text:
|Home | About | Journals | Submit | Contact Us | Français
Fetal alcohol spectrum disorders (FASD) are a leading cause of neurodevelopmental disability. The mechanisms underlying FASD are incompletely understood, and biomarkers to identify those at risk are lacking. Here, we perform metabolomic analysis of embryoid bodies and neural lineages derived from human embryonic stem (hES) cell to identify the neural secretome produced in response to ethanol exposure.
WA01 and WA09 hES cells were differentiated into embryoid bodies, neural progenitors or neurons. Cells along this progression were cultured for four days with 0%, 0.1% or 0.3% ethanol. Supernatants were subjected to C18 chromatography followed by ESI-QTOF-MS. Features were annotated using public databases and the identities of four putative biomarkers were confirmed with purified standards and comparative MS/MS.
Ethanol treatment induced statistically significant changes to metabolite abundance in human embryoid bodies (180 features), neural progenitors (76 features) and neurons (42 features). There were no shared significant features between different cell types. Fifteen features showed a dose-response to ethanol. Four chemical identities were confirmed; L-thyroxine, 5'-methylthioadenosine, and the tryptophan metabolites L-kynurenine, and indoleacetaldehyde. One feature with a putative annotation of succinyladenosine was significantly increased in both ethanol treatments. Additional features were selective to ethanol treatment but were not annotated in public databases.
Ethanol exposure induces statistically significant changes to the metabolome profile of human embryoid bodies, neural progenitors and neurons. Several of these metabolites are normally present in human serum, suggesting their usefulness as potential serum FASD biomarkers. These findings suggest the biochemical pathways that are affected by ethanol in the developing nervous system and delineate mechanisms of alcohol injury during human development.
Fetal alcohol spectrum disorders (FASD) are a leading cause of neurodevelopmental disability and are associated with deficits in learning, motor skills and executive functioning. The basis of alcohol injury to neurons includes altered proliferation, differentiation, and synaptogenesis and is incompletely understood (reviewed in Miller 2006). Deeper understanding of the mechanisms underlying alcohol's toxicity could facilitate prevention, earlier detection or repair the resulting damage. However, implementation of such strategies is challenged by the limited availability of diagnostic biomarkers that could identify at-risk pregnancies and those with FASD.
The human embryo and the regulatory mechanisms directing its development are challenging to study. Human embryonic stem (hES) cells permit this study using an in vitro model. hES cells are pluripotent, self-renewing cells isolated from human embryos (Thomson et al., 1998). They give rise to all differentiated cell types in the human body and recapitulate embryonic development in vitro, including the faithful replication of neurodevelopment and differentiation of hES cells into neural precursors, neurons and glia (Reubinoff et al., 2000; Zhang et al., 2001). Because they function similarly to in vivo counterparts (Ben-Hur et al., 2004; Zhang et al., 2001), hES cell-derived models are an ideal research tool to elucidate biochemical mechanisms underlying disease, including alcohol's effects on human neural development.
The human metabolome is the comprehensive set of endogenous small molecules. It is a product of health or disease states and can be measured on every level of complexity e.g., tissues, biofluids and cells (Fiehn, 2002). Abnormal metabolite production in response to environmental, dietary or disease insult provides mechanistic insights into these atypical processes. Metabolite composition of a biological sample is very sensitive to environmental effects (Lindon et al., 2003). We have used hES cells to both discover biomarkers and establish predictive models of chemically-induced developmental toxicity (Cezar et al., 2007; West et al., 2010).
Specific changes to metabolites identified in response to alcohol exposure could serve as biomarkers for FASD. Such biomarkers also outline the mechanisms underlying alcohol's pathogenicity, leading to novel treatment opportunities. The analysis of biomarkers in serum and amniotic fluid is an established approach to predict birth defects and premature births. The presence of fatty acid ethyl esters (FAEEs) in meconium is employed to identify alcohol exposure during pregnancy. However, FAEEs can be present in the meconium of unexposed infants (Chan et al., 2003) and do not provide information on the timing, duration and dosage of exposure (Litten et al., 2010). Additionally, FAEEs do not provide information on the damage that may have occurred specifically to the developing brain and the mechanism of injury. Thus, there is a significant need for reliable biomarkers that can be used for FASD prevention, diagnosis and treatment.
This study tested the hypothesis that alcohol exposure induces statistically significant changes to the abundance of small molecule metabolites in human neural progenitors and neurons. To test this, we treated hESC-derived embryoid bodies, neural progenitors and neurons with 0, 0.1% or 0.3% ethanol. We employed liquid chromatography coupled with electrospray-ionization time-of-flight mass spectrometry (LC-ESI-QTOF-MS) to identify and measure changes to secreted metabolite abundance in response to alcohol exposure. Here, we describe the metabolome of hES cell derivatives exposed to ethanol during three stages of neurogenesis.
WA01 and WA09 hES cells, obtained from WiCell Research Institute (Madison, WI), were exposed to 0%, 0.1% and 0.3% ethanol for four days at one of three stages of neurogenesis: days 0–4 (embryoid bodies, EBs), days 17–21 (neural progenitors) and days 28–32 (neurons) (Zhang et al., 2001) (Outlined in Figure 1). Ethanol doses were selected to mimic fetal exposure to binge-like low and high ethanol levels, respectively. Each cell line, time point and treatment included five biological replications of three samples per treatment per replication. Additionally at each time point, media controls were included containing appropriate media exposed to the plate conditions without cells.
hES cells grown in mTeSR1 media (Stem Cell Technologies, Inc., Vancouver, BC) were dispersed following treatment with dispase (2 mg/ml; Invitrogen, Carlsbad, CA) and grown in suspension. EBs were cultured in EB formation media, containing Iscoves's Modified Dulbecco's Medium (1×; Invitrogen), supplemented with 15% heat inactivated certified fetal bovine serum (Invitrogen) for four days in 90 mm low-binding dishes (Nalge Nunc International, Rochester, NY). EB formation media was changed every other day for neural progenitor and neuron differentiation experiments. For treatment, ~50 EBs were placed in each well of 24-well low-binding microplates (Nalge Nunc International) 24 hours after formation. During treatment, media and compound were replaced daily.
On day 5 of suspension culture, media was switched to neural precursor media, which consisted of DMEM/F12 (1:1) supplemented with 1% N2 Supplement, 0.1 mM MEM non-essential amino acids (NEAA), 1% Insulin-Transferrin-Selenium-X Supplement, 20 ng/ml FGF2, 20 ng/ml EGF (all from Invitrogen), 2 µg/ml heparin, 0.5 µM retinoic acid, and 60 µM putrescine (all from Sigma-Aldrich, St. Louis, MO). Neurospheres were maintained in neural precursor media for 11 days, with media changed every other day.
On day 17 of neural differentiation, neurospheres were dissociated into a single cell suspension using 0.25% trypsin-EDTA (Invitrogen) containing 2% chick serum (Sigma-Aldrich). The cells were plated on pre-coated poly-L-ornithine and laminin 24 well plates (BD Biosciences, San Jose, CA) at 2×106 cells/well. Neural progenitors were maintained in neural progenitor media, which consisted of DMEM/F12 (1:1) supplemented with 1% N2 supplement, 0.2 mM MEM-NEAA, 500 ng/ml sonic hedgehog (R&D Systems, Minneapolis, MN), 100 ng/ml FGF8 (Invitrogen), and 10 ng/ml FGF4 (Invitrogen). Media was changed every other day during routine maintenance. Treatment began on day 18 and media was replaced and fresh compound added daily during the exposure period. Treated neural progenitors were fixed on day 22 for nestin and caspase-3 analysis.
Beginning on day 22, neural progenitors were differentiated into neurons. Cells were cultured in neural differentiation media consisting of neurobasal medium supplemented with 1% N2 supplement, 0.1 mM MEM-NEAA, 0.5 mM L-glutamine, 1 µg/ml laminin, 20 ng/ml BDNF, 10 ng/ml GDNF (all from Invitrogen), 1 µM cAMP, 30 µM serotonin (both from Sigma-Aldrich), and 50 ng/ml sonic hedgehog. Starting on day 28, neurons were treated for 4 days and media was replaced daily. Neurons were fixed on day 32 for caspase-3, synaptogenesis and differentiation immunocytochemistry analysis.
Immunostaining of paraformaldehyde-fixed cells was performed using standard techniques. Primary antibodies included anti-nestin (MAB5326. Millipore, Billerica, MA), anti-β-III-tubulin (T8660, Sigma-Aldrich), anti-synaptophysin (ab52636, Abcam, Cambridge, MA), anti-SV2A (sc-28955, Santa Cruz Biotechnology, Santa Cruz, CA), anti-tyrosine hydroxylase (AB152, Millipore), and anti-cleaved caspase-3 (9661, Cell Signaling Technology, Danvers, MA). Secondary antibodies were conjugated with Alexa Fluor 488 (A11001, Invitrogen) or Alexa Fluor 594 (A21207, Invitrogen). For synaptophysin and SV2A, Triton X-100 was omitted to maintain the integrity of membrane structures. Positive staining of post-mitotic neurons was defined as cells that co-expressed β-III-tubulin and an additional neuronal marker: synaptophysin, SV2A, or tyrosine hydroxylase. At least 300 cells/sample and 6 samples/treatment were counted. Statistical analysis was performed using a one-way ANOVA with statistical significance defined as p ≤ 0.05.
On the fourth day of treatment, spent media was collected from each sample, quenched with LC-MS grade acetonitrile (final acetonitrile concentration = 40%), and stored at −80°C until preparation. For mass spectrometry analysis, equal amounts of quenched sample and LC-MS grade water were added to a Microcon centrifugal filter with a molecular weight cut-off of 3 kDa (Millipore). Each sample was centrifuged at 13,000 × g for three hours at 4°C. The flow-through was retained and dried using a vacuum concentrator and stored at −80°C. Samples were reconstituted in 50 µl of 0.1% formic acid in water prior to mass spectrometry analysis.
Liquid chromatography-electrospray ionization-quadrupole time of flight mass spectrometry (LC-ESI-QTOF-MS) was performed at Stemina Biomarker Discovery (Madison, WI) using an Agilent QTOF LC-MS system consisting of a 1200SL HPLC system and a G6520AA QTOF high resolution mass spectrometer (Agilent Technologies, Wilmington, DE) capable of exact mass MS and MS/MS ion fragmentation. The flow through of each sample was analyzed using a 2.1 × 50mm Zorbax 1.8 um C18-SB column (C18, Agilent Technologies). Ultra performance liquid chromatography (UPLC) analysis was performed using a 20 minute gradient starting with ratio of 95% 0.1% formic acid in water (Solvent A) to 5% 0.1% formic acid in acetonitrile (Solvent B) and ending with 100% Solvent B at a flow rate of 500 µl/min. Electrospray ionization was employed using a dual ESI source, with an Agilent isocratic pump continuously delivering an internal mass reference solution into the source at approximately 0.01 ml/min for mass correction. For positive-ion ESI the reference solution consisted of 7H-purine (m/z 121.0509) and HP-0921 (m/z 922.0098). The reference solution for negative-ion ESI contained trifluoroacetic acid (TFA, m/z 112.9856) and the TFA adduct of HP-921 (m/z 1033.9881). The mass range of the instrument was set at 70–1600 Da. The sample order was randomized and 5 µl of each sample was injected. A solvent blank (0.1% formic acid) was run after every ten samples. All three time points were analyzed with positive-ion ESI. Additionally, negative-ion ESI was performed on the EB experiment samples. Data acquisition was performed with Agilent MassHunter Acquisition software using high-resolution exact mass conditions.
Prior to data analysis, the total ion chromatogram (TIC) of each sample was carefully inspected for quality and reproducibility of the MS signal. If a sample's TIC abundance deviated by more than 25% from the median across the LC-MS gradient, the LC-MS analysis was repeated for that sample. The data was deisotoped and then converted into the open source mzData format. Data analysis was performed using the open source statistical programming and analysis software, R. The XCMS package (Smith et al., 2006) was used to analyze the LC-ESI-QTOF-MS resulting files using the Centwave algorithm for peak peaking (Tautenhahn et al., 2008). Retention time deviations across EB LC-MS samples were corrected using retcor loess regression and the obiwarp method for neural precursor and neuron samples. After retention time correction, the features were grouped using the density based functions in XCMS. After the grouping function was performed features missing in LC-MS samples were iteratively integrated using m/z and retention time windows based on the range of the feature group. Contaminant ions were removed by comparing spent media extracts with blank extraction samples. Statistical significance of individual mass features was performed under the null hypothesis that no difference exists in feature abundance between control and treated samples. Differential small molecule metabolites, or features, were determined using a complete block design ANOVA with the model "log2(abundance) ~ treatment + replicate + cell line." Features were considered statistically significant if they had a p-value ≤ 0.05 in the treatment factor of the ANOVA model. Additionally, to be considered authentic, each feature was required to show a statistically significant alteration in both cell lines evaluated. The extracted ion chromatogram (EIC) of each statistically significant feature was then visually evaluated to confirm an observable difference between treated and control samples and to reduce the inclusion of spurious results.
For feature annotation, the neutral exact mass of each feature was queried against the public searchable databases METLIN (http://metlin.scripps.edu), The Human Metabolome Database (http://www.hmdb.ca), and the Kyoto Encyclopedia of Genes and Genomes (www.genome.jp/kegg/) for candidate identities. Measured mass features were considered a putative match to a small molecule present in the databases if their exact masses were within 20 ppm of the annotated database molecule (0.00002 × mass). Confirmation of statistically significant annotated features was carried out using tandem mass spectrometry (MS/MS).
Analytical grade chemical standards for thyroxine, 5'-methylthioadenosine, L-kynurenine, and indoleacetaldehyde were purchased from Sigma-Aldrich for comparative mass spectrometry. Chemical references were evaluated using identical sample preparation and chromatographic methods employed in the analysis of the original samples. Additionally, the three original samples with the highest abundance for each feature of interest were re-prepped and analyzed for comparison with the standard. Chemical references were dissolved in the appropriate basal media at three concentrations: 1 mM, 0.1 mM and 0.01 mM. Additionally, a 0.1 mM solution was prepared for each standard in 0.1% formic acid, with the exception of thyroxine, which was solubilized in a 0.1 mM solution of 50/50 methanol and dichloromethane. Data acquisition for targeted MS/MS was performed by inputting the m/z value of the precursor ion of interest into the acquisition software with a value for the collision-induced dissociation (CID) collision energy and expected retention time range. A putative annotation was considered confirmed if the retention time, measured exact mass and fragmentation pattern of the metabolite reasonably match these data from chemical references. If published MS/MS data was available, the fragmentation pattern of the sample must additionally match the published MS/MS spectra.
In culture medium from human embryoid bodies, we identified 3246 total features using C18 chromatography followed by positive ionization ESI-QTOF. Of these features, 510 were considered secreted molecules, as defined by having at least a 10% increase in abundance in the cell samples compared to the media controls. This standard was used in defining secreted molecules for all data sets. In 0.1% and 0.3% ethanol treated EBs, 70 and 62 secreted features were statistically significant, respectively, from control-treated cells (p<0.05, Supplemental Table 1). There were a total of 9 significant features in common between the two ethanol treatments (Table 1). One of the most challenging aspects of metabolomics is that >50% of the features identified do not have annotation in public databases yet. Of these 9 features, two had molecular weights consistent with a putative annotation of diethylphosphate and heptacarboxylporphyrin III. Three were tentatively assigned identities as tripeptides and four additional small molecules were unannotated. Several of these identities remain putative given the unavailability of a commercially available chemical standard. We confirmed the identity of 5'-methylthioadenosine (MTA, exact mass 297.0895) using MS/MS (Figure 2, Table 2). MTA participates in polyamine synthesis and methionine metabolism. MTA levels were significantly decreased in 0.3% ethanol-treated EBs (p=0.0275). MTA was also decreased in 0.1% ethanol-treated EBs, although the reduction was not statistically significant (p=0.1251).
Using C18 chromatography and negative ionization ESI-QTOF, 1009 total features were measured of which 295 were secreted. Of the secreted features, 21 and 27 were significantly altered in 0.1% and 0.3% ethanol-treated EBs, respectively, when compared to controls (p<0.05, Supplemental Table 2). Two secreted features were significantly altered in both ethanol treatment groups, and based on their exact mass and retention time they were putatively identified as the methylated purine 7-methylinosine (exact mass 283.1060) and succinyladenosine (exact mass 383.1075, Table 1). Succinyladenosine levels were significantly increased in both 0.1% and 0.3% ethanol-treated EBs compared with untreated controls (p=0.0380 and 0.0387, respectively). Succinyladenosine is a biomarker for adenylosuccinate lyase deficiency but it has also been measured in the cerebrospinal fluid of children without this condition, using HPLC-negative ion ESI mass spectrometry on a C18 column (Krijt et al., 1999). Unfortunately, no chemical standard was commercially available for chemical identity confirmation. Using negative ionization ESI-QTOF MS/MS, we also confirmed the identity of thyroxine (exact mass 776.6867; Figure 3, Table 2). Thyroxine was significantly increased in 0.1% ethanol-treated EBs compared with controls (24% increase, p=0.0011).
At the neural progenitor differentiation stage, we measured 927 total features with C18 chromatography and positive ionization ESI-QTOF. Nearly half of the total features were considered secreted (n=434). In comparison to untreated neural progenitors, 56 and 20 features were significantly different in 0.1% and 0.3% ethanol treatment groups, respectively (p<0.05, Supplemental Table 3). Between the two ethanol treatment groups, there was one significant feature in common, which had a decreased abundance in ethanol treatment (Table 1). It could not be assigned an identity and had neutral mass of 746.0645 Da. In addition, L-Kynurenine (exact mass 208.0848), a tryptophan metabolite, was significantly increased in 0.1% ethanol-treated neural progenitors (p=0.0200). The identity of kynurenine was confirmed using MS/MS (Figure 4).
In terminally differentiated neurons, 1268 total features were measured by C18 chromatography and positive ionization ESI-QTOF. The total number of secreted features was 582. In 0.1% and 0.3% ethanol-treated neurons, 23 and 19 features, respectively, were statistically significant when compared with control samples (p<0.05, Supplemental Table 4). Three significant features were common to both ethanol treatment groups (p<0.0001 for all vs. control neurons, Table 1). The EIC of the primary feature is shown in Figure 5. These three features were tentatively identified as a tripeptide containing cys-his-lys (exact mass 386.1734), and its respective sodium (exact mass 408.1558) and potassium adducts (exact mass 424.1308). The amino acid sequence order could not be determined. We confirmed the identity of indoleacetaldehyde (IAA, exact mass 159.06842), another metabolite in the tryptophan pathway, with MS/MS (Figure 6). IAA was significantly increased in the media of 0.1% ethanol-treated neurons compared to controls (p=0.0493). IAA levels were also increased in 0.3% ethanol-treated EBs compared to controls, although the increase was not statistically significant (p=0.47).
To determine if ethanol exposure affected the differentiation of these neural populations, we examined the expression of neural markers at the end of the four-day alcohol exposure period. For the neural progenitors, we found no alcohol-related differences in their expression of nestin, an intermediate filament protein expressed in mitotic neural progenitors (Table 3). Control, 0.1% and 0.3% ethanol treatments all exhibited greater than 95% nestin expression in the progenitor cultures, confirming their neural progenitor identity. For the neurons, we observed no significant differences in the percentage of cells that co-expressed β-III-tubulin, demarcating neuronal populations, and tyrosine hydroxylase, a marker of dopaminergic neurons (Table 3, Supplemental Figure 1). Similarly, there were no significant differences in the percentage of cells that co-expressed β-III-tubulin and synaptophysin or SV2A; the latter are both integral membrane glycoproteins present on presynaptic vesicles.
We found modest ethanol effects on apoptosis of human neural cultures (Table 3, Supplemental Figure 2). For the neural progenitors there was no statistically significant difference in the percentage of cleaved caspase-3-positive cells in controls and those treated with 0.3% ethanol (p=0.19); there was a non-significant trend to reduced number of cleaved caspase-3-positive cells following treatment with 0.1% ethanol (p=0.06). In the neurons (D32), we detected a statistically significant decrease in the percentage of caspase-3-positive cells in 0.1% ethanol-treated neurons compared to controls (p=0.03), but not in those treated with 0.3% ethanol (p=0.10).
In this study, hES cells were differentiated into neuronal lineages and exposed to either 0, 0.1 or 0.3% ethanol at three developmental stages, EBs, neural progenitors and neurons, to model the effects of ethanol exposure on early human brain development. This in vitro model was coupled with metabolomics to test the hypothesis that ethanol exposure induces statistically significant changes to endogenous small molecules. We also sought to define specific small molecules and biochemical pathways that are altered in alcohol-induced developmental neurotoxicity, leading to FASD. Neural differentiation of hES cells is an important model to study the earliest effects of alcohol on human development given that the timing of neuronal and glial differentiation from hES cells is similar to lineage allocation in vivo (Zhang, 2006). Alcohol exposure during embryogenesis and early neural differentiation, a period when pregnancy may be unknown, leads to a higher incidence of craniofacial defects and mental disabilities (Ernhart et al., 1987; Guerri, 2002). There is a significant need to identify the biochemical pathways that play a causal role in alcohol-induced developmental neurotoxicity. Our study confirms that the exposure of hES cell-derivatives to ethanol results in significant changes to the abundance of human endogenous metabolites.
No significant features were shared between the three cell types following ethanol treatment. This likely reflects the disparate maturation states of these differentiating cells. Approximately half of the features showed similar responses at both ethanol concentrations, although the magnitude of change did not always achieve statistical significance. Other features, such as T4 and kynurenine, were increased at 0.1% ethanol but had decreased abundance at 0.3%. This differential response likely reflects the cells' adaptive responses to the higher ethanol level over the four-day culture period (e.g. Buck and Harris, 1991; Yakovleva et al. 2011). This interpretation is endorsed by the finding that 0.1% ethanol treatment produced appreciably more significant features (154) than did 0.3% ethanol (112), of which 15 were significant in both exposures. Many individuals with FASD experience chronic alcohol exposure and biomarkers that reflect those adaptive mechanisms are clinically relevant.
Embryoid bodies are comprised of the three primary germ layers and thus model the earliest stages of differentiation. We identified statistically significant changes in multiple developmentally important metabolites between 0, 0.1% and 0.3% ethanol-treated EBs. The abundance of 5'-methylthioadenosine (MTA) was significantly decreased in 0.3% ethanol-treated EBs compared to untreated controls. MTA is produced in all mammalian cells during the synthesis of polyamines putrescine, spermidine and spermine from decarboxylated S-adenosylmethionine (SAM), and its levels directly reflect the rates of polyamine synthesis (Avila et al., 2004; Sufrin et al., 1989). Inhibition of polyamine synthesis impedes embryo growth (Fozard et al. 1980; Mendez 1989) whereas exogenous polyamines protect against embryotoxicants (Chirino-Galindo et al., 2009). Both acute and chronic ethanol exposure suppress polyamine synthesis and its dysregulation was identified as a potential mechanism contributing to FASD (Desiderio et al., 1987; Sessa and Perin, 1997; Shibley et al., 1995; Thadani et al., 1977). The decline in MTA is consistent with those reports and thus secreted MTA may be a useful biomarker for alcohol's toxicity. MTA also supplies the sulfur atom for methionine synthesis within the methionine salvage pathway. Reductions in MTA might also contribute to the depleted methionine and SAM levels observed in chronic ethanol consumption (Finkelstein et al., 1974; Walcher and Miller, 2008). Importantly, because MTA is produced from SAM, its alteration may reflect a larger disruption of methyl group homeostasis within ethanol-exposed cells, an interpretation affirmed by the increased folic acid and polyglutamate content found in ethanol-treated EBs and neural progenitors (Supplemental Table 1 and 2). Methyl group supplements in the form of choline, betaine and SAM can prevent or reverse ethanol's developmental toxicity both in vitro and in vivo (Seyoum and Persaud, 1994; Thomas et al. 2009, 2010). To our knowledge there are no previous reports specifically linking MTA to ethanol exposure or FASD. Its metabolic links with polyamine and methyl metabolism make MTA an excellent potential biomarker in addition to offering mechanistic insights into FASD.
We additionally confirmed thyroxine as a putative biomarker of prenatal ethanol exposure during the embryogenesis stage. Thyroxine abundance was significantly increased in 0.1% ethanol-treated EBs compared to untreated controls. Thyroxine (T4) is one of the two hormones known as thyroid hormone secreted by the thyroid gland. In these cultures T4 likely originated from the serum in the culture medium. Thyroxine signaling through its nuclear receptors is critical for normal central nervous system development and contributes to multiple processes including cell migration, dendrite/axon outgrowth and synaptogenesis. A precise balance of thyroid hormone is necessary for proper brain development and both deficiency and hyperthyroidism can cause significant and irreversible brain dysfunction (Bernal and Nunez, 1995; Lauder, 1977; Pharoah et al., 1971). Our studies show a 24% increase in exogenous thyroxine levels, suggesting an ethanol-induced perturbation to thyroxine metabolism and/or its uptake by the early embryo. This is consistent with prior demonstrations of altered thryroid hormone metabolism in animal models and infants with FASD (Cudd et al. 2002; Hannigan and Bellisario 1990; Kornguth et al. 1979). In rat models, prenatal alcohol exposure (PAE) also alters the levels of iodothyronine deiodinase III and thyroid hormone receptor alpha-1 (Shukla et al., 2010, 2011). Thus the significant increase in thyroxine detected herein suggests that ethanol exposure is likely to impair proper thyroid hormone metabolism in the early embryo, corroborating a proposed mechanistic pathway that ethanol may produce neurodevelopmental disorders by negatively affecting maternal-fetal hormonal homeostasis. Our study is the first to indicate a differential uptake or processing of maternal thyroxine by human embryos as a result of PAE. It is plausible to speculate that this detrimental effect may be mediated by functional impairment of the thyroid hormone-specific transporters Oatp1c1 (organic anion transporting polypeptide 1c1), monocarboxylate transporter (MCT) 8 and MCT10. Mutations to MCT8 actually increase the extracellular abundance of thyroxine in vivo, producing severe psychomotor retardation (Visser et al. 2008; Grijota-Martinez et al., 2011). There are currently no published studies examining the direct effects of ethanol exposure on thyroxine-specific transporters, which will be a focus of future studies.
Ethanol exposure at two different stages of neurogenesis produced statistically significant changes to tryptophan metabolism. These changes are summarized in Figure 7. These differences were not attributed to trivial effects of ethanol upon differentiation states, as the cellular type distribution between treatments did not differ. Tryptophan is metabolized primarily along the kynurenine pathway and kynurenine levels were increased in neural progenitors treated with 0.1% ethanol, as were the levels of the tryptamine catabolite indoleacetaldehyde in neurons exposed to 0.1% ethanol. Dysregulation of tryptophan metabolism is reported in several models of alcohol exposure. Kynurenine levels are increased in human plasma following acute ethanol consumption (Badawy et al. 2009) and tryptophan levels are significantly reduced in the plasma, brain and liver of rat pups following gestational ethanol exposure (Lin et al. 1990). Serum levels of kynurenine are also perturbed in several cognitive disorders such as Down syndrome, bipolar disorder and postpartum depression but had not been previously described in FASD. Kynurenine has additionally been identified in hES cells treated with valproate, a known inducer of neurodevelopmental defects (Cezar et al. 2007). Kynurenine was recently reported to activate the aryl hydrocarbon xenobiotic receptor (AhR) with an apparent Kd ~4 µM (Optiz et al. 2011). AhR activation adversely affects brain development and behavior (Latchney et al. 2011; Seo et al. 1999). Our finding of elevated Kyn in ethanol-treated neural progenitors is a novel insight into the mechanisms underlying FASD, and confers this metabolite a translational role as a candidate biomarker of FASD.
Our study is the first to indicate a direct effect of ethanol exposure on the human endogenous metabolite IAA. Tryptophan is critical for normal neurogenesis as the precursor to the neurotransmitter serotonin (5HT) and its availability has a direct effect on 5HT synthesis (Fernstrom and Wurtman, 1971). Development of the serotonergic system occurs earlier than other neurotransmitter systems and its sensitivity to disruption by PAE is well documented (Druse et al., 1991; Maier et al., 1996; Sari et al., 2010). Elevated IAA decreased the activity of tryptophan hydroxylase, the rate-limiting enzyme in serotonin synthesis, by as much as 33% (Nilsson and Tottmar, 1987). Thus, elevations of kynurenine and IAA detected here could be biomarkers for a metabolic diversion of tryptophan away from serotonin synthesis and toward these alternate metabolic pathways and may explain the reduced tryptophan and 5HT content of the ethanol-exposed fetal brain (Maier et al., 1996; Sari et al., 2010). In fact, an increase in the IAA-related 5-hydroxylindoleacetic acid (5-HIAA) was measured in the urine of rats chronically exposed to ethanol (Bonner et al., 1993) and IAA itself is amenable to detection in urine, which renders it a translational application in biofluids. Alternatively, we note that kynurenine also lies along the synthetic pathway of nicotinamide from its tryptophan precursor. Ethanol oxidation places a high requirement for niacin and NADPH reducing equivalents, and one possibility is that ethanol's effects on cellular redox potential might be driving these changes in tryptophan metabolites. Whether one or both of these hypotheses underlie increased kynurenine levels in ethanol-exposed neural progenitors, serving as the cause of serotonergic dysfunction, will be elucidated in future mechanistic studies. Nonetheless, it is noteworthy that significant perturbations to the tryptophan pathway and its metabolites were consistent across different timelines of neural differentiation, neural precursors and neurons.
In addition to the four confirmed metabolites with marked changes resulting from ethanol exposure, we identified multiple unknown compounds with statistically significant changes in abundance. Further experiments are required to determine the chemical formula of such compounds by ion fragmentation and other analytical platforms such as NMR. The fact that statistically significant small molecules are not annotated in public databases does not preclude their potential application as candidate diagnostic biomarkers for FASD. Given the highly sensitive, high resolution nature of the mass spectrometric detection system employed in this study, a measurable endpoint (exact mass) is determined which can be used for continued chemical structure determinations. An important limitation to the lack of a chemical annotation is that these unannotated small molecules cannot be mapped to a biochemical pathway for mechanistic elucidation.
No changes in the overall cell differentiation ability of hES cells were measured in response to 0.1% or 0.3% ethanol (Table 3), at least for the culture conditions employed in this study and for the relatively short ethanol exposure period. There was a non-significant trend for an increased percentage of neurons expressing synaptophysin and SV2A following the ethanol treatment of mature neurons. Together with the multiple changes in the levels of tryptophan metabolites, these data may indicate an overall trend of increased synaptogenesis in response to ethanol exposure. In a finding that seemed counterintuitive, we found modest non-significant decreases in apoptosis frequency following ethanol treatment of neural precursors and neurons. Prock and Miranda (2007) reported a similar resistance to apoptosis in ethanol-treated mouse neural progenitors, a population substantially similar to those studied herein. Our findings endorse their interpretation that apoptosis in response to ethanol may be a differentiation state-specific response. Further study is required to understand why this population is resistant to apoptosis upon exposure to ethanol.
In conclusion, exposing hES cell-derived EBs, neural progenitors and neurons to ethanol identified marked changes in biochemical pathways and metabolites critical for proper neurodevelopment. Our findings not only provide novel candidate diagnostic biomarkers for FASD, but also corroborate independent studies that have proposed alterations to methionine, methyl group, thyroid hormone and tryptophan metabolism as potential mechanisms underlying FASD. Although some of these metabolites, i.e. kynurenine, have been described as putative biomarkers for other neurodevelopmental disorders, it is the integration of several of these endogenous small molecules from independent biochemical pathways, that may be used as a specific fingerprint test set or specific fingerprint for the diagnosis of FASD in biofluids. Metabolomics is able to measure simultaneous changes to multiple unrelated biochemical pathways, providing not only a biochemical signature specific to FASD but also mechanistic insight into the effects of PAE on early human embryogenesis. The identification of differential biomarkers in our in vitro model prior to analysis of biofluids allows for a more targeted approach in an in vivo model, which has multiple confounding factors, i.e. maternal-fetal interactions. Biofluids are complex mixtures of systemic byproducts influenced by both endogenous and exogenous factors e.g., genetics, diet and environment. Using our strategy, we can design specific analytical protocols to examine biofluids based on our confirmed metabolites. Our future efforts will examine whether molecules detected here are subject to similar changes in in vivo models and children diagnosed with FASD. The biochemical pathways revealed by metabolomics of hES cell derivatives may also present new targets for the identification and development of novel therapies for FASD.
Supported by NIH awards AA16958 (G.G.C.) and MERIT award AA11085 (S.M.S.).
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.


text:
Cshun Utz
- Size: 640px
- Quality: High
- Size: 5.72 MB 118.96 KB
This Gif entitled Cshun Utz was created from the youtube video 3 years ago. It uses 18 frames per second and is displayed at 640px resolution. This animated image plays for 2 seconds, was built with high quality and an estimated weight of 5.72 MB for download. This animation was originally created from the Taking off my shoes and socks video from 00:46 to 00:48.
Flag This GifSend

subset: commoncrawl


text:
Download presentation
Presentation is loading. Please wait.
Published byMarissa Purkiss Modified over 2 years ago
Mauritius Case Study Nawsheen Hosenally 5 th September 2012
Country Background 2045 km2 Population: 1,3 million Economy based on different sectors Agriculture Textile Financial Tourism ICTs
Youth Demographics Youth is defined as a person between 14 and 29 years of age Youth population between 15-29 years old was estimated to be 306,532 (CSO, 2010) 23.9% of total population 154,863 (12.1%) males and 151,669 (11.8%) females
Youth Demographics Youth and Education Literacy rate is 88% Primary & secondary education free Youth and Employment Minimum legal working age is 16 years old Unemployment rate is 7.9% 38% below 25 years of age Urban and Rural youth Difference not very clear-cut Pockets of poverty in north and eastern coast
Agriculture Background Contributes to 4% of GDP Arable land: 43% Sugarcane, tea and tobacco (33%), Food crops and others (28%), Livestock and Poultry (22%), Government services (13%), Fishing (4%) CAADP Status: About to sign Challenge: High Imports (>70% - Approx 1billion USD) Ageing farming population & lack of youth interest in Agriculture
Youth Perceptions Generally perceived negatively by youths Different categories of youths interviewed Youth of different field of studies from University of Mauritius Youth engaged in Agriculture (producers and entrepreneurs) Each category has its own perception
Youth Perceptions Agriculture can help solve world issues like poverty and food security No opinion. Its just not meant for me I am studying engineering and intend to get a job in the same field, but I see Agriculture as a good business opportunity, and it will be my part-time activity. I have been in Agriculture for years and despite all challenges, this is where I belong
Youth Perceptions Agriculture does not pay because it a luck business, Ramadhani Sigareti, young horticultural farmer in Morogoro region Agriculture takes time to earn money and it is always unpredictable, Elizabeth Singu, youth member UVIKIUTA I have never seen a successful young farmer in my entire life; there are no role models in agriculture, Elizabeth Singu, youth member UVIKIUTA.
Youth Perceptions Although I am doing Bsc in Agricultural Economics and Agribusiness, I would like to work in a bank or telecommunication industry because agriculture doesnt pay, Kijagwa Gerald, Bsc. Agribusiness, second year student SUA, Morogoro. If you look at the conditions of the farmers, there is no way you can be attracted to be a farmer, Samson Simangwi, Bsc. Agribusiness, third year student SUA.
Youth Perceptions Negative Old-fashioned activity for the elderly and poor people (Subsistence v/s commercial, farm to eat v/s farm for income) A field for residual youth (Failure – lowest class student) Difficult job – too laborious Low income generating sector (No money in Agriculture, issue: it takes too long to get results)
Youth Perceptions Positive High opportunities in Agribusiness and entrepreneurship Leisure (Health, Exercise, Diet Issue) Address poverty – eliminate climate change Discipline
Challenges Lack of enabling environments Society Schools Governments Incentives
Opportunities Changing the negative perception of youth on Agriculture can help solve many issues Food Security Poverty Unemployment
Recommendation Re-brand Agriculture Through ICTs National campaigns Magazines Introducing Agriculture in school
Similar presentations
© 2017 SlidePlayer.com Inc.
All rights reserved.


text:
my other money pit in front of some nice artwork...the guy playing the trumpet is all spray paint.
Here's some I saw on a local trail ride.
If you blow the next one up to 'original size' you can see a lot of graffiti in different layers in the distance, including some with dates back to the 1930s. This is from a trail that goes under a local bridge, not many ride it but it's good.
Check out the dog walking the donkey too.

· MTBR Hiatus UFN ·

I win.
Originally Posted by lidarman
Good Stuff. Graffiti is one of my favorite photo subjects.
What kind of camera are you using? Really nice photos
Florence Nightingale's Stormtrooper
broadcasting from
"the vinyl basement"
build trail!
ABQ south foothills
Suitably themed wall ride in the Vortex pit, Ocala, FL...
Spotted just south of Phoenix, Alice Cooper makes perfect sense. But Foghat? Guess Casa Grande's a slow ride... take it easy...
And your reward for climbing 2500' to reach Reddish Knob in VA is...
Last edited by FoldsInHalf; 02-11-2010 at 12:00 PM.
"Rollin' on 20s and 27.5s" ride reports and more at WWW.DEBCAR.COM : Debbie and Carey's RV Travel Website
A little different than the urban-style graffiti more typical in the cities.....This stuff kind of looks "indie" to me (whatever that means), which matches the vibe of Athens, GA.
Photos taken under the Loop 10 highway, where Chase Street and Loop 10 trails meet.
PS- The dorky rear rack was used for commuting during the week. Now that I have another job, which I ride my motorcycle to, I don't ride dirt with that thing on.
I saw tons of graffiti when I was interning in Detroit 2 and 3 years ago.

This one is at the stage at Hart Plaza. It isn't really grafitti but it looks like it could be.

'08 Specialized Rockhopper 29er (modified)

The bicycle; the noble invention that saves my health, inside...and out.
Sarah Palin done up Warhol style. (From last year)
By Zoltron, outside Fairfax, Cali
....but saw this mural in Burlington Vermont Saturday.....
There are two paths you can go by but in the long run........
Somewhere in New Mexico...
I prefer the more "straight to the point" approach
Crankfire.com / New Englandish Mountain Biking Community
+1 Rip Dime
When I started scrolling through this, I was waiting for the Santa Cruz tanks. Always wished I had a camera on me when I rode up there.
Pleasantly surprised by the Reddish Knob pic in VA...that was one of my first rides.
Moving to Montana soon, gonna be a dental floss tycoon.
Various photos from the last 10 or so years of riding:
Costa Rica:

New Jersey:

Abandoned PA Turnpike:

.....looking down into a granite quarry at Millstone Vermont....great place to ride!
i took these over at the abandoned High School where my kid's Lacrosse team practices
a little Physical Graffitti
I hit this guy's blog once a week to check out his graffitti photos
theses are some of his
Last edited by brado1; 06-23-2010 at 06:23 AM.
Fat, Drunk and Stupid is No Way to Go Through Life... But it's Worked Out Pretty Well for Me!
Some of the better stuff found on the Waterworks Trail near Emory University in Atlanta.
....saw a few more cars with some new markings:
Live Free and RIDE Hard!!!!
Defiant MTB
Here's some more of the "straight to the point" approach.
More from Millstone VT
I'm Better known as Splat
For the Vintage, Retro, Classic crowd, this is the finest Graffiti that you will see.
If you are interested in graffiti more similar to what others have posted. Here is some that I saw in Toro Muerto in Peru in May. It's a desolate valley with several thousand of these things.
Originally Posted by victim
I agree with you but that hack graffiti is on a cess pool near a junk yard so no biggy in this case. Great riding spot however that alot of people here will recognize.
I think if you write on anything that isn't your property without permission it's an act of vandalism. Vandalism if an act committed by people with little respect for anything, including themselves.
Originally Posted by zrm
I think if you write on anything that isn't your property without permission it's an act of vandalism.
Rhetorically; Do you own this server?
Devil's Advocate
Terrible, yet funny graffitti:
Same place, different wall. Nothing spectacular though:
Originally Posted by victim
If you are going to show one of the Milford quarries , at least show them in use.
.....Millstone Quarry in Vermont....this was above Sunrise lookout.
Originally Posted by brado1
This is pure art.
Canadian distribution: X-Fusion Shox, Rand Momentum lube and Canadian Shield fenders.
This photo was taken while exploring the unofficial catacombs in Paris.
here is my contribution
Brother Seamus?
Like an Irish monk?
Comic relief in a discussion does no harm..
CS Lewis
Originally Posted by zrm
I think if you write on anything that isn't your property without permission it's an act of vandalism. Vandalism if an act committed by people with little respect for anything, including themselves.
Do you work for the fuzz?
In most cases this art work let's us imagine and see past the shittyness of the urban sprawl!
some town above keystone
Bicycle-works FB
flat repair man.
oh yeah the place was Montezuma,Co.
Near a rail-to-trail I ride once or twice a week:
Playing with the macro setting, I kinda like how this pic turned out:
Last edited by heartland; 02-20-2011 at 05:39 PM.
Reason: resized pics
"Never trust a man in a blue trench coat. Never drive a car when you're dead." -- Tom Waits
The Nurburgring is notorious for grafitti on the track. Here is the official press release Ferrari sent out of their fastest car yet, the 599xx. Notice anything particular??
this just popped up in my neighborhood
Nothing better than some good street art ... even on trails.
Last edited by Pabs; 02-20-2011 at 07:33 PM.
"Fact is only what you believe; fact and fiction work as a team." Jack Johnson
Originally Posted by whos that guy
The Nurburgring is notorious for grafitti on the track. Here is the official press release Ferrari sent out of their fastest car yet, the 599xx. Notice anything particular??
Found about 12 of these under the I-40 bridge crossing the Wolf River Trails, best trails in Memphis....
Originally Posted by JeffSkisMontana
....but saw this mural in Burlington Vermont Saturday.....
ha i usually pass that every day.
Forum Rules


text:
Kentuckians For The Commonwealth
is a community of people, inspired by
a Vision, building New Power and
a better future for all of us. Together,
we organize for a fair economy, a
healthy environment, new safe energy
and an honest democracy. Join us.
Tell Congress to keep the promise to coal miners
Health care benefits will be taken away from more than 22,600 coal miners and their families in just a few days. Call Senator McConnell now.


text:
"The defendant argues that defective construction lacks the element of 'fortuity' necessary for an accident. This suggests that a foreseeable event can never be an accident under the terms of the commercial general liability policy. Insurance policies, however, are designed to cover foreseeable risk, including negligent acts. Accordingly, because negligent work is unintentional from the point of view of the insured, we find that it may constitute the basis for an 'accident' or 'occurrence' under the plain terms of the commercial general liability policy." Capstone Building Corp. v. American Motorists Ins. Co. 67 A.3d 961 (Conn. 2013)"
As construction defect litigation continues to progress, the trend appears to be in favor of coverage, albeit in various forms. Three of the supreme courts corrected prior precedent. Of particular note is the commentary of the Supreme of West Virginia regarding the need to revisit its prior rulings:
To learn more about how AmWINS can help you place coverage for your clients, reach out to your local AmWINS broker. If you do not have a contact at AmWINS, please click here.
Legal Disclaimer. Views expressed here do not constitute legal advice. The information contained herein is for general guidance of matter only and not for the purpose of providing legal advice. Discussion of insurance policy language is descriptive only. Every policy has different policy language. Coverage afforded under any insurance policy issued is subject to individual policy terms and conditions. Please refer to your policy for the actual language.
(c) 2017 AmWINS Group, Inc.
The Federal Motor Carrier Safety Administration mandate which requires nearly all U.S. truck operators to use electronic logging devices (ELDs) to track duty status has been upheld in court and will take effect December 16, 2017. The mandate will impact not just the trucking industry, but the trucking insurance sector as well.
For the insurance industry, a major hurricane making landfall means an enormous number of claims, often resulting in rate increases for insureds and changes to catastrophe models. Check out this infographic which highlights the 10 most costly hurricanes in U.S. history, ranked by estimated insured loss.
Additional insured endorsements are not a universal remedy and coverage is contingent on making sure a written agreement or contract is in place, with the named insured specifically adding the people or organizations included.
Liquor liability is a complex coverage that is becoming increasingly difficult to procure, but with a proper understanding of the type of risk, venue and location, you can more effectively position your clients for success with underwriters.
The resurging construction industry means that builder's risk submission activity is on the rise. As such, it's important to understand this line of business. Here's an overview of some things to consider on a builder's risk policy.
Property damage doesn't always mean there was an alteration to structure or contents. In this article, we review a court case in which harmful air quality was ruled as causing "direct physical loss of or damage to" the company's property.


text:
Poverty and Inequality
List of References
- Chenery,.H.,M. S. Ahluwalia, J. H. Duloy and R. Jolly, [1974] Redistribution with Growth, Oxford University Press
- Drèze, J. and A .K. Sen[1989] Hunger and Public Action, Oxford :Clarendon Press.
- Kuznets, S ,[1955] "Economic Growth and Income Inequality", American Economic Review, XLV(1) March, pp.1-28.
- Oman, P. Charles and Ganeshan Wignaraja[1991] The Postwar Evolution of Development Thinking, St. Martin's Press.
- Sen, A.K.[1981] Poverty and Famine, Oxford: Clarendon Press
- Sen, A., K [1985] Commodities and Capabilities, Elsevire Science Publisher.
- Sen,A.K.[1992] Inequality Reexamined, Oxford, Oxford University Press.
- Sen, A.K.[1999] Development as Freedom, Oxford University Press.
- Stewart, Frances [2000] Civic Prevention: Tackling Horizontal Inequalities, Oxford Development Studies, Volume 28, No.3, October, pp.245-262.
- Streeten, P. with S.J. Burki, M.ul Haq, N. Hicks and F. Stewart.[1981] First Things First: Meeting Basic Human Needs in the Developing Countries, Oxford University Press.
- UNDP (various years) Human Development Report, New York, Oxford University Press
- World Bank[1990] World Development Report 1990, Oxford University Press.
- World Bank[2000] World Development Report 2000/2001: Attacking Poverty, Oxford University Press.
- List of References
- Links to Relevant Sites
- Research Projects


text:
Oh boy. Looks like the sale of 1UP to Hearst hasn't gone as smoothly as you'd expect. Rather than simply move house, it seems most of the site's most experienced/popular writers have instead been let go.
You may also like
Recent from Luke Plunkett
Kinja is in read-only mode. We are working to restore service.


text:
Event: Arjun Rampal, Imtiaz Ali & Prateik Babbar graced the launch of Elite Calender with their warm presence at Olive Bar, Mumbai.
Guest List: Roshni Chopra, Vida Samadzai, Arjun Rampal, Imtiaz Ali, Prateik Babbar, Rocky S
Venue: Olive Bar, Mumbai.
Date: 16-Dec-2009
Bollywood actress Malaika Arora Khan was snapped at Bandra.
Bollywood actress Shraddha Kapoor was snapped at Bandra.
Bollywood actor Sanjay Dutt's wife Manyata Dutt along with kids were snapped at Bandra.
MERI PYAARI BINDU stars Ayushmann Khurrana and Parineeti Chopra were snapped at filmistaan studio to promote their upcoming film MERI PYAARI BINDU.
Sizzling HOT Bollywood actress Priyanka Chopra was snapped at Mumbai International Airport.


text:
Flashback from Brittany Soares, who uses the following chart to help articulate a vital, long-forgotten truth: … [Read more...]
Meme Reveals Leftist HYPOCRISY On Liberty, Security, and the American Founders
Robert Gehl reports that an MSNBC bobblehead made the suggestion that Donald Trump might start murdering journalists soon. … [Read more...]
The One Good Thing about the Obama Presidency….
There is a very dangerous assumption today, one guaranteeing a loss of freedom. Even conservatives generally fall victim to it, as evidenced by a … [Read more...]
What Democrats and Slave Owners Have in Common BRUTALLY Summed Up
C.E. Dyer reports that a ridiculous, race-baiting bill keeps cropping up in each Congress over at least the last twenty years courtesy of Rep. John … [Read more...]
How To Respond To Your Annoying Liberal Friends In One HILARIOUS Meme
C.E. Dyer writes that a new survey confirmed that Democrats/liberals are significantly less tolerant than Republicans/conservatives on social media -- … [Read more...]
Map Shows Where Americans Go If They Crave Freedom and Liberty
The John Locke Foundation has released the "First in Freedom Index" which ranks states by how much personal freedom it's residents enjoy. The ranking … [Read more...]
BOOM: Classical Liberalism Versus Progressivism Brilliantly Compared
C.E. Dyer notes that the left has had an epic meltdown over President-elect Donald Trump's historic victory, along with their shellacking in Congress, … [Read more...]
Justice Alito Issues Dire Warning on Freedom
Over the past 8 years, Freedom of Religion and Freedom of Speech have been two areas the Obama Administration has trampled over on a regular basis. … [Read more...]
JURY NULLIFICATION: How Citizens Can Fight Back Against Big Government
There's little doubt that the seven militia members who occupied the wildlife refuge in the Oregon wilderness were de facto guilty of a federal crime. … [Read more...]
Bush's 9/11 Emergency Status Extended… 15 Years Later
On September 11, 2001, the world would be forever changed with the infamous attack on the World Trade Center and Pentagon. Nearly 3,000 people were … [Read more...]
Cartoon Perfectly Explains What Guns Can Do
The firearm is one of the greatest inventions in world history. It has enabled people around the world to defend themselves against those who seek to … [Read more...]
The Simple FACT About "Rights" That Liberals Will Never Understand
Despite labeling themselves "pro-choice" on abortion, liberals have never respected Americans' right to make their own personal decisions. … [Read more...]
For the Sake of our Safety, It's Time to Abolish the TSA
Incompetence, negligence, theft, sexual assault, harassment. To what entity am I referring? The Transportation "Security" Agency, one of the most … [Read more...]
Samuel Adams Warned Us About Demagogues
Conservatives often praise the Founders for writing the greatest political document in human history: the United States Constitution. However, in … [Read more...]
What's a Greater Threat to Liberty, Threat of Terrorism or….
BRUTAL Meme Captures Why We're Right To Fear For The Future of Our Republic
Meme Drops Epic TRUTH BOMB On Anti-Gun Democrats
What It Means To Be A Liberal Today Summed Up In EPIC Meme
If a Flag Burns in a Park and no one is Around to See it, Does it Really Make a Statement?
The NY Post reported yesterday that "[c]op haters plan flag-burning rally at NYC park" this evening, July 1. A group called Disarm NYPD initially … [Read more...]
IOWA: It's Official, You Can Now Be Drunk on Your Own Porch!
Believe it or not a woman in Iowa back in 2013 was arrested for being drunk in public on her own front porch. … [Read more...]
Silly Cartoon Perfectly Illustrates How Big Government Operates
Personally I think Benjamin Franklin got it right when he wrote: … [Read more...]
Map Shows Americans Where to Go If They Crave Freedom and Liberty
The John Locke Foundation has released the "First in Freedom Index" which ranks states by how much personal freedom it's residents enjoy. The ranking … [Read more...]
John Stossel: Government Controls On Gambling and Liberty (Video)
Stossel's take on why all voluntary gambling should be legal. He claims that laws against betting create a black market and puts criminals in charge. … [Read more...]
The Government Versus The American Character
Over the past 50 years, the purpose of the American government has radically transformed. Whereas it's main goal in domestic matters used to be to … [Read more...]
Allen West: For What Does Our Republic Stand?
Every American needs to read this and ponder what this country stands for, how we lost our way, and then commit to helping us restore this great … [Read more...]
Ayn Rand – Liberty vs Socialism (Video)
In this classic interview Mike Wallace interviews Ayn Rand regarding socialism and individual liberty. What is really interesting is how many of … [Read more...]
(Video) Walter E. Williams on Libertarians and Liberty
In this lecture given at a Libertarian Party of Georgia event Williams talks about libertarianism generally and relates his own moral arguments … [Read more...]
John Wayne on Liberty, Freedom, and the Role of Government
Hollywood used to be for America before they were against it. Watch John Wayne at his finest as he talks about liberty, freedom and the proper role of … [Read more...]
Rand Paul: Americans Trading Liberty for False Security
Via Breitbart: We have been too lax in giving up our privacy. We are trading our liberty for some sort of sense of ostensible security. Look at how … [Read more...]
The Law by Frederic Bastiat
Get a FREE copy of "The Law" by Frederic Bastiat: Anyone building a personal library of liberty must include in it a copy of Frédéric Bastiat's … [Read more...]
Samuel Adams, America will maintain her freedom
No Character appears with a stronger Luster in my Mind, than that of a Man, who nobly perseveres in the Cause of public Liberty, and Virtue, through … [Read more...]
Is The American Spirit Alive?
"Let Freedom's land rejoice! Our ancient bonds are riven; Once more to us the eternal choice Of good or ill is given." -Rudyard Kipling "The … [Read more...]


text:
The right side of this image reveals the portion of the crater edge that descends into the mouth of Ma'adim Vallis, a channel that opens into Gusev Crater. Spirit is currently traveling toward the informally named "Columbia Hills," which lie to the left of the region pictured here.
This image is similar to a panoramic camera image taken on sol 68, but Gusev's ridge is more visible here because the atmospheric dust caused by winter dust storms has settled. Scientists expect to get even clearer images than this one in upcoming sols.
This image has been modified to make the crater rim more visible.


text:
Kmart Complaints
Kmart.com SnapShot
Last Updated On: March 14, 2017
Kmart RatingsAverage Rating: 1.44 out of 5
Based On: 75 Reviews
Number Of Comments: 61
Kmart ReportsReported Losses: $3,920.93
Average Reported Losses: $52.28
Most Recent Kmart Complaints
Is Kmart a Scam, Rip Off, or Fraud? WARN OTHER CONSUMERS By SharingCategory: Department Stores Directory ListingSears Brands LLC Listing
New returns policy bad for customers
I've been shopping at Kmart for many years. I will never shop there or Sears again because they have changed the returns and exchanges policy to a 30 day limit.
Kmart refused to refund or exchange items I bought totaling about $40 for a gift that was not usable. Now I am stuck with children's clothes.
I am not the only person who sometimes waits until I have more than one reason to drive to a town to shop. That is why I waited to return these items. Times must be pretty bad for them to need to keep customers' money this way.
They will lose far more than $40 from me in the future sales. I am posting this to as many places as I can.
Kmart – 1000 Montauk Highway West Babylon NY 11704
15 Most Recent Reviews About Kmart
Rude cashier
Complaint Filed By: Lucy
On: March 14, 2017
I sent my son Osmani to pick up an order I placed online at Kmart located in 3825 N.W 7th...
Costs Me Way More Than My Purchase
Complaint Filed By: Mike K.
On: November 13, 2016
Rating: 1 out of 5
Sunday 7/17/16 my wife and I went into the K-mart on Brice Rd. in Reynoldsburg, OH to shop. After making...
A month of a nightmare for a crib bumper!
Complaint Filed By: Amanda Riley
On: November 13, 2016
Rating: 1 out of 5
I ordered a Minnie Mouse 4 piece crib set that couldn't be shipped directly to my home, so I had...
Return policy at Kmart is not customer friendly
Complaint Filed By: Carol
On: May 17, 2016
Rating: 1 out of 5
Reported Losses of $8.00
I made a purchase of a female garment several months ago and it did not fit. I had the receipt...
Overcharged by $400
Complaint Filed By: vlaroue
On: March 27, 2016
Rating: 1 out of 5
Reported Losses of $400.00
I went to the fort pierce florida store on Sunday February 29th 2016. I went to the front desk the...
Payment to the wrong company
Complaint Filed By: Karen
On: March 27, 2016
Rating: 1 out of 5
Back in Jan 2016 went to Kmart to have my utility bill paid and the staff member took my money...
Horrible smell
Complaint Filed By: Polly Adams
On: March 27, 2016
Rating: 5 out of 5
Reported Losses of $5.00
Something needs to be done about the horrible odor that hits you when you walk in the door of the...
Bait and switch
Complaint Filed By: Angie
On: March 27, 2016
Rating: 1 out of 5
K-Mart of Hollywood Florida will advertise a product on sale and it appears too good to be true, but when...
Check out is ridiculous
Complaint Filed By: Jennie Osborne
On: March 27, 2016
Rating: 1 out of 5
Every time I go to K Mart, there is a problem at checkout. Today, I stopped in for a few...
Racial profiling
Complaint Filed By: josefsmom
On: February 26, 2016
Rating: 1 out of 5
On February 15th,I went shopping with my 14 year old son, who left my side briefly to look for a...
- Click Here To Search For More Kmart Complaints
Submit a Complaint or Review For Kmart
Leave a Reply
61 Comments on "Kmart"
Boycott KMart ladies. They do business with the Trump products/family. Why enrich their coffers? Shop Target instead. Boycotts are highly effective.
Contact the manufacturer, they should have a warranty as well!
They should have been able to do a sales adjustment right at the Cust Service desk and credit you back the difference to your card. Ask for Managers whenever you run into these types of issues. I find it makes a difference, always. No way I wouldn't have been screaming for a manager waiting in line for three hours!
I would like to know why KMart has NO AUSTRALIANS in their TV commercials what do Australians NOT wear Hoodies?????? Your shop has inferior cheaply made products and I NEVER shop there but KMart who is your target market certainly NOT AUSTRALIANS shame on you!
BROCKTON,MASS. STORE EMPLOYEES VERY RODE AND DO NOT CARE IN THE LEAST ABOUT CUSTOMER SERVICE.Really bad experience.Even dealt with some manager on the floor who was rude also.When you'r on the Titanic of department stores you should try a little harder.It was my 1st visit to kmart in 10 years Now my last.
had a $10 coupon with a new Rx (I was going to buy coffee maker there) and was told with M'care you can't use it–transferring "All" my Rx's to Publix !!–you are losing "all my business" –being punished because I am old !!–
I have worked hard all my life–never depended on the gov't for anything until now–thks for making me feel like I am getting something for nothing !!!
What a sad sad store. Dirty disorganized rude. Terrible marketing terrible commercials no target market. Just shutdown. "Where members get more". wHAT MEMBERS. The store is horrible. Every site not just one.
The Kmart in Bed ford In. is closing and have a ton of toys and if no one buys them on Sunday they are going to throw the rest away, instead of giving them to children in need or to Riley for Christmas presents, now if that don't say Scrooge then I don't know what does.
The Human Resource person at the Steger Kmart in Illinois is an idiot and should be fired. When she hires you she lies about hours wages and work you will be doing. The turn over of people is horrible. Nobody wants to stay. Every week there is a new face in that store. She's been at her job too long and needs to go!
Who is Butch?
Bought a Singapore Chain reg Price 499.00 on sale @70% off Was then 160.00.got home went on Amazon they were selling exact same one for 45.00.isnt this considered Price gouging or at least bad buisness.kmart You Have Lost my buisness and since store is closing i Cant return it.
I shop at kmart in Olean,New York and there is only one power chair which hardly works am handicap and can onley walk so far I thjnk they should have more than one chair so Im gone to have to shop at a store nthat sit up for handicap people that has more than one power chair gonnnne to miss shopping akmart unless they get moree piower chairs FOR THE DISABLE PEOPLE.
I work there and it is suppose to be 75% off the discounted price. If it's original price is $10. and it is marked $8. You would get it for $2.
I'll never shop at K-Mart or buy Joe Boxer products again.
The Joe Boxer commercial was the worst, disgusting and shameful ad I have seen in a long time… Shame on you…for the love of money or for whatever reason you did this…it was terrible…little children with all their innocence and love of the Christmas Holiday were exposed to this…I will never shop at your store again…even the men that would take part in such a disgusting display of bad taste ought to be ashamed…forever ashamed…and again…SHAME ON YOU!!!!


text:
deck bridge
Definition of deck bridge
: a bridge whose supporting elements (as trusses, girders, arches) are below the track or roadway — compare through bridge
Love words? You must — there are over 200,000 words in our free online dictionary, but you are looking for one that's only in the Merriam-Webster Unabridged Dictionary.
Join MWU now and get access to America's largest dictionary, with:
- 300,000 words that aren't in our free dictionary
- Expanded definitions, etymologies, and usage notes
- Advanced search features
- Ad free!
Seen and Heard
What made you want to look up deck bridge? Please tell us where you read or heard it (including the quote, if possible).


text:
Natalie Calls Victor Out On 'Big Brother 18' For His Sexist Comments & The World Rejoices
My, oh my. How does it feel, Victor? Being called out by someone on national television? Not so great? On Thursday's live eviction episode of Big Brother, Natalie called Victor out for his sexist comments while in the house, and basically it was everything that needed to happen. If you don't remember, Victor made some pretty absurd comments during his short-lived Big Brother life. But, Natalie didn't take those comments lying down. Instead, she used her goodbye message to Victor as the perfect platform to suggest a better treatment of women, and was seriously awesome.
On a previous episode, Victor was seen criticizing her makeup, clothing, and even what she ate. A.k.a., it was not cool. Twitter took notice, too, that his comments weren't OK. Natalie increasingly became upset about Victor's attitude toward her, saying that the now evicted houseguest has an opinion on everything. Well, now was Natalie's time to give her opinion on Victor, and hopefully he takes some tips from it. While it was brief, the words that Natalie spoke were poignant and powerful, and certainly things that Victor will be reminded of as he exits the house (even if he potentially returns). As a reminder, here is that epic goodbye message.
Natalie's goodbye- I really hope you take away a lot from this experience and learn to be a little nicer to people, especially women #BB18— Big Brother Updates (@BB_Updates) July 8, 2016
Fans on Twitter applauded Natalie for her short, but oh so powerful goodbye, noting that she not only stood up for women, but also put Victor in his place.
natalie standing up for women in her goodbye message tho >>>>>— jenny (@majorkeyalert) July 8, 2016
Natalie's goodbye message to Victor haha YESS drag him #BB18— Jess • 7/27 (@JaureCuttie) July 8, 2016
Damn Natalie, speaking the truth girl! Her goodbye speech was. #BB18— kaytie (@kaytiebeth_) July 8, 2016
Natalie dragged Victor so hard!!!! #bb18 pic.twitter.com/L8ppNbu3KM— nadia (@nedaskaIantar) July 8, 2016
Natalie's goodbye message to Victor was a solid A+. Love her. The show is messing up by making her non-existent. #BB18— Andy Herren (@AndyHerren) July 8, 2016
Can I talk about how much I loved Natalie's goodbye message? Stand up for the women in the house!! #BB18— Lizz The Fabulous (@lizzthefabulous) July 8, 2016
Ouch. Natalie sent Victor home with a slap upside the head. #BB18— The BB President (@TheBBPresident) July 8, 2016
It wasn't all about the shade throwing, though. Fans also noted that while Natalie had her moment to shame Victor for his comments, she also proved to be very kind, regardless of her clapback.
Natalie was so sweet— reh (@ZAKlYAHS) July 8, 2016
"I wish you all the health in the world." - Natalie. I LOVE HER SO MUCH HOLY COW. #BB18— Katie (@katielll) July 8, 2016
Basically, Natalie came out on top in this case, and it was amazing. You go, girl!


text:
Asked by pinkdragon36 at 1:05 PM on Jan. 9, 2011 in Just for FunLevel 40 (117,668 Credits)
Answer by san78 at 1:36 PM on Jan. 9, 2011
Answer by djdiddles62 at 1:11 PM on Jan. 9, 2011
Answer by Hazelnutkin at 7:59 PM on Jan. 9, 2011
Next question in Just for Fun
look at your hand. everyone else have a M line on their palm?
Next question overall
(Teens (13-17))
14 Year old son with the flu.............
Recently Bumped in Fun & Games
36 more days!
Featured Posts in All Groups
Already Joined? LOG IN
© 2017 CMI Marketing, Inc. All rights reserved.


text:
Vidyatech Solutions Private Limited
A(z) Vidyatech Solutions Private Limited profilja Magyar nyelven nem érhető el. Az információ English (India) nyelven olvasható.
Vidyatech, in its solutions and/or projects has used a variety of Azure products/services like Virtual Machines, Web Roles, Data Management, Data Storage, and Data Services. Vidyatech realizes that for an IT business to run successfully globally, it has to systematically and securely store data. With support for Linux, Windows Server, SQL Server, and Azure Virtual Machines allowed Vidyatech the flexibility of virtualization for a wide range of computing solutions—development and testing, running applications, and extending its data center. Virtual Machines were particularly useful when Vidyatech had to create a security lab for educational institutions, which otherwise could not afford to get a hand on real machines to run those security labs.
KapcsolatKeresse fel a webhelyet


text:
download PDF
All Pages
Browse Issues
by clicking the arrows at the side of the page, or by using the toolbar.
by clicking anywhere on the page.
by dragging the page around when zoomed in.
by clicking anywhere on the page when zoomed in.
web sites or send emails by clicking on hyperlinks.
Print Page
Email this page to a friend
Search this issue
Index - jump to page or section
Archive - view past issues
GP Week : Issue 169
MARK-DOWN For his part, Mark would have been ropeable after Singapore. There was Seb, qualifying P3 and inheriting another win. And here was Mark, qualifying P7 (not helped by a free-practice brush with the wall) and finishing nowhere thanks to the pit perch making difficult calls at what turned out to be the wrong times. Mark re-signed with RBR partly in order to maximise his chances of winning the 2012 World Championship. Now he must focus on Suzuka, Korea, India and all points west. He's too good not to be looking at another couple of wins (minimum). BURNING BRUNO I suspect Bruno Senna is so accustomed to having a rough ride this year that he didn't think twice about the massive heat build-up that engulfed his race from about half- distance onwards. It was only later, on the massage table, that he realized that he'd incurred mild back burns during the evening from what turned out to be an overheating electric loom. Bruno thus drove exceptionally well under the circumstances: late in the race his KERS unit failed and took with it the Renault engine. Mechanics were astonished to find that the front of the V8 had actually melted as a result of the seizure. SUPERB SEB Seb Vettel reminded us just how good he and the Adrian Newey Red Bull can be on street circuits by heading the times in FPs 1, 2 and 3. Qualifying was slightly less convincing, with Seb finding a reduction in grip as Q2 gave way to Q3: "I just don't know what that happened," he said later (about a groove that for most others was quicker as the night developed). "It's just something to do with the temperature and the tyres." He was quickly up to P2 in the race thanks to Pastor's moment at Turn 1 – and was living in the turbulence of Lewis Hamilton when – grip still not quite right – he suddenly realized he had braked too late for the Singapore Sling. Neatly, he steered right, out of trouble. Lewis's retirement handed him the race on a golden platter but there would be another moment of drama when Seb was leading the race behind the Safety Car on lap 38. With the re-start looming, he accelerated sharply towards Turn 16, then braked hard into the apex, attempting (as several drivers are now doing in this year of elusive tyre temperatures) to 'heat-soak' the brakes and thus the wheel rims (and, thus, the tyres on this grip-sensitive evening). Jenson, weaving behind the Red Bull, almost rammed the leader... A few drivers complained after the race that the Safety Car speeds were abnormally low in Singapore, ruining tyre pressures and temperatures. I guess the answer is that street circuits are notoriously difficult to subjugate – which in turn probably explains why no official action was taken over the Vettel-Button near-miss. 26 GPWEEK.com // F1 >>> SPA 26 GPWEEK.com // PARTNERS: XXXXXXX
Issue 168
Issue 170
Previous Page
Next Page


text:
24″ × 36″, acrylic on canvas, 2007Gas station in Seal Beach, CA, circa 1975. Painted from a slide I took.
gas california tire station sale
great work!
Unbelieveable! Realism at its best!
Thats an awesome old shot
Congrats on the redbubblehomepageTHURSDAY, MAY 20, 2010
ABSOLUTELY WONDERFUL… i love this work..
Who would have thought a self service station such an exciting subject. You have made it look like a very intriguing and inviting place. Great colors and contrasts and composition. Nice vibrant colors. Interesting and creative.
Fabulous image!!
This is simply amazing! Unbelievable amount of detail…WOW!
Very nice. A lot of work. great detail.
Loading more work by Michael Ward...
10% off
for joining the Redbubble mailing list
Receive exclusive deals and awesome artist news and content right to your inbox.
Free for your convenience.


text:
They just simply are a must have. I thought I would just share with you a few inspirational photos featuring a mirrored shade.
Personally I love this trend (the trend being mirrored shades) and the great thing is there are so many style options and varied price points so this trend is definitely one for the masses.
*Images in this post are not my own.


text:
INTRODUCING: Weirds
April 17th, 2017
We're putting the spotlight on the hottest and most exciting new bands around with a brand new Introducing feature, ready to highlight the artists with the biggest potential to make an impact on their scene. This time, we're taking a look at Leeds newcomers Weirds who are preparing for the release of their debut record 'Swarmculture', which is out on May 12th via Alcopop! Records. We caught up with guys to talk about their formation, influences and their upcoming tour with The Wytches.


text:
Contact your Engagement Executive to begin the acquisition process.
Specialty engines on the Integrated Facility for Linux Mainframe allow the mainframe to run Linux workloads. What does this mean to our partners? DISA's Linux on System z environment offers many of the benefits of the traditional mainframe environment combined with the flexibility of Linux. The benefits include:
Our partners save money several ways.
DISA will host DoD IBM Linux on System z applications using DISA-provided hardware, operating systems (OSs), and labor. The z/Linux processing closely follows the server model. The Mainframe staff maintains the z/VM (virtual machine) layer and defines the guest partitions. Server staff supports the z/Linux OS itself.
System Administration (SA): IBM Linux on System z technical support including, but not limited to, the following. DISA will:
Data Communications:
ESM Software:
Level 2 Service Desk Support
Assured Computing/IT Service Continuity:
Mainframe Internet Access Portal (MIAP):
Capacity Management:
Because all our partners require different levels of support, DISA provides choices from the following supplemental features. Each feature has its own set of rates, priced per OE on the Linux on System z platform. However, if a partner has a uniquely large workload, DISA will work with the partner to develop an agreeable labor support cost method outside of these rates. Optional features include:
Database Administration (DBA):
If our partners serve as their own database administrators, DISA must still ensure that their databases comply with the DoD's security guidelines. The charge for this service is 10 percent of the full DBA rate per database OE. The security fee includes the following services:
If our partners serve as their own database administrators, DISA must still ensure that their databases comply with the DoD's security guidelines. The charge for this service is 10 percent of the full DBA rate per database OE. The security fee includes the following services:
Note : If DISA is serving as the partner's database administrators, there is no security surcharge as it is included in the DBA rate.
Web Administration:
Note: This service only applies to the OEs that are functioning as web servers, not to application servers, database servers, domain name servers, etc.
24 x 7 SA:
24 x 7 Application Support:
Local Operational Recovery:
DISA will use a Linux on System z rate to charge our partners by measuring the number of CPU hours their workloads use. DISA will charge for storage in the Linux on System z environment using server storage rates.
View DoD-approved FY16 | FY17 rates for this service.
This service can be obtained by working with your Mission Partner Engagement team to complete a Mainframe Service Request Form (SRF) that will identify the specifics of your requirement.
Technical Support
If you are experiencing technical difficulties, please contact the applicable service desk for support.
For a description of service desk support and service desk levels and tiers, please visit the Service Desk Support page.
New Service Requests
Mission partners interested in learning more about this service should contact their Mission Partner Engagement team.
Mission Partner Portal
For additional information, please visit the Mission Partner Portal.
Performance Standards
DISA has standard performance level data available for our partners to view. Additional data can also be provided as requested. All performance data to be provided will be documented in the service level agreement (SLA), which will be executed when the service is ordered.
Terms and Conditions
Please review the Terms and Conditions for information governing the overarching agreement with our mission partners.


text:
- Teen Haircare is now part of the AKS Hair Product Family!
Celebrating 20 years of styling and color care success in 2017! To commemorate the milestone, AKS is adding to their signature Salon AKS hair care range with the launch of a Teen Shampoo and Conditioner, available at www.shopaks.com.
Aida Angjeli
Click to see Aida at work!
"Beyond looks and style, effortless elegance is powerful!"
Aida embraces the opportunity to meet the highest standards in the beauty industry, and specializes at Salon A·K·S
® as an eyebrow designer. She is passionate about creating perfect brows. She's a strong believer in the incredible power of the right brow shape to enhance a woman's entire look.
For perfect skin in no time, Aida will execute the ideal Dr. Dennis Gross peel, individualized to each clients specific skincare needs.


text:
Thanks for Tuning In to The Prosperity Show
To subscribe to the podcast, use the links below:
Click Here to Subscribe via iTunes!
Click Here to Subscribe via RSS (non-iTunes feed)!
While you are on iTunes, I would appreciate it if you would click the Ratings & Reviews tab and leave a rating and review. This really helps the show's rankings in iTunes!
Podcast: Play in new window | Download (9.5MB)
Subscribe: iTunes | Android | RSS
The Identity Factor is a mechanism that kicks in each time you attempt to make a major change in your life. In this episode you'll learn why the Identity Factor causes resistance and so-called self-sabotage and how to get beyond it's effects. Read more
Podcast: Play in new window | Download (16.0MB)
Subscribe: iTunes | Android | RSS
Highlights from Interview with George Huang
- Entrepreneurs have a lot of fear that causes them to avoid dealing with their money.
- Fear of the unknown, running out of money, not knowing what to do.
- What you need to have a business that is balanced.
- The advantages of keeping track of your money. Read more
Podcast: Play in new window | Download (10.6MB)
Subscribe: iTunes | Android | RSS
Show Highlights:
- Uncertain times exacerbate fear
- Fear is about the future.
- Financial fear is not dependent on how much money you have.
- Financial fear is never about money, but about the consequences of not having money.
- At the base of the fears are emotions such a shame, guilt, deprivation, and the fear of being alone. Fear of losing face or losing your position.
- Another base for fear is behaviour learned from parents.
- People worry because they think if they stop worrying, everything will fall apart. Read more
Podcast: Play in new window | Download (7.7MB)
Subscribe: iTunes | Android | RSS
Tip of the Week- Roboform
Great utility for remembering passwords that syncs across devices. www.Roboform.com (Affiliate Link)
Highlights from Today's Episode
Key points:
- You can't build a successful business if you have a lousy relationship with money!
- Signs of both a good and bad relationship with money
The 5 Keys are:
- Count your money
- Be aware of your thoughts, beliefs, and emotions about money and wealth Read more
Podcast: Play in new window | Download (8.3MB)
Subscribe: iTunes | Android | RSS
Tip of the Week:
If you use a Kindle or read Kindle books on the Kindle App, you may have tried to copy your highlights and discovered that you can't do this. However, there is a way to copy your highlights and notes. Here's how:
Go to https://kindle.amazon.com and sign into your account.
On the top Navigation bar is a link that says Your Highlights. Click this link and you will see all of your Kindle books and the highlights you have made. You can now copy the text and use however you wish.
I head this on Ray Edwards Podcast
Podcast: Play in new window | Download (10.1MB)
Subscribe: iTunes | Android | RSS
This is the first episode The Prosperity Show Podcast. Lots of new and interesting information.
The show presents a holistic approach to business success and financial health. There will be a combination of my sharing information along with periodic guests, tips to help you grow your business and much more.
What you'll learn from this podcast series: Read more


text:
Roberto Martinez looks set to ask Swansea for permission to speak to Celtic over their managerial vacancy after apparently expressing interest in the post.
Swansea turned down an initial request from Celtic but chairman Huw Jenkins admitted he would not stand in Martinez's way if the Spaniard insisted on talks.
The 35-year-old appears destined to do so when he returns from holiday tomorrow.
"Managing Celtic is one of the top jobs in European football," he said in the Scottish Daily Express.
"Any young, aspiring manager with ambition would be interested in a job like that.
"It gives you the chance of being involved in Champions League football and the opportunity to win silverware every season in Scotland.
"Anyone would be interested in a job like this. However, everything would have to be sorted with Swansea first.
"I have an agreement in place with the Swansea chairman as to what would happen and I'd want that to be honoured."
But the likelihood of Celtic facing competition from Wigan grew today as Latics boss Steve Bruce broke off from his holiday to discuss personal terms with Sunderland.
Martinez enjoyed adulation from Wigan fans during his playing career and sources at Swansea believe that would be his most likely destination if he were to leave Wales.
Jenkins has also admitted Real Sociedad have made enquiries about the Spanish manager.
However, Martinez has four years left on his contract and City may demand £2million compensation for a manager who led them to the Coca-Cola League One title in his first full season and eighth place in the Championship this season.
Celtic have refused to comment on the approach but other names are still in the frame to replace Gordon Strachan.
West Brom have repeatedly stated there has been no official approach for Tony Mowbray while Motherwell manager Mark McGhee and Dundee United boss Craig Levein have consistently been linked with the post.
Owen Coyle was thought to be Celtic's first choice and, although he pledged his commitment to Burnley before going on holiday at the weekend, he is yet to sign an extended contract at Turf Moor.
Everton manager David Moyes has ruled himself out but Charlie Nicholas insists his former Celtic team-mate is the calibre of appointment the club should be making - and not Martinez.
"I would be surprised if Celtic fans would be captivated by the news that Celtic are interested by Martinez," Nicholas told BBC Sport Wales.
"I have to say I'm quite surprised about it. Celtic is a club that is obviously close to my heart but they are a club who seem to be going from manager to manager in terms of budget management.
"And unfortunately I think whoever the manager is, he is going to be controlled and asked to be a glove puppet and financial monitor rather than anything else."
Nicholas, who had two spells with Celtic, has been impressed by Martinez's football philosophy but questioned his ability to attract big-name players to Parkhead.
"He plays a lovely, passing, European style and Celtic fans would like that," the former Arsenal player added.
"But Celtic fans are looking for a big name to bring big-name players and the first reaction, in my mind, would be the Celtic fans saying he would be just controlled by the financial guys."
- More about:
- Celtic F.c.
- Motherwell F.c.
- Swansea City
- Wigan Athletic


text:
Current vacancies
Preference will always be given to an applicant who has lived in Africa or who comes from Africa. Applicants must be numerate, articulate, enjoy wildlife and have a passion for Africa. This is a wonderful career opportunity to join one of the UK's leading specialist safari companies.
CONTACT: Jonathan Drew on email@example.com or Nicola Shepherd on firstname.lastname@example.com


text:
Nanotech Europe 2017
21-23 June, Valletta, Malta
Meet the researchers, entrepeneurs and investors who are shaping the future of nanotechnologies and advanced materials
The Nanotech Europe 2017 Exhibition runs in parallel with the conference and offers a great opportunity for exhibitors to showcase their activities to leading research institutes, key industrial actors, high-tech SMEs, policy makers, funding organisations and media.
Nanotech Europe and EuroNanoForum 2017 together create the biggest European marketplace for nanotechnology and materials applications, products and research. It is not just another nanotechnology exhibition – we offer a unique programme to make you seen and heard!
Book your stand
Click on the image for a larger view


text:
ChasePart of J.P. Morgan
ChasePart of J.P. Morgan
Chase offers great bonuses. Competitive base pay. Good advancement opportunities for bankers trying to become managers. Training is awesome. Easily the best I've ever received. Going thru their management training program rivals MBA level knowledge. Good mobility and ability to grow in your position. I love the branding mentality. They have the best branch support system of any bank. You call they pick up in less than 45 seconds and they got the answer to most anything.
Phenomenal training but some of the former wamu employees didn't get the same training as people hired under chases ownership. A lot of the problems around me stem from former wamu employees. The two banks were a bad culture fit. Some bad customer service on some of their credit cards.
Advice to Management
Let California employees do loans in the branches! Get rid of any disgruntled former wamu employees.
Helpful (3)
Competitive salary
Sales compensation is the best in the industry
Full selection of health and wellness benefits to fit individualized needs
Amazing life insurance offerings
Thankful for the 401k Plan and in branch financial advisors to walk each person through choices
Pension plan is a gift from above
Management, in all levels, competent and willing to help/listen
Training and development is structured and easy to understand
Retail banking side requires employee uniforms - professionalism is a must
SENIOR Management at the top, i.e. Jamie Dimon, Charles Scharf, Heidi Miller are ethical and moral people that employees can look up to and stand by
Recognition for a job well done is given immediately and rewarded by higher-ups
I will agree with some (and I mean some-very few) of the cons mentioned in other posts that the balance of work/life is a bit rough. Concessions must be made to work in the retail banking sector; our clients need us to be there for them which requires expanded work hours. It is a 40 hour schedule, but it is different from the normal 9 to 5.
It truly is a numbers game for bankers- not so much a "con" for me, but if you are not sales oriented, do not apply.
***** I am rather insulted by the posts I've read that say we do not care about our clients. That is simply not true. You must understand the logistics of banking to understand why we are compensated for simple things and try so hard to get our clients to use our products. It is simply the way banking as a whole is moving and it began many many years ago. Many times I have met a client that only uses us in a traditional sense, just a bank to cash checks. Think about it though; if I help that customer setup his/her paycheck as a direct deposit then they do not have to wait to get paid or bother with the antiquated "show up to the bank to cash my check" system - it is available immediately. 15K plus ATMs/5K plus branches nationwide give them immediate access to their funds. If I help this same person setup and pay bills using the Chase online banking/bill pay service then I helped this person make sure that their bills are paid in a efficient and timely fashion. If the average person pays 10 bills per month then the stamp cost alone is $4.40 per month/ $52.80 per year. That service is FREE at Chase and we back up our bill pay with guarantees. If my client has a credit card with no rewards and even a normal interest rate, I can roll over those balances to a 0% card and save them money and set them up for rewards. If that client has no savings, I can set them up with a free savings account that they can manage so the nestegg can begin. If that same client left a job many years ago and left behind a 401k, I can setup a meeting with a Chase Financial Advisor that is in-house and free. They can take control of their finances on the spot. If they need a mortgage, I have a mortgage specialist on staff - bam there's your mortgage! And ALL OF IT is available to manage and view through the Chase online banking system. All in-house, all easy to understand, easy to contact your professional financial team, and available nationwide. What more could a client possibly ask for from their bank? Furthermore, what more could a banker ask for from their employer? It is the sweetest possible setup imaginable!!! On top of it, I am compensated to do things I would have done for my clients anyway! How could anyone not see the benfits of working for Chase bank/banking with Chase bank.
For those people that made us sound like mindless drones- you are wrong! We don't try to hurt people, why try to help. If you are not succeeding is this atmosphere then you should consider going to a financial institution that doesn't mind just being the "check casher." Then you can watch the flow of traffic in and out of the branch and never really do anything all day long. That's a great way to "help" people (note the sarcasm).
It is richly rewarding to do my job everyday. Not every day is easy, some people come in and are not happy or want to get more than is afforded to them with our packages, but it is my job to help those people as well- and make sure they leave happy. We can't always help, but we are willing to try.
Advice to Management
Feel the passion of the team that holds you up! We are your foundation - and are proud to be there! You keep doing what you do - and we'll keep the foot on the gas pedal!
This is an amazing company and I appreciate the fact that our good name is not dragged through the mud because of senior management. I respect and appreciate your ethics and morals- and I am thankful for my employment at Chase Bank.
The company provides clear direction of job requirements. Opportunity to help employees advance and learn. opportunity to make incentive compensation.
One is not encouraged to be creative or to think of ways to make the job more interesting and challenging. Following a set scrips is all the job requires.
Advice to Management
Pay more attention to cultural values. Promote innovation and encourage out of the box thinking. Empower employees to take ownership of their job families.


text:
GTA Vice City
Category page
11,680pages onAdd New Page
this wiki
this wiki
This category contains article relating to Grand Theft Auto: Vice City.
Pages in category "GTA Vice City"
" This page shows all the available cheats in Grand Theft Auto: Vice City. They are activated by a...
Cheats in GTA Vice City
" All the radio stations in GTA Vice City feature publicity breaks. Some advertised products are...
Commercials in GTA Vice City
" A garage is generally an area in the game where the player's car can interact with. It can be a...
" Item placement files, known by the extension.ipl, are map files used to place objects into the...
Item Placement
" Grand Theft Auto: Vice City Official Soundtrack Box Set is a collection of seven CDs, each a...
Grand Theft Auto: Vice City Official Soundtrack Box Set