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Neuronal apoptotic death generally requires de novo transcription, and activation of the transcription factor c-Jun has been shown to be necessary in multiple neuronal death paradigms. Caspase-2 has been implicated in death of neuronal and non-neuronal cells, but its relationship to transcriptional activation has not been clearly elucidated. In the present study, using two different neuronal apoptotic paradigms, β-amyloid treatment and NGF (nerve growth factor) withdrawal, we examined the hierarchical role of caspase-2 activation in the transcriptional control of neuron death. Both paradigms induce rapid activation of caspase-2 as well as activation of the transcription factor c-Jun and subsequent induction of the pro-apoptotic BH3 (Bcl-homology domain 3)-only protein Bim (Bcl-2-interacting mediator of cell death). Caspase-2 activation is dependent on the adaptor protein RAIDD {RIP (receptor-interacting protein)-associated ICH-1 [ICE (interleukin-1β-converting enzyme)/CED-3 (cell-death determining 3) homologue 1] protein with a death domain}, and both caspase-2 and RAIDD are required for c-Jun activation and Bim induction. The present study thus shows that rapid caspase-2 activation is essential for c-Jun activation and Bim induction in neurons subjected to apoptotic stimuli. This places caspase-2 at an apical position in the apoptotic cascade and demonstrates for the first time that caspase-2 can regulate transcription.
- β-amyloid
- Bcl-2-interacting mediator of cell death (Bim)
- caspase-2
- nerve growth factor (NGF)
- neurons
- RIP (receptor-interacting protein)-associated ICH-1 [ICE (interleukin-1β-converting enzyme)/CED-3 (cell-death determining 3) homologue 1] protein with a death domain (RAIDD)
Widespread neuron death occurs during normal development, after trauma, and in neurodegenerative diseases. Understanding the mechanisms of such death may provide the basis for useful therapies. Caspases, cysteine aspartate proteases, are key mediators of neuronal apoptotic death [1]. In vertebrates, caspase-2 is the most highly evolutionarily conserved member of the caspase family and the closest in sequence to Caenorhabditis elegans ced-3 (cell-death determining 3) [2]. Yet, there is much uncertainty about the extent to which caspase-2 participates in apoptotic death, the mechanism by which it does so and its hierarchical position in apoptotic cascades [3,4]. The present study addresses these issues in two different paradigms of neuron death: β-amyloid (Aβ1–42) treatment and NGF (nerve growth factor) withdrawal.
Although caspase-2 has been less studied than most other caspase family members, it has been implicated as a required constituent in a variety of apoptotic cell death paradigms by a substantial body of evidence from silencing and knockout experiments in non-neuronal cells [5–8]. Moreover, silencing of caspase-2 expression rescues neurons from apoptotic death triggered by NGF withdrawal and β-amyloid (Aβ1–42) treatment [9–12]. Additional studies indicate that caspase-2 siRNA protects retinal ganglion cells from death evoked by optic nerve transection [13], whereas in grey and white matter, injury in response to hypoxia/ischaemia or excitotoxic stress is reduced in caspase-2-deficient neonatal mice [14]. Dopaminergic neuron loss in an MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease is also significantly decreased in caspase-2-null mice [15]. Such findings support a role for caspase-2 in apoptotic death of neurons and other cell types.
The JNK (c-Jun N-terminal kinase)/c-Jun pathway has been identified as a central element in apoptotic death mechanisms and has been shown to lead to induction of pro-apoptotic molecules including the BH3 (Bcl-homology domain 3)-only protein Bim (Bcl-2-interacting mediator of cell death) [16]. In this context, transcriptional up-regulation of Bim is required for apoptotic neuron death in response to NGF withdrawal and exposure to β-amyloid [16–19]. Placing caspase-2 within the death pathway has been problematic. Findings that caspase-2 is activated by dimerization induced by interaction with signalling platforms that include the caspase-2-binding adaptor protein RAIDD {RIP (receptor-interacting protein)-associated ICH-1 [ICE (interleukin-1β-converting enzyme)/CED-3 homologue 1] protein with a death domain} [20,21], have indicated that it is an initiator caspase. However, other findings identify caspase-2 as an effector that is downstream of other caspases [22,23]. These issues have not been systematically addressed in neurons.
Our past work has shown that caspase-2 is required for apoptotic neuronal death associated with NGF withdrawal and Aβ1–42 exposure. In the present study, we have examined the function and hierarchical role of caspase-2 in the death signalling pathways triggered by these two apoptotic stimuli. We show that caspase-2 is rapidly activated in response to apoptotic stimuli and, surprisingly, promotes induction of Bim mRNA and protein. Moreover, we find that this action is mediated by caspase-2-dependent activation of the transcription factor c-Jun. These findings causally associate caspase-2, c-Jun and Bim in the same apoptotic pathway and provide a novel mechanism by which activated caspase-2 triggers neuron death.
For all animal experimentation, Institutional and National guidelines for the care and use of laboratory animals were followed.
Primary hippocampal neuron cultures
Neurons were cultured as described previously [24]. Briefly, E18 (embryonic day 18) rat fetuses were removed from CO2-killed pregnant Sprague–Dawley rats (Charles River). The hippocampus was dissected from surrounding tissue and the meninges removed. Pooled hippocampi were mechanically dissociated in a serum-free defined medium. Medium consisted of a 1:1 mixture of Eagle's MEM (minimal essential medium) and Ham's F12 (Invitrogen) supplemented with glucose (6 mg/ml), insulin (25 μg/ml), selenium (30 nM), progesterone (20 nM), transferrin (100 μg/ml), putrescine (60 μg/ml), penicillin (0.5 units/ml) and streptomycin (0.5 μg/ml). Dissociated cells were grown on poly-D-lysine-coated plates or 8-well chamber slides. Neurons were cultured for 7 days prior to experimental treatments.
Primary sympathetic neuron cultures
Neurons were cultured as described previously [11]. Briefly, sympathetic neurons were dissected from 1-day-old wild-type and caspase-2-null [5] mouse pups. Cultures were maintained in RPMI 1640 medium supplemented with 10% horse serum and mouse NGF (100 ng/ml) on collagen-coated 24-well dishes. For cells that were subjected to microscopic imaging, Matrigel-coated 8-well chamber slides were used. At 1 day after plating, uridine (10 μM) and 5-fluorodeoxyuridine (10 μM) were added for 3 days to eliminate non-neuronal cells. Experiments were conducted after 6 days of culture.
Neuronal viability
Hippocampal or sympathetic neuron survival was scored as reported previously [12]. For hippocampal neurons, culture medium was removed by aspiration and 100 μl of detergent-containing lysis solution was added to the well. This solution dissolves cell membranes providing a suspension of intact nuclei. Intact nuclei were quantified using a haemacytometer. Triplicate wells were scored and values reported as mean±S.E.M. Significance was calculated by Student's t test. For sympathetic neurons, each culture was scored as numbers of living phase-bright neurons counted in the same field at various times. Three replicate cultures were assessed for each condition, and data are normalized to numbers of neurons present in each culture at the time of Aβ1–42 addition or NGF deprivation and reported as mean±S.E.M.
β-Amyloid preparation
Lyophilized and HPLC-purified β-amyloid1–42 (Aβ1–42) was purchased from Dr David Teplow (UCLA, Los Angeles, CA, U.S.A.). Peptides were prepared according to Fa et al. [25] except that monomerized Aβ1–42 was reconstituted in DMSO to 1 mM. To form Aβ1–42 aggregates stocks of 1 mM peptide were resuspended in PBS to a concentration of 100 μM and incubated at 37°C for 24 h. A concentration of 3 μM Aβ1–42 was used in all experiments.
siRNA conjugation and use
siRNAs against caspase-2 and RAIDD were generated (Dharmacon) and their sequences were GCCAUGCACUCCUGAGUUU and CCACAUUCAAGAAAUCAAA respectively. The siRNAs were customized with a thiol group attached to the 5′ ends of the sense strands and carrying 3′ poly(dT) overhangs. Prior to use each siRNA sequence was conjugated to Pen1 (penetratin 1) [26]. Pen1-linked siRNA allows efficient delivery of siRNA into cells with minimal toxicity. For experiments all Pen1–siRNA were used at 80 nM.
Western blotting
Hippocampal neurons or sympathetic neurons were lysed in CHAPS lysis buffer [150 nM KCl, 50 mM Hepes, 0.1% CHAPS and a protease inhibitor tablet (pH 7.4)]. Protein concentration was determined using the Bio-Rad Laboratories protein assay reagent. Equal amounts of protein were loaded on to 10% or 12% polyacrylamide gels. The proteins were then transferred on to nitrocellulose transfer membranes (Millipore). Subsequently, the membranes were blocked in 5% (w/v) non-fat dried skimmed milk powder for 1 h. The primary antibodies used for Western immunoblots include anti-caspase-2 (affinity purified, 1:250 dilution), anti-[phospho-c-Jun (Ser63)] (Cell Signaling Technology, 1:750 dilution), anti-c-Jun (Cell Signaling Technology, 1:750 dilution), anti-ERK1 (extracellular-signal-regulated kinase; Santa Cruz Biotechnology, 1:10000 dilution), anti-α-Tubulin (Abcam, 1:10000 dilution) and anti-Bim (Cell Signaling Technology, 1:1000 dilution). Proteins were detected using either enhanced chemiluminescence (Thermo Scientific) or fluorescence using the Odyssey infrared imaging system (LI-COR Biosciences). The relative densities of immunopositive bands were analysed using ImageJ (http://rsbweb.nih.gov/ij/).
RT-PCR (real-time PCR)
Hippocampal or sympathetic neuron cultures grown in 24-well plates were harvested using ice-cold 100% TRIzol reagent (Invitrogen). cDNA was transcribed from RNA using Superscript RT II (Invitrogen). Equal amounts of cDNA were used for each PCR for Bim, normalized to β-tubulin mRNA expression. The sequence for the rat-specific Bim primer was published previously [18]. cDNA was added to 25 μl of reaction mixture containing OmniMix HS master mix (Cepheid) and SYBR Green I (Invitrogen) together with appropriate primers. qPCR (quantitative PCR) was performed using a Cepheid SmartCycler according to the manufacturer's directions.
Caspase-2 activity assay
This unbiased caspase-2 activity measurement was adapted from [21]. bVAD-fmk (biotin-Val-Ala-DL-Asp-fluoromethylketone; 50 μM), a biotinylated pan-caspase inhibitor that traps active caspases, was added to neuronal cultures 2 h prior to Aβ1–42 or NGF deprivation. Cells were lysed in CHAPS buffer. Active caspase–bVAD-fmk complex was pulled out with streptavidin-coated beads (Invitrogen). Active caspase-2 was detected by Western blotting using affinity-purified polyclonal anti-caspase-2 antibody [12].
E18 rat hippocampal neurons and P1 (postnatal day 1) mouse sympathetic neurons were cultured on 8-well chamber slides (Nunc) for 1 week. Cultures were then fixed in a solution of 3.7% formaldehyde and 5% sucrose at 37°C for 20 min. The cells were rinsed in TBS. Cultures were blocked in TBS supplemented with 3% NGS (normal goat serum) for at least 1 h at room temperature (25°C). The primary antibodies used include anti-[phospho-c-Jun (Ser63)] (1:100 dilution) and anti-βIII-Tubulin (1:1000 dilution, Abcam).
Frozen mouse and human brain tissue sections were washed in PBS for 15 min and then blocked in PBS with 1% BSA, 10% NGS and 0.5% Triton X-100 for 1 h. The primary antibodies used include anti-Bim (1:100 dilution) and anti-caspase-2 (1:100 dilution, affinity purified). To prevent autofluorescence 1% Sudan black (prepared in 70% ethanol) was used to treat the sections for 5 min prior to coverslip mount.
Fluoro-Jade B labelling
Frozen mouse brain sections were air dried at 45°C for 20 min. Slides were incubated in 1% NaOH and 80% ethanol for 5 min, followed by a 2 min rinse in 70% ethanol and a 2 min rinse in double-distilled water. Sections were then incubated in 0.06% potassium permanganate for 10 min, and subsequently incubated with 0.0004% Fluoro-Jade B (made in 0.1% acetic acid) for 30 min. Finally, they were rinsed with double-distilled water for 1 min, followed by air drying for 5 min at 50°C and then mounted with permount.
Mouse hippocampal Aβ1–42 infusion
Adult wild-type or caspase-2-null male mice were used. Mice were anaesthetized with xylazine (7 mg/kg of body mass) and ketamine (95 mg/kg) and then placed on to a stereotaxic frame. At a point 2.45 mm AP (anteroposterior), 1.5 mm ML (mediolateral) and 1.7 mm DV (dorsal–ventral) from the Bregma a hole was drilled into the skull and then a Hamilton syringe was inserted into the right CA1 region of the hippocampus. Aβ1–42 (4 μl, 100 μM) was infused via convection-enhanced delivery at a rate of 0.2 μl per min. Following injection, the head wound was closed using Vetbond (3M) and the animals were maintained for 2 weeks.
Mouse brain processing and sectioning
After the 2 week survival period animals were anaesthetized with xylazine (7 mg/kg of body mass) and ketamine (95 mg/kg of body mass) and transcardiacally perfused with 4% paraformaldehyde. Brains were removed and post-fixed with 4% paraformaldehyde for 24 h at 4°C, followed by 30% sucrose infiltration. The brains were embedded in Optimal Cutting Temperature embedding medium (Tissue-Tek) and stored at −80°C. For immunohistochemistry, brains were sectioned on a cryostat at 15 μm thickness and mounted on to SuperfrostPlus slides.
AD (Alzheimer's disease) and control brain samples
AD and control brains were obtained at autopsy by the New York Brain Bank at Columbia University (Dr J.-P. Vonsattel, Director) as described on their website (http://nybb.hs.columbia.edu/index.htm). Detailed descriptions of the brains used are in [18]. Human tissue was obtained using a protocol approved by the Institutional Review Board.
Both in vitro and in vivo immunostainings were visualized at room temperature using a PerkinElmer Ultraview spinning disc confocal system with a Hamamatsu ORCA-ER digital camera, Nikon inverted TE-200 microscope, and 20×/0.75, 40×/1.30 or 60×A/1.40 objectives. Images acquired using Volocity 6.0.1. Images were prepared in Adobe Photoshop CS3.
Bim and caspase-2 proteins are elevated and co-expressed in neurons from AD patients
We have previously shown in cellular models of AD that Aβ1–42 induces Bim transcripts and that Aβ-induced neuronal death requires Bim as well as caspase-2 [10,18]. Additionally, we observed that Bim expression is elevated in more than 80% of entorhinal cortical neurons of AD patients [18], a brain region that shows early degeneration in AD. To determine whether caspase-2 might also be dysregulated along with Bim in AD, we co-immunostained brain sections from six AD patients and six age-matched non-demented controls for both proteins. Representative images are shown in Figure 1. We confirmed the increased expression of Bim in entorhinal cortical neurons, and consistently found an increase in caspase-2 (Figure 1), with AD patients' brains containing more than 200 caspase-2 positive cells/field and the controls containing less than 20. Moreover, Bim and caspase-2 co-localized almost completely within the same entorhinal cortical neurons in AD brains (Figure 1). In contrast, there was no increased expression in either protein in cerebellum, an area spared of AD pathology (results not shown).
Caspase-2 is rapidly activated by exposure to Aβ1–42 and by NGF deprivation
The required roles of Bim and caspase-2 for neuron death in cellular models of AD and their increased expression and co-localization in AD neurons raised the possibility that they may function in the same apoptotic pathway. To assess a potential functional interaction between Bim and caspase-2 in neuron death, we utilized primary cultures of rat hippocampal neurons. Hippocampal neurons undergo apoptotic death in response to treatment with Aβ1–42, which requires both Bim [18] and caspase-2 [10]. We also used a second well-studied model of neuron death: cultured sympathetic neurons deprived of NGF. In this system, NGF deprivation induces elevation in Bim transcripts and protein and the subsequent neuron death requires both Bim [17] and caspase-2 expression [11,12]. We first aimed to detect caspase-2 activation in these death models and to determine the time at which it occurs. To achieve this, we used an unbiased caspase-activity probe that we previously adapted for use in neurons [24,27]. This approach employs a biotinylated pan-caspase inhibitor, bVAD-fmk, that irreversibly binds and inhibits active caspases within cells and permits their subsequent isolation and identification by Western immunoblotting [21]. When cells are pre-treated with bVAD-fmk and then exposed to a death stimulus, bVAD binds to proximal caspases (usually initiator caspases) and inhibits their activation, usually that of initiator caspases. All subsequent events dependent on activity of the proximal caspases are blocked. The specificity of the polyclonal caspase-2 antiserum used in the present study was confirmed by using brain lysates from wild-type and caspase-2-null animals (Figure 2A).
The levels of activated caspase-2 in cultured hippocampal neurons significantly increased within 30 min of treatment with 3 μM Aβ1–42 (Figure 2B). Moreover, NGF withdrawal from sympathetic neurons elicited an increase in activated caspase-2 within 2 h (Figure 2C). We have reported that sympathetic neurons undergo apoptotic death in response to Aβ1–42 and do so by a caspase-2-dependent mechanism [10]. Consistent with this, Aβ1–42 also activated caspase-2 in sympathetic neurons within 2 h (Figure 2C). Total caspase-2 levels remained unchanged in all models at these times (Figures 2B and 2C). Taken together, these data show that caspase-2 is rapidly activated in neurons in response to death stimuli, consistent with our previous findings that neuronal apoptosis caused by Aβ1–42 or NGF deprivation requires caspase-2.
Bim induction by Aβ1–42 occurs after caspase-2 activation
If Bim and caspase-2 have the potential to function in the same apoptotic pathway, then it is important to determine the temporal relationship of Bim induction and caspase-2 activation. In other models Bim elevation precedes activation of caspases such as caspases-9 and -3 [28,29]. Our previous studies with hippocampal neurons indicated that Aβ1–42 induces Bim mRNA within 1 h, with a maximal effect at 3–6 h, and that Bim protein is elevated within 4 h of treatment [18]. We therefore examined Bim protein expression in hippocampal neuron cultures at relatively early times after Aβ1–42 exposure, starting at 30 min (Figure 2D). This revealed no change in Bim expression at 30 and 60 min, but a significant increase by 2 h. These data (Figures 2B and 2C) indicate that caspase-2 activation occurs prior to Bim protein elevation and suggest that caspase-2 activation is independent of Bim induction. They also raised the possibility that activated caspase-2 may be upstream of Bim regulation.
Caspase-2 activation by Aβ1–42 does not require Bim induction
Although our temporal data suggest that caspase-2 activation could be upstream of Bim induction, the canonical pathway places Bim upstream of caspase-2. To determine if Bim induction is required for caspase-2 activation, we utilized a Bim-specific siRNA (siBim) conjugated to the cell-penetrating peptide Pen1 for highly efficient low toxicity delivery into neurons [26]. We previously used this sequence with an shRNA [17] to suppress Bim expression and to provide protection from apoptotic stimuli and, as shown in Figure 3(A), found that a 4 h treatment of cultured hippocampal neurons with Pen1–siBim yielded substantial knockdown of Bim expression (Figure 3A). Cultures of hippocampal neurons were pre-incubated with Pen1–siBim for 3 h and then bVAD-fmk was added for 2 h followed by the addition of Aβ1–42. After 4 h of Aβ1–42 exposure, the neurons were harvested and activated caspase-2 was detected by Western immunoblotting. This revealed that knockdown of Bim did not alter the activation of caspase-2 by Aβ1–42 (Figure 3B), thus indicating that Bim is not upstream of caspase-2 activation in this model.
Bim induction by Aβ1–42 is blocked by a pan-caspase inhibitor
Since Bim does not appear to act upstream of caspase-2 activation, we next considered that it may act downstream. As a first step to determine whether caspase activity is required for Bim induction, we utilized the pan-caspase inhibitor bVAD-fmk, which we have shown above captures active caspase-2, and should inhibit any subsequent action of caspase-2 and any other captured caspases. Cultured hippocampal neurons were pre-treated with bVAD-fmk for 2 h, followed by the addition of Aβ1–42 for another 4 h, and then assessed for Bim mRNA and protein levels. Caspase inhibition by bVAD-fmk blocked induction of both Bim transcripts and protein (Figures 3C and 3D). Western immunoblotting confirmed the capture of activated caspase-2 under these conditions (Figure 3E). In contrast, when bVAD-fmk was added 2 h after Aβ1–42, a time at which caspase-2 has already been activated (Figure 2B), induction of Bim mRNA and protein still took place (Figures 3C and 3D). These findings thus support the idea that caspase activation occurs upstream of Bim induction.
Induction of Bim mRNA and protein by apoptotic stimuli requires caspase-2 expression
We next examined whether caspase-2 is specifically required for Bim induction by apoptotic stimuli. We used a caspase-2-specific siRNA (siCaspase-2) conjugated to Pen1. In cultured hippocampal neurons knockdown of caspase-2 with this reagent was evident within 2 h and appeared to be maximal by 4 h (Figure 4A). Knockdown of caspase-2 in these neurons completely blocked the induction of Bim mRNA that occurs after 4 h of Aβ1–42 treatment (Figure 4B). Similarly, Pen1–siCaspase-2 fully inhibited the capacity of Aβ1–42 to induce Bim transcripts in cultured sympathetic neurons (Figure 4C). Finally, caspase-2 knockdown repressed Bim mRNA induction caused by NGF withdrawal from sympathetic neurons (Figure 4C). Taken together, these data suggest that caspase-2 expression is required for transcriptional regulation of Bim in two different apoptotic models and two different neuronal types. To further support this conclusion, we utilized sympathetic neurons cultured from caspase-2-null mice. Bim mRNA was measured following 4 h of Aβ1-42 treatment or NGF deprivation. In both paradigms, in contrast with sympathetic neurons from wild-type mice (Figure 4C), Bim transcripts were unchanged in the caspase-2-null neurons (Figure 4D).
Parallel experiments were carried out to examine the role of caspase-2 in the elevation of Bim protein levels by Aβ1–42 and NGF deprivation. Pen1–siCaspase-2 fully inhibited the capacity of Aβ1–42 to increase Bim protein expression in cultured hippocampal neurons (Figure 4E) after 8 h of treatment. As a control, we employed a Pen1–siRNA targeting the firefly luciferase gene. In contrast with Pen1–siCaspase-2, this construct had no effect on the increase in Bim protein levels caused by Aβ1–42 treatment (Figure 4F). We also compared the effects of apoptotic stimuli on Bim protein expression in wild-type and caspase-2-null sympathetic neurons. To achieve this, cultures (with and without Aβ1–42 treatment or NGF deprivation for 5 h) were immunostained for Bim expression and scored in a blinded manner as described previously [17] for the proportion of neurons with high Bim staining. In cultures from wild-type animals, there was a substantial increase in the proportion of neurons that showed high Bim staining and this response was completely blocked by pre-treatment with Pen1–siCaspase-2 (Figure 4G). In contrast, apoptotic stimuli caused no significant change in Bim expression in caspase-2-null neurons (Figure 4G). Collectively, these findings indicate that caspase-2 is required for induction of Bim mRNA and protein in neurons after Aβ1–42 exposure and NGF deprivation.
Bim induction by Aβ1–42 requires RAIDD expression/caspase-2 activation
The above studies indicate that caspase activity and caspase-2 expression are necessary for Bim induction by Aβ1–42 exposure or NGF deprivation. We next specifically addressed whether such induction requires caspase-2 activation. To do so, we took advantage of prior findings that caspase-2 activation requires the death adapter RAIDD [30–32]. RAIDD expression is also necessary for neuron death caused by NGF deprivation [31,32] and Aβ1–42 treatment [31]. We used a Pen1-linked RAIDD siRNA (Pen1–siRAIDD) that effectively knocks down RAIDD mRNA and protein levels in cultured hippocampal neurons (Figure 5A) and that protects hippocampal neurons from death induced by Aβ1–42 [31]. Simultaneous treatment with Pen1–siRAIDD suppressed the induction of Bim protein elicited by 8 h of exposure to Aβ1–42 (Figure 5B). Thus, it appears that RAIDD expression and therefore caspase-2 activation are required for Bim induction by Aβ1–42.
Caspase-2 acts upstream of Bim induction by enabling activation of c-Jun
Bim induction by apoptotic stimuli requires transcriptional activation that can be mediated by a variety of transcription factors [17,33–35]. We therefore next examined whether caspase-2 might function upstream of transcription factor activation. Here, we focused on c-Jun which is reported to be elevated in neurons from AD patients and which is activated in response to a cascade of phosphorylation events set in motion by Aβ1–42 treatment [11] or by NGF deprivation [17,33–35]. Inhibition of this phosphorylation cascade blocks both Aβ1–42- and NGF deprivation-mediated Bim induction as well as neuron death [11,17].
To assess the potential role of caspase-2 in c-Jun activation, we treated hippocampal neurons with Aβ1–42 for 8 h in the presence or absence of Pen1–siCaspase-2 and carried out Western immunoblotting to detect phospho-c-Jun. Aβ1–42 caused robust phosphorylation of c-Jun on Ser63 leading to nuclear translocation, which was significantly inhibited by Pen1–siCaspase-2 (Figure 6A), and by Pen1–siRAIDD (Figure 6B). Complementary experiments were conducted to assess the effects of Pen1–siCaspase-2 and Pen1–siRAIDD on Aβ1–42 induction of nuclear phospho-c-Jun. Hippocampal neuron cultures were treated with or without the two Pen-1 siRNAs and with or without Aβ1–42 for 8 h, immunostained with anti-phospho-c-Jun and then blindly assessed for the proportion of neurons with strong nuclear staining for phospho-c-Jun. Aβ1–42 alone elicited a large increase in the proportion of neurons exhibiting strong nuclear phospho-c-Jun immunostaining, which was fully blocked in the presence of Pen1–siCaspase-2 or Pen1–siRAIDD (Figure 6C). To assess the effect of Pen1–siCaspase-2 on the appearance of nuclear phospho-c-Jun in response to NGF deprivation, sympathetic neuronal cultures were treated with or without Pen1–siCaspase-2 and maintained with or without NGF. The cultures were then analysed for strong nuclear phospho-c-Jun immunostaining as described above for the Aβ1–42 treatments. NGF deprivation induced a large increase in the proportion of neurons with strong nuclear phospho-c-Jun immunostaining and this was fully blocked in the presence of Pen1–siCaspase-2 (Figure 6D). These findings indicate that caspase-2 expression, as well as caspase-2 activation, is required for c-Jun phosphorylation/activation and nuclear localization in response to Aβ1–42 and to NGF deprivation. These observations illustrate at least one mechanism by which caspase-2 promotes Bim induction, and demonstrate that activated caspase-2 participates in c-Jun activation in response to apoptotic stimuli.
Caspase-2 is required for Bim induction in an animal model of AD pathology
To extend our in vitro studies, we determined whether caspase-2 also regulates Bim in an in vivo animal model of AD pathology. Previous studies have shown that infusion of Aβ1–42 into the hippocampus causes neurodegeneration by 2 weeks [36]. We infused Aβ1–42 (0.4 nmol) or vehicle alone via convection-enhanced delivery (Figure 7A) [24] into the right hippocampi of 16-month-old wild-type (n=4) and caspase-2-null (n=4) mice. The animals were killed 2 weeks later and the brains prepared for Fluoro-Jade B and Bim immunohistochemical staining. Consistent with the previously described role of caspase-2 in neuron death caused by Aβ1–42 in vitro [10], Fluoro-Jade B staining revealed evidence of neurodegeneration in wild-type, but not caspase-2-null, brains; staining increased 15-fold in wild-type mouse brains injected with Aβ1–42, but there was no change in the caspase-2-null brains (Figure 7B). Immunostaining of brains from wild-type mice showed a 3-fold increase in Bim expression within cortical neurons near the site of infusion for animals receiving Aβ1–42 compared with those infused with vehicle alone (Figure 7C). No such increase occurred in the brains of caspase-2-null mice infused with Aβ1–42 (Figure 7C). Taken together, these findings confirm Bim induction in an animal model of AD pathology and indicate that, in this model, caspase-2 and Bim up-regulation are required for neurodegeneration.
In the present study we examined the mechanism by which caspase-2 participates in neuron death and its position in the hierarchy of apoptotic signalling cascades triggered by β-amyloid exposure and NGF deprivation. Surprisingly, our findings place caspase-2 upstream of the transcription-dependent induction of Bim, a BH3-only protein that participates in neuron death in a variety of apoptotic paradigms (Figure 8) [17,37–41].
Although gene silencing and knockout studies have strongly supported the participation of caspase-2 in multiple apoptotic paradigms in both neuronal and non-neuronal cells, the absence of a strong developmental phenotype in caspase-2-null mice has led to questions about its physiological functions in vivo [5]. However, compensatory changes in gene regulation have been observed when apoptosis-related genes are deleted [42] and, in this regard, we have reported that both caspase-9 and Diablo [direct IAP (inhibitor of apoptosis)-binding protein with low pI]/Smac (second mitochondrial-derived activator of caspase) levels are elevated in neurons isolated from caspase-2-knockout animals [11]. Moreover, recent studies with caspase-2-null animals show significant protection of neurons from apoptotic stresses including optic nerve transection, hypoxia/ischaemia, excitotoxic stress and MPTP treatment [14,15]. We additionally report in the present study that neurons in caspase-2-null mice are protected from in vivo β-amyloid exposure.
Several different types of evidence in the present study support the positioning of caspase-2 upstream of Bim induction. First, bVAD-fmk trapping shows rapid activation of caspase-2 by apoptotic stimuli and, at least in the β-amyloid model, this occurs well before induction of Bim mRNA or protein. Secondly, the addition of bVAD-fmk before apoptotic stimulation, which will both trap and inhibit initiator caspases, blocks Bim induction. Thirdly, Bim induction by apoptotic stimuli is abolished in neurons in which caspase-2 is either silenced or deleted. Fourthly, knockdown of RAIDD, an adapter protein required for caspase-2 activation and neuron death in the apoptotic paradigms that we employed, suppresses Bim induction. Fifthly, silencing of either caspase-2 or RAIDD inhibits phosphorylation/activation and nuclear translocation of c-Jun, a transcription factor required for efficient induction of Bim in the apoptotic paradigms that we studied. Lastly, neurons of caspase-2-null mice are resistant to Bim induction and to degeneration promoted in vivo by infusion of β-amyloid.
In addition to placing caspase-2 upstream of Bim induction, our findings indicate that this is a cell-autonomous mechanism. The Pen1-linked siRNAs that we used to silence caspase-2 and RAIDD efficiently enter essentially all treated cells [26], thus arguing against cell-extrinsic and bystander mechanisms.
Our bVAD-fmk capture experiments indicate that caspase-2 is activated soon after neurons encounter apoptotic stimuli; within 30 min for β-amyloid treatment and 2 h for NGF deprivation. This is well before any evident sign of neurodegeneration occurs (8–12 h) and is consistent with the role of caspase-2 as an apical regulator of a pathway that requires downstream activation of genes such as Bim. The rapidity of caspase-2 activation places this event close to the initial responses of neurons to apoptotic stimuli. In this regard, it may be relevant that capase-2 activity can be regulated by phosphorylation/dephosphorylation [43].
Since Bim has been described to act upstream of a caspase cascade [19,37], we also considered the possibility that Bim might function upstream of caspase-2 activation. However, knockdown of Bim, which is protective in the apoptotic paradigms used in the present study had no apparent effect on caspase-2 activation as monitored by bVAD-fmk capture.
Positioning caspase-2 to a proximal role in the apoptotic cascade has raised the major question of how it might influence mitochondrial release of apoptotic substances. Our observation that caspase-2 activation leads to Bim induction addresses this question. An alternative mechanism that has been raised by which activated caspase-2 may affect mitochondria is by cleavage of the BH3-only protein Bid [44–46]. When cleaved, the truncated form of Bid (tBid) contributes to cell death by promoting mitochondrial release of apoptosis-inducing proteins such as cytochrome c [47]. However, it has been questioned whether the efficiency with which caspase-2 cleaves Bid would be sufficient to promote death [44]. In any case, it remains to be seen whether caspase-2-dependent Bid cleavage occurs in the systems examined in the present study, and whether this plays a role in the cell death process along with elevated Bim.
In many death paradigms, including NGF deprivation and β-amyloid exposure, apoptotic stimuli initiate signalling pathways that culminate in activation of transcription factors, leading to the induction of death promoting genes such as Bim. A number of these transcription factors have been described as influencing Bim expression including CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein], FoxO (forkhead box O), c-Jun, B- and C-myb, NF-Y (nuclear transcription factor Y) and Egr1 (early growth response 1) [16,17,33–35,48]. In the present study, we focused on c-Jun which is phosphorylated/activated in response to NGF deprivation and β-amyloid treatment and is required for Bim induction and neuron death in both paradigms [11,18]. We found that β-amyloid and NGF deprivation stimulated c-Jun phosphorylation/activation and nuclear translocation and that these effects were suppressed by knockdown of either caspase-2 or RAIDD. Thus, it appears that at least one upstream mechanism by which caspase-2 mediates Bim induction is by stimulating c-Jun transcriptional activity. An important next step is to determine whether caspase-2 is involved in regulating additional transcription factors including those listed above.
The mechanism by which activated caspase-2 promotes c-Jun phosphorylation is currently unknown. Apoptotic regulation of c-Jun in neurons requires a cascade of events that includes successive activation of upstream protein kinases including MLKs (mixed-lineage kinase), MKKs (mitogen-activated protein kinase kinases) and JNKs as well as the involvement of scaffold proteins such as POSH (plenty of SH3s) and JIPs (JNK-interacting proteins) [49,50]. A parsimonious model for stimulation of this pathway by activated caspase-2 is that caspase-2 cleaves a suitable substrate. This cleavage may lead to either activation of a required initiator of the pathway or removal of an inhibitory regulator. In this light, it has been reported that caspase-2 participates in doxorubicin-induced apoptotic death of non-neuronal cells by cleaving/activating PKCδ (protein kinase Cδ) that in turn stimulates JNK signalling and c-Jun phosphorylation [51]. However, we were unable to detect any cleavage of PKCδ in either of our apoptotic paradigms (results not shown).
c-Jun has been shown to regulate several other proteins in addition to Bim that mediate death of neurons including the BH3-only protein DP5/HRK (activator of apoptosis harakiri) [52] and the prolyl hydroxylase SM20/EGLN3 (Egl nine homologue 3) [53]. It thus seems possible that induction of these and potentially other apoptotic proteins will also require caspase-2 activation. Furthermore, JNK/c-Jun signalling and Bim induction have been implicated in a variety of cell death paradigms in addition to those examined in the present study [54,55], raising the possibility of caspase-2 involvement in a wide range of apoptotic conditions.
Previous studies have indicated that caspase-2 expression and activation, JNK/c-Jun signalling and Bim induction are required for neuron death promoted by NGF deprivation and β-amyloid exposure [10,16–18]. We further show in the present study that these are hierarchically and causally linked in the same apoptotic pathway. Bim induction and neuron death were also found to be downstream of caspase-2 in an in vivo model of AD. These findings may be directly relevant to AD in which β-amyloid [56,57] and defective trophic factor signalling [58] have been implicated in the neurodegeneration and death that are characteristic of this disorder. We observed that both Bim and caspase-2 are elevated and co-expressed in a vulnerable population of neurons in post-mortem brains of AD patients. Immunostaining for c-Jun [59] and phospho-c-Jun [60] is also enriched in AD neurons. Taken together, these observations indicate that each of the elements of the apoptotic pathway described in the present study are present and abnormally expressed in AD neurons. Given the apical position of caspase-2 in this pathway and its apparently limited role in unstressed cells, this caspase and its activation mechanism may be suitable targets for clinical intervention in AD.
Ying Jean, Elena Ribe, Lloyd Greene and Carol Troy conceived the study and designed the experiments. Ying Jean, Elena Ribe, Maria Elena Pero, Marina Moskalenko, Zarah Iqbal and Lianna Marks performed the experiments. Ying Jean, Elena Ribe, Lloyd Greene and Carol Troy analysed the data and wrote the paper.
This work was supported, in part, by the NIH [grant numbers 2RO1NS043089 (to C.M.T.) and 5R01NS033689 (to L.A.G.)].
We thank Rebecca Goldstein and Nathalie Celcis for excellent technical assistance.
Abbreviations: AD, Alzheimer's disease; BH3, Bcl-homology domain 3; Bim, Bcl-2-interacting mediator of cell death; bVAD-fmk, biotin-Val-Ala-DL-Asp-fluoromethylketone; ced-3, cell-death determining 3; E18, embryonic day 18; JNK, c-Jun N-terminal kinase; ERK, extracellular-signal-regulated kinase; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NGF, nerve growth factor; NGS, normal goat serum; P1, postnatal day 1; Pen1, penetratin 1; PKCδ, protein kinase Cδ; qPCR, quantitative PCR; RAIDD, RIP (receptor-interacting protein)-associated ICH-1 [ICE (interleukin-1β-converting enzyme)/CED-3 homologue 1] protein with a death domain
- © The Authors Journal compilation © 2013 Biochemical Society


text:
Sarcasm(which is a weird juxtaposition with honesty)
Attempting to be really good at cooking(constantly learning)
and I'm pretty good with a bowstaff
Favorite movie: I absolutely love movies, my favorite is the Usual Suspects
Favorite Music: I love live music of any kind, if i had to choose its folk music is my favorite kind but I love Dave Matthews as well.
Favorite food: anything. Trying to teach myself how to cook many different things
my phone(although I am trying to cut the tie)
A red sox hat
My dogs
my pictures

subset: commoncrawl


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Borrowing a bridging loan will be a good option for you, if you are searching for finance to buy a new property, but you have not sold the existing one. People frequently get stuck in such circumstances when they find their perfect property and don't want to loose it due to lack of adequate funds. Fast bridging loans makes it feasible for you to buy the property of your interest even prior to selling your old property.
Bridging Finance are short term loans. These loans are available for those who want to buy a new property but are not in a position to sell their present property immediately. It helps the borrower to bridge the monetary gap and fulfill his/ her specifications until he/ she is able to arrange the essential finance. Therefore by borrowing a bridging loan you will be able to meet the monetary specifications which come up between promoting your present property and purchasing a new one.
You can borrow a bridging loan from a principal lender by supplying collateral. You can offer your house or any other property as the collateral while borrowing the loan. The quantity approved as the loan is based on the value of the collateral supplied. These are short term loans, consequently, the repayment period in brief and the rate of interest is greater compared to the other types of loans. Nevertheless, there are lenders with low price of interest. The maximum limit of quantity offered as loan by the lender, varies from 1 lender to another. In case you fail to repay the loan within the fixed time period, the collateral placed by you is repossessed. It is always advisable to do a correct research on the amount of loan offered and the rate of interest, prior to you lastly borrow a loan. You can find a lot of information on bridging loans on the internet itself.
You can avail a bridging loan even if you have a poor credit history. For you the rate of interest can be considerably higher, but you also get a opportunity to enhance your credit score.
Bridging loans can be borrowed to fulfill different monetary requirements whether or not it is for personal factors such as vacation, marriage or buying a new property for personal requirements or for industrial reasons such as buying an office premises, purchasing resources etc.
The principal lender offers two kinds of bridging loans - closed bridging loans and open bridging loans. Closed bridging loans are for those borrowers who have currently sold their current property. A closed bridging loan is usually for a set period of time. Open bridging loans on the other hand are for borrowers who want to buy a new property but have not sold their present property.
In short, a bridging loan is the very best solution to bridge your financial gap in times of emergency. It will help you to buy your ideal property even if you are not able to sell your current property in time. Whether the property is for your personal require or commercial specifications, it will assist you to cover the monetary gap. But whilst borrowing a bridging loan you require to keep in mind that the repayment period is short and the price of interest are comparatively greater.


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DENVILLE TWP. - Ernest Palestis, currently superintendent of the Mine Hill School District, will be appointed superintendent of the Morris Hills Regional District at the Monday, April 30 meeting of the Board of Education, officials announced Friday, April 20.
Palestis, 51, has been superintendent of the grade K-6 Mine Hill District for the past 12 years.
He will assume duties at the 9-12 regional district on July 1.
An educator with some 28 years of experience in New Jersey public schools, including 14 years in high schools, will replace James McNasby.
McNasby, who announced his retirement last Fall, effective Dec. 31, later agreed to stay on as an interim superintendent until his replacement was found.
Palestis earned both a bachelor's and a master's degree at Jersey City State College. He also earned a second master's degree at William Paterson College and his doctorate in education from Nova University in Florida.
Mine Hill students go to the Dover Schools for middle school and high school.
There are about 400 students in the K-6 Mine Hill elementary schools.
Residents of Rockaway Township, Rockaway Borough, Wharton and Denville attend the two-high School Morris Hills Regional District. Current enrollment is about 2,200.
The Canfield Avenue School in Mine Hill was named one of the 10 choice schools in the state and the only such school in this county. Choice schools are allowed to accept children from other districts on a tuition basis.
Canfield was recognized last year by then-State Commissioner of Education David Hespe as one of the state's best schools. It has Internet access in every classroom and new science and computer laboratories. The honor was memorialized in a resolution by the state Legislature.
Hespe, however, rejected a request from the Mine Hill District regarding its seventh and eighth graders. The Mine Hill Board of Education sought to have its middle school students withdrawn from the Dover district citing tuition hikes from Dover that could affect its own ability to effectively deliver its program at Canfield School.
That request was rejected but the Mine Hill school board is considering a possible appeal of that ruling.
Residents of Rockaway Township, Rockaway Borough, Wharton and Denville attend the two-high School Morris Hills Regional District. Current enrollment is about 2,200.


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What is JotForm?
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At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
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Did I just cancel my subscription?Asked by william_bernhard on April 24, 2016 at 12:08 PM
I was trying to downgrade to the free plan and I am concerned I canceled my subscription—I didn't mean to do that
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text:
Poison Ivy is Gotham City's resident eco-terrorist. A unique human/plant hybrid, she cares only for her beloved plants and their ultimate dominion over the planet. Poison Ivy's once-human body has been chemically altered and can create any number of pheromones and toxins to entice or destroy.
Her unique bond with plants means she can swiftly generate new breeds or plant explosive seed pods on her enemies.
Pale, gangly and unremarkable, Pamela Isley was a shy botanist when she went to work for famed scientist Dr. Jason Woodrue. Hoping to create a human/plant hybrid, Woodrue experimented on Isley, creating the beautiful-but-deadly Poison Ivy. Now a physical beauty with chlorophyll instead of blood and capable of exuding various pheromones and natural toxins, Ivy began a criminal career to fund her cause as a champion for the world's diminishing flora species.
During the War of the Gods incident, caused by Circe, Ivy stumbled upon Circe's stronghold in the Amazon and was captured by the Bana Mighdall. Hung up and about to be tortured, she was saved when the Suicide Squad, led by the Black Adam, co-incidentally attacked the stronghold to stop Circe.
Combat StatisticsEdit
- Poison Ivy (The Greenhouse)
- Poison Ivy (Arkham Asylum)
- Villainy United
- Poison Ivy (Harley's Heist)
- Poison Ivy (Wayne Manor Gala)
- She is also the center of attention of the Spring Seasonal event where Heroes must stop her plants from taking over Gotham.
- Poison Ivy has released her legions of plants to spread across the Giordano Botanical Gardens area. Robin has gone ahead to investigate. You must clear the area of her plant pods and locate Robin inside Poison Ivy's lair.
- She is one of the bosses in Arkham Asylum (Alert). She, Scarecrow, and Mr. Freeze have taken over Arkham Asylum and Heroes must fight them.
- Heroes must stop her, Harley Quinn and Catwoman during Harley's Heist.
- Heroes must stop her, Harley Quinn and Catwoman at the Iceberg Lounge.
- Heroes must stop her, Harley Quinn and Catwoman at the Wayne Manor Gala.
- She is also the center of attention of the Spring Seasonal event where Villains must help her plants from taking over Gotham. She's located in The Pit where she gives villain players their quest.
- She is one of the bosses in Arkham Asylum (Alert). She, Scarecrow, and Mr. Freeze have taken over Arkham Asylum and Villains must fight them.
- Villains must help her, Harley Quinn and Catwoman during Harley's Heist.
- Villains must help her, Harley Quinn and Catwoman at the Iceberg Lounge.
- Villains must help her, Harley Quinn and Catwoman at the Wayne Manor Gala.
- Poison Ivy first appeared in Batman #181 (June 1966).
- Poison Ivy is voiced by Cyndi Williams.
- When discussing her origins, Ivy originally stated that she was a botanist who assisted Marc LeGrande in stealing ancient Egyptian poisonous herbs. When he attempted to kill her with them, she survived and discovered her current powers. However, she has since admitted that this origin was a lie.
- Ivy's best friend is the Joker's sidekick, Harley Quinn.
- Ivy's skin is green due to the deadly toxins that flow though her body. As such she is immune to most toxins and possesses a "poison kiss" which can paralyze and kill her foes. By manipulating the amount of toxins in her veins, Ivy can return her skin colour to it's previous "human pink" hue.
- Ivy's physiology is dependent on receiving a certain amount of sunlight, which she absorbs and converts to energy in much the same way plants do. She becomes greatly weakened if deprived of sunlight for an extended period of time.
- When fighting in The Greenhouse, her stance is that of Martial Arts. Yet in the Arkham Asylum (Alert) her stance is that of Hand Blaster.
- One year after the initial decommissioning of the Suicide Squad and Ivy's release, Ivy became the lover of General Vaca, the president of Rio Brava; the capital city of Pueto Azul, South America. Ivy took advantage of her status to plunder the nation's treasury, by the time Batman had come to recruit her for the new Squad she had driven the country's populace into rioting for her and the General's execution.
- Both self professed "Guardians of The Green", Poison Ivy and Swamp Thing are considered to be mirror images of each other; where the latter is plant life convinced it is still a man, while the more humanoid Ivy will willingly sacrifice the lives of humans in favour of her beloved plants.
See alsoEdit
External linksEdit
- Poison Ivy (comics) Wikipedia
- Poison_Ivy DC Database
|v · e · d
|Amanda Waller - Bane - Black Adam - Captain Boomerang (George Harkness) - Captain Boomerang (Owen Mercer) - Captain Cold - Deadshot - Doctor Light - Duchess - Harley Quinn - Killer Frost - King Shark - Mirror Master - Oracle - Parasite - Penguin - Poison Ivy - Power Girl - Solomon Grundy - Speedy - Steel - Superboy - Vixen
|Batman - Lois Lane
|Chemo - Vicki Vale
|Bombshell Belle Reve's Poster
|Expert Marksman - Punchline
|Belle Reve Federal Penitentiary
|Belle Reve Sanitarium
|Checkmate - United States of America - Justice League of America - Justice Society of America


text: Yuri Sergeyevich Sergeyev (1843? - March 28, 1911) was an Alaskan politician and soldier, best known for his service in the Alaskan War as one of the chief aides to Boris Anasenko, primarily due to his leadership of the cavalry of the Army of the Pacific. After the war, Sergeyev declined a civilian office under the new leadership formed out of the Dmitrov Coup, feeling weary after years of war and disgusted by the terms of the Treaty of Sofiyagrad. Sergeyev remained a cavalry officer in the army, twice declining more significant promotions, and retired to Siberia in 1904, disillusioned with the Anasenko regime. He died of pneumonia in 1911 at the age of 68.


text:
From the Publisher
When the ESF/ AGAST war broke out both sides would use any angle to their advantage even assassins. As the war continued they to were forced to choose a side. However when one assassin rose up with a gift the two sides tried to not let the other one have him. However he too was forced to choose a side. In the year 2173, Teymos became not only the most powerful assassin in the galaxy but also the most wanted. As the bounty rises old friendships will be tested and new bonds will be forged in blood.
Published: Xlibris on Apr 26, 2013


text:
Still @ the lake. But, what was it? Yup, it's one of the creeks that flowed right into the lake, I suppose. Yet, the garden itself was one of those gardens that surely quintessentially Korean. It's a typical Korean garden that surely captivated those who devoured such a thing. It's summer, and yet..it's good to look at. I mean..I am really into autumn/spring..and the fact that this garden is indeed a sight to behold means only one thing--> it's beautiful.
Or was it because we rarely see this kind of garden around our neighborhood? dunno. But, it would've been a vain regret had we not taken a picture right in front of it.^^
and..let's do ONE selfie so as to have 'ME' in the pic.^^


text:
Country Living Kit: Belle of the Ball
- Available in 2 sizes: 12×12″ Classic Kit & 16×16″ Big Kit
- Includes: Genuine canvas on natural wood floating frame, acrylic paints, brushes, instructions.
- Available for shipping within the US only.
Officially Licensed Product of Country Living magazine.
Additional Information
12×12″ Classic Kit, 16×16″ Big Kit
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text:
Removal & Installation
- Before servicing the vehicle, refer to the Precautions Section.
- Check to be sure that the park brake is released.
- Raise and support the vehicle.
Remove the tire and wheel assembly.
NOTESupport the brake caliper with heavy mechanic's wire, or equivalent, to prevent damage to the flexible brake hose.
- Remove the brake caliper and the caliper mounting bracket as an assembly from the suspension knuckle.
- Matchmark the position of the brake rotor to the wheel studs.
- Remove the brake rotor mounting screw.
- Remove the brake rotor. Do not force the rotor off. If the rotor is difficult to remove, ease it off by gently rotating it as you pull outward.
To install:
If installing a new brake rotor, adjust the clearance of the parking brake shoe to the drum-in-hat portion of the brake rotor.
- Install the brake rotor to the hub/axle flange. Use the matchmark made prior to removal for proper orientation to the flange.
- Install the brake rotor mounting screw tighten to 124 inch lbs. (14 Nm).
- Install the brake caliper and the brake caliper bracket as an assembly to the suspension knuckle.
- Install the tire and wheel assembly.
- Lower the vehicle.


text:
What is employee efficiency? In accordance with a Whatis.com guide, "Productivity could be examined with regards to a member of staff in a particular period of time's productivity. Typically, the given worker's productivity will be considered relative to a typical for personnel performing similar work. Because a lot of the achievement of any firm relies upon the efficiency of its staff, worker output is definitely an important consideration for businesses. "Why should you desire a cell phone monitoring software?
From portable phone technology's inception, interaction in operation options has actually improved. However, the release of cell phones is a double edged sword. Similarly, it has the advantages all of US know of and to the other; it has manufactured corporations to undergo, where it's employed without control. When you have witnessed that your personnel are misusing the phones, or they're currently doing badly, you are able to install monitoring application. By doing this, you'll be able to understand what the staff is performing constantly. Check out the Highster mobile site, it can help track all of the staffs' activities and offers you using a complete record.
Tracking your employee's efficiency
The ultimate aim of any enterprise is to make gains, and also this has to be your purpose too. There has to be excellent input from the individuals in case a company must produce earnings. Using the mobile phones' distractions, this desire may be from being achieved far. Specifically, for agencies that have workers inside the field, the method to gauge whether they are currently working is by using spy software.
When it is becoming a nightmare monitoring your employees, it's greatest in the event that you consider Mobile. Does Highster Mobile Software work? It is really a stress direction method that is free. You may not need to enjoy hide-and-seek together with your personnel; only install the cell phone monitoring software and leave it at that. But remember, it's only appropriate to put in the spyware about the phones that are company's; otherwise, you'll be slapped with substantial penalties. Together with the software, you are ready to learn if they employed the automobile for their individual opportunities, or whether that distribution was built by the merchant on time. This will allow you to evaluate if the staff is currently working towards reaching the business' definitive goal or not. Using this, you possibly can make choices that are informed.
Advantages your business security
Having a competitive advantage is the imagine every organization. Once the company has some key methods for functioning, which their opponents are not alert to this comes. To ensure your special limitations of functions remain secret, be sure to deploy Mobile. From selling off your organization tips to competitors with this spy software, you'll be able to avoid employees. Additionally, additionally you reach capture the individuals whose operations can be a threat for the company.
Makes exchange of data easy.
Cell phone monitoring application may allow the people to share necessary information where employees are ATEAM. Whether or not the employee is traditional or online Mobile permits other employees to gain access to the phone documents and acquire the things they desire.
Surveillance doable.
You certainly don't have time for you to maintain eavesdropping on your own personnel because they make calls. One other option is befriending work gossiper, that you do not need to take. That is why nothing may monitor much better a mobile phone better than highstermobile com.
The circulation of connection among the team members is fantastic, when your personnel will work as expected so when the stability are at its best, efficiency is assured. Highster Mobile cell phone spy App claims every one of the aforementioned benefits to you. Ensure that you install it, you may be assured that you will see no regrets.


text:
Sure 48h invisible black & white
£1.60 (£3.20 per 100ml)
Product Summary
Women Crystal Invisible Black & White Roll-On Anti-Perspirant Deodorant
Aqua, Aluminum Zirconium Pentachlorohydrate, Glycerin, HelianthusAnnuus Seed Oil, Steareth-2, Parfum, Steareth-20, Caprylic/Capric Triglyceride, Gelatin Crosspolymer, Cellulose Gum, Sodium Benzoate, Hydrated Silica, Disodium EDTA, Pentaerythrityl Tetra-Di-t-Butyl Hydroxyhydrocinnamate, Potassium Lactate, Alpha-Isomethyl Ionone, Benzyl Alcohol, Citronellol, Geraniol, Hexyl Cinnamal, Limonene, Linalool
Please note that while we take every care to make sure the product information displayed on our website is correct, product recipes are regularly changed. This may affect nutrition and allergen information therefore you should always check product labels and not rely solely on the information presented here.If you require specific advice on any Waitrose branded product, please contact our Customer Care Team. For all other products, please contact the manufacturer.This information is supplied for personal use only. It may not be reproduced in any way whatsoever without the prior consent of Waitrose Limited nor without due acknowledgement.


text:
|This article or section is a stub. You can help by expanding it.
|Type 38
|Torpedo boat
|No. of positions
|Position 1
| Helmsman
Torpedo tubes
|Position 2
|20mm gun
The Type 38' is a Japanese torpedo boat.
Withhin the Japanese Navy, small vessels for near-coast duties like Motor Torpedo Boats (MTB) or Gun Boats played no significant strategic or tactical role prior and through WW2. Development and production of such vessels had no much priority. As a result, Japanese MTB development was far behind other navies. The Japanese boats especially lacked powerful engines which made them slow and seriously reduced combat value. They also suffered from bad supply and maintanance. 248 torpedo and gun boats where build by the Sasebo shipyard for the IJN in WW2, and among the most numerous with 80 examples build was the Type 38 (B) Motor Torpedo Boat (H-10 class). The boats were 18m long and 4.30 wide, powered by air-cooled radial engines with 1075 hp. Maximum speed was 27,5 kts. This gave the boats little offensive capacity, so the Type 38 boats served mainly as coastal patrol and harbour guard.
In Battlegroup42, the Type 38 torpedo boat is armed with two torpedoes on its sides and mines at the back for anti-shipping purposes. The gunner has access to a 20mm Oerlikon gun, which is useful against small surface targets, as well as low flying aircraft.
It can be found on a number of maps in the Pacific theatre, such as 4506-Oboe Six and 4209-Bloody Ridge.
The model is modified from the original Battlefield 1942's Type 38.


text:
Simplicity 2778; ca. 1938; Misses' Dress. Peter Pan collar and buttons down the front. Gathers from the shoulder. Three-quarter or short draped sleeves. May also be made with a short tailored sleeve. Skirt joins blouse at high waistline and has flared godet.
Links to reviews/blog postsEdit
Add a photo to the gallery by clicking the "modify" button below.
[insert your username, and make sure your preferences allow for people to contact you via e-mail. Do not list your email address here!]


text:
Choice modelling approaches: a superior alternative for environmental valuation?
Hanley, N. and Mourato, S. and Wright, R.E. (2002) Choice modelling approaches: a superior alternative for environmental valuation? OECD Economic Surveys, 15 (3). pp. 435-462. ISSN 0376-6438Full text not available in this repository. Request a copy from the Strathclyde author
In this paper, we examine some popular 'choice modelling' approaches to environmental valuation, which can be considered as alternatives to more familiar valuation techniques based on stated preferences such as the contingent valuation method. A number of choice modelling methods are consistent with consumer theory, and its focus on an attribute-based theory of value permits a superior representation of many environmental management contexts. However, choice modelling surveys can place a severe cognitive burden upon respondents and induce satisficing rather than maximising behavioural patterns. In this framework, we seek to identify the best available choice modelling alternative and investigate its potential to 'solve' some of the major biases associated with standard contingent valuation. We then discuss its use in the light of policy appraisal needs within the EU. An application to the demand for rock climbing in Scotland is provided as an illustration.
|Item type:
|ID code:
|international environmental agreements, game theory, political economy, economics, econometrics, Economic Theory, Environmental Sciences, Public Finance
|Social Sciences > Economic Theory
Geography. Anthropology. Recreation > Environmental Sciences
Social Sciences > Public Finance
|Strathclyde Business School > Economics
|Depositing user:
|Strathprints Administrator
|Date deposited:
|02 Oct 2008
|Last modified:
|23 Jul 2017 00:01
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text:
Summer Gone
A paperback edition of Summer Gone.
|David Macfarlane
|Cover artist
|Tamara Reynolds (photograph), CS Richardson (design)
|Alfred A. Knopf, Canada
Publication date
|Media type
|Print (Hardback & Paperback)
|266 pp
|Preceded by
|The Danger Tree
|Followed by
Summer Gone is the first novel by Canadian writer David Macfarlane. Published in 1999 by Knopf Canada, Summer Gone was a national bestseller in Canada. It was nominated for the Giller Prize, and won the Books in Canada First Novel Award.
Plot summary[edit]
The book deals with the life of Bailey Newling and his three lost summers. It tells the story of a divorced Bailey and his young son Caz, where on one fateful canoe trip, they share a remarkable night of truth and love.
Macfarlane set this novel among the cottage country in northern Ontario, the Waubano Reaches. Bailey, nicknamed Bay, tells of the three summers in his life: the summer he was 12 and attended the camp where he met his camp instructor Peter Larkin, the summer where he, his wife Sarah and 6 year old son rented a cottage near his old campsite and, the summer where he and his 12-year-old son shared their extraordinary night.
Macfarlane uses a notable technique in the writing of Summer Gone, where he would start the story of one summer and drift into another. It may start with Bay telling of his tale at camp and then shift onto another thought which may have occurred decades later involving his wife or his son. This technique ties all of Bay's summer stories together into one when he tells it to his son. The narration of this story is told by Caz's half brother, from a one night stand of Bailey's, as an adult, retelling what Caz had told him.
|This article about a 1990s novel is a stub. You can help Wikipedia by expanding it.
See guidelines for writing about novels. Further suggestions might be found on the article's talk page.
|This article about a Canadian novel is a stub. You can help Wikipedia by expanding it.
See guidelines for writing about novels. Further suggestions might be found on the article's talk page.


text:
Centura Health has more experts in more places throughout the region. Start your health journey by finding the right one for you.
The multidisciplinary inpatient acute stroke rehabilitation program provides intensive physical therapy, occupational therapy and speech therapy to give patients the strengths and skills they need to return to their highest level of function.
A referral can be made by a local physician, hospital case managers or by personally calling 720-321-1502 . An evaluation by a pre-admission screening liaison is required (provided at no charge) to determine rehabilitation needs.
Health insurance benefits will be verified prior to admission. Most major insurance carriers, including Medicare and Medicaid, cover inpatient rehabilitation programs.
Learn more about our Acute Inpatient Rehabilitation Program.
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Founded in 1997 in Taiwan, HTC (High Tech Computer Corporation) started its business by manufacturing notebooks and smartphones. It was the first to manufacture a Microsoft-powered smartphone in 2002) and to produce the HTC Dream, an Android-powered commercial smartphone in 2008.HTC re-entered the tablet market with the Google Nexus 9 in 2014.
If you want to sell online your old HTC tablet for top dollar in order to save money for your new electronic device, don't hesitate, you can always do it at iGotOffer.
Meet Nexus 9. Source of the photograph: HTC
Produced by HTC and designed for work and play, the Android-based Nexus 9 features a 8.9" screen with front-facing speakers for rich music and audio. 64-bit processor drives productivity and play to new levels, with intuitive voice commands and automatic Android updates that keep you on the cutting edge. The device integration makes it mobile and access to over a million apps makes it amazing.


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Filmmakers always come up with interesting ways to bring attention to their films. Here comes director Anurag Basu who made movies like Life In A Metro and Barfi, wooing our attention again.
"Jagga Jasoos" is an upcoming movie of brokeup couple Ranbir Kapoor and Katrina Kaif, which is directed by Basu and has Pritam scoring the music. In this film Pritam confirms, there are altogether 29 songs for the hero and heroine. Some are full length songs, some are bit songs, but total of 29 has come out.
That's stuns everyone for this romantic CG based film that takes us a trip to a fantasy land.


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Listen to free music by Dot Flash Line
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TerraSense - Model SMT2 - Soil Moisture and Temperature Sensor
From Sensors
Parameters under measurement: Soil Water volume content (WVC) (WVC), Soil temperature. Compatible with: MeteoSense base station/MeteoSense PRO, AgriSense/VineSense, Wireless unit AgriSense/VineSense, HandySense reader. Item Code, PS-0077-DD.
Means of Operation
- The TerraSense SMT2 sensor carries out the water volume content measurement, by measuring the soil dielectric constant.
- The specific implementation features make the sensor insensitive to soil temperature and conductivity variations.
- Furthermore, the internal analysis algorithms and the digital output allow high accuracy with all soil kinds, thus not requiring manual calibration.
Technical Specification
Soil moisture sensor
- accuracy: ± 3 %
- Output: from 0 VWC to field saturation
Soil temperature sensor
- Accuracy: 1 %
- Resolution: 1 ° C
- Output: - 20 + 50 ° C
- Operative range: - 30 + 60 °C
- International Protection: IP68
- Standard operative cable: 180 cm
- Power supply: standard 3-5VDC,on request 5-18 VDC
- Output: serial protocol over RS485 interface.
Customer reviews
No reviews were found for TerraSense - Model SMT2 - Soil Moisture and Temperature Sensor. Be the first to review!


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So on valentines day I gave this card to this girl I really like. We work together and she's really lovely, I've liked her for awhile now since round November last year now and she's known I liked her. When she recieved my card she said it was really nice and lovely it was, but when I tried to strike up a convosation with her she blanked me like nothing was said after that and its like she didn't wanna talk all of a sudden. So should I give up with her and just move on or keep trying and give it a few more days/weeks? .
Should I just give up and move on?
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Nervous System is a generative design studio that works at the intersection of science, art, and technology. Drawing inspiration from natural phenomena, they create computer simulations to generate designs and use digital fabrication to realize products.
Kinematics is a system for 4D printing that creates complex, foldable forms composed of articulated modules. The system provides a way to turn any three-dimensional shape into a flexible structure using 3D printing. Kinematics combines computational geometry techniques with rigid body physics and customization. Practically, Kinematics allows you to take large objects and compress them down for 3D printing through simulation. It also enables the production of intricately-patterned wearables that conform flexibly to the body.
This is how the Kinematic dress was born. The first Kinematics Dress was acquired by the Museum of Modern Art for its permanent collection.
Kinematics Dress by Nervous System – 3D Printed by Shapeways from Nervous System on Vimeo.


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Download Family Medical Info apk 1.9 free for Android smartphone
Posted by Appventive in Medical | Sept. 13, 2013 | 59 Comments
Apk file size: 1.3 MB
Family Medical Info
Stores medical info for the entire family for use in emergencies, doctor visits, filling out school & camp forms for the kids, etc.
*** Optionally share some or all family info with our companion app "ICE: In Case of Emergency" to make the info available even when the phone is locked. ***
For each family member you can enter as much or as little info as you like for the following categories:
+ Personal info (photo, date of birth, height, weight, blood type)
+ Allergies
+ Conditions (e.g., diabetes, heart disease, high blood pressure, etc)
+ Medications (including optional Rx numbers)
+ Insurance policies (you can call your insurance company directly from the app)
+ Doctors (you can call doctors directly from the app)
+ Medical History (e.g., surgeries, hospitalizations)
+ Vaccinations
+ Notes w/attachments (any other info you want, including attached images of insurance cards, test results, etc.)
Optionally email some or all information to your doctor or others.
Note: if you already use ICE: In Case of Emergency then you can import your ICE profile into Family Medical Info.
Optionally export profiles for use on other devices, or to save an off-device backup. Exported profiles can also be used by ICE.
*** NEW: optionally use the (FREE) web version of Family Medical Info to create & edit profiles on your computer. From Google Chrome on your computer, go to http://goo.gl/N80BQ to install the web app (currently, this only works
with the Chrome browser). Or, just search for Appventive in the Chrome Web Store. Profiles created with the web version of FMI can be imported into either FMI or ICE. ***
If you have any questions, suggestions or problems, PLEASE email us at [email protected]
Whats new
v1.9: reorder profiles + support for Greek
v1.8: new icon. Please let us know what you think.
v1.7: use the (FREE) web version of Family Medical Info to create & edit profiles on your computer that can be imported into either FMI or ICE on your Android device.
v1.6: fixes a problem some were having with entering dates
v1.5: provides auto-complete suggestions when entering medicine name in the Medication section.
Appventive part of our Medical and have average installs from 1000 to 5000. Last Update Sept. 13, 2013. Google play rating is 89.9115. Current verison is 1.9. Actual size 1.3 MB.
Similar applications
Medical Doctor: Reference Tool119 Comments
59 Comments
A Google User
Must-have app! Simple, organised interface with all the information EMTs or doctors would need in most situations. Would be great to have a direct print feature so you can share copies with medical staff.
Catherine Simpson
Great medical App!! This is a great medical App that you can use to put all your medical info in (i.e ailments, medication taken & allergies). It works side by side with ICE by the same developer, it's definitely worth having both Apps. HTC One X
Patti Stone
Great App What I was looking for. A good way to have everything in one place. My husband has an long list of medical issues & this makes it easier to find what I need in case of an emergency.
Jaisen Mahne
Does the job Happy with the software. It's simple to set up and useful in those emergencies.
A Google User
A Must Have for Anyone. This medical app allowed me to capture all the necessary information about my Family that should be sufficient for any medical institution in case of emergency. I have not experienced any problems and trust that the developer/s keeps it updated for optimum performance and to cater for everyone's needs. Thank you and blessings.
Mimi Nichols
Family medical information It's great. And has saved my life.
Tia Penrod
Great App This app is amazing help. Being a mom of a child with special needs it gives me a little piece of mind knowing EMS would know helpful information to help our daughter.
Chelesse Risley
Dropbox Gone? I was able to save profiles to Dropbox and now I cannot. Bring back that feature and I will gladly give more stars.
Debbie Little
I have been looking for an app like this for a long time. It is phenomenal!
tony nisbet
You need this Who knows who will think of looking at your phone, but your information is available to the emergency services and that could save your life.
Larry L. Wilson
Great For Storing Your Medical Information I think we will see improvements in this program over time. If you need help Tech Support is right there, prompt and friendly to help. I would recommend to others.
Joyce Weeks
Good App I really like this app. The only reason I didn't give it 5 stars is because I haven't had it that long and don't know all the ins and outs yet . I like that I can have the whole family's info in 1 place and I'll have it at my fingertips if I have an emergency. That's one less thing to worry about!
Wade Hampton
Good enough Could use more detailed forms. Otherwise ok.
Joyce Crager
Not what I expected When it mentioned helping follow conditions, I thought it was going to allow me to input daily blood pressure readings, menstrual cycles, and illnesses, things like that. Instead it is a useful tool to gather information like doctors and previous conditions but it's more of a note taking app
Chuck Decker
Crashed while entering a date. I am a prorammer with 38 years experience. There is no excuse for lack of quality control. This is an example of where 5 stars means nothing. All I did was press the "Set" button while entering a date, when it crashed. Strange that the author says in the history that this same bug was fixed. This bug has been raised from the dead.
Deanna Chavez
Easy to use I love the ease of the app and I can use real pics of family.
Rachael Sandrock
Helps with all family medical stuff and helps a lot when needing quick medical information. Its all in one place. Great app!
ConnieSue Pruitt
Love it. Is there a way to add contacts pics for law enforcement, hosp, ect.? Would provide excellent identification. If so, I'll give 10 stars!!! Thanks!!!
Vicki Shaw
Family medical info Great way to keep important info at your fingertips when you really need it
Inez Arakaki
It's ok It's ok so far, so good. At lease it helps
John Tocher
Great med info app. Good for keeping track of my medical information. Doctors don't seem able to do it here in Canada where you have medical clinics that have 6 or more doctors running around. Needs syncing with web edition for 5 stars.
Nikki Anderson-Laraway
Does exactly what I was looking for. Keeps excellent records as we move from doctor to doctor. Easy to use, and great price.
Don Coates
. Great app but needs to be movable to the SD card.
Brian Lloyd
Good, Handy App Dont use it much, but its nice to have it there to speak for me if I cant.
Mary Campbell
Family Medical Info Easy to use. Less tedious to enter data than similar apps. A couple things need tweaking but I really like it.
Vanessa Troche
REALLY GOOD! Very customizable, user friendly and you can add detailed medical info of other family members in case of emergencies.
Gary Runningbear
Nice app Best of the free ones. But direly needs to be able to back up data to sd card AND the cloud for changing to new phone.
Leon Russell
Used it for years, would not be without it.
Betty Tanner
You want me to rate this before getting used to it after using the one I had on my iPad. It's OK, does what I need it to do, but I liked my iPad one better.
ron harrison
Ron Harrison Very good easy to use and helpful.
Bob Miner
Great App This is what emergency responders look for
Tiffanie Fauer
Awesome! Can cover the whole family in one place! Priceless!!!
Barbra Fitzgerald
Great for keeping important medical info. Wonderful app for use in emergency!
Gary Sousa
Works for all the purposes I need
CJ Fattig
Very handy! I love how this app integrates with ICE. It needs the ability to photograph insurance cards.
Jan Bester
Great App Love it, I can keep track of all family members medical Thanks
Pamela Corkum
Great app Love the fact that I have everyone medical info in one place
Debra Bernal
An Added Plus I've used ICE for years already but needed a place to add the rest of my family. Thank you
Angela King
Really good app
John Johnson
John Transferred from old phone to new phone using Bluetooth. Great App. Have years of information for Wife & Myself
Karen Richard
Great App! This app is so useful when going to Dr's appointments or in the case of a medical emergency. I would highly recommend it!!
Chris de Kock
A Must Have for Anyone. This medical app allowed me to capture all the necessary information about my Family that should be sufficient for any medical institution in case of emergency. I have not experienced any problems and trust that the developer/s keeps it updated for optimum performance and to cater for everyone's needs. Thank you and blessings.
A Google User
Ok, but out of date There are situations where this info would be very useful. Holds much-needed medical information. No updates since 2013.
Michael Harber
Very good Easy to use and understand. Great interface. I do wished it had a listing for emergency contact phone numbers...like ICE. Nevertheless, five stars for sure.
A Google User
Dropbox Gone? I was able to save profiles to Dropbox and now I cannot. Bring back that feature and I will gladly give more stars. Can export to drop box again! Yay!
Shirley Boyes
Such an easy way to have your medical history with you all the time in case of an emergency.
Mary Williams
Great app I love being able to make a profile for each family member and being able to find what I need so quickly.
Gayle Mcintosh
Awesome Great job making this helpful app.
Debbie Thomas
This is one of the best apps anyone could have created. It also helpful when you go to a doctor's appointment all you have to do is give them your phone to get all your medical history
T Antz
Wasted my money All the information here can be kept on a notecard. Calling directly from the app is ok, but worthless on tablet. It gives me a whole page to input allergies (I have none), and nowhere to input my doctor's address. Definitely not for someone who really NEEDS this.
John Johnson
John Transferred from old phone to new phone using Bluetooth. Great App. Have years of information for Wife & Myself
Brandy Kelly
Great It's what any person should have. It just lacks ICE contact info.
Brandy Kelly
Great It's what any person should have. It just lacks ICE contact info. UPDATE: all updates have deleted all info, have to re-enter days after every update. NOT GOOD.
James Macko
I was able to back my data up at one time, however the newer ver. you can only back up to a SD card. My phone doesn't have SD cards. At one time you could email your data to yourself, however now you can try to save links to your profile, but when you do this the link goes nowhere. (broken link) I paid for this app 2 times due to new phones. The two stars are for the medicine part of the app.
Nancy Preston
Family health I love the app. I just wish you could record running lists of blood pressures and weight.
Sherry Genauer
Serious problem, crappy program! All my family medical info is missing!
Sharon Bibb
Dose The Job Dose The Job. The only little thing I would like is too be able to rotate photos when attached to name. Some how they apply counter clockwise.
Elise Albano
Hasn't been updated in 3 years - no longer an active app. Put in all my info before finding this out.
Tim Chetney
Awesome app I was pleasantly surprised that this app was so easy to use and will be incredibly useful to use in times of need.


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In 2013, legends of Ukrainian rock "Okean Elzy» recorded eighth Studio album, "Earth" and went to a large stadium tour in the cities of Ukraine, Russia and Belarus to present new concert program. On 22 and 23 November "Okean Elzy" will perform at Stadium Live in Moscow.
the leader of the group Sviatoslav Vakarchuk proud of the new album, recorded in Belgium with the participation of the English producer, winner of three Grammy awards Ken Nelson (Coldplay, Gomez, Kings Of Convenience, etc.). Svyatoslav Vakarchuk considers that the group was able to act in the best form and combining the highest level of performance, powerful drive and song form, typical of the best the world of rock. No wonder Ken Nelson called the new song "Okean Elzy» "classic".
the album has twelve songs including the song "Stra», which is familiar to the public as a solo project by Svyatoslav Vakarchuk "Brussels". On the cover of the album "Earth» – a blend of beans. Svyatoslav Vakarchuk says of them: "Here are the twelve kinds of seeds, including hemp. Each seed is a separate song. Because the land is valuable primarily because it gives new life".
for 2013 "Okean Elzy» get a new program parks and the sports palaces in 27 cities and more than twenty Russian cities. Svyatoslav Vakarchuk confessed in an interview that the hype surrounding the concerts were a surprise to him: "Everywhere more people coming than we expected".
Concerts in Stadium Live close Russian tour "Okean Elzy", or rather – the first part. In 2014, the group will continue touring, but their theme of to a greater extent will have the upcoming 20th anniversary of the group.
Billboard Magazine has calculated which stars last year joined your wallet more than the rest of their colleagues. And the undisputed leader in this "race" was country music singer Taylor swift. In 2013, this young artist has earned $ 39.7 million. According to the publication, much of this income provided her world tour in support of her fourth Studio album Red. However, the star is no stranger — in 2012 she was the winner of this rating.
Second place went to Kenny chesney, country musician. He last year was able to earn a little less impressive sum — 32.9 million dollars.
«Bronze» went to Justin Timberlake, who in that year after a seven-year break returned to the music scene and has released a very successful album. He earned $ 31.5 million.
the Fourth was Bon Jovi with an income of 29.5 million dollars.
And on the fifth line is the band The Rolling Stones, earned $ 26 million.
Otar Kushevskiy:
PROLOGUE. Now every air of iconic "PARTY ZONE» I'm going to comment in the corporate style, which is seventy-five million fans, which includes Galkin and Portuguese loggers. Accustom who do not know, to my refined and fierce style; cheering is not forbidden.
INTERCHANGE. What brilliant my pogrugenie LERA KUDRYAVTSEVA better than any proinfra, known and without me. But the fact that it is Number of Times at work live, we need to discuss.
live in one shimmering vibrato does not hold, there is need for great style and sense of timing. You should not feel that the time goes out, you have to know when it comes out, and manage to raise humanity mood. The most that neither is a job for pragmatic romantics, and I don't know anyone better to Read on the job.
EPILOGUE. It is authentically reported that "PP" one of my favorite programs, and knows about it, but Armand Davletyarova, the whole country, especially "VEGAS".
Stay tuned to the blog Otar Kushanashvili on our website.


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Rialto Beach Trail is a 13.6 mile heavily trafficked out and back trail located near Forks, Washington that features beautiful wild flowers and is good for all skill levels. The trail offers a number of activity options and is best used from March until October. Dogs are also able to use this trail but must be kept on leash.
dogs on leash
kid friendly
nature trips
trail running
wild flowers
This hike is probably one of the most beautiful hikes in the area The 1st half is an easy hike along the beach, after you pass the whole in the wall it becomes more of a rocky hike where you may need to use your hands to get pass the rocks. The rocks can be slippery unless you walk close to the wall to your right. You can camp at the first stop if you are running low on daylight, but there is a better camp spot around the second piece of land going into the sea. There's not too much drift wood since the many campers going there use it all, but you may be able to collect enough for a small fire.
Beautiful pebble beach with a lot of drift wood. Not easy to walk for a long time because your feet sink in the pebbles. Wear good shoes if walking the beach. Very picturesque and parking is free. Waves are intense, not for swimming.
Great time with the kids in 2001.
We only went to Hole in the Rock, but even that was very enjoyable, plenty of tide pools & photo ops. If you only have time for a few beaches I would put this on your list, it had everything you expect to see, not sure I would call this easy, I never find it too easy walking on sand, unless you can stay on the sand down by the waves
Tiffany N W.
This is where the forest meets the ocean. Easy, short walk through beautiful driftwood paths to the beach. Stunning view.
Year round A+++. Used to spend Christmas Eve's out there. Take your hammock and camp out in the tree line down by hole in the wall. Have a nice beach fire with the driftwood. Take a deep breath of the sea air and love life.
Hiked out to the Chilean site and overnighted. It was a fairly easy trip though some areas were very slick and you had to climb over quite a few rocks and logs. You have to be mindful of the tides and be sure to get a ride schedule from the ranger station. Camp site where we camped used to be pretty large but has eroded away. We Had two tents but it was a pretty tight sqeeze.
Abigail S.
This was the first west cost beach I've been on and it was awesome; we went on a cloudy, rainy day, but the sun was peaking out in a very ominous way. The washed up trees and the choppy waves proved to us just how unrelenting nature is. We didn't complete the whole trail, however we walked south until a sign stated not to go beyond a certain point as it was tribal land. We managed to see 2 bald eagles. Climbing over the rocks and trees was pretty difficult, but worth it. Part of the trail near the parking/picnic/camping area was completely flooded. The haystacks were amazing.
My favorite beach in this amazing corner of our country. It's truly magical here!
Beautiful day at Rialto. It is quite busy during the weekend, parking was full. Also many people camping up higher on the beach the entire way up the north end of the beach. The tides were too high to get to the hole and see any tide pools but it was beautiful nonetheless. If it is low tide, I would recommend actually checking out the south side of the beach were the large haystack is. We caught the end of low tide and the sand bar allows you to walk to tide pool areas on the north side of the haystack. Just be careful you don't let your guard down and get stranded!
I had an amazing time here! Though, I recommend wearing pants if you want to scale the driftwood--learned that the hard way. Also, look into tide schedules, it'll be harder to hike along the water if you constantly have to dodge waves/floating driftwood--also learned the hard way.
Trail is on the beach (and small stones) to Hole in the Wall rock formation. Beautiful views and plenty of starfish and urchins to see. Hard walking...like snow-showing at some locations. Unique beach-to-forest views, washed up Sitka giant spruce trees and rock formations.
After easily building fire, and cooking breakfast, we ventured off at low tide to access hole in the wall and it's wonderful shell fish beds. The other side of hole in the wall is completely different terrain, I am excited to make many return trips to discover more of what this has to offer. Maybe even trekking from Rialto to Lake Ozette, for week or two. Highly recommend this backpacking/camping trail to anyone! Easy access, lots of fun, wonderful experience, just respect the tides, noon and night!!!
Catch a late Ferry ride, then drive the 101 across the peninsula, which put us in late to the Rialto Beach parking lot. With headlamps and backpacks in tow, we set off up the beach at 2am. Following the cresting low night tide we reached the designated campsites after crossing a cottonwood over a washout and maneuvering the driftwood scattered beach. Upon waking the next morning with the crashing tides, pillars of ocean crafted rocks stood like beacons holding back the coming waters against the rising sun, revealing the this areas magnificent beauty.


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I'm at last on the 3rd book of the Trilogy that my Niece has lent me. This last book is rather big and heavy so has been sat on my table for weeks now un read, the problem being that i like to carry my books around with me so i can sneak a quick glance at any moment, and this one is not very handbag friendly.
But i took it with me while we went away for the Bank Hol and i actually made a fair dent in it due to the terrible weather conditions.. (piccies of that weekend to come!)
Anyway these books are recommended, they are totally not my kind of thing (or so i thought) but i've got quite into them and think i'll be raiding my nieces bookshelves again in the future!!


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It only takes a minute to get started. Fill out our uncomplicated sign up form and login to the website.
This step is up to you but will drastically raise your chances of getting interest from other members on the site. Write a little about yourself, your pasttimes and upload some good photos of yourself.
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Discovered someone who looks like a good match? Send a letter to them or just a wink and see what happens next. With a little luck you'll be on your way to uncover the love of your life.
It's really quick


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Job Title: Project Field Officers: National Institutional Development (ID) Experts (2 Employment Placements)
Organisation: BTC Uganda
Duty Station: Arua and Kaliro, Uganda
About US:
BTC Uganda is implementing the 'Improve Teachers Secondary Education in the National Teachers' Colleges Project in Muni and Kaliro in collaboration with the Ministry of Education and Sports (MoES).
Job Summary: The National Institutional Development (ID) Experts will be assigned to work full-time in the National Teachers' Colleges and focus predominantly on institutional capacity building in the NTC, supporting management and staff of the college in the delivery of project objectives. The national ID expert will have specific responsibility for the achievement of result of the project, which relates to strengthening the organisational and financial management capacity of the college.
Key Duties and Responsibilities:
- The National Institutional Development Expert will coordinate and supervise the implementation of project activities at the level of the NTC in accordance with the approved project work plans;
- Provide technical expertise in institutional development in areas related to strategic planning, financial management, human resource management, academic management and asset management;
- Work in close cooperation with the college management, develop capacity building strategies for all level of NTC staff aligned to the NTCs strategic plans and the project objective;
- Support and provide on-the-job training and coaching to the college management, with a view to improving the institutional effectiveness of the NTC in the achievement of its objectives and results;
- Support the dissemination by the college of successes and good practices to other colleges and invite where possible other colleagues from other schools.
Qualifications, Skills and Experience:
- The applicant must hold a university degree - relevant to the objectives of the project (education management, public administration, business administration, human resource management);
- A minimum of seven years' experience in school management and/or institutional development preferably in the education sector;
- Proven expertise in modern public sector management approaches and public sector reform in Uganda;
- Knowledge of and experience with previous interventions supported by BTC is an advantage;
- Good command of IT (text processing, spread sheets, database & internet communication software);
- Good communication and facilitation skills
How to Apply:
All candidates should send their application letters and copies of updated CVs with the names and contact information of 3 work related referees should be sent to BTC - Lower Kololo terrace Plot 1B - Kampala (mention "Field Officer ID" on the application envelope!)
Deadline: Friday, 19th August 2016 at 17hrs
For more of the latest jobs, please visit http://www.theugandanjobline.com or find us on our facebook page https://www.facebook.com/UgandanJobline


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Fairless Hills, PA
MATH is the only subject that COUNTS!80/hour
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