samples were analysed by SDS–PAGE and Coomassie blue staining. Digested products were identified by Edman N-terminal sequencing and electrospray mass sprectrometry (Proteomic platform, Institut de Microbiologie de la Méditerranée, Marseille, France). Size-exclusion chromatography (SEC) was performed on an Alliance 2695 HPLC system (Waters) using KW803 and KW804 columns (Shodex) run in Tris-HCl 20 mM pH 8.0, NaCl 150 mM at 0.5 ml per min. MALS, UV spectrophotometry, QELS and RI were monitored with MiniDawn Treos (Wyatt Technology), a Photo Diode Array 2996 (Waters), a DynaPro (Wyatt Technology) and an Optilab rEX (Wyatt Technology), respectively, as described12. Mass and hydrodynamic radius calculation were performed with the ASTRA software (Wyatt Technology) using a dn/dc value of 0.185 ml g−1. Steady-state interactions were monitored using a BIAcore T200 at 25 °C12. All the buffers were filtered on 0.2 μm membranes before use. The HC200m sensor chip (Xantech) was coated with purified Hcp, VgrG, TssE or TssBC complex, immobilized by amine coupling (∆RU = 4,000–4,300). A control flow-cell was coated with thioredoxin immobilized by amine coupling at the same concentration (∆RU = 4,100). Purified TssA, TssA N-terminal and TssA C-terminal domains (six concentrations ranging from 3.125 to 100 μM) were injected and binding traces were recorded in duplicate. The signal from the control flow cell and the buffer response were subtracted from all measurements. The dissociation constants (K ) were estimated using the GraphPad Prism 5.0 software on the basis of the steady state levels of ∆RU, directly related to the concentration of the analytes. The K were estimated by plotting on x axis the different concentration of analytes and the different ∆RU at a fixed time (5 s before the end of the injection step) on the y axis. For K calculation, nonlinear regression fit for xy analysis was used and one site (specific binding) as a model which corresponds to the equation y = B × x/(K + x). Different combinations of plasmids were transformed in BL21(DE3): (i) pRSF-TssJLM + pIBA37(+); (ii) pRSF + pIBA37-FLTssA; (iii) pRSF-TssJLM + pIBA37-FLTssA; and (iv) pRSF-TssJM + pIBA37-FLTssA. Transformed BL21(DE3) cells were grown at 37 °C in 200 ml LB medium supplemented with kanamycin and ampicillin until A ≈ 0.6 and gene induction was achieved by the addition of IPTG (1 mM) and anhydrotetracycline (0.02 μg ml−1) during 15 h at 16 °C. After cell lysis through three passages at the French press, total membranes were isolated as described previously13. Membranes were solubilized by the addition of 1% Triton X-100 (Affimetrix). Solubilized membrane fractions were purified on a 1 ml Streptactin column (GE Healthcare). The column was washed with buffer S (HEPES 50 mM pH 7.5, NaCl 50 mM, Triton X-100 0.075%) and bound proteins were eluted with buffer S supplemented with desthiobiotin (2.5 mM) and visualized by Coomassie blue staining and immunoblotting. For electron microscopy (EM) analyses, BL21(DE3) cells producing TssJLM and Flag-tagged TssA were grown and the TssJLM-A complex was purified as described for the TssJLM membrane core complex13. After the two consecutive affinity columns (His- and Strep-Trap-HP), the pooled fractions were injected onto a Superose 6 10:300 column equilibrated in HEPES 50 mM pH 7.5, NaCl 50 mM supplemented with 0.025% DM-NPG. Nine microlitres of the purified TssJLMA complex (~ 0.01 mg ml−1) were incubated to glow-discharged carbon-coated copper grids (Agar Scientific) for 30 s. After absorption, the sample was blotted, washed with three drops of water and then stained with 2% uranyl acetate. Images were collected on an FEI Tecnai F20 FEG microscope operating at a voltage of 200 kV, equipped with a direct electron detector (Falcon II) at 50,000 magnification. Nine microlitres of the purified full-length TssA protein (~ 0.01 mg ml−1) were incubated on a glow-discharged carbon-coated copper grid (Agar Scientific) for 30 s. After absorption, the sample was blotted, washed with three drops of water and then stained with 2% uranyl acetate. Images were recorded automatically using the EPU software on a FEG microscope operating at a voltage of 200 kV and a defocus range of 0.6–25 nm, using a FEI Falcon-II detector (Gatan) at a nominal magnification of 50,000, yielding a pixel size of 1.9 Å. A dose rate of 25 electrons per Å2 per second, and an exposure time of 1 s were used. A total of 100,000 particles were automatically selected from 500 independent images and extracted within boxes of 180 pixels × 180 pixels using EMAN2/BOXER38. The CTF was estimated and corrected by phase flipping using EMAN2 (e2ctf). All two- and three-dimensional (2D and 3D) classifications and refinements were performed using RELION 1.3 (refs 39, 40). The automatically selected data set was cleaned up by three rounds of reference-free 2D class averaging, and highly populated classes displaying high-resolution features were conserved and a final data set of 20,000 particles was assembled. An initial 3D-model was generated in EMAN2 using using 30 classes. 3D classification was then performed in Relion with five classes. The particles corresponding to most populated class (~18,000) were used for refinement. The Relion auto-refine procedure was used to obtain a final reconstruction at ~19 Å resolution after masking and with D6 symmetry imposed. Reported resolutions are based on the gold-standard Fourier shell correlation (FSC) 0.143 criterion; the FSC curve was corrected for the effects of a soft mask on the FSC curve using high-resolution noise substitution (Extended Data Fig. 5o)41. All density maps were corrected for the modulation transfer function of the detector and then sharpened by applying a negative B-factor (−1000) that was estimated using automated procedures. The electron microscopy map of the EAEC TssA full-length protein has been deposited in the Electron Microscopy Data Bank under accession number EMD-3282. Small-angle X-ray scattering (SAXS) analyses were performed at the ID29 beamline (European Synchrotron Radiation Facility, Grenoble, France) at a working energy of 12.5 keV (λ = 0.931 Å). Thirty microlitres of protein solution at 1.6, 3.7, 7.1, 9.8 and 14.9 mg ml−1 in Tris-HCl 20 mM pH 8.0, NaCl 150 mM were loaded by a robotic system into a 2-mm quartz capillary mounted in a vacuum and ten independent 10-s exposures were collected on a Pilatus 6M-F detector placed at a distance of 2.85 m for each protein concentration. Individual frames were processed automatically and independently at the beamline by the data collection software (BsxCUBE), yielding radially averaged normalized intensities as a function of the momentum transfer q, with q = 4πsin(θ)/λ, where 2θ is the total scattering angle and λ is the X-ray wavelength. Data were collected in the range q = 0.04–6 nm−1. The ten frames were combined to give the average scattering curve for each measurement. Data points affected by aggregation, possibly induced by radiation damage, were excluded. Scattering from the buffer alone was also measured before and after each sample analysis and the average of these two buffer measures was used for background subtraction using the program PRIMUS42 from the ATSAS package43. PRIMUS was also used to perform Guinier analysis44 of the low q data, which provides an estimate of the radius of gyration (R ). Regularized indirect transforms of the scattering data were carried out with the program GNOM45 to obtain P(r) functions of interatomic distances. The P(r) function has a maximum at the most probable intermolecular distance and goes to zero at D , the maximum intramolecular distance. The values of D were chosen to fit with the experimental data and to have a positive P(r) function. Three-dimensionnal bead models that fit with the scattering data were built with the program DAMMIF46. Ten independent DAMMIF runs were performed using the scattering profile of TssA, with data extending up to 0.35 nm−1, using slow mode settings, assuming P6 symmetry and allowing for a maximum 500 steps to grant convergence. The models resulting from independent runs were superimposed using the DAMAVER suite47 yielding an initial alignment of structures based on their axes of inertia followed by minimisation of the normalized spatial discrepancy (NSD)48. The NSD was therefore computed between a set of ten independent reconstructions, with a range of NSD from 0.678 to 0.815. The aligned structures were then averaged, giving an effective occupancy to each voxel in the model, and filtered at half-maximal occupancy to produce models of the appropriate volume that were used for all subsequent analyses. All the models were similar in terms of agreement with the experimental data, as measured by DAMMIF χ parameter and the quality of the fit to the experimental curve (calculated ). The SAXS data parameters are provided in Extended Data Table 1. Seleno-methionine (SeMet)-labelled TssA and TssA crystallization trials were carried out by the sitting-drop vapour diffusion method in 96-well Greiner crystallization plates at 20 °C, using a nanodrop-dispensing robot (Cartesian Inc.). Crystals of SeMet-labelled TssA grew in a few days after mixing 300 nl of protein at 4.7 mg ml−1 with 100 nl of 20% PEG 8000, 0.2 M calcium acetate, 0.1 M MES pH 6.8. Crystals of SeMet-labelled TssA grew in a few days after mixing 300 nl of protein at 4.7 mg ml−1 with 100 nl of 29% PEG 3350, 0.1 M HEPES pH 7.5. Crystals were cryoprotected with mother liquor supplemented with 20% polyethylene glycol and flash frozen in liquid nitrogen. Data sets were collected at the SOLEIL Proxima 1 beamline (Saint-Aubin, France). After processing the data with XDS49, the scaling was performed with SCALA and the structures were solved using the SHELXD program50. The structure was refined with AutoBUSTER51 alternated with model rebuilding using COOT52. The final data collection and refinement statistics are provided in Extended Data Table 2. The Ramachadran plots of the TssA and TssA structures exhibit 90.7/3.3 and 91.8/2.9 residues in the favoured and outlier areas, respectively. Figures were made with PyMOL53. The tail sheath modelling was performed using the Vibrio cholerae VipAB (TssBC) complex as starting structure23 (PDB: 3J9G) and the contracted tail sheath structures of Vibrio cholerae23. To date, however, the molecular structure of the extended (non-contracted) sheath is not available. In a recent paper, a low-resolution model of the extended VipAB sheath was modelled using the low-resolution EM map of the extended T4 phage tail sheath22. By superimposing the VipAB EM map to the gp18 bacteriophage T4 sheath protein structure, gross features of the sheath structure were obtained22. A similar approach was applied with Chimera54 using the VipAB molecular model in the extended T4 phage tail sheath instead of using the low-resolution VipAB EM map, yielding a model similar to that of Kube et al.22, but with molecular details. The sheath internal channel diameter shrinks from 110 to ~95 Å, and the external diameter from ~290 Å to ~190 Å. The internal diameter of the tail sheath makes it possible to fit stacked Hcp hexamers that are in contact with the tail sheath internal wall. Both extended and contracted tail sheath conformations were used to explore the faisability of sheath complexes with TssA using its EM map. TssA being at the distal end of the sheath, the polarity of the sheath was taken into consideration. It was suggested that the polarity of T6SS tail sheath is similar to that of bacteriophage T4 and therefore that the VipA (TssB) N-terminal and VipB (TssC) C-terminal helices point to and contact the baseplate31. TssA was therefore docked at the opposite extremity of the tail sheath using Chimera54. Hcp release11, 25 and fractionation assays11, 19, 25 have been performed as previously described. SDS-polyacrylamide gel electrophoresis was performed using standard protocols. For immunostaining, proteins were transferred onto 0.2-μm nitrocellulose membranes (Amersham Protran), and immunoblots were probed with primary antibodies, and goat secondary antibodies coupled to alkaline phosphatase, and developed in alkaline buffer with 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium. The anti-TolB polyclonal antibodies are from our laboratory collection, while the anti-Flag (M2 clone, Sigma Aldrich) and anti-EFTu (Roche) monoclonal antibodies and alkaline phosphatase-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Beckman Coulter) have been purchased as indicated. Coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 4YO3 and 4YO5 for TssA and TssA , respectively. Electron microscopy map for full-length TssA has been deposited in the Electron Microscopy Databank (EMDB) under accession code EMD-3282.
News Article | November 2, 2015
"EuPRAXIA will define the missing step towards a new generation of plasma accelerators with the potential for dramatically reduced size and cost," said EuPRAXIA coordinator Ralph Assmann from DESY. "It will ensure that Europe is kept at the forefront of accelerator-based science and applications." The EuPRAXIA consortium includes 16 laboratories and universities from five EU member states. In addition, it includes 18 associated partners from eight countries, involving leading institutes in the EU, Japan, China and the United States. Particle accelerators have evolved over the last 90 years into powerful and versatile machines for discoveries and applications. Today some 30,000 accelerators are operated around the world, among those some of the largest machines built by human mankind. A new technology for particle acceleration has emerged and has demonstrated accelerating fields a thousand times beyond those presently used: Plasma acceleration uses electrically charged plasmas, generated by strong lasers, instead of the usual radio frequency used in conventional accelerators, to boost particles like electrons to high energies. By the end of 2019, EuPRAXIA will produce a conceptual design report for the worldwide first five Giga-Electronvolts plasma-based accelerator with industrial beam quality and dedicated user areas. EuPRAXIA is the required intermediate step between proof-of-principle experiments and versatile ultra-compact accelerators for industry, medicine or science, e.g. at the energy frontier of particle physics as a plasma linear collider. The study will design accelerator technology, laser systems and feedbacks for improving the quality of plasma-accelerated electron beams. Two user areas will be developed for a novel free-electron laser, high-energy physics and other applications. An implementation model will be proposed, including a comparative study of possible sites in Europe, a cost estimate and a model for distributed construction but installation at one central site. As a new large research infrastructure, EuPRAXIA would place Europe at the forefront of the development of novel accelerators driven by the world's most powerful lasers from European industry in the 2020's. The EuPRAXIA consortium has the following participants: Centre National de la Recherche Scientifique (CNRS), Commissariat à l'Énergie Atomique et aux énergies alternatives (CEA) and Synchrotron SOLEIL from France, DESY and the University of Hamburg from Germany, Istituto Nazionale di Fisica Nucleare (INFN), Consiglio Nazionale delle Ricerche (CNR), Agenzia nazionale per le nuove tecnologie, l'energia e lo sviluppo economico sostenible (ENEA) and Sapienza Universita di Roma from Italy and Instituto Superior Técnico (IST) from Portugal, Science & Technology Facilities Council (STFC), University of Manchester, University of Liverpool, University of Oxford, University of Strathclyde and Imperial College London from the UK. Associated partners are: Jiaotong University Shanghai and Tsingua University Beijing from China, Extreme Light Infrastructures - Beams (ELI-B) in Czech Republic, University of Lille in France, High Energy Accelerator Research Organization (KEK), Kansai Photon Science Institute, Japan Atomic Energy Agency, Osaka University and RIKEN Spring-8 Center from Japan, Helmholtz-Institut Jena, Helmholtz-Zentrum Dresden-Rossendorf and Ludwig-Maximillians-Universität München from Germany, Wigner Research Center of the Hungarian Academy of Science in Hungary, University of Lund in Sweden, European Organization for Nuclear Research (CERN) in Switzerland, Center for Accelerator Science and Education at Stony Brook University & Brookhaven National Laboratory (BNL), Lawrence Berkeley National Laboratory (LBNL), SLAC National Accelerator Laboratory and University of California at Los Angeles (UCLA) in the U.S.
Spaulding D.K.,CEA DAM Ile-de-France |
Spaulding D.K.,Harvard University |
Weck G.,CEA DAM Ile-de-France |
Loubeyre P.,CEA DAM Ile-de-France |
And 3 more authors.
Nature Communications | Year: 2014
New topochemistry in simple molecular systems can be explored at high pressure. Here we examine the binary nitrogen/hydrogen system using Raman spectroscopy, synchrotron X-ray diffraction, synchrotron infrared microspectroscopy and visual observation. We find a eutectic-type binary phase diagram with two stable high-pressure van der Waals compounds, which we identify as (N 2) 6 (H2)7 and N2 (H2)2. The former represents a new type of van der Waals host-guest compound in which hydrogen molecules are contained within channels in a nitrogen lattice. This compound shows evidence for a gradual, pressure-induced change in bonding from van der Waals to ionic interactions near 50 GPa, forming an amorphous dinitrogen network containing ionized ammonia in a roomerature analogue of the Haber-Bosch process. Hydrazine is recovered on decompression. The nitrogen-hydrogen system demonstrates the potential for new pressure-driven chemistry in high-pressure structures and the promise of tailoring molecular interactions for materials synthesis. © 2014 Macmillan Publishers Limited.
Bahout M.,CNRS Chemistry Institute of Rennes |
Tonus F.,CNRS Chemistry Institute of Rennes |
Prestipino C.,CNRS Chemistry Institute of Rennes |
Pelloquin D.,National Engineering School of Caen |
And 3 more authors.
Journal of Materials Chemistry | Year: 2012
The structural and redox stability of the n = 1 Ruddlesden-Popper (RP) oxide Pr 0.5Sr 1.5Cr 0.5Mn 0.5O 4-δ, synthesized by the citrate-gel method, has been investigated over the temperature range 25-700 °C under reducing (5% H 2 flow) and oxidizing (O 2 or air flow) conditions by means of in situ neutron powder diffraction (NPD) and X-ray absorption near-edge structure spectroscopy (XANES). Sequential Rietveld refinement of the NPD patterns collected under hydrogen revealed de-intercalation of oxide ions from the equatorial anion positions with retention of I4/mmm symmetry. The reduction from Pr 0.5Sr 1.5Cr 0.5Mn 0.5O 4.00(2) to Pr 0.5Sr 1.5Cr 0.5Mn 0.5O 3.81(2) is accompanied by an expansion of both the a and c lattice parameters. When the reduced sample is heated in air, oxygen refills the equatorial sites and the unit cell contracts; the interlayer interstitial site remains unoccupied. XANES showed the oxidation states in the as-prepared composition to be Pr 3+, Cr 3+ and Mn 4+. When the material is heated under dilute hydrogen, the oxidation states Pr 3+ and Cr 3+ are retained whereas Mn 4+ is reduced to Mn 3+. These observations constitute the first direct evidence that the d-block element, and not praseodymium, is responsible for the electrocatalytic activity of Pr-containing RP oxides. When the reduced material is heated under oxygen, Mn 3+ is reoxidised to Mn 4+ and a low concentration of tetrahedrally-coordinated Cr(vi) forms, suggesting a possible poisoning mechanism in fuel-cell applications. © The Royal Society of Chemistry 2012.
News Article | November 15, 2016
Photograph of the MR2 archaeological site at Mehrgarh occupied from 4 500 to 3 600 BC, where the amulet was found. Credit: C. Jarrige, Mission archéologique de l'Indus At 6000 years old, this copper amulet is the earliest lost-wax cast object known. Now, researchers have finally discovered how it was made, using a novel UV-visible photoluminescence spectral imaging approach. All the parameters of elaboration process, such as the purity of the copper, and melting and solidification temperatures, are now accurately known. This work has enabled the scientists to solve the mystery of the invention of lost-wax casting, a technique that led to art foundry. Resulting from a collaboration1 between researchers from the CNRS, the French Ministry of Culture and Communication and the SOLEIL synchrotron, the work is published on 15 november 2016 in the journal Nature Communications. The researchers examined a copper amulet, discovered in the 1980s at a site that was occupied 6,000 years ago and had been a focal point for innovation since Neolithic times: Mehrgarh, in today's Pakistan. The shape of the object shows that it was designed using the earliest known precision casting technique, lost-wax casting (still in use today). The process begins with a model formed in a low melting point material such as beeswax. The model is covered with clay, which is heated to remove the wax and then baked. The mould is filled with molten metal and then broken to release the metal object. This was all that was known about the process used to make the copper amulet until it was subjected to a novel photoluminescence approach, which revealed that it had an unexpected internal structure. Although the amulet today consists mainly of copper oxide (cuprite), it emits a non-uniform response under UV-visible illumination. Between the dendrites formed during initial solidification of the molten metal, the researchers found rods that were undetectable using all other approaches tested. The shape and arrangement of the rods enabled the team to reconstruct the process used to make the amulet with an unprecedented level of detail for such a corroded object. 6,000 years ago, following high-temperature solidification of the copper forming it, the amulet was made up of a pure copper matrix dotted with cuprite rods, resulting from the oxidizing conditions of the melt. Over time, the copper matrix also corroded to cuprite. The contrast observed using photoluminescence results from a difference in crystal defects between the two cuprites present: there are oxygen atoms missing in the cuprite of the rods, a defect that is not present in the cuprite formed by corrosion. This innovative imaging technique, with high resolution and a very wide field of view, made it possible to identify the ore used (extremely pure copper), the quantity of oxygen absorbed by the molten metal, and even the melting and solidification temperatures (around 1072 °C). The discovery illustrates the potential of this new analytical approach, which can be applied to the study of an extremely wide range of complex systems, such as semiconducting materials, composites and, of course, archaeological objects. Comparison of high spatial dynamics-photoluminescence (PL, top), and optical microscopy (bottom) images. The area imaged corresponds to part of one of the spokes of the amulet. The PL image reveals a eutectic rod-like structure that is undetectable using all other tested techniques. The image at last made it possible to explain the process used to make the amulet. Credit: T. Séverin-Fabiani, M. Thoury, L. Bertrand, B. Mille, IPANEMA, CNRS / MCC / UVSQ, Synchrotron SOLEIL, C2RMF Explore further: Voltammetry of microparticles used to date archeological artifacts made of copper and bronze More information: M. Thoury et al. High spatial dynamics-photoluminescence imaging reveals the metallurgy of the earliest lost-wax cast object, Nature Communications (2016). DOI: 10.1038/ncomms13356
News Article | March 31, 2016
Abstract: The detector group at the Swiss Light Source at PSI has been one of the pioneers in the development of custom-made hybrid pixel array detectors (HPADs) for synchrotron applications. In a paper published recently [Jungmann-Smith et al. (2016). J. Synchrotron Rad. 23, 385-394; doi:10.1107/S1600577515023541], this group shows that it is now possible to develop HPADs with sufficient low noise to allow single-photon detection below 1 keV as well as to perform spectroscopic imaging. A commentary has also been written about the work [Graafsma (2016). J. Synchrotron Rad. 23, 383-384; doi:10.1107/S1600577516002721]. For decades, detectors have been a limiting factor in experiments at synchrotron radiation facilities. Even though imaging detectors evolved over time, the evolution of the source always outran the evolution of the detector. This situation started to change with the introduction of the so-called hybrid pixel array detectors, which contain a pixelated readout chip custom-designed for a well-defined experiment or technique. One of the revolutionising advantages offered by this technology is that every single pixel contains all necessary electronics, including for instance counters, for X-ray detection. This massive parallelisation increased the overall efficiency of the detector by several orders of magnitude as compared with the charge-coupled-device-based system. There are now various examples of HPADs, specifically developed for X-ray experiments at storage-ring synchrotron sources, as well as various spin-off companies commercialising them. Most of these systems are so-called photon-counting detectors, where each incoming photon is processed by the readout electronics in the pixel and individually counted. The advantage of photon counting is that electronic noise, present in any system, can be efficiently discriminated against, yielding `noise-free' detectors. An application for such low-noise systems is in energy-dispersive measurements. The researchers show in their paper that, with the use of a proper mask to shield the edge regions between pixels, very good fluorescence spectra can be obtained. This capability was subsequently used for multi-colour imaging at the SOLEIL synchrotron. The innovative aspect of the work contained in this paper does not lie in the spectroscopic results obtained as they could very well have been obtained with other detectors. But what is truly impressive is that these results were obtained with an HPAD using a standard planar diode array as sensor. This means that the system uses relatively standard and thus easy-to-manufacture components, making it possible to envision building larger and/or further-optimised systems in the near future. And with that, low-noise HPADs have entered a field formally reserved for silicon drift detectors and complementary metal-oxide semiconductor imagers. For more information, please click If you have a comment, please us. Issuers of news releases, not 7th Wave, Inc. or Nanotechnology Now, are solely responsible for the accuracy of the content.
News Article | November 21, 2016
The drying process is a critical final stage in various manufacturing processes – it influences the quality of many a product and has many industrial applications, particularly in the food and pharmaceutical sectors. Freeze drying (lyophilization) is a drying method where the solvent is frozen prior to drying and is then sublimed. In addition to providing an extended shelf-life, successful freeze-drying should yield a product that has a short reconstitution time with acceptable potency levels. The process should be reproducible with well defined temperature, pH and time parameters for each step. Visual and functional characteristics of the dried product are also important for many applications. In pharmaceuticals, freeze drying is commonly used to preserve the integrity and bioactivity of protein drugs, minimising chemical and physical degradation during their shelf life. Unfortunately for such an integral process, storage in freeze-dried states does not guarantee long-term stability, and aggregation is often observed after thawing or reconstitution of freeze-dried powder samples. A particular issue is the formation of ice which is known to have a destabilising effect on protein molecules during freezing. Therefore an essential step in optimising the conservation and stabilisation of samples is to understand the biophysical mechanisms involved during the freeze-drying process – a knowledge gap that a recent a Institut Laue Langevin (ILL) study addressed. This work was a collaboration between ILL, Soleil, CEMHTI laboratory in Orleans and the University of Palermo. The study focused on the structural evolution of protein solutions up to supersaturation conditions, created by partial evaporation of the solvent, to mimic drying process conditions. This enabled structural analysis to be obtained at a series of time intervals at different drying conditions of the sample, therefore enabling data to be collected during the process itself and mimicking realistic lab conditions. Container interactions and contamination of the sample can affect greatly the freeze-dried process and thereby, the quality of the final product – there is no universal 'safe' choice of container. Therefore, in order to ensure optimum understanding of the freeze-drying process, container-free or 'contactless' techniques must be used. In this study, acoustic levitation was the chosen technique – solid and liquid samples positioned in the surrounding medium (ambient air or defined gas) by means of a stationary ultrasonic field creating a pressure gradient in the medium. A single droplet is held in a node of a standing acoustic wave, avoiding any contact with a thermally-conducting holding-device. The droplet size can be controlled in a wide range up to 5 mm in diameter. The surrounding drying-gas can be conditioned such that its temperature, relative humidity, and flow rate past the droplet are accurately controlled. Evaporation of the solvent during levitation gradually decreases the volume of the droplet and therefore increases the corresponding concentration of the solute. Thus, formation of aggregates and processes of crystallisation could be followed in situ in order to identify suitable crystallising conditions. This paper shows the capabilities of the levitation technique once integrated into small angle neutron scattering (SANS) and x-ray scattering (SAXS) beamlines. Despite the small quantities of sample that it is possible to suspend (just few tens of nanoliters in volume), the high intensity of neutron instruments make possible in-situ monitoring of fast containerless reactions, providing detailed molecular and structural information. Changes in the SANS and SAXS signal intensities provide information on particle distances and morphology of proteins, possible formation of larger domains, as clusters or heterogeneities, directly linked to the drying condtions. It was observed that, considering a lysozyme dissolved in D2O solution at low concentration, the center-to-center distances between proteins become smaller due to the evaporation inducing an increase in concentration. For high concentration solutions, the distance between molecules changes little during evaporation. The acoustic levitation device shows its potential when used in combination with synchrotron radiation circular dichroism. Preliminary results on myoglobin in aqueous solution allow to follow up the evolution of the secondary structure of the protein as a function of concentration, revealing an increase of α-helices content and the full loss of parallel β-sheets. The results prove that the acoustic levitator can be used as a tool for structure analysis and that it easily permits the contactless study of many kinds of samples. Neutrons are the ideal tool for this type of experiment due to their non-destructive properties so data can be collected at room temperature, closer to physiological temperatures and resulting in the determination of 'damage-free' structures – essential for studying biological molecules. SAXS experiments were carried out at the SOLEIL synchrotron's SWING beamline while SANS was conducted on the ILL's D16 and D33 instruments. This study allows us to validate levitation methodology for investigating bio-based materials during the drying process. To our knowledge, this SANS investigation combined with acoustic levitation is the first study of its kind. Using these advanced analytical methods for characterisation of various pharmaceuticals including small molecules and proteins, drug substances and products, we open up the way for novel insights into aggregation and crystallisation phenomena. A better understanding of the biochemical mechanisms of these classes of bio-materials is necessary to improve the long-term stability of pharmaceutical formulations. These studies could underpin improvements in industrial processes such as spray-drying and spray-freeze-drying and the process development of pharmaceuticals and biopharmaceuticals. The behaviour of protein solutions at low temperature and the role of cryo- and lyo- protectant agents added to a protein solution using neutron (SANS) techniques will be the main topic of future work in this area. Explore further: Researchers develop environmentally friendly process to improve storage stability of probiotics More information: Viviana Cristiglio et al. Combination of acoustic levitation with small angle scattering techniques and synchrotron radiation circular dichroism. Application to the study of protein solutions, Biochimica et Biophysica Acta (BBA) - General Subjects (2016). DOI: 10.1016/j.bbagen.2016.04.026