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CD40, a tumor necrosis factor (TNF) receptor (TNFR) family member, conveys signals regulating diverse cellular responses, ranging from proliferation and differentiation to growth suppression and cell death. The ability of CD40 to mediate apoptosis in carcinoma cells is intriguing given the fact that the CD40 cytoplasmic C terminus lacks a death domain homology with the cytotoxic members of the TNFR superfamily, such as Fas, TNFR1, and TNF-related apoptosis-inducing ligand (TRAIL) receptors. In this study, we have probed the mechanism by which CD40 transduces death signals. Using a trimeric recombinant soluble CD40 ligand to activate CD40, we have found that this phenomenon critically depends on the membrane proximal domain (amino acids 216 to 239) but not the TNFR-associated factor-interacting PXQXT motif in the CD40 cytoplasmic tail. CD40-mediated cytotoxicity is blocked by caspase inhibitors, such as zVAD-fmk and crmA, and involves activation of caspase 8 and caspase 3. Interestingly, CD40 ligation was found to induce functional Fas ligand, TRAIL (Apo-2L) and TNF in apoptosis-susceptible carcinoma cells and to up-regulate expression of Fas. These findings identify a novel proapoptotic mechanism which is induced by CD40 in carcinoma cells and depends on the endogenous production of cytotoxic cytokines and autocrine or paracrine induction of cell death.
CD40, a member of the tumour necrosis factor (TNF) receptor (TNFR) superfamily, is expressed on a plethora of different cell types, including B cells, macrophages, dendritic cells, endothelial cells, and fibroblasts, and this widespread expression is likely to account for the central role of CD40 in the regulation of humoral immunity and host defense (54). Studies from our and other laboratories have shown that CD40 is also expressed in normal basal epithelial cells in stratified squamous epithelium and in a number of carcinomas, including ovarian, nasopharyngeal, bladder, and breast, where its precise role remains elusive (15, 55, 74, 75). The ligand for CD40 (CD40L) (gp39 or CD154) is a 39-kDa type II integral membrane protein with homology to TNF which can be induced on T cells following their activation via the T-cell receptor (54). CD40L expression has also been reported in B cells, monocytes, and NK cells, and a soluble form of this molecule has been detected in the serum of patients with hematological malignancies (73).
The central role of CD40-CD40L interactions in orchestrating immune responses is emphasized by studies of mice lacking CD40 or CD40L. In these knockout animals, thymus-dependent responses to foreign antigens, such as immunoglobulin production, isotype switching, and somatic hypermutation are impaired (39, 72). A similar phenotype (HIGMX) is observed in patients with hyperimmunoglobulin M syndrome, a genetic disease which results from mutations in the CD40L gene (6). Interestingly, HIGMX individuals also appear to be prone to development of tumors of the pancreas and liver (30). Our recent work also implicates the CD40 pathway in hepatocyte death during liver allograft rejection through a cooperative interaction with Fas, another member of the TNFR superfamily (1).
In vitro studies have shown that while CD40 ligation provides an antiapoptotic and proliferative signal for normal resting B cells (26), CD40 stimulation in lymphoblastoid and Burkitt's lymphoma cells induces growth inhibition (2, 22). CD40 ligation in carcinoma cell lines also results in growth inhibition and sensitizes these cells to apoptosis induced by a variety of agents, including TNF-α, anti-Fas, and cytotoxic drugs (15). Furthermore, when exogenously expressed, CD40 has been shown to transduce apoptotic signals in certain cell lines of epithelial or mesenchymal origin (31), but the mechanism of this phenomenon is unknown. In agreement with these in vitro findings, a recombinant soluble form of CD40L has been found to inhibit the growth of breast carcinoma cells in xeno-transplanted SCID mice (32), an observation which underlines the potential therapeutic use of CD40L for the treatment of carcinomas. In addition to its growth-regulatory properties, CD40 ligation in cell lines of epithelial or B-cell origin induces homotypic cell adhesion, up-regulation of various cell surface markers, and cytokine production (2, 11, 18, 25).
The signalling pathways that are activated by CD40 stimulation and thereby control its diverse effects on cellular phenotype have been the subject of intense investigation. While the cytoplasmic C terminus of CD40 lacks intrinsic kinase activity, adapter proteins of the TNFR-associated factor (TRAF) family, most notably TRAF2 and TRAF6, appear to mediate the activation of CD40 signalling cascades such as the cJun N-terminal kinase (JNK) and NF-κB (53, 58, 66). A TRAF2- and TRAF6-dependent extracellular signal-regulated protein kinase (ERK) mitogen-activated protein kinase signal is induced by CD40 ligation in cells of epithelial but not of B-cell origin (37,61). Other pathways activated by CD40 stimulation include the JAK3-STAT3 (29) and phosphatidyl inositol 3-kinase–Akt (57), which may contribute to the antiapoptotic properties conferred by CD40L in B cells. Further insight into the differential activation and integration of these signals is required to explain the diverse phenotypic consequences of CD40-CD40L interactions in different cell types.
In this study, we have probed the mechanism by which CD40 transduces death signals in carcinoma cells. We have identified the membrane-proximal domain of CD40 as being important for apoptosis induction and shown that this phenomenon occurs through a crmA-sensitive, caspase-dependent pathway involving activation of cytotoxic ligands of the TNF family.
DNA constructs.The pcDNA3-based CD40 expression vector pc-CD40 has been previously described (19). A PXQXT254→PXQXA mutation was generated from pc-CD40 using the Quick Change site-directed mutagenesis kit of Stratagene and mutated primers 5′-GCTCCAGTGCAGGAGGCTTTACATGGATGCC-3′ and its complementary primer. To generate CD40 deletion mutant Δ(216-239), CD40 amino acids (aa) 1 to 215 were PCR amplified using a forward primer with an artificial HindIII site upstream from the start codon (5′-CTGGTCTAAGCTTGCCATGGTTC-3′) and a reverse primer with an artificial Asp718 site (5′-GCTTCTTGGTACCCTT TTTGATAAAG-3′) and introduced intoHindIII/Asp718-digested pcDNA3. CD40 aa 240 to 278 were then PCR amplified using a forward primer with anAsp718 site (5′-GGAGATCAATGGTACCGACGATC-3′) and a reverse primer with a NotI site downstream from the CD40 stop codon (5′-ACCCACCGCCGGCGGAGTGA-3′). This PCR fragment was digested with Asp718 and NotI and inserted in frame in CD40 aa 1 to 215 in pcDNA3 to give pc-CD40Δ[216-239]. The presence of the above mutations was verified by sequencing.
Cell culture and treatment.Carcinoma cell line MG75 was generated from an epithelial ovarian solid tumor, and MG79 was established from tumor cells present in the ascitic fluid of a patient with adenocarcinoma of the ovary (24) (kindly provided by M. Gilligan, Institute for Cancer Studies, University of Birmingham, Birmingham, United Kingdom). These carcinoma cell lines together with HeLa cervical and A2780 ovarian carcinoma cells were maintained in RPMI supplemented with 10% fetal calf serum. To generate stable HeLa clones expressing wild-type or mutated CD40, 2 × 106 cells were electroporated (125 μF, 450 V) with 10 μg of plasmid DNA in 0.8 ml of ice-cold phosphate-buffered saline, and subsequent selection was performed in growth medium supplemented with 600 to 650 μg of G418 (Geneticin; Gibco BRL) per ml.
For induction of apoptosis, carcinoma cell lines were plated on a 96-well plate at 8,000 cells per well in 0.2 ml of complete medium in triplicate. The following day cells were treated for 6 h with 1 μg of trimeric recombinant soluble (rsCD40L) (52) (kindly provided by Immunex Corporation, Seattle, Wash.) per ml or with 1 μg of a soluble CD40L with enhancer cross-linking antibody (Alexis Corporation) per ml and then cocultured with cycloheximide (CHX) (Sigma) for an additional 24 h time period. Alternatively, cells were treated with CH11 anti-Fas monoclonal antibody (MAb) (Immunotech), soluble Fas ligand (FasL) (Alexis Corporation) or TNF-related apoptosis-inducing ligand (TRAIL) with enhancer (Alexis Corporation). In these experiments, HeLa cells were treated with 50 μg of CHX per ml as previously described (31), MG75 and MG79 cells were exposed to 8 μg of CHX per ml, and A2780 cells were exposed to 10 to 15 μg of CHX per ml. Under these conditions, CHX caused very limited or no toxicity in these cell lines. For neutralization experiments, cells were pretreated for 1 h with anti-CD40L MAb (Ancell Corporation), NOK1 anti-FasL MAb (Pharmingen), TRAILR1:Fc (R&D Systems), neutralizing anti-TNF-α (R&D Systems), or neutralizing anti-interleukin 6 (IL-6) antibody (R&D Systems) and then cocultured with rsCD40L and CHX as described above. Purified OX34 anti-CD2 MAb was kindly provided by M. Rowe, University of Cardiff, United Kingdom. For apoptosis inhibition experiments using caspase inhibitors, cells were pretreated for 30 min with 50 μM zVAD-fmk (Calbiochem) in 0.1 ml of complete medium before addition of rsCD40L and CHX; final concentration of zVAD was then 25 μM and was maintained throughout the assay. The cell-permeating and irreversible caspase 3 and caspase 8 specific inhibitors zDEVD-fmk and zIETD-fmk, respectively, were purchased from Calbiochem.
Apoptosis assays.Following treatment, cells were trypsinized and fixed in 1% paraformaldehyde. Cell suspensions were stained with propidium iodide (5 μg/ml), and apoptotic cells were identified on the basis of nuclear condensation and degradation and counted. A minimum of 300 cells were counted in each experiment, and assessments were performed independently by two of the authors (A.G.E. and C.D.). In some experiments, apoptosis was determined using electrophoretic analysis of DNA fragmentation (17) or in situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay (Promega), according to the manufacturer's instructions.
Coimmunoprecipitations.Human kidney embryonic 293 cells were plated on 100-cm2 dishes and allowed to adhere overnight. The following day cells were transfected with 8 μg of CD40A or Δ(216-239) expression vectors in the presence of pCMV-TRAF2 (14), Flag-tagged pME-TRAF6 (66), or control vectors using calcium phosphate. Thirty-six hours later, the cells were lysed in 0.5 ml of lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 3% glycerol, 1.5 mM EDTA, 0.5% NP-40) supplemented with protease inhibitors, and 500 μg of cell lysates was incubated overnight at 4°C with 2 μg of EA5 anti-CD40 MAb (Ancell). Following a 2-h incubation with protein G-Sepharose beads, immunoprecipitates were resolved by sodium dodecyl sulfate–10% polyacrylamide gel electrophoresis and immunoblotted for TRAF2 (C20; Santa Cruz Biotechnology), TRAF6 (M2 anti-Flag; Kodak), and CD40 (C20; Santa Cruz Biotechnology) accordingly.
JNK in vitro kinase assays.Following stimulation, cells were washed twice in ice-cold phosphate-buffered saline and lysed in 1 ml of kinase lysis buffer (20 mM Tris [pH 7.6], 0.5% Triton X-100, 250 mM NaCl, 3 mM EGTA, 3 mM EDTA, 2 mM sodium vanadate, aprotinin (10 μg/ml), leupeptin (10 μg/ml), and 1 mM dithiothreitol) for 20 min on ice. JNK was immunoprecipitated from 200 μg of total protein extracts using 5 μl of anti-JNK1 antibody (Santa Cruz Biotechnology) for endogenous JNK and 0.8 μg of anti-hemagglutinin MAb (Boehringer/Roche) for transfected hemagglutinin-tagged JNK. In vitro kinase assays were subsequently performed as previously described (16, 19).
Generation of crmA recombinant adenovirus.The crmA cDNA was PCR amplified from a crmA expression vector (kindly provided by Chris Gregory, University of Nottingham, Nottingham, United Kingdom) using primers with artificial BglII sites: 5′-CAAAATAGATCTCCATGGATATCTTC-3′ (crmA forward) and 5′-GA ATGAGATCTAATTAGTTGTTG-3′ (crmA reverse). The PCR product was digested with BglII, cloned inBamHI-digested pMC3 adenovirus transfer vector (4), and sequenced. A recombinant crmA adenovirus was generated by methods first described by McGrory and coworkers (49). Briefly, recombinant virus was obtained following cotransfection in 293 cells of pMC3/crmA and pJM17 (containing the entire Ad5dl309 genome). As a result of homologous recombination between these two plasmids, crmA under the control of the cytomegalovirus immediate-early promoter was inserted into the adenovirus genome in place of E1 gene sequences. Progeny virus was taken through several rounds of plaque purification before expansion of viral stocks.
Caspase 3 and caspase 8 activity assays.Caspase 3 activity in cell extracts from carcinoma cells exposed to various agents was determined using the caspase 3 cellular activity colorimetric assay kit from Calbiochem. Briefly, 106 cells were lysed in 30 μl of a lysis buffer containing 50 mM HEPES (pH 7.4), 1 mM dithiothreitol, 0.1 mM EDTA, and 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), supplemented with 0.1% NP-40 (Sigma). Protein concentration was determined using the Bio-Rad protein assay kit and 30 μg of total cell lysates were then incubated with 200 μM caspase 3 substrate Ac-DEVD-pNA in assay buffer at 37°C for 5 h. Absorbance at 405 nm was determined on a microtiter plate reader. Recombinant caspase 3 (30 U) was used as a positive control. For inhibition studies, cell lysates were preincubated for 15 min with the caspase inhibitors Ac-DEVD-CHO and zVAD-fmk at final concentrations of 0.1 μM before the addition of caspase 3 substrate. Activation of caspase 8 was detected by immunoblot analysis. After the indicated treatment, cells were lysed in JNK kinase lysis buffer supplemented with protease inhibitors, and 100 μg of cell lysates was resolved by sodium dodecyl sulfate–15% polyacrylamide gel electrophoresis. Immunoblotting was performed with an anti-caspase 8 antibody (C20; Santa Cruz Biotechnology) followed by enhanced chemiluminescence (ECL) (Amersham).
Reverse transcription (RT)-PCR.cDNA synthesis from 3 μg of total RNA was performed as previously described (16). Primers used were as follows. For FasL, the forward primer was 5′-GGTCCATGCCTCTGGAATGG-3′, the reverse primer was 5′-CACATCTGCCCAGTAGTGCA-3′, and the probe was 5′-ATGAGGAACTCTAAGTATCC-3′. For TRAIL (Apo-2L [56]), the forward primer was 5′-AGACCTGCGTGCTGATCGT-3′, the reverse primer was 5′-GACCAGTTCACCATTCCTC-3′, and the probe was 5′-GTAGCAGCTCACATAACT-3′. For CD40L, the forward primer was 5′-AGAATCCTCAAATTGCGGC-3′, the reverse primer was 5′-TGTGGGTATTTGCAGCTCTG-3′, and the probe was 5′-ATGCCCAAGTCACCTTCTGT-3′. Conditions for 38 cycles of PCR amplification were as follows: for FasL, 94°C for 45 s, 54°C for 50 s, and 72°C for 50 s; for TRAIL, 94°C for 40 s, 48°C for 40 s, and 72°C for 50 s; for CD40L, 94°C for 45 s, 49°C for 60 s, and 72°C for 60 s. TNF-α primers and PCR conditions have been previously described (27). The oligonucleotide used as a TNF-α-specific probe was 5′-TGAGGCCAAGCCCTGGTAT-3′. FasL, TRAIL, CD40L, and TNF-α products (25 μl) and positive controls for FasL and CD40L (5 μl) were analyzed on a 1.5% gel before transfer onto a nylon membrane (Hybond N+; Amersham) and hybridization. For PCR amplification of CD40, primers 5′-ATGGTTCGTCTGCCTCTG-3′ (CD40-forward) and 5′-TCACTGTCTCTCCTGCACTGA-3′ (CD40-reverse), which amplify the entire CD40 cDNA, were used. Conditions for 30 cycles of PCR amplification were 94°C for 40 s, 51°C for 45 s, and 72°C for 60 s. Products (5 μl) were analyzed on a 0.8% agarose gel.
rsCD40L induces apoptosis in carcinoma cells.Previous work has demonstrated that membrane-anchored CD40L induces apoptosis in CD40-transfected HeLa cells when de novo protein synthesis is inhibited (31). To assess the effects of CD40 engagement on carcinoma cell death we have used an rsCD40L molecule in which the extracellular domain of CD40L is linked to an isoleucine zipper trimerization motif (52). This soluble trimeric molecule mimics membrane-bound CD40L function and retains full biological activity in resting B cells. HeLa clones stably expressing CD40 (Table1) were treated for 6 h with 1 μg of rsCD40L ml and then cocultured with the protein synthesis inhibitor CHX for an additional 24 h time period. Cell death was quantitated using propidium iodide staining and fluorescence microscopy. These experiments demonstrated that 40 to 50% of HeLa/CD40 cells undergo apoptosis following treatment with rsCD40L and CHX (Fig.1A). The ability of CD40 ligation to transduce apoptotic signals in CD40-transfected HeLa cells was verified by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and electrophoretic DNA fragmentation assays (data not shown). Induction of cell death, although to a lesser degree, was also observed using similar concentrations of an alternative source of monomeric rsCD40L coupled with a cross-linking antibody which allows dimerization of soluble ligand monomers (Fig. 1B).
This phenomenon was not restricted to carcinoma cells expressing exogenous CD40, as MG75 and MG79 ovarian tumor cell lines which naturally express CD40 (Table 1) also responded to rsCD40L treatment by induction of apoptosis (Fig. 1A). This effect on viability was specific for CD40, as a neutralizing anti-CD40L MAb blocked rsCD40L-induced apoptosis in MG79 cells (Fig. 1C). However, CD40-positive A2780 ovarian carcinoma cells were resistant to CD40L-induced cell death even when high amounts of recombinant ligand (5 μg/ml) were used (Fig. 1A). These experiments demonstrate that exposure of some but not all carcinoma cell lines to soluble trimeric CD40L induces apoptosis when de novo protein synthesis is inhibited, a phenomenon which is reminiscent of the effects of anti-Fas treatment on carcinoma cell survival. Indeed, treatment of HeLa/CD40 clone 14 cells with low concentrations (10 ng/ml) of CH11 anti-Fas MAb in the presence of CHX induced cell death in a time-dependent manner, but anti-Fas alone had no effect (Fig. 1D and data not shown). This apoptotic response was more potent and rapid compared to that following CD40 ligation. In addition, treatment of this HeLa/CD40 clone with 1 μg of soluble FasL per ml and 50 μg of CHX per ml for 18 h induced approximately 90% cell death, which is considerably higher than the effect of rsCD40L at the same time point (data not shown). This was not particular to CD40-transfected HeLa cells, as MG79 ovarian carcinomas were also more sensitive to Fas-induced cytotoxicity (Fig. 1E). Furthermore, A2780 cells, which do not respond to CD40 ligation by apoptosis, were found to be susceptible to anti-Fas treatment (Fig.1E). Thus, CD40 transduces a delayed and reduced apoptotic response in carcinoma cells compared to Fas.
A membrane-proximal domain but not a PXQXT motif in the CD40 cytoplasmic tail is critical for CD40L-induced apoptosis in carcinoma cells.To identify the CD40 domains responsible for apoptosis induction, we have generated two mutations in the cytoplasmic tail of CD40, which are known to abrogate TRAF binding and influence signalling and phenotypic changes. Thus, using site-directed mutagenesis, a Thr254→Ala mutation (PXQXT→PXQXA) was introduced in the cytoplasmic tail of CD40 (CD40A). This point mutation is known to abrogate CD40 interaction with TRAF2 and TRAF3 but not TRAF6 (66) and represses at least some of the phenotypic consequences of CD40 stimulation, such as growth inhibition, induction of homotypic cell adhesion, and up-regulation of the costimulatory molecule B7.1 (11, 25, 33). In addition, we have constructed a CD40 deletion mutant lacking aa 216 to 239 [CD40Δ(216-239)], which are critical for interaction with TRAF6 but not TRAF2 (35,66).
HeLa cells stably transfected with a cytomegalovirus promoter-driven CD40A or CD40Δ(216-239) expression vector were obtained [HeLa/CD40A clones 1 and 17 and HeLa/CD40Δ(216-239) clones 8, 9, and 10, respectively], and expression was verified using flow cytometry (Table1) and RT-PCR (Fig. 2A). PCR-amplified CD40 cDNA from HeLa/CD40Δ(216-239) cells demonstrated a slightly higher electrophoretic mobility than the wild type or CD40A transfected clones, consistent with the presence of a 23-aa deletion in its CD40 cytoplasmic tail (Fig. 2A, lane 3). The effect of rsCD40L and CHX treatment on the viability of HeLa/CD40A and HeLa/CD40Δ(216-239) cells was assessed. Interestingly, it was found that HeLa/CD40A clones responded to CD40L-induced cytotoxicity to a similar degree as wild-type CD40-transfected cells (Fig. 2B). However, exposure of HeLa/CD40Δ(216-239) clone 8, 9, or 10 to rsCD40L and CHX failed to induce cell death, suggesting that the TRAF6-interacting domain of CD40 is critical for apoptosis induction (Fig. 2B).
To demonstrate that the Thr254→Ala mutated CD40 is functionally defective, the effects of CD40 engagement on JNK activation in CD40A versus CD40-transfected HeLa cells were examined. For this purpose, two clones which express similar levels of CD40 (HeLa/CD40 clone 14 and HeLa/CD40A clone 17 [Table 1]) were selected. Following CD40 stimulation, cell lysates from these cultures were immunoprecipitated with an anti-JNK1 antibody and assayed for kinase activity using glutathione S-transferase–cJun (aa 1 to 79) as substrate. CD40 ligation was found to induce a delayed and reduced JNK activation in HeLa/CD40A compared to HeLa/CD40 cells (Fig. 2C), in agreement with an important role for TRAF2 in CD40-mediated JNK induction (44, 53). Furthermore, transient expression of an N-terminally deleted dominant-negative TRAF6 inhibited CD40 and CD40A but not CD40Δ(216-239)-mediated JNK activation in HeLa cells, verifying that this CD40 membrane-proximal deletion mutant is functionally inactive with respect to TRAF6 signalling (data not shown). In addition, coimmunoprecipitation experiments in human embryonic kidney 293 cells verified the ability of CD40A to interact with TRAF6 but not TRAF2, while CD40Δ(216-239) was found to strongly associate with TRAF2 but not TRAF6 (data not shown). We therefore conclude that the membrane-proximal domain but not the PXQXT motif of the CD40 cytoplasmic tail is critical for CD40L-induced cell death in carcinoma cells.
CD40 ligation in apoptosis-susceptible carcinoma cells leads to caspase activation.To probe the mechanism of CD40L-induced apoptosis, CD40-expressing carcinoma cells were exposed to rsCD40L and CHX in the presence of zVAD-fmk, a broad-spectrum caspase inhibitor. zVAD-fmk is known to inhibit anti-Fas or TNF-induced activation of caspases 1, 3, and 8, thereby preventing Fas and TNFR-mediated cytotoxicity (63). Interestingly, zVAD-fmk was also found to diminish CD40L-induced cell death in HeLa/CD40 cells as well as in HeLa/CD40A and MG79 cells (Fig. 3A). However, treatment of HeLa/CD40 clone 14 cells with this caspase inhibitor did not influence their ability to engage the JNK pathway in response to CD40 stimulation, suggesting that JNK is not critical for CD40-mediated apoptosis (Fig. 3B).
The cowpox virus-encoded protein crmA is a potent inhibitor of proapoptotic and proinflammatory caspases and also blocks anti-Fas and TNF-induced apoptosis (65). To determine if crmA also suppresses CD40-induced cell death, we constructed a crmA-expressing recombinant adenovirus (RAd-CrmA) and used it to infect MG79 ovarian carcinoma cells. RAd-CrmA infected MG79 cells maintained viability when challenged with rsCD40L in the presence of CHX, but infection with control virus delivering lacZ (RAd35 [70]) did not inhibit CD40L-induced cell death in these cells (Fig. 3C). Importantly, although crmA expression had a pronounced effect on viability, it did not influence the ability of CD40L to induce JNK activation in MG79 cells (Fig. 3D). We therefore conclude that caspase inhibitors block CD40-induced apoptotic but not JNK signals.
To verify that caspase activation occurs during CD40L-induced cell death, we used a colorimetric assay for the measurement of caspase 3 activity. Lysates from rsCD40L- and CHX-treated HeLa/CD40 clone 14, HeLa/CD40A clone 17 and MG79 cells demonstrated a three- to ninefold increase in enzymatic activity compared to untreated controls, which correlated with the extent of apoptosis in these cells (Fig.4A). Treatment with rsCD40L alone had no effect, while CHX induced marginal increases in enzymatic activity (Fig. 4A and data not shown). Exposure of vector-transfected HeLa (HeLa/neo clone 1) or HeLa/CD40Δ(216-239) clone 10 cells to rsCD40L and CHX did not induce caspase 3 activity above background, in agreement with their inability to apoptose in response to CD40 stimulation (Fig. 4A). As a positive control, treatment of HeLa/CD40 clone 14 cells with 10 ng/ml CH11 anti-Fas MAb in the presence of CHX induced a 16-fold induction in caspase 3 activity (Fig. 4B), consistent with the ability of Fas to confer more potent apoptotic signals than CD40 in this cell line (Fig. 1D). These measurements were specific for caspase 3, as peptide inhibitors such as zVAD-fmk and the more selective Ac-DEVD-CHO blocked caspase 3 activity in lysates from rsCD40L- and CHX-treated HeLa/CD40 cells (Fig. 4C).
We then investigated the effects of CD40 ligation on the activity of caspase 8, which functions upstream of caspase 3 in the Fas death pathway. Immunoblot analysis using an antibody specific for the active, cleaved form of caspase 8 (p18) demonstrated that treatment of HeLa/CD40A clone 17 cells with rsCD40L and CHX induced a reduction in pro-caspase 8 levels and formation of the fast-migrating p18 cleaved form (Fig. 4D, lanes 3 and 4). This caspase 8 activity was absent in lysates from CHX-treated cultures and was diminished in the presence of zVAD-fmk (Fig. 4D, lanes 2 and 5 respectively). The contribution of caspase 3 and caspase 8 activation to CD40-mediated apoptosis was verified by the ability of peptide inhibitors which specifically target these caspases, such as zDEVD-fmk and zIETD-fmk, respectively, to inhibit cell death in response to CD40L and CHX treatment (data not shown). Overall, these data provide the first demonstration that CD40-mediated apoptosis involves a crmA-sensitive, caspase 3- and caspase 8-dependent pathway.
CD40 stimulation induces the expression of Fas and cytotoxic ligands of the TNF superfamily in apoptosis-susceptible carcinoma cells.The similarities between Fas and CD40-activated cell death pathways, coupled with the lack of a death domain sequence in the cytoplasmic tail of CD40, suggest that CD40-mediated apoptosis occurs through an indirect mechanism which may involve the Fas pathway. To examine this possibility, the effects of CD40 ligation on Fas expression in carcinoma cells were first assessed using flow cytometry. MG75, MG79, and HeLa/CD40 cells treated for 24 h with rsCD40L (1 μg/ml) demonstrated a small but significant and consistent increase in Fas cell surface expression (Table 1). Significant levels of Fas induction were also observed in HeLa cells carrying a Thr254→Ala mutation in the cytoplasmic tail of CD40 but not in HeLa/CD40Δ(216-239) cells (Table 1). However, treatment of HeLa/CD40Δ(216-239) cells with rsCD40L induced the expression of the cell surface marker ICAM1 (CD54, data not shown), suggesting that deletion of the membrane-proximal domain of CD40 may affect some but not all the phenotypic effects of CD40 stimulation. In addition, A2780 cells, which do not respond to CD40 stimulation by apoptosis, failed to induce Fas expression even when prolonged incubations with rsCD40L (36 or 48 h) were performed (Table 1 and data not shown).
We then investigated the effects of CD40 ligation on FasL expression. For this purpose, RNA isolated from control untreated HeLa/CD40A clone 17 or from cells exposed to rsCD40L (1 μg/ml) for 2 or 12 h was subjected to semiquantitative RT-PCR using primers specific for FasL. RNA expression levels were normalized to those of GAPDH. As shown in Fig. 5A, CD40 ligation induced an increase in FasL expression levels in these cells, which was evident at 2 h and sustained at 12 h of rsCD40L treatment. Importantly, CD40 ligation was also able to induce the expression of other cytotoxic members of the TNF superfamily, such as TNF and TRAIL (Apo-2L). Strong induction of TNF RNA was observed as early as 2 h following CD40 stimulation, in agreement with a recent report (27), and levels then declined. The kinetics of TRAIL up-regulation was relatively slow, with only low levels of induction observed at 2 h and higher levels at 12 h of treatment (Fig. 5A). However, unlike the effects of rsCD40L on FasL, TNF, and TRAIL expression, CD40 engagement did not affect CD40L RNA, confirming the specificity of the observed phenomena (Fig. 5A).
Interestingly, exposure of HeLa/CD40Δ(216-239) clone 10 or A2780 cells to rsCD40L failed to induce FasL expression (Fig. 5B and data not shown). In addition, while significant up-regulation of TRAIL RNA was observed in HeLa clones carrying wild-type or Thr254→Ala mutated CD40, CD40 ligation in HeLa/CD40Δ(216-239) clone 8 or clone 10 cells had no effect (Fig. 5C). We therefore conclude that CD40 stimulation induces the expression of Fas and cytotoxic ligands of the TNF superfamily in apoptosis-susceptible carcinoma cells.
Involvement of Fas or FasL and other cytotoxic ligands in CD40-mediated cell death in carcinoma cells.To demonstrate that the observed CD40-mediated induction of Fas and FasL expression is functional and contributes to CD40L-induced apoptosis, HeLa/CD40 clone 14 cells were incubated with the neutralizing anti-FasL MAb NOK1 in the presence of rsCD40L and then exposed to CHX before cell death was assessed. It was found that NOK1 was able to suppress CD40-mediated apoptosis by approximately 30%, and similar results were obtained with the HeLa/CD40A clone 17 cell line (Fig.6A). Treatment with OX34 anti-CD2 isotype control antibody had no effect, confirming the specificity of the observed phenomenon (data not shown).
As TRAIL is also induced in apoptosis-susceptible carcinoma cells following CD40 stimulation, we first examined whether CD40-expressing HeLa cells are responsive to the cytotoxic activity of this ligand. We have found that treatment of HeLa/CD40A clone 17 cells with soluble TRAIL, coupled with a cross-linking reagent (Alexis Corporation), induced a concentration-dependent apoptotic effect (Fig. 6B). This was specific for TRAIL, as it was inactivated in the presence of TRAILR1:Fc, which acts as a soluble decoy receptor. We then examined the ability of the TRAILR1:Fc hybrid molecule to neutralize the proapoptotic ability of CD40 ligation and found that treatment of HeLa/CD40A clone 17 cells with TRAILR1:Fc had only a small but consistent inhibitory effect (15 to 20%) on CD40L-induced cell death (Fig. 6C), a phenomenon which could be probably attributed to the slower kinetics of TRAIL induction compared to those of TNF or FasL. Recent work has demonstrated that neutralizing anti-TNF antibodies inhibit CD40- as well as TNFR2- and CD30-mediated cell death (27). We have verified the ability of neutralizing anti-TNF to partially inhibit CD40L-induced apoptosis (Fig. 6D) and in addition, we have found that this effect can be augmented by anti-FasL MAb treatment. Thus, while NOK1 or anti-TNF alone induced only a partial (30 to 40%) decrease in CD40-mediated apoptosis, combination treatment inhibited this effect by approximately 60 to 65% and was further enhanced in the presence of TRAILR1:Fc (Fig. 6E). These data verify that even in the presence of CHX, CD40 ligation is able to induce up-regulation of cell surface expression of cytotoxic ligands. This may be accounted for by translation of these molecules during the 6-h CD40L incubation period prior to CHX treatment and/or by the presence of preexisting pools of the cytotoxic ligands.
FasL and TNF up-regulation occurs rapidly upon CD40 ligation, suggesting direct transcriptional control. To verify that the ability of CD40 to regulate the expression of FasL and TNF-α is not the result of an autocrine or paracrine cascade involving these ligands and their receptors, HeLa/CD40A clone 17 cells were treated for 2 h with rsCD40L (1 μg/ml) in the presence or absence of NOK1 (1 μg/ml) or anti-TNFα antibody (0.5 μg/ml), and extracted RNA was subjected to RT-PCR for FasL, TNF, or GAPDH expression. As shown in Fig.7A, inhibition of FasL did not influence the ability of CD40 ligation to activate TNF RNA, and conversely, neutralization of TNF had no effect on CD40-mediated induction of FasL. Furthermore, when CD40-expressing HeLa cells were treated for 12 h with rsCD40L in the presence of anti-FasL or anti-TNF antibody, induction of TRAIL also remained unaffected (Fig. 7B). Therefore, CD40 ligation can independently induce the expression of FasL, TNF, and TRAIL. This observation is further reinforced by the inability of TNF treatment to up-regulate FasL or TRAIL expression. Indeed, when HeLa/CD40 clone 14 or HeLa/CD40A clone 17 cells were exposed for 2 or 12 h to 80 ng of recombinant human TNF-α per ml, TNF mRNA was rapidly activated, in agreement with a recent report (27), but no induction of FasL or TRAIL expression was observed (data not shown).
TNFR family members convey signals leading to the regulation of diverse cellular responses, ranging from proliferation and differentiation to growth suppression and apoptosis (12, 46,64). Among these receptors, TNFR1, Fas, TRAIL-R1, TRAIL-R2, and DR3 share death domain homology in their cytoplasmic tails, through which they transduce apoptotic signals. Paradoxically, other members of the TNFR superfamily which lack the death domain in their cytoplasmic regions, such as TNFR2, CD30, and CD40, have also been reported to suppress growth and survival in a number of carcinoma cell lines (15, 27, 31, 67, 75).
In this study, we have investigated the mechanism by which CD40 induces cell death in carcinomas. For this purpose, we have exposed human carcinoma cell lines to recombinant soluble forms of CD40L and found that induction of cell death depends on the oligomerization status of CD40L. Thus, treatment with CD40L monomers had no effect on survival, but apoptosis was induced following antibody-induced monomer cross-linking, which leads to ligand dimerization. Cell death was even more pronounced following treatment with trimeric rsCD40L (Fig. 1A and B), a phenomenon which probably reflects differences in the efficacy of these molecules to aggregate CD40 (20). Consistent with our findings, previous studies, including the recent identification of the crystal structure of the CD40-TRAF2 complex, have emphasized the significance of ligand-mediated trimerization for efficient CD40 signalling (7, 20, 50). While oligomerization of CD40 is necessary for transduction of signals which activate the cell death machinery in carcinoma cells, its execution also requires inhibition of protein synthesis by CHX, in common with the effects of anti-Fas or TNF treatment in carcinoma cell lines (9, 51). It is possible that CHX blocks the production of protective antiapoptotic proteins, thereby unmasking the cytotoxic potential of CD40 activation. Indeed, the interactions of CD40 with its ligand have been shown to induce the transcriptional up-regulation of a number of negative regulators of cell death, such as Bcl-xL, Bfl1, and A20 (10, 42, 62).
The requirement of protein synthesis inhibition for efficient killing also unveils a mechanism by which tumor cells, through the activation of antiapoptotic programs such as the reported constitutive activation of phosphatidyl inositol 3-kinase–Akt and Bcl-2 overexpression in a subset of ovarian tumors (8, 17), may escape CD40-mediated cytotoxicity. While the ability of these pathways to block CD40-mediated cell death remains to be verified, previous work has implicated Bcl-2, Bcl-xL, and Akt in suppression of TNF- and Fas-induced apoptosis in certain cell types (5, 36, 40, 63). Furthermore, CD40 is absent in a proportion of tumors of the breast and cervix as well as in a number of tumor cell lines, such as the cervical HeLa and ME180, the MCF7 breast, and the 2780CP ovarian carcinoma cell lines, suggesting possible selection for CD40-negative cells (15, 32; Eliopoulos and Young, unpublished observations). An alternative mechanism of resistance to CD40-mediated cell death may occur through disruption of CD40L-induced signals. This is exemplified by the inability of A2780 ovarian carcinoma cells to respond to CD40-mediated cell death (Fig. 1A). In these cells, NF-κB but not JNK activation in response to CD40 stimulation appears to be impaired (N. J. Gallagher, A. G. Eliopoulos, et al., unpublished data). This is not peculiar to A2780 cells, as CD40 ligation fails to induce NF-κB-dependent transcription in Hodgkin's cell lines (71) and similar deficiencies have been identified in a mouse pre-B-cell line (13). While NF-κB has been implicated in the generation of protective responses against TNF- and drug-induced cell death (68, 69), its contribution as a proapoptotic signal has also been noted (3). Indeed, anticancer drugs are known to induce FasL, as well as Fas expression, through a mechanism which critically involves activation of NF-κB (38). In addition, inhibition of NF-κB by a constitutively active IκBα has been recently shown to suppress phorbol myristate acetate- and ionomycin-induced FasL expression and apoptosis in Jurkat T cells (45), and NF-κB is a positive regulator of serum withdrawal-induced apoptosis in 293 cells (28). Interestingly, while A2780 cells do not undergo apoptosis in response to CD40 ligation, we have previously shown that their long-term exposure to CD40L in the absence of CHX leads to growth inhibition (15). This phenomenon is reminiscent of the antiproliferative properties of CD40 ligation in Burkitt's lymphoma cell lines, in the absence of an effect on viability (2). Therefore, CD40 engagement in tumor cells may activate two distinct pathways, leading to inhibition of proliferation or induction of cell death. The signalling cascades which regulate these CD40 pathways will be an interesting area for future studies.
Mutational analysis of the CD40 cytoplasmic tail demonstrated that the TRAF2- and TRAF3-interacting PXQXT motif, a major CD40 signalling effector site, is not critical for induction of apoptosis but death signals are transduced through its membrane-proximal domain. This region binds TRAF6, which is a known regulator of NF-κB, JNK, and ERK signals by CD40 (35, 37). While a role for TRAF6 in modulating cell death has not been described, TRAF6 but not TRAF2 or TRAF3 interacts with the NF-κB and apoptosis-inducing protein RIP2 (48). The contribution of TRAF6 and RIP2 to CD40-mediated cytotoxicity is currently under investigation. Intriguingly, CD40 signals generated from its membrane-proximal, TRAF6-interacting domain appear to be qualitatively different from those engaged by the PXQXT motif. Thus, ERK activation by the membrane-proximal region is Ras independent, whereas that by the PXQXT is Ras dependent (37), and recent evidence suggests differential regulation of NF-κB by these two CD40 domains (66). Furthermore, JAK or STAT signalling is engaged exclusively from the membrane-proximal region (29), and our work provides further evidence for differential signalling emanating from these two domains.
The ability of CD40 ligation to confer a reduced and delayed apoptotic response compared to Fas stimulation, coupled with previous reports demonstrating a synergistic role for CD40 in anti-Fas-induced cytotoxicity (15, 23, 59), prompted us to investigate the possibility that CD40-mediated apoptosis in carcinoma cells occurs indirectly via a mechanism involving Fas and/or its ligand. Three pieces of evidence corroborate this hypothesis. Firstly, we have shown that CD40L-induced cell death occurs through a crmA-sensitive, caspase-dependent pathway (Fig. 3 and 4), in keeping with the ability of Fas to engage a proapoptotic cascade that leads to caspase activation and is suppressed by crmA (63, 65). Furthermore, we have found that CD40 ligation induces the expression of Fas and FasL in apoptosis-susceptible cell lines (Fig. 5). Finally, we have demonstrated that CD40L-induced apoptosis in carcinoma cell lines is partially inhibited by reagents which neutralize FasL (Fig. 6).
The inability of neutralizing anti-FasL antibodies to completely abolish CD40-mediated cytotoxicity implies the contribution of additional death signals. Indeed, exposure of HeLa/CD40 cells to rsCD40L was also found to mediate transcriptional activation of other cytotoxic members of the TNF family, such as TRAIL (Apo-2L) and TNF, although with different kinetics. Consequently, we have found that a neutralizing anti-TNF antibody (27) or a soluble TRAILR1:Fc partially protects against CD40-mediated cell death and that combination treatment with these neutralizing reagents has an additive effect on the survival of CD40L-treated cells. Therefore, CD40 ligation may induce apoptosis in susceptible carcinoma cell lines via an indirect pathway targeting more than one cytotoxic ligand of the TNF family. Whether CD40 ligation regulates the redistribution of intracellular cytoplasmic pools of these cytotoxic ligands, in addition to their de novo transcription and translation remains to be elucidated. Interestingly, our preliminary results indicate the presence of a preexisting cytoplasmic FasL pool in HeLa/CD40 cells which is significantly enriched following treatment with rsCD40L. This is consistent with previous studies in human carcinoma cells, monocytes, and T cells demonstrating the presence of high intracellular levels of FasL or TRAIL which rapidly translocate to the cell surface in response to various stimuli (41, 47). These observations may explain the ability of neutralizing FasL or TRAIL to suppress CD40L-induced apoptosis in carcinoma cells even in the absence of de novo protein synthesis.
The function of many TNF/TNFR family members appears to be tightly controlled in vivo partly through regulation of their expression. For example, Fas and CD40 are widely expressed, but their ligands are restricted to activated T cells and sites of immune privilege. Conversely, TWEAK is expressed in a number of tissues, but its receptor, DR3, is found only in lymphoid cells. The restricted expression of CD40L in vivo, coupled with its antiproliferative and preapoptotic properties when applied as a soluble form, makes it a suitable candidate for tumour therapy. This is supported by the ability of CD40 ligation alone to reduce growth and survival in early-passage ovarian carcinoma cells cultured in vitro (our unpublished observations) and by a recent study demonstrating significant breast tumor regression and apoptosis in xeno-transplanted SCID mice treated with rsCD40L (32). While the mechanism of CD40-mediated carcinoma cell death in vivo is currently unknown, it is likely to involve activation of FasL and/or other death receptor ligands. In addition to regulating CD40-mediated cytotoxicity, these ligands and/or their receptors are also important in apoptosis induced by a broad spectrum of stimuli, including chemotherapy, radiation, ectopic c-myc expression, and anoikis (21, 34, 60), further emphasizing their extensive and central role in programmed cell death.
We are grateful to Immunex Corporation for the gift of soluble trimeric CD40L and to Elliot Kieff and Jun-Ichiro Inoue for providing us with plasmids.
This work was generously supported by the Cancer Research Campaign, United Kingdom, grant SP2091/0501. A.G.E. is a Medical Research Council (United Kingdom) research fellow.
- Received 15 December 1999.
- Returned for modification 8 February 2000.
- Accepted 8 May 2000.
- Copyright © 2000 American Society for Microbiology


text: Lucius Mimic List (50ss) Look, Mimics. Most of the crew sticks around Lucius, to empower Mr. Graves and Doppelganger to beat face and cripple the opponent's ability to hit back, while Vogel runs schemes and eats other runners. Candy (and the Terracotta Warrior by shunting damage) keeps the main group healthy, and acts as a layer of protection for the Lucius command bubble with her control abilities. Doppelganger will just need to stick close to The Scribe for defense. Terracotta Warrior and Mr. Tannen share th Masquer February 1, 2018 7

subset: commoncrawl


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Efficiency and safety to store liquefied gases.
Made to store gases in liquid phase under low pressure, cryogenic tanks must ensure constant low temperatures to keep evaporation of the product to a minimum. There are a number of important factors to consider in their design, including use of proper thermal insulation techniques, knowledge of materials and their behavior under thermal gradient and application of the best control systems. Optimal tank operation and performance depends on all these elements.
In this sense Maraldi is able to ensure design experience and precision in the construction phase of single or double-walled tanks for containing ammonia, oxygen, nitrogen, ethylene, ethane, propane and other hydrocarbons.
Double-wall cryogenic tank, Plock (Poland).
Erection of dome roof.
32,000 m3 ethylene tank, Bandar Assaluye (Iran).


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I spent time this weekend playing in my parents' garden with my little niece. She's four and a half, full of energy and apparently fearless. She loved rolling down the slope where my brother and I used to do the same thing several decades ago. She revelled in doing a run-up to perform multiple forward... Continue Reading →
80 years
Numbers again. This time celebrating a birthday -- my mum's 80th. Not that she's counting. She says this feels ridiculous, that she simply can't believe she's 80. And she doesn't look it, I tell her, and I mean it. We zipped down recently to spend a weekend with her and my dad. Time always flies... Continue Reading →
Deep compassion
I've been avoiding someone from my past life for a while now. They hurt me a lot, in ways I'm only just coming to recognise, let along come to terms with. For the past few months these memories, long suppressed, have come out in my yoga practice. That's my laboratory and place of experimentation. How... Continue Reading →
Yoga with the dentist
So I've finally had my troublesome tooth taken out. It's been grumbling for a long time and I consulted with various experts to see if it could be saved... Turns out not. So facing one of my greatest fears, I went for an extraction. The last time I had a tooth out it was agonising,... Continue Reading →
Phoenix rising
Again a quick word from my TT whirlwind, to capture a moment, although I am too tired for careful words. Stay with me, my beloved readers. At the end of the day on my mat, a circle of yoginis around me, I don't know how many. My eyes were blinded by tears and I was hanging... Continue Reading →


text:
Offering exclusive content not available on Pornhub. Please Sign In.
You can register for the site in one easy step and you can start a gay chat as soon as you've registered. By becoming a Fan, you are supporting this model to continue creating amazing content and you may even get additional exclusive content that would be listed below. Fucking my little brother on his 18th birthday
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Job type: Full-time
Experience level: Mid-Level, Senior
Role: Backend Engineer
Industry: Mobile music apps
Company type: Startup
Are you interested in working for a dynamic fast growing company?
Deplike is developing a mobile app that replaces complex amplifiers and effects processors of guitar players. Those equipment are very heavy, expensive and not portable. Unlike them, using our app, guitar players can use their mobile phones to create the same effects, everywhere anytime. Also, we've reached almost one million guitar player and mostly from the US.
Deplike is seeking an experienced Backend Engineer to join our growing team. Means, you will work closely with developers, strategists, and project manager to solve music world problems for our users through innovative product design.
You will make a direct impact on a variety of interesting projects, drive change and improve processes. We embrace big picture thinkers who also enjoy the finer details of day-to-day activities. You will be given the opportunity to apply your abilities to meaningful work. If you're the type of person who says: "I have a better way to do this", then we want to hear it. Join a world-class engineering-driven company which is located ODTU Teknokent and moving its to HQ to in London. So, you'll have an instant impact from day one. Deplike has an open culture and flat organization.
Who you are:
- A perceptive, patient, and hardworking self-starter who loves working on collaborative delivery teams.
- A positive team player who thrives in a fast-paced, ever-changing environment.
- A lifelong learner who craves constructive feedback.
- Sensitive to long-term project goals and business objectives.
- Considerate of budgets, timelines, deadlines, and external constraints.
Primary Responsibilities:
- Continuously develop and expand APIs for the platform.
- Architect a scalable backend with high-concurrency, low-latency, and multi-threaded to support our products.
- Collaborate with other Engineers on code reviews, infrastructure, and process enhancements.
- Take the existing product and work with team members to redesign/refactor/develop the product.
- 3+ years of application development with Python, Java, Erlang, Go etc.
- Strong experience in database management.
- Industry experience at a large-scale internet service.
- B.S. in Computer Science or related engineering field.
- Backend architecture experience.
- Knowledge and experience building mobile applications.
- Experience building on cloud platforms such as AWS.
- Go-Get'er attitude and a team player mentality.
Join Our Team
If you want to apply to this position, please contact us through email@example.com and submit your resume


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LAWYER: Here's How Trump's Legal Team is Likely Preparing Him for 'Mueller Showdown'
When asked if he expects to sit down for an interview with special counsel Robert Mueller, President Trump told Fox News reporter John Roberts that he believes the event is "unlikely," to occur. However, just because Trump doesn't foresee the interview taking place, it doesn't mean the special counsel won't pull a fast one and request a one-on-one with the President.
Remember, we are talking about Robert Mueller. Trump's legal team has discussed the prospect of the meeting and according to lawyer Bradley P. Moss, here's how they are likely preparing for the 'Mueller showdown.'
Moss writes in POLITICO Magazine:
…[T]he president's lawyers must prepare him to address any number of potential topics, and also thoroughly coach him on how he will talk about them. They'll do this with mock interviews. A standard mock session could consist of multiple lawyers jumping in with questions at different times, placing documents in front of Trump and seeking to trip him up on the facts. It would be advisable—although it is debatable whether the president would agree to it—for several mock interview sessions to be conducted before the president sits down with Mueller. […]
He needs to be able to recall with a reasonable measure of clarity what (if anything) he knew about the June 2016 Trump Tower meeting between Donald Trump Jr. and a Russian lawyer, when he learned of it and the context of his involvement in crafting the official White House response when the media started reporting on the meeting in July 2017. […]
They need to make sure he is able to stick to the proverbial script when pressed. […] [Trump] naturally relies upon simple verbiage and is not prone to getting caught up in complicated or nuanced explanations. That's good. He should keep it simple.
As The Gateway Pundit's Cristina Laila reported, President Trump's legal team is seeking to avoid a sit-down interview with Robert Mueller and may request a written affidavit instead, declaring he did not collude with the Russians during the 2016 election.
According to the report, Justice Department veterans doubt Mueller will accept a written response or an affidavit in lieu of a sit-down interview with Trump.
Chuck Rosenberg, former U.S. attorney for the Eastern District of Virginia and chief of staff to then-FBI Director Comey, told NBC News that "Prosecutors want to see and hear folks in person. They want to probe and follow up. Body language and tone are important."
Only time will tell if Mueller will request an interview with President Trump.


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Lýtingsstaðir Cottages
Just 20 km away from Varmahlíð on road #752 you will find our peaceful farm where we share our home with the Icelandic horse.
Lýtingsstaðir offers accommodation in three cozy farm cottages with self-cooking facilities and private bathroom. Two of the cottages are ideal for a family and can fit up to 5 people. One cottage fits for 6 people in two rooms. Equipped kitchen, TV, coal barbecue and deck furniture are provided in all cottages. Available all year around.
You´ll be at the heart of Iceland's nature, take a ride or walk, watch horses, sheep and other animals. Relax to the sound of the horses hoof and explore the valleys and breathtaking mountains.
Horse riding tours for beginners and experienced riders all year around.
On Lýtingsstaðir you find THE OLD STABLE, which is a built from turf and let you experience how horses where kept in farms in the old days.
Lýtingsstaðir is a fully licensed riding holiday company, operating since the year 2000, welcoming you with warm hospitality and a personal atmosphere.


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- About
- Academics
- Outreach & Training
- Facilities
- Research
- People
- Beaver Classic™ Cheese
- FST in Action!
- Alumni and Friends
- COVID-19 Resources
Dave Takush
BS 2007, Fermentation Science
MS 2009, Food Science
Co-owner & Cider Maker
2 Towns Ciderhouse
Every day at 2 Towns Ciderhouse we focus on producing quality local craft cider. Most days are spent monitoring fermentations: checking brix levels and evaluating taste and aroma attributes. We spend a good chunk of time doing sensory evaluation and barrel work, but most of our time is spent cleaning tanks and bottling cider!
Coming up with a new style of cider and bringing it from a crazy idea to reality. Seeing your pet projects actually hit the shelf, and having the freedom to do so is the best part of this job. Having the resources and work environment that allows us to push the bounds of what cider is, that is always fun too.
Fermentation is the best blend of science and art. I have always wanted to become a scientist but I always loved cooking and working with things you can smell, taste, and drink. I started my career home-brewing, and eventually got a job in a brewery. I then moved on to winemaking and when I had the opportunity to become a cidermaker, I jumped at the chance. My first batch of hard cider was a 10 gallon batch that I pressed on my parent's farm when I was still an undergrad at OSU. We still have that old press hanging out in the tasting room at the Ciderhouse.
I really believe that a firm understanding of the science behind fermentation is important to succeed in today's industry. Not only has my education at OSU helped me excel in my current job as a cidermaker, but it exposed me to new techniques, technologies, and career opportunities along the way.
Finding new and unique apple varieties is a really fun process. Usually it means visiting an orchard or grower and trying a bunch of different apples off of the tree. Just like winemakers searching for the best grape cultivars, we search for specific old world French and English cider varieties that have the best taste and aroma properties for traditional hard cider. We actually seek out apples that contain high levels of acid, tannins, and other phenolic compounds. I spend a lot of time looking at how to blend these apples after fermentation, and how much barrel aging they should see.
Do you have any advice for people starting out in the craft fermentation world?
Find your passion and follow it. You spend too much time working to not love what you do. Every day I think, "If I didn't have a job, this is what I would be doing anyway."


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Yael Cohen
The brazenly defiant name Yael Cohen gave her organization perfectly encapsulates her conviction that most cancers, when caught early, are curable. Fuck Cancer pushes young people to get their parents screened early and often to detect cancer before it's too late.
Learn More
- letsfcancer.com
- Twitter: MsFuckCancer
- Twitter: letsfcancer
- Facebook: letsfcancer
- Fast Company: Most Creative People
- TEDxVancouver: Yael Cohen
Similar SIRs
"Cancer's no longer a fatal disease, it's a chronic disease – and that's a pretty exciting thought."
Yael Cohen has "built a platform of early detection" at Fuck Cancer using "new technology, humor and celebrity." As a result, Yael says, "We've gotten kids talking about something that's not only uncomfortable but just really not in their realm of our day-to-day discussion. It's a conversation that we've been lacking for far too long." But opening that dialogue can be a transformative moment where the participants are, "uniquely positioned to make a huge and real change." In addition, "We're layering on prevention and communication and really rounding out this unbelievable community and building assets for our supporters — that aren't campaigns. They can have longevity."
It's surprising that the fact that "Ninety percent of cancers are curable in stage one" isn't common knowledge — yet. But for Yael, learning that piece of information "fundamentally shifted my perspective." The ability to be "actively looking for it and finding it when it's most curable — that was a really positive thought. It was really exciting to learn there's so much I can control when it comes to cancer."
Yael expresses excitement that the technology today can have such an immediate effect, stating that, "for the first time in history, our generation can change their world with a few clicks of the mouse — they can send a message around the world in a matter of minutes." That power has "the potential to be harnessed or tapped into when it comes to a huge issue like cancer."
The organization grew from a T-shirt emblazoned with the phrase Fuck Cancer that Yael had created for her mom and herself. She says, "I wouldn't want her to have to go through that alone." The T-shirts resonated with a lot of cancer survivors, so much so that it eventually grew into a small business. Not long after, Yael expanded the business into something even more useful to her community. She says, "The resources that I wish we'd had — I want to build those so that other people don't have to look for them." With a fresh round of supporters and funding, Yael anticipates this to be one of their best years, expanding those resources and their reach.


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Today Labour launched their Manifesto for the 2010 General Election.
Labour provided us with a late (and, frankly, slightly unexpected) lurch towards the post-bureaucratic agenda in recent months. Data.gov.uk, digital inclusion, and Brown's 'digital economy' speech have largely outweighed the Digital Economy Bill saga, and positioned Labour as a nascent post-bureaucratic party.
Their manifesto also contains a few hints in right direction. First, as William Heath noted a small step in the direction of Vendor Relationship Management (VRM) over at MyDex:
"We will explore how to give citizens direct access to the data held on them by public agencies, so that people can use and control their own personal data in their interaction with service providers."
There were a number of other very positive soundbites for the PBA agenda, even if they were light on detail. A post-bureaucratic agenda should be adaptive, flexible and localised, so a lack of detail isn't of itself 'wrong'. How far you believe any government will act in pursuit of laudable aims is subject to personal interpretation.
Bits we liked:
"We will now publish a Domesday Book of all non-personal datasets held by gov't…with a default assumption that these will be made public" This is very similar to the the Tory position, but does not go so far as our call for a Freedom of Data Act.
"Citizens should be able to compare local services, demand improvements, choose between providers, and hold government to account." This is absolutely fundamental to moving government into the post-bureaucratic age. It is interesting that Labour have chosen to frame the policy in terms of competition and accountability, when they might just have easily pointed at the PBA's potential to empower people to combine local services and develop the role of the third sector – rather than just extracting the maximum value from suppliers.
"We will open up government, embedding access to information and data into the very fabric of public services." This is perhaps the most encouraging language we have seen from Labour that they have come to terms with what the PBA really means (or could really mean) for government. The phrase "embedding access to information and data into the very fabric of public services" is an elegant explanation of the first step of post-bureaucratic government.
Bit we didn't like:
"We will update the intellectual property framework that is crucial to the creative industries and take further action to tackle online piracy." You can almost hear the record industry's lobbyists' whispers in this line. The Digital Economy Bill fiasco was bad enough, but to validate and then threaten to build upon it suggests that Labour may only support an agenda as far as a union or vested interest will let them. Unsavoury.
"Digital government also demands digital inclusion. Sowe will build on our network of UK Online centres and public libraries to spread free internet access points within the community, and develop new incentives for users to switch to online services." The single occurrence of 'digitial inclusion' within the 76-page manifesto; no commitment to spending; no suggestion about the kind of 'incentives' that might be used. This simply does not stack up for a party that also wants to "save money for taxpayers as we switch services over to digital-only delivery". The 'digitial divide' is very, very real, and Labour have moved digital inclusion into the 'nice-to-have' category. Internet access is now a fundamental utility, without which a child growing up the UK will suffer. As usual, the poorest and least-educated (who need and could benefit from it most) are in danger of being left behind.
Ali Unwin ( @aliunwin)


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We have two different types of bungalows: for groups of 4 or 5 people. They are all fully equipped with kitchen utensils, dishes, cutlery, heating, bed linen, towels, TV, microwave, fridge...
Pets are allowed, but they must be kept on a leash and supervised by their owners at all times. Pets must never be left alone in the bungalow. Pets will not be allowed when booking a bungalow with the special offer for two people. We welcome you to come along and check out our campsite. We will be delighted to show you our bungalows and all of the services and facilities we can offer you.
This is the perfect place to spend time in the countryside, but with every convenience a city has to offer.
Rúa Vintecinco de Xullo, 35
15704 Santiago de Compostela (A CORUÑA – ESPAÑA)
Teléfono: (+34) 981 580 266 | Fax: (+34) 981 580 266
e-mail: email@example.com


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Terrier Dog Cufflinksby FrenchCuffed
Collection | Animals Cufflinks
At FrenchCuffed we just want to make affordable cufflinks that will make our customers proud and these Terrier Dog Cufflinks are sure to bring a crazy envy from anyone that gets the chance to see them. These Terrier Dog Cufflinks are categorically cool and the black and white face of these cufflinks will add something different to any french cuffed shirt. These cufflinks are sensational as a Secret Santa for a loving husband, whether that be for a entire bridal party, something a touch corporate or for no specific reason at all.
If you are a fan of a man's best friend, dogs, or simply terriers, what could be more perfect? At the end of the day, these unique black and white cufflinks make your french cuff a show stopper, which sure is better than cufflinks that blend into the background.
Make ANY Changes
All of our FrenchCuffed cufflinks can be made to order - which means that if you would like a different background color, or a slightly different image, or have any other questions / changes - please reach out to us and we can tailor you the perfect cufflink email@example.com.
Customer Testimonials
With thousands of happy customers from all over the globe, we wanted to share their comments!


text: We hold expertise in manufacturing and exporting high quality range of Level Sensor. In accordance with set industry norms, this is manufactured using quality approved materials and pioneering technology. It is basically a device used for determining the level or amount of fluids, liquids or other substances that flow in an open or closed system. It detects the level of liquids and other fluids and fluidized solids, including slurries, granular materials, and powders. This Level Sensor is made available at market leading rates.


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Please come play in Ann Arbor, Michigan at the Ark...or anywhere near here. Moved here from Dallas and miss you guys at Lee Harvey's. They would love you here.
We would love to play for the people of Ann Arbor. Only problem, at this time, is you're probably one of the only folks in the area that know about us. Hopefully, in the near future, we will have enough friends in Ann Arbor to make it possible for us to come up there.
Have a great day,
Tejas Brothers
Hi Tejas Brothers. I am John's cousin Cora. Had a great time at your Gun Barrel City Show. Hope you come back someday. I have one request. I love Elvis and maybe someday you could include one of his songs.Tejas Brothers responded on 05/26/2013 Next question
where can I buy the CD's that have I Just Can't Sleep, Boogie Woogie Mammacita? Thanks... Like your sound!Tejas Brothers responded on 06/06/2013


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CALIFORNIA – The Florin Resource Conservation District (FRCD), which governs the Elk Grove Water District, faces an uncertain future…Board members instead voted, 4-1, to dedicate their entire budget to their water service. Activities not related to water services such as conservation, community outreach, and public will be cut from the budget.
Source: Water district officials face budget challenge | News | egcitizen.com
Categories: District News


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Wheat Flour
There are several varieties of wheat produced in order to meet the requirements of food processors and wheat mills. The companies carefully select the wheat class to come up with wheat flour for processing wheat foods & products, as well as selling to grocery stores, super markets, homes, hotels and restaurants. The whole wheat flour is considered as the most healthiest variety as it comprises vitamin B rich bran, healthy fats and other vitamins rich germ, and proteins & vitamins rich endosperm. In total, whole wheat flour is a rich source of minerals, vitamins, fiber and antioxidants. Aashirvaad is a famous brand in Indian market. Our company supplies wheat flour of this brand in two different varieties.
L N STORE All Rights Reserved.(Terms of Use)
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When it comes to measuring a credit union's financial stability, capital is valuable. It acts as a cushion against losses and as protection for members when a credit union is experiencing economic instability. When looking at safety and soundness, the higher the capital, the better.
COMMUNITY HEALTHCARE finished below the national average of 15.65 on our test to measure capital adequacy, racking up 6 out of a possible 30 points.
COMMUNITY HEALTHCARE's capitalization ratio of 6.00 percent in our test was worse than the average for all credit unions, an indication that it's on less solid financial footing than its peers.
In this test, Bankrate tries to estimate the effect of problem assets, such as past-due loans, on the credit union's capitalization and allocated loan loss reserves.
Having lots of these kinds of assets could eventually force a credit union to use capital to absorb losses, decreasing its equity cushion. Many of those assets are also likely to be in non-accrual status and no longer earning money, resulting in lower earnings and potentially more risk of a failure in the future.
COMMUNITY HEALTHCARE scored above the national average of 38.09 on Bankrate's test of asset quality, racking up 40 out of a possible 40 points .
A below-average ratio of problem assets of 0.00 percent in our test was potentially indicative of greater financial strength than other credit unions.
A credit union's earnings performance has an effect on its safety and soundness. Earnings can be retained by the credit union, giving a boost to its capital cushion, or be used to deal with problematic loans, likely making the credit union better prepared to withstand economic trouble. Losses, on the other hand, diminish a credit union's ability to do those things.
COMMUNITY HEALTHCARE received below-average marks on Bankrate's test of earnings, achieving a score of 10 out of a possible 30.
COMMUNITY HEALTHCARE had an earnings ratio of 0.00 percent in our test, better than the average for all credit unions, a sign that it's doing better than its peers in this area.
Bankrate.com's Safe & Sound Ratings provide a star rating system to evaluate the current financial status of financial institutions. The information gathered about banks, credit unions and thrifts is updated as set forth in the Terms of Use of Safe & Sound Ratings and Reports. The Safe & Sound Ratings information is grouped by categories of banks, thrifts and credit unions.
Bankrate.com evaluates the financial condition of institutions and assigns a one- to five-star rating for each with five stars representing the highest rating. Institutions with satisfactory performance will generally receive a rating of three or more stars. The majority of institutions fall into the three- to four-star range. An institution with an "NR" rating may be too new to rate or may have limited the publicly available information in their regulatory filings. The "NR" is not an indication of financial strength or weakness. The Safe & Sound rating is believed to be reliable, but the information is not guaranteed. In addition, events since the information was collected may have altered the institution's financial condition.


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Dr. Evans has dedicated years to embracing and studying dentistry that stays true to what is represented in nature. He is among a small group of dentists whose goal is to treat causes, not symptoms, while offering treatment solutions that address a range of dental concerns.
Some clients present with debilitating headaches, while others may have a history of failed dentistry and repeat work. Jaw Joint discomfort, neck and shoulder tension and/or grinding and clenching of teeth may also be the reason(s) clients seek Dr. Evan Evans for dental care.
Treating a client's dental issues consists of listening and understanding the client's concerns while providing proven clinical methods for treating symptoms.
Dentistry is an effective treatment for serveral conditions
Patient's who experience dental care by Dr. Evans and his team also experience improved health in their gum tissue, esthetics of their smile, jaw joint discomfort and overall level of comfort. They also experience individualized relationships that are based on mutual trust and shared goals – they experience a level of care that enables them to view their dental health in a positive nurturing environment.


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Determine how much you need.
This is a really simple step. First, you take the monthly tuition rate and subtract the amount of scholarship money you have received.
Determine which site(s) you want to use for your crowdsourcing.
There are several different crowdsourcing websites available today to raise funds, however, not all sites are the same. You want to make sure the site you select best fits your needs; for instance, is it focused on social missions, how much does it charge, or what percentage of donations does it take, etc... We have provided a list of websites you should take a look at GoFundMe.com, Fundly.com, and Snap-raise.com
Make sure you have a bank account set up to transfer the funds into.You can also go to your bank or use their mobile app to set up either a one-time or recurring monthly wire transfer. Some banks will charge a fee for the service so make sure you ask your bank before using this option.
Write out your reason for asking people to donate. Be honest and include how you can change your life by earning a diploma.
Write a description that clearly defines a specific, timely need. Write as though you're talking to friends and family, don't expect people who have no personal connection to you to donate.
Reach out personally to close friends and family first in order to get the ball rolling, and then encourage sharing once you have some momentum.
Sign up and then share your message.
First, make a list of people that you want to share your campaign with directly.
Then most websites will allow you to share your donation campaign via your social media pages.


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I am doing research for my website and am having a hard time finding the best spots to hire a boat in Monte Cristi to visit Isla Cabra, La Plataforma, and Cayos Siete Hermanos? Sadly, when we visited Monte Cristi we never got a chance to see these places.
I have found one tour company who offers these tours, but nothing about hiring a boat yourself once in the city. Is it possible to find a private captain to take you near the other side of the river from the Nautical Club?
The one tour company I have found offers private tours for 2 people (which is not preferred for me as I'm looking for the cheapest ways to visit) for the following prices:
Isla Cabra - 2000
Mangroves/Plataforma/Isla Cabra: 3800
Mangroves/Plataforma/Natural Pools/Isla Cabra: 5400
7 Hermanos/Visit 2 islands: 8000
I am looking for the most budget friendly options and am mostly interested in Isla Cabra and the Plataforma, however would love to get information on 7 Hermanos if there is a cheaper way to see them.


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Ideal Pet Aluminum Side Sliding Window Pet Door – Perfect For Mobile Home, Lanai and RV– FREE SHIPPING! (Continental U.S. Only)
Product Description
Fits Into The Inside Frame Of An Aluminum Side Sliding Window
Pet Door Styles Available: Cat Flap 6-1/4"x6-14", Chubby Kat 7-1/2"x10-1/2", Small 5"x7" and Medium 7"x11-1/4" Flap Sizes
Custom Built For Window Heights From 30" to 72" (Measure flush to top track to inside of bottom track)
The Aluminum Side Sliding Window Pet Door is just like a patio insert for sliding glass doors; but made shorter for side sliding windows. The top of the unit is spring loaded to give you a 2.5" adjustment range for easy installation and removal. A locking pin is included that you may install on the frame of your sliding window. All weather stripping and bumper seal are included.
How to Measure Your Ideal Pet
To determine the correct size door simply measure the width (A) and height (B) of your pet.
(A) Measure the widest point of your pets shoulder
(B) Measure from the top of your pets back (just behind his head) to the bottom of chest.


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Happy birthday to Alyson Hannigan. She turns 41 today.
As do I. Got some nice chocolates.
March 24 2015
Happy birthday to both of you! Hard to believe little Willow is 41! On the other hand, it seems like she's done really well with her life.
... but who is Satya?
... but who is Satya?
Satyana: People - Mar 31, 2009
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About membership.


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A.C. Moore, in partnership with the Alicia Rose Victorious Foundation (ARVF), is proud to support a mobile art cart program for children's hospitals. The arts & crafts cart, named Alicia's Art Cart, is stocked with age-appropriate products from A.C. Moore and is designed to give chronically ill teens receiving medical treatment an opportunity for creativity and expression during their hospital stays.
Alicia Rose Victorious Foundation's co-founders Gisele & Mario DiNatale established ARVF in honor of their daughter, Alicia Rose DiNatale, who died in October 2002 after a 13-month battle with cancer. "During Alicia's treatments for cancer, both as an in-patient and out-patient 16-year-old, there were not a lot of 'grown up' things to do to occupy her time," said Gisele DiNatale. "I found myself at our local A.C. Moore to get her a craft kit or activity which she loved so much. The activity kits and projects provided on Alicia's Art Cart, named in her honor, help to pass the time and there's a sense of accomplishment and purpose just by being creative."
A.C. Moore & ARVF deliver a mobile art cart and provide each hospital's Child Life Specialist team with a gift card to A.C. Moore. The Child Life Specialists are then able to shop at their local A.C. Moore to stock their cart with appropriate art supplies, kits, and activities for the hospital's teen patients.
"At A.C. Moore, we know that art is therapeutic. Making art relaxes us, reduces stress, gives us an outlet for personal expression, and helps to distract us for a while. Most importantly, it's fun! We hope that the art activities on the art cart will inspire children and teens battling illness in our communities – making the tough days a little bit brighter." Pepe Piperno, owner & CEO
A.C. Moore & ARVF currently support art carts in the following hospitals and cities:
Want to learn more about Alicia Rose Victorious Foundation?


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Our customers have spoken.
The word is out, and people are loving their custom Borah Teamwear products! We love to showcase our customers stories of inspiration and triumph. It is what drives us forward as a company. Here are just a few of the many success stories, photos and testimonials sent in from people around the world.
Team Apex P/B Crankworx
"This was probably the greatest ride Thomas and I have done together. For 100% sure Thomas's greatest performance. Absolutely untested over this duration and he simply knocked it out of the park. Such an epic day. The DK special kit was perfect for me.Thanks guys." – Chris Evans
Featured Product: OTW SS Skin Suit
Fueled by Friendship
"The girls all love their jerseys! One of my friends is a seamstress and she specifically noted the "pattern match" of the stars on the back of the jersey where it meets the pockets! She was very impressed. One gal, who didn't originally order, now wants to order a jersey. My husband is on the board for our local race team and he keeps telling me "I wish we had ordered from Borah!" – Jody Hofstra
Featured Product: Women's Club SS Jersey, Women's Team SS Jersey
Oregon State University Cycling
"Borah is the first technical clothing provider that treated our small team like true customers. They provided with a plan and service to fit our needs at a price that allowed us to operate at the highest level of collegiate cycling possible. Our kits turned out better than expected and were the envy of many teams when we attended nationals." – Brandon Pendergrass
Featured Product: Pro SS Skin Suit
Moose Club
"Our Moose Club jerseys are fantastic! We could not be happier with the design and quality of the jerseys. They have made "international" appearances in Spain (Mallorca), Sweden (Stockholm), and Italy (Tuscany)." – Kevin Moran
Featured Product: Team SS Jersey
Action Bikes
"We love the look and feel of our Borah products. One teammate said it makes him feel like a "Super Hero!" – Bob Cornes
Featured Product: OTW SS Tour Jersey, OTW Cycling Bib
Team Hill Hunters
"Had tons of compliments on how good the kits looked. All of our support crew really loved how they looked as the colors and design made it super easy for them to find us. Thanks again for all your and the team's effort." – Sean Dunn
Featured Product: Pro Tri Top, Pro Tri Short
Our mission is to inspire healthy and positive lives. We do it by delivering quality, style, and comfort to teams and individuals with our running, cycling, skiing, and triathlon apparel.
Great design separates your team from the rest. Our design pros provide any level of design support you need.
Every garment made by Borah Teamwear comes with our Titanium Pledge Warranty for lifetime product performance.
Mon-Fri, 8am-5pm CT
Live Chat
Mon-Fri, 8am-5pm CT


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"I love to massage people and make them feel better!"
A big beautiful smile and friendly personality coupled with magnificent massage hands, Rina is sure to make your aches, pains and stresses seem like they were never even there. Start your session by relaxing into the warm, flowing touches of a classic Swedish style treatment before progressing, if you wish, into a more intensive massage by asking Rina to massage stronger and deeper, which she certainly can. A luxury treatment from renowned cruises now brought to your home!
Unique Mobile Massage™ offers therapeutic massage services and does not by any means offer services of erotic nature!


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930 Yishun Avenue 2
#B1-184 Northpoint City South Wing
Singapore 769098
11:00am - 10:00pm
firstly, i love this place. i always book it on eatigo at 50% off which only amounts to $11/pax after adding gst and service charge and let me say there is not a single more worth it steamboat buffet out there!! not only is it extremely affordable, i personally find the quality and taste of their meat to be very good, especially the "tender meats" on their menu. those are marinated with egg before serving to you and once cooked, the egg thoroughly enhances the flavour of the meats, not to mention how silky smooth the meat is as well. their pork belly has a really good fat to meat ratio and the beef and mutton are yummy too 😭 for spice lovers you can try the "spicy meats" on the menu - they come marinated in chili flakes and i can assure you your lips will be (satisfyingly) on fire after eating it!
for the soup base my favourite is the pork bone one, it is really flavourful and fragrant. the mala soup is also a good option as you can choose the spiciness level just like you do at a mala xiang guo shop!
additionally, they also have ice creams, side dishes like nuggets and potato wedges, fruits and even cake on some occasions! once i had this really yummy matcha&chocolate cake 😋
all in all, this restaurant's extensive variety of food and tasteful meat at such an affordable price really impresses me!
rating: 4/5
Nestled in Northpoint City South Wing, this conveyor belt hotpot restaurant offers lunch & dinner buffet with a catch, which is free-flow xiao long baos during dinner.
If you are looking for a full conveyor belt steamboat experience, this is probably not the right place as most ingredients still have to be ordered through them. There was also a variety of finger food available but sadly, they were not readily replenished. For the health-conscious ones, I felt that MSG was used in their soups.
Here's wishing everyone a Happig Chinese New Year 🎇!
Rating: ⭐⭐⭐
Honestly this is one of the worst steamboat I've eaten. Even those at bugis liang seah street are way better. I'm not sure why there are so many positive review for them.
Firstly, the utensils and cutlery were basically dirty. You can still see the stain on the bowls. After we wiped, we could see the red water stain marks on the tissue. Freaking disgusting. The whole shop was manned by China Chinese (no offense but you can gauge the hygiene standard). But we already sat inside and ordered, so bo bian lor.
Food selection was very limited. XLB was not available unlike what they advertised. The quality of food was also bad, pig intestine tested weird (definitely didn't clean properly), pork liver tasted weird (like not fresh). Tomato soup is simply thick ketchup soup, not worth the additional $3 paid.
The staff was not attentive either. Ice cream out of the 6 tubs placed, 3 tubs were empty. Will never return even if there is promotion.
This is probably our most frequently visited spot after trying out all its other competitors. On top of their sweet hospitality, they genuinely pride themselves in serving the freshest shabu-shabu plates, quality seafood, & a wide variety of other usual steamboat ingredients! & Unlike some places that charge you $1-3 per item, this buffet style dining certainly won't give you a bill shock at the end of the day. Psst! Best bonus is you don't have to pay extra for premium ingredients, order all you want! ❤
Get a healthy selection of 10 soup bases ranging from lighter ones like herbal chicken, pork bone, collagen, mushroom or tomato, to heavier options like mala, boiled fish with Szechuan pickles, laksa, tomyum or curry!
Lunch $19.80+ | Dinner $26.80+
Feel free to DM me if you want a copy of their ingredients list (too big to post as multiple photo). P.S. not sponsored at all.
📍Yi Qian Ling Yi Ye (一千零一✌🏻)
#B1-184, Northpoint City South Wing
1 Northpoint Drive, S769098
Daily: 11am-11pm
Buffet Hotpot restaurant at @northpointsg
Opening lunch promotion at S$16.80++ per person, which included selected soups and free flow of soft drink, soft serve and condiments. Ingredients were served conveyor belt style. Discount voucher for next visit.
As we were at the far end of the belt, most of the meat dishes were gone by the time the plates reached our side, but the staff who was serving us made an effort by serving us with trays of meat 🥩 straight from the kitchen without us requesting.
Yi Qian Ling Yi Ye Steamboat Buffet 一千零一夜自助火锅店
Address 🏠 : 1 Northpoint Drive, # B1-184 Northpoint City South Wing, Singapore 🇸🇬 769 098
Tel ☎ : 8720 2600
Open 💈 : 11am - 10pm
MRT 🚇 : Yishun (NS13)
Note 📝 : Opened on 24 Dec 2017
Apart from the conveyor belt concept, they added in the Genki Sushi train to deliver meat and carbs and they uses smartphone QR CODE to order.... The best deal? There's 50% off on Eatigo, rounding it to $10.... though I feel that the meat could be better esp lamb and beef (dissapointing much)... but the rest was alright.
#singapore #food #singaporefood #hotpot #hotpotbuffet #chinesefood #chinesefoodlover #相機食物 #新加坡必吃 #新加坡美食 #新加坡 #火锅 #火锅🍲 #foodporn #foodstagram #whati8today #8dayseat #globetrot #steamboat #吃貨 #instafood #instafoodsg #rollingoohinSG #burpple


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PiPES delivers a 6 week intervention to all 10th grade students to promote postsecondary education and STEMM careers. A copy of the PiPES Intervention Manual (Gibbons & Hardin, 2017) is available for your use by clicking on the image below.
The flagship campus of the University of Tennessee System and partner in the Tennessee Transfer Pathway.


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Death lurks around every corner. No matter how safe you think you are, something terrible can happen at any time, putting your life in danger. Your house *might fall into a sinkhole. You could be dragged into the woods by rabid beavers. Or you could be hit by a flaming toilet seat from space.
In If I Stay, the accident that claims the lives of Mia's family is a little more mundane than extraterrestrial bath fixtures, but it is no less devastating. Mia learns how precious life is when hers is almost taken away.
*(If in Florida, change "might" to "will any day.")
If Mia experienced a warm and fuzzy afterlife—like a light at the end of a tunnel—she would be more tempted to die instead of live.
After waking, Mia will live every day to her fullest, because she knows there is no happy afterlife.
Sleeping Beauty might have been literature's first coma patient, unconscious until woken by true love's kiss. Surprisingly, her story isn't entirely in the realm of fiction, either. In 2009, a woman left unconscious after a heart attack was woken by a kiss from her husband (source).
For Mia, it's a terrible car accident instead of a magic spell or cardiac arrest that puts her under, but it's still love that brings her back to life.
Mia thinks she isn't a hard-rocking punk chick, and she isn't. But she is totally metal. Yes, we're putting the cello and heavy metal in the same sentence. Why? Because science.
Scientists say that heavy metal music and classical music—and the people who love them—are very similar. Their listeners are both sensitive and dramatic, which is the perfect way to describe If I Stay, a book about a violent car accident that causes a teen girl to ponder life and death. What's more metal than that?
Mia's real love is music, not Adam. She will always choose music over a man.
Music creates another community for Mia. She has her family, her friends, and people she would never have associated with were it not for their shared love of music.
There are many ways to express your identity, whether it's with your hairstyle, your clothes, or the music you have blaring from your earbuds. All announce to the world, "This is who I am!" And if who you are is a mulleted man in a kimono listening to Celine Dion, more power to you.
In If I Stay, the characters aren't quite that eclectic, but they're all trying to find out where they belong in this great big symphony that is life. They do it through the music choices, their wardrobes, and who they hang out with. But identity seems futile when it's wiped out in the blink of an eye in a terrible accident. Be whoever you want to be, because eventually you'll just be dead, right?
Mia struggles with her identity. She's trying to find a balance between being herself and being who she thinks Adam wants her to be.
In If I Stay, clothes and music are used to define a person's identity, and almost every character is obsessed with looking cool or listening to cool music.
In lots of young adult novels, the protagonist either doesn't associate much with his or her family or outright despises them. But in If I Stay, Mia's family is practically perfect. Their biggest disagreement is over what to listen to first in the car—NPR or classical music. A lot about Mia's life has a fairy-tale quality to it—she's got a perfect music career; she's got a perfect boyfriend who also has a perfect music career; she's basically Sleeping Beauty. And her perfect family enhances the ethereal quality even more.
Let's hope Mia doesn't have to live with wicked godparents in the sequel.
Mia likes her family so much because her mom and dad are more like friends than parents.
If Mia had a damaged family, she wouldn't be glad they were dead, but it would make the choice of living or dying much easier. She would choose to live immediately, because she would be okay living on her own without them.
When you're a teenager, you have a lot of different choices to make. Choosing extracurricular activities, finding the right part-time job, and figuring out what college to attend can all be tough decisions. But none of them are life-and-death situations. In If I Stay, Mia does have a life-or-death decision to make—the choice between life and death. It's a choice very few people expect to make, especially at such a young age, but it provides the book's big conflict. Which will she choose?
Mia lives a privileged life and a privileged death. Not only does she have a wealth of choices in life—where to go to college, what instrument to play, which fairy-tale ending she'd like for her life—she can also choose to live or die. Every aspect of her own life is under her control.
Mia's parents were often left without a choice. Both their kids were surprises, and their own death was also a surprise. They had to adapt to what life dealt them, and Mia must do the same after her accident.
There are many movies and books that imagine an afterlife. Patrick Swayze discovered what it was like to be a Ghost. Heaven Is For Real is a popular non-fiction memoir about a boy who finds out that, well, heaven is for real. And Robin Williams found out What Dreams May Come when he lost his fictional family, as Mia loses hers.
You can add If I Stay to the pantheon of afterlife fiction, but this one is the most understated and mundane of the bunch. Mia doesn't get to fly around, see God, or even walk through walls. Her supernatural power is merely to listen in on things her friends say in private. But maybe that's because she's not quite dead. Yet.
As a spirit, Mia gets proof that people are waiting for her to wake up, like Jimmy Stewart's character does from It's a Wonderful Life.
The supernatural aspect of the book is primarily used as a plot device. If the book were written in third person, we as readers could still find out what happens while Mia is unconscious, but she wouldn't. She would be distant from the action, and the book would feel less emotionally charged.
People form lifelong friendships during childhood, so it's no surprise that young adult novels often feature iconic pairs of BFFs. Harry and Ron. Tom and Huck. Bella and… Okay, Bella didn't really have any friends.
If I Stay's Mia and Kim aren't going to be joining the Sisterhood of the Traveling Pants, but they are good friends, even if Mia sometimes veers into Bella's territory of choosing her man above anything else. Kim gives Mia something else to live for. At least that's what Kim tells herself.
Kim is an important foil for Mia. She is spiritual, while Mia isn't. She puts Mia's relationship with Adam in perspective. She enables Mia to make informed decisions.
Mia doesn't place as high a value on her friendship with Kim as she does her relationship with Adam. She never wonders about Kim's post-high-school plans, so Mia will likely go off on her own path without considering Kim at all.


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Cholera is an infectious disease caused by the bacterium Vibrio cholerae, and is transmitted in contaminated water. Cholera is prevalent in many tropical regions and produces almost continuous, watery diarrhea often accompanied by vomiting and muscle cramps that can lead to severe dehydration. Untreated, it can be fatal. But proper treatment, including fluid replacement and antibiotics, result in a high recovery rate.
The fear of cholera is called Cholerophobia. A person who suffers from the fear may have experienced the loss of a loved one, or watched terrifying reports about the disease on television and when they are exposed to this factors they are likely to manifest some symptoms which includes nausea, syncope and stricken with terror. Some might have also experienced being sick with cholera or severe diarrhea that their unpleasant experience in the hospital triggered this fear.
Education is the key in treating cholerophobic person. Teaching them about cholera, letting them understand that it is highly treatable and in the event that they are about to experience an outbreak, giving them enough information will surely help them to conquer their fears. If fear is uncontrollable despite of all the information, panic treatments can be of great help as well for this will teach the brain to respond differently thus reducing panic and physical symptoms.


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A Teaching Topic in Ethics and Accountability
Trust, Accountability and Legitimacy
This topic considers how governments build and lose trust from their citizens, and how governments establish legitimacy. This topic examines the role of accountability, transparency and due process in generating public trust in government. Students are also introduced to the concept of regulatory capture and how this phenomenon can undermine government performance and public trust in government.