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Bacterial Lipopolysaccharide and IFN-γ Induce Toll-Like Receptor 2 and Toll-Like Receptor 4 Expression in Human Endothelial Cells: Role of NF-κB Activation
Emmanuelle Faure, Lisa Thomas, Helen Xu, Andrei E. Medvedev, Ozlem Equils and Moshe Arditi
J Immunol February 1, 2001, 166 (3) 2018-2024; DOI: https://doi.org/10.4049/jimmunol.166.3.2018
Emmanuelle Faure
Division of Pediatric Infectious Diseases, Ahmanson Department of Pediatrics, Steven Spielberg Pediatric Research Center and
Lisa Thomas
Helen Xu
Division of Cardiology, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA 90048; and
Andrei E. Medvedev
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814
Ozlem Equils
Moshe Arditi
Toll-like receptor (TLR) 4 has been identified as the primary receptor for enteric LPS, whereas TLR2 has been implicated as the receptor for Gram-positive and fungal cell wall components and for bacterial, mycobacterial, and spirochetal lipoproteins. Vascular endothelial cell (EC) activation or injury by microbial cell wall components such as LPS is of critical importance in the development of sepsis and septic shock. We have previously shown that EC express predominantly TLR4, and have very little TLR2. These cells respond vigorously to LPS via TLR4, but are unresponsive to lipoproteins and other TLR2 ligands. Here we show that LPS, TNF-α, or IFN-γ induce TLR2 expression in both human dermal microvessel EC and HUVEC. Furthermore, LPS and IFN-γ act synergistically to induce TLR2 expression in EC, and LPS-induced TLR2 expression is NF-κB dependent. LPS and IFN-γ also up-regulate TLR4 mRNA expression in EC. These data indicate that TLR2 and TLR4 expression in ECs is regulated by inflammatory molecules such as LPS, TNF-α, or IFN-γ. TLR2 and TLR4 molecules may render EC responsive to TLR2 ligands and may help to explain the synergy between LPS and lipoproteins, and between LPS and IFN-γ, in inducing shock associated with Gram-negative sepsis.
Lipopolysaccharide, or endotoxin, a major component of the outer surface of Gram-negative bacteria, is a potent activator of cells of the immune and inflammatory systems, including macrophages, monocytes, and endothelial cells (EC),3 and contributes to the systemic changes seen in septic shock (1, 2, 3, 4). The endotoxic shock syndrome is characterized by systemic inflammation, multiple organ damage, circulatory collapse, and death (1, 2, 4). Sepsis and septic shock cause ∼200,000 deaths annually in the United States (3).
The endothelium, although initially envisioned as a passive, inert vascular lining, is now considered important in the regulation of vascular tone, coagulation and fibrinolysis, cellular growth, differentiation, and immune and inflammatory responses (5, 6, 7). Furthermore, the vascular EC, like macrophages, are critical targets for LPS and many cytokines (5, 7, 8, 9, 10). Activation of vascular endothelium by LPS results in EC production of various proinflammatory molecules, including leukocyte adhesion molecules, as well as soluble cytokines and chemokines (5, 6, 7).
Janeway and coworkers (11) have hypothesized that the innate immune system senses invading pathogens by virtue of germline-encoded pattern recognition receptors that interact with microbial structures and deliver a danger signal to the host cell (12). Vertebrates and invertebrates initiate a series of defense mechanisms in response to infection by various microorganisms by sensing the presence of conserved pathogen-associated molecular patterns, such as bacterial LPS or lipoproteins (5, 13). Host organisms have developed a set of receptors, referred to as pattern recognition receptors, that specifically recognize pathogen-associated molecular patterns (14, 15). The family of human Toll-like receptors (TLRs) has recently been shown to be involved in innate immune recognition and cellular activation in response to microbial Ags (13, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27). TLR family members are type I transmembrane proteins with leucine-rich repeat extracellular domains and conserved cytoplasmic domains with homology to the mammalian IL-1R (27, 28). Ligand engagement of TLRs results in activation of NF-κB and induction of the cytokines and costimulatory molecules required for the activation of the adaptive immune response (27, 28, 29, 30, 31, 32). At least eight human homologues of Drosophila Toll have been cloned and designated TLRs 1–8 (Refs. 27, 29 , and 33 , and GenBank accession number NM 016562 for TLR7 and NM 016610 for TLR8), but only two TLRs, TLR2 and TLR4, have known functions. It is now well established that TLR4 is the primary signaling receptor for enterobacterial LPS (16, 23, 26, 31, 34), a conclusion further substantiated by recent work by Hoshino et al. (35), who showed that mice in which the tlr4 gene has been deleted fail to respond to LPS. Recent reports have shown that mice lacking TLR2 respond normally to enterobacterial LPS and that neither human nor murine TLR2 plays a role in LPS signaling in the absence of contaminating endotoxin proteins (23), further supporting the concept that TLR4 is the transducing subunit of the receptor for enteric LPS (23, 26). However, current data do not rule out a role for TLR2 in signaling induced by LPS of nonenterobacterial origin. TLR2 has been implicated mainly as the signaling receptor for Gram-positive cell wall components; bacterial, mycobacterial, and spirochetal lipoproteins; and fungi (19, 21, 22, 23, 24, 25, 36, 37, 38). Therefore, a concept of "division of labor" between these two receptors in recognizing diverse microbial pathogens and alerting the immune system has emerged (39). Bacterial lipoprotein, a TLR2 ligand, is one of the most abundant proteins in the outer membrane of Gram-negative and Gram-positive bacteria (40) and acts synergistically with LPS to induce proinflammatory cytokine production and lethal shock (41).
We have reported recently that human dermal microvessel EC (HMEC) and HUVEC express TLR4 predominantly (42). HMEC and HUVEC express little TLR2 and respond vigorously to Escherichia coli LPS via TLR4, but not to Mycobacterium tuberculosis 19-kDa lipoprotein, a TLR2 ligand (42). Transfection of TLR2 confers to HMEC responsiveness to 19-kDa lipoprotein (42). Because several microbial Ags, e.g., Gram-positive cell wall fragments; bacterial, spirochetal, and mycobacterial lipoproteins; and fungal Ags, require TLR2 to activate cells, the regulation of TLR2 mRNA expression in vascular EC could determine EC responses to these TLR2 ligands and influence the overall immune responses. We now describe the regulation of TLR2 expression in EC in response to inflammatory stimuli such as LPS, TNF-α, and IFN-γ. To investigate whether the transcriptional events that lead to LPS-mediated TLR2 mRNA up-regulation in EC are mediated by NF-κB, we studied the effects of various NF-κB inhibitors and NF-κB p65 antisense oligonucleotide. Our results indicate 1) that LPS, TNF-α, and IFN-γ up-regulate TLR2 expression in vascular EC, 2) that LPS-induced TLR2 expression and up-regulation in EC is NF-κB dependent, and 3) that LPS and IFN-γ synergize to induce expression of TLR2 in EC. LPS and IFN-γ also induced TLR4 up-regulation in EC. Induction and up-regulation of TLR2 in response to inflammatory stimuli such as LPS, TNF-α, or IFN-γ in vascular EC may have important implications in host defense against bacterial, mycobacterial, and spirochetal infections and may help explain the well-known synergy between LPS and lipoproteins in the induction of septic shock. The up-regulation of TLR4 by IFN-γ may represent a novel mechanism for the well-described synergy between IFN-γ and LPS.
Cells and reagents
The immortalized HMEC (43), a generous gift of Dr. Candal (Center for Disease Control and Prevention, Atlanta, GA), were cultured in MCDB-131 medium supplemented with 10% heat-inactivated FBS, 2 mM glutamine, and 100 μg/ml penicillin and streptomycin in 24-well plates and used between passages 10 and 14. Tissue culture reagents were obtained from Life Technologies (Gaithersburg, MD). HUVEC were isolated and cultured as previously described (44) and were cultured in Medium 199 with 20% heat-inactivated FBS, 2 mM glutamine, and 100 μg/ml penicillin and streptomycin. PBMC from healthy human volunteer donors were isolated by Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, NJ) density gradient centrifugation . PBMC (25 × 106) were resuspended in RPMI 1640 medium supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES, 0.3% sodium bicarbonate, and 10% FBS. Highly purified, phenol-water-extracted E. coli K235 LPS (<0.008% protein), which was prepared according to the method of McIntire et al. (45), was obtained from Stefanie N. Vogel (Uniformed Services University of the Health Sciences, Bethesda, MD). The purity of this LPS preparation has been previously demonstrated (46, 47), and this preparation of E. coli LPS is active on TLR4-transfected HEK 293 cells and not on TLR2 transfectants (Stefanie N. Vogel, unpublished observation). Human TNF-α and IFN-γ were purchased from R&D Systems (Minneapolis, MN), the 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) and proteasome inhibitor I from Cayman Chemicals (Ann Arbor, MI), and cycloheximide from Sigma. The anti-human TLR2 mAb (TL2.1), which recognizes a TLR2-associated epitope and was generated by immunization of BALB/c mice with Chinese hamster ovary-TLR2 cells (46), was kindly provided by Terje Espevik (The Norwegian University of Science and Technology, Trondheim, Norway). The specificity of this Ab for TLR2 has been reported elsewhere (48). Phosphorothioate-modified oligodeoxynucleotides for NF-κB p65 sense (5′-GCCATGGACGAACTGTTCCCC-3′) and antisense (5′-GGGAACAGTTCGTCCATGGC-3′) were obtained from Isao Kitajima (Kagoshima, Japan) (49).
RT-PCR analysis
Total RNA was isolated from resting and LPS-, TNF-α-, or IFN-γ-stimulated HMEC and HUVEC using an RNA Stat60 isolation reagent (Tel-Test, Friendswood, TX) following the manufacturer's instructions and treated with RNase-free DNase I. For reverse transcription reaction, the Moloney murine leukemia virus preamplification system (Life Technologies, Gaithersburg, MD) was applied. PCR amplification was performed with Taq polymerase (Perkin-Elmer, Foster City, CA) for 32 cycles at 95°C for 45 s, 54°C for 45 s, and 72°C for 1 min (for TLR2 and TLR4), as described earlier (42). The TLR2 and TLR4 oligonucleotide primers used for RT-PCR have been described elsewhere (42). GAPDH primers were obtained from Clontech (Palo Alto, CA). The TLR2 and TLR4 RT-PCR fragments were purified and sequenced to confirm the identity of the fragments. Real-time quantitative PCR was done on the iCycler (Bio-Rad, Hercules, CA) using PE Applied Biosystems (Foster City, CA) SYBR Green PCR kit and the TLR primers described above. The semiquantitative RT-PCR experiments were repeated with cells pretreated for 1 h with 15d-PGJ2 (20 μM), proteasome inhibitor I (100 μM), or cycloheximide (10 μg/ml). EC were pretreated with NF-κB p65 antisense and sense oligonucleotides (30 μM) for 24–48 h three times before LPS stimulation (50 ng/ml) as described earlier (49, 50). For densitometry analysis, the intensity of the bands were measured by the Kodak Digital Science 1D Program with Kodak (Rochester, NY) camera DS40-D2120 and normalized with GAPDH intensity.
Conditioned EC or PBMC were lysed for 30 min on ice in a lysis buffer containing 10 mM Tris-HCl (pH 7.8), 50 mM NaF, 1% Triton X-100, 2 mM EDTA, 100 mM NaCl, 1 mM sodium orthovanadate, 0.1 mM quercitin, and 1 μg/ml each of the protease inhibitors pepstatin, leupeptin, aprotinin, antipain, and chymostatin, 30 μg/ml TPCK, and 1 mM PMSF. Following lysis, cell debris was removed by centrifugation (14,000 × g, 4°C, 15 min), and supernatants were collected and stored at −80°C until use. Protein concentrations were determined using the Bio-Rad assay kit. One-hundred-thirty micrograms of total protein was added in Laemmli buffer, boiled for 5 min, resolved by SDS-10% PAGE in Tris/glycine/SDS buffer (25 mM Tris, 250 mM glycine, 0.1% SDS), and blotted onto Immunobilon P transfer membranes (Amersham Pharmacia Biotech) (100 V, 1.5 h, 4°C). After blocking for 2 h in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20) containing 5% nonfat milk, membranes were washed three times in TBST and probed for 18 h at 4°C with anti-TLR2 mAb TL2.1 (final concentration, 1 μg/ml) in TBST. After washing three times in TBST, membranes were incubated with secondary HRP-conjugated goat anti-mouse IgG (1:2500 dilution; Bio-Rad) and washed five times in TBST, and bands were detected using ECL reagents (Amersham Pharmacia Biotech) according to the manufacturer's description.
Immunohistochemistry staining
HMEC were cultured on eight-chamber microscope slides. Cells were fixed in 4% paraformaldehyde and without drying were incubated with anti-TLR2 mAb TL2.1 (10 μg/ml) or with mouse IgG and an irrelevant IgG2a control Ab for 1 h. Primary Ab was detected with FITC-conjugated goat anti-mouse antiserum following the instructions from the manufacturer (ABC vector kit) (Vector Laboratories, Burlingame, CA). The cells were counterstained with hematoxylin. Samples were viewed and photographed using a Zeiss (Oberkochen, Germany) Axiophot microscope.
Transfection and NF-κB-luciferase assay
HMEC were plated at a concentration of 50,000 cells/well in 24-well plates and cotransfected the following day with reporter genes pCMV-β-galactosidase (0.1 μg) and ELAM-NF-κB-luciferase (0.5 μg) using the FuGene 6 Transfection Reagent (Boehringer Mannheim, Indianapolis, IN) as described earlier (31). After overnight transfection, HMEC were stimulated for 5 h with 50 ng/ml LPS with and without pretreatment (for 60 min) with the proteasome inhibitor I or 15d-PGJ2. Cells were also pretreated with NF-κB p65 antisense oligonucleotide (30 μM) for 24 h three times before LPS stimulation. Cells were lysed in 60 μl of reporter lysis buffer (Promega, Madison, WI), and luciferase activity was measured with a Promega kit and a luminometer (Promega), and β-galactosidase activity was determined by the calorimetric method to normalize transfection efficiency as described earlier (28).
To investigate whether TLR2 expression in EC is regulated by inflammatory stimuli, we first analyzed the effects of LPS, TNF-α, and IFN-γ on TLR2 mRNA and protein expression in HMEC and HUVEC by RT-PCR, Western blot analysis, and immunohistochemical staining. HMEC were stimulated with E. coli LPS (50 ng/ml), TNF-α (20 ng/ml), or IFN-γ (20 ng/ml or 200 IU/ml) for 1, 2, 4, and 5 h, and semiquantitative and real-time RT-PCR were performed by using human TLR2 primers as described earlier (42). TLR2 mRNA expression was significantly up-regulated by LPS, TNF-α, and IFN-γ in HMEC (Fig. 1⇓A). LPS-induced TLR2 mRNA up-regulation was apparent by 30 min (data not shown) and maximal at 2 h. Similar observations were made from primary HUVEC stimulated with LPS (Fig. 1⇓B) or IFN-γ (data not shown). Real-time quantitative PCR confirmed the results obtained by semiquantitative RT-PCR (data not shown). To determine whether LPS-mediated TLR2 mRNA up-regulation in EC was dependent on new protein synthesis, we preincubated EC with cycloheximide (10 μg/ml) for 60 min before LPS stimulation. Cycloheximide pretreatment inhibited LPS-induced TLR2 up-regulation, suggesting the need for new protein synthesis, i.e., that TLR2 is not an immediate early gene or primary response gene for LPS activation (Fig. 1⇓C). To test whether LPS stimulation of TLR2 synthesis also requires de novo transcription, HMEC were preincubated with the transcription inhibitor actinomycin D before LPS stimulation. Actinomycin D also inhibited LPS-induced up-regulation of TLR2 mRNA (data not shown), suggesting that LPS-TLR4 activation triggers both transcriptional and translational events in EC that lead to TLR2 induction. Although our data suggest that the regulation of TLR2 at the transcriptional level may be important, these preliminary experiments about the mechanism of TLR2 mRNA up-regulation do not rule out additional possible mechanisms, including mRNA stabilization.
Up-regulation of human TLR2 mRNA in EC in response to LPS, TNF-α, and IFN-γ by RT-PCR. A, Semiquantitative RT-PCR for human TLR2 (347 bp) in HMEC was performed. HMEC were stimulated with E. coli LPS (50 ng/ml), TNF-α (20 ng/ml), or IFN-γ (20 ng/ml) for 1, 2, 4, and 5 h. GAPDH expression was used as control. Data are from an experiment representative of six independent experiments yielding similar results. Graphs indicate relative TLR2 mRNA expression (densitometric quantification of TLR2 mRNA transcript over the GAPDH mRNA transcript) for each time point, and constitutive expression is expressed as 100%. B, HUVEC were stimulated with LPS (50 ng/ml) for 1, 2, and 5 h. Semiquantitative RT-PCR was performed. GAPDH expression was used as control. C, HMEC were pretreated with medium or 10 μg/ml of the protein synthesis inhibitor, cycloheximide (CHX), for 1 h and then stimulated with LPS for 4 h. Total RNA were extracted, and RT-PCR was performed. GAPDH expression was used as control.
LPS-induced TLR2 protein expression and up-regulation in HMEC was also observed by Western blot analysis (Fig. 2⇓A) and by immunohistochemistry (Fig. 2⇓B) using an anti-human TLR2 mAb (TL2.1). Unstimulated HMEC exhibited a low expression of TLR2 protein that was markedly up-regulated in response to LPS stimulation for 4 h (Fig. 2⇓A). Likewise, following 4 h of LPS (50 ng/ml) stimulation, cellular expression of TLR2 protein was significantly enhanced on fixed HMEC, whereas unstimulated HMEC showed very weak TLR2 expression (Fig. 2⇓B). An isotype-matched control Ab (data not shown) and IgG control stainings were negative. Vascular EC can be activated by LPS in one of two ways: a direct pathway where LPS-sCD14 (soluble CD14) complexes directly activate ECs, and an indirect pathway through various inflammatory mediators induced by LPS-activated immune cells (i.e., IL-1β, TNF-α, IFN-γ) (reviewed in Ref. 51). Induction and up-regulation of TLR2 in response to inflammatory stimuli such as LPS, TNF-α, and IFN-γ in vascular endothelium may have important implications in host defenses against bacterial, mycobacterial, and spirochetal infections by rendering these cells responsive and more sensitive to microbial ligands that specifically use TLR2, and may help explain the well-known synergy between LPS and lipoproteins in the induction of shock (41). This study provides the basis for further investigating the functional correlation of TLR2 up-regulation in EC by various inflammatory mediators. Our observations also suggest that the induction of TLR2 expression is not limited to TLR4 activation by LPS. TNF-α and IFN-γ stimulation also result in a similar level of TLR2 up-regulation, suggesting that multiple signaling pathways induced by various inflammatory mediators converge to induce TLR2 expression.
LPS up-regulates TLR2 protein expression in HMEC. A, Western blotting. Confluent HMEC monolayers were treated for 4 h with either medium or 50 ng/ml LPS, rinsed with ice-cold PBS, detached with trypsin/EDTA, and centrifuged, and total cellular extracts were prepared as described (31 ). As a positive control for human TLR2 expression, cellular extracts from 25 × 106 human PBMC were used. Protein samples were subjected to SDS-PAGE (130 μg/ml of total protein per lane) followed by blotting with anti-human TLR2 mAb TL2.1. The signal was detected with a secondary HRP-labeled Ab by application of standard ECL techniques. The positions of the m.w. standards are shown on the left. B, Immunostaining. HMEC were incubated for 4 h with LPS (50 ng/ml), fixed in 4% paraformaldehyde/PBS, and stained with anti-human TLR2 mAb or control mouse IgG. Panel A, LPS-stimulated HMEC stained with control mouse IgG; panel B, resting HMEC stained with anti-human TLR2 mAb TL2.1 (10 μg/ml); panel C, LPS-stimulated HMEC with anti-human TLR2 mAb TL2.1 (10 μg/ml).
We observed that, in addition to TLR2, LPS also induced the up-regulation of TLR4 expression in HMEC (Fig. 3⇓) and in primary HUVEC (data not shown). To investigate whether IFN-γ also induces TLR4, we incubated HMEC with IFN-γ. We show that, in addition to TLR2, IFN-γ also induces the up-regulation of TLR4 mRNA in EC (Fig. 3⇓). It is well known that IFN-γ enhances both sensitivity and the magnitude of cellular responses to LPS and causes synergistic activation of cells (52, 53, 54). In the presence of IFN-γ, otherwise nontoxic doses of LPS become highly lethal for mice (55). The mechanisms of this synergistic toxicity are not well understood. In addition, the combination of the direct and indirect pathways of LPS-induced EC activation would provide a much more realistic representation of the LPS-triggered inflammatory stimuli that the endothelium is exposed to in the course of response to infection and might reveal changes that are much more potent. To investigate whether LPS and IFN-γ can act synergistically to induce TLR2 expression in EC, we incubated HMEC with suboptimal concentrations of LPS (1 or 5 ng/ml), IFN-γ (1 or 5 ng/ml), or various combinations of LPS plus IFN-γ and assessed TLR2 mRNA expression by RT-PCR. When used together, LPS and IFN-γ acted synergistically to induce significant up-regulation of TLR2 expression in HMEC at concentrations that were ineffective when each was used alone (Fig. 4⇓). We postulate that IFN-γ-induced up-regulation of TLR4 and TLR2 may transform a small amount of LPS or lipoprotein into a lethal stimulus capable of causing shock. This mechanism may also enable the host to respond quickly to relatively low doses of LPS and lipoproteins, thereby enhancing the innate immune response and antibacterial defenses.
LPS and IFN-γ up-regulate TLR4 mRNA in HMEC. HMEC were stimulated either with LPS (50 ng/ml) for 1 and 2 h or with IFN-γ (20 ng/ml) for 1 and 4 h. Expression of human TLR4 (548 bp) in HMEC was analyzed by semiquantitative PCR following reverse transcription. RT-PCR analysis of GAPDH expression was used as control (lower panel, 983 bp). Data are from an experiment representative of three independent experiments performed with similar results.
LPS and IFN-γ act synergistically to up-regulate TLR2 expression in HMEC. HMEC were stimulated for 4 h with suboptimal concentrations of LPS (1 or 5 ng/ml) or IFN-γ (1 or 5 ng/ml) and with different combinations of LPS/IFN-γ concentrations. Expression of human TLR2 (347 bp) was analyzed by semiquantitative PCR following reverse transcription. GAPDH expression was used as control. Data shown are from an experiment representative of three independent experiments performed with similar results.
We have previously shown that human microvascular ECs, which are one of the first lines of defense against invading microbial agents including Gram-negative bacteria, actively participate in innate immune responses and that enterobacterial LPS-induced responses in EC are specifically mediated via TLR4 (31, 42). TLR4 in human vascular EC participates in sensing LPS by a mechanism that is sCD14 dependent (42). After detecting the LPS molecules, TLR4 transmits the information across the cell membrane via the IL-1R intracellular signaling molecules, leading to the activation of NF-κB and regulation of several responsive genes (42). To investigate whether LPS-induced TLR2 up-regulation in EC is dependent on NF-κB activation, we pretreated HMEC with various chemical inhibitors of the transcription factor NF-κB and with sense and antisense p65 NF-κB oligonucleotides and analyzed their effects on LPS-mediated TLR2 mRNA up-regulation by RT-PCR. We observed that pretreatment of HMEC with proteasome inhibitor I, an NF-κB inhibitor (56), or with antisense phosphorothioate oligodeoxynucleotide against p65 NF-κB inhibited LPS-induced TLR2 up-regulation (Fig. 5⇓A) and NF-κB-luciferase activity (Fig. 5⇓B), whereas NF-κB p65 sense oligonuclotide had no significant effect on the mRNA TLR2. The housekeeping enzyme, GAPDH, was unaffected in these cells (Fig. 5⇓A). These inhibitors did not result in EC death as measured by trypan blue uptake. Collectively, these results suggest that LPS-induced TLR2 mRNA expression in EC is NF-κB dependent. Our findings are consistent with a recent report that mouse TLR2 promoter contains NF-κB binding sites (57). Another study, investigating the putative 5′-proximal promoter of the human TLR4 gene, did not find any NF-κB binding site, at least in the proximal portion of this promoter (58). Because LPS-mediated NF-κB activation in EC is sCD14 dependent (42), as expected, the LPS-induced TLR2 up-regulation in EC was sCD14 dependent as well (data not shown). Of interest, 15d-PGJ2, a cyclopentenone PG (cyPG), also inhibited LPS-induced TLR2 up-regulation (Fig. 5⇓A) and NF-κB-luciferase activity (Fig. 5⇓B). It has been suggested that cyPGs exert anti-inflammatory activity through the activation of peroxisome proliferator-activated receptor-γ (PPAR-γ) (59, 60). The bioactive cyPG, 15d-PGJ2, which is physiologically formed by dehydration and isomerization of the cyclooxygenase metabolite PGD2, can activate PPAR-γ, a nuclear receptor that interferes with NF-κB transcriptional activity (59, 60). A recent study demonstrated a novel mechanism of anti-inflammatory activity of cyPGs that is PPAR-γ independent and based on the direct inhibition and modification of the IκB kinase (IKK) β subunit of IKKs (61). Elevated cyPG synthesis has been detected in late phases of inflammation (59) and is associated with resolution of inflammation (62). Concentrations of cyPG that are sufficient to inhibit IKK may occur locally during the late phase of inflammation, and down-regulation of TLR2 in response to LPS or cytokines may be part of the resolution of inflammation. Our results would also suggest that cyPG, and possibly more potent derivatives, may have therapeutic value in the treatment of sepsis and inflammatory diseases in which inhibition of NF-κB activity may be desirable, and where LPS-induced TLR2 up-regulation may synergistically augment the inflammatory responses.
Up-regulation of TLR2 is NF-κB dependent. A, RT-PCR. HMEC were pretreated for 1 h with either proteasome inhibitor I (100 μM) or 15d-PGJ2 (20 μM) and stimulated with LPS (50 ng/ml) for 4 h. NF-κB antisense and sense oligonucleotides (30 μM) were added to the medium three times during 48 h before stimulation with 50 ng/ml LPS. Expression of human TLR2 (347 bp) was analyzed by semiquantitative PCR following reverse transcription. GAPDH expression was used as control. Data shown are from an experiment representative of three independent experiments performed for each inhibitor with similar results. B, NF-κB-luciferase activity. HMEC were transiently transfected with NF-κB-luciferase and β-galactosidase reporter vectors overnight. Cells were then stimulated with LPS (50 ng/ml) for 5 h with and without pretreatment with proteasome inhibitor I, p65 antisense oligos, and 15d-PGJ2, and luciferase and β-galactosidase assays were performed as described under Materials and Methods. Data shown are mean + SD of three or more independent experiments and are reported as a percentage of LPS-stimulated NF-κB promoter activity.
Our data indicate that TLR2 and TLR4 expression in EC can be regulated at sites of infection and inflammation, either directly by LPS or indirectly by inflammatory cytokines such as TNF-α and IFN-γ. Our observations are consistent with and extend the findings of two recent reports showing that LPS induces TLR2 in mouse adipose cells (39) and TLR4 mRNA expression in rat coronary EC (63). In the latter study, the investigators showed that NF-κB is involved in LPS induction of TLR4 mRNA, but found no accompanying increase in TLR4 protein (63). Although LPS was found to inhibit the expression of TLR4 mRNA in two earlier studies using RAW 264 mouse macrophage cell lines (26, 64), a more recent study reported that LPS and proinflammatory cytokines increase TLR4 mRNA expression in human monocytes and polymorphonuclear cells (65). This divergence most likely reflects differences in cell types and differentiation stages. The observation that EC express the receptor for LPS and induce the receptor for lipoproteins and Gram-positive, spirochetal, and fungal cell wall components in response to LPS suggests that the eventual responsiveness of EC to various TLR2 ligands may be modulated by external stimuli. Furthermore, a recent study showed that TLR2 can also function as a "death receptor" that mediates apoptosis in addition to NF-κB activation in response to bacterial lipoproteins (66). Therefore, TLR2 expression and up-regulation in EC cells by LPS and/or various inflammatory cytokines may also augment and contribute to endothelial cell apoptosis in bacterial sepsis and septic shock leading to vascular collapse. Indeed LPS has been shown to induce disseminated endothelial apoptosis resulting in endotoxic shock (67).
Our data also indicate that EC are involved at the earliest stages of the immune response and are capable of directly sensing the presence of bacterial cell wall components. These observations underline the importance and direct role of the vascular endothelium as an integral component of the innate immune response.
We thank Drs. Harvey Herschman (University of California, Los Angeles, CA) and Stefanie Vogel (Uniformed Services University of Health Sciences, Bethesda, MD) for critical reading of the manuscript.
↵1 This work was supported by National Institutes of Health Grants AI-40275 (to M.A.) and AI-18797 (to A.M.).
↵2 Address correspondence and reprint requests to Dr. Moshe Arditi, Department of Pediatrics, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Room 4400, Los Angeles, CA 90048. E-mail address: moshe.arditi{at}cshs.org
↵3 Abbreviations used in this paper: EC, endothelial cells; HMEC, human dermal microvessel EC; TLR, Toll-like receptor; sCD14, soluble CD14; cyPG, cyclopentenone PG; 15d-PGJ2, 15-deoxy-Δ12, 14-PGJ2; PPAR-γ, peroxisome proliferator-activated receptor-γ; IKK, IκB kinase.
Accepted November 3, 2000.
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Emmanuelle Faure, Lisa Thomas, Helen Xu, Andrei E. Medvedev, Ozlem Equils, Moshe Arditi
The Journal of Immunology February 1, 2001, 166 (3) 2018-2024; DOI: 10.4049/jimmunol.166.3.2018
Anti-inflammatory and anti-bacterial effect of polyacetylene compound from Cirsium japonicum var. ussuriense (54.19)
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A woman taps her husband on his forehead with every decision he believes to be healthy. He tells her that despite her efforts, he doesn't like drinking vegetable juice. V8 also delivers a full serving of vegetable and fruits with its V-Fusion drinks, so even you can have a V8.


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text:
Ohlen's Restaurant and Bakery
Ohlen's Restaurant and Bakery (HMP2Y)
Location: Baxter Springs, KS 66713 Cherokee County
Baxter Springs Historic Walking Tour
Henry and Louisa Ohlen purchased this property in February 1878 and opened Ohlen's Restaurant and Bakery. Among their offerings were ice cream and oysters which were advertised "in their proper seasons." The Ohlen's [sic] also offered a day boarding house so those who wished could "freshen up" before their meal. The restaurant was also known as Delmonico's.
In the fall of 1889, the Ohlen's [sic] tore down their frame restaurant and constructed the present brick building in an effort to "modernize." The grand opening was held in January 1890. The Ohlen's [sic] sold their business in 1914 after 36 years, bringing an end to one of the favorite eating spots of early-day Baxter Springs.
HMP2Y
Industry and Commerce
Baxter Springs Heritage Center & Museum
Friday, October 24th, 2014 at 12:37am PDT -07:00
37° 1' 31.68" N, 94° 44' 7.26" W
At or near 1011 Military Ave, Baxter Springs KS 66713, US
Show me all markers in: Baxter Springs, KS | Cherokee County | 66713 | Kansas | United States of America
Polster Dry Goods Store
Bischosberger Hotel
Drovers' and Farmers' Bank
Murder of C. M. Taylor
Chicago Drug Store
Opperman Harness and Saddle Shop
Phillips 66 Petroleum Company Gas Station
Illinois Cash Store
Caf? on the Route
Crowell Bank
This marker can also be identified by its HM code. You can use the hashtag #HMP2Y in tweets, and any other place where you might refer to this marker, including when searching on this site.

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text:
University of Louisiana at Lafayette
#3 Best Public Universities in Louisiana
Lafayette, Louisiana
University of Louisiana at Lafayette overview
Location Lafayette, Louisiana
Online louisiana.edu
Find out how likely you are to get accepted at UL Lafayette based on your GPA, test scores, and even extracurricular activities. Then, get custom recommendations for how to improve your odds - completely for free.
Located in Lafayette, Louisiana, University of Louisiana at Lafayette is a mid-size four year public college offering both undergrad and graduate programs. In the most recent statistical year 9,138 prospective students applied, and 6,206 were granted admission. Of those admitted 2,536 enrolled in the school. 13,484 students are enrolled on a full time basis, and 3,639 attend part time. In-state tuition for 2019/2020, excluding room and board, is $5,407 plus fees of $4,975. UL Lafayette is ranked 635 nationally in the CollegeSimply.com 2020 U.S. Colleges Ranking.
UL Lafayette is rated within the lower 47% of four-year colleges nationally and earns a CollegeSimply grade of 5 out of 10. Learn how ratings are calculated
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Rankings for University of Louisiana at Lafayette within the 2021 CollegeSimply Best Colleges Lists.
#5 in Best Colleges in Louisiana
#3 in Best Public Colleges in Louisiana
#35 in Best Value Colleges in Louisiana
SAT 1020/1268
GPA 3.2
Applications Due Jun 1, 2022
Applicant Competition Low
Louisiana Resident Tuition
In State Costs for University of Louisiana at Lafayette
Out of State Costs for University of Louisiana at Lafayette
Popular subjects studied by University of Louisiana at Lafayette students
Liberal Arts and Humanities 15%
Engineering 6%
credit: Visit Tuscaloosa / license
The University of Alabama
The University of West Florida
Primary data source, U.S. Department of Education https://nces.ed.gov/collegenavigator/?id=160658 IPEDS survey data for University of Louisiana at Lafayette.
Current students may use the University of Louisiana at Lafayette GPA calculator to determine their current college GPA.


text:
Rick Rubin is an American record producer who holds eight Grammy Awards, including Album of the Year which he won with the Dixie Chicks and Adele in 2007 and 2012, respectively. He has also won numerous producer of the year awards. This is a list compiling his production discography.

1980s

 1981: The Pricks – The Pricks
 1983: Hose – Hose
 1984: T La Rock & Jazzy Jay – "It's Yours"
 1984: LL Cool J – "I Need a Beat"
 1985: Beastie Boys – "Rock Hard"
 1985: Run-DMC – King of Rock (mixing)
 1985: Jazzy Jay – "Def Jam/Cold Chillin' in the Spot"
 1985: Jimmy Spicer – "This Is It / Beat the Clock"
 1985: Hollis Crew – "It's the Beat"
 1985: LL Cool J – Radio
 1986: Run-DMC – Raising Hell
 1986: Slayer  – Reign in Blood
 1986: Beastie Boys – Licensed to Ill
 1986: Original Concept – Can You Feel It?
 1986: Junkyard Band  – "The Word" / "Sardines"
 1987: Public Enemy – Yo! Bum Rush the Show (exec.)
 1987: The Cult – Electric
 1987: Run-DMC – "Christmas in Hollis"
 1987: Various – Less than Zero (soundtrack)
 1987: LL Cool J – "Jack the Ripper"
 1988: Public Enemy – It Takes a Nation of Millions to Hold Us Back (exec.)
 1988: Slayer  – South of Heaven
 1988: Danzig – Danzig
 1988: Run-DMC – Tougher Than Leather
 1988: Original Concept – Straight from the Basement of Kooley High! (exec.)
 1989: Masters of Reality – Masters of Reality
 1989: LL Cool J – Walking With a Panther
 1989: Wolfsbane – Live Fast, Die Fast
 1989: Andrew Dice Clay – Dice

1990s

 1990: Andrew Dice Clay – The Day the Laughter Died
 1990: Danzig – Danzig II: Lucifuge
 1990: Slayer – Seasons in the Abyss
 1990: Geto Boys – The Geto Boys (supervisor)
 1990: Trouble – Trouble
 1990: Wolfsbane – All Hell's Breaking Loose Down at Little Kathy Wilson's Place
 1991: Red Hot Chili Peppers – Blood Sugar Sex Magik
 1991: Slayer – Decade of Aggression
 1991: Andrew Dice Clay – Dice Rules
 1991: The Four Horsemen – Nobody Said It Was Easy
 1991: Dan Baird – Love Songs for the Hearing Impaired
 1992: Sir Mix-a-Lot – Mack Daddy (exec.)
 1992: Trouble – Manic Frustration
 1992: Danzig – Danzig III: How the Gods Kill (exec.)
 1992: The Red Devils – King King
 1992: Andrew Dice Clay – 40 Too Long
 1992: Red Hot Chili Peppers – What Hits!?
 1993: Flipper – American Grafishy (exec.)
 1993: Mick Jagger – Wandering Spirit
 1993: Raging Slab – Dynamite Monster Boogie Concert (exec.)
 1993: The Cult  – The Witch (CD, Single)
 1993: Danzig – Thrall-Demonsweatlive
 1993: Joan Jett and the Blackhearts – Flashback
 1993: Tom Petty and the Heartbreakers – Greatest Hits
 1993: Messiah – 21st Century Jesus (exec.)
 1993: Andrew Dice Clay – The Day the Laughter Died, Part II
 1993: Digital Orgasm – Do It (exec.)
 1993: Barkmarket – Gimmick (exec.)
 1993: Various – Last Action Hero: Music From The Original Motion Picture
 1994: Johnny Cash – American Recordings
 1994: Sir Mix-a-Lot – Chief Boot Knocka (exec.)
 1994: Slayer – Divine Intervention (exec.)
 1994: Danzig – Danzig 4
 1994: Tom Petty – Wildflowers
 1994: Andrew Dice Clay – Dice Live at Madison Square Garden
 1994: Deconstruction – Deconstruction (exec.)
 1994: Milk – Never Dated (exec.)
 1994: Lords of Acid – Voodoo-U (exec.)
 1994: Red Hot Chili Peppers – Live Rare Remix Box
 1994: Red Hot Chili Peppers – The Plasma Shaft
 1995: God Lives Underwater – God Lives Underwater (exec.)
 1995: Nine Inch Nails – Further Down the Spiral
 1995: Red Hot Chili Peppers – One Hot Minute
 1995: AC/DC – Ballbreaker
 1995: God Lives Underwater – Empty (exec.)
 1995: Jazz Lee Alston – Jazz Lee Alston (exec.)
 1996: Slayer – Undisputed Attitude (exec.)
 1996: Tom Petty and the Heartbreakers – She's the One
 1996: Sir Mix-a-Lot – Return of the Bumpasaurus (exec.)
 1996: Donovan – Sutras
 1996: LL Cool J – All World: Greatest Hits
 1996: Johnny Cash – Unchained
 1996: Barkmarket – L. Ron (exec.)
 1996: Raging Slab – Sing Monkey Sing (exec.)
 1996: Kwest tha Madd Ladd – This Is My First Album (exec.)
 1997: System of a Down - Demo Tape 3
 1997: Various – Private Parts: The Album
 1998: Slayer – Diabolus in Musica
 1998: Johnny Cash & Willie Nelson – VH1 Storytellers
 1998: System of a Down – System of a Down
 1998: Lucinda Williams – Car Wheels on a Gravel Road (mixing)
 1998: Various – Chef Aid: The South Park Album
 1998: Sheryl Crow – The Globe Sessions
 1999: Kula Shaker – Peasants, Pigs & Astronauts
 1999: Tom Petty and the Heartbreakers – Echo
 1999: Red Hot Chili Peppers – Californication
 1999: Various – Loud Rocks
 1999: Melanie C – Northern Star
 1999: Mr. Hankey Poo – Mr. Hankey The Christmas Poo
 1999: Various – Mr. Hankey's Christmas Classics (exec.)

2000s

 2000: Johnny Cash – American III: Solitary Man
 2000: Rage Against the Machine – Renegades
 2000: Eagle-Eye Cherry – Living in the Present Future
 2000: Paloalto – Paloalto
 2001: Saul Williams – Amethyst Rock Star
 2001: American Head Charge – The War of Art
 2001: System of a Down – Toxicity
 2001: Slayer – God Hates Us All (exec.)
 2001: Macy Gray – The Id (exec.)
 2001: Nusrat Fateh Ali Khan – The Final Studio Recordings
 2001: Krishna Das – Breath of the Heart
 2002: Aerosmith – O, Yeah! Ultimate Aerosmith Hits
 2002: Red Hot Chili Peppers – By the Way
 2002: Johnny Cash – American IV: The Man Comes Around
 2002: Audioslave – Audioslave
 2002: System of a Down – Steal This Album!
 2002: Rahat Nusrat Fateh Ali Khan – Rahat
 2003: The Jayhawks – Rainy Day Music (exec.)
 2003: The Mars Volta – De-Loused in the Comatorium
 2003: Limp Bizkit – Results May Vary
 2003: To My Surprise – To My Surprise (exec.)
 2003: Joe Strummer & The Mescaleros – Streetcore
 2003: Jay-Z – "99 Problems"
 2003: Rage Against the Machine – Live at the Grand Olympic Auditorium
 2003: Johnny Cash – Unearthed
 2003: Paloalto – Heroes and Villains
 2003: Krishna Das – Door of Faith
 2003: Manmade God – Manmade God (exec.)
 2004: Johnny Cash – My Mother's Hymn Book
 2004: Slipknot – Vol. 3: (The Subliminal Verses)
 2004: The (International) Noise Conspiracy – Armed Love
 2004: Lil Jon & the East Side Boyz – Crunk Juice
 2004: T.H. White – More Than Before (keys)
 2005: Weezer – Make Believe
 2005: System of a Down – Mezmerize
 2005: Audioslave – Out of Exile
 2005: Shakira – Fijación Oral Vol. 1 (exec.)
 2005: Limp Bizkit – Greatest Hitz
 2005: Neil Diamond – 12 Songs
 2005: Johnny Cash -The Legend of Johnny Cash
 2005: System of a Down – Hypnotize
 2005: Shakira – Oral Fixation Vol. 2 (exec.)
 2006: Red Hot Chili Peppers – Stadium Arcadium
 2006: Dixie Chicks – Taking the Long Way
 2006: Johnny Cash – American V: A Hundred Highways
 2006: Slayer – Christ Illusion (exec.)
 2006: Justin Timberlake – FutureSex/LoveSounds
 2006: God Dethroned – The Toxic Touch
 2006: U2 – U218 Singles
 2006: Johnny Cash The Legend of Johnny Cash Vol. II
 2007: Linkin Park – Minutes to Midnight
 2007: Poison – Poison'd!
 2007: Luna Halo – Luna Halo (exec.)
 2007: Gossip – Live in Liverpool (exec.)
 2007: Dan Wilson – Free Life (exec.)
 2007: Kanye West, Nas, KRS-One, Rakim – "Classic (Better Than I've Ever Been)"
 2007: Vanessa Carlton - "Heroes & Thieves"
 2007: Coheed and Cambria - "Good Apollo, I'm Burning Star IV, Volume Two: No World for Tomorrow"
 2008: Ours – Dancing for the Death of an Imaginary Enemy
 2008: Neil Diamond – Home Before Dark
 2008: Weezer – Weezer (Red Album)
 2008: Jakob Dylan – Seeing Things
 2008: Metallica – Death Magnetic
 2008: The (International) Noise Conspiracy – The Cross of My Calling
 2009: Gossip – Music for Men
 2009: Pete Yorn – Back & Fourth (exec.)
 2009: The Avett Brothers – I and Love and You
 2009: Brandi Carlile – Give Up the Ghost
 2009: Slayer – World Painted Blood (exec.)
 2009: Type O Negative – Bloody Kisses "Top Shelf" edition (2009 re-release) "Summer Breeze" (Rick Rubin Mix)

2010s

 2010: Johnny Cash – American VI: Ain't No Grave
 2010: Gogol Bordello – Trans-Continental Hustle
 2010: Linkin Park – A Thousand Suns
 2010: Josh Groban – Illuminations
 2010: Kid Rock – Born Free
 2011: Adele – 21
 2011: Red Hot Chili Peppers – I'm with You
 2011: Metallica – Beyond Magnetic
 2011: Linkin Park – A Thousand Suns+
 2012: Linkin Park – Living Things
 2012: Howlin' Rain  – The Russian Wilds (exec.)
 2012: ZZ Top – La Futura
 2012: The Avett Brothers – The Carpenter
 2012: Lana Del Rey – Paradise
 2012: Red Hot Chili Peppers – Rock & Roll Hall of Fame Covers EP
 2013: Linkin Park – Living Things +
 2013: Black Sabbath – 13
 2013: Kanye West – Yeezus (exec.)
 2013: Eminem – The Marshall Mathers LP 2 (exec.)
 2013: Jake Bugg – Shangri La
 2013: Linkin Park – Recharged
 2013: Lady Gaga – Artpop
 2013: The Avett Brothers – Magpie and the Dandelion
 2013: Red Hot Chili Peppers – I'm Beside You
 2014: Jennifer Nettles – That Girl
 2014: Ed Sheeran – X
 2014: Angus & Julia Stone – Angus & Julia Stone
 2014: Damien Rice – My Favourite Faded Fantasy
 2014: Linkin Park & Alec Puro - Mall
 2014: Wu-Tang Clan – A Better Tomorrow
 2014: Yusuf – Tell 'Em I'm Gone
 2015: GoldLink – And After That, We Didn't Talk
 2016: Kanye West – The Life of Pablo
 2016: Various – Star Wars Headspace
 2016: James Blake - The Colour in Anything
 2016: The Avett Brothers – True Sadness
 2017: Billy Corgan  – Ogilala
 2017: Jovanotti  – Oh, vita!
 2017: Eminem – Revival
 2018: Ruen Brothers – 'All My Shades Of Blue'
 2018: The Smashing Pumpkins  – Shiny and Oh So Bright, Vol. 1 / LP: No Past. No Future. No Sun.
 2019: Santana – Africa Speaks
 2019: Jovanotti – Jova Beach Party
 2019: Jovanotti – Lorenzo sulla Luna
 2019: Kae Tempest – The Book of Traps and Lessons
 2019: The Avett Brothers – Closer Than Together

2020s

 2020: The Strokes – The New Abnormal
 2021: Imagine Dragons –  Mercury – Act 1
 2021: Santana - "Peace Power", "America for Sale", "Mother Yes"
 2022: Jovanotti - Mediterraneo
 2022: Red Hot Chili Peppers - Unlimited Love
 2022: Imagine Dragons - Mercury - Act 2
 2022: Red Hot Chili Peppers - Return of the Dream Canteen
 2022: Neil Young and Crazy Horse - World Record

References

Production discographies
Discographies of American artists
Rock music discographies
Pop music discographies
Hip hop discographies
Rick Rubin

redpajama_set_name: RedPajamaWikipedia


text:
Home Big Biz Rohit Sharma Becomes FIRST-ever Indian Cricketer To Play 100 T20I Matches
Rohit Sharma has become the FIRST-ever Indian cricketer to play 100 Twenty20 International matches.
Indian International Cricketer Rohit Sharma has become the first-ever Indian cricketer and second overall after Pakistani cricket Shoaib Malik to play 100 Twenty20 International (T20I) matches.
Sharma attained the feat after taking the field against Bangladesh in the second T20I in Rajkot on 7th November.
As of 27 October 2019, Sharma has scored 37 international centuries: 6 in Test Cricket, 27 in One-day Internationals and four in Twenty20 internationals.
Sharma is also ranked joint 24th among players with the most centuries in international cricket.
He holds the world record for the highest individual score by a batsman playing in a one-day international match, having scored 264 against Sri Lanka at Eden Gardens, Kolkata, on 13 November 2014.
Sharma is the only player to have scored three double-centuries in this form of international cricket.
In Twenty20 international cricket, he currently holds the world records for most career runs and most career centuries.
On 5 October 2019, during a Test match against South Africa, he became the first batsman to score two centuries in his first appearance as an opening batsman.
Sharma also plays for Mumbai in domestic cricket and captains Mumbai Indians in the Indian Premier League as a right-handed batsman and an occasional right-arm off break bowler.
He is the vice-captain of the Indian national team in limited-overs formats.
What's The Market Size of India's Chemicals & Petrochemicals Sector? – Get The Info Here


text:
Episode 1 Episode 2 Episode 3 Episode 4 Episode 5 Episode 6 Episode 7 Episode 8 Episode 9 Episode 10 Episode 11 Episode 12 Episode 13 Episode 14 Episode 15
Samantha Newly has been a bad, bad girl. But thanks to an 8-day coma, she can't remember a thing. Fortunately her best friend Andrea is by her side with a cocktail and the 4-1-1 on every dirty deed. But the more Sam pieces together her past, the less she likes her old self. Maybe this is a chance to start over and do it all again.
Director: Cecelia Ahern, Donald Todd
Actors: Barry Watson, Christina Applegate, Jean Smart, Jennifer Esposito, Kevin Dunn, Melissa McCarthy, Tim Russ
Networks: ABC
Keywords:Series 2007
High school mathlete Lindsay Weir rebels and begins hanging out with a crowd of burnouts (the "freaks"), while her brother Sam Weir navigates a different part of the social universe…
What if you could use time travel to save your life? Welcome to "LIFELINE," the new YouTube Red Original Series about the Lifeline insurance agency, whose specially trained agents jump…
Dating Rules from My Future Self
A girl gets romantic advice from herself ten years in the future via text message.
During an experiment gone bad, radiation turns a scientist into a raging green behemoth whenever he becomes agitated. Unable to control his transformations, David Banner searches for a cure as…
Jungle Gold
Jungle Gold is an American reality television series on the Discovery Channel that debuted on October 26, 2012.
The Late Show with Stephen Colbert
Improv actor and comic Stephen Colbert leaves his "The Colbert Report" character behind as he makes his long-awaited return to television. As the host of The Late Show franchise redux…
Genre: Comedy, Talk
The story of how Detective Superintendent Steve Fulcher deliberately breached police procedure and protocol to catch a killer, a decision that ultimately cost him his career and reputation.
Living Single
Living Single is an American television sitcom that aired for five seasons on the Fox network from August 22, 1993, to January 1, 1998. The show centered on the lives…
MasterChef Australia is a Logie Award-winning Australian competitive cooking game show based on the original British MasterChef. It is produced by Shine Australia and screens on Network Ten. Restaurateur and…
Genre: Reality
Two siblings – an idealistic teenage boy and his naïve younger sister – are forced to pilot an abandoned Jaeger across a hostile landscape in a desperate attempt to find…
Fast N' Loud
Motor mastermind Richard Rawlings and mechanical prodigy Aaron Kaufman search for forgotten and neglected vintage cars. They need big projects and big profits to keep the doors open at Gas…
Deadly Cults
Dive into unhinged murder cases in which investigators uncover and expose the dark world of cults that kill. The series explores how power and manipulation can lead to extreme actions.


text:
class CustomVariable < ApplicationRecord
  has_paper_trail

  VARIABLES = {
    "single_advisor_points" => :text,
    "multiple_advisor_points" => :text,
    "program_level" => :text,
    "identity_issuing_country" => :text,
    "class_schedule_text" => :text,
    "redirect_email" => :text,
    "notification_footer" => :text,
    "minimum_grade_for_approval" => :text,
    "grade_of_disapproval_for_absence" => :text,
    "professor_login_can_post_grades" => :text,
  }

  validates :variable, :presence => true
  validate :check_constraints_between_variables
  before_destroy :validate_destroy
  before_validation :validate_create, :on => :create

  def self.single_advisor_points
  	config = CustomVariable.find_by_variable(:single_advisor_points)
  	config.nil? ? 1.0 : config.value.to_f
  end

  def self.multiple_advisor_points
  	config = CustomVariable.find_by_variable(:multiple_advisor_points)
  	config.nil? ? 0.5 : config.value.to_f
  end

  def self.program_level
    config = CustomVariable.find_by_variable(:program_level)
    config.nil? ? nil : config.value.to_i
  end

  def self.identity_issuing_country
    config = CustomVariable.find_by_variable(:identity_issuing_country)
    Country.find_by_name(config.nil? ? "Brasil": config.value)
  end

  def self.class_schedule_text
    config = CustomVariable.find_by_variable(:class_schedule_text)
    config.nil? ? "" : config.value
  end

  def self.redirect_email
    config = CustomVariable.find_by_variable(:redirect_email)
    config.nil? ? nil : (config.value || '')
  end

  def self.notification_footer
    config = CustomVariable.find_by_variable(:notification_footer)
    config.nil? ? "" : config.value
  end

  def self.minimum_grade_for_approval
    config = CustomVariable.find_by_variable(:minimum_grade_for_approval)
    if config.nil? or config.value.blank?
      return 60
    else
      return ((config.value.tr(',','.').to_f)*10.0).to_i
    end
  end

  def self.grade_of_disapproval_for_absence
    config = CustomVariable.find_by_variable(:grade_of_disapproval_for_absence)
    if config.nil? or config.value.blank?
      return nil
    else
      return ((config.value.tr(',', '.').to_f)*10.0).to_i
    end
  end

  def self.professor_login_can_post_grades
    config = CustomVariable.find_by_variable(:professor_login_can_post_grades)
    if (!config.nil?) && (!config.value.nil?)
      formatted_value = config.value.strip.downcase
      if (formatted_value == "yes") || (formatted_value == "yes_all_semesters")
        return formatted_value
      end
    else
      return "no"
    end
  end

  def to_label
    "#{self.variable}"
  end

  private

  def check_constraints_between_variables
    case self.variable
    when "minimum_grade_for_approval"
      self.errors.add(:value, I18n.translate("activerecord.errors.models.custom_variable.minimum_grade_for_approval_not_gt_grade_of_disapproval_for_absence", :minimum_grade_for_approval=>(self.value.blank? ? 6.0 : self.value.tr(',','.')).to_s, :grade_of_disapproval_for_absence=>(CustomVariable.grade_of_disapproval_for_absence.to_f/10.0).to_s)) if (not CustomVariable.grade_of_disapproval_for_absence.nil? ) and (   ( (self.value.blank?) and ( CustomVariable.grade_of_disapproval_for_absence >= 60 ) )   or   ( (not self.value.blank?) and ( CustomVariable.grade_of_disapproval_for_absence >= ((self.value.tr(',','.').to_f)*10.0).to_i  )    ) )
    when "grade_of_disapproval_for_absence"
      self.errors.add(:value, I18n.translate("activerecord.errors.models.custom_variable.grade_of_disapproval_for_absence_not_lt_minimum_grade_for_approval", :grade_of_disapproval_for_absence=>self.value.tr(',','.'),:minimum_grade_for_approval=>(CustomVariable.minimum_grade_for_approval.to_f/10.0).to_s)) if (not self.value.blank?) and ( ((self.value.tr(',','.').to_f)*10.0).to_i >= CustomVariable.minimum_grade_for_approval )
    end
  end

  def validate_destroy
    case self.variable
    when "minimum_grade_for_approval"
      self.errors.add(:base, I18n.translate("activerecord.errors.models.custom_variable.validate_destroy_of_minimum_grade_for_approval")) if (not CustomVariable.grade_of_disapproval_for_absence.nil?) and ( CustomVariable.grade_of_disapproval_for_absence >= 60 )
    end
    self.errors.blank?
  end

  def validate_create
    case self.variable
    when "minimum_grade_for_approval", "grade_of_disapproval_for_absence"
      if not CustomVariable.find_by_variable(self.variable).nil?
        self.errors.add(:base, I18n.translate("activerecord.errors.models.custom_variable.check_duplicate", :variable=>self.variable))
      end
    end
  end

end


Holi is just around the corner and the weather is just perfect to gear up for the most vibrant and fun festival of the year. This year the festival of colours would be celebrated on 2nd March 2018. Painting people in hues of rainbow to getting drenched in water or dodging the attacks of water guns and balloons, playing Holi definitely makes for a festival which is not just fun, but high on adrenaline too. This year, Holi falls on 2nd March 2018 and preparations for it have begun in full swing….


text:
Tynnered is one of 21 boroughs in the city of Gothenburg, Sweden. It is located in the western part of the city, about 8-10 kilometres from the inner city, and it has a population of 27,787 (2010) on 29.85 square kilometres (11.53 mi2). Alike several other boroughs of the city, Tynnered has a fairly high percentage of immigrants. On January 1, 2011, Tynnered became part of the district (stadsdelsnämnd) Western Gothenburg (Västra Göteborg). In 2021 the stadsdelsnämd districts were replaced by larger stadsområde districts and Tynnered became part of South-West Gothenburg (Sydväst Göteborg).

Travelling by tram from Gothenburg city centre to southern Tynnered takes about 25 minutes. The two trams that go to Tynnered are the tram routes 1 and 7, and they stop at Briljantgatan, Smaragdgatan and Opaltorget.  To northern Tynnered the bus number 50 is the fastest, taking about 20 minutes from the city centre. The shopping mall Frölunda Torg is located a couple of minutes away from Tynnered when going by foot.

Tynnered is also a parish, with approximately the same area. The church was built 1973.

History
In the middle of the 60's, the area around Opaltorget and several other apartment blocks in Tynnered were started to be built. This venture, which is called Million Programme (Miljonprogrammet), was made in order to furbish up the area and make it more friendly to live in.

During the fall 2009, the local district publicly published new plans for Tynnered. Approximately 500-600 new apartments, premises for shops and refurbishing of streets are included in the project. Opaltorget is also to be developed into a vibrant town square for service and local shopping. One of the reasons to this project is that Opaltorget has been seen as an unsafe and dark square, according to Gothenburg municipality. Later in the same year, the real estate committee approved of the proposal. The project is expected to start being built in 2013.

In its 2017 report, Police in Sweden placed the district in the most severe category of urban areas with high crime rates. In 2021, it moved to the middle category of Risk area.

Demographics
The population has increased with only circa 500 people the last ten years. In Tynnered, almost 34% of the inhabitants are younger than 25 years old. That percentage is much higher compared to the average in the city of Gothenburg. In principle have the inhabitants of Tynnered generally work, income and health like the rest of the people in Gothenburg. Due to the lack of workplaces in the borough, most employees work in other parts of the city. The turnout during Swedish general election 2006 was higher than in Gothenburg in general.

Education
The following six schools are located in Tynnered:
Tynneredsskolan
Kannebäcksskolan
Grevegårdsskolan
Ängåsskolan
Vättnedalsskolan
Drakbergsskolan
During 2005 the percentage of students who continued with tertiary education was notably lower compared to the average in Gothenburg. However, 89% of the students had eligibility to gymnasium as of 2010. There are a number of kindergartens in Tynnered.

References

Boroughs of Gothenburg


text:
Bureau of Homeowner Protection v. Myles Incorporated
OAL Dkt. No. CAF 4799-99 (Bureau of Homeowner Protection 2000)
Opinion Date: January 12, 2000
CONTRACTORS; WARRANTY REGISTRATION—An arbitrator under the New Home Warranty program has the power to suspend a builder's registration for failure to remedy violations.
A homebuilder constructed a house and the buyer of the house subsequently identified several deficiencies that required repair. The parties entered into arbitration to settle their disputes. Thereafter, the Bureau of Homeowner Protection conducted a follow-up inspection of the property and found that many of the deficiencies had not been repaired and were not in compliance. The homebuilder was given thirty days to remedy the deficiencies. A second inspection of the home, after the thirty days had passed, revealed that none of the deficiencies were remedied. The Bureau then solicited bids for the completion of the work and paid the homeowners the amount necessary for them to have the work completed. The State filed a notice of violation against the homebuilder and ordered suspension of its New Home Builder's Registration. The homebuilder filed suit and the case was transferred to the Office of Administrative Law (OAL) for a hearing. The OAL held that because the parties submitted their disputes to arbitration, the decision of the arbitrator to suspend the builder's registration was permissible. It held further that because the arbitrator found in favor of the homeowner, the arbitrator correctly prescribed that the builder correct the defects or make necessary replacements. Finally, it held that in the event that the builder defaulted, the homeowners could request payment from the State New Home Warranty Security Fund to have the repairs made.


text:
Mxit	• 11 Aug 2009
Furious MXIT lashes out at media
Read nextBloggers now eligible for major journalism award
MXIT is a world-wide mobile instant messaging tool (now evolving into a social network) with around 15-million users (claimed). It's based in South Africa but has offices around the world and substantial numbers in countries like the Phillipines.
Every time there is an incident via their platform, journalists with scant understanding of the technology tend to focus on MXIT as the source of the problem, rather than the real issues. Mainstream media, in particular, tend to get it wrong. It's part of an ongoing problem with journalism and media here, which sees a lack of seniority and too many junior reporters in newsrooms. An offshoot of this is that we don't see enough specialisation in newsrooms, ie: we see general reporters covering specialist, technical beats requiring in-depth knowledge, in context.
It boils down to a lack of understanding of the environment. Blaming MXIT is akin to calling for a ban on telephones or email, because reprehensible elements of society (eg: pedophiles) happen to use them.
It rather misses the point. It's a classic case of "shoot the messenger": the problem isn't MXIT, which is merely a communication enabler. The issue is education and common sense. The broader problem is society itself, but this, I suspect, is a harder one to solve than educating users.
In a rather long (and angry) press release, which I have shortened for your reading pleasure, MXIT singles out local media for "sensational" and "unprofessional" reporting. I'm inclined to agree:
"MXit is calling on the media to stop misusing its name in what the company believes is a dangerous trend to sensationalise headlines… is concerned about the ongoing misleading and inaccurate use of its name in media reports across all media platforms, including television, print and online. MXit is currently consulting its lawyers to determine whether the most recent example is a breach of the South African Press Code, or indeed if it amounts to defamation.
In the latest example, media reports claim that MXit is responsible for a teenage girl from Johannesburg disappearing for 48 hours after telling her school that she would not be attending classes. Her parents allege that she may have met someone on MXit. The school is considering disciplinary action on the girls return and although the case is being investigated, there is no proof that a conversation with an unknown person on MXit led to the girl's disappearance…
…MXit is not the problem. We offer a system that allows people to communicate at a fraction of the cost of sending sms or voice calls. Our users send approximately 35 000 messages per second during peak times and the MXit community visits our platform more than 20 million times a day. Even if it does emerge that she accepted a friend request from a stranger, it is not fair to condemn a technology of close to 15 million users for bad choices made by one user," says Juan du Toit, international marketing manager for MXit…
MXit has an obligation to ensure that its users understand that media reports are not entirely true in this case and has sent a message to its community which reflects the facts…
…We condemn the unprofessional and sensation-seeking journalism displayed by e-TV and The Star newspaper this last week…
…We want to warn all users again to enjoy our technology with the necessary responsibility and level of maturity; and never to reveal personal information. We challenge both media owners to get these simple facts accurate, and to properly understand our technology. Can we trust what is reported?
…MXit has always stressed the importance of education and responsible online behavior in a technological advancing world. In addition to the safety regulations that is constantly reminding its users of, it has also developed a set of guidelines for parents, available on its website: http://www.mxit.com"
Posted in Mxit, News, Social networkingTagged Instant Messaging, journalism standards, Mxit, South African Press Code
Bloggers now eligible for major journalism award
Blogs • 17 Aug 2009
The best films set in South Africa
It is undeniable that the United States and the United Kingdom dominate the silver screen. However, especially in recent years, South Africa has increasingly…
Wiehahn Diederichs TV & Movies • 13 Jan 2022


text:
Q: Too many local variables in ASP.NET I wrote a control, and use a tool to do the code analysis. There is a test that I didn't pass. Avoid excessive locals, http://msdn.microsoft.com/library/ms182263(VS.90).aspx.
In my CreateChildControls function, I built a big table with lots of field. I need to create a  lot of TableRow and TableCell to construct the table. But these  are not field or property of control. Thery are local variables in the function, which are created dynamically.
Should I make these TableCells and TableRows as fields of the control? Or I just keep them as the local variables in the CreateChildControl function?
Best Regards,

A: First of all, I would suggest that you consider using a Repeater control instead of dynamically creating lots of rows and cells in the backend code. By using a Repeater, you can more easily modify the actual markup without having to go through hundreds of lines of code to find it.
But if that's not an option, then consider the question: Do you actually need to have a unique variable for each one? Could you instead do something like this:
TableRow tr;
TableCell tc;

tr = new TableRow();
tc = new TableCell();
tc.Text = "whatever";
tr.Cells.Add(tc);
table.Rows.Add(tr);

tr = new TableRow();
tc = new TableCell();
tc.Text = "whatever # 2";
tr.Cells.Add(tc);
table.Rows.Add(tr);

That is, reuse the same variable for multiple rows/cells.

A: Code analysis rules should always be used as a guideline - if you're satisfied with a solution that violates a rule, add an exception and move on to harder problems.
That said, eliminating local variables is often as easy as extracting methods (and classes if appropriate) to implement the desired logic. In your case, you may have a method to construct a TableRow which calls other methods to construct each TableCell. This has the additional benefit of making your CreateChildControls method easier to read and test.
Update: To answer your question about promoting the variables to fields, I would suggest avoiding fields unless they are absolutely necessary. In this case, they don't appear to be, so you would be better off keeping them as local within the relevant method.

redpajama_set_name: RedPajamaStackExchange


text:
Lizzo Gets Candid on the Struggle of 'Hating' Her Body in Emotional TikTok Videos
By Desiree Murphy 8:59 AM PST, December 10, 2020
David M. Benett/Dave Benett/Getty Images for Warner Music
Lizzo admits there's plenty of days when she's not "feeling good as hell."
In a series of emotional videos shared to TikTok this week, the 32-year-old singer gets raw and real about the struggles she often faces with body image.
In the first video, Lizzo explains that she came home, took her clothes off to take a shower and "just started having all these really negative thoughts" about herself.
"Of like, 'What's wrong with me? Maybe all the mean things people say about me are true,'" she shared. "And, you know, 'Why am I so disgusting?' and hating my body. Normally I would have some positive thing to say to get me out of this but I don't. And that's OK too."
@lizzo
A little tmi for ur fyp... for anyone who has these thoughts too I hope you have a better day tomorrow
♬ original sound - lizzo
"I think these are normal [thoughts], they happen to everybody. They happen to the best of us. We are the best of us," she continued. "I just have to know that tomorrow, how I feel in [my head] is going to change, and I can only hope it changes for the better. I know I'm beautiful. I just don't feel it, but I know I'm going to get through it."
Lizzo added in the description, "For anyone who has these thoughts too I hope you have a better day tomorrow."
In the second video, Lizzo rocked a black sports bra and Batman briefs, showing off her figure while hugging and rubbing her arms, shoulder and stomach.
"Woke up feeling better," she explained. "Not 100% but I'm getting there. Gave the parts of me I hated last night a rub & a hug ❤ issss a new day."
#duet with @lizzo woke up feeling better.. not 100% but I'm getting there. Gave the parts of me I hated last night a rub & a hug ❤ issss a new day
Back in June, Lizzo also took to TikTok to call out body shamers. "Hey, so I've been working out consistently for the last five years and it may come as a surprise to some of y'all that I'm not working out to have your ideal body type," she explained in a video of her riding a bike, doing squats and jumping rope. "I'm working out to have my ideal body type, and you know what type that is? None of your f**king business. Because I am beautiful, I am strong, I do my job, and I stay on my job."
"So next time you want to come to somebody and judge them, whether they drink kale smoothies or eat McDonald's or workout or not workout, how about you look at your own f**king self and worry about your own god-d*mn body because health is not just determined on what you look like on the outside, health is also what happens on the inside," she added. "And a lot of y'all need to do a f**king cleanse for your insides. Namaste, have a great day."
The "Truth Hurts" singer also revealed her new vegan diet and everything she eats in a day. She usually starts her mornings with a dark green breakfast smoothie made of coconut water, kale or spinach and frozen fruit, following it up with a fresh salad consisting of kale, red cabbage, broccoli, a slice of avocado, onions and carrots.
For a snack, Lizzo said she recently traded in hot Cheetos for garlic hummus and paleo puffs. "These are a Cheeto-alternative I love that's vegan and I dip them in hummus," she raved. "Badda-boom badda-snack."
The singer said her dinner consists of mushroom balls with truffle and chickpeas, quinoa and leftover salad from lunch. She also drinks vegan diet cola because she "needed some sparkles in [her] throat," ending her day with a peanut butter and jelly smoothie consisting of oats, oat milk, vanilla protein powder and "love from Jesus."
Hear more in the video below.
Lizzo Shuts Down Body Shamers, Says She's Been Working Out for 5 Years
Lizzo Poses Nude With American Flag on Election Day
Lizzo Slays Rihanna's Savage X Fenty Show With Solo Dance
Lizzo Shuts Down Body Shamers and Says She's Been 'Working Out Consistently for the Last 5 Years'
Must-See Celeb Sightings: December 2020
'All American' Clip: What Happened With Spencer & Olivia Over Summer?


Wendys World: "LATE LATE LATE"
And the words go like this "One out of Three ant Bad"
MEANWHILE in Wendy's World I have been to Perth here in Australia, off to the USA, over to England, and few other trips here and there to drop some little thank yous to all of you, so if you haven't heard from me yet! Well as they say tomorrow is another Day my friends.
The most beautiful Shane in the Whole World was so excited today as he was blessed with some new "Babies" well that's what he called them, Ya just gotta love him! they are really called, of course just between you and me girlfriends, "ORCHIDS" now this bit is very important, he has been growing this ONE yes One plant for TWO YEARS, OK and this is the very first time it has flowered, and flower it did, it is really beautiful, and if I was a great blogger I would send you all a wee little photo, so maybe you should put your hands together in the praying position and say a prayer that it doesn't take me TWO Years to show you the photo!LOL not!! This is where you tell me it does get easier!!
JOLLY JUMBO JETS WHAT A WOMAN!!! Though it did turn out at the top of the page LOL just a small detail really!!
Well their you have it Shane's babies!!
You know what?? I'm going to do that again, again and again just cause I can!!
Well no wonder Shanes excited, thats one pretty perfect Orchid.
You are not alone in the 'jumping out of bed to go to your blog' department. I do it quite often! Sometimes you just gotta!


text:
Les Halocypridina sont un sous-ordre de crustacés de la classe des ostracodes, de la sous-classe des Myodocopa et de l'ordre des Halocyprida.

Liste des super-familles
 Halocypridoidea Dana, 1853
 Thaumatocypridoidea Müller, 1906

Références

  Morphology and Musculature of a New Species of Thaumatocypris (Ostracoda, Halocyprida, Thaumatocypridoidea). Louis S. Kornicker, Journal of Crustacean Biology, Volume 24, Issue 1, 1 January 2004, Pages 54–71, .

Liens externes

Myodocopa
Ostracode (nom scientifique)
Sous-ordre d'arthropodes (nom scientifique)