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Neurons preconditioned with non-injurious hypoxia or the anesthetic isoflurane express different genes but are equally protected against severe hypoxia/ischemia. We hypothesized that neuroprotection would be augmented when preconditioning with isoflurane and hypoxic preconditioning are combined. We also tested if preconditioning requires intracellular Ca2+ and the inositol triphosphate receptor, and if gene expression is similar in single agent and combined preconditioning. Hippocampal slice cultures prepared from 9 day-old rats were preconditioned with hypoxia (95% N2, 5% CO2 for 15 min, HPC), 1% isoflurane for 15 min (APC) or their combination (CPC) for 15 min. A day later cultures were deprived of O2 and glucose (OGD) to produce neuronal injury. Cell death was assessed 48 hr after OGD. mRNA encoding 119 signal transduction genes was quantified with cDNA micro arrays. Intracellular Ca2+ in CA1 region was measured with fura-2 during preconditioning. The cell-permeable Ca2+ buffer BAPTA-AM, the IP3 receptor antagonist Xestospongin C and RNA silencing were used to investigate preconditioning mechanisms. CPC decreased CA1, CA3 and dentate region death by 64–86% following OGD, more than HPC or APC alone (P<0.01). Gene expression following CPC was an amalgam of gene expression in HPC and APC, with simultaneous increases in growth/development and survival/apoptosis regulation genes. Intracellular Ca2+ chelation and RNA silencing of IP3 receptors prevented preconditioning neuroprotection and gene responses. We conclude that combined isoflurane-hypoxia preconditioning augments neuroprotection compared to single agents in immature rat hippocampal slice cultures. The mechanism involves genes for growth, development, apoptosis regulation and cell survival as well as IP3 receptors and intracellular Ca2+.
Preconditioning is a potent method of inducing ischemic tolerance in the brain and other tissues that involves a non-injurious stress (mild hypoxia, brief ischemia, etc.) that induces resistance to injurious ischemia. Ischemic preconditioning can be induced by a wide variety of stimuli and has been demonstrated in both in intact animals and in cells and tissues in culture. Cerebral preconditioning was first demonstrated with hypoxia (Dahl and Balfour, 1964; Gidday et al., 1994) and later with heat stress, excitotoxins such as glutamate, bacterial endotoxins, oxidative stress, volatile anesthetics and neuromodulators such as adenosine and nerve growth factors (Chopp et al., 1989; Gidday, 2006; Shpargel et al., 2008; Simon et al., 2007). Preconditioning can be achieved in heart, kidneys, lever and other organs. Isoflurane preconditioning of the myocardium is effective in humans (Landoni et al., 2008) but cerebral protection with isoflurane or hypoxia remains an experimental procedure that has not yet been tested in human clinical trials.
Preconditioning involves changes in gene expression. The phenotype of preconditioned brain includes up-regulated stress response genes, intracellular signaling genes, hypoxia-inducible genes, and others (Shpargel et al., 2008; Simon et al., 2007). The specific pattern of gene expression depends on the type of preconditioning stimulus. For example, the volatile anesthetic isoflurane increases genes regulating growth and development, while hypoxic preconditioning increases expression of genes involved in the regulation of cell survival and apoptosis (Bickler and Fahlman, 2009). Differences in gene expression with isoflurane and ischemic preconditioning have also been described in the myocardium (da Silva et al., 2004).
Combinations of preconditioning agents may produce greater neuroprotection than single agents, have fewer side effects and require briefer periods for induction of preconditioning protection. However, few studies have tested combinations of preconditioning agents. Studies of combined hypoxic and anesthetic preconditioning have involved the heart but rarely the brain (Inoue et al., 2004; Inoue et al., 2006). The purpose of this study was to determine, in a rat hippocampal slice model, if combined hypoxic and isoflurane preconditioning is more neuroprotective than either type of preconditioning alone. We also wished to define differences in the pattern of gene expression in single-agent and combined preconditioning. Because increased intracellular calcium is an upstream signal common to different types of preconditioning(Bickler and Fahlman, 2004), we measured intracellular calcium ([Ca2+]i) in cells in the CA1 region during preconditioning. Further, we tested whether preventing increases in [Ca2+]i with a cell permeable Ca2+ buffer (BAPTA-AM) prevents preconditioning neuroprotection in this cellular region. In addition, because endoplasmic reticulum IP3 receptors are responsible for the increase in cytosolic Ca2+ required for induction of neuroprotection in both isoflurane and hypoxic preconditioning (Gray et al., 2005), we tested if this is also true for combined preconditioning by using siRNA against the IP3 receptor.
The effects of preconditioning duration on cell death in CA1, CA3 and dentate regions was evaluated following hypoxic (HPC) and isoflurane preconditioning (APC). Fifteen min of either preconditioning agent did not increase cell death 24 hr later, but 30 min of APC increased death in all cell regions and 30 min of HPC increased death in CA3 and dentate (Fig. 1, panel A). Accordingly, a preconditioning duration of 15 min was used for the remaining studies.
Preconditioning with a combination of isoflurane and hypoxia (CPC) for 15 min yielded greater neuroprotection than did 15 min of HPC or APC alone (Fig. 1, Panel B). This protection was observed in all cell regions (CA1, CA3 and dentate) of the cultures.
Increases in intracellular Ca2+ in CA1 neurons of approximately 50 nM above pre-exposure baseline were observed with APC and HPC. A moderately larger increase (79±13 nM, p<0.05 compared to APC and HPC) was found with combined preconditioning (Fig. 2 panel A). When added to the cultures 30 min before preconditioning, the cell-permeable Ca2+ buffer BAPTA-AM prevented the increase in [Ca2+]i.
Fig. 2, panel B shows that chelation of intracellular Ca2+ with BAPTA-AM and antagonism of IP3 receptors with Xestospongin C during CPC prevents neuroprotection. The reagents used, BAPTA-AM and Xestospongin C, had no effect on survival when present during a mock preconditioning protocol. Further, slice cultures treated with RNA silencing reagents directed against IP3 receptors were not protected against OGD by preconditioning with a combination of isoflurane and hypoxia (Fig. 3). The RNA silencing protocol resulted in a ~90% decrease in ITPR1 mRNA levels at the time of preconditioning, as determined by quantitative PCR.
Table 1 presents the fold-changes in the expression of signal transduction genes 24 hr following HPC, APC, CPC and CPC with Ca2+ chelation pre-treatment. In this table, only genes exhibiting significant changes in expression (1.5 fold increase or decrease) following one of the types of preconditioning are included. A total of 40 of 119 genes on the array were significantly increased or decreased by at least one type of preconditioning. Of these, 30 genes were significantly changed following CPC, 23 by HPC and 25 by APC.
HPC induced genes regulating apoptosis and survival, whereas APC increased the expression of genes regulating growth and development. Of 12 apoptosis or cell survival-associated genes showing a significant change in expression following at least one type of preconditioning, HPC increased 10 genes compared to control. In contrast, APC increased the expression of only 2 genes in the apoptosis/survival regulation group (Bax and Mdm2), and decreases in one (Birc3).
Growth and development genes were preferentially increased by APC. Fourteen genes in this category were changed by preconditioning, with 12 of the 14 genes increasing following APC, and only 3 following HPC. Combined preconditioning increased both survival/apoptosis regulation and growth/development groups of genes.
Genes in the intracellular signaling and stress response groups were activated by both HPC and APC. Many of both gene groups were activated with CPC; that is, with CPC the pattern of gene response was generally the additive of HPC and APC expression patterns.
Treatment of slice cultures with BAPTA-AM before preconditioning prevented the increased expression of mRNA of almost all genes normally increased with preconditioning. Only Ptgs2 and Myc were still increased in these cultures.
Quantitative PCR was used to validate the gene array methodology for a genes in several different groups. Table 2 demonstrates good general agreement between the qPCR and array methods.
Combined preconditioning of rat hippocampal slice cultures with hypoxia and isoflurane increased survival compared to single agent preconditioning and involved a unique pattern of gene expression. The pattern of gene expression following combined preconditioning is a combination or amalgam of HPC and APC, in that both survival and apoptosis regulating genes are increased. Whereas hypoxia alone increased 10 survival/apoptosis genes, and isoflurane increased 12 development/growth genes, the combination increased 19 genes from both groups. This pattern is seen in Table 1. We also found that combined preconditioning required the involvement of IP3 receptors and moderate increases in intracellular Ca2+, the same as with hypoxic preconditioning (Bickler et al., 2009) or isoflurane preconditioning (Gray et al., 2005).
Differences in signal gene expression with similarly neuroprotective paradigms of isoflurane and hypoxic preconditioning were reported previously (Bickler and Fahlman, 2009). The present study involved a combination of hypoxia and isoflurane that increased the neuroprotective outcome and also involved a larger increase in intracellular Ca2+ during the preconditioning period. Because of this outcome, it is unknown whether the unique pattern of gene expression observed in this study was a result of the greater neuroprotective signaling or if it is related to the separate effects of each type of preconditioning. For example, it could be that a larger preconditioning “dose” (concentration or duration of isoflurane or hypoxia) would have produced gene changes, increases in [Ca2+]I or survival benefits similar to the combined regimen studied here. This seems unlikely however, because longer preconditioning with either isoflurane or hypoxia increased cell death, probably from the inherent toxicity of hypoxia and anesthetics (Wei et al., 2008).
Changes in gene expression in hippocampal slice cultures during hypoxia or isoflurane preconditioning are similar to patterns in studies that have analyzed whole brain responses to preconditioning in intact animals. For example, increased expression of heat shock protein genes, cell signaling genes, and other stress response genes have been noted (Hamaya et al., 2000; Pan et al., 2006; Rampil et al., 2006; Sakamoto et al., 2005) and are similar to our general findings. The overall response of the brain transcriptome remains controversial, with recent reports (Stapels et al., 2010; Stenzel-Poore et al., 2003) suggesting that widespread transcriptional suppression is key to the conditioned state in post-ischemic brain. Our data would argue otherwise, although without genome-wide expression analysis, and analysis of gene expression after simulated ischemia, this cannot be verified. The genomic responses of the brain to anesthesia exposure also has been reported to vary substantially between studies, with some reports (e.g. (Rampil et al., 2006)) reporting widespread changes in multiple gene families (including some of the signaling genes found to be altered in this study), whereas other studies reported few changes(Sakamoto et al., 2005). The data in Table 1 would suggest relatively broad effects. This impression may be biased because although a small percentage of the total genome may be altered by anesthesia, many of the altered genes are in the signaling pathways examined in the present study.
It is important to note that this study is limited because only signal transduction genes were examined; i.e. we did not quantify expression of all genes in the rat hippocampus transcriptome. The main value of our study design is the generation of specific hypotheses concerning the mechanisms involved in preconditioning. While we have described correlations between gene expression and selected changes in gene expression during preconditioning, it was beyond the scope of the study to prove that changes in any gene or group of genes is related mechanistically to neuroprotection. Our analysis did show that the pattern of gene expression normally seen after preconditioning was not present when cultures were treated with a cell permeable Ca2+ chelator, but because a broad collection of genes was affected by chelation, it remains uncertain which of the genes are key to the preconditioned phenotype. Another limitation of the study is that we measured gene response and not changes in the levels of proteins encoded by those genes. Because many signaling pathway proteins (e.g. protein kinases) are regulated by phosphorylation, a comprehensive understanding of the effects of altered gene expression on function would in addition require analysis of phosphorylation state and the functional effects of the activated phospho-proteins. An analysis of the consequences of increased expression of specific genes is required to understand the neuroprotective basis of preconditioning. Blocking the expression of specific genes, for example with RNA interference, is one approach that could be used to obtain that information. Finally, our results may only apply to the immature brain. The effect of development and aging on the capacity of the brain to be preconditioned against ischemic damage is important, but little studied.
Although we did not analyze the entire genome’s response to preconditioning, the 119 signal transduction genes is a sufficient sample to indicate broad patterns of responses, i.e. that both growth and development genes and apoptosis/survival regulation genes are part of the pattern of gene expression following combined isoflurane and hypoxic preconditioning. We suggest that measuring whole genome responses is unnecessary to define such important patterns in gene expression, especially when the focus is on a narrow question such differences in signaling gene activation between different types of preconditioning (Hess et al., 2001).
Differences in upstream initiators of gene expression during HPC, APC and CPC, except for the broadly important role of increases in intracellular Ca2+, remain undefined. One may ask how a common upstream signal (increases in [Ca2+]i) can elaborate different gene responses depending on the stimulus. We speculate that Ca2+ signals arising during preconditioning with isoflurane and hypoxia are actually separate, as they arise from different mechanisms causing the increases in intracellular Ca2+, and cause different patterns of gene expression. Hypoxia releases Ca2+ from the endoplasmic reticulum as does isoflurane(Bickler and Fahlman, 2004) but hypoxia involves changes in mitochondrial and cytosolic redox balance (Mayevsky and Rogatsky, 2006), resulting in a broad range of responses including cellular stasis such a spindle checkpoint arrest in development (Fischer et al., 2004) and a diverse group of cell defense mechanisms(Bickler and Donohoe, 2002). In contrast, signaling involving increases in intracellular Ca2+ produced by isoflurane preconditioning may be more similar to developmental signaling such as that following growth factor receptor activation, cell fate/differentiation decisions, and synaptic strengthening in the developing nervous system (Berglund and Augustine, 2008). It is also important to note that IP3 receptor-mediated processes can have deleterious effects as well as beneficial ones. Several studies have shown that IP3 receptor-mediated Ca2+ release can contribute to neuronal death following prolonged exposure to volatile anesthetics (Wei et al., 2008; Yang et al., 2008); this may be a function of exposure duration or anesthetic concentration.
Combined preconditioning increased the expression of numerous signaling genes. Combined preconditioning was associated larger numbers of genes expressed (40 of 119), rather than increases in the magnitude of gene expression compared to single agent preconditioning (data not presented). This increase in the total number of genes upregulated may be the basis for enhanced neuroprotection following combined preconditioning.
A greater number of cell survival and apoptosis regulating genes were increased following combined preconditioning than by single-agent preconditioning. These genes included Bcl2, Bcl2a1, Fn1 and p53. These responses are, on balance, consistent with pro-survival and anti-apoptosis signaling following preconditioning, even though p53 can be pro-apoptotic by up-regulating Bax. The relative levels of these proteins complexly influence survival or apoptosis (Basu and Haldar, 1998). Bcl2, p53 and Mdm2 were all increased 24 hr after CPC. Bcl2 is an important survival signal following hypoxic preconditioning (Meller et al., 2005). In intact rodents, isoflurane preconditioning increases Bcl2 levels (Li et al., 2008). The p53 gene product regulates apoptosis by interacting with a number of different proteins (Banasiak and Haddad, 1998). p53 mRNA increased following HPC and CPC, but not after APC. One of the genes induced by p53 is Bax, which is pro-apoptotic; translocation of Bax to mitochondria is a crucial step in p53-mediated apoptosis. Bax mRNA levels increased following APC, HPC and CPC. The pro-apoptotic actions of p53 and Bax must therefore be countered by the anti-apoptotic actions of other genes (e.g. Bcl2) or signals because, on balance, preconditioning enhances cell survival. Our preconditioning model could be used to determine the relative importance of these proteins in neuroprotection or injury.
Tank and Ppia were both increased by combined preconditioning. Tank is a scaffolding protein that binds TRAF proteins, and is a key activator of NfκB (Bonif et al., 2006; Pomerantz and Baltimore, 1999), which promotes survival. Similarly, Ppia, which encodes a widely expressed scaffolding/protein folding gene (Colgan et al., 2000), was upregulated by HPC and CPC. This could suppress apoptosis following preconditioning, since deletion of unfolded of proteins is an adaptive response believed to increase cell survival following hypoxic endoplasmic reticulum stress (Feldman et al., 2005).
Isoflurane increased more genes associated with regulation of cell proliferation and development than did hypoxia, and CPC also increased the mRNA levels of these genes. Genes in this group included Egr1 (an early growth response gene), Pten (a tumor suppressor gene associated with developmental regulation) Rbp1 (retinol binding protein, an important developmental regulator), the Irf1 (interferon regulatory factor), Ccnd1 (cell cycle protein cyclin d1), Egfr (epidermal growth factor receptor), Igfbp3 insulin like growth factor receptor and Cdkn1 (cyclin dependent kinase inhibitor, significant because it decreased after isoflurane). However, bone morphogenic protein (Bmp4) was not increased following CPC.
All three types of preconditioning increased the expression of Myc, a gene regulating both growth and survival. The Myc-Max heterodimer binds to the promoter of ornithine decarboxylase (ODC1) a growth/cell metabolism gene (Dang, 1999; Grandori et al., 2000). ODC1 was increased by CPC. An unexplained finding was that Myc continued to be up-regulated (as was Ptgs2) by preconditioning in Ca2+-chelated cultures. This illustrates that important Ca2+-independent processes probably are normally involved in preconditioning processes.
Expression of genes in the NfκB pathway also showed greater quantitative expression following combined preconditioning. This may be significant for neuronal apoptotic/anti-apoptotic responses: NfκB has both pro and anti apoptotic functions, activating genes with death-inducing properties like p53, c-myc, Fas and the survival genes Bcl-2, Bcl-x, and MnSOD. NfkB induction of these survival genes may play a role in excitatory, chemical and ischemic preconditioning (Marini et al., 2007). In contrast, acutely inhibiting NfκB delays p53 induced death. NfκB therefore has a dual role, maintaining neuron survival under baseline conditions, but signaling death after DNA damage. The Jnk/JunD pathway works with NfκB to increase expression of anti-apoptotic genes (Lamb et al., 2003). Gadd45 is involved in cellular response to DNA damage or oxidative stress. CPC increased Gadd45a mRNA.
Combining hypoxia with isoflurane in a preconditioning model with hippocampal slice cultures produces greater neuroprotection than either agent alone and a pattern of gene expression that is a unique amalgam of the two. Intracellular Ca2+ may be involved in the neuroprotective actions of this type of preconditioning. While the complete mechanistic basis for these observations was not revealed in this study, the findings have significant implications for the long terms effects of anesthesia and for the use of hypoxia or isoflurane as preconditioning agents against cerebral ischemia.
The studies were approved by the University of California San Francisco Committee on Animal Research and conform to relevant National Institutes of Health guidelines for the use of animals in research.
Organotypic cultures of the hippocampus were prepared by standard methods (Laake et al., 1999; Stoppini et al., 1991) and slightly modified by our laboratory (Sullivan et al., 2002). Briefly, 7 to 9 day old Sprague Dawley rats (Charles River Laboratories, Hollister, CA) were anesthetized with 2–5% isoflurane until they did not move in response to a vigorous tail pinch. Following decapitation, the hippocampi were quickly removed and placed in 4 °C Gey’s Balanced S alt Solution (GBSS) with 20 mM glucose. The isolated hippocampi were transversely sliced (400 μm thick) with a tissue slicer (Siskiyou Design Instruments, Grants Pass, OR), and returned to GBSS at room temperature. The slices were placed on 30-mm diameter membrane inserts (Millicell-CM, Millipore, Billerica, MA) in 6-well culture trays with 1.2 ml of slice culture medium per well. The slice culture medium consisted of 50% Eagle’s Minimal Essential Medium, 25% Earle’s balanced salt solution), 25% heat inactivated horse serum (all from the UCSF cell culture facility) with 6.5 mg/ml glucose and 5mM KCl. After 2 days in culture the serum concentration was decreased to 10%. 7–10 days later the slices were preconditioned.
Slice cultures were preconditioned by placing them in a Billups-Rothenberg modular incubator chamber (Del Mar, CA) filled with humidified 95% N2/5% CO2 (HPC), or 1% isoflurane in humidified air and 5% CO2 (APC) for 15 min. Combined preconditioning (CPC) was done similarly with 94% N2, 5% CO2 and 1% isoflurane, also for 15 min. RNA was extracted for gene array analysis 24 hr after preconditioning.
Ischemia was simulated in vitro by one of 2 techniques: 1) immersion of cultures for 10 min into glucose-free media bubbled with 95% N2/5% CO2 (oxygen/glucose deprivation, OGD); 2) transfer of cultures to glucose free media and exposure to humidified 95% N2/5% CO2 for 30 min. The later was used with cultures treated with RNA interference or BAPTA, as these reagents sometimes caused slices to float off the culture membranes. The temperature of the media was 37°C, measured with a thermocouple thermometer. The partial pressure of oxygen, measured with a polarographic oxygen electrode, was 0–0.2 mmHg. After this insult, the cultures were returned to standard slice culture media. The percentages of dead and living neurons in CA1, CA3 and dentate was measured 48 h after OGD.
Cell death was measured with propidium iodide (PI) fluorescence. PI, a highly polar dye, penetrates damaged plasma membranes and binds to DNA. 2.3 μM PI was added to the wells of the culture trays 30 min before imaging. After 30 minutes digital images of PI fluorescence 9excitation light 520 nm, emission 600 nm) were taken with and inverted microscope and a SPOT Jr. Digital Camera (Diagnostic Instruments, Sterling Heights, MI). The camera sensitivity and the excitation light intensity were identical from day to day. Slices were discarded if they showed more than slight PI fluorescence in after 7–10 days in culture. PI fluorescence images were recorded in the dentate gyrus, CA1, and CA3 regions of the hippocampal slices prior to OGD (signal assumed to represent 0 % cell death), and 48h following OGD. In previous studies, we found that maximum post-OGD death consistently occurs at about day 2 or 3, and declines over the next 11 days (Sullivan et al., 2002). Serial measurements of PI fluorescence intensity were made in pre-defined areas (selecting CA1, CA3 and dentate separately) for each slice using NIH Image-J software (U.S. National Institutes of Health). After the measurement of PI fluorescence on the 2nd post-OGD day, all the neurons in the slice were killed to produce a fluorescence signal equal to 100% neuron death in the regions of interest. This was done by adding 100-μM potassium cyanide and 2 mM sodium iodoacetate to the cultures for 20 minutes. One hr later, final images of PI fluorescence (equated to 100% cell death) were acquired. Percent of dead cells 48 hr after OGD were then calculated based on these values. PI fluorescence intensity is a linear function of cell death (Laake et al., 1999; Newell et al., 1995).
The percentages of living and dead neurons in the different regions of the slices sometimes is not be normally distributed. Therefore, the Kruskal-Wallis test followed by the Mann-Whitney U-test (JMP, SAS Institute, Cary, N.C.) was used to compare the medians of different treatment groups. T-tests or ANOVA were used to compare the means of normally distributed data, and allowance was made for multiple comparisons (Tukey-Kramer multiple comparison or Dunnett’s test). Differences were considered significant for P<0.05.
[Ca2+]i in CA1 was measured before, during and after preconditioning. Estimates of [Ca2+]i in CA1 neurons in slice cultures were made using the indicator fura-2-AM and a dual excitation fluorescence spectrometer (Photon Technology International) coupled to a Nikon Diaphot inverted microscope. Slice cultures were incubated with 5–10 μM fura-2 AM plus 1% pleuronic acid for 30 min before measurements. Cultures for these measurements were grown on Nunc Anopore (Nalge Nunc) membranes because of their low ultraviolet fluorescence. Slit apertures in the emission light path were adjusted to restrict measurement of light signals to those coming from the CA1 cell body region. Calibration of [Ca2+]i was performed using the KD of fura-2 determined in vitro with a Ca2+ buffer calibration kit (Invitrogen). The calibration process involved using the same light source, optical path and filters as used with the slice culture measurements. The KD for fura-2 was 311 nM, similar to published values (Hyrc et al., 1997). Background fluorescence (i.e. fluorescence in the absence of fura) was subtracted from total fluorescence signals prior to calculation of [Ca2+]i as described previously (Bickler and Hansen, 1998). Estimates of [Ca2+]i with this technique are accurate to about ±10 nM(Grynkiewicz et al., 1985). Measurements of [Ca2+]I were made briefly at discrete periods during the preconditioning, to avoid photobleaching of fura-2. These were at baseline, at mid-point and termination of preconditioning, and after 10 min washout of preconditioning medium. Peak [Ca2+]i always occurred at the end of the preconditioning period.
In some studies, the Ca2+ chelator BAPTA in the cell permeant form BAPTA-AM (10–25 μM) was added to cultures 30 min before preconditioning and remained during preconditioning. In others, the IP3 receptor antagonist Xestospongin C was included in the media during preconditioning. The cultures were transferred to fresh media after the preconditioning period.
Pools of interfering RNA (Custom smart pool RNAi, Dharmacon, Lafayette, CO) directed at the IP3 receptor subtype 1 (ITPR1) were used to transfect slice cultures with the Trans IT-neural transfection reagent (Mirus Bio Corp, Madison, WI). Three treatment groups were studied: target RNAi, nonsense (nontargeting) RNAi, and transfection reagents only. Preliminary studies defined time of maximal knockdown of IP3 receptors; RT-PCR was used to quantify transcript levels at 12, 24 48, 72, and 96 hours after addition of transfection reagents to slice culture medium. Maximal reduction in mRNA and IP3 receptor protein was at 72 hr after transfection in both cases. Slices were transfected on days 7, 8 and 9 in culture. To assess the effectiveness of target mRNA depletion, slices were transferred into −80°C RNAlater-ICE (Ambion, Austin, TX) and stored until RNA was extracted with Trizol (Invitrogen, Carlsbad, CA). DNA was synthesized using an OmniScript Reverse Transcriptase kit (Qiagen, Valencia, CA). Quantitative PCR was run using a QuantiTect SYBR Green PCR kit (Qiagen) and the following primers, created using Primer3 online software (Roizen and Skaletsky, 2000): IP3r1 Forward: tcgtggatgttctacacaga, IP3r1 Reverse: agctgcttggtgtgttttat
(Product size: 112 bp, Accession number 1054962) and for β-actin forward: acagctgagagggaaatcgt β-actin reverse: ttctccagggaggaagagg
(Product size: 107 bp, Accession number: 031144).
RNA for microarray analysis was extracted from slice cultures 24 hrs after mock preconditioning (control), hypoxic preconditioning, isoflurane preconditioning and combined preconditioning as follows. Pooled tissue slices (12–18) were homogenized in 1 ml of TriZol Reagent. The RNA was precipitated from the aqueous phase with isopropyl alcohol, rinsed with 75% ethanol, and then re-suspended in DEPC-treated water. RNA was further purified by means of the ArrayGrade™ Total RNA isolation kit (SuperArray, SA Biosciences, Frederick, MD), and concentrated down to a final volume of 50 μl in RNASE-free water.
cDNA was synthesized using 0.1 to 2 μg of total RNA, by means of the TrueLabelingTM LinearRNA Amplification Kit (SuperArray). From this cDNA an amplified Biotin-Labeled cRNA was synthesized. Biotinylated UTP was obtained from Roche Applied Science. The cDNA synthesis reaction was incubated overnight at 37° C. The cRNA was then purified using spin columns from SuperArray’s cRNA Cleanup Kit. Quality and concentration of cRNA was determined by absorbance of 260nm and 280nm light.
The cRNA was hybridized onto Oligo GEArrays at 60 ° C overnight with continuous agitation. The arrays used were Rat Signal Transduction Pathway FinderTM Microarrays ORN-14, ORN-14.2, and Rat Apoptosis Microarray ORN-12 from SuperArray. Table 1 contains a listing of all the genes on the arrays. After rinsing in wash buffers the arrays were probed using a chemiluminescence method. Arrays were exposed to high performance chemiluminescence film (HyperfilmTM ECL, Amersham, South San Francisco, CA) and developed in a mechanical darkroom developer. Films were scanned at the highest pixel density (1200 dpi output resolution) for analysis.
Array scans were analyzed using the internet-based GEArray Expression Analysis Suite provided by SuperArray. All genes were normalized to a series of “housekeeping” gene expression levels and a group of synthetic control sequences included on the array by the manufacturer. For background normalization, a pair of blank spots and local background correction for each tetra spot was employed. Gene expression was considered significant if there was a minimum 1.5-fold increase or decrease compared to the control level.
Supported by a grant (RO1 GM 52212) from the USA National Institutes of Health (Washington, D.C.) to PEB.
We thank Pablo Gabatto, BS, Will McKleroy, BS, and Maren Gregerson, BS, Staff Research Associates, Dept. Anesthesia, UCSF, for technical assistance.
Reprints will not be available from the authors
Conflicts of interest: None
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New Delhi, India (CNN) -- U.S. President Barack Obama started his second day in India on a lighter note: pulling a few dance moves and celebrating a major religious festival with local students.
Obama and first lady Michelle Obama spent Sunday morning at a local school, where students were celebrating Diwali. Youngsters performed dances in colorful saris to mark the festival of lights. The president bobbed his head to the music and clapped.
Young girls danced with baskets on their heads as the room erupted in cheers.
At one point, students pulled the first lady on stage and taught her how to twirl.
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Students later extended the offer to the president, who joined them and towered over the youngsters as he danced.
Obama also visited students at Holy Name High School, where students in school uniforms explained their projects to the president. One girl described her "eco-friendly village" with a large windmill.
"Did you guys get that -- a tree a day keeps global warming away," Obama told journalists.
Obama also attended an agriculture exposition and took part in a town hall-style meeting with students at St. Xavier's College in Mumbai. He and the first lady then visited Humayun's Tomb, a magnificent architectural precursor to the Taj Mahal, before heading to New Delhi for a private dinner Sunday night with Indian Prime Minister Manmohan Singh.
At the meeting with the students, Obama faced some difficult questions including his thoughts about jihad and why the United States doesn't consider Pakistan -- India's neighbor and rival -- a terrorist state.
The president said it was necessary to isolate those who distort religion as a means for war, and said young people have a role in rejecting violence as a way to mediate differences.
On Pakistan, Obama called it a country of enormous potential that had extremist elements. He acknowledged that progress against militants there was "not as quick as we'd like," particularly in the difficult terrain of the northwest territories along the border with Afghanistan.
Obama also said that India has the biggest stake in its neighbor's success, and that he hoped over time that the two nations develop further trust and cooperation.
"The United States stands to be a partner in that process, but can't impose that," Obama said.
The relatively light agenda on the second day of Obama's 10-day, four-nation Asian trip followed the president's unveiling Saturday of about $10 billion in contracts for U.S. exports to India.
"The United States sees Asia, and especially India, as a market of the future," Obama said at a meeting with business leaders from both countries. "For America, this is a jobs strategy."
Promoting broader trade relations with India is a delicate balancing act for Obama, given American frustration with the outsourcing of jobs to call centers in the country.
But Obama said the notion of Indian outsourcing being a net drain on the U.S. economy is part of a "caricature of India as a land of call centers and back offices that cost American jobs."
Obama's three-day visit to India, Asia's third largest economy and one of the world's few growth markets, also will include bilateral talks with Singh and an address to the the nation's parliament.
After India, Obama will travel to Indonesia, then on to the G-20 meeting in South Korea and APEC (Asia-Pacific Economic Cooperation) summit in Japan.

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seeing our customers each week and we are endlessly appreciative of their continued support.
The summer squash and zucchini pulled through and we had enough winter squash for a few each but not the mountains of pumpkin and winter squash we have enjoyed in previous seasons. The third planting of cucumbers finally produced. The answer to the squash we think is two fold. The first part is a prayer to the heavens for a cold ass winter to kill off some of the overwintering pests that plague us in years when the winter is mild and the populations of pests don't get knocked back.
Secondly, we will plan to invest in a substantial amount of row cover and use this as a barrier to deter the interaction of the bugs with the squash plants. Finally, we are going to abandon the acre of landscape fabric that we have invested in over the years. We think it might harbor squash bugs, that they may like it. If we have another squash crop failure next season, it certainly won't be for not trying.
We had a majorly successful year for Solanaceous crops such as peppers, eggplants and tomatoes. We are still getting some ripe tomatoes out of the hoophouse! The peppers and eggplants loved growing on black plastic, making the soil temps warmer and allowing for the bell peppers to change from green to red. It was fantastic for them, all around. We hope folks weren't overwhelmed by too much eggplant and too many peppers. It's hard not share when there is an abundance.
Sometime close to the New Year, we will start our membership drive for next year. In the meantime, we'll clean up the fields, pack equipment away for the winter, start interviews for next year's crew....already the resumes are rolling in! We'll spend a little time updating the website and evaluating whether or not to use the same software program as we did this year to manage memberships. We will evaluate how to grow the business, what to do more or less of and what makes us happy. We'll talk to wedding couples about flowers. And when the snow comes, we'll ski! Next month, we'll head the MOFGA Farmer to Farmer conference to, ironically, present about squash. And then, we'll travel to Tacoma, Washington for the Association of Specialty Cut Flower Grower's conference. I can hardly wait to talk flowers with other growers and designers and to be on the West coast for a stretch. That said, if you have any input you would like to share about how the season went for you as a customer, please do. We welcome feedback as it helps direct our energy towards improving the farm experience for our customers.
We'll try to keep you all filled in as the winter moves in on us. I have been saving a few stories to share about barn renovations and hoophouses and gorgeous wedding flowers so you won't be continually subjected to photos of my icy dooryard and my dark root cellar all winter. Till then...
Blessings on the meal,

domain: agronomy


text:
Free Blackjack Games For Excitement AS WELL AS FOR Winning REAL CASH
Free of charge Blackjack games for excitement and rest is exactly what everybody needs in these modern times. Enjoying online flash games are usually thus much fun than participating in in casinos and thus everyone really wants to try it out for themselves. You can find so several free internet games for all age brackets. From very easy jackpot game titles to multi-player video games. Here’s set of top five almost all downloaded no cost Blackjack online 우리카지노 games for everyone to enjoy.
This is among my in history favorite free blackjack games. This activity gives you an opportunity to learn to develop and apply your personal blackjack approach and tactics. This is one particular real money video games but is absolve to play. The basic strategy would be to always bet once you expect to come to be paying full residence.
Ace greeting card counting is one of those free blackjack games for online fun and entertainment. It is a game with a straightforward concept. The player counts cards, while the dealer bargains them out. If the cards need to be dealt out to you, do the keeping track of just before and after dealing out the credit cards. This way, you’ll get to know when to improve or fold in addition to count cards precisely.
Blackjack in the casino game offers its own rules and strategies. One of these is called dealer blind. Within this, the dealer will keep all the cards hidden from players so that they cannot find out the actual dealer has waiting for you for them. In other words, they cannot start to see the cards in their hand and hold playing, counting credit cards blind.
There are also other variants on blackjack games for online modern casino play. There is Texas Holdem, No Limit TEXAS HOLD EM, Five Cards Stud, seven-card stud and other variations. In most cases, it would depend on the sort of poker you are going to play whether you will try your good luck in a gambling establishment game with a residence edge or definitely not.
There are free demo options for these online casinos. There are many free demo options available to players to test different blackjack versions free. There are many poker games online casinos that offer free demo video games for their people. They invite fresh players to test the game free of charge and in return, these players will have the opportunity to see the game using special features and benefit rounds. Many players find these free of charge demos very exciting given that they can training their game strategies and strategies against the poker algorithms in the casinos prior to going directly to real cash games.
It is possible to find a very good free video games for pleasure and profit in a variety of casino websites. They provide free video games for texas holdem, blackjack, craps, slots and other game titles. All you need to do is to find one webpage that provides these free game titles. You must enroll first before you can start playing the game. Once you’ve registered, you can begin playing the free game titles.
You should note that most of the free games offered by the web casinos are played for pleasure and recreation purposes only. This is the reason why most of the websites usually do not provide any real money play options. On the other hand, there are some websites that allow players to place a maximum choice as high as one percent of these bankroll at any stage of time. The main reason why these casinos offer free games is to attract new players and retain their clientele. Members will be drawn to these casinos, because they can play free of charge games without worrying about losing any money.
On the other hand, you should remember that you must never play your favorite blackjack games for real money until you are completely comfortable with the software provided by the casino. The first thing that you ought to look for in an online casino may be the terms and conditions, which state evidently the sort of gaming provided in the gambling house. You must become absolutely sure about the software you will be using inside the casino prior to deciding to make a initial deposit. A number of the casinos require which you make your first of all deposit in combination with another currency such as credit card or debit greeting card.
You also need to discover more about the free of charge blackjack games offered in the gambling house. There are various types of no cost blackjack games which are being offered today. You’ll find out what type of variant will greatest suit your style of play. For instance, you might find some free blackjack video games that enable you to play for money, without allowing you to use any real cash. Which means that you will be playing simply for fun, and not for any feasible chance at receiving real money. In this case, you would probably be participating in against real individuals, and you have to produce your blackjack approach accordingly.
If you have been enjoying playing free games on the net before, you might think that there is you don’t need to build up any blackjack strategy at all. But there are some things that you need to know about the very best blackjack game. When you can easily enjoy online for free games, it generally does not mean that you do not need to build up any strategies by any means. You need to ensure that you are acquainted with the guidelines of the overall game, to enable you to maximize the probability of winning real money.

domain: game


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Mark Zuckerberg to testify in shareholder suit on voting rights
San Francisco: Mark Zuckerberg is heading back to court. Facebook Inc.’s chief executive officer is expected to testify later this month in a shareholder lawsuit over voting power, according to a person familiar with the plans.
The trial, set to start on 26 September, will explore whether the board had shareholder interests in mind when it approved a new class of non-voting shares. The move lets Zuckerberg keep voting control of Facebook even if he sells shares. He asked for the change so he could give away most of his wealth to charities.
But shareholders alleged conflict of interest in a complaint that disclosed an embarrassing episode: Director and venture capitalist Marc Andreessen worked with Zuckerberg behind the scenes to pass the change, even though he was on a committee that was supposed to look out for shareholders.
“Facebook is confident that the special committee engaged in a thorough and fair process to negotiate a proposal in the best interests of Facebook and its shareholders,” the company said in a statement on Friday.
This will be Zuckerberg’s second court appearance this year. He testified in a case about whether the company’s Oculus virtual reality headset maker used stolen computer code. Zuckerberg said the claims were false, but Facebook lost, with a jury awarding $500 million to plaintiff ZeniMax Media Inc. Bloomberg
- Donald Trump pressures US senators to back Republican healthcare bill
- India to send 700 tonnes of relief material for Rohingya refugees in Bangladesh
- Sushma Swaraj slams Pakistan at UNGA, asks its leaders to introspect
- Mexico jittery after new earthquake of 6.1 magnitude
- Sushma Swaraj calls for early start of negotiations for UNSC reforms

domain: law


Posts Filed Under: cycling
Today BIXI celebrates its one millionth ride in Toronto. What's next for the successful bike-sharing program?
New cycling infrastructure, new problems.
A group of passionate bike couriers, friends, and family say they refuse to be spun by Michael Bryant's PR power.
Ever wondered how Toronto bike thieves do what they do? This video has answers.
A cycling-advocacy group installed a memorial for a cyclist who died after catching his wheel in a streetcar track.
Study includes recommendations to improve cycling conditions in the province.
For a second year, cyclists take to Jarvis street to protest the planned removal of its bike lane.
Cycle Toronto cried foul, then hastily retracted its claim that Mayor Ford wants the lanes gone.
The Toronto Cyclists Union's members will vote on dropping the "Union" from the group's name.
Stenciled bike posts where real ones were once installed—removed by Astral Media to make way for information pillars.
The third annual Blessing of the Bicycles married religion, environmentalism, and free bike maintenance.
Urban Repair Squad strikes again, with a never-ending bike lane as part of an AGO exhibit.
If you drive, and like Jarvis Street the way it is, Dave Meslin wants to hear from you.
At this year's Toronto International Bike Show, attendees spent big bucks on nice rides.
Friday is your last opportunity to tell the City what you think of its new bike-trail plans.
Studs on ice! Fearless riders took to the rink at Dufferin Grove this weekend, for the annual Ice Bike Races.
New campaign promotes a brand of bikes called Ghost, but also inadvertently evokes ghost bike memorials.
Monday's "Coldest Day of the Year Ride" was neither very cold nor very long, but it was fairly well attended.


The Tilley jersey can be worn all year round. This jersey is ideal in cool weather and will keep you warm, and can almost replace a light wool sweater.
Our fast-drying Tilley `Travel` Tank Top is a welcome addition to our collection of the finest, most practical and comfortable travel underwear you`ll ever own. Fast drying and sweat defying, it will neither chafe nor bind. The patented fabric wicks away perspiration better than cotton to keep you cooler and drier in the summer and warmer in the winter. Wash in a hotel sink and hang to dry by morning! Unisex style for Men and Women. See chart below.
o Stain, odour and bacteria resistant fabric
o Dries fast - wash at night, it`s dry in the morning
o Electrostatic wicking action speeds evaporation
o Lightweight and long-wearing - it keeps its shape and appearance
A patented lightweight polyester fibre that wicks away moisture and dries quickly. Stain, odour and bacteria resistant. It will keep its shape and appearance longer than traditional cotton.
S 34" - 36"
M 38" - 40"
L 42" - 44"
2XL 48" - 50"
No special-care required, simply wash and wear, or hand-wash in a hotel sink, twist in a towel and hang to dry. Ready to wear by morning.


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The Kingsford Smith International Airport is Australia’s busiest and one of the oldest continuously operated commercial airports in the world.
The Kingsford Smith International Airport, named after an Australian aviation pioneer, started taking regular flights in 1924, making it one of the world’s oldest continuously operated commercial airports. Australia’s busiest, it handles over 42 million travellers a year. After years of seemingly endless renovations, its international terminal has settled to become a much-improved and more inviting space.
Located at Mascot, south of the city centre, with runways jutting into Botany Bay, where James Cook first landed in Australia in 1770. Distance to CBD 15 kilometres
Taxis & hire cabs
Taxi stands are located outside arrivals at both the international and domestic terminals. Fares are metered. The city centre is 25 minutes away and costs $AUD45–55. Terminals also have Avis, Budget, Hertz, Europcar and other car-rental outlets.
The train (airportlink.com.au) stops at both international and domestic terminals; follow signs from arrivals. Trains run about every 10 minutes at peak times. Public buses (transportnsw.info) also operate from T1 and T3.
There are three terminals, international (T1) and domestic (T2 and T3). You can walk between domestic terminals. A complimentary shuttle bus connects the international terminal.
Air Niugini’s Gold Executive Club members have access to the Qantas Club International Lounge, which has a barista and bar counter, buffet food, work stations, relaxation areas and showers.
International fashion and cosmetic outlets, sunglasses, watches and duty free are complemented by shops selling Australian goods, including wine, chocolates, merino-wool clothing, and boots from UGG and iconic outback company R.M. Williams.
Food & beverage
The international terminal has sushi, informal cafe-style and fast food, Chinese dining, barbecued chicken and gourmet food from The Bistro by Wolfgang Puck, the celebrity Austrian-American chef.
Sydney Airport has free Wi-Fi. Select ‘Free SYD Wi-Fi’ from your connection options and register your details to obtain access.
Pullman Sydney Airport (pullmansydneyairport.com.au) is the only five-star hotel in the airport area and has elegant art-hung spaces, chic rooms, meeting facilities and flexible dining times.
Rydges Sydney Airport (rydges.com) is right beside the international terminal and has runway views and good soundproofing. It has several dining options, conference facilities and a gym. The surprisingly stylish Ibis Sydney Airport (ibissydneyairport.com.au) is a good economy option a kilometre from the domestic terminal, at Mascot train station.
You’ll also find a pharmacy, meeting and conference rooms, shower facilities, prayer room and a post office. There are two interactive kids’ zones with game options and touch-screen technology.
Hard to resist a browse at the Lonely Planet shop in the departures area at T1. There are shelves of enticing travel guides and maps, and even the odd globe or two – enough to set you dreaming of your next destination.
This story was first published in the January-February edition of Paradise, the in-flight magazine of Air Niugini.
Credit: Source link

domain: geography


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Back To Element 63 Periodic Table

domain: chemistry


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I love Joe Scarano’s 2d work, so I was beyond excited when he sent in some images of 3D work that will be debuting at Stranger Factory in March!
He’s been casting up these brilliant little faces of his character Grimley, and hand painting them as wall plaques. They’ll exist in unique colourways, but he’ll have somewhere between 10-15 done for the show, so there’s a good chance that you’ll be able to snag one of these impish masks for your collection!
A bit more about Grimley from Joe:
Grimley lives in the trees, he lives in the walls, the ground, the clouds… he appears like smoke to lead you astray. You had planned on walking through the woods to get to Granny’s house, but he has other plans for you. You will happily follow him anywhere he leads you.
Deadeyes runs March 7th – 30th, with an opening reception on Friday, March 7th from 6 – 9 PM.

domain: artistic


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SAVAGE POSSESSION – MARGARET TANNER
A sweeping tale of love’s triumph over tragedy and treachery in frontier Australia.
A mistaken identity opens the door for Martin Mulvaney to take his revenge on the granddaughter of his mortal enemy.
An old Scottish feud, a love that should never have happened, and a series of extraordinary coincidences traps two lovers in a family vendetta that threatens to destroy their love, if not their lives.
Don’t forget to call back and read Juliet Waldron’s blurb on 27th January


Apple remains the company’s largest vendor, contributing 27 percent of the revenue.
Buy / Sell Redington share
Recommended ArticlesView All
'Elephant in the room' — Finance Minister red-flags gold smuggling
IST2 Min(s) Read
Redington Ltd., India’s largest reseller of Apple products, reported its best ever half-year revenue growth of 25 percent between April-September. Consequently, shares ended over 8 percent higher, marking their best intraday gain since August.
For the September quarter, the company's consolidated profit increased 26 percent to Rs 387 crore from Rs 307.2 crore in the same period a year ago.
The company’s revenue grew by 25 percent year-on-year. Apple remains the company’s largest vendor, contributing 27 percent of the revenue.
Of its total revenue, 74 percent was from the IT sector while mobility contributed 25 percent of the revenue. Singapore, India, and South Asia contributed 48 percent of the revenue, the same as last year.
The company is present in 30 countries and serves 38 markets, with product lines like electronics (PCs, desktops, laptops, iPads, MacBooks, smartphones, printers, consumables, and accessories), services (Digital printing, Industry 4.0, Cloud resell, managed services, Enterprise security solutions) and more.
Shares of Redington India ended 8.6 percent higher at Rs 149.20.
(Edited by : Rukmani Krishna)


By Cindi Stowell of Cindi’s BJD Designs
Step 1: Gather supplies
10″ x 10″ (25cm x 25cm) wool felt square
white glue, stiffening agent like Aileen’s Stiffen Quick
Small bowl around the size of doll’s head
Step 2: Boil water
Step 3: Immerse your 10 x 10 piece of 100% wool felt in the hot water for 2 minutes
Step 4: After 2 minutes, using tongs, turn off the heat, lift the wool from the pot. Allow it to drip and cool.
Step 5: When the wool piece of felt is hot but not too hot to handle, place it over your small bowl.
Step 6: Mold the hot felt piece evenly over the small bowl.
Step 7: Place a rubber band around the bottom edge and let dry.
Step 8: Spray the felt piece with a stiffener like Aleene’s Stiffen Quick spray. Let dry. After your felt piece is completely dry, spray again with Aleene’s Stiffen Quick spray. Let dry again. Repeat this process for your desired stiffness. I usually do it about 3 or 4 times.
Step 8: Remove your hat from the small bowl. Take a ruler and mark your desired width for your hat brim. In this picture I did 2 inches, but I also like 1 inch brims.
Step 9: Cut around the dots.
Step 10: Your hat should look like this now after you have cut the brim of the hat.
Step 11:Fold up the front of the hat brim and glue in place. Let glue dry thoroughly.
Step 12: Glue on your applique on the center front of the hat.
Step 13: Add your other embellishments to the hat.


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A 60 Minutes investigation in 2010 uncovered that dogs being produced were often not purebreds as people expected, but cross-bred so called ‘designer’ dogs such as ‘labradoodles' (a labrador crossed with a poodle), or ‘spoodles' (a spaniel crossed with a poodle).
Footage showed dogs living in mud and filth, without bedding, as well as sick puppies in need of urgent veterinary attention. One dog was dragging around a tyre attached to his collar by a chain (to prevent escape), and another had a cruel anti-barking device attached to her neck (which emits an electric shock when the dog barks).
The 60 Minutes programme linked at least one farm in Gisborne to supplying young dogs to the largest pet store chain in the country.
With dogs being retailed for around $1,500 each, it’s an industry driven by profit at the expense of the dogs.

domain: pet


What is a Megapixel?
Answering the question "What is a Megapixel" is really pretty simple because a megapixel is simply one million pixels. But then that leads to the next question where things get a little more interesting and that is "What is a Pixel"?
Understanding Pixels and Megapixels
Digital cameras use an electronic image sensor to take pictures instead of film. These image sensors convert the optical image (picture) into an electric signal which is then processed and stored electronically in some type of external (a removable memory card such as a SD or Compact flash card) or internal memory (memory built into the camera). Each image sensor is made up of individual optical components known as pixels (picture elements). One million of these pixels is referred to as a megapixel.
An image sensor is made up of individual optical components known as pixels (picture elements). The term megapixels simply means one million pixels with a pixel being a single point or dot that makes up part of the picture or image.
Now that we have a basic idea of what a pixel is we know the simple answer to our question "What is a Megapixel" but we still need to understand why megapixels matter and how important they are to overall picture quality.
Why are Megapixels Important?
Megapixels are important because they are an indication of camera's image quality or resolution. Higher megapixel cameras will produce higher resolution images which generally equals better quality photographs.
Important Note: There are other factors that influence picture quality as well; especially the size of the image sensor so be wary of falling into the Megapixel Myth trap and automatically equate more megapixels with better pictures.
In film cameras the image resolution was determined by the size of the film however in today's digital cameras it determined by the size of the camera's image sensor and the number of megapixels it has. To determine the number of megapixels you multiply the number of pixels the camera's image sensor has vertically by the number of pixels horizontally. That gives you the total pixel count for the camera which is expressed in megapixels.
Avoiding the Megapixel Myth Trap
Now that we have a better understanding of "What is a Megapixel" we need to make sure that we avoid falling prey to the Megapixel Myth trap. The megapixel myth is the assumption that "more megapixels are better" when evaluating digital cameras.
The number of megapixels a camera has is important because it indicates the image resolution that camera can capture, but it is just one of many factors that play a role in the overall quality of the picture. There are simply too many other related factors such as the size of the image sensor that affect overall image quality to automatically assume that just because one camera has more megapixels you will get a higher quality image than another camera.
While the number of megapixels is important when it comes to how large of an image can be printed there are other factors that contribute to the overall picture quality. Therefore when considering a digital camera do not assume that a higher megapixel camera will always be the best choice.
How many megapixels are needed for different print sizes?
As we continue to learn about what is a megapixel we will look at the important relationship between the number of megapixels and maximum print size. The general rule is that the more megapixels you have the larger your print can be without noticeable distortion.
The number of megapixels needed for different print sizes is determined by the print resolution which is normally listed in dots per inch with 300 dpi being a common standard for typical prints. Therefore determining the maximum print size is as easy as dividing the number of horizontal and vertical pixels by 300. For example a 5 megapixel camera might have a resolution of 2580 pixels by 1944 pixels or 5,015,520 total pixels. This means you can easily print an image up to 8.53 inches by 5.4 inches with a 5 megapixel camera.
To determine the maximum print sizes for cameras with a different number of megapixels please see the table below.
|Camera Resolution In Megapixels||Maximum Print Size At 300 dpi|
|5MP (2,580 x 1,944)||9x7|
|6MP (3,008 x 2,000)||10x7|
|7MP (3,072 x 2,304)||10x8|
|8MP (3,264 x 2,488)||11x8|
|9MP (3,488 x 2,616)||11x9|
|10MP (3,648 x 2,736)||12x9|
|11MP (3,830 x 2,872)||13x10|
|12MP (4,000 x 3,000)||13x10|
|14.6 MP (4416 x 3312)||15x11|
|24.6 MP (6048 x 4032)||20x13|
*Horizontal and vertical measurements can vary depending on the format of the image sensor, etc. These are general guidelines not absolute standards.
How many pictures can my memory card hold?
In this last part of our discussion of "What is a Megapixel" we will briefly cover the relationship of megapixels and file size. The more megapixels a camera has means the larger the size of the standard JPEG picture file. This means that to store the same number of photos you will need a larger memory card, an important fact to keep in mind when considering replacing your digital camera with one that has more megapixels.
Below is a chart that shows an average file size for different megapixel sizes and the number of standard JPEG images that can be stored on different sizes of memory cards.
|Memory Card Capacity|
|Camera||File size||2GB||4GB||8GB||16 GB||32 GB|
|5 MP||1.5 MB||1200||2400||5000||10000||20300|
|6 MP||1.7 MB||1000||2000||4300||8700||17300|
|8 MP||2.3 MB||700||1500||3200||6400||12900|
|10 MP||2.9 MB||600||1200||2500||5000||10100|
|12 MP||3.4 MB||500||1000||2000||4300||8600|
|16 MP||4.6 MB||300||700||1600||3200||6400|
|21 MP||6.4 MB||200||550||1100||2300||4600|
While many people associate a higher megapixel camera with better quality photos there are too many other related factors to make that type of sweeping generalization.
There are many other factors than just the amount of Megapixels a camera has that must be given careful consideration when purchasing a digital camera. For more information on the different types of digital cameras available checkout our digital camera comparison page. I hope you have found this page on what is a megapixel helpful.


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How do you sneak healthy food to a 2 year old, who is a picky eater?
My daughter HATES most fruit as soon as she touches it she freaks out and won't eat it. She pretty much just loves pizza, milk, cheese, chicken noode soup, and rice. She does like yogurt with fruit but that's about it.
The easiest way would be juicing. All kids usually enjoy juice because it's sweet. That would probably be your best bet. Also, sneak veggies into the juice..She won't be able to tell there is even veggies in the juice.
We juice at least 3 times a day and I've gotten my daughter whose 5, to "eat" kale, and other veggies by juicing them with a couple apples for sweetness. She loves it. (insert evil muhahaha laugh here...)
Let her go hungary until she is not spoiled , if there is no junk food in the house, she can't eat. I have to think, who got her into the habit of only eating foods that, are not as heallthy as other stuff.
Kids & dips are a natural match! Since she likes the yogurt/fruit mixture, try offering small portions (about 1 T.) of caramel sauce or even chocolate and let her dip the foods in that. Ranch dip seems to be another big favorite with toddlers. Cut the fruits into interesting sizes and shapes, set out several dips, and let her choose which to use.
by Charlu6 years ago
Is your pet a picky eater or do they just gobble it down in no time?
by Peeples5 years ago
Ideas for healthy food for a 1 year old who is picky?My daughter (13mths old) was started on veggies but now hates them. Her diet consists of fruit, whole grain crackers and bread, cheese, yogurt and milk. Outside of...
by sunbun1436 years ago
What are some ideas or recipes for a picky eater (toddler)?He likes rice but does not like proteins aka meat.
by Maureen Macleod15 months ago
My 9 month old French Bulldog is so picky with her foodShe is perfectly healthy but has always been a fussy eater. I have tried raw food, and have a load in the freezer after buying several portions because for a whole...
by Beata Stasak5 years ago
Are you ready to grow your own food?Will Allen believes that everybody regardless of their economic means should have access to the same healthy, safe and affordable food that is grown naturally.For some poor urban...
by Susan Britton6 years ago
Can you disguise vegetables enough so a meat eater will eat them?How do you introduce or hide vegetables to a meat eating family so they will eat and benefit from veggies.
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Written by Josiah Ariyama
Supervised by Dr. Asato Ikeda
A Third Gender: Beautiful Youths in Japanese Prints, exhibited at the ROM from May until November, 2016 offers but a glimpse into the lives of Wakashu, or “young companions” living in Edo period Japan (1603-1868). The exhibition not only features a plethora of great woodblock prints, but exacerbates the viewer’s imaginary journey into this time through the use of film, screens, and sartorial artefacts such as armour, kimono and hair ornaments.
With his talk at the upcoming 11th annual Eva Holtby Lecture on Contemporary Culture, part of the ROM Speaks series, constitutional lawyer and civil liberties advocate Jameel Jaffer will focus on the phenomenon of official secrecy. Zeroing in on the legal, political, and social repercussions of allowing democratic governments to withhold information about national security policy from the public. The ROM's Ann Webb recently talked with Jaffer about the interssection of art and secrecy.
In September, 1971, the ROM opened the landmark exhibition Keep Me Warm One Night, a kaleidoscopic display of over 500 pieces of Canadian handweaving. It was the culmination of decades of pioneering research and collecting by the ROM curatorial powerhouse duo ‘Burnham and Burnham’, aka Dorothy K. Burnham and Harold B. Burnham.


Polyethylene terephthalate (PET)/nano-hydroxyapatite (nHAp) composite granules were obtained using twin-screw extruder. Preforms were prepared by injection molding and then PET/nHAp bottles were produced by blow molding. For PET bottles with nHAp, the migration amounts of carboxylic acid (COOH), acetaldehyde (AA), diethylene glycol (DEG), and isophthalic acid (IPA); glass transition temperature (T-g); melting temperature (T-m); and the maximum crystallization temperature (T-cry) were measured. The load-carrying capacity, burst strength, stress cracking, and regional material distribution tests were carried out on the bottles. X-ray diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and ultraviolet transmittance analyses were conducted to explain the changes in mechanical, chemical, physical properties, and light transmission of bottles. It was found out that the COOH amount increased and the AA content decreased with increasing nHAp amount. On the other hand, no change was observed in the amounts of DEG and IPA. Although the mechanical properties such as load-carrying capacity and burst strength of the bottles have improved, it has been determined that the standard environmental stress crack resistance test procedure cannot be applied to such a composite. Experimental findings indicate that nHAp disrupts the chemical structure of PET and it isolates harmful chemicals such as AA by forming intermolecular bonds. Moreover, with the addition of up to 0.8% nHAp, PET bottles block the light transmission approximately 80% within 400-700 nm wave length zone. The study demonstrates that the PET/nHAp composite bottles can be used in the food industry, particularly in the packaging of milk and milk products which are vulnerable to light exposure.


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January 4th 1928 - Astrain Antonio
Spanish Historian of the Order
† in Loyola
Born on November 17th 1857 in Undiano (Navarra), he entered very young into the Society of Jesus. After special studies in literature and rhetoric he studied - since 1895 exclusively - the history of the Society of Jesus in the countries of the Spanish Assistence, and had therefore to undertake many journeys.
His researches found their expression in many articles and essays, particularly however in the seven volumes of his chief work 'Historia de la Compaņía de Jesús en la Asistencia de Espaņa', which were published 1902-1925 in Madrid.
His Ignatius Biography appeared 1924 by E. Weber also in German language (publishing house Hermann Rauch in Wiesbaden).
Astrain deserved well of the publication of the sources of the Order's history (Monumenta Historica SJ), which he led from 1921 up to his death.
His plastic representation, his critical sense, his matter-of-fact objectivity and his thorough knowledge of the sources make his Order History, which goes up to 1773, valuable also for the general church history of the Spanish countries.
Astrain died at an age of 71 in the college in Loyola.
[back to Otto Syre's SJ-Calendar]

domain: history


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Duck -- here comes another one! Close on the heels of Richard Egarr's superb recording with the Academy of Ancient Music -- one of my 2009 Blue Note choices -- we now have this enlivening set bearing the name of perhaps the most decorated period-instrument conductor of our time. Given Gardiner's prolific discography, I was actually surprised to find that he had never before recorded this landmark set of concerti grossi, although just about everyone else has. But after one time through these concertos, it was clear that this is far from a "me too" recording. In his booklet note, Gardiner explains that he conducts only the First and Second Concertos from the podium; he acted more as a coach for the remaining pieces, which are led in performance by violinist Kati Debredzeni.
The Egarr recording used one player per part, and was captured in a rather close-up perspective. This new version has a slightly larger ensemble, and consequently a bit more instrumental weight, but it is recorded with a somewhat more distant perspective. The two are similar in projecting a most engaging sense of energy and commitment to this marvelous music. Under Gardiner's guidance, fast tempos are consistently crisp and exciting, and the slow movements are shaped with affecting expressiveness. A choice between these two really comes down to personal preference; I would not want to be without either of them. There are of course many fine recordings of the Brandenburgs, but in my opinion fans of historically informed Bach performance need look no further than these gorgeously recorded recreations.


Security has been beefed up in Osogbo, the Osun capital as the Peoples Democratic Party (PDP), holds its zonal congress.
The News Agency of Nigeria (NAN), reports that there is heavy security presence at Women and Children Development Initiative Foundation (WOCDIF), venue of the congress on Monday, in Osogbo.
Men of the Nigeria Police, Department of State Security Service (DSS), and the Nigeria Security and Civil Defence Corps (NSCDC), were sighted at the entrance and around the venue.
Some of the security personnel were also strategically located few kilometres away from the venue of the congress.
Supporters of the candidates were also seen arriving the venue for accreditation.
Though accreditation of delegates was fixed for 9: 00 a.m., however, as at the time of filling this report, it was yet to commence.
NAN reports that 42 aspirants were cleared by the PDP screening committee on Sunday, to contest for the various positions in the zone.
The congress would be attended by 734 delegates across the six western states.
According to the list released by Retired Col. Austin Akobundu, the PDP National Organising Secretary, Oyo has 171 delegates, Osun -130, Lagos 101, Ogun- 112, Ondo 114, and Ekiti with 115 delegates .
NAN also reports that the chairman of the screening committee Sen. Dino Melaye, had said on Sunday that the screening of aspirants was very peaceful.
A former deputy governor of Oyo State, Taofeek Arapaja, and the immediate zonal chairman of the party, Dr Eddy Olafeso, are contesting for the chairmanship position.
While Gov. Seyi Makinde of Oyo Sate, is supporting Arapaja for the chairmanship position, Mr Ayo Fayose , a former Ekiti governor is supporting Olafeso. (NAN)


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So I'm sure most of you know by now that I'm a huge nerd. The hoopla-along this month was for mythological things, so I chose one of the Daedric Princes from the Elder Scrolls. I always thought Azura's statue was pretty, so I stitched her. I was stupid enough to set my color palette to 30 colors when I made the pattern so there was a lot of confetti stitching. She fits in a 6" hoop.
And here's the image I used
Even with all the colors she stitched up fairly quickly. I might do Meridia next, but I'll remember to set the color palette to less colors.


Undercover investigation exposes illegally long working hours and low wages for staff at factory producing toys for Disney
She sings. She sparkles. And she’s made by women paid just 1p for each doll that shimmers off shop shelves.
This Christmas, tens of thousands of children the world over will excitedly tear the wrapping paper off an Ariel doll – Disney’s Little Mermaid – secure in the knowledge that it was made for them by Santa’s happy elves at the north pole.
Link : Revealed: Disney’s £35 Ariel doll earns a Chinese worker 1p


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Just as in the human world, some dogs are naturally more dominant than others. This behavior can be influenced by factors such as how a dog is raised, but from the time puppies are just a couple of weeks old, dominant traits can be recognized.
Dominant behavior isn’t necessarily a “bad” thing, but it is something that must be managed before it gets out of control. First, it is important to understand that there is a huge difference between a dominant dog and an aggressive dog. While a dominant dog wants to be in charge (be the leader of the pack), an aggressive dog wants to do harm to other animals or to people. A dominant dog is not necessarily a dangerous dog, but it is important to recognize, understand and tame his dominant behavior. The dog must understand that YOU are the leader of the pack and that HE is a follower. A dog that is dominant is not always aggressive. If you are unsure about where your dog fits, get the advice of an expert, such as a vet or a professional dog trainer.
In the dog world, there is a social structure based on ranking, starting with the leader of the pack on down. Some dogs have no desire to be the leader of the pack while others are willing to fight their way to the top. In your household, a dominant dog may want to be the leader, not only of other animals in the house, but of you as well.
Dominant dogs can be large or small, and are found in every breed. There are degrees of dominance. Some dogs will be happy being the leaders of the other animals in the house while submitting to you. Other dogs will keep vying for power until they think that you are submissive to them.
Many dog owners do not even recognize that their dog is displaying dominant behavior as most of these behaviors are in no way violent. It’s very important to learn the traits of a dominant dog, so that you can recognize if you have one. You may be living with a dominant dog right now and not even realize it. Learning the behaviors of a dominant dog is an important step in helping your dog become submissive to you and other family members.
Below are some behaviors often found in dominant dogs.
- The dog will try to push through a door before you. He will knock you out of the way to get outside first.
- The dog will try to prevent you from petting him on the top of his head.
- The dog will attempt to move you out the way when sitting or laying with you.
- While playing, the dog will growl or bark at you.
- The dog will not release food or toys when you command him to do so.
- When the dog wants something that you have, such as a treat or toy, he will bark at you until you give it to him.
- The dog will jump on the furniture before you have given your permission.
- The dog will not obey basic commands such as “no”, “sit”, or “get down”.
- Instead of obeying when you give a command, the dog may try to begin to play.
- The dog will mark your personal items, such as clothing or shoes.
- The dog will mouth you. Even during play this is a dominant behavior.
The above list includes some examples of dominant (NOT aggressive) behavior, but is not an exhaustive list. If your dog displays any of the above behaviors, he may be dominant. Again, it’s not necessarily a bad thing, just something that must be managed.
You have to take control back from your dog. He must know that YOU are the leader of the pack, and that he is a follower. The techniques you use to accomplish this should never be physical, and should not put the dog in a position where he feels the need to become aggressive.
The first step in taking back control from a dominant dog is basic obedience training. This training is, of course, a good idea for ALL dogs, but is particularly necessary with a dominant dog. In addition to basic obedience classes, below are some tips to help you reestablish your role as leader of the pack. None of these tips should be applied with anger towards your dog. Do everything in a calm, non-confrontational manner.
- Recognize one of your dog’s favorite places. This could be his bed or a favorite chair etc…Stand in that place for a couple of minutes many times during the week.
- Do not pet your dominant dog unless he has done something, such as obeying your command, to earn your attention.
- If you play games with your dog, especially a game with a clear winner, such as tug of war, YOU must always be the final winner.
- Eat before you feed your dog, and make sure that the dog sees that you are eating first.
- Do not allow your dog on the furniture without permission. Also, the dog should get off of the furniture on command. If he doesn’t, pull the dog off, but do not use unnecessary roughness.
- If your dog sleeps in your bed, move him to the floor or to a dog bed in your room. Or, move him from the bedroom completely.
Again, DOMINANCE IS NOT AGGRESSION. The tips here will only work with a dominant dog, not an aggressive one. If you think that your dog is going to harm you or another person or animal, it’s important to seek professional help right away.
A dog that thinks he is the leader of the pack is not going become to submissive to you in one day. You must be consistent in your training to reestablish your dominance. As long as you are consistent, you will soon be viewed as the leader of the family by your dominant dog. Rather than being a negative thing, this will help your dog to feel more secure as he will be crystal clear about where he stands within the family.


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UPMC Western Maryland opens Center for Hope and Healing
CUMBERLAND – On Wednesday, September 2, 2020, UPMC Western Maryland officially opened its Center for Hope and Healing, a 24-hour residential treatment facility that will house patients with substance abuse and behavioral health issues.
The center is located on Leslie Lane, adjacent to the UPMC Western Maryland main campus, and was built on land gifted to UPMC Western Maryland from the state. Funding for the project was largely attributed to a $1,000,000 pledge from the WMHS Auxiliary, including an initial gift of $400,000 made in May of 2019. Ground broke on the facility last August.
At a ribbon cutting ceremony held on August 6, UPMC Western Maryland President Barry Ronan said, “Today we celebrate the opening of this historic behavioral health and substance abuse treatment center. It will benefit so many patients that face these challenges in our region.”
Auxiliary President Julie Davis, who works as the Fire Administration Officer for the City of Cumberland Fire Department, said at the time of the initial gift, “I see the impact this crisis has on a daily basis. I see how it taxes our emergency service providers. We were all unanimously in favor of facing this challenge and supporting a valuable asset that will effectively address the need.”
“The community has said loud and clear that there is a tremendous need for behavioral health, which includes both mental health and substance abuse treatment services,” said Jeff O’Neal, Executive Director, Clinics, Practices, and Behavioral Health Services. “This is something special for our community, and I’d like to thank everyone that made this possible. We are now the envy of every other hospital-based program in the state.”
The center will serve patients who are suffer from chronic mental illnesses and those with opioid addiction. It has 8 beds, a communal area where group and one on one outreach will be provided, and a kitchen, along with a working area for staff. Patients will stay roughly 10 days and the option to extend the stay 10 more days will exist.
“This residential treatment center will provide a bridge to the gap between patients who present at our emergency department in acute crisis that may not need to be admitted,” O’Neal said. “It will also be used as a step down for those patients coming out of the hospital, so we can provide them stability that will allow them to maintain in the community and not go back into the hospital.
“This facility provides a safe, residential environment where they will have counseling and physician services. Twenty days in a facility like that can absolutely make a difference between a patient needing to go back to the emergency department and being able to sustain and maintain in the community. While in this facility, individuals will have all of their basic needs met, while receiving treatment and linkage to the additional resources in the community they may need,” O’Neal said.
Sherry Schmitt, Immediate Past President of the Auxiliary, said the center will be a much-needed resource. “Everyone we talked to finds it to be such a worthwhile and positive thing, she said at the time of the groundbreaking. “We have all had people in our lives who are dealing with addiction or have behavioral health problems. We can connect this with the other programs at UPMC Western Maryland and, as a result, we have a shot at making a real impact. Speaking personally, though I imagine most who work in health care would agree, we all seek to find that one thing that will help just one person.”


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The Boathouse Restaurant at Lake Daylesford
Located on the banks of Lake Daylesford and with exquisite water views from any vantage point, The Boathouse Restaurant offers a sophisticated dining venue for lunch and dinner. You can take in the beautiful views from the warm and cosy indoor dining area for winter catch ups and the outdoor deck and seating area during the warmer months. The Boathouse Restaurant provides year-round comfort with stylish food and a relaxed yet professional style of service, making it the perfect place for a glass of wine and a meal with family and friends. The Boathouse Restaurant does not take bookings for lunch but welcomes guests on a walk in basis. Reservations are available for dinner. There is a pet-friendly courtyard just to the left of the main entrance, and your well-behaved dogs are very welcome. Please note that the The Boathouse Restaurant does not own or operate the Paddle Boats on Lake Daylesford.


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December 27, 2001
Quick Pizza By meetapatel by meetapatel
Quick pizza is a simple and quick recipe to recreate the magical Italian delight, pizza at home. The pizza base is topped with sauces and topping of choice and sprinkled with a generous dose of grated cheese.
December 14, 2001
Quick Pizza By Mehta5 by mehta5
Quick pizza is a tempting, mouthwatering and an appealing pizza recipe with select vegetables as topping. The pizza is created within minutes and makes for a lovely way of adding veggies and cheese in a child's diet.
December 23, 2001
Quick Pizza! by maniarjayshree
A wealth of recipe tips, methods and list of ingredients for quick pizza recipe, bread pizza recipe and many more at tarladalal.com
January 03, 2002
Quick Pizzas by maithilie
A simple and quick pizza recipe which can be prepared in minutes and in big quantities , especially for kid's birthday parties and family gatherings.
June 29, 2001
Rolli Polli Stuffed Bread Piece by mushu
Rolli polli stuffed bread piece is a delicious and crispy snack item. The bread slices are cut into squares and then stuffed with a mixture comprising of semolina, chopped vegetables and chillies. The stuffed bread are then deep fried and served hot with tomato sauce.


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This beautiful lighthouse at the coast of Punta de Capdepera is the easternmost point of Mallorca. It's about 55 meters above the ocean and not far away from Cala Ratjada, a pretty popular city for tourists that visit the island.
There is a winding and narrow road up to the lighthouse. Beware of oncoming traffic.
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Lighthouse at Punta de Capdepera (Cala Ratjada / Mallorca), Spain by Dirk Wiemer is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
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domain: travel


Hire Rat Pack & Swing Singer in Ascot
Browse Rat Pack & Swing Singers
Recent review: Jess was amazing and exactly the type of singer we needed for our care home residents Tea Party. All of them loved it and many sang along. Thank you ever so much.
Recent review: "A big thank you to Vintage Ruby for attending our Dignity Day Event. Our residents, friends and family enjoyed her performance, her play list was excellent, she interacted with the audience really well and made our event sparkle."
Recent review: "Hi Anna, Thank you so much for singing at our event yesterday! Evertyhing you sung was perfect and fit really well with our theme. We had some really great feedback about you! Thank you so much again and if we ever do something similar would hire you again in a heartbeat! It was really lov
About: So Sinatra, is one of the leading Event Frank Sinatra impersonators 15 years experience performing solely as Sinatra for just about every conceivable event from London, Singapore , Palm Springs and Sinatra’s Birthplace of New York, Frank is probably the most experienced and capable ‘Sinatra guy’
About: Linley Weir will provide exquisite vocals accompanied by a Duo/Trio/Quartet or Quintet for your wedding, private party, or a corporate event. The musicians are of the highest quality providing a sharp backdrop whether they are playing Swing, Samba rhythms, hip groovy beats to get your feet tapping o
About: Helen the Vintage Singer specalises in 1940's jive and swing, with 1950's rock n roll and a little 1960's motown magic. Singing to custom tracks played through her own PA equipment, she can turn up to any event and bring it to life with her vintage music.
Recent review: Thanks Tony for making my Mother- in- Law's party a huge success! Your music was enjoyed by all ages and encouraged many to get up and dance. Your singing sounded very professional and you listened to and followed our requests. You were very pleasant and flexible and certainly good value for money!T
Recent review: Rodney was absolutely brilliant - everyone said how good he was. He has a very good voice and sang a wide range of songs.
About: A tribute to classic Frank Sinatra along with other famous rat pack songs. Freddie recreates "Sinatra" with his uncanny likeness to the original singer. He has performed throughout the Uk and abroad , working for some prestigious events .
Recent review: I hired Daniel for my parents surprise 70th birthday party. I cannot recommend him enough. A true professional, nothing was a problem and all guests said how brilliant and talented he is. Will definitely be recommending Daniel again as Elvis and a great DJ.
Recent review: Chris sang for us at a corporate Christmas event, he has a fantastic voice and his choice of music was perfect for the occasion. Chris couldn't have been more helpful, and we would definitely hire him again!
Recent review: "We thoroughly enjoyed Blue Orchid's entertainment during ladies night at Lark in the park. The set was just right for the tone of the evening and thank you for being a part of the night, wonderful performance. (10 / 10) Carmela"
Recent review: I volunteer as a driver for Contact the Elderly where we have "host's" who cater a "tea party" for our fabulous elderly guests once a month many of whom rarely get out their home any other time and as a driver I pick up the same person every month. Dave sang for our lovely guests and it bought tears
Recent review: Thank you The Night & Day Collective for your professional attitude - you all arrived with plenty of time to set up etc and were all very considerate. Everyone at the party really enjoyed the music and the 3 x 30 minute sets were a great hit - great music for a garden party!
Recent review: I am so glad I booked Anthony for my husband’s birthday party. He was a great singer, all our guests were impressed with how much he sounded like Sinatra, Elvis etc. but also a great entertainer. He took time to make sure the set list was exactly what we wanted and was flexible in suggesting changes
Recent review: "In one word "" wow"" Laura is an amazing singer and is very friendly, Complete value for money I will definitely be booking Laura again for future events Thank you Laura ,you are a star!"
Recent review: Just to say a BIG thank you for your great performance on our Christmas Friends and Family Event. Your performance was thoroughly enjoyed and the homeowners said they would love to have you again, so I shall be contacting you again in the near future
Recent review: We booked Daniel for our annual Christmas party. His performance and engagement with our guests was above our expectations. He was truly amazing!!!! Taking requests from Sammy Davis to The Smiths. All of this was delivered with great humour. Would recommend him for any function, he made ours truly m
Recent review: "we wanted to say thank you for your performance here on the 5th. We’ve had some super feedback and one member even said “best tribute act we’ve had so far”. Hopefully I will be in touch for any future performances, and thanks for a great show from everyone at Camberley Heath."
About: Hi, I am Leo Mann, a singer-songwriter performing as a solo with guitar and a looper, in a duo (with a guitarist) or in a 3p band (guitarist-drummer). We are performing our compositions and a repertory of covers (up to 150covers, that we can extend with songs of your choice). Our style goes from sou


text:
Kids reenact Richard Sherman's postgame interview with Erin Andrews
Watch this adorable reenactment.
Admirers Flock to Dole During Kansas Homecoming
Raw: Kangaroo Rescued From Swimming Pool
New Pictures of Ship That Sank in 1888
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domain: entertainment


lhotse is a natural range of ethically-produced yoga clothing.
Embodying the values of yoga, all designs keep healthful body and planet in mind.
Handmade in New Zealand using eco-dyed, organic cotton, lhotse is kind to the environment and the wearer. We source our organic cotton from Australia, with fabric printed in Melbourne using water-based, colour-fast pigments.
The lhotse brand and ethos is inspired by Lhotse peak in Nepal.


Eley 22LR AMMO
Eley 22LR Match Competition Ammunition for Sale
Eley Ammunition is a long established brand for
all rimfire disciplines. A consistent winner in competition, this quality
ammunition will meet all your shooting needs.
For more than 150 years, the ELEY brand has been synonymous with quality,
consistency and medal winning accuracy. ELEY’s research and development
team consistently strives to set new standards in engineering innovation. We
break boundaries by continuously improving our ammunition always aiming to lead
the way in .22LR performance.
Our forward looking approach has led to new
products such as ELEY edge. An all-black cartridge which features a
scientifically developed surface treatment, increasing the friction between the
case and bullet to give a more consistent propellant burn and most importantly


Oct 26, 2016
commented on Vote "Approved" on ST3 on November 8!
I voted no solely because of 12. I've voted yes on every other mass transit system (including the monorail - what was it? 3 times? - but this one seems to mainly connect suburbs to other suburbs. I just don't think we'll get another chance at light rail after we pay for this one so it's important that it's useful.
Nov 21, 2009
commented on Late Returns
"But in Boston's defense, that project undid a snarl of highways to open up a most unique city's divided heart, on arguably the most vital road corridor in the world, I-95"
Interesting point except that I-95 loops around Boston. The road in question is I-93 which connects Boston to Canada and is mainly vital to Bostonians who want to go to Vermont. Fact checking is your friend.
Oct 26, 2009
commented on Constantine Pulling Ahead of Hutchison
The cell phone thing didn't make a difference in 2008. Don't get excited over leaks of internal polling because we have no idea if that is really what the poll said and, for that matter, if this is one of 7 polls this week...
Oct 22, 2009
commented on Well, That Was Close.
14 - the crosswalk light doesn't change at all though and the other cars are slowing down even before the pedestrian is visible.


text:
Coming-of-age story set in the 1950's stars Harvey Keitel as a small-time hustler/dreamer, recently widowed, who tries his best to care for his two daughters, 17-year-old Sonya, and 12-year-old Greta, while Sonya's rapid disulisionment with her father puts her at odds with him more times than nessessary. Written by
When a father doesn't live up to his dreams... a daughter has to stand up for hers.
Did You Know?
Never has a man less equipped for parenthood tried so hard.
Performed by Sons of the Pioneers
Written by Bob Nolan
Courtesy of RCA Records Label of BMG Music See more


text:
The President of the Association of Bureau De Change Operators of Nigeria (ABCON ), Alhaji Aminu Gwadabe, has called on the Central Bank of Nigeria (CBN), to widen sources of forex to members of the association.
Gwadabe on Monday said that for the CBN to improve access to Forex for BDCs, it should register more International Money Transfer Organisations and make BDCs to access export proceeds.
He said that the apex bank should allow the application of BDCs rates for foreign investors’ inflow at the point of entry and exit.
Gwadabe also wanted the Monetary Policy Committee of the CBN to reduce the benchmark interest rate and to settle importers foreign obligation.
He said that for the economy to move forward the CBN should “clean the toxic balance sheets of banks and extend agricultural borrowers scheme like that of rice in the north to cocoa in the west and palm oil in the South East.

domain: finance