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X-ray data for coupons: AM1_Ply16_011, AM1_Ply16_013, AM1_Ply16_014 and AM1_Ply16_015 (IMPACT)
No full textDetails about this dataset are available from the main record: https://doi.org/10.15125/BATH-0019
Coupon AM1_OI16_021 (IMPACT)
No full textStacking sequence: [(45/0)_2/(-45/90)_2]_s
Material: T800/M21
Ply-thickness: 0.2540
Number of plies: 16
Impact energy (J): 12Details about this dataset are available from the main record: https://doi.org/10.15125/BATH-0019
Coupon BM1_Lam16_034 (IMPACT)
No full textStacking sequence: [(+45/-45/0/90)_2]_s
Material: T800/M21
Ply-thickness: 0.2574
Number of plies: 16
Impact energy (J): 12Details about this dataset are available from the main record: https://doi.org/10.15125/BATH-0019
Coupon BM1_Ply16_042 (IMPACT)
No full textStacking sequence: [45_2/-45_2/0_2/90_2]_s
Material: T800/M21
Ply-thickness: 0.2554
Number of plies: 16
Impact energy (J): 6Details about this dataset are available from the main record: https://doi.org/10.15125/BATH-0019
Coupon BM1_Ply16_043 (IMPACT)
No full textStacking sequence: [45_2/-45_2/0_2/90_2]_s
Material: T800/M21
Ply-thickness: 0.2532
Number of plies: 16
Impact energy (J): 12Details about this dataset are available from the main record: https://doi.org/10.15125/BATH-0019
Dataset for research paper: α-Synuclein Increases β-Amyloid Secretion by Promoting β-/γ-Secretase Processing of APP
No full textIncludes the original data used for all main figures and supplementary data figures, and data used in Table 1.Compounds
TAPI-1, Amyloid Precursor Protein β-Secretase Inhibitor (βSI), and β-Secretase Inhibitor IV (βIV) were purchased from Merck Millipore. DAPT was purchased from Tocris. TAPI, βSI, βIV, and DAPT were made up as 1000x concentrated stocks in DMSO (Sigma).
DNA Constructs
Plasmids encoding wildtype α-synuclein (RefSeq accession number XM_011532204) and β-synuclein (RefSeq accession number NM_001001502) in a pcDNA 3.1 (+) vector were previously described (1,2). A pCI-neo-APP695 plasmid was kindly donated by Prof. Chris Miller, Kings College London (3). Steap3 plasmid was generated by cloning the human Steap3 sequence (RefSeq accession number NG_042823.1) into a pcDNA 3.1 (+) vector. pFR-Luciferase reporter vector (pLuc) containing firefly (Photinus pyralis) luciferase gene, with a synthetic promoter of yeast Gal4 upstream activation sequence in 5 tandem repeats upstream of a minimal TATA box, was from Promega. phRL thymidine kinase vector (pTK) containing sea pansy (Renilla reniformis) luciferase gene, under control of the herpes simplex virus-TK promoter, was also from Promega. pRC-CMV-APP695-Gal4 (APP-Gal4) and pSec-Tag2-Notch3Gal4 (Notch-Gal4) were kindly provided by Dr Robert J. Williams, University of Bath (4,5). Human BACE1 promoter luciferase reporter, a pGL3-Basic vector containing a 4.3kb fragment of BACE1 promoter from -4372 to -1 of the promoter sequence, was previously described (6).
Cell Cultures and Transfection
SH-SY5Y neuroblastoma cells (obtained directly from ATCC, cat no. CRL-2266) were grown in 1:1 DMEM (high glucose with L-Glutamine, Lonza) and Ham’s F 12 (Lonza), supplemented with 10% fetal bovine serum (Sigma), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma). Growth conditions were maintained at 37°C and 5% CO2 in a humidified incubator. Cells were stably transfected with pcDNA 3.1 (+)-α-synuclein, or mutations thereof. The mutants included truncations Δ2-9, at the extreme N-terminus, and Δ71-82, in the NAC domain of α synuclein. PD-associated point mutations A30P, E46K and A53T were also included. Additionally two substitutions of S129, a phosphorylation site in the C-terminus of α-synuclein. S129A blocks phosphorylation, whereas S129D aims to mimic permanent phosphorylation. Transfection was achieved using FuGene HD lipid reagent (Promega) according to the manufacturer’s instructions. Stable selection was performed with 0.8 mg/ml G418 (Sigma) 24 hours after transfection, and cells maintained in 0.4 mg/ml G418. Successful transfections were assessed by western blotting to ensure over-expression of the protein.
Antibodies
Rabbit monoclonal anti-α-synuclein (MJFR1, Abcam, immunogen human α-synuclein 1-150) was used for human α synuclein detection at a dilution of 1:4000. Mouse monoclonal anti-α-synuclein (610787, BD Biosciences, immunogen rat α-synuclein 15-123) was used for rat α-synuclein detection at a dilution of 1:2000. Mouse monoclonal anti-α tubulin (T5186, Sigma, immunogen acetylated tubulin from Strongylocentrotus purpuratus sperm axonemes) was used at a dilution of 1:10,000. Rabbit monoclonal anti-APP C-terminus (Y188, Abcam immunogen human APP750 C-terminus) was used at a dilution of 1:2000. Rabbit monoclonal anti-BACE1 (D10E5, Cell Signaling Technology immunogen human BACE1 residues around His490) was used at a dilution of 1:1500.
Western Blotting
Cells were lysed in 0.5% Igepal CA-630 and ‘complete’ protease inhibitor cocktail (Roche), sonicated 3 x 3 seconds on ice, and centrifuged 10 000 xg for 3 minutes to remove insoluble membranes. Protein concentration was determined with a Bradford protein assay (Bio-Rad), according to the manufacturer’s instructions. Supernatant protein concentrations were normalized and boiled for 5 minutes with 1 x Laemmli SDS-PAGE buffer. To determine levels of α-synuclein, full-length APP, or BACE1: samples were loaded into a 12% acrylamide SDS-PAGE gel, with a buffer of Tris (250 mM) + Glycine (1.92 M) + SDS (0.1% w/v), run at 250V for 45 minutes. To resolve bands of C99 and C83 APP: samples were loaded into a 16% acrylamide + 10% glycerol SDS-PAGE gel, with an anode buffer of Tris-HCl (20 mM, pH 8.9), and a cathode buffer of Tris (100 mM) + Tricine (100 mM) + SDS (1% w/v). The 16% gel was electrophoresed at 100 V, 4 °C, for several hours. Separated proteins were transferred to a PVDF membrane by a semi-dry transfer apparatus, run at 25V for 1.3 hours. Membranes were blocked in 5% w/v non-fat milk powder dissolved in TBS-T (0.05% Tween-20, 10 mM Tris, 100 mM NaCl) for 30 minutes, incubated with primary antibody for 1-2 hours, and washed 3 x 5 minutes in TBS-T. Membranes were blocked again and incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour. A further 3 x 10 minute washes were performed, and the membranes developed with Luminata Crescendo or Luminata Forte ECL substrate (Thermo Scientific), and imaged with a Fusion SL CCD imaging system (Vilber Lourmat).
Measurement of Aβ40 and Aβ42 by Meso Scale Discovery Assay
Fresh serum-free B-27-supplemented DMEM, ± compounds, was added to SH-SY5Ys a day after seeding in 24-well plates. Conditioned media was collected after 72 hours and immediately assayed without further manipulation, using the V-PLEX Plus Aβ Peptide Panel 1 (6E10) Kit from Meso Scale Discovery, according to the manufacturer’s instructions. The plate was read with a Sector Imager 6000 (Meso Scale Discovery). Peptide concentrations (pg/ml) were calculated by Meso Scale Discovery Workbench software, with reference to a standard curve, and were normalised to the mean concentration for each experiment.
Dual Glo Luciferase Reporter Assay
SH-SY5Ys in 24-well plates were co-transfected with plasmids complexed with 1.5 µl/well FuGene HD (Promega). For the APP-Gal4 reporter assay for amyloidogenic processing, cells were transfected with 50 ng each of APP-Gal4 plasmid, pLuc, and pTK. For measuring human BACE1 promoter activity, cells were transfected with 200 ng of BACE1 promoter luciferase reporter plasmid, and 50 ng pTK. For the Notch-Gal4 reporter assay for γ-secretase activity, cells were transfected with 100 ng of Notch-Gal4 plasmid, and 50 ng each of pLuc and pTK. Luciferase activity was measured 20-22 hours post-transfection using the Promega Dual-Luciferase Reporter Kit according to the manufacturer’s instructions. Raw data was normalised by division with the mean firefly or Renilla luminescence for that experiment. Relative Luciferase Units (RLU) were calculated for each well by division of the firefly signal by the Renilla signal. The average RLU for 3-5 replicate transfections was calculated in each experiment.
Oxidative Stress Assay
Cells were seeded at a density of approximately 1x106 cells/ml onto a poly-D-lysine-coated 48-well plate. After 48 hours, cells were incubated with 10 µM CM-H2DCFDA probe in HEPES-buffered media (20 mM HEPES, 140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1.2 mM Na2HPO4, 1.2 mM CaCl2, 5.5 mM glucose) for 20 minutes. The probe was removed and 300 µl of HEPES-buffered media added. Fluorescence intensity (Ex/Em= 488/534 nm) was measured every 5-10 minutes for 60 minutes. The linear rate equation was determined from a kinetics plot using MS Excel, and the rate at 60 minutes calculated. For each experiment, rates were normalised to the average.
Viral Vector Production and Titration
Serotype 6 adeno-associated viral (AAV6) vectors were produced and titrated as previously described (7). The number of transducing units (TU) was determined by infecting HEK293T cells. The number of S1-nuclease resistant vector genome copies was measured by real-time PCR at 48 hrs post-infection. The AAV6-α-syn vector encodes expression of full-length wild-type human α-syn under the control of the constitutive mouse pgk-1 promoter. The titre of the vector suspension was 7.9E10 TU/ml.
Animal Experiments
All procedures were performed in accordance with Swiss legislation and the European Community Council directive (86/609/EEC) for the care and use of laboratory animals. Female adult Sprague-Dawley rats (Charles River Laboratories, France), weighing 180-200 g, were housed in a 12-hour light-dark cycle, with ad libitum access to water and food. Rats were injected with empty or α-synuclein-encoding AAV6 vectors in the right substantia nigra, with the non-injected left acting as an internal control. The total injected dose for each animal was 2.8E7 TU in a volume of 2 µl. The general procedure for vector injection was as previously described (7). We used the following stereotaxic coordinates: 5.2 mm anterior and 1.9 mm lateral to bregma point, 7.9 mm ventral from the skull surface. The injected rats were culled one month after injection. For biochemical analysis, fresh striatal tissue was obtained from each brain hemisphere separately. Neuropathology and motor deficits of the rat model have been previously characterised (7, 8, 9). Additionally, brain α-synuclein expression and aggregation were visualised by immunohistochemistry. For immunohistochemistry, the rats were sacrificed three months post-injection, and the brains were perfused with 4% paraformaldehyde (1.5 h post-fixation), before being transferred to 25% sucrose solution. Immunostaining for tyrosine hydroxylase (Millipore, #AB152; 1:1000), total α-syn (Millipore #AB5334P; 1:1000) and aggregated α-syn (clone 5G4, Millipore #MABN389; 1:1000) were performed on striatal sections (25 µm) from the rat striatum and substantia nigra, using standard procedures.Statistical analysis was performed using MS Excel. With the exception of the rat material, all data were analysed by unpaired two-tailed Student’s t-tests, with an assumption of equal variance. Rat striata were analysed by a paired two-tailed Student’s t-test. Differences were defined as statistically significant when p < 0.05.1. Wang X, Moualla D, Wright JA, Brown DR. Copper binding regulates intracellular alpha-synuclein localisation, aggregation and toxicity. J Neurochem. 2010;113(3):704–14.
2. Wright JA, McHugh PC, Pan S, Cunningham A, Brown DR. Counter-regulation of alpha- and beta-synuclein expression at the transcriptional level. Mol Cell Neurosci. 2013;57:33–41.
3. Lau K-F, McLoughlin DM, Standen C, Irving NG, Miller CCJ. Fe65 and X11β co-localize with and compete for binding to the amyloid precursor protein. Mol Neurosci. 2000;11(16):3607–10.
4.Cox CJ, Choudhry F, Peacey E, Perkinton MS, Richardson JC, Howlett DR, et al. Dietary (-)-epicatechin as a potent inhibitor of βγ-secretase amyloid precursor protein processing. Neurobiol Aging. 2014;31(1):178–87.
5. Hoey SE, Williams RJ, Perkinton MS. Synaptic NMDA receptor activation stimulates α-secretase amyloid precursor protein processing and inhibits amyloid-β production. J Neurosci. 2009;29(14):4442–60.
6. McHugh PC, Wright JA, Williams RJ, Brown DR. Prion protein expression alters APP cleavage without interaction with BACE-1. Neurochem Int. 2012;61(5):672–80.
7. Gaugler MN, Genc O, Bobela W, Mohanna S, Ardah MT, El-Agnaf OM, et al. Nigrostriatal overabundance of α-synuclein leads to decreased vesicle density and deficits in dopamine release that correlate with reduced motor activity. Acta Neuropathol. 2012;123(5):653–69.
8.Azeredo da Silveira S, Schneider BL, Cifuentes-Diaz C, Sage D, Abbas-Terki T, Iwatsubo T, et al. Phosphorylation does not prompt, nor prevent, the formation of alpha-synuclein toxic species in a rat model of Parkinson’s disease. Hum Mol Genet. 2009;18(5):872–87.
9. Pino E, Amamoto R, Zheng L, Cacquevel M, Sarria JC, Knott GW, et al. FOXO3 determines the accumulation of α-synuclein and controls the fate of dopaminergic neurons in the substantia nigra. Hum Mol Genet. 2014;23(6):1435–52
Dataset for Sox10 contributes to the balance of fate choice in dorsal root ganglion progenitors
No full textThe development of functional peripheral ganglia requires a balance of specification of both neuronal and glial components. In the developing dorsal root ganglia (DRGs), these components form from partially-restricted bipotent neuroglial precursors derived from the neural crest. Work in mouse and chick has identified several factors, including Delta/Notch signaling, required for specification of a balance of these components. We have previously shown in zebrafish that the Sry-related HMG domain transcription factor, Sox10, plays an unexpected, but crucial, role in sensory neuron fate specification in vivo. In the same study we described a novel Sox10 mutant allele, sox10baz1, in which sensory neuron numbers are elevated above those of wild-types. Here we investigate the origin of this neurogenic phenotype. We demonstrate that the supernumerary neurons are sensory neurons, and that enteric and sympathetic neurons are almost absent just as in classical sox10 null alleles; peripheral glial development is also severely abrogated in a manner similar to other sox10 mutant alleles. Examination of proliferation and apoptosis in the developing DRG reveals very low levels of both processes in wild-type and sox10baz1, excluding changes in the balance of these as an explanation for the overproduction of sensory neurons. Using chemical inhibition of Delta-Notch-Notch signaling we demonstrate that in embryonic zebrafish, as in mouse and chick, lateral inhibition during the phase of trunk DRG development is required to achieve a balance between glial and neuronal numbers. Importantly, however, we show that this mechanism is insufficient to explain quantitative aspects of the baz1 phenotype. The Sox10(baz1) protein shows a single amino acid substitution in the DNA binding HMG domain; structural analysis indicates that this change is likely to result in reduced flexibility in the HMG domain, consistent with sequence-specific modification of Sox10 binding to DNA. Unlike other Sox10 mutant proteins, Sox10(baz1) retains an ability to drive neurogenin1 transcription. We show that overexpression of neurogenin1 is sufficient to produce supernumerary DRG sensory neurons in a wild-type background, and can rescue the sensory neuron phenotype of sox10 morphants in a manner closely resembling the baz1 phenotype. We conclude that an imbalance of neuronal and glial fate specification results from the Sox10(baz1) protein’s unique ability to drive sensory neuron specification whilst failing to drive glial development. The sox10baz1 phenotype reveals for the first time that a Notch-dependent lateral inhibition mechanism is not sufficient to fully explain the balance of neurons and glia in the developing DRGs, and that a second Sox10-dependent mechanism is necessary. Sox10 is thus a key transcription factor in achieving the balance of sensory neuronal and glial fates
Dataset for "Phase transition for quenched coupled replicas in a plaquette spin model of glasses"
No full textSimulation results for "Phase transition for quenched coupled replicas in a plaquette spin model of glasses"see Robert L. Jack and Juan P. Garrahan, "Phase transition for quenched coupled replicas in a plaquette spin model of glasses", Phys. Rev. Lett. 116, 055702 (2016).
(http://doi.org/10.1103/PhysRevLett.116.055702
Dataset for Future probabilistic hot summer years for overheating risk assessments
No full textFuture hot summer years to be used for assessing risk of overheating and heat stress under a changing climate. They were created in two alternative ways: one is based on Weighted Cooling Degree Hours, the other is based on Physiologically Equivalent Temperature.Future weather data was generated by UKCP09 weather generator. Weather data in 2050s under high emission scenario was generated for fourteen UK locations.UKCP09 weather generator provides future weather data which were used for creating new future weather files to be used in building simulation packages.The future weather files are in the format of epw which can be used in building simulation packages such as EnergyPlus, DesignBuilder and IES. Any software which opens text file can be used to view the data
Data for paper: Initiating and imaging the coherent surface dynamics of charge carriers in real space
No full textThe tip of a scanning tunnelling microscope is an atomic-scale source of electrons and holes. As the injected charge spreads out it can induce adsorbed molecules to react. By comparing large-scale `before' and `after' images of an adsorbate covered surface, the spatial extent of the nonlocal manipulation is revealed. Here we measure the nonlocal manipulation of toluene molecules on the Si(111)-7x7 surface at room temperature. Both the range and probability of nonlocal manipulation have a voltage dependence. A region within 5 to 15 nm of the injection site shows a marked reduction in manipulation. We propose that this region marks the extent of the initial coherent (i.e., ballistic) time-dependent evolution of the injected charge carrier. Using scanning tunnelling spectroscopy, we develop a model of this time-dependent expansion of the initially localized hole wavepacket within a particular surface state and deduce a quantum coherence (ballistic) lifetime of about 10 fs.Please see main paper methods section. For radial distributions we present the thermally corrected N/N0 data, again see methods section for details.SJB Custom Low Temperature ST
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