Approximately one-third of the AIDS cases in the United States have been attributed to the use of injected drugs, frequently involving the abuse of opioids. Consequently, it is critical to address whether opioid use directly contributes to altered susceptibility to HIV-1 beyond the increased risk of exposure. Previous in vitro and in vivo studies addressing the role of μ-opioid agonists in altering levels of the co-receptor CXCR4 and subsequent HIV-1 replication have yielded contrasting results. The bone marrow is believed to be a potential anatomical sanctuary for HIV-1.
The well-characterized CD34+CD38+ human bone marrow–derived hematopoietic progenitor cell line TF-1 was used as a model to investigate the effects of the μ-opioid receptor–specific peptide DAMGO (D-Ala2,N-Me-Phe4, Gly5-ol-enkephalin) on CXCR4 expression as well as infection of undifferentiated human hematopoietic progenitor cells.
The results revealed the presence of the μ-opioid receptor-1 isoform (MOR-1) on the surface of TF-1 cells. Furthermore, immunostaining revealed that the majority of TF-1 cells co-express MOR-1 and CXCR4, and a subpopulation of these double-positive cells express the two receptors in overlapping membrane domains. Three subpopulations of TF-1 cells were categorized based on their levels of surface CXCR4 expression, defined as non-, low-, and high-expressing. Flow cytometry indicated that treatment with DAMGO resulted in a shift in the relative proportion of CXCR4+ cells to the low-expressing phenotype. This result correlated with a >3-fold reduction in replication of the X4 HIV-1 strain IIIB, indicating a role for the CXCR4 high-expression subpopulation in sustaining infection within this progenitor cell line.
These experiments provide insight into the impact of μ-opioid exposure with respect to inhibition of viral replication in this human TF-1 bone marrow progenitor cell line model.
In addition to several studies linking chronic opioid use to immunomodulation and increased susceptibility to bacterial infections, the role of opiates as potential cofactors in HIV-1 pathogenesis and disease has also been proposed. In vitro experiments that involve treatment of peripheral blood mononuclear cells with morphine prior to HIV-1 exposure resulted in increased viral replication. It is now known that prolonged treatment with morphine or the selective μ-opioid receptor agonist D-Ala2,N-Me-Phe4,Gly5-ol-enkephalin (DAMGO) enhances the percentage of T cells and monocytes expressing the HIV-1 co-receptors CXCR4 and CCR5, respectively, thereby increasing the number of infected cells and the overall amount of infectious virus produced in subsequent experiments. More directly, morphine treatment increases HIV-1 infection of blood monocyte–derived macrophages by upregulating CCR5 expression and inhibiting production of β-chemokines, endogenous CCR5 ligands.
Ongoing in vivo studies performed in the simian immunodeficiency virus (SIV)-infected rhesus macaque/model have yielded a better understanding of the impact of prolonged morphine exposure on HIV-1 pathogenesis. Prolonged morphine exposure increased viral replication[6, 7], increased the number of SIV-infected T cells, accelerated disease progression and neuropathogenesis, increased the amount of plasma virus[6, 7], and increased the incidence of mortality. Despite these numerous studies, a direct link between an alteration in CXCR4 or CCR5 surface expression levels and quantity of plasma virus has not been established.
The μ-opioid receptor-1 isoform (MOR-1), the best characterized isoform of the μ-opioid receptor family, has been found on cellular subsets of the immune system, as well as cells of the central nervous system, including but not limited to neurons[9–11]. It is possible that reported inconsistencies in the literature regarding the expression profile of CXCR4 may be attributable to a cell type–specific regulation of this chemokine co-receptor by μ-opioids. This process in turn might translate into the differential ability of μ-opioids to modulate HIV-1 replication in divergent cellular populations. To investigate the effect of μ-opioids on CXCR4 expression in human bone marrow progenitor cells, the TF-1 cell line was used; it represents a model of susceptible CD34+/CD38+ human hematopoietic progenitor cells that are blocked at an early stage of differentiation. To begin experimentation in the TF-1 cell line, experiments were performed to assess levels of MOR-1 in these cells by western immunoblot analyses, flow cytometry, and immunofluorescence microscopy. To analyze the relative surface distribution of MOR-1 and CXCR4, immunofluorescence microscopy studies were also performed. Alterations in total CXCR4 protein levels in DAMGO-treated TF-1 cells were determined using western immunoblot analyses and surface expression levels were examined using flow cytometry. We have previously demonstrated that, in addition to CXCR4, TF-1 cells express the primary HIV-1 receptor CD4 on their cell surface, thereby supporting productive infection by the HIV-1 X4-utilizing (X4) IIIB strain. This observation prompted studies examining the consequence of DAMGO-mediated perturbation in CXCR4 levels on HIV-1 X4 replication in this human bone marrow–derived progenitor cell population.
Western immunoblot analysis confirmed the presence of MOR-1 protein within TF-1 cells, clearly demonstrating the existence of a specific protein species at approximately 50 KDa, the expected molecular mass of human MOR-1 (Figure 1A). As expected, the levels of MOR-1 in undifferentiated SH-SY5Y neuroblastoma cell lysates (positive control) were much higher than those observed in TF-1 lysates. In addition, the detection of MOR-1 was abrogated by preincubating the primary antibody with the MOR-1 blocking peptide. Blotting the membrane for β-actin confirmed equal loading in all lanes. Immunostaining of nonpermeabilized cells analyzed by flow cytometry (Figure 1B) and fluorescent microscopy (Figure 1C) clearly demonstrated that MOR-1 was expressed at the cell surface.
MOR-1 is expressed on the surface of TF-1 cells. (A) Western immunoblot analyses performed with the human neuroblastoma cell line SH-SY5Y (positive control) and TF-1 cell line whole cell lysates demonstrate the presence of μ-opioid receptor-1 protein (MOR-1) at ~50 KDa. Abrogation of the 50-KDa band with preincubation of the polyclonal antibody with a MOR-1 blocking peptide confirms the specificity of reaction. The membrane was stripped and reblotted for human β-actin (43 KDa) as the endogenous loading control. The blot is representative of results obtained from three independent experiments. (B) Flow cytometry of nonpermeabilized TF-1 cells stained with an antibody directed against a region mapping within the N-terminus of MOR-1 (20 μg/ml) shows expression on the cell surface. A fluorescein isothiocyanate (FITC)-conjugated secondary antibody directed against the primary host was used in combination with primary staining (blue) or alone (red) as a negative control. (C) The left panel shows a subpopulation of paraformaldehyde-fixed, nonpermeabilized TF-1 cells expressing MOR-1 on the surface (detected in green with a FITC-tagged antibody directed against a region mapping within the N-terminus of MOR-1). The right panel shows a merge with DAPI (4',6-diamidino-2-phenylindole) nuclear staining of the same field. The images were taken at 60× with an Olympus IX81 deconvolution microscope.
To analyze the relative distribution of MOR-1 and CXCR4 at the cellular level, immunofluorescence studies on nonpermeabilized TF-1 cells was performed. CXCR4 and MOR-1 were shown to be co-expressed on the surface of a number of TF-1 cells. The majority of the cells quantitated (87%) expressed both MOR-1 and CXCR4 at the cell surface (Figure 2). Far fewer cells expressed MOR-1 or CXCR4 exclusively (2% and 3% of counted cells, respectively), and 8% of cells quantitated did not express either receptor.
TF-1 cells express MOR-1 and CXCR4 in overlapping domains. (A) TF-1 cells were cultured and stained as described above. Briefly, TF-1 cells were grown on poly-D-lysine coated coverslips and stained, nonpermeabilized, with primary antibodies directed against MOR-1 and CXCR4, and with DAPI (4',6-diamidino-2-phenylindole) staining to show nuclear morphology. The upper left panel demonstrates a nuclear reaction with DAPI (in blue). The upper right panel demonstrates a surface reaction pattern for MOR-1 (in green). The bottom left panel also demonstrates a surface reaction pattern for CXCR4 (in red). The bottom right panel is an overlay showing the relative expression of MOR-1 and CXCR4. These images are representative of all fields captured. All images were obtained at 60× with an Olympus IX81 deconvolution microscope. (B) TF-1 cells were quantified by counting a total of 1000 cell nuclei, and these cells were then characterized based on MOR-1 and CXCR4 staining as single-positive for either protein, double-positive, or double-negative. This analysis shows that the majority of TF-1 cells are double-positive for surface expression of the two proteins.
Interestingly, on the surface of double-positive cells, the staining for the two receptors appears to overlap, suggesting the possibility of physical proximity. A proportion of these double-positive cells co-expressed CXCR4 and MOR-1 in distinct domains on the surface that traced the circumference of the nucleus. Additionally, double-positive cells that exhibited uniform distribution of expression of the two receptors across the cell surface differed in nuclei morphology. A similar co-localization pattern of MOR-1 and CXCR4 has been previously reported in cortical neurons. Given the observed changes in nuclear morphology, we proceeded to determine whether these discrete domains were specific to MOR-1 and CXCR4 or resulted from general TF-1 cellular topography. To distinguish between these two possibilities, fluorescently tagged amphipathic molecules that embed within regions of high lipid density were used to visualize the plasma membrane. This procedure clearly demonstrated that the observed discrete domains were regions of high lipid density (Figure 3A), likely owing to membrane thinning and nuclear dispersion during M phase of the cell cycle.Together these observations suggest a possible link between cell cycle and the observed MOR-1 and CXCR4 surface localization patterns. To better understand the influence of the phase of the cell cycle on MOR-1 and CXCR4 cell surface localization, TF-1 cells were treated with aphidicolin (10 μg/ml) for 16 hours to induce an arrest in early S phase. Following treatment, MOR-1 expression was analyzed on the surface of TF-1 cells in S phase using fluorescent microscopy and flow cytometry. TF-1 cells in S phase expressed MOR-1 in a punctate pattern across the cell surface (Figure 3B) and expressed MOR-1 at a higher mean fluorescent intensity (MFI) than the untreated, heterogeneous population of TF-1 cells (Figure 3C). These observations suggested that the diverse cell surface expression patterns of MOR-1 in conjunction with CXCR4 are the result of a specific phase of the cell cycle. While the two receptors are expressed in overlapping membrane domains, suggesting the possibility of proximity, the localization in discrete membrane domains surrounding the nucleus may not be due to regulated trafficking of the two receptors.
MOR-1 surface localization varies with the phase of the cell cycle. (A) TF-1 cells were grown on poly-D-lysine coverslips and left nonpermeabilized. Cells were then stained with a fluorescently tagged amphipathic molecule that embeds within the lipid bilayer. The molecule embeds preferentially into regions of high lipid density, and therefore these regions are represented by high fluorescence. (B) TF-1 cells were grown on poly-D-lysine coverslips and then treated with aphidicolin (10 μg/ml) for 16 hours to induce arrest in S phase. Following treatment, cells were left nonpermeabilized and stained for MOR-1 and with DAPI (4',6-diamidino-2-phenylindole). MOR-1 puncta are located diffusely throughout the cell. All images were obtained at 40× with an Olympus IX81 deconvolution microscope. (C) TF-1 cells were left untreated or arrested in S phase and stained for MOR-1 as described above, then analyzed by flow cytometry. Analysis shows an increase in MOR-1 expression in S phase, as indicated by an increase in mean fluorescent intensity.
To study the effects of MOR-1 signaling on CXCR4 expression, the levels of total as well as surface CXCR4 was examined in DAMGO-pretreated TF-1 cells. Western immunoblot analyses were performed on whole cell lysates prepared from TF-1 cells, in the absence or presence of two different concentrations of DAMGO (1 and 10 μM), a specific MOR-1 agonist. Total levels of CXCR4 protein (glycosylated CXCR4 monomer detected at 45 KDa) remained unchanged, suggesting that DAMGO treatment does not affect overall expression or stability of the CXCR4 protein within the cell (Figure 4A). This observation prompted studies to determine whether the subcellular localization of CXCR4 was affected by DAMGO treatment. Immunostaining for surface CXCR4 was performed on nonpermeabilized TF-1 cells and analyzed by flow cytometry. Untreated TF-1 cells displayed a distribution among a range of MFIs, with three distinct cellular populations: a large population of CXCR4 non-expressing cells, a low-expressing cell population, and a smaller population of CXCR4 high-expressing cells (Figure 4B). This is consistent with the heterogeneity in CXCR4 expression observed in the human hematopoietic stem cell compartment and is consistent with levels of CXCR4 surface expression in primary human bone marrow cells. DAMGO (1 and 10 μM) pretreatment for 24 hours resulted in an alteration in the relative proportion of CXCR4+ cells toward the low-expressing phenotype, which was evident from an increase in the low-expressing (midrange) MFI peak and a reduction in both the non-expressing and high-expressing MFI peaks. Collectively, these studies show that total protein levels of CXCR4 remain unchanged, while surface expression levels of CXCR4 are altered following DAMGO exposure.
DAMGO alters surface expression of CXCR4 on TF-1 cells. (A) Total levels of CXCR4 remain unaltered in TF-1 cells upon DAMGO treatment. Western immunoblot analyses performed with TF-1 cell line whole cell lysates demonstrate the presence of glycosylated CXCR4 monomer at 45 KDa. In comparison to the untreated cells, DAMGO (1 and 10 μM)-treated cells exhibited a similar density of CXCR4, indicating that CXCR4 expression at the protein level does not change. The membrane was stripped and reblotted for human β-actin (43 KDa) as the endogenous loading control. (B) Treatment of TF-1 cells with DAMGO results in a shift in CXCR4+ cells to the low-expressing phenotype. Immunofluorescence flow cytometry analysis of nonpermeabilized TF-1 cells was performed to detect a change in surface expression of CXCR4 following DAMGO treatment. Immunofluorescence analysis for surface CXCR4 by flow cytometry demonstrated that TF-1 cells were distributed among three populations, non-expressing cells, low-expressing cells (lower mean fluorescence intensity [MFI]), and high-expressing cells (higher MFI). DAMGO pretreatment (1 and 10 μM) for 24 hours resulted in a shift in the relative proportion of CXCR4+ cells to the low-expressing phenotype, as is evident from the increase in the lower MFI peak and the reduction in the higher MFI peak. CXCR4 antibody isotype controls are shown at the bottom.
Given the observed changes in the level of CXCR4 surface expression following DAMGO treatment and the understanding that co-receptor expression levels correlate with cell susceptibility to HIV-1 infection, the level of impact DAMGO would have on HIV-1 replication was of interest. HIV-1 core antigen (p24) assays were used to assess HIV-1 replication in TF-1 cells in the presence of DAMGO. In comparison with untreated cells, DAMGO pretreatment for 24 hours resulted in a dose-dependent decline in p24 production (Figure 5). Specifically, p24 levels dropped from 885 ng/ml in untreated cells to 386 ng/ml in cells treated with DAMGO at 1 μM (p < 0.001) and 254 ng/ml in cells treated with DAMGO at 10 μM (p < 0.001). This decline, which ranged from 2.3- to 3.5-fold, was partially reversed upon CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH) pretreatment to 763 ng/ml (p < 0.001). Altogether, the results suggested that exposure to μ-opioids might render bone marrow progenitor cell populations less susceptible to HIV-1 infection.
DAMGO treatment inhibits the replication of HIV-1 X4-utilizing strain IIIB in TF-1 cells. Analyses involving the detection of HIV-1 p24 capsid protein were performed on cellular supernatants 24 hours after infection to assess HIV-1 IIIB replication in response to DAMGO treatment of TF-1 cells. Absolute values of p24 levels are denoted in ng/ml on the Y axis. A 2.3-fold decrease was observed with DAMGO treatment (1 μM), which further declined to 3.5-fold at 10 μM. CTAP pretreatment reversed DAMGO-mediated decline in p24 levels. Samples were assayed in triplicate and results shown are the average of two independent experiments. *p = 0.00012, **p = 0.00000076, and *** p = 0.000002.
Because HIV-1 has been reported to penetrate the bone marrow[17, 18] of infected patients, it has been suggested that cellular subsets of this important compartment may serve as reservoirs for the virus. We aimed to determine the effects of the specific MOR-1 agonist DAMGO on viral replication in human bone marrow progenitor cells. The human CD34+/CD38+ TF-1 bone marrow–derived hematopoietic progenitor cell line was used as an in vitro model because it is known to express the HIV-1 receptor CD4 as well as the co-receptor CXCR4.
MOR-1 is the most extensively characterized receptor subtype of the family and it has been identified on terminally differentiated mature immune cell populations, including human CD4+ cells, cells of the monocyte-macrophage lineage, and more primitive CD34+38− peripheral blood and cord blood stem cells. Its presence on more terminally differentiated progenitor cells and the concomitant underlying significance of its presence have, however, not been fully elucidated. In this regard, we have clearly demonstrated the presence of MOR-1 in a TF-1 human bone marrow progenitor cell population.
MOR-1 has also been shown to oligomerize with other G protein–coupled receptors (GPCRs) such as the chemokine co-receptor CCR5 with an adverse impact observed on the G-protein coupling of the other oligomerization partner. Alterations in G-protein coupling, differential affinity of specific agonists, and enhanced internalization have also been reported for the μ-δ opioid receptor (MOR-DOR) hetero-oligomeric complex. Therefore, the presence of MOR-1 and CXCR4 in overlapping domains on TF-1 cells leaves open the possibility of dimerization between these receptors. Such localization of MOR-1 has also been reported in neuronal membranes of the rat caudate putamen wherein μ-opioid receptors were found in cholesterol and sphingolipid-rich membrane subdomains known as lipid rafts, where they seem to be important in regulation of GPCR signaling, receptor phosphorylation, and membrane trafficking. This observation raises interesting questions concerning the biological significance of MOR-1 localization in TF-1 progenitor cells. The results reported herein have also revealed MOR-1-mediated alteration in surface CXCR4 levels, in line with our previous studies[10, 11]. It has been previously reported that CXCR4 signaling is impeded by DAMGO in rat cortical neurons[10, 11]. Therefore, future studies will be focused on investigating whether molecular events triggered by MOR-1 signaling affect CXCR4 receptor recycling and/or CXCR4 signaling in bone marrow progenitor cells.
These studies also provide a unique insight into the role of μ-opioids in modulating HIV-1 replication in bone marrow progenitor cells. In the setting of HIV-1 infection, downregulation of CXCR4 might translate into a reduction in HIV-1 replication under the influence of μ-opioids, thereby serving a protective function, as suggested in studies performed in macaques. As shown here, DAMGO exposure resulted in a decrease in HIV-1 replication. While this decrease may be the result of a decrease in viral entry involving CXCR4, the possibility remains that this decrease is the result of inhibition of post-entry events in the viral life cycle. Paradoxically, because the SDF-1–CXCR4 axis is vital for survival of progenitor cells, a reduction in levels of CXCR4 might render them more susceptible to apoptosis in vivo. This result might provide a plausible explanation for the hematopoietic dysregulation observed in animal models of long-term morphine administration[26–29]. These results warrant further studies aimed at dissecting the divergent and dichotomous effects of μ-opioids on terminally differentiated cell populations in the peripheral blood and bone marrow.
These studies have demonstrated the presence of both MOR-1 and CXCR4 on the cell surface of the hematopoietic progenitor cell line TF-1. Additionally, these receptors are localized to regions of high hydrophobicity within the membrane, indicative of high lipid density. As such, MOR-1 and CXCR4 are localized in proximity in this cell population. Most importantly, these experiments demonstrate the alteration in surface expression of CXCR4 as a result of DAMGO stimulation, and reduction in HIV-1 replication of a CXCR4-utilizing (X4) viral strain.
The potent cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine was obtained from Sigma-Aldrich (St. Louis, MO). DAMGO, a selective μ-opioid peptide, and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), a selective MOR antagonist, were purchased from Sigma-Aldrich. Amicon Ultra-0.5, Ultracel-3 Membrane, 3-kDa centrifugal filters for protein purification were obtained from Millipore (Bellerica, MA). Whole cell lysates were prepared for protein studies in radioimmunoprecipitation assay (RIPA) buffer (Pierce ECL; Thermo Fisher Scientific, Rockford, IL), and protein concentrations were calculated using the bicinchoninic acid (BCA) protein assay as described by the manufacturer (Pierce ECL).
The TF-1 CD34+38+ cell line (American Type Culture Collection [ATCC], Manassas, VA) was grown in TF-1 media which is composed of RPMI-1640 medium (ATCC; Mediatech, Inc., Manassas, VA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone, Thermo Fisher Scientific, Logan, UT), penicillin (100 U/ml), streptomycin (100 μg/ml), and recombinant human granulocyte/macrophage colony-stimulating factor (2 ng/ml) (eBioscience, San Diego, CA). The SH-SY5Y human neuroblastoma cell line (ATCC) was propagated in a 1:1 mixture of Eagle's minimal essential medium with Earle's salts, L-glutamine, and F-12 K nutrient mixture (Mediatech) supplemented with 10% heat-inactivated FBS (HyClone), penicillin (100 U/ml), and streptomycin (100 μg/ml). The cells were maintained at 37°C in 5% CO2 at 90% relative humidity.
To identify MOR-1 at the protein level, an N-terminus-specific antibody was used that recognizes a region mapping within the extracellular domain of human MOR-1 (N-20, Santa Cruz Biotechnology, Santa Cruz, CA). To demonstrate the specificity of interaction, assays were performed under nonblocking and blocking conditions. Whole cell lysates were prepared in RIPA buffer (Pierce ECL) and used to determine protein concentrations utilizing the BCA protein assay as described by the manufacturer (Pierce ECL). An equal amount of protein was run on a 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to a 0.45-μm Immobilon-P polyvinylidene fluoride membrane followed by probing with the MOR-1 polyclonal antibody and detecting with a horseradish peroxidase (HRP)-conjugated antigoat IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). As a negative control, the primary antibody was preincubated with a MOR-1-specific blocking peptide (1:1) (Santa Cruz Biotechnology) before probing for the receptor protein. Because SH-SY5Y neuroblastoma cells are known to constitutively express high levels of MOR-1, they were used as a positive control in the experiments. Equal loading was confirmed by stripping the membrane using the Restore Western Blot Stripping Buffer (Thermo Scientific) and reblotting for β-actin with a mouse monoclonal antibody from Sigma-Aldrich. To study the total CXCR4 protein expression resulting from prolonged DAMGO exposure, TF-1 cells were first starved for 1 hour. After washing in 1× phosphate-buffered saline (PBS), aliquots were kept untreated or treated with DAMGO (1 or 10 μM) for 24 hours followed by lysis and processing for western immunoblot analysis using a polyclonal anti-CXCR4 antibody (H-118) (Santa Cruz Biotechnology) and an HRP-conjugated antirabbit IgG antibody (Jackson ImmunoResearch Laboratories). All immunoblots were visualized using a chemiluminescent detection procedure as described by the manufacturer (Pierce ECL) on a ChemiDoc acquisition/analysis station (Bio-Rad Laboratories, Hercules, CA).
To examine cellular distribution of MOR-1 and to compare the expression pattern of MOR-1 with that of CXCR4 on the surface of TF-1 cells, immunofluorescence studies were performed. Cells were incubated on poly-D-lysine-coated coverslips. Where indicated, cells were treated for 16 hours with aphidicolin (10 μg/ml) to arrest cells in early S phase. Cells were subsequently fixed with 2% followed by 4% paraformaldehyde. So that we could visualize the surface expression of the two receptors, TF-1 cells were not permeabilized prior to staining. Sequential staining began with primary labeling with an N-terminus-specific MOR-1 antibody, N-20 (Santa Cruz Biotechnology), followed by secondary labeling with a fluorescein isothiocyanate-conjugated antigoat IgG antibody. Subsequently, CXCR4 was labeled with an N-terminus-specific phycoerythrin-conjugated antihuman CXCR4 monoclonal antibody, 1D9 (Pharmingen, BD Biosciences, San Jose, CA), followed by secondary staining with a rhodamine (TRITC)-conjugated antirat IgG antibody (Jackson ImmunoResearch Laboratories). Each step was accompanied by washing three times with PBS. Negative controls (not shown) consisted of "no primary" and "isotype primary" antibody conditions. Cells were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and observed under an Olympus IX81 deconvolution microscope at 60× magnification. A total of 1000 cells were counted following three independent staining experiments, and the percentage of cells in each phenotype was calculated based on the total number of cells counted. Averages reported were calculated based on the three independent experiments, and error bars represent the standard deviation of the results.
To determine mean fluorescence intensity of surface CXCR4 on TF-1 cells, the cells were serum starved for 1 hour, then resuspended in TF-1 serum free media. Aliquots of 5 × 105 cells were incubated in the absence or presence of two different concentrations of DAMGO (1 and 10 μM) for 24 hours. These concentrations were selected because 10 μM represents the upper range of morphine observed in the serum of morphine-dependent animals. With respect to the MOR-1 analysis, where indicated, cells were incubated in the absence or presence of aphidicolin (10 μg/ml) for 16 hours to arrest cells in early S phase. Collected cells were then washed with FACS wash buffer (Hanks balanced saline solution [Mediatech], 3% FBS, and 0.02% NaN3). So that we could visualize the surface expression of the two receptors, TF-1 cells were not permeabilized prior to staining. Cells were then reacted with a titrated amount of a monoclonal phycoerythrin-conjugated antihuman CXCR4 antibody 12G5 (R & D Systems, Minneapolis, MN) or MOR-1 (N-20, Santa Cruz Biotechnology) on ice for 30 minutes. Concentration-matched IgG2A isotype or whole IgG control antibody preparations were used as negative controls. Subsequently, cells were again washed with FACS wash buffer and fixed with 1% paraformaldehyde. We performed flow cytometry using a FACSCalibur Flow Cytometer (BD Biosciences) and analyzed the results using FlowJo version 6.1.1 software (Tree Star, Ashland, OR).
To assess the effects of DAMGO pretreatment on HIV-1 replication in TF-1 cells, p24 assays for measuring viral core antigen were performed. Cells were seeded into 12-well plates at a density of 0.5 × 106 cells/ml and pretreated for 24 hours with DAMGO (1 or 10 μM) alone or DAMGO (10 μM) and CTAP (1 μM) in TF-1 serum free media. After 24 hours, treated cells were seeded in a new 12-well plate at a concentration of 0.5 × 106 cells/ml/well. This step was followed by exposure to the X4-utilizing (X4) HIV-1 IIIB strain at a titer of 105 median tissue culture infective dose (TCID50) (Advanced Biotechnologies, Inc., Columbia, MD). Two hours after infection, cells were washed with PBS, resuspended in TF-1 media containing serum, and reseeded in 12-well plates. Following incubation for 24 hours, the supernatant was collected and assayed for p24 core antigen using an Alliance p24 ELISA procedure as described by the manufacturer (Perkin Elmer, Waltham, MA). All samples were assayed in triplicate and p24 values were normalized for 1 × 106 cells/ml.
Significance was determined using the student t-test, considering p values of <0.05 significant.
These studies were funded in part by the Public Health Service, National Institutes of Health, through grants from the National Institute of Mental Health Comprehensive NeuroAIDS Core Center (CNAC) Developmental Grant, 1P30 MH-092177-01A (Nonnemacher, principal investigator), National Institute of Neurological Disorders and Stroke, NS32092 and NS46263, the National Institute of Drug Abuse, DA19807 (Wigdahl, principal investigator), DA15014 and DA32444 (Meucci, principal investigator), and under the Ruth L. Kirschstein National Research Service Award 5T32MH079785 (Rappaport, principal investigator NIH Award to Temple University and Wigdahl, principal investigator of subcontract from Temple University to Drexel University). The contents of the paper are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
MS performed the experiments in Figures 1B and C,2, and3, participated in the design of the study, and drafted the manuscript. AB performed experiments in Figures 1A and4A and participated in design of the study and in writing the first draft of the manuscript. AA performed experiments in Figure 4B. SRP performed experiments in Figure 5. OM participated in the design of the study. VP performed experiments in Figure 5 and participated in the design of the study. BW and MRN participated in design and coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript.


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Celestus macrotus är en ödleart som beskrevs av  Thomas och HEDGES 1989. Celestus macrotus ingår i släktet Celestus och familjen kopparödlor. Inga underarter finns listade i Catalogue of Life.

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We've fallen in love with our beloved mobile apps, but how can we use them to solve our real social challenges?
The start of 2017 saw many of South Africa's Northern provinces hit by a deluge of rain as typhoon Dineo ravaged the northerly parts of the country, and the effects of La Nina continue to dump millions of litres of water from the sky. It may be a welcome relief from the drought of 2016, but it wreaks havoc with our daily commute: motorists are forced to navigate broken traffic lights and potholes appearing overnight, and many roads turn into concrete rivers. Ironically, while this was happening, the Southern provinces continued to battle sustained drought, with wildfires raging, and emergency teams on constant high-alert.
Devastating though these natural world crises may be, they do give us an opportunity to apply our ideas and our technology to solve some of society's most pressing issues: from disaster management to service delivery, from crime prevention to social grant payments, and much more.
Yes, Candy Crush and Angry Birds may be fun apps, but how can we leverage the power of millions of connected smartphones, 'Connected Citizens', to address the real problems of our nation?
In the economic heartland of Johannesburg, the first signs of this are already appearing. We now have mobile apps for motorists to report faults on the city's 7000 kilometres of road, instantly report crimes and suspicious activity, check for scheduled power cuts, find bus routes and schedules, and see upcoming public events.
nance team, and funnels the request into the JRA's workflow systems.
With Find 'n Fix, we've proven the success of such a tool – but now we can start imagining the broader possibilities. Citizens could quickly and easily report fires breaking out, overflowing refuse bins, burst water pipes, fallen trees and debris swept onto roads after a storm.
We could start reporting crimes or suspicious activity, capturing camera footage and GPS coordinates that aid investigators in solving crimes. It's an unfortunate reality that high crime levels always feature prominently in the national agenda – and each of us has a vested interest, and a responsibility, to play our part in tackling the endemic.
By leveraging an army of citizens on the ground, City officials can literally borrow the eyes and ears of everyone around them.
The concept of 'Smart Cities' entails using new technology to connect and enhance many of the City's basic services: like electricity, water, transportation, road networks, waste management, crime prevention, local government voting, billing services, general information, and more.
But to truly realise the Smart City vision, our thinking needs to evolve, to embrace the concept of 'Smart Citizens'. Millions of urban residents must start adopting new digital and mobile eGovernment solutions, and start positively engaging with officials, government departments, and the broader urban community.
As with the introduction of any new technology, the catalyst for adoption is always the end-user. It's only through widespread adoption that City authorities can start benefiting from those 'eyes and ears on the ground'.
So, just what's needed for us to create 'Smart' cities, and truly harness the power of millions of connected citizens – reporting issues, contributing information, crowdsourcing solutions, and participating meaningfully in their local environment?
1. End-to-end digitisation of City services… Having sleek digital interfaces on the surface must be accompanied by transformed and digitised operational processes at the core of any government department or service. For instance, you may create an app that allows you to request title deeds to a property by mapping your location, scanning transfer papers and ID books, but unless that app connects to modern services within the Deeds Office, the effect will be muted.
3. The Internet of Things… Of the astonishing 20 billion connected devices that Gartner predicts by 2020, a vast number of these will be deployed in the service of local governments – tracking everything from the movement of trains on the railway tracks, to the levels of power-loss on the electricity lines. Sensor-based information can be merged with the data being streamed by citizens, to give city officials deep visibility into key aspects of urban infrastructure and services.
4. Predictive Analytics… Of course, all this data needs to be processed by analytics engines – to reveal meaningful insights. As our intelligence engines grow in sophistication, we're able to start predicting when certain faults or incidents might happen, based on things like the surrounding conditions or the average lifespan for certain infrastructure components. For instance, crews could be despatched to electricity lines before they suffer an impending fault.
5. Great user services… The Johannesburg Road Agency's Find 'n Fix app touched a nerve among the city's motorists, and quickly became a runaway success. The consumer apps and digital services that we choose to develop need to address these 'hot topics', and quickly enable the user to get involved and start to help in solving the problem.
The example of the Find 'n Fix app has shown that when we're empowered with the tools to engage more meaningfully with local government, we will indeed make the effort to contribute to the greater good.
Let's see how far that kind of nation-building culture can carry us. Let's see the results when we can quite literally pull on the insights from millions of those eyes and ears on the ground.


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Q: redis reconnect to db0 after connection error I run several programs on the same redis server using dbs from 1 to 8. I handle redis errors and all programs are working fine and reconnects to given db in most cases but when restarting redis server errors 110 (timed out) and 113 (no route) occur all programs reconnects to db0. Is there any way in which I can protect myself from that? as i said in case of error 111 (connection refused) all programs reconnect to given db and there is no problem. Example:

r = redis.Redis('127.0.0.1', 6379, 3)

redpajama_set_name: RedPajamaStackExchange


Use this tool to explore dockless mobility travel patterns 🛴 🚲 🔌.
When you place a point on the map, shaded hexagons will indicate where trips from that region ended. If you switch the flow to origin, the hexagons indicate where trips to that region started.
We welcome your feedback at 📫 transportation.data@austintexas.gov. Visit the open-source project on Github.
This map has been produced by the City of Austin for the sole purpose of geographic reference. No warranty is made by the City of Austin regarding specific accuracy or completeness.
The open data feed that powers this map is under maintenance and this site will be temporarily unavailable. Please check back soon or reach out to us at transportation.data@austintexas.gov for more information. Thanks!


Vote on Courtney_Holm as a helpful ChristiaNet blogger by clicking this link. Currently Courtney_Holm has 3 votes. The higher the number of votes the more helpful this blogger is considered by the ChristiaNet community.
It also depends on if your consuming alcohol brings glory to Jesus. Well...Does it?


text:
2015 General Election Losing Elections
Is It A Good Election To Lose?
No Comments on Is It A Good Election To Lose?
It hasn't been a good week to be a Conservative.
There are several points which could be picked up on, and these are my favourites. Or these are possibly my socialist enemies favourites.
Firstly the rather unedifying way that it was pointed out that Ed Miliband is a backstabber and could stab the United Kingdom in the back. This is not something I want to hear prominent Conservatives saying – what I wanted to hear was the commitment to renewing our nuclear defence. Which was in there but was rather overshadowed. I've had enough of hearing Conservatives talk about what we can all conclude are the negatives of Ed Miliband as Prime Minister. I want positive election pledges.
Secondly a rather confusing policy about train fares being frozen. Excuse me? Why are we copying socialist failed policies? I appreciate that there is a difference – the rail operators are only allowed to increase prices at a government set percentage plus inflation, as per legislation. Energy firms are part of the private market. But it is a really bad signal to be suggesting price freezes are ok for one industry and not for others. Economic theory is reasonably proven that price controls cause supply failure in markets – Venezuela is an absolute perfect example at the moment.
There could be an argument for freezing rail prices given the large government subsidy to the railway and the monopoly position most rail companies enjoy. It is after all a totally different market due to the network inflexibilities.
One remembers the very large increases in rail fares under the Labour government. And council tax, and national insurance, and petrol taxes and Gordon's beloved stealth taxes. Do you remember the tax?
I am also concerned that the anti-markets mood seems to be winning and gaining ground at the moment. Do we want to be like North Korea? Do we want to emulate Greece? It seems to be that many people do. Why do people think a return to the days of British Rail is a good idea? It was a disgraceful service. Yet people also seem to favour nationalising energy companies, water companies and various other industries, How has this gained ground? How has Thatcher's revolution started to become degraded?
Where are Conservative ministers when it comes to explaining and promoting the economic theory behind market privatisation, who is promoting the benefits of free markets? Who is defending capitalism? I do worry sometimes that there is a vacuum of leadership.
And how are UK businesses going to pay to give employees 3 days off to go volunteering?!
Finally for this week, the polls.
Yes I know polls come and go but yesterday's were fairly unedifying for a Conservative. One poll had Labour at 37% to a Conservative 31%. Two polls also had Labour ahead – two had the Conservatives ahead by a point – but unlikely to be enough to win the election – we know the electoral system is biased towards Labour as it stands.
But should I be bothered?
Whoever wins the next election is going to have a very tough 5 years.
The European Union will feature prominently next term, especially if the Conservatives win, but Labour will not be able to brush this under the carpet. It could again tear the Conservative party apart, but ignoring it could cause huge problems for Labour, especially in working class territory.
Immigration will continue to be high (until Labour's next recession) – the NHS will need more money pouring in and there will be more problems in the health service on occasion. And housing – is anyone going to build the houses we need?
The great bear to our east under Putin will continue to prod and poke us. Who know's what Russia's next move is. What is the next government going to do? Who is going to stand up to Russia? Not to mention ISIS and the next wave of terrorists, whom have not formed yet. And what about if Argentine invade the Falklands? Or what if, heaven forbid, Saudi Arabia came under ISIS control?
And then of course the economy. How are we going to handle the next recession with this great big debt around our necks? Because it will come. Despite what Labour promised in the past, boom and bust has not been defeated and never will. The economy works in cycles and I will be shocked if there is no form of recession between 2018 and 2021.
But when you have a debt as large as ours and a deficit that not even the Conservatives have been able to control – who is going to be the one to make massively difficult decisions in the face of panicking markets (by the way there are already nerves in the gilts markets by the prospect of an uncertain election).
With a debt our size then privatising the NHS might have to happen. Or massive cuts to it. Or huge cuts for pensions. The economic situation is still fucking serious. We need to be running a budget surplus soon.
So maybe us Conservatives should just sit back and let Labour deal with all the problems.
Maybe it is about time they cleared some of their own mess up for a change. About time they got all of the unpopularity for making difficult decisions.
I really am sick to the back teeth of socialists lying, deceiving and denying what went wrong. Denying their involvement, nay, creation of the greatest recession in 80 years. Labour are the party of economic irresponsibility and they piss me off so much, How dare they destroy my country?
Plus many of us Conservatives are not 100% reconciled to David Cameron. He makes a decent leader and I fully support his leadership. But I do really want Boris as leader, He would be an inspirational leader. He would really take our country forward and I feel that he would be able to deliver a two-term Conservative majority. He has that common touch.
Clearly though it would be selfish to not want to finish the job of repairing Labour's mess.
Historically the Conservatives have to pick up the pieces from Labour and recreate the country – just look at the United Kingdom prior to Margaret Thatcher – known as the sick man of Europe, and begging to the IMF for funding. Controlled by the unions instead of the elected governments. It was a shambles.
It is our job to fix the mess. To restore the pride of the country. To ensure we return as many people to employment as possible. To ensure our country has a future.
The United Kingdom needs a Conservative government,
← Milimania → Those That Are Too Bloody Lazy To Vote…

redpajama_set_name: RedPajamaCommonCrawl


I like a room with a view; a breakfast nook with lots of windows and a table set for looking. Not that I'm really looking for anything in particular, but right after I wake up, when the clock says it's time to get started but my body says "you're nuts" I like to stare outback and give things time to warm-up.
I've often sat, as I tried to shake the cobwebs of sleep, and thought what would be the ideal view. It's an individual preference of course. Some folks like a view of the city from high up in their apartment, but a concrete jungle doesn't appeal to me. Water is attractive to some. The river with morning fog or a lake with ducks clears the morning mind.
I like my mountains, perhaps a field or pasture. I like to watch the animals as they search for a morning morsel. I could sit for hours but life's schedule never permits. We live a life of timekeeping, so much so that we identify retirement as freedom from a schedule – liberation.
Well, it's 7:00 a.m., time to get a shower, some breakfast, and then baseball practice. I have a bike ride planned after that and then some chores I've long promised Lisa. It's Saturday and I've a schedule to keep…..
Good read, Jimmy. I have a covered patio myself. Out there every morning, rain or shine, hot or cold. Neighbors think I am a little "strange" when they see me out there in a camo duck hunting coat with steaming coffee and it's thirty degrees. I wouldn't miss it for a minute. Strange how people think they have to have a "schedule". Let the day go as it goes… things will get done and it is far less stressful. Keep at it Jimmy, you bring a lot of peace to those who are seemingly stressed by everyday life. Are you still beating the air into submission for a living? Good on ya!
Yes, actually I flew yesterday. It was my first flight in 2 1/2 years but I'm back in the saddle for a good while now. It was great to get up and see what's changed on Mother Earth.
Wish I had the time for a contemplative start in the mornings. I'm up at 4:15, drive 30 miles and at my desk at 5:30. My parents had a house on Cross Lake in Minnesota while I was home; I'd sure like to live where I can see the water and woods again.
That's about my timeline during the week. I'm up at 4:30 most days, 4:00 on some, and running or on my bike 6:00 – 9:00 for physical training (Army).


We booked a flight from Koln Bonn airport via Vienna to Thessaloniki, Greece- ref MQOLGN for two people.
Cologne, and will have to again pay extra for our luggage??
luggage.This is a very bad experience & a first degree rip-off! I emailed the above complaint last week to [email protected], but have had null response! So now what to do?!
Bag charges are done on an each way / segment basis.
Yes, the above still does not explain anything or help me in any way.
In my view terms & conditions such as 'Eco Light' or 'Full Economy' etc should be clearly apparent at the beginning of the booking form so that travellers are not rudely surprised at the airport!
Pretty much every airline I know with the exception of SouthWest in the US charges extra for bags. Some give a discount if you pay on line before the airport. But they ALL charge extra for bags. I cant see how you managed to miss it when you booked. Did you book on line?
"This has never happened to us with any other flight" - I'd be interested to know what other airlines you fly with that do NOT charge extra for bags.
"It goes without saying that passengers have luggage" that is absolutely not a true statement. Close to 50% of all passengers have carry on bags only.


Members of the Virginia Ship Repair Association (VSRA) strive to provide quality products and services to the maritime industry. The challenge of meeting and exceeding expectations with regard to quality can become more daunting if you are a service provider, because service quality may be intangible and based on a customer's perception.
What criteria might our customers use to form an opinion about a service provider? According to the Handbook for Measuring Customer Satisfaction and Service Quality: "Customers expect a consistent, reliable and timely provision of service, a service provider who possesses the skills necessary to provide the service and an accessible service provider who is polite and respectful, and who communicates with the customer in a language the customer is able to comprehend." The authors of this publication suggest there are six components of service quality: tangibles, service reliability, responsiveness, assurance, safety and empathy.
Tangibles are the physical assets that a customer may see, feel or touch. A good example is replaced structural metal in the fuel tank of the USS Carter Hall.
The service reliability dimension is the ability to work within the time constraints that the customer imposes during the negotiation period. If a decommissioning or dry-docking is scheduled for two months, the customer has a reason for that timeline. Maybe it's based on operating funds available or maintaining a certain amount of fleet readiness. Either way, completing the work within the required timeline will put VSRA members in a favorable position with its customers that will result in more follow- up work.
The responsiveness dimension in service quality is the ability to provide flexible service when job parameters or timelines change. For instance, if the customer decided that they wanted to remove a ship from dry-dock a week earlier or open additional tank work to a company, the VSRA members would have to respond in a collaborative way to meet their customer's needs.
The assurance dimension is based on providing the right materials, worker certifications, paperwork and inspections necessary to prove that the completed work meets the required material and customer specifications and can be used as objective quality evidence. This is a very time-consuming process but one that is necessary to provide the guarantees the customer uses to justify the cost of the repairs.
The safety dimension provides for personnel and asset protection against damage or injury. These aspects are a major concern to our customers' insurance costs and overall sustainability. Not being able to provide proper safety or assurance services provides no customer value and no competitive advantage for the VSRA's members.
The last service quality dimension is empathy. Empathy relates to our understanding of the customer's needs, budgets and responsiveness for the negotiated jobs. Sometimes our customers' scopes of work, budgets or timelines are changed based on national defense measures or emergent problems. We have to understand how the problems affect the customers and collaborate with the customers to provide effective, efficient and sustainable solutions.
By focusing on these concepts and course-correcting as necessary, service providers may be able to enhance how they are perceived by customers.
Eric Strickland is a retired U.S. Navy Chief Petty Officer who spent 20 years in the Navy on nuclear submarines. He earned a Master's in Business Administration (MBA) from Regent University in 2014, and is married with four children. Eric currently works for Advanced Integrated Technology (AIT) as their Safety Manager.
 Eric J. Miller, Ricardo Byrd and David S. Kriger, A Handbook for Measuring Customer Satisfaction and Service Quality, (National Academy Press, 1999).


Jammu and Kashmir government has started the recruitment process for 2014 posts in border battalions.
It adds that the proposal was forwarded to Ministry of Home Affairs, Government of India.
According to a government order No. 1534 dated Nov 6, 2018, Home department in consultation with PHQ has formulated a proposal to raise two border battalions of Jammu and Kashmir Police.
It said that sanction for raising two border battalions of Jammu and Kashmir Police has been granted but the recruitment for the posts should be strictly from the youth living in the areas of 0-10 km from IB/LOC/AGPL/LAC and should also be equally divided amongst the 10 border districts of J&K.
As far as recruitment for the posts from Leh and Kargil is concerned this criteria may be suitably relaxed if sufficient number of youths are not available from 0-10 km of AGPL/LAC/LOC.


text:
No More Drama: Mashonda's New Book Reveals How She Made Peace With Swizz & Alicia Keys After Divorce
The singer stressed that all three adults had to "check their egos" in order to be successful co-parents for their children.
Written By: Kellee Terrell
Source: Getty Images/Daniel Zuchnik/Mireya Acierto / Getty
For Mashonda, a lot has changed in the past decade since she and her ex-husband Swizz Beatz divorced and he married Alicia Keys.
Yes, initially the mother of one was upset and even bitter, but in her new book, Blend: The Secret to Co-Parenting and Creating a Balanced Family, she details her journey of forgiveness and finding peace, while co-parenting her 11-year-old son Kasseem "KJ" Dean, Jr. with the Grammy-winning couple.
The singer recently told PEOPLE that "time" has played a huge role in where they are as a family now.
"Time has a way of healing things, but we also had to heal ourselves," Mashonda shared.
She admitted to the publication that writing this book, "reopened old wounds."
"Many weeks I'd drop my son off at school, come home, hibernate, write and cry."
On Wednesday night (Oct. 3), the three co-parents took to the 92nd Street Y to talk about the book and to share their story as a united front.
Our family is a beautiful example that families can blend and coexist in LOVE!! And now the @theblendbook written by my golden girl @mashondatifere is in the world to share the blueprint of how it works!!! Congratulations to us @mashondatifrere & @therealswizzz !!!!!!! We are living it!! Everybody read it and love it!! 🌈🌈🔥💕🙏🏽💜
A post shared by Alicia Keys (@aliciakeys) on Oct 2, 2018 at 1:42pm PDT
Page Six noted that at the event, Mashonda talked about the importance of the trio "checking their egos" in order to successfully blend their families, which also includes Keys two sons, but it wasn't until Kaseem Jr. had a serious accident while vacationing in Thailand with Keys and Beatz did she feel that they really bond.
Apparently, her son had smashed his finger in a door and required an emergency surgery. While Swizz texted her to let her know of the incident, it was Alicia that gave her constant updates to put her at ease.
I just felt like, 'What if we didn't have that bond?' I would be sitting on the couch not knowing what to do completely lost." the 40-year-old said, thanking her son's "bonus mom." "Moments like that let me know all the work paid off… It was the perfect demonstration of blended."
The women also shared details on their first one-on-one meeting that went down a reserved a table at a quiet restaurant so they could meet in private, Page Six wrote.
"I prayed and I meditated the day before. The day of. Up until the meeting pointing," Mashonda said. "I didn't want to go in with a bunch of emotions … I wanted to see her as a soul."
We love this song! Glad to see that this family is going strong!
Be Yourself! Alicia Keys Is Bare-Faced And Beautiful On Four New ELLE Covers
Swizz Beatz Thinks Mashonda Is Living Her Best Life On The Sidelines
5 Simple Rules For Parenting Blended Families
Celebrating Stars That Wrote Children's Books
17 photos Launch gallery
1. Terrell Owens
Source:	1 of 17
2. Terrell Owens wrote 'Little T Learns To Share'.
3. George Foreman wrote 'Let George Do It'.
4. Jada Pinkett Smith
5. Jada Pinkett Smith wrote 'Girls Hold Up This World'.
6. Spike and Tonya Lee
7. The couple wrote 'Giant Steps To Change The World'.
8. Alex Rodriguez
9. Whoopi Goldberg
10. Whoopi wrote 'Sugar Plum Ballerinas'.
Source:	10 of 17
11. Bill Cosby
12. Garcelle Beauvais wrote 'I Am Mixed' and 'I Am Living in Two Homes'.
13. Will Smith
14. Jay Leno
15. Madonna
16. Monyetta Shaw wrote 'The Adventure of Maddie'
17. Alicia Keys wrote 'Blue Moon'
Continue reading Celebrating Stars That Wrote Children's Books
No More Drama: Mashonda's New Book Reveals How She Made Peace With Swizz & Alicia Keys After Divorce was originally published on hellobeautiful.com


text:
TWO TIME GRAN FONDO WORLD CHAMPION FOUND GUILTY OF DOPING
South African Institute for Drug-Free Sport (SAIDS) has announced that 2012 UCI Gran Fondo World Champion and elite cyclist Lynette Burger, has been found guilty of intentionally violating World Anti Doping Agency (WADA) rules.
Burger won both the 2012 Road and Time Trial UCI Gran Fondo World Championship titles (formerly the UCI World Cycling Tour Finals) when the event was held in South Africa. After winning the she told The Oakley Dispatch, "…to wear the rainbow stripes of world champion is simply the best accomplishment for me."
Burger winning 2012 Worlds
Now a guilty doping verdict has tarnished her accomplishments.
On 1 November 2019, Burger provided an out-of-competition sample after a whistle-blower had alerted SAIDS of potential doping violations.
SAIDS submitted the sample for analysis to the South African Doping Control Laboratory (SADoCoL). Their analysis confirmed the presence of Nandrolone, a banned substance listed in the SAIDS Anti-Doping Rules and the World Anti-Doping Prohibited List.
Nandrolone is associated with dramatic improvements in the levels of muscle mass, strength and endurance in athletes. Its reputation as an incredibly effective anabolic steroid is perhaps only surpassed by its reputation for being easy to detect. It's one of eighteen steroids that every standard doping test screens for.
Burger did not request an analysis of her "B" sample. Though she initially denied using the prohibited substances, she alluded to the possibility that the substance was administered after an accident in 2018.
Legal advisers for Burger argued that she suffered from PTSD, depression, anxiety, and physical pain, as a result of her accident. They further argued that she used the substances to improve her mental state and not to gain an unfair performance advantage.
Comprehensive medical research was conducted by SAIDS to determine if the substances provided any mental health benefits, but SAIDS could not find any medical evidence of Nandrolone improving mental health.
Burger admitted to receiving anti-doping education and the requirement to apply for a Therapeutic Use Exemption (TUE) if taking banned substances for medical reasons. However, Burger never applied for a TUE.
SAIDS rejected Burger's defense and explanations in addition to denying her request for a reduced sanction. The 40 year-old cyclist received a four-year ban effective from 26 November 2019 and may return to competition on 26 November 2023.
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According to the PwC Healthcare Research Institute, two of the top 12 industry impacting trends for 2018 are the increasing investments in and development of artificial intelligence and the security surrounding the many connected devices in healthcare facilities, also known as their "internet of things" or IoT.
Given that a breach into a facility's artificial intelligence or IoT could mean access to HIPAA protected patient information and organization finances or even grant control to connected devices and machines vital for the wellbeing of patients, healthcare IT and cybersecurity services have never been more vital.
It stands to reason then that if healthcare IT companies are needed more than ever to reduce the risk of potentially devastating network breaches, they need to proactively partner with an agency experienced in health IT public relations.
An experienced health IT public relations agency will understand the nuances of the industry, be able to speak to the right audiences, and ultimately perform two critically important actions for the IT business.
Positive PR and top-of-mind brand awareness takes time to cultivate, and a good public relations agency knows how to gain momentum while operating within niche healthcare media markets. Through press releases and pitching stories to the media, the agency can position your IT business as an expert in the field, work toward or reinforce a positive brand image and help generate new business leads as a result.
No one likes the word "crisis," but when a network fails or is breached under the watch of your health IT business, a crisis of leaked information may realistically come to fruition. To err is human, and since even the best security and IT services are still put in place by humans, mishaps are eventually inevitable. However, a PR agency can work with your IT business to put a plan in place for those mishaps, and the plan will establish an easy to follow set of guidelines for remaining calm and getting through the situation with professionalism and grace.
Considering the evolving needs of the healthcare industry and the increasing responsibilities of health IT companies, we highly recommend finding an agency that will help you increase brand awareness, build and "bank" positive PR, and quickly react when you need crisis-assistance the most.
To learn more about what health IT public relations can do for your business, contact Ripley PR at 865-977-1973 or visit www.ripleypr.com.


text:
Allegheny Health Network Innovates IV Medication Management with IV Robotic Technology
Podcast Episode 4
Like many health systems, Allegheny Health Network was challenged by the cost, compliance, and complexity of compounding sterile products (CSPs). Learn how Allegheny Health Network innovated its CSP program using advanced robotics and expert technicians, leading to enhanced safety, improved practices, and cost savings.
Host: Ken Perez, Vice President, Healthcare Policy and Government Affairs, Omnicell
Guest Expert:
Arpit Mehta, PharmD, MPH, MHA, Director of Pharmacy, Allegheny General Hospital
Arpit Mehta, PharmD, MPH, MHA
Director of Pharmacy,
What were some of the supply chain issues you experienced with compounded sterile products (CSPs)?
Arpit Mehta: Similar to other facilities across the country, we outsourced a lot of our ready-to-use medications from 503B vendors, primarily critical care drips, such as norepinephrine, phenylephrine, and vasopressin. Also some OR syringes like ephedrine, neostigmine, phenylephrine, succinylcholine, and others.
The challenge was balancing demand with inventory on hand to reduce waste.
For example, during the winter months, our vasopressin and norepinephrine usage would increase significantly but we couldn't get the supply that we need. We would compound the medications in our clean rooms, but were constrained by beyond-use dating. This prevented us from putting medications at room temperature in automatic dispensing cabinets or anesthesia carts.
How did these supply chain issues impact operational and clinical decisions?
Arpit Mehta: This became a huge inventory management concern. It was a constant struggle to keep up with the demand that we had. Certainly, shortages played a huge role in in hindering our day to day operations—trying to balance inventory versus what we needed, when we needed it.
When our 503B vendor went out of business, we switched to another 503B vendor. That company also ultimately went out of business.
I would go to our hospital nursing huddle on a daily basis and have to discuss the shortages. I remember seeing the look on their faces when I told them, "Today we have fentanyl, tomorrow we have dilaudid."
We knew we needed to get ahead of the problem, that we needed to get control over our inventory. After conversations with the VP of Pharmacy and the hospital CEO, we started exploring solutions.
What other challenges did you uncover during your analysis?
Arpit Mehta: It's important for us to have standardization in our cleanrooms, so that we are consistently using the same compounding processes, not only in the hospital, but across the health system. That we follow the same beyond-use dating procedures, completing all documentation the same way, and so forth. Standardization allows us to run reports and do data analytics on the back end to improve efficiency, and to ensure safety and quality for all preps across the network.
Why did you choose the IV robotics service solution?
Arpit Mehta: We were struggling to fill our regular technician positions. We didn't want an automation solution that required us to rotate a group of technicians through to learn the technology and to compound the medications. We decided on the Omnicell service program that provides the staff to help run the robotics compounding program.
The experience has been amazing. It really helped us focus, prioritize, and have technicians dedicated to the robot. They know the robot inside and out. They know the capacity, they can push the capacity to the limits. It's helped us really expand the program quite a bit.
How did your operational processes change with IV robotics?
Arpit Mehta: "If you don't measure, you don't know." That's the challenge with manual compounding – there's no way to measure accuracy. With our automated solutions, each prep is measured with gravimetric / volumetric verification. It gives us 100% confidence that everything we compounded in cleanrooms is accurate.
Another key is having a dedicated cleanroom supervisor and a well-trained team. It's helped us to ensure appropriate processes are followed for checking the medications for robot compounding, for the quarantine process, for controlled substance management, and for tracking all production, minimizing waste, and things of that sort.
What has IV robotics solution enabled you to do?
Arpit Mehta: The operational benefits have been tremendous. We're saving a great amount of time because we no longer are trying to figure out what we have on hand and what to compound. Just the shortage management piece has been huge. We've been able to produce what we want, when we want, and to reduce waste.
From a financial perspective, it's been a huge success for us. We used to spend about $2.5 million with 503B vendors. By insourcing with the robot and service technicians, just in the last year—and really, it's only six months of production—we've saved almost $700,000. And that's after paying for the robot and service.
Clinically, it's great because we don't have to decide whether we were going to use fentanyl today or dilaudid. The clinical team can make that decision, without operational challenges dictating that patient decision.
The Future of Pharmacy Podcast is produced and distributed by Pharmacy Podcast Network. The views and opinions expressed in this podcast are those of the authors and do not necessarily reflect the official policy or position of any other agency, organization, employer or company. Assumptions made in the analysis are not reflective of the position of any entity other than the author(s). These views are always subject to change, revision, and rethinking at any time and may not be held in perpetuity.
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text:
Q: Twitter Collection Grid does not work AngularJS I am trying to use a Twitter Collection Grid (like this: https://dev.twitter.com/web/embedded-timelines/collection) on the site that I'm building, using an angularJS directive. The widget shows on the site but the grid does not work. Instead of showing in a grid formation the pictures stack on top of each other, all with the width of the widget. Resizing the widget does not work for getting them to show as a grid. The weird thing is that some times when I reload the page it is working but I cannot get it to work consistently. Edit: it loads the right way like 25% of the times... i counted...
This is the html I'm using:

  <twitter-collection></twitter-collection>

And this is the directive:
.directive("twitterCollection", [ function() {
  return {
  restrict: 'E',
  template: 'Tweets with pictures',
  link: function(scope, element, attrs) {
    function run() {
      (! function (d, s, id) {
        var js, fjs = d.getElementsByTagName(s)[0],
          p = /^http:/.test(d.location) ? 'http' : 'https';
        if (!d.getElementById(id)) {
          js = d.createElement(s);
          js.id = id;
          js.src = p + "://platform.twitter.com/widgets.js";
          fjs.parentNode.insertBefore(js, fjs);
        }
      }(document, "script", "twitter-wjs"));
      console.log('run TwitterCollection script');
    }
    run();
   }
  };
}])

Have someone experienced this problem? Or just gotten the Twitter Collection Grid to work and how did you do it? Or could someone help me find out why this is not working? Thanks!

A: Got it to work by ditching the directive and just going with html, like the code below. Now it shows in a grid formation every time i reload.

  A Collection on Twitter

The reason I tried to use a directive for showing the collection is that the pure html code (that you get from twitter) for a "normal" timeline widget did not work properly. When you navigated to another page of the site and then back again the widget did not reload. Therefor I use a directive for the timeline, and that works.

A: you can use that :
customized twitter feed script - `https://jsfiddle.net/reimeister/9x9fc/`

https://github.com/StanScates/Tweet.js-Mod
hope is helping you


Let say you've never had a problem with online activity with your tween, and she is very mature for her age (you think). Now, at the tender age of 11, she desperately wants to have a Facebook account. All her friends have one, which is never reason enough to do something in the parenting realm, and you know the "official" age limit is 13, but you're inclined to agree. Is there a way to do it safely?
Set her account to "private" and see that it stays that way.
Have an in-depth discussion about what is appropriate to post, and which friend requests to accept.
Ensure that she does not post personally identifying information, including address, phone number or too many photos.
She should not post photos of her friends without their permission.
She shouldn't post negative comments about other people, ever.
She shouldn't "check in", or do anything else to reveal her exact location. You should also turn off the GPS settings on the phone's camera.
She should only post things that make her look like a fine, upstanding young lady.
Next, go to the Privacy Settings page on her account (Click on the Lock Icon at the upper right, then click "See More Settings"), pictured at right. For every section where "Friends" is an option, choose that. Don't choose "friends of friends", "public" or "everyone".
If you've followed the list above, you're in good shape. You've made all the right moves, except for maybe one thing.
You should look from time to time and see how many Facebook friends she has. If it's a large number, or increases quickly over a short period of time, some aspects of her online activity may require your attention.
In real life, the definition of the word "friend" is pretty straightforward. You know your child's friends when you see them, literally. In real life, unless there's a serious problem, kids are encouraged to interact with their friends. In the age of social media, the online definition of friend is not so clear.
By a large number of friends, we mean any number that is much greater than the number of real world friends she has. According to Pew Internet Research, the median teen Facebook user has too many Facebook friends – 300 on average, and 20% have more than 600. That could be a problem.
Why is it a problem? Let's say your daughter is a Facebook Friend Collector, and is in the 600+ friends category. Since there is no way she knows all of those 600 people in real life, you might want to ask her why she has so many online "friends". If she and her friends are treating their online life as if it's a popularity contest, they may be doing other things that aren't altogether safe or healthy.
Popularity is a common goal for teens, but until now, hasn't really been quantifiable. Now with the help by social networks it can become a game, and an area of too much focus. Without any other red flags there may be no issue, but be aware that she may be doing other riskier things online to gain popularity.
There is a safety issue that comes with having too many friends as well. A network of 600 people is absolutely not private, as her "friends" are the ones who have access to her personal information, posts, photos and who can privately message her. Even if you are Facebook friends with her, you don't know what she is sending/receiving via private message. If she is being cyberbullied on a private Facebook account, the bully will be one of those "friends".
Facebook would like to think that is has changed the definition of "friend", but they haven't. The closer your teen's friend group online resembles her friends and family in real life, the safer she will be.
Again, we don't recommend ignoring Facebook's official age limit, but if you do with your tween, take steps to make sure she stays as safe as possible.


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You are here: Home ∼ Honoring 'the Preacher's Preacher' after 65 years
Honoring 'the Preacher's Preacher' after 65 years
jock February 20, 2013
By Brian Walker
Carrboro Commons Staff Writer
Despite the near freezing temperatures and wintery mix falling from the sky, it was warm inside the First Baptist Church of Chapel Hill the morning 88-year-old the Rev. John Ruffin Manley accepted the honor of being named Pastor Emeritus for his years of service to the church and community.
Rev. Manley accepts the title of Pastor Emeritus from Rev. Harris, Deconess Trevaughn Eubanks and Deacon Catharyne Butler.
"When you can come through the snow and the rain like you have today, that sure means we're on our way," Manley told the audience after accepting the position.
The position of Pastor Emeritus is an honorary title generally given in retirement to recognize an individual's work in the church over the years. It is intended to show the church's love for its pastor and holds no official duties or responsibilities. This marks the first time the title has been given to a pastor at First Baptist Church of Chapel Hill.
Manley served as the church's pastor for 65 years before retiring in 2011. Aside from his work with the church, Manley also became an important figure in the community, helping to guide members of his congregation though all aspects of their lives.
"He's a very unique individual," the Rev. Norris Trice, an associate minister at First Baptist, said. "Wisdom, leadership; you name it. We would refer to him as 'the Dean' when it comes to many things in our lives. You could go to him and he would get you on the right track."
"He baptized me, he ordained me, preached at my mother's funeral, preached at my father's funeral…he did my marriage. He was a daddy, he was a father," Trice said.
Manley was born in Murfreesboro in 1925, the second oldest of six children. He graduated from Shaw University in Raleigh, and was later awarded an honorary doctorate. Additionally, he attended UNC-CH and earned a master's degree in theology from Duke's Divinity School. He is the father of one son, J.R. Manley Jr., a grandfather of two, and a great-grandfather of four.
The Rev. Chancy Rudolph Edwards, former president of the General Baptist State Convention of North Carolina and a fellow Pastor Emeritus in Fayetteville, spoke at the ceremony about the first time he saw Manley on the campus of Shaw University.
"I saw this young man on campus with his head high, with his shoulders erect walking across the campus," Edwards said. "Anybody who saw him move about the campus knew that he was a serious student and that he was on his way somewhere."
Edwards wound up being right, of course; Manley was on his way. Manley would later become president of the General Baptist State Convention of North Carolina; vice-president of the National Baptist Convention; president of the local Interdenominational Ministerial Alliance of Chapel Hill-Carrboro; president of the local chapter of the NAACP, and was one of the founders of the South Orange Black Caucus, to name just a few of his many accomplishments.
Manley was also the first African-American to be elected to the Chapel Hill-Carrboro City School Board of Education and founded the First Baptist and Manley Estates in Chapel Hill, which provides affordable housing for low-income seniors.
"Dr. Manley felt that his duty and his calling was to love people, and in loving people he became involved in this community, and I mean involved," Edwards said. "It was in this community where he put down his bucket, and he found not only water but he found himself some gold."
Rev. J.R. Manley addresses the audience from the pulpit at the First Baptist Church of Chapel Hill. "God knows there is a good way ahead when people love each other and love the Lord," he said.
And it nearly didn't happen at all. When Manley first arrived in Chapel Hill, he was not given the warmest of welcomes.
Manley already had a job preaching when in 1946 when he was asked to come preach at First Baptist. However, when he arrived, Manley found that another preacher had come as well who wound up preaching instead of Manley.
"I said, 'What in the world have I gotten myself into?' Afterwards I told them 'If you want me to come back I will," Manley said, even though at the time, the church he was preaching at near Greensboro had offered to send him to school for free.
"The problem with that was their headquarters were right across from Bennett College where all those pretty girls were and that's too much temptation for a preacher, no question about that," Manley said, laughing.
So when First Baptist called again, Manley decided to give Chapel Hill another shot. "I said, 'Well the Lord knows best,' and so we've been right here ever since."
Manley said Chapel Hill has supplied him with plenty of work over the years.
"This building (on Roberson Street) was not built here. I built this building and I built another building, the Hickory Road Baptist Church, where I pastored (simultaneously) for 46 years," Manley said. "So it's been one thing after the other. I haven't had to look for any work; it just looks for me."
"Dr. Manley can preach," Edwards said. "Anybody who can hold down two station churches as long as he did, that meant he could preach. God gave him the strength to do two station churches preaching twice a Sunday to different congregations for many years, but John Manley didn't wear out, he just kept on preaching…he preached the gospel so that men and women boys and girls might be saved. That's the primary reason for it all."
Besides ordaining Trice, Manley has licensed and ordained a dozen members of his congregation over the years.
"Most of us can talk about him as our spiritual minister, as the one under whom we accepted our call," said the Rev. Josephine Harris, the church's interim pastor. "Many ministers in the local ministry see him as the preacher's preacher. He's really there to provide that mentoring; be it spiritual be it theological, or be it son-or-daughter-I'm-giving-you-advice kind of stuff; just nurturing you spiritually and keeping us in our lane."
"Definitely keeping us in our lane," Harris added, laughing.
"I thought I'd heard and seen it all, but I tell you right now I feel as if I'm in a dream," Manley told the audience after accepting the position.
"I know you have a lot of people calling on you but don't send them to me, send them to God," he joked.
At the reception after the ceremony, Manley said the day had been "beautiful, superb…I couldn't have dreamed of anything better," and then added with a glimmer in his eye, "Now I'm looking to take it easy for a while."
Posted in Church and Community, History


text: Q: Masking the input to model as a graph I was wondering if it is possible to mask the input in order to get attention like in GNNs. Specifically, if there was a way to send an attention mask in 2D we could tell a token to attend just to its neighbours and not the other tokens. Is this behaviour possible for example using a pretrained language model from Huggingface?


Kroger is making it easy for customers to fight hunger in the Blue Ridge region in September, Hunger Action Month™.
Kroger is partnering with the Blue Ridge Area Food Bank and the Great American Milk Drive to invite shoppers to Feed a Childhood by buying a $1, $3 or $5 milk voucher donation at checkout. All donations collected will help local families get the milk they need.
Milk provides nine essential nutrients, including 8 grams of high-quality protein in each 8-ounce serving.
Now in its fourth year, Kroger's annual Hunger Action partnership with The Great American Milk Drive delivers nutrient-rich milk to the Blue Ridge Food Bank. Over the last three years, Kroger shoppers have donated more than 4 million servings of milk to children and families in need across the country.
The Great American Milk Drive has delivered more than one million gallons—16 million servings—to food banks across the country. To learn more or donate online, visit milklife.com/give. By entering your zip code, you can ensure that the milk is delivered to a local food bank in your community.
America's milk brands and dairy farmers are dedicated to fighting hunger in our communities. Through this partnership with Feeding America, the dairy industry is committed to providing 50 million servings of milk to kids and families in need by 2020.


Computer security is difficult (maybe even impossible), but imagine for a moment that we've achieved it. Strong cryptography where required; secure protocols are doing whatever needs to be done. The hardware is secure; the software is secure. Even the network is secure. It's a miracle. Unfortunately, this isn't enough. For this miraculous computer system to do anything useful, it is going to have to interact with users in some way, at some time, for some reason. And this interaction is the biggest risk of them all. People often represent the weakest link in the security chain and are chronically responsible for the failure of security systems. – Bruce Schneier (in Secrets & Lies book).
I agree with Bruce (well, it's difficult to disagree with him on cyber security affairs anyway). People – the end user and the security practitioner alike – represent the weakest link in the security chain. When they work well together, the organization is better for it. When they don't, it's worse off. Of these two classes of employees, however, the cyber security practitioner has the duty of strengthening that weak link through prompt action and well informed decision making when required.
FUD is short for Fear, Uncertainty and Doubt. In cyber security, a FUD guy is a fall guy in the making. That title is not cool so I don't think anybody wants it. Apathy is on the other end of the scale. The apathetic cyber security practitioner, on the other hand, is popular because s/he tends to "let things run". As a professional, how much of a struggle is this for you? Do you always scream "time out" or do you always want the business to run by default?
Any one of FUD and apathy is a sin.
The most important (and arguably, the most difficult) aspect of cyber security is risk-based decision making. If you're guilty of constantly rushing your decisions then chances are high that you're either a FUD guy or an apathetic professional.
The FUD guy says "there's a problem" almost every time while the other guy hardly ever sees any problem. One of the things I learned in the early part of my cyber security career is how unhealthy false negatives and false positives are. This lesson comes to mind each time I think of these two categories of professionals.
False Positive: The FUD guy is like an intrusion detection system (IDS) that lights up like Christmas tree, spewing up thousands of high priority alerts on daily basis. Analysts typically find most of these alerts to be mere noise, which are then suppressed through various tuning techniques. This is a false positive situation. Here the security tool falsely sends the kind of alert that the analyst is concerned about to the console. This results in waste of valuable time as the analyst needlessly gets busy doing so much without getting any meaningful thing done.
False Negative: The apathetic guy, on the other hand, is like an IDS that fails to do its primary job thus denying an analyst the much needed visibility into events of concern. In a false negative situation, the adversaries' activities fly under the radar of the security analyst because his/her trusted tool fails to do its job.
Knowing when to flash the red or green light is a function of several factors, chief among which are the professional's level of experience, the endangered asset, corporate culture, and the organization's appetite for risk.
The FUD guy presses the panic button nearly every time a security issue is identified and tries to scare people into actions that may introduce bigger risk while his/her apathetic version hardly ever sees a problem in the identified issue. Both these professionals commit the same sin of not duly considering aforementioned decision making factors.
In Andrew Jacquith's Security Metrics book, he wrote about the Hamster Wheel of Pain as thus (paraphrasing): identifying security issues is easy, because that is what highly specialized, domain-specific diagnostic security tools are supposed to do. Quantifying and valuing risk, however, is much harder because diagnostic tool results are devoid of organizational context and business domain knowledge.
What is the value of the asset at risk to the business and the bad guy alike?
What other mitigating controls are in place and how effective are they?
What could go wrong should the realistic worst case scenario come to fruition?
What is the cost (in time, money, human resource) of the proposed control in relation to the realistic worst case scenario?
What other risks could be introduced if we treat this one? Are they higher or lower?
While the foregoing list of questions is not exhaustive by any stretch, it represents a good starting point for the practitioner to arrive at a well informed and carefully thought out decision point.
One thing to always bear in mind is that as a security practitioner paid to keep an organization secure (enough), senior management relies on you to make the right call in the best interest of the organization. If your priority is to be popular always, then you're in the wrong field.
Regardless of your position in the field – whether you're an associate or a CISO – you're a leader simply because you're relied upon. More often than not, the calls you make will be unpopular in most quarters but as long as you fully understand why you're making a call and can duly justify it, you would have been earning your keep.
To Cloud or Not to Cloud? Meltdown and Spectre Flaws: Time to Activate Panic Mode?


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If you aid and abet terrorists...
Dec. 13, 2012 8:34 am
By louisehartmann
You'll end up dead in a drone strike or indefinitely detained in Gitmo. But, if you're a big Wall Street bank and you finance terrorist activities, then guess what? You get a slap on the wrist fine and then…well…that's it.
Now that Governor Rick Snyder signed Right-to-Work-for-less...
Into law in Michigan, the focus shifts to how working people can overturn the legislation. Unfortunately, Republicans purposefully designed the law to make this task more difficult. By attaching Right-to-Work-for-less to a budget appropriations bill, Republicans made it harder for Michigan voters to repeal the law down the road. According to the Michigan Constitution, voter referendums do not apply to budget appropriations bill.
Massive labor protests are expected in Michigan today...
As Republicans lawmakers and Republican Governor Rick Snyder give their final approval to the state's new right to work for less law. Michigan will join 23 other states in the nation that have gone right to work for less – but it is by far the most unionized state to suffer this fate, which should be very troubling to the organized labor community.
Republicans just tipped their hand...
They're going to use the debt ceiling to hold senior citizens on Medicare hostage. In an interview with Fox News on Sunday, prominent Senate Republican Bob Corker said Republicans plan to use the upcoming debt ceiling debate as leverage to force cuts to insurance programs like Medicare.
Ground zero in the labor battle has moved to the Wolverine State
Dec. 7, 2012 2:43 pm
In a sudden move on Thursday, Michigan's Republican Governor Rick Snyder called on his allies in the state legislature to immediately introduce and pass a Right-to-Work FOR LESS law in the state. These laws have been passed in a number of states around the nation allowing union members to opt out of paying union dues, which cripples organized labor and turns workers against each other.
If at first you don't succeed, bribe, bribe again...
Dec. 6, 2012 9:35 am
The Republicans' top oligarch, foreign casino magnate Sheldon Adelson, is already plotting how he can buy the next election and protect himself from criminal prosecution. Adelson spent $150 million on Republicans in the 2012 election, more than anyone in any American election in history.
Paranoid delusions swept across the floor of the United States Senate...
On Tuesday as a group of Republican Senators blocked ratification of a UN treaty to help disabled people around the planet. The Convention on the Rights of Persons with Disabilities requires signatory nations to provide for disabled populations in the same way the 1990 Americans with Disabilities Act does. The treaty would require absolutely no changes to current U.S. law. It was originally signed by President George W.
Hey GOP... In case you hadn't noticed, YOU LOST!!
Someone needs to remind Republican Speaker of the House John Boehner and his Party that they lost the election in November. On Monday, House Republicans presented their proposal to avoid the so-called fiscal cliff and – as you'd expect – it's a giant kick in the gut to working Americans.
Is fracking responsible for killing off an alarming number of livestock around the nation?
Authors of a new report looked into 24 different case studies in six different states where hydraulic fracking is taking place, to find out why livestock is getting sick and dying. Their conclusion: it's the fracking chemicals! For example in Louisiana, the study found 17 cows that died after being exposed to spilled fracking chemicals for only one hour.
President Obama fired the opening salvo on Thursday...
Nov. 30, 2012 8:29 am
In negotiations over the so-called "fiscal cliff." In a plan presented to Republicans by Treasury Secretary Tim Geithner, the President is asking for $1.6 trillion in new revenue by letting the Bush tax cuts expire on the richest 2% of Americans, and by increasing capital gains and estate taxes, which are mostly paid by very wealthy Americans like Mitt Romney.
Who rejected United States-North Korea peace talks?
There were conflicting reports on Sunday regarding a recent proposal for United States-North Korea peace talks which was allegedly made before North Korea"s recent nuclear test
U.K. Pound Falls As Markets Get Brexit Jitters
Bloomberg said on Monday the pound had sustained its biggest fall against the dollar in 11 months
Clinton: I'll defend Israel but push for 'two-state solution
Hillary Clinton believes both Republican candidates Donald Trump and Ted Cruz "missed the mark" with their approach to the Israel-Palestinian Arab conflict
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Great comebacks if someone calls you a loser
When a hater calls you a loser, don't listen and don't let it define who you are. We could all be called a loser anytime we are going through a hard time or trying something new.
Michael Jordan, Walt Disney, Steve Jobs and Tony Hawk and many others have all felt like losers during moments in time when things weren't going well. They have all had ups and downs in their life when they were at the top of their game and other times they hit rock bottom. I'm sure J. K. Rowlings wasn't feeling super sparkly when she was on welfare writing Harry Potter.
If a hater insults you and calls you a loser, shut them up with one of our great comebacks.
Vote for the best comeback when somebody calls you a loser
The zoo called, you are due back by 6.
Looks like your mama had an epic fail. YOU!
Whoever told you to be yourself gave you really bad advice!
I'm not a Proctologist but I know an asshole when I see one.
Gee, I have always wondered who was in charge of deciding who is, and who is not a loser ...
More snappy comebacks you might like
Comebacks if someone calls you ugly
Great replies if you are called fat
More bully comebacks
Famous Skateboarder Tony Hawk went through hard times when he must have felt like a loser
Tony Hawk started skateboarding at a young age, by the time he was a high school senior he was earning more money than some of
By Tinou Bao – originally posted to Flickr as Bob tony hawk, CC BY 2.0, https://commons.wikimedia.org/w/index.php?curid=6011911
his teachers from sponsorship royalties.He even managed to by his own house before graduating from high school. During the 80's, he traveled around the globe living an amazing life, competing in skateboarding contests.
In 1991, the sport of skateboarding tanked and all of a sudden the sponsorship money disappeared, along with Tony's income. He gave himself an allowance of $5 a day to eat at Taco Bell.
When skateboarding was uncool, many skaters moved on to traditional careers while Tony was hopeful that the skating industry would rebound. Tony refinanced his home and started his own skateboard company Birdhouse Projects. At the beginning, Birdhouse wasn't making a lost of cash and Tony had a very uncertain financial future.
In the late 90's, the popularity of skateboarding surged, and there was Tony Hawk front in centre with his skateboarding company. He was offered many endorsement deals and his company expanded, and he became the legend he is today.
We could all be called losers at specific moments in time when things aren't going well. Don't listen to haters, keep following your dreams and get on with your life.
Once you find your passion, run with it. Ignore what peers or career counselors say. Whatever you pick, as long as you truly love it, get really good at it. That means spending a lot of time at it. Tony Hawk
Got any comments, questions or tips for dealing for dealing with someone who calls you a loser? Share them in the comments below.
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rILEY ok says:
I LIKE ANSWER NUMBER ONE.
Sista says:
Jazzabel herrera says:
I'll just say oh I'm sorry I didn't hear u I thought u we're talking about yourself
Narwhal says:
Speaking about competition… i'm winning.
Navjot says:
I'm not a loser, I just take my time to win, making victory all the more sweeter over punks like you.
That sound good? 😛
'That sound good :P' isn't part of the comeback, btw.
faith says:
here's a better one "I'm a loser? you're the one who keeps losing things"
Bob the builder says:
You can say
The only thing I lose at is being uglier than you
Adam Arnold says:
Your mom lost when she had you
Tanya says:
you're a winner at being a loser
Roast god says:
Nice, I got bullied by my best friends and they just like dumped me. Gonna use this on them!
Myra says:
I'm going to use number 3, it fits me well.
Lillee says:
It would be easier to eat the crap you talk if you would just cut it!
Mahika says:
Here's one: Yep I am a loser at being a loser and you for sure are a winner at being a loser
Bully: Sorry, I don't speak loser.
Me: So.. You don't speak your own language?
Nobody says:
Were you born on the highway? Since that's where accidents happen.