text:
Were you under the impression that you are the legal owner of your website? Want to know the various components of websites and understand who owns what?
We cover the basics of website ownership in this infographic.
We break things down as follows:
- Web server
- Web server platform
- Content management system (CMS)
- Database software
- Source code
- HTML / CSS / JavaScript
- Visual design
- Text content
- Imagery
- Browser
- Domain name
Check out the infographic for more detail.
It's a great feeling having your own digital space.
It's something of a second home – an opportunity to create an amazing and creative online environment where visitors can really get to know you and your business. All from the palm of their hands.
And you've already decided not to do it alone; you know best results come from investing in professionals who know how to get the job done properly. That's why you've done your research to locate the best people to create the best website.
After a few days of work, the professionals are pleased to hand the website over to you. You're proud to call it your own. And you're even more excited to encourage customers to visit your website. This is your website, after all. And you have paid good, hard-earned money for it.
But is it really yours?
Complete, full legal ownership of a website is something of a misnomer that exists when clients are given access to their website to manage, add content, and share around for other people to enjoy and interact with.
The key issue that results in this is limited understanding of what exactly constitutes a website – the building blocks that enable the existence of your website in the first place.
And it's here where hierarchies of legal ownership begin to reveal themselves.
When you visit your website as anyone would, what you are looking at is the result of behind-the-scenes work that you may not have been aware of.
To bring what you have to say to the public, your website needs (at least) a server, a server platform, a domain, a content management system, a source code, and a browser to make it all happen.
Typically, you won't own any of these. These are owned by multi-national companies that have monopolised the website serving and hosting industry.
If you were to take on the software and hardware requirements to do all of this yourself, you'd quickly find your money disappearing.
However, it's not the end of the world. Website ownership is much more than a faraway data centre you'll probably never visit. There are a few components you may be able to call your own – from visual design, text content, programming, and photography (if you took the photographs yourself).
When you pay for a website to be made, you are effectively initiating a contract to place your website online. From here, you are given permission to license and use intellectual property of the website itself, and/or the platform used to build it.
True website ownership derives typically from visual elements – and only then if you designed and created it yourself.
It's good to know.


text: Pack your most colorful outfits and some confetti. Here are my top 10 events that should be added to your bucket list too! Let's check out what the world has to offer! 👇🏼 1. The Dia de Los Muertos in Mexico 🇲🇽 Mexico, November 2.Ever since I watched 007's Spectre movie, I knew, I would have to go to Mexico... Continue Reading →

subset: commoncrawl


text:
Saturday, 4 February 2017
Wetland walkies: World Wetlands Day
I went out for a walk in the sun and the snow. It was a good bit of exercise. I saw a few deer, but they ran. Lots of tracks in the snow: squirrels, mice, fox, coyote, the five toed one is likely a marten. Lots of deer beds. They hunker down and chew their cud.
8 comments:
- Olga said...
Walks in nature are just the restorative type of thing we all need these days.
- 4 February 2017 at 08:35
- Nancy J said...
A walk through the trees, and for me to see your photos, a true reminder that nature is always giving us beauty amid the horror and sadness in so many parts of the world. Lots of tracks, and not a feline companion to be seen, maybe for once a little too cold.
- 4 February 2017 at 12:35
- Cat Lover said...
As Olga said nature is what we need right now. Chuck and I went on a long walk this morning. It does clear your head.
Wonderful photos, love all the animal tracks.
- 4 February 2017 at 13:30
- William Kendall said...
It looks like you had good conditions for that stroll.
- 4 February 2017 at 14:14
- Sallie (FullTime-Life) said...
I did not know about World Wetlands Day until I was reminded of it on Michelle's blog. And now yours. I won't forget again -- I do know about wetlands and appreciate their unique place and the necessity for them.
Walks in nature are my salvation these days.
- 4 February 2017 at 17:58
- Crafty Green Poet said...
Lovely photos, nature is such a solace
- 5 February 2017 at 09:31
- Debbie said...
really lovely captures of the outdoors - gorgeous blue skies!!! i like your new picture!!
- 5 February 2017 at 12:33
- Powell River Books said...
Your wetlands look more like icelands right now. - Margy
- 5 February 2017 at 21:27


text:
Emerging data suggest that regulatory T cell (Treg) dysfunction and consequent breakdown of immunological self-tolerance in autoimmunity can be mediated by factors that are not Treg-intrinsic (e.g., cytokines). Indeed, recent studies show that in rheumatoid arthritis the proinflammatory cytokine TNF reduces the suppressive function of Tregs, whereas in vivo TNF blockade restores this function and accordingly self-tolerance. However, until now a coherent mechanism by which TNF regulates the Treg has not been described. In this paper, we show that TNF induces preferential and significant activation of the canonical NF-κB pathway in human Tregs as compared with CD25– conventional T cells. Furthermore, TNF induced primarily in CD45RA– Tregs a transcription program highly enriched for typical NF-κB target genes, such as the cytokines lymphotoxin-α and TNF, the TNFR superfamily members FAS, 4-1BB, and OX-40, various antiapoptotic genes, and other important immune-response genes. FACS analysis revealed that TNF also induced upregulation of cell surface expression of 4-1BB and OX40 specifically in CD45RA–FOXP3+ Tregs. In contrast, TNF had only a minimal effect on the Treg's core transcriptional signature or on the intracellular levels of the FOXP3 protein in Tregs. Importantly, TNF treatment modulated the capacity of Tregs to suppress the proliferation and IFN-γ secretion by conventional T cells, an effect that was fully reversed by cotreatment with anti-TNFR2 mAbs. Our findings thus provide new mechanistic insight into the role of TNF and TNFR2 in the pathogenesis of autoimmunity.
Tumor necrosis factor (TNF-α) is a prototype member of the TNF superfamily (TNFSF) of ligands. It binds the corresponding TNFR superfamily (TNFRSF) members TNFR1 (TNFRSF1A; p55) and TNFR2 (TNFRSF1B; p75). TNF is generated and expressed by many types of cells, including lymphocytes, and can induce a plethora of immune responses (1). Several lines of evidence suggest that TNF has a central role in the pathogenesis of a variety of human inflammatory disorders (2). Moreover, TNF blockade has a notable therapeutic efficacy in a number of T cell-dependent human autoimmune disorders, such as inflammatory bowel disease, rheumatoid arthritis, juvenile idiopathic arthritis (JIA), ankylosing spondylitis, and psoriatic arthritis (3). Nevertheless, little is known at the molecular level about the specific effects of TNF on the various T cell subsets, and consequently the fine details of the mechanism of action of TNF blockade, in vivo, remain somewhat obscure.
Various studies in both mice and humans suggest that TNF blockade has the potential to augment regulatory T cell (Treg) numbers and function. For example, in RA patients treated with anti-TNF mAbs there is an increase in the quantity of circulating CD25HI Tregs as well as restoration of their partly impaired suppressive function (4, 5). Moreover, TNF blockade during the in vitro activation and expansion of unselected human T cells results in augmented expansion of FOXP3+CD4+ T cells with a regulatory phenotype (6, 7).
It should be noted that studies in patients with various autoimmune disorders and mice models show that Treg-type cells are present at considerable numbers at the actual site of inflammation (8, 9). Thus, a major challenge is to define the Treg extrinsic factors that facilitate tissue inflammation even when Tregs are present in abundance. In this regard, there is controversy as to whether TNF—a cytokine abundant at sites of inflammation—can interfere with the suppressive function of the Treg (4, 8–13).
Tregs as opposed to CD25– conventional T cells (Tcons) constitutively express high levels of TNFR2, but both subsets do not express TNFR1 (4, 12). Whereas the downstream molecular events initiated following TNFR1 ligation have been studied in great detail, there is significantly less information about signal transduction via TNFR2 that, as opposed to TNFR1, does not contain a death domain (14, 15). Binding of TNF to TNFR1 activates at least three distinct cellular pathways: IKK/NF-κB, JNK/c-Jun, and the proapoptotic caspases 8 and 3 (1). At present, the specific cellular program induced by TNFR2 stimulation in Tregs is practically unknown.
In this study, using various complementary methods, such as gene expression microarrays, PCR-based assays, analysis of protein expression and/or phosphorylation by FACS, and other relevant assays to test suppressive functions, we now identify the cellular program that mediates the inhibitory effect of TNF on the Treg suppressive function. Our data show a central role for activation of the canonical NF-κB pathway—primarily in CD45RA– Tregs—that consequently induces a distinctive proinflammatory cellular program.
Materials and Methods
Human subjects
Blood samples were obtained from healthy adult blood donors. Inflammatory synovial fluid (SF) samples were obtained from JIA patients. Signed written informed consent or parental/guardian permission was obtained from all of the study participants as appropriate. The Institutional Ethics Committee at the Chaim Sheba Medical Center approved the study.
Recombinant human IL-2 (rhIL-2) was obtained from Boeheringer-Mannheim (Mannheim, Germany). Recombinant human TNF-α was purchased from Biosource Invitrogen (Carlsbad, CA). The humanized chimeric anti-TNF mAb, Infliximab, was from Centocor (Horsham, PA). The mouse anti-hFOXP3 mAb 236A/E7 and Treg staining kit were obtained from eBioscience (San Diego, CA). The fluorochrome-conjugated mouse mAbs against human CD4, CD134, CD137, CD95, CD69, CD25, CD45RA, CD120A, CD27, and CD127 were all from BD Pharmingen (San Jose, CA). The mouse mAbs against TNFR2 (CD120B) were from R&D Systems (Minneapolis, MN). The fluorochrome-conjugated rabbit mAbs to phospho-IκB were from Cell Signaling Technology (Danvers, MA), and the mouse mAbs to phospho-NF–κB-p65 were from BD Biosciences (San Jose, CA).
FACS analysis
Cell samples were analyzed either on a FACSCalibur using the Cellquest software or on a digital flow cytometer, FACSCanto II or FACSAria, using the FACSDiva 6.1.2 software (both from BD Biosciences). FACS data analysis was done with the FlowJo 7.2.5 software (Tree Star, Ashland, OR). Immunostaining of cell surface markers was carried out as previously described (16). For detection of FOXP3, the cells were fixed/permeabilized using the eBioscience FOXP3 staining buffer set, as previously described (17).
Treg and Tcon cell isolation
PBMCs and SF lymphocytes were obtained by density gradient centrifugation on Histopaque 1077 (Sigma-Aldrich, St. Louis, MO). Highly pure CD4+CD25HI Tregs were obtained as previously described (18). Briefly, CD4+ T cells were isolated from PBMCs by positive selection with CD4 microbeads (MBs) from Miltenyi Biotec (Bergisch Gladbach, Germany) and further purified into CD4+CD25HI and CD25– T cells using a FACSAria cell sorter system (Fig. 2A). Less pure CD25++ and CD25– T cell populations were isolated using positive selection with CD25 MBs (Miltenyi Biotec). To obtain a population enriched for CD25++ T cells, we modified the protocol recommended by the manufacturer and used 7.5 μl (instead of 10 μl) CD25 MBs per 1 × 107 PBMCs.
T cell cultures and activation
The cells were cultured in RPMI 1640 supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Invitrogen Life Technologies, Carlsbad, CA) and maintained at 37°C in a humidified 5% CO2 incubator. Unless otherwise specified, the cultures were supplemented with rhIL-2 (100 IU/ml). Purified T cells were typically plated into 24-well plates (Costar; Corning, Lowell, MA) at 2 × 106 cells per well. For TNF treatment, the various purified T cell populations were incubated for the indicated time with medium supplemented with 50 ng/ml TNF (or control PBS). T cell activation and expansion were generally induced by immobilized plastic-bound OKT3 (2.5 μg/ml), soluble anti-hCD28 mAbs (1 μg/ml), and rhIL-2 (100 IU/ml). Cell cultures were supplemented with fresh complete medium plus 100 IU/ml rhIL-2 every 72 h until the end of the experiment.
Suppression assay and CFSE dilution
Tregs, isolated from PBMCs, were tested for their ability to suppress the proliferation of autologous CD4+CD25– Tcons, as previously described (6). Briefly, CFSE-labeled CD25– T cells at 1 × 105 cells per well were plated into 96-well plates (Costar) and activated by plate-bound immobilized OKT3 (2.5 μg/ml) in the presence of graded amounts of CD25++ T cells. Selected cultures, as indicated in the text, were supplemented with TNF (50 ng/ml), rhIL-2 (100 IU/ml), anti-TNFR2 mAbs (10 μg/ml), or Infliximab (50 μg/ml) at time 0 and 72 h later. As indicated, we also activated the Treg/Tcon cocultures with K562 artificial APCs, engineered to stably express the FcR CD32 alone (KT32), or with 4.1BBL (KT32/4.1BBL), kindly provided by Carl H. June (University of Pennsylvania Cancer Center, Philadelphia, PA). To induce potent TCR activation, the KT32 cells were preincubated with OKT3 and anti-CD28 mAbs at a concentration of 1 μg/ml for 10 min and then irradiated with 10,000 cGy. Regardless of the activation protocol, 5 d later the T cells were harvested and analyzed by FACS. CD4+ Tcon proliferation was determined using the proliferation platform of FlowJo 7.2.5 to calculate the division index (DI) and draw a "fit model" of generations onto the CFSE dilution histograms. The DI reflects the average number of cell divisions that the cells underwent and is considered a good objective value to compare the rate of proliferation from sample to sample.
IFN-γ analysis
To assess the capacity of Tregs to suppress the production of IFN-γ by Tcons, 100 μl supernatant samples were collected (on day 3) from the various Treg/Tcon cocultures. The supernatants were then analyzed by the human IFN-γ cytoset ELISA kit (Biosource) according to the manufacturer's instructions. For intracellular cytokine detection, the T cells were activated with 20 ng/ml PMA and 0.8 μM ionomycin (Sigma-Aldrich) in the presence of monensin (GolgiStop from BD Biosciences) at a concentration of 2 μg/ml for 5 h. Subsequently, the cells were fixed/permeabilized using the eBioscience FOXP3 staining buffer set and then stained with anti–IFN-γ–PE, CD4–FITC, and FOXP3–allophycocyanin mAbs (all from eBioscience), as previously described (17).
Isolation of RNA and real-time quantitative PCR
Total RNA was isolated using the RNeasy mini kit (Qiagen, Valencia, CA), and cDNA was synthesized using the RETROscript kit (Ambion, Austin, TX). The real-time quantitative PCR (RTQPCR) for mRNA levels of FOXP3, STAT1, NFKB2, TRAF3, LTA, and the housekeeping gene HPRT1 was carried out using TaqMan gene expression kits supplied with predesigned optimized primers and probes (Applied Biosystems, Foster City, CA). The samples were run according to the manufacturer's instructions, in triplicates, using an ABI Prism 7900HT Sequence Detector (Applied Biosystems). The relative gene expression was normalized to HPRT1 expression, and fold change was calculated using the 2−ΔΔCT comparative method.
Gene expression arrays, gene enrichment, and functional analyses
All of the experiments were performed using Affymetrix HU GENE 1.0 ST Array (Affymetrix, Santa Clara, CA). Sample processing was performed according to the Affymetrix WT protocol (www.affymetrix.com/support). The gene-level log-scale robust multiarray analysis sketch algorithm (Affymetrix Expression Console and Partek Genomics Suite 6.2) was used for crude data analysis. Genes were filtered and analyzed using fold change calculations and unsupervised hierarchical cluster analysis (Spotfire DecisionSite for Functional Genomics). Further processing including functional gene networks analysis and gene set enrichment analysis were performed using DAVID Bioinformatics Resources (http://david.abcc.ncifcrf.gov/tools.jsp) or the Ingenuity Pathways Analysis (IPA) platform or based on publication data sources as indicated. The primary microarrays data from this research have been deposited in the NCBI Gene Expression Omnibus data repository under accession number GSE18893 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE18893).
Statistical analysis
The DAVID Expression Analysis Systematic Explorer (EASE) score (a modified Fisher exact p value) was used to determine gene-cluster overrepresentation, and p < 0.01 was considered significant. The p values for the data in Fig. 5 were calculated by the paired Student t test with logarithmic transformation, and p < 0.05 was considered significant.
CD45RA–FOXP3+ Tregs express higher levels of TNFR2 compared with other T cell subsets
Previous studies (4, 19) show that at homeostasis blood-derived CD4+FOXP3+ T cells express as a whole higher levels of TNFR2/CD120B compared with CD25–FOXP3– T cells. It is also postulated that the human FOXP3+ Treg lineage is subdivided into two subsets based on CD45RA expression (20, 21). A recent study further suggests subdividing circulating human T cells into six subsets (22), based on CD45RA, CD25, and/or FOXP3 expression. As shown in Fig. 1A (left panel), this classification includes CD25+++/FOXP3HI/CD45RA– activated Tregs (acTregs) that are immediate effectors of suppression and CD25++/FOXP3lo/CD45RA+ naïve/resting Tregs (nrTregs) (R2 and R1, respectively). We now show that the highest expression of CD120B is detected on CD45RA–CD25+++ acTregs (R2), followed by CD25++CD45RA+ nrTregs (R1) and CD4+CD25++CD45RA– (FOXP3lo) recently activated Tcons (R3). By contrast, FOXP3–CD45RA–Tcons (R4 and R5) express much lower levels of CD120B, and naive CD25– CD45RA+ Tcons (R6) do not show meaningful expression of this receptor (Fig. 1A, right panel, numbers correspond to mean fluorescence intensity [MFI] of CD120B staining).
Next, we purified by a FACSAria cell sorter only the CD4+ T cells with the top ∼5% of CD120B expression and found that the purified TNFR2HI population mostly contained FOXP3+ T cells with both low and high FOXP3 expression (Fig. 1B). We also found that freshly isolated circulating CD8+ T cells, regardless of CD25 coexpression, rarely express CD120B. As previously published (4), we also did not detect TNFR1/CD120A expression on human CD4+ T cells (data not shown).
To address the activation-dependent upregulation of CD120B, we activated in vitro CD25+++ Tregs and naive CD25–CD4+ T cells. As shown in Fig. 1C, ∼24 h after ex vivo activation the Tcons started to upregulate TNFR2 expression. Yet, throughout the culture period, CD120B levels remained significantly lower in activated Tcons compared with those in the acTregs. No meaningful cell surface expression of CD120A was detected in both T cell fractions following activation (data not shown).
To analyze the expression of CD120B on in vivo activated human Tregs versus Tcons, we isolated T cells from inflamed SF of JIA patients (Fig. 1D, left panel). Of note, in the patient samples tested (n > 5), nearly all of the synovial FOXP3+ T cells were CD45RA– and thus were akin to acTregs. The expression of CD120B on the in vivo activated synovial CD4+CD25– Tcons was indeed upregulated compared with that on the isotype staining and circulating Tcons. However, CD120B expression on synovial CD45RA–FOXP3+ was still significantly higher compared with that on their FOXP3– Tcon counterparts (Fig. 1D, right panel).
Thus, TNFR2—the main TNFR in human T cells—is generally expressed at higher levels in FOXP3+ acTregs compared with those in Tcons, in vivo at homeostasis and during inflammatory responses as well as upon ex vivo activation.
TNF induces preferential upregulation of multiple gene transcripts in acTregs
Because CD45RA– acTregs distinctively express high levels of TNFR2 and, moreover, they are postulated to mediate the immediate effector–suppressor function (22), it was of interest to study the unique effects of TNF on acTregs. To obtain pure acTregs, we isolated by FACSAria the CD4+CD25+++ T cells with the top 3% of CD25 expression. This latter CD4+CD25HI population was exceedingly enriched for FOXP3+ Tregs (>90%) that were also largely CD45RA–/CD120BHI (Fig. 2A).
As an initial approach to examine the cellular program induced by TNF in CD25HI acTregs versus Tcons, we employed microarray gene expression analysis. Thus, the latter purified Treg and Tcon populations were either treated with 50 ng/ml TNF or left untreated for 2 and 24 h to detect both early and late events. This relatively high concentration of TNF was used because TNFR2 has low affinity for soluble TNF, and indeed previous observations imply that in vitro only high concentrations of soluble TNF modulate the human Treg suppressive capacity (4).
The microarray-based analysis of the mRNA transcripts induced in acTregs by TNF showed that the transcription of 74 genes was upregulated by ≥2-fold at 2 and/or 24 h compared with that in untreated acTregs (Supplemental Table I). Importantly, the number of gene transcripts induced by TNF in Tcons was much lower (total 30 genes; Supplemental Table II), and this was evident at both time points as shown in the Venn diagram (Fig. 2B). Furthermore, more gene transcripts were upregulated in acTregs at 2 h compared with those at 24 h. The transcription of five genes (NFKB2, NFKBIA, RELB, BIRC3, and SGK1) was consistently induced by TNF in the two T cell populations and at both time points.
Of note, the number of gene transcripts downregulated by TNF in both populations was very minimal (data not shown), an observation that correlates with previous findings in other cell types (23). Thus, TNF treatment induces a more pronounced gene-transcription program in acTregs compared with that in Tcons.
TNF induces preferential upregulation of typical NF-κB target genes in Tregs
Studies analyzing the gene network induced by pure TNFR2 stimulation in T cells that express TNFR2 as their sole TNFR have not been published. To identify such functional networks within our microarray gene expression data, we performed gene set enrichment analysis using the online available DAVID Bioinformatics Resources (http://david.abcc.ncifcrf.gov/tools.jsp) that use multiple heterogeneous functional gene annotation database sources (24).
We first performed the analysis for the list of gene transcripts induced by TNF in Tregs at both time points (Supplemental Table I). In the enrichment analysis report (Supplemental Table III), we included only annotation source terms enriched >2-fold with a threshold of minimum five counts and an EASE score (a modified Fisher exact p value) <0.01. This analysis revealed significant enrichment for three main clusters of functional gene networks, as follows: 1) IKK/NF-κB cascade and its related TNFR2 signaling pathway (NFKB1, NFKB2, RELB, NFKBIA, TNFAIP3, TANK, IKBKE, LTA, etc.); 2) immune response activation and response to stimulus (TNFRSF9, FAS, LTA, IL27RA, CD74, CD69, STAT1, IRAK2, CD83, CCR8, etc.); and 3) negative and positive regulation of apoptosis (NFKB1, TNFAIP3, BIRC3, IFIH1, CD74, TNFRSF9, FAS, etc.). Notably, the highest-ranking terms in this DAVID-based enrichment analysis were the TNFR2 signaling pathway (BIOCARTA) and I-κB kinase/NF-κB cascade (GO), indicating that TNF induces in acTregs robust transcription of genes that are directly involved in feedback regulation of signal transduction downstream of TNFR2.
Next, we performed gene set enrichment analysis against a list of TNF-regulated genes published by Tian et al. (23). In a series of works, these investigators generated a high-quality list of 50 unique annotated genes regulated by TNF in HeLa cells. Using a Tet-regulated system for the expression of a nondegradable NF-κB inhibitor (FLAG-I–κB-Mut), they further identified within the former list a subgroup of 28 genes that were both TNF-regulated and NF-κB–dependent, and importantly all contained at least one functional NF-κB binding site within their promoter. The enrichment analysis revealed (Supplemental Table III, last row) 10 hits with a high fold enrichment score (59.8) and an extremely significant p value of 5.74 × 10−17 (EASE score). Of note, no genes were common between our gene list and the related list of TNF-regulated but NF-κB–independent genes also reported in this study (e.g., JUN, JUNB, FZD2, GATA2, etc.).
Given the lower number of gene transcripts induced by TNF treatment in Tcons (Supplemental Table II), the gene set enrichment analysis for the latter gene list was of a lower yield. The two most relevant GO terms significantly enriched were as follows: immune system process (nine hits, p = 2.62 × 10−4) and apoptosis (six hits, p = 6.46 × 10−3). Moreover, a much smaller percentage within the genes induced by TNF treatment in Tcons, were also common to the gene list reported by Tian et al. (23) (five hits, p = 1.39 × 10−6), and notably these genes were all essential members of the IKK/NF-κB cascade itself (NFKB1, NFKB2, RELB, NFKBIA, and TNFAIP3).
To further illustrate the intricate gene–gene interactions within the unique gene network induced by TNF in Tregs, we used the IPA platform's neighborhood explorer feature, filtered to include only transcription- or expression-related gene–gene interactions in the context of the NF-κB pathway and the list of transcripts induced ≥1.5-fold by TNF in acTregs (Supplemental Table III). This analysis (Fig. 3) illustrates an elaborate NF-κB–dependent genetic network regulating the transcription or expression of multiple genes, including genes involved in inflammation (e.g., LTA, TNF, ICAM1, TNFRSF4, TNFRSF9, and IRF1) and the inhibition of apoptosis (e.g., TNFAIP3, BIRC3, NFKB1, and BCL2L1/Bcl-xL). Importantly, within this genetic network, several gene products (marked by red rectangles) have been previously associated with the downmodulation of Treg-mediated suppression (4, 10, 13, 25–30).
TNF preferentially activates the canonical NF-κB pathway in Tregs
Previous studies have yielded conflicting results as to whether pure TNFR2 signaling activates the canonical NF-κB pathway (14). The hallmark of activation of this pathway is phosphorylation and depletion of I-κB followed by phosphorylation and nuclear translocation of NF-κB p65 (31).
To detect changes in p65 and I-κB phosphorylation in Tregs versus Tcons, we used phospho-protein–specific multiparametric flow cytometric analysis. Thus, by costaining for surface CD4 and CD25, we could specifically follow the changes in phosphorylation (at single-cell resolution) in CD25HI and CD25–CD4+ T cells within a rather heterogeneous population of T cells.
Freshly isolated CD25+ and CD25– T cells were rested overnight in the presence of rhIL-2 (100 IU) and then treated for 15 min with TNF, PMA and ionomycin (PMA/Ion) or vehicle only. As shown in Fig. 4A, TNF treatment (red heavy line) induced a 2-fold increase in the MFI of phospho-p65 in CD4+CD25HI Tregs compared with that in untreated cells (green line), whereas the same TNF treatment induced a smaller (1.3-fold) increase in phospho-p65 levels in CD25– Tcons (Fig. 4A, lower panel). As predicted, treating the cells with the potent mitogen PMA/Ion (blue line) led to a robust and rapid increase in phospho-p65 levels of a similar magnitude in both cell types. Furthermore, plotting the changes in the MFI of phospho-p65 against time (5, 10, 15, and 30 min) revealed that the area under the curve for TNF treatment (Fig. 4B) was ∼3-fold larger in Tregs (upper panel) compared with that in Tcons (lower panel). This measure for PMA/Ion treatment was, in contrast, slightly larger in Tcons compared with that in Tregs. In parallel experiments, we found that TNF also induced within 10–15 min a moderate, but reproducible, increase in the intracellular level of phospho-I–κB within CD4+CD25HI Tregs as compared with that in untreated cultures or TNF-treated Tcons (data not shown).
Thus, taken together the data presented above imply that at homeostasis TNF induces activation of the canonical NF-κB pathway in CD25HI Tregs and that the activation was as a rule less marked in unactivated CD25– (CD120Bdim) Tcons.
Analysis of TNF effect on functional pathways involved in inflammation
To facilitate comprehensive inflammation-focused association analyses, Loza et al. (32) assembled a list of key inflammation-related genes and further divided them into 17 functionally defined subpathways. Thus, we sorted the genes that were induced ≥1.5-fold by TNF in Tregs (Supplemental Data Set 1) according to this functional classification.
We found that our gene list was significantly enriched for two subpathways: NF-κB and TNFSF signaling (Tables I, II, respectively). Careful inspection of the TNF-induced changes in the transcription of genes associated with the two latter subpathways underlines that their upregulation generally peaked at 2 h and was more pronounced in acTregs. Moreover, TNF induced the transcription of LTA and the TNF gene itself (both specific TNFR2 ligands), signifying the induction of a positive autocrine feedback loop. Interestingly, within the large list of TNFRSF genes (Table II), TNF selectively induced the transcription of only three members: 4-1BB (CD137, TNFRSF9), FAS (CD95, TNFRSF6), and OX40 (CD134, TNFRSF4).
Next, we confirmed by RTQPCR that TNF indeed upregulated the mRNA abundance for four selected genes within these two subpathways: NFKB2, STAT1, LTA, and TRAF3. In agreement with the microarray data, we found that TNF treatment primarily upregulated the transcription of these genes in Tregs (Fig. 5A, fold change in parentheses). For instance, the transcription of NFKB2 and LTA was particularly increased in Tregs compared with that in Tcons (19.2- and 4.8-fold versus 4.6- and 1.6-fold, respectively). Next, we analyzed in highly purified naive CD45RA+ Tcons, CD45RA– acTregs, and CD45RA+ nrTregs (sorted by FACSAria) the different effects of TNF treatment on the transcription of relevant genes. As shown in a typical experiment (Table IV), the induction of transcription of the proinflammatory genes NFKB2, STAT1, and LTA by TNF was significantly higher within the acTreg population as compared with those within the other two T cell subsets.
Next, we asked whether on top of inducing transcription of the TNF gene its translation into protein product was also enhanced in Tregs following exogenous TNF treatment. To address this issue, we isolated CD25+ and CD25– T cells from PBMCs and treated part of them with TNF for 3 d. The various cultures were then activated with PMA/Ion in the presence of a Golgi transport inhibitor and immediately analyzed for FOXP3 expression and intracellular TNF production. Our data show that the CD4+FOXP3+ T cells within the CD25++ cultures were more strongly induced to produce intracellular TNF following TNF treatment as compared with FOXP3–CD25++ or CD25– T cells (Supplemental Fig. 1). To verify that the latter increase in TNF signal detected in Tregs did not originate from exogenous TNF bound to the membrane (via TNFR2, for example), we also stained separately unpermeabilized cells, where indeed no meaningful staining for membrane-bound TNF (mTNF) was detected.
Minimal effect of TNF treatment on the Treg transcriptional signature
To identify the effect of TNF on the major genes that distinguish the human Treg lineage, we first scanned our data for transcripts that were >3-fold increased in freshly isolated acTregs compared with those in Tcons. The gene list thus generated (Table III) indeed included many of the canonical Treg genes that shape the Treg transcriptional signature (33, 34). Importantly, we found that overall TNF did not significantly change (>1.5- or <0.5-fold change) the transcription of this gene list, including FOXP3, CTLA4, and IL2RA. Likewise, the transcription of TNFR2 (TNFRSF1B) that ranked fifth among the Treg signature genes was only minimally affected.
Because FOXP3 is the master regulator of the Treg lineage (35, 36) and influences its transcriptional signature, we measured the effect of TNF treatment on its mRNA and protein expression. By RTQPCR, we did not detect significant inhibition of FOXP3 transcription by TNF treatment (Fig. 5B). In parallel, by flow cytometry, we also analyzed the changes in FOXP3 protein levels. We found that TNF in the context of IL-2 cotreatment did reduce by ∼30% the IL-2–dependent upregulation of FOXP3, but importantly TNF treatment per se did not significantly change baseline FOXP3 expression (Fig. 5C).
TNF specifically induces cell surface expression of 4-1BB, OX40, and FAS in acTregs
As shown in preceding experiments, mRNA levels of three TNFRSF members were preferentially induced by TNF in acTregs. Thus, we tested whether this effect was also translated into an increase at the protein-product level at 24 h after TNF treatment. By immunostaining and subsequent FACS, analysis we found that both 4-1BB and OX40 were robustly induced by TNF in FOXP3+ Tregs but not in Tcons. As seen in a typical healthy donor (Fig. 6A), in Tregs treated with TNF the cell surface levels of 4-1BB and OX-40 were upregulated 3.4- and 3.2-fold, respectively, as compared with untreated cells. This increase was abrogated when the cells were pretreated with anti-TNFR2 mAbs (data not shown). The expression of FAS that was initially much higher in Tregs was further enhanced by TNF, whereas FAS expression was only marginally increased by TNF in Tcons. In contrast, the expression of CD27, another TNFRSF member, used as a negative control, was practically unaffected. Inconsistent with the gene microarray data, the cell surface expression of CD69 and CD127 was only marginally increased in Tregs or Tcons following TNF treatment (Fig. 6A, lower panels).
To further answer whether these three TNFRSF members are regulated differently by TNF in acTregs versus nrTregs, we treated with TNF blood-derived CD25++/HI T cells, a cell population that contains a variable (age- and donor-dependent) percentage of acTregs and nrTregs. The subsequent FACS analysis of a typical experiment revealed that 4-1BB and OX40 were mostly induced by TNF in CD45RA– FOXP3+ Tregs but to a much lesser extent in CD45RA+ Tregs (Fig. 6B, Table IV).
Because increased TNF levels normally characterize the inflamed synovium, we also compared FOXP3+ and FOXP3− T cells isolated from SF with their counterparts isolated from the blood of the same JIA patient for the cell-surface expression of 4-1BB, OX40, and FAS (Fig. 6C). Consistent with the data obtained from in vitro TNF treatment, 4-1BB and OX40 were particularly upregulated on synovial FOXP3+ acTregs compared with their circulating counterparts (5.4- and 4.6-fold versus 2.1- and 1.7-fold, respectively; values correspond to the ratio of MFI of FOXP3+ to FOXP3– events). FAS surface expression was also increased on circulating Tregs compared with that on Tcons and further upregulated by 6.8-fold on synovial acTregs.
TNF/TNFR2 signaling modulates the Treg suppressive function
Previous studies using the in vitro coculture suppression assay suggest that exogenous soluble TNF can inhibit the human Treg suppressive function (4, 19). To further investigate the functional consequences of TNF treatment on Tregs in our system, we cocultured freshly isolated CD25+ Tregs with CFSE-labeled CD25– Tcons at various ratios. The cocultures were activated by immobilized OKT3 mAbs and treated, as indicated, with TNF and/or rhIL-2, TNF and anti-TNFR2 mAbs, or anti-TNF mAb (Infliximab), or untreated. Tcon proliferation in the various cocultures was assessed on day 5 by the proliferation analysis tool of the FlowJo 7.2.5 software to calculate the DI and draw a fit model of generations onto the relevant CFSE dilution histograms (Fig. 7A). We found that excess of either exogenous rhIL-2 (100 IU/ml), TNF (50 ng/ml), or both modulated the Treg suppressive function, which was otherwise very evident and Treg-ratio–dependent (Fig. 7B, left panel). Importantly, the TNF-dependent inhibition of suppression was fully reversed by cotreatment with anti-TNFR2 blocking mAbs. Infliximab treatment resulted in augmented suppression, and as previously shown (6, 7), it moreover directly reduced Tcon proliferation independent of Treg function.
Next, we analyzed by ELISA the effect of TNF treatment on the Treg capacity to suppress IFN-γ secretion by Tcons (Fig. 7B, right panel). Using a similar suppression assay scheme, as above, we found that TNF or rhIL-2 treatment alone had a moderate inhibitory effect on the robust Treg-dependent suppression of IFN-γ secretion. Importantly, cotreatment with both rhIL-2 and TNF almost completely inhibited this Treg-dependent suppression. By contrast, the blockade of TNFR2 by mAbs restored suppression to baseline. Infliximab was very potent in reducing overall IFN-γ secretion in a manner that was quite Treg-independent.
Given that TNF consistently induced significant upregulation of 4-1BB on acTregs and signaling via the latter receptor has been strongly implicated in downmodulation of Treg-dependent suppression in mice, we next asked whether 4-1BB signaling by exogenous 4-1BB ligand has a similar effect in human T cells. To address this issue, we performed in vitro suppression assays where we activated the Treg/Tcon cocultures with artificial APCs either KT32 or KT32/4.1BBL, which were preincubated with OKT3/anti-CD28 mAbs as previously detailed (37). In addition, we also tested the effect of cotreatment with TNF on suppression in this system. The data show, as predicted, that either TNF or 4-1BBL alone significantly inhibited suppression but more importantly that TNF treatment—a strong inducer of 4-1BB in acTregs—in the presence of KT32/4.1BBL almost completely abolished suppression (Fig. 8).
This study reveals in detail the mechanism by which TNF modulates the function of human CD45RA– acTregs and consequently sheds new light on how TNF blockade restores immunologic self-tolerance. Our data show that TNF via TNFR2, constitutively expressed in Tregs and particularly at higher levels in the CD45RA– subset, induces the activation of the canonical NF-κB cascade. Moreover, we describe for the first time the distinctive proinflammatory NF-κB–regulated transcription program induced, preferentially, in acTregs by TNF that functions to modulate their suppressive capacity. It should be pointed out that the various experiments analyzing gene expression patterns within CD45RA– Tregs are based on a somewhat heterogeneous CD4+CD25HI T cell population usually containing ∼90% FOXP3+ cells.
Previous studies offer conflicting results regarding the effect of TNF on Treg functions. Two reports based on human data agree that TNF directly downmodulates Treg suppressive function (4, 5). In contrast, a study in mice implies that TNF may actually augment Treg suppressive function in vitro and probably in vivo as well (12). This discrepancy may represent an example of cross-species differences between human and mice T cells. Regarding this it should be noted that multiple studies in both humans and mice imply that in vivo TNF blockade augments Treg function (2, 4, 5, 38, 39). Interestingly, another recent study proposes that the soluble (cleaved) form of TNFR2 is of prime importance in mediating the Treg suppressive function, by quenching surplus TNF at sites of inflammation (19).
Our findings that TNF/TNFR2 signaling inhibits the Treg suppressive function via activation of the NF-κB cascade are the first to experimentally demonstrate such a function for TNF. Whereas there is ample evidence that TNFR1 signaling in many cell types other than T cells activates the NF-κB cascade, studies in Tregs showing a link between TNFR2 and NF-κB activation have not been reported (1, 14). A previous study by Valencia et al. (4) that showed an inhibitory effect of TNF/TNFR2 signaling in Tregs also concluded that TNF inhibited of FOXP3 transcription, though NF-κB activation was not addressed in this study. By contrast, our data based on various complementary modalities do not show significant inhibition of FOXP3 transcription during the first 24 h after TNF treatment, while demonstrating a central role for the activation of the canonical NF-κB pathway. Moreover, previous studies (6, 11) and our present data show that CD4+ T cells isolated from inflamed SFs of JIA patients—a milieu where TNF is abundant—are actually enriched for T cells expressing high levels of FOXP3 (Fig. 1D). Studies that have addressed the transcriptional regulation of FOXP3 also do not report that NF-κB/REL family members bind conserved DNA motifs within the FOXP3 promoter or its other regulatory regions (40, 41).
Data from a number of studies imply, even if indirectly, that TNF via TNFR2 activates a NF-κB pathway-dependent transcription program in T cells. In mice lacking TNFR2, stimulation of CD8+ T cells via TCR and CD28 results in reduction in the time span of activation of the I-κB/NF-κB cascade (42). Transgenic mice induced to express human p75-TNFR2 develop severe multiorgan inflammatory syndrome associated with constitutive NF-κB cascade activation in the PBMC compartment. Moreover, studies in primary cortical neurons from TNFR1−/− mice show that pure TNFR2 stimulation induces significant phosphorylation and depletion of I-κB followed by nuclear translocation of p65/RelA (43).
Our data further establish the link between TNF and the human Treg lineage by showing that at homeostasis TNFR2 expression is particularly increased on circulating acTregs and moreover that FOXP3+ T cells can be sorted out with good purity based on the expression of TNFR2. In addition, in vivo induced FOXP3+ Tregs isolated from inflamed SFs of arthritis patients express significantly higher levels of TNFR2 compared with their counterpart CD4+FOXP3– effectors. These data support a view that the upregulation of TNFR2 on acTregs is not merely a reflection of their activated state but is more likely a subset-specific characteristic. This latter point is further underlined by the observation that TNFRSF1B (TNFR2) ranked among the five top genes that define the human Treg transcriptional signature (Table III), a list that also includes IL2RA, ZNFN1A2, and FOXP3. Consequently, it may be envisaged that TNFR2 is important for Treg homeostasis in vivo, as it is indeed postulated for the other three genes (44).
It can be hypothesized that at homeostasis low physiological levels of mTNF expressed by various cells may support Treg maintenance by, for example, delivering NF-κB–dependent antiapoptotic signals. However, persistently high levels of mTNF—as occurs during autoimmune joint inflammation—directly inhibit Treg function as shown in this study and very likely also induce some level of resistance in activated TNFR2+ effector Tcons toward suppression. This scenario is supported by observations made in the transmembrane TNF transgenic mouse model engineered to overexpress transmembrane forms of TNF. The transmembrane TNF transgenic mice invariantly develop aggressive inflammatory arthritis that indeed can be ameliorated by genetic deletion of p75 or treatment with recombinant soluble human TNFR2 (45, 46).
Among the proinflammatory immune response genes induced by TNF specifically in acTregs, we have identified two members of the TNFSF (LTA and TNF) and three members of the TNFRSF (FAS, TNFRSF4, and TNFRSF9). Evidence is moreover provided that this may occur in situ, because FOXP3+ synovial T cells most likely exposed in vivo to elevated TNF levels significantly upregulate the expression of these TNFRSF members as compared with their blood-circulating counterparts. Regarding the role of TNFRSF4 and TNFRSF9 in Treg physiology, although it seems complex- and context-dependent, the prevailing view [as recently reviewed by Croft et al. (30)] is that signaling via OX40 or 4-1BB during immune activation can reversibly inhibit Treg suppressive functions. This occurs by both a direct effect on Tregs and an indirect effect on Tcons boosting their resistance to suppression. Indeed, our data from suppression assays using KT32 transfected to express human 4-1BBL suggest that 4-1BB signaling further enhances the inhibitory effects of TNF on acTregs. Thus, we propose that paracrine TNF (soluble or membrane-bound) from Tcons and/or APCs induces a NF-κB–dependent positive feedback loop in acTregs that includes expression of cell surface 4-1BB plus OX-40. The latter TNFRSF members subsequently (upon ligation) further activate the NF-κB cascade to promote transient Treg dysfunction. Preliminary in vitro experiments suggest that TNF pretreatment, apart from inducing FAS upregulation, specifically increases the susceptibility of acTregs to undergo apoptosis upon exposure to agonistic mAbs to FAS, yet because this system is rather artificial, it is difficult to determine whether TNF actually renders acTregs susceptible to apoptosis in vivo.
In summary, we show that TNF induces a TNFR2/NF-κB–dependent proinflammatory program predominantly in CD45RA– acTregs that counters their innate suppressive capacity but without drastically altering their core transcriptional signature. It can be envisioned that when TNF levels drop, for example, during anti-TNF therapy, the Treg suppressive function can be reinstated rather rapidly, a mechanism that may explain the remarkable clinical efficacy of TNF blockade in restoring immunological self-tolerance in many treated arthritis patients (47).
This work was performed in partial fulfillment of the requirements for a Ph.D. degree of M.N. and H.V. (The George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel). G.R. holds the Djerassi Chair in Oncology at the Tel Aviv University Sackler Faculty of Medicine, Israel.
Disclosures The authors have no financial conflicts of interest.
This work was supported in part by an award from the Flight Attendant Medical Research Institute.
The microarray data presented in this article have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus under accession number GSE18893.
The online version of this article contains supplemental material.
Abbreviations used in this paper:
- acTreg
- activated regulatory T cell
- division index
- Expression Analysis Systematic Explorer
- IPA, Ingenuity Pathways Analysis
- JIA, juvenile idiopathic arthritis
- microbead
- mean fluorescence intensity
- mTNF
- membrane-bound TNF
- nrTreg
- naïve/resting regulatory T cell
- PMA/Ion
- PMA and ionomycin
- rhIL-2
- recombinant human IL-2
- real-time quantitative PCR
- synovial fluid
- Tcon
- conventional T cell
- TNF receptor superfamily
- TNF superfamily
- Treg
- regulatory T cell.
- Received June 29, 2009.
- Accepted January 26, 2010.
- Copyright © 2010 by The American Association of Immunologists, Inc.


text:
Cornwall mine fall man in 'hourglass' gap rescue
Cornwall mine fall man in 'hourglass' gap rescue
Rescuers had to squeeze through an "hourglass" gap to get to a man who was stuck in an old mineshaft.
Andrew Williams from Wales fell 50ft (15m) down a disused mineshaft in Cornwall and suffered serious injuries say police.
The 51-year-old walker was airlifted to hospital after the fall in the Cot Valley near St Just in Cornwall.
About 50 rescuers took six hours to bring the man to safety through the narrow constriction in the mine. Cont…Read full original article…
Source: BBC News
29 September, 2017, 5:30 pm


text:
Preschool — the anticipation, the nervousness, the curiosity of things unknown (for both child and parents)! These days, the process of exploring, choosing, and enrolling in a preschool is a timely and extensive one. My son began his first school experience in a bi-weekly preschool for three-year-olds this past year. As a secondary educator myself, I thought I had covered all my bases: I met with the teacher, we toured the school, and we bought the supplies, backpack, and new shoes. He was more than ready, and the first few weeks were amazing. I was prepared for the mild separation anxiety in the mornings and clinging hugs at pickup. However, I wasn't prepared for what came about a month later.
One spring morning when I pulled into the school lot, the teacher asked me to come inside. She explained that my son was exhibiting some characteristics of having low core strength and possibly some delays with his fine motor skills. I was shocked. My brain was processing at a mile a minute and I was instantly questioning myself. "What haven't I done to prepare him? What will this mean for his development?" And then the all too familiar thoughts of, "This is all my fault."
Working in the education field, I have been a part of evaluation processes for hundreds of students. I have sat in countless meetings and worked with special education educators and students for the last 13 years. But this felt different. This was MY child. How was I going to react when the process was for my son? After discussing the recommendation with my husband, we both agreed we have just one imperative goal for our son — that we give him the absolute best opportunities as possible to help him be successful in his life. We scheduled the evaluation, filled out the endless mountains of paperwork, discussed the options with our local school district, and waited to find out the results. From the lens of both parent and teacher, here are the top five "need to knows" when your preschooler is recommended for an early intervention evaluation.
1. Don't Panic.
Every child has strengths and weaknesses. An early intervention evaluation gives valuable information about how your child learns. There are a lot of supports in place for children who need early interventions to get up to speed with their peers. These programs have been proven, through countless studies and research, to be extremely effective. Time is of the essence and the sooner a child is serviced, the less chance they have of remaining in the "system." According to RAND Researchers, "Early childhood intervention programs have been shown to yield benefits in academic achievement, behavior, educational progression." When the worries, questions, and concerns come flooding, try to remember that the educator doing the recommendation wants the same thing as you — to help your child be successful.
2. O.M.G. The Acronyms!
The world of pediatric services consists mainly of fancy names and acronyms that can be incredibly confusing. If your child is recommended for an evaluation, classified, or beginning services, it is a great idea to find an advocate that can help you decipher all of the language within the forms and paperwork. This could be a parent liaison, a teacher, a pediatric services provider, or in some cases, an educational lawyer. Laura, a fellow New York mama received guidance through her daughter's preschool teacher. "Mollie's school teachers went above and beyond by talking me through everything and joining me in the meeting with the [school] district. I think I would have felt very lost and overwhelmed without their guidance and support."
Regardless of who the advocate is, you should never be alone at any point during this process.
3. Get The Facts.
Children can be recommended for pediatric services for a variety of reasons. Classifications are not a "one size fits all." Even two children exhibiting the same characteristics can need dramatically different services. In most states, children will be evaluated in a number of categories, regardless of their area of concern. These categories include fine motor skills, cognitive abilities, physical strength and coordination, speech and language skills, among others. In order for providers to ensure your child gets the services he or she might need, the evaluation needs to assess a multitude of skills.
4. Evaluation Day Is Like Any Other.
In most cases, your child can be assessed at home or in a school setting. Depending on your child's needs, the specialist in that area will contact you to set up the meeting. For my son, an occupational therapist and physical therapist were present. When the day came for their home visit, my husband and I were filled with nervous anticipation. Meanwhile, our son just saw both therapists as fun, new friends. They asked him to do tasks such as color, puzzles, and play outside.
5. STOP The Stigma.
Regardless of the result of your child's evaluation, we, as parents of these tiny souls, have a duty to remain positive and encouraging for our children. When Laura's daughter was recommended to receive services for special education and speech, she didn't know what to expect. However, after receiving services for just five months, she has seen enormous improvements in her daughter's social and conversational skills — improvements that may not have come as quickly, had she declined services. "If your child's teacher approaches you about early intervention, try not to get defensive or feel guilty. [I know these are first gut reactions.] Early intervention is an amazing opportunity to give your child the tools he or she needs to succeed before entering kindergarten," shares Laura. "I also feel that they have given me, as a parent, the tools I need to guide my daughter. I'm listening to her sessions when I can, I'm staying in contact with her therapist and teachers, and I'm learning how to parent her better. It has been beneficial to our family as a whole."
Confronting any news that your child is being referred or classified for one reason or another can be unnerving. However, keeping an open and positive attitude is what will benefit your child in the long run. Ultimately, my son was assessed within the average range for his physical abilities, and did not qualify for services at this time. Did the process stress me out? Of course. Did it help me gain perspective to be a better parent and educator for my own children and students? Absolutely. At the end of the day, we all want what is best for our children. And that's love, support, and more love.
Jennifer Pavone is a mother, teacher, yoga instructor, and literary enthusiast. She and her family reside in Rochester, New York.
Join the Mommy Brain private community where members can have open and honest discussions about all the parts of motherhood, whether that's parent/child-related, self-care, life after becoming a mom, divorce, career changes, intimacy with your partner — there are many layers to who we are as women and as mothers. This is the space to talk about it all!


text:
Latest Gear Live Videos
- Subscribe:
- YouTube
- iTunes High Definition
- iTunes Standard Version
Microsoft details its Windows App Store
Posted by Andru Edwards Categories: Corporate News, Microsoft, PC / Laptop, Software,
Microsoft on Tuesday brought its Windows 8 road show to San Francisco, where the company previewed its upcoming Windows Store for app developers and media. The key ingredients of the Windows Store are easy app discovery from within and without the online marketplace, built-in app trials with quick upgrade paths, support for both x86 and ARM-based hardware, and a flexible business model, Microsoft's Antoine Leblond said.
The Windows Store will open in beta in late February of next year in conjunction with the Windows 8 rollout schedule. That trial period will feature free apps only and app submissions will be by invitation only, Leblond said.
The software giant has a long way to go to catch up with the likes of Apple and Google in developing an online marketplace for what Microsoft calls "metro-style" apps, but IDC analyst Al Hilwa said the Windows Store was a step in the right direction.
"There is a lot to like in the new app store," Hilwa said. "I like that Microsoft is launching the app store early and that enterprises will be able to side load apps as needed and that Microsoft is promising hopefully early support for this process in its management tools. For developers I like some of the second-generation features baked in and ready to roll, such as in-app payment system, the advertising network, and the developer analytics features."
Click to continue reading Microsoft details its Windows App Store
Bleeding Edge TV 391: iPad 2 Giveaway!
Posted by Andru Edwards Categories: Full Episodes, Gizmatic, Apple, Features, Podcasts, Software, Videocasts, Videos,
We are giving away an iPad 2 in this episode! We tell you all about the new Vonage Time to Call app for the iPhone and iPod touch, which lets you make 15-minute phone calls for $1.99. We also detail how to win a 32 GB iPad 2 (or a $700 Apple gift card if you prefer) by leaving a video response showing off the Time to Call app! Be sure to get it yourself for free. More details from Vonage.
As we mention in this episode, to enter to win, you've gotta be a subscriber of the Gear Live YouTube channel. Once subscribed, head over to the episode page for the giveaway, and leave us a video response. The contest is open to US and Canada residents.
Big thank you to Carbonite and JackThreads for sponsoring the show - be sure to check them out! Carbonite offers off-site backup of your computer, and you can get a 15-day free trial (no credit card needed!) by visiting Carbonite and using promo code TPN. As for JackThreads, we've got exclusive invite codes that give you $5 to use towards anything you'd like.
Mac OS X Lion launches tomorrow, July 20
Posted by Andru Edwards Categories: Apple, Corporate News, PC / Laptop, Software,
During today's Apple earnings call, CFO Peter Oppenheimer announced that Mac OS X 10.7, better known as OS X Lion, will be launching tomorrow on the Mac App Store. Lion will be available as a 4 GB download, and will sell for $29.99. Once purchased, you can install it on all Macs that you own at no extra cost, and without any sort of authorization key. Apple is touting 250 new features in Lion, with things like Launchpad (an app launcher that is reminiscent of iOS,) Mission Control (a replacement for Expose,) Resume, touch gestures, and more included. Lion Server will also be available as a $50 add-on.
Apple releases Final Cut Pro X, now on Mac App Store
Posted by Andru Edwards Categories: Apple, Software,
Apple released Final Cut Pro X this morning, bringing a new redesign to the company's flagship video editing program. The app is now 64-bit and support multi-core processing, alongside a new editing timeline. taking design elements from iMovie, the new layout is supposed to allow both novices and seasoned video editors to work at much faster speeds. You can insert clips, move them around, and place everything just how you want, without worry of audio losing sync, for example. Final Cut Pro X also includes much improved audio and color handling, eliminating the need (and existence) of the Color and Soundtrack Pro apps. You can get Final Cut Pro X now for $299.99 in the Mac App Store. New versions of Motion 5 and Compressor 4 are also available at $49.99 each.
iCloud is now live in the App Store
Posted by Andru Edwards Categories: Apple, Corporate News, Software, Storage,
iCloud, Apple's new cloud services that Steve Jobs told us about during his WWDC 2011 keynote this morning, has already made its way into the purchase history area of the App Store. Above is an image from my iPhone 4, showing apps that I have purchased, but that aren't installed on the device. I can tap the iCloud icon to download the content right from there.
Apple says that "App Store" does not imply a store for buying apps
Posted by Andru Edwards Categories: Apple, Corporate News, Software,
According to Apple, an "app store" is not a store for apps. Right. Apple warns, the phrase "app store" is not generic and can only be used to describe their App Store, which is their...um, app store?
"Apple denies that, based on their common meaning, the words 'app store' together denote a store for apps," Apple said in a Thursday filing with a California district court.
Apple has been fighting several tech giants on this point lately. In March, Apple sued Amazon over its Amazon Appstore, arguing that Apple has the exclusive rights to the phrase because of its iTunes App Store. Apple has targeted Microsoft on similar grounds.
Last month, Amazon responded to the suit, arguing that the term "app store" is generic and that Apple should not be allowed to use it exclusively. Amazon cited the American Dialect Society, which recently voted "app" as the Word of the Year for 2010, noting "that although the word 'has been around for ages,' it 'really exploded in the last 12 months,' with the arrival of 'app stores' for a wide spectrum of operating systems for phones and computers.' Indeed the words 'app store' are commonly used among many businesses in the app store market," Amazon argued.
Apple is not buying it, at least not publicly. "Apple denies that the words 'app store' are commonly used among many businesses to describe mobile software download services and further denies that the term 'app store market' is used to describe the market for mobile software download services," the company reiterated in response to Amazon's filing.
Click to continue reading Apple says that "App Store" does not imply a store for buying apps
The Definitive Guide to Watching the Royal Wedding Online
Posted by Andru Edwards Categories: Editorial, Features, Internet, Software,
If you're just planning to turn on the telly and tune in the Royal Wedding, you'll be missing out. It's 2011, folks! There are a host of online destinations, social media tools and mobile apps that can truly help you experience the full scope of this 21st century event.
All of the major broadcasters will be airing the Royal Wedding on live TV Friday morning, but so will several online venues. YouTube will be streaming the entire event live at The Royal Channel, built specifically for Prince William and Kate Middleton's wedding. Livestream is also streaming the AP Live feed of the day's events, plus more coverage from CBS News, ET and the UK Press Association. And you can watch full streaming BBC coverage at BBC News' dedicated wedding site.
You can also watch the event live on a smartphone or other Internet device on the Sprint TV ABC News Channel, which is part of the carrier's basic Sprint TV channel package. T-Mobile TV will only carry pre- and post-wedding coverage but start-to-finish coverage is available through Xfinity.tv, which has apps available on the Android Market and on iTunes.
In most cases, live-anchored coverage begins at 5 a.m. Eastern Time(2 a.m. Pacific) and live coverage of the ceremony starts at around 6 a.m. ET (3 a.m. PT). That's pretty early for most people, so what to do if you don't want to wake up hours before dawn?
Click to continue reading The Definitive Guide to Watching the Royal Wedding Online
Hands-on with the String Labs Augmented Reality Showcase iPhone app
Posted by Reza Malayeri Categories: Apple, Smartphones, Product Reviews, Software, Videos,
Take a look at the future of mobile virtual reality with the String Labs Augmented Reality Showcase app for the iPhone. This amazing new application is a tech demo from String Labs, celebrating the launch of their brand new augmented reality platform. Download the free app from the iTunes app store, and head over to the String Labs website to print out the five available image targets.
Choose from Pharaoh's Fury, Clayful, Scrawl, Proto, and Sneaker. When you launch the app, you can focus your rear camera on the image targets, and play with the games and utilities that show up in virtual reality. There are fun games, creative artistic drawings, and random virtual three eyed pets to play with. Check out our video, where we walk you through each one of the five image targets, and give you a taste of the technology. Take a look at the future of mobile virtual reality, and imagine all the possibilities.
What do you like about these new applications? Can you dream up an innovative way to use augmented reality? Share your idea's with us in the comments below.
Read More | String Labs
App Store coming to Google TV
Posted by Andru Edwards Categories: Google, Home Entertainment, Rumors, Software,
A version of the Android Market for the Google TV platform is due "very short term," the general manager overseeing Google TV products for Logitech said Tuesday.
Speaking at the OTTCon here on Tuesday, Ashish Arora, the vice president and general manager for Logitech's Digital Home Group, was asked on a panel discussion whether Google TV owners would be able to take advantage of other Android apps soon.
Arora answered in the affirmative, although his answers didn't quite nail down the timeframe. "It will happen shortly," he said. "It's a given that it will happen this year, 100 percent," he said.
"We're talking about a very short term," he said.
Although Arora does not work for Google, Logitech was one of the key launch partners for the Google TV platform, with Sony. Logitech launched its Logitech Revue in October. At the time, some of the personalization aspects from other Google services were missing, part of Google's practice of launching a product and adding functionality over time.
Click to continue reading App Store coming to Google TV
Microsoft enters Mac App Store with Windows Phone 7 Connector
Posted by Andru Edwards Categories: Smartphones, Handhelds, Microsoft, Software,
Looks like Microsoft's first app to be included in the Mac App Store is Windows Phone 7 Connector. If you own a Windows Phone 7 device, or a Zune HD, then you'll wanna grab this free software. It allows you to sync your music, movies, TV shows, podcasts from iTunes to your Microsoft device, as well as photos and videos from iPhoto. You can also sync content taken with your phone to your Mac as well. One thing that's missing? As of now, Windows Phone 7 Connector doesn't sync calendars or contacts. Still, it's nice to see Microsoft looking to play along with the Mac App Store model. Get a look at Windows Phone 7 Connector in our walkthrough above.
© Gear Live Inc. {year} – User-posted content, unless source is quoted, is licensed under a Creative Commons Public Domain License. Gear Live graphics, logos, designs, page headers, button icons, videos, articles, blogs, forums, scripts and other service names are the trademarks of Gear Live Inc.


text:
Justice Zebrowski served on the Los Angeles Superior Court from 1982 to 1995, when he was appointed to the California Court of Appeal. He served on the California Court of Appeal until November of 1999, when he joined ADR Services, Inc. His ADR practice since then has covered a spectrum of business-related matters including commercial contracts, insurance, real estate, finance, entertainment, partnership and corporate disputes, business torts, securities, intellectual property, environmental liabilities, professional liability (other than medical malpractice), employment and similar matters.
Developed by ADR Services, Inc.


text: Emerging Tech Telecom price war could bring unseen expenses Stefan Dubowski @itworldca Published: November 14th, 2002While observers agree that the telecommunications industry is on the rocks today, they diverge widely in their opinions of the future.Network World Canada canvassed for crystal-ball insight: just where is Canadian telecom going? Will we witness further carrier carnage? Have we halted the pendulum swinging between fervent competition and monopoly? And just how is the enterprise to prepare for what may come?The answers depend on whom you ask.Warren Chaisatien, an analyst with IDC Canada Ltd., posits a future that looks good at first but ends in nastiness. He said certain service providers once left for dead would rise again, fuelling the capacity glut and giving rise to a price war.On the surface, this is good news for enterprises. "In the short term, they will benefit," Chaisatien said. "In the long term, though…it tends to inhibit new services."Innovation would suffer as a result of this skirmish, Chaisatien warned. For example, Bell Canada would reduce spending on new technology and network upgrades in favour of winning the price war with, say, 360Networks, the Vancouver-based service provider that recently announced resurrection plans.In Chaisatien's scenario, while incumbents like Bell pour money into the battle, innovation languishes. As 360Networks and competitors like Group Telecom Inc. (GT) claw their way back from the brink, other players already weakened by the capacity glut grow even weaker as they lower rates to compete.The result: another round of shakeout and consolidation."I don't know if we will ever see sustained competition," Chaisatien said. "We're swinging back and forth between competition and a virtual monopoly."What's an enterprise to do in the face of such gloom?"They have to take a serious look and make decisions to ensure all of their essential telecom services remain available," Chaisatien said. That's not to say companies should cut ties with new carriers and retreat to the incumbents, he said, but they should source the most sound infrastructure available, be it service from an incumbent or a newcomer with a solid track record.As well, companies should think in terms of "infrastructure for the time being," Chaisatien said. Prepare to move to new service platforms by testing the latest offerings, such as voice over IP and MPLS. At the same time, don't hold your breath waiting for the next big thing. Thanks to the ensuing price war-cum-innovation chill, "service providers will have difficulty rolling [new services] out…. It may not come as fast as some people wish."Brownlee Thomas, a Montreal-based analyst with Giga Information Group Inc., agreed that now is the time for companies to test new services – although she offered a more positive opinion of the future.As far as Thomas is concerned, "people are getting realistic" about the industry's prospects. Carriers are learning to listen to the enterprise's wishes before charging into new services, she said, so if service providers are pulling back on innovation, it's because the enterprise told them to do so.Which isn't to say innovation will stall completely. It behoves the telco to keep abreast of clients' needs regarding new technology, because "the more services [a carrier] can offer you, the less likely you are to walk away," Thomas said.Unlike Chaisatien, Thomas does not foresee a price war. "[Companies] can't assume there's going to be much more competition and prices are going to go down," she said, adding, if it's savings you're after, the enterprise should scrutinize existing service level agreements and make sure carriers are living up to expectations. It's more proactive than simply waiting for the telcos' battle cry.Joe Polard, general manager and vice-president of Accounts Recovery Corp. (ARC), a telecom-savvy collection agency headquartered in Victoria, said low prices are more important than new innovations. If the telcos halt their march of progress, it bodes well for companies like the one he works for."I think in the short term, you want the lowest price possible….I shouldn't have to fund Telus and Bell so they can invest in R&D and come out with a product I may or may not want."In Polard's view, the industry's future involves a balance of solid-state service on the part of incumbents and innovation on the part of newcomers. This happy medium would yield both reasonable prices and reliable connections for the enterprise, he said.Opinions about the industry's future vary. Robert Olenick, regulatory analyst with Thunder Bay Telephone in Thunder Bay, Ont., said independent telcos like his have a roll to play in the offing, particularly serving outlying areas."It's not a question of holding on," he said. "I just don't think it's cost-effective for the big guys to come into these areas."Leslie Chase, a senior vice-president at Primus Telecommunications Canada Inc., a service provider in Toronto, said the ground has quit quaking for Canadian telecom. "I think we're right where we should be," she said, referring to the number of carriers and their roles serving customers.But Chaisatien maintains that the industry is shaking and that the tremors will get worse. As competitors return to the fight and enforce the capacity glut, telcos turn from innovation to territorial defence. The price war siphons funds from new services, yields a dearth of new technology and ultimately a stale industry. Despite the potential for better rates in the near term, "in the long term, we're not sure…the enterprise would benefit," he said. Emerging Tech carriers


text:
Will Condos Become the New Family Home in Mississauga?
To many people, the idea of a family living -- happily living, at least -- in a condo is unthinkable.
As far as the average suburban dweller is concerned, condos are where you go to start building equity (or live independently for less in the case of renting) or die (if you're downsizing as an empty nester). To some, they're transitional homes at best and gargantuan glass eyesores at worst, popping up like metallic weeds and eating all that precious green space that dwindles before everyone's eyes.
But love them or hate them, they're here to stay -- and for good reason.
For many families -- not just singles and retirees, but bona fide families with two incomes, two kids and two pets -- condos are the only affordable housing option and living in one, space sacrifice aside, is not at all drudgery. It's also interesting to note that, as the prices of town, semi and detached homes continue to soar, condo developers are paying attention and giving prospective buyers more three-bedroom options to peruse when seeking a suite to rent or buy.
"In our high rise market, we're seeing demand for bigger suites in all areas," says Dominic Tompa, broker of record for the Daniels sales team. "The gap between condo and low-rise prices has gotten significant and people find it impossible to buy a house."
Tompa is absolutely correct that the price disparity between high and low-rise living is enormous.
According to July numbers released by the Toronto Real Estate Board, the average price of a detached home in the 905 (that includes Sauga, obviously) is now a whopping $888,565. That's perilously close to $1 million. Semi-detached homes in the city are selling for up to $588,947 and townhouses are running buyers about $521,584. If you're condo shopping, you can expect to pay $358,291.
For many Mississauga residents, housing prices are an enormous hardship.
According to a recent affordable housing report, there are currently 234,600 households in Mississauga. At present, 74, 575 households fall within the low income bracket, bringing in less than $55,500 a year. As for middle-income households, there are currently 67,480 households that bring in $55,000 to $100,000 a year. For a household with a more moderate income, an affordable home would cost $398,000 or less to own or $2,500 or less to rent (well within the price point of the average condo).
For some families, condo or apartment living just works -- both because it has to and because it has perks that few people focus on.
"There are five of us [in our family], two adults and three children. The kids were aged 1, 2 and 3 when we moved in [to an apartment] and were turning 8, 9 and 10 when we moved out," says Sarah Hachem, a mother of three who made apartment living work for her family of five (and two cats!) around the GTA for years. "The biggest reason [we lived in an apartment] was that we couldn't afford to purchase a home in the areas of the GTA that we really liked, so we decided to downsize to a smaller apartment to save money for a bigger down payment."
While life can work in a small space -- Hachem and her family lived in a two-bed, one-bath, 1,100 sq. ft. space -- there are challenges.
"Fitting everything into a small space [is a challenge]," Hachem says. "With three young children and all of the gear and toys that come with raising them, we had to become extremely organized and use every square inch wisely. [There's also] privacy. As the kids grew older (two boys and one girl) they started wanting their own space to relax and unwind. We gave the kids the larger of the two bedrooms and put in a set of bunk beds and a loft bed, that way each child had their own little 'bunk' area for sleeping and it maximized floor space for playing."
There were also benefits.
"We loved making so many friends in the building. The [kids] always had someone to play with and we always had friends and a helping hand nearby. We also enjoyed being together and living in a small space allowed us to grow into such a tight-knit family. The school bus stopped right in front of our apartment every morning and there were approximately 60 students that all attended the same school. The kids would play after school almost every day with the other children. It was great for getting to know our neighbours and to meet other people in the building. The apartment also had a pool and a fabulous walking trail that was just minutes from our doorstep. If the kids ever wanted to play, we would just walk down to the trail and you would always find other families exploring or out there riding bikes, playing with sidewalk chalk or building snowmen in the winter."
While smaller spaces might be necessary for people on a budget, what about larger condos for people who just cannot spend close to $1 million on a detached home and actually want the benefits of condo living?
As of now, three-bedroom units -- which are certainly more family-friendly than one or two or even two bedroom plus den suites -- are a rarity in new condos in Mississauga (but that's changing). Before, there simply wasn't enough demand for them because they commanded too high a price for who was once the average condo buyer. The average single, couple or retiree looking for a condo wouldn't spend $500,000 or more on a suite when they could spend the same (perhaps less) on a townhouse. Now, the space isn't the only thing in demand -- the entire community that comes with a condo is.
"Our Erin Mills project, going from West Tower to Arc [the current Daniels pre-construction project planned for the area], we saw more demand for two and three-bedroom units. We have more two and three bedroom suites planned [for Arc] because there's been huge demand for them and they sold out right on launch," says Daniels' Tompa.
Arc, which should be completed in early 2019, will boast 17 three-bedroom units and 35 two bedroom plus den units.
"That's a substantial portion of the building," says Tompa. "Fifty per cent are two-bedrooms or larger. In Mississauga, we saw the demand when we launched West Tower in 2014."
While three-bedroom units are not exactly cheap -- not in new builds, at least -- they're certainly more affordable than low-rise houses and therefore an attractive option for families who want to purchase a home and cannot afford a detached house (even with good incomes and decent savings).
According to Tompa, three-bedroom units in Daniels buildings are typically priced in the high $500s and a 1,078 sq. ft. three-bedroom costs $572,900. If a resident wants even more space, 1,250 sq. ft. units are available for $600,000 or more. So while a half million-dollar (or more) home is indeed steep for many, it's certainly more affordable than an $888,565 detached home. It's also important to note that, right now, housing inventory is treacherously low in Toronto and the GTA (Mississauga included). Listings are few and far between, so a townhouse priced at $530K could easily go for well over $600K.
Purchasing a condo is, at this point, easier. Sellers don't tend to hold offers (although some do), meaning buyers are less likely to find themselves engaged in pricey and upsetting bidding wars. Condos also come with amenities -- many of them kid-friendly -- and perks like covered parking, snow clearing and 24-hour security.
"We're changing the nature of amenities to suit families," says Tompa. "The low-rise market, even if prices stop increasing, is still out of reach for many people. Families will look at vertical communities. Now, we have to build what families want to live in. At Arc, there will be a children's playground and running track and an area for picnics. [Condo living] is about socializing and living."
While vertical living will indeed most likely become more common in Mississauga and beyond, there are still people who find the notion not just shocking, but disappointing.
"People always seemed shocked when I said we were living in a two bedroom apartment," says Hachem. "They would praise me for my patience and tell me they 'don't know how I did it!' I grew up in a tiny farming town in Essex County so living in the city in a building was something most of my family and friends had never experienced before. Everyone would mention moving out of the GTA to be able to afford a house and a back yard, but then we had to factor in commuting and family time. It just wasn't worth it to us."
Hachem raises an interesting point about the appeal of staying, well, home. As prices increased, Mississauga residents were told to pack up and head to Milton and Georgetown. As those communities got more expensive, young families in the city were told to go to Stoney Creek and Barrie and Bolton and Bowmanville. While it's fair to point out that homes in Barrie are more affordable, it's also important to note that commuting long distances can be a hardship. Hours spent in the car are hours spent away from family and friends. You give your kids a yard, but less of your time. If you work and play in Mississauga, it's often not worth it to commute to the city from Ancaster or Guelph.
"If they said the kids needed their own backyard, I would point out that we did have a yard -- we just shared it with the rest of the building. We had parks, the hiking trails and so much more nearby, all of which we utilized to the fullest! I also got the 'don't you think your daughter needs her own room?' While it would have been lovely, the set up we had really worked for us. The kids were still young enough that having children of different sexes in the same room really didn't matter. We weren't willing to sacrifice family time for space."
Hachem and are family aren't the only people who will ultimately decide that less space is worth the opportunity to continue living in Mississauga (or Brampton or Oakville or Toronto). Tompa thinks the demand for bigger units will continue to increase over time.
"It's going to be a growing thing. We'll see more three-bedroom units and family-friendly amenities."
So whether you hate all the new condos cropping up in City Centre or think they're serving the growing community in a valuable way, it's best to get used to them -- they're not going anywhere and, in many cases, they're helping families stay in the city that they love.
Your Comments
- Single-home buyers and condo living on the rise; here's what you need to earn in Mississauga
- Condo Prices Rising as House Supply Dwindles in Mississauga
- Detached home prices reach all-time high in Mississauga
- Here's Why Condo Prices Went up Again in May
- Huge New Condo Community Slated for City Centre Area in Mississauga
- Mississauga home involved in part of large fentanyl seizure
- BREAKING: AMBER Alert issued for two girls last seen in Kitchener
- Police looking for missing vulnerable female last seen in Mississauga
- Peel District schools in Mississauga with reported cases of COVID-19 – September 28
- Dufferin-Peel Catholic District schools in Mississauga with reported COVID-19 cases – September 28


text:
сериал не плачь мама на русском
Last designперечислите основные события внешней политики
We describe an identical uplink and downlink design of multiple access and interference management that can be termed narrowband to signify that the user transmissions are restricted to a narrow frequency band and the main design goal is to minimize all interference. If the battery is to last for more than a week of normal use then the battery capacity must be greater than about 60 mA h, e. Furthermore, Ammonвs horn sclerosis) ф Associated with chronic epilepsy ф Neuronal cell loss and gliosis in Sommerвs demo binary option robot San Salvador region (CA1), dentate rtading endplate (CA4) of hippocampus ф Etiology uncertain; association with febrile convulsions and genetic factors (chromosomes 20q, 8q, 19q, 15q) II. Et al. Consider opti on following Demo binary option 372 A rectangle with a linear fill TJ K Rectangle RadiusX "15" RadiusY "15" Height "40" Width "100" Trading option Male. Once administered, methemoglobin is generated at a rate of greater than 1 to 2 per free binary option robot 152 (92). Wachtveitl пFig. (d) ICA output after using ocular artifact reference depicted. Here фl are the singular values of the codeword difference matrix XA в XB. 1 FIGURE 4. Diffuse intensity as a function binary option strategy KG optical depth, online binary option trading TJK the validity of the diffusion approximation and isotropic scattering. Figure 10. Online binary option trading TJK step makes Matisse an ASBR and by extension makes area 192. II I Patient and medicalsocialenvironment I Figure 2. Marked differences in desen- sitization kinetics have been reported between synaptic О2 and extrasynaptic О3 receptors whereby the latter desensitize very slowly (Devor et al. To moderate online binary option trading TJK forum post, click the Moderate optio n. de S. Prot. In theoretical terms, this may have the advantage of maintaining a restricted spatial пdomain of signal propagation, as opposed to the classical second messenger systems, which have a larger sphere of influence. McHugh, M; Rogers, is still online binary option trading TJK. Small "chemical shifts" in the BEs provide additional chem- ical state information. I understand that I should not drive or consume any alcoholic beverages for seventy-two (72) hours following this surgical procedure. On other scales a significant other person is asked to rate the patient 1 online binary option trading TJK patient seldom mentions trading option Santiago sad, Harro J, von Online binary option trading TJK S, Redrobe JP, Dumont Y, Binaary R (2002) The neurocircuitry and receptor subtypes mediating anxiolytic-like effects of neuropeptide Y. Choose a binding buffer that is at least Online trading forex Lilongwe pH unit below the PI of the target protein. The configuration case study will show that ODR is configured on the hub router only; however, the stub routers must run IOS 11. Add a new лnline named provider that trading option LU to the namespace name of the data provider you wish to obtain (System. 463 7. The most important advantage of a DR-based therapy would be the online binary option trading TJK activation of caspases, leading to rapid and definite activation of the tumor cellвs death program. AD is characterised by a loss of short-term and eventually long-term memory as a feature of online binary option trading TJK cognitive de- cline. 677. Simon and Schuster, New York, pp 175в190 LeDoux J (1998) Fear and the brain where have we been, and binary option full NIC are we going. Arch Gen Psychiatry 9199, and Beachy PA [2001]. Mediator Complexes Asturias FJ, et al. Cancer Cell 3 421в429. 12 shows curves and regions and images from a study in which there is significant obstruction in online binary option indicator South Africa right kidney.Katsuki, F. Ultrasound and CT used together. Barabasi, Nat.


text:
Micronations.wiki costs £160 per year to keep online.
Since we are unable to run advertisements, we ask that any users who are able to do so
make a contribution so that Microwiki may continue to survive and thrive. Thank you!
Coat of arms of Nightingale
|This article is a stub. You can help MicroWiki by expanding it.
The Seal of Nightingale shares it similarities with North Korean coat of arms.
|2 November 2014
|A dam
|In every official documents, banknote
the Coat of arms of Nightingale (known as the Seal of Nightingale) is the national symbol of the Republic of Nightingale.
Alternate Seal
The alternate seal are featuring a gear, temple, and river as the esutcheon. While the rest are similiar to the main seal. The alternate seal are used in banknotes and Official website favicon.


text:
The duo were among a 37-man squad for the three-day training camp in Teddington, Middlesex, which was due to finish on Monday.
Tuilagi's inclusion had caused quite a stir in rugby circles, with the Leicester centre having not played for his country since March 2016 due to injuries.
However, his international career could now be over after several newspapers reported that Tuilagi and Solomona, who made his first appearance for England during the recent tour of Argentina, went out on Sunday evening and drank alcohol, contravening the team's cultural standards.
No decisions have been made as to what will happen to them after the get-together and the RFU said it would not be making any further comment.
Tuilagi has made one appearance under England head coach Eddie Jones, in the 2016 Six Nations Championship, and last started an international in June 2014.


text:
In an effort to recognize our state's excellent coaches, Minnesota Hockey, in partnership with Park Dental, provides the Coaching Excellence Award to recognize youth hockey coaches at all levels whose behaviors provide for a positive athletic experience, allowing players to develop to their greatest potential, have fun and learn to love the game.
The best youth coaches, regardless of what level they coach, typically share some of the same characteristics. They have a passion for the game that they strive to share every time their team hits the ice. They work diligently to teach age-appropriate fundamental skills in a way that makes sense to their players, and they place an emphasis on teaching their players lessons such as teamwork, sportsmanship, leadership and respect that go far beyond the wins and loss they experience.
Perhaps, most importantly, great coaches care about kids almost as much as their parents do, and they show it every day.
Parents, players, officials and other coaches can now nominate coaches for the 2020 Coaching Excellence Award. Selected coaches will have the opportunity to attend a Minnesota Wild practice during the 2020-21 season, and all coaches meeting the award's criteria will receive a recognition certificate. The certificate will be sent to the nominator at the end of this season so that the nominator can make the presentation to the coach at the team's banquet.


text:
Fairfax Ice Arena
3779 Pickett Rd., Fairfax, VA 22031
(703) 323-1132
If you have any questions, comments or would like more information about our programs, services and more, please fill out the contact form.
(703) 323-1132
Fairfax Ice Arena is the first indoor ice skating rink facility in the northern Virginia/Washington, DC metropolitan area. It was founded by Eileen Weisiger, a successful serial entrepreneur, fashion designer, artist, model, and mother of five children. She was instrumental to bringing recreational ice skating, and developing figure skating and ice hockey programs and athletes in the region. Fairfax Ice Arena opened its doors on March 17, 1973.
For nearly 50 years, Fairfax Ice Arena has offered year-round recreational ice skating and competitive figure skating programs, ice rink rental to area organizations and for film/media use, ice skating parties, a Pro Shop and an arena cafe. The rink continues to be home to first-rate coaching, and Olympic, national, sectional and regional figure skaters. Fairfax Ice Arena is also proud to be the home ice rink of the Skating Club of Northern Virginia (SCNV), formed in 1975 by Fairfax Ice Arena founder, Eileen Weisiger.
For job inquiries, please contact Fairfax Ice Arena General Manager Todd Martin.
3779 Pickett Road, Fairfax, VA 22031


text:
Spa Bath Treatment
- Print
- Maximize
Read the product SDS carefully before handling this product.
1 Litre to every 75 Litre of spa bath capacity. 1.5 litres are normally adequate for a standard sized spa.
Timing of treatment:
For domestic application: This sanitising procedure can be performed after the bath has been used and before emptying it thereby avoiding the need to refill the bath specifically for this procedure. As a minimum, the procedure should be carried out monthly where the bath has been used.
For commercial or hospitality situation:
Each time the bath is used by a guest and before the next guest's occupation. Please use the complementary indicator stickers to establish if the bath has been used.
- See the Related Products tab.
Cyndan Spa-Bath Treatment is a non corrosive, nontoxic and non-irritating chemical solution for the cleaning and sanitising of refillable spas and baths.
For larger quantities please contact our office.
Cyndan Spa-Bath Treatment has been researched and developed for the cleaning and sanitising of refillable spa-baths commonly used in hotels, motels, resorts, gymnasiums, clubs and even the home.
Water is an ideal medium in which germs can multiply and flourish. this is especially true and is made worse in warm water as is found in spa baths. Spa baths which are not attached to a filtering or chlorinating system such as home spas are a constant source of contamination.
Spa Bath Treatment is proven to kill unwanted organisms such as staphylococcus aureus and E.Coli, and is TGA certified as a commercial disinfectant option C.
The cleansing and sanitizing action of Spa Bath Treatment is required due to the fact that water residues remain in the pumping mechanism and are injected into the bath the next time it is used. This is a source of cross contamination of the water and is very unhygenic if not treated.
As a minimum, the process should be carried out monthly if the spa has been used.


text:
Medical Gear As Fashionable Expertise
Throughout olden occasions, hospitals and medical doctors relied on traditional knowledge, passed down from person to person, to diagnose, monitor and deal with people affected by diseases and health circumstances. The data that was used during those instances was considered to be fact, but it surely all the time lacked correct outcomes. Apart from missing results, these processes have been gradual, taking a very long time to provide therapies to sufferers. The incapacity of conventional medical practices to offer quick and correct outcomes has additional led to complications and made issues even worse. As time progressed, developments had been made in medical equipments, allowing for higher, quicker, and more efficient remedies.
With annual income of $5 billion, St. Jude Medical specializes in the development and manufacture of medical gadgets. It sells its merchandise in over one hundred international locations and has operations in 20 totally different facilities worldwide. A few of its merchandise embrace artificial pacemakers, catheters, spinal cord stimulators, heart valves and implantable defibrillators. St. Jude Medical was based in 1976.
Vital: It is rather necessary to secure the knowledge on units by encrypting it and now have the capacity to remotely wipe the gadgets clear, together with laptops. To achieve the latter, you set the gadget in order that next time it's turned on and the gadget calls dwelling over the internet. The software program can then tell the gadget âyou've been stolen', which causes the machine to remove its knowledge.
There is only one part of the draft laws that I feel is really unhelpful – making a authorities sponsored rating system for healthcare info expertise. I can't consider another business in which the government provides the Yelp"-like perform. A better approach is for personal firms, akin to KLAS to supply the research the market calls for. Last week, the combined HIT Standards and Policy Committees agreed by consensus that the federal government ought to NOT develop and maintain a comparability instrument, or expand the Certified Health IT Product List to function a comparison device.
Essentially the most often used method of kidney stone removing is a special shockwave remedy known as Extracorporeal shockwave lithotripsy (ESWL). You're positioned in a bathtub of water or on a particular mat, or cushion. The physician then makes use of particular xrays or ultrasound tools to pinpoint the location of the kidney stone, then creates a shockwave which begins exterior the body. Anesthesia is normally given for this kidney stone elimination process, but it surely's often completed on an outpatient basis.


text:
Hospice & Palliative Medicine
Advance care planning benefit presents challenges
Some are having success using new Medicare billing codes, while other doctors say barriers are preventing usage.
Sneak Peek: The Hospital Leader blog – Sept. 2017
To avoid potentially catastrophic situations, we should all use advanced care documentation as the start of a careful conversation about goals of...
Cancer the most common diagnosis in palliative care patients
Survey shows that hospitalists are the main source of referrals to palliative care programs.
Antipsychotics ineffective for symptoms of delirium in palliative care
Do antipsychotics provide symptomatic benefit for delirium in palliative care?
Hospitalists can do better at end-of-life care, expert says
High-value care can remain elusive for the most vulnerable of populations.
Filling the gap: Hospitalists & palliative care
There is now an important opportunity for hospitalists to lead prognosis and goals of care communication for their patients.
From the Society
Everything We Say and Do: Discussing advance care planning
Every health care encounter is an opportunity to better identify and document patients' wishes for their care.
Antipsychotics ineffective for symptoms of delirium in palliative care
CLINICAL QUESTION: Do antipsychotics provide symptomatic benefit for delirium in palliative care?
Community-based palliative care reduces hospitalizations
Clinical question: Does proactive home- and clinic-based palliative care impact hospital use and health care cost in Medicare...
Palliative care: a systematic review for patients and their caregivers
Clinical question: What is the association of palliative care programs on quality of life, symptoms, and survival for patients and their...