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EXPERIMENTAL CLINICAL INVESTIGATION
Effects of Calorie Restriction on Polymicrobial Peritonitis Induced by Cecum Ligation and Puncture in Young C57BL/6 Mice
Dongxu Sun, Alagar Raju Muthukumar, Richard A. Lawrence, Gabriel Fernandes
Dongxu Sun
Department of Medicine, Division of Clinical Immunology, University of Texas Health Science Center at San Antonio, San Antonio, Texas
Alagar Raju Muthukumar
Richard A. Lawrence
Gabriel Fernandes
DOI: 10.1128/CDLI.8.5.1003-1011.2001
Calorie restriction (CR) is known to prolong the life span and maintain an active immune function in aged mice, but it is still not known if rodents under CR can respond optimally to bacterial infection. We report here on the influence of CR on the response of peritoneal macrophages to lipopolysaccharide, splenic NF-κB and NF–interleukin-6 (IL-6) activities, and mortality in polymicrobial sepsis induced by cecal ligation and puncture (CLP). Macrophages from 6-month-old C57BL/6 mice on a calorie-restricted diet were less responsive to lipopolysaccharide, as evidenced by lower levels of IL-12 and IL-6 protein and mRNA expression. Furthermore, in vitro lipopolysaccharide-stimulated macrophages from mice under CR also expressed decreased lipopolysaccharide receptor CD14 levels as well as Toll-like receptor 2 (TLR2) and TLR4 mRNA levels. In addition, the phagocytic capacity and class II (I-Ab) expression of macrophages were also found to be significantly lower in mice under CR. Mice under CR died earlier (P < 0.005) after sepsis induced by CLP, which appeared to be a result of increased levels in serum of the proinflammatory cytokines tumor necrosis factor alpha and IL-6 and splenic NF-κB and NF–IL-6 activation 4 h after CLP. However, mice under CR survived significantly (P < 0.005) longer than mice fed ad libitum when injected with paraquat, a free radical-inducing agent. These data suggest that young mice under CR may be protected against oxidative stress but may have delayed maturation of macrophage function and increased susceptibility to bacterial infection.
Calorie restriction (CR; level of restriction, 25 to 50%) is known to increase the life span in rodents by 30 to 40% (23, 63). One of the well-known protective effects of CR is enhanced T-cell-mediated immune function including the prolonged maintenance of naive T cells and the delay of the early rise of memory cells (16, 26, 58). Numerous studies have reported that CR significantly delays the onset of malignancy and autoimmune renal disease in autoimmune-susceptible and -resistant mice and rats (27, 37, 58, 64, 65). Furthermore, CR is known to increase the numbers of cytotoxic T cells, increase the level of resistance to influenza viral infection, reduce the incidence of ulcerative dermatitis, and preserve spleen interleukin-2 (IL-2) production (16, 26, 28, 43, 45, 58). There is evidence that calorie-restricted rodents are more resistant to a variety of stress-inducing agents or insults (37) including surgical trauma (38), heat shock (32), and the toxicities of a variety of drugs (20). Despite the wealth of most supportive evidence describing the cellular and molecular changes that occur with CR, it is still not known whether rodents on CR can initiate a protective response against bacterial infection. There are also no studies that have assessed the effects of CR in an experimental model that mimics the clinical disease symptoms of polymicrobial sepsis and septic shock. The studies of CR with bacterial pathogens carried out so far are also inconclusive due to variability in experimental conditions such as diet and strain variations (19, 44).
Macrophages constitute the first line of defense against infection and are primarily responsible for the clearance of gram-positive or -negative bacteria from the blood via phagocytosis and intracellular killing. In addition, macrophages are central to both immune effects and autoregulatory function and are critical to defense mechanisms. Also, macrophages play an important role in the pathogenesis of sepsis and septic shock (11). However, the effects of CR on sepsis and/or macrophage function have not yet been fully addressed. We first reported on the impaired antigen-presenting function of macrophages and decreased antigen-specific T-cell proliferation in C57BL/6 mice fed calorie-restricted diets (17); Shi et al. (54) later confirmed these effects in energy-restricted BALB/c mice. We therefore formulated a hypothesis that although CR increases T-cell-mediated cellular immune function against viral infection (25), it may impair antigen presentation and/or phagocytosis by macrophages. A recent review by Weindruch et al. (62) on CR has pointed out the paucity of studies in the area of CR and infection. Since studies of CR are already being carried out with primates (14, 30, 34, 60, 62) and plans are under way to initiate studies of CR with humans (49), it is urgent that the effects of CR on the immune response to commonly occurring pathogens be addressed as soon as possible to prevent any infection-related fatalities in humans during CR or weight-reduction studies.
In light of this, it was the aim of this particular study to examine first the effects of CR on the responsiveness of peritoneal macrophages to lipopolysaccharide (LPS), a major cell wall constituent of gram-negative bacterial organisms in vitro. Additional studies were conducted to examine the effects of CR on splenic NF-κB and NF–IL-6 activities and mortality in a murine model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). Since oxidative damage has been implicated in the pathogenesis of septic shock (9, 73) and animals fed calorie-restricted diets have been reported to have enhanced antioxidant enzyme capacity (13, 69), we have also determined the susceptibility to free radical-induced death by paraquat poisoning of mice fed ad libitum (AL) and mice fed calorie-restricted diets.
Materials.Fetal bovine serum, RPMI 1640 medium, and penicillin-streptomycin were purchased from GIBCO (Great Island, N.Y.). LPS (Escherichia coli O111:B4) was supplied by Sigma Chemical Company (St. Louis, Mo.). All fluorescence-labeled monoclonal antibodies for CD14, I-Ab, tumor necrosis factor alpha (TNF-α), IL-6, and IL-12 were obtained from Pharmingen, San Diego, Calif. Perm-wash buffer was purchased from Becton Dickinson, San Jose, Calif. All other chemicals were reagent grade and were purchased from Sigma Chemical Co. Double-stranded consensus binding site oligonucleotides for NF-κB and NF–IL-6 (CCAT enhancer-binding protein) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.).
Animals.Inbred, weight-matched, female C57BL/6 weanling mice were obtained from Taconic Farms (Germantown, N.Y.) at 4 weeks of age. The mice were grouped and housed at five mice per cage in a temperature-controlled room at 24°C and were maintained on a 12-h dark and 12-h light cycle in the laboratory animal care facility at the University of Texas Health Science Center at San Antonio. The mice were fed a nutritionally adequate semipurified diet (AIN76 formula) containing 5% (wt/wt) corn oil either AL or in a gradually restricted manner (CR) until the number of calories in the diet was 40% of the calories fed to the mice fed AL from 6 weeks of age, as described earlier (57). Briefly, the mice were initially fed 5 to 10% less than mice fed AL for the first 2 weeks and were then fed 10 to 20% less for the 3rd and 4th weeks and 40% less thereafter. The composition of the diet was 20% casein, 50% dextrose, 15% starch, 5% cellulose, 5% corn oil, 3.5% AIN (American Institute of Nutrition) salt mixture, 1% AIN vitamin mixture, 0.3%dl-methionine, and 0.2% choline chloride. Mice fed a calorie-restricted diet were not given additional vitamin or mineral mixture supplements (48), primarily to prevent excess vitamin and mineral intake per gram of body weight, which may alter gene expression between mice fed AL and mice fed a calorie-restricted diet. After 20 weeks of feeding, the mice were used for the experiments. All animal care and surgical procedures were approved by the Institutional Animal Care and Use Committee.
Peritoneal macrophage isolation and culture.Ninety-six hours before the mice were killed, the mice received an intraperitoneal injection of 2.0 ml of thioglycolate broth (Sigma Chemical Co.). The mice were killed by carbon dioxide inhalation. The macrophages in the peritoneal exudate were harvested by lavage with 10 ml of ice-cold phosphate-buffered saline (PBS) under aseptic conditions. After extensive washing in PBS, the cells were resuspended in RPMI 1640 (Gibco, Grand Island, N.Y.) supplemented with 1% penicillin-streptomycin, 2 mM l-glutamine, 10 μM β-mercaptoethanol, and 10% heat-inactivated fetal bovine serum and counted. After incubation for 2 h in 5% CO2at 37°C in a plastic tissue culture dish, nonadherent cells were removed by vigorous washing. The resultant adherent cell population consisted of 95% macrophages, with viability being >97%, as demonstrated by trypan blue exclusion. Viability remained >97% at all times in culture.
Phagocytosis assay with latex beads.Macrophage monolayers were preincubated for 30 min in a buffered salt solution (140 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES ]pH 7.5[). Macrophages were incubated with the latex beads (Sigma Chemical Co.) at a density of 107/dish for 30 min at 21 to 30°C. The monolayers were then washed five times with cold PBS to remove uningested, freely suspended particles. The contents of each dish were then incubated in PBS containing 0.25% trypsin for 0.5 h at 37°C on a shaker to detach the macrophages from the bottom of the culture dishes and to remove uningested particles attached to the macrophage's membrane surface. The macrophages were then fixed with 1% paraformaldehyde (PFA).
Phagocytosis assay with zymosan.Macrophages were incubated with zymosan (Molecular Probes) at a ratio of 1:10 at 37°C for 15 min and were then washed with 0.9% NaCl–0.02 M EDTA (pH 5.9) and fixed with 1% PFA at 4°C for 1 h. The macrophage phagocytic function was analyzed with a FACScan (Becton Dickinson) flow cytometer.
Flow cytometry analysis.Macrophages obtained from thioglycolate-treated mice were stimulated with LPS at 1 μg/ml for 12 h. Both LPS-stimulated and nonstimulated macrophages were harvested and washed with PBS. The Fc receptor was blocked with anti-CD16/CD32 by incubation for 10 min at room temperature with shaking. After two washes in wash buffer, 105cells were incubated in 200 μl of wash buffer for 30 min at room temperature with either phycoerythrin (PE)–anti-CD14 or PE–anti-I-Ab antibody. The samples were analyzed with a FACScan (Becton Dickinson) flow cytometer with CellQuest software, as described earlier (1).
Intracellular cytokine staining.Intracellular cytokine analysis was carried out as described previously (22). Briefly, peritoneal macrophages were preincubated with LPS (1 μg/ml) for 18 h, and then brefeldin A (10 μg/ml) was added and the incubation was continued for 6 h. The cells were harvested and fixed with 1% PFA at 4°C for 1 h and then permeabilized by incubation with PBS containing 0.5% saponin for 30 min at room temperature to allow the antibodies to penetrate the cell membrane. The cells were then incubated with PE-conjugated anticytokine antibodies for 30 min at room temperature. Stained cells were analyzed on the FACScan flow cytometer with CellQuest software.
Cytokine and other protein mRNA expression.Total cellular RNA was isolated with the Trizol reagent (Gibco BRL) by the protocol recommended by the manufacturer. Aliquots of 2 μg of RNA were transcribed in a total volume of 20 μl with random primers and superSCRIPT II reverse transcriptase. Aliquots of 2 μl of the product obtained by reverse transcription were taken as substrates for PCR, as described previously (35, 42). The other PCR components were added as a master mixture containing 10 mM Tris · HCl (pH 8.0); 1.5 mM MgCl2; 50 mM KCl; dATP, dCTP, dTTP, and dGTP, each at a concentration of 200 mM; 100 ng of primer; sterile water; and 2.5 U of Taq polymerase in a final volume of 50 μl. The sequences for the primers for Toll-like receptor 2 (TLR2), TLR4, TNF-α, IL-6, IL-12, and β-actin were obtained from published literature (39, 46, 52). β-Actin was used as a housekeeping gene to control for the differences in mRNA quantity and/or level of degradation.
Aliquots of 10 μl of PCR product were analyzed by electrophoresis in a 1.5% agarose gel in TBE (Tris-borate-EDTA) buffer (pH 8.0) containing ethidium bromide and were visualized by UV illumination. Densitometric analysis for semiquantitation of the PCR products was performed with an Alpha Imager 2000 analyzer.
Preparation of nuclear extracts.Spleens were homogenized in solution A (0.6% Nonidet P-40, 10 mM KCl, 10 mM HEPES [pH 7.9], 0.1 mM EDTA, 0.1 mM EGTA, 0.1 mM dithiothreitol [DTT], 1.0 μg of leupeptin per ml, 5.0 μg of pepstatin ml, 0.5 mM phenylmethylsulfonyl fluoride, 5.0 μg of trypsin per ml, 5.0 μg of aprotinin per ml, 0.1 mM benzamidine). After transfer to a 15-ml tube, the debris was pelleted by centrifugation at 360 × g for 30 s. The supernatant containing intact nuclei was incubated on ice for 10 min and then centrifuged for 10 min at 2,240 × g. The nuclear pellets were then resuspended in solution C (10 mM HEPES [pH 7.9], 450 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1 mM DTT, 1.0 μg of leupeptin per ml, 5.0 μg of pepstatin l per ml, 0.5 mM phenylmethylsulfonyl fluoride, 5.0 μg of trypsin per ml, 5.0 μg of aprotinin per ml, 0.1 mM benzamidine) and incubated on ice for 30 min. The nuclei were then centrifuged for 5 min at 17,560 ×g. Supernatants containing nuclear proteins were aliquoted and stored at −70°C. The protein concentration was determined by the Bradford assay.
Electrophoretic mobility shift assay.Oligonucleotides for NF-κB and NF–IL-6 were end labeled by treatment with T4 kinase in the presence of [γ-32P]ATP. Labeled oligonucleotides were purified on a Sephadex G-25 M column. Aliquots of 2.5 μg of nuclear protein were added to a binding reaction mixture containing 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5 mM DTT, 1 μg of bovine serum albumin per μl, and 0.1 μg of poly(dI-dC)(dI-dC) per μl; and the mixture was incubated on ice for 10 min. After incubation, 30,000 to 50,000 cpm of labeled double-stranded oligonucleotide was added to each sample. The mixtures were incubated for 10 min at room temperature and separated on a 5% polyacrylamide gel in 0.5× TBE buffer. The gels were vacuum dried and subjected to autoradiography. To eliminate nonspecific binding, cold competition was done by adding 100 ng of unlabeled double-stranded NF-κB and NF–IL-6 oligonucleotides to the reaction mixture before adding the labeled probes. Nonspecific competition was done by adding 100 ng of unlabeled double-stranded Sp-1 oligonucleotide before the addition of the labeled probes.
Cytokine immunoassays.Plasma TNF-α and IL-6 levels were measured by standard enzyme-linked immunosorbent assay (ELISA) techniques. In brief, each well of flat-bottom 96-well microtiter plates were coated with 50 μl of purified anti-TNF-α and anti-IL-6 antibodies (4 μg/ml in binding solution) overnight at 4°C. The plates were rinsed four times with washing buffer, and diluted serum was added, followed by incubation for 2 h at room temperature. The plates were washed four times with washing buffer, followed by the addition of biotinylated anticytokine antibodies. The plates were incubated in room temperature for 1 h and then washed four times with washing buffer. Streptavidin-alkaline phosphatase conjugate was added, and the plates were incubated for 30 min at room temperature. The plates were again washed four times with washing buffer, and the chromogen substrate was added. The plates were then incubated at room temperature to achieve the desired maximum absorbance and were read at 410 nM in an ELISA reader.
Model of polymicrobial sepsis.Polymicrobial sepsis was induced by using a CLP model described for mice by Baker et al. (7) and Ayala et al. (6). In brief, after anesthesia with methoxyflurane, a midline incision was made. The cecum was isolated and ligated and was then punctured twice with a 25-gauge needle. A small amount of the bowel contents was extruded through the puncture holes to maintain their patency. Upon returning the bowel to the abdomen, the midline incision was closed in layers with 5-0 ethicon sutures. The animals were resuscitated with saline (40 ml/kg of body weight) administered subcutaneously. Mice that underwent a sham operation underwent the same procedure but without ligation and puncture of the cecum. The animals that were not subjected to anesthesia or surgery served as negative controls. Spleens and blood were harvested 4 h after the operation for assessment of NF-κB and NF–IL-6 activation and for analysis of the levels of cytokine TNF-α and IL-6 mRNA expression. To examine the mice for mortality after CLP, the mice were observed four times daily over a period of 72 h.
Paraquat injection.Oxidative stress was induced as described by Migliaccio et al. (40). Briefly, mice were injected intraperitoneally with paraquat (methylviologen; 1,1′-dimethyl-4,4′-bipyridinium dichloride) prepared in PBS at a dose of 70 mg/kg of body weight, and their health status was observed every 2 h.
Statistics.All data were analyzed by a factorial analysis of variance (ANOVA) with NCSS software (version 5.01; NCSS, Kaysville, Utah) or one-way ANOVA with Bonferroni's posttest with GraphPad Prism software (version 3.00; GraphPad Software, San Diego, Calif.). A P value of <0.05 was considered significant. Survival analysis was done with NCSS statistical software (version 5.5). The data were fit to two different survival distributions, and the parameters for the two groups were compared by the Peto-Wilcoxon test and Gehan's Wilcoxon test for the Weibull distribution and by the log rank test and the Cox-Mantel test for the exponential distribution. Results for the distribution of best fit were used to determine significance. Data for mice that had not died at the end of the observation period were treated as censored datum points.
Animal weights.Body weight was significantly affected by calorie restriction. Mice in the group fed AL tended to gain weight with age, whereas animals fed the calorie-restricted diet gained very little body weight. The data are shown in Fig.1.
Effect of CR on body weight. Six-month-old C57BL/6 mice were fed a semipurified diet containing 5% (wt/wt) corn oil AL or the same diet whose calories were restricted 40% (CR).
Peritoneal macrophage responsiveness to LPS ex vivo.Since macrophages are the first line of defense during the innate immune response and play an important role in the pathogenesis of sepsis and septic shock, we assessed the responsiveness of macrophages from animals fed a calorie-restricted diet and AL to LPS ex vivo. Figure 2 shows the result of flow cytometric analysis of peritoneal macrophages stained for intracellular cytokines. After 24 h of LPS stimulation ex vivo, peritoneal macrophages from mice fed a calorie-restricted diet produced significantly less IL-6 and IL-12 than macrophages from mice fed AL. Although the percentage of TNF-α-positive cells in mice fed a calorie-restricted diet was less, there was no statistically significant difference between these two groups. To determine the effect of CR on inflammatory cytokine gene expression, reverse transcription-PCR was carried out with the isolated RNA. Cytokine TNF-α, IL-6, and IL-12 mRNAs were present at low basal levels in unstimulated macrophages; and there were no differences between mice fed AL and mice fed a calorie-restricted diet. After LPS stimulation, TNF-α, IL-6, and IL-12 mRNA levels were lower in peritoneal macrophages from animals on a calorie-restricted diet than those isolated from mice fed AL (Fig.3). These results show that the responsiveness to LPS of macrophages from mice fed a calorie-restricted diet was lower than that of macrophages from mice fed AL.
Effect of CR on macrophage cytokine production after LPS stimulation. Peritoneal macrophages from 6-month-old C57BL/6 mice fed a calorie-restricted diet were stimulated with LPS ex vivo. Twenty-four hours after stimulation, macrophages were incubated with fluorescent antibodies to TNF-α, IL-6, and IL-12. The cells were analyzed by flow cytometry. Each bar represents the mean ± standard error of the mean of six independent measurements. ∗, significantly different from controls fed AL (P < 0.05).
Effect of CR on cytokine, TLR2, and TLR4 gene expression in peritoneal macrophages of C57BL/6 mice after LPS stimulation. Peritoneal macrophages were isolated as described in Material and Methods and were stimulated with LPS (1 μg) for 12 h in vitro. Total RNA was isolated and transcribed to cDNA and was then amplified by PCR with primers specific for TLR2, TLR4, IL-12, TNF-α, IL-6, and β-actin, as described in Materials and Methods.
Peritoneal macrophage phagocytic function.In order to define the effects of CR on the phagocytic function of peritoneal macrophages, the levels of ingestion of fluorescence-labeled latex beads and zymosan were measured by flow cytometry. Figure4 presents these measurements. Both before and after LPS stimulation, macrophage phagocytic function in mice fed a calorie-restricted diet remained lower than that in the group fed AL.
Peritoneal macrophage phagocytosis in mice fed a calorie-restricted diet. Six-month-old C57BL/6 mice were fed a semipurified diet containing 5% (wt/wt) corn oil AL or the same diet whose calories were restricted 40% (CR). Peritoneal macrophages were harvested 96 h after injection of thioglycolate intraperitoneally. Phagocytosis of latex beads and zymosan was assessed before and 4 h after LPS stimulation (1 μg/ml) (n = 6). ∗, significant differences between the group fed AL and the group fed a calorie-restricted diet (P < 0.05).
Peritoneal macrophage CD14 and I-Ab antigen expression.The constitutive expression of LPS receptor CD14 (mice fed AL, 26.9% ± 2.8%; mice fed a calorie-restricted diet, 19.9% ± 3.2% [P, not significant]) and major histocompatibility complex class II (MHC II) antigen I-Ab (mice fed AL, 53.2% ± 0.9%; mice fed a calorie-restricted diet, 37.6% ± 6.7% [P < 0.05]) on macrophages from mice fed a calorie-restricted diet was lower than that on macrophages from mice fed AL. After stimulation by LPS in vitro, CD14 expression (mice fed AL, 35.3% ± 3.1%; mice fed a calorie-restricted diet, 24.3% ± 3.5% [P < 0.05]) and I-Abexpression (mice fed AL, 85.2% ± 0.9%; mice fed a calorie-restricted diet, 69.1% ± 3.0% [P < 0.05]) on macrophages increased in the group of mice fed a calorie-restricted diet, but to a lesser extent than that on macrophages from the group fed AL.
Expression of TLR2 and TLR4 mRNAs.Our primary data showed that TLR2 and TLR4 mRNA expression peaked at 12 h after LPS stimulation (data not shown). As shown in Fig. 3, after LPS stimulation, TLR2 and TLR4 mRNA expression in mice fed a calorie-restricted diet was significantly lower than that in mice fed AL.
Survival trends of mice fed AL and mice fed a calorie-restricted diet with CLP-induced polymicrobial sepsis.Figure5 shows the 48-h survival pattern of C57BL/6 mice with CLP-induced sepsis. The first death was observed at 18 h in the group of mice fed a calorie-restricted diet, and all of the animals in the group of mice fed a calorie-restricted diet died within 36 h. Mice fed AL began to die later and survived significantly longer than mice fed a calorie-restricted diet (P = 0.004).
Effects of CR on the survival of C57BL/6 mice after CLP. Six-month-old C57BL/6 mice fed AL and a calorie-restricted diet were subjected to polymicrobial sepsis induced by CLP. The survival rate was monitored 72 h after CLP. There is a significant difference between these survival curves (P = 0.004 by the log rank test).
Splenic NF-κB and NF–IL-6 activation after polymicrobial sepsis induced by CLP. The effect of CLP on splenic NF-κB activation is presented in Fig. 6. Since splenic NF-κB nuclear binding activity peaked at 3 to 6 h after CLP (data not shown), we took the spleens at 4 h after CLP. The magnitude of the increase in NF-κB-binding activity was much greater in the group of mice fed a calorie-restricted diet than in the mice fed AL. Figure 7 shows the effect of CLP on splenic NF–IL-6 activation in the mice fed AL and the mice fed a calorie-restricted diet. Polymicrobial sepsis induces splenic NF–IL-6 activation in both the group fed AL and the group fed a calorie-restricted diet, but the NF–IL-6-binding activity was much higher in the group fed a calorie-restricted diet than in the group fed AL.
Electophoretic mobility shift assay of NF-κB-binding activity. Nuclear extracts were isolated as described in Materials and Methods. Nuclear extracts (2.5 μg) were incubated with radiolabeled probes specific for NF-κB, separated on a 5% polyacrylamide gel, and then exposed to film. The gel is representative of gels from five independent experiments. Band intensity was measured by densitometry and expressed as the mean ± standard error of the mean. ∗, significantly different from the corresponding control group (P < 0.05); †, significantly different from the corresponding group that underwent a sham operation (P < 0.05); §, significantly different from the group that underwent CLP and that was fed AL (P < 0.05).
NF–IL-6 activation and binding activity in murine spleen 4 h after CLP. Nuclear extracts were isolated as described in Materials and Methods. Nuclear extract (2.5 μg) was incubated with radiolabeled probes specific for NF–IL-6 and separated on a 5% polyacrylamide gel and then exposed to film. The gel is representative of gels from five independent experiments. Band intensity was measured by densitometry and is expressed as the mean ± standard error of the mean. ∗, Significantly different from corresponding control group (P < 0.05); †, significantly different from the corresponding group that underwent a sham operation (P < 0.05); §, significantly different from the group that underwent CLP and that was fed AL (P < 0.05).
Effects of CR on TNF-α and IL-6 mRNA expression in splenic tissue of septic mice.The levels of TNF-α and IL-6 mRNA expression in splenic tissue were examined 4 h after CLP. We selected only these cytokines because of their potential role in sepsis, and they are known to be transcriptionally regulated by NF-κB and/or NF–IL-6. TNF-α and IL-6 mRNA levels were found to be significantly higher in all mice that underwent CLP but were increased more in mice fed a calorie-restricted diet than in mice fed AL (Fig.8).
Effects of CR on cytokine gene expression after polymicrobial peritonitis induced by CLP puncture. Polymicrobial peritonitis was induced by CLP in C57BL/6 mice. Four hours after the operation total RNA was isolated from the spleens, as described in Material and Methods. The RNA was transcribed to cDNA and was then amplified by PCR with primers specific for TNF-α, IL-6, or β-actin, as described in Materials and Methods. The resulting samples were subjected to electrophoresis on agarose gels and stained with ethidium bromide. Band intensity was quantitated by densitometry. The values are the means ± standard errors of the means for five gels. ∗, significantly different from corresponding control group (P < 0.05); †, significantly different from corresponding group that underwent a sham operation (P < 0.05); §, significantly different from group that underwent CLP and that was fed AL (P < 0.05).
Generation of TNF-α and IL-6 in sera after CLP.Previous investigations demonstrated that the levels of certain cytokines, such TNF-α, IL-1β, and IL-6, are elevated in serum during the evolution of CLP-induced peritonitis and peak at 3 to 6 h after sepsis. In the present studies, serum TNF-α and IL-6 levels were higher in mice that underwent CLP than in control mice and mice that underwent a sham operation (Fig. 9). Interestingly, 4 h after induction of polymicrobial sepsis by CLP, the levels of TNF-α and IL-6 in mice fed a calorie-restricted diet, and especially the level of IL-6, were extremely high. These data indicate that TNF-α and IL-6 levels in systemic tissues in general were augmented much more during the evolution of CLP-induced peritonitis in mice fed a calorie-restricted diet than in mice fed AL, which may have caused earlier deaths due to sepsis in mice fed a calorie-restricted diet.
Effect of calorie restriction on serum cytokine TNF-α and IL-6 levels of 6-month-old C57BL/6 mice fed a calorie-restricted diet. Mice were treated as described in the legend to Fig. 8. Serum TNF-α (A) and IL-6 (B) levels were quantitated by ELISA. Each bar represents the mean ± standard error of the mean of five independent measurements. The means for bars labeled with different letters are significantly different (P < 0.05).
Effect of CR on oxidative stress.It is known that the numbers of oxidative stress-induced deaths are higher in mice fed AL than in mice fed a calorie-restricted diet. Since death from sepsis was found earlier in mice fed a calorie-restricted diet than in mice fed AL, we chose to compare oxidative stress-induced death in mice fed AL and mice fed a calorie-restricted diet. To examine the ability of mice fed a calorie-restricted diet to resist oxidative stress in vivo, animals were treated with paraquat, which generates superoxide anions upon cellular metabolism. Groups of six 6-month-old mice were injected intraperitoneally with 70 mg of paraquat per kg. All of the mice fed AL died within 12 h after injection, whereas of the six animals fed a calorie-restricted diet and injected with paraquat, only one died within 12 h, four died within 48 h, and one survived for 3 days after injection (Fig. 10). These differences between the two survival curves were found to be significantly different (P = 0.0048). Furthermore, these data also suggest that mice fed a calorie-restricted diet were not malnourished and that their antioxidant defense mechanisms were elevated compared to those of mice fed AL.
Fig. 10.
Survival of mice fed a calorie-restricted diet and mice fed AL after challenge with the oxidizing agent paraquat. Six-month-old C57BL/6 mice fed a calorie-restricted diet and mice fed AL were injected intraperitoneally with paraquat at 70 mg/kg. Mice fed AL died significantly earlier than mice fed a calorie-restricted diet (P = 0.0048).
In the present experiments, we first examined the effects of CR on the responsiveness of peritoneal macrophages to LPS ex vivo. The data indicated that the peritoneal macrophages of young mice fed a calorie-restricted diet show a significantly suppressed response to LPS. Intracellular cytokine analysis revealed that the percentage of Il-12-, IL-6-, and TNF-α-positive cells from mice in the group fed a calorie-restricted diet is lower after stimulation with LPS ex vivo. Similarly, macrophages from mice fed a calorie-restricted diet had significant reductions in IL-6, IL-12, and TNF-α mRNA levels.
Surprisingly, very few studies have attempted to investigate the effects of CR on the responsiveness of macrophages to LPS. Dong et al. (19) addressed the effects of 25% CR on rat alveolar macrophages. In vitro, LPS-stimulated alveolar macrophages from mice fed a calorie-restricted diet generated less NO and TNF-α as well as less TNF-α and IL-6 mRNA than animals maintained on a diet fed AL. From these results, it seems that the response to LPS of macrophages from mice on calorie-restricted regimens depends on various factors including the duration and extent of CR, the strain of animal used, and the anatomical site from which the macrophages are isolated.
It is well known that LPS mediates many of the pathophysiologic events in sepsis by stimulating the release of host-derived proinflammatory cytokines (18). In response to endotoxin exposure, TNF-α, IL-1β, and IL-6 are produced by tissue macrophages and peripheral monocytes. These cytokines have been shown to produce many of the hemodynamic and inflammatory changes of sepsis either directly or indirectly. It has only recently been reported that CD14 participates in the mediation of LPS responsiveness by high-affinity binding of LPS during the innate immune response (67). We therefore analyzed the CD14 expression in peritoneal macrophages by fluorescence-activated cell sorter analysis and found no difference between the groups fed a calorie-restricted diet and AL. However, after LPS stimulation, the level of CD14 expression in the macrophages of both groups was increased, but the percentage of CD14+ cells was less in mice fed a calorie-restricted diet than in mice fed AL, which may be one of the mechanisms involved in the impairment of macrophage function in mice fed a calorie-restricted diet.
Along with the CD14 receptor, several mammalian Toll homologues have recently been identified. Toll was first identified as a protein that controls dorsoventral pattern formation in the early development ofDrosophila (10). Since that time it has been shown to participate in the antimicrobial immune response. Two of these human Toll homologues, TLR2 and TLR4, have been found to be involved in LPS signaling (36, 70). Because of a lack of availability of TLR2 and TLR4 antibodies for mice, we chose to analyze the expression of mRNA for these two receptors by reverse transcription-PCR and found it to be significantly lower in macrophages from mice fed a calorie-restricted diet than from mice fed AL. These results suggest that the animals fed a calorie-restricted diet are likely to respond less to bacterial pathogens, whereas the response to an external insult is known to be intact in animals fed a calorie-restricted diet (53). Furthermore, the macrophages from mice fed a calorie-restricted diet showed hyporesponsiveness to the acute stress, as evidenced by down regulation of the expression of mRNAs for the LPS receptors TLR2 and TLR4 and the cytokines TNF-α, IL-6, and IL-12 as well as the release of decreased levels of TNF-α, IL-6, and IL-12 peptides.
It was established that IL-12 plays a crucial role in both the innate and the acquired immune responses. Although first recognized as a cytokine critical for activation of cytotoxic lymphocytes, the most important biological significance of IL-12 secretion relates to its effects on T-helper cells. IL-12 is known to drive Th1 cytokine production during physiological immune responses, mediated principally by its ability to stimulate proliferation and gamma interferon production in T cells and natural killer (NK) cells (29,33). Furthermore, clinical evidence reveals a critical role for IL-12 in early resistance to postoperative infection (31). We also noted that macrophages from mice fed a calorie-restricted diet express less MHC class II antigen than those from mice fed AL. LPS-induced augmentation of MHC class II antigen I-Ab was also found to be less in macrophages from mice fed a calorie-restricted diet. In addition to decreased levels of CD14, TLR2, and TLR4 expression, MHC class II also modifies LPS responsiveness. A lack of or a low level of expression of MHC class II molecules results in diminished secretion of proinflammatory cytokines by macrophages following stimulation with LPS (47). These results are consistent with those described in our first report that CR leads to impaired antigen processing and presentation by macrophages and/or defects in recognition of antigen in vivo by T cells in young C57BL/6 mice (17).
Thus, in support of our previous observation, our present results also show that CR either suppresses the macrophage phagocytic function or delays macrophage maturation. We used two different approaches to assess the macrophage phagocytic function: phagocytosis of latex beads and phagocytosis of zymosan. Both approaches showed similar trends. These results, however, do not agree with the findings of Dong et al. (19), who examined alveolar macrophages in rats fed a calorie-restricted diet. They found that CR enhanced phagocytic function in both control and ozone-exposed rats. The discrepancy may be due to two factors. First, the duration of CR and the species of animals used were different. They restricted calories only by 25% for 21 days, whereas we restricted calories by 40% for 5 months. Second, macrophages in different anatomical sites behave differently (5).
In addition to studying the functional activity of macrophages, we also used the CLP model to assess the effect of CR on septic shock. The CLP model in rodents is analogous to perforated appendicitis in humans (7). It produces peritonitis that leads to 95% mortality within 48 to 72 h (15). This is a widely used model that has been found to mimic the clinical disease symptoms of polymicrobial sepsis and septic shock (15). Our present results show that CR renders young C57BL/6 mice more susceptible to sepsis than mice fed AL. Animals in the group fed a calorie-restricted diet died earlier than animals in the group fed AL. Peck et al. (44) investigated the effects of CR and protein restriction on mortality in A/J mice subjected to salmonella infection. They showed that after 3 weeks of feeding of a calorie-restricted diet, CR improved the survival rate. This discrepancy may again be due to strain differences and differences in the duration of CR. For instance, the mice fed a calorie-restricted diet were supplemented with additional levels of minerals and a vitamin mixture to maintain their intake equal to that of the mice fed AL, whereas in the present study the mice fed a calorie-restricted diet were not supplemented with additional mineral and vitamin mixtures, which may cause increased numbers of deaths among the mice fed a calorie-restricted diet. However, this needs to be confirmed in studies with and without vitamin supplements to determine the protective effects of vitamins and minerals in preventing the immune deficiency in sepsis and/or other bacterial infections.
In our present studies, the earlier deaths due to sepsis in mice fed a calorie-restricted diet appear to be due to increased levels of TNF-α and IL-6 mRNA expression in the spleen and activation of the transcriptional factors NF-κB and NF–IL-6, whose levels we measured 4 h after induction of CLP in both groups. These results are in agreement with previous observations made in CLP studies by other investigators (12, 66). The levels of both TNF-α and IL-6 mRNA expression and NF-κB and NF–IL-6 activation in the spleen were found to be higher in the group fed a calorie-restricted diet than in the group fed AL. Several lines of evidence show that activation, nuclear translocation, and binding of NF-κB or NF–IL-6 are pivotal steps in controlling the transcription of genes coding for proinflammatory and immunoregulatory cytokines (41, 61, 68,72). Activation of NF-κB and NF–IL-6 in the early stages of sepsis accompanies bacteremia, cytokine expression, and mortality (12, 66). Furthermore, we observed that systemic TNF-α and IL-6 levels were significantly increased after CLP. Of greatest interest is that systemic TNF-α and IL-6 levels were much higher in mice fed a calorie-restricted diet than in mice fed AL. This finding is somewhat in contrast to the findings of Spaulding et al. (56), who reported that CR prevents the rise in serum TNF-α and IL-6 levels during aging. Those investigators, however, did not measure TNF-α and IL-6 levels after stimulation either in vivo or in vitro to establish the differences between young and old mice fed a calorie-restricted diet and young and old mice fed AL. TNF-α and IL-6 are the typical proinflammatory mediators, and it is well known that they are released during polymicrobial sepsis (2,4). High levels of IL-6 in the circulation have been reported to be one of the more consistent markers associated with increased rates of morbidity and mortality in traumatic injury and/or sepsis (8). Salkowski et al. investigated the production of cytokines and chemokines during the early phase of sepsis induced by CLP (51). The data indicate that polymicrobial sepsis stimulates a broad range of cytokines during the early periods of sepsis (51). In the present study, we did not examine cytokine expression in other organs, which needs to be carried out. Other studies have shown that, besides macrophages from the spleen, macrophages from the liver are another predominant source of proinflammatory cytokines during polymicrobial sepsis (3).
The results of our present CLP study suggest that CR, initiated at 6 weeks, increases the susceptibility of mice to polymicrobial sepsis and suppresses macrophage function, as evidenced by a decrease in the levels of expression of the LPS receptors CD14, TLR2, and TLR4 after stimulation by LPS and suppression of macrophage phagocytic function and MHC class II antigen expression. After infection is established, it is quite possible that macrophages in animals fed a calorie-restricted diet are unable to function adequately to prevent the rise in the number of invading pathogens, and the pathogens may spill over into the system due to the impairment of the activation of immune cells (T cells, NK cells, neutrophils). Furthermore, it is also possible that the regulation of Th-1 and Th-2 cytokines may be altered by CR, thereby releasing more proinflammatory cytokines, which leads to failure to confine the infection and, consequently, induction of more severe sepsis, septic shock, and/or multiorgan dysfunction.
The earlier deaths from sepsis in mice fed a calorie-restricted diet are quite surprising since CR is known to prolong the life span in virus-infected mice (25). We and others have reported higher cytotoxic T-cell function in mice fed a calorie-restricted diet (21, 24). We speculate that CR initiated at 6 to 8 weeks of age may have a selective immunosuppresive effect on macrophages due to increased levels of cortisol, which are known to increase in animals fed a calorie-restricted diet (50). It is possible that if CR is started in adult mice aged 4 to 6 months, the macrophage function may be less impaired compared to that in 6- to 8-week-old mice, although the survival rate is known to be much less in adult animals fed a calorie-restricted diet than in 6- to 8-week-old animals fed a calorie-restricted diet (63). However, this needs to be studied in detail soon.
Oxidant damage is known to be increased in septic shock and endotoxemia and has been implicated in the pathogenic mechanism (9,73). Several previous studies showed that CR can increase the levels of antioxidant enzymes superoxide dismutase and catalase and decrease the levels of free radicals (13, 55, 71). In order to determine the effect of CR on oxidative stress in vivo, both animals fed AL and animals fed a calorie-restricted diet were also treated with paraquat. In contrast to the deleterious effects of CR on mice with sepsis, the results from paraquat administration show that CR enhances or maintains the increased level of resistance to oxidative stress in vivo. Thus, the results of the present studies are similar to those reported earlier by others and show that mice fed a calorie-restricted diet have increased resistance to oxidative stress (53); the greater susceptibility to infection is not due to dietary deficiency and/or the effect of young age.
Although our present findings suggest that CR increases susceptibility to CLP in young mice, experiments are urgently needed to establish the role of CR during aging. It is quite possible that with age mice fed a calorie-restricted diet may develop increased resistance to bacterial infection, whereas older mice (24 months or older) fed AL may become more susceptible than mice fed a calorie-restricted diet due to the age-related decline in immune function (23, 59). We and several other investigators have shown a decline in IL-2 production with age in mice fed AL (23, 26, 43, 58), whereas CR maintains better cell-mediated immune function during aging. Thus, additional studies with aged mice fed a calorie-restricted diet and mice fed AL are needed to measure the response to CLP as well as resistance to well-known gram-negative and gram-positive bacterial pathogens.
This work was supported in part by Public Health Service grants AG14541 and AG13693 from the National Institute on Aging.
Received 18 January 2001.
Returned for modification 30 March 2001.
Accepted 30 May 2001.
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Clinical and Diagnostic Laboratory Immunology Sep 2001, 8 (5) 1003-1011; DOI: 10.1128/CDLI.8.5.1003-1011.2001
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Members of our Joint Coordination Group come together to discuss how we can best enhance our efforts to deliver and deploy vaccines by addressing challenges related to the research and development, regulation, stockpiling, and delivery of these products. In particular, while the group focuses on advancing our vaccine portfolio, its work also informs the broader field of vaccine development and preparedness.
Members also have a role in planning for rapid response to a priority pathogen or an unknown pathogen. Our Joint Coordination Group consists of 10 to 15 member organisations, which in turn appoint individuals to represent them at meetings.
From time to time, the group may choose to invite other organisations and experts to participate in meetings.
The current members of the Joint Coordination Group include: WHO, GAVI, EMA, FDA, MSF, UNICEF, IFRC, AVAREF, NIBSC, and Wellcome.


text:
Dopo aver messo in atto un golpe militare il 18 febbraio 2010, spodestando il presidente in carica Mamadou Tandja e formando una giunta militare a comando di un governo ad interim, il tenente Djibo si è insediato come presidente del Niger, divenendo così il leader de facto dello stato africano. Djibo ha espresso la volontà di portare il Niger verso la democrazia, istituendo il Consiglio Supremo per la Restaurazione della Democrazia.

Onorificenze

Onorificenze nigerine

Onorificenze straniere

Note

Altri progetti

redpajama_set_name: RedPajamaWikipedia


What is a Distinguished Ruffian?
Places – Travel, Night Life, Outdoors, Restaurants etc..
As this site has grown so has our idea of what to means to be a Distinguished Ruffian. With our members we stand tall as a group of growing men and women who want to be more than just ordinary…We are Ruffians BUT Distinguished. As Distinguished Ruffians, our desire is to inspire. Inspire thought. Inspire action. Inspire change. This is the seed, and the idea that the merging of distinction and ruthlessness can be achieved (without being a politician). We believe one can be a Ruffian and be Distinguished. The question now is, DO YOU?
How long did it take to get appoved?
Send me your contact information so I can help you out with this.
How does one join the Distinguish Ruffian?
On the website here we have a tab titled "Want To Be An Official Distinguished Ruffian." On that page you will find all the details you need for joining. We look forward to your application sir.
Thanks for my acceptance into this awesome group of badassdom, for I Am DISTINGUISHED AND A RUFFIAN.
I would like to be a ruffian.
Just found this most exellent place. All the things I love here…..well done.
I want to be a distinguished ruffian.But I m from Serbia. Little country in europe. End I speak bad english. My instagram name is krle_fuckerzz .
I see I am not on the worldwide map of DR's…this change needs to happen…Please.
Just submitted my application to become a DR and anxiously awaiting to be approved! This site/blog is amazing and encompasses everything I and my close friends subscribe to.
I'm located in SE Wisconsin and host a monthly pipe club meeting: Refer to our Facebook page: Kenosha Pipe Club and if you're in the neighborhood please stop by and share some good beer, smoke and conversation with us. Would love to meet you.
Just wondering… sent my application in…how long to be approved?
Thank you very much for the approval! cheers!
how long does it take to receive official letter and patch after approval and can merchandise be ordered before receiving official letter of membership?
Thank you for the addition! I've been a DR for a few months now, but not on the member map yet. How long does it usually take? Thanks so much and Merry Christmas everyone!
It takes a bit of time for me to get everyone loaded onto the map. Unfortunately there is no set amount of time it's just when I am able to have the time to do it. Hope to have it fully updated soon though.


Apple has made its March 7 iPad event official with an invite to the press, promising "something you really have to see. And touch."
The most likely feature for Apple's iPad 3 is a "Retina" display, or at least a screen with double the resolution of the iPad 2, at 2048 pixels by 1536 pixels. The upgraded display, combined with 4G LTE connectivity, may actually result in a slightly thicker frame than that of the iPad 2. For a processor, Apple watchers expect to see either a quad-core A6 processor inside the iPad 3, or a dual-core A5X.
The invite Apple sent for its event, which shows a small section of an iPad, has some tech bloggers in a tizzy because there's no home button on the iPad's bezel. But supposed pictures of the iPad 3's front glass panel shows the round home button intact, and it's possible that the image Apple sent is showing the side or the top of the device. I think the home button is sticking around.
A report last week on Apple.pro, a Chinese blog, suggested that Apple will bring the Siri virtual assistant to the iPad 3. This hasn't been a recurring rumor, but it seems plausible.
Best Buy has already dropped the price of the iPad 2 by $50 for all models, but last year, Apple slashed the original iPad's price by $100 after releasing the iPad 2. Once the new iPad launches, I'm guessing last year's model will get its own $100 price drop at all retailers--while supplies last.
Other Stuff: New Apple TV, iPods?
Earlier this month, the Apple TV set-top box sold out at Best Buy's Website, and it was no longer listed at Walmart. That led to speculation that a new Apple TV will replace the old set-top box, possibly with 1080p video support. The Apple TV box is not to be confused with the rumored Apple television, which probably isn't anywhere close to being finished.
I also noticed that Best Buy is having a sale on iPods. While there's not much buzz about an iPod refresh in the tech world, Apple hasn't updated its line of media players since September 2010. They're overdue for a facelift.


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OneSearch
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Library research guides
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CredoReference Topic Page: Poetry
Related Research Guides
Catalog Resources
Poet Biographies and Resources
Poetry Recommended Reading
MLA Citations, 8th Edition
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Hayley Bommarito
St. Clair County Community College
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Welcome! This library research guide provides an introduction to library resources related to both American and International poets and poetry.
This guide will assist you in finding books, e-books, scholarly journal articles, streaming videos, websites, and more that will help you research topics in this subject.
Poetry is defined as:
"an arrangement of words in verse, always rhythmical, sometimes rhymed, expressing facts, ideas, or emotions in a style more concentrated, imaginative, and powerful."
Poetry 101: From Shakespeare and Rupi Kaur to Iambic Pentameter and Blank Verse, Everything You Need to Know About Poetry
Become a poet and write poetry with ease with help from this clear and simple guide in the popular 101 series. Poetry never goes out of style. An ancient writing form found in civilizations across the world, poetry continues to inform the way we write now, whether we realize it or not--especially in social media--with its focus on brevity and creating the greatest possible impact with the fewest words. Poetry 101 is your companion to the wonderful world of meter and rhyme, and walks you through the basics of poetry. From Shakespeare and Chaucer, to Maya Angelou and Rupi Kaur, you'll explore the different styles and methods of writing, famous poets, and poetry movements and concepts--and even find inspiration for creating poems of your own. Whether you are looking to better understand the poems you read, or you want to tap into your creative side to write your own, Poetry 101 gives you everything you need!
Poetry & Short Story Reference Center Database
A collection of hundreds of thousands of classic and contemporary poems, as well as short stories, biographies and authoritative essays on such topics as poetic forms, movements and techniques
The Cambridge Introduction to Twentieth-Century American Poetry
The Cambridge Introduction to Twentieth-Century American Poetry is designed to give readers a brief but thorough introduction to the various movements, schools, and groups of American poets in the twentieth century. It will help readers to understand and analyze modern and contemporary poems. The first part of the book deals with the transition from the nineteenth-century lyric to the modernist poem, focussing on the work of major modernists such as Robert Frost, T. S. Eliot, Ezra Pound, Wallace Stevens, Marianne Moore, and W. C. Williams. In the second half of the book, the focus is on groups such as the poets of the Harlem Renaissance, the New Critics, the Confessionals, and the Beats. In each chapter, discussions of the most important poems are placed in the larger context of literary, cultural, and social history.
The Best Poems of the English Language: From Chaucer Through Frost
The great critic presents his personal selection, with commentary, of the finest poems in the English language. This comprehensive anthology attempts to give the common reader possession of six centuries of great British and American poetry. The book features a large introductory essay by Harold Bloom called "The Art of Reading Poetry," which presents his critical reflections of more than half a century devoted to the reading, teaching, and writing about the literary achievement he loves most. In the case of all major poets in the language, this volume offers either the entire range of what is most valuable in their work, or vital selections that illuminate each figure's contribution. There are also headnotes by Harold Bloom to every poet in the volume as well as to the most important individual poems.
The Princeton Handbook of World Poetries
The Princeton Handbook of World Poetries provides a comprehensive and authoritative survey of the history and practice of poetry in major regional, national, and diasporic literatures and language traditions around the globe.
Understanding Poetry: Rhythm and Meter, Rhyme
This program explores both the history and the essential components of poetry. After viewing, students will understand how poets utilize rhythm/meter and rhyme in various forms to achieve the desired effect. Classic works of poetry are presented along with detailed graphics, engaging examples, and exciting video. On-screen multiple-choice reviews at the end of each segment reinforce important concepts and make learning fun. Wonderscape Entertainment.
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URL: https://esearch.sc4.edu/poetry
Subjects: Communications, English and Literature, Humanities
Tags: beat poetry, blank verse, eng 207, english, epic poem, haiku, harlem renaissance, iambic pentameter, poems, poetry, poets, sonnet

redpajama_set_name: RedPajamaCommonCrawl


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Q: SELECT the oldest of the most recent lines I have a table storing the scores (with the date) of players they did at each game.
Example:
john  154  10/02/2014
mat   178  09/02/2014
eric  270  08/02/2014
mat   410  07/02/2014
john  155  06/02/2014

In this example I want "eric 270 08/02/2014" because thins is the oldest of the most recents.
Which request must I do to retrieve that ?

A: As I understand it, you request the oldest entry among the set containing the most recent one of each user.
In such a case, you can deal with your problem using a subquery given the last date for each user, then used in the main query to select and sort only the most recent entry of each user.
SELECT scores.*
FROM scores
INNER JOIN
(
  SELECT max(date) last, name
  FROM scores
  GROUP BY name
) last_temp_table
ON scores.name = last_temp_table.name
AND scores.date = last_temp_table.last
ORDER BY scores.date ASC LIMIT 1;

More info in different SO threads such as MySQL order by before group by

A: The question as worded doesn't make much sense unless you define most recent.
If I assume that you have some criteria like: "Give the oldest event that happened within the last 3 days" then that is a simple matter of ordering and limiting across a date range.
select * from events where ts >= CURDATE() - 3
 order by ts asc
  limit 1

redpajama_set_name: RedPajamaStackExchange


When you're planning a holiday with your family, where you want to go can be affected by the entire group. They need to generally agree on a destination or someone will feel left out, hurt, and angry. That person – or those people – may not have a very good time. The good news is that there are plenty of places that have something for everyone. These include London, Torquay in Devon, and the Isle of Wight.
All three are very family friendly and they have a wide range of things that they can offer to people who want to be pampered, people who want to play, and people who just want to see the sites and relax.
The chances are, especially if you have a big or extended family, that you're taking a lot of people with you and they all have different ideas about what a holiday should really involve. When they realize that they all can get what they want, making sure that they have a good time and picking a destination for your holiday can be a lot easier than it otherwise would have been. London has so much to offer, and Torquay in Devon does, as well, but the Isle of Wight often stands out as the most family-friendly place on the list because it has so many different things for people to do.
On the Isle of Wight there are a lot of popular towns like Shanklin. Hotel rates there are very reasonable and the accommodations are some of the best in the area. That doesn't mean that there aren't other great places to stay on the Isle of Wight, but only that Shanklin is one of the highest rated. There are archaeological areas to explore, and you can find fossils and artifacts from long ago. You can also go to all kinds of places to play games and enjoy time with your children. You can visit the coastline and walk along the rugged shore, or you can hike inland a bit and enjoy the natural beauty. You can also go to the spa and be pampered with saunas, steam rooms, and various treatments.
No matter what you decide that you want to do while you're on the Isle of Wight, there is truly something for everyone there, and that means that you and your family can all have a great time without being too terribly worried about pleasing the other members of your group. It makes for a great holiday for everyone.
For an ideal location on the Isle of Wight why not try a Shanklin Hotel for a great break away.


This wig fully wefted with adjusters at the back for a more secure and comfortable fit. The fringe is approx 3" and the whole look is feathered and light. The back is approx 1.5" at the nape so this really is a very short style for those looking for a pixie cut. This wig is average fitting.


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Poetry Old Pen
Pilot scheme giving professional artists & writers easier access to social welfare supports launched
Creative / Cultural Development
Positive step for professional artists and writers as Ministers Humphreys and Varadkar launch pilot scheme to make it easier to access social welfare supports.
The Minister for Social Protection, Leo Varadkar TD, and the Minister for Arts, Heritage, Regional, Rural and Gaeltacht Affairs, Heather Humphreys TD, have today, 12 June, announced the details of new arrangements to make it easier for artists and writers to access social welfare supports. The Ministers made the announcement at Poetry Ireland's new headquarters on Parnell Square, Dublin 1.
The initiative, which is a key commitment under the Creative Ireland Programme, will assist self-employed artists who apply to the Department of Social Protection for Jobseeker's Allowance. The pilot initiative, which will be reviewed after one year, will apply to visual artists and writers. Under the new mechanism, the Department of Social Protection will provide for the classification of self-employed professional artists. Such artists would not be subject to the activation process for 12 months.
Speaking today's launch Minister Humphreys said:
"Artists are at the very centre of the Creative Ireland programme, and I have been particularly keen for some time now to do more to recognise the income challenges faced by artists. This pilot scheme is not a panacea, but it is a clear sign that the Government recognises the vital role that artists play in Irish society and that we respect and value their contribution.
"I would like to thank Minister Varadkar for his consistent support for this initiative. We first met to discuss this idea early last year, and Leo has been very supportive ever since. I would also like to thank the Arts Council which has provided invaluable support and advice as to how the scheme should operate. I would also like to thank the Irish Writers' Centre and Visual Artists Ireland for their input and guidance. Getting this far has been a team effort.
"This is a pilot initiative which will initially be available to writers and visual artists. The issue of income for artists is something that has been raised with me on a regular basis, so I hope this pilot initiative can be seen as a very positive step for the arts community. We will closely monitor the implementation of the pilot initiative, before considering whether extending the arrangements to professional artists in other disciplines."
Speaking at the announcement Minister Varadkar said:
"Artists are central to Irish society, and I believe it is only right that we allow for some flexibility within the social welfare system to allow artists to access social welfare supports when they need them. Up to now, artists have found it difficult to access social welfare and of course many artists take on extra jobs to support their livelihoods.
"Following extensive work between both Departments, with input from the Arts Council, this new mechanism will allow professional self-employed artists to be classified as such for the purposes of accessing social welfare supports. I welcome the involvement of Visual Artists Ireland and the Irish Writers Centre in this process. The normal checks and balances will apply to ensure the initiative is not open to abuse, but it is my hope that this will make it much easier for professional artists to access social welfare supports when they need them.
"I am very supportive of the Creative Ireland programme, which has the potential to be transformative in terms of public policy. Creative Ireland aims to increase both access and participation in artistic and cultural activities; we need to also ensure that artists are adequately supported and I believe today's announcement is a very positive development in this regard."
Social Welfare Pilot Scheme For Artists & Writers
Map Irish Design project explores the role of design in shaping Irish society


I helped a father obtain primary custody of his daughter and prevent the mother from moving away to Texas with her new husband and the child. The father was African American, the mother Filipino, and the new husband Caucasian. Race was absolutely not a factor in the court's decision, nor in my approach to the case, but they wanted to move with the child to a small town in Texas, where the new husband's family lived. What was a factor in the case and the decision was the mother's and new husband's belittlement of the father's relationship with the child, their passive-aggressive interference with that relationship and their flaunting of court ordered visitation details. My client won primary custody after a four-day trial. Rather than move without the child and limit her contact further, the mother and her new husband stayed in California, and remain here to this day.
I helped a mother obtain primary custody of her child, whom she had not seen for over one year. The mother and father lived in different counties, about an hour apart by car. The mother had left the relationship because of domestic violence. She later heard from her son that the father might be abusing him, at least psychologically, and attempted to protect him by leaving the jurisdiction with him, although she kept him in the same school, commuting three hours a day. As a result, the father obtained sole custody and supervised visitation for the mother, with the help of an attorney. The mother, at the time was self-represented, and did not know what to do. For the next year the mother concentrated on her older child by a different father, who was having teenage problems, and was diagnosed bi-polar. When her daughter was stabilized the mother retained me. By this time she was at risk of losing her parental rights to her son, not having had any contact with him for over one year and not paying any child support. We went to court and enforced the supervised visitation order, gradually moving to unsupervised visitation after about six months. After another six months, we obtained an order for a custody evaluation by a psychologist, and the court also appointed an attorney for the child, who informed the court that the child wanted to live with his mother. The psychological evaluation favored primary custody for the mother, and revealed some psychological problems with the father, including depression. With opposing counsel, we negotiated a settlement, giving my client primary custody during the school year.
I helped a father obtain primary custody of his son and successfully fought false domestic violence allegations against him, including a referral to a criminal defense attorney who successfully fought the same allegations in criminal court. I also referred my client to a civil rights attorney who settled a lawsuit against the police department for injuring him during his arrest. I settled the divorce and property division aspects of his case, and continued on to fight for custody. The mother had undiagnosed psychological issues, from the father's description of her behavior. We obtained orders for a psychological evaluation, which favored primary custody to my client. The mother's attorney asked for appointment of an attorney for the child, as a last resort. The child's attorney revealed even more disturbing behavior by the mother in her report, which resulted in an order for sole custody of the child to my client, and supervised visitation to the mother. The mother is finally getting psychological help, and after a year of no contact, the mother and child are having supervised visits.
I helped a couple negotiate a full settlement of their divorce, including joint custody of their son. The husband was a winery executive, who formerly worked in the dot com industry, as did his wife, a talented programmer. After moving to Napa and starting his new job in the wine industry, the wife stopped working, and lost interest in everything. I believe she was suffering from depression, though we never got a formal diagnosis. The husband believed that if the mother would simply go back to work – she could earn close to six figures working from home – she would regain interest in life. She kept refusing to work, so he asked her for a divorce. He was very generous in his settlement offer, giving her more than half their substantial assets, and allowing her to stay living in their home with him and their son, so long as the boy was school-aged. We finalized the marital settlement agreement and judgment documents, and filed everything. The husband came to see me six months later, and told me that they still lived together, his wife was back working and enjoying life again, and they had never been happier, continuing on in their full relationship and parenting their child together … just not married!
I helped obtain orders for the return of a child to her father in the U.S., after the child was abducted by her mother and taken to her native country, France, against court orders. I obtained a sole custody order in Napa County, and, with the assistance of the Napa County District Attorney's Child Abduction Unit, was able to have the order enforced in France by French counsel through the Hague Convention, including two appeals the mother filed in France. While awaiting the results in France, I successfully fought an attempt by the mother's pro bono international law attorney, whom she found in San Francisco, to have the Napa sole custody order rescinded. The attorney then had the case removed to federal court, but, with the help of a federal practitioner, whom I associated into the case, I prepared and filed a brief opposing the removal and got the case remanded back to Napa Superior Court. It took two and a half years, but the child is now in her father's primary custody in the United States, and the mother's contact is limited to school vacations. To assure the mother's compliance with the visitation orders, I negotiated a bond which would forfeit her parents' house in France should she re-abduct the child.
If you or someone you know in Northern California needs the assistance of an experienced Napa Family Attorney, call Christopher Loizeaux today at 707-200-1293, or complete the contact form provided on this site to schedule your free phone consultation.


text:
Flyers beat Penguins, 5-1, to tie first-round series
Published 1:49 am Saturday, April 14, 2018
By Post Sports
Pittsburgh Penguins' Sidney Crosby (87) leaps out of the way of a shot from the point against Philadelphia Flyers goaltender Brian Elliott, left, during the second period in Game 2 of an NHL first-round hockey playoff series in Pittsburgh, Friday, April 13, 2018. (AP Photo/Gene J. Puskar)
PITTSBURGH (AP) — Sean Couturier had a goal and two assists, Brian Elliott stopped 34 shots and the Philadelphia Flyers beat the Pittsburgh Penguins 5-1 on Friday night to even the first-round Eastern Conference series at a game apiece.
Shane Gostisbehere, Travis Konecny, Andrew McDonald and Nolan Patrick also scored for the Flyers. They bounced back from an embarrassing 7-0 loss in the opener to beat the Penguins for the first time this season. Game 3 is Sunday in Philadelphia.
Patric Hornqvist scored Pittsburgh's lone goal with just over 5 minutes left to avoid the shutout, but the Penguins looked ordinary for long stretches after looking unstoppable in Game 1.
Matt Murray's shutout streak dating to the 2017 Stanley Cup finals ended at 226:49 when Gostisbehere scored on the power play late in the first period. Murray finished with 16 saves to lose for just the third time in 17 home playoff starts in his career.
JETS 4, WILD 1
WINNIPEG, Manitoba (AP) — Paul Stastny, Andrew Copp and Patrik Laine scored in the third period and Winnipeg beat Minnesota to take a 2-0 lead in the Western Conference first-round series.
Defenseman Tyler Myers had a goal in the second period and also had an assist. Rookie Jack Roslovic, replacing injured Mathieu Perreault, had two assists in the game that ended with a series of fights.
Zach Parise had his second goal of the series when he deflected Mikko Koivu's shot past Connor Hellebuyck to end the goalie's shutout bid with 45 seconds left. The goal came on the power play after Jets forward Brandon Tanev was called for hooking with 59 seconds left.
Hellebuyck made 16 saves. Devan Dubnyk stopped 39 shots for Minnesota.
Winnipeg won 3-2 on Wednesday night. Game 3 is Sunday at Minnesota.


"Go On Treading Difficult Path" of success because a professional journey of any exemplary successful individuals, persons, people, teams, organisations or any type characteristics is not a bed of roses but full of thorns."Go On Treading Difficult Path" of achievements because a professional journey of any high-end achievers, individuals, persons, people, teams, organisations or any type characteristics is not a bed of roses but full of thorns. "Go On Treading Difficult Path" of achievements because a professional journey of any commendable go-getter, individuals, persons, people, teams, organisations or any type characteristics is not a bed of roses but full of thorns."Go On Treading Difficult Path" of achievements because a professional journey of any laudable performer, individuals, persons, people, teams, organisations or any type characteristics is not a bed of roses but full of thorns. I am neither demotivating nor giving you an injection of negativity rather preparing you to face and find solutions in your professional journey to convert these thorns into a bed of roses. Either just by throwing away and rejecting the thorns out of roses or breaking their sharp and hurting tip to make them harmless and redundant. So anyway you have to face the hurdles, difficult situations, persons who create stumbling blocks in your way of professional success and you have to win over it.
To be successful in Business, in a Job, in Projects you have undertaken or wherever you are in and to be number one in business and to be number one in job amongst your colleagues, amongst your coworkers, amongst your employees, amongst your employer "Go On Treading Difficult Path" and cross it and win over it with your passion for facing and overcoming difficult situations and influence and persuading people to work for you.
To be successful in Business, in a Job, in Projects you have undertaken or wherever you are in and to be number one in business and to be number one in job amongst your colleagues, amongst your coworkers, amongst your employees, amongst your employer "Go On Treading Difficult Path" and cross it and win over it only when you develop your confidence , dependability and truthfulness on people working for you giving them a message that is you are trustworthy.
To be successful in Business, in a Job, in Projects you have undertaken or wherever you are in and to be number one in business and to be number one in job amongst your colleagues, amongst your coworkers, amongst your employees, amongst your employer "Go On Treading Difficult Path" and cross it and win over it only when you do not compromise with core values of business like quality, performance, durability etc and many more core qualities and be flexible and keep on changing your plan and strategies as per customer's demands and requirements.
To be successful in Business, in a Job, in Projects you have undertaken or wherever you are in and to be number one in business and to be number one in job amongst your colleagues, amongst your coworkers, amongst your employees, amongst your employer "Go On Treading Difficult Path" and cross it and win over it with your fearlessness and boldness to win over failures with totally new policies and programmes.
To be successful in Business, in a Job, in Projects you have undertaken or wherever you are in and to be number one in business and to be number one in job amongst your colleagues, amongst your coworkers, amongst your employees, amongst your employer "Go On Treading Difficult Path" and cross it and win over it with your prompt and timely decisions to take full advantage of any opportunity knocking at the door of your organisation.
To be successful in Business, in a Job, in Projects you have undertaken or wherever you are in and to be number one in business and to be number one in job amongst your colleagues, amongst your coworkers, amongst your employees, amongst your employer "Go On Treading Difficult Path" and cross it and win over it by taking lot of care of health and finance of you and your staff, colleagues, coworkers, employees more than the machinery and software of the company.
SO GO ON TREADING DIFFICULT PATH TO BE VICTORIOUS AND CONQUEROR.
" MERITORIOUS PEOPLE ALWAYS TREAD THE MOST DIFFICULT PATH AND WIN OVER IT."
Maniparna thanks for your comments.I am really sorry for responding late.I am in total agreement with you that treading on different paths can only make you well versed with highs and lows, ups and downs of various paths.Though we tread smoothly on comfort zones but that makes us sleepy and easy goer.Your comment are thought provoking.Thanks.


text:
Q: Google App Engine Java: how to remove unused indexes? If I found information about removing unused indexes, like in Uploading and Managing a Python App / Deleting Unused Indexes, it was only for the Python environment...
Any way to tag an index in the [~project]/war/WEB-INF/datastore-indexes.xml file?

A: Since version 1.4.2 the Java SDK supports the vacuum_indexes action.
Run this command to delete unused indices:
./<appengine-java-sdk-path>/bin/appcfg.sh vacuum_indexes <myapp-path>/war

On Windows with the Eclipse SDK it's something like:
<eclipse-path>\plugins\com.google.appengine.eclipse.sdkbundle_1.4.3.v201103311225\appengine-java-sdk-1.4.3\bin\appcfg.cmd vacuum_indexes <myapp-path>\war

A: The only way you currently can remove an index is using the Python SDK as described here.
The missing vacuum_indexes functionality from the Java SDK is logged as a bug, but not yet fixed.


text:
Repositories Index Register
L1 - Educational Studies in Languages and Literature
L1 Educational Studies in Language and Literature
Publications Top 10
Matre, S. & Solheim, R. (2015). Writing education and assessment in Norway: Towards shared understanding, shared language and shared responsibility. Contribution to a special issue Paradoxes and Negotiations in Scandinavian L1 Research in Languages, Literatures and Literacies, edited by Ellen Krogh and Sylvi Penne. L1-Educational Studies in Language and Literature, 15, pp. 1-33. http://dx.doi.org/10.17239/L1ESLL-2015.15.01.05
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MC-D250RTC-B 250cc Ninja-style Sports Bike- Exclusive Limited Edition!
250cc Street Legal Full-Size Motorcycle!
The new Ninja-Streetfighter-Style MC-D250RTC-B offers pure motorcycling thrills thanks to its perfect combination of Rock Solid 250cc 4-stroke engine and a new light weight naked style framing. Outstanding control and precision make the RTC-B irresistible.
The RTC-B is upgraded and modified to be lighter, more powerful and with a more aggressive look.
This well-mannered middleweight Streetfighter sports bike is simple to use, cheap to buy and cheap to run. If you take this RTC-B out on a twisting back road and let its class-leading handling, brakes and rock solid engine performance shine. Do that a few times and like us, you'll be hooked too.
The chassis complements the engine well and it feels light on its feet. The suspension offers a good balance between sport and comfort.
The RTC-B derived 4-cylinder engine of 250cc capacity supplies plenty of relaxed grunt everywhere in the rev-range. High performance Mikuni carburetor supplies the unleaded to the donk via a 3.5 gallon tank.
Cooling is taken care of by the well proven air cooling system that served the 250RTC-B so well for many years and ensures this aggressive look bike never gets too hot and bothered.
The 5-speed gearbox is as positive as they come and a sheer joy to use.
Final drive is via the normal chain drive set-up. Chain lubrication is convenient by the easy to use centre-stand.
Instrumentation is acceptable with both a large Digital Speedo and tachometer. An extremely welcome addition that is not seen on enough bikes these days is the reliable and easy to read fuel gauge.
Seat height is quite reasonable at 31.5 inches and ground clearance is more than adequate at 5.15 inches. The seat is quite supportive even for medium to long distances.
The front forks give the rider enough confidence to push the bike reasonably hard in to tighter corners. Riders that would push this bike over its limits should really be riding something a bit sportier.
With front and rear disc braking system, the Streetfighter is not only efficient and powerful, but safe too. 270mm disc is arrested by two-piston calipers at the front, whilst rear 220 mm discs adopt a twin-piston caliper for precise control.
The suspension features preload forks and a conventional monoshock rear end that is adjustable for both rebound damping and preload. These work very well for a bike so reasonably priced.
Handling is quite good with light steering and quite good feel for a bike of this class. The upright bars and smooth power delivery make rear-end drifts out of tight corners quite easily controlled.
You are probably wondering how we can afford to sell these scooters at such low prices. We don't hand pick products from different factories; we design, we select the best quality parts, and we build our own scooters! Factory direct! We ship directly from our 96,000 sq. ft. warehouse here inChinoCalifornia. We are here 7 days a week to assist you with your every need.
Race-developed & lightweight conventional forks delivers superb feedback and consistent performance.
This Motorcycle/Scooter is registerable with the department of motor vehicles and legal to own and operate in the public road in all states except for CA, PA and NJ. Title, stickers, and other registration documents will be included with your purchase.