text:
- Research article
- Open Access
Signal pathways JNK and NF-κB, identified by global gene expression profiling, are involved in regulation of TNFα-induced mPGES-1 and COX-2 expression in gingival fibroblasts
© Båge et al; licensee BioMed Central Ltd. 2010
- Received: 22 October 2009
- Accepted: 15 April 2010
- Published: 15 April 2010
Prostaglandin E2 (PGE2) is involved in several chronic inflammatory diseases including periodontitis, which causes loss of the gingival tissue and alveolar bone supporting the teeth. We have previously shown that tumor necrosis factor α (TNFα) induces PGE2 synthesis in gingival fibroblasts. In this study we aimed to investigate the global gene expression profile of TNFα-stimulated primary human gingival fibroblasts, focusing on signal pathways related to the PGE2-synthesizing enzymes prostaglandin E synthases (PGES), as well as the upstream enzyme cyclooxygenase-2 (COX-2) and PGE2 production.
Microarray and western blot analyses showed that the mRNA and protein expression of the inflammatory induced microsomal prostaglandin E synthase-1 (mPGES-1) was up-regulated by the cytokine TNFα, accompanied by enhanced expression of COX-2 and increased production of PGE2. In contrast, the expression of the isoenzymes microsomal prostaglandin E synthase-2 (mPGES-2) and cytosolic prostaglandin E synthase (cPGES) was unaffected by TNFα treatment. Using oligonucleotide microarray analysis in a time-course factorial design including time points 1, 3 and 6 h, differentially expressed genes in response to TNFα treatment were identified. Enrichment analysis of microarray data indicated two positively regulated signal transduction pathways: c-Jun N-terminal kinase (JNK) and Nuclear Factor-κB (NF-κB). To evaluate their involvement in the regulation of mPGES-1 and COX-2 expression, we used specific inhibitors as well as phosphorylation analysis. Phosphorylation analysis of JNK (T183/Y185) and NF-κB p65 (S536) showed increased phosphorylation in response to TNFα treatment, which was decreased by specific inhibitors of JNK (SP600125) and NF-κB (Bay 11-7082, Ro 106-9920). Inhibitors of JNK and NF-κB also decreased the TNFα-stimulated up-regulation of mPGES-1 and COX-2 as well as PGE2 production.
In the global gene expression profile, the enrichment analysis of microarray data identified the two signal transduction pathways JNK and NF-κB as positively regulated by the cytokine TNFα. Inhibition of these TNFα-activated signal pathways reduced the expression of mPGES-1 and COX-2 as well as their end product PGE2 in gingival fibroblasts. The involvement of the signal pathways JNK and NF-κB in the regulation of PGE2 induced by TNFα may suggest these two pathways as possible attractive targets in the chronic inflammatory disease periodontitis.
- Differentially Express
- PGE2 Production
- Gingival Fibroblast
- Gingival Tissue
The chronic inflammatory disease periodontitis is characterized by tissue and bone destruction. The current concept of the etiology of periodontitis is that bacterial components of the biofilm initiate the inflammatory cascade, including infiltration of immune cells and production of inflammatory mediators in the periodontal tissue. The initial inflammation, gingivitis, may then develop into a chronic inflammatory state of the gingiva causing destruction of the gingival tissue as well as the alveolar bone resulting in decreased support for the teeth, and ultimately tooth loss [1–3].
Among inflammatory mediators involved in periodontitis, prostaglandin E2 (PGE2) has been associated with periodontitis as a potent stimulator of bone resorption, and increased PGE2 levels have been reported in gingival tissue and gingival fluid from patients with periodontitis [4–9]. Moreover, administration of nonsteroidal anti-inflammatory drugs (NSAID), inhibitors of PGE2 production, has been shown to reduce the progression of alveolar bone resorption in patients with periodontitis, implying that PGE2 is a key mediator in the pathogenesis of periodontal disease [10, 11].
The proinflammatory cytokine TNFα is also reported to play an important part in the pathogenesis of periodontitis [12, 13]. For instance, it has been shown that attachment loss is decreased in periodontitis patients after anti-TNF treatment, and subcutaneous administration of recombinant TNFα is demonstrated to exacerbate experimental periodontitis in rats [14, 15]. Also, the chronic inflammatory condition rheumatoid arthritis, which shares many characteristics with periodontitis, has been successfully treated using TNFα blockers, further highlighting TNFα as a key inflammatory mediator in chronic inflammatory conditions [16–18]. We have previously shown that TNFα enhances the production of PGE2 in gingival fibroblasts, but the intracellular signal pathways regulating PGE2 production and PGE2-synthesizing enzymes have still not been completely elucidated [4, 19].
The biosynthesis of PGE2 from membrane lipids is catalyzed by three groups of enzymes acting sequentially. First, phospholipase A2 converts membrane lipids to arachidonic acid [20, 21]. The cyclooxygenases (COX-1 and COX-2) then convert arachidonic acid to prostaglandin H2[22]. Finally, the third and most recently identified group of isoenzymes is the prostaglandin E synthases (PGE synthases) which catalyze the conversion of COX-derived prostaglandin H2 to PGE2[23, 24]. Up to date, three PGE synthases have been described: The microsomal and inducible mPGES-1, the constitutively expressed cytosolic cPGES and the most recently discovered microsomal mPGES-2 [25–29]. We have previously reported that mPGES-1 and COX-2 are up-regulated by TNFα and IL-1β in gingival fibroblasts [4, 30, 31].
The inflammatory mediator PGE2 as well as the PGE2-regulatory enzymes COX-2 and mPGES-1 have been shown to be up-regulated by inflammatory stimuli such as lipopolysaccharides, IL-1β and TNFα also in other cell types, including gastric fibroblasts, synovial fibroblasts, cardiac fibroblasts and gastric cancer cell lines [32–37]. Various intracellular signaling pathways have been reported to be involved in inflammatory-induced PGE2 production and in the expression of PGE2-sythesizing enzymes, although these pathways seem to be both cell- and stimulus-specific. For example, in gingival fibroblasts, we have previously reported that the signal pathways PKC and p38 mitogen-activated protein kinase are involved in the regulation of the cytokine-induced COX-2 expression but not in the regulation of mPGES-1 expression [19]. In contrast, these two signal pathways are demonstrated to be involved in cytokine-induced mPGES-1 expression in colonocytes and orbital fibroblasts [19, 38, 39]. The differences between cell types and between stimuli make it imperative to study the intracellular regulation of mPGES-1 as well as COX-2 using a cell type and stimulus which is relevant to the disease of interest. In periodontal tissue, the most ubiquitous residential cells are gingival fibroblasts. These cells, by producing inflammatory mediators such as cytokines, prostaglandins and proteolytic enzymes, participate in the inflammatory response and contribute to disease persistence [40–44].
In this work, we therefore aim to explore the signal transduction pathways involved in the regulation of PGE2-regulating enzymes mPGES-1 and COX-2 in TNFα-stimulated primary gingival fibroblasts through a global gene expression approach, using microarray technology. Our results show that the intracellular signal transduction pathways JNK and NF-κB are involved in the regulation of TNFα-induced mPGES-1 and COX-2 expression, as well as PGE2 production, in gingival fibroblasts.
Effect of TNFα on protein expression of PGE synthases and COX-2 and production of PGE2
Effect of TNFα on mRNA expression of PGE synthases and COX-2
Differential expression between TNFα-treated cells and control cells
Gene Ontology analysis of differentially expressed genes
Enrichment analysis was performed to identify overrepresented gene ontology (GO) categories among the differentially expressed genes in the different comparisons. The gene ontology categories represent classes of genes in which more of the individual genes, included in the particular class, are differentially expressed in this data set than would be expected by a random distribution of differentially expressed genes. Complete lists of differentially expressed genes and significant GO categories are presented in the additional material (Additional files 1, 2, 3, 4, 5, 6, 7, 8 and 9).
PGE2-synthesizing enzymes and differentially expressed genes in selected significant GO categories (1-3 h).
Gene Name
Fold Change
p value
PGE 2 -synthesizing enzymes
microsomal prostaglandin E synthase-1 (probe 1)
2.4 × 10-4
microsomal prostaglandin E synthase-1 (probe 2)
microsomal prostaglandin E synthase-2
cytosolic prostaglandin E synthase (probe 1)
cytosolic prostaglandin E synthase (probe 2)
GO category: Positive regulation of I-kappaB kinase/NF-kappaB cascade
apolipoprotein L, 3
1.2 × 10-8
inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon
5.1 × 10-4
inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon
3.9 × 10-4
filamin A, alpha (actin binding protein 280)
nucleoporin 62 kDa
1.0 × 10-5
nucleoporin 62 kDa
9.5 × 10-3
toll-like receptor adaptor molecule 2
1.6 × 10-3
CD40 molecule, TNF receptor superfamily member 5
3.6 × 10-3
toll-like receptor 3
TRK-fused gene
coagulation factor II (thrombin) receptor
receptor-interacting serine-threonine kinase 2
1.7 × 10-6
tumor necrosis factor receptor superfamily, member 10b
1.5 × 10-3
tumor necrosis factor (TNF superfamily, member 2)
2.1 × 10-3
solute carrier family 20 (phosphate transporter), member 1
1.4 × 10-3
tripartite motif-containing 38
baculoviral IAP repeat-containing 2
4.8 × 10-3
gap junction protein, alpha 1, 43 kDa
v-rel reticuloendotheliosis viral oncogene homolog A, nuclear factor of kappa light polypeptide gene enhancer in B-cells 3, p65 (avian)
1.6 × 10-4
GO category: Positive regulation of JNK cascade
ectodysplasin A2 receptor
1.3 × 10-3
toll-like receptor 3
TAO kinase 3
TAO kinase 3
9.0 × 10-3
homeodomain interacting protein kinase 2
3.4 × 10-5
tumor necrosis factor (TNF superfamily, member 2)
2.1 × 10-3
Differential expression of PGE2-synthesizing enzymes when comparing 1 h and 6 h of TNFα treatment.
Gene Name
Fold Change
p value
microsomal prostaglandin E synthase-1 (probe 1)
6.2 × 10-7
microsomal prostaglandin E synthase-1 (probe 2)
1.8 × 10-3
microsomal prostaglandin E synthase-2
cytosolic prostaglandin E synthase (probe 1)
cytosolic prostaglandin E synthase (probe 2)
Since the focus of this study was on signal transduction pathways, we proceeded to further explore the potential role of the two positively regulated intracellular signaling pathways NF-κB and JNK, identified in the 1 h to 3 h comparison, in the regulation of TNFα-induced mPGES-1 and COX-2 expression. For this purpose, we investigated the effect of specific inhibitors of the JNK and NF-κB signal pathways on the expression of mPGES-1 and COX-2 as well as on the phosphorylation of these two pathways.
Involvement of JNK in the regulation of mPGES-1 and COX-2
Involvement of NF-κB in mPGES-1 and COX-2 regulation
PGE2 is involved in numerous inflammatory associated diseases including the chronic inflammatory disease periodontitis. The aim of this study was to investigate the global gene expression profile of TNFα-stimulated human gingival fibroblasts with a focus on signal transduction pathways related to the expression of mPGES-1 and COX-2. We used a time-course factorial design for the oligonucleotide microarray hybridizations, which enabled us to statistically discern the interaction effect between the TNFα treatment and time aspect in the experiments studied. To our knowledge, this is the first study investigating the global gene expression profile of TNFα-stimulated gingival cells as an in vitro model of gingival inflammation. In the current study, we demonstrate a TNFα-stimulated up-regulation of mPGES-1 and COX-2 expression, both at mRNA and protein levels, accompanied by enhanced PGE2 production. In addition, our microarray results showed a regulation of immune response, apoptosis and signal transduction gene categories in response to TNFα treatment of gingival fibroblasts, including positive regulation of the signal pathways JNK and NF-κB.
The TNFα up-regulated mPGES-1 and COX-2 expression, accompanied by increased PGE2 production, is in line with our previously published results on gingival fibroblasts [30]. In contrast to the mPGES-1 and COX-2 enzymes, the expression of the PGE synthases mPGES-2 and cPGES was not affected by TNFα treatment, highlighting the importance of mPGES-1 and COX-2 in the regulation of inflammatory-induced PGE2 production. However, when considering the similar kinetics of PGE2 production and COX-2 expression, as well as the magnitude of COX-2 induction compared to mPGES-1, COX-2 seems to be the more important enzyme driving the TNFα induced PGE2 production in gingival fibroblasts. Increased PGE2 production, via induction of the PGE2-synthesizing enzymes mPGES-1 or COX-2, has also been reported in other cell types stimulated with TNFα, including synovial cells, chondrocytes and colonocytes [38, 50, 51].
Various intracellular signaling pathways have been reported to be involved in inflammatory-induced PGE2 synthesis, mainly through regulation of COX-2, which is the most widely studied enzyme of the PGE2 synthesis chain. In contrast, limited reports exist regarding the regulation of the PGE synthases downstream of COX-2. To further explore the regulation of mPGES-1 and related PGE2-synthesizing enzymes in gingival fibroblasts, we here used a global gene expression profiling approach to achieve a broader view of the genes and signal pathways related to the regulation of mPGES-1, in parallel with COX-2, using microarray analysis of TNFα-treated gingival fibroblasts. The effect of TNFα on global gene expression profiles has previously been investigated in synovial fibroblasts, preosteoblasts and HeLa cells, but not in gingival cells [52–56]. With regard to periodontal disease, microarray analysis of gingival tissue has been used in an attempt to define subclasses of periodontitis and to evaluate the effect of periodontal therapy [57, 58]. In addition, blood cell gene expression profiling has been performed in subjects with aggressive periodontitis [59]. Concerning gingival fibroblasts, microarray studies have been performed on unstimulated cells from healthy and inflamed gingival tissue, and on IL-1β-stimulated immortalized cells [60, 61]. However, a majority of the abovementioned microarray studies on cell cultures use only one time point of RNA analysis after stimulation. In this work, using primary gingival fibroblasts, we employed a time-course factorial design to extract as much relevant information as possible from our data set. Commonly, when using a simpler design for a time series experiment, it becomes difficult to relate the gene expression differences at the different time points to each other. This is greatly facilitated by the use of a time-course factorial design, which allows for the identification of the genes that are differentially regulated between the time points due to TNFα treatment [45].
Our results from the microarray analysis indicated the JNK and NF-κB pathways as possible targets for interrupting the TNFα-induced signal transduction leading to increased expression of the PGE2-synthesizing enzymes mPGES-1 and COX-2. Thus, we next further investigated the involvement of JNK and NF-κB in the TNFα-stimulated mPGES-1 and COX-2 expression. By using inhibitors specific for these signal pathways, we could demonstrate that JNK and NF-κB are partly involved in the complex network of intracellular signal transduction pathways leading to increased expression of mPGES-1 and COX-2, as well as PGE2 production, in response to the inflammatory cytokine TNFα. To our knowledge, this is the first study pointing out the involvement of JNK in up-regulation of mPGES-1 expression in TNFα-stimulated cells. However, previous studies have shown that mPGES-1 is stimulated by IL-1β through the JNK pathway in cardiac myocytes, cardiac fibroblasts and A549 human lung epithelial cells [36, 62, 63]. In this study we also show that JNK is involved in TNFα-induced COX-2 expression in gingival fibroblasts, suggesting that JNK-dependent decrease of mPGES-1 may not be the major event whereby the inhibition of this signal pathway exerts its effect on PGE2 production. The finding that COX-2 expression was somewhat more influenced by inhibition of the JNK pathway, suggests that the JNK pathway may be more significant for COX-2 induction by TNFα in gingival fibroblasts. The involvement of JNK in TNFα-induced COX-2 expression is in line with results obtained from human alveolar epithelial cells and murine osteoblasts [64, 65]. Similarly, IL-1β- and lipopolysaccharide-induced COX-2 has been shown to be mediated through JNK in other cell types [66, 67]. In contrast, it has also been reported that COX-2 expression induced by epidermal growth factor is unaffected by JNK inhibition in astrocytes, emphasizing the differences between cell types as well as the inflammatory stimuli used for investigation of signal transduction pathways [68].
The JNK pathway has been implicated in chronic inflammatory disorders such as rheumatoid arthritis and inflammatory bowel disease [69, 70]. Moreover, JNK-1 deficiency as well as chemical JNK inhibition has been demonstrated to prevent joint destruction in rodent models of rheumatoid arthritis [69, 71, 72]. Thus, our novel finding that JNK is partly involved in the regulation of TNFα-induced mPGES-1 expression, which in concert with COX-2 regulates PGE2 production in gingival fibroblasts, indicates that this signal pathway may also be of importance in the pathogenesis of periodontitis.
In addition to the JNK pathway, we also found that the NF-κB pathway was involved in the regulation of mPGES-1 and COX-2 in gingival fibroblasts stimulated with TNFα. Our results pointing out the NF-κB pathway in the regulation of mPGES-1 are in line with our previous observations [19]. Induction of mPGES-1 expression using another inflammatory mediator, IL-1β, has been reported to be mediated by NF-κB in A549 cells [73]. In regard to COX-2 regulation, the involvement of the NF-κB pathway in the signal transduction of TNFα-induced COX-2 expression has been observed in other cell types, including endothelial cells and astrocytes [74, 75]. Concerning PGE2 production, no decrease was observed in TNFα-stimulated PGE2 production in 24 h cultures, in contrast to the inhibition observed in 6 h cultures. One reason for the lack of inhibition of PGE2 in 24 h cultures by the NF-κB inhibitor Bay, in contrast to mPGES-1 and COX-2 expression, may be a toxic effect of the inhibitor, affecting the release of PGE2 although no visual signs of cellular toxicity were observed. However, when using the NF-κB inhibitor Ro, reported to inhibit NF-κB via selective inhibition of TNFα-induced IκBα [49], it decreased the TNFα-stimulated expression of mPGES-1 and COX-2 as well as the production of PGE2 in 24 h cultures. Furthermore, both the NF-κB inhibitors Bay and Ro as well as the JNK inhibitor SP decreased the basal expression of mPGES-1 in cells not treated with TNFα, which might be due to slightly raised basal levels of mPGES-1 expression resulting from a lingering effect of the serum present in growth medium before the start of cell culture experiments.
A time-course factorial microarray analysis, like the one performed in this work, yields massive amounts of data. In this study we have focused on the signal transduction aspect, especially the JNK and NF-κB pathways, in order to investigate the regulation of mPGES-1 and COX-2 expression in relation to PGE2 production. Inhibition of JNK and NF-κB signal pathways by SP and Ro abolished the production of PGE2, although the induction of mPGES-1 and COX-2 by TNFα was not completely abrogated. One explanation for this might be that other enzymes may contribute to the production of PGE2 stimulated by TNFα. For instance, it is known that the signal pathways JNK and NF-κB, in addition to the COX-2 and mPGES-1 enzymes, are also involved in the regulation of cPLA2, an upstream key enzyme of the PGE2 synthesis reported to be induced by TNFα in gingival fibroblasts [76–78]. Another explanation for the strong inhibition of PGE2 production, in contrast to the partial reduction of mPGES-1 and COX-2 expression, may be a synergistic effect of the concerted inhibition of these two enzymes, since they are functionally coupled and responsible for the coordinated PGE2 synthesis [79]. In addition, one has to be aware that the JNK and NF-κB pathways activated by TNFα may not be entirely responsible for the increased expression of mPGES-1 and COX-2. There are many additional TNFα-regulated genes and pathways involved in the regulation of inflammatory conditions, including PGE2 regulatory enzymes, that merit further study, and investigations are ongoing to continue charting the genome-wide effect of TNFα on gingival fibroblasts.
We here present for the first time a gene expression profiling approach to explore the signal pathways involved in the TNFα-stimulated PGE2 production and mPGES-1 and COX-2 expression in gingival fibroblasts. In the global gene expression profile, the enrichment analysis of microarray data identified the two signal transduction pathways JNK and NF-κB as positively regulated by the inflammatory cytokine TNFα. Inhibitors of the JNK and NF-κB pathways reduced the TNFα-stimulated expression of mPGES-1 and to a higher extent COX-2, accompanied by abolished PGE2 production. Altogether, the microarray and phosphorylation data provide insight into the regulatory network of signal pathways related to PGE synthase, COX-2 and PGE2 production in gingival fibroblasts. The involvement of JNK and NF-κB in the regulation of PGE2 production induced by TNFα in gingival fibroblasts suggests these two signal pathways as key elements in the inflammatory-induced PGE2 production in gingiva, and also as possible attractive targets in the chronic inflammatory disease periodontitis.
Cell culture of human gingival fibroblasts
Human gingival fibroblasts were established from gingival biopsies obtained from 3 patients, 3 to 12 years of age, with no clinical signs of periodontal disease. The protocol, including the collection of gingival biopsies, was approved by the Regional Ethics Board in Stockholm (Dnr 377/98 and 2007/114-31/4). Minced pieces of gingival tissue were explanted to 25 cm2 Falcon tissue culture flasks containing 5 ml of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 50 units/ml penicillin, 50 μg/ml streptomycin (50 μg/ml) and 5% fetal calf serum (FCS, Invitrogen Life Technologies, Scotland, UK). Gingival fibroblasts were obtained by trypsinization of the primary outgrowth of cells. The cells were grown at 37°C with 5% CO2 and routinely passaged using 0.025% trypsin in phosphate-buffered saline (PBS) containing 0.02% EDTA. The fibroblasts used in the experiments proliferated in the logarithmic phase between the 9th and 14th passage. Gingival fibroblasts were seeded in Petri dishes in DMEM supplemented with 5% FCS and cultured for 24 h at 37°C. The cell layers were then rinsed with serum-free DMEM followed by the addition of DMEM with or without the inflammatory mediator TNFα (20 ng/ml, according to previous dose-response studies [19, 30]) and in the absence or presence of the inhibitors SP600125 (SP; Sigma-Aldrich, St. Louis, MO, USA), Bay 11-7082 (Bay, Sigma-Aldrich) or Ro 106-9920 (Ro, Tocris Bioscience, Bristol, UK). After different incubation periods, as indicated in the figure legends, culture medium was removed and stored at -20°C for subsequent PGE2 determination. The cell monolayer was washed twice with ice-cold PBS and the cells were used for oligonucleotide microarray, flow cytometry or phosphorylation specific luminex analyses.
RNA extraction
Gingival fibroblasts were seeded and grown as described above. For each of the three cell lines, two 100-mm Petri dishes were used for each treatment. After incubation for 1, 3 or 6 h with or without TNFα (20 ng/ml), the cells were immediately frozen in liquid nitrogen and then stored at -70°C for subsequent isolation of total RNA. Total RNA was isolated from fibroblasts using the commercially available RNeasy kit (Qiagen Inc., CA, USA) and quantified spectrophotometrically at 260/280 nm. The RNA from each pair of dishes with identical cell line and treatment were pooled and treated as one sample throughout further analyses. The RNA quality was assessed using the RNA 6000 Nano LabChip kit of the Bioanalyzer system (Agilent Technologies, Palo Alto, CA, USA).
Labeling and cDNA synthesis for microarray analysis
The synthesis and labeling of cDNA was performed as previously described by Lindberg et al. 2006[80] Briefly, cDNA was synthesized using random hexamer primers (Operon, Alameda CA, USA) and Superscript III (Invitrogen, San Diego CA, USA). The reaction was terminated and the RNA was hydrolyzed with EDTA and HCl. NaOH was then used to restore pH before proceeding with cDNA purification using MinElute Reaction Cleanup Kit (Qiagen, Hilden, Germany). For washing and elution of the cDNA, 80% EtOH and NaHCO3 pH 9 was used instead of supplied buffers due to the presence of TRIS, which would affect the labeling reaction. Labeling was performed using Cy3 and Cy5 mono-reactive esters from Amersham-Biosciences (Little Chalfont, Bucks, UK). The labeling mixture was then purified using MinElute Reaction Cleanup Kit (Qiagen) according to the manufacturer's protocol. A Nanodrop instrument (Nanodrop Technologies, Wilmington, DE USA) was used to confirm labeling success and to measure fluorophore concentrations. The fluorophore concentrations of the labeling reactions were used to balance the sample volumes. To obtain equal fluorophore amounts within each hybridization, a smaller volume of the sample with a higher fluorophore concentration was hybridized with the entire volume of the sample with a lower fluorophore concentration.
Oligonucleotide microarray
The oligonucleotide microarrays used in this study were printed at the KTH microarray core facility [81]. The 70-mer oligos originate from version 3.03 of Operons Human Genome Oligo Set, and the microarray contains 35344 features representing 28948 Entrez Gene ID:s [82] of which 17972 are unique. Additional information regarding the oligonucleotide microarray can be found in additional files 10 and 11.
Hybridization was performed as previously described [80]. Briefly, the microarrays were put in a trough and prehybridized with a bovine serum albumin based buffer for 30 minutes in a 42°C water bath. After washing the microarrays, the corresponding Cy3 and Cy5 labeled samples were pooled and hybridization buffer was added to the mixture. Lifter slips (Erie Scientific Company, Shelton, CT, USA) were used to contain the hybridization mixture on the array during hybridization. The arrays were then hybridized over night in a 42°C water bath. After subsequent washing the arrays were immediately scanned.
Scanning and image processing
Scanning was performed on an Agilent G2565BA scanner (Agilent Technologies, Palo Alto, CA, USA) using a scanner resolution of 10 μm, as previously described [80]. The software GenePix 18.104.22.168 (Axon Instruments, Foster City, CA, USA) was used to extract the raw signals from the TIFF images and to assign each spot an ID.
Statistical methods and low level analysis of microarray data
The data was analyzed using different packages in the software R [83]. All packages except the KTH package [81] are available in the Bioconductor open source software project for analysis of genomic data [84]. First, the raw data from GenePix was read into R. Thereafter, four filters were used to remove spots with abnormal physical properties, as previously described [80]. On average, 75% of all spots passed the filters for each slide. After filtering, the slides were normalized using print tip Lowess normalization [85]. A linear model in the Bioconductor package LIMMA (Linear models for microarray data) was set up to estimate the M-value and variance for each gene. The M-value is the log2 of the fold change (e.g. gene expression of TNFα treated cells/gene expression of control cells). Subsequently, differentially expressed (DE) genes were identified in the different contrasts of interest by using a moderated t-test where information is borrowed across all features present on the microarray to obtain a better variance estimate [86]. A false discovery rate algorithm was then applied to the calculated p values to correct for multiple testing [87]. Thereafter, differentially expressed genes were defined as genes with a q value < 0.05 (the false discovery rate analog of a p value), meaning that the proportion of false positives among the differentially expressed genes was 0.05. The Gene Ontology database was then used to assign functions to genes [88]. Utilizing the Gene Ontology annotation, enrichment analysis was performed to discover biological themes among the differentially expressed genes in the different comparisons [89]. Basically, a conditional hypergeometric test is performed for each GO-term where each gene is counted only in the most specific statistically significant GO-term to decorrelate and facilitate the interpretation of the results. False discovery rate was also used here to correct for multiple testing.
Design of microarray experiments
The experimental design of the microarray study was set up as a time-course factorial design, to best observe the TNFα-induced gene expression changes over time [45]. A C++ program (provided by the authors of reference [45] on request) was used to determine the exact layout of the design in order to estimate the interaction effect between treatment and time, i.e. genes that are differentially expressed over time, with optimal statistical efficiency [45]. The experimental design is illustrated in Figure 3A, where each arrow represents one hybridization. Thus, each sample was measured four times in this design, and the design was repeated for each of the three cell lines.
Accession codes
The microarray data set has been deposited at Gene Expression Omnibus (National Center for Biotechnology Information), accession number GSE13903 [90].
Flow cytometric analysis
Cells were seeded and grown in 60-mm petri dishes as described above. After 24 h of treatment, the cells were collected by trypsinization, and washed three times with PBS. Thereafter, the cells were fixed in 2% paraformaldehyde for 15 min at room temperature (RT) and washed with PBS prior to permeabilization with SAP buffer containing 0.1% Saponin (Sigma-Aldrich) in PBS (15 min, RT). The cells were then incubated with primary antibodies for mPGES-1 (monoclonal mouse, Cayman) mPGES-2 (polyclonal rabbit, Cayman), cPGES (polyclonal rabbit, Cayman) or COX-2 (monoclonal mouse, Cayman) for 40 min in the dark (RT). Antibodies were titrated in preliminary experiments. After washing with SAP-buffer, the cells were incubated with a secondary goat anti-mouse Fluorescein Isothiocyanate-labeled antibody (DakoCytomation, Glostrup, Denmark) or sheep anti-rabbit Phycoerythrin-labeled antibody (Serotec, Oxford, UK) in the dark for 40 min at 4°C. Thereafter, the cells were washed with SAP-buffer, resuspended in PBS and analyzed in a FACSCalibur™ flow cytometer using CellQuest software (Becton & Dickinson, San Jose, CA, USA). For each sample, 10 000 events were acquired and cells were analyzed regarding the expression of mPGES-1, mPGES-2, cPGES or COX-2. The results obtained are illustrated as histograms of cell counts, drawn using the program R, together with the software package rflowcyt [83, 84].
Western blot analysis
Cells were seeded and grown in 60-mm petri dishes as described above. To isolate the total protein, the cells were washed, resuspended in PBS and centrifuged. Thereafter, the pellet was resuspended in 100 μl lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT and protease inhibitors: 1 mM PMSF, pepstatin, aprotinin and leupeptin at 1 μg/ml). The protein concentration of the cell lysates was determined using the Bradford method (Protein Assay; Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin (Sigma-Aldrich) as standard. Equal amounts of the obtained protein were separated by electrophoresis on a 4-15% linear gradient polyacrylamide tris-HCl gel (Bio-Rad) and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was then immersed in blocking buffer (Tris buffered saline with 0.1% Tween 20, TBST, pH 8.0 with 5% defatted dry milk; Bio-Rad) for 1 h at RT and incubated at 4°C over night in blocking buffer with primary antibody diluted 1:200 for mPGES-1 (monoclonal mouse IgG, Cayman), 1:250 for mPGES-2 (polyclonal rabbit IgG, Cayman), 1:150 for cPGES (polyclonal rabbit IgG, Cayman), 1:1000 for COX-2 (monoclonal mouse IgG, Cayman) and 1:20000 for the loading control actin (polyclonal rabbit IgG, Sigma-Aldrich). Following primary antibody incubation, the membranes were washed in TBST and incubated for 1 h in RT with horseradish peroxidase-conjugated secondary antibody, diluted in blocking buffer (1:1000 goat anti-mouse or 1:2000 swine anti-rabbit; Dako Corporation, A/S, Denmark). Finally, the membranes were washed in TBST, developed using enhanced chemiluminescence (ECL) (Amersham Biosciences, Bucks, UK) and exposed to hyperfilm-ECL (Amersham Biosciences).
Analysis of JNK and NF-κB phosphorylation
Cells were seeded and grown in 60-mm petri dishes as described above. After an incubation period of 1, 3, 6 or 24 h, the cells were scraped in PBS and centrifuged. The pellet was then resuspended in the lysis solution of the Bio-Plex Cell Lysis Kit (Bio-Rad) and frozen at -20°C. The samples were then thawed and centrifuged at 4500 g for 20 min at 4°C, and the supernatant was collected. The lysate protein concentration was determined using the Bradford method (Protein Assay; Bio-Rad) with bovine serum albumin (Sigma-Aldrich) as standard, followed by addition of an equal volume of assay buffer from the Bio-Plex phoshpoprotein detection kit (Bio-Rad). The samples were frozen in -20°C until determination of the amount of phosphorylated JNK (T183/Y185) or NF-κB p65 (S536) using Luminex technology on a Bio-Plex Suspension Array System (Bio-Rad) with the Bio-Plex phoshpoprotein detection kit (Bio-Plex, Bio-Rad) according to the manufacturer's instructions.
Prostaglandin E2 determination
The amount of PGE2 in the culture media was determined using Luminex technology on a Bio-Plex Suspension Array System (Bio-Rad) using a commercially available enzyme immunoassay (EIA) kit (Cayman), and a conventional EIA kit (Cayman).
Statistics for non-microarray analyses
All non-microarray experiments were analyzed in triplicates and reproducible data representing one of at least three independent experiments is demonstrated. Results are expressed as the mean ± s.d. Student's t test (two-tailed) was used in the statistical analysis and p values less than 0.05 were considered statistically significant.
This work has been supported by grants from the Swedish Research Council, project no. 73XD-15005, Knut och Alice Wallenberg Foundation, the Swedish Patent Revenue Fund, and Karolinska Institutet.
Authors' Affiliations
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Considerations To Know About 菲律賓夏令營
菲律賓夏令營One particular Finnish custom also arose quickly after WWII, was affirmation camps. Confirmations camps, religious camps for thirteen??16 decades aged youths organized because of the local churches, aimed to mix the normal idea of affirmation college plus the more recent notion of summertime camps to be able to fight secularization of the Culture.
When the island was initial settled, it was thoroughly forested. Inside the late 12th century, Ari the Wise described it during the Íslendingabók as "forested from mountain to sea shore".[sixty eight] Everlasting human settlement significantly disturbed the isolated ecosystem of thin, volcanic soils and confined species range. The forests ended up greatly exploited above the centuries for firewood and timber.[65] Deforestation, climatic deterioration during the Little Ice Age, and overgrazing by sheep imported by settlers brought about a loss of critical topsoil due to erosion.
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The southwest corner of Iceland is among the most densely populated area. Additionally it is The placement in the money Reykjavík, the northernmost national cash on the globe.
The ideal sports camps do way more than just improve a camper's soccer, tennis, lacrosse, or wrestling abilities; they help Each and every boy or girl become a a lot more skillful athlete, a far more gracious competitor, a more dedicated staff player, and a far more self-confident individual.
The oldest Activity association in Iceland will be the Reykjavík Taking pictures Affiliation, founded in 1867. Rifle taking pictures grew to become very popular from the 19th century with the encouragement of politicians and nationalists who ended up pushing for Icelandic independence. To this day, it continues to be a big pastime.[224]
Iceland is volcanically and geologically Energetic. The inside consists of a plateau characterised by sand and lava fields, mountains and glaciers, although quite a few glacial rivers move to the sea throughout the lowlands. Iceland is warmed via the Gulf Stream and has a temperate local climate, Even with a superior latitude just outside the Arctic Circle. Its higher latitude and marine affect continue to retains summers chilly, with the majority of the archipelago possessing a tundra local weather. a long time and therefore the majority attend university) to go to the camps whilst however finishing normal education.|Almost all of the summertime camps are sponsored by the instructional bureau. Nevertheless, at present, you can find more privately held camp systems. The standard camps are only open up to the selected students in individual university district.|Each multi-Activity and single-Activity camps are usually operate by knowledgeable teachers and coaches (who generally have summers off from their college tasks). Cabin workers, instructors, and counselors are typically faculty athletes. The most effective sports camps triumph at difficult aspiring athletes both equally mentally and physically, when also advertising and marketing their social and Management expertise.}
Only if the social gathering leaders are not able to reach a summary by themselves within just a reasonable time span does the president work out this energy and appoint The cupboard personally. This has not took place since the republic was Launched in 1944, but in 1942 regent Sveinn Björnsson, who had been set up in that posture via the Althing in 1941, appointed a non-parliamentary government. The regent had, for all useful reasons, the situation of the president, and Sveinn would afterwards come to be the country's initial president in 1944.
The redistricting here adjust was produced to balance the burden of various districts of your state, due to the fact Formerly a vote cast during the sparsely populated parts across the country would depend A great deal over a vote Forged within the Reykjavík town location. The imbalance amongst districts has become minimized by The brand new technique, but nevertheless exists.[forty nine]
The most crucial music festival is arguably Iceland Airwaves, an annual occasion within the Icelandic audio scene, the place Icelandic bands in conjunction with overseas kinds Enjoy from the golf equipment of Reykjavík for weekly. Digital musicians incorporate ones for instance Thor and GusGus.
It is usually a single of some international locations presently able of manufacturing hydrogen in satisfactory quantities at a reasonable cost, as a consequence of Iceland's plentiful renewable resources of energy.
Activity is a vital A part of Icelandic society, given that the inhabitants is usually pretty Energetic.[218] The main common sport in Iceland is Glíma, a sort of wrestling imagined to have originated in medieval periods.
Icelanders are noted for their deep sense of Neighborhood: an OECD study located that 98% believe that they know anyone they might rely upon in the time of need, better than in every here other industrialised state. Likewise, only six% documented "almost never" or "never ever" socializing with Other people.
seven%, not including the quite a few deductions that are available.[one hundred fifteen] The corporate tax fee is a flat 18%, amongst the lowest on the globe.[115] There may be also a value extra tax, Whilst a Internet prosperity tax was eradicated in 2006. Work rules are rather versatile as well as more info labour market place is one of the freest on earth. Assets rights are potent and Iceland is among the several countries wherever 菲律賓夏令營 They're placed on fishery management.[one hundred fifteen] Like other welfare states, taxpayers pay back numerous subsidies to one another, but with spending check here staying under for most European countries.}

subset: commoncrawl


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Sunday, May 14, 2017
Go get some
Remember this nice little 60s showbike featured on here a while ago? Well, it needs to make room for another project, so it is up for sale! (or trade for a 30s Ford coupe ;-) Completely sorted Triumph pre unit 500, located in the north of the Netherlands. Contact me here so I can forward you to the seller. More info on the build can be found here
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Have you anytime thought of visiting Dubai? Visiting Dubai is straightforward when you have a mind-boggling vehicle rental organization to rent a car Dubai. This is to make your stay a serene one and a…Read More
Author: kuldeep
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There can be a parcel of various explanations behind leasing a vehicle. It tends to be an uncommon event like work excursion, work, get-away with relatives and a standout amongst the most valuable event weddings….Read More
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Will the guardians jump on? Who will the bridesmaids be? Will a ruler's payment be sufficient to pay for the enormous day? These are for the most part addresses that will unavoidably come up when…Read More


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For most people, purchasing art is quite informal. They don't plan and budget to buy a piece of art; they just bump on a lovely piece at the store and they abruptly make a purchase. Hardly do they make any effort to know more about the quality of the art. What is the history behind the art? Is it an original art or is it just any other reproduction? These are questions that most home owners should be asking when buying a piece of art for their homes. Asking these questions is so important because you will know exactly what kind of art you are buying before you decorate a space. That way you are bound to make the right decision. In this article we shed light on why you should buy original art over reproductions.
What is the difference between original art and reproductions?
Original art is that piece of art that is made by the artist himself ; a handcrafted masterpiece from an artist. This content is not only original but also very creative. It is one of a kind. A masterpiece!
A reproduction on the other hand is a duplicate of the original piece of art usually duplicated by machines in large quantities. These duplicates are of low quality and are cheaper than original art.
Why You Should Buy Original Art Over Reproductions
It is unique and scarce
Original art is a rare commodity. You will hardly find other versions of the artwork. When buying a piece of art, the last thing you want is to have imitations of art. You will want something that is rarely seen, a piece of art that will look attractive to your visitors. Something special and valuable. Reproductions are duplicated contents that are owned by thousands of people hence are less valuable.
Is of a higher Quality
In creating original artwork an artist puts in a lot of effort. He may spend a long period to make the final piece of artwork. This efforts ensure the artwork is finely made; in such a way that they meet high quality standards. That is why they are sold at a higher price. On the other hand reproductions are made in large quantities hence are made using cheap materials. They use commercial inks rather than acrylic paints. Due to this, the quality is always compromised. It appears to have a much "flatter" look when compared to the original art.
Has a Deeper Sense of Wealth and Personality
Having a unique piece of art in your living room will add that depth and richness that you have been longing for. It will evoke feelings of wealth and personality in your home. An original art will fill your room much better as compared to a flat poster reproduced by machines.
They have a rich story behind
Buying an original art, brings with it an adventure. If you are lucky you may meet the artist in person and listen to their story behind the artwork. This is because the process of making an original artwork is mind boggling for the artist. As you identify the story behind the original art, you will be able to explain this to your guests. This help you interact well with your guests.
As seen above, original pieces of artworks can add a lot of bloom to a room. They will not only help you fill the empty wall space in your home, but also bring a sense of depth of richness. Therefore, if you are embarking on a project to decorate your house, don't run for cheap reproductions rather, spend some resources in looking for original pieces of art. Just know what art you want, allocate a budget and identify where you want to place the art and you are good to go!


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Thursday, July 29, 2010
Simply Primitives Update and "That Fall Feeling!"
With temperatures dipping into the low 50's at night, things have been feeling a bit like Fall around here in the Pacific Northwest! It naturally makes me get into the Harvest and Halloween state of mind and so my offerings for Simply Primitives are just that, some adorable sock cats, a prim crow, a small hanging pumpkin head and a prim patch pumpkin, to get you and me into the Autumn spirit!! If I could package up the cool weather and send it to those of you dealing with the scorching temps. right now, believe me, those packages would be on your doorstep!! Since I can't do that, I hope my offerings and those of my other fellow artists will bring to mind the smell of woodsmoke, the taste of warm apple crisp, the fragrance of a wonderful candle and the coziness that comes this time of year!
Wishing you all "That Fall Feeling!"
Blessings my dear friends~
Friday, July 23, 2010
Finally Organized....sort of!!!!
Do you remember the big as in B I G file cabinet that my wonderful dear husband Jeff found for me? I've had several of you ask me about it, and now that it is finally filled to capacity, I will show you some pictures!
It has a PERMANENT home in my large walk-in closet in my bedroom!! I know that sounds like a strange place for it, but sadly, there is NO room in my actual work room!! The closet seemed to be the only place left to put it and so it proudly resides there, with easy access, but hidden as it is not really very attractive! Ok, it is just UGLY, but so very helpful to keep my clutter contained!! We are definitely not going for the primitive, country colonial look here!!
The dimensions of the cabinet are quite impressive! It stands at over 5' tall, x 19" deep x 36" wide, with 5 very large drawers, complete with rails for hanging files, so that is a LOT of room to store things!! What things, you ask?
Well, first of all, on the very top are lots of magazines that I collect, read and use for inspiration and they sit in a large wooden box that holds them nicely.
Now moving on to the drawers, we have the very top one which actually works differently from the others. Not only does it pull out, but the front lifts forward and slides up and in. I'm not sure why that one does it and none of the rest of them do, but it is a nice feature anyway. This drawer holds more magazines....Country Sampler, EAL, old Country Living, and Country Home, old Country Marketplace, old Create and Decorate, Mercantile Gatherings, The Simple, Life, and various random other ones. Maybe I should start a lending libary?
The next 3 drawers hold all my primitive patterns, from A to Z, and it is the first time I have had them all in one place, neatly organized and right at my fingertips!! The file cabinet was full of new file folders and my super organized DH actually labelled each file and put all my patterns away for me when I was at work! There are NO WORDS for that!!!
The final drawer holds even MORE magazines!! I did not realize I had so many!! Funny but Jeff said he did!!! It was probably good he was the one putting them away, as I would have started seeing articles I wanted to read and looking at the pictures and.....you get the idea!!!
So there you have it.....my AMAZING FILE CABINET!!! All organized and in one place! Now if I could just find that pattern I saw in that magazine....which one was it, Country Living 1986???
Blessings and Happy Organizing...
Monday, July 12, 2010
Waste Not, Want Not
I'm sure all of you are familiar with this famous proverb that some have credited to Benjamin Franklin, and have no doubt heard it or even quoted it many times! Growing up as I did in a large family with limited resources, it was a daily motto and practice that I have inherited, and hopefully passed on to my 2 children! Of course at the time, the words "waste not, want not","making do", and "hand me downs" were not appreciated and I can actually remember being chased and made fun of for a mustard colored corduroy jumper ( a size too large) I wore to school that was not considered fashionable!! Oh the sweet memories (and not so sweet ones) of childhood!! Now it seems anything goes that you choose to wear and thankfully with age comes wisdom!! Is that another famous quote or proverb? I digress..back to my original thought...
I still practice these same principles in my life as well as in my art and LOVE, LOVE, LOVE to find objects that no longer are usable for their specific purpose and reinvent or reuse them for a new one! When I find that small funnel, oil can, bobbin or spool, candlestick, salt shaker, glass insulator, old silver creamer or sugar, piece of vintage linen or fabric...you get the idea....my heart goes pitter patter and I have visions of make dos dancing in my head!!
When I first heard the title of a little book called, WASTE NOT WANT NOT, The Art of the Make-Do, I was intrigued and have been fortunate enough to now own my own copy! It is full of wonderful examples of early found objects, re-purposed into unique, sometimes humorous, and inventive repairs to create new purposes and to preserve the life of a former loved treasure! This book makes my heart sing!!
I'm sharing some pictures of make-dos I have made and I hope you will be inspired, and encouraged to begin your own quest for discarded objects and begin creating some special re-purposed treasures!! Perhaps you might even find a vintage old mustard colored corduroy jumper!!!
Happy Creating my friends!
The Possible's slow fuse is lit
By the Imagination.
~ Emily Dickenson
Sunday, July 4, 2010
Celebrate Liberty!!
I can still remember as a child, standing up in school, putting my hand over my heart and saying the following:
" I pledge allegiance to the flag, of the United States of America,
And to the republic for which it stands, One nation, under God, indivisible,
With liberty and justice for all!"
I still believe those important words, and still stand up, put my hand over my heart and say the words proudly because I love my country, I love my God, I love my flag, and I love my freedoms!!
Wishing you a very happy and safe Independence Day, filled with laughter, love, good food and a thankful heart as you Celebrate Liberty, the land of the free and the home of the brave!!
Blessings and Peace~
Thursday, July 1, 2010
We Have A Winner!!
The winner for my June Giveaway is.........
Gina of Cat Nap Inn Primitives!!
Thank you to ALL of you for taking part in my first giveaway, and I have to say I wish we could have picked all your names!! I feel like such a winner myself, as I look at all the new friends I have made! I have been truly amazed and humbled by your response and am so grateful for all the wonderful comments as well!!
A Big Congratulations to you Gina, and please send me your address so I can get your gifts mailed to you!!
Again, Thank you my friends and now that I have caught the "Giveaway Fever" I may just have to do another one in the near future!!!
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Built in about 1485, the Old Palace provides excursions as well as being a venue for Weddings,company and personal parties and way more. With only common home goods, a Swiss Army knife, and somewhat bit of duct tape, MacGyver may foil the evil plans of any villain to cross his path. If you are not snug promoting your own home on this financial system, and simply want to watch for a better time to sell you may just rent it instead. Fall Harvest Festival October 5, 2013 from 10 am to five pm on the Historic Village Green in Pendleton SC – Bring a Chair and benefit from the entertainment! Elected by their peers, certain representatives hold positions that mix institutional, administrative and partisan roles.
The guidelines in the USA state that ALL the air in a prefab (or equivalent) dwelling should be absolutely vented (utterly exchanged) each couple of minutes by a special system built into the house. The Anderson House Office Building is open to the general public Monday by means of Friday, 8:00 AM – 5:00 PM, excluding state and federal holidays. With Sell My Own Home, you're taking your individual photographs and add the details of your home yourself. Those with many planets in The Tenth House will go far in life, whether for good or ill. The research of many philosophies and religions are additionally a matter of curiosity on this house.
Where the ecliptic intersects the meridian overhead, at the prime of the chart, we have now the Midheaven or Tenth House cusp. While we do our best to verify our information is correct, please contact a professional tiny home builder for info. The Seventh House is ruled by Libra, and has to do with marriage, partnerships, contracts, unions, open enemies, lawsuits and wars. Don't promote your own home prefer it's a resort and in the event you do keep in mind even resorts have a check out time. EA: No, we have a home proper now, however taking a look at these designs, I would completely take into account them!
But I even have liked all of the ladies through the years and I suppose that Porshia is an efficient addition to this season I suppose she is as cute as a button but I'm not fairly positive about Kenya, I suppose she is somewhat beside herself. If I had been you I'd write to the Council (write, not telephone) and point out to themm that there's three' of water below the floor and that it will result in structural problems in the home. In the entrance garden of the house is a Mango tree, a bougainvillaea, and or a Frangipani. Restored within the Sixties, the home maintains it's unique stairway and ornate ceiling moldings in the parlor and eating room.
I would really love to show you a full dwelling tour with plenty of amazing Halloween decor but alas the home is in bit of a tizzy in the intervening time and so I will default to the front door and the little little bit of fall decor I may muster up in this chaotic mess of issues! Where the ecliptic crosses the western horizon, we've the Descendant or Seventh House cusp.


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We can practically call them our neighbours and I don't think that's extending the truth too much. Perched within the clouds 3,000 feet above sea level lies 23-million-years of ancient Gondwana Rainforest that we discovered from O'Reilly's Rainforest Retreat (in a whirl-wind 22-hour overnight stay).
We arrived upon dusk after a day's work and were ecstatic to pull into our very own architecturally designed Villa, with a cantilevered driveway and beautiful (solid) timber door.
The Retreat is dotted with 66 rainforest retreat accommodation rooms (which are TV and phone free) and 48 self-contained, privately owned (strata titled in 2007) eco villas set within 20,000ha of rainforest. (This is where we stayed, nice).
Scanning the (enormous) Villa and absorbing the layout and décor (as you do), who could miss the enormous raised Jacuzzi that's perched on the deck. It made for the perfect spot for a glass of bubbles (O'Reilly's Lona Sparkling Blush in fact) before bed.
Keeping to schedule we dined (in style) in the Retreat Restaurant which (totally) reminded us of a rustic timber chalet in the mountains of Canada with soft music, a crackling fireplace and local produce presented by Executive Chef Mus.
We were then whisked away (in the rain) to one of the most sacred spots on the property – The Glow Worm grotto. A surreal experience (and MUST do).
We rose (nice 'n early) to the sound of birds knocking on the Villa glass and warm sunlight streaming through the curtains (ahh). We didn't want to miss the signature guided Bird Walk Tour along the elevated timber boardwalk, where we met the likes of Mr Regent Bowerbird (featured below).
Our taste buds were (extremely) satisfied after a delicious buffet breakie with all the trimmings, including (real) fluffy eggs and brewed coffee as good as any fancy urban café.
To be honest, I was (extremely) nervous (and prolonging breakie) as the day's activities fast approached. However, can I tell you how much fun we had (rain and all).
Have you heard of off-road Segway? Well, it's a two wheeled toy (you can check out here) that silently manoeuvres through the rainforest using your (forward) body weight with absolutely no emissions (and at a decent 9 kilometers per hour).
We scooted through bush trails and creek crossings and were rewarded with views over (the famous) Moran Falls (featured below).
Strapping on a harness is quite possibly the most unflattering outfit you'll ever wear. My ego may have been bruised however the freedom of launching yourself from a perfectly safe platform down a 180 meter whizzing zip line does make up for the humility.
It's. So. Much. Fun. (You can even (try) pushing your limits and rotating up-side-down).
On our next visit (to our neighbours Retreat, ha) we plan to explore the 23-kilometre Border Trail (from Binna Burra), followed by swim in the Infinity Pool and couples vinotherapy hot tub (oh yes); where therapists use the wine grapes grown down the road at the Canungra Valley Vineyards (literally, near our house).
// We were lucky enough to stay as guests of O'Reilly's and all pictures (excluding me on the Zip Line) are courtesy of O'Reilly's Rainforest Retreat (as ours in the rain weren't much chop).


text: Marcelo M. Garcia wrote: > Hi > > I tried to install the kernel Red Hat 5.3 x86_64 (2.6.18-128.el5.x86_64) > with the command: > rpm -Uvh kernel-2.6.18-128.el5.x86_64.rpm > but there is problem with dependecy: ecryptfs-util < 44. How to solve > problems with depencies when installing a beta kernel? > > I'm trying this kernel to see if I get CentOS working with the wireless > iw4965. I tried the drivers from Intel, but the compilation failed. It seems that ecryptfs-util is filesystem specific, and that's not a default filesystem in linux, so you should be safe in forcing the rpm to install (--nodeps --force or something). Another option may be to get the .src.rpm and build it from source, it may detect that you don't have ecryptfs-util and not build in support for it or something. nate


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14 Then Ahimelech answered the king, and said, And who is so faithful among all thy servants as David, which is the king's son in law, and goeth at thy bidding, and is honourable in thine house?
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The Chaotic Kristy Baby Shower...it's chaotic.
To celebrate the impending birth of Chaotic Kristy's baby boy, we here at BCE are throwing a virtual baby shower. It's the online kind, a cyber, digital extravaganza of fun, happiness and mirth!
(Did we mention, it'll be fun?)
Following on from the critically-acclaimed, worldwide success of the Mommie Daze Virtual Baby Shower, this new event promises to be bigger and better, with lots of games to play, prizes to win* and party favors for you to...well, do whatever you do with party favors.
You don't have to be pregnant, a mom or dad to enter and enjoy. Everyone is welcome, no matter who you are or where you are!
So, tell your friends, block out the dates on your calendar and get ready: the Chaotic Kristy Baby Shower is
Link with us!
*Please note some competitions/giveaways will only be available to residents in certain countries.


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Keystone XL: Obama the Pragmatist
On April 3rd and 4th, President Obama spoke at several San Francisco fundraisers. While he didn't specifically mention the Keystone XL pipeline, the tenor of his remarks indicated that he's likely to approve the controversial project. Obama seems to be most influenced by his inherent political pragmatism.
I've heard Barack Obama speak on several occasions. The first was February 19, 2007, at a San Francisco ore-election fundraiser with a lengthy question and answer session. Towards the end of the event a woman asked then presidential-candidate Obama what his position was on same-sex marriage. For an instant, Obama seemed surprised; then he gathered himself and responded he was aware of strong feelings on both sides of this issue and his position was evolving. Five years later, in May of 2012, President Obama announced his support for same-sex marriage.
What took Obama so long to make up his mind? No doubt he needed to clarify his own moral position – although the Protestant denomination he was baptized into, the United Church of Christ, announced its support for same-sex marriage in 2005. But I'm sure the President carefully weighed the political consequences and, last May, thought the timing was right.
Over the last six years I've realized Barack Obama has several personas. On occasion he moves us with stirring oratory; that's Reverend Obama, the rock star. Once in a while, he turns philosophical; that's Professor Obama, the student of American history. On April 3rd, I saw Politician Obama, the pragmatic leader of the Democratic Party. -more-


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Though Hungary is not in the Mediterranean area which is supposed to have a suitable climate for growing vines, the fact is that because of their strong adaptation capability are planted in other countries with different weather conditions, being Hungary one of the main producers in Europe since the Roman Empire.
Hungary has seven large regions that include 22 smaller regions over the country. The large regions are:
- Balaton, with sub-regions
- Eger, with sub-regions which is mostly known about its full red wine called Egri Bikavér
- Észak-Dunántúl, with sub-regions
- Pannon, with sub-regions
- Sopron, with sub-regions
- Tokaj, which is the most famous wine region in Hungary
- Duna, with sub-regions, the biggest region of all, where Kiskunság takes place.
Orgoványi Gazdaszövetkezet (OGSZ) owns land and works in Kiskunság (Duna region), in the southern part of the country, between the most important rivers: the Danube and the Tisza. In this area it is absolutely necessary to grow robust types of grape, because weather can be really extreme, so the most common types of grapes are kövidinka, kékfrankos, cserszegi fűszeres, izsáki sárfehér and bianka. Wines in Duna are light and fresh
Egri Bikavér is a red wine made from Blaufränkisch (Kékfrankos in Hungary) and various proportions of other grapes such as Cabernet Sauvignon, Cabernet Franc, Merlot and Menoire.
Tokaj-Hegyalja, in the northeast of Hungary, is the most famous wine-growing region due to its association with the Tokay wine, made from the botrytised Furmint, Hárslevelű, Muscat Blanc à Petits Grains, Kövérszőlő and/or Zéta grapes. The distinguing characteristic of this wine is that the ripened grapes are infested by the Botrytis fungus and when picking them at a certain point the wine produced is fine and sweet and pairs with desserts.
In the last few years, the measure of vineyards has drastically reduced because of the economic and agrarian crisis. The full size of Hungary's vineyards used to be around 100 000 Has, but today it is only around 60 000 Has. Thanks for the support of the European Union, this tendency seems to stop and turn: the planting spirit is just coming back to viticulture.
Because of the effort of the EU, Hungarian farmers are able to modernise their technology, by using metal stake systems, mechanised cultivation and controlled fermentation. This leads to higher quality of wine, and lower costs of production.