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Reactive oxygen species and p47phox activation are essential for the Mycobacterium tuberculosis-induced pro-inflammatory response in murine microglia
- Chul-Su Yang†1,
- Hye-Mi Lee†1,
- Ji-Yeon Lee†1,
- Jeong-Ah Kim2,
- Sung Joong Lee3,
- Dong-Min Shin1,
- Young-Ho Lee2,
- Dong-Seok Lee4,
- Jamel El-Benna5 and
- Eun-Kyeong Jo1, 6Email author
© Yang et al; licensee BioMed Central Ltd. 2007
Received: 28 August 2007
Accepted: 26 November 2007
Published: 26 November 2007
Activated microglia elicits a robust amount of pro-inflammatory cytokines, which are implicated in the pathogenesis of tuberculosis in the central nervous system (CNS). However, little is known about the intracellular signaling mechanisms governing these inflammatory responses in microglia in response to Mycobacterium tuberculosis (Mtb).
Murine microglial BV-2 cells and primary mixed glial cells were stimulated with sonicated Mtb (s-Mtb). Intracellular ROS levels were measured by staining with oxidative fluorescent dyes [2',7'-Dichlorodihydrofluorescein diacetate (H2DCFDA) and dihydroethidium (DHE)]. NADPH oxidase activities were measured by lucigenin chemiluminescence assay. S-Mtb-induced MAPK activation and pro-inflammatory cytokine release in microglial cells were measured using by Western blot analysis and enzyme-linked immunosorbent assay, respectively.
We demonstrate that s-Mtb promotes the up-regulation of reactive oxygen species (ROS) and the rapid activation of mitogen-activated protein kinases (MAPKs), including p38 and extracellular signal-regulated kinase (ERK) 1/2, as well as the secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-12p40 in murine microglial BV-2 cells and primary mixed glial cells. Both NADPH oxidase and mitochondrial electron transfer chain subunit I play an indispensable role in s-Mtb-induced MAPK activation and pro-inflammatory cytokine production in BV-2 cells and mixed glial cells. Furthermore, the activation of cytosolic NADPH oxidase p47phox and MAPKs (p38 and ERK1/2) is mutually dependent on s-Mtb-induced inflammatory signaling in murine microglia. Neither TLR2 nor dectin-1 was involved in s-Mtb-induced inflammatory responses in murine microglia.
These data collectively demonstrate that s-Mtb actively induces the pro-inflammatory response in microglia through NADPH oxidase-dependent ROS generation, although the specific pattern-recognition receptors involved in these responses remain to be identified.
Mycobacterium tuberculosis (Mtb) infection of the central nervous system (CNS), particularly in cases of meningitis, accounts for 1 to 10% of all cases of tuberculosis (TB). It is the most severe form of systemic TB because of its high mortality rate and possible serious neurological complications. In the CNS, where the function of neurons is protected by the maintenance of an anti-inflammatory environment [1], infection with Mtb leads to catastrophic, inflammatory tissue destruction [2]. The mechanisms behind this phenomenon are currently unknown. Unlike pulmonary TB, which has been intensively investigated in numerous clinical trials, the pathogenesis, diagnosis, and treatment of CNS-TB have received little attention. A better understanding of CNS-TB pathogenesis is urgently required to improve existing therapies, which still leave over half of those affected dead or paralyzed [3].
The CNS-resident macrophages, microglia, are productively infected with Mtb and may be the principal cellular target in the CNS [4, 5]. Activated microglia release a number of cytokines/chemokines that contribute to both defense against and the neuropathogenesis of CNS infection [6]. Upon activation, microglia produce and secrete potentially neurotoxic pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α/β, interleukin (IL)-1 α/β, and IL-6 [7]. Both TNF-α and IL-1β have been found at increased concentrations in the cerebrospinal fluid (CSF) of patients with CNS-TB [8–10]. Upon mycobacterial infection, mitogen-activated protein kinases (MAPKs) play important roles in promoting anti-mycobacterial activity and the production of immune effector molecules, including TNF-α [11–15]. There is increasing evidence that reactive oxygen species (ROS) also function as second messengers to regulate several downstream signaling molecules, including MAPKs or the NF-κB pathway [16–18].
ROS are produced in mammalian cells in response to the activation of various cell surface receptors [17]. In brain-resident immune cells, the generation of free radicals plays important roles in anti-microbial defense as well as in pro-inflammatory signaling [19, 20]. Activation of the NADPH oxidase pathway initiates an intracellular ROS signaling pathway that amplifies the production of pro-inflammatory cytokines, such as TNF-α [21]. Intracellular ROS mediate β-amyloid peptide-induced microglial activation [22]. In addition, microglia-mediated neurotoxicity is influenced by the release of microglial NADPH oxidase-mediated ROS [23–25]. Previous studies indicate that p47phox, an essential component of the phagocyte NADPH oxidase, is required for superoxide anion release from microglia [26]. To date, the roles of NADPH oxidase-derived ROS and the intracellular regulatory mechanisms by which these pro-inflammatory responses are induced in microglial cells during mycobacterial infection are poorly understood.
Activated microglia express Toll-like receptors (TLRs) [27], CD14 [4], and mannose receptors [28]. TLRs play an important role in the activation of immune cells by pathogens such as Mtb. Receptors other than TLRs, including C-type lectins, are also involved in mediating host responses to Mtb. Recently, Yadav et al. [29] reported that the β-glucan receptor dectin-1 works with TLR2 to mediate Mycobacterium-induced pro-inflammatory responses in macrophages. To date, no attempt has been made to identify the specific mycobacterial antigens that interact with specific TLRs or other pattern-recognition receptors (PRRs) in microglia. To better understand the Mtb-induced molecular signaling pathways in microglia, we selected BV-2 cell lines that retain the characteristics of activated microglial cells, and we confirmed our results in murine primary mixed glial cells. We investigated the role of ROS and MAPK signaling in the regulation of pro-inflammatory cytokine expression in response to sonicated Mtb (s-Mtb). We found that s-Mtb activates inflammatory mediators in microglial cells and primary mixed glial cells through NADPH oxidase-dependent ROS generation. In addition, p38 and extracellular signal-regulated kinase (ERK) 1/2 signaling is essential for the expression of TNF-α, IL-10, and IL-12p40 in s-Mtb-stimulated microglia. Furthermore, we investigated the potential roles of PRRs, such as TLR2 and dectin-1, in microglial cells.
Murine mixed glial cells, and cell lines
Mice with a targeted deletion in the TLR2 gene (homozygous mice and their homozygous litter mates) were kindly provided by Dr. S. Akira (Osaka University, Osaka, Japan). All animals were maintained under standard laboratory conditions on a 12-h light/dark cycle, with free access to food and water. All of the animal procedures were conducted in accordance with the guidelines of the institutional Animal Care and Use Committee, Chungnam National University.
Primary mixed glial cultures were prepared from 1- or 2-day-old neonatal C57BL/6 mice. The cerebral cortices were dissected, carefully stripped of their meninges, and digested with 0.25% trypsin for 25 min at 37°C. Trypsinization was stopped by adding an equal volume of culture medium (Dulbecco's modified Eagle medium-F-12 nutrient mixture, fetal bovine serum 10%, penicillin 100 U/mL, streptomycin 100 μg/mL, and amphotericin B (Fungizone® 0.5 μg/mL) to which 0.02% deoxyribonuclease I was added. The solution was pelleted, resuspended in culture medium, and brought to a single cell suspension by repeated pipetting followed by passing through a 105-μm-pore mesh. Cells (at a density of 250,000 cells/mL) were cultured at 37°C in humidified 5% CO2/95% air. Medium was replaced every 5–7 days. Cultures were used between 12 and 15 days in vitro. At this point they typically consisted of 75% type-I astrocytes and 25% microglia (data not shown). Separately, to obtain astrocyte-enriched cultures, non-astrocytes (microglia) were detached from the flasks by shaking, and removed by changing the medium [30]. We confirmed that astrocyte-enriched cultures consisted of > 95% astrocytes (data not shown).
The BV-2 murine microglial cell lines were kindly provided by Dr. Sung Joong Lee (Seoul National University, Seoul, Korea). The cells were grown and maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco, U.K.) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco) at 37°C in a humidified incubator under 5% CO2.
Cultures of Mtb and preparation of s-Mtb
Cultures of Mtb H37Rv were grown to mid-log phase in Middlebrook 7H9 liquid medium supplemented with oleic acid/albumin/dextrose/catalase (Difco, Becton-Dickinson, Palo Alto, CA, USA), washed three times in sterile saline, and resuspended in RPMI 1640 medium at the various concentrations. Separate culture suspensions were sonicated for 10 min on ice, in order to obtain noninfective cell lysates, as described previously [31]. S-Mtb was pooled and applied to an immobilized polymyxin B column (Detoxi-Gel endotoxin removing gel; Pierce Chemical Co.). Preparations of the s-Mtb used in experiments were tested for lipopolysaccharide (LPS) contamination by a Limulus amebocyte lysate assay (BioWhittaker) and contained less than 50 pg/ml at the concentrations of the s-Mtb used in experiments. In order to show that the stimulatory capacity of the s-Mtb was not the result of contamination with LPS, experiments were performed with the addition of the specific LPS-inhibiting oligopeptide polymyxin B (10.0 μg/ml) before s-Mtb stimulation.
Reagents and antibodies (Abs)
LPS (Escherichia coli 026:B6) and peptidoglycan (PGN, Staphylococcus aureus) was purchased from Sigma (St. Louis, MO, USA). BLP, a synthetic bacterial lipopeptide (Pam3Cys-Ser-Lys4-OH) derived from the immunologically active N-terminus of bacterial lipoproteins, was purchased from Invitrogen (Carlsbad, CA, USA). NAC, DPI, allopurinol and rotenone were purchased from Calbiochem (San Diego, CA, USA). Dimethyl sulfoxide (DMSO; Sigma) was added to cultures at 0.1% (vol/vol) as a solvent control. Specific inhibitors of p38 MAPK, SB203580, and specific inhibitors of MEK, U0126, and PD98059 were purchased from Calbiochem. The expression plasmids that encode p47phox WT and DN, and TAT-Ser345 peptide were kindly provided by Dr. J. El-Benna (Université Paris 7-Denis Diderot, Paris, France). Cells were transfected using LipofectAMINE as indicated by the manufacturer (InvitroGen, Carlsbad, CA, USA). Specific Abs against ERK1/2, phospho-(Thr202/Tyr204)-ERK1/2, p38, phospho-(Thr180/Tyr182)-p38 Abs were purchased from Cell signalling Technology (Beverly, MA, USA). Anti-Dectin-1 mAb (clone 2A11, IgG2b) was from Serotec (Oxford, UK). Abs to p47phox (H-195), and α-actin (I-19) were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). The anti-phospho-(Ser345)-p47phos Ab was used, as previously described [32]. Anti-IL-1β mAb (clone 303311, IgG1) and isotype mAb were purchased from R & D system (Minneapolis, MN, USA).
Measurement of intracellular ROS
Intracellular ROS levels were measured by 2',7'-Dichlorodihydrofluorescein diacetate (H2DCFDA) and dihydroethidium (DHE) assays, as previously described [32]. Briefly, BV-2 or primary mixed glial cells were stimulated with s-Mtb or LPS for 30 min. The cells were incubated with either 10 μM H2DCFDA or 2 μM DHE (Molecular Probes, Eugene, OR, USA) for 15 min at 37°C in 5% CO2. The cells were then washed and examined with a laser-scanning confocal microscope (model LSM 510; Zeiss, Germany) and the mean relative fluorescence intensity for each group of cells was measured with a Zeiss vision system (LSM510, version 2.3) and then averaged for all groups.
Determination of NADPH oxidase activity
NADPH oxidase activities were measured by lucigenin (bis-N-methylacridinium nitrate) chemiluminescence assay (5 × 10-6 mol/L lucigenin, Sigma) in the presence of its substrate NADPH (10-4 mol/L, Sigma) as described previously [33]. In brief, BV-2 or primary mixed glial cells were incubated with s-Mtb or LPS for 30 min in the presence or absence of DPI. Lucigenin-enhanced chemiluminescence assay was performed to analyze the level of superoxide production as previously reported [33]. The cells were transferred into scintillation vials containing Krebs-HEPES buffer (100 mM NaCl, 4.7 mM KCl, 1.9 mM CaCl2, 1.2 mM MgSO4, 1.03 mM K2HPO4, 25 mM NaHCO3, 20 mM Na-HEPES, pH 7.4) with 5 μM lucigenin. The chemiluminescence, which occurred over the ensuing 1 min in response to the addition of 100 μM NADPH, was recorded using a luminometer (Lumet LB9507; Berthold Technologies, Bad Wildbad, Germany). The emitted light units, after subtracting a blank, were used as a measure of superoxide production. Values are expressed as relative light units per 1 × 105 cells.
Enzyme-linked immunosorbent assay and Western blot
A sandwich enzyme-linked immunosorbent assay (ELISA) was used for detecting TNF-α, IL-6 and IL-12p40 (PharMingen, San Diego, CA, USA) in culture supernatants. Assays were performed as recommended by the manufacturers. Cytokine concentrations in the samples were calculated using standard curves generated from recombinant cytokines, and the results were expressed in picograms per milliliter.
For Western blot analysis, total cell lysates were prepared after treatment with s-Mtb or LPS during the time indicated (0~480 min). Abs to phospho-ERK1/2, phospho-p38, total ERK1/2, total p38 and α-actin were used at 1:1,000 dilutions. Membranes were developed using a chemiluminescence assay (ECL; Pharmacia-Amersham, Freiburg, Germany) and subsequently exposed to chemiluminescence film (Fuji Film, Tokyo, Japan)
Statistical analysis
For statistical analysis, data obtained from independent experiments are presented as the mean ± SD and they were analyzed using a Student's t test with Bonferroni adjustment or ANOVA for multiple comparisons. Differences were considered significant for p < 0.05.
S-Mtb stimulation induces intracellular ROS generation and MAPK (ERK1/2 and p38) activation in murine microglial BV-2 cells and primary cultures of mixed glial cells
ROS may serve as intracellular signaling molecules [34]; however, ROS generation in response to mycobacterial antigens is poorly understood in microglia. We examined whether s-Mtb stimulation caused ROS generation in murine microglial BV-2 cells (Fig. 1A) and primary mixed glial cells (Fig. 1B) using the oxidative fluorescent dyes H2DCFDA and DHE to detect H2O2 and superoxide production, respectively. LPS treatment activated ROS generation in microglia. The chemiluminescent signal intensities attributable to H2O2 and superoxide production increased markedly in BV-2 microglial cells stimulated with s-Mtb within 30 min (data not shown). The antioxidant NAC and the NADPH oxidase inhibitor DPI significantly attenuated s-Mtb-induced H2O2 and superoxide production (Fig. 1A and 1B). When NADPH oxidase activity was measured in cultured microglial BV-2 cells via lucigenin chemiluminescence, the s-Mtb-stimulated cells showed increased NADPH oxidase activity compared to resting cells (Fig. 1C). The stimulatory effect of lucigenin on NADPH consumption in microglial cells was nearly abolished by pre-treatment with DPI.
S-Mtb stimulation induces pro-inflammatory cytokine production in murine microglia
The ERK1/2 and p38 pathways are critical for the s-Mtb-induced production of TNF-α, IL-6, and IL-12p40 in murine microglia
Intracellular ROS formation is essential for MAPK activation and pro-inflammatory cytokine production
We examined whether intracellular ROS formation plays a role in MAPK activation and cytokine release in microglia using various inhibitors of ROS generation. As shown in Fig. 4A, S-Mtb-induced ERK1/2 and p38 activity in BV-2 microglial cells was substantially attenuated in the presence of such ROS scavengers as NAC (a general ROS scavenger), DPI (an NADPH oxidase inhibitor), and rotenone (a mitochondrial electron transfer chain subunit I inhibitor) in a concentration-dependent manner.
Activation of the cytosolic NADPH oxidase component p47phox and MAPK is mutually dependent on s-Mtb-induced inflammatory signaling in murine microglia
It was reported that p47phox phosphorylation at Ser345 serves as a point of convergence for various MAPKs to induce the priming of ROS production [40]. To explore the possible role of MAPK upstream from the NADPH oxidase in microglia, we examined the effects of MAPKs inhibitors on the phosphorylation of p47phox and ROS production in BV2 microglial cells. Pretreatment with inhibitors of MEK1 (U0126) or p38 (SB203580) significantly downregulated the phosphorylation of p47phox in BV2 cells in a dose-dependent manner (Fig. 5C and 5D). In addition, superoxide production by BV-2 cells was substantially inhibited by pretreatment with inhibitors for MEK1 (U0126) and p38 (SB203580). Combined, these findings indicate that p47phox phosphorylation and MAPK (ERK1/2 and p38) activation are mutually dependent on s-Mtb-mediated inflammatory signaling pathways in microglial cells.
Neither TLR2 nor dectin-1 is involved in s-Mtb-induced inflammatory mediator expression in murine microglia
Neither astrocytes nor indirect stimuli such as IL-1β adversely affected the s-Mtb-induced ROS release and cytokine production by primary mixed glial cells
Because IL-1β affected ROS generation by astrocytes [41], we also investigated whether the s-Mtb-induced cytokine and ROS production by primary mixed glial cells resulted from indirect stimuli such as IL-1β. To investigate this hypothesis, we examined the cytokine and ROS production from primary mixed glial cells in the absence or presence of anti-IL-1β Ab (Fig. 7C and 7D). Both superoxide and H2O2 were robustly produced by primary mixed glial cells in response to s-Mtb, regardless of treatment with anti-IL-1β Ab (Fig. 7C and data not shown). In addition, s-Mtb-induced TNF-α and IL-6 production was not affected by pretreatment with anti-IL-1β Ab (Fig. 7D). Thus, neither astrocytes nor indirect stimuli such as IL-1β adversely affected the overall findings for primary mixed glial cells.
Given that human microglia are productively infected with Mtb and may be the principal cellular target in the CNS [4, 5, 42], understanding the molecular mechanisms of microglial activation and the anti-microbial response is required to develop targets for therapeutic intervention in CNS-TB. Rabbits are an excellent in vivo model for the study of CNS infection and pathogenesis because of their sensitive inflammatory response and their similarity to humans in terms of the clinical and histological symptoms of disease [43, 44]. Mice are also used to study host immune responses to TB meningitis [45] because of the advantages in terms of genetic manipulation and the availability of commercial immunological reagents. We demonstrated that murine microglia produces pro-inflammatory cytokines in response to s-Mtb, and revealed the important roles of MAPK signaling and ROS production in this process. Although ROS signaling controls a broad range of physiological and pathological processes, including cellular proliferation, inflammation, and apoptosis [46, 47], our study is the first to demonstrate its role in microglial activation in response to Mtb.
LPS activates both macrophages and microglial cells, which have specific roles in microbial defense within the peripheral and central nervous systems, respectively. Previously, Watters et al. [38] investigated the mechanism of LPS signaling in murine macrophages and microglial cells, and revealed different roles for MAPK signaling in these two cell types. We also demonstrated that LPS stimulates the production of TNF-α, IL-6, and IL-12p40 in murine BV-2 cells and in primary cultures of mixed glial cells, which is consistent with previous studies using primary cultures of human, murine, and rat microglial cells [5, 48, 49]. In contrast, very little research has been conducted regarding the mechanisms of recognition and intracellular signaling that induce the initial immune response to Mtb in microglia. In this study, we prepared non-infective Mtb lysates, as described previously by Netea et al. [31], and used them throughout the study. We found that s-Mtb strongly activated the inflammatory response and ROS generation in BV-2 microglial cell lines, as in those infected with live Mtb (data not shown). In addition, the astrocyte-enriched cultures did not play a major role in the s-Mtb-induced cytokine production and ROS generation by primary mixed glial cells. These findings are supported by previous findings that the tubercle bacillus preferentially infects human microglia, rather than astrocytes [5]. The same study also reported that microglial Mtb infection elicited the production of a variety of cytokines, including TNF-α, IL-1β, and IL-6 [5]. Because IL-1β affected the ROS generation from astrocytes [41] and because it might be released by activated microglia, we examined whether IL-1β affects the s-Mtb-induced ROS production by primary mixed glial cells. Pretreatment with anti-IL-1β Ab did not affect the s-Mtb-induced ROS generation or cytokine production, suggesting that the results for primary mixed glial cultures were specific to s-Mtb.
The mechanisms resulting in tissue destruction in TB meningitis are currently unclear. However, growing evidence suggests that inflammatory responses in the brain lead to tissue destruction in the distinct immunological setting of the CNS [50]. Roles for Mtb-induced proinflammatory cytokines and chemokines in CNS-TB have been suggested because dexamethasone treatment suppresses the production of pro-inflammatory cytokines and chemokines in Mtb-infected human microglia [5]. It may explain the beneficial effects of this adjunctive therapy with steroids on the outcome of TB meningitis [3]. Furthermore, recent studies by Harris et al. [51] showed that treating human astrocytes with conditioned medium from Mtb-infected monocytes significantly up-regulated matrix metalloproteinase-9, which suggests that Mtb may increase the activity of tissue-destructive matrix metalloproteinases. Therefore, pro-inflammatory mediators or tissue-destructive enzymes could contribute to the neurological damage observed in CNS-TB.
Our data clearly demonstrate that the activation of p47phox and MAPK is mutually dependent on inflammatory signaling in s-Mtb-stimulated microglia. The results indicate that ROS formation occurs immediately after s-Mtb stimulation and that ROS act as signaling molecules in MAPK activation and subsequent processes. Possible redox-dependent signaling leading to MAPK activation includes the H2O2-mediated inactivation of phosphatase and the deletion of the tensin homologue on chromosome 10 [52]. In addition, ROS mediate calcium release, as all three MAPKs are downstream of calcium-dependent processes [53]. These studies and our data suggest that the NADPH oxidase-derived ROS operate upstream of MAPKs. In addition, the data demonstrate that MAPK activation is required for the phosphorylation of p47phox and ROS production in microglial cells. The phosphorylation of p47phox at several serine residues within the polybasic region of the protein is an essential step in the activation of the NADPH oxidase complex [54]. Previous studies have shown that p47phox is a good in vitro substrate for ERK2 and p38 MAPK [55, 56], and that the phosphorylation of p47phox on Ser345 is directly related to GM-CSF- and TNF-α-induced priming of ROS production [40]. Taken together, the crosstalk between p47phox and MAPK activation may play a pivotal role in the induction of ROS-dependent inflammatory responses by microglial cells.
Although they play different roles, both IL-12 and TNF-α are critical factors in the defense against mycobacteria. IL-12 is crucial for the differentiation of IFN-γ-producing Th1 cells [57]. Because mycobacteria are strong inducers of IL-12, mycobacterial infection can skew the response to a secondary antigen toward the Th1 phenotype [58]. We previously demonstrated that Mtb-induced IL-12 (p40 and p35) expression is negatively regulated by ERK1/2 signaling, whereas TNF-α is induced by ERK1/2 at both the transcriptional and translational levels in human monocyte-derived macrophages (MDMs) [13]. In the present study, we found that IL-12p40 was negatively regulated by p38, but not by ERK1/2. This is inconsistent with previous findings [59] showing that ERK1/2 suppresses the production of IL-12, whereas p38 promotes IL-12 production. This discrepancy may be the result of the differences between microglia and MDMs. Our results strongly suggest that macrophages and microglia have distinct regulatory machinery for the modulation of IL-12 proteins. Additional studies are required to clarify the precise regulatory mechanism of IL-12 production and its role in microglia.
It is important to identify the PRR that triggers microglial activation after Mtb stimulation. The TLR family recognizes a diverse spectrum of microbial ligands. TLR2 is classically recognized as a principal inducer of the pro-inflammatory signal, TNF-α, in response to Mtb [60]. In addition, it has been suggested that the soluble, heat-stable mycobacterial fraction signals mainly through TLR2, whereas the heat-labile components signal through TLR4 [61]. However, we showed that live, sonicated, or heated Mtb elicited robust amounts of cytokines in TLR2 knockout mixed glial cells, indicating that TLR2 is not essential for activation of the pro-inflammatory response. Our data also demonstrate that s-Mtb-induced pro-inflammatory cytokine production in microglia was not dependent on dectin-1. These results are partly consistent with previous studies [29] showing that TNF-α production in response to virulent M. avium 724 and M. tuberculosis H37Rv was not dependent on dectin-1 in macrophages, although dectin-1 was required for TNF-α secretion in macrophages infected with M. smegmatis and other avirulent mycobacterial strains. Therefore, s-Mtb may be recognized through other PRRs or an as yet uncharacterized signaling pathway. To understand the neuropathogenesis of CNS-TB infection, additional studies are required to identify the PRRs that detect pathogen-derived molecules and lead to the development of both innate and adaptive immunity.
In conclusion, our results provide important insight into microglial biology. First, s-Mtb is a potent inducer of ROS generation, pro-inflammatory cytokine production, and MAPK signaling. Second, intracellular ROS play an essential role in the regulation of s-Mtb-activated pro-inflammatory cytokine production in murine microglia, which is mediated via MAPK activation. Our data also emphasize the key roles of crosstalk between p47phox and MAPK activation in the pro-inflammatory response to s-Mtb in microglia.
Bacterial lipopeptide
Central nervous system
Enzyme-linked immunosorbent assay
Extracellular signal-regulated kinase
2',7'-Dichlorodihydrofluorescein diacetate
- MAPKs:
mitogen-activated protein kinases
- Mtb:
Mycobacterium tuberculosisNACN:-acetyl-L-cysteine
- NFκB:
Nuclear factor kappa B
Polymerase chain reaction
Reactive oxygen species
Pattern-recognition receptor
Reverse transcription
Toll-like receptor
- TNF-α:
Tumor necrosis factor-α
We thank Dr. S. Akira (Osaka University, Osaka, Japan) for kind provision of TLR2 knockout mice. This research was supported by the Korea Science & Engineering Foundation through the Infection Signaling Network Research Center (R13-2007-020-01000-0) at Chungnam National University.
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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George Soros might be one of the most well-known names when it comes to philanthropy. After fleeing communist Hungary in 1947, he attended the London School of Economics. After graduation, he found himself in the United States of America. In 1979, he began to provide money to those in apartheid South Africa who wished to attend Cape Town University. His active philanthropist lifestyle started with that simple act, and has grown to the point where he is one of the biggest contributors to American political campaigns.
With the 2016 political campaigns running in full swing, the presidential candidates are strong and many. Noteworthy candidates – for one reason or another – include Donald Trump, Hillary Clinton, and Bernie Sanders. Soros has made where his political alliances lie quite clear this year.
He donated $6 million to a PAC that supports presidential hopeful Hillary Clinton at the end of 2015. In total, Forbes billionaire Soros donated $8 million to Clinton's funds for the presidential race. All of the money went to Priorities USA Action. The donation meant that for 2015, the PAC raised $41 million, and had $36 million to start off with in 2016.
This donation is quite a shocker. In 2004, he donated over $20 million to support George W. Bush. When that effort ended in a disastrous failure, he dialed back how much he gave to political people by a considerable amount. In 2012, Priorities USA Action received only $1 million from Soros in support of Obama instead of Clinton. Later on, he was heard telling an ally – who supported Clinton in the 2008 presidential race – that he regretted supporting Obama over Clinton. He even praised Clinton for allowing him to discuss policy with her. Emails released in December 2015 confirm at least this much.
As the 2016 presidential election race comes into full swing, Soros' donation is sure to be taken as a green light for other philanthropists to donate to Priorities USA Action. The gateways for over $1 billion more in donations has been opened, but only time will tell if that flood will come.

subset: commoncrawl


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Give us a call to get our secret deals 1-800-468-3578
Property Location
With a stay at Petit Palace Arturo Soria in Madrid (Arturo Soria), you'll be minutes from Alcala Norte and close to Arturo Soria Plaza Shopping Center. This 4-star hotel is within close proximity of Las Ventas and Centro Comercial Campo de las Naciones.
Make yourself at home in one of the 44 air-conditioned rooms featuring minibars and plasma televisions. Complimentary wired and wireless Internet access keeps you connected, and digital programming provides entertainment. Bathrooms have showers and hair dryers. Conveniences include phones, as well as laptop-compatible safes and desks.
Make use of convenient amenities such as complimentary wireless Internet access, concierge services, and babysitting/childcare (surcharge). Additional amenities at this hotel include wedding services and a television in a common area.
Buffet breakfasts are available daily for a fee.
Business, Other Amenities
Featured amenities include complimentary high-speed (wired) Internet access, a business center, and express check-out. Planning an event in Madrid? This hotel has 2001 square feet (186 square meters) of space consisting of conference space and a meeting room. Limited parking is available onsite.


text: Item is not available Find Another One Others Looked At... SALE! Walther G43 Semi-Automatic Rifle i... SELLER: Two Wright Arms Co 1 $4,245.00 MOSIN NAGANT RUSSIAN RIFE 762X54 CAL.- SELLER: LLEWELLYN 0 $325.00 WW2 ARISAKA PARATROOPER RIFLE VET BRING ... SELLER: The Hunters Shop 1 $2,395.00 Mosin Nagant, Finland, M39 SELLER: Vicari Gun Sales 1 $449.00 ARISAKA TYPE 99 SHORT RIFLE 7.7 CAL SELLER: JohnCT 1 $395.00 ARISAKA 6.5MM SPORTER RIFLE SELLER: JohnCT 1 $165.00 Guns Rifles Military Misc. Rifles Non US Other CARCANO BERETTA CALVARY CARBINE . MM | 455 | 98 | Lc | 250.0021ms CARCANO BERETTA CALVARY CARBINE 6.5MM For Sale from JohnCT | Positive feedback: 97% View | Verified Seller | 2371 Completed Sales View Sellers Items This item is no longer available. /UserImages/5611/980495074 7985206.jpg /UserImages/5611/980495074 7985207.jpg /UserImages/5611/980495074 7985208.jpg /UserImages/5611/980495074 7985209.jpg /UserImages/5611/980495074 7985210.jpg /UserImages/5611/980495074 7985211.jpg /UserImages/5611/980495074 7985212.jpg Description Shipping and Payment More Info Very clean Carcano, carbine with a folding bayonet. The bore is very clean and shiny, with only mild roughness in the grooves, but far above average.The bluing is about complete 96%, gun functions very well with Gardone, VT (Beretta) maker SN SX5113, in CAL 6.5mm. Stock is very good, with a different number number, and dents, and dings from field use. Overall a very clean wartime gun.We don't have a web site .I prefer money orders or a check. Item ships when funds come in. If you can only pay with a card let me know. I'm sorry I don't list my phone number, did that for 30 days, never again.. I can't talk on the phone all day…who will ship the guns? I'm just one person, and it's very bust here! I do a brisk volume! I hope you like my wide selection of unusual guns, knives and gun parts. Shipping price determined by zip code. I try to keep the prices affordable, which means I make less money than most dealers. You won't get altitude sickness with my prices! I consistently have the best selection of older guns If you BUY IT NOW you must still contact us for details buy it now doesn't end the sale, it just sends an email telling us you want to buy this gun. A phone call won't secure a sale! Not home many times, the first buyer by email buys the gun !This web site doesnot close down a sale when you do a BUY IT NOW like Ebay!I can get six buy it nows in an hour, first buyer to email gets the guns, others are in second place! Best to send another email . Sometimes not home to get calls. Some guns are required shipment to a gun dealer near you for a transfer to you when it arrives. With the widest selection of older guns at great prices, We don't have a store, and sell on the internet only. SHIPPED BY CHOICE Trades Accepted: No Use the Ask Seller a Question link to contact me. I am an estate dealer, and buy whole contents of homes, mostly gun smith's estates. Been doing this for 40 years. Never know what I'll find next.We look for the unusual, we don't buy new guns from wholesalers, and resell! !KEEP CHECKING MY INVENTORY. We don't do feedback this isn't EBAY! Thank you. Condition: Used - See Item Description Caliber: 6.5 mm (all) Location: CT Share Item Item #: 980495074 Stock No.: CARCANO BERETTA CALVARY CARBINE 6.5MM This item is no longer available. Price: Shipping: See Description (or Arrange local transfer) Accepted Payment Methods: Returns: 3 Days JohnCT Seller Since: Dec. 2005 Seller Level: Gold Seller + This seller is an FFL City: East Granby State: CT Your Recently Viewed Items


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Lot 565: A Maison Bagues style brass two tier trolley, W.2ft 8in. H.2ft 8in.
Gorringes Winter Fine Sale
by Gorringes
November 29, 2016
Lewes, United Kingdom
Live AuctionSold
Looking for the realized and estimated price?
Description: A Maison Bagues style brass two tier trolley, with ropetwist frame, W.2ft 8in. H.2ft 8in.
Condition Report: Descriptions provided in both printed and on-line catalogue formats do not include condition reports. The absence of a condition statement does not imply that the lot is in perfect condition or completely free from wear and tear, imperfections or the effects of aging. Interested bidders are strongly encouraged to request a condition report on any lots upon which they intend to bid, prior to placing a bid. All transactions are governed by Gorringes Conditions of Sale.


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Off Road Taster for Two
Off Road Taster for Two Product Description
To have a well-developed sense of adventure is a true gift. Now you can treat someone to one of the best experiences of their life! Enjoy some of the most heart-thumping, adrenaline-pumping thrills in the world! These adventure days away are the perfect extravagances you could possibly want yourself and your best friends to enjoy. Get in on the action-packed adventures now!
Head for the hills across some rough, tough terrain in a hardy 4-wheel drive vehicle. You'll be amazed by what a four wheel drive vehicle can do. Mud, holes, gullies, steep slopes - the most rugged terrain can be crossed if you have the skills. We have carefully selected the best off road driving locations in the country. All will give you some seriously challenging driving in some beautiful locations. We have also made sure the quality of driving tuition is the best.
Your session begins with an introductory briefing on off roading techniques, together with an explanation of 4WD systems. The instructor will do a demonstration drive, and then it's time for you to take turns at the wheel! 15 minutes are spent behind the wheel by each participant but the whole programme is spent in the car.
The person driving the car will be required to sign a disclaimer accepting liability in the event of damage to the vehicle. The driver will be given an option to purchase damage waiver cover when booking to minimise their financial exposure should there be an accident. Drivers are under no obligation to purchase this optional waiver. Full details are available at the time of booking.
When can you go?
This experience is available on selected Saturdays and Sundays, throughout the year.
How long does this activity last?
Your experience will last for approximately 1 to 1½ hours.
You need to know
The minimum age for this experience is 17 years old.
You need to be fit to drive. No alcohol or drugs prior to the event. This may not be suitable for people suffering from diabetes, epilepsy or angina pain. If in doubt please check before booking.
You are required to hold a full valid UK or European driving licence
This is for a voucher entitling the holder to any of the activities supplied by Activity Superstore up to the value stated.
You and how many others?
There will be three people per car, you will take it in turns to drive.
Family and friends
Unfortunately, this experience is not suitable for spectators.
- London - London - Northolt
- Midlands - Rutland - Greetham
- Midlands - Warwickshire - Shipston-on-Stour
- North - Durham - Bishop Auckland
- North - Durham - Rushyford
- North - Yorkshire - Ferrybridge
- Scotland - East Lothian - Pencaitland
- Wales - Swansea - Pontardawe
- Wales - Wrexham - Wrexham
Valid until: 01/03/2017
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Lucky 7 Design
Im the mama of 5 & a REAL New Jersey Housewife. My husband says I have a decorating disorder because I change my mind so much, I say that Im frequently inspired! I take vintage items & give them new life. I blog about my craigslist finds and...
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Based on a 2,000 calorie diet
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Excellent summer meal or a great appetizer for those summer parties. I made this is a gallon sized glass jar to transport to a party. I tossed all the ingredients into the jar as I was preppin...


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Sci fi/Fantasy image by
Joseph ShopsinThere are according to many myths, 7 layers of hell. Neer the upper layers, lesser demons scour the barren.. cavernliek terrain. Amongst these lesser demons exist the Imps. Small hairy, and reddish-brown in appearance with long floppy ears these demons are. They are no taller than 4 feet, and hunt in packs on the smaller demons of the landscape. they tend to mate often and so their numbers are great. There is no earthly tounge in hell but they do communicat though simple gegstures and language above that of grunts and snarls. Quite cunnning and able to use their suroundings to the fullest extent, they can make quite the adversary if faced in numbers.. which is most often the case.
Published More than a year ago
Category Fantasy
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Welcome to Cincinnati Software Craftsmanship's first Code Retreat!
The event is for software developers from different paradigms to learn new techniques, languages, and practices from each other by pairing to solve a pre-determined problem. Participants must choose a new partner for each session and new programatic constraints will be introduced with each session.
The format is as follows:
5:30 - 6:00 PM: Introductions, pizza
6:00pm - 6:45pm: Session 1
6:45pm - 7:00pm: Session 1 Review
7:00pm - 7:45pm: Session 2
7:45pm - 8:00pm: Session 2 Review
8:00pm - 8:45pm: Session 3
8:45pm - 9:00pm: Session 3 Review
Make sure to bring a laptop and have your development environment ready for the type of coding you'd like to be doing.
Check out our new Meetup group
Meetup members, Log in
By clicking "Sign up" or "Sign up using Facebook", you confirm that you accept our Terms of Service & Privacy Policy


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This is a fun place to eat. The atmosphere cool and service has always been great. They have enough gf options to make it worth while, especially when looking for places that non gf friends will also enjoy.
Not GF kitchen but knowledgable staff on celiac. Helpful staff, waiting to fond out if get sick or not.
I have celiac and did not get sick eating here, but it is not a GF facility so I had to say no on the celiac friendly. The food was very good. Great hamburger buns. They get 3 stars for having a poor selection of cider.
cool green building and business model. good beer (they don't make gf beer but they have some in bottles). decent food. They do have a small kid play area, a little square of carpet worth some toys, but it points to the coolest part: they really are kid friendly. There are kids running all over the place. It's great.
This restaurant has gluten free buns for any sandwich and also has dairy free cheese options. My daughter loves the kids play area. Gluten free beer too! Yummy!
Monday | 11:00 am - 11:00 pm |
Tuesday | 11:00 am - 11:00 pm |
Wednesday | 11:00 am - 11:00 pm |
Thursday | 11:00 am - 11:00 pm |
Friday | 11:00 am - 12:00 am |
Saturday | 11:00 am - 12:00 am |
Sunday | 11:00 am - 11:00 pm |
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Amazing Experience
Breast Augmentation
Doctor Review
14 Oct 2016
Updated, 1 day ago
Amazing Experience
I think Dr. Watts is an amazing person. She is down to earth and is great at what she does. I'm very pleased with my breast augmentation and she came highly recommended by my girlfriend. From pre to post surgery, it truly was a great experience.


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Mountain lion carcass found near Dodge City
The recent discovery of a dead mountain lion in western Kansas brings to five the number of mountain lion confirmations in the state since A…
Sponsored by:
The recent discovery of a dead mountain lion in western Kansas brings to five the number of mountain lion confirmations in the state since A…
The Royals won 5-4 to take a 1-0 lead in the series.
The Royals outfielder was immediately intrigued.
Kansas officials won't learn for weeks whether a six-week tax amnesty program raised the $30 million that legislators anticipated.
The Douglas County Sheriff's Office wants the county to commit to reducing the number of jail inmates who are seriously mentally ill.
As victories have mounted, interest in the Royals has expanded from the core die-hards to a whole new generation of fans who weren't around …
The Wichita school district has disabled a computer system while the district investigates a recent hacking attempt.
A man has been charged in the death of a 17-year-old Wichita boy who was shot in the head at a private birthday party.
Alcides Escobar sped into second, turned and saw third base coach Mike Jirschele jumping and waving him toward third. And Jirschele kept wav…
A De Soto man has pleaded guilty in the death of a 21-year-old Prairie Village man.
Two jazz museums are getting in on the World Series action.
Kill worked through his health issues to build a roster full of NFL talent, especially on the defensive side of the ball.
Game #2 tonight from Kansas City, on FOX KTMJ at 7 p.m.
The City and the U.S. Army Corps of Engineers want to hear from homeowners about proposals to tame the creek when heavy rains come.
You could say Kansas City Royals fans are coming out of the woodwork for the team's second post season run in two years.
His son was informed of Volquez's passing on the way to the ballpark, where he was scheduled to make his World Series debut.
Royals start off with inside the park HR in bottom of 1st inning.
A segment of the show was taped on The University of Kansas campus, for the World Series.


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Builders looking for all-in-one, back-office software faced a major disappointment last summer when i4Synergy put development of i4Builder on hold and said it would not be delivering product this year.
The company's process-oriented approach held broad appeal. Leading builders Hovnanian Enterprises and John Wieland Homes were among i4's seven early adopters.
i4Synergy promised the next generation in back-office software. The goal was to deliver a Web-based product that let customers, builders, and trade partners work collaboratively across every home building process.
An i4 employee who requested anonymity said the company planned to get the early adopters running by fourth-quarter 2003 and start delivering product industrywide by first-quarter 2004. But when it became clear that i4 would not meet its deadline, the company halted development, started discussing options with the early adopters, and gave back cash deposits to a handful of other companies that were on a waiting list. At press time, the early adopters were deciding whether to continue development work independently or collaboratively among themselves.
Officially, i4 is still in business, but it's unlikely the company can deliver product for at least a year--if ever.


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Amity High School Theater Department presents: Fiddler on the Roof March 31st-April 8th
Fiddler on the Roof is the heartwarming story of fathers and daughters, husbands and wives, and life, love and laughter. This classic musical is rich with Broadway hits, including "To Life (L'Chaim!)," "If I Were A Rich Man," "Sunrise, Sunset," "Matchmaker, Matchmaker," and "Tradition."
Visit the AHS Theater Department's website for tickets and other information: http://www.amitytheaterdepartment.com/ .


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Crowd-Pleaser Crab Appetizers Recipe
chives, cocktail sauce, +rye bread
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Kate Wright - James Ingham's Jog on to Cancer Party at Kensington Roof Garden, London at Kensington Roof Gardens - London, United Kingdom - Thursday 7th April 2016 (2 Pictures)
Kate Wright
Kate Wright and Danielle Armstrong
View All: James Jog on to Cancer
The long-awaited fourth season of 'Peaky Blinders' debuts later this year, with Tom Hardy now confirmed to reprise his role as Alfie Solomons.
For the sci-fi horror thriller Life, Jake Gyllenhaal and Ryan Reynolds team up to play astronauts trying to contain an aggressive alien creature.
Deadpool creator Rob Liefeld says that the chemistry between Ryan Reynolds and Zazie Beetz in upcoming Marvel movie 'Deadpool 2' needs to be...
The British actress opened up about the disorder she's been battling since she was a teenager.


text: Search Results for "F" FLAGRANS CRIMEN This, among the Romans, signified. that a crime was then or had just been committed for example, when a crime has just been committed and the... more FLAGRANTE DELICTO The act of committing a crime, when a person is arrested flagrante delicto, the only evidence required to convict him, is to prove that fact. ... more FLEET punishment, Eng. law, Saxon fleot. A place of running water, where the tide or float comes up. A prison in London, so called from a river or ditch... more FLETA The title of an ancient law book, supposed to have been written by a judge who was confined in the Fleet prison. It is written in... more FLIGHT crim. law. The evading the course of justice, by a mans voluntarily withdrawing himself. 4 Bl. Com. 387. Vide Fugitive from justice. ... more FLORIDA The name of one of the new states of the United States of America. It was admitted into the Union by virtue of the act... more FLORIN The name of a foreign coin. In all computations of customs, the florin of the southern states of Germany, shall be estimated at forty cents, the... more FLOTSAM, or FLOTSAN A name for the goods which float upon the sea when a ship is sunk, in distinction from Jetsam, (q. v.) and Legan. (q. v.) Bract.... more FLUMEN civ. law. The name of a servitude which consists in the right of turning the rain water, gathered in a spout, on anothers land., Ersk. Inst. B. 2,... more FOEDUS A league, a compact. ... more FOENUS NAUTICUS . The name given to marine interest. (q. V.) 2. The amount of such interest is not limited by law, because... more FOETICIDE med. jur. Recently, this term has been applied to designate the act by which criminal abortion is produced. 1 Becks Med. Jur. 288, Guy, Med. Jur. 133.... more FOETURA civil law. The produce of animals, and the fruit of other property, which are acquired to the owner of such animals and property, by virtue of his... more FOETUS med. jur. The unborn child. The name of embryo is sometimes given to it, but, although the terms are occasionally used indiscriminately, the latter is more frequently employed... more FOLCMOTE The name of a court among the Saxons. It was literally an assembly of the people or inhabitants of the tithing or town, its jurisdiction extended... more FOLD-COURSE Eng. law. By this phrase is understood land used as a sheepwalk, it also signifies land to which the sole right of folding the cattle of others... more FOLK-LAND Eng. law. Land formerly held at the pleasure of the lord, and resumed at his discretion. It was held in villenage. 2 Bl. Com. 90. ... more FOOT A measure of length, containing one-third of a yard, or twelve inches. See Ell. Figuratively, it signifies the conclusion, the end, as, the foot of the... more FOOT OF THE FINE estates, conveyancing. The fifth part of the conclusion of a fine. It includes the whole matter, reciting the names of the parties, day, year, and place, and before... more FOR THAT pleading. It is a maxim in law, regulating alike every form of action, that the plaintiff shall state his complaint in positive and direct terms, and not by... more |


text:
Your Cyber Minute: What to keep in mind as a litigator
FCPA Compliance Report-Episode 318-Erica Byrne on Ethisphere's 2017 World's Most Ethical Companies
Day 19 of One Month to Operationalizing Your Compliance Program-Operationalizing Compliance Through Leadership From The Top
Day 18 of One Month to a Operationalizing Your Compliance Program-Through Management of 3rd Party Relationships
Everything Compliance-Episode 9, DOJ Evaluation of Corporate Compliance Programs, Part II
FCPA Compliance Report-Episode 316-Kristy Grant-Hart & a debate on ISO 37001 Certification
FCPA Compliance Report-Episode 317-Susan Divers on LRN 2016 Compliance Program Effectiveness Report
Compliance Report-International Edition, Carlos Ayres
LendIt USA 2017 Recap Discussion
This Week in FCPA-Episode 44-the St. Patrick's Day Edition
Your Cyber Minute: Lessons learned from the automotive sector
Everything Compliance-Episode 8, DOJ Evaluation of Corporate Compliance Programs, Part I
Day 11 of One Month Operationalizing Your Compliance Program-Internal Control Objectives
Compliance into the Weeds-Episode 32
FCPA Compliance Report-Episode 315-James Koukios
Day 10 of One Month to Operationalizing Your Compliance Program-the Role of Payroll in Compliance
Your Cyber Minute: Intersection of cybersecurity and the automotive industry
FCPA Compliance Report-Episode 314-John Champion on 50th Anniversary of Star Trek
Day 9 of One Month to Operationalizing Your Compliance Program-Connected Compliance
Day 7 of One Month to Operationalizing Your Compliance Program-Risk Based Monitoring
Partner Harriet Pearson and Head of our Privacy and Cybersecurity practice in Europe Eduardo Ustaran discuss the Global Data Protection Regulation and what companies need to know now. ...more
In this week's episode, Jay Rosen and I discuss the following: Show notes for this week's edition: 1. Tom's week long series on Well's Fargo scandal. 2. Christina Muehl's two part series on Corruption, Crime and...more
In this special Brexit episode, I visit with UK lawyer Jonathan Armstrong about the potential impacts of the British exit from the EU for the FCPA compliance practitioner around issues relating to data privacy and data...more
Introducing ALM's cyberSecure — A two-day event designed to unite business leaders and the entire risk management team. Sessions and workshops will provide more than 1,500 attendees the insights and connections necessary to...more
In this episode I visit with Debra Bruce, Principle at Lawyer-Coach. She discusses the recent IPO of the English law firm Gately and alternative funding vehicles available to UK law firms. She explains how these new models...more
Jan. 21, 2015 (Mimesis Law) -- Houman Shadab, professor at New York Law School, talks with Lee Pacchia about the concept of Bitcoin 2.0 and the types of opportunities the crypto-currency presents for law students. ...more
Amber Dolman and Robert Bishop came to Goodwin Procter in much the same way: Both started at the firm right out of law school, after having worked there as summer associates. While Bishop began in the REIT practice, he soon...more
Mar. 26 2014 (Mimesis Law) -- David Perla, co-founder of Pangea3 and current CEO of Matterhorn Transactions, talks with Lee Pacchia about the rise of a 'startup culture' in legal technology. According to Perla, approximately...more
The Association of Business Trial Lawyers recently asked Cogent Legal to prepare the graphics for a presentation on mock trials. The panel consisted of some of the best jury consultants in the country exploring the factors...more
I always like to share examples of what I consider powerful graphics to give attorneys ideas on what's possible to create for use in litigation. This morning, I was struck by a video going viral that describes economic...more
The American College of Trust & Estate Counsel (ACTEC) has just released its newest tool, and it is free. The ACTEC Wealth Advisor application for iPad® is now available for complimentary download via the Apple App Store....more
A discussion of the latest counterfeit scam targeting lawyers....more
Understanding Track Changes in Microsoft Word 2010....more
LOS GATOS, CALIFORNIA (February 7, 2011) – Chapter11Cases.com today launched a database of over 5,000 of the most important bankruptcy court pleadings filed since the beginning of 2008 with each document available to anyone...more
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Learn through educational play. With our range of educational toys, you can discover the power of magnetism, gryoscopic forces, electricity, solar power, kinetic energy and other scientific principles.
Learn about the solar system, volcanos and nature by getting involved 'hands-on' with our fun range of educational kits.
40 Fridge Magnetic Letters
A set of 40 magnetic letters.This colourful set of lower case magnetic letters are education fridge magnets.… |
£2.99 | ||
Alarmed Safe Kit
Construct your own alarmed safe money box.Childern will learn how connect a wire circuit whilst making this safe.Build and configure the digital lock and install an alarm buzzer.… SORRY - OUT OF STOCK
£19.99 | ||
Bubble Stomp Rocket
This stomp rocket comes complete with bubble mixture.Pour the mixture into the tray and sit the rocket on top.Stomp on the pump and the rocket will then produce a shower of bubbles… SORRY - OUT OF STOCK
£12.99 | ||
Bug Box
Go out and capture real life bugs and insects to study in this bug - viewing box with 2 levels of magnification.This bug viewer has two lenses.The basic lens magnifies by 2x, the f… |
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Lightweight binoculars with a detachable strap perfect for the little explorer to take out and about.These binoculars are an educational toy for children to view birds and wildlife… SORRY - OUT OF STOCK
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Crystal Powered Radio
All the components including a sturdy mounting board are included to make a working radio which requires no batteries or AC power supply.Clear instructions and diagrams are include… |
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Explorer Walkie Talkies
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Explorers Magnet Kit
A set of various sized magnets to experiment with and learn whilst playing.… SORRY - OUT OF STOCK
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A geode mining kit with hammer.This kit contains two geodes to smash apart to real the crystals inside. Children can have fun hammering the geodes and then learn about the way they… SORRY - OUT OF STOCK
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Giant Horseshoe Magnet
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Glow in the Dark Galaxy
Glow in the dark stars and planets to stick that look great stuck on bedroom ceilings.Nine glow planets and shooting stars with sticky pads to stick them where ever you choose.… |
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New Educational Toys for March |
Snail Calendar Blocks |
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Explorer Walkie Talkies |
Stone Polishing Kit |


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Many industrial control systems use programmable logic controllers (PLCs) since they provide a highly reliable, off-the-shelf hardware platform. On the programming side, function blocks (FBs) are reusable components provided by the PLC supplier that can be combined to implement the required system behaviour. A higher quality system may be realized if the… (More)
- Linna Pang, Chen-Wei Wang, Mark Lawford, Alan Wassyng, Josh Newell, Vera Chow +1 other
- 2015
A critical step towards certifying safety-critical systems is to check their conformance to hard real-time requirements. A promising way to achieve this is by building the systems from pre-verified components and verifying their correctness in a compositional manner. We previously reported a formal approach to verifying function blocks (FBs) using tabular… (More)
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Benecos Natural Quattro Eye Shadow - Coffee & Cream - 8g
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Product code: 356537List price
Now £5.56
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Product Description
For all your eyeshadow needs, this eye shadow quad highlights, contours, lines and defines your eyes as well as keeping the delicate skin around your eyes moisturised and cared for. Made with BDIH certified natural, vegan and organic ingredients, including macadamia and sunflower oils, this ethically made eyeshadow is perfect for day or evening looks.
Also available in Beautiful Eyes (lilac, pink, ivory and chocolate).
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About Benecos
Benecos products are BDIH-certified natural products, and do not contain mineral oils or derivatives, silicones, parabens, synthetic emulsifiers or PEG derivatives, synthetic colorants, synthetic fragrances or aromatic substances.
Benecos cosmetics are natural with beneficial skin ingredients whenever possible from controlled organic agriculture. It's a natural, cruelty free and beautiful treat for your skin.
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TALC, MICA, CAPRYLIC/CAPRIC TRIGLYCERIDE, MACADAMIA TERNIFOLIA SEED OIL*, RICINUS COMMUNIS (CASTOR) SEED OIL*, MAGNESIUM STEARATE, KAOLIN, GLYCERYL CAPRYLATE, p-ANISIC ACID, TOCOPHEROL, HELIANTHUS ANNUUS (SUNFLOWER) SEED OIL, [+/- (MAY CONTAIN) TIN OXIDE, CI 77499 (IRON OXIDES), CI 77491 (IRON OXIDES), CI 77492 (IRON OXIDES), CI 77891 (TITANIUM DIOXIDE), CI 77742 (MANGANESE VIOLET), CI 77007 (ULTRAMARINES)


text:
MONTREAL, April 13, 2016 /CNW Telbec/ - Air Canada will hold a conference call for analysts on Friday, April 29, 2016 to present first quarter 2016 results. Air Canada President and Chief Executive Officer, Calin Rovinescu, Executive Vice President and Chief Financial Officer, Michael Rousseau, and President, Passenger Airlines, Benjamin Smith will be available for analysts' questions. Following the analysts' questions, Michael Rousseau and Pierre Houle, Managing Director and Treasurer, will be available to answer questions from holders of Air Canada bonds and term loan B lenders. Media and the public may access this call on a listen-in basis. Details are as follows:
Date: |
Friday, April 29, 2016 |
Time: |
09:00 a.m. ET |
By telephone: |
1-866-225-0198 toll free or (416) 340-2216 for the Toronto area. Please allow 10 minutes to be connected to the conference call. |
Webcast: |
Note: This is a listen-only audio webcast. Media Player or Real Player is required to listen to the broadcast; please download well in advance of call. | |
Replay: |
Instant replay will be available beginning approximately one hour after the call and until midnight ET Thursday, July 28, 2016. Dial 905-694-9451 or 1-800-408-3053 toll free and enter the passcode 2984015#. |
First quarter 2016 results will be released prior to the opening of markets and the conference call on Friday, April 29, 2016.
SOURCE Air Canada
For further information: Isabelle Arthur (Montréal), (514) 422-5788; Peter Fitzpatrick (Toronto), (416) 263-5576; Angela Mah (Vancouver), (604) 270-5741; Internet: aircanada.com
RELATED LINKSAir Canada
Air Canada - Corporate - Finance
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