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The apoC-III proteoform containing two sialic acid residues (apoC-III2) has different in vitro effects on lipid metabolism compared with asialylated (apoC-III0) or the most abundant monosialylated (apoC-III1) proteoforms. Cross-sectional and longitudinal associations between plasma apoC-III proteoforms (by mass spectrometric immunoassay) and plasma lipids were tested in two randomized clinical trials: ACT NOW, a study of pioglitazone in subjects with impaired glucose tolerance (n = 531), and RACED (n = 296), a study of intensive glycemic control and atherosclerosis in type 2 diabetes patients. At baseline, higher relative apoC-III2 and apoC-III2/apoC-III1 ratios were associated with lower triglycerides and total cholesterol in both cohorts, and with lower small dense LDL in the RACED. Longitudinally, changes in apoC-III2/apoC-III1 were inversely associated with changes in triglycerides in both cohorts, and with total and small dense LDL in the RACED. apoC-III2/apoC-III1 was also higher in patients treated with PPAR-γ agonists and was associated with reduced cardiovascular events in the RACED control group. Ex vivo studies of apoC-III complexes with higher apoC-III2/apoC-III1 showed attenuated inhibition of VLDL uptake by HepG2 cells and LPL-mediated lipolysis, providing possible functional explanations for the inverse association between a higher apoC-III2/apoC-III1 and hypertriglyceridemia, proatherogenic plasma lipid profiles, and cardiovascular risk.
apoC-III is increasingly recognized as an important contributor to dyslipidemia and CVD risk in the setting of insulin resistance and type 2 diabetes (1). apoC-III is a major protein moiety of VLDL and chylomicron particles, and is also present in HDL and LDL (1, 2). In vitro, apoC-III inhibits LPL, reduces receptor-mediated uptake of triglyceride-rich lipoproteins, and facilitates VLDL production (3–5). In mice, reduced expression of the gene for apoC-III (APOC3) is associated with low plasma triglycerides, whereas APOC3 overexpression is associated with high triglyceride levels (6, 7). In humans, increased apoC-III on lipoproteins is associated with reduced hepatic uptake of apoB-containing lipoproteins and increased conversion of the light and more buoyant LDL to small dense LDL (2). Consequently, high plasma apoC-III levels in humans are associated with hypertriglyceridemia and a high prevalence of pro-atherogenic small dense LDL particles (8, 9).
In addition to promoting pro-atherogenic blood lipid profiles, apoC-III may facilitate atherosclerosis via actions on blood monocytes and vascular cells (10). It enhances binding of LDL particles to proteoglycans and, thereby, their retention in the vascular wall, and stimulates production of inflammatory cytokines and adhesion molecules in monocytes and endothelial cells (11–13). In humans, higher apoC-III concentrations on lipoprotein particles are associated with both atherosclerosis progression and incident coronary artery disease (14–17). Naturally occurring loss-of-function mutations in APOC3 are associated with decreased coronary artery calcification and a reduced risk of ischemic CVD (18, 19). Importantly, APOC3 expression is upregulated by glucose and downregulated by insulin (20, 21). The latter may be impaired in the setting of insulin resistance and diabetes, and together with hyperglycemia, may aggravate diabetes dyslipidemia, further increasing cardiovascular risk (20).
Mature apoC-III polypeptide undergoes glycosylation in the Golgi apparatus prior to incorporation in lipoproteins. The sugar moiety consists of galactose, N-acetyl-galactosamine, and either zero, one, or two molecules of sialic acid, resulting in three major proteoforms, apoC-III0, apoC-III1, and apoC-III2, respectively (22, 23). Sialylation of apoC-III is catalyzed by sialyltransferases, whereas desialylation of apoC-III is mediated by lysosomal neuraminidase (24). apoC-III proteoforms show substantial functional differences in regulating cellular VLDL uptake in vitro (25). The few small studies in humans also indicate distinct effects of major apoC-III proteoforms on metabolic phenotypes (12, 22, 26). The scarcity of available data characterizing relationships between apoC-III proteoforms and human lipid phenotypes is partly explained by the limitations of using the relatively labor intensive isoelectric focusing approach for studies in larger cohorts.
We recently developed a high throughput apoC mass spectrometric immunoassay (MSIA) (27) that can simultaneously measure both major and minor proteoforms of apoC-III, and applied it to a small group of obese youth participants without type 2 diabetes (28). Our results revealed a strong inverse association between the ratio of apoC-III2 to the most abundant apoC-III1 and fasting plasma triglycerides. In the present study, we validate and extend these findings by demonstrating the unique inverse association between the relative abundance of apoC-III2 and proatherogenic plasma lipids in individuals with impaired glucose tolerance and type 2 diabetes participating in two large clinical trials. We also evaluated the effect of a higher apoC-III2/apoC-III1 ratio on VLDL cellular uptake and LPL activity ex vivo, and investigated the relationships of higher relative apoC-III2 with pro-atherogenic lipoprotein subclasses and risk of developing cardiovascular events in patients with type 2 diabetes.
Clinical trial blood samples and data were obtained from participants of the Actos Now for Prevention of Diabetes (ACT NOW) study and the Risk Factors, Atherosclerosis, and Clinical Events in Diabetes (RACED) study (29, 30). Plasma samples for in vitro studies were obtained from a subset of participants in the University of Southern California Obesity Study (28). All studies were approved by the Institutional Review Boards of all study centers and all participants gave written informed consent.
ACT NOW was a prospective, randomized, double-blind, placebo-controlled trial to examine the effectiveness of pioglitazone in the prevention of type 2 diabetes (29). Participants were overweight adults with impaired glucose tolerance and at least one other risk factor for type 2 diabetes who were randomized to up to 45 mg pioglitazone or matching placebo per day. Participants returned every 2 months during the first year of the study and once every 3 months thereafter for up to 48 months, with a mean and median follow-up time of 2.3 years. The present analyses include plasma samples and matching data from the baseline visit and the latest available follow-up visit.
The RACED study included type 2 diabetes patients that were participating at seven study sites of the Veterans Affairs Diabetes Trial (VADT). Briefly, the VADT investigated the effect of tight glucose control versus standard therapy on cardiovascular events in veterans with uncontrolled diabetes, while the RACED substudy evaluated the effect of the VADT intervention on novel cardiovascular risk factors (30, 31). The primary VADT outcome was the time to the first occurrence of any one of a composite of macrovascular events including: myocardial infarction; stroke; death from cardiovascular causes; new or worsening congestive heart failure; surgical intervention for cardiac, cerebrovascular, or peripheral vascular disease; inoperable coronary artery disease; and amputation for ischemic gangrene. The current analysis includes plasma measurements at baseline and approximately 9 months after randomization.
apoC-III proteoforms were measured by MSIA, as previously described (27). After rapid thawing on ice, samples were centrifuged for 5 min at 3,000 rpm and then aliquoted into 96-well microplates and stored at −80°C until measured. In brief, after sample processing and preparation of the polyclonal anti-apoC-III antibody (Academy Biomedical Co., Houston, TX) derivatized pipettes, apoC-III protein was captured from the analytical samples during repeated aspiration and dispensing cycles. Captured proteins were then eluted directly onto a 96-well formatted MALDI target using a sinapinic acid matrix. Linear mass spectra were then acquired from each sample spot using Bruker's Ultraflex III MALDI-TOF instrument (Bruker, Billerica, MA) in positive ion mode. Mass spectra were internally calibrated using the protein calibration standard-I, and further processed with Flex Analysis 3.0 software (Bruker Daltonics). All peaks representing apolipoproteins and their proteoforms, along with a lysozyme control (internal reference standard) peak, were integrated baseline-to-baseline using Zebra 1.0 software (Intrinsic Bioprobes Inc.), and the obtained peak area values were tabulated. To distinguish between noise and low intensity signals, the peak areas were corrected individually with baseline noise-bin signals. The corrected apolipoprotein peak areas were then divided by the lysozyme peak area. Relative abundance of each apoC-III proteoform was expressed as relative peak area (RPA). To reduce the variation due to less precisely captured low-abundant proteoforms, major proteoforms were also expressed as ratios to the most abundant proteoform.
In the ACT NOW study, total plasma cholesterol and triglycerides were measured using the CHOD-DAOS method (WAKO, Richmond, VA) and an enzymatic assay (Stanbio Laboratory, Boerne, TX). HDL cholesterol was measured after precipitation of apoB-containing lipoproteins with dextran sulfate-Mg2+, using the CHOD-DAOS method (WAKO). LDL cholesterol was calculated by the Friedewald equation. In the RACED study samples, standard lipids and lipoprotein subclass concentrations were measured by the Vertical Auto Profile II method (VAP II; Atherotech, Inc., Birmingham, AL), as previously described (32). Total plasma apoC-III concentrations were measured in baseline samples from the RACED study by sandwich ELISA using an anti-apoC-III antibody (identical Academy Biomedical antibody as in the MSIA) and apoC-III protein standard obtained from Academy Biomedical, as previously described (33). The individual concentrations of apoC-III proteoforms were then computed for this subset by multiplying the total apoC-III concentration by the relative abundance of each proteoform.
Plasma samples from overweight and obese youth participants in the University of Southern California Obesity Study (28) with either high (upper quartile) or low (lower quartile) apoC-III2/apoC-III1 ratios were used to isolate VLDL and apoC-III complexes from the VLDL. These plasma samples for VLDL isolation were selected based on MSIAs performed on a total 72 available plasma samples.
Plasma was overlaid with a density solution of 1.006 g/ml (11.40 g of NaCl plus 0.1 g of EDTA-2Na) in a 2:1 ratio. Samples were centrifuged at 100,000 rpm (8°C) for 2 h (Beckman Optima TLX 120.1). The top third layer (VLDL) was isolated and dialyzed for 2 h against PBS solution using Slide-A-Lyzer mini dialysis unit (Thermo Fisher Scientific, Waltham, MA).
Dynabeads (Life Technologies, Grand Island, NY) were conjugated with an anti-apoC-III antibody per the manufacturer's instructions. VLDL (150 μl) was added and gently pipetted to resuspend the Dynabeads-antibody complex and incubated for 45 min to allow VLDL binding. The supernatant was removed and the Dynabeads complex sample was washed three times using 200 μl phosphate buffered saline with Tween-20 (PBST) for each wash. After the third wash, the Dynabeads complex was resuspended in 100 μl PBST. After removal of the supernatant, apoC-III was eluted by adding 20 μl elution buffer [50 mM glycine (pH 2.8)] and gently pipetted and incubated with rotation for 2 min to dissociate the complex. The supernatant containing apoC-III was transferred to a clean tube, and this step was repeated until complete elution of all the protein. The protein was dialyzed against PBS using Slide-A-Lyzer MINI dialysis unit in the cold room.
HepG2 cells were plated in 24-well (15 mm) culture plates in 1 ml of MEM containing 10% FBS and grown for 4 days. At 80% confluence, cells were incubated in MEM supplemented with 10% lipoprotein-deficient serum for 24 h.
Isolated VLDL cholesterol (50 μg/ml) was added to the cells and incubated for 16 h at 37°C in 5% CO2 in the presence of 0.25 units/ml bovine milk LPL (Sigma-Aldrich, St. Louis, MO) (34). The cells were then washed twice with PBS and cell lipids were extracted in hexane:isopropanol. Total triglycerides in the cells were measured using a colorimetric assay and normalized to cell protein concentration.
Twenty micrograms of DiI-labeled VLDL cholesterol were added to HepG2 cells and incubated for 4 h at 37°C. The cells were preincubated for 30 min with 10 ng/ml recombinant human leptin (Life Technologies) for maximized expression of lipolysis-stimulated receptor (LSR) (25). Based on preliminary experiments (supplementary Fig. 1), the incubation was performed in the presence of 0.5 mM oleic acid (Sigma-Aldrich) (35), and isolated apoC-III complexes containing 5 μg/ml of apoC-III were used to inhibit DiI-VLDL uptake. The cell monolayers were washed twice with PBS (pH 7.4). DiI-fluorescence was measured at an excitation/emission of 513/550 nm. The cells were then fixed with 4% paraformaldehyde for 10 min. The DiI-values were normalized by staining the cells with 4′,6-diamidino-2-phenylindole (DAPI) (1:10,000, 10 min) followed by three washes with PBS. DAPI was measured at an excitation/emission 358/461 nm. VLDL uptake was defined as the ratio of DiI to DAPI fluorescence values.
A modification of a previously published protocol was used (36). Dialyzed VLDL was heated at 56°C for 1 h to inactivate any residual endogenous lipase activity. VLDL substrate (2 mM triglycerides) and 30 μg/ml apoC-III were added to the assay buffer [4% BSA, 180 mM Tris HCL, 100 mM NaCl, 1 mM EDTA, 0.01% NaN3, 66.7 μg/ml sodium heparin (pH 9)] with a final total volume 50 μl, which was then preincubated at 37°C on a shaker for 20 min. The dose of apoC-III was based on Wang et al. (37) and our initial studies with pure apoC-III (Academy Biomedical). The reaction was then initiated by adding 2 units/ml bovine milk LPL. At the beginning of the reaction, one tube was placed on ice and served as control, while the other tubes were incubated at 37°C on a shaker for 40 min. The reaction was stopped by placing the tubes on ice. NEFA release was assessed by a colorimetric assay at wavelength 550 nm and normalized to the reading from the control tube.
Depending on data distribution, differences in apoC-III measures were tested by Wilcoxon rank-sum test for two categories, and by Kruskal-Wallis test for three or more categories. Paired Student's t-test or Wilcoxon signed rank test was used to determine differences from baseline within each treatment group. Relationships between continuous variables were evaluated using Spearman correlations. General linear models were used to test the relationships between apoC-III proteoforms and metabolic variables after adjustment for potential covariates, including age, gender, BMI, ethnicity, and use of lipid-lowering therapy. Data were log10 transformed if not normally distributed. All longitudinal analyses were adjusted for treatment assignment. Cox proportional hazards regression was used to determine the association between apoC-III proteoform measures and development of cardiovascular events in RACED in the models adjusted for treatment assignment. To control for cardiovascular risk, a United Kingdom Prospective Diabetes Study (UKPDS) risk engine coefficient representing a composite index of traditional cardiovascular risk factors [age, hemoglobin A1c (HbA1c), sex, blood pressure, race/ethnicity, smoking, and total and HDL cholesterol] was used. The interaction of apoC-III proteoform measures with the randomized treatment assignment was also tested in the Cox proportional hazard model along with the main effects, with P < 0.15 as threshold for stratified analyses. Statistical analyses were performed using SAS Institute software (version 9.2; Cary, NC). P < 0.05 was considered statistically significant.
Baseline plasma samples for measurements of apoC-III proteoforms were available for 531 participants with impaired glucose tolerance from the ACT NOW study and 296 participants with type 2 diabetes from the RACED study (Table 1). ACT NOW participants included more women and individuals of minority race, and were on average more obese than RACED participants. RACED participants were on average older predominantly white males with poor glucose control, and a high percentage were receiving lipid-lowering therapy and had a history of hypertension or CVD.
Consistent with our initial description of the assay (27, 28), 12 apoC-III proteoforms were detected by MSIA in both cohorts (supplementary Table 1). In both cohorts, the most abundant form (RPA) was apoC-III1, followed by glycosylated apoC-III0 (apoC-III0b), apoC-III2, and native apoC-III (apoC-III0a), while other forms were substantially less abundant compared with the four major proteoforms (Fig. 1A, B).
The apoC-III proteoforms' distribution was related to several demographic and clinical characteristics. In the ACT NOW cohort, African Americans had lower apoC-III0b and higher apoC-III2, whereas Hispanics had higher apoC-III0b and lower apoC-III2 compared with non-Hispanic whites (Fig. 1C). In the RACED study, African Americans had higher apoC-III0a and lower apoC-III1 than non-Hispanic whites (Fig. 1D). In both cohorts, apoC-III2 was higher in those receiving lipid-lowering medications (Fig. 1E, F). In the ACT NOW study, lipid-lowering therapy was almost exclusively statins (one participant was on fibric acid). Most RACED patients were receiving statins (n = 149), but a number of patients were on fibric acid alone (n = 24) or in combination with statins (n = 27). The relative abundance of apoC-III2 was higher with use of statins, fibrates, or their combination (median RPAs were 0.14, 0.15, and 0.16, respectively, vs. 0.12 with no lipid-lowering drugs, P < 0.05 for all).
Follow-up plasma samples were available in 362 ACT NOW and 253 RACED participants. In the ACT NOW cohort, relative amounts of apoC-III0a and apoC-III2 were higher, and apoC-III1 was lower after pioglitazone (Fig. 1G). In the RACED cohort, apoC-III2 increased and apoC-III1 decreased similarly in both the standard and intensive glucose lowering groups (Fig. 1H). The increases in apoC-III2 after pioglitazone or after glucose lowering [both RACED treatment groups had significant declines in HbA1c from baseline at 9 months (32)] were the most pronounced (P < 0.05 for all) among the proteoforms (Fig. 1G, H).
At baseline, higher relative apoC-III2 abundance was associated with lower triglycerides and total cholesterol, and higher HDL cholesterol concentrations in both cohorts, and with lower BMI in the RACED study (Table 2). In contrast, relative abundances of other major proteoforms showed mostly positive associations with BMI, triglycerides, and total cholesterol concentrations and negative associations with HDL cholesterol concentrations (Table 2). In the RACED study, apoC-III0a negatively correlated with fasting glucose and HbA1c concentrations (Table 2).
As apoC-III2 demonstrated different relationships with metabolic variables than the most abundant proteoform apoC-III1, we also examined relationships of the apoC-III2/apoC-III1 ratio in plasma. The apoC-III2/apoC-III1 showed a strong inverse association with plasma triglycerides in both cohorts (Fig. 2A, B). In the multivariate analyses, this association remained significant (P < 0.0001) after adjustment for age, gender, BMI, race, and use of lipid-lowering medications. Variation in apoC-III2/apoC-III1 in the ACT NOW cohort also accounted for approximately 23 and 30% of the difference in plasma triglyceride concentrations between African Americans and whites, and African Americans and Hispanics (African Americans, 91 ± 38 mg/dl; whites, 126 ± 59 mg/dl; Hispanic, 132 ± 57 mg/dl; P < 0.0001). Higher apoC-III2/apoC-III1 was also associated with lower total cholesterol and higher HDL cholesterol in both cohorts and with lower LDL cholesterol in the RACED cohort (Table 3).
Longitudinal changes in apoC-III2/apoC-III1 also inversely correlated with changes in fasting triglycerides (Fig. 2C, D) and total and LDL cholesterol in both cohorts, and positively correlated with changes in HDL cholesterol in the RACED cohort (Table 3). The association between changes in apoC-III2/apoC-III1 and changes in plasma triglycerides was independent of changes in body weight and fasting glucose (P < 0.001 in both cohorts). Notably, adjusting for changes in apoC-III2/apoC-III1 appeared to account for 38% of the decline in plasma triglycerides after pioglitazone therapy in the ACT NOW cohort.
The ratios of other major apoC-III proteoforms to apoC-III1 in both a cross-sectional and a longitudinal setting tended to have positive associations with triglyceride levels and less favorable associations with other lipid values (Table 3).
Total plasma apoC-III concentrations were measured in the baseline plasma samples of the RACED cohort. Total apoC-III was not associated with apoC-III2/apoC-III1 (r = −0.06, P = 0.3). Plasma triglycerides positively correlated with total apoC-III (r = 0.19, P = 0.002), and total apoC-III-derived concentrations of apoC-III0a (r = 0.15, P = 0.01), apoC-III0b (r = 0.23, P = 0.0001), and apoC-III1 (r = 0.22, P = 0.0001). In contrast, triglycerides negatively correlated with apoC-III2 concentrations (r = −0.14, P = 0.02). Adjustment for total plasma apoC-III concentrations had no effect on the negative associations between apoC-III2 RPA or apoC-III2/apoC-III1 and plasma triglycerides (P < 0.0001 for both).
LDL cholesterol subclasses were measured in plasma samples of the RACED cohort. Higher apoC-III2/apoC-III1 at baseline was associated with lower LDL1 (largest LDL) and LDL4 (smallest LDL), and higher LDL2 (Fig. 3). After adjusting for plasma triglycerides, apoC-III2/apoC-III1 was still negatively associated with LDL1 (P < 0.0001) and LDL4 (P = 0.002), but not significantly associated with LDL2. Association between apoC-III2/apoC-III1 and LDL4 remained significant after adjusting for lipid-lowering therapy or total apoC-III concentrations (P < 0.0001 for both).
Longitudinally, changes in apoC-III2/apoC-III1 were also inversely associated with changes in LDL1 and LDL4, and positively associated with changes in LDL2 (Fig. 3). Among individual proteoforms, higher LDL4 was associated with lower RPAs of apoC-III2 and higher apoC-III1 both at baseline and longitudinally, and with higher apoC-III0b at baseline (supplementary Table 2).
To examine whether the uniquely favorable associations of apoC-III2 with metabolic risk factors may have an impact on CVD, we explored its association with major adverse cardiovascular events (MACEs; myocardial infarction, stroke, or CVD death) in the RACED cohort. A total 40 MACEs occurred during the 5 year median follow-up.
Among demographic and clinical characteristics, a baseline UKPDS risk score was the strongest predictor of incident MACEs (supplementary Table 3). Incident MACEs were also associated with baseline HbA1c in the whole group, baseline fasting triglycerides in the standard group, and older age in the intensive group (supplementary Table 3).
Incident MACEs trended negatively with both the RPA of apoC-III2 and the apoC-III2/apoC-III1 ratio in treatment-adjusted models (Fig. 4A). In the treatment-stratified analysis, a significant association of apoC-III2 (P = 0.02), and a trend for a significant association of apoC-III2/apoC-III1 (P = 0.06) with incident MACEs was found in the standard glucose-lowering group (Fig. 4B, C). In contrast, total apoC-III or apoC-III1 showed no association with incident MACEs (Fig. 4A).
The association between apoC-III2/apoC-III1 and MACEs in the standard group was significant after adjusting for the UKPDS risk score {hazard ratio (HR) 0.54 [95% confidence interval (CI) 0.32–0.90], P = 0.02}, was slightly weaker after adjusting for HbA1c (HR 0.69 [95% CI 0.45–1.05], P = 0.08) or age (HR 0.66 [95% CI 0.42–1.04], P = 0.08), and disappeared after adjusting for fasting triglycerides (HR 0.88 [95% CI 0.54–1.46], P = 0.6).
To explore potential mechanisms underlying the inverse association between relative apoC-III2 abundance and plasma triglycerides, VLDL was isolated from pooled plasma from nondiabetic participants whose plasma apoC-III2/apoC-III1 was within the upper or lower quartile. VLDL from subjects with a higher apoC-III2/apoC-III1 contained less total apoB, apoC-III, apoE, triglycerides, and cholesterol than VLDL from subjects within the lower quartile of plasma apoC-III2/apoC-III1 (Table 4). The differences in apoC-III, apoE, triglycerides, and cholesterol were less prominent after normalization to apoB concentrations.
HepG2 cells incubated with VLDL from the upper quartile of plasma apoC-III2/apoC-III1 had higher triglyceride content than HepG2 cells incubated with VLDL from the lower quartile of plasma apoC-III2/apoC-III1, indicating increased triglyceride delivery (Fig. 5A).
To further delineate the specific effect of apoC-III on VLDL uptake and LPL-mediated lipolysis, apoC-III was immunoprecipitated from VLDL of both high and low apoC-III2/apoC-III1 plasma pools. The difference in apoC-III2/apoC-III1 between these two pools was confirmed by MSIA (Fig. 5B, C). The apoC-III complex from the upper quartile of plasma apoC-III2/apoC-III1 also showed a more prominent intensity peak of the native apoC-II (Fig. 5B, C).
A previous study by Mann et al. (25) has shown that apoC-III sialylation may influence VLDL uptake via the LSR, a receptor for triglyceride-rich lipoproteins that is activated in the presence of fatty acids. We established an assay for LSR activity in HepG2 cells, measured as DiI-VLDL uptake in the presence of oleic acid, and confirmed that pure apoC-III inhibited DiI-VLDL uptake (supplementary Fig. 1). Using the apo-CIII isolated from VLDL, we found that the inhibition of DiI-VLDL uptake in the presence of oleic acid was less pronounced with the apoC-III isolated from VLDL obtained from those within the upper quartile of the plasma apoC-III2/apoC-III1 ratio, indicating a less inhibitory effect of higher relative apoC-III2 on VLDL uptake, as shown by immunofluorescence (Fig. 5D) and confirmed by quantitative fluorimetric analysis (Fig. 5E).
Inhibition of LPL activity was measured by incubating VLDL with the distinct apoC-III complexes containing 30 μg/ml of total apoC-III. The amount of NEFA released from VLDL was significantly higher after incubation with apoC-III prepared from plasma from individuals in the upper quartile of apoC-III2/apoC-III1 versus apoC-III from the lower quartile, indicating less prominent inhibition of LPL-mediated lipolysis (Fig. 5F).
In two separate large cohorts, we found strong cross-sectional and longitudinal inverse relationships between the relative abundance of apoC-III2 proteoform and plasma triglyceride concentrations. Importantly, the associations were very similar in the two current cohorts and our recently reported cross-sectional study (28), despite differences in their demographic and clinical characteristics (e.g., age, gender, ethnicity, glucose tolerance status, and prevalence of CVD). The association between relative apoC-III2 abundance and triglycerides remained present after adjustment for relevant demographic factors and even lipid lowering medications, suggesting that apoC-III2 abundance may be an independent determinant of plasma triglyceride levels. Furthermore, the inverse relationship between apoC-III2 abundance and plasma triglyceride levels remained whether apoC-III2 was expressed as the relative abundance among all apoC-III proteoforms, the ratio to the most abundant proteoform apoC-III1, or, in the RACED cohort, as the absolute apoC-III2 concentrations. In both cohorts, higher apoC-III2 relative abundance was also associated with a more favorable overall lipid profile, including lower total and LDL cholesterol and higher HDL cholesterol.
Increased prevalence of proatherogenic small dense LDL particles is commonly reported in patients with type 2 diabetes, particularly in those with poor glycemic control (38). apoC-III has been previously shown to facilitate production of small dense LDL (2). The current analyses in the RACED cohort showed distinct associations between relative amounts of apoC-III proteoforms and the smallest LDL4 particles. LDL4 was positively correlated with relative abundances of apoC-III0b and apoC-III1, and negatively correlated with apoC-III2 abundance. Higher relative apoC-III2 abundance was also associated with higher (buoyant) LDL2. The association between the apoC-III2/apoC-III1 ratio and LDL4 persisted after adjusting for plasma triglyceride concentrations, suggesting potentially favorable effects of apoC-III2 on LDL4 production that were separate from the effects on triglyceride levels. Interestingly, the relative abundance of apoC-III2 was negatively associated with the most buoyant, but very low abundant, LDL1. This negative association may reflect preferential removal of the larger LDL1 particles, which are most rapidly cleared, as previously noted in studies with LDL cholesterol-lowering agents (39). Importantly, these associations between relative apoC-III2 abundance and LDL subclasses were generally consistent between cross-sectional and longitudinal analyses, providing further confirmation of the validity of these relationships and suggesting that they may be causally related.
Given these favorable associations between apoC-III2 abundance and the major lipid categories or LDL cholesterol subclasses, we examined the relationship between apoC-III2 and incident MACE outcomes during the VADT study. A reduced hazard ratio for apoC-III2 was most evident in the standard group, suggesting the possibility that improvement in other risk factors or the specific effects of diabetes medications in the intensive treatment group might have masked the association between the apoC-III2 relative abundance and MACEs. Although the number of MACEs was relatively small in this subcohort, we were able to find the expected increase in MACEs with higher values of standard risk factors represented by the UKPDS risk coefficient. Importantly, the association between the relative abundance of apoC-III2 and MACEs in the standard group was independent of the UKPDS risk coefficient, but not of fasting triglyceride concentrations, indicating that this effect was largely mediated by changes in triglyceride metabolism.
Several previous studies showed association between apoC-III concentrations on lipid particles and coronary artery disease (14–17). In the present study, we did not find a significant association between total apoC-III concentration in plasma and MACEs. There are several potential explanations of our results. First, total apoC-III in plasma may be a less sensitive biomarker of cardiovascular risk compared with apoC-III on specific lipid particles. In fact, no studies, to date, have shown a relationship between total plasma apoC-III concentrations and incident cardiovascular events. Second, our VADT subset cohort was modest in size, participants were relatively homogenous in their high cardiovascular risk, and were on multiple medications that could alter either cardiovascular risk and/or total apoC-III concentrations. Third, our data suggest that apoC-III proteoforms vary in their association with, and potential contribution to, plasma lipids and cardiovascular risk. Thus, a measure of total apoC-III in plasma may not reflect the overall risk of all the individual proteoforms.
This is the first analysis of the relationships between apoC-III proteoforms and plasma lipids, LDL-cholesterol subclasses, and cardiovascular outcomes in larger cohorts. This type of analysis has previously been limited by the time- and labor-intensive nature of the isoelectric focusing methodology that was used in previous studies. In two earlier small cross-sectional studies, the concentrations of sialylated apoC-III proteoforms in triglyceride-rich lipoproteins and their production rates correlated more strongly with plasma triglycerides than apoC-III0 (22, 26). The production rate of apoC-III2 also negatively correlated with LDL peak particle size (26). However, in both studies, the regression slopes between apoC-III proteoforms and plasma triglycerides were greater for apoC-III1 than for apoC-III2, indicating a less harmful relationship between apoC-III2 and triglycerides. The reasons for differences between results of these smaller studies and our findings are unclear, but may be explained by differences in study participants and methodologies. Participants in our cohorts demonstrated moderate or advanced levels of glucose intolerance, and had a high prevalence of hypertension and use of glucose-lowering agents. In addition, isoelectric focusing does not distinguish among the many nonsialylated proteoforms. For example, two major nonsialylated proteoforms showed distinct associations with several demographic and metabolic outcomes in our study.
apoC-III inhibits both lipolysis in vitro and cellular uptake of triglyceride-rich lipoproteins (3, 4). Prior in vitro studies indicated that apoC-III sialylation may have a substantial functional effect on both VLDL uptake and LPL-mediated lipolysis (25, 40). Of particular relevance to our study, Mann et al. (25) reported that, compared with apoC-III1 or apoC-III0, apoC-III2 is a less effective inhibitor of VLDL uptake by the hepatic LSR. The LSR has been proposed as a rate-limiting factor for the clearance of triglycerides during the postprandial period (41, 42). LSR heterozygous mice display increased postprandial triglyceride levels, decreased clearance of lipid particles, and increased levels of proatherogenic lipoproteins following a Western diet (35). Silencing of hepatic LSR in mice has also been shown to lead to hypertriglyceridemia (43). We observed an increase in DiI-VLDL uptake in HepG2 cells in the presence of oleic acid that was inhibited by apoC-III. This inhibition was less pronounced with apoC-III isolated from subjects with high plasma apoC-III2/apoC-III1, as compared with that observed with the same amount of apoC-III isolated from subjects with low apoC-III2/apoC-III1. This is consistent with the previously demonstrated effect of apoC-III proteoforms on uptake of triglyceride-rich lipoproteins (25). As the conditions of the VLDL uptake experiments favored the LSR pathway, i.e., including pretreatment with leptin, shorter duration of cells starvation time, and coincubation with oleic acids, these experiments did not permit exploration of other important mechanisms contributing to clearance of triglyceride-rich lipoproteins. These additional pathways include the LDL-receptor (44), LDL receptor-related protein 1 (45), and heparan sulfate proteoglycans (46, 47).
Our ex vivo data also showed less efficient inhibition of LPL-mediated lipolysis for apoC-III with higher apoC-III2/apoC-III1 ratio. This may be in contrast with older studies showing either no effect (48, 49) or greater inhibition of LPL activity (40, 50) with increased apoC-III sialylation. The discrepancy may be due to methodological differences in the isolation of apoC-III isoforms, the effect of neuraminidase used for desialylation in some studies, the distinct action of the two sialylated proteoforms rather than sialylation in general, differences in the amount of apoC-III used in the experiments, and differences in the clinical characteristics of apoC-III donors. The effective amount of apoC-III to inhibit LPL-mediated lipolysis in our study and in some previous studies (37, 40) was also substantially higher than apoC-III concentrations inhibiting VLDL uptake. Thus, it is possible that inhibition of lipolysis by apoC-III may contribute to increased triglyceride levels only in those individuals with very high apoC-III levels.
apoC-III glycosylation may affect metabolism of triglyceride-rich lipoproteins by several mechanisms. Glycosylation alters multiple apolipoprotein properties, including conformational stability, resistance to proteolysis, charge, water-binding, and biological recognition in protein targeting and cell to cell interactions (51). Greater sialylation may stabilize apoC-III complexes with other apolipoproteins, including apoA-II, apoB, apoC-I, apoC-II, and apoE, and thus potentially enhance their effects on lipid metabolism, including clearance of triglyceride-rich particles (33, 52–54). The higher native apoC-II abundance noted in association with precipitated apoC-III from individuals with higher apoC-III2/apoC-III1 ratios may represent one example of such a scenario. It is possible that greater activation of LPL-mediated lipolysis by increased amounts of apoC-II could help counter the inhibitory effect of apoC-III on these events.
There were several other notable findings related to glucose- and lipid-lowering therapy in this study. First, relative apoC-III2 abundance was increased after pioglitazone treatment in the ACT NOW patients. Relative apoC-III2 abundance also increased in the RACED cohort in both treatment groups, and they both received rosiglitazone. This suggests the possibility of a PPAR-γ agonist class effect on apoC-III2, which could be related to their known triglyceride lowering action. The positive cross-sectional association of apoC-III2/apoC-III1 with insulin sensitivity in our previous study (28) indicates that this effect may be related to the insulin-sensitizing action of PPAR-γ agonists. Second, the relative distribution of apoC-III proteoforms was modified by the use of lipid-lowering drugs, and there was evidence of proteoform-specific effects. Whereas the use of both fibrates and statins was associated with lower native apoC-III, the increase in apoC-III2 was more evident with fibrates. Relative apoC-III proteoform abundance also significantly differed between the races or ethnicities. In the ACT NOW cohort, higher relative apoC-III2 in African Americans appeared to account for more than 20% of the differences in plasma triglyceride concentrations between African Americans and both non-Hispanic whites and Hispanics, suggesting a possible contribution of this proteoform to the well-described lower triglyceride concentrations in African Americans (55). Aggressive use of lipid-lowering therapy per protocol in the entire cohort and a smaller number of African Americans may explain why we did not observe significant racial differences in the relative apoC-III2 abundance in the RACED cohort. These observations need to be validated in larger populations where lipid-lowering medication use is relatively low.
Several limitations of the study deserve mention. The study was a post hoc analysis of baseline and follow-up samples from two previously completed studies. Although the studies were well powered to examine associations between apoC-III and both baseline and longitudinal lipid values, the analysis relating apoC-III proteoforms and incident MACEs was not, and must be considered exploratory. We also did not measure apoC-III proteoforms in different lipoprotein fractions, which may permit further insight into their role in lipid metabolism. Samples for these analyses were stored for several years at −80°C before MSIA was performed. Previous investigation of the effects of storage, time, and freeze/thaw cycles on these assays has indicated that the measurements are relatively stable (27). Moreover, despite differences in the storage time of the samples in the two different cohorts, there was a striking consistency in the relationships between apoC-III proteoforms and plasma lipids. Because the apoC-III complexes in our ex vivo studies contained other apolipoproteins, we could not distinguish to what extent the observed effects resulted from modification of apoC-III action per se or effects on the amount and activity of other apolipoproteins. Thus, further studies are needed to identify the effects of individual apoC-III proteoforms. While the RACED cohort showed a weak but significant negative association between absolute apoC-III2 concentrations and plasma triglycerides, our previous study in obese adolescents showed no association between these two variables. The discrepancy may be explained by differences among the cohorts, with the RACED cohort larger in size, comprised of diabetes patients, and having a broader range of triglyceride concentrations. Importantly, the associations of plasma triglycerides with total apoC-III or absolute concentrations of other major apoC-III proteoforms were positive in both the RACED cohort and the previous study, further supporting a different relationship of apoC-III2 with plasma triglycerides.
In conclusion, the present analyses provide the first evidence, from two independent large cohorts, of a strong and inverse association between the relative amounts of a disialylated apoC-III proteoforms and proatherogenic plasma lipid profiles in individuals with abnormal glucose metabolism. These relationships are distinct from those of total apoC-III and other major apoC-III proteoforms, and, together with our ex vivo data, support the concept that apoC-III proteoforms may have different effects on lipid metabolism. Measuring relative amounts of apoC-III variants may add to the risk assessment that can be obtained through measurement of total plasma apoC-III. Importantly, gender, race, and several classes of medications appear to influence the relative amounts of the potentially more favorable apoC-III2 proteoform, and this may help account for some of their effects on triglyceride and other lipid levels. These novel results also emphasize the need for further study of posttranslational modifications of apolipoproteins to clarify their role in lipid metabolism and cardiometabolic risk.
This work was supported by the National Institutes of Health Grants R24-DK090958 (R.W.N./P.D.R.), R01-067690 (P.D.R.), R01-HL94775 (P.D.R.), R01-DK082542 (R.W.N.), and K23-HL107389 and 15BGIA25690024 from the American Heart Association (H.Y.), the Cooperative Studies Program of the Department of Veterans Affairs Office of Research and Development (#431), and Takeda Pharmaceuticals. The contents of this article do not represent the views of the Department of Veterans Affairs or the United States Government. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
[S]The online version of this article (available at http://www.jlr.org) contains a supplement.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Poll: Poll: Is human nature constant?
This Poll:
- Votes: 420
- Comments: 1
- Added: November 2004
All Polls:
- Polls: 1,164
- Votes: 684,866
- Comments: 4,835
Simple and confusing question at the same time. What is this all about?. To understand the question? Because the answer is quite clear, isn't it? o_O
You must vote before you can post a comment.

subset: commoncrawl


text:
Meri Pyaari Bindu Trailer 2: Ayushmann Is Sambha To Parineeti's Gabbar
After releasing the interesting trailer of Samosa aur chutney wala pyaar in the 1st chapter, the makers of Meri Pyaari Bindu released another funny trailer through their 2nd chapter which is called Gabbar aur Sambha.
While in the 1st chapter, the lead characters were shown in their childhood, the 2nd chapter showcases the fun-filled relationship around their college times.
The promo has an exam scene where Ayushmann aka Abhi is busy in completing his answer sheet, while Parineeti aka Bindu is busy in making deals with other students to sell the answers. Students offer her 50 bucks, a pair of sunglasses and even a CD player for the answers.
Watch the 2nd chapter right here:
The 2nd chapter is called Gabbar aur Sambha, because the relationship between the lead characters is much more like these two popular characters from Sholay. Abhi needs to and has to listen whatever Bindu says, whether it's right or wrong.
The film is set in Kolkata and Parineeti Chopra portrays the character of an aspiring singer in the film.
The trailer of the film will be released through 5 chapters in 5 days and the 3rd chapter will be released tomorrow.
Directed by Akshay Roy and produced by Maneesh Sharma and Yash Raj Films, Meri Pyaari Bindu is scheduled to release on 12th May. The film will clash with Ram Gopal Varma's Sarkar 3 which features the ensembled cast of Amitabh Bachchan, Jackie Shroff, Manoj Bajpayee, Ronit Roy, Amit Sadh and Yami Gautam.
Post Meri Pyaari Bindu, Ayushmann will be seen in Bareilly Ki Barfi along with Kriti Sanon and Rajkummar Rao. The romantic comedy is being directed by Ashwiny Iyer Tiwari, who previously helmed the critically acclaimed film Nil Battey Sannata featuring Swara Bhaskar in the lead role.
While Parineeti will be seen in Takadum along with Sushant Singh Rajput and Irrfan Khan. The 28-year old actress will also feature in the 4th instalment of the Golmaal series. Golmaal Again is being directed by Rohit Shetty and boasts an ensemble cast including Ajay Devgn, Tabu, Arshad Warsi, Tusshar Kapoor, Neil Nitin Mukesh, Shreyas Talpade, Kunal Khemmu, Johny Lever, Sanjai Mishra and Mukesh Tiwari.
The post Meri Pyaari Bindu Trailer 2: Ayushmann Is Sambha To Parineeti's Gabbar appeared first on Koimoi.
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Union University Accelerated BSN Program Spring 2018
Is any one applying for the Accelerated BSN program at the Germantown campus???Nov 21I subbmitted everything through Nursing CAS as instructed, and received an email once my application was verified. After Nursing CAS verifies your application, it's sent over to nursing admissions. You'll receive an email then as well, then you wait for an admissions decision
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Question about Samsung BDC6900 3D Bluray Disc Player
Posted by Alistair Cooke on
Please read the instructions below Fully BEFORE following them. If you read and do at the same time, you will not be able to enter the code fast enought and you have to start over.
1. Ensure that there is no disk in the tray.
2. Switch the player off and then back on. This is necessary to clean-boot the firmware.
3. Wait until the main menu page appears and the player stops doing anything.
4. Press the eject button to open the tray. (The button is between the "Power" and "TV Power" buttons at the top of the controller.)
5. Press the eject button to close the tray.
6. Wait while the player searches for a disk.
7. As soon as the "No Disk" message appears at the top left corner do the following.
8. Press the REPEAT button - just above the TV channel selector at the bottom right of the controller.
Step 8.5 You need to know the current region the player is locked to and in step 9 enter the correct code for your current region
region1 - 2 9 3 3 4
region2 - 5 7 5 3 8
region3 - 5 6 7 3 2
region4 - 7 6 8 8 4
region5 - 5 3 8 1 4
region6 - 2 4 4 6 2
9. Enter X X X X X on the number pad. (SEE STEP 8.5)
STEP 9.5 Note that REPEAT is part of the code, you must press repeat and then enter the 5 digits. If you wait after pressing repeat it won't work. This has to be entered before the no disc message dissapears from the screen, do it fast but be accurate.
10. Your region code should appear at the top left corner of the screen.
11. Enter a "9" to make the player region free.
12. Power down and power up again.
13. Play a foreign DVD to verify the change.
Posted on Oct 04, 2010
This worked fine for me on my BDC6900 3D blueray player with region code 5 (india). Now Iam able to view all region DVDs. Is there a way to change/switch Blueray regions too? (A, B, C) ?
Thanks a ton for this post.
i have tried it several times on samsung bd-c6900 and it didn't work...
is there anything else to do ?
you must be doing something wrong, try again, if you are not sure about the current region try all of the 6 codes offered on step 8.5
i tried it on samsung bd-c59000 and it didnt work (i tried all zones)
this is not for 5900
is there anything else to do with this module bd-c5900 ?
I also tried it to no avail... I have a region 1 player and wheni hit repeat and the code, nothing happens, it doesn't display anything and it doesn't allow me to hit 9 to change anything... any clues?
i replaced my blue rey to the 6900 and its still not working..
i tried all codes
probably the firmware has been updated or is different. so it doesn't work, its probably been changed or removed. I can not help you with that, keep trying, try to update or downgrade your firmware etc and see if it starts working.
SOURCE: I have freestanding Series 8 dishwasher. Lately during the filling cycle water hammer is occurring. How can this be resolved
a 6ya expert can help you resolve that issue over the phone in a minute or two.
Best thing about this new service is that you are never placed on hold and get to talk to real repairmen in the US.
the service is completely free and covers almost anything you can think of.(from cars to computers, handyman, and even drones)
click here to download the app (for users in the US for now) and get all the help you need.
Posted on Jan 02, 2017
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After repairing the mirror, Link enters the Twilight Realm once more. This stage represents the first area-- with the Twilight Mirror, and the two moving platforms that led to the areas with the Sol and the Zant Hands.
A very simple stage, you've entered my realm, better make yourself at home.
Recommended number of players: Any


text:
This property has been removed by the agent. It may be sold or temporarily removed from the market
4 bedroom terraced house for saleHarrowdene Road, Knowle, BRISTOL, BS4
Sold STC £400,000
Property Description
Key features
- Harrowdene Road
- 4 double bedrooms
- en-suite
- upstairs bathroom
- separate receptions
- kitchen
- breakfast room
- rear garden
- no chain
- tenure - freehold
Full description
A double bay fronted period terrace on this popular road in Knowle close to local amenities and transport links. Downstairs the property features a lounge with feature fireplace, separate dining room, a breakfast room and kitchen that leads out to the garden. Upstairs are 3 double bedrooms and a family shower room. Up again in the converted dormer loft conversion you will find the master bedroom with velux window, air conditioning, full-height picture window with southerly views over Dundry and Ashton Court, an en-suite bathroom and built-in storage. Outside is a well kept garden with a south west aspect as well as rear access. Additionally there is gas central heating, many remaining period features and no chain.
Lounge - 15'4 (into bay) x 11'3 (4.67m ( into bay) x 3.43m) -
Dining Room - 16'11 (into bay) x 9'5 (5.16m ( into bay) x 2.87m) -
Breakfast Room - 9'11 x 9'7 (3.02m x 2.92m) -
Kitchen - 10'2 x 9'7 (3.10m x 2.92m) -
Bedroom 2 - 17'2 x 15'4 (into bay) (5.23m x 4.67m ( into bay)) -
Bedroom 3 - 12'8 x 8'4 (to chimney breast) (3.86m x 2.54m ( to -
Bedroom 4 - 12'5 x 9'7 (3.78m x 2.92m) -
Shower Room - 6'11 x 6'2 (2.11m x 1.88m) -
Master Bedroom - 19'6 x 9'3 (5.94m x 2.82m) -
En-Suite Bathroom - 8'3 x 4'9 (2.51m x 1.45m) -
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Disclaimer - Property reference 26360015. The information displayed about this property comprises a property advertisement. Rightmove.co.uk makes no warranty as to the accuracy or completeness of the advertisement or any linked or associated information, and Rightmove has no control over the content. This property advertisement does not constitute property particulars. The information is provided and maintained by Ocean, Knowle. Please contact the selling agent or developer directly to obtain any information which may be available under the terms of The Energy Performance of Buildings (Certificates and Inspections) (England and Wales) Regulations 2007 or the Home Report if in relation to a residential property in Scotland.
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Did anyone else go see Wonder Woman this summer? The superhero biopic has broken records and glass ceilings all over the place, making it the highest-grossing live-action film directed by a woman, the highest-grossing opening weekend from a female director, the highest-grossing woman-led superhero film, the first Marvel or DC Comics film directed by a woman, and the biggest domestic earnings for a DC Comics film (take that, Batman and Superman!) Records aside, I was totally smitten with the film. I walked out of that movie theatre bubbling with energy, feeling inspired and ready to take on the world. My husband described me as "amped up."
Wonder Woman's creator, Dr. William Marston, once described Wonder Woman as "psychological propaganda for the type of woman who, I believe, should rule the world."
A recent Forbes article by Kathy Caprino discussed gender bias on display with regard to the repeated interruptions, and characterization of dogged questioning of then-nominee Attorney General Jeff Sessions by U.S. Sen. Kamala Harris (D-Calif.) as "hysterical." The
point of the Caprino article was the need to change our differing perceptions, reactions, and inherent or explicit bias that occurs when women and men behave in the same manner. She challenges readers to "Flip It To Test It" (#FlipItToTestIt), which is an excellent method of raising awareness of personal biases. She also notes that many women are punished, reprimanded, and even scolded, by men and women when they conduct themselves in the same way a man does. But, she advises that women "cannot stop or change their behavior to avoid it. They need to continue speaking up and advocating with self-assuredness and authority, in order for the world to change."
While I do not personally disagree with the need to develop change in judgments and attitudes, I found Caprino's encouragement to speak "forcefully," curious in light of the study she cites in the same article:
A new study by New York Times bestselling authors, Joseph Grenny and David Maxfield revealed that gender bias in the workplace is real, finding that women's perceived competency drops by 35% and their perceived worth falls by $15,088 when they are judged as being "forceful" or "assertive." Compare this with the drops in competency and worth that men experience when being judged as forceful: their competency drops by 22% and their worth falls by $6,547. This significant difference reveals a true gender bias that prohibits women from succeeding fully in leadership and management roles where assertiveness is, of course, a crucial behavior.
No one really needed to tell the women attorneys at Constangy that they were #1 in the best law firms for female attorneys, but it is nice to get public recognition like we did today in Law360.
Women make up more than 40 percent of law school classes, but comprise only about 35 percent of attorneys in private practice, and only 23 percent of partners. Law360's 2017 Glass Ceiling ...
The Florida Bar has taken steps to recognize parental leave as a valid reason for a continuance of trials and significant court activity for lead attorneys in cases—including both women and men attorneys. Recently, the Florida Bar Board of Governors voted unanimously to recommend a new Rule of Judicial Procedure to allow lead attorneys to obtain a three-month continuance for ...
Mark and Michelle are hired at the same time, by the same law firm in the same city, to do the same job. They are both third-year associate attorneys; they went to the same law school; they graduated with the same GPA; and they have the same skill set and job experience. Now, let's assume the law firm, their new employer, offers them two different salaries. Mark is offered $10,000 a ...
May is Mental Health Awareness Month. This annual campaign aims to raise awareness for mental illnesses and break the stigma associated with mental health issues. According to the National Alliance on Mental Illness, 1 in 5 Americans will be affected by a mental illness in their lifetime, and every American is affected through friends and family who have mental illnesses.
Thus, it ...
In the last two weeks, I posted Part 1 and Part 2 of this blog series which discussed what women want from the workplace, why many women are leaving the workforce when it does not accommodate their need for flexibility, the desire for businesses to recruit women, and developing business trends and needs requiring flexibility. This final blog will discuss how the Fair Labor ...
Last week we published Part 1 of How the FLSA Hurts Women which discussed what women want from the workplace, and some reasons why they are leaving the workforce when it does not accommodate their work-life balance needs. In this part, we will be discussing business trends and business needs in general. Part 3 will discuss how the FLSA works against businesses' being able to provide women with attractive work options, thus hurting women and businesses.
What?! Blasphemy!
Our politicians regularly debate how high the minimum wage should be, and which workers should earn overtime pay, but in my 30+ working years, I cannot recall any significant push to rid ourselves of the nearly untouchable dinosaur known as the Fair Labor Standards Act (FLSA). Women in particular, however, should take a fresh look ...
What do the following have in common?
- Americans who can drive a stick shift;
- Female equity partners in AmLaw 200;
- Projected sales growth for smartwatches in 2017;
- Americans who have seen a ghost.
Answer: 18%
In 2016, women made up 18% of the female equity partners in the AmLaw 200. Am I happy with that statistic? Eh, no. (But I'm pretty excited that 18% of Americans claim to have seen a ghost ...
We recognize the immeasurable value of women leaders supporting other women in the workplace through example, mentorship, education and empathy. We hope you enjoy our tidbits of legal and practical information, wisdom, and humor. Thanks for joining the conversation!


text:
Antonio Rucinski
I have a teenage low kind of voice, but if needed to can make my voice go high. Im very talented when it come to dramatic scenes as I've been told. And to some it up I always have feeling with how my voice sounds.
English (North American), English, English (British)
Child, Teen, Young Adult
Special Skills
Audiobooks, Business, Cartoons, Internet, Movie Trailers, Podcasting, Radio, Telephone, Television, Videogames
I've never voice acted in anything before. I have done live performances for my school and have done two years of improv.
I was in an improv class for two years that was ran and put together by the LOL Superstars. Which is an improv group in the inner city of Rochester NY.
Service Description
I'm a young actor looking for anything that will put me on the map. So any client that is willing to take me doesn't have to look to far. Also I'm young and can learn from my client.
Payment Terms
Hire Antonio as a voice actor. Payment is made through Voices.com SurePay, an escrow service that guarantees your work is done to your satisfaction. Send payment securely by Visa, Mastercard or PayPal through Voices.com then download your files from the website.
Watch a video to see how to make a payment
Next Steps
To contact Antonio Rucinski about your project, request a quote today. We'll walk you through the process step-by-step. It's fast and easy.
This talent has not uploaded any demos yet.
Hire Antonio Rucinski as a voice actor. Working with Antonio Rucinski is fast and easy. We'll walk you through the process step-by-step.
Request a quote for your project today.


text:
Summer is fun again with this Banzai Slide 'N Soak Splash Park. It's a whole water park adventure right in your back yard and consists of a slide, pool area and water shower. Regularly $599.99 at Kohl's, it's on sale for $359.99, but drops to just $244.79 after codes SPLASH20 and SUMMERDAYZ are applied at checkout. Happen to be a Kohl's charge card carrier? Use codes SPLASH20 and PICNIC instead and score this splash park for only $230.39. Both offers include free shipping. Deal ends 06/29/2016.
* Hot deals expire quickly. This deal may no longer be valid.
Get in on a special Black Friday offering from Amazon right now. They're offering the Fire HD 10 32GB for just $99.99 with free shipping, a savings of $50 off the regular $149.99 retail price. Features include a 10.1" 1080p HD display, a 1.8GHZ quad-core processor, 2GB RAM and up to 10 hours of battery life. Deal ends 11/24/2017.
Stores: 20x200, 6pm, Amazon, Apple Store, American Eagle, Banana Republic, Best Buy
Sections: Automotive, Computers, Electronics
Follow the Bargainist on Twitter!
Become a Fan of The Bargainist on Facebook!
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text:
Item# 1008-2288
Sweet Baby Shower Invitations
$4.99 $3.49
Ships To: United States Only
This item is currently not available.
Product Actions
Additional Information
- Pretty baby design sends a sweet message.
- Invitations: 5.5 x 8.5 in; envelopes 5.75 x 8.75 in.
Return Policy:
Don't worry! Returns and exchanges are easy. All returns and exchanges must be received within 180 days of the purchase date. And, since every order is shipped with an itemized invoice as well as complete return instructions, simply follow the detailed instructions for a quick, easy no-hassle return.
Except to the extent required by law, perishable items cannot be returned.


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Go Back
Learning Assistant
Posted 6th Nov 2017
The Role
A leading Dubai based Education provider is currently seeking a Learning Assistant.
The person should be someone who enjoys working with children and helping them learn and grow.
The Learning Assistant position is a fulfilling job which gives you the opportunity to help with students learning and development.
The Company
A leading Dubai based Education provider
The Person
- Minimum High School education in English and Maths, degree preferred
- Experience or aptitude for working with students, previous teaching or teaching assistant experience preferred
- Good levels of written and spoken English
- Enthusiasm and aptitude for working with students
- Good team player, able to work well as part of a team
Permanent Contract
Full Time
Apply Now...
Is this the job for you? Don't waste any time:


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10€ off Ambassador membership during Black Friday and Cyber Monday! On special now for 39€ lifetime until 27th Nov 2017 23:59 CET.
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About cjung
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Real Name
Carsten Jung
- Website
Did that but there is no change. I use the . as decimal seperator in all prices. Otherwise I can't save the product without a warning. When I use the , and save the product, the price will be set to 0.00 after saving. But as well as I use the . in my prices, they are all displayed with , in the shop .... Maybe there is a generel mistake in it.... hummm
Hello, no changes. Even if I use , instead of . the price is transfered with . This shurely is defined in a script. But in which and where?....
cjung posted a topic in PayPalHello, I'm using the contribution PayPal IPN v2.3.3. It all works properly but there is one little mistake and I don't know where I can find it. When I check out of my cart all the prices are handled in osC with ',' (e.g. 14,99). The PayPal-Site is opening but the price you see ist 0.00. So it's transfered with points rather than commas? When I enter the price at the paypal-site all works well. I cant't find the problem right now. Any ideas where to look? kind regards .carsten


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Question about Orange SPV M3100 Smartphone
Posted by eben20087 on
internet problem,my network card is not connecting,
SOURCE: I have freestanding Series 8 dishwasher. Lately during the filling cycle water hammer is occurring. How can this be resolved
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Coronado, California
$15,0003 Beds4 BathsActive
$15,0003 Beds4 BathsActive
Coronado, California 92118 - $12,000-$15,000 per month. Seasonal rental -26 night minimum stay, furnished down to the linens. Rental rates vary depending on desired month and length of stay. Check on availability. Gorgeous home with 3 master suits. Views of Downtown & the San Diego Bay from second level as well as roof top deck, elevator & pool! Beds: Master-1 King, Master 2-2 double beds, 3rd room- two sets of bunk beds. Security deposit/reservation fee due at signing. Rent & cleaning fee due 60 days prior to move in.
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Minimum wage for workers aged 20 and under to be frozen at under £5-an-hour 'to create more jobs for them'
- Minimum wage for 18 to 20-year-olds currently £4.98 per hour; £3.68 for 16 and 17-year-olds
- Adult hourly minimum wage of £6.08 likely to rise by 2%
- Ministers believe employers will be deterred from taking on new staff if rate goes up
The minimum wage for workers aged under 20 is likely to be frozen at under £5-an-hour in a bid to create more jobs for them, it was revealed today.
Vince Cable, the business secretary, has decided not to raise pay above the minimum of £4.98 in an attempt to persuade companies to take on more young workers.
With more than one million 18 to 24-year-olds out of work, the Department for Business, Innovation and Skills, hope the move will boost youth employment.
Freeze: Business Secretary Vince Cable, pictured speaking last night, is set to hold the minimum wage for 18 to 20-year-olds at £4.98 per hour
A freeze would represent a real terms wage cut for 18 to 20-year-olds as inflation is currently running at 4.2 per cent.
The minimum wage for 16 and 17-year-olds is also likely to be frozen at £3.68-an-hour, the Independent reported.
A final decision on the minimum wage will be taken later this month, around the time of the March 21 Budget.
However, the adult minimum wage, currently £6.08-per-hour, is likely to increase by 2 per cent in line with the rise in average earnings.
Brendan Barber, the Trade Union Congress general secretary, said that increasing the pay of young workers would boost the economy.
has been much debate about the minimum wage and young people, mainly
amongst those who have always wanted it scrapped, but there is no
evidence that it has had any adverse impact on jobs,' he said.
'The real reason why firms have not been hiring new workers is because they lack confidence that we have embarked on a sound economic recovery.
'Boosting consumer demand is vital for our economic prospects but this will not be achieved by cutting pay in real terms.
'We know that minimum wage workers
tend to spend 100 per cent of any increase, so a well-judged increase
would have a beneficial effect on the UK economy.'
Worrying rise: The number of jobless 16-24-year-olds has passed the one million mark
A spokesman for the Department of Business, Innovation and Skills said: 'The Low Pay Commission delivered its report to the Government in February.
'We are carefully considering its recommendations and will announce the Government's response in due course.'
Mr Cable last night launched a new onslaught on the banks, accusing them of 'imperilling' Britain's economic recovery.
He used his annual set-piece address to the City to denounce the support offered by the banks to enterprising small and medium businesses as 'second division'.
Speaking at the Lord Mayor of London's trade and industry banquet at Mansion House, he said thousands of firms were trapped in a 'valley of death' because they could not get the credit they needed to develop new products.
Mr Cable made clear his frustration at the unwillingness of the banks to lend, saying that having been responsible for the financial crash in 2008, they were now holding back the recovery.
He said: 'Britain's recovery is being imperilled by the parlous state of the very institutions that cause the crisis in the first place.'
He said there was a 'yawning mismatch' between the needs of what he called 'productive business' and the ability of the UK financial sector to service those outside the world of the 'big corporates'.
He added: 'I would go so far as to say that we have a financial services industry in London that plays in the Champions League, with overseas owners to match, and a British business finance system which struggles in the second division.
'We have some of the world's smartest, most creative, financiers, doing brilliantly in the City or Canary Wharf while there are also smart creative British entrepreneurs - or even run-of-the-mill smaller businesses - still struggling to raise finance to operate and expand.'
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What's new in Game Connect?
Game Connect
New Sort Order screen
June 30th, 2015
Just over a week ago, we introduced a new design for the Columns screen. Today, we moved the new design over to the Sort Order screen, so that both have a consistent user interface.
Just drag your desired Sort Field to the right, then select Ascending or Descending using the toggle buttons below the field.
Next up: multi-field sorting!
Recent changes
New design and layout for game details page October 31st, 2017 New: Manage your pick list entries September 29th, 2017 New fields added to sort and/or group your games on August 7th, 2017 Improved "Edit Multiple" screen with new fields August 2nd, 2017 New: Set a default view for friends June 22nd, 2017 New: Easier ways to share your collection or a single item with friends! June 21st, 2017 Private Cloud Sharing June 13th, 2017 Improved searching in Connect and Cloud June 13th, 2017 New: Improved Edit Screen and Duplicate feature June 9th, 2017 Add by Barcode: Now directly connect to your CLZ Barry barcode scanning app! June 2nd, 2017 New: Improvements to "Selection Mode" and added "None" folders, May 22nd, 2017 Faster editing, updated Statistics and other tweaks May 16th, 2017 New: Print your list to a PDF file May 1st, 2017 New "Completeness" field: Loose vs CIB vs New April 10th, 2017 New: Back up / Restore your database March 27th, 2017 New: Flexible import from Text/CSV files March 14th, 2017 New: "Search-As-You-Type" suggestions when adding games by title January 12th, 2017 New: Import text / CSV files January 3rd, 2017 New Collectorz.com Help Center December 14th, 2016 New Collectorz.com shop at Shop.CLZ.com November 8th, 2016 Export to Text feature now available in CLZ Cloud viewer September 7th, 2016 Game Connect: improved "Add by Title" searches April 28th, 2016 No more database limits during free trial April 13th, 2016 Security update: Connect and Cloud sites now running on HTTPS November 9th, 2015 Further performance optimizations November 6th, 2015


text: Beautiful day sat outside The Crown at Hutton Le Hole Ryedale North Yorkshire. Great choice of Lunchtime menu of which we chose the Ploughman's lunch. We were pleased we did as it is the best ploughmans I have ever seen, Salad, Large piece of Boiled Ham, 3 Cheeses generous portions. Large sausage roll. Pot of Coleslaw made with red onion.... More


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Houston, TXUSA
January 14, 2013
CEO of medical device company and entrepreneur in residence at HTC and RedHouse Associates
I am involved on lots of start ups in the Houston area.
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Niles (IL) condo blaze sends 4 residents, firefighter to hospital
Several people, including a firefighter, were injured during a fire that broke out on the fifth floor of a six-story condominium building in the 9700 block of North Dee Road Friday morning, according to Niles Fire Chief Steve Borkowski. Read the article………….
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Benzer to plead guilty in Las Vegas (NV) HOA scheme
Leon Benzer, the alleged mastermind of the massive scheme to take over homeowners associations in the Las Vegas area, is


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Welcome to the Trailsend Volleyball Club,
Feel free to look around.
Any Questions? Call Leilani 310-940-9784 or Email trailsendvbc@gmail
Welcome to the Trailsend Volleyball Club,
Feel free to look around.
Any Questions? Call Leilani 310-940-9784 or Email trailsendvbc@gmail
Congrats to all Trailsend players whom made a Trailsend Team this year. We are all looking forward to a fun and passionate season!!!!!
Here is an update of things that should already be done or should be worked on over Thanksgiving break!
Faith Bufferale
Allie Collins
Sydney Fogelman
Kennady Grant
Amiyah Halford
Avery Lanz
Ellie Low
Madeline O'brein
Hailey Parks
Kylee Pittam
Sarah Trusty
Emma Vandecoevering
Please change membership over to $55 through CEVA and USAV. I can not put you on an official roster until this is done.
-Other unknown Players need to change their club name on membership to Trailsend club. These players also can not be on a roster without this being done.
-Calendar is updated till January. Please continue to check for updates in the following weeks.
-Email will be sent out by each team parent shortly about the Dec. 9th and 10th friendship tourney coming up at DSK.
18-1/16-1/15-1 Players and families headed down to Eugene in January. We are not going to block rooms this year for this event due to lack of Hotel space in Eugene and many families drive to and fro from this event in the past. So please feel free to book rooms as you all see fitter this event. Most of us will all play at the football field! BEST WESTERN in Springfield has great reviews from our 18-1 families in years past!
Players should have:
-Jersey Deposits $150
-Club Deposits $450 or $550
-Parent/Player agreement
-Jump Ropes
-Sweats (Optional)
-Long sleeve shirt
-2 Jersey's are being sorted and will be given out shorty to each player.
-Players should start working on Ref/Scoring certifications.
-Team parents and Coaches should be planning team bonding event in the next 3 weeks!
Thank you for everyones support and encouragement for Trailsend! We are so lucky to have the best families in the Region at our club! Have a great Thanksgiving! We are thankful for you!