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The clinical hallmark of Alzheimer's disease (AD) is impairment of cognition associated with loss of synapses, accumulation of amyloid β (Aβ) both within neurons and as extracellular deposits, and neurofibrillary degeneration composed of phospho-tau. Neurons in the hippocampus are among those that are most vulnerable. The purpose of this study was to investigate the expression of genes associated with cognition, synapse, and mitochondrial function in hippocampal neurons of AD compared to normal individuals. Neurons from the hippocampus with intraneuronal Aβ immunoreactivity were captured with laser microdissection; RNA was extracted; and levels of brain-derived neurotrophic factor (BDNF), TrkB (BDNF receptor), dynamin-1 (DYN), and cytochrome C oxidase subunit II (COX2) were assessed with quantitative real time-polymerase chain reaction. We found no significant differences in the expression of these genes in AD between neurons associated with Aβ compared to those lacking Aβ immunoreactivity. Double immunofluorescence microscopy showed the number of hippocampal neurons coexpressing Aβ or phospho-tau and either BDNF, TrkB, or DYN was similar in AD and controls. Our results suggest that intraneuronal Aβ or phospho-tau do not have obligatory effects on reducing the expression of genes important for memory and cognition in hippocampus of AD.
The clinical hallmark of Alzheimer's disease (AD) is impairment of memory and cognition that correlates with accumulation of Aβ within neurons as well as in extracellular deposits referred to as senile plaques [1–3]. Aβ is derived from amyloid precursor protein (APP) by regulated intramembranous proteolysis and eventually is broken down or eliminated [4]. In AD, these processes are disrupted. Accumulation of soluble forms of Aβ in the hippocampus and amygdala appears to be one of the earliest events that lead to cognitive impairment [5, 6]. More recently, injection of a natural soluble form of Aβ into rat brain rapidly and potently impaired cognitive function without inducing permanent neurological deficits [7]. These results led to the hypothesis that low amounts of diffusible forms of Aβ can directly reduce expression of neuronal genes involved in memory and cognition.
Neuronal synaptic dysfunction is a significant factor contributing to memory loss in AD [8–12]. Evidence suggests that Aβ may affect hippocampal synaptic efficacy before pathological structural degeneration [13, 14]. For example, treatment of hippocampal neurons in vitro with Aβ results in decreases in expression of dynamin-1 (DYN), a protein important in synaptic function [15]. Similarly, mitochondrial genes are up-regulated in APP transgenic mice because of oxidative damage at the earliest stage, indicating impairment of mitochondrial energy metabolism by Aβ [16]. Aβ peptides also cause leaks in the mitochondrial membrane, resulting in the release of cytochrome c, activation of caspase-3, and apoptotic death of neurons [17, 18]. It is possible that intraneuronal Aβ has direct effects on expression of synaptic or mitochondrial genes as an early event in the molecular pathogenesis of AD [3, 13, 16, 19, 20].
We hypothesize that if intraneuronal Aβ directly reduces the expression of neuronal genes associated with neuronal growth and survival, as well as synaptic and mitochondrial functions, hippocampal neurons containing Aβ immunoreactivity will have lower expression of these genes compared to neurons not associated with intraneuronal Aβ. Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are representative genes important for memory and cognition that were analyzed in this study [21–23]. We also analyzed DYN, important for synaptic vesicle recycling and acquisition of memory, and cytochrome C oxidase subunit II (COX2), important for mitochondria function [24, 25]. It is clear from previous studies that the expression of BDNF, TrkB, and DYN is lower in brains of AD compared to controls [26–31]. Cytochrome oxidase activity is decreased, but the expression of mitochondrial gene, such as COX2, is increased, possibly due to compensatory effects on surviving neurons [19, 25, 32, 33]. At present, it is not clear to what extent the changes in the expression of these genes are due to direct effects of intraneuronal Aβ.
In the present study, we used laser capture microdissection to isolate hippocampal neurons associated with intraneuronal Aβ immunoreactivity, as well as neurons without Aβ, in brains of AD and normal controls and determined the levels of BDNF, DYN, and COX2 by qRT-PCR. We found no significant differences in the expression of these genes in neurons with or without Aβ immunoreactivity in AD. Using double immunofluorescence microscopy (DIF), we confirmed there were hippocampal neurons with Aβ immunoreactivity that express BDNF, TrkB, or DYN in both AD and controls. Similar to previous reports, we found lower levels of BDNF, TrkB, and DYN in AD that were correlated with an overall increase in intraneuronal Aβ.
We then investigated whether hippocampal neurons with phosphorylated tau (P-tau) immunoreactivity express lower levels of BDNF, TrkB, and DYN than neurons without P-tau immunoreactivity. Tau is a microtubule-binding protein that becomes abnormally phosphorylated in AD and accumulates to form neurofibrillary tangles [34, 35]. Our results using DIF were similar to Aβ, with no significant difference in gene expression for BDNF or DYN in neurons with and without P-tau immunoreactivity in AD and controls. We conclude that neither Aβ nor P-tau has obligatory intracellular effects on reducing the expression of genes important for memory and cognition in hippocampus of AD.
Both cryosections and paraffin-embedded hippocampal tissue sections (10 μm thick) were obtained from AD and age-matched controls. At the time of autopsy 0.5-cm thick pieces of hippocampus were placed in OCT medium and flash frozen in isopentane chilled with liquid nitrogen. Sections were cut at 10 μm thickness and stored at −80°C before analysis. Paraffin sections were obtained from additional AD cases and controls that had been fixed for 10–14 days in neutral buffered formaldehyde before embedding. All cases were confirmed by standardized clinical and neuropathologic methods that included quantitative assessment of senile plaques and neurofibrillary tangles with thioflavin-S fluorescent microscopy in multiple cortical sections and the hippocampus. Tissue was obtained from Mayo Clinic, Jacksonville, FL. Pathological information on the cases is summarized in Table 1. The time interval from death to freezing or fixation of tissue was between 2 and 11 hours.
All procedures were carried out in an RNase-free environment, and all solutions contained placental RNase inhibitor (40 U/100μl) from Invitrogen (Carlsbad, CA). Slides were processed singly with rapid staining and washed briefly with phosphate-buffered saline (PBS) after each step. For immunostaining prior to laser capture, we followed the procedures of Fend F, et al. and D'Andrea, et al [36, 37]. The procedure is as follows: cryosections of AD or controls were thawed, fixed with 75% ethanol for 30 sec, and then immunostained for 5 min at room temperature for Aβ using a rabbit polyclonal antibody (0.83 μg/μl) from Invitrogen diluted to 1:625 with 1.5% goat serum in PBS. Labeled cells were identified by incubating for 2 min at 37°C with fluorescein isothiocyanate (FITC)-labeled swine anti-rabbit secondary antibody (DAKO, Carpinteria, CA) diluted to 1:10 with 1.5% goat serum in PBS. Slides were pretreated with 0.05% pontamine and 0.1% Triton X-100 to minimize background. Subsequently, neurons were morphologically identified by staining with 1% Nissl (Sigma, St. Louis, MO; ref. 38) for 30 sec, dehydrated with xylene, and stored in a vacuum desiccator until microdissection. Sections were microdissected using a Leica laser capture microdissection (LCM) unit at the Imaging Laboratory, University of Miami Miller School of Medicine (Miami, FL). From an AD case, Aβ positive and negative Nissl stained neurons were collected into separate tubes (Fig. 1). Neurons from normal controls often displayed only Nissl staining. On average, ~500 neurons of each category were collected per slide for AD and ~1000 neurons per slide for the controls. Neurons from six slides for each case were pooled. Three AD cases (AD1, AD2, AD3) and four controls (C1, C2, C3, C4) were processed (Table 1).
RNA from pooled neurons for each case was extracted using an Absolutely RNA Nano kit from Stratagene (La Jolla, CA) and treated with RNAse-free DNase I (Invitrogen) to remove DNA. The concentration for each RNA preparation was determined using the Ribogreen kit (Invitrogen). RNA was asymmetrically amplified through two cycles using a Message Amp aRNA kit from Ambion (Austin, TX). In addition, total RNA from an age-matched normal case from the hippocampus tissue was obtained from Ambion.
Four µg or less of RNA isolated from LCM neurons or hippocampus tissue was reverse transcribed to cDNA with random primers using the First Strand cDNA Synthesis kit for RT-PCR from Roche Applied Sciences (Indianapolis, IN) in a thermo-cycler GeneAmp 9600 PCR from Perkin-Elmer (Waltham, MA). Primer-pair sequences specific for BDNF (Qiagen, Chatsworth, CA), DYN (Operon Huntsville, AL), and COX2 (TIB Mol Biol, Adelphia, NJ) were designed using published literature or the Primer 3 program (http://frodo.wi.mit.edu) (Table 2). Ribosome elongation factor 1α (EF1α) was used as the reference gene for the calculation of Ro [39] (Table 2). Quantitative RT-PCR was performed using the Light Cycler DNA Master SYBR Green I kit with varying amounts of cDNA (1 to 200 ng) and a Light Cycler system from Roche Applied Sciences (Indianapolis, IN). The qRT-PCR and melting point (Mp) profiles were obtained. Each product was confirmed for the expected size by agarose gel electrophoresis. For EF1α and COX2, PCR was started with denaturation at 95°C, 30 sec, followed by amplification for 45 cycles at 95°C (0 sec, 20°C/sec), annealing at 50°C (10 sec, 5°C/sec), and elongation at 72°C (10 sec, 2°C/sec),and linked to detection at 80°C (0 sec, 20°C/sec). For BDNF and DYN, target genes with small copy number, PCR was started with a two-step denaturation at 50°C for 2 min, 95°C for 2 min, followed by amplification for 45 cycles at 95°C 15 sec, 58°C 30 sec, 72°C 45sec, and detection at 80°C [40]. The qRT-PCR profile was obtained by the instrument software using the fluorescent intensity at the detection temperature for each cycle. Negative controls were without reverse transcriptase (RT), resulting in no PCR product. The EF1α reference gene was always analyzed using the same conditions as the target gene for calculation of Ro. Age-matched hippocampus RNA was also included in each run and used as a reference (Ro=1). Each sample was analyzed at least three times from at least three independent experiments.
Ro, the initial ratio of target RNA of a specimen to E1Fα RNA, was obtained by the equation derived by Pfaffl [41]. Ro for each specimen was averaged from at least three independent experiments. The amplification efficiency (E), whose log is the slope for the logarithmic phase of each amplification curve, was obtained by the PSI-plot software from Poly Software International (Salt Lake City, UT) or by the Microsoft Excel program [42]. Crossing point (Cp) or the threshold of logarithmic amplification was obtained as the second derivative maximum using the instrument software. Statistical analyses as described by Fujimura, et al. [43] showed that the ratios of target genes to the reference gene EF1α (Ro) for each set of specimens were independent of cDNA concentrations. Therefore, the significant differences observed in Ro between specimens were due to differences in amounts of target gene RNA. For each target gene, the analysis of variance was performed to compare Ro values between AD (n=3) and controls (n=4). Significance level was set at 0.05 for all tests. A post-hoc power analysis showed that with a two-sided 0.05 significance level and assuming no interclass correlation, there is an 80 percent and 88 percent power to detect an effect size of 1.75 and 2.00, respectively.
For DIF, additional paraffin-embedded hippocampal sections from AD and controls were used (Table 1). Endogenous peroxidase activity was blocked using 3% H2O2 in methanol for 5 min. For antigen retrieval, sections were incubated in hot 10 mM citrate buffer (pH 6.0) for 20 min. Immunofluorescence for Aβ (Abcam, Cambridge, MA; ab10148), P-tau (clone AT8; Pierce Biotechnology, Rockford, Il), BDNF (Santa Cruz Biotechnology, Santa Cruz, CA; H-117), TrkB (Cell Signaling, Beverly, MA; clone 80G2), and DYN (BD Biosciences, San Jose, CA; clone 41) was done using a 1:50 dilution of rabbit polyclonal or mouse monoclonal antibodies for 30 min at room temperature. After incubation with antibodies to either Aβ or P-tau, antibody binding was detected with biotinylated anti-rabbit or mouse IgG (1/200 dilution; 20 min at room temperature; Vector Laboratories, Burlingame, CA), a 1:300 dilution of FITC-avidin DCS (Vector Laboratories) for 5 min, followed by avidin/biotin blocking for 15 min, and then immunostaining with BDNF, TrkB, or DYN and detection with biotinylated anti-rabbit or mouse IgG and Texas Red DCS for 5 min. Psuedocolor images were captured using a Nikon fluorescence microscope with FITC and Texas Red filters using IP Lab software (Fairfax, VA) and merged using Adobe Photoshop (San Jose, CA). The number of Aβ, P-tau, BDNF, TrkB, and DYN positive cells from 3–16 high-power fields (x200) were chosen from the pyramidal layer in areas with the highest density of immunostaining for three AD (AD5, AD6, AD7) and three controls (C5, C6, C7). The number of cells coexpressing Aβ/TrkB, Aβ/DYN, P-tau/BDNF, and P-tau/DYN was determined by counting yellow cells from the merged images. For negative controls, we used the same concentration of mouse or rabbit IgG (Santa Cruz Biotechnology) instead of specific primary antibodies, resulting in lack of immunostaining.
Total human hippocampus tissue lysates from normal and AD and from normal prostate was purchased from ProSci, Inc. (Poway, CA). After separation of 5!10 μ-g protein by SDS-PAGE, proteins were transferred by electrophoresis to Immobilon-P membrane and incubated in 5% nonfat dry milk, TBS, and 0.1% Tween-20 for 1 hour. The same antibodies used for DIF were diluted 1/1,000 in 5% nonfat dry milk, TBS, and 0.1% Tween-20 and incubated overnight at 4EC. Membranes were washed in TBS and 0.1% Tween-20 and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (1/2,000 dilution; Santa Cruz) for 1 hour, washed in TBS and 0.1% Tween-20, and analyzed by exposure to X-ray film using enhanced chemiluminescence plus (ECL plus, GE Healthcare Bio-Sciences Corp., Piscataway, NJ). Staining of total protein with Coomassie blue was used as protein loading controls. X-ray films were scanned using an Epson Perfection 2450 Photo scanner.
We hypothesized that Aβ-associated neurons would have lower expression levels of genes essential for memory, as well as synaptic and mitochondrial functions, compared to Aβ-free neurons. To test this hypothesis, we obtained frozen sections of hippocampal tissue from AD and controls (Table 1). Aβ-positive and Aβ-negative Nissl-positive neurons were obtained by LCM from three AD and four control cases and analyzed by qRT-PCR for the expression of BDNF, DYN, and COX2 (Figs. 1, ,2).2). The results showed that for each AD case, there were no significant differences in the levels of BDNF, DYN, or COX2 between Aβ-positive and Aβ-negative neurons (Fig. 2). In addition, the changes in expression of COX2 and DYN were not necessarily linked to changes in expression of BDNF. For BDNF, a gene essential for memory and cognition, the expression in Nissl-positive neurons of the controls was much higher (Ro = 10) compared to total hippocampal RNA, while in AD the expression in Nissl-positive neurons was much lower (Ro = 0.5) (Fig. 2A). For TrkB, a receptor for BDNF, the expression in AD cases was also very low (Ro ≤ 0.01) (data not shown). For DYN, a gene essential for synapses, the expression was similar between AD and controls, but much lower than in total hippocampal RNA (Ro ≤ 0.1) (Fig. 2B). For mitochondrial COX2, the expression was similar in controls compared to total hippocampal RNA (Ro = 1), with substantial variation in AD (Fig. 2C). Thus the changes in the expression of these three target genes did not appear to be linked. We conclude that there may not always be a direct relationship between memory and cognitive impairment and mitochondrial or synaptic dysfunction in AD, contrary to the expectation from the literature [3, 6, 8–13, 18–20].
We used DIF to determine if hippocampal neurons with Aβ immunoreactivity that expressed BDNF, TrkB, or DYN were present in both AD and controls. As expected, we found more neurons with intracellular Aβ immunoreactivity in AD compared to controls and more neurons immunoreactive for BDNF, TrkB, and DYN in controls compared to AD (Figs. 3, ,4,4, ,7).7). The number of hippocampal neurons double immunostained for Aβ and TrkB or Aβ and DYN (yellow cells in the merged images) was similar in AD and controls (Fig. 7B). These results support the qRT-PCR analysis, in that expression of BDNF and DYN is similarly detected in hippocampal neurons of AD cases that are both Aβ-positive and Aβ-negative.
In addition to Aβ, P-tau, the major structural component of neurofibrillary tangles, has been hypothesized to have an important role in neuronal dysfunction in AD [31, 34, 35]. We used DIF to determine if hippocampal neurons that were immunoreactive for P-tau also expressed BDNF or DYN. Our results showed, as predicted, more neurons with P-tau immunoreactivity in AD compared to normal controls (Figs. 5, ,7A).7A). Similar to the results with Aβ immunoreactivity, the number of hippocampal neurons double-immunolabeled for P-tau and BDNF or for P-tau and DYN (yellow cells in the merged images) was similar in AD and controls (Figs. 5, ,7B).7B). There were fewer double-labeled neurons for P-tau and DYN (0.9–1.3 cells/hpf) compared to Aβ and DYN (2.3–2.5 cells/hpf) in both AD and controls (Fig. 7B); however, the differences were not statistically significant (P = 0.1). The specificity of the antibodies used for DIF were confirmed by Western blot analysis of human hippocampus protein lysates from normal and AD compared to proteins from human prostate (Fig. 6). Overall, these results suggest that intraneuronal accumulation of Aβ and P-tau protein in hippocampal neurons does not have a strong negative effect on expression (mRNA and protein) levels of BDNF, TrkB, or DYN in either AD or controls.
The underlying molecular mechanisms of AD remain to be elucidated. Two prevailing hypotheses for the progressive neurologic deterioration in AD are increases in Aβ in senile plaques and P-tau in neurofibrillary tangles [2, 3, 35, 44]. More recent evidence in transgenic mouse and rat models suggest that the soluble oligomers of Aβ are the toxic species in AD [5–7]. It is not clear in human cases of AD if the increases in Aβ or P-tau in hippocampal neurons directly decreases the expression of genes important for memory and cognition, such as BDNF and TrkB, or genes important in synaptic and mitochondrial function, such as DYN and COX2, respectively. In the present study, our results using LCM and qRT-PCR analysis of RNA showed no differences in the expression of these genes (i.e., BDNF, DYN, and COX2) in neurons of the hippocampus of AD and controls that displayed intraneuronal Aβ immunoreactivity compared to neurons without Aβ immunoreactivity (Fig. 2). Utilizing DIF in AD and controls, we also found a similar number of hippocampal neurons coexpressing Aβ/TrkB, Aβ/DYN, P-tau/BDNF, and P-tau/DYN (Figs. 3–5, ,7B).7B). Therefore, our data suggest that the accumulation of intracellular Aβ and P-tau in hippocampal neurons does not always play a major role in directly reducing expression of BDNF, TrkB, DYN, or COX2.
Data suggesting that Aβ can directly reduce the expression of DYN come from experimental treatment of fetal rat hippocampal neurons grown in culture [15]. A small but significant decrease in DYN protein in hippocampal extracts isolated from the Tg2576 transgenic mouse model of AD has also been demonstrated [15]. These mice contain a double mutation in APP that increases production of Aβ and produces some of the characteristics of AD pathology, in particular, senile plaque formation [45]. The reduction of DYN protein in cultured rat hippocampal neurons and possibly in the Tg2576 transgenic mice may be due to Aβ-mediated increases in calpain degradation of DYN [15]. This potential mechanism of Aβ-mediated increase of calpain degradation of DYN protein may explain why our data in human hippocampal tissue sections from AD show no difference in DYN mRNA levels compared to control cases, whereas our results utilizing DIF show reduced levels of DYN protein in AD compared to controls (Figs. 2, ,3B,3B, ,7A).7A). However, if the Aβ-mediated increase in calpain degradation of DYN protein is a major mechanism, it is difficult to explain why our results show similar numbers of dual-labeled Aβ/DYN hippocampal neurons in AD and controls (Figs. 3B, ,7B7B).
Additional evidence in fetal rat cultured cortical neurons suggests that Aβ can interfere with the ERK and AKT signaling pathways that activate BDNF transcription factors [46]. Another study found that treatment of a human neuroblastoma cell line with soluble Aβ peptides decreases phosphorylated cAMP response element-binding protein (CREB), a regulator of BDNF transcription, and BDNF total mRNA [40]. In the present report, our data are similar to those of other previously published reports showing that BDNF mRNA and protein levels are reduced in AD compared to controls [21–23]. Because our data showed that the expression level of BDNF mRNA was similar in hippocampal neurons with intraneuronal Aβ compared to neurons free of Aβ immunoreactivity (Fig. 2A), it is likely that intraneuronal Aβ-mediated reduction of transcription factors regulating BDNF is not a major feature of AD.
Our results in hippocampal tissue sections are contrary to the hypothesis that intraneuronal Aβ directly reduces the expression of genes important in memory and cognition. However, it is possible that the hippocampus in AD is permeated with soluble oligomeric Aβ peptides that are not visible with immunostaining. We also investigated by DIF whether P-tau was associated with reduced levels of BDNF, TrkB, or DYN in hippocampal neurons of AD and controls. The abnormal accumulation of P-tau is hypothesized to disrupt transport of proteins and growth factors by interfering with microtubule-dependent axoplasmic transport [35, 44]. The results with DIF, using the AT8 monoclonal antibody that specifically recognizes P-tau but not nonphosphorylated tau protein, were similar to the results with DIF for Aβ (Figs. 5, ,7).7). However, in view of our negative findings, future studies with greater sample sizes are warranted.
There are no reports that the overexpression of P-tau has a direct effect in reducing the levels of DYN or BDNF, as has been reported for Aβ in cultured neuronal cells [15, 40, 46]. In contrast, one report shows that BDNF stimulation of differentiated P19 mouse embryonic carcinoma cells results in a rapid decrease in tau phosphorylation, suggesting that BDNF activation of its receptor, TrkB, has a role in lowering P-tau protein [47]. Therefore, one possibility is that reduction of BDNF protein increases P-tau, while P-tau does not directly reduce BDNF. However, if the reduction of BDNF is correlated with increased P-tau, it is difficult to explain why our results show similar numbers of hippocampal neurons coexpressing P-tau and BDNF in both AD and controls (Figs. 5A, ,7B).7B). There were fewer P-tau labeled neurons coexpressing BDNF and these cells were present in the same proportion in the AD and control cases. It would be of interest to determine if these cells are the same cells that coexpress Aβ and BDNF.
Perhaps a better way to determine whether Aβ and P-tau protein are maintained at harmless levels in healthy controls is to study animal models. Injection of a natural soluble form of Aβ peptide into rat brain rapidly and potently impairs cognitive function without inducing permanent neurological deficits, although the effect on genes important in cognitive function was not reported [7]. Numerous transgenic mouse models are available that overexpress mutated APP or mutated tau that develops amyloid plaques and neurofibrillary tangles, respectively [48, 49]. The advent of inducible transgenic mouse models that target neuronal cells with APP or tau in a controlled and timed manner may be required to determine whether genes important for memory and cognition, like BDNF, TrkB, and DYN, are reduced [48–51]. Although the neuropathology of transgenic mouse models appears similar to that in AD, synaptic changes at the molecular level may not completely recapitulate the pathology in humans [48].
To analyze the expression levels of genes associated with memory and cognition, we obtained hippocampal tissue from AD and controls 2 to 11 hours after death. This analysis reduces variables associated with cultured neuronal cells and the differences observed in animal models compared to human AD. Our results show that in AD and controls, the reduction in intracellular levels of BDNF, TrkB, DYN, and COX2 does not correlate with intraneuronal Aβ and P-tau. There may be an important indirect effect of Aβ and P-tau that reduces the levels of the expression of these genes. An alternative proposal is that Aβ and P-tau are not the primary cause of AD but that increased oxidative stress caused by other pathogenic factors may lead to the increases in Aβ and P-tau, and possibly decrease the expression of neural genes [16, 52].
This project was initiated while R.K.F. was in Dr. Paul Shapshak's laboratory (Department of Psychiatry, University of Miami Miller School of Medicine). We thank Dr. Carol Petito (Department of Pathology, University of Miami Miller School of Medicine) and Brigitte Shaw (Imaging Laboratory, Diabetes Research Institute, University of Miami Miller School of Medicine) for advice and guidance with LCM. We thank David Vasquez and Blanca Rodriguez for excellent technical assistance and Virginia Roos for editorial assistance. This work was supported in part by the South Florida Veterans Affairs Foundation for Research and Education and by the Department of Veterans Affairs. G.A.H. is the recipient of a VA Senior Research Career Scientist award.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Saturday night's 10-7 loss to Washington State was a disappointing Coliseum debut for USC freshman Su'a Cravens.
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In the past few days, the Atlanta Braves have signed free agent closer Billy Wagner and now the Philadelphia Phillies have agreed to terms with infielder Placido Polanco.
The Phillies were known to be in the market for a third baseman this offseason, but signing Polanco (who played a Gold Glove second base last season for the Tigers) to a three-year deal is a bit of a head-scratcher.
Apparently free agent Chone Figgins was going to be too expensive for the Phillies' pocketbook. But Polanco hasn't played third regularly since 2002. He's still decent offensively and will get an upgrade in his fantasy value by gaining multiple position eligibility and moving into the Phillies potent offense. Polanco's a prototypical No. 2 hitter and should score plenty of runs by hitting in front of Chase Utley and Ryan Howard.
It's just that he's 34 years old and his OPS has declined each of the past two seasons. Be careful not to overvalue him in mixed leagues, but with the lack of quality second basemen in the NL, he could give the Phils two of the league's top five at the position.
The Braves move makes a lot of sense with both Mike Gonzalez and Rafael Soriano testing the free agent waters. The 38-year-old Wagner was quite effective last season after returning from elbow surgery -- posting a 1.72 ERA and 1.02 WHIP in 15 2/3 innings.
Say what you will about small sample size, but Wagner was throwing gas out of the Red Sox pen (26 K's) and has had only one season in his 15-year career with an ERA above 2.86.
As Aaron Gleeman of RotoWorld.com points out, the Red Sox will end up getting a pair of draft picks as compensation for Wagner signing ... which goes to show how much smarter the Red Sox are than the team that traded Wagner to them -- the New York Mets.
With Atlanta re-signing Tim Hudson and also adding reliever Takashi Saito to its stockpile of pitching, a trade for another bat could even further improve the team's chances to contend in the NL East.
Ummmm ... New York Mets, the ball is in your court. Do they dare go after what they need most, a power hitter such as Matt Holliday or Jason Bay?
(Oops. Almost forgot. They did sign Chris Coste.)
Steve's been with USA TODAY in one form or another since 1993. He started at USATODAY.com in 1996, becoming the website's baseball editor in 1999, and has written the fantasy baseball column for Sports Weekly since 2006. Follow him on Twitter: @sgardnerUSAT.
Sign up for Fantasy Windup e-mail alerts


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Diving In British Columbia
Any British Columbia diver will tell you that diving the emerald waters off our coast is the greatest experience on earth. Many will quote Jacques Cousteau, proclaiming that ours is the best cold water diving in the world.
Indeed, scuba and travel magazines consistently rate British Columbia diving as top notch when it comes to biodiversity, shore diving, photography, and the presence of BIG critters.
In addition to great diving, we are fortunate to have many excellent retailers, charters, training professionals and ocean conservation groups who make diving in BC easy, fun and rewarding.
A few Diving Locations throughout British Columbia
- The Underwater Council of British Columbia has put together a list of a few diving locations in BC, dive clubs, dive shops and resorts members of UCBC as an easy reference for all divers.
For more details on dive locations, please see the UCBC Diving Map.
- • Caulfield Cove: Enter from the shore or jump off the dock.
- • Larson Bay: From the parking lot, follow the path down to the water.
- • Whytecliff Park:
• The Cut
• Whyte Islet
- • Lookout Point
- • Kelvin Grove: Drop off gear in the cul-de-sac, then move your car into one of the 5 the "Visitor" spots in the upper parking lot.
- • Porteau Cove Provincial Park:
• Porteau Wall: Only accessible with permission of the park ranger, during the winter. Park near the entrance to the walk-in campsites then walk to the far end of the walk-in sites to enter the water.
- • Furry Creek
- • Copper Cove
- • Whiskey Cove
- • Tuwanek
- • Wall Beach Parking
- •Tyee Beach
- • Coopers Green
- • Grey Rocks
- • Mt. Gardner: Enter from dock then surface swim toward "Elephant Rock.
- • Seymour Landing
- • Madrona Point
- • Smuggler's Cove: Must ask permission from strata to enter area.
- • VT100 Parking: Park on shoulder beyond "No Parking" signs.
- • Cates Park
- • Woodlands
- • Ogden Point Breakwater
- • McMicking Point
- • Ten Mile Point
- • Clover Point


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Note: This message is displayed if (1) your browser is not standards-compliant or (2) you have you disabled CSS. Read our Policies for more information.
The Indiana State Excise Police offer statewide public information programs targeted at increasing awareness to the public and the industry regarding alcohol and tobacco sales. State Excise Officers conduct server training programs to educate those who serve alcoholic beverages and/or sell tobacco products. The server training program provides an overview of the criminal, civil and administrative liabilities connected with the sale of alcoholic beverages and tobacco products. Recognizing false or altered identification, refusing service to intoxicated patrons and giving the employee a good working knowledge of all Indiana laws regarding alcohol and tobacco sales is a major part of the Server Training Program.


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The Smart Business Newsletter is full of useful tidbits this month! I've been having internal conversations with people at Microsoft arguing that small businesses can have mature IT practices and fit on the IO model – in addition to using Small Business Server or Essential Business Server, here's another example…
How to stop worrying about IT security
When you're confident that you can find the information you need, quickly and simply; that you can retrieve 'accidentally deleted' files, and that your systems are being backed up regularly and automatically, you can stop worrying about technology and focus on growing your business.
– Why it's time to upgrade to Windows Vista SP1
– How do you stay safe, not sorry?
– Get a free 90-day trial of Windows Live OneCare


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|279 sq. ft.
|Property type
House 8970 in the town of Mlini, Dubrovnik - South Dalmatia contains accommodation units of type Apartment (3) and is 500 m away from the sea. A beach is nearby (pebble). The house is categorized as "For families with kids". Other guests will most likely be present in the house during your holiday, as it contains several apartments. The owners will be present in the house during your stay. The house owner is under no obligation to accept additional persons and pets that were not stated in the reservation request and it is necessary to report them in advance.
Apartment can accommodate 4 guests. Beds are situated in 1 sleeping rooms, but also in the dining room, within 26m2. Guests can enjoy a sea view from the private terrace. Food can be prepared using barbecue which is shared with other guests too. Other guests or hosts pass through the terrace of the accommodation unit. Apartment has rooms which are not connected with the rest of the unit.
INCLUDED IN THE PRICE: tourist tax, final cleaning, air-conditioning, parking, internet, laundry, baby cot, iron, ironing board, hairdryer, sunchairs.
OBLIGATORY ADDITIONAL COSTS: short stay surcharge.
The availability of additional services for your chosen dates of stay, as well as their prices (if none are listed) must be checked ahead of time. Any additional services not shown in the price calculation are payable directly to the property owner and only if used by the visitor.
Dining room (10 m2): Sofa (2)
Bedroom (10 m2): Double bed (2)
Kitchen (3 m2): Kitchenette: Refrigerator, Sink, Additional equipment: Stove, On request: Water kettle, Coffee machine
Bathroom (3 m2): Shower
Terrace (10 m2): Sea view, Sun protection, Other guests pass through the room, The room is not part of the accommodation unit
Infield (Well-equipped), Courtyard (800m2, Area suitable for children, Sitting area), Parking lot (Number of parking spaces: 3, Distance from the house: In the courtyard), Barbecue (shared), WiFi Internet, Laundry service, Hairdryer, Iron, Ironing board, Sunchairs, Baby cot
The facility is not located near the road
There is no road between the property and the beach
Number of stairs from the property to the beach: 166
Car access possible: Yes, Macadam road: 50 m
The house is located near a potential source of noise
The property is surrounded by greenery
Sea: 500 m
Beach: 500 m
Supermarket: 370 m
Restaurant: 150 m
Emergency room: 10 km
Post office: 1.4 km
Disco: 1.1 km
Diving Centre: 750 m
Clinic: 1.5 km
Airport: 10 km
Pharmacy: 1.7 km
Internet cafe: 600 m
Exchange office: 1.4 km
Tourist info: 600 m
Adriatic.hr is the leading agency for private accommodation in Croatia. The agency was founded in 2000 and has been developing and growing ever since. We have expanded our private accommodation offer, we have gained new foreign partner agencies, and we have a growing number of customers. Our main goal is a satisfied customer- before, during and after using our services. We are committed to finding and managing a pleasant stay for our customers in the destination of choice. The agency supports the client from the moment they submit a booking till the end of their stay.
*Plus compulsory or optional charges based on actual occupancy or consumption. The payment has to be made in the currency offered by the property manager, before use of conversion tool. Payments in other currencies have to be negotiated beforehand with the property manager. The currency conversion tool is provided for your convenience only and is non-binding.


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|For some of the finest real estate representation in Richmond, Virginia, you've come to the right place. Mike Curley of RE/MAX Commonwealth is a real estate professional who does business in the Richmond, Virginia area. Mike Curley was one of few real estate agents chosen as a member of the Real Estate Professionals directory to represent the consumers real estate needs for the Richmond, Virginia area. Mike Curley may be found in the foreclosed homes for sale category of the Real Estate Professionals web site. View this members profile below and decide for yourself if Mike Curley is the one to help you with your real estate needs.
Mike Curley, GRI, CRS, CRB
RE/MAX Commonwealth
7201 Glen Forest Drive
Suite 104
Richmond, VA 23294
Contact: click here
Business Speciality: foreclosed homes for sale, relocation services, foreclosed homes for sale
Years in Business: 26
Bio: Mike provides a superior level of informed service delivered in a courteous and efficient manner.
Professional Memberships: NAR, VAR, RAR
Awards and Achievements: 20 Year Distinguished Achiever Award Recipient
Areas Served: Richmond, Henrico County, Hanover County, New Kent County, Goochland County, Chesterfield
Key Search Words: residential, richmond, CRS, CRB, relocation, experienced, henrico, hanover, chesterfield
To contact a real estate professional in the Richmond, Virginia area, please click here
To continue viewing other real estate professionals in Richmond, Virginia, click on the button below.


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Member Spotlight — Zachary Calev of Fluxco Design Talks Strategy
Krrb is now part of the Apartment Therapy family! Check out the Marketplace for an even wider selection of furnishings and home decor.
Visit the Apartment Therapy Marketplace »
When you want something, you go out and get it. And when you can't find exactly what you're looking for? You make it yourself. That's the mentality behind Fluxco Design, a small business powered by the husband and wife team of Zachary and Lindsay Calev. The pair work hard to source gorgeous vintage finds, as well as repurposing and handcrafting original pieces for their clients in Los Angeles. I got to know Zach and chat about his design strategy below.
Hi Zach! Tell us a bit about yourself. Where are you from and where do you currently live?
I was born and raised in Phoenix, Arizona but moved to California 12 years ago. My wife and I have lived up and down the coast, but settled in Los Angeles a little over 5 years ago.
What is the best thing about living in your neighborhood?
The best thing about living in our neighborhood is that it's quiet and I have space for all my toys, but it's still easily accessible to the best parts of LA.
Can you explain the origin of the shop name "Fluxco?"
The name Fluxco was born out of a nerdy love for sci-fi and time travel. Since I'm also in the business of vintage furniture, I'm inherently attracted to and inspired by items from other time periods so it only felt natural to combine the two themes of time travel and furniture. And what makes time travel possible? The flux capacitor, of course.
We hear you got into the furniture business after growing up building and experimenting with design. What was the first real furniture piece you built?
The first piece of furniture I built was a dining table for me and my wife.
Describe the original design you're the most proud of.
I'm most proud of the Mid-century inspired breakfast nook I made for a client in Eagle Rock. The benches and table base were crafted from solid walnut and designed to fit in the corner their newly remodeled kitchen.
When repurposing a piece, how do you decide what it's final form will be?
I look at the material and shape of the item to see what its strengths and best features are and then examine how it can lend itself to other uses. Sometimes the original function is closely related to the repurposed item and other times it's not related at all.
Which furniture piece that you repurposed underwent the biggest transformation?
The pendant lamp I made out of jukebox parts. I was able to use the repurposed parts to create a lighting experience that you can't get from traditional fixtures.
In addition to building and repurposing furniture, you also source and sell vintage. Where do you go locally to find the best vintage furniture?
I enjoy going to the Long Beach Flea Market to see the best mix of vintage furniture and good deals in one place. However, when I need to get a quick fix of vintage shopping I usually head over to Lankershim Boulevard in North Hollywood.
Describe your dream piece of vintage furniture you'd love to stumble upon.
I'm currently on the hunt for a matching set of four original Eames DKR chairs with orange bikini covers.
Lastly, what's the most rewarding thing about owning your own business? And the most challenging?
The most rewarding thing is seeing an idea you've put on paper come to life. The most challenging is figuring out where to keep all the furniture.
For more on Fluxco, check out their website and Instagram to see if you don't end up coveting a new piece for yourself. Don't forget to head over to the Fluxco Krrb corner to find your new favorite piece!


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Added on 18-Oct-2010
Brian is set to receive a special award in recognition of his campaigning on behalf of animals from the International Fund for Animal Welfare at a ceremony hosted by Baroness Gale, at the House of Lords on October 19.Brian has always been passionate about animals and devotes much of his time to animal welfare issues, particularly the protection of British wildlife. Brian set up a soft release site in Surrey nine months ago so that rehabilitated foxes, badgers, owls and other wildlife can be returned to the wild. He has also been a vocal supporter of many animal welfare campaigns. In 2006 he was involved in a successful campaign against the culling of hedgehogs in Uist, Scotland and has also campaigned for the banning of snares and pushed for greater protection for UK seals.More recently, Brian launched his Save-Me campaign to promote decent treatment for animals with the belief that every creature deserves a decent life and a decent death. The campaign opposes badger culs and urges politicians to keep the Hunting Act and protect foxes, deer, hare and mink from being chased and killed by dogs.Robbie Marsland, UK Director of IFAW, said: "We are delighted to be able to reward Brian's dedication to protecting animals over many years with our Campaigner of the Year Award.â€Brian said: "I was very touched when I heard I was to be honoured by IFAW. Fighting for the welfare of our wild animals is not a task that any of us want to have to do. We do it because these magnificent animals live, breathe, see, hear, and feel pleasure and pain like we do, but they do not speak our language, so in the world of humans, they have no voice. "They have no way to speak out against the abuse being perpetrated against them. I am proud and honoured to receive this medal today from one of the world's greatest animal welfare organisations – recognition that I, and all those good souls who have supported the SAVE-ME campaign, have joined the dedicated band of people who care enough to try to give the animals their voice; and one day to put an end to the cruelty.â€To find out more about Brian's Save-Me campaign visit www.save-me.org.uk Source: http://www.queenonline.com/news/1843/
Submitted by:
Queenzone Ghost


text:
It only takes a minute to get started. Fill out our uncomplicated sign up form and login to the site.
This step is optional but will dramatically enhance your chances of your profile being viewed by interested members. Type a little about yourself, your hobbies and upload your best photo.
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Software Developer ( 2 - 6 yrs)
Lalima Jobs
RS 2,25,000 To 4,00,000 Patna
Design and develop software tools for printing designing
Key Skills : embedded software developer,senior software developer,senior software engineer
Urgent Opening for Automation Lead Patna Bihar ( 0 - 1 yrs)
Somya Info Edge Services Pvt. Ltd.
RS 1,25,000 To 1,50,000 Patna
An Automation Resource Will Be Responsible for the Ongoing Quality Assurance of a Portfolio of Systems. the Tester Participates in Qa Activities Across Various Phases of the Software Development Lifecycle (sdlc) Depending On the Requirements of the P...
Key Skills : automation lead,automation engineer,uality utomation engineer,test automation engineer,\r\nelectronic automation engineer
Global Tech Serve
RS 2,00,000 To 4,00,000 Patna
Graduation in It/computer Science/mca with 5 Years of Experience with 2 Years Experience in Power Sector. Candidate Must Be Able to Handle the It Department. Salary Will No Bar for the Right Candidate.
Key Skills : software engineer,senior software engineer,embedded software engineer,trainee software engineer,associate software engineer,sr. software...
Urgent Position for It Service Assurance Patna Bihar ( 0 - 1 yrs)
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RS 1,00,000 To 1,50,000 Patna
1. Extensive experience with working on Synthetics and Application Performance Monitoring including New Relic, BMC TM ART , BMC Trusight and New Relic and its integration with Event Management processes and Tools. 2. Experience with working on Co...
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Computer & Soft Skill Trainer ( 2 - 5 yrs)
Centre of Technology and Entrepreneurship Development (CTED)
RS 1,25,000 To 1,50,000 Bhabua, Buxar, Gaya, Patna
Job Description: Centre Of Technology And Entrepreneurship Development (Cted) Requires Computer & Soft Skill Trainer For Its Bihar Skill Development Centre Established At Buxar And Kaimur (Bhabua) District Different Blocks. Candidates Should Have ...
Key Skills : teaching,soft skill education,training,counseling,software engineer,senior software engineer,embedded software engineer
Aren't you a member yet ?
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Naukri Jobs
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We have urgent vacancies in MNC companies for software engineers. 2014, 2013, 2012 batches can apply. Freshers can apply. Candidate must have min 60% of aggregate without any backlogs. Companies: INVESCO, IBM, WIPRO, GENPACT, TCS.. Immedi...
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Angulo-Ortiz for IBA belt
By Felipe Leon
On Saturday night, an interesting clash of light weltweights will take place when Fernando "La Fiera" Angulo (23-7, 15KO) of Caracas, Venezuela, travels to the hometown of Silverio "Chamaco" Ortiz (20-13, 9KO) of Merida, Yucatan, for the vacant IBA lightweight welterweight title over twelve rounds. In the semi main event, David Lopez (38-12, 23KO) of Nogales, MX, goes against Cesar Alberto Leiva (40-11, 20KO) of Mar de Plata, Argentina, for the also vacant IBA middlweight strap. The fight card will take place at the Poliforum Zamna of Merida, Yucatan. "We have two exciting bouts for titles and I sure it will be a great night of boxing," Jorge Marron, the attending IBA supervisor, stated. "Soon we will have two new champions that I am sure will reprensent the association with honor."


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Classroom Volunteer - Empower young people to achieve a successful tomorrow! Fun & rewarding!
Cause Areas
WhenIt's flexible! We'll work with your schedule.
Wherevarious schools12 counties, NY 12110
Do you remember having a mentor when you were young? We all remember that one person who inspired us or changed our lives in some way. Now YOU can be that person for a student in your own community.
Junior Achievement is a nonprofit organization with the mission of empowering young people to own their economic success. We achieve this mission by recruiting volunteers to present our programs in grades K-12 during the school day.
Our programs center around promoting skills in work readiness, financial literacy, and entrepreneurship. JA uses hands-on, experiential activities to teach young people about business, economics, how to manage money, how to live within their means, and how staying in school will have a direct impact on their future.
How does it work?
Volunteering for JA is fun, flexible and rewarding. JA programs are easy to teach, require a modest time investment, and provide immediate feedback and personal satisfaction. JA provides volunteers with everything they need to be successful in the classroom, including:
Choice of elementary, middle or high school close to home or work
All classroom materials needed to teach the program including a step-by-step lesson plan
Training on how to use the JA materials as well as teaching and classroom management tips (The teacher will remain in the classroom at all times to assist you!)
Connection to a teacher to coordinate when you teach the JA program. (Each program consists of about 5-7 lessons, ranging from 30 minutes - one hour each, depending on grade level. You may schedule these lessons with the teacher however best fits your schedules.)
Good Match For
Teens People 55+
Requirements & Commitment
- Must be at least 18
- Orientation or Training


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Home >
Michael Dalius
Sales Associate
Family: Mom, Dad, Matt and Mark
Pets: Logan
Hobbies/Interests: Building sweat lodges, wood.
Favorite Books: Ursula K. Le Guin
Favorite Bands: Destroyer
Favorite TV: Game of Thrones
Favorite Movies: Spirited Away
Favorite Food: Mayo
Favorite Drink: Whiskey
Favorite Sports/Games: Chess, Boxing
Favorite Apple Products of All Time: iPhone
Favorite Thing About Small Dog: The dogs!
Favorite Quote: "I'm Nick Cage" - Unknown
Favorite Apps
Run Keeper


text:
The 24-year-old has reportedly agreed personal terms on a five-year contract after passing a medical.
Hearts are expected to receive a fee of around £7million for Gordon, with add-ons taking the deal up to £9million and making him potentially the most expensive goalkeeper in British league history.
Gordon's agent Andy Evans was unavailable for comment on Tuesday night, however it is known that the shot-stopper has passed a medical on Wearside.
Hearts assistant boss Stephen Frail does not expect Gordon, who came through the club's youth ranks, to play again for the Jambos.
"If a bid has been accepted, it's just about personal terms," Frail told BBC Sport.
"It's very sad and comes on the back of us losing our goalkeeping coach, Jim Stewart, so it's a difficult time. But it just shows how good the youth policy is when one person moves for £9million."
Villa were believed to have had a bid of £7million accepted for Gordon as manager Martin O'Neill is desperate to bring in a new keeper with last season's number one Thomas Sorensen currently sidelined with a hamstring injury.
Manchester City were also linked with Gordon after losing Swedish international Andreas Isaksson for six weeks with a thumb injury.
However, it appears Gordon has decided to join Roy Keane's Premier League newcomers on Wearside.
- More about:
- Healthcare
- Young People


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Canberra (Australia) Aug 17 (ANI): Australian Prime Minister Julia Gillard, whose Labour Party has long held that the country should become a republic and stop having the British monarch as its head of state, has said that although citizens have a "deep affection" for Queen Elizabeth II, ceremonial royal rule over Australia should end once her reign is over.
"What I would like to see as Prime Minister is that we work our way through to an agreement on a model for the republic. I think the appropriate time for this nation to move to be a republic is when we see the monarch change," News.com.au quoted Gillard, as saying.
"Obviously, I'm hoping for Queen Elizabeth that she lives a long and happy life, and having watched her mother, I think there's every chance that she will," she added.
However, her comment has been criticised by the opposition.
Liberal Party leader Tony Abbot, Gillard's opponent in national elections on Saturday, has disagreed with Gillard, saying he sees no need to change status quo.
"I think that our existing constitutional arrangements have worked well in the past, and I see no reason whatsoever why they can't continue to work well in the future," he added.
Victorian Opposition leader Ted Baillieu has expressed deep concern over Gillard's remarks and added that the Queen's life expectancy should not be part of a debate about an Australian republic.
"I think it's an unfortunate reflection to consider the life of the Queen now, it's not something I would wish to do," he said. (ANI)


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Illini QB racks up 431 yards, 4 TDs to beat Michigan
There are 22 comments on the Chicago Tribune story from Oct 4, 2008, titled Illini QB racks up 431 yards, 4 TDs to beat Michigan. In it, Chicago Tribune reports that:Juice Williams beat Michigan with an accurate arm, quick feet and a cool head. Williams threw for 310 yards and two touchdowns and ran for 121 yards and two scores without making a mistake, leading Illinois to ...
Join the discussion below, or Read more at Chicago Tribune.
#1 Oct 4, 2008
Thanks for a great game and HAPPILY it was on here in GA, so I could watch it.
Will help ease the pain of the pending Cubs loss.
#2 Oct 4, 2008
Nice to hear the "I-L-L I-N-I" chant in the Big House at the end of the game!
#3 Oct 4, 2008
Fortunately, I'm a much bigger Illini fan than a Cubs fan, so my weekend is set.
Great win in Ann Arbor!
#4 Oct 4, 2008
Hail to the Orange!
United States
#5 Oct 4, 2008
Wow. Where did THAT come from?
First couple of drives, and the D was tackling (or not) like the Bears.
Maybe they've finally figured it out.
Now, about that kick coverage...
#6 Oct 4, 2008
I here ya, I'll take Ilinois winning @ mich over the Sox winning tomorrow
United States
#7 Oct 4, 2008
#8 Oct 4, 2008
I couldn't agree more with everything you said as an Illini fan, and to a much lesser extent, a Cubs fan. Baseball is fun for a while, but matters little to me once football season starts.
Even if the Wolverines are down, getting out of Ann Arbor with a win is not easy and is truly special. I'm feeling much better about the Illini season now.
#9 Oct 4, 2008
Great game, Illinois could've put up 60.
#10 Oct 4, 2008
First of all, I am happy for Illinois. I hate Michigan with a passion, and I enjoy a good whooping on them. I wanted to point out more fine reporting by the Trib:
"While Michigan (2-3, 1-1) is not among the Big Ten's elite this year, and showdowns against Top 25 teams like Wisconsin and Ohio State remain, a game like this gives the Illini hope their quarterback is becoming more and more of a catalyst."
I hate to tell them, but Michigan beat Wisconsin last week in the largest comeback in Michigan Stadium history.
#11 Oct 4, 2008
I always enjoy our once-a-decade win over Michigan. The team still isn't where it was last year, but they're still likely bowl-bound.
I still wish Juice would run more. He's passing up easy five-yard rushing gains for passing opportunities. That said, he's making way better decisions this year when he passes. So it kind of evens out.
Brit Miller did an admirable impression of J Lehman today. Keep it up, Brit!
#12 Oct 4, 2008
Return to glory!
#13 Oct 4, 2008
We collected that rent at the big house! Just got back in Chicago. Juice was on today!
#14 Oct 5, 2008
The 100 year curse on America and the Cubs is over after October 14th.
Keep the Faith, stay warm this winter and stay close to your loved ones.
#15 Oct 5, 2008
Now that was JUICE! Gotta go. My boyfriend is calling.
United States
#16 Oct 5, 2008
Great win Illini !!! This takes much of the sting out of the Cubs collapse.
As an Illinois alumn living in MI, I don't face much teasing because the Michigan fans don't see the Illini as being worthy (IL record vs MI shows they are right). But after hearing all their announcers expecting an easy win due to the past record of Illinois and Michigan's big comeback win against Wisconsin - This feels especially sweet. A 25 point pasteing - WAY TO GO ILLINI !!!
#17 Oct 5, 2008
The Illini won because Chris Duvalt was not out there dropping every pass Juice threw to him.
#18 Oct 5, 2008
Football is the only cure for Cub disappointments. I was a freshman in Champaign when the Bears beat the Redskins 73 to 0 in 1941. Maybe they
will help this year too.
United States
#19 Oct 5, 2008
I saw him for the first time and now I intent to watch his games every saturday. He was amazing against Michigan. Williams is putting up better numbers than Payton Manning.
#20 Oct 5, 2008
The last time a quarterback single-handedly manhandled the UM defense, Vince Young. The last time at home, Dononvan McNabb and Syracuse. I was a college junior that year (1998-99), the year after UM 'split' the national championship. I never thought Williams had it in him to do likewise, but he did. Irony of all ironies is that UM's new system demands a QB similar to Juice, and I bet that wasn't lost on Rodriguez. Enjoy Illnois fans, it won't happen again for a while, but at least Juice will likely be in the NFL.
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text: I have xenforo running in my domain. My client has it in her domain. Both of our licenses are current and active. Do I need to download the upgrade from her customer area or can I just download it from mine and install it in both places? I have her license validation token, but I don't know if that matters. Thank you.


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HURRY! Agents available now! 4:15:36
HughesNet in Fairbury
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When you work on your own, it's hard to ignore the financial challenges that come along with it- especially when you're just starting out. There are currently millions of Americans who work for themselves, and many of them find themselves in debt without a safety net. Not only is this an uncomfortable situation, but it can significantly impair your ability to do the kind of work you want to do.
You don't have to let debt get in the way of your dream of striking it out on your own. Here are three things you can do to keep your debt to a minimum as you go about building your income stream:
1. Pay attention to your cash flow. You need to be very clear about the flow of money coming in and going out of your business. This is particularly important if you are bootstrapping your activities. Consider purchasing an accounting suite, such as Quickbooks, or if your budget is limited, you can also check out the free opensource option GnuCash. These programs generally offer a full range of features, such as financial reporting and billing- all of which will help you to stay on top of your cash flow. Once you are in touch with your income and expenses, you can make financial decisions that stay within your means.
2. Know when and how to hire. As a self-employed professional, you'll likely reach a point where outsourcing some of your responsibilities will make sense. But when you do, here's a rule you want to stick to: never bring anyone on board unless by doing so you can anticipate gaining twice that person's wages in new revenue- whether directly or indirectly. For example, you may hire a freelance virtual assistant or social-media expert who can free up your time for more direct revenue generation. One way to ensure that your hired help will deliver on the additional revenue is to be very clear about this person's goals and job summary.
3. Use credit and financing responsibly. Even if you are a conscientious boostrapper, there will likely be moments when you will need to tap into outside financing- whether it is in the form of a business or person credit card or even a small loan from friends, family, or peers. Deciding if and how much to take in financing is similar to the decision of whether or not to hire above. You want to be as certain as you can that the financing you receive will either be paid back quickly or will be used to expand your business in a way that very likely will lead to an increase in revenues.
In short, working on your own doesn't have to leave you drowning in debt. With a little forethought and discipline, you can turn your self-employed work into a lean, mean operation.
Susan Brown
Growing Your Biz- Helping new small and home-based business owners survive and thrive in the first years of operation.
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Antique Beadwork Purse, Silk Embroidered, 2 Sided- NICE
- Sold for: Start Free Trial or Sign In to see what it's worth.
- Item Category: Textiles, Clothing & Accessories
- Source: eBay
- Sold Date: Dec 21, 2008
- Channel: Auction House
Antique Beadwork Purse, with Silk Embroidery. A 2 Sided beadwork purse. No metal frame or liner. Beadwork in very good condition. It is 5" by 9 1/2". AWESOME!!! Shipping charges are postal fees plus $1 for a sealed plastic bag and a new box, no extra handling fee's. --COMBINED SHIPPING DISCOUNTS AVAILABLE FOR MORE THAN ONE PURCHASE. MAD MAGGIE'S MONTAGE HAS BEEN IN BUSINESS FOR OVER 25 YEARS. DEALING IN VINTAGE CLOTHES, VINTAGE AND RARE TEXTILES, ANTIQUES AND ART PRODUCED BY "SAMI", THE OWNER OF MAD MAGGIE'S. SAMI IS GONE NOW BUT LEFT AN INCREDIBLE INVENTORY FOR US TO SHARE WITH YOU. PLEASE ADD US TO YOUR FAVORITE SELLERS LIST- WE HAVE MUCH MORE TO OFFER! INTERNATIONAL PURCHASES ARE WELCOME BUT PLEASE, CHECK SHIPPING CHARGES BEFORE BIDDING. MONEY ORDERS AND CASHIER'S CHECKS ACCEPTED BUT SHIPPING WILL BE DELAYED UNTIL CHECKS CLEAR. ---THANKS AGAIN FOR LOOKING!


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Great Cruise
Sail Date: November 13, 2011
Reviewed: 5 years ago
Traveled As: Couple
Room Type: Oceanview
Pros: Rooms were nice and roomy. Entertainment was great. Food was to die for. People and staff were very helpful.
Cons: no trays were given to hold your plates and bowls with food to get to the tables at the buffets.
Recommends Carnival Freedom? Yes!
Category Ratings
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|It would have been better to have a tray to carry your food to the tables at the buffets.
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Question about 1995 Toyota Tacoma
Posted by bmseymour on
PLugs have a strong orange spark but not a blue fire. Will tach up but not under a load, it will stumble
Is there a sensor or module I need to look into?
Thanks I apppreciate it.
This am when it was cold (15) it flooded, 1st time that has happened.
I have just replaced the plugs, wires were replaced approx 6 months before the trouble started. The problem started & has progressively gotten worse. Starts out smooth then when tacs up starts falling on its face.
Just to bring you up to date. We changed the fuel filter, no chg. Took it today to put on a machine. So far, come to find out we are running the wrong plugs, we should be running double electrobe plugs & not single electrobe. ( but this is what was given to us from the auto stores).. As soon as we get those in, I will update you. Per machine test I have a misfire on #2 cylinder.. Also they said if the plugs don't fix the trouble it is possible I may need to replace the mass air flow sensor. thanks for your help & as I said I will update, once the plugs are changed. Any other info you may have would be greatly appreciated.
will get that done. Thanks appreciate all the help.
If tune-up, wires etc, haven't been done in a while, it would be a good place to start. When were they last done?
Posted on Mar 04, 2009
Usually the parts stores like autozone & PEP boys will scan for free, which should tell you if you have a faulty sensor, but if you want to try a fuel filter replacement, if not done recently, then that is also a good inexpensive place to start.
If you can talk someone in to a fuel pressure test, that would also help with diagnosis.
I'm still leaning towards plugs & wires, but lets see what happens.
O.k. now that answer has come in, lets try the fuel pressure & filter change, and get it scanned while your there getting your parts.
Don't for get to erase codes once that's done to Give everything a fresh start.
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We have devised a savings calculator which provides a basic return on investment calculation based on your number of lines of code, your approach for this code - are you MISRA compliant, or only use a subset of the language ? Do you have legacy code to handle? - as well as the loaded cost for one of your developer/engineer. The return on investment is demonstrating the savings made by deploying the PRQA static analysis solutions.
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I know this isn't the best episode of House ever, but why does it deserve such a low score compared to the others? From a 8.8 to a 7.4? Doesn't make sense to me..
That being said, this episode sort of reinforced my idea of this season, which is that a lot of the cases are less interesting than the personal stuff going on in the characters' lives. Not to say that the personal stuff is good material, but the cases feel ripped off from previous seasons.. with the Joy to the World episode, Foreman being described as House, the case involving the chubbier girl being teased all the time.. it just felt rehashed. And this episode was basically the opposite of the episode of the girl who could feel no pain, except much less interesting.
However, House was on point once again. He seems to save the show when it starts to lose focus. Kutner also is an interesting character, although Taub and Thirteen leave something to be desired. If they could've just started getting Chase and Cameron involved in the show much earlier on than this, I would've been happier. It's too bad. Either way, the episode wasn't nearly as bad as the score indicates. It was a pleasant enough episode that was just a little average.


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A stunning force of nature. This is large format at over a metre in length, inspirational, full of intensity and emotion. Whoever decides to purchase this is purchasing a part of my soul...
Where the sky meets the sea, even on a stormy day, I find absolute peace. I have named this piece Pure Union because the view is just that. A silent space, a peaceful reflection from sky to sea, rendered mute by memories, thoughts and dreams. As part of the Artfinder Mute Collection, for me a mute place is where I can find solace. A place for rejuvenation from a busy life. This is why I have chosen the very simple palette, mostly monochrome, with a dash of teal - simple.
Pure emotion on canvas.
Painted with professional acrylics, this artist quality, deep, beautiful cotton canvas will provide a striking focal point for your room.
With painted sides and varnish it is ready to go with no need for a frame. There is a signature on the front and on the reverse is the Title, a date and signature. All my works are accompanied with a unique Certificate of Authenticity.
All my works are gently and individually packaged with commercial bubble wrap, and their protection is reinforced with strong cardboard packaging to secure safe passage. Paintings are sent tracked, and a signature is requested upon delivery.
Many thanks for looking :)
Slimline canvas, Acrylics
$503.12 Sold
Acrylic paintingby Mel Graham
Acrylic paintingby Mel Graham
Acrylic paintingby Mel Graham
Acrylic paintingby Mel Graham


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An Introduction to Philosophy
This new textbook is a lively and highly accessible introduction to philosophy. From the fundamental issues of philosophical thought to the latest theories in the philosophy of mind, An Introduction to Philosophy provides clear and incisive discussion of the key areas of philosophy for students new to the subject. * Provides the tools new students need to tackle philosophical arguments themselves * Clearly presents and explains contemporary issues and current debates * Covers the key areas of philosophy, including perception, epistemology, metaphysics, the mind, philosophy of religion, ethics and political philosophy * Contains numerous learning features such as introductions, summaries, questions and further reading An Introduction to Philosophy is an ideal text for AS level, A level and first-year undergraduate students or anyone studying the subject for the first time.
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Target: U.S Congress
Goal: Don't require farmers to verify legal immigration status of workers until adequate legal workforces are in place.
Farmers across the United States depend considerably upon a workforce of undocumented immigrants to keep up with food production and economic demands. Recent crackdowns on illegal immigration including a growing force behind the legal verification system E-verify are producing significant shortages even as unemployment remains high. Until farmers have a secure legal workforce, congress should not rush to mandate universal verification of workers' legal status.
The verification system receiving support in the federal government, E-verify, allows employers to check the legal immigration status of workers. Though not foolproof, it has become more accurate since its adoption, and several states have required that all employers must use it. Farmers adamantly oppose the bill because if E-verify were to be required they would very likely not survive, and in turn, dependence on imported food would increase. Illegal immigrants make up 50 to 70% of the agricultural workforce in the U.S, are largely willing to do the demanding work required of the industry, and are migrant workers willing to move as the agricultural industry often necessitates. Even with high unemployment among American citizens, there are few Americans willing to take such agricultural jobs, and those that attempt to statistically do not usually last long in the industry.
Though heavy dependence on the exploitation of illegal immigrants is not sustainable, there is not another workforce able to replace it right now. Enforcing a universal verification system would crush many farm businesses and food production as well as reinforce discrimination and inequality. Congress should instead work towards facilitating citizenship, and establishing rights to health care and education for the immigrants who supply the majority of U.S agricultural labour.
Please request that U.S Congress not require farmers to verify workers' legal status with the damaging E-verify system. Instead, encourage a shift towards increased amnesty towards immigrants and support for American farmers.
Dear U.S Congress,
Washington state and other states across the U.S have been facing farm-labour crises, largely due to recent crackdowns on illegal immigration in the form of a legal verification system called E-verify. By requiring employers to verify the legal status of workers, farms lose a huge portion of their workforce which has no ready replacement, and thus many farms are at serious risk of failure. Please take steps to provide farmers with a sufficient legal workforce before mandating that they strictly limit their current workforce with E-verify.
Farmers in the U.S depend on illegal immigrants to provide up to 70% of labour in the agricultural industry. Regardless of who supplies it, this work is necessary for farms and agricultural communities to survive, and to produce sufficient food for America. Although unemployment is high, very few Americans are willing to do such debilitating work, especially as it often necessitates a migrant lifestyle and can barely support a family.
Since structures now in place create such a heavy dependence on the work of illegal immigrants, it is not practical to immediately cut that workforce with a verification system like E-verify. Instead, restructuring should be employed first to ensure that the farms supplying America with food have a sufficient legal workforce. Please take steps to support immigrants by providing citizenship and rights, and thus support American farmers.
[Your Name Here]
Photo credit: Alex E. Proimos


text:
From the Department of Surgery (Dr Nelson), the Division of Hematology/Oncology (Dr Jackson), the Division of Gastroenterology and Hepatology (Dr Darling), and the Department of Surgery, Lineberger Cancer Center (Dr Gerber), University of North Carolina Hospitals, Chapel Hill, NC.
Correspondence to: Jennifer S. Nelson, MD, Department of Surgery, University of North Carolina Hospitals, 4024 Burnett-Womack, Campus Box 7211, Chapel Hill, NC 27599; e-mail: email@example.com
Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (http://www.chestpubs.org/site/misc/reprints.xhtml).
© 2011 American College of Chest Physicians
1KatoYTanakaNKobayashiKIkedaTHattoriNNonomuraAGrowth of hepatocellular carcinoma into the right atrium. Report of five casesAnn Intern Med19839944724742HanflingSMMetastatic cancer to the heart. Review of the literature and report of 127 casesCirculation1960224744833CokkinosDDDourakisSPAlexopoulouACardiac metastasis of hepatocellular carcinomaEur J Radiol200556391924GoudieRBSecondary tumours of the heart and pericardiumBr Heart J19551721831885LongoRMociniDSantiniMUnusual sites of metastatic malignancy: case 1. Cardiac metastasis in hepatocellular carcinomaJ Clin Oncol20042224501250146BabaHAEngersRHeintzenMPRight atrial metastasis as primary clinical manifestation of hepatocellular carcinomaInt J Cardiol19954732812847OnegliaCNeuhausPSimoncelliUGraffeoMRusconiCCardiac metastasis of a hepatocellular carcinoma after liver transplantation [in French]Presse Med1994232612248LynchMClementsSDShanewiseJSChenCCMartinRPRight-sided cardiac tumors detected by transesophageal echocardiography and its usefulness in differentiating the benign from the malignant onesAm J Cardiol19977967817849HayashiPHTrotterJFEversonGTHepatocellular carcinoma extension into the right atriumLiver Transpl20039111225122610ChuaSOChiangCWLeeYSLinSHLiawYFMoving right atrial mass associated with hepatoma. Two cases detected by echocardiographyChest198689114815011KooJFungKSiuKFRecovery of malignant tumor cells from the right atrium during hepatic resection for hepatocellular carcinomaCancer198352101952195612EngberdingRDanielWGErbelREuropean Cooperative Study GroupDiagnosis of heart tumours by transoesophageal echocardiography: a multicentre study in 154 patientsEur Heart J19931491223122813RahmanianPBCastilloJGSanzJAdamsDHFilsoufiFCardiac myxoma: preoperative diagnosis using a multimodal imaging approach and surgical outcome in a large contemporary seriesInteract Cardiovasc Thorac Surg20076447948314ShyuKGChiangFTKuanPLLienWPChenCLHowSWCardiac metastasis of hepatocellular carcinoma mimicking pericardial effusion on radionuclide angiocardiographyChest1992101126126215DasDKCytodiagnosis of hepatocellular carcinoma in fine-needle aspirates of the liver: its differentiation from reactive hepatocytes and metastatic adenocarcinomaDiagn Cytopathol199921637037716ChuMWAboguddahAKrausPADewarLRUrgent heart surgery for an atrial mass: metastatic hepatocellular carcinomaAnn Thorac Surg200172393193317SaïsseJHardwigsenJCastellaniPCausTLe TreutYPBudd-Chiari syndrome secondary to intracardiac extension of hepatocellular carcinoma. Two cases treated by radical resectionHepatogastroenterology2001483983683918LlovetJMBustamanteJCastellsANatural history of untreated nonsurgical hepatocellular carcinoma: rationale for the design and evaluation of therapeutic trialsHepatology1999291626719ChernMCChuangVPChengTLinZHLinYMTranscatheter arterial chemoembolization for advanced hepatocellular carcinoma with inferior vena cava and right atrial tumorsCardiovasc Intervent Radiol200831473574420FukudaSOkudaKImamuraMImamuraIEriguchiNAoyagiSSurgical resection combined with chemotherapy for advanced hepatocellular carcinoma with tumor thrombus: report of 19 casesSurgery20021313300310
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Floplast Half Round Gutter Downpipe Clip (Dia)68 mm, Black, Pack of 1
- Guarantee 10 year
- 10. Pipe Clip
- Next Day Delivery . Order by 7pm (2pm Sunday) for next day delivery. (note that this will be subject to different terms and conditions)
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text:
Over the next few days, opinion polls are expected to show the first signs of an upturn in Tory fortunes after months of trailing badly behind Labour. All the parties have started to move into election planning mode, amid rumours the Prime Minister could decide to go to the polls as soon as next month.
His senior aides were moving to dampen down election fever this weekend, insisting that a date next year was still preferred, but the speculation has seen a burst of activity in Tory ranks in preparation for any early campaign.
Cameron will this week encourage his party's modernisers by shifting the stress onto social policy and away from the more traditional Conservative strong suits such as immigration and crime.
A long-awaited report on health and education, co-chaired by the former Health Secretary Stephen Dorrell, will be published on Tuesday. It will focus on how the poorest children are most exposed to disease because public health measures are not being implemented effectively. It will also point out that a child born in Newcastle is twice as likely to die before its first birthday as a child born in Winchester.
The Public Services Improvement report will also say that children growing up in areas with failing schools need financial help to flourish. New measures to build more social housing will also be included.
Polls in recent months have shown Labour maintaining an eight- to 10-point lead over the Tories, with the Lib Dems languishing at about 16 per cent. But an Ipsos Mori poll last week for the Sun showed a five-point lead, with 41 per cent of people saying they would vote Labour, and 36 per cent saying Tory.
Two further polls this week are expected to show that the gap is narrowing, with Labour just three to four points ahead. Cameron's messages about Britain's 'broken society' and his tougher lines on immigration and crime appear to have helped his standing.
Cameron's aides said their latest policy review would be used to remind the country of his 'modernising' message. Education proposals include a plan to provide extra central government funding for schools linked to accepting disadvantaged pupils.
In an article in today's Sunday Telegraph, the Tory leader launches a proposal to require poorly performing primary school pupils to re-sit their final year in order to reduce failures at secondary level.
Later this month, another Tory policy group, the Quality of Life review, will call for a series of 'green taxes' and a moratorium on new airport runways in London and the South East. In an interview for today's GMTV Sunday Programme , John Redwood will say: 'Over the last 10 years, the big success area has been the colossal growth in business and financial services and you wouldn't be able to achieve that without sufficient airport capacity.'
A short election campaign would make it possible for Labour to go to the polls as early as October. But the timing might not be advantageous, as the Intergovernmental Conference (IGC) in Lisbon takes place on 18 October, when the EU reform treaty will be debated.
Brown is likely to face renewed calls for a referendum on the treaty at the conference. The government's spending review, setting out the targets for the next three years, is also due to be produced that week.


text:
As the Opposition stuck to its stand, government sought their cooperation for passage of the "most progressive reform" at a time when penetration of insurance was very low.
Parliamentary Affairs Minister M Venkaiah Naidu told PTI that government was ready to accept any meaningful suggestion from the opposition and was awaiting their response after yesterday's meeting to thrash out consensus.
"Country needs investment and in larger interest of the country and for betterment of economy the Bill is needed as people's interest are paramount," he said, making a pitch for early passage.
Informal consultations took place today between Naidu, Finance Minister Arun Jaitley and Congress leader Anand Sharma. Naidu said the Bill has already been through the Standing Committee, which had deliberated on it for long.
He said the government would not have clamoured for foreign investment if resources were available domestically. When told that parties like Congress have problems over the FII provision in the Bill, Naidu said, "Whatever is their concern they should say it. I hope they will come back and there would be meeting of minds".
Naidu's remarks came even as opposition parties insisted on referring the first major economic reform initiative of the Narendra Modi government to a select committee.
The Insurance Laws Amendment Bill was introduced in the Rajya Sabha in 2008. The Bill seeks to raise foreign investment cap in the sector to 49 per cent, with management control in the hands of Indian promoters. At present 26 per cent FDI is allowed in insurance.


text:
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Centered on a bright white backdrop this white iPhone 4 is the perfect place for your mockup. The iPhone 4 is set to landscape view and holds an image of 960×640. The iPhone has a subtle shadow and reflection on the stark white surface for a bit of depth. Your app mockup would fit perfectly here.
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text:
Laxman rattled up an unbeaten 109 as India reached 254 for four in their second innings after following on 274 behind. Still 21 runs short of avoiding an innings defeat, India were all out for 171 in their first visit to the crease.
Promoted to No3 after hitting 59 in the first innings, Laxman reached his ton, which boasted 17 fours, off 166 balls. Australia - going for a 17th straight Test victory, but seeking their first series win in India since 1969 - lead the three-match series 1-0.
Meanwhile, West Indies lead by 258 runs going into the final day of their First Test against South Africa in Guyana after reaching 286 for four at the close.
After South Africa hit 332 in reply to the Windies' first innings of 304, Ramnaresh Sarwan reached 71 not out and Carl Hooper 31 not out at stumps.
- More about:
- Caribbean Islands (west Indies)
- Cricket
- Guyana
- India
- South Africa


text:
All Android applications categories
Donation version (Version without ads)
The application should have all Swiss on his phone.
This can still be used in emergencies.
Just always run the application and click on the image of the desired emergency service for emergency calls happen immediately.
Thank you not to call these numbers only in emergencies.
Hoping that you did not often need. ;)
Features of ER (CH) 2.0:
* Call emergency numbers (Switzerland)
* Call emergency numbers border to Switzerland
* Call emergency veterinary
* Send position via Google Maps
* SD card support
Also available in German (Notfälle (CH) PRO in the market)
For owners of tablet, please buy the version developed for them that is in the market (Emergencies (CH) 2.0 Tab)
Thank you to buyers of Emergencies (CH) PRO. Thanks to them if that version 2.0 is available today!
And we would like to thank you again for having us send us your feedback, we've all read and feel free to continue to let us know.
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text:
Backgammon was a board game at which the First Doctor was adept. In one 13th century session, he won from Kublai Khan: thirty-five elephants with ceremonial bridles, trappings, brocades and pavilions; four thousand white stallions; twenty-five tigers; the sacred tooth of Buddha; and the entire commerce of Burma for one year. Khan's wife disapproved of her husband's playing the game for stakes. She claimed that it aggravated his gout. (TV: "Assassin at Peking")
In July of 1778, Shardlow lost a game of backgammon to the Celestial Toymaker at the Hellfire Club and was forced to become his slave. (PROSE: The Nightmare Fair, AUDIO: The Nightmare Fair)
Ashildr played backgammon during her time as a medieval Queen. (TV: The Woman Who Lived)