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Currently, only symptomatic therapy is available for Parkinson's disease. The zebrafish is a vertebrate animal model ideally suited for high throughput compound screening to identify disease-modifying compounds for Parkinson's disease. We have developed a zebrafish model for Parkin deficiency, the most commonly mutated gene in early onset Parkinson's disease. The zebrafish Parkin protein is 62% identical to its human counterpart with 78% identity in functionally relevant regions. The parkin gene is expressed throughout zebrafish development and ubiquitously in adult zebrafish tissue. Abrogation of Parkin activity leads to a significant decrease in the number of ascending dopaminergic neurons in the posterior tuberculum (homologous to the substantia nigra in humans), an effect enhanced by exposure to MPP+. Both light microscopic analysis and staining with the pan-neuronal marker HuC confirmed that this loss of dopaminergic neurons is not due to general impairment of brain development. Neither serotonergic nor motor neurons were affected, further emphasizing that the effect of parkin knockdown appears to be specific for dopaminergic neurons. Notably, parkin knockdown zebrafish embryos also develop specific reduction in the activity of the mitochondrial respiratory chain complex I, making this the first vertebrate model to share both important pathogenic mechanisms (i.e. complex I deficiency) and the pathological hallmark (i.e. dopaminergic cell loss) with human parkin-mutant patients. The zebrafish model is thus ideally suited for future drug screens and other studies investigating the functional mechanisms underlying neuronal cell death in early onset Parkinson's Disease. Additional electron microscopy studies revealed electron dense material in the t-tubules within the muscle tissue of parkin knockdown zebrafish. T-tubules are rich in L-type calcium channels, therefore our work might also provide a tentative link between genetically determined early onset Parkinson's disease and recent studies attributing an important role to these L-type calcium channels in late onset sporadic Parkinson's disease.
Autosomal recessively inherited, homozygous or compound heterozygous mutations in the parkin gene on chromosome 6q25–27 are the most common identifiable genetic cause for early onset parkinsonism (Kitada et al., 1998). There is also an ongoing debate whether a single heterozygote parkin mutation may be a susceptibility factor for late onset Parkinson's disease (Kay et al., 2007; Klein et al., 2007; Langston et al., 2007). The parkin gene (PARK2) encodes a ubiquitin E3 ligase (Shimura et al., 2000). Impaired mitochondrial function is currently seen as an important mechanism leading to neuronal cell loss in both genetically determined forms of early onset Parkinson's Disease and late onset, apparently sporadic Parkinson's disease (Schapira, 2008). Data on mitochondrial function in post-mortem brain tissue of patients with two parkin mutations are not available yet, but we and others have identified isolated complex I deficiency in peripheral tissue of parkin-mutant patients (Muftuoglu et al., 2004; Mortiboys et al., 2008). The results from the different post-mortem studies of parkin-mutant brains are somewhat variable, but share the core histopathological feature of neuronal cell death in the ascending nigrostriatal dopaminergic system with sporadic Parkinson's disease (Cookson et al., 2008).
Zebrafish (Danio rerio) are vertebrates and therefore more closely related to humans than other model organisms such as Drosophila or Caenorhabditis elegans. They have a short generation time (3 months) and breed prodigiously (hundreds of offspring per female per week). Embryos develop externally, can readily be manipulated genetically and are transparent. Micro-injection of morpholino (MO) antisense oligonucleotides into zebrafish embryos at the single cell stage can be used to suppress the translation of a particular gene transiently (Ekker and Larson, 2001). The dopaminergic (DA) system is well characterized in both embryos and the adult zebrafish. Dopaminergic neurons are first detected at ∼19-h post-fertilization (hpf) in a cluster of cells in the posterior tuberculum of the ventral diencephalon. These neurons represent the dopaminergic system ascending to the striatum, comparable to the nigrostriatal system in humans (Holzschuh et al., 2001; Rink and Wullimann, 2001, 2002b). Zebrafish homologues with typically widespread expression pattern already at early stages of embryonic development have been identified for several firmly established Parkinson's disease genes, namely alpha-synuclein, PINK1 and DJ-1 (Bretaud et al., 2007; Anichtchik et al., 2008; Flinn et al., 2008; Sun and Gilter, 2008).
We have identified a zebrafish orthologue of the human parkin gene, analysed its expression and used the MO antisense strategy to determine the effect of Parkin deficiency in zebrafish embryos. parkin knockdown (k/d) results in impaired mitochondrial function without concomitant abnormal mitochondrial morphology. parkin knockdown also leads to selective loss of dopaminergic neurons with increased susceptibility to the Parkinson's disease neurotoxin 1-methyl-4-phenylpyridinium (MPP+, the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP) without impairment of overall brain development and sparing of other neuronal sub-populations such as motor neurons which are also unaffected in human Parkinson's disease patients. Intriguingly, EM analysis of muscle tissue in parkin knockdown zebrafish suggests protein aggregation in transverse (t)-tubules. T-tubules are invaginations of the surface membrane which are of crucial relevance for the calcium (Ca2+) homeostasis of the muscle tissue (Brette and Orchard, 2007). Of note, they are particularly rich in L-type Ca2+ channels. Our data therefore suggest a tentative link between mechanisms leading to early onset Parkinson's Disease and impaired Ca channel function which has recently been implicated in late onset, sporadic Parkinson's disease (Chan et al., 2007).
Materials and Methods
Fish strains and maintenance
The London wild-type (LWT) strain of zebrafish was used for all experiments. Embryos were collected after natural spawning, staged according to standard criteria, and synchronously raised at 28.5°C. All zebrafish husbandry and experimental procedures were performed in accordance with the UK. Animals (Scientific Procedures) Act.
Isolation and sequencing of zebrafish parkin
Human Parkin protein sequence (accession number NP_004553) was blasted to the zebrafish genome using Ensembl (http://www.ensembl.org/Danio_rerio/blastview). The conceptual translation product of a single annotated transcript (ENSDARG00000021555, zgc:112390; accession number NP_001017635) was identified as the top match. The predicted zebrafish protein sequence was aligned with the human protein using ClustalX (www.clustal.org), and the predicted transcript aligned with the zebrafish genome in order to determine exon–intron boundaries and splice site locations. To confirm the in silico data, the coding sequence of parkin was amplified from reverse-transcribed zebrafish cDNA, using overlapping parkin-specific primer sets and experimental sequences aligned with in silico predictions.
RT–PCR analysis of parkin mRNA expression
Total RNA was extracted from whole embryos at different stages of development and from various adult zebrafish organs, using Trizol reagent (Invitrogen, Paisley, UK). Up to 3 μg of total RNA per sample was used to synthesize first-strand cDNA using reverse transcriptase (Superscript II kit; Invitrogen). The resulting cDNA (2 μl) was amplified using primers for parkin, 5′-GCGAGTGTGTCTGAGCTGAA-3′ (forward) and 5′-CACACTGGAACACCAGCACT-3′ (reverse); for β-actin, 5′-AAGCAGGAGTACGA-TGAGTCTG-3′ (forward) and 5′-GGTAAACGCTTCTGGAATGAC-3′ (reverse) were used as a positive control to confirm consistent quality and quantity of cDNA.
MO-mediated knockdown of Parkin
Anti-sense oligonucleotides (MO, GeneTools LLC, Philomath, OR, USA) were designed by GeneTools to target the splice donor site of exon 9 and splice acceptor site of exon 10 of the zebrafish parkin transcript. The MO sequences were: e9i9 5′-TGATTTGGTTCTCTTACCCCACAGC-3′ and i9e10 5′-ACACAAACTGACA-CAAACAGCAAAT-3′. Control MOs with five mismatches were used in order to distinguish phenotypic effects specific to the knockdown of parkin from the effects of non-specific MO toxicity. MOs were re-suspended in sterile water at 2 mM stock concentration. Immediately prior to injection, e9i9 and i9e10 MOs were diluted to 0.75 mM and 1% phenol red (Sigma, Poole, UK) added to monitor injection efficiency. MOs were injected into the yolk of one- to two-cell stage embryos. Both parkin and control MOs were injected at 1.5 μM final concentration. To confirm efficacy of the splice-blocking MOs, 2 μl of cDNA were used in PCR amplifications using primers 5′-ATCGCCTGCACAGACATCAT-3′ (forward) and 5′-GAGCCGCTTCTTCATCTACAA-3′, spanning exons 6–11 of the parkin mRNA.
Mitochondrial respiratory chain function assays
Embryos harvested at 72 hpf (15–30 per sample) were homogenized in 200 μl buffer (250 mM sucrose, 20 mM HEPES, 3 mM EDTA, pH 7.5), then stored at −80°C until ready for use. All spectrophotometric assays were performed at 30°C in 200 µl final volume using a Fluostar Omega spectrophotometer/plate reader, samples were diluted to 0.7 mg protein/ml and subjected to three cycles of liquid N2 freeze thawing. Complex I activity was determined by following the oxidation of nicotinamide adenine dinucleotide reduced disodium salt (NADH) at 340 nm with a reference wavelength of 425 nm (ε = 6.22 mM−1 cm−1). The assay buffer contained 25 mM KH2PO4, 5 mM MgCl2, (pH 7.2), 3 mM KCN, 2.5 mg/ml BSA, 50 µM ubiquinone Q1, 2 µg/ml antimycin A and zebrafish homogenate, the reaction was started with 125 µM NADH and inhibited with 3 µg/ml rotenone. Complex II activity was measured by following the reduction of 2,6-dichlorobenzenone-indophenol sodium salt (2,6-DCPIP) at 600 nm (ε = 19.2 mM−1 cm−1). The assay buffer contained 25 mM KH2PO4, 5 mM MgCl2, (pH 7.2), 3 mM KCN, 20 mM succinate and zebrafish homogenate and was incubated for 10 min before the addition of 50 µM 2,6-DCPIP, 2 µg/ml antimycin A and 3 µg/ml rotenone, the reaction was started with 50 µM ubiquinone Q1. The specific activity was normalized to that of citrate synthase.
Complex III was measured by following the reduction of cytochrome c at 550 nm with a reference wavelength of 580. The assay buffer contained 25 mM potassium phosphate, 5 mM MgCl2, 3 mM KCN, 2.5 mg per ml BSA, 15 µM cytochrome c, 3 µg/ml rotenone and 50 µM ubiquinol-2. The non-enzymatic reduction of cytochrome c by ubiquinol-2 was measured for 1 min, after which x µl zebrafish homogenate was added and the reaction followed for a further 4 min, finally a few grains of ascorbate were added to maximally reduce the cytochrome c. The rates were calculated as a first order constant and the results are expressed as K/min/citrate synthase activity. Complex IV activity was measured by following the oxidation of reduced cytochrome c at 550 nm, with a reference wavelength of 580 nm. The assay buffer contained 20 mM potassium phosphate, (pH 7.2), 150 µg/ml n-dodecyl-β-d-maltoside, 15 µM reduced cytochrome c and mitochondrial extract. The reaction was followed for 3 min after which time a few grains of potassium ferricyanide were added to fully oxidize the cytochrome c. The rates were calculated as a first order constant and the results are expressed as K/min/citrate synthase activity.
Toxin exposure using MPP+
WT (uninjected), control MO-injected and parkin knockdown embryos were raised for 48 hpf before exposure to toxins. At 24 hpf, embryos were transferred in groups of 20 to 12-well plates containing E3 embryo media. At 48 hpf, media was replaced in the treatment groups with 3 mM MPP+ (Sigma, Poole, UK) in E3 embryo media. Propylthiouracil (Sigma) was used at 0.2 mM concentration to prevent the development of pigmentation in all experiments other than the behavioural analysis. Embryos were harvested at 72 hpf, fixed overnight at 4°C in 4% paraformaldehyde, then washed into 100% methanol and stored at −20°C. All experiments were undertaken in triplicate.
In situ hybridization for quantification of dopaminergic neurons
Whole-mount in situ hybridization was undertaken as previously described (Thisse and Thisse, 2008). Briefly, a digoxigenin-labelled antisense RNA probe was synthesized from pBS-tyrosine hydroxylase (TH) plasmid linearized by XhoI and transcribed using T3 RNA polymerase (Invitrogen, Paisley, UK). Embryos were fixed at 3-day post-fertilization (dpf) overnight in 4% paraformaldehyde in phosphate-buffered saline (PBS), then transferred to 100% methanol for storage at −20°C for at least 24 h before undergoing hybridization. After the hybridization procedure was performed as described (Thisse and Thisse, 2008), embryos were washed extensively in 0.1% Tween 20 in PBS, re-fixed in 4% paraformaldehyde in PBS, then transferred to 80% glycerol. Embryos were mounted in glycerol and TH-positive diencephalic neurons counted under a Zeiss microscope. In order to avoid introducing unintended bias, all embryos were coded and scored blind. The results are expressed as percentage of neurons present in WT embryos (uninjected), which were staged and treated in parallel with parkin knockdown embryos.
Combined in situ hybridization and antibody immunofluorescence
Digoxigenin-labelled antisense RNA probe was synthesized from pCRIItopo-aromatic amino acid decarboxylase (AADC) plasmid, linearized by XhoI and transcribed using Sp6 RNA polymerase (Invitrogen). A modified in situ hybridization protocol was performed in which the anti-digoxigenin-POD antibody (Roche) was co-incubated with anti-tyrosine hydroxylase (TH) primary antibody (Millipore, UK) at 1:1000 final concentration overnight at 4°C. Embryos were extensively washed, stained with TSA-Cy3 (Perkin Elmer, Beaconsfield, UK) for AADC detection, again washed and then incubated with goat anti-rabbit AlexaFluor 488 antibody (Invitrogen, 1:500) for detection of TH. Whole-mount embryos were imaged using an Olympus laser scanning confocal microscope. Z-stack images were generated by scanning through the brain using a 20× objective at an optimal image slice distance. All Z-stack images of control and parkin knockdown embryos were captured using identical settings, determined empirically for optimal imaging of control MO embryos.
Whole-mount antibody immunofluorescence
For whole-mount antibody immunofluorescence detection of neuronal HuC/HuD proteins, embryos at 30 hpf were manually dechorionated, fixed at 4°C in 4% paraformaldehyde in PBS overnight, then stored at −20°C in 100% methanol. Embryos were serially washed into water, acetone shocked for 7 min at −20°C, then blocked for 2 h in 1% bovine serum albumin, 2% sheep serum in PBS containing 1% DMSO, 0.1% Tween 20 and 0.5% Triton X-100 (PBDTT). After blocking, embryos were incubated overnight at 4°C in blocking solution containing monoclonal 16A11 anti-Huc/D (Invitrogen) at 1:500 final concentration, followed by washing several times with PBDTT and incubation with secondary antibody (goat anti-mouse AlexaFluor 488, Invitrogen). Whole-mount embryos were imaged on an Olympus laser scanning confocal microscope as described. For visualization of Islet-1 positive motor neurons, primary anti-Islet1 antibodies (Developmental Studies Hybridoma Bank, IA, USA) were used in the same procedure (Higashijima et al., 2000).
Behaviour analysis of parkin knockdown embryos
Embryos were tested for behavioural consequences of parkin knockdown by measuring swimming behaviour at 5 dpf. WT and parkin knockdown embryos were raised in E3 and at 5 dpf screened for inflated swim bladder formation. Fully developed larvae were arrayed 1 per well in six-well plates containing 4.5 ml of E3 and placed on a light stage with both a transmitted and infrared light source. The larvae were allowed to acclimate to the dish for 5 min, and then recorded for 5 min using Ethovision Pro digital video recording to track movement (Tracksys, Nottingham, UK). All digital tracks were analysed for total distance moved, using an input filter of 0.06 cm as the minimum distance to be considered 'movement'. Each experiment was performed in triplicate, and the results expressed as mean total movement ± SEM.
Electron microscopic analysis
Larvae injected with parkin MO and WT larvae were fixed in 1× PBS containing 2% paraformaldehyde and 2% glutaraldehyde at 5 dpf. Embedding, sectioning, and image acquisition were undertaken as previously described (Rieger and Koester, 2007).
Statistical analysis
All experiments were undertaken in triplicate, each with n = 10 per treatment group. Data represent mean ± SEM. Each treatment group was normalized to the appropriate WT, uninjected control group and results expressed as percentage of WT mean. One-way ANOVA and t-tests of significance were used when applicable as measures of significance (GraphPad Prism software).
The zebrafish parkin orthologue is highly conserved
BLAST searching of the zebrafish genome identified a single gene homologous to human parkin, ENSDARG00000021555, on chromosome 13. This 1 455 base transcript encodes a peptide sequence of 458 amino acids, of which 62% are identical and 75% similar to the sequence of the human gene. Within the E3 ubiquitin ligase domain (residues 1–76 in human parkin protein sequence), the human and zebrafish amino acid sequences display 61% identity and 78% similarity, respectively. In the functionally relevant RING domain regions (residues 238–294 and 418–450), the amino acid identity rises to 75–78% identity and 89–93% similarity. We confirmed the in silico results by amplifying reverse-transcribed zebrafish cDNA using parkin-specific primers and then sequencing the PCR products. Overlapping sequences were aligned and the consensus compared to in silico annotation. The genomic structure of zebrafish parkin is identical to the human gene, with a total of 12 translated exons. The sequence data we obtained from RT-PCR largely confirmed the annotated Ensembl sequence, with the exception of several single nucleotide polymorphisms (SNPs). Most of these single nucleotide polymorphisms were synonymous at the amino acid level (SNPs at positions 12, 27, 63, 69, 162, 210, 787, 1119, 1324 if the A in the ATG is +1). The only discrepancy between the in silico data and our own sequencing data resulting in a change of the protein sequence was a G substitution at position +112 in the highly conserved E3 ubiquitin ligase (UBL) domain, resulting in an alanine residue at codon 38. In contrast, the published Ensembl sequence at this position is a T, which would encode a serine. Of note, the corresponding residue in the human Parkin protein sequence is also an alanine.
Parkin is expressed throughout zebrafish embryonic development and in adult tissues
We used RT–PCR to characterize the expression pattern of parkin in zebrafish embryos at various stages and in different adult tissues. parkin mRNA was detectable throughout embryogenesis, from ∼5 hpf until 5 dpf (Fig. 1A). Similarly, parkin is expressed ubiquitously in adult zebrafish, with variable levels of transcript detectable in all organs tested (Fig. 1B).
MO targeting of exon splice sites partially inactivates parkin in zebrafish embryos
Injection of the MOs e9i9 and i9e10 (see Materials and Methods section) resulted in complete (1 dpf) or partial (3 dpf) abolishment of full-length WT PCR product (Fig. 1C). Sequencing of the smaller band on the PCR gel revealed the primary product was indeed missing MO-targeted exon 9 from the mRNA, which results in deletion of 51 amino acids from the in-between ring (IBR) domain of Parkin. The amount of this product was greatly reduced in the knockdown embryos as compared to the WT, uninjected mRNA extracts, suggesting that nonsense-mediated decay may also affect the amount of mature parkin mRNA in MO-injected embryos.
Complex I activity is reduced in parkin knockdown embryos
We next measured mitochondrial respiratory chain complex I activity to determine whether this abnormality of energy metabolism, currently considered to be of crucial relevance in the pathogenesis of human parkin-linked Parkinson's disease, is also manifest in parkin knockdown zebrafish embryos. Spectrophotometric assessment of complex I activity was lower by an average of 45% in parkin knockdown zebrafish compared to either WT embryos or control MO embryos (Fig. 2; P < 0.05). In contrast, there was no significant difference in complex II activity in parkin knockdown embryos relative to both WT and control MO embryos (mean ± SD, WT 0.26 ± 0.1, control MO 0.31 ± 0.09, parkin MO 0.19 ± 0.03). Likewise measurement of complexes III and IV showed no significant difference in activity of either complex in the parkin knockdown embryos relative to both WT and control MO embryos, although there was a trend towards a reduction in complex IV activity (mean ± SD, Complex III: WT 85.75 ± 19.7, control MO 67.62 ± 16.4, parkin MO 74.7 ± 5.7, Complex IV: WT 9.32 ± 3.7, control MO 9.55 ± 3.6, parkin MO 5.87 ± 1.5). These findings are thus in good agreement with the current view that it is complex I which is specifically affected in parkin-related Parkinson's disease as well as in the more common, sporadic form of Parkinson's disease.
Knockdown of zebrafish parkin results in dopaminergic neuronal cell loss and increased susceptibility to MPP+
Using in situ hybridization with an antisense RNA probe for TH, we investigated the effect of parkin knockdown on the number of diencephalic dopaminergic neurons. Dopaminergic neurons ascending to the striatum (populations 1, 2, 4 and 5) according to terminology developed by Rink and Wullimann (2002a) were counted in WT uninjected embryos, control MO-injected embryos and parkin MO-injected embryos at 3 dpf. We calculated the mean number of these diencephalic dopaminergic neurons for WT embryos in each of three experiments and used this mean as the standard number of dopaminergic neurons, which was set to 100% in order to normalize the data. The number of ascending dopaminergic neurons present in each treatment group was then calculated as the percentage of the number of these dopaminergic neurons in WT zebrafish. parkin knockdown embryos showed a significant reduction in the number of diencephalic dopaminergic neurons by ∼20% (P < 0.01) already at 3 dpf. In contrast, the number of dopaminergic neurons in control MO-injected embryos did not differ significantly from WT uninjected embryos (Fig. 3A).
Knockdown of zebrafish parkin results in increased susceptibility to MPP+
We next determined whether parkin knockdown results in an increased susceptibility to the mitochondrial neurotoxin MPP+. Treatment of parkin knockdown zebrafish embryos with MPP+ resulted in a significantly greater reduction in the number of diencephalic dopaminergic ascending neurons by ∼50% (P < 0.001), suggesting a synergistic effect of parkin knockdown and MPP+ treatment (Fig. 3B). In contrast, MPP+ exposure of WT and control MO-injected zebrafish embryos only resulted in a reduction by ∼25% (P < 0.01).
The effect of parkin knockdown on dopaminergic neurons is specific
We next undertook double-staining with a TH antibody and an in situ probe for aromatic amino acid decarboxylase (AADC). This allowed us to discriminate between catecholaminergic and serotonergic neurons, respectively. While both neuronal sub-populations express AADC (red in Fig. 4), only catecholaminergic neurons express TH (green in Fig. 4) as well. Co-expression of both markers (yellow in Fig. 4) is thus specific for catecholaminergic neurons such as the ascending diencephalic dopaminergic neurons (Fig. 4A). These co-expression studies confirmed that the marked reduction of diencephalic neurons in parkin knockdown zebrafish embryos was specific for these dopaminergic neurons and further supported our assumption that the TH in situ based cell counting results were not simply reflecting down-regulation of TH transcription. In contrast, the (red) serotonergic neurons of the Raphe nuclei appeared similar in WT and parkin knockdown zebrafish embryos.
To exclude the possibility that the effect we observed was due to generalized impairment of brain development (either due to a specific effect of parkin knockdown or due to a non-specific toxic effect of the MO injections) rather than being specific to dopaminergic neurons we next undertook staining with anti-HuC/D antibody, an early pan-neuronal marker for post-mitotic neurons (Park et al., 2000). The HuC staining resulted in a similar staining pattern and intensity in WT uninjected and parkin knockdown embryos at 30-hpf (Fig. 4B). To assess further the specificity of the parkin knockdown effect we also undertook staining of spinal motor neurons, using anti-Islet 1 antibodies. The staining pattern was identical in WT uninjected and parkin knockdown embryos at 30 hpf (Fig. 4C). Overall, our morphological studies suggest that parkin knockdown has a largely selective effect on the number of ascending dopaminergic neurons rather than leading to gross morphological changes or widespread neuronal cell loss within the zebrafish brain.
Parkin deficiency does not alter larval swimming behaviour
Because of the locomotor defects observed in Parkinson's disease patients, we next tested whether parkin knockdown results in changed swimming behaviour of 5 dpf larvae. We compared spontaneous swimming behaviour at 5 dpf in WT uninjected, control MO-injected, and parkin MO-injected larvae. The mean distance moved over three experiments for WT, control MO and parkin MO-injected larvae did not differ significantly (Fig. 5), suggesting no obvious differences in swimming ability between groups. We noted a trend towards reduced total distance moved in both control MO and parkin MO-injected larvae, but this result was not significant (P > 0.05).
Electron microscopy studies reveal morphologically normal mitochondria, but electron-dense material in the t-tubules in parkin knockdown zebrafish
Drosophila parkin mutants develop apoptotic muscle necrosis and swollen mitochondria with broken cristae (Greene et al., 2003). In contrast, both muscle tissue as such and the mitochondria in particular are morphologically normal in parkin knockout mice (Palacino et al., 2004). We therefore undertook TEM of fast muscle cells in the somites of the zebrafish trunk to determine whether abnormalities similar to those found in Drosophila are manifest in the absence of Parkin function. TEM did not reveal any obvious morphological abnormalities of the mitochondria in parkin knockdown zebrafish embryos. In particular, the mitochondria were not swollen and the cristae remained intact. Furthermore, there was no evidence for apoptotic muscle necrosis (Fig. 6A–D). Surprisingly, however, electron-dense material was observed in the t-tubules of the parkin MO-injected zebrafish (Fig. 7A–F). These invaginating tubules allow for fast depolarization of the interior of the cell and may thus play a role in Ca2+ mediated muscle contraction. In initial swimming tests parkin MO-injected larvae did not display obvious locomotive defects (see above). As the temporal activity of the MO is limited though this t-tubule alterations may be tolerated at larval stages but could become more severe in mutants with permanent Parkin depletion.
We have developed a new vertebrate model to study the biochemical and morphological consequences of Parkin deficiency using MO-mediated knockdown of the zebrafish orthologue of the parkin gene. Comparison with embryos injected with a mismatch MO give us high confidence that the defects we observe are attributable specifically to the loss of Parkin activity rather than being caused by non-specific off target effects of the MO antisense oligonucleotide.
parkin knockdown zebrafish embryos share a lower activity of the mitochondrial respiratory chain complex I with parkin-mutant human patients (Muftuoglu et al., 2004; Mortiboys et al., 2008). Mitochondrial impairment is currently considered to be of crucial importance in the pathogenesis not only of parkin-related Parkinson's disease, but also in sporadic Parkinson's disease (Schapira, 2008). Furthermore, there is some evidence that Parkin itself may also be involved in the pathogenesis of sporadic Parkinson's disease: Parkin is clearly present in Lewy bodies and its transcription is upregulated in the substantia nigra of patients with sporadic Parkinson's disease (Schlossmacher et al., 2002; Moran et al., 2007). Parkin protects against the toxicity associated with mutant alpha-synuclein in vitro and in vivo (Lo Bianco et al., 2002; Petrucelli et al., 2002).
Post-mortem tissue of parkin-mutant patients has not as yet been assessed for morphological abnormalities of the mitochondria (Cookson et al., 2008). The normal shape and integrity of mitochondrial cristae in the parkin knockdown zebrafish embryos that manifest impaired mitochondrial function could indicate that the latter leads to impaired mitochondrial morphology, but further studies are needed to clarify this issue. The MO antisense approach only causes transient loss of Parkin function. Other strategies such as targeted gene mutagenesis using zinc finger nucleases could be used to develop stable parkin knockout zebrafish lines which would then permit serial assessment of mitochondrial morphology at different ages (Woods and Schier, 2008). Similarly, t-tubule morphology and function could be assessed at different ages.
Morphological analysis revealed selective loss of dopaminergic neurons in parkin knockdown zebrafish embryos, a finding that contrasts both with the more widespread defects observed in parkin knockout Drosophila, and also with the absence of any robust morphological changes in parkin knockout mice (Greene et al., 2003; Perez and Palmiter, 2005). Such selective dopaminergic neuronal cell loss together with lower complex I activity mirrors the human disease better than any other currently available animal model for parkin disease. The future development of stable parkin knockout zebrafish lines (see above) will help to determine whether the observed loss of dopaminergic neurons is progressive. The increased susceptibility to MPP+ observed in our parkin knockdown zebrafish model is in keeping with data from in vitro studies, but contrasts with the lack of increased susceptibility to this neurotoxin in parkin knockout mice (Hyun et al., 2002; Thomas et al., 2007). We had previously demonstrated increased susceptibility to a different mitochondrial toxin, rotenone, in parkin-mutant patient tissue (Mortiboys et al., 2008).
In Drosophila, Parkin and PINK1 appear to act in the same mitochondrial pathway, resulting in the same phenotype when either (or both) of these genes is mutated (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). PINK1 knockdown in zebrafish results in a severe developmental phenotype (Anichtchik et al., 2008). In contrast, our morphological studies, in particular the normal staining pattern with the pan-neuronal marker HuC and the motor-neuron specific marker Islet-1, suggest a considerably more selective effect of parkin knockdown than PINK1 knockdown. The MOs used in our study resulted in a 51 amino acid deletion within the in-between ring domain of parkin. The in-between ring domain augments binding of E2 proteins and the subsequent ubiquination of proteins such as synphilin-1 (Beasley et al., 2007). The resulting partially deleted Parkin protein in our study may thus have decreased enzymatic activity as a ubiquitin 3 ligase, but could nevertheless have some remaining functional activity since other crucial parts of the protein such as the ubiquitin ligase (UBL) domain and the two RING domains remained intact. In contrast, MOs directed against the ATG translation initiation site were used in the zebrafish PINK1 knockdown studies (Anichtchik et al., 2008). This might have led to a more complete suppression of PINK1 function. It is nevertheless difficult to understand why PINK1 deficiency results in such marked impairment of gross brain development in zebrafish embryos, but does not result in any morphological abnormalities in PINK1 knockout mice (Zhou et al., 2007; Gautier et al., 2008). Post-mortem studies have not as yet been undertaken in human patients with PINK1 mutations, but the clinical phenotype is obviously only suggestive of regionally selective neuronal cell loss rather than gross brain dysmorphology. Both the PINK1- and our parkin knockdown zebrafish studies nevertheless suggest that the encoded proteins may have a crucial role for the development and/or survival of neurons at early developmental stages. This may appear counter-intuitive for an adult-onset neurodegenerative condition, but is in keeping with recent observations describing abnormalities in key developmental mechanisms, namely microRNA signalling, in late-onset sporadic Parkinson's disease (Kim et al., 2007).
parkin knockdown zebrafish embryos did not display an overt behavioural phenotype. It is currently thought that human patients have to lose at least two thirds of their dopaminergic neurons in the substantia nigra before they develop any motor symptoms of Parkinson's disease. The loss of dopaminergic neurons observed in our model system (∼25%) may therefore not have been sufficient enough to result in a motor phenotype. Complete and longer lasting loss of Parkin function in stable parkin knockout lines will reveal whether dopaminergic neuron loss becomes more pronounced both during embryonic development and at later stages which may in turn lead to a more marked behavioural phenotype.
The observation of electron-dense material in the t-tubules of the parkin knockdown zebrafish embryos was a serendipitous finding. T-tubules are rich in L-type Ca2+ channels (Brette and Orchard, 2007). Dopaminergic neurons in the substantia nigra pars compacta (SNpc) rely on L-type Ca2+ channels for pacemaking with a much larger Ca2+ influx in these neurons than in other cells (Surmeier, 2007). This increased Ca2+ load of the dopaminergic neurons could in turn lead to a considerably higher energy demand to transport Ca2+ back out of the cells, consuming ATP produced by oxidative phosphorylation in the mitochondria. Future studies are needed to determine whether the observed impairment of mitochondrial function in parkin-linked Parkinson's disease contributes to neuronal cell death not only due to its effect on overall energy production, but also because of the important role of mitochondria in the maintenance of intracellular Ca2+ homeostasis (Szabadkai and Duchen, 2008).
Zebrafish embryos are highly amenable to high throughput drug screening and might therefore be a useful new tool to work towards the identification of disease-modifying therapy for Parkinson's disease. We have already provided proof of principle data in our DJ-1 knockdown studies that zebrafish embryos can be a useful tool to investigate compounds for their putative neuroprotective effect (Bretaud et al., 2007). Similarly, we will now be able to use parkin knockdown zebrafish embryos to rapidly assess promising substances in vivo in this new vertebrate animal model of Parkin deficiency. The recent development of an enhancer trap transgenic zebrafish line, ETvmat2:GFP, with green fluorescent monoaminergic neurons will further facilitate the rapid assessment of drugs for a putative neuroprotective effect on dopaminergic neurons in high throughput compound screens (Wen et al., 2008). It has been suggested that small molecules should be tested for their ability to induce parkin transcription to identify disease-modifying treatment for Parkinson's disease (Butcher, 2005). Genetically modified zebrafish embryos may also be an ideal tool to undertake such studies.
Parkinson's Disease Society (PDS, G-0608); Research into Ageing (RiA, 282 to O.B. and P.W.I.); Medical Research Council (MRC Centre Grant to P.W.I.); Bundesministerium fuer Bildung und Forschung (BMBF, BioFuture Grant 0311889 to K.V. and R.W.K.); Wellcome Trust (GR077544AIA) for the University of Sheffield Light Microscopy Facility.
We would like to acknowledge the contribution of Dr S. Bretaud to the early stages of this project. We would also like to thank the aquarium staff at CDBG for zebrafish maintenance and Luise Jennen for excellent help with TEM recording.
- Dpf
day post-fertilization
- Hpf
hour post-fertilization
single nucleotide polymorphisms


text:
Hi CAGs,
Wanted to let you all know that we're having a friendly contest this weekend in Supersonic Acrobatic Rocket-Powered Battle-Cars (PSN).
We're giving away a ton of BAWLS energy drinks and other BAWLS swag.
For the full contest details and rules check out our official forums here:
- Official contest page:
- FAQ Thread:
- Official Facebook Page:
Have fun and hope to see some of GAGs in SARB!
Edited by Psyonix-DavidB, 21 May 2010 - 11:45 PM.

subset: commoncrawl


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Lake Como
Just when we thought Venice was the most beautiful place we would visit in Italy, Varenna may have beaten it into second place. Neither of us can get our heads round the fact that we are actually seeing what's around us. The apartment is amazing and the views from the balcony alone are worth the trip. Claire has also befriended a blind cat so we keep having to think of excuses to go and see the lady so Claire can rub the cat.
Weather ...
Treasured time in Italy
... little villages connected by an amazingly beautiful pathway. We stayed in that area for three days. We did a lot of hiking and really enjoyed the beauty. My favourite moment started when we realised, to our dismay, that the pathways connecting the first three villages were closed down because of some landslides. We looked in chagrin at the gate that locked us out from the path thinking "how possible would it be to climb over"?? Just then a group of five ...
Lugano Easter Saturday
On the bus to Lagura Switzerland, free time for coffee and shopping in the markets. Weather is very cold here. We took the bus to the cable car up the mountain called Monte Tamaro. Beautiful views on the way up of the valley below, snow on the alps here. We had a set lunch menu and a walk around at the top, threw ...
Day 24. Travelling day. Saas Grund to Locarno
... and in spite of the roads, at times, being narrow, windy and steep,
Corinne performed well as a driver and we arrived safely without any dings or
We booked into a campsite on the banks of Lake Verbano, fed
the local ducks and braaied in a slight rain storm.
Hiking and biking in Locarno
... and Higher- maximum assistance. The bike also has 8 mechanical gears, all of which can be used with no or any level of assistance. Battery power is limited to about 100 km, depending on the time in the various modes. Obviously, you have no distance limitation in the "no assistance" mode.
I chose a route that would take me to Ascona, another lakeside town west of Locarno- the Lake is oriented more or less east-west in the area of Locarno (i.e., ...


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- 08-13-2014, 09:35 PM Thread Author #1
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Calendar issues with Xperia Tablet Z (but not other 4.4 device)
OK - this must be something simple, but I've been banging my head against the wall for a week - to no avail. Cannot seem to find a way to properly search, so here goes. I have an iCal shared calendar set up using Chrome on a PC. On my Phone (moto X) I can go into setting and see Calendars to show. I check it - it shows up. On my table (also running 4.4), it's nowhere to be seen. I get a similar page in the settings, but the calendar isn't there. I've tried refreshing, rebooting, waiting, cursing - all without success. What am I missing??? Gotta be something simple I guess...
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Re: Calendar issues with Xperia Tablet Z (but not other 4.4 device)
I am curious if this is a bug for the Xperia. Have you emailed the developer to see what they might suggest?
- 08-14-2014, 03:28 AM Thread Author #3
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Golden Gate Park
Transverse Dr., San Francisco, CA 94121
FeaturesFree, Popular with locals, Family-friendly
This 1,017-acre landmark might be world famous, but it's still full of hidden gems. Visit the bison paddocks on the park's west end, where urban buffalo graze the fields (seriously), stroll a traditional Japanese tea garden, row across Stow Lake, or just get lost walking the many trails that wind through the botanical garden and beyond.
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Top Definition
to "play for bossie" means to bat for the other team, be on the other bus, butter bread on the wrong side. "to play for bossie" means to be homosexual.
eg. "that ogre plays for bossie"
eg. "he used to play for bossie but now he likes girls"
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Premier Inn Chichester Hotel
No prices found through our partners. Please contact the business directly or check some of our recommended alternatives.
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London to Chichester
... direction to last night. We found we wer right next to West,oyster Cathedral beautiful and another Facebook photo. Mum armed with the newly discovered PIN number - it worked - yay! We could see signs to Victoria station but there was lots of construction Nd we were confused about how to get there, so mum decides to ask another Italian with no English. After going around I. A complete circle and crossing back over the road we found the way. Into the station for a rekke ...
Wonderful Wales
... particular pub already impressed us in London, being a franchise they are found all over England and Wales (and I hope Ireland and Scotland!), and they have some of the most delicious foods! We had the most delectable Irish stew served with warm soda bread and thyme cobblers (like little dumpling things) as well as heaps of tea in my case. It was delicious, and of course we lingered yet again. We then decided it was prudent to head back to the hostel to book a beach tour ...
Back to the Mother Country.
... But coming back (even in the driving rain on the A3 in a hire car an hour after landing) it looked lovely. All green. A verdant oasis, So different to the dusty, sun blasted desert we call home these days. So what did we do for the two weeks. Well looking back we tried to see as many people as possible and still have time for the kids to play in the townhouse. We juggled days out at theme parks with days at regular parks with family and friends. The ...
Off to Brighton
... very friendly and helpful, so we're very happy with it. As soon as we drop our bags off, we're down to the seaside. Its great – just what you would expect from an English seaside – vast beech of rocks and pebbles; seriously bloody cold water; no waves (at all – could have been a pond!); and loads of white bodies stretched out in rented striped deck ...
Fourteen - that's how many days there are to go before we set out. So much has happened leading up to today that it seems like a huge task just to try to describe it. The blog starts today but the planning started over a year and a half ago. Every move we have made, every life decision we have taken has somehow been informed by this thing. Many evenings spent agonising over routes on our map of ...


text: OverviewBack to top Brew La La - Organic Tea Strawberry Chocolate - 50 Tea Bags Item# :132440UPC# :879855004170 Brand:Brew La La Size/Form:50 Tea Bags Ship Weight:0.45 Flavor:Strawberry Chocolate Brew La La - Organic Tea Strawberry Chocolate - 50 Tea Bags Brew La La Organic Tea Strawberry Chocolate is is a perfect beverage for your health, and for your taste buds. It's the rich seduction of chocolate with a subtle hint of strawberries in an organic tea. Brew La La Tea is made with the finest tea leaves from across the globe and every tea bag is packed with love, joy and passion. Sip, savor and soothe your soul with Brew La La Organic Tea Strawberry Chocolate! USDA Certified Organic 50 String & Tag Tea Bags Kosher Certified Imported from Sri Lanka Rooibos Tea About Brew La La Ooh La La Candy started in business with a sweet and chic greeting card line known as Candy Care. What started as a creative endeavor of attaching candy onto a greeting card and incorporating it into the message evolved into a full blown sweet shop full of creative, delicious and stylish products ranging from candy to coffee to tea to potpourri and so much more! All of their products are created with what they like to call their ingredients for success: love, passion, creativity, joy and a great sense of humor all mixed into one. Their products taste great, look gorgeous, and have the power to change an attitude, lift a mood and hopefully make the world a sweeter place. After all, thinking big and setting high goals is how it all begins!


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|Message view
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|« Date » · « Thread »
|Justin Erenkrantz <jus...@erenkrantz.com>
|Re: mod_dav inconsistent behaviour for GET requests
|Sun, 31 Jan 2010 00:06:59 GMT
On Sat, Jan 30, 2010 at 2:04 AM, Stefan Fritsch <email@example.com> wrote: > The recommended setup is to map separate URLs for DAV and script > execution to the content. It has been like this since at least 2.0. > > The patch intentionally breaks existing configs that rely on the > ability to use the same URLs for DAV and script execution. Is this not > an acceptable change from 2.2 to 2.4 (if properly documented), as it > makes life a lot easier for people who use the recommended setup? I don't see how your patch would "intentionally" break - the failure mechanism is that the source scripts are served - not that a configuration error stops the server from running. -- justin
|View raw message


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> It's just that usually when I click the Daves link from Google it turns
> out to be incomplete info. It's unfullfilling to come across an entry
> for which most sections say "Unknown-Tell us". And I don't really know
> these people or how accurate they are. Anyone can put anything they want
> on there - doesn't mean it is true.
That's one of the problems I was struggling with on hort.net -- how do I
ensure that the data I'm getting is worthwhile?
I just settled it through inaction, I guess...
To sign-off this list, send email to firstname.lastname@example.com with the
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ATLANTA and WASHINGTON (November 17, 2009) – Sutherland Asbill & Brennan LLP announces that three partners have been named Best Lawyers' "2010 Lawyers of the Year." After more than a quarter of a century in publication, Best Lawyers is designating "Lawyers of the Year" in high-profile legal specialties in large legal communities. Only a single lawyer in each specialty in each community is being honored as the "Lawyer of the Year."
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|the most handsome man under 3 feet tall
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Flooring Replacement - Internet Area (Main)
The Internet, word processors and express terminals will not be available today at the Main Library.
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Have fun with all things Dr. Who! Play a trivia game, make duct tape bow ties and other crafts. Have a photo op with the Tardis, compete in an angel staring contest, enjoy snacks and much more! For teens ages 13-18.
This program will take place in the Lenski Program Room at the Main Library, 201 S. Fountain Ave., Springfield, OH. Call (937) 328-6905 for more information.


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text: Another month gone! I don't know where the time goes. Am I the only one who thinks time speeds up the older you get? Well, I guess the one good thing is this gives Suzy and I another opportunity to hand out a desktop freebie!! For the month of April we have included 2 versions in the download. One with a calendar and one without. Last month there were a few comments wishing for a calendar, and I'm happy to oblige. I personally don't like a calendar, so I'm still going to offer that version too! I'm making it for myself anyway. As always feel free to leave a comment with thanks, praise, or requests if there is something you feel we're missing. I hope you enjoy this month's desktop freebie using SuzyQ Scraps digital scrapbooking kit, Sunshine.


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Screenwriter Hayes's action-packed debut thriller introduces Scott Murdoch, a burnt-out, upper-echelon spy known as Pilgrim, who is drawn out of retirement when a Middle Eastern terrorist launches a devastating biological attack on the U.S. The voice that British-trained American actor Ragland uses is, on first hearing, surprisingly soft and youthful for a brilliant, world-weary ex-agent. But as the story progresses on a twisty, neatly crafted journey from the U.S. to Afghanistan, Lebanon, the Gaza Strip, and, eventually, Turkey, this almost-nerdy narration develops a harder, determined edge. There is a particularly grueling, gruesome sequence in which Murdoch is battered, beaten, and waterboarded, nearly to death. Ragland turns the spy's very human reaction to the torture into a performance piece. An Atria/Emily Bestler hardcover. (May)
I Am Pilgrimby Terry Hayes
Critics are calling I AM PILGRIM:
"Unputdownable." —Booklist
"The best book of 2014." —Suspense Magazine
"The next Girl with the Dragon Tattoo." —The New York Post
A breakneck race against time…and an implacable enemy.
An anonymous young woman murdered in a run-down hotel, all identifying/i>/i>/i>/i>
Critics are calling I AM PILGRIM:
"Unputdownable." —Booklist
"The best book of 2014." —Suspense Magazine
"The next Girl with the Dragon Tattoo." —The New York Post
A breakneck race against time…and an implacable enemy.
An anonymous young woman murdered in a run-down hotel, all identifying characteristics dissolved by acid.
A father publicly beheaded in the blistering heat of a Saudi Arabian public square.
A notorious Syrian biotech expert found eyeless in a Damascus junkyard.
Smoldering human remains on a remote mountainside in Afghanistan.
A flawless plot to commit an appalling crime against humanity.
One path links them all, and only one man can make the journey.
Editorial Reviews
Screenwriter Hayes's action-packed debut thriller introduces Scott Murdoch, a burnt-out, upper-echelon spy known as Pilgrim, who is drawn out of retirement when a Middle Eastern terrorist launches a devastating biological attack on the U.S. The voice that British-trained American actor Ragland uses is, on first hearing, surprisingly soft and youthful for a brilliant, world-weary ex-agent. But as the story progresses on a twisty, neatly crafted journey from the U.S. to Afghanistan, Lebanon, the Gaza Strip, and, eventually, Turkey, this almost-nerdy narration develops a harder, determined edge. There is a particularly grueling, gruesome sequence in which Murdoch is battered, beaten, and waterboarded, nearly to death. Ragland turns the spy's very human reaction to the torture into a performance piece. An Atria/Emily Bestler hardcover. (May)
Screenwriter and producer Hayes (Payback) makes his fiction debut with an exceptional thriller that boasts an utterly credible narrator who has had so many covert identities he can barely remember his original name. Soul-weary Scott Murdoch (aka the Pilgrim) has retired from the top echelon of ultrasecret espionage, but duty and faith in the human spirit call him back into service. A lone-wolf Middle Eastern native whom the Pilgrim code names "the Saracen" has a sure-fire bioterrorist plot to destroy the United States. In the cinematic chase that ensues, the action traverses the globe, from the Oval Office to the dusty trails of Afghanistan, each scene fleshed out in the smallest resonating detail (e.g., a Down syndrome child's laughter, the endless nausea of waterboarding). Like many pilgrimages, this one is painfully long and packed with unexpected menace, its glimpses of the goal fitful and far between, but readers will agree that this journey of body and soul is well worth the effort. Agent: Jay Mandel, WME. (May)
Pick of the Week: The Boston Globe
Nominated: Goodreads Choice Award
One of Amazon's Best Books of 2014
One of the Top 16 Books on The Wall Street Journal'sSpring Break Reading List
This hefty debut from longtime screenwriter Hayes was 2014's most memorable thriller, filled with cinematic twists and turns. Retired CIA operative Pilgrim connects a seemingly random murder in a New York hotel room to the case of a young, brilliant Saudi doctor who synthesizes an incurable strand of smallpox with designs of releasing it in the United States as a biological weapon. (LJ 1/1/14)
Tom Clancy meets Robin Cook in a thriller that should find a place in many beach bags this summer.Debut novelist Hayes brings well-refined storytelling chops to the enterprise: He's written numerous screenplays, including Mad Max Beyond Thunderdome. Indeed, while reading this novel, one gets the sense it was written to turn into a screenplay or perhaps began life that way, what with its shifting points of view and a narrator who may or may not be reliable. Whatever the case, Hayes gets us into the thick of things right away: Pilgrim, a federal agent, is a brilliant student of the human psyche who just happens to have awesome killing skills that he's practiced on several continents; in Moscow, for instance, he recounts, "even though I was young and inexperienced I killed my boss like a professional." Don't give him a bad performance review, then. He finds plenty of scope for his talents when put up against a former mujahedeen ominously code-named The Saracen, who's resolved to wreak all kinds of havoc on the West for its offenses against Islam. He's a bad, bad man—the fact that he wasn't killed in the war along with a million other Afghans, Hayes writes, "would make most people question if not God's existence at least His common sense." Hayes is a master of the extremely gruesome scene—the opening involves an acid bath, and later we get popped eyeballs, beheadings and all kinds of grisliness. The story does go on a hundred pages too long and gets sidelined here and there, but it has considerable strengths, and the author gets points for avoiding at least some clichés and putting a few Arabs into key good-guy (or good-girl) positions.Two psychos enter, and one psycho leaves. Good entertainment for readers with a penchant for mayhem, piles of bodies and a lethal biochemical agent or two.
Product Details
- 9781442369443
- Publisher:
- Simon & Schuster Audio
- Publication date:
- 05/27/2014
- Edition description:
- Unabridged
- Pages:
- 19
- Sales rank:
- 334,065
- Product dimensions:
- 5.10(w) x 6.00(h) x 1.90(d)
Read an Excerpt
I Am Pilgrim
There are places I'll remember all my life—Red Square with a hot wind howling across it, my mother's bedroom on the wrong side of Eight Mile, the endless gardens of a fancy foster home, a man waiting to kill me in a group of ruins known as the Theater of Death.
But nothing is burned deeper in my memory than a walk-up in New York—threadbare curtains, cheap furniture, a table loaded with tina and other party drugs. Lying next to the bed are a handbag, black panties the size of dental floss, and a pair of six-inch Jimmy Choos. Like their owner, they don't belong here. She is naked in the bathroom—her throat cut, floating facedown in a bathtub full of sulfuric acid, the active ingredient in a drain cleaner available at any supermarket.
Dozens of empty bottles of the cleaner—Drain Bomb, it's called—lie scattered on the floor. Unnoticed, I start picking through them. They've all got their price tags still attached and I see that, in order to avoid suspicion, whoever killed her bought them at twenty different stores. I've always said it's hard not to admire good planning.
The place is in chaos, the noise deafening—police radios blaring, coroner's assistants yelling for support, a Hispanic woman sobbing. Even if a victim doesn't know anyone in the world, it seems like there's always someone sobbing at a scene like this.
The young woman in the bath is unrecognizable—the three days she has spent in the acid have destroyed all her features. That was the plan I guess—whoever killed her had also weighed down her hands with telephone books. The acid has dissolved not only her fingerprints but almost the entire metacarpal structure underneath. Unless the forensic guys at the NYPD get lucky with a dental match, they'll have a helluva time putting a name to this one.
In places like this, where you get a feeling evil still clings to the walls, your mind can veer into strange territory. The idea of a young woman without a face made me think of a Lennon/McCartney groove from long ago—it's about Eleanor Rigby, a woman who wore a face that she kept in a jar by the door. In my head I start calling the victim Eleanor. The crime-scene team still have work to do, but there isn't a person in the place who doesn't think Eleanor was killed during sex: the mattress half off the base, the tangled sheets, a brown spray of decaying arterial blood on a bedside table. The really sick ones figure he cut her throat while he was still inside her. The bad thing is—they may be right. However she died, those that look for blessings may find one here—she wouldn't have realized what was happening, not until the last moment anyway.
Tina—crystal meth—would have taken care of that. It makes you so damn horny, so euphoric as it hits your brain that any sense of foreboding would have been impossible. Under its influence the only coherent thought most people can marshal is to find a partner and bang their back out.
Next to the two empty foils of tina is what looks like one of those tiny shampoo bottles you get in hotel bathrooms.
Unmarked, it contains a clear liquid—GHB, I figure. It's getting a lot of play now in the dark corners of the web: in large doses it is replacing rohypnol as the date-rape drug of choice. Most music venues are flooded with it: clubbers slug a tiny cap to cut tina, taking the edge off of its paranoia. But GHB also comes with its own side effects—a loss of inhibitions and a more intense sexual experience. On the street one of its names is Easy Lay. Kicking off her Jimmys, stepping out of her tiny black skirt, Eleanor must have been a rocket on the Fourth of July.
As I move through the crush of people—unknown to any of them, a stranger with an expensive jacket slung over his shoulder and a lot of freight in his past—I stop at the bed. I close out the noise and in my mind I see her on top, naked, riding him cowgirl. She is in her early twenties with a good body and I figure she is right into it—the cocktail of drugs whirling her toward a shattering orgasm, her body temperature soaring, thanks to the meth, her swollen breasts pushing down, her heart and respiratory rate rocketing under the onslaught of passion and chemicals, her breath coming in gulping bursts, her wet tongue finding a mind of its own and searching hard for the mouth below. Sex today sure isn't for sissies.
Neon signs from a row of bars outside the window would have hit the blond highlights in her three-hundred-dollar haircut and sparkled off a Panerai diver's watch. Yeah, it's fake but it's a good one. I know this woman. We all do—the type anyway. You see them in the huge new Prada store in Milan, queuing outside the clubs in Soho, sipping skinny lattes in the hot cafés on the Avenue Montaigne—young women who mistake People magazine for news and a Japanese symbol on their backs as a sign of rebellion.
I imagine the killer's hand on her breast, touching a jeweled nipple ring. The guy takes it between his fingers and yanks it, pulling her closer. She cries out, revved—everything is hypersensitive now, especially her nipples. But she doesn't mind—if somebody wants it rough, it just means they must really like her. Perched on top of him, the headboard banging hard against the wall, she would have been looking at the front door—locked and chained for sure. In this neighborhood that's the least you could do.
A diagram on the back shows an evacuation route—she is in a hotel but any resemblance to the Ritz-Carlton pretty much ends there. It is called the Eastside Inn—home to itinerants, backpackers, the mentally lost, and anybody else with twenty bucks a night. Stay as long as you like—a day, a month, the rest of your life—all you need is two IDs, one with a photo.
The guy who had moved into room 89 had been here for a while—a six-pack sits on a bureau, along with four half-empty bottles of hard liquor and a couple of boxes of breakfast cereal. A stereo and a few CDs are on a nightstand and I glance through them. He had good taste in music, at least you could say that. The closet, however, is empty—it seems like his clothes were about the only thing he took with him when he walked out, leaving the body to liquefy in the bath. Lying at the back of the closet is a pile of trash: discarded newspapers, an empty can of roach killer, a coffee-stained wall calendar. I pick it up—every page features a black-and-white photo of an ancient ruin—the Coliseum, a Greek temple, the Library of Celsus at night. Very arty. But the pages are blank, not an appointment on any of them—except as a coffee mat, it seems like it's never been used and I throw it back.
I turn away and—without thinking, out of habit really—I run my hand across the nightstand. That's strange, no dust. I do the same to the bureau, bed board, and stereo and get the identical result—the killer has wiped everything down to eliminate his prints. He gets no prizes for that, but as I catch the scent of something and raise my fingers to my nose, everything changes. The residue I can smell is from an antiseptic spray they use in intensive care wards to combat infection. Not only does it kill bacteria, but as a side effect it also destroys DNA material—sweat, skin, hair. By spraying everything in the room and then dousing the carpet and walls, the killer was making sure that the NYPD needn't bother with their forensic vacuum cleaners.
With sudden clarity I realize that this is anything but a by-the-book homicide for money or drugs or sexual gratification. As a murder, this is something remarkable.
Meet the Author
Terry Hayes is the New York Times bestselling author of I Am Pilgrim and the award-winning writer and producer of numerous movies. His credits include Payback, Road Warrior, and Dead Calm (featuring Nicole Kidman). He lives in Switzerland with his wife, Kristen, and their four children.
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I Am Pilgrim is a deeply engaging thriller by Terry Hayes. It's filled with everything I love about thrillers - great characters, a frightening scenario, the slow and unrelenting build of suspense, and a deeply layered story. Pilgrim is brilliant but so is Saracen, the person he is trying to track down to avoid a disaster of epic proportions. Watching Pilgrim's mind work is a real joy. He's clever and inventive. He is the first to admit to his shortcomings. He does make mistakes. He's not omniscient, but when it comes to investigating he has no peer. He puts things together is ways that are remarkable yet explainable (and are explained). This adds a great deal of depth and veracity to the novel. We get to see how Pilgrim thinks. He also works hard. He hunts down clues. He's a man of action when necessary. The Saracen is also very intelligent. His life history is slowly shown to the reader throughout the novel. The reader is privy to why he's become single-minded in his quest to bring down the United States. The Saracen is very, very patient. He is one man, more or less off the grid, planning unimaginable destruction. The plan he eventually concocts appears frighteningly real which heightens the intensity of the novel. In addition to trying to stop the Saracen, Pilgrim is also figuring out a couple of murders that are unrelated to the Saracen plot. I enjoyed this part of the story as well because it gave me additional opportunities to see Pilgrim in action and see how his mind works. I Am Pilgrim is a beautifully written novel with excellent pacing, wonderful twists and turns and plenty of action. The story not only switches back and forth between Pilgrim and Saracen, but there are flashbacks to Pilgrim's past. The reader gets to learn quite a bit about Pilgrim's life and who he is. Hayes handles this back and forth and the shifting story lines skillfully. There is a very strong cast of well-developed supporting characters who help or hinder Pilgrim in various ways. I Am Pilgrim is a nail-biter. Even after 600+ pages I was disappointed that the novel ended. I wanted keep reading about Pilgrim and to be in his mind a bit more. If you enjoy well-crafted, exciting espionage thrillers this is an absolute must read.
I have never written a review until i read this book. Must have!! Finally a real novel with depth. No skipping of pages like i have done with baldacci, cussler, and even clancy. I will be buying his next book as soon as it comes out. Kris
Unbelievable novel. If you like Ludlum or early Tom Clancy - you have found a new author to follow. I recommend you purchase this book and take a couple days off to read it. An astounding read and one of the best. Exceptional work and I cannot wait for the author's next novel!
I haven't read a page-turner this gripping for a long time. I neglected other priorities, stayed up past my usual bed time and read over lunch at work a couple days in a row just to find out what would happen next. Great read! There are lots of specifics about the book I really liked, but those would be spoilers. So, just pick up a copy and enjoy the ride.
I tried to make this book last but I could not stop reading it; will recommend to everyone and look foward to other books by this new author
I have never written a book review before. I read over 300 books a year. This is the best book I have ever read.
I read a lot of books. So I have gotten a little picky over the years. So when I find a book that is pretty remarkable, I have to say something about it. Especially when it is a first novel. I Am Pilgrim by Terry Hayes is such a book. Oh My Gosh! I remember seeing this book in an airport bookstore in Denver a couple months ago. The cover wasn't spectacular but for some reason I picked it up and read the blurbs and the synopsis and filed it away in the "to be read" box in my head. Fast forward to a week ago. I am in my little local library just seeing if there is anything new that would keep me busy and I noticed in the large print new releases, I Am Pilgrim. My memory kicked in and I remembered that I had wanted to give it a try. Large print didn't bother me, my eyes probably need it. So, I took it home. I was in the middle of reading another favorite author, Terry Brooks, but right before bed I picked up Pilgrim and read the first page. And the second, and the third and realized very quickly that I had found something special. It is a rare thing for this to happen. For me, only a handful of times. The Unlikely Spy by Daniel Silva, Hyperion by Dan Simmons, a very little known book by Martin Davies titled, The Conjurer's Bird, The Name of the Wind by Patrick Rothfuss. Now, I add Mr. Hayes to the list. There is a depth to this book that rises it above most thrillers. A book I will actually remember as opposed to many that are just so much fluff. James Patterson comes to mind. There are some great thriller authors out there, Ben Coes, Marc Cameron, the late Vince Flynn, Brad Thor, Matthew Dunn, Daniel Silva, Richard Doetsch, Greg Rucka. I read everything that is produced by these fine authors, but the only one that comes close to Hayes is Silva.Pilgrim begins with a brutal murder scene in New York City, a emotionally and physically wounded homicide detective and a brilliant investigator who has no name. From this beginning we learn all about this man, what he has done and where he has been. The action ranges from the dark days of 9/11 to the corridors of power in Washington DC, a farmhouse in North Dakota and to the mountains of the Hindu Kush in Afghanistan as well as a city square and a beheading in Saudi Arabia which leads to a birth of a new kind of terrorist that will be hunted to prevent the deaths of millions of people. Hayes brilliantly weaves the histories and locales of all of these disparate entities into a novel that at times you long to put down, but can't. Watching the matching of wits between the Pilgrim and the Saracen is a thing to behold. This book and Mr. Hayes are the real deal. I am ready for the next one.
Terry Hayes is a well known screenwriter and this is his first novel. And what a debut. The Pilgrim (a name adopted by our hero in the book) has "retired" from a super secret department of intelligence known only as The Division. The Division was disbanded after 9/11 and this man of many names is living in Paris when a series of events causes him to return to the U.S. to protect his former identity. While in NY, he is observing the investigation of a murder in lower Manhattan. He quickly realizes that the murder is somehow tied to a book he authored under a pen name dealing with modern investigative techniques. Among the forensic evidence is a charred piece of paper with a set of numbers that don't make any sense. And the murder is somehow connected to the main threat posed by the other main character in the book, code named the Saracen, which means Arab, or a Muslim who fights Christians, or simply, nomad. His formation begins watching his father executed in Saudi Arabia and progresses through the Afghan-Russian war to current post-9/11 terrorism. The Saracen has devised a massive bio-terrorism threat, and the development of this thread is detailed and disturbing. When the threat is recognized by the government, the Pilgrim is recruited back into service. He is not the perfect hero, and there are many twists and turns that will get your pulse beating rapidly. Despite being over 600 pages, the development of the story does not pause, coming to a heart pounding conclusion, and of course, the possibility of a sequel (although I would not bet my life on it). Don't miss this one.
Way too convoluted of a story. Only made it partway through and you too frustrated by the complex story within a story within a story, with pointless details and overly dramatic vague statements. I literally have no idea where the story is going because I get invested in one thing and then we have to go deeper into the back story... which then has its own back story, which then has it's own back door. I'm not reading 700+ pages of this.
I loved this book. The writing was a bit trite at times. The wry comments made by the main character at the end of almost every chapter got a bit old. However, the book was very suspenseful with lots of twists and turns. It also took you across several countries investigating a crime (and a potential major crisis). I love the international flavor of the book. Overall - I highly recommend it as a vacation or weekend read.
Couldn't put this book down -- storytelling at its best!
This is one of the best most thorougb books i have ever read,it rates up there with steven king in the details.I cant wait for the next one
I liked the synopsis when this book popped on my list of recommendation and I preordered it knowing that is not a quick read. What an awesome and fascinating book, well researched and mesmerizing. Before long the pages flew and I was left wanting more. Highly recommended.
Great read!!! This is an exceptional book. I haven't read a spy thriller in years. I'm not even sure why I picked it. But boy am I glad I did. All of the characters are finely drawn. This is a complicated, deep plot. But the writing is so good that it just carries you along. I couldn't put it down. And I had a knitting project I needed to get done. Didn't matter. The knitting had to wait. I'm recommending this book to,everyone I know. Can't wait for Terry Hayes' next book. I wish I could tell him personally how much I enjoyed his first book.
I found this book extraordinarily entertaining and I'm not even a fan of the genre, but as far as spy thrillers go, Terry Hayes' I am Pilgrim is better than what I've read from Clancy and DeMille. What made the story so enthralling was the attention to detail at every turn and inside of every character back story. But Hayes doesn't try to overdo it he gives you just enough for you to intelligently lend your empathy to whoever you're rooting for. Obviously you'll be rooting for his main protagonist. With just the right amount of testosterone his character is only a few steps beyond beleivable, maybe this is his trick. Perhaps this is his send-up of the genre. As I said, I'm not a huge fan. But I am Pilgrim left me wanting more while leaving me afraid to start on another spy thriller for fear of it coming up short by comparison.
Excellent book.. Do not use as a sleep inducer! Too thrilling.
I can't wait for this to be in theaters! If it's anything like the book, it will be a Blockbuster!
Once I started reading, I was hooked. He is a genius at tying together stories within stories, until he hands you a neat package all tied up neatly at the end.
Read this book when it first came out and loved it then. Just re-read it and loved it just as much. So many twists and turns that it is impossible to put down. Eagerly awaiting a sequel!!
One of the best books I have read in a long time. Wish i could read it again for the first time.
Fast paced & well written, hard to put down!
I truly enjoyed this book. The story is very timely and at times reads like fact.
Excellent book. It was hard to put down to the very end. Its also frightening to think about the real possibiillity of this happening.


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Closeouts. A warm winter boot with an outsole that excels on snow and ice, Lowa's Tirolina Gore-Tex® snow boots have a double-sided sheepskin collar and warm Partelana fleece insulated lining.
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What do you think of the city's settlement of the FDNY lawsuit?
Mayor Bill de Blasio's administration settled a civil-rights lawsuit dating back to 2007 that accused the Fire Department of discriminating in its hiring practices. The deal includes $98 million in back pay to black and Hispanic applicants who took civil service exams in 1999 and 2002 and also requires the department to make changes to its recruiting policies. What do you think of the city's settlement of the lawsuit?
It puts too much burden on the FDNY. It's about time the city settled this matter. It won't change anything at the FDNY.


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Lord Of The Rings
May 25th, 2011
Believe it or not, he does his own makeup.
(submitted by Scott)
honestly, who wears stripes with rings after Labor Day???
This was the worst case of cooties his physician had ever seen.
Marker Boy looks eerily like a younger version of my exboyfriend. And my ex's stepdad was named Scott. Hmm…
Possibly he was a seaQuest fan if this photo was from the mid-to-late 90s? Looks almost like G.E.L.F/Dagger makeup.
Kids can come up with some of the best (more permanent in this case) costumes. Great photo!!! lol
I think he may be the Thing from Fantastic Four.
that's what I was thinking
Is he a fish? Bubbleboy? Hmmm awkward indeed. lol
All that is missing is the Colgate ring of confidence!!
I like the Apple logo on his forehead best.
Um…what is that thing on the wall just to the left (our left) of the group?
Okay, who took the sharpie to the Down's Syndrome kid again??
The glasses are a nice touch
"No,honey….'Permanent' market actually means it rubs right off in water"…..*snigger*
…just like "quarantine" means, "come on in, kids, free ice cream!!!"
So, did they also use permanent marker for the little girl's whiskers???
ha, now I wonder too
He was told he could not get any earrings, so he went with an alternative
The kids are sending him in circles.
the strangest thing is the fact that he is presumably covered in his arms and torso as well. this leads me to believe he had an accomplice. or maybe he is a victim in a horrible sharpie attack.
Please tell me this is the result of passing out first at football party. Crazy!
Maybe he's Ray Bradbury's 'Illustrated Man'
That is awesome!
ok, so it's halloween and the little girl is dressed as ralphie from christmas story, but what the h#ll is dad trying to look like? i want to hear the story behind this.
My guess is he is a turtle.
Offspring of The Thing and Lizard Man? hmmm…
'Costume? Oh, Halloween is this week?'
Thing ring do your thing!!!
Fantastic 4-Eyes
What the french, toast?!


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Ibanez RG421 Electric Guitar
Ibanez RG421 Electric Guitar
The RG is the most recognizable and distinctive guitar in the Ibanez line. Three decades of metal have forged this high-performance machine, honing it for both speed and strength. Whether you favor a hardtail (fixed) bridge or our industry-leading locking tremolo system, the RG is a precision instrument.
Neck type Wizard III Maple neck
Body Mahogany body
Fretboard Rosewood fretboard w/White dot inlay
Fret Jumbo frets
Bridge Fixed bridge
Neck pickup Quantum (H) neck pickup (Passive/Ceramic)
Bridge pickup Quantum (H) bridge pickup (Passive/Ceramic)
Factory tuning 1E, 2B, 3G, 4D, 5A, 6E
String gauge .010/.013/.017/.026/.036/.046
Hardware color Cosmo black
Scale 648mm/25.5"
a : Width at Nut 43mm
b : Width at Last Fret 58mm
c : Thickness at 1st 19mm
d : Thickness at 12th 21mm
Radius 400mmR
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What is Cultural History?
2nd Revised edition
By (author) Peter Burke
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What is Cultural History? by Peter Burke
Book DescriptionWhat is Cultural History? has established itself as an essential guide to what cultural historians do and how they do it. Now fully updated in its second edition, leading historian Peter Burke offers afresh his accessible guide to the past, present and future of cultural history, as it has been practised not only in the English-speaking world, but also in Continental Europe, Asia, South America and elsewhere. Burke begins by providing a discussion of the 'classic' phase of cultural history, associated with Jacob Burckhardt and Johan Huizinga, and of the Marxist reaction, from Frederick Antal to Edward Thompson. He then charts the rise of cultural history in more recent times, concentrating on the work of the last generation, often described as the 'New Cultural History'. He places cultural history in its own cultural context, noting links between new approaches to historical thought and writing and the rise of feminism, postcolonial studies and an everyday discourse in which the idea of culture plays an increasingly important part. The new edition also surveys the very latest developments in the field and considers the directions cultural history may be taking in the twenty-first century. The second edition of What is Cultural History? will continue to be an essential textbook for all students of history as well as those taking courses in cultural, anthropological and literary studies.
Buy What is Cultural History? book by Peter Burke from Australia's Online Bookstore, Boomerang Books.
Book DetailsISBN: 9780745644097
(220mm x 140mm x 19mm)
Pages: 168
Imprint: Polity Press
Publisher: Polity Press
Publish Date: 1-Aug-2008
Country of Publication: United Kingdom
Other Editions...
Books By Author Peter Burke
What is the History of Knowledge?, Paperback (November 2015)
What is the history of knowledge? This engaging and accessible introduction explains what is distinctive about the new field of the history of knowledge (or, as some scholars say, knowledges in the plural ) and how it differs from the history of science, intellectual history, the sociology of knowledge or from cultural history.
Year-Round Indoor Salad Gardening, Paperback (October 2015)» View all books by Peter Burke
"The low-tech, no grow lights approach to abundant harvest"--Cover.
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Author Biography - Peter Burke
Peter Burke is Professor Emeritus of Cultural History at the University of Cambridge.
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Today's Devotional
What's ImportantHow has God been training you lately?
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I spent over 20 years enjoying the opportunity to express myself through music and songwriting. I spent three years pursuing a career in music and another five years in a bluegrass gospel music ministry. I am now involved with a music ministry at my church, The Torch Church of God. I continue to write, and always will. Once a writer always a writer I guess. Every one has a song insode them. Some people just don't understand how to write it.


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- 07-11-2014, 03:49 AM Thread Author #1
Galaxy Note 3 no media playing through Bluetooth
I was at work listening to a podcast on my Note 3 through a Bluetooth headset when all the audio stopped. Calls worked fine and settings showed media playback through Bluetooth checked, but still no sound from music or the podcast.
I later tried on the Bluetooth in my car(2013 Nissan Altima and use the Bluetooth to listen to music from my phone everyday.) there was still no audio. The settings were the same, both boxes checked. Calls worked fine again.
I tried restarting the phone, pulling the battery, un-pairing and re-pairing both the headset and car.. still didn't work. Any ideas or tips to the cause?
I haven't even had the phone 4 months.
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Re: Galaxy Note 3 no media playing through Bluetooth
Welcome to Android Central! Try booting into Recovery Mode and clearing the system cache partition: Reset: Samsung Galaxy Note 3 | T-Mobile Support
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Re: Galaxy Note 3 no media playing through Bluetooth
as of today I have the same problem; I had been happily listening to TTS and music via bluetooth on this headset, and today I installed the Navigate gps maps app, and when I set it's TTS engine to use Google text-to-speech, the media channel to bluetooth ceased to make any sound -- apps still believe there is a channel, the Music app appears to play and Utter appears to speak, but no sound comes through the headset.
I have restarted, unpaired and repaired, unselected and reselected the media, still no sound on the media channel via BT, yet when I switch off the headset or if, in the Music player I select Sound On Device, the sound now plays through the phone speaker. Only the BT headset is silent and it is only silent on some apps, I even get voice prompts still from Voice Notify (that says it also uses Media for TTS) even though from that same setup menu I can access the Text-To-Speech options and play Listen To An Example and hear no sound!
Again, this is a new issue, the hardware all worked for months up until installing Navigate and setting it's TTS to use the system Text-To-Speech.
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Re: Galaxy Note 3 no media playing through Bluetooth
ok, now I feel stupid: somehow I must have set the media-channel volume to zero for only bluetooth!!
when I increased the volume with bluetooth activated, the volume slider showed it increasing from zero, and sure enough, first the music became audible, and the TTS checks also worked.
something to remember next time it goes silent.
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East Windsor Hill Historic District
From Wikipedia, the free encyclopedia
East Windsor Hill Historic District
The 1788 John Watson House
|Roughly bounded by the Scantic River, John Fitch Blvd., Sullivan Ave., and the Connecticut River, South Windsor, Connecticut
|41°51′23″N 72°36′45″W / 41.85639°N 72.61250°WCoordinates: 41°51′23″N 72°36′45″W / 41.85639°N 72.61250°W
|535 acres (217 ha)
|Architectural style
|Greek Revival, Georgian, Federal
|NRHP Reference #
|Added to NRHP
|May 30, 1986
East Windsor Hill Historic District is a historic district located in the northwestern corner of the town of South Windsor, Connecticut. It was listed on the National Register of Historic Places in 1986.[1] The district runs along both sides of Main Street from the Scantic River south to the Edwards Cemetery. The district also includes areas west of Main Street to the Connecticut River, including properties along Ferry Lane. The district is located directly north of another historic district, Windsor Farms Historic District. The district encompasses a neighborhood of largely folk vernacular buildings erected between about 1700 and 1860.[2]
See also[edit]
- ^ a b National Park Service (2009-03-13). "National Register Information System". National Register of Historic Places. National Park Service.
- ^ "NRHP nomination for East Windsor Hill Historic District" (PDF). National Park Service. Retrieved 2014-12-13.
|This article about a property in Connecticut on the National Register of Historic Places is a stub. You can help Wikipedia by expanding it.
- Neighborhoods in Connecticut
- Historic districts in Hartford County, Connecticut
- Georgian architecture in Connecticut
- Federal architecture in Connecticut
- Greek Revival architecture in Connecticut
- South Windsor, Connecticut
- National Register of Historic Places in Hartford County, Connecticut
- Historic districts on the National Register of Historic Places in Connecticut
- Connecticut Registered Historic Place stubs


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Publisher's Description+
From LEMICO Soft: Oil tycoon 2 is a fun pipe puzzle game, perfect for those situations where you need to kill time. You have to connect all sources to objects. Rotate the items you will need to reconnect each object. At first glance, it seems simple, but with each level of play becomes more difficult. There are a total of 400 levels available. You are challenged by leaderboard system as well as timed against the clock. The controls for game use simple tap gestures that are very responsive. Pipes rotate a full 360 degrees and can be activated at any time.Features: - A lot of themes;- A lot of levels;- Sync your progress through iCloud, between devices and games with a different theme design;
What's new in this version:
- New UI- New skins- New levelsBest update!!!
read more +


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Showing Results: Most Recent
This review is from: GIGABYTE G1 Gaming GA-Z97X-Gaming 7 LGA 1150 Intel Z97 HDMI SATA 6Gb/s USB 3.0 ATX Intel Motherboard
Pros: Great motherboard. Things installed easily, efficiently and the layout is great. Work with all my gear and still room for expansion. Everything seemed sturdy, in-place and the addition of the BIOS boot-button and error display is a good kick for trouble-shooting.
Cons: There's a constant 'noise' from any audio-jack whenever the computer is processing at any rate above an average of 5-7%. The heavier the process, the louder the 'ringing' is and its rather annoying. Sound is coming through almost too loud as well, I had to turn down my system to 10% or below to keep it at moderate levels, and even with the system down to that, other programs don't abide by it? Started up a game to check the temperatures and the sound was catastrophic; clipping, no regards to the previous volume controls I had set before launch. Not quite sure what to think of it, but I've only seen one other person review about the 'noise' as there aren't many writings on this product.
Other Thoughts: Two out of 17(current reviews thus far) is a high percentage for this sound issue, I'd shoot for a better quality motherboard from Gigabyte that'll cost a bit more, but hopefully you'll steer clear of this and avoid losing your hearing.READ FULL REVIEW
This review is from: ASUS VX229H Black 21.5" 5ms (GTG) HDMI Widescreen LED Backlight LCD Monitor AH-IPS 250 cd/m2 80,000,000:1
Pros: Lovely design, sleek and attractive. Good use of desk space and solid stand.
Cons: An earlier review said they got no cables for the video input. I got some, but not all of them that were stated on the products page, which, I needed and HDMI cable, and it says there are supposed to be two, but there's none in the box. Now I can't enjoy this monitor till I go out and get one. Huge, inconvenient hassle.READ FULL REVIEW