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Bacterial DNA and Lipopolysaccharide Induce Synergistic Production of TNF-α Through a Post-Transcriptional Mechanism
Jian Jun Gao, Qiao Xue, Christopher J. Papasian and David C. Morrison
J Immunol June 1, 2001, 166 (11) 6855-6860; DOI: https://doi.org/10.4049/jimmunol.166.11.6855
Jian Jun Gao
Department of Basic Medical Sciences, University of Missouri, and
Qiao Xue
Christopher J. Papasian
David C. Morrison
Department of Basic Medical Sciences, University of Missouri, and Saint Luke's Hospital, Kansas City, MO 64111
LPS is well recognized for its potent capacity to activate mouse macrophages to produce TNF-α, an important inflammatory mediator in bacterial infection-related diseases such as septic shock. We demonstrate here that while inducing only low levels of TNF-α alone, DNA from both Gram-negative and Gram-positive bacteria synergizes with subthreshold concentrations of LPS (0.3 ng/ml) to induce TNF-α in the RAW 264.7 macrophage-like cell line. The bacterial DNA effects are mimicked by synthetic CpG-containing oligodeoxynucleotides, but not non-CpG-containing oligodeoxynucleotides. Pretreatment of macrophages with either DNA for 2–8 h inhibits macrophage TNF-α production in responses to DNA/LPS. However, when pretreatment was extended to 24 h, DNA/LPS synergy on TNF-α is further enhanced. RT-PCR analysis indicates that mRNA levels of the TNF-α gene, however, are not synergistically induced by bacterial DNA and LPS. Analyses of the half-life of TNF-α mRNA indicate that TNF-α message has a longer half-life in bacterial DNA- and LPS-treated macrophages than that in bacterial DNA- or LPS-treated macrophages. These findings indicate that the temporally controlled, synergistic induction of TNF-α by bacterial DNA and LPS is not mediated at the transcriptional level. Instead, this synergy may occur via a post-transcriptional mechanism.
Recent data indicate that purified bacterial DNA can activate macrophages and other inflammatory cells (reviewed in Refs. 1 and 2). Macrophages stimulated with bacterial DNA reportedly produce proinflammatory cytokines such as TNF-α (3, 4), IL- 1 (4), IL-6 (5), IL-12 (6, 7, 8), IFN-αβ (9, 10), IFN-γ (9), and the reactive nitrogen intermediate, NO (11, 12). Subtle structural differences between bacterial and eukaryotic DNA apparently account for the ability of bacterial DNA to serve as an immune-activating agent. Specifically, bacterial DNA is thought to activate inflammatory cells because of its high content of short sequences with unmethylated CpG dinucleotides (13). In mammalian DNA, CpG-containing sequences occur at a much lower frequency than in bacterial DNA, and the cytosine present in CpG dinucleotides of mammalian DNA is usually methylated (14, 15).
In vivo studies support the concept that bacterial DNA is an important proinflammatory stimulus, as bacterial DNA has been shown to trigger septic shock in d-galactosamine treated mice. (16). Also of interest is the finding that bacterial DNA acts synergistically with LPS to induce TNF-α production in vivo, resulting in lethal shock in mice (11, 17). The molecular mechanism(s) by which bacterial DNA acts synergistically with LPS to induce TNF-α production remains to be determined. LPS, a constituent of the Gram-negative bacterial cell wall, is well known as a potent inducer of mouse macrophage activation, resulting in production of many inflammatory mediators, including TNF-α, that play a key role in the development of septic shock (reviewed in Refs. 18 and 19). The induction of TNF-α secretion from mouse macrophages in response to LPS stimulation is controlled at transcriptional, post-transcriptional, and translational levels (17, 20, 21).
In this communication we explore the mechanisms by which LPS and bacterial DNA act synergistically to activate macrophages. We present in vitro data demonstrating that bacterial DNA acts synergistically with substimulatory concentrations of LPS to enhance TNF-α secretion by the murine RAW 264.7 macrophage-like cell line. The observed synergy depends upon the presence of unmethylated CpG residues in DNA and is also dependent upon the temporal order of treatment by LPS and bacterial DNA. Enhanced TNF-α secretion by RAW 264.7 cells simultaneously exposed to bacterial DNA and LPS is not accompanied by enhanced transcription of the TNF-α gene. Analyses of the half-life of TNF-α in differentially treated macrophages suggest that bacterial DNA and LPS act synergistically to enhance TNF-α production through a post-transcriptional event.
Purified LPS from Escherichia coli O111: B4 was obtained from List Biological Laboratories (Campbell, CA). E. coli strain B genomic DNA, salmon sperm genomic DNA, and RNase-free DNase I were purchased from Sigma (St. Louis, MO). Synthetic oligodeoxynucleotides (ODNs;3 T3, 5′-AACGTT AACGTT AACGTT-3′; C3, 5′-CCATGGCCATGGCCATGG-3′) were obtained from Sigma-Genosys (The Woodlands, TX). The endotoxin levels in these ODNs are <0.01 ng/μg of DNA based upon the Limulus amebocyte lysate assay.
Culture of macrophages
The murine macrophage-like cell line RAW 264.7 (American Type Culture Collection, Manassas, VA) was used in all the studies described here. Macrophages were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 10% heat-inactivated FBS (endotoxin content of <0.06 ng/ml; Sigma) at 37°C in a humidified, 5% CO2 environment. Before being stimulated, macrophages were seeded into culture plates and cultured overnight.
E. coli DNA and salmon sperm DNA (Sigma) were further purified by two-step CsCl ultracentrifugation. DNA from Staphylococcus aureus was extracted exactly as described by Dyer and Iandolo (22). DNA digestion was performed using RNase-free DNase I (2 U/μg of DNA) in buffer (pH 7.6) containing 20 mM Tris-HCl and 20 mM MgCl2 at 37°C for 3 h. The endotoxin levels in these DNA preparations were <0.001 ng/μg of DNA according to the Limulus amebocyte lysate assay.
TNF-α analysis
After 20 h of stimulation, TNF-α production in macrophage culture supernatants was analyzed using the ELISA Duoset kit (purchased from R&D Systems, Minneapolis, MN). The protocol from the manufacturer was followed exactly for the assay. All data for TNF-α represent the average of duplicate samples ± SEM. Each experiment was repeated at least twice.
RNA isolation and RT-PCR analysis
RAW 264.7 macrophages were stimulated for various periods of time with different combinations of stimuli as described in Results. Total RNA from macrophages was isolated using TRIzol reagent (Life Technologies) according to the manufacturer's instructions. A total of 1 μg of RNA from each sample was used for RT-PCR using the One-Step RT-PCR kit from Qiagen (Valencia, CA) according to the manufacturer's protocols. The sequences of the specific primers used in these studies are: mouse TNF-α sense, 5′-GGC AGG TCT ACT TTG GAG TCA TTG C-3′; mouse TNF-α antisense, 5′-ACA TTC GAG GCT CCA GTG AAT TCG G-3′; mouse β-actin sense, 5′-TGT GAT GGT GGG AAT GGG TCA G-3′; and mouse β-actin antisense, 5′-TTT GAT GTC ACG CAC GAT TTC C-3′. PCR products were analyzed using agarose gel electrophoresis, stained with ethidium bromide, and photographed. The photographs were scanned using Adobe PhotoShop software (Adobe Systems, San Jose, CA) and analyzed using a GelPro Analyzer (Meyer Instruments, Houston, TX).
It has been reported that LPS and bacterial DNA act synergistically to enhance TNF-α production in vivo, which contributes to septic shock in mice (3, 23). Because macrophages are a major source of TNF-α in vivo, we endeavored to reproduce this phenomenon in vitro with cultured mouse macrophages. RAW 264.7 macrophages were exposed to various concentrations of purified E. coli DNA with or without LPS at a subthreshold concentration (0.3 ng/ml; Fig. 1⇓). At this subthreshold concentration LPS by itself either fails to induce TNF-α secretion or induces a minimal response (data not shown). RAW cells stimulated with E. coli DNA alone produced relatively low levels of TNF-α (<2000 pg/ml) even at the highest DNA concentration tested (30 μg/ml) in this study (Fig. 1⇓A, ▴). However, in the presence of a subthreshold LPS stimulus the TNF-α response to E. coli DNA was dramatically enhanced. For example, TNF-α production induced by 3.0 μg/ml E. coli DNA was increased ∼14-fold by adding 0.3 ng/ml LPS (Fig. 1⇓A, ▴). In the presence of LPS, TNF-α production was dose dependent for E. coli DNA stimuli between 1.0 and 10 μg/ml, then subsequently declined. Importantly, TNF-α secretion in response to E. coli DNA plus a subthreshold LPS stimulus was almost completely abrogated by treatment with DNase I, indicating that the response is specific to DNA (Fig. 1⇓A, ▪). Salmon sperm DNA, which contains highly methylated CpG dinucleotides, induced a weak TNF-α response by itself and failed to synergize with LPS, suggesting that the macrophage-activating ability of DNA is dependent upon nonmethylated CpG dinucleotides (Fig. 1⇓A, • and ○).
Induction of TNF-α by E. coli DNA, S. aureus DNA, and synthetic ODNs. RAW 264.7 macrophages were stimulated with E. coli DNA (A), S. aureus DNA (B), or synthetic CpG-containing ODN (C) in either the absence (▴) or the presence (▵) of 0.3 ng/ml LPS for 20 h before culture supernatants were collected for TNF-α ELISA. Controls include salmon sperm DNA (A and B, •), salmon sperm DNA plus LPS (A and B, ○), DNase I-treated E. coli DNA plus LPS (A, ▪), DNase I-treated S. aureus DNA plus LPS (B, ▪), synthetic non-CpG-containing C3 (C, •), and C3 plus LPS (C, ○).
To determine whether the ability of E. coli DNA to act synergistically with LPS was applicable to other bacterial species, we repeated the above experiments with DNA purified from the Gram-positive bacteria, S. aureus. S. aureus DNA alone induced RAW cells to produce only low levels (<2000 pg/ml) of TNF-α (Fig. 1⇑B, ▴). However, in the presence of a subthreshold LPS stimulus (0.3 ng/ml), S. aureus DNA induced a greatly enhanced TNF-α response that was dose dependent (Fig. 1⇑B, ▵). This synergistic induction of TNF-α by S. aureus DNA plus LPS was also abolished by treatment with DNase I (Fig. 1⇑B, ▪). Control salmon sperm DNA did not stimulate macrophages to produce significant levels of TNF-α, either alone or in the presence of LPS (Fig. 1⇑B, • and ○).
The stimulatory activity of E. coli and S. aureus DNA could be reproduced by a synthetic nonmethylated CpG-containing ODN (termed T3; see Materials and Methods). Although T3 by itself was a more potent stimulus than either E. coli or S. aureus DNA alone, it still induced only low levels of TNF-α secretion (peaking at ∼4000 pg/ml) from RAW macrophages (Fig. 1⇑C, ▴). However, as with E. coli and S. aureus DNA, T3-induced TNF-α secretion was markedly enhanced (peaking at ∼12,000 pg/ml) in the presence of a subthreshold LPS stimulus (Fig. 1⇑C, ▵). In marked contrast to the results with T3, treatment of macrophages with a non-CpG-containing ODN (C3) resulted in minimal TNF-α secretion (<1000 pg/ml) in either the absence or the presence of LPS (Fig. 1⇑C, • and ○).
It has been reported that TNF-α production by mouse macrophages in response to LPS stimulation is controlled at both transcriptional and post-transcriptional levels (17, 20, 21). However, the mechanism by which bacterial DNA plus LPS act synergistically to induce macrophage TNF-α production remains to be determined. To assess whether synergistic induction of macrophage TNF-α by bacterial DNA and LPS is controlled at the level of gene transcription, macrophages were treated for 2 h with E. coli DNA or LPS alone or with a combination of E. coli DNA plus LPS (along with appropriate controls) as described in Fig. 2⇓. After stimulation, total RNA was extracted from macrophages and subjected to RT-PCR analysis for detection of TNF-α mRNA. TNF-α mRNA levels in macrophages treated with either E. coli DNA (1.0 μg/ml) or LPS (0.3 ng/ml) were elevated compared with TNF-α mRNA from control macrophages treated with cell culture medium; recall that LPS at this concentration fails to induce a TNF-α response, and E. coli DNA induced low levels of TNF-α secretion (<2000 pg/ml). Simultaneous treatment of macrophages with both E. coli DNA and LPS did not further enhance TNF-α mRNA expression compared with treatment with either E. coli DNA or LPS alone despite the finding that this combined stimulus markedly enhanced TNF-α secretion. Treatment of E. coli DNA with DNase I essentially abrogated the TNF-α mRNA response to E. coli DNA alone, but did not alter the TNF mRNA production observed in response to simultaneous E. coli DNA plus LPS treatment. Finally, although salmon sperm DNA by itself failed to induce significant TNF-α mRNA production, TNF-α mRNA levels induced by simultaneous treatment with salmon sperm DNA and LPS were comparable to those induced by simultaneous E. coli DNA and LPS treatment. Together these data all support the conclusion that E. coli DNA and LPS do not act synergistically to enhance TNF-α mRNA production.
Induction of TNF-α mRNA by E. coli DNA and LPS. Macrophages were stimulated with culture medium alone, medium containing 1 μg/ml E. coli DNA, DNase I-treated E. coli DNA, or salmon sperm DNA in the absence or the presence of 0.3 ng/ml of LPS for 2 h before RNA isolation for RT-PCR analysis of TNF-α message (20 PCR cycles), as described in Materials and Methods. mRNA of the β-actin gene amplified under the same conditions as the internal controls.
In addition to the experiments presented above in which macrophages were stimulated for 2 h before RNA extraction, we also performed experiments in which RNA was extracted after 0.5, 1, and 4 h of stimulation. Varying the period of stimulation did not alter the findings. Simultaneous treatment of macrophages with both E. coli DNA and LPS did not further enhance TNF-α mRNA expression compared with treatment with either E. coli DNA or LPS alone for any of the stimulation periods tested (data not shown).
The data presented above indicate that although a subthreshold LPS stimulus (0.3 ng/ml) synergizes with bacterial DNA to enhance TNF-α secretion from RAW 264.7 macrophages, LPS and bacterial DNA do not synergize to enhance TNF-α mRNA expression. It has previously been shown that TNF-α gene expression is regulated at both transcriptional and post-transcriptional levels when LPS alone is the stimulus (17, 20, 21). Consequently, it is reasonable to hypothesize that enhanced TNF-α production in response to a combined LPS and bacterial DNA stimulus results from altered post-transcriptional controls. To test this hypothesis, we determined the half-life of TNF-α mRNA in macrophages treated with E. coli DNA (Fig. 3⇓A) or LPS alone (Fig. 3⇓B), E. coli DNA combined with LPS (Fig. 3⇓C), or salmon sperm DNA combined with LPS (Fig. 3⇓D). To determine the half-life of TNF-α mRNA, transcription was inhibited by treating macrophages with actinomycin D (5.0 μg/ml) after they had been exposed to specific stimuli for a period of 2 h. Total RNA was isolated from macrophages at various time points after actinomycin D treatment and subjected to RT-PCR analysis to assay for TNF-α mRNA. The half-lives of TNF-α mRNA in macrophages stimulated with bacterial DNA alone, LPS alone, bacterial DNA plus LPS, or salmon sperm plus LPS are estimated to be 36, 45, 100, and 45 min, respectively (Fig. 3⇓). Thus, the half-life of TNF-α mRNA in macrophages stimulated with a combination of LPS plus bacterial DNA is 2–3 times that of TNF-α mRNA from macrophages exposed to any of the other stimuli tested. This prolonged half-life of TNF-α mRNA could account at least in part for the synergistic production of TNF-α by macrophages exposed to a combination of bacterial DNA and LPS.
TNF-α mRNA stability in macrophages treated with different combination of stimuli. Macrophages were stimulated with E. coli DNA (3 μg/ml; A), LPS (0.3 ng/ml; B), E. coli DNA (3 μg/ml) plus LPS (0.3 ng/ml; C), or salmon sperm DNA (3 μg/ml) plus LPS (0.3 ng/ml; D) for 2 h, at which time new transcription was stopped by treatment with transcription inhibitor actinomycin D (5 μg/ml). At increasing times total RNA was extracted, and the presence of TNF-α mRNA was analyzed by RT-PCR. E, The density of each TNF-α band in A and B was expressed as the fraction of that observed for the controls (A and B, zero time points). Cell viability was not affected by treatment with 5 μg/ml actinomycin D, as judged by the >95% cellular exclusion of trypan blue in parallel cultures.
The sequence and timing of exposure to two different stimuli often impact the ability of these stimuli to synergize, presumably because pertinent activating signals must be initiated in a permissive temporal pattern. When multiple stimuli were used in the experiments presented above, macrophages were simultaneously exposed to these stimuli. For the experiments presented in Fig. 4⇓, the sequence and timing of exposure to bacterial DNA and LPS were altered. Pretreatment of macrophages with E. coli DNA for 2–8 h before adding E. coli DNA plus LPS (0.3 ng/ml) resulted in a time-dependent reduction of TNF-α secretion (Fig. 4⇓, •). The maximum observed effect in this experiment was when macrophages were pretreated with E. coli DNA for 8 h before adding LPS; TNF-α secretion was reduced more than 3-fold compared with macrophages that were not pretreated. Interestingly, when pretreatment with E. coli DNA was extended to 24 h, TNF-α secretion was comparable to levels attained in macrophages that were not pretreated (Fig. 4⇓, compare 24 and 0 h points). Macrophages pretreated with LPS for 2–8 h before adding E. coli DNA plus LPS also displayed a time-dependent reduction in TNF-α secretion compared with macrophages that were not pretreated (Fig. 4⇓, ○). However, the reduction in TNF-α secretion was not as extensive as that observed with E. coli DNA pretreatment (Fig. 4⇓, compare ○ and •). As with DNA pretreatment, when pretreatment with LPS was extended to 24 h, TNF-α secretion was comparable to levels attained with macrophages that were not pretreated (Fig. 4⇓, compare 24 and 0 h points).
Effects of E. coli DNA and LPS pretreatment on induction of TNF-α from macrophages in response to E. coli DNA plus LPS. Macrophages were preincubated with 3 μg/ml E. coli DNA or 0.3 ng/ml LPS for various amounts of time as shown. They were then washed twice with culture medium and continually incubated with 3.0 μg/ml E. coli DNA plus 0.3 ng/ml LPS for 20 h before the supernatants were collected for TNF-α ELISA.
To determine whether the suppressive effect of E. coli DNA or LPS pretreatment was attributable to reduced TNF-α mRNA transcription, macrophages were pretreated with E. coli DNA, LPS, or medium (control) for 8 h before stimulation with E. coli DNA plus LPS for 2 h. After stimulation, total RNA was extracted from macrophages and subjected to RT-PCR analysis for detection of TNF-α specific mRNA. TNF-α mRNA levels in macrophages pretreated with either LPS or E. coli DNA before stimulation with E. coli DNA plus LPS were comparable to TNF-α mRNA levels in macrophages pretreated with medium (Fig. 5⇓), results comparable to those observed earlier regarding the relative quantity of TNF-α secreted by macrophages and the quantity of mRNA produced by macrophages. Pretreatment of macrophages with either LPS or E. coli DNA reduces TNF-α secretion in response to a subsequent LPS plus E. coli DNA stimulus, but appears not to alter transcription of TNF-α mRNA. These data lend additional support to the conclusion that post-transcriptional events account for the synergistic interactions among bacterial DNA, LPS, and macrophages that lead to altered patterns of TNF-α secretion.
Effects of E. coli DNA and LPS pretreatment on TNF-α mRNA expression in macrophages stimulated with E. coli DNA plus LPS. Macrophages were either unpretreated or pretreated with 3 μg/ml E. coli DNA or 0.3 ng/ml LPS for 8 h before exposure to E. coli DNA (3 μg/ml) plus LPS (0.3 ng/ml) for 2 h. Total RNA was then isolated for RT-PCR analysis of TNF-α mRNA as described in Fig. 2⇑.
In the current study we have shown that treatment of RAW macrophages with either bacterial DNA or a subthreshold concentration of LPS (0.3 ng/ml) induced the secretion of relatively low levels of TNF-α protein and enhanced expression of TNF-α mRNA (Figs. 1⇑ and 2⇑). When macrophages were simultaneously treated with bacterial DNA plus LPS, secretion of TNF-α protein was dramatically enhanced, while levels of TNF-α mRNA remained comparable to those attained with either bacterial DNA or LPS stimulation alone (Figs. 1⇑ and 2⇑). However, the half-life of TNF-α mRNA in macrophages treated simultaneously with bacterial DNA plus LPS was substantially longer than that in macrophages treated with bacterial DNA or LPS alone. Additionally, pretreatment of macrophages with either bacterial DNA or LPS alone caused a time-dependent suppression of TNF-α secretion induced by subsequent stimulation with bacterial DNA plus LPS. This suppression of TNF-α secretion was not accompanied by reduced expression of TNF-α mRNA. These findings suggest that synergistic interactions among bacterial DNA, LPS, and macrophages leading to enhanced TNF-α secretion are controlled by post-transcriptional events. A synthetic oligonucleotide rich in nonmethylated CpG residues also acted synergistically with LPS, whereas both salmon sperm DNA, which contains highly methylated CpG residues, and a synthetic non-CpG-containing oligonucleotide, failed to act synergistically with LPS to enhance TNF-α secretion. These data support the conclusion that nonmethylated CpG residues are critical to bacterial DNA's capacity to act synergistically with LPS to enhance TNF-α secretion by macrophages.
The data we present are consistent with the results of a recent in vivo study by Schwartz et al. (24). Schwartz et al. reported that i.v. pretreatment of mice with CpG-containing oligonucleotides suppressed production of TNF-α and macrophage inflammatory protein-2 in lung lavage fluid in response to inhaled LPS. However, analysis of total lung mRNA indicated that TNF-α and macrophage inflammatory protein-2 mRNA levels were not reduced by pretreatment with CpG oligonucleotides. Thus, this in vivo study shows discrepancies between TNF-α production and TNF-α mRNA transcription following interactions between LPS, CpG DNA, and macrophages. This suggests that modifications in TNF-α production attributable to these interactions in vivo are controlled by post-transcriptional events.
We have previously reported that bacterial DNA and LPS interact synergistically with macrophages to enhance production of NO, another important inflammatory mediator (12). Although the combination of bacterial DNA plus LPS dramatically enhanced the production of both NO and TNF-α, the mechanisms responsible for the synergistic induction of these two inflammatory mediators appear to differ. The synergistic induction of NO is regulated primarily at the transcriptional level, because a combination of bacterial DNA plus LPS enhanced the production of mRNA for the inducible NO synthase gene, which accounts for the synthesis of NO in mouse macrophages (25). In contrast, levels of TNF-α mRNA in macrophages treated with a combination of bacterial DNA plus LPS were similar to those in macrophages treated with either bacterial DNA or LPS alone, suggesting that transcriptional controls do not account for enhanced TNF-α secretion under these conditions. However, the half-life of TNF-α mRNA in macrophages treated with a combination of bacterial DNA plus LPS is 2–3 times that of TNF-α mRNA in macrophages stimulated with either bacterial DNA or LPS alone. This suggests that post-transcriptional controls are responsible for the synergistic induction of TNF-α by bacterial DNA plus LPS.
Expression of the TNF-α gene in mouse macrophages is regulated at multiple levels. Transcriptional (20, 21), post-transcriptional (17, 20), and translational (17, 26, 27, 28) controls have all been reported to contribute to regulation of TNF-α gene expression. Stimulation of RAW 264.7 macrophages with LPS resulted in increased transcription and translation of the TNF-α gene, although no apparent change in TNF-α mRNA stability has been observed (20, 28). Transcriptional regulation of the TNF-α gene is mediated primarily by binding of transcription factors (e.g., NF-κB) to the promoter region of the TNF-α gene (29, 30), whereas post-transcriptional and translational regulation is related to the 3′ untranslated region (UTR) of TNF-α mRNA. It has been reported that the UA-rich 3′ UTR of TNF-α mRNA contributed to instability as well as translation repression of TNF-α mRNA (17, 26, 27, 28). Whether the prolonged half-life of TNF-α mRNA in macrophages treated with a combination of bacterial DNA plus LPS is mediated by the 3′ UTR of TNF-α mRNA and whether the 3′ UTR-mediated translational mechanism also plays a role in the synergistic induction of TNF-α remain to be investigated.
The available experimental evidence suggests that bacterial DNA and LPS may use different signaling transduction pathways, even though both activate common transcription factors such as NF-κB and AP-1 (31, 32, 33, 34, 35). Although LPS uses TLR4 as its receptor (36), bacterial DNA uses TLR9 as its receptor that might be dependent on the adaptor protein MyD88 (37, 38, 39). Our finding that a temporally controlled synergy exists between bacterial DNA and LPS lends further support to the idea that these two microbial components use different signaling pathways. Whereas LPS alone induces macrophages to produce TNF-α primarily through transcriptional and translational signaling (20, 28), a combination of bacterial DNA plus LPS may activate a signal(s) directed toward post-transcriptional controls, which might contribute to the synergistic production of TNF-α.
Collectively, the data presented in this communication indicate that nonmethylated, CpG-rich, bacterial DNA synergizes with LPS to enhance TNF-α secretion from the RAW 264.7 macrophage cell line cultured in vitro. The synergistic production of TNF-α induced by a combination of bacterial DNA plus LPS appears to be controlled at post-transcriptional levels, as these conditions enhance the half-life of TNF-α mRNA, but do not enhance the transcription of TNF-α mRNA. Our data also showed that pre-exposure of macrophages to either bacterial DNA or LPS resulted in a time-dependent reduction in TNF-α secretion in response to a combined bacterial DNA plus LPS stimulus. This inhibition also appears to be controlled at the post-transcriptional level, as pretreatment with bacterial DNA or LPS reduced TNF-α secretion without impacting TNF-α mRNA levels. These findings provide a mechanistic explanation for previous reports indicating that bacterial DNA and LPS acted synergistically to enhance TNF-α production in vivo, leading to lethal shock in mice (3, 23).
We thank MeiGuey Lei and Shu Ouyang for their constructive advice, Chia Lee for generously providing S. aureus, and Eleanor Zuvanich and Kathy Rode for administrative assistance during the process of manuscript preparation.
↵1 This work was supported by National Institutes of Health Grants AI23447, AI44936, and AI46493, and an unrestricted Medical Research Grant from Merck & Co. (West Point, PA). D.C.M. is supported in part by a Westport Anesthesia Service/State of Missouri Endowed Chair in Research.
↵2 Address correspondence and reprint requests to Dr. David C. Morrison, Office of Research Administration, Room 3112, Main Hospital, Saint Luke's Hospital of Kansas City, 4401 Wornall Road, Kansas City, MO 64111. E-mail address: dmorrison{at}saint-lukes.org
↵3 Abbreviations used in this paper: ODN, oligodeoxynucleotide; UTR, untranslated region.
Received February 2, 2001.
Accepted March 21, 2001.
Copyright © 2001 by The American Association of Immunologists
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Ulevitch, R. J., P. S. Tobias. 1995. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu. Rev. Immunol. 13: 437
Sweet, M. J., D. A. Hume. 1996. Endotoxin signal transduction in macrophages. J. Leukocyte Biol. 60: 8
Yi, A. K., R. Tuetken, T. Redford, M. Waldschmidt, J. Kirsch, A. M. Krieg. 1998. CpG motifs in bacterial DNA activate leukocytes through the pH-dependent generation of reactive oxygen species. J. Immunol. 160: 4755
Yi, A. K., A. M. Krieg. 1998. Rapid induction of mitogen-activated protein kinases by immune stimulatory CpG DNA. J. Immunol. 161: 4493
Beutler, B.. 2000. Tlr4: central component of the sole mammalian LPS sensor. Curr. Opin. Immunol. 12: 20
Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto, K. Hoshino, H. Wagner, K. Takeda, S. Akira. 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408: 740
Schnare, M., A. C. Holtdagger, K. Takeda, S. Akira, R. Medzhitov. 2000. Recognition of CpG DNA is mediated by signaling pathways dependent on the adaptor protein MyD88. Curr. Biol. 10: 1139
Hacker, H., R. M. Vabulas, O. Takeuchi, K. Hoshino, S. Akira, H. Wagner. 2000. Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6. J. Exp. Med. 192: 595
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text:
Explosives & fireworks
Mines & quarries
Resources policy & legislation
Media & public statements
Mining safety and health
Petroleum and gas safety
Explosives safety and security
Persons' appropriateness for access to explosives
Print notice
Explosives information bulletin no. 41 | 02 November 2020 | Version 6
References and further information
A. Explosives Act 1999
This Information Bulletin is to inform any person who employs the services of another, who may have access to explosives, that they have a duty under section 33 of the Explosives Act 1999 to ensure that the person having access to those explosives is old enough, qualified, experienced and appropriately security cleared, and to provide guidance on how such duty may be exercised.
The Explosives Act 1999, Section 33 sets out the employer's obligation about employees.
Explosives Act 1999 Section 33 - employer's obligation about employees.
An employer must not allow an employee to have access to an explosive unless—
the employee is the age prescribed by regulation; and
for an employer who holds a security sensitive authority—
the employee holds a security clearance; or
the access is within the course of the employee's employment and in the presence, and under the direct supervision, of a person who holds a security clearance.
Before an employer asks or allows an employee to carry out an activity involving the handling of explosives, the employer must be reasonably satisfied the employee has the qualifications, experience and expertise prescribed by regulation for the carrying out of the activity.
This Information Bulletin applies to those explosives where potential risks are high i.e. security sensitive explosives (SSE).
Schedule 2 of the Explosives Act 1999 sets out the definition of SSE: Security Sensitive Explosive means—
ammunition, other than small arms ammunition, that contains an explosive; or
a blasting explosive; or
a firework, other than a firework prescribed by regulation; or
a propellant powder; or
a pyrotechnic substance used in a firework; or
another explosive prescribed by regulation that—
if used, could cause the death of a person or serious personal injury; or
could be readily adapted for use in a device that, if used, could cause the death of a person or serious personal injury
Explosives are essential tools for the mining and construction industries in Queensland. However, they also are an inherently dangerous class of materials which present risks to the community. Such risks arise primarily as a result of their use and handling, including transport, storage and sale, but also because they are attractive to an undesirable element within the community. As such, explosives need to be treated within a safety and security management system which :
minimises the probability of incidents,
minimises the potential consequences of any incident, and
imposes adequate controls on access, availability and stock records.
Under normal 'duty of care' requirements (refer Section 32 of the Explosives Act 1999) any employer who employs someone to do something would be required to ensure, among other things, that the person was competent to carry out the tasks. This would include:
training appropriate to the tasks,
physical and mental ability to carry out the tasks,
an understanding of the hazards involved and the safety measures necessary for the tasks,
exposure to the written procedures, if any,
assessment of the above, and
maintenance of the above.
Section 33 of the Explosives Act 1999 imposes a further obligation requiring an employer to only allow an employee who is 18 years old or over and who holds a security clearance to have unsupervised access to explosives. Where an employee does not hold a security clearance they are not allowed access to explosives unless the employee is in the presence of, and under the direct supervision of, a person who holds a security clearance.
Hence, in addition to a person's competence as outlined above, the employer is also required to consider, when the nature of the explosives requires; Does the person:
satisfy the statutory requirements, e.g. age, licence, qualifications?
demonstrate satisfactory physical and mental abilities ?
have a satisfactory prior work history which would indicate the person could reasonably be expected to comply with work procedures? e.g. acceptable attitude, no prior relevant disciplinary issues, satisfactory references,
have a security clearance? (holding a security clearance ensures the person has a satisfactory police clearance and satisfactory politically motivated violence check - ASIO check)
continue to satisfy the above requirements?
To enable a proper consideration of the above the employer should:
limit the number of persons having access to explosives as far as practicable,
advise those having access to explosives that the employer has a statutory obligation to check their suitability and appropriateness as outlined above,
implement health checks as considered appropriate,
implement effective security controls over explosives under the control of the employer,
require those needing unsupervised access to explosives to provide to the employer their security clearance,
notify those having access to explosives that any changes to their situation in regard to criminal history or domestic violence orders, need to be advised to the employer immediately.
While considerations of this type, carried out by an employer, are necessarily subjective in nature, an auditable process such as this would be considered as satisfactory evidence that an employer was seeking to meet relevant obligations under Section 33 of the Explosives Act 1999.
Authorised by Chief Inspector of Explosives
Contact: explosives@rshq.qld.gov.au
Issued by Resources Safety & Health Queensland
General: This information is a guide only. It is not to be taken as a statement of law and must not be construed to waive or modify any legal obligation.
Find more safety notices
Search the hazards database
© Resources Safety & Health Queensland 2021

redpajama_set_name: RedPajamaCommonCrawl


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" It concerns me that your idea of freedom is being able to walk outside and piss wherever you want." - my friend Larry to me the other day.
I definitely grew up in the suburbs, outside of Washington, DC, and about 20 miles from Baltimore, Maryland, but I was fortunate enough to have access to at least a hundred acres of woods behind my house. Within these woods was a natural trout steam, and this stream also contained bluegill, suckers, snapping turtles, and various types of snakes. Also the woods contained deer, raccoon, fox, etc. I spent many days down there swimming and fishing, and I did all of my running for football in both high school and college in those woods, performing a cross-country/sprinting/hill training all in each session.
And in the summer my family and I went to Ocean City, Maryland and fished for flounder.
When I entered college, I got hooked on bass fishing in North Carolina and grew to love it. In the summertime, back home in Maryland, I would fish everyday, waking up at 3:30 AM and pounding coffee before heading to farm ponds or reservoirs.
I came into hunting later in life, in college. I started when I bought my first Labrador Retriever, but it is easily my favorite activity. A perfect day would be to wake up before sunrise, perform some squats and then go duck hunting with my Labs.
Growing up doing all of these activities, I just learned how to do stuff: How to bait a hook, what bait to use for each species of fish, where to cross a creek at the right place, how to chop wood, build a garden, navigate trails. I thought that everyone knew how to do those things, and I was just lucky to have my father teach me about these things, and to recognize the importance of the outdoors in a boy's life.
Now I live in the suburbs again, and I am far away from any woods that are worth a damn. Here ponds and lakes are filled with litter and pollution and there are too many people around, irritating me. I bolt to my buddy Steve's farm in Maryland whenever I can. Those brief respites save me from serious depression brought on by too many people and pollution. That kind of stuff changes you, and I get angry at the closeness of everything.
Given my citified life now, when I was given the opportunity to take my seven year old son to a cabin on the Eastern Shore of Maryland a few weeks ago, I jumped at the chance. I firmly believe that a kid should be well rounded; well read but should know how to handle himself in the outdoors.
The cabin is owned by my sister's boyfriend and he told my sister that I was welcome to go down there anytime that I wanted. He didn't have to say it twice. I have always loved that part of Maryland. It is different there, with the wildlife and the farms and the the creeks and rivers.
So James and I took off early one morning and in a few hours, we arrived.
To get to the cabin, you must open two locked gates and drive down a narrow dirt road. When we first drove in, I noticed a small pond sitting on the left. Being a duck hunter, I always look for birds when I see a body of water. I am guessing that six or seven wood ducks flew out of the small body of water as we drove by. My heart raced. This cabin turned out to be a paradise for us. It sits on 22 acres of woods with a river flowing out in front of it, literally 20 yards from the front porch. The cabin was a huge man cave complete with bones of animals prominently displayed, and antlers and plastic snakes and whiskey bottles on the shelves. It had one bedroom upstairs , and had a small kitchen and living room and a screened in porch downstairs. There were pines surrounding it, and the ground was covered with pine needles.
We walked to the river and started to fish immediately. I had stopped at The new Cabelas in Delaware on our way and bought night crawlers. We went to the river and it was high tide. I swear, for a solid hour, we either got a bite or caught a fish on every cast. Now that is the way to get a kid to love fishing. The fish were bluegill and had some fight to them and I believe that one was around a pound or so. It was so damn perfect, catching fish and watching the wildlife and literally not seeing one other person for hours.
After some retrieving work with the dogs, we drove around 20 minutes to a seafood restaurant that sat right on the Choptank River. This place had an amazing view, with boats on the docks and a truly scenic river. Maryland crabs were selling for 55 dollars for 12 mediums, so I passed and settled on the soft shell crabs. James thought that he wanted them also, but when he took a bite, they were definitely not his cup of tea. I don't think that he knew that you had to eat the whole crab. The crab's eyes where staring up at him and he was a little freaked out, I think.
Back at the cabin, it was now dark and James and the dogs and I sat on the front porch and listened to the geese, herons, ducks and crickets. We couldn't hear a car or truck or another person talking.
The next day, I woke up and did 200 kettle bell swings in the yard. I had to get those in or I wouldn't be able to enjoy the day. And then we made a trip to a Walmart about 20 miles away to buy a new rod for James before we went to the river. Nothing was biting. We then went to a lake to fish. We literally had about a 20 acre lake all to ourselves. This place was unreal, a crystal clear quarry with white sand beaches. James caught his first bass, and by this time he was learning how to bait a hook and cast without getting tangled in a tree.
We had so much fun, just being outside. Not seeing anyone else, no traffic, no worries. To see my son get so excited when he saw that bobber start to go under the surface was priceless. And I was getting as excited as he was every time that it happened.
I did a few more swings when we got back to the cabin, and then we trained the dogs, and I was shooting the shotgun as we did some retrieving drills. You can not do that in the suburbs, for sure.
As night fell we drove to the closest town and had a steak and some fries.
The next morning we woke up early. A few retrieves with the dogs and we were off. I had to get back to Philly for work. We both hated to leave. It was truly a magical few days for us.
These type of trips are important, I think. It is fine to have the suburbs , the city, and Little League. But just as important is to start a connection with the the outdoors, to begin learning the skills to pass on to your kids from generation to generation. With everyone panicking about the absence of "real men" and the drugging of our kids and the video games and all of that stuff, maybe one of the answers to their well being is to get outside and learn the ways of the woods and water.


text:
Archbishop Viganò – Calls Out Agenda 2030 (Italian Audio; English Subtitles)
Patrial transcript:
Dear friends, you have gathered so square in Turin as many hundreds of thousands of people around the world are expressing their opposition to the establishment of a global tyranny. Millions of citizens of every nation in the deafening silence of the media have been shouting their "No" for months.
No to pandemic madness. No to lock downs and curfews and to the imposition of vaccinations.
No to health passports from the blackmail a totalitarian power enslaved to the elite; a power that shows itself as inherently evil, animated by an infernal ideology and moved for criminal purposes.
A power that now declares that it has broken the social pact and considers itself above its citizens. Slaves of a dictatorship. Today health and tomorrow ecological.
This power so convinced of being now successful in his silent coup d'état that it shoves in our faces with impudence. It's not only the ideology that moves them but also the religion. This same day on the Quirinal Hill, the palace that was the residence of the High Pontiffs in the city of Rome an exhibition is inaugurated with the emblematic title "Inferno," whose centerpiece is Auguste Rodin's sculpture, The Gates of Hell,….
See also The Great Reset: Siemens Chairman Calls for 'Billion People to Stop Eating Meat' at the Globalist Elites' World Economic Forum
More from 15:14 – 16:25 – We don't have a Pope who will defend those who do not want to have the experimental gene serum. Nor bishops who will raise their voices to condemn the use of aborted fetuses to produce these so-called vaccines. On the contrary, the shepherds kick us out of churches if we don't have the Green Pass. They remove the priest who does not yield to the pandemic delirium, and in the Vatican, the employees of the Holy See are fired, including the Swiss guards who refuse to get branded by rejecting the diktat of a courtly hierarchy of the New World Order.
I express here my total solidarity with priests outlawed by their bishops, and with the former Swiss Guards; in particular to Pierre-André Udressy, who had the courage to publicly denounce the abuses he suffered by the Vatican authorities.
See also World eminent Cardiologist Prof.Abdullah Alabdulgader calls for suspension of mRNA jab because of cardiac harm concerns
h/t RoxLife
Tags agenda, archbishop, audio, calls, english, italian, vigano Post navigation
ROBOTS have already TAKEN our JOBS! Robots in a warehouse filling GROCERY orders!
Escape From LA II? Corelogic Home Price Index UP 18% YoY, But Forecast To Slow To 1.9% YoY In 2022


text:
Janneh Commission
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Home Videos Latest News Tanzania lowers legal age for HIV/AIDS self-testing to 15
Tanzania lowers legal age for HIV/AIDS self-testing to 15
EYEAFRICA TV: Dar es Salaam, Tanzania: Tanzanian parliament's recent constitutional amendments designed to allow HIV/AIDS self-testing and lowering the age of HIV testing without parental consent to 15 years mark a big milestone in the fight against the disease.
The bill allows its citizens to buy HIV test kits at pharmacies without a doctor's prescription. Medical experts say the move can help the country meet the 90-90-90 goals, an ambitious treatment target to help end the AIDS epidemic.
It states that by 2020, 90 percent of all people living with HIV will know their HIV status, 90 percent of all people with diagnosed HIV infection will receive sustained antiretroviral therapy and 90 percent of all people receiving antiretroviral therapy will have viral suppression.
"This will increase the penetration in the number of people who avail themselves for testing for the HIV virus," said Omar Sherman, head of pathology in Aga Khan Hospital.
Statistics from the Health Ministry show that new infections on youth aged 15 to 19 are high, about 65,000 yearly and out of them 80 percent are girls and only 20 percent are boys.
Tanzania has over 1.4 million people living with HIV while only 62 percent of the population know their status. Now the government hopes that with this new testing method that number could increase up to 90 percent.
Before the bill, testing was only done in labs, using rapid tests and enzyme-linked immunosorbent assay (ELISA).
The new tests have not been made available yet but doctors say users have to know that the virus also has an incubation period, meaning it's important to take the test every three months.
"With certain tests, you might have contracted the disease five days ago and the fact that your result is negative does not necessarily mean that you are HIV negative," said Sherman.
The new law will allow 15 years old children to undergo HIV/AIDS self testing, but insists the process should be voluntary. It also requires a person assisting another one to undertake HIV self-testing to comply with principles of confidentiality.
Young people currently account for 40 percent of new infections. It's hoped that the nation can use this testing method to save the lives of its youth who account for 65 percent of its population.
It is estimated that over 3,000 patients died in sub-Saharan Africa on a daily basis due to HIV in 2015. Ten countries in Africa carry 80 percent of the total HIV burden. They are South Africa, Kenya, Malawi, Ethiopia, Mozambique, Tanzania, Nigeria, Uganda, Zimbabwe and Zambia.
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BAKOTEH DUMP-SITE REOPENS


First week of December is gone, and work has been crazy hectic. The calculation is that 80 percent of people in the world are jiggling their toes planning christmas/new year/chinese new year holiday, where my line of work is the one that is ought to be the busiest and most prosperous sector. Bright side of the story is how I really learn to understand the whole process of travel industry. Not that I say there's nothing left to learn, well i'll be dayumed.
Let's talk a bit about work, next week on 18th, I am working for 3 full months alrighty. It's not a long time, but it's also not a short time. I have passed 25% of my internship contract. My friends, families they know I am working instead of studying here, but sometimes I will still be asked : "What kinda job you are doing actually?" "How come you are working?" "What about school?" "Do you get paid?". The questions are of similar thing, then again I will need to explain myself all over again from the Milky-Way galaxy. pooh.
But yesterday a friend; surprisingly just an average of my normal rank of buddyness asked me; "Do you like your work?". And I was like whoa. Do i like my work? If you put like and hate side by side, I think i will slide between them and cover a little of my foot under "Like". I remembered the first few weeks i am at office, I sometimes felt lost and hopeless. Everything was new, raw to me, that I became overwhelmed in learning. But now, all i have to say is time changed people, yes in good an bad way. I mean to think that i actually come this far and I will still be out of my bed by 9,30 tomorrow, and stay at office for whole day; and come back home again. I was surprised i had the audacity to move along with my new flow of life, while i am always filled with doubt and insecurities. I know I will still have plenties plenties of things to learn and know about. And since I am now stepping near to finish my remaining 75% of intern, I hope i can still go on this path fearlessly until the end.
I know the writings are a bit random and off topic, the days are near to be twenty and i think i shall really start to see ahead my future. Sorting priorities, sorting life and sorting self. Well at least if anything, I know i will always have a coin in my pocket to remind me of following my own heart; not others.


text:
Gustav von Pfuel (vollständiger Name Felix Bertram Gustav von Pfuel; * 24. März 1829 in Berlin; † 7. März 1897 auf Schloss Wilkendorf) war ein preußischer Gutsbesitzer, hoher Staatsbeamter sowie Politiker als Mitglied des Herrenhauses.

Leben

Nach dem Besuch des Gymnasiums in Neisse und Stettin studierte Pfuel in Bonn und Berlin Jura. 1848 wurde er Mitglied des Corps Borussia Bonn. 1851 trat er in den staatlichen Justizdienst ein, den er jedoch 1855 bereits wieder verließ. Nach ausgedehnten Kavaliersreisen durch Europa und den Orient widmete er sich der Bewirtschaftung seiner Güter Wilkendorf, Gielsdorf und Eichenbrandt, die er zu einem Fideikommiss zusammenfasste. In Wilkendorf ließ er zwischen 1852 und 1855 ein Jagdschloss im Tudorstil errichten, Schloss Wilkendorf war fortan sein Wohnsitz. Auf Schloss Wilkendorf entwickelte Theodor Fontane die Idee für seinen Roman Effi Briest.

Am Krieg gegen Österreich 1866 beteiligte er sich als Mitglied, seit 1867 als Rechtsritter, des Johanniterordens. Anschließend trat er erneut in den Staatsdienst ein und nahm Verwaltungsaufgaben im besetzten Königreich Hannover wahr. Von 1868 bis 1872 war er Kreishauptmann und Polizeidirektor des Verwaltungsgebiets Amt Celle. Während des Deutsch-Französischen Kriegs wurde Pfuel der Präfekt des Departements Seine-Inférieure in Rouen sowie Zivilkommissar in den Departements Aisne und Ardennes. 1872 schied er diesmal endgültig und wurde 1874 Hauptritterschaftsdirektor der Kurmark und der Neumark. 1879 berief ihn der preußische König Wilhelm auf Präsentation des Verband des alten und befestigten Grundbesitzes im Landschaftsbezirk Mittelmark (Barnim) in das Herrenhaus, dem er bis zu seinem Tode 1897 angehörte. Der Fideikommiss Wilkendorf wurde vor 1896 aufgelöst und verkauft.

Familie

Gustav von Pfuel entstammte dem seit 926 in der Mark Brandenburg ansässigen Uradelsgeschlecht der Pfuel, das über ausgedehnten Grundbesitz vor allem nördlich von Berlin (Pfuelenland) verfügte.

Er war der Sohn des preußischen Generalleutnants Friedrich Heinrich Ludwig von Pfuel aus dessen Ehe mit Klara von Rochow sowie der Enkelsohn des Hofmarschalls Ludwig von Pfuel. Der preußische Premierminister Ernst von Pfuel war sein Onkel.

Pfuel heiratete Elise von Reventlow (* 8. September 1842; † 30. Mai 1865), Tochter von Theodor von Reventlow aus dessen erster Ehe mit Sophie Gräfin von Bernstorff. Das Paar hatte zwei Töchter:
 Klara Sophie (* 1. September 1863)
 Martha (* 20. April 1865; † 11. Mai 1914) ⚭ 1889 Theobald von Bethmann Hollweg (* 29. November 1856; † 2. Januar 1921), Verwaltungsbeamter, später Reichskanzler

Elise starb sechs Wochen nach der Geburt der gemeinsamen Tochter Martha. In zweiter Ehe heiratete Pfuel 1879 Agnes Amalie Charlotte Clementine Burggräfin und Gräfin zu Dohna-Schlodien (1847–1911), eine Tochter des Abgeordneten Alfred zu Dohna-Mallmitz. Dieser Ehe entstammten fünf weitere Kinder:
 Bertram (* 22. Februar 1880; † 25. August 1880)
 Klementine Agnes (* 5. Juni 1881)
 Freda Agnes Gabriele (* 1. Januar 1883; † 1920) ⚭ 1914 Waldemar von Mohl (* 6. September 1885; † 1. März 1966), Jurist und Landrat
 Agnes Gustava (* 10. Mai 1884; † 1964) ⚭ 1922 Waldemar von Mohl (1885–1966)
 Richard Gustav (* 6. August 1889)

Literatur
 Hermann Krüger (Hrsg.): Chronik des preußischen Herrenhauses. Ein Gedenkbuch zur Erinnerung an das dreißigjährige Bestehen des Herrenhauses. Berlin 1885, S. 191 f.
 Theodor Fontane: Wanderungen durch die Mark Brandenburg II. Das Oderland Gielsdorf
 G. G. Winkel: Biographisches Corpsalbum der Borussia zu Bonn 1821–1928. Aschaffenburg 1928.
 Genealogisches Taschenbuch der Ritter- u. Adels-Geschlechter 1880, S.325

Einzelnachweise

Politiker (Deutsches Reich)
Mitglied des Preußischen Herrenhauses
Corpsstudent (19. Jahrhundert)
Gustav
Adliger
Deutscher
Geboren 1829
Gestorben 1897
Mann

redpajama_set_name: RedPajamaWikipedia


text:
Balanced effort moves William & Mary into tie for first in the CAA (VIDEO)
By Raphielle JohnsonJan 24, 2015, 7:20 PM EST
William & Mary entered Saturday's game against Northeastern one game behind the Huskies in the CAA standings, and after their loss at Delaware on Wednesday this was a game Tony Shaver's team had to get. And thanks to a balanced offensive effort the Tribe picked up the win, beating Northeastern 78-62 with Omar Prewitt leading four starters in double figures with 19 points.
Sean Sheldon and Tony Tarpley added 15 points apiece, with Tarpley also grabbing eight rebounds, and senior guard Marcus Thornton scored 13 as William & Mary pulled even with the Huskies at 6-2 in CAA play. Quincy Ford led Northeastern with 17 points and Scott Eatherton added 12, but the Huskies' struggles from beyond the arc proved too much to overcome.
Northeastern, which is shooting 38 percent from beyond the arc for the season, made just two of its twenty attempts on the afternoon. By comparison, William & Mary (the CAA's best three-point shooting team) made six of its nineteen attempts.


text:
Q: Using a variable in D3 data method to pull JSON data So I'm pulling data from JSON code, but I want the data I pull to be a variable (that I would let the user define by using select menus) and not a direct reference.
I was just wondering if it was possible for what follows the 'd.' to be a variable? If not I would love to hear some proposed workarounds. Here is an example of a solution along the lines of what I'm hoping to achieve.
canvas.selectAll("circle")
.data(finaldataset)
.enter();

var userBuiltVariable1 = someStringMadeViaSelectMenus;
var userBuiltVariable2 = someStringMadeViaSelectMenus;
var userBuiltVariable3 = someStringMadeViaSelectMenus;

circles.transition()
.duration(1000)
.attr("cx", function(d){ return d.userBuiltVariable1})
.attr("cy", function(d){ return d.userBuiltVariable2})
.attr("r", function(d){return d.userBuiltVariable3});

The JSON code looks like so;
"Country":"Wales",
"BoyEveryday1102":"0.9",
"BoyOnceWeek1102":"1.2",
"BoyLessThanOW1102":"1.9",
"BoyDont1102":"96.0"

this shorthand means Gender - How often they smoke - how old they are - what year the data was taken. So the second line is showing Boys, who smoke everyday, aged 11, in the year 2002. Other examples include "GirlOnceWeek1510" or "GirlDont1306" so this variable would be built by the user with select menus. Any help greatly appreciated - thank you

A: Yes, this is quite easily done. The solution would be nothing to do with d3 per se, just Javascript basics.
If you have a Javascript object, there are a number of ways you can query its properties.
The first, and most common one, is the dot notation, which you are already using in your code above.
var myObject = { foo: 'bar' };
console.log( 'dot notation', foo.bar );

Another, less common opne, is to use square brackets. With this, you may use any variable (but it only makes sense to use strings).
var myObject = { foo: 'bar' };
var myProperty = 'foo';
console.log( 'square brackets', foo[myProperty] );

Now applying this to your example code:
var userBuiltVariable1 = someStringMadeViaSelectMenus;
var userBuiltVariable2 = someStringMadeViaSelectMenus;
var userBuiltVariable3 = someStringMadeViaSelectMenus;

circles.transition()
.duration(1000)
.attr("cx", function(d){ return d[userBuiltVariable1]})
.attr("cy", function(d){ return d[userBuiltVariable2]})
.attr("r", function(d){return d[userBuiltVariable3]});

... should give you what you want.
HTH!

redpajama_set_name: RedPajamaStackExchange


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text:
The Queen of Nothing (The Folk of the Air (3))
"Holly Black weaves a compelling spell that's certain to ensorcell her readers.
Young Adult, Bullying, Fantasy
The Wicked King (The Folk of the Air)
"Like the faerie folk whose wicked doings it depicts, The Wicked King is truly spellbinding, with an ending that will leave its readers panting for the next installment."
Author Holly Black calls her Curse Workers series "mobster fantasy," an unusual description for a most engaging trilogy.
"Ms. Black has created a deliciously dark and edgy world quite unlike anything else in today's teen literature.
Holly Black, Justine Larbalestier
Young Adult, Sci-Fi, Short Stories, Humorous
Zombies vs. Unicorns
Courtney Webb
In 2007 an intense debate heated up on the blogs of young adult authors Holly Black and Justine Larbalestier.


text:
Tekken all games download for android
27.09.2021 in 14:57| Paul Kadam
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1 thoughts on "Tekken all games download for android"
Jeremy Sandifer :
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Brb, heading to the grocery store.
It's always fascinating to find out what people eat when no else is around. It's particularly interesting when it involves a woman who owns her fair share of restaurants. So what does Lisa Vanderpump turn to when it's time for a little snack? She was more than happy to reveal in this exclusive video.
It all starts out very normally. A little granola, a little fruit (don't you just love the way she pronounces "berries"?), but it's what she tops it with that's suprising: not milk, not yogurt, not a dollop of whipped cream, but chocolate ice cream.
Even vanilla ice cream wouldn't be so terribly strange, but it's the chocolate that mixes things up. As Lisa says, she doesn't put in too much, so "it's still kind of healthy, but it's a little naughty."
So when you get that 3 PM craving and want something that isn't "too well behaved," reach for the fruit, granola and chocolate ice cream...but maybe don't share it with your dog.
Is Lisa V. Ready to Run for Office (Again)?
What Was Lisa Like Before She Was a Housewife?


On January 16, the Commission adopted an EU-wide strategy on Plastics as part of the 2015 Action Plan on Circular Economy. The strategy lays the foundations for a new plastics economy that respects reuse, repair and recycling needs and includes development of more sustainable materials.
It presents key commitments for action at EU level to boost demand for recycled plastics, curb plastic waste and microplastics and to support global action. Legislative measures will be proposed to the Council and Parliament throughout this and next year. For the full list of planned actions and their timeline see Annex I of the Communication.
The private sector also has a central role in this strategy, as the Commission also calls upon the companies to mobilise through a number of activities. Among others, the EC asks companies to contribute through public consultations, commit to voluntary pledges and cross-sector collaboration. From more information please note that CSR Europe is currently preparing an issue insight on this topic for its members.
We would also like to draw your attention to the ongoing public consultation that will be closing soon, to provide input, opinions and data for preparation of follow-up to the Plastics Strategy in relation to a.o. single-use plastics. Companies can contribute until February 12, 2018.
Intertwined with the Plastics Strategy, the Commission also published a communication on the interface between chemical, product and waste legislation that identifies the four most critical issues that hamper recycling, such as lack of information on presence of substances of concern and harmonisation on EU's end-of-waste rules. For more information please see the dedicated EC staff working document, which lays out several options to address these issues that will be subject to a public consultation in the next 12 weeks, said to be published this week.
To update you on the plastics strategy and the communication on the interface between chemical, product and waste legislation, CSR Europe will organise an EU update webinar on February 27. If you want to register, please send an email to Laura Natrop (csr16@csreurope.org).


text:
Petraeus Personally Investigated Benghazi Attack
Petraeus went to Libya to be prepared for congressional testimony.
ByJONATHAN KARL
Nov. 13, 2012 -- In late October, Petraeus traveled to Libya to conduct his own review of the Benghazi attack that killed four Americans, including U.S. Ambassador Christopher Stevens.
While in Tripoli, he personally questioned the CIA station chief and other CIA personnel who were in Benghazi on Sept. 11 when the attack occurred.
The Libya stop was part of a six nation trip to the region. Petraeus intended the review as a way to prepare for his upcoming testimony before Congress on Benghazi.
"He was looking forward to testifying," a Petraeus friend told ABC News. "He wanted to be fully prepared."
Petraeus' friend described his investigation as Petraeus' "personal review" of both the Sept. 11 attack and the events leading up to it.
Get more pure politics at ABCNews.com/Politics and a lighter take on the news at OTUSNews.com
The trip was so recent that the CIA has told the Congressional Intelligence committees that the trip report has not yet been completed.
Petraeus' personal involvement in this investigation is one reason some in Congress are likely to insist he testify on Benghazi.
But now Petraeus is telling friends he does not think he should testify.
Petraeus has offered two reasons for wanting to avoid testifying: Acting CIA Director Morell is in possession of all the information Petraeus gathered in conducting his review and he has more current information gathered since Petraeus' departure; and it would be a media circus.
Police search for 3 rappers who went missing after canceled event
Andrew Tate to appeal second 30-day detention


text:
American Sign Language Word Recognition with a Sensory Glove Using Artificial Neural Networks
Cemil Oz, Missouri University of Science and TechnologyFollow
Neelesh Sarawate
Ming-Chuan Leu, Missouri University of Science and TechnologyFollow
An American Sign Language (ASL) recognition system is being developed using artificial neutral networks (ANN) to translate the ASL words into English. The system uses a sensory glove CybergloveTM and a flock of Bird 3-D motion tracker to extract the gesture features. The finger joint angle data obtained from strain gages in the sensory glove defines the handshape while the data from the tracker describes the trajectory of hand movement. The data from these devices is processed by two neural networks, a velocity network and a word recognition network. Our goal is to continuously recognize ASL gestures using these devices in real time. We trained and tested our ANN model for 50 ASL word for different number of samples. Our test results show that the accuracy of recognition is 94%.
C. Oz et al., "American Sign Language Word Recognition with a Sensory Glove Using Artificial Neural Networks," Intelligent Engineering Systems Through Artificial Neural Networks, (ANNIE '04), American Society of Mechanical Engineers (ASME), Jan 2004.
National Science Foundation (U.S.)
University of Missouri--Rolla. Intelligent Systems Center


The former chief executive of Aberdeen University's student union has pleaded guilty to defrauding the charity of £16,000.
Jacqueline Williams, formerly McKay, 49, from Dollar in Clackmannanshire, pleaded guilty on 23 January to running a £16,000 fraudulent scheme while at Aberdeen University Students' Association.
The scheme, which ran from 19 August 2014, led to Williams's departure in 2016.
She will be sentenced on 19 February at Aberdeen Sheriff Court.
Aberdeen University Students' Association declined to comment on the case.


The first kind arises if one used a lot of drugs or alcohol in former lives. Then in this life, one will be born without enough neurological connections in the brain—so the "machine" works with some defects.
Second, there are malfunctions that result from using a lot of drugs or alcohol in this life.
The third type appears when a person has lived so unhappily, brought so little joy into his life, and let himself go so much that he is no longer protected by the good impressions in the mind of a healthy person. Then various strange energies can come in and work inside a person. This is called schizophrenia.
And the fourth possibility is that a person has allowed so many negative and disturbing impressions to accumulate in the store consciousness that every time the mind comes to these points, it can't stand it and tries to escape into something else. These would be states of paranoia.
After death, problems and disturbances associated with the body fall away, and one has new possibilities again. On the other hand, when one has built up very strong disturbing energies within oneself, then these also go along into the next life.


Getting a loan for people with bad credit is not anymore uncommon these days. In fact, more and more people resort to these kinds of short-term loans to help them tide over till the next payday, help pay off emergency expenses, or even consolidate their loans.
If you're one of the many thinking of getting a loan for people with bad credit, we've rounded up a simple game plan to help you make the most out of your loan and at the same time, avoid getting into a major loan headache.
Compare loan offers from various lenders before signing anything. Not all loans for people with bad credit are equal, and some are more borrower-friendly than others in terms of interest rate, penalties and charges. Find the one that's best suited to your needs and your financial capacity.
See if you can pay off other existing loans before taking out a new one. One short-term loan may not seem bad, but multiple loans can be detrimental for your credit score, not to mention, your peace of mind. Before trying to get a new loan for people with bad credit, try to see if you can pay off any existing short-term loans first. This will be easier for you on all fronts.
Devise a payment scheme. When it comes to loans for people with bad credit, you would want to make sure that you can make the payments on time as much as possible. Delayed and missed payments can mean high penalty fees and you'd rather spend this money on other things. Create a payment schedule where you plan how much money you'll be setting aside from your savings or your next paycheck to pay off your loan. Make sure too that this budget leaves you with enough money for your other needs, otherwise if you're strapped for cash again, you'll likely take out another loan in no time.
Pay the loan no matter what. There's really no better, smarter way of putting it across. The wisest advice if you have a loan is always to make sure to meet your payments. If this means scrimping a bit on the personal luxuries, then be committed than suffer through exorbitant interest rates and charges.
Loans for people with bad credit are not necessarily bad, and many have benefitted from the temporary relief that they offer especially during emergencies. The bottom line always is to be a responsible borrower.


English Gypsies are certain groups of nomads, and may mean: Romanichals, or English Gypsies of Romani origin. Historically, people have both romanticized and formed negative steretypes views of Gypsies.
Some would characterize them as lazy and lawless, along with being unsanitary and a host of other stereotypes. However, such views do no justice as to who English Gypsies really are.
Romanichals are a Romani sub-group from the United Kingdom and other parts of the English-speaking world.
They are closely related to the Welsh Kale and to other Romani groups in the United Kingdom and continental Europe. Romanichal are found across the United Kingdom, particularly England, Lowland Scotland and Wales.
They claim Christianity as their religious adherence.
Pray God will work through and in the lives of the English Gypsies to grow in their understanding Jesus Christ.
Pray that evangelical English Gypsies they will be light to the least reached peoples in North America.


If the tree widget's Parent is a base widget, the tree widget becomes the root of a new tree widget hierarchy. The tree widget itself is not displayed, but it becomes the parent widget for other tree widgets that are displayed. This type of tree widget is referred to as a root node. Note that a tree widget that is a root node cannot be the parent widget for any widget except another tree widget.
If the FOLDER keyword to WIDGET_TREE is present, the node becomes a branch node. Tree widgets that are branch nodes can become the parent widget of other tree widgets (but not of any other type of widget).
If the FOLDER keyword to WIDGET_TREE is not present, the node becomes a leaf node. Leaf nodes cannot serve as the parent widget for any other widget.


Primo: How do you do that again?
Primo: You are so much better at this than I am.
Me: Well I've been doing it since I was a little kid.
Me: Wait. When did you start making your bed?
Me: You mean your mom MADE YOUR BED FOR YOU? UNTIL YOU WENT TO COLLEGE?
Primo: I had to do other chores!
Primo: I had to help with the dishes.
Me: But your mom. Made your bed. Every. Morning.
Really - why would you reward people for showing up unexpectedly at your house?
I don't mind cooking and baking things to take to a sick friend, but I can drop stuff off and not have to socialize.
If the doorbell rings and I'm not expecting anyone, I ignore it.
Doris has not come back from the dead. She is still dead. Still ashes. Still not ordering product. It is safe to say, I think, that she will never order from you again. If you wish to keep wasting money by sending your catalog to her at my address, that's up to you, but I do not buy stuff.
Please remove her from all your mailing lists.
Thank you for contacting Smithsonian.
We're sorry to hear that you wish to cancel your subscription, however we have canceled your subscription as requested.
We are processing a refund of $39.00 for the unmailed issues. Please allow three to four weeks to receive the refund.
Because our mailing labels are preprinted, you may receive one or two more issues. Please discard them or share them with a friend. Again, we are sorry that you are canceling and hope that you will consider ordering with us in the future.
We appreciate this opportunity to be of service.
Thank you for supporting Smithsonian.
Please cancel Doris' subscription to Smithsonian magazine. She died in May.
My uncle died. He was 81. We went to the memorial service, which was a fish fry at my aunt and uncle's lake place. There were about 150 people there. There was a ton of food - my cousins fried fish and got some BBQ and then of course people brought food.
The only formal aspect of the event, which lasted all afternoon (because it takes a while to eat all those rhubarb bars)(the event, not the formal part), was when my cousin's husband spoke, reading the stories that my eight cousins had written about their father.
Not one single story started with, "He could be difficult." Instead, they all mentioned what a happy person my uncle was and how much he loved his family.
It was what a memorial service should be.


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text:
Name: johanson2 -- Witherington-Widdrington family tree-a speculated line from MD to Northumberland, England
Updated: 04-04-2019 15:04:69. Owner: SUSAN
Rudolf von HABSBURG (Albrecht IV /von HABSBUEG/, Rudolf /von HABSBURG/) was born 01 MAY 1215 in Limburg, Upper Rhine, and died 15 JUL 1291 in Germersheim, Germany. He married Gertrud von HOHENBERG, daughter of Mechtild /von TUBINGEN/ and Burchard /von ZOLLERN/ V. She was born ABT 1235 in Austria, and died 16 FEB 1281 in Vienna, Austria.
Children of Rudolf /von HABSBURG/ and Gertrud /von HOHENBERG/ are:
Mechtild von HABSBURG was born 1253 in Rheinfelden, Baden-W�erttemberg, Germany, and died 1304.
Jutta von HABSBURG was born 13 MAR 1271 in Habsburg, Switzerland, and died 18 JUN 1297.
Search for Rudolf von HABSBURG in Fold3 Records
Search for Rudolf von HABSBURG in Newspapers
Mechtild von HABSBURG was born 1253 in Rheinfelden, Baden-W�erttemberg, Germany, and died 1304. She married LUDWIG , son of Agnes /von BRAUNSCHWEIG/ and OTTO // II Duke of Bavaria. He was born 13 APR 1229 in Heidelberg, Baden-W�rttemberg , Germany, and died 02 FEB 1294 in Heidelberg, Baden-W�rttemberg , Germany.
Children of Mechtild /von HABSBURG/ and LUDWIG // II Duke of Upper Bavaria are:
LUDWIG was born ABT 1282 in Frankfurt, Hessen, Germany, and died 11 OCT 1347.
Jutta von HABSBURG was born 13 MAR 1271 in Habsburg, Switzerland, and died 18 JUN 1297. She married WENCESLAS , son of PREMYSL OTAKAR // King of Bohemia II and Kunegunda /ROSTISLAVNA/ of Kiev. He was born 17 SEP 1271 in Prague, Bohemia, and died 21 JUN 1305.
Children of Jutta /von HABSBURG/ and WENCESLAS // King of Hohemia II are:
Elizabeth de BOHEMIA was born 20 JAN 1292 in Prague, Bohemia, and died 28 SEP 1330 in Wischehred, Bohemia.
LUDWIG was born ABT 1282 in Frankfurt, Hessen, Germany, and died 11 OCT 1347. He married Margaretha d'Avesnes on 26 FEB 1324, daughter of Jeanne /de VALOIS/ and William /d'AVESNES/ Count of Hainaut III. She was born ABT 24 JUN 1310 in Mons, Hainault, Belgium, and died 23 JUN 1356 in Le Quesnoy. He married Beatrix von SCHWEIDNITZ. She was born 1290 in Schweidnitz, Poland, and died 24 AUG 1322 in Munich, Bavaria, Germany.
Children of LUDWIG // IV Emperor of Bavaria and Margaretha /d'Avesnes/ Countess of Holland are:
William V de Bavaria was born 12 MAY 1330 in of Frankfurt, Hessen, Germany, and died 15 APR 1388 in Valenciennes, Nord, France.
Children of LUDWIG // IV Emperor of Bavaria and Beatrix /von SCHWEIDNITZ/ are:
Stephen von BAYERN was born 1319 in Munich, Bavaria, Germany, and died 19 MAY 1375 in Landshut, Bavaria, Germany.
Elizabeth de BOHEMIA was born 20 JAN 1292 in Prague, Bohemia, and died 28 SEP 1330 in Wischehred, Bohemia. She married Jean de LUXEMBOURG on 01 SEP 1310, son of Henry /de LUXEMBOURG/ VII Holy Roman Emperor and Marguerite /de BRABANT/. He was born 10 AUG 1296 in Luxembourg, and died 26 AUG 1346.
Children of Elizabeth /de BOHEMIA/ and Jean /de LUXEMBOURG/ I King of Bohemia are:
Judith de BOHEMIA was born 20 MAY 1315 in Prague, Bohemia, and died 11 NOV 1349 in France.
Charles was born 14 MAY 1316 in Prague, Bohemia, and died 29 NOV 1378 in Prague, Bohemia.
William V de Bavaria was born 12 MAY 1330 in of Frankfurt, Hessen, Germany, and died 15 APR 1388 in Valenciennes, Nord, France. He married Maud de Lancaster on 1352 in Westminster, London, Middlesex, England, daughter of Henry /de Lancaster/ of Grosmont Duke Lancaster and Isabel /de Beaumont/. She was born 04 APR 1340 in Stafford, Staffordshire, England, and died 1362.
Stephen von BAYERN was born 1319 in Munich, Bavaria, Germany, and died 19 MAY 1375 in Landshut, Bavaria, Germany. He married ISABELLA , daughter of FREDERICO // II King of Sicily and Lenore /d'ANJOU/. She was born 1310 in Sicily, Itlay, and died 21 MAR 1338.
Children of Stephen /von BAYERN/ II Duke of Bavaria and ISABELLA // of Sicily are:
Stephen von BAYERN was born ABT 1337 in Lanshut, Niederbayern, Bavaria, and died 26 SEP 1413 in Germany.
Judith de BOHEMIA was born 20 MAY 1315 in Prague, Bohemia, and died 11 NOV 1349 in France. She married Jean de VALOIS, son of Philip /de VALOIS/ King of France VI and Jeanne /de BURGUNDY/. He was born 16 APR 1319 in Chateau de Gube-de-Maulny, France, and died 08 APR 1364 in London, Middlesex, England. He was buried in Saint Denis, �le-de-France, France.
Children of Judith /de BOHEMIA/ and Jean /de VALOIS/ King of France II are:
Charles de VALOIS was born 21 JAN 1337 in Chateau de Vincennes, Val-de-Marne, France, and died 16 SEP 1380 in Chateau de Vincennes, Val-de-Marne, France.
Isabel de France was born 01 OCT 1348 in Vincennes, Paris, France, and died 11 SEP 1372.
Charles was born 14 MAY 1316 in Prague, Bohemia, and died 29 NOV 1378 in Prague, Bohemia. He married Blanche de Valois, daughter of Charles /de VALOIS/ III Count of Valois & Alencon and Mahaut /de CHATILLON/ Countess of St. Pol. She was born ABT 1316 in Valois, Bretagne, France, and died 01 AUG 1348.
Stephen von BAYERN was born ABT 1337 in Lanshut, Niederbayern, Bavaria, and died 26 SEP 1413 in Germany. He married Taddea VISCONTI. She was born ABT 1351 in Milan, Italy, and died 28 SEP 1381.
Children of Stephen /von BAYERN/ Duke of Bavaria and Taddea /VISCONTI/ are:
Isabelle de BAVARIA was born 1371 in Ingolstadt, Oberbayern, Bavaria, and died 29 SEP 1435 in Paris, Ile-de-France, France.
Charles de VALOIS was born 21 JAN 1337 in Chateau de Vincennes, Val-de-Marne, France, and died 16 SEP 1380 in Chateau de Vincennes, Val-de-Marne, France. He married Jeanne de BOURBON on 08 APR 1350 in Lyon, Rhone, France, daughter of Isabelle /de VALOIS/ and Pierre /de BOURBON/ I. She was born 03 FEB 1339 in Vincennes, Paris, France, and died 06 FEB 1378 in Paris, Ile-de-France, France.
Children of Charles /de VALOIS/ King of France V and Jeanne /de BOURBON/ are:
Charles de VALOIS was born 03 DEC 1368 in Paris, Ile-de-France, France, and died 22 OCT 1422 in St. Pol, Paris, France.
Isabel de France was born 01 OCT 1348 in Vincennes, Paris, France, and died 11 SEP 1372. She married Gian Giangaleazzo Visconti on JUN 1360. He was born NOV 1351 in Italy, and died 1402.
Isabelle de BAVARIA was born 1371 in Ingolstadt, Oberbayern, Bavaria, and died 29 SEP 1435 in Paris, Ile-de-France, France. She married Charles de VALOIS, son of Charles /de VALOIS/ King of France V and Jeanne /de BOURBON/. He was born 03 DEC 1368 in Paris, Ile-de-France, France, and died 22 OCT 1422 in St. Pol, Paris, France.
Charles de VALOIS was born 03 DEC 1368 in Paris, Ile-de-France, France, and died 22 OCT 1422 in St. Pol, Paris, France. He married Isabelle de BAVARIA, daughter of Taddea /VISCONTI/ and Stephen /von BAYERN/ Duke of Bavaria. She was born 1371 in Ingolstadt, Oberbayern, Bavaria, and died 29 SEP 1435 in Paris, Ile-de-France, France.


Hi! Im new to minecraft and have a minecraft server. IP: MineLock.qc.to I would love help to improve my server. I need to make kitpvp and learn to make my server run 24/7. If you can help that would be great!
What are you using to run your server? are you running it off your home comp or through a hosting company?
you will not be able to run your server 24/7 if you ever want to turn your computer off. I recommend getting your server professionally hosted. I use OVH because it gives you the greatest value and lets you setup whatever you want yourself. However, it isn't as easy as going through a dedicated minecraft host.
how do they run it forever then?
FI have a machine or two dedicated to my servers. They run full time. I have configured the router to send all MC traffic from the internet to them. I use my regular PC to connect the servers and friends join from their PCs. You need to know a little bit about computers and routers, though.