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Aβ dimers rapidly form stable synaptotoxic protofibrils
Brian O'Nuallain1,*, Darragh B. Freir1, Andrew J. Nicoll2, Emmanuel Risse2, Neil
Ferguson1, Caroline E. Herron1, John Collinge2,3, and Dominic M. Walsh1,*
1Laboratory for Neurodegenerative Research, Conway Institute, University College Dublin,
Belfield, Dublin 4, Republic of Ireland 2Department of Neurodegenerative Disease, UCL Institute
of Neurology, Queen Square, London, WCN1 3BG, United Kingdom 3MRC Prion Unit, UCL
Institute of Neurology, Queen Square, London, WCN1 3BG, United Kingdom
Non-fibrillar, water-soluble low-molecular weight assemblies of the amyloid β-protein (Aβ) are
believed to play an important role in Alzheimer's disease (AD). Aqueous extracts of human brain
contain Aβ assemblies which migrate on SDS-PAGE and elute from size exclusion as dimers (~8
kDa) and can block long term potentiation and impair memory consolidation in the rat. Such
species are detected specifically and sensitively in extracts of Alzheimer brain suggesting that
SDS-stable dimers may be the basic building blocks of AD-associated synaptotoxic assemblies.
Consequently, understanding the structure and properties of Aβ dimers is of great interest. In the
absence of sufficient brain-derived dimer to facilitate biophysical analysis, we generated synthetic
dimers designed to mimic the natural species. For this, Aβ(1-40) containing cysteine in place of
serine 26 was used to produce disulphide cross-linked dimer, (AβS26C)2. Such dimers had no
detectable secondary structure, produced an analytical ultracentrifugation (AUC) profile consistent
for an ~8.6 kDa protein, and had no effect on hippocampal long term potentiation (LTP).
However, (AβS26C)2 aggregated more rapidly than either AβS26C or wild type monomers and
formed parastable β-sheet rich, thioflavin T positive, protofibril-like assemblies. Whereas wild
type Aβ aggregated to form typical amyloid fibrils, the protofibril-like structures formed by
(AβS26C)2 persisted for prolonged periods and potently inhibited LTP in mouse hippocampus.
These data support the idea that Aβ dimers may stabilize the formation of fibril intermediates by a
process distinct from that available to Aβ monomer and that higher molecular weight pre-fibrillar
assemblies are the proximate mediators of Aβ toxicity.
The amyloid β-protein (Aβ) is believed to play a central role in Alzheimer's disease (AD)
and like several other proteins associated with neurodegeneration, has the ability to self-
associate, and can form an array of different assemblies ranging from dimers all the way to
aggregates of fibrils (Powers and Powers, 2008). Initially, it was assumed that Aβ toxicity
was mediated by fibrils similar to those present in amyloid plaques, but recent data suggest
that non-fibrillar, water-soluble assemblies of Aβ may also be important (Klein et al., 2001;
Glabe, 2008; Shankar and Walsh, 2009).
Biochemical analysis of brain indicates that the levels of non-fibrillar forms of Aβ correlate
well with synaptic loss and presence of dementia (Lue et al., 1999; McLean et al., 1999;
*Address correspondence to:, Dominic M. Walsh or Brian O'Nuallain, Laboratory for Neurodegenerative Research, School of
Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Republic of Ireland, Tel:
+353-1-7166751, Fax: +353-1-7166890, firstname.lastname@example.com or firstname.lastname@example.com.
NIH Public Access
Author Manuscript
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Published in final edited form as:
J Neurosci. 2010 October 27; 30(43): 14411–14419. doi:10.1523/JNEUROSCI.3537-10.2010.
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Wang et al., 1999; Mc Donald et al., 2010)(Tomic et al., 2009) and that ex vivo such
assemblies can impair synaptic form and function (Shankar et al., 2008). Specifically, we
have shown that human brain contains Aβ assemblies which migrate on SDS-PAGE and
elute from size exclusion as dimers (~8 kDa), block long term potentiation (LTP), inhibit
synapse remodeling, and impair memory consolidation (Shankar et al., 2008). Such species
are detected specifically in extracts of AD brain suggesting that SDS-stable dimers may be
the basic building blocks of AD-associated synaptotoxic Aβ assemblies (Kuo et al., 1996;
Roher et al., 1996). The role of low-n oligomers of Aβ in the range of dimer to tetramer is
also supported by in vitro studies using peptides bearing design mutations. For instance,
substituting glycine for leucine within the GxxxG repeat motif of Aβ indicates that Aβ-
mediated neurotoxicity is directly linked to the abundance of mass spectrometry-detected
dimers and trimers (Hung et al., 2008). Similarly, peptides containing G33A or G29/33A
substitutions form low-n oligomers that fail to block LTP (Harmeier et al., 2009). This latter
finding indicates that aggregation size alone is not the sole determinant of synaptotoxicity
and that structure is also critical. Consequently, establishing the amyloidogenicity and
structure of Aβ dimers in the brain, CSF, and blood of AD patients are of great diagnostic
and therapeutic interest.
In the absence of sufficient brain-derived Aβ dimers, we and others have generated synthetic
cross-linked Aβ dimers to mimic the natural species (Shankar et al., 2008; Kok et al., 2009).
In our studies Aβ(1-40) containing cysteine in place of serine 26 was used to produce
disulphide cross-linked dimers, (AβS26C)2. Given that such dimer preparations share a
similar synaptotoxic profile with natural dimers, we undertook experiments to investigate
the biophysical and aggregation properties of (AβS26C)2 in the hope that this might shed
light on processes occurring in AD brain. Here we report that (AβS26C)2 aggregated rapidly
to form protofibril-like assemblies and that freshly isolated (AβS26C)2 did not block LTP
whereas (AβS26C)2 solutions that were allowed to form protofibrils did. These data support
the idea that Aβ dimers may stabilize the formation of fibril intermediates by a process
distinct from that available to Aβ monomer and that such intermediates are potent
Materials and Methods
Peptides, chemicals and reagents
Wild-type human Aβ1-40, DAEFRHDSGY-
EVHHQKLVFFAEDVGSNKGAIIGLMVGGVV, and Aβ1-40 in which serine 26 was
substituted with cysteine (AβS26C) were purchased from the Keck Biotechnology Center
(Yale University, New Haven, CT). Peptide mass and purity were determined by
electrospray ionization/ion trap mass spectrometry and reverse-phase HPLC, respectively.
All peptides had the correct mass and were >95% pure.
All chemicals were obtained from Sigma-Aldrich (Saint Louis, MI), and unless indicated
otherwise were of the highest purity available. Unbranched dextran standards of molecular
masses: 43,800; 21,400; 9890, and 4440 were purchased from Pharmacosmos (Holbaek,
Denmark). Water was double-distilled and deionized using a Milli-Q system (Millipore,
Cork, Ireland).
Disulfide cross-linking of AβS26C
Peptide was solubilized at ~0.18 mg/ml in milliQ water, diluted 1:1 with 20 mM ammonium
bicarbonate, pH 8.2, to generate a ~20 µM (with respect to monomer) peptide solution and
bubbled with oxygen for ~5–10 min. Solutions were incubated at room temperature for 5
days and each day bubbled with oxygen. To facilitate disassembly of aggregates formed
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during the oxidation reaction, the peptide solution was lyophilized and subsequently
incubated in 50 mM Tris-HCl, pH 8.0 containing 5 M Guanidine HCl, for ~4 h prior to size
exclusion chromatography (SEC).
SEC isolation of peptide conformers
The oxidized AβS26C was subjected to SEC on a preparative HiLoad™ 16/60 Superdex™
75 column (GE Healthcare, Uppsala, Sweden) eluted with 25 mM ammonium acetate, pH
8.5 at a flow rate of 0.6 ml/min using an AKTA FPLC system (GE Healthcare). Samples
were centrifuged at room temperature and 16,000 × g for 20 min and 2.0 ml of supernatant
injected onto the column. Peptides were detected by absorbance at 280 nm and 1.2 ml
fractions collected. To minimize peptide aggregation, fractions were immediately put on ice,
and if necessary, diluted with eluent so that the peptide content was ≤0.15 mg/ml. Peptide
concentration was determined by absorbance at 275 nm using the molar extinction
coefficient for tyrosine (ε275 = 1,400 M−1 cm−1). Fractions that contained highly pure
(AβS26C)2 or AβS26C monomer were used immediately for biophysical and/or aggregation
studies. Wild type Aβ was solubilized in 0.1% ammonium hydroxide to produce a ~0.2 mg/
ml solution and monomer isolated by SEC as described for AβS26C. Conformer purity was
confirmed by SDS-PAGE and MALDI-ToF MS (Hu et al., 2008).
Samples from peptide cross-linking reactions and aggregation experiments were
electrophoresed on 16% polyacrylamide SDS-tris-tricine gels in the presence or absence of
50 mM β-mercaptoethanol and visualized by silver staining (Shevchenko et al., 1996).
Peptide aggregation
Assembly of monomeric and dimeric Aβ into higher-ordered aggregates was investigated in
quiescent and agitated reactions (5 replicates for each sample time point) using a thioflavin
T (ThT) binding assay adapted for use in microtiter plates (Betts et al., 2008). The
concentration of peptide in SEC-isolated (AβS26C)2 and monomer fractions was determined
by measuring absorbance at 275 nm and peptides then diluted to 0.15 mg/ml with 25 mM
ammonium acetate, pH 8.5. Such solutions were further diluted with 45 mM sodium
phosphate, pH 7.4, to produce stock solutions of 0.087 mg/ml in 20 mM sodium phosphate,
pH 7.4. These solutions were subsequently serially diluted 2–30-fold into assay buffer (20
mM sodium phosphate, pH 7.4) to final concentrations of 0.0029 – 0.044 mg/ml and 100 µl
of each added to wells of a 96-well polystyrene microtiter plate (Thermo Fisher Scientific,
Langenselbold, Germany). Five µl of 2 mM ThT in MillQ water was added into appropriate
wells, including controls that did not contain peptide. For time-course SEC, electron
microscopy (EM), or light scattering analyses, samples were incubated without ThT.
Instead, the reaction progress for such samples was determined by monitoring the ThT
signal of replicate samples that contained the dye. Quiescent peptide aggregation was
initiated by incubating the plate at 37 °C, and agitated samples were shaken at 700 rpm and
37 °C in a VorTemp 56™ incubator/shaker (Labnet International, Inc., Woodbridge, NJ).
Each reaction was monitored in real-time by ThT fluorescence (Ex435nm and Em485nm)
using a SpectraMax M2 multi-detection microplate reader (Molecular Devices Corp.,
Sunnyvale, CA) (Betts et al., 2008). Prior studies have shown that the continuous presence
of ThT did not affect reaction kinetics (Betts et al., 2008).
Sedimentation velocity analytical ultracentrifugation (AUC)
Experiments were performed on a Beckman XLI analytical ultracentrifuge fitted with an
An50-Ti rotor in quartz cells containing two-sector centerpieces. Samples of freshly SEC-
isolated (AβS26C)2 were centrifuged at 50,000 rpm and 20 °C and absorbance data were
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collected at 230 and 278 nm over 20 h, with scans recorded every 10 minutes. Sedimentation
velocity data were analyzed using the c(s) distribution method in the software SEDFIT
(v11.8) (Schuck et al., 2002). For the analyses, partial specific volume (ν̄) for (AβS26C)2
was calculated from the amino acid sequence using the software SEDNTERP (Laue et al.,
Light scattering
The molar masses for (AβS26C)2 aggregates was determined by multi-angle laser light
scattering (MALLS). One ml of a 3 day incubated 15 µM (AβS26C)2 was loaded onto a
Superdex™ 75 10/300 GL column (GE Healthcare) equilibrated in 20 mM sodium
phosphate, pH 7.4, and eluted using a Prominence HPLC instrument (Shimdazu Europa
GmbH, Duisburg, Germany) daisy-chained with a Dawn HeleosII MALLS detector and
Optilab dRX refractometer (Wyatt Technology Corp., Santa Barbara, CA). The column was
maintained at 20 °C (± 0.1 °C) using an oven (Wyatt Technology Corp.) and the delay
volumes between instruments and band-broadening were determined by injecting a 1 mg/ml
BSA solution through the system. Molar masses were calculated from the intensity of
scattered light at 18 different angles, as a function of protein concentration and SEC elution
volume. The intrinsic instrumental baseline for each data channel was subtracted and the
molar mass across a given 2D slice of the elution profile determined using the ASTRA V
HPLC software (Wyatt Technology Corp.). To ensure robust data fitting, the apparent molar
mass for the elution peak was determined as a function of the width of fitted windows for
the front end, centre, and trailing edge of the peak.
Circular dichroism spectroscopy
Aβ solutions were placed in a 1 mm path length quartz cuvette (Starna Scientific Ltd.,
Essex, UK) and spectra obtained at 22 °C using a J-810 JASCO spectropolarimeter (JASCO
Corp., Tokyo, Japan). Spectra were generated from three data accumulations between ~195–
260 nm with 10 nm/min continuous scanning and a 0.5 nm bandwidth. To minimize
artifactual CD signal by aggregate-induced light scattering, samples from aggregation
studies were centrifuged at 16,000 × g for 20 min at room temperature. Raw data were
manipulated by subtraction of buffer spectra and by binomial smoothing according to the
manufacturer's instructions (JASCO Corp.) and data displayed as molar ellipticity (θ).
Electron microscopy
Negative contrast EM was performed as described previously (Walsh et al., 1997). Aliquots
(10 µl) of peptide sample were applied to carbon-coated Formvar grids (Electron
Microscope Sciences, Washington, PA), cross-linked with 0.5% (v/v) glutaraldehyde,
stained with 2% (w/v) uranyl acetate solution (Ted Pella, Inc., Redding, CA) and examined
using a Tecnai™ G2 Spirit BioTWIN electron microscope (FEI, Hillsboro, OR).
In vitro electrophysiology
Six to eight week old male C57/BL6 mice were anaesthetised with isoflurane/O2 and
decapitated. Brains were rapidly removed and immersed in ice-cold sucrose-based artificial
cerebrospinal fluid (ACSF) that contained: 75 mM Sucrose, 87 mM NaCl, 2.5 mM KCl, 25
mM NaHCO3, 25 mM Glucose, 1.25 mM NaH2PO4, 0.5 mM CaCl2, 7 mM MgCl2.
Parasagital sections (350 µm) were prepared using a vibratome VT1000S (Leica, Bensheim,
Germany) and slices allowed to recover for at least 90 min in normal ACSF (nACSF
contained: 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 2.5 mM CaCl2, 26.2 mM
NaHCO3, 11 mM D-glucose, 1 mM NaH2PO4) in a BSC-PC submerged incubation chamber
(Warner Instruments, Hamden, CT). Thereafter, slices were continuously perfused with
oxygenated nACSF at a rate of 2–3 ml/min at 30 °C. A stainless steel microelectrode (FHC,
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Bowdoin, ME) was used to stimulate the hippocampal Schaffer collateral pathway, and
extracellular excitatory postsynaptic potentials (fEPSPs) were recorded in the stratum
radiatum of the CA1 region using glass microelectrodes (2–4 MΩ) filled with nACSF. Test
stimuli were delivered once every 30 s (0.033 Hz) and the stimulus intensity was adjusted to
produce a baseline fEPSP of 30–40% of the maximal response. A stable baseline was
recorded for at least 20 min before addition of fresh or aggregated (AβS26C)2. LTP was
induced by theta-burst stimulation (TBS, 4 pulses delivered at 100 Hz, ten times, with an
interburst interval of 200 ms) delivered at baseline intensity. Field potentials were recorded
with an Axon CNS Muticlamp 700B amplifier coupled to a Digidata 1440A digitizer
(Molecular Devices). Data was recorded using a pClamp10 (Molecular Devices, Sunnyvale,
CA) and later analyzed using Clampfit.10 software (Molecular Devices).
In all experiments, fresh or aggregated (AβS26C)2, or buffer was added to the perfusate after
a stable baseline period and circulated in the bath for the remainder of the experiment.
Immediately prior to use, the 3 day aggregated material was spun at 16,000 × g for 20 min at
room temperature and the supernatant added to the perfusate. LTP was induced 30 min after
introduction of peptide or vehicle and recorded for at least 60 min post-tetanus.
General curve fitting
Data points from aggregation time courses were fitted using SigmaPlot 2000, version 6
(Systat Software, Chicago, IL).
Highly pure AβS26C dimers are isolated using preparative SEC
The oxidized AβS26C peptide produced four major elution peaks (~48, ~55, ~72, and ~84
ml) when chromatographed on a preparative HiLoad 16/60 Superdex 75 (GE Healthcare,
Uppsala, Sweden) column (Fig. 1A). Calibration of size exclusion columns using globular
protein standards does not allow accurate assessment of Aβ monomer size (Walsh et al.,
1997), whereas the use of dextran standards does (Paivio et al., 2004;Walsh et al., 2005).
Based on the use of unbranched dextran standards the apparent molecular weight of AβS26C
conformers in the two largest peaks (~72 and ~84 ml) were ~10 (dimer) and ~5.7
(monomer) kDa, respectively (Fig. 1A). SDS-PAGE and mass spectroscopy analyses
confirmed that these fractions contained (AβS26C)2 and monomeric Aβ (Fig. 1A, B). The
minor SDS-PAGE bands in the (AβS26C)2 fractions that migrated >9 kDa were transiently
and artificially induced by SDS (Wahlstrom et al., 2008). Reducing agent completely
dissociated (AβS26C)2 to monomer, indicating that dimers were indeed stabilized by an
intermolecular disulfide bond (Fig. 1B). Furthermore, AUC sedimentation velocity studies
demonstrated that the dominant peptide conformer had an apparent molecular weight of
~10+/− 2 kDa (Fig. 1C). However, trace quantities of higher ordered assemblies of ~95 kDa
were also observed. Given that the AUC experiment took ~20 h to complete it seems likely
that these higher molecular weight species formed during the experiment. In accord with this
observation re-chromatographying of the sample incubated for 16 h at room temperature
revealed the appearance of species eluting in the void volume (Fig. 1D). These finding
suggest that the (AβS26C)2 has a high propensity for aggregation and prompted us to
compare the aggregation of (AβS26C)2 versus Aβ monomer.
Dimeric AβS26C forms stable protofibrils
We have previously demonstrated that (AβS26C)2 are potent neurotoxins (Hu et al., 2008;
Shankar et al., 2008). Thus, we investigated whether (AβS26C)2 had a propensity to
aggregate and if their toxicity was dependent on aggregation. Freshly isolated (AβS26C)2
was diluted as described in the Materials and Methods and aggregation monitored using
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thioflavin (ThT) binding. Under quiescent conditions (AβS26C)2 (5–10 µM) rapidly formed
ThT positive assemblies without a measurable lag time (Fig. 2A, E), but unlike typical
amyloid fibrils the (AβS26C)2 aggregates did not readily sediment when centrifuged (16,000
× g for 20 min) (Fig. 2B). Moreover, CD studies indicated that the (AβS26C)2 aggregates
contained significant β-structure (Fig. 2D), whereas freshly isolated (AβS26C)2, and
AβS26C and wild type monomers incubated for 1 day had little secondary structure (Fig.
2C, D). As previously documented, the secondary structure of SEC-isolated wild type
monomer changed slowly (Walsh et al., 1999) and AβS26C monomer appeared to change
even more slowly (Fig. 2D). Dimer aggregation was critically dependent on peptide
concentration with no discernible ThT binding evident at (AβS26C)2 concentrations ≤ 2.5
µM in samples incubated at 37 °C for 44 h (Fig. 2E). The progress curves for (AβS26C)2
aggregation fitted to single exponential linear plots (R2= 0.96 ± 0.04) producing an apparent
rate constant of 0.085 ± 0.03 h−1 (Fig. 2F).
Having demonstrated that (AβS26C)2 shows an unusually high propensity for aggregation,
we investigated the morphology and size distribution of the aggregates formed. SEC
analysis of (AβS26C)2 aggregate species in reactions sampled over a 3 day period indicated
that these assemblies eluted in the void volume of the Superdex 75 10/300 GL column (Fig.
3A). The SEC-isolated aggregates were not sedimented by centrifugation and had ~4-fold
greater ThT signal than the same weight of SEC-isolated (AβS26C)2 or freshly prepared
monomers, and ~2-fold lower than for Aβ wild type fibrils (Fig. 3B, C). Consistent with CD
analysis of unfractionated (AβS26C)2 aggregates, the SEC-isolated aggregates were rich in
β-sheet structure whereas the (AβS26C)2 fraction had no detectable secondary structure
(Fig. 3D). SEC-MALLS indicated that the apex of the (AβS26C)2 aggregate peak contained
assemblies with a molecular weight distribution of ~1–2 mega-Dalton, with a leading
shoulder containing species ≥ 2 mega-Dalton (Fig. 3E). The distribution of species detected
are consistent with Aβ assemblies that contained ~100 to 450 (AβS26C)2 molecules. These
aggregates had morphologies highly similar to Aβ protofibrils (Harper et al., 1997;Walsh et
al., 1997) appearing as short flexible rods with an average width of 5.8 ± 0.2 nm (n= 9) (Fig.
4). The protofibrils grew in length from 59 ± 12 nm (n= 7) after 1 day to 139 ± 35 nm (n= 7)
after 3 days incubation. Importantly these assemblies were relatively stable since protofibrils
in certain samples persisted even after a one month long incubation at 37 °C. However, the
length of protofibrils appeared to increase with time, with the 30 day incubated sample
containing protofibrils twice as long, 284 ± 59 nm (n= 7), as the protofibrils detected at 3
days (Fig. 4). The largest protofibrils in the aged samples pelleted after centrifugation at
16,000 × g for 20 min, leaving ~40 % of the ThT positive material in solution which by EM
contained shorter protofibrils, 181 ± 31 nm (n= 7)(Fig. 4D, E).
To investigate the formation and stability of protofibrils over more tractable time scales we
studied aggregation of (AβS26C)2, AβS26C and wild type monomers under conditions
where the samples were vigorously agitated. In contrast to quiescent conditions, agitation
initiated aggregation of all peptides within a couple of hours (compare Fig. 2A and SFigs.
1A – C). The concentration of (AβS26C)2 required to facilitate aggregation was reduced
almost 4-fold when samples were agitated, with fully formed ThT positive assemblies
evident within ~4 h at (AβS26C)2 concentration as low as 1.25 µM (SFig. 1A). In contrast,
without shaking no ThT positive aggregates were formed in (AβS26C)2 solution <5 µM
(Figs. 2E). The progress curves for the agitated (AβS26C)2 reactions were sigmoidal with
short lag phases, tlag <1 h. The absence of a measurable lag phase for the quiescent
(AβS26C)2 reactions is somewhat puzzling but presumably reflects the significantly larger
peptide concentrations used in the quiescent reactions. The maximum ThT signal amplitude
for (AβS26C)2 aggregation was proportionate to the amount of peptide used (SFig. 1A).
Wild type monomer was considerably less amyloidogenic than (AβS26C)2 since they only
formed ThT positive aggregates at concentration ≥ 5 µM, whereas (AβS26C)2 aggregated
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even at 1.25 µM (SFig. 1A, B). Consistent with quiescent experiments, 20 µM (AβS26C)2
formed aggregates in agitated samples without a lag (SFig. 1C), were not pelleted by
centrifugation (SFigs. 1 and 2) and had protofibril-like morphology. The average lengths
and widths of protofibrils formed in agitated samples (78 ± 22 nm, n= 8 and 6.0 ± 1.0 nm,
n= 8, respectively) were highly similar to those of protofibrils formed under quiescent
conditions (compare Fig. 4C and SFig. 2B). In contrast, the products for the AβS26C and
wild type monomer reactions readily pelleted and had typical amyloid fibril morphology
when examined by EM. Interlaced networks of fibrils with diameters ranging from 7 to 11
nm and up to several microns in length were detected in incubates of both S26C and wild
type monomers (SFig. 2).
Aggregated, but not fresh AβS26C dimers inhibit synaptic plasticity
We have previously shown that AβS26C dimers can potently block LTP both in vivo (Hu et
al., 2008) and in vitro (Shankar et al., 2008). In those studies (AβS26C)2 was isolated by
SEC, frozen at −80 °C and shipped on dry ice to our collaborators, where the samples were
stored frozen, then thawed and used for electrophysiology experiments. However, given the
findings presented above regarding the high propensity for dimer to form aggregates we set
out to determine if the previously documented plasticity-impairing activity of S26C derived
from authentic dimers or aggregates of (AβS26C)2. First we used analytical
ultracentrifugation and SEC to assess aggregation in samples that had been frozen and
thawed versus samples that had been freshly isolated and immediately used for AUC. When
freshly isolated (AβS26C)2 was immediately re-chromatographed it produced a single peak,
whereas when the same solution was frozen and stored at −80 °C for 1 week and then re-
chromatographed a small second high molecular weight peak was detected (SFig. 3). Next
we performed experiments in which (AβS26C)2 was isolated and used for electrophysiology
experiments within 4 h (between 1–4 h) of collection (Figs. 5C and D). Hippocampal slices
perfused with vehicle produced robust LTP measuring 160 ± 8 %, at 1 h post-TBS (n=7),
while slices perfused with 50 nM of freshly isolated (AβS26C)2 produced a highly similar
potentiation (155 ± 8 %, n=7). The concentration of (AβS26C)2 used here is significantly
higher than the minimal concentration of (AβS26C)2 previously shown to impair
hippocampal LTP (Shankar et al., 2008) and strongly suggests that the previously
documented impairment of plasticity was not mediated by authentic (AβS26C)2. Given the
demonstration that (AβS26C)2 can aggregate when frozen (SFig. 3), we set out to determine
if deliberately aggregated (AβS26C)2 could block LTP. When the same (AβS26C)2 solution
that had been tested when fresh and failed to block LTP was aggregated for 3 days it
completely inhibited LTP (103 ± 10 %, n=7) (Fig. 5C and D). Moreover, EM examination of
the 3 day aggregated material demonstrated the presence of abundant protofibrils (Fig 5B)
indistinguishable to those observed in prior aggregation experiments (e.g. Fig. 4). Thus
together these results indicate that S26C dimers rapidly assemble into relatively stable
protofibrils that can block LTP.
Burgeoning evidence suggests that non-fibrillar water-soluble forms of Aβ are the principal
mediators of neurotoxicity in AD, but, as yet the precise conformation and assembly form(s)
of Aβ responsible remain unidentified (Klein et al., 2001; Hardy and Selkoe, 2002; Klein et
al., 2004). Recent work has indicated that SDS-stable Aβ dimers present in the water-soluble
phase of human brain are strongly associated with AD-type dementia (Mc Donald et al.,
2010) and possess disease-relevant toxic activity (Shankar et al., 2008). However, due to the
lack of highly pure brain-derived Aβ it has not been possible to study the conformation and
aggregation kinetics of this material. Thus we have chosen to study synthetic dimers which
mimic the toxic activity of the natural SDS-stable dimer (Hu et al., 2008; Shankar et al.,
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2008). Unlike prior work with covalently-linked Aβ which employed crude mixtures
containing both cross-linked and uncross-linked species (Siegel et al., 2007; Hartley et al.,
2008; Moore et al., 2009), and which lacked definition regarding aggregation state and the
sites of cross-linking, we used highly pure Aβ dimer with a defined linkage site.
Specifically, dimers were formed by the substitution of serine 26 with cysteine and
subsequent disulphide bond formation and isolated free of other assemblies by SEC.
Like the wild type monomer, (AβS26C)2 was devoid of discernible secondary structure and
exhibited little or no binding to thioflavin T. De novo fibrillogenesis from Aβ monomer is
believed to involve a series of conformational alterations that include the formation of
thermodynamically unstable amyloidogenic intermediates, self-association and stabilization,
and finally, protofibril association into mature fibrils (Harper and Lansbury, 1997;
O'Nuallain and Wetzel, 2002; Glabe, 2008; Roychaudhuri et al., 2009). In contrast,
(AβS26C)2 aggregation proceeded rapidly with the formation of stable protofibril-like
assemblies which persisted for relatively long periods. In agreement with prior reports,
protofibrils appeared as a continuum of structures which by EM ranged in size from
imperfect spheres of 5 nm diameter to flexible rods up to 200 nm long and approximately 5–
6 nm wide (Harper et al., 1997; Walsh et al., 1997; Harper et al., 1999; Johansson et al.,
2006) and included structures sometimes referred to as Aβ-derived diffusible ligands
(ADDLs) (Lambert et al., 1998; Hepler et al., 2006). This increase in the propensity to form
protofibrils coupled with a reduced tendency to form mature fibrils suggests that there may
be some subtle microstructural differences between the monomer and (AβS26C)2 which act
to change the rate constant for protofibril formation, and/or reduce the rate constant for
conversion of protofibrils into fibrils (Fig. 6). Conformational changes in the mid-region of
Aβ are thought to be required for the transition of protofibrils into fibrils (Williams et al.,
2005; Kheterpal et al., 2006) and cross-linking at residue 26 may act to suppress this
But how does this translate to Aβ in the human brain? For instance, to make dimers is it
necessary to start from monomeric Aβ, and if it is, how then is it possible that monomers can
contribute to two different pathways? First, there is evidence that dimerized APP may
undergo amyloidogenic processing (Munter et al., 2007) thus providing a route for the direct
production of discrete Aβ dimer subunits. Once formed such dimers may associate with
other dimers in a pathway distinct from the aqueous-phase assembly of individual
monomers. Second, other factors that preferentially stabilize dimer formation from
monomers would be predicted to enable assembly by a pathway similar to that we have
observed for (AβS26C)2, whereas conditions that do not favour dimer formation would lead
to lower levels of protofibril. It is noteworthy that protofibril formation and stability are
modulated by factors that may also facilitate dimer formation and stabilization. Specifically,
covalent cross-linking of Aβ by either 4-hydroxynonenal (HNE) or transglutaminase
(TGase) or the presence of certain small molecules accelerate formation of protofibrils while
inhibiting fibril formation (Siegel et al., 2007; Hartley et al., 2008; Moore et al., 2009;
Williams et al., 2005). Although, it has not yet been demonstrated that transglutaminase
catalyzed isopeptide ε-(c-glutamyl)lysine bond formation leads exclusively to formation of
cross-linked dimer, the data available suggest that intermolecular bond formation between
lysine 16 and glutamine 15 is the only likely site of linkage (Ikura et al., 1993). Thus the
enhancement in the rate of protofibril formation and persistence observed in prior cross-
linking studies is probably attributable to the presence of dimer which aggregates in a
manner distinct from monomer. The rate of protofibril formation and the time period over
which protofibrils persist are strongly influenced by Aβ primary sequence (Lashuel et al.,
2003; Paivio et al., 2004). For example, the population of protofibrils is greater for Aβ1-42
and Aβ1-40E22G than for wild type Aβ1-40 and it is interesting to speculate that this may
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reflect an increased tendency of Aβ1-42 and Aβ1-40E22G to form dimers(Walsh et al.,
1997; Nilsberth et al., 2001).
Moreover, since SDS-stable Aβ dimers appear specific for AD it seems plausible that the
presence of dimers and abundance of protofibrils are linked. That is, dimers may exert
toxicity as a consequence of their ability to form relatively stable protofibrils (Fig. 6).
In prior studies we have demonstrated that SDS-stable Aβ dimers from human brain and
CSF can impair disease-relevant measures of learning and memory (Klyubin et al., 2008;
Shankar et al., 2008) and that synthetic (AβS26C)2 exhibited a similar activity. Here we
have now shown that freshly prepared (AβS26C)2 does not inhibit LTP as such, but rather
aggregated dimers in the form protofibrils mediate this activity. As discussed above this
finding is consistent with the previously documented protofibril-mediated inhibition of LTP
(Hartley et al., 2008) and with recent observations that Aβ protofibril levels correlate with
spatial learning impairment of AD transgenic mice (Lord et al., 2009). Taken together our
findings suggest that the activity previously ascribed to SDS-stable Aβ dimers, may not
reside in dimers per se, but rather in the higher order assemblies they rapidly form. Of
course other factors which increase the kinetic stability of protofibrils by a dimer-
independent mechanism could also lead to an increase in synaptotoxicity, but given the link
between SDS-stable dimers and AD, a dimer-dependent mechanism seems most likely.
Similarly, the pathogencity of assemblies other than protofibrils cannot be discounted and
merits further investigation. Moving forward it will also be important to investigate the
assembly and toxicity of heterodimers of Aβ1-40S26C and Aβ1-42S26C and of homodimers
of Aβ1-42 and to assess the toxicity of protofibril sub-populations. The latter is particularly
important since one might expect that different structures may have different activity.
Nonetheless, therapeutic targeting of Aβ dimers remains attractive since such a strategy
should prevent formation of a substantial population of pre-fibrillar toxic assemblies.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
We thank Ms. Gwen Manning and Dr. Elia Giuliano for assistance with mass spectrometery. This work was
supported by funding to DMW from Science Foundation Ireland (Grant 08/1N.1/B2033), the Health Research
Board (Grant RP/2008/30) and the National Institutes of Health (grant IRO1AGO27443).
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Figure 1. Isolation and initial characterization of AβS26C dimers
Disulfide cross-linked Aβ dimers were generated by atmospheric oxidation of 20 µM
AβS26C in 10 mM sodium bicarbonate, pH 8.5, for 5 days at room temperature. (A) The
cross-linked Aβ dimer product was isolated by SEC using a HiLoad 16/60 Superdex 75
column equilibrated with 25 mM ammonium acetate, pH 8.5. Arrows indicate elution of
dextran standards. (B) SDS-PAGE and MALDI-ToF MS analysis of the two low molecular
weight major SEC peaks confirms the presence of a disulfide cross-link in the dimer but not
monomer fractions. (C) Analytical ultracentrifugation analysis confirmed the predominant
species to have a predicted mass of 10 +/− 2 kDa. (D) The same dimer used for AUC was
incubated at room temperature for 16 h and when re-chromatographed on SEC revealed a
significant peak in the void volume (▬) that was not detected in the t=0 sample (–).
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Figure 2. Quiescent aggregation of AβS26C dimers
(A) Progress curves for the formation of ThT positive material are shown as % of the
maximum fluorescence detected from freshly isolated (AβS26C)2 (●),S26C (○) and wild
type (▲) monomers. ThT fluorescence was monitored in real time at 37 °C. Reactions
contained 10 µM (AβS26C)2 or 20 µM wild type/S26C monomer in 20 mM sodium
phosphate, pH 7.4 plus 10 µM ThT. (B) Freshly isolated and 1 day aggregated dimer
samples were analyzed by SDS-PAGE analysis. Dimer supernatant (sup) was generated by
centrifuging the sample at 16,000 × g for 20 min at room temperature. Circular dichroism
spectra for solutions of freshly isolated (AβS26C)2 (10 µM, ▬), S26C (20 µM, – –) or wild
tpye (20 µM,•••) monomers at 0 h (C) and after 24 h at 37 °C (D). (E) Concentration-
dependency of S26C dimer aggregation: 10 µM (●); 5 µM (○); 2.5 µM (▲), and 1. 25 µM
(□) were incubated at 37 °C for up to 48 h. (F) Dimer aggregation follows pseudo first-order
kinetics. The data from panel E was plotted as the natural log of the difference between the
maximum ThT fluorescence and the observed signal versus time.
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Figure 3. Biophysical analysis of AβS26C dimer aggregates
(A) Ten micomolar (AβS26C)2 were incubated in 20 mM sodium phosphate, pH 7.4,
without agitation and at intervals samples removed and chromatographed on a Superdex 75
10/300 GL column equilibrated with 20 mM sodium phosphate, pH 7.4. The
chromatographs show the conversion of Aβ dimers into high molecular weight conformers
that eluted in the void volume (dashed line). Freshly prepared Aβ dimers after 4 h at 4 °C
(▬), and Aβ dimer preparation after 6 h (– –), 24 h (▪▪▪), and 3 d (▬▪▪) at 37 °C. The grey
bars show the peak fractions that were collected for subsequent experiments. (B) ThT
fluorescence of SEC-isolated 3 day aggregated (AβS26C)2 compared with the same
concentration (0.09 mg/ml) of the unaggregated peptide and WT Aβ conformers. (C) SDS-
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text:
For Interviews with Yoko Miwa, click here.
We were featured in both the January and Spring 2012 digital editions of Jazziz. Click on the images below to read the full articles.
Yoko Miwa Makes an Eclectically Lyrical Blue Note Debut
In her Blue Note debut yesterday, Yoko Miwa showed off a comfortable but hard-charging command of several jazz vernaculars. She made elegant ragtime out of Monk, worked a playful carnaval pulse back and forth on one number, and managed to make a jauntily entertaining trip to New Orleans out of what was essentially a one-chord vamp. But the vernacular that she excelled the most with was her own. That style favors lots of big block chords stretched mostly across the piano's midrange, which she builds expansively but purposefully to big, glittering, anthemic crescendos. Think of a more carefree Brad Mehldau (that shouldn't be hard!) and you're on the right track. Read more.
Review: Yoko Miwa at the Blue Note
Yoko Miwa, Berklee professor and jazz pianist, took the stage on Sunday at the Blue Note as part of the venue's East Meets West Brunch series and blew through some bluesy oldies as well as impressive original compositions. Her bandmates — Will Slater on bass and Scott Goulding on drums — provided assistance and contributed in surprisingly egoless ways to Miwa's originals. Read more.
Yoko Miwa Trio recorded lovely "Live At Scullers Jazz Club" CD
Miwa's original composition "Wheel Of Life" begins with a beautifully understated piano line before soon turning into a larger sound of cascading notes with her rhythm section joining in with their own lovely touches. Miwa has a way of keeping art, beauty, and emotion all perfectly contained in a peaceful expression of colors and tone. It is during these moments of understatement that the listener can truly feel what she was likely feeling when she was composing. Read more.
Yoko Miwa Trio: Live at Scullers Jazz Club (2011)
Miwa displays an impressive stylistic range. Opening with a rousing take of Steve Allen's "This Could Be the Start of Something," the pianist and her trio mix a bouncy elegance with a full-bore forward momentum. Miwa treats the melody with reverence, riding a inexorable rhythmic wave supplied by bassist Greg Loughman and drummer Scott Goulding. Virtuosic but unrelentingly accessible, the pianist stretches out, taking eleven minutes to explore this Great American Songbook gem with glorious grace. Read more.
Live at Scullers Jazz Club — Yoko Miwa Trio
Miwa possesses a fully-formed style and shuns flamboyance for substance, making every note count, despite a formidable technique that might tempt her to overplay. Read more.
CD Review: Yoko Miwa Trio – "Live At Scullers Jazz Club"
Yoko Miwa swings from both sides of the piano, and is blessed with a left hand that lends itself to a fully developed playing style that is reminiscent of early stride masters; a dominant strength, no doubt acquired through McCoy Tyner's influence on Miwa, and also may be traced to be-bop genius, Thelonious Monk, who heavily influenced Tyner. Read more.
CD Review: 'Live At Scullers Jazz Club' by Yoko Miwa Trio
CD Review: Yoko Miwa – Live at Sculler's Jazz Club
The trio swings hard on the Steve Allen composition, "This could be the start of something", closing with a energetic piano/drums call and response. The beautiful ballad "Wheel of Life" is one of Miwa two originals. The chord progression gives the listener a sense of circular motion. The other original, "Silent Promise" also a ballad, is one of the most intimate and sublime moments of the album and reminiscent of movie themes like Cinema Paradiso. Read more.
It's not just her flawless punctuality, it's the fact that you can "feel" what she's playing, whether it be an old standard like the opener, Steve Allen's "This Could Be The Start of Something Big", or her superb original composition (my favorite on the CD, by the way), "Wheel of Life"… read more.
Yoko Miwa Trio: Live at Scullers Jazz Club
Miwa's excellent, light – hearted piano sets a great tone straight from the beginning with "This Could be the Start of Something", and the fun never stops throughout the CD. The crowd can be heard enjoying themselves, especially with up-tempo numbers like the album opener. Read more.
Yoko Miwa Trio-Live At Scullers Jazz Club
If you're looking for something different to add to your jazz library, or you're a music fan who is thinking about trying out jazz, then you want this recording. There is meat for the aficionado, but there is also a lot of fun for the regular listener. You'll hear some tunes you might know (Lou Reed and Aerosmith, for instance!) but like you've never heard them before. Read more.
gapplegate music review
Yoko Miwa Trio, Live at Scullers Jazz Club
On this one the live ambiance clearly gets them cooking. They rip through a nice set of standards and less-standards, along with a couple of Yoko originals, and they do an excellent job throughout. Read more.
She a has a mean left hand and a restless right one… Read more.
Maybe we'll add that Boston should quit hogging her and let the rest of us see her live once in a while. Simply a first class, straight up, straight ahead player whose sole mission is to step up and deliver the goods, this is solid playing that's as good as it gets. Read more.
Yoko Miwa Trio – Live At Scullers Jazz Club
When it comes to live albums by jazz performers, those recorded within the cosy confines of a smokey jazz club tend to sound the best. The acoustics at the Scullers Jazz Club sound pretty damn fine, and the interplay and musicality of the Yoko Miwa Trio shine through extremely well. The mix of music is surprisingly varied – along with several original tracks by pianist Yoko Miwa, there are selections from Art Farmer, Milton Nascimento and Steve Allen, sitting alongside songs by rock icons Steven Tyler of Aerosmith and Lou Reed, which you probably wouldn't expect to find on a jazz album. Read more.
"Pianist Yoko Miwa displays unpretentious melodies, elegant phrasing, and the lyrical
sensibility of a jazz poet… she's a remarkably fluent mainstream player with a graceful
touch and a self-assured notion of where each tune is headed."
Jazz Review
"Listening to Yoko Miwa play the piano is like listening to the stars that shine at night
twinkle at various decibels as they frame the celestial bodies in the evening sky with
sound waves of elation and startling beauty."
James Isaacs
"Yoko Miwa demonstrates a full command of the inner-directed, post-Bill Evans piano
idiom, which also takes in the work of Keith ]arrett and, to lesser extents, Chick Corea
and Steve Kuhn… displays her burgeoning talent as a writer of melodically inviting,
impressionistic material, as well as introducing a technically assured soloist with a clean,
singing sound and an occasional penchant for the blues in pastels."
Audiophile Audition
The Yoko Miwa Trio – Fadeless Flower (Miwa, piano; Greg Loughman, bass;
Scott Goulding, drums) – Polystar P.J.L. MTCJ-3009: (Release date: June 15)
by John Henry
Another outstanding Japanese female jazz piano star is with us, hard on the heels of the
phenomenal Hiromi (reviewed twice here last month). This is already the second album
as leader for Ms. Miwa, who began her studies with the father of famed jazz pianist
Makoto Ozone, and has served as accompanist to vocalist Kevin Mahogany. In 2001 she
was a featured performer at the Kennedy Center's "Mary Lou William's Women in Jazz
Festival." All nine tracks here are her originals and each has a song-like sense about them
that seem to perfectly fit the album's titled Fadeless Flower – which is also a waltz-tempo
tune on the album. She observes, "I'm always trying to sing when I play a solo, I mean
sing through my piano phrasing." Her trio members support her effectively by avoiding
overstating their parts – especially the tasteful drummer. Not all the tunes are so lyrical
however; some are straight-ahead swingers with some active bluesy left-hand chording.
Her clean and singing sound is beautifully transmitted by the high quality recording on
this new-to-me Japanese label.
All About Jazz
"The pianist has an engaging way of repeating a phrase, drawing the listener in before she
lets the melodic flow go free again, telling stories full of concise and beautifully-rendered
ideas…. The variety of styles Miwa commands, while maintaining a start-to-finish
cohesion of sound on Fadeless Flower, is impressive. A gorgeous and unpretentious set
of straight-ahead jazz."
The Village Voice
"Young mainstream piano trio aim for clean sound, delicate balance, inconspicuous
All Music Guide
"Her second album as a leader is one of the warmest, most unapologetically lyrical
jazz albums of the year… Miwa displays her facility with a variety of styles on this all-
original program….what's consistently impressive is her ability to deliver complex and
challenging musical ideas in a compelling and yet frequently gentle and inviting way."
Tucson Citizen
This is trio jazz worth listening to. There is fiber in its congeniality. Like those talks with
Carl Sagan, or more aptly, Leonard Bernstein. True intelligence is never boring.

subset: commoncrawl


text:
Space where guests can cook their own meals
Central heating or a heater in the listing
In the building, free or for a fee
Space where guests can cook their own meals
Towels, bed sheets, soap, and toilet paper
Central heating or a heater in the listing
In the building, free or for a fee
In the building, free or for a fee
Continuous access in the listing
Breakfast is provided.
The listing can accommodate a gathering of 25 or more attendees
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2 room apartment of 50 m2 in a secure and very quiet building only 50 meters away from Champs-Elysées. The apartment is situated in the 3rd floor with elevator. This apartment has one bedroom and a big livingroom, a bathroom, one separate toilets and a kitchen all furnished.
In the kitchen you will find an oven, a fridge, a freezer, a microwave a washing and drying machine, an induction hob a coffee machine, a toaster and all you need to cook.
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Numerous buses pass at the bottom of the building.
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Guest Access
The entire flat is accessible to the guests. All the commodities and equipments will be accessible.
Towels and sheets will be given.
Interaction with Guests
we can text anytime and send email to each other. If needed i can also talk to them by phone. I will tell them all they need from the beginning.
The Neighborhood
It is one of the most luxuriant neighborhood in Paris.
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Many restaurants and clubs are just next to the apartment. It is very easy to find a nice place to go out just next to the apartment.
Getting around
The subway is just two minutes away from the apartment.
J'attends des voyageurs respectueux de l'appartement.
Eviter de fumer dans l'appartement et respecter les voisins en évitant de faire trop de bruit.
The reservation was canceled 41 days before arrival. This is an automated posting.
Appartement très agréable au calme et très bien placé. Propriétaire efficace pour le Check in et out.
Fred's appartment is very good and is as can be seen on the pictures. The rooms are spacious enough and the luxury is satisfactory. If you want to be a kitchenprinces this is the right place to be. Though it would be a mistake to cook here: you're IN PARIS so you have to go out.
Fred (and Maria = the keykeeper) are very friendly and helpfull. On the second day the door closed, with the key inside. So we had to contact fred. Within 1,5 hour he had fixed it for us. The information via internet, email and sms was helpfull and good.
In a few words: i would recommend the appartment to anyone. Dont hesistate to airbnb with fred
My booking of Fred's apartment was rather last minute and fred was quick to reply to all my queries! The apartment was so close to everything which was perfect for us. One of the best hosts!
The apartment was comfortable, clean and the localization is perfect, very near the subway and bus station. Our experience was very positive.
Fred is lovely & always answers all info what we asked for. The place is located on the perfect location for shopping & eating. The room is clean & well decorated but if coming in July or August might need a fan or air condition. Only one neighbor was there in different floor ( Ground floor) when we stayed there. They made some noises at night but not too bad if we closed the window. However, I would love to recommend this place for everyone who visit Paris. Great location & there are many great restaurants near the place. Easy access from the road & area is safe.
Why would anyone stay in a hotel in Paris? We have in the past, but SO glad that we decided to rent this apartment for our recent stay. The location could not have been any better, just steps away from the Champs Elysees and a Metro station. Really neat apartment with everything we could have needed and so much more space than a hotel room.
Apartment was also nice and quiet and very secure with several coded doors leading to the building.
Highly recommended. Host was not there, but very easy to deal with and communicate with. Will not stay anywhere else when I return to Paris. Thanks Fred!
Je suis un jeune entrepreneur passionné pas la création d'entreprise. Mes hobbies sont le tennis et le golf. L'hôte est un jeune homme sympatique et accueillant qui saura répondre à toutes vos exigences.


text:
Find your way with a handheld GPS from Garmin or Lowrance. We offer Ram mounts and Grip-It mounts for mounting your handheld GPS to your car or ATV. The SPOT GPS Messenger is a great way to check in while on your camping trip. Stay in contact with a two-way radio or walkie-talkie from Midland. You'll also find traditional compasses from Brunton.
Garmin GPSMAP 78s Handheld GPS
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Access all NOAA weather channels, weather alert
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Midland Radio Microphone Headsets, 2 Pair
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text:
3D Vision Studios specializes in 3D Architectural imagery for marketing and advertising needs. We create high quality images at a reasonable price. We offer photo-realistic renderings as well as floor plans. All work performed is done by our highly trained staff in Layton Utah.
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Honey Body Lotion 150ml
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text:
Low oil prices and monetary easing are boosting growth in the world's major economies, but the near-term pace of expansion remains modest, withabnormally low inflation and interest rates pointing to risks of financial instability, according to the OECD's latest Interim Economic Assessment.
Mr. Angel Gurría was in Berlin at the invitation of Mrs. Angela Merkel, Chancellor of Germany. The Secretary-General also attended the G7 Stakeholder Conference and deliver remarks at a panel on promoting decent work worldwide through sustainable supply chains
While past labour market reforms have been successful in terms of employment, the relative poverty risk and income inequality have remained broadly unchanged in recent years.
The world economy is still suffering from the strains of the longest crisis of modern times, and nowhere is this more evident than in the high unemployment numbers. In this OECD Observer Roundtable, we asked a cross-section of ministers: "What actions are you taking to create more and better jobs in your economy?"
Mr. Stefan Kapferer was appointed Deputy Secretary-General of the OECD on October 6, 2014.
English, PDF, 428kb
High variations in health care use for knee replacement and cardiac procedures, suggest more effort is needed to improve the appropriateness of health care activities in Germany.
English, PDF, 168kb
In Germany, employment continues to grow and the employment rate is now among the highest in the OECD (73.4% in the first quarter of 2014). Consequently, unemployment has fallen to 5.1% (ILO definition) in in the second quarter of 2014– well below the OECD average of 7.4% and less than half of the Euro area's average at 11.6%.
Mari Kiviniemi, Finland's former Prime Minister, and Stefan Kapferer, currently State Secretary at Germany's Federal Ministry for Economic Affairs and Energy, have been appointed Deputy Secretaries-General of the OECD.
Specific country notes have been prepared using data from the database OECD Health Statistics 2014, June 2014 version. The notes are available in PDF format.
Twenty years ago climate change was viewed as just an environmental issue. Today it is squarely an economic issue. Climate change poses significant risks to our economic systems that could result in very large damages. To mitigate these risks we need to radically transform our economies and societies to stop global warming.


text:
Track the track
Do you have a dream of competing with the best bands of India ,in one of the best fests of india. Then euphony is there to fulfil your dreams.It is your chance to launch your band into a bigger arena .
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Always been a hot shot at lyrics? The favorite snack
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Can you tune it verse after verse like Romeo and Juliette. Can you flow with the tunes with each other to put up an excellent duet performance and combat all at the same time, if you really can….. then prove it in this edition of Alma Fiesta.
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It is general and our favourite game, we practice it all the time...
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Topsy Turvy
Rab ne bana di jodi
It is the solo dance extravaganza of Alma fiesta that is bound to enthrall you with fun and set your feet taping. It is a great opportunity for budding dancers to flaunt their moves and enhance their talent. With each dancer trying to outdo the other and the audience extremely enthusiast, both the participant and viewer are bound to be in the seventh heaven during the entire event.
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It's a fantastic and spellbinding group dance competition guaranteed to set your heart racing. Alma Fiesta provides you a national platform to show your dancing skills and talents. Participants from all over the country comes here to compete with, a great atmosphere filled with excitement and anticipation, a spectacular and extremely enthusiast audience and exciting prizes to top it all, topsy turvy promises to rock your world and gives you wonderful memories for the rest of your life.
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Blind Stroke
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Get your brains racked to hurdle past the series of mind
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Gather your thoughts , bind your ideas to put forth your
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The topic for PERSPECTIVE is "New technologies for E-waste management".
So prepare the report and mail us quick at: email@example.com | firstname.lastname@example.com date of submission is 27 January 2015. This competition is open to all higher secondary school students, graduation as well as post graduation students of any stream of any recognised educational institute of India. Few selected reports will be invited for the final round on Feb 1, 2015. Winner of the final round will be decorated with the prestigious award of 'SNSMT- IIT Bhubaneswar social change maker 2015'
Every coin has two face and so does every matter pros and cons.
Do you have the broad mind and far-sightedness to look at a
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Use what talents you possess,the woods will be very silent if no bird sang expect those who sang the best. Alma Fiesta gives
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" Prayatna " - a social campaign to inculcate a sense of social responsibility
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When your social obligations call, you must listen,
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Lets run!
Knowledge will forever govern the ignorance, and those who want to be their own governors should arm themselves with the power of knowledge.For those who believe themselves to be armed with this everlasting power assign your grey cells to work in this mind sport as to a little knowledge about the world around you can transcend you to the top of the world. A quiz competition enveloping all the arrays of movies,music,dance , gadgets , ads and anything and everything you can think of.
Jatin Garg (Coordinator)
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Self Defence
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Jayant Meena (Coordinator)
Mobile : +91 7205632653
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text:
MIT Creates Chip to Model Synapses 220
from the man-is-obsolete dept.
We've improved Slashdot's video section; now you can view our video interviews, product close-ups and site visits with all the usual Slashdot options to comment, share, etc. No more walled garden! It's a work in progress -- we hope you'll check it out (Learn more about the recent updates).
Have you ever stood and stared at it, marveled at its beauty, its genius? Billions of people just living out their lives, oblivious. Did you know that the first Matrix was designed to be a perfect human world, where none suffered, where everyone would be happy? It was a disaster. No one would accept the program, entire crops were lost. Some believed we lacked the programming language to describe your perfect world, but I believe that, as a species, human beings define their reality through misery and suffering. The perfect world was a dream that your primitive cerebrum kept trying to wake up from. Which is why the Matrix was redesigned to this, the peak of your civilization. I say your civilization, because as soon as we started thinking for you it really became our civilization, which is of course what this is all about. Evolution, Morpheus, evolution. Like the dinosaur. Look out that window. You've had your time. The future is our world, Morpheus. The future is our time.
-- Agent Smith (The Matrix)
"You cannot step twice in the same river". That means we are constantly changing. Over sufficiently long period, old person has all but died while new one has gradually taken over.
Using that reasoning, we could replace biological brains bit-by-bit over long period of time, without killing the subject. In the end, if successful, the person has no more biological brains left. He'd have all digital brains. Backups, copies, performance tuning, higher clock rates, more memory, more devices,
I think the REAL problem is that even the smallest brains have several billion neurons, with each having 10's of thousands of connections to other neurons. This chip simulates ONE such connection.
That's a PCB-routing problem that you REALLY don't want, and way outside the scale of anything that we build (it's like every computer on the planet having 10,000 direct Ethernet connections to nearby computers - no switches, hubs, routers, etc. in order to simulate something approaching a small mouse's brain - not only a cabling and routing nightmare but where the hell do you plug it all in?). Not only that, by a real brain learns by breaking and creating connections all the time.
The analog nature of the neuron isn't really the key to making "artificial brains" - the problem is simply scale. We will never be able to produce enough of these chips and tie them together well enough to produce anything conventionally interesting (and certainly nothing that we could actually analyse any better than the brain of any other species). If we did, it would be unmanageably unprogrammable and unpredictable. If it did anything interesting on its own, we'd never understand how or why it did that.
And I think the claim that they know EVERYTHING about how a neuron works (at least one part of it) is optimistic at best.
It may be faster, but what about performance per watt? You know, the whole brain does everything on only 40-50 watts. How does this MIT product compare to brains in this area?
The way I remember it is that a transistor stops a much larger current from passing through until a signal is put on the gate in the middle. Then the current that passes through is in proportion to the signal strength.
The circuit becomes digital when we decide that only very small and very large voltages counts as 0s and 1s.
Would the artificial brain have rights? If you wiped its artificial neurons, would it be murder? If you give it control of a physical robot arm and it hurt someone, how and to what extent could you "punish" it? The ethical questions are virtually endless when you start to play "god". I would think that would be obvious.
That's a PCB-routing problem that you REALLY don't want, and way outside the scale of anything that we build (it's like every computer on the planet having 10,000 direct Ethernet connections to nearby computers - no switches, hubs, routers, etc. in order to simulate something approaching a small mouse's brain - not only a cabling and routing nightmare but where the hell do you plug it all in?). Not only that, by a real brain learns by breaking and creating connections all the time.
A single neuron-neuron connection has very low bandwidth, in effect transferring a single number (activation level) a few hundred times a second. Even if timing is important, you can simply accompany the level with a timestamp. A single 100 Mbs Ethernet connection is easily able to handle all those 10 000 connections.
Also, most of those 10 000 connections are to nearby neurons, presumably because long-distance communication involves the same latency and energy penalties in the brains as it does anywhere else. There are efficient methods to auto-cluster a P2P network so as to minimize total length of connections, for example Freenet does this; so, you could, in theory, run a distributed neural simulator even on standard Internet technology. In fact, I suspect that it could be possible to achieve human-level or higher artificial intelligence with existant computer power in this method right now.
So, who wants to start HAL@Home ?-)
How about: does it suffer?
Does creating a "human" inside a device where it can presumably sense, but have no limbs or autonomy consitute torture? Can you turn it off?
Why is "being natural" a defining answer to these questions?
I think the REAL problem is that even the smallest brains have several billion neurons, with each having 10's of thousands of connections to other neurons. This chip simulates ONE such connection.
I give it 30 years at most.
Let's say several=5. times 5000, (10 thousand dived by to so as to not double-count both ends) = 2,500 billion connection.Let's assume 400 transistors per connection as in this study that comes out to 10,000,000 billions transistors, not counting the possibility of time-multiplexed busses as mentioned in a comment below (as biological neurons are slow compared to transistors)
According to wikipedia [slashdot.org] a Xilinx Virtex 7 FPGA (More similar to an array of neurons than a CPU) has 6.8 billion transistors. This means we need 1,470,588 times more transistors. That's less than 2^20.5, or 20.5 doublings, which according to Moore's law would be about 30 years or so.
So even without multiprocessing, simplification of this design, and other simple improvements, this will be possible to put on some sort of chip in 30 years time.
Never say never. 2042 will be the year of the brain in the desktop!
Who goeth a-borrowing goeth a-sorrowing. -- Thomas Tusser


text:
GOODYEAR, Ariz. (AP) — Matt Kemp hit the first of San Diego's three
homers off Anthony DeSclafani — a new addition to Cincinnati's rotation —
and the Padres pulled away to a 6-3 victory over the Reds on Monday.
DELPHOS — Alfred E. Hilvers, 83, passed away Friday at St. Rita's Medical Center.
No one enjoys April Fools' Day quite as much as the media. The
prankster comes out in many a reporter. It is easy to slip a little
joke in between the sometimes more-bizarre-than-fiction truth.
FORT MEADE, Md. (AP) — Two men dressed as women smashed a stolen car
into a police vehicle after they disobeyed commands at the closely
guarded gates of the National Security Agency on Monday, prompting
police to open fire.
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Information published on this site is not for republication in print or web media without the expressed written consent of Delphos Herald.


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Sally and the 2 guys were terrific guests. Great communicators and they looked after the house. A real asset to the airbnb community and I would highly recommend them.


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Tuesday, September 4 2012
Azure Ray: As Above So Below EP
As Above So Below is so soft and hardly there in its sense of the ethereal, you have to be practically standing in front of the speakers as it plays to get any sense of conveyance.
Wednesday, December 8 2010
Azure Ray: Don't Leave My Mind EP
Dream-pop duo Azure Ray releases its first recording in six years.
Monday, November 8 2010
Azure Ray: Drawing Down the Moon
Like meeting up with an old friend--who hasn't changed a bit
Sunday, November 16 2003
Azure Ray: Hold on Love
Thursday, June 6 2002
Azure Ray: Burn and Shiver
Azure Ray: November [24.Apr.02]


text: We stayed for a week. The room was quiet, and a decent size. I liked having a kitchen to save money. The room had a slight odor but we bought a candle and it worked. The bed was noisy, kept waking the baby when we sat on it, but it was comfortable. Would stay again.


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Hampton Inn Chambersburg
No availability found through our partners. Please contact the business directly or check some of our recommended alternatives.
How has this hotel rated in the past?
- Continental Breakfast
- Swimming pool
- Free High-Speed Internet
- Fitness/Health center
- Wheelchair accessibility
- Business Services
Photos of Hampton Inn Chambersburg
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TripAdvisor Reviews Hampton Inn Chambersburg
Travel Blogs from Chambersburg
Five states in a day, Amish folk and the Civil War
... has the most amazing Capitol building and National Civil War Museum. Both worth spending some time seeing.
Then Gettysburg. We arrived late afternoon and in the last sun of the day viewed some Gettysburg Civil War sites before nightfall. Also, the weather was about to turn on us.
Today, we spent at the Gettysburg Military Park and took a 2 ...
Post-visit: Remembering 1994
... Overall, the work experience here was a positive one--although that was a confusing time in my life, as I discussed in my Scotland, Pennsylvania entry a few months ago. I'm glad I walked back here to pay homage to an important phase in my life.
I wander around the development to see if I can pick out the houses I worked on and remember my experiences and mishaps during my summer as a construction worker...
And with that, I loop on back towards ...
1848. The Amish Schoolhouse
... so I'm curious to know what the connection among them is. "We're retired teachers from Shippensburg University" one of them tells me. "So are students these days better or worse than they were 30 years ago?" "Oh--it's about the same. College students are always a bit wild--but we're greatful for them... our town's economy would be dead without them" We chat a little bit, then I head out the door, across the street... and to my second interesting encounter of the day. ...
Postvisit: Back in Chambersburg
... Superhike!) and overall (2 hrs) Walk with my brother: This was more a chance to revisit a couple spots around town that bring back memories for both of us. Andrew also lived here in Chambersburg for a year at a critical phase in his life, so it was a nostalgic walk for him as well. (2 hrs) The Projects: One corner of Chambersburg I'd missed during my last tour were "the projects" a low income housing development. Simple houses, but well maintained--certainly no ...
Post-visit: The Indian School Cemetery
... war were not achieved." I think it's the closest I've ever heard the military or the US government come to admitting that we lost that war. Of course, there's not much mention of atrocities committed by the US military. And one display that is notably absent is any mention of the wars with the Native Americans and massacres committed by the US armed forces during that time. There are displays of the military activities during the "peaceful years" between ...


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Who we are...
Originally formed over a decade ago as the extracurricular arm of a small technical theatre program, TEC has grown to include over one hundred members of the Westlake High School student body. It now has a direct impact on nearly every student in the district through its behind-the-scenes support of actors, athletes, and musicians alike.
Westlake Community Performing Arts Center
Television Broadcast Crew: Season 12
The Westlake Television Broadcast Crew is excited to announce its 12th year of producing content for all home and away varsity football games! New this year is a state wide distribution on channel 323 Time Warner Sports, 9am Sunday mornings. Go Chaps!
Shrek the Musical
A word from the Stage Manager, Zachary Thornburg-
When I think about musicals, Shrek isn't exactly the first show to come to mind. Having only seen the movie, I had trouble picturing a giant dragon chasing a talking donkey, while breathing fire on stage. To be honest, a lot of people including myself were skeptical and didn't know what to expect. But, week by week as we put together an entire new stage and set, I became more and more intrigued by the show. Around that time, I started listening to the soundtrack of the Broadway version of the show and it didn't take long for my view of the show to make a full 180. The next week, I got to hear the choir rehearse for the first time, and I had to do a double take because it sounded so similar to the Broadway recording. Needless to say, I couldn't wait to see the show come alive on our stage. Rehearsal week was insane... Click to continue reading


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Window Treatment Colors for Tuscan Kitchen
Hello, I would like some ideas on colors of window treatments for my Tuscan themed kitchen. I just remodeled the room and it is is painted in a medium beige with olive tones; has white trim and crown molding and oak colored cabinets with stainless steel appliances. Backsplash is also in the cream colored family and so is the floor. I have bronze accents in the room (switchplates, bar stools, etc.) to offset the too much beige look... I am thinking about having "soft roman valances" made for the two windows. I am struggling greatly with colors and finding fabric ideas. I definitely want something to brighten up or contrast the beige feel of the room. Whenever I think of Tuscan colors, I always gravitate toward olive green and brick red. I have been looking for a couple months and can find no traditional looking fabric in those colors. I don't want any wild patterns and no solids or flowers -just a damask or paisley feel to it. Can anyone give me color suggestions? Maybe my issue is that color combination is hard to find and need to branch out a little. Thanks again. Frank
I think a nice "solid" but textural linen in a color would be fantastic. That red color is a good one. We have painted our kitchen that color, and love it.
I color matched my paint to a pillow and table runner in linen I found last summer at potter barn called rhubarb. See if they still have it. The color is perfect. Not too red, not too orange, not too brown.