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Cluster of differentiation 36 (CD36) is a transmembrane glycoprotein involved in many biological processes, such as platelet biology, angiogenesis and in the aetiopathology of atherosclerosis and cardiovascular diseases. Toll-like receptors (TLRs) are one of the most important receptors of the innate immune system. Their main function is the recognition of conserved structure of microorganisms. This recognition triggers signaling pathways that activate transcription of cytokines and co-stimulatory molecules which participate in the generation of an immune response against microbes. In particular, TLR2 has been shown to recognize a broad range of ligands. Recently, we showed that CD36 serves as a co-receptor for TLR2 and enhances recognition of specific diacylglycerides derived from bacteria.
Methodology/ Principal Findings
Here, we investigate the mechanism by which CD36 contributes to ligand recognition and activation of TLR2 signaling pathway. We show that the ectodomain of murine CD36 (mCD36ED) directly interacts with negatively charged diacylglycerol ligands, which explains the specificity and selectivity of CD36 as a TLR2 co-receptor. We also show that mCD36ED amplifies the pro-inflammatory response to lipoteichoic acid in macrophages of wild-type mice and restores the pro-inflammatory response of macrophages from mice deficient in CD36 (oblivious), but not from mice deficient in cluster of differentiation 14 (CD14) (heedless).
Conclusion/ Significance
These data indicate that the CD36 ectodomain is the only relevant domain for activation of TLR2 signaling pathway and that CD36 and CD14 have a non-redundant role for loading ligands onto TLR2 in the plasma-membrane. The pro-inflammatory role of soluble CD36 can be relevant in the activation of the immune response against pathogens, as well as in the progression of chronic diseases. Therefore, an increased level of soluble forms of CD36, which has been reported to be increased in type II diabetic patients, could accelerate atherosclerosis by increasing the pro-inflammatory response to diacylglycerol ligands.
Citation: Jimenez-Dalmaroni MJ, Xiao N, Corper AL, Verdino P, Ainge GD, Larsen DS, et al. (2009) Soluble CD36 Ectodomain Binds Negatively Charged Diacylglycerol Ligands and Acts as a Co-Receptor for TLR2. PLoS ONE 4(10): e7411. https://doi.org/10.1371/journal.pone.0007411
Editor: Derya Unutmaz, New York University School of Medicine, United States of America
Received: May 19, 2009; Accepted: September 18, 2009; Published: October 22, 2009
Copyright: © 2009 Jimenez-Dalmaroni et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by NIH grants AI42266 (IAW), CA58896 (IAW), AI070167–01(BB), and by a Joint Scripps/Oxford Graduate Scholarship. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
CD36, the prototype of scavenger receptor class B, is a multifunctional transmembrane glycoprotein, involved in the aetiopathology of several biological processes and diseases, including cardiovascular diseases, Alzheimer's disease, Plasmodium falciparum (P. falciparum) infection, diabetes, angiogenesis, platelet biology, atherosclerosis [1]–[3], and anti-tumor responses [4]. The role of CD36 in these processes is attributed to its ability to bind a broad range of ligands [5]. Among these are long-chain fatty acids [6], advanced glycosylation products [7], [8], oxidized Low Density Lipoproteins [9]–[11], oxidized phosphocholines [12], collagen [13], growth-hormone releasing hormone (GHRH) peptides hexarelin and EP80317 [14], and thrombospondin-1 (TSP-1) [15].
Recently, we demonstrated that mice carrying a missense mutation in CD36, termed oblivious, had a reduced response to specific TLR2 ligands, lipoteichoic acids (LTA) and macrophage activating lipoprotein-2 (MALP-2), but responded normally to the synthetic lipopeptides, Pam3CSK4 and Pam2CSK4 [16]. Moreover, CD36obl/obl mice were unable to clear Staphylococcus aureus (S. aureus) bacteria and showed higher mortality compared to C57BL/6 mice. Cd36−/− mice were also more prone to develop abscesses [17]. A recent report also revealed that CD36-deficient mice showed impaired tumor necrosis factor-alpha (TNF-α) secretion in response to glycosylphosphatidylinositol (GPI) from P. falciparum and higher parasitemia levels and mortality when infected with Plasmodium chabaudi chabaudi AS (P. chabaudi chabaudi AS) [18]. Although these data highlight the role of CD36 in controlling infection with S. aureus and P. falciparum, the mechanism by which CD36 contributes to TLR2 signaling pathway activation has been elusive. Recent reports have attempted to address this issue, but with contrasting findings. One study [17] initially demonstrated requirement of the CD36 C-terminus for endocytosis and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, whereas other reports showed that internalization of LTA is not necessary for signal transduction [19], [20].
Here, we address the role of CD36 in the TLR2-dependent activation of the myeloid differentiation protein 88 (MyD88) signaling pathway. To investigate the role of CD36 as a TLR2 co-receptor, the ectodomain of murine CD36 (mCD36ED) was cloned and expressed using a baculovirus expression system. The interaction of mCD36ED with TLR2 ligands and the effect of mCD36ED on the pro-inflammatory response to LTA by primary macrophages isolated from mice with non functional CD36 (oblivious) [16], CD14 (heedless) [21], TLR6 (insouciant) [22], knock-out mice (Tlr2−/−, Tlr1−/−), and wild-type (C57BL/6) mice were determined.
mCD36ED expresses in insect cells as a monomer with α-helical and β-sheet secondary structure
mCD36ED DNA, amplified by PCR with a hexa-histidine tag at its carboxyl-terminus (C-terminal His-tag), was cloned in a baculovirus transfer vector and mCD36ED protein was expressed using a baculovirus/insect cell expression system. Protein expression was confirmed with an anti-His-tag Western blot and peptide identification using mass spectrometry (Supplementary Figure S1). mCD36ED ran as a monomer on size exclusion chromatography (Figures 1A, 1B) and its CD spectrum at 25°C revealed a broad minimum from 207 to 230 nm and a maximum at 195 nm, indicative of a folded protein containing α-helices and β-sheets in its secondary structure [23]. Typical CD spectra of proteins rich in α-helical, β-sheet, and mixed α/β secondary structure are also shown for comparison (Figure 1C).
(A) mCD36ED is an apparent monomer in size exclusion chromatography. mCD36ED and molecular weight standards (depicted in red) were run on a Superdex 200 10/30 gel filtration column. mCD36ED elutes at 14.7 ml which corresponds to the retention volume for a protein of ∼52 kDa and roughly corresponds to a molecular weight of 58.3 kDa of mCD36ED as determined by MALDI TOF MS. (B) Fractions of mCD36ED after size exclusion chromatography were run on an SDS PAGE. The position of purified mCD36ED, which runs with an apparent molecular weight between 50 and 75 kDa, is indicated. (C) mCD36ED is folded. The CD spectrum of mCD36ED was recorded at 25°C. Triplicate measurements were averaged, the CD spectrum of the buffer subtracted, and the CD data were converted from millidegrees to molar ellipticity per residue. Typical CD spectra of proteins rich in α-helical, β-sheet, and mixed α/β secondary structure are shown for comparison. α-helical proteins typically display a CD spectrum dominated by a strong maximum around 190 nm and two minima at 208 nm and 222 nm, while β-sheet proteins show a less pronounced maximum at 195 nm and a minimum around 215 nm. The CD spectrum of mCD36ED with its broad minimum between 207-230 nm and a maximum around 195 nm is thus indicative of the presence of both α-helices and β-sheets in its secondary structure.
mCD36ED has intramolecular disulfide bonds and N-linked glycans
Under reducing condition, the electrophoretic mobility of mCD36ED is altered which indicates the presence of intramolecular disulfide bonds (Supplementary Figure S2A). N-linked glycosylation can be partially removed from mCD36ED by treatment with Peptide-N-glycosidase F (PNGase F) in native conditions to the same extent as in denaturing conditions, revealing that its N-glycosidic linkages are accessible for enzymatic cleavage (Supplementary Figure S2B). However, PNGase F was not able to release all glycans from mCD36ED (i.e., the theoretical molecular weight of mCD36ED is 47,424 kDa, but after digestion with PNGase F, mCD36ED runs at a molecular weight above 50 kDa). mCD36ED were expressed in Hi-5 insect cells, which have been reported to modify recombinant glycoproteins by adding a core (α 1→3) fucose [24], [25]. Therefore, the resistance to PNGase F treatment could be explained by the presence of core fucosylation in N-linked glycans of mCD36ED. The position of disulfide bonds based on bovine CD36 [26] and predicted N-linked glycosylation sites are depicted in Supplementary Figure S2C.
mCD36ED binds to LTA, FSL-1, Pim2, and Pim4
We then demonstrated by native PAGE assays that mCD36ED binds to LTA from S. aureus, synthetic phosphatidyl-myo-inositol mannosides (Pims) with two (Pim2) and four mannoses (Pim4) from mycobacteria, synthetic fibroblast stimulating lipopeptide-1 with a fluorescein tag (FSL-1-fluorescein) from Mycoplasma salivarium, but not to Pam3CSK4 (Figures 2A, 2B). Binding of mCD36ED to FSL-1-fluorescein was also confirmed by fluorescence (Figure 2B). As a negative control, we employed influenza hemagglutinin from the H5N1 Viet 04 strain [27], which lacks diacylglycerol moieties and was unable to interact with human TLR2 ectodomain (data not shown). Moreover, although Pam2CSK4 and Pam3CSK4 did not alter the retention volume of mCD36ED, binding of both Pim2 and LTA produced a shift to higher molecular weights in size exclusion chromatography (Figure 2C). Dot blot experiments using an antibody specific to the polyglycerophosphate moiety of LTA confirmed its presence in the mCD36ED-LTA complex (Figure 2D). Furthermore, we employed a T-cell receptor (TCR) termed KRN [28], which has a C-terminal His-tag, to discard the possibility of an non-specific interaction between the C-terminal His-tag and TLR2 ligands. The lack of binding of Pam3CSK4, FSL-1 and LTA by KRN TCR excluded the possibility that the C-terminal His-tag is involved in the binding of TLR2 ligands (Figure 2E).
mCD36ED was incubated overnight with TLR2 ligands or influenza hemagglutinin (H5N1 Viet 04 strain) and subsequently run on a native 4–20% PAGE for 4 hours at 100 volts. Fluorescence was determined with a Versadoc imaging system. Subsequently, the gel was stained with Coomassie Blue. (A) Native gel stained with Coomassie Blue. (B) Fluorescence from the native gel determined using a Versadoc imaging system. Lanes: 1- 1 µg of Pam3CSK4- fluorescein (Pam3CSK4f) ; 2- 1 µg of FSL-1-fluorescein (FSL-1f); 3- 1 µg of Pim4; 4- 1 µg of Pim2; 5- 1 µg of LTA; 6- 10 µg of hemagglutinin Viet 04; 7- 10 µg of mCD36ED incubated with 10 µg of hemagglutinin Viet 04; 8- 10 µg of mCD36ED incubated with 1 µg of Pim4; 9- 10 µg of mCD36ED incubated with 1 µg of Pim2; 10- 10 µg of mCD36ED incubated with 1 µg of Pam3CSK4f; 11- 10 µg of mCD36ED incubated with 1 µg of FSL-1f 12- 10 µg of mCD36ED incubated with 1 µg of LTA; 13- 10 µg of mCD36ED. (C) The retention volume of mCD36ED-LTA and mCD36ED-Pim2 is decreased as compared to uncomplexed mCD36ED. An appreciable shift in molecular weight is seen after incubation of mCD36ED with LTA or Pim2, but no shift is observed with Pam2CSK4 or Pam3CSK4. Molecular weight standards were run in parallel to determine the apparent change in molecular weight of mCD36ED. (D) The presence of LTA in the mCD36ED-LTA size exclusion chromatography fractions was confirmed by dot blot analysis using an anti-LTA antibody. Lanes 1- PBS; 2- Pam2CSK4; 3- anti-LTA antibody; 4- LTA; 5- mCD36ED; 6- mCD36ED- LTA. (E) KRN TCR with a C-terminal His-tag does not bind ligands of TLR2. Lanes 1- 1 µg of Pam3CSK4; 2- 1 µg of LTA; 3- 1 µg of FSL-1, 4- 1 µg of Pim2, 5- 1 µg of Pim4, 6- 10 µg of KRN TCR; 7- 10 µg of KRN TCR incubated with 1 µg of Pam3CSK4; 8- 10 µg of KRN TCR incubated with 1 µg of LTA, 9- 10 µg of KRN TCR incubated with 1 µg of FSL-1, 10- 10 µg of KRN TCR incubated with 1 µg of Pim2, 11- 10 µg of KRN TCR incubated with 1 µg of Pim4.
Lipomannan, FSL-1-fluorescein, and LTA activation of the MyD88 pathway is dependent on TLR2, CD36, and CD14
To determine which receptors are important for the biological activity of lipomannan, FSL-1 and LTA, we employed wild-type C57BL/6, insouciant (TLR6int), oblivious (CD36obl), heedless (CD14hdl), Tlr1−/− and Tlr2−/− mice. Insouciant, oblivious and heedless were generated by germ-line, random mutagenesis using ENU (N-ethyl N-nitrosourea) where their phenotypes result from loss of functional TLR6, CD36 and CD14, respectively [22], [16], [21]. Macrophages isolated from heedless, oblivious, insouciant and Tlr1−/− mice were incubated with different concentrations of LTA for 4 hours. While macrophage secretion of TNF-α by FSL-1-fluorescein and LTA, were dependent on TLR6, macrophage activation by lipomannan from M. smegmatis was dependent on TLR6 and TLR1. Furthermore, TNF-α secretion by macrophages at low concentrations of the ligands was dependent on CD36, while the absence of TLR2 and CD14 impaired TNF-α secretion by macrophages even at higher ligand concentrations of LTA, FSL-1-fluorescein and lipomannan (Figure 3).
(A) lipomannan from M. smegmatis, (B) FSL-1 fluorescein, and (C). LTA from S. aureus. Macrophages from C57BL/6 mice, heedless, insouciant (int), oblivious (obl), Tlr2−/−, Tlr1−/− were exposed to different concentrations of lipomannan from M. smegmatis, LTA from S. aureus and FSL-1 fluorescein for 4 hours at 37°C. The amount of TNF-α secreted to the supernatant was determined by an L929 cell cytotoxic assay. Values are expressed as mean values +/− SEM (n = 2 mice).
mCD36ED amplifies the response to LTA in wild-type macrophages and restores the response to LTA in CD36-deficient, but not in CD14-deficient macrophages
To further explore the role of CD36 in activation by TLR2 ligands, we examined the effect of mCD36ED on increasing amounts of LTA in vitro by measuring TNF-α production in primary cultured peritoneal macrophages. Addition of mCD36ED (50 and 100 ng/ml) increased macrophage sensitivity in C57BL/6 mice at low concentrations of LTA and restored the LTA activity to wild-type levels in CD36obl/obl mice (Figures 4A, 4B). Moreover, denaturation of mCD36ED, by heating for 2 hours at 100°C, abolished restoration of the response of oblivious mice to LTA (Figure 4C). In contrast, addition of mCD36ED only slightly increased TNF-α secretion by insouciant macrophages, and no significant increase of TNF-α secretion was observed for Tlr2/− and heedless macrophages (Figure 5).
(A) mCD36ED increases secretion of TNF-α by low LTA concentrations in C57BL/6 macrophages. (B) mCD36ED restores secretion of TNF-α in oblivious (obl) macrophages. (C) Denatured mCD36ED did not restore secretion of TNF-α by oblivious (obl) macrophages. Macrophages from C57BL/6 and oblivious were exposed to different concentrations of LTA plus a constant amount of mCD36ED, or heated denatured mCD36ED (for oblivious macrophages) for 4 hours at 37°C. The amount of TNF-α secreted to the supernatant was determined by L929 cytotoxic assay. Values are expressed as mean values +/−_SEM (n = 3 mice).
Macrophages from tlr2−/−, heedless and insouciant macrophages were exposed to different concentration of LTA plus a constant amount of mCD36ED for 4 hours. The amount of TNF-α secreted to the supernatant was determined by an L929 cytotoxic assay. Values are expressed as mean values +/− SEM (n = 3 mice).
mCD36ED does not interact directly with mTLR2ED
We investigated by immunoprecipitation whether direct interaction of mCD36ED occurs with a murine TLR2 ectodomain fused with an Fc domain of human IgG1 (mTLR2ED/Fc). We incubated mCD36ED with mTLR2ED/Fc and subsequently with magnetic protein G beads. After washing, samples from the immunoprecipitation were run on an SDS-PAGE. No immunoprecipitation of mCD36ED with mTLR2ED/Fc was observed (Figure 6A), suggesting that mCD36ED does not interact directly with mTLR2ED.
(A) mCD36ED does not interact with an mTLR2ED/Fc chimera. SDS PAGE of the samples from immunoprecipitation using protein G beads. Lanes: 1- Protein G incubated with mTLR2ED/Fc chimera and mCD36ED; 2- Protein G incubated with mTLR2ED/Fc chimera alone; 3- Protein G incubated with mCD36ED; 4- mCD36ED; 5- mTLR2ED. (B) Model of CD36-dependent activation of the TLR2 signaling pathway. CD36 binds LTA (step 1) which is transferred to CD14 (step 2). Alternatively, the soluble ectodomain of CD36 binds LTA (step 1a), and transfers it to CD14 (step 2a). Subsequently, CD14 transfers LTA to TLR2/TLR6 (step 3) and the MyD88 pathway is activated (step 4).
CD36 is a type III transmembrane glycoprotein composed of an extracellular domain, N-and C-terminal anchors and two short intracellular domains [29]. Although other members of scavenger receptors class B, SRB-I and SRB-II, have been implicated in the uptake of Mycobacterium fortuitum into non-phagocytic cells [30], no role has been assigned to CD36 in innate immunity. We previously showed that CD36 is a selective co-receptor of TLR2 [16]. The role of CD36 as a co-factor for transmembrane proteins appears to be evolutionary conserved among species. Sensory neuron membrane protein (SNMP), a Drosophila melanogaster CD36 homolog, has a role in pheromone detection and acts as a co-factor for the OR67d/OR83 receptor [31]. However, the mechanisms underlying these interactions remain unclear.
In this study, we addressed the mechanism by which CD36 cooperates with TLR2. To investigate the role of CD36 as a TLR2 co-receptor, mCD36ED was cloned and expressed using a baculovirus expression system. Highly purified mCD36ED was obtained after three purification steps: immobilised metal affinity chromatography (IMAC), cationic exchange chromatography and size exclusion chromatography. Purified mCD36ED behaved as a monomer in gel filtration chromatography and its CD spectrum was consistent with folded protein with α-helical and β-sheet secondary structure. We also showed that recombinant mCD36ED contains N-linked glycans and disulfide bonds. We employed native PAGE, which has been successfully applied to investigate the binding of toll-like receptor 4/ myeloid differentiation factor 2 (TLR4/MD-2) with LPS (lipopolisaccharide) [32] and CD14 with LTA [33], to show that mCD36ED binds Pims, LTA, FSL1-fluorescein, but not Pam3CSK4. This selective binding displayed by mCD36ED explains the enhanced NF-κB activation by specific TLR2 ligands, but not others. Thus, the monomeric form of mCD36ED can bind TLR2 ligands in the absence of any other receptors or co-receptors. Furthermore, it was recently reported that monomeric transmembrane rat CD36 binds acetylated and oxidized low density lipoproteins [34].
The substantial increases in electrophoretic mobility shown by LTA-CD36 and Pim2-CD36, and several bands shown for the Pim4-CD36 complex, indicate that CD36 is able to bind more than one molecule of each of these ligands which was also confirmed by shifts in size exclusion chromatography for mCD36ED-Pim2 and mCD36ED-LTA complexes. These results are consistent with the role of CD36 as a scavenger receptor that sequesters glycolipid ligands. This selective binding displayed by mCD36ED explains the enhanced NF-κB activation by specific TLR2 ligands, but not others. Comparison of TLR2 ligands that were able to bind mCD36ED, versus those that were not, reveals that mCD36ED ligands have negatively charged moieties (polyglycerophosphates and myo-inositol phosphate for LTA and Pims, respectively), in agreement with reports that CD36 binds negatively charged ligands, such as phospholipids and phosphocholine [12]. These combined results are consistent with the role of mCD36ED as a scavenger receptor that sequesters polyanionic ligands [35]. Although CD36 binds Pim2 and Pim4, these synthetic phosphatidylinositol mannosides could not stimulate secretion of TNF-α by macrophages from C57BL/6 mice. Lipoarabinomannan (LAM) and its precursors have been reported to activate cells via the TLR2/TLR1 heterodimer [36]. Thus, this lack of activity for Pim2 and Pim4 may arise from the lack of the third acyl chain in these synthetic Pims that is necessary for interaction with TLR1 [37]. Although these synthetic Pims were not active, lipomannan from Mycobacterium smegmatis could induce TNF-α secretion in a TLR2- and CD36-dependent manner.
Because of the importance of TLR2 in the control of mycobacterial infections [38], [39], it will also be important to evaluate the role of CD36 in the pathogenesis of Mycobacterium tuberculosis and Mycobacterium leprae. In CD36 binding studies, we employed the same synthetic Pim2 that was loaded onto CD1d for determining the crystal structure of the Pim2-CD1d complex [40] and the same Pim4 structure that was reported to be a natural ligand of CD1d [41]. Furthermore, CD36 may also play a role in the endocytosis of Pims for presentation by CD1d, as with mannose receptor in the presentation of LAM by CD1b [42]. Pim2 shares the phosphatidylinositol diacylglycerol structure with the glycosylphosphatidylinositol (GPI) anchors from Toxoplasma, Plasmodium, Leishmania and Trypanosome, that could result in binding of GPI to mCD36ED. In fact, a recent report showed that secretion of TNF-α by GPI from P. chabaudi chabaudi AS is impaired in Cd36−/− macrophages, compared with wild-type macrophages. Cd36−/− mice also showed a higher level of parasitemia and mortality than wild-type mice [18]. Therefore, it is highly possible that CD36 cooperates with TLR2 in the recognition of GPI by binding and accumulating GPIs from parasites. It would be relevant to investigate if Cd36−/− mice infected with Leishmania major, Toxoplasma gondii and Trypanosoma cruzi, display higher levels of parasitemia and/or mortality, such as for P. chabaudi chabaudi AS.
Although the relevance of CD36 in the control of infection has been demonstrated, little is known about the mechanism by which CD36 enhances the response to TLR2 ligands. Our data reveal that response to lipomannan, LTA and FSL-1-fluorescein is dependent on the presence of TLR2, CD14, and CD36. In the case of LTA, loss of expression of functional CD36, CD14 or TLR6 caused a decrease in the ability of macrophages to activate the MyD88 signaling pathway. Furthermore, addition of either 50 or 100 ng/ml of mCD36ED increased the sensitivity of C57BL/6 macrophages to low concentrations of LTA , and restores the secretion of TNF-α by LTA from oblivious macrophages, but failed to restore the secretion of TNF-α from either heedless or insouciant macrophages. Increase in TNF-α secretion by LTA in the presence of mCD36ED is TLR2-dependent, as no increase in TNF-α secretion was observed in macrophages from Tlr2 −/− mice. Recently, soluble CD36 in plasma was reported to be increased in type II diabetic patients and patients with polycystic ovarian syndrome [43], [44]. Because our data indicate enhancement of TNF-α secretion in C57BL/6 mice by mCD36ED at low concentrations of LTA, the presence of high levels of soluble CD36 in plasma from type II diabetic patients could contribute to exacerbation of the inflammatory response of macrophages in the presence of negatively charged diacylglycerol ligands and, therefore, may play a role in atherosclerosis development in diabetic patients.
Interestingly, our data indicate that mCD36ED is not able to restore TNF-α secretion in the absence of CD14. Thus, the main function of CD36 is to bind and transfer diacylglycerol ligands onto TLR2, in a CD14-dependent manner. This transfer of ligands onto CD14 by CD36 is also consistent with our immunoprecipitation results which show that mCD36ED does not directly interact with mTLR2ED/Fc.
Furthermore, CD14 also binds LTA, as well as LPS [34], [45]. The crystal structure of the murine CD14 ectodomain shows a possible hydrophobic pocket in its N-terminal region, which could be involved in ligand binding [46]. Soluble CD14 or transmembrane CD14 can bind monomers of LPS and transfer them to TLR4/MD2 complex [47]-[49]. In a similar way, CD14 could be a "shuttle" that takes monomers of diacylglycerol ligands, which are bound to CD36, and transfer them to TLR2/TLR6. Addition of mCD36ED did not restore TNF-α secretion by insouciant macrophages, indicating that CD36 cannot replace the function of TLR6 as a TLR2 co-receptor. In summary, we show that the TLR2 co-receptor role of CD36 is dependent only on the ectodomain of CD36, but not dependent on either signaling or endocytosis mediated by the intracellular domains of CD36. Therefore, our data are consistent with activation of the TLR2-dependent MyD88 signaling pathway by LTA from the plasma membrane [19], [20]. Similarly, activation of the MyD88 signaling pathway by LPS was also reported to be independent of LPS internalization [50], [51]. Based on our data, we propose a model in which CD36 binds diacylglycerol ligands, transfers them to CD14, which then loads these ligands onto TLR2/TLR6 (FSL-1, MALP-2, and LTA), or TLR2/TLR1 (lipomannan). Whether the transfer of pheromones from SNMP to OR67d is a direct or an indirect process remains to be shown.
Materials and Methods
Ligands, H5N1 Viet04 hemagglutinin and KRN TCR employed in native PAGE
Pam2CysSK4, Pam3CysSK4, Pam3CysSK4-fluorescein, FSL-1 -fluorescein were obtained from EMC Microcollection (Tubingen, Germany). Purified LTA from S. aureus, lipomannan and FSL-1 was obtained from Invivogen (San Diego, CA, USA). Influenza hemagglutinin from the H5N1 Viet04 strain was a gift from Dr. James Stevens in our laboratory. The hemagglutinin precursor, corresponding to the H5N1 Viet04 strain, with a C- terminal His-tag was cloned in pAcGP67A (BD Biosciences, USA) for expression in insect cells. A thrombin site was included to release the C-terminal His-tag and a foldon was also included to help in the trimerization process. H5N1 Viet04 hemagglutinin was expressed using a baculovirus/ insect cell expression system and purified from culture supernatants, as previously described [27]. Synthetic phosphatidylinositol mannosides (Pim2 and Pim4) were synthesized as described previously [52]. cDNA for the α and β chain of KRN TCR were individually subcloned into the fly TCR expression vector pRMHa3. The final constructs code for the α1α2 and the β1β2 domains, respectively, followed by a linker sequence (SSADL), a thrombin site (LVPRGS), a leucine zipper (acidic for the α chain, basic for the β chain) and a hexa-histidine tag (C-terminal His-tag). Vectors were co-transfected into Drosophila Scheneider-2 insect cells along with a vector encoding a puromycin-resistance gene and stable cell-lines were established. Soluble TCRs were purified from culture supernatants, as previously described [53].
Cell culture and cell lines
Sf-9 and Hi-5 insect cells were purchased from Invitrogen (USA) and employed for baculovirus generation and protein expression, respectively. Cell lines were cultured in suspension cultures in serum-free HyQ media (Hyclone, USA) at 27°C shaking at 225 rpm. Drosophila Scheneider-2 insect cells (Invitrogen, USA) were cultured in serum-free SFX insect express media (Hyclone, USA) in roller bottles. DMEM supplemented with 5% FCS and 2% penicillium streptomycin was used for culturing macrophages.
C57BL/6, insouciant (TLR6int), oblivious (CD36obl), heedless (CD14hdl), Tlr1−/− and Tlr2−/− mice were maintained and bred in The Scripps Research Institute Vivarium under the supervision of the Department of Animal Resources. All studies involving mice were performed in accordance with the rules of Institutional Animal Care and Use Committee of The Scripps Research Institute. All experiments were performed according to the US National Institutes of Health guidelines.
Cloning and expression of mCD36ED
The mCD36ED was amplified by PCR from a vector harboring the complete murine CD36 DNA (CMV CD36-pCDNA3.2/V5-DEST2) and the following primers:
Forward primer: GTG TGT GGA TCC CGG AGA CAT GCT TAT TGG GAA GAC AAT CAA AAG GG and Reverse primer: GTG TGT GAA GCGGCC GC TCA GTG ATG ATG ATG A TG ATG CTTG ATT TTC CCA GTC ACT TGT GTT TTG AAC. In the forward primer, a BamHI restriction enzyme site was included (indicated in bold), while in the reverse primer a NotI (indicated in bold) restriction site and a stop codon (indicated by underline) was inserted. PCR amplification was performed using GC- rich DNA polymerase (Roche, USA) and the following steps were carried out: initial denaturation at 95°C for 4 minutes, denaturation for 35 cycles of 45 seconds at 95°C, annealing for 1 minute at 65°C, polymerization for 4 minutes at 72°C, final elongation for 7 minutes at 72°C.
The PCR product was purified by gel extraction, cloned into XL PCR Topo cloning vector (Invitrogen, USA) and subsequently subcloned into the BamHI and NotI sites of the baculovirus transfer vector pAcGP67A (BD Biosciences, USA) for expression of mCD36ED with a C-terminal His-tag (Gen Bank accession number: GQ227601). The correct mCD36ED DNA sequences were confirmed at each step by sequencing both DNA strands. 2 µg of mCD36ED pAcGP67A plasmid DNA was co-transfected with 2 µg of Profold ER1 baculovirus DNA (AB vector, USA) in Sf-9 cells. After 5 rounds of viral amplification, a titer of 1×109 virus/ml was obtained as determined by an end point dilution assay. Baculovirus infection was monitored by GFP expression, which was encoded in the Profold ER1 baculovirus DNA. For large scale mCD36ED protein expression, 3 liters of Hi-5 cells were infected with mCD36ED Profold ER1 baculovirus at an MOI of 3 and harvested after 7 days. The first purification was done by IMAC using Ni-NTA beads (Qiagen, USA) and a Western blot was performed to confirm the presence of mCD36ED in the elution from the Ni-NTA beads. After SDS PAGE, the proteins were transferred to a PVDF membrane by electro-blotting for 1 hour at 100 volts. The membrane was blocked with 1 % BSA, 0.1% Tween 20 for 1 hour at room temperature and incubated overnight at 4°C with an anti penta-His tag antibody (1/100 dilution; Calbiochem, USA). A second incubation with an anti-mouse IgG-peroxidase conjugated antibody (1/5000 dilution; Pierce, USA) was performed for 1 hour at room temperature. After developing the Western blot with ECL (Pierce, USA), a band between 50 and 75 kDa was observed.
CD spectroscopy of mCD36ED
mCD36ED at a concentration of 0.14 mg/ml in 50 mM NaCl, 10 mM TrisCl pH 8 was used for circular dichroism (CD) experiments. CD spectra of mCD36ED were recorded at 25°C using an AVIV 202 spectropolarimeter (Hellma, Mullheim, Baden, Germany). The scans were performed in triplicate from 260 to 195 nm with 1 nm resolution. The spectra from triplicate scans were averaged and the final spectra were obtained by subtracting the buffer measurement spectra obtained under identical conditions. Results are expressed as molar ellipticity per residue. Protein concentrations were determined using the Bradford method (Bradford solution, Pierce, USA). Typical CD spectra of proteins rich in α-helical (Che a 3, [54]), β-sheet (human TLR-2 ectodomain, [55]), and mixed α/β secondary structure (Bet v 1, [56]) are shown as reference.
Determination of N-linked glycosylation and disulfide bonds in mCD36ED
To investigate the presence of disulfide bonds in mCD36ED, 20 µg of mCD36ED was incubated at 100°C in SDS buffer with or without β-mercaptoethanol for 5 minutes and the samples were subjected to SDS PAGE for 1 hour at 200 volts. To demonstrate the presence of N-linked glycosylation, 10 µg of mCD36ED was deglycosylated with 50 units of PNGase F under native conditions. The difference in electrophoretic mobility was indicative of the presence of N-linked glycans in mCD36ED.
Native PAGE and gel filtration experiments
To determine binding of mCD36ED to different ligands, 10 µg of mCD36ED was incubated overnight at 37°C with 1 µg of synthetic Pim4, 1 µg Pim2, 1 µg LTA, 1 µg of Pam3CSK4 fluorescein, 10 µg of Viet 04 hemagglutinin, or 1 µg FSL-1 fluorescein. The molar ratio of trimeric hemagglutinin to mCD36ED is approximately 0.3 and the molar ratio of the monomeric HA to mCD36ED is approximately 1. In the case of the lipid ligands, it was added an excess of ligands respect to mCD36ED. The molar ratios ligand/mCD36ED were: 3.9 (Pam3CysSK4), 5.6 (FSL-1 fluorescein), 3.2 (Pim2), 3.9 (Pim4), 2.5 (LTA). For negative control, 10 µg of KRN TCR was incubated for one hour at 37°C with 1 µg of synthetic Pim4, 1 µg Pim2, 1 µg LTA, 1 µg of Pam3CSK4, or 1 µg FSL-1. The molar ratios of ligand/ KRN were: 4.0 (Pam3CysSK4), 5.8 (FSL-1), 3.3 (Pim2), 4.0 (Pim4), 2.6 (LTA). The samples were run for 4 hours at 100 volts in a native 4–20% polyacrylamide gel (Criterion Tris HCl gel, Biorad) at room temperature with a Glycine Tris running buffer at pH 7.4. Fluorescence was determined using a Versadoc imaging system and the gel stained with Coomassie Blue. To determine if binding altered the retention volume of mCD36ED, 100 µg of mCD36ED were incubated overnight at 37°C with 1 µg of LTA, Pam3CSK4 or Pam2CSK4. The samples were loaded on a Superdex 200 10/30 column (Pharmacia). The gel filtration chromatography was performed at room temperature using PBS pH 7.4 as running buffer at a flow rate of 0.5 ml/min and a fractionation volume of 0.5 ml
Dot blots experiment to detect LTA in mCD36ED-LTA complex purified by size exclusion chromatography
The collected fractions of CD36-LTA and CD36 from Superdex 200 10/30 were used for dot blots with an anti LTA antibody (clone 55, Hycult, Netherland) that detects the LTA polyglycerophosphate moiety. The first step was to blot 3 µL CD36 LTA, 3 µL of CD36 in a PDVF membrane, previously activated with 100% methanol. The membrane was blocked with 1 % BSA, 0.1% Tween 20 for 1 hour at room temperature and incubated overnight at 4°C with a 1/1000 dilution of an anti-LTA antibody (1/1000 dilution; clone 55, Hycult, Netherland). A second incubation with a goat anti-mouse, IgG -peroxidase conjugated antibody (1/5000 dilution; Pierce, USA) was performed for 1 hour at room temperature. The dot blot was developed with ECL (Pierce, USA). The anti-LTA antibody and LTA were employed as positive controls, while Pam2CSK4 and PBS were employed as negative controls in the dot blot experiment.
Determination of secretion of TNF-α by macrophages
Macrophages from the different mutant mice were induced by addition of 3% thyoglycollate. After 3 days, peritoneal macrophages were harvested and 50,000 macrophages per well (macrophages harvested from the same mouse were seeded in duplicate wells) were incubated in DMEM with 5 % FCS and 2 % penicillium streptomycin at 37°C, after which culture media were discarded and replaced with 100 microliters of fresh media and incubated for 4 hours with different concentrations of bacterial ligands or different concentrations of LTA plus a constant amount of active mCD36ED (50 or 100 ng/ml), or heated denatured mCD36ED (100 ng/ml). Subsequently, the supernatants were harvested and the values of TNF-α were determined by an L929 cytotoxic assay. An average value of TNF-α was obtained for each duplicate wells (TNF-α duplicate wells) and the reported TNF-α values were the average values of the TNF-α duplicate wells. Because the data of each duplicate wells represent the secretion of TNF-α from macrophages of a particular mouse, the duplicate wells are independent of each other. Therefore, the number of independent replicates per experiment is the number of mice per experiment. In order to determine the effect of the different mutants in the response to lipomannan, FSL-1-fluorescein and LTA, and for determination of the activity of mCD36ED, macrophages were extracted from two and three mice, respectively. Values are expressed as mean values +/− SEM. All experiments were repeated three times. Therefore, the data are representative from three independent experiments.
To determine any direct interaction between the ectodomains of murine CD36 and TLR2, 24 µg of mCD36ED was incubated with 10 µg of mTLR2ED/FC chimera overnight at room temperature in PBS pH 7.4, and then incubated with 50 µl of magnetic protein G beads (NEB, USA) for 1 hour at 4°C. The supernatant was removed and the protein beads were washed 5 times with PBS. The proteins were eluted from the beads by heating for 5 minute at 100°C with reducing loading buffer and run on an SDS PAGE.
Supporting Information
Figure S1.
Characterization of recombinantly expressed mCD36ED. (A) Western blot showing the presence of mCD36ED after Ni-NTA purification of supernatants of Hi-5 cells infected with mCD36ED ER1 Profold baculovirus. Lanes: 1- Molecular weight markers; 2- Supernatant of non-infected Hi-5 cells; 3- Elution from Ni-NTA beads of supernatant from Hi-5 cells infected with mCD36ED ER1 Profold baculovirus. (B) The identity of mCD36ED was confirmed by mass spectrometry. To confirm that the purified protein was indeed mCD36ED, the gel bands from figure 1B were excised, reduced with DTT (10 mM), digested with trypsin overnight before being analyzed by nano LC-MS/MS (TSRI Center for Mass Spectrometry). The 6 peptides, which were identified using MASCOT, are depicted in red.
Figure S2.
mCD36ED has intramolecular disulfide bonds and N-linked glycosylation. (A) SDS PAGE of mCD36ED under reducing and non-reducing conditions. Lanes: 1- mCD36ED under reducing conditions; 2- mCD36ED under non-reducing conditions. (B) 10 µg of purified mCD36ED (native and denatured) was digested with 50 units of PNGase F overnight at 37°C. The samples were reduced and run on an SDS PAGE.
We thank Dr. James Stevens for providing recombinant hemagglutinin (Viet 04) and Julie Vanhnasy for technical support.
Author Contributions
Conceived and designed the experiments: MJJD NX KH BB IAW. Performed the experiments: MJJD NX ALC PV. Analyzed the data: MJJD KH BB IAW. Contributed reagents/materials/analysis tools: GDA DSL GFP. Wrote the paper: MJJD NX KH. Revised the manuscript: MJJD IAW ALC PV GFP PMR RAD KH BB.
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Hey all! We are an American couple in the process of moving to Taiwan from Tokyo!
If you're in Tokyo and like ramen, please feel free to check out our ramen blog, for recommendations ( http://ramenloving.blogspot.com/ ) If you want a good bowl of ramen, we know a lot of places!
We also like to run so we've been trying to do short races whenever we can. Micah loves guitar and Brenna loves dancing. We used to do awa-odori, a traditional Japanese dance. Most recently Brenna has done some Bollywood-style dancing though.
NOTE: Sorry, but due to the high volume of requests we get, if it looks like you've sent a copy and pasted request, it's likely we'll decline your request!
Be excellent to each other.
Why I'm on Couchsurfing
We've been hosting couchsurfers since September 2010, and it's been a real delight meeting people from all over the world! We do our best to respond to every request and email we get, so if we can help y'all out, either with a couch, directions to a good but cheap dive, or if you just want to meet someone local for drinks and karaoke, let us know!
Currently we cannot host, but please contact us for hanging out! We're in a very central location, Shinjuku, so if you're in that part of Tokyo, we'd love to meet up!
So far we've only hosted, but it's been a blast! Going to karaoke, drinking and eating with our guests... it's been great!
Hiking, camping, cooking, animal rescue, reading, playing guitar, dancing, listening to music, drinking, photography, karaoke, art and design, crafts, dancing, yoga, making things, gardening, having fun!
- animals
- arts
- culture
- design
- photography
- dancing
- cooking
- yoga
- running
- drinking
- crafts
- gardening
- reading
- socializing
- blogging
- drawing
- survival
- music
- guitar
- karaoke
- hiking
- camping
Music, Movies, and Books
Movies: Star Wars (eps 4 through 6), Princess Bride, Zoolander
Music: Electronic, acoustic, folk, pop, rock, show tunes, most anything with a good danceable beat
Books: Elizabeth Peters, JK Rowling, Terry Pratchett, Neil Gaiman, Philip K Dick, Stephen Baxter, Ayn Rand
Television: The Venture Bros, Community, 30 Rock, Parks and Recreation, Archer, Adventure Time, Game of Thrones, Sleepy Hollow, Bee and PuppyCat, The Walking Dead, True Blood, Breaking Bad
One Amazing Thing I've Done
Sunset over Ashinoko in Hakone, Japan
Teach, Learn, Share
Teach: English and survival Japanese, art (drawing), cooking (Japanese and Western)
Countries I've Visited
Countries I've Lived In


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Panels explore how major metropolitan areas pose serious environmental woes
March 8, 2011
Phil Brozynski
(630) 829-6094
Lisle, Illinois ~ Hong Kong. Mexico City. Stuttgart. Istanbul, Varanassi, India – major metropolitan areas that pose major environmental problems. As more and more people are drawn to megalopoles around the world, city governments are confronted with challenging moral, ethical and scientific problems.
The Years for the Environment initiative and the Faith and Reason Symposium series at Benedictine University will examine the issues of climate change and the environmental pressures facing major metropolitan areas during a daylong forum featuring Gregory A. Petsko, D.Phil. and a panel of distinguished faculty from 8:00 a.m.-4:30 p.m. on Friday, April 1 in Birck Hall, Room 112.
Petsko, who earned a Ph.D. from Oxford University, is an internationally known biochemist and chemist and past president of the 12,000-member American Society for Biochemistry and Molecular Biology.
Benedictine faculty addressing the global cities issue from 9:00-11:00 a.m. include: Rita George-Trtkovic, Ph.D., assistant professor of Theology; Christine Isom-Verhaaren, Ph.D., instructor of History; Ovid Wong, Ph.D., associate professor of Education; and Margarete Roth, Ph.D., professor emeritus of International Business and Economics.
Petsko will speak during lunch from 12:00-1:15 p.m., followed by a climate change panel from 1:30-3:30 p.m. featuring Al Martin, Ph.D., professor, department of Biology; Tim Marin, Ph.D., associate professor of Chemistry; Philip Hardy, Ph.D., instructor, Department of Political Science; and Ralph Feeney, M.S., M.B.A. of Environmental Resources Management.
The climate change panel discussion is the capstone event for students participating in the Climate Change semester.
The symposium is free and open to the public. For more information, contact Tonia Rucker at (630) 829-6250.
Benedictine University is an independent Roman Catholic institution located in Lisle, Illinois just 25 miles west of Chicago. Founded in 1887, Benedictine provides 53 undergraduate majors, 13 graduate and four doctorate programs. Benedictine University is ranked as a Top School in the Midwest (11th in Illinois) for Master's Universities, 12th in the Midwest (and sixth in Illinois) for Racial Diversity, and eighth in Illinois for Freshmen Retention for 2011 by U.S. News & World Report.


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He's a regular parlor-room magician. But instead of pulling a never-ending trail of colorful handkerchiefs or a bouquet of flowers from his sleeves, we are retrieving remote controls, spoons, wads of tissues, beads, toy cars, small stuffed animals and hairbrushes.Yesterday, when I announced bed time to him, he stood up sleepily and started to stagger upstairs. In true Lil D style of late, he was wearing two hoodies over an undershirt.(He just finds them around the house and piles them o … [Read more...]
The Magician Who Can't Make Autism Numbers Disappear
March 29, 2013 by Leave a Comment


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A twisted shape offers striking visual appeal to this coastal-styled giant tower floor lamp. In a rich green hue with cocoa leaf material on a metal frame. Design by Eangee.
Our team has over 100 years combined experience with contemporary lighting.
Every EuroStyleLighting.com order is backed by our 100% Satisfaction Pledge.
Shipping and Delivery Information - For item EU8P146 if ordered by 1 pm Pacific Time


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Steals & Deals
On Sale!
Baratza Vario-W Burr GrinderTake the effort out of espresso, or any other brew method, with the Baratza Vario-W coffee grinder! Designed to reduce your coffee-brewing steps in the morning, the Vario-W offers built-in weight-based dosing and three programmable dose options for multiple brew methods. Adjusting your grind has also never been easier, thanks to the Vario's unique dual-adjust macro and micro stepped dials, which give you over 230 unique grind settings to choose from. Learn More$559.00


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City police want to hit former police dispatcher Beth Fike, 26, of 1738 S. Liberty Ave. in the pocke
Current subscribers have full access to all online news content as part of their subscription. If you are a current subscriber, please log in with your website account information for full access. If you are a current subscriber, but have not yet registered for access to the website, please call our Circulation Department at (330) 821-1200


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UnPoetia talk:Nobody reads UnPoetia!
From Uncyclopedia, the content-free encyclopedia
Feature! -RAHB 09:49, 20 February 2009 (UTC)
- I agree. --The Dit 09:57, 20 February 2009 (UTC)
edit Scrap the replies?
I wrote a reply to the original rant, but then had to write another to confer the reality that UnPoetia wasn't in fact a permament fixture to the main page. Now it's long and cumbersome, so would it be better to scrap the replies altogether? Sigh, it would be easier if UnPoetia actually was on the main page... --The Dit 16:10, 22 February 2009 (UTC) Okay, no mind.


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The objective of the Gordon Research Conference on Biomineralization is to explore the biological, chemical and physical controls on biomineralization in vertebrate, invertebrate, plant and biomimetic systems. Particular attention is paid to the consequences of interactions at the organic-inorganic interface. The interest in this topic stems from the important role that biomineralization processes play in biology, biotechnology, medicine, chemistry, interfacial science, materials science, and nanotechnology. Over the years attendees of this conference have witnessed the spectacular advances made in the field of biomineralization, which have been, in part, due to the interactions and collaborations stimulated by this GRC. The goal of this year's GRC is to bring together chemists, physicists, biologists, physicians and engineers, who would otherwise not overlap on the conference circuit, in a setting that promotes cross-disciplinary fertilization of new ideas. In doing so, our intent is to create the premier venue to discuss fundamental and applied aspects of biomineralization that intersect biology, chemistry and materials science.
We anticipate an exciting meeting with invited oral sessions that will cover cellular and genetic controls on mineral formation, in vitro models of mineralization, interactions and molecular contacts between macromolecular and mineral components, pathological mineralization, macromolecular scaffolds for materials synthesis and organization, new directions in tissue engineering, biomineral architecture in marine systems, the link between architecture and function, and biomineralization through time. Special emphasis will be placed on novel unpublished results, new experimental techniques, and the role of simulation and modeling.
The oral sessions will be followed by the highly successful afternoon poster sessions. The poster sessions provide all conferees with the opportunity to present their latest research and to exchange ideas in a more informal setting. As in the past, this opportunity will continue during the nightly social gathering that takes place in the poster hall following the evening lectures. As with the lectures, the posters should present work that has not been previously published to a significant extent. All conferees are encouraged to submit and present posters. We will select 8-10 of the best poster abstracts for short oral presentations.
We recommend early application to the Biomineralization GRC as attendance is limited and the conference is regularly over-subscribed. We welcome applications from graduate students and young postdoctoral scientists to attend and present their research findings. A number of travel awards will be made based on submitted poster abstracts.


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Volume 15, Issue 3: Stauron
Gary Hagen
Christ reigns over His kingdom. As the Apostle's Creed states, He sits at the right hand of the Father, and will
come again to judge the earth. The outworking of His redemption of the world involves past, present, and future events.
But when the Scriptures speak of the salvation of the world, it does not do so in terms of the signs we used to see under
the Golden Arches (1 million sold, 10 million sold, etc). There are no cosmic widget counters in heaven to let God
know when that last soul comes in "just as he am."
Back to top
Back to Table of Contents
Copyright © 2012 Credenda/Agenda. All rights reserved.


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Dog Is Not A Julia Roberts Fan
Don't try to take this pooch to see a Julia Roberts flick because he's not a fan. He must be having a delayed reaction to "Larry Crowne." or maybe he's seen the new photos of her as the Evil Queen in "Snow White." She does look terrifying in feathers. [Newslite]


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London (Oct. 13)
An agreement has been concluded between the Jewish National Fund and the Prudential Assurance Company, the J.T.A. is informed, under which a special Assurance Department for Great Britain and Ireland is being established in London at the Jewish National Fund headquarters. Life and Endowment policies of all descriptions issued by the Prudential can be taken out through this Department, enabling supporters of Palestine to assist the Fund without any extra cost to themselves in taking out life or endowment policies.
For a number of years past the Jewish National Fund has been co-operating on similar lines with the Phoenix Insurance Company of Vienna, and as a result of such co-operation, policies to a total amount of £10,000,000 have been issued through the National Fund Insurance Departments on the Continent of Europe and in Palestine. The net profits to the Jewish National Fund under the Phoenix agreement have exceeded £45,000.
The agreement with the Prudential Assurance Company, it is added, also provides possibilities for the investment of capital in Palestine.


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I was invited to present my views on the IPv6 deployment in enterprise networks during the local IPv6 summit. Instead of joining the cheering few or the dubious crowds, I'm trying to present a realistic view answering questions like "what do I have to do", "when should I start" and "where should I focus my efforts".
Here's the outline of my presentation, any feedback, additional thoughts or insightful critique is most welcome.
Background information
Scenario: Enterprise network connected to the Internet. No need for internal IPv6 (RFC 1918 is good enough).
Question: where shall I focus my IPv6 efforts?
Facts of life:
- IPv6 is a reality, get used to it.
- Migration is supposed to be easy, but you will get stuck on details.
- Start small, but start now.
Phases of public IPv6 deployment:
Phase#1: Dual stack content (starting now)
Phase#2: IPv6-only Internet clients (in a few years)
Phase#3: IPv6-only major content providers (10+ years from now)
Obviously this is just my perception of the critical milestones, as they apply to enterprise network deployment.
Proposed action plan
Phase#1 has already started, get ready for it:
- Establish IPv6 connectivity with all the upstream providers
- Deploy IPv6 on your public servers. Start with small, non-critical applications to get hands-on experience.
- Change your whole DMZ into dual-stack DMZ.
As an enterprise network, you don't care about Phase #2:
- Your content is reachable over IPv4 and IPv6
- Interesting content is reachable over IPv4.
- Use this time to plan your internal IPv6 deployment.
When the public content becomes available only over IPv6 (phase #3) you might be in a morass if your internal network is not yet dual-stack (you'll have to face ugly 4to6 NAT). Deploy dual-stack throughout your network:
- RFC1918 + 4to4 NAT
- Public IPv6 address space


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Darkroom Familia: The Darkroom Video Collection
This collection of music videos from Darkroom Familia features an assortment of great hip-hop artists performing your favorite songs, including "I Got Five on It Too," "For Love of Money," "The Drunks Song" and "Cuddy What U Need?" Artists include Duke, Crooked, Sir Dyno, Oso, Dub, Young Ant, Big Dru, Unknown, Venomous, Young Crayze, Barney Bumble, Sapo, J-Loc and more. Bonus features include Oaktown side shows, movie trailers and more.


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In most polymorphic genera some of the species have fixed and definite characters.
But it is polymorphic variability in the strictest sense of the word.
Multiform, mul′ti-form, adj. having many forms, polymorphic.
We now understand the process by which a polymorphic race maintains its polymorphism.
polymorphic species may now be regarded as the link which unites ordinary variability with the historical production of species.
The variations have lead authors to divide this polymorphic species into several species and varieties.
The general rule which correlates this polymorphic change is that its direction changes at a given temperature.
The models of a polymorphic mimic are almost always closely related species.
Passant oultre—one of Rabelais' favourite and most polymorphic expressions.
This interesting form is polymorphic and mimics different species of Amauris.
"organism of several forms," 1828, from Greek polymorphos "of many forms" (see polymorphous).


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Bugzilla: Close a bug report
Questions should be directed to Web Development, email@example.com
Bug reports generally go through these stages:
Report created
Fixed by an IT-WD programmer
Tested and verified by the bug reporter
Closed by the bug reporter
If you have tested the resolution provided by the IT-WD programmer, and are satisfied that the bug is fixed as you specified, then you need to change the bug status to Mark bug as CLOSED. For compliance with our auditing procedures, all Bugzilla reports need to be marked as closed by the person who opened the report.
On the bug report that you are editing, find the radio buttons just above the Commit button.
Click the radio button for Mark bug as CLOSED.
You may want to enter Additional Comments to note that you have tested and verified that the bug is fixed.
Click the Commit button.
No further action will be taken on the bug, unless it is reopened.
Next step:
After you click Commit, the Bug Processed page will be presented. To edit your report, click the bug number in the box at the top of the page. To change pages, click on a link in the yellow box at the bottom of the screen.


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Quartet® Cork Bulletin Board, 6' x 4', Silver Aluminum Frame
Item #: 901-2307
Model #: 2307
Brand: Quartet
$185.49 Each
When you buy 3+
Buy more & save!
Product Highlights
- Surface Material: Cork
- Bulletin Board & Sign Type: Bulletin Board
- Frame Finish: Aluminum
Heavy-gauge aluminum frames this cork bulletin board with a self-healing surface. Use with push pins for all your document display needs for years to come. Conveniently mounts to the wall in either vertical or horizontal orientation. Quartet® Cork bulletin board with aluminum frame measures 6' x 4' and has high-density backing for better pin-holding power. Bulletin board quickly secures documents with provided quick clips.
- Durable natural-cork bulletin board with heavy-gauge aluminum frame secures documents with push pins
- Low use - break rooms or factory floors
- 6'W x 4'H
- Self-healing surface
- Heavy-duty, factory-installed mounting system mounts to walls easily
- Modern aluminum construction with satin finish frame complements any interior
|Bulletin Board & Sign Type : Bulletin Board
|Frame Finish : Aluminum
|Surface Material : Cork
|Board Size (H x W) : 48" x 72"
|Frame Color : Silver
|Height (in.) : 48"
|Width (in.) : 72"
|Brand : Quartet
|Weight : 28.35 lbs. per Each
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Search our blog
Recent Posts
Category Archives: Welding Tips & Tricks
Suggested Welding Material for Rebuilding
At Welding & Welder our technical team just love to help customers with specific wleding scenarios that they have encountered in the past, or are currently trying to solve. We recently had this question asked: "Currently We are using Feeding … Continue reading
Posted in Welding Tips & Tricks Tagged cobalt, cromium carbide, electrode welding, electrodes, rebuilding, welding Leave a comment
Choosing The Right Electrode
We were asked recently, "what should I consider before choosing the correct electrode?". To help answer we came up with 10 (actually 11) basic steps, prior to making your weld; 1. What is the analysis of the base material? 2. … Continue reading
Posted in Guides, Welding Tips & Tricks Tagged electrodes, mma welding, stick welding, welding electrodes Leave a comment
Weld Purging Tips
Put Argon in too fast and from the wrong place and ….. For those people weld purging with Argon, consider that Argon, although a gas, will move to some extent as though it were a fluid. A previous tip described … Continue reading
Posted in Welding Tips & Tricks Tagged weld purging tips, weld purging with argon Leave a comment


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Via Rancho Parkway near Del Dios Highway in the North County is closed in both directions Thursday morning after an injury crash that took out a utility pole and left power lanes draped across the roadway.
A driver in a black Toyota 4Runner slammed into the pole about 6:20 a.m. just east of Lake Drive, the California Highway Patrol said. The driver suffered minor injuries, the CHP said.
Crews with SDG&E are working on repairing the power lines. The utility said the work is expected to last several hours and that commuters should expect a day-long closure. Ninety-seven customers in the area are without power, according to the utility's web site.
Via Rancho Parkway is shut down from Del Dios to Highland West Drive, the CHP said.


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Consumer Goods»Consumer Goods & Retailing»Apparel»Women's Apparel
Spain: Women's Or Girls' Clothing (Knitted Or Crocheted) - Market Report. Analysis And Forecast To 2020The report provides an in-depth analysis of the market for Women's Or Girls' Clothing (Knitted Or Crocheted) in Spain. It presents the latest data of the market size and volume, domestic production, exports and imports, price dynamics and turnover in the industry. The report shows the sales data, allowing you to identify the key drivers and restraints. You can find here a strategic analysis of key factors influencing the market. Forecasts illustrate how the market will be transformed in the medium term. Profiles of the leading companies and brands are also included.Countries coverage: SpainProduct coverage: Women's or girls' overcoats, car-coats, capes, cloaks, anoraks (including ski-jackets), wind-cheaters, wind- jackets and similar articles, suits, ensembles, jackets, blazers, dresses, skirts, divided skirts, trousers, bib and brace overalls, breeches and shorts (other than swimwear), of wool or fine animal hair, of cotton, of synthetic fibers and of other textile materials, knitted or crocheted.Data coverage:
Learn how to effectively navigate the market research process to help guide your organization on the journey to success.


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Brain Traffic Lands the Quad!
At the last Winter Olympics, figure skater Evan Lysacek won the gold medal without a quad jump, much to the chagrin of the Russian favorite, Evgeni "The-KGB-stole-Steve-Perry's-mullet-for-me" Plushenko.
Afterwards, Evgeni glowered and ranted. He briefly stood on the gold medal platform at the medals ceremony. His official website declared him the "platinum" medal winner. He made it clear that you need a quad to compete. Vladimir Putin agreed.
I don't know about you, but we at Brain Traffic got the message. I mean, seriously, Evgeni and Vladimir are not guys you want mad at you. So, we got to work.
The content strategy quad
As of today, the Brain Traffic team has been landing its own quad regularly for more than a year. But our quad isn't an ice-skating feat—it's an infographic describing the critical components we consider in every content strategy.
What it all means
At the center is the core content strategy, the central idea for using content to achieve an organization's business goals. To achieve that strategy most effectively, we look at four closely related components (the four areas of the quad):
Content-focused components
1) Substance—What kind of content do we need (topics, types, sources, etc.), and what messages does content need to communicate to our audience? 2) Structure—How is content prioritized, organized, formatted, and displayed? (Structure can include communication planning, IA, metadata, data modeling, linking strategies, etc.)
People-focused components
3) Workflow—What processes, tools, and human resources are required for content initiatives to launch successfully and maintain ongoing quality? 4) Governance—How are key decisions about content and content strategy made? How are changes initiated and communicated?
So far, it's getting high marks
Our quad will probably never be discussed by Dick Button and Scott Hamilton, but over the past year it has received consistently high marks from clients and seminar attendees. The quad helps people quickly understand the complexity of content strategy and puts their content challenges into perspective.
Bonus quad-related craft project
Want to look like a content strategy Olympian? Here's how:
1. Print this page2. Cut out the quad3. Poke a hole in the middle4. Put it on a string around your neck
Presto, you're just like Evgeni.


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As a frequent visitor to Waubedonia Park during most of his 88 years, Fredonia resident Norman Jagow probably knows the park along the banks of the Milwaukee River as well as anybody.Jagow, who has lived in the area since 1933, has become fond of the towering trees, intimate green spaces and the remnants of artifacts from decades past.That is why he is objecting to a county plan to remove a small dam that has created a quarter-acre pond once considered the focal point of a charming setting."I want to protest the ridiculous waste of money to remove the dam," Jagow said.The county's Planning and Parks Department is pursuing funding for removing the two-foot-tall, 33-foot-wide stone and cement dam that environmentalists feel is impeding fish passage from the river to seasonally flooded spawning grounds in the park and wetlands to the north.Andrew Struck, director of Ozaukee County's Planning and Parks Department, said the dam removal is part of a more comprehensive Ozaukee Fish Passage Program that is part of the Great Lakes Restoration Initiative."The program reconnects naturally occurring high-quality habitats in the Milwaukee River watershed by modifying or removing obstructions and impediments to fish and aquatic life passage," Struck said.Tapping federal and state funding, he said the effort has identified and removed more than 175 waterway impediments on the river and 24 tributaries, opening more than 100 miles of streams to fish passage.That work has been covered by $5.2 million from the National Oceanic and Atmospheric Administration through the American Recovery and Reinvestment Act and $2 million from the U.S. Environmental Protection Agency.The county contends the source for the Waubedonia Park pond is Crystal Springs Creek, which connects to nine acres of wetlands deemed suitable fornorthern pike spawning north of Highway A.Jagow contends the water that fills the pond comes from an artesian well located about 100 yards north of the dam.When Crystal Springs Lake overflows during the spring melt, the resulting runoff follows the same creek bed."I walk the park every day and have never seen fish in that stream," he said.What Jagow has seen, he said, are laughing children who enjoy the pond, playing with the polliwogs and frogs that live in the pool safely removed from the river.The 40-acre park property was purchased by the county in 1926 from Peter Jung for $300.Although details are sketchy, Jagow said the pond dates to the 1930s, when it was part of an idyllic setting that included a small stone windmill and archway.He said those quaint structures were demolished by rambunctious teenagers in the 1970s.The DNR contends the dam is starting to show signs of deterioration, and must be repaired or removed.Struck said the county's plan is to remove the dam, rebuild the natural creek channel and restore the pond area. In all, four separate restoration projects are planned for the Crystal Springs Creek waterway.Jagow will take his protest to the county's Natural Resources Committee, which meets at 8:30 a.m. Thursday, Feb. 7, in the Administration Center in Port Washington.
SNOW COVERS THE small dam in Waubedonia Park which is scheduled to be removed to open the area for native fish and aquatic life.


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And now we present, the Domestic Enemies of the Gay Mom(s):
When our daughter was a baby we had fun with these questions (and when we were foster parents of babies of different races we really had fun with them). But there comes a time when our little big-eared darlings don't need to hear me tell the IHOP waitress that we had the tests done, but we're waiting for our Maury Povich taping next week to hear the results.
So please, don't make me pull out that line again until all of the children are at least 12 and I can enjoy the inevitable mortification it would cause, because I am already forced to have the weekly awkward conversation where I interpret whatever strange story Dear Daughter has decided to divulge during Sunday School without adding THAT to the mix.
Das Authorityski
If you've ever had your authority as a parent questioned by anyone, it is an express train ride to The Rage. And if your kids are present when it happens, it's kicked up a few notches to Hungry Mama Bear on a Bad Spring Day.
But it is a common occurrence for at least one half of our parenting team. Only one of us is allowed to be listed on the birth certificate because it would cause some pin-headed bureaucrat's head to explode if we crossed out Father and put Parent on the form (see Forms below). Which leaves one of us with all of the parental legal authority as a 15 year old babysitter, but with less pay.
Enter here our well paid and friendly attorney, who drafts reams of legal documents that no one understands (not even me, and I'm an attorney), giving the Non-Legal Parent "approximate parental rights". This is all fine and good, but those rights only exist on paper and our diaper bag does not have a waterproof compartment large enough to carry the equivalent of Shakespeare's complete works unabridged.
|The TSA is awesome. Everyone knows that.
The Freaking Forms
I touched on this a bit in the prior section, but every mother-lovin' form in existence helpfully has information lines for a Mother and a Father, and Maude help you if you cross out the words that were printed on the page and insert a generic Parent #1 and Parent #2.
Want to have fun the next time you are filling out papers at a new doctor's office/school/hospital/job? Where it says Marital Status and has boxes for Married, Widowed, Divorced, Never Married ... draw your own box at the bottom and write None of the Above. See how that works out for you.
Or, failing that, just put a big smear of peanut butter across that part. Marital Status? We're just Skippy, thanks...
(c)Herding Turtles, Inc. 2009 - 2011


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We continue the discussion about the distinctiveness of Catholic charitable work with a commentary from Fr. Andrew Small, who is the Director of the Committee for the Church in Latin America at the USCCB.
Small looks at the issue of Catholic identity, which is so integral to the new report issued this morning by the USCCB regarding the work of the Catholic Campaign for Human Development.
The question: What is distinctive about Catholic charitable work?
Fr. Small: Without action, nothing changes. But without identity, the action is not Catholic, regardless of who performs it.
How do activity and identity combine to express the virtue of Christian charity? When the external change of activity carries with it the internal change of religious conversion.
A necessary element in social change is a sense of the self that is structured towards an engagement with others over and against certain obstacles and that seeks transformation. However, such action can equally reveal a view of the world that flows from the Gospel as one that does not. Activism prioritizes external change over internal transformation. The reason for such action is secondary.
Identity, on the other hand, suggests a relatively stable core that can be recognized in times of change. Periods of identity crisis challenge a collective understanding of what constitute the defining qualities of a person or their actions, in light of the dynamic interplay between the internal and external.
As outside pressure impinges on an acting subject, identity that is channeled or manifested in some essential way through observable realities – mainly though words and actions– becomes open to interpretation or even reinterpretation. Hermeneutics, then, becomes an essential tool in the identification process.
After the Council, it became popular to distinguish charity from justice, with the latter a more considered version of the former. But, it would be a mistake to view justice as a mature version of charity.
Reading closer, we see how the Council began with and concluded with the notion of charity to ground the nature of the Church's work in behalf of the poor. The notion of charity that emerged from the Council was a more comprehensive vision of the Christian vocation. The globalizing nature of poverty demanded an equally globalizing concept of charity, one that incorporates and surpasses the modern concepts of social justice, solidarity and human development.
In this sense, charity is not distinct from justice, rather charity perfects justice.
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|Add Comment
By Benjamin Chapman
From: Small Town, USA
Jun 29, 2013
|Wow. As "The Ole 6" has a mixed rating and apparently WI, as well, that must have been an exciting lead under that roof with all that gear. Nice job, Erik.
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|5.0 from 3 votes
|Your Score