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AIRLIE WARD: Anyone watching Tasmanian politics needs little reminder it's an election year. As speculation continues, the Premier will call an early poll. I'm joined by three MPs, David Bartlett, Nick McKim and Brett Whiteley, who will debate the issues during each week of the campaign.
Thanks very much for joining us. David Bartlett, first to you. Shouldn't Paul Lennon have declared a conflict of interest, as per the code of conduct, concerning his brother?
DAVID BARTLETT: Well, the important thing about this - what I'd say is a non-story, Airline - is that all due process has been followed. There are very strict processes for letting of contracts within the public service, and I'm confident, from the evidence that I've seen - - -
AIRLIE WARD: It is not just about due process, though, is it? It's about perception, isn't it?
DAVID BARTLETT: No, it is about due process. Just let me finish. There's two things. All due process has been followed. The process that those public servants, diligent public servants, that, I'm sure, would have followed, the getting of three quotes for any contract under 50,000, and the going to tender of any contract over $50,000, including at least one Tasmanian company, because, when the Labor Party came to office in 1998, we changed the procurement rules to ensure that Tasmanian companies had the best opportunity to bid.
The other important thing about this non-story, Airlie, is that if The Australian, or any other journalist, had been tracking – and the opposition parties, for that matter, had been tracking estimates for the last three years, they would have seen that all government contracts let and all the names of people who – sorry, the companies who received those contracts are tabled in estimates.
AIRLIE WARD: Nevertheless, it's the Premier's brother?
DAVID BARTLETT: So there's nothing to hide, it's open. Yes, but - - -
AIRLIE WARD: Why not declare it?
DAVID BARTLETT: I think Tasmanians deeply understand that the six degrees of separation, if you like, in Tasmania, is one degree of separation, and if we rule out anybody in this state who has any connection to a political family from doing business here, I think we're going to be running a, well, fairly odd sort of regime, aren't we?
AIRLIE WARD: Brett Whiteley, you yourself breached the code of conduct before entering parliament. It is a bit rich for you to be making accusations.
BRETT WHITELEY: Look, those matters have been dealt with. What I want to say about this issue is quite clear. This Premier has form. Not only is he arrogant but he does special deals with special friends. He needs, tomorrow, to come out and actually table every piece of documentation in relation to this, the relationship to his brother. We don't want to see any relatives disadvantaged by procurement issues but they certainly shouldn't be given any special advantage.
The Premier needs to come forward with all notations of discussions - - -
DAVID BARTLETT: All those notations have been tabled - - -
BRETT WHITELEY: - - - when he's been involved with local government, where it it's all been local government involvement.
DAVID BARTLETT: All contacts have been tabled - - -
BRETT WHITELEY: He needs to come clean, because he has form on this and he needs to come clean with the Tasmanian people.
DAVID BARTLETT: All contacts have been tabled recently.
AIRLIE WARD: In terms of principles, Nick McKim, I mean, you've turned around policies, the Greens have turned around policies, on the Spirit of Tasmania 3 and also on drugs, and admitted that it's a vote - it is a grab-catching exercise. How can you convince the electorate of your sincerity?
NICK MCKIM: I'll come to that in a moment, but just on David's argument, the very smallness of Tasmania is the reason why we need strict accountability and the disinfectant of sunlight on the deals that the Lennon government has been caught out doing, a number of times, now behind closed doors. When I go into the electorate, people tell me they're sick of government behind closed doors, for the benefit of corporate mates of the Lennon government; and they want openness and transparency. It is why we need an ICAC in this state.
Now, in relation to our policies, of course, we've acted to clarify our policy on drugs because it was so badly misrepresented by our political opponents in the last senate election.
AIRLIE WARD: You've changed your tune, though.
NICK MCKIM: We've certainly changed some aspects. We've certainly changed some aspects of our drug's policy, and now it's effectively in line with the drugs position which the state Labor Party has. I thought you would be happy with that, David.
DAVID BARTLETT: Well, I'm always amused when you hear people like Will Hodgeman, over the development at Ralphs Bay earlier, and people like Nick McKim, just last week, when they come out and say we're doing this because it's about votes. I think people are very cynical about politicians, saying this, they are doing this about votes. I think that - - -
AIRLIE WARD: David Bartlett, what about Recherche Bay? Isn't that the government's own grab for votes? I mean, if it helps sure up your own seat.
DAVID BARTLETT: Let me say this, Airline. No Tasmanian who has a sense of Tasmanian history should be at all surprised that Labor has moved and, together with many others - - -
NICK MCKIM: Voted by the Greens.
DAVID BARTLETT: - - - moved with many others to save Recherche Bay, and I'll tell you why. Let me say, every single – every single world heritage area and National Park, bar one, in the last 100 years, has been declared by a Labor government. At the heart - - -
NICK MCKIM: (indistinct)
DAVID BARTLETT: - - - by a Labor government - - -
NICK MCKIM: (indistinct)
DAVID BARTLETT: At the heart of Labor is an environmental core but we always - - -
NICK MCKIM: Oh rubbish.
DAVID BARTLETT: Unlike my colleague on my left here, Mr McKim, we always believe we need to balance that with what's good for working Tasmanians, what's good for our economy and what keeps Tasmanians in work.
AIRLIE WARD: Brett Whiteley, in terms of what's good for Tasmanians, I mean, you argue that it's not the PLP, but how can you convince the electorate of your credibility on health, when your party accepts money from tobacco companies?
BRETT WHITELEY: That's a matter for the organisation. Well, the reality is we distance ourselves from organisational matters. We make, David and Nick - - -
DAVID BARTLETT: (indistinct)
NICK MCKIM: (indistinct)
BRETT WHITELEY: We make decisions totally in isolation from the organisation. We were the people first and foremost that wanted the tougher smoking laws. Don't tell me - - -
DAVID BARTLETT: No.
NICK MCKIM: That's not right.
BRETT WHITELEY: That is true. The reality is here - what we've seen this week is no problems with the Liberal Party. What we've seen are two parties making a bed together, ready to lie in it – bedfellows of Labor-Green accord. Political history will show you, that in '89 and '93, through those years, this is exactly, exactly, what was happening. We have the Greens moving closer to Labor, and Labor moving close tore the Greens, and it's going to happen.
AIRLIE WARD: And, just finally, did you all have a good summer, any work, any eyes, you know, hair, new colour, or anything along those lines?
NICK MCKIM: Not for me, Airlie. I sat on the beach, drank a few beers and listened to the cricket on the radio.
DAVID BARTLETT: Do you think we need it?
AIRLIE WARD: Thanks for joining us.


text:
About the Book
Pictures of two adult sons taking their father into the high country of the Washington Cascades for his 72nd birthday present.
Features & Details
- Category Biographies & Memoirs
Small Square, 7×7 in, 18×18 cm
70 Pages
- Publish Date Jan 03, 2011
- Language English
- Tags cascade mountains, Pacific Crest Trail, family, brothers, father, PCT, Washington, hiking, trail, men, outdoors, active, people
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About the Creator
Tim McGuire Images
Seattle, WA USA
Commercial and Editorial Photography on Location around the World
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subset: commoncrawl


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Oct 4, 2016
Mumford and Sons
Isleta Amphitheater - Albuquerque, NM
Isleta Amphitheater Ticket Information
The Albuquerque, N.M., Isleta Amphitheater has hosted countless concerts, festivals, and theater events over the years. From Ozzfest to Vans Warped Tour, Alan Jackson to Alice Cooper, this outdoor venue is a haven for all manner of live entertainment. Scan the list above to see the diverse events on tap for 2016, or select criteria—like genre, date, or a specific performer—from the filter tool to narrow down the schedule. Advance to the next page, where you can click through highlighted sections of the venue map to locate the seats that fit your needs. Make sure the quantity you require is shown before you click "Buy." At checkout, provide us with accurate billing and shipping information to ensure we can process your order for Isleta Amphitheater tickets as quickly as possible. After you verify all the details you have included in the order form, click "Place Order."
Isleta Amphitheater Seating Information
On the Isleta Amphitheater seating chart, you'll see this venue provides both reserved and general admission seating for shows. Occasionally there may be VIP options available as well. For assistance, you can call 866.848.8499. You can also connect with one of our representatives with the online Live Chat service.


text:
Killer cell lectin-like receptor G1
- Nrp1:
- Treg cell:
Regulatory T cell
Frontline: Neuropilin-1: a surface marker of regulatory T cells
CD4+CD25+ regulatory T cells (Treg cells) control immune responsiveness to a large variety of antigens. The isolation and therapeutic manipulation of Treg cells requires the use of reliable surface receptors that are selectively up-regulated in Treg cells. On the basis of global gene expression studies, we identified neuropilin-1 (Nrp1) as a specific surface marker for CD4+CD25+ Treg cells. Nrp1, a receptor involved in axon guidance, angiogenesis, and the activation of T cells, is constitutively expressed on the surface of CD4+CD25+ Treg cells independently of their activation status. In contrast, Nrp1 expression is down-regulated in naive CD4+CD25– T cells after TCR stimulation. Furthermore, CD4+Nrp1high T cells express high levels of Foxp3 and suppress CD4+CD25– T cells. Thus, Nrp1 constitutes a useful surface marker to distinguish Treg cells from both naive and recently activated CD4+CD25+ non-regulatory T cells.
1 Introduction
Strong evidence is emerging that immune responsiveness to self- and allo-antigens can be controlled by naturally occurring CD4+CD25+ regulatory T cells (Treg cells) 1, 2. The key role of Treg cells in controlling self-reactive T cells has been established for CD4+CD25+ T cells in transfer models, where they were shown to suppress multi-organ-specific autoimmune disease induced either by depletion of Treg cells, reconstitution of T cell deficient mice with CD4+CD25– Tcells or thymectomy at day 3 after birth 3. Treg cells are anergic under certain culture conditions and exhibit in vitro and in vivo suppressor activity. The mechanisms of suppression include currently unknown cell surface molecules or cytokines operating most likely in a cell contact dependent manner. Although Treg cells are able to produce IL-10 and TGF-β, both cytokines are not necessarily required for the regulatory function of the cells in some systems 4–7. Recently, the transcription factor Foxp3 has been identified as being essential for the development and function of regulatory CD4+CD25+ Treg cells 8–10. Although it remains unclear which genes are induced by Foxp3 and mediate the regulatory activity, ectopic expression of Foxp3 in non-regulatory CD4+CD25– T cells generates Treg cells that are similar to naturally occurring CD4+CD25+ Treg cells; they are anergic and able to suppress T cell proliferation, and function both in vitro and in vivo8–10.
Characterization and manipulation of Treg cell function in vivo would be greatly facilitated by the identification of a specific cell surface receptor that should optimally be highly expressed exclusively in Treg cells independently of their activation status. Treg cells exhibit constitutive expression of cell surface proteins such as CD25, CTLA-4 and GITR 5, 11–14 that are commonly used to identify Treg cells. However, these receptors are highly induced upon TCR ligation of non-suppressive T cells thereby making it difficult to distinguish Treg from conventional CD4+ T cells activated in response to antigen; therefore these markers can not be considered as specific for Treg cells.
On the basis of global gene expression profiling, we identified neuropilin-1 (Nrp1), a semaphorin III receptor originally described to be involved in axon guidance and more recently as essential component of the immunological synapse in humans 15, 16, as a potent surface marker for Treg cells. In contrast to other genes up-regulated in Treg cells but also expressed following activation of naive T cells, Nrp1 expression is significantly down-regulated after T cell activation. Moreover, Nrp1 expression on Treg cells correlates with the expression of Foxp3 and in vitro suppressor function, thus representing a suitable new surface marker for the identification of Treg cells and their discrimination from recently activated T cells.
2 Results and discussion
2.1 Gene expression profiling reveals Nrp1 as marker for Treg cells
To identify a reliable marker that distinguishes Treg cells from non-regulatory naive T cells and activated CD4+ T cells, and to characterize these cells in more detail at the molecular level, we compared the gene expression profiles of naturally occurring polyclonal CD4+CD25+ Treg cells with their naive CD4+CD25– counterparts. In addition, monoclonal CD4+CD25+ Treg cells and naive CD4+CD25+ T cells of known antigen specificity as well as CD4+ T cells recently activated with their specific antigen were included in our analysis. RNA was isolated from the different cell pools and analyzed by microarrays (Affymetrix MOE430A).
Among the 22400 genes and expressed sequence tags (EST) analyzed, we identified a set of regulated genes that belong to a wide variety of gene classes encoding molecules involved in cell cycle, apoptosis and survival, signaling, general metabolism, transcription and translation factors as well as surface receptors (data not shown). Co-regulated genes were combined in clusters. By this approach we intended to identify those genes that are highly expressed on monoclonal and polyclonal Treg cells without being up-regulated upon T cell activation. As the transcription factor Foxp3 is well established as being a key regulator necessary for the function of Treg cells, we further focused on the analysis of genes that follow the same expression profile as Foxp3. Fig. 1 shows the cluster containing Foxp3 and in addition 86 additional genes that are over-expressed in Treg cells but exhibit low expression levels in naive T cells and are down-regulated in activated T cells (Fig. 1, and Supporting Information at http://www.wiley-vch.de/contents/ jc_2040/2004/24799_s.html). Among these genes, cell surface receptors are of special interest for the isolation, characterization and manipulation of Treg cells.
Two surface molecules identified by this approach were considered as candidate markers for Treg cells — Nrp1 and the killer cell lectin-like receptor G1 (KLRG1) (Fig. 1B). KLRG1 is an inhibitory C-type lectin previously described to be expressed on NK cells and activated CD8+ T cells 17. This molecule was highly expressed in CD4+CD25+ Treg cells, but was down-regulated 16 h as well as 4 days after T cell activation (Fig. 1B and data not shown). However, FACS analysis revealed that only a minor subpopulation (about 3%) of Treg cells expressed KLRG1 on the cell surface. Since depletion of KLRG1+ T cells from CD4+CD25+ Treg cells did not change their in vitro suppressive capacity (data not shown), KLRG1 cannot be considered as a marker for Treg cells and therefore was not considered further.
Nrp1 is a receptor for class III semaphorin subfamily and the heparin-binding forms of vascular endothelial growth factor (VEGF), known to be involved in axon guidance and angiogenesis 15, 16, and was more recently described as mediating homophilic interactions between DC and T cells in humans 18. Being co-regulated with Foxp3, Nrp1 represents a candidate marker for Treg cells, as it exhibits a high expression level in both polyclonal and monoclonal Treg cells but not in antigen-stimulated non-regulatory T cells (Fig. 1B). Moreover, Nrp1 expression was even significantly down-regulated following antigen-specific activation of naive T cells.
Extending our analysis to activated polyclonal CD4+CD25– T cells revealed that Nrp1 expression is down-regulated 16 h or 4 d after anti-CD3 + anti-CD28 stimulation (Fig. 2A), and even lost at day 11 post-stimulation (Fig. 2B). Since the expression of a suitable marker for Treg cells should be independent of activation, we examined the kinetics of Nrp1 expression in CD4+CD25+ Treg cells after in vitro stimulation. As shown in Fig. 2C, the mRNA level of Nrp1 in CD4+CD25+ Treg cells remains constantly high after in vitro stimulation with anti-CD3 + anti-CD28. The same results were obtained when stimulating the cells in the absence of CD28 or with the addition of high amounts of IL-2 to the cultures (data not shown).
Finally, Nrp1 expression on CD4+CD25+ Treg cells was confirmed by antibody surface staining and FACS analysis. To prove the specificity of the polyclonal anti-Nrp1 antibody, control staining on the hemagglutinin (HA)-specific T cell hybridoma 16.2.11, which was negative for Nrp1 in RT-PCR, was performed. Infection of 16.2.11 cells with an Nrp1-expressing retrovirus, but not with a control virus expressing GFP alone, leads to specific surface staining for Nrp1 as documented in Fig. 3A. Antibody staining of CD4+ T cells revealed that only CD4+CD25+ Treg cells express elevated levels of Nrp1 at their surface, whereas Nrp1 surface expression of CD4+CD25– T cells did not exceed background staining (Fig. 3B).
2.2 Nrp1 expression correlates with in vitro suppressor function
It has been reported that blocking of Nrp1 inhibits primary activation of human T cells 18. Therefore, we analyzed the influence of antibodies to Nrp1 on the stimulation of murine naive CD4+ T cells. Thus, sorted CD4+CD25– T cells were stimulated in vitro in the presence or absence of the anti-Nrp1 antibody H-286. In contrast to results obtained by Tordjman et al. in the human system 18, we found that anti-Nrp1 treatment strongly interferes with primary activation of murine CD4+ T cells (Fig. 4A). However, preincubation of CD4+CD25+ Treg cells with anti-Nrp1 did not affect suppressor function of CD4+CD25+ Treg cells (data not shown).
To address the question of whether Nrp1 expression on CD4+ T cells correlates with their regulatory phenotype, CD4+Nrp1high and CD4+Nrp1low T cells were isolated by cell sorting and tested for their capacity to inhibit proliferation of naive CD4+CD25– T cells. CD4+CD25+ Treg cells were included as an internal control. As shown in Fig. 4B, only CD4+Nrp1high T cells suppress proliferation of naive CD4+CD25– T cells, whereas CD4+Nrp1low cells lack the capacity to inhibit the growth of activated CD4+CD25– T cells. Thus, we could demonstrate that Nrp1 expression on CD4+ T cells correlates with in vitro suppressive function.
2.3 Linked expression of Nrp1 and Foxp3
Treg cells have been recently shown to specifically express the forkhead transcription factor Foxp3, which represents a critical regulator for the development and function of these cells 8–10. Since Nrp1 and Foxp3 follow the same expression pattern in regulatory, naive and activated CD4+ T cells (Fig. 1) and CD4+Nrp1high T cells have regulatory properties, we addressed the question of whether the level of Nrp1 expression in Treg cells correlates with elevated levels of Foxp3 mRNA. Analysing sorted Nrp1high and Nrp1low T cells for the expression of Foxp3 by RT-PCR revealed that Foxp3 message correlates well with Nrp1 expression in the CD4+Nrp1high T cells (Fig. 5A). Also, the expression of other Treg cell markers such as PD-1, αEβ7 (CD103) and CTLA-4 was found to be increased in CD4+Nrp1high cells (data not shown).
As CD4+Nrp1high Treg cells exhibit high levels of Foxp3 mRNA, we next investigated whether ectopic expression of Foxp3 in non-regulatory CD4+CD25– T cells would induce Nrp1 expression in these cells. To this end, naive CD4+CD25– T cells were infected with a Foxp3-encoding retrovirus and analyzed for the expression of Nrp1. It has been described before that Foxp3 expression confers regulatory function to the infected T cells 10. This was confirmed in our hands (data not shown). Moreover, RT-PCR analysis (Fig. 5B) indeed revealed elevated Nrp1 levels in Foxp3-transduced T cells. Thus, Foxp3 expression is accompanied with suppressor function as well as up-regulation of Nrp1 expression in CD4+ T cells, further corroborating Nrp1 as selective marker for Treg cells.
Taken together, the aim of this study was the identification of a surface marker on Treg cells, because the currently known cell surface receptors for these cells such as the B7-family members CTLA-4, PD1, and ICOS, as well as the members of the TNF receptor superfamily GITR (TNFRSF18) or the αEβ7 integrin 11–14, 19, 20, are also up-regulated in activated non-regulatory CD4+ T cells. On the basis of gene expression profiling, we identified genes that are co-regulated with Foxp3 — known to be a key regulator for Treg cells. Our major finding was that Nrp1 is over-expressed in the subpopulation of CD4+CD25+ Treg cells independently of their activation status. Moreover, in conventional T cells Nrp1 is expressed at a significantly lower level and is further down-regulated upon T cell activation. Our data further suggest that the expression of the Nrp1 gene is regulated by the transcription factor Foxp3.
Nrp1 was initially described as semaphorin III and VEGF165 receptor, being essential for axonal guidance and vascularization, respectively 15. Recently, Nrp1 has been also identified on hematopoietic cells and shown to be involved in the formation of the immunological synapse, as it co-localizes with CD3 at the naive T-cell–DC interface in human T cells 16. Also, some Nrp1 antibodies inhibit DC-induced proliferation of human resting T cells 18. The latter result was elaborated in murine polyclonal CD4+CD25– T cells stimulated with anti-CD3 antibodies in spite of the fact that these cells express only tiny amounts of Nrp1 (Fig. 4). One might speculate that because of the much higher expression in polyclonal CD4+CD25+ Treg cells (Fig. 2 and 3), Nrp1 is involved in the functional control of these cells. However, further studies are required to support or refute such a hypothesis.
In the nervous system, Nrp1 is able to either repel or attract different axons and thus Nrp1 might have distinct roles in the immune system as well. Interestingly, agrin, a glycoprotein important in the formation of neuronal synapses, has also been found on T lymphocytes, consistent with some molecular similarities of the neurological and immunological synapses 16. Although the detailed immunological functions of Nrp1 in immune regulation remain currently unclear, we conclude that Nrp1 represents an additional surface marker to distinguish Treg cells from naive and activated T cells which in concert with other Treg cell markers will greatly facilitate the characterization and manipulation of Treg cell function in vivo.
3 Materials and methods
3.1 Mice and cell lines
BALB/c mice were obtained from Harlan (Borchen, Germany). TCR-HA transgenic mice, expressing a TCRα β specific for peptide 110–120 from influenza HA presented by I-Ed, have been described previously 21. Mice aged 6–16 weeks were used for experiments and were maintained under specific pathogen free conditions. The HA-specific and MHC class II-restricted T cell hybridoma 16.2.11 was kindly provided by W. Gerhard (Wistar Institute, Philadelphia, PA).
3.2 Antibodies and flow cytometry
The monoclonal antibody 6.5 (anti-TCR-HA) was purified from a hybridoma supernatant and used in FITC-labeled form. Anti-CD4 (GK1.5) and anti-CD25 (PC61), from BD Biosciences (San Jose, CA), were used as biotin, FITC or PE conjugates. Anti-Nrp1 (H-286, rabbit polyclonal IgG) and the corresponding isotype-control antibody (normal rabbit IgG, control Ig) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). PE–streptavidin, cychrome–streptavidin and FITC-labeled goat anti-rabbit-Ig (mouse, rat and human absorbed), from BD Biosciences, were used as secondary reagents. Flow cytometric analyses were done on a FACSCalibur with the CellQuest software (BD Biosciences). Cells were sorted with a MoFlow cell sorter (Cytomation, Fort Collins, CO).
3.3 Cell isolation
For cell sorting experiments, red blood cell-depleted splenocytes from TCR-HA and BALB/c mice were labeled with anti-CD4, anti-TCR-HA (6.5) and anti-CD25 in variable combinations. For separation of cells expressing high levels of Nrp1, splenocytes were stained with anti-Nrp1, and goat anti-rabbit-Ig as the secondary antibody. Labeled cells were separated with a MoFlow and purity was >97%. Isolated cells were either used for RNA purification or for T cell proliferation experiments.
3.4 Proliferation assay
Sorted naive CD4+CD25– T cells, CD4+CD25+ Treg cells, CD4+Nrp1high cells, or CD4+Nrp1low cells (5×104), alone or in addition to CD4+CD25– T cells at a ratio of 1:1, were stimulated with 1 μg/ml soluble anti-CD3 (2c11) without the addition of APC. Alternatively, 10 μg/ml anti-Nrp1 or the corresponding isotype-control antibody was added together with the anti-CD3 antibody. Background proliferation was quantified by [3H]thymidine incorporation in cells incubated without anti-CD3. Proliferation assays were done for 72 h in 96-well flat-bottom plates in a final volume of 200 μl IMDM medium that contained 10% fetal calf serum. 1 μCi/well [3H]thymidine was added for the last 6 h of the experiment and thymidine incorporation was measured by scintillation counting.
3.5 In vitro T cell activation
Splenic CD4+CD25– T cells or CD4+CD25+ T cells from BALB/c mice were sorted and cultured in 12-well plates in the presence of 0.75 μg/ml anti-CD3 (platebound) and 1 μg/ml anti-CD28 (soluble). At different times after stimulation, cells were recovered for RNA preparation and FACS analysis. For antigen-specific stimulation, red blood cell-depleted spleen cells from TCR-HA mice were stimulated with 10 μg/ml of the MHC class II-restricted HA peptide HA110–120. After 16 h of culture, cells were harvested and labeled with anti-CD4, 6.5 (anti-TCR-HA) and anti-CD25. CD4+6.5+CD25+ T cells, representing in vitro-activated T cells, were sorted and used for RNA preparation.
3.6 Retroviral infection and analysis of transduced CD4+CD25+ T cells
Foxp3 cDNA (amino acids 1–429) was amplified by RT-PCR from sorted CD4+CD25+ cells using specific primers (5′-CTG-ACA-AGA-ACC-CAA-TGC-CCA-ACC-3′ and 5′-CTC-CCC-CGC-CCA-CCT-TTT-CT-3′). After cloning into pCR2.1 TOPO (Invitrogen, Karlsruhe, Germany), the Foxp3 cDNA was inserted into an MCSV-based retroviral vector encoding eGFP under control of an IRES site. Nrp1 cDNA (amino acids 1–924) was isolated from pB II SK-m.nrp1 EP (kindly provided by H. Fujisawa, Tokio, Japan) and cloned into the MCSV-based retroviral vector described above. The GPE86+ ecotropic packaging cell line was transfected as described previously 10. Retrovirus-containing culture supernatant was collected after 24 h and passed through 0.45 μm filters. Sorted CD4+CD25– splenocytes were activated as described above (see Sect. 3.5) and, after 48 h of culture, cells were incubated with virus-containing supernatant supplementedwith 20 mM Hepes, 8 μg/ml polybrene and 100 U/ml recombinant IL-2 (not in the case of 16.2.11) following centrifugation for 60 min at 5000×g. Twenty-four hours post-infection, 50% of the culturesupernatant was removed and fresh medium was added. GFP+ cells were sorted after 7–10 days.
3.7 Real-time RT-PCR
Total RNA was prepared from sorted T cells using the TriFast FL reagent from PeqLab (PeqLab Biotechnology, Erlangen, Germany). The cDNA was synthesized using oligo-dT primers and MLV RT polymerase (Invitrogen), following the manufacturer's recommendations. Alternatively, RNA was isolated with the RNeasy kit (Qiagen, Hilden, Germany) following cDNA synthesis using the Superscript II Reverse Transcriptase (Invitrogen). Quantitative real-time RT-PCR was performed in an ABI PRISM cycler (Applied Biosystems) using a SYBR Green PCR kit from Applied Biosystems or Stratagene and specific primers optimized to amplify 90–250 bp fragments from the different genes analyzed. A threshold was set in the linear part of the amplification curve, and the number of cycles needed to reach itwas calculated for every gene. Relative mRNA levels were determined by using included standard curves for each individual gene and further normalization to RPS9. Melting curves established the purity of the amplified band. Primer sequences are: Nrp1 (5′-GCC-TGC-TTT-CTT-CTC-TTG-GTT-TCA-3′, 5′-GCT-CTG-GGC-ACT-GGG-CTA-CA-3′); Foxp3 (5′-CTG-GCG-AAG-GGC-TCG-GTA-GTC-CT-3′,5′-CTC-CCA-GAG-CCC-ATG-GCA-GAA-GT-3′); and RPS9 (5′-CTG-GAC-GAG-GGC-AAG-ATG-AAG-C-3′, 5′-TGA-CGT-TGG-CGG-ATG-AGC-ACA-3′).
3.8 DNA microarray hybridization and analysis
The quality and integrity of the total RNA isolated from 5×104 cells was controlled by running all samples on an AgilentTechnologies 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). For RNA-amplification, the first round was done according to Affymetrix without biotinylated nucleotides using the Promega P1300 RiboMax Kit (Promega, Mannheim, Germany) for T7 amplification. For the second round of amplification the precipitated and cleaned aRNA was converted to cDNA using random hexamers (Pharmacia, Freiburg, Germany). Second-strand synthesis and probe amplification was done like in the first round, except for an incubation with RNAse H before first-strand synthesis to digest the aRNA and for the use of the T7T23V oligo for initiation of the synthesis of the second strand. The concentration of biotin-labeled cRNA was determined by UV absorbance. In all cases, 12.5 μg of each biotinylated cRNA preparation was fragmented and placed in a hybridization cocktail containing four biotinylated hybridization controls (BioB, BioC, BioD, and Cre) as recommended by the manufacturer. Samples were hybridized to an identical lot of Affymetrix MOE430A for 16 h. After hybridization the GeneChips were washed, stained with SA–PE and read using an Affymetrix GeneChip fluidic station and scanner.
3.9 Data analysis
Analysis of microarray data was performed using the Affymetrix Microarray Suite 5.0, Affymetrix MicroDB 3.0 and Affymetrix Data Mining Tool 3.0. For normalization, all array experiments were scaled to a target intensity of 150, otherwise using the default values of the Microarray suite. Filtering of the results was done as follows: genes are considered as strongly regulated when their fold change is greater than or equal to 2, or less than or equal to –2, the statistical parameter for a significant change is less than 0.01 [change p-value for changes called increased (I)] or greater than 0.99 [change p-value for changes called decreased (D)]. Additionally, the signal difference of a certain gene should be greater than 100. Genes are considered as weakly regulated when their fold change is greater than or equal to 1.5, or less than or equal to –1.5, the statistical parameter for a significant change is less than 0.001 or greater than 0.999 and the signal difference of a certain gene should be greater than 40.
We thank Patricia Gatzlaff, Tanja Toepfer and Silvia Prettin for excellent technical assistance as well as Lothar Gröbe for cell sorting. We thank H. Fujisawa for kindly providing us the plasmid pB II SK-m.nrp1 EP encoding the Nrp1 cDNA. This work was supported by the Deutsche Forschungsgemeinschaft, the Deutsche Krebshilfe and the VolkswagenStiftung.
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Briquetting Systems Companies
Category: Waste & Recycling ×
Subcategory: Briquetting Systems ×
Industry: Construction & Construction Materials ×
Harsco Metals & Minerals
based in Leatherhead, UNITED KINGDOM
Harsco Metals & Minerals provides logistics and resource recovery services for the steel industry, operating at more than 160 customer locations across 30 countries. Working on site under multi-year contracts, our customers rely on us to deliver critical services on a daily basis, while demonstrating the service excellence that our company was founded on. Harsco began ...
Husmann Umwelt-Technik GmbH
based in Dörpen, GERMANY
Manufacturers of equipment for waste disposal / environmental technology in Europe. Since more than 50 years the brand Hussman stands in the disposal branch for liability, quality and competence. We use our experience for finding solutions that fit exactly our customers' requirements. This as well as our innovative technology and our wide range of products for the ...
Pentek Inc.
based in Coraopolis, PENNSYLVANIA (USA)
Pentek, Inc. is the industry leader in manufacturing, engineering and designing specialty surface decontamination technologies and processes for various commercial, industrial and nuclear applications. We offer turnkey solutions for all of your decontamination needs, and our tools are used by contractors and commercial markets around the world. During the 1979 accident at ...


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FORT WAYNE, INDIANA -- My wife enjoys Wendy's chili so I have gone to Wendy's with her 6 times. I just order a double cheese burger with just lettuce, tomato and onion. They have yet to get it correct, the problem is usually someone assumes that mayonnaise is part of lettuce tomato and onion. Normally it takes one more try to get it correct. Although my wife and I were traveling and stopped at a Wendy's in Iowa. I ordered my usual. My wife ordered some food also and we paid for our food. The young man working the counter gave us cups for our soft drinks and we went to fill them while waiting for our order.
We returned to the counter and continued to wait while several other people ordered and received their food. My wife asked when our order would be ready. The young man looked confused and called the manager over. Apparently somehow our order had been removed from the system or marked as done. The manager had our order hurried through and we sat down to eat. My wife's order was fine. My cheeseburger had mayonnaise on it. I was just going to wipe off all of it that I could but my wife took the cheeseburger, went back to the counter and complained.
The manager again stepped in and said he would get it taken care of. He returned in a few seconds with the cheeseburger. They had scraped the mayonnaise off, put it back on the bun and re-wrapped it. When I unwrapped it I told my wife I could have done that, she got really pissed. Took the food back to the counter. The manager said he would have a new one made. And when he brought it to the table this time it was correct.
I should mention also that there were 3 local uniformed police officers sitting a few tables away, watching intently. At first they must have thought I was a trouble maker as my wife and I were traveling by motorcycle. By the time the manager got the cheeseburger correct, the police understood what was happening and said nothing but I did notice one of them was grinning and shaking his head...
ALLENTOWN, PENNSYLVANIA -- Yesterday I was in an urban area I am unfamiliar with, and needed a quick bite to eat. The only place visible was a Wendy's, and the last time I was in one was July 2007. Had a totally disgusting Baconator and the usual horrible customer service and said I'd never return.
I decided to give it a try, figuring it is only a $1 hamburger, how bad can it be? The counter experience was just as I recalled. 3 CSRs walking back and forth and standing around looking stupid. **, the Employee of the Month according to the picture prominently posted in the area, was flirting with the register person and/or looking like she didn't know what to do. I recognized her by her earbud, which was also in the picture. I was thinking she could use one of those TVs like in McD's that shows the pending orders.
A patron walked up with his receipt and stood waiting to speak with one of the people who were ignoring him. He patiently stood for 4 minutes, and finally someone saw him after ** walked away, right past him, and out of the field of view. They hadn't given him his soda with his order. A "family" walked in, and the 5 year old insisted 5 times in 15 seconds that he had to have bacon on his burger. The future of America. His mom said she wanted "the largest soda you can give me." The apple and the tree, in the same place. I waited 8 minutes for my hamburger, and was amazed that ** was able to locate it and hand it to me.
I left the restaurant so as to not hear the outcome of whether Junior got his artery clogging waistline expanding bacon burger. As I stood in the parking lot, I sampled the merchandise. The first thing I noticed was fresh red onion peeking out. Then the square burger. I bit into it, and the sweet flavor of the onion and pickles hit, followed by the tender burger. It was most enjoyable - far better at a glance than my usual McD's burger. It was actually "prepared" medium, and not cooked to death. It appeared more like meat than what I usually get at McD's.
As I considered this sequence of events, It occurred to me that the customer service was just as lousy as I remembered at Wendy's. Then I thought "if I had one of these burgers regularly, the novelty might wear off and I'd also have to deal with the customer service." So, it's back to McD's for me, but maybe I'll switch up once in a while for a change. I'm usually one to focus on quality of the goods, but I find it interesting that customer service is at least as important to me when it comes to fast food.
PARMA HEIGHTS, OHIO -- Wendy's is the only place I buy my French Fries. Even if I buy the rest of my meal somewhere else, I always stop at Wendy's to get my fries. Today, I stopped at Wendy's and went inside to order my fries. I was told they were just being made. I like that, that means they are going to be nice and hot. This Wendy's ALWAYS has nice hot fries, they really are the bestest!
Anyway, I got my order to go and the bag was steaming hot. However, those great fries that I'm so used to, were completely ruined by so much salt that they put on them. Now, I have always been one that salts everything, so for me to say something was too salty is really a surprise to others.
After eating just a few fries, my tongue and the roof of my mouth were actually sore and felt raw. My Dad sat down to eat his and he had the same problem. I was so disappointed because they were so fresh and hot and just enough crispiness on the outside yet hot and soft on the inside, which is the way I would describe the PERFECT Fry! I tried to ignore the salt for as long as I could. Then, I even took my fingers and rubbed the salt off each fry before I ate them. It was really hard for me to just throw them away, because as I said, they were cooked so perfectly... but the salt was just too much to handle.
I don't know if one employee salted them, then maybe another came along and salted them, not knowing they were already salted... but why-o-why would you ruin such a beautiful, perfectly cooked fry like that? WHY??? WHY??? WHY???? My Dad and I just couldn't finish them because our mouth and tongue were so sore. It's now been about two hours since I ate them and the inside of my upper lip feels a bit swollen.
I hate to write a complaint because this Wendy's location have always served perfect food for as long as I've been going there... but I just needed to bring this to their attention. Yeah, I'll still go there and yeah, I'll still buy my fries there, hoping this was just a one time thing.
PANAMA CITY BEACH, FLORIDA -- I had to wait through traffic to get into the location on Thomas drive in PC Florida taking probably 10-15 minutes to start this complaint. I ordered our drinks 5 of us. 2 sprites, 1 coke, and 2 large sweet teas. I ordered 2 #4's, 1 #7, 2 grilled chicken sandwich meals. I got to the window I got 1 small tea, 1 large tea, 3 cokes. I told the man the drinks were wrong and that both teas were supposed to be large and he smarted off "She read you your order back didn't she!" I said, "Yes sir she did but when she read it back it was what I ordered and this is not right."
He said, "We didn't charge you for that" and I told him, "I didn't care what it cost." I wanted the large teas and I gave him money. He got aggravated like he didn't want to exchange the small tea. Then I told him the cokes were supposed to be sprite I thought he would stroke out. Then to the food order. He handed the bag out the window and I just counted the sandwiches and we were 2-short the chicken sandwiches.
Well he repeated the statement about she read your order and you should have listened. "She read your order out" and I told him again that "she read it out right but the order was wrong." I paid 36 or 37 dollars. Needless to say I was furious this manager had belittled me, he talked down to me as if I were stupid after I had gone out of my way. Waited for 15 minutes in traffic finally pulling into some amusement park next to it and having to wait to get in the entrance then for me to have such terrible service. Not to mention my husband and I didn't get our meals.
I will never go to that Wendy's again and I suggest you get someone down there that has a few manners to run your store. Figure out a better entrance for the store. Get order takers who know how to key in what they repeat to you. I hate to complain but that night was such a bomb because I had gotten so upset. We went to the room and I had potato chips for supper.
GAINESVILLE, FLORIDA -- Wendy' 33 Feb.7, 2012 at 1:37 pm Reg# 1 Cashier **. Outside they seemed to be very clean. No one was in the drive through, and there was no big line it seemed inside. We went in and there was no one ordering but there was some customers waiting for their order. We ordered a #1 with no cheese, no pickle, large fries, no salt and 1 med. drink. My son mentioned the mess in the cook area. It was a mess, I could see it if they were slammed but they were not. We got our drink and was waiting when the Burger cook came over to ** and said to him if the burger had no salt. He said no the fries have no salt.
My burger was made and sat at the station before the customers in front of us even got their food. One order of fries just got done but instead of pulling mine out they dumped it and salted it. There was one man behind us and he got his food before either of us did. They finally gave the customer in front of us their food. We were still waiting. The fries' cook told ** to offer us their apology because of the wait. He told her he did but he did not say anything to us at all.
As we waited, the fries' cook finally pulled up the second order of fries and loaded them into a large box and put both my burger and fries in a bag and handed it to me. We decided to just sit so I could eat. I needed something. I was not feeling well. So we sat down and that is when I found that my hamburger was cold and my fries were pulled out before they wear done. So, I ate my hamburger and threw away the fries, and the fries were the reason I went to Wendy's, but they left a bit to be desired. As we left a customer from the drive through came in and complained about the fries.
CITRUS HEIGHTS, CALIFORNIA -- I would like to say that the Wendy's in Citrus Heights Ca, across from the Sunrise Mall is a joke. I went through the drive through on date 01/17/2012 and the service I received was just terrible the people working the late shift including the manager were very rude and inconsiderate. I ordered a classic single meal and 3 dollar burgers. I received fries that had looked like they had been cooked twice over. They were hard and brown and not even hot when I tried them. I noticed this when I got to the street light just down from the Wendy's I purchased them from.
So I went back drove through the drive through stopped at the window they did not see me. So I went back a second time there was a car in front of me. The people working at the Wendy's turn the lights off and said they were closed. I said "I am not happy with the fries would you please give me my money back or replace my order." And this is what I got ''NO'' SORRY we are closed you will have to come back in the morning.
''THAT'' IS TOTAL CRAP! I paid for fries yes but fresh fries and if they could not replace my order they should have right there given me my money back but instead I had to drive home with fries I could not eat. ''So'' half of the order I got it was not a good experience the workers should be fired. There is ''no'' excuses for that kind of service and the attitudes sucked. They are getting paid and they are not even doing their jobs! Part of working and running a business involves treating the consumer with respect which I did not receive. I should not have to come back in the morning when I got there just 1 minute before they closed.
''Also'' anyone that does not care enough about their job to see that the customer leaves the store with the product they paid for should not be working for the company. I saw a lack of caring and respectful attitudes. I do not recommend Wendy's in Citrus Heights Ca, across from the Sunrise Mall to anyone who cares to receive what they paid for. The store and the workers should be inspected and replaced.
MEMPHIS, TN -- I went to the drive thru tonight and the girl taking the order was cussing at her 2 children who she brought to work with her (around 7-8 yrs old) while she was taking my order. I pulled to the window for my total b/c she couldn't even get that right. While she was standing there yelling at her kids, and stuffing french fries down her throat, she took my money and gave me my change (without a receipt of course) then instructed me to reverse my car and back away from the window.
There was another car behind me who also had to back up to make room for me. She finally then waves me forward to pick up my food. She hands me the bag and slammed the window shut and continues with her fries and drink. Her children stood there pulling on her shirt as she ignored them, to tell her that I was still sitting at the window. She opens it up with "yea, what you need?" (How rude!) I told her that she didn't give me my drink. She hands me the drink and without thinking I pull away... Once I got at my destination, I opened my bag only to realize that there was no salad dressing for my salad. Which just made me even more angry.
I called the store and was told to come by tomorrow and talk to a manager. But how can I even prove I was there without a receipt? Completely unprofessional, unorganized and unsanitary. There were 2 kids in the back behind the counter... are these children been made to wash their hands, or taught not to sneeze or cough on food? They weren't wearing any hairnets, gloves or anything.
Children DO NOT belong behind the counter at a fast food restaurant. Well in this case they were the only ones who seems to be paying attention to what was going on. I will never eat there again and will tell anyone who will listen not to either. I will be stopping by tomorrow to speak with a store manager and not just some little girl they have answer the phone and say she was. There is no way a true manager would let any of this happen.
CINCINNATI, OHIO -- I think that this was the worst experience I have ever dealt with at a company. I am very empathetic towards people who work at underpaying jobs. I worked in customer service for a few years and know just how great people are towards you while you're on the clock even though the pay still is not enough to deal with unnecessarily rude people for 8+ hours.
Therefore, I was surprised when the cashier was rude when asking for my order, rude after taking it, giving me the wrong change, telling me I can have my receipt "when she's done", telling me I can have my change since she "has to open the drawer anyway to put in a roll of pennies", giving me a wrong item in my order, asking my sister with an eye roll "well which one do you want," when discussing frosty or frosty floats. I mean it went on and on.
I asked her who the manager was and she said his name with a nasty tone. I complained on the company website and never received a response (after I had contacted the manager who rushed me off the phone). So I decided to write another review almost three months later and they got back to me the same day. Now I am just waiting on a phone call and we'll see where it goes. People like that cashier do not deserve to keep their job.
I could see a reason for her to continue being rude to me if she was having a bad day and I was also rude, but I stayed nice the entire conversation and every single reply whether verbal or not, was incredibly rude. I don't even write complaints or care when I have the normal poor service, which tells you how bad this case was.
Commentary on the new Natural Fries from Wendy's: Well, my place was one of the test markets, and I've had nearly a month to test. Glad to say that these fries are good. Now, they're just fries. A side order for most, a take it-or-leave-it food that all burger joints have. But you have to hand it to them, these fries are different for fast food.
These new fries have the skin on (a certain percentage of it), sea salt, and has a special blend of oils that it's fried in, rather than the regular stuff they would used to fry that chicken piece). Nothing else is supposed to be added.
The result? Tastes like something I would have made at home. I do indeed make my own fries, but results vary from time to time. I do love leaving the skins on.
So if these are "Natural", and only contain 3 ingredients, potato, salt and oil, what were they before? What do the other places sell? Well one of the major ingredients that a lot of folks find surprising that other fries have is sugar. Sugar is sprayed on to fries from Mc'D's and BK. Makes them a bit more rigid, and of course, makes them taste better. Sodium acid pyrophosphate may be added for color, and whatever stuff they add to the regular salt on the regular fries. Of course, there are those waffle fries over at places like Chick-fil-A that are great chunky things, that are probably just as good, but that's for another review.
But is it healthier? Nah. It might have less sugar, but these "Natural" fries are just cut potatoes, just like the other places. But they sure taste good. It could be the skins. It could be the oil blends they use. It could be the bigger chunks of salt. And don't give me that about salt being bad. Salt is very good for you. There's no such thing as too much salt on fries. I'm diabetic. But I want to live! Bottom Line: It's better than the regular stuff. Try it when it comes to your town. Right now, I have half a mind to stop over a drive-through and order just the natural fries.
JACKSONVILLE, FLORIDA -- I am fortunate enough to have a few dozen options for fast food close to me yet the closest one to my home is Wendy's in Jacksonville on Post and Cassat Street. I like a few of their items so I occasionally make an attempt to get a meal there. I say "attempt" as in the 10 most recent attempts, the order has never been correct. It doesn't matter what time you go, the line in drive through never moves even if you're the second car in line and if you go inside, you have to set off a flare to catch anyone's attention to wait on you. I was the first one in line on 3 of those 10 occasions and have waited 15 minutes only to get a wrong order.
The staff is lazy, shiftless and has no direction and the managers seem to be changed weekly. The staff and management are always so engrossed in their personal conversations they barely acknowledge you are there to buy food!! One fool threw me attitude when I had to interrupt his personal conversation to ask for my food which was sitting at the window. He told me it wasn't ready and pushed the bag back out of sight. Then 5 minutes later presented the same bag and said sorry for the wait. Of course the order was wrong so it came back anyway. Invariably, all the employees have bad attitudes and often challenge you when you complain your order is wrong.
I have been shorted sandwiches, shorted change, told I ordered the sandwich wrong... you name it. I don't even bother pulling out of the drive through because when I check my sandwich, I know it's wrong so I always ask for my money back. When I go out of town I never have a problem so that's how I get my Wendy's fix since in 10 attempts in the last year I still haven't had a correct order taken. Fast food isn't fast in this town as a rule and I hope this Wendy's closes and that something, anything will take its place. It can't possibly have any poorer service!!


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tgoma turns your Springfree into the first outdoor interactive digital game system designed for a trampoline.
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Video: Hockey Night in Canada's playoff montage is pretty awesome
I live in the United States and I love it. I'm proud to be an American.
But man do I feel like I miss out on the cool stuff you Canadians get to see such as Hockey Night in Canada's annual playoff montage after the Stanley Cup is hoisted.
However, thanks to the all-mighty Interwebs, I found it!
It's almost as good as One Shining Moment for the NCAA basketball tournament. Sorry, but Luther Vandross > Coldplay.
Outside of that though, this is pretty awesome. It has some of more memorable single images of the playoffs including the agony of Panthers GM Dale Tallon when Florida lost in double OT of Game 7 and the excitement of Mamma and Poppa for son Braden Holtby. Of course, Raffi Torres' hit on Marian Hossa made an appearance, too.
I believe missing altogether from the montage were the San Jose Sharks, who unceremoniously went out in just five games to the St. Louis Blues in the first round.
And was it just me or was that awfully heavy on the Los Angeles Kings? I mean, what did that team do to deserve the lion's share of the highlight package? Oh, right.
H/t to Seth Rorabaugh, Pittsburgh Post-Gazette, he's the one who really found it
For more hockey news, rumors and analysis, follow @EyeOnHockey and @BrianStubitsNHL on Twitter and like us on Facebook.


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British Wildlife
Have you seen a frog or a toad? Pat Morris explains how you can tell them apart.
Visit a UK meadow this summer to enjoy beautiful wildflowers.
Discover 6 fascinating facts about the BTO June Bird of the Month.
Nature blogger Will Harper-Penrose enjoys a wildlife-packed day at his local reserve and encounters his spirit animal.
Discover fascinating facts about this small non-native species.
These little brown mustelids are both fast and ferocious, with sinuous bodies and short legs. So how do you tell them apart?
Reserve warden Paul Green explains why you should visit RSPB Minsmere, Suffolk, this summer.
Visit one of these puffin hotspots in the UK for a wildlife encounter to remember.
Attract pollinators to your wildlife garden with these plants.


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Marketing Principles report
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Four tourists from Britain and two from Australia were among seven people killed Saturday when a helicopter crashed on a scenic New Zealand glacier, police said.
Belgium's capital entered a security lockdown Saturday as the government warned of a threat of a repeat of Paris-style attacks
Hundreds of people gathered outside a Minneapolis police precinct on a cold Friday night to call for peace and release of videos after a black man was fatally shot by an officer.
Republican presidential rivals rushed Friday to condemn Donald Trump's support for a government database to track Muslims in the United States
A central Pennsylvania woman accused of running a prostitution service out of her home says she charged only for massages, and that any sex that occurred with clients afterward was just "free fun."
A professional dancer who was injured in the Boston Marathon bombing says American Airlines lost her luggage, which included her prosthetic leg.
Federal prosecutors want the trial of Joey Meek, the friend of the man charged in the Charleston church shootings, to be held in the city where the shootings occurred
Horry County is considering a proposal to base all new tour operations at public airports.
South Carolina's unemployment rate has fallen for the fifth month in a row.
A third suspect is charged in connection with a fatal summer shooting.
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Researchers developed a mathematical model to forecast metastatic breast cancer survival using techniques usually reserved for weather prediction and financial forecasting. They looked at 25 years of data regarding 446 patients at Memorial Sloan Kettering and focused on a subgroup who were diagnosed with localized disease but later relapsed with metastatic disease. The model shows cancer metastasis is neither random nor unpredictable.
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How To Drive A Stick Shift In Ten Easy Steps
Only 6% of new cars sold stateside have a manual transmission, but every one of the Fiesta Movement cars is a stick. We taught one winner, Jill Hanner, how to drive a manual transmission car.
Only 6% of new cars sold stateside have a manual transmission, but every one of the Fiesta Movement cars is a stick. We taught one winner, Jill Hanner, how to drive a manual transmission car.
Ken Block did what Ken Block does in his terrifying rally Fiesta: delivering the donuts to this year's Goodwood Festival of Speed.
Up and coming World Rally Championship driver Hayden Paddon rolled his Hyundai off the Ponte de Lima stage of this weekend's Rally Portugal, starting a fire in the dry brush past the road. Ford Fiesta driver Ott Tänak rolled minutes later at the same spot.
'One owner, only driven on weekends' has never been more amazingly misleading.
Nobody can shut up about how awesome the new AWD, 350 hp "Drift Mode" Ford Focus RS is, including us. Ford probably thinks that's great because it's the only hyper-hatch the brand plans on making, as it has now been all but confirmed that there are zero plans for a Fiesta RS.
Even front-wheel drive Fiestas go sideways on dirt. Here's what happens with AWD and a pro WRC driver.
Heh heh heh FIRE! Mmmmmm FIRE! Heh heh heh huh huh heh heh heh cool. Heh heh FIRE! FIRE! FIRE! FIRE!
Poor Elfyn Evans didn't have the best time at Rally de Portugal. First, his M-Sport Ford Fiesta RS WRC experienced throttle problems which plagued him all throughout Day 1. Then his car just gave up the ghost on SS8, and guess what? That ghost was a fire spirit.
Crashing is bad enough. Rolling down a hill into a lake is probably the worst case scenario, ever. Here's the onboard footage from the crash where driver Ott Tänak and codriver Raigo Mōldar plowed nose-forward off the track and rolled into a reservoir during the World Rally Championship's Rally Mexico.
Rally Mexico has not gotten off to a good start. Ford's WRC team lost contact for 17 minutes with their crew after Estonian driver Ott Tänak rolled off the road into a lake.
The Col de Turini is one of the most famous stretches of road in the world, curling along the Alps close to the French/Italian border. Watch this video and you'll understand why it's so well known.
Ken Block isn't just content to jump cars into the air. He's going to space.
Estonian Ott Tänak was absolutely hounding has way through the Rallye Monte Carlo over the weekend until he slid his Festa WRC car of the road and down the side of a mountain. Luckily, the entire French nation showed up to push him back into the race.
You can't really drift a front-drive car on pavement. On ice, though? No problem for a rally driver.
Slow car fast. It's a mantra of the car enthusiast that we hear constantly. What's more fun than being on the limit and not actually breaking any laws? But what's the best: The fastest or pretty much the slowest Ford Fiesta?
The Ford Fiesta, even with the 1.0-liter three cylinder, isn't the perfect car for everything. For instance, there are times when a Jeep would be better.
Former F1 driver Robert Kubica crashed his way out of Rally France this past weekend, but not before setting the fastest time out of anyone in the event on Special Stage 15. It appears Special Stage 15 takes place in a Grimm fairy tale.
[British sprinter Marcia Richardson practices in 1993 by running behind a Ford Fiesta. This does not look safe. Photo Credit: Getty]
Because Ford Fiesta pickup trucks are better than you might think.
Have you ever been driving a hot hatch and asked yourself — I wish my car was smaller, and had fewer seats, but also a pickup bed? Ford built a car just for you!


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Department of Language and Literature
The primary mission of the Department of Language and Literature is to meet the education and research related needs of the diverse cultural populations of the region by exploring global languages and literatures.
We seek to communicate, to describe our surroundings with precision, to move the heart, to explore the ways of knowing, and to discover how culture is encoded through language. We aspire to grow intellectually and to share knowledge and culture with those beyond our four walls.
The department makes a major effort to provide life-long learning opportunities in modern languages including English, Spanish, and French, to residents of all ages and to serve as the linguistic and literary center for the region.
Degrees We Offer
Special Events
The Department is proud to publish a new volume of Writer's Bloc, featuring poetry, fiction, and art. Copies available in Fore Hall, room 110, price $10.00.
For information about submitting poetry, fiction, or art to the next Writer's Bloc, please see Ms. Aletha Eyerman.
The English Club Booksale is September 21-25. Held on the first floor of Fore Hall, the sale offers bargain books for every reader.
This page was last updated on: January 14, 2016


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|Reviews for YuYuGiDigiMoon: Gaiden
| Guest chapter 23 . 1/2
the pendulum can vecome the final power up of the duelist PLEASE!
| Belletiger BT chapter 24 . 11/8/2015
That was a pretty cool chapter. I loved the part with the Kuiper flashback. It was very cute. Now the return of Seal of Orichalcos card was a big surprise for me. I thought these cards are gone for good. Things are becoming complicate for our heros. I cant wait to see more!
| JNaegi chapter 24 . 11/4/2015
Ok finally got to this chapter.
My such a little scandalous setting for Vivian to have such a lewd moment with Ken (Prince Sedna) in the past. Gotta comment Ken being the typical "only guy with a hand of girls as friends" concept (like Tenchi/Ranma/Negi) except he wouldn't peak on any of them.
Duel was sweet and Kaiba having a boner? Kind of OC but again I can see him having that charge for seeing a nice, big dragon who hits the spot (Lyn should get some advice from the Goddess of Love to keep her man happy XD).
So Ken being Santa for Yusei and Viv? Cool!
I did enjoy this colab chap! Still amazing that Ken gets so flustered with the ladies. And how can I forget the Terumi ref? You can't keep a good troll down!
GJ with this fellas!
Till next time!
| Jacob9594 chapter 24 . 11/4/2015
I love this chapter. It. Would. Pretty coolKai is if you added the charcters from Yugioh Zexala no Arc-V to the Sotry. That would be sweep. And Deadppol of possible for comedic effects. What do you think Kainus?
| Chaosblazer chapter 24 . 11/4/2015
Vivian scars Ken in the one way that really hurts, too bad brain bleach doesn't exist, but perhaps getting smashed in the head by his partners will cleanse his mind...if they don't kill him in the process.
Had a lot of fun with this, the stuff with the Paradias trio was interesting (I like what Ford said in his review about them) and the Kuriboh's are funny.
The duel had many options and various ways to be done, decided to do things that way in the end, and yes I did reference Yugioh abridged as well as the recent DBZ abridged movie to an extent with the Terumi line
Well Ken likes to help people improve their decks cause he loves the game probably as much as Yugi and Jaden do, but he is abit more serious when he fights.
I am surprised at how quick you edited and got this up, especially with that scene with the Grandra's leaving, thanks for working with me dude.
It's always fun and sometimes chaotic for the characters lol, let's keep it up for the future.
| Ford1114 chapter 24 . 11/4/2015
That concludes this decent gaiden. To be honest, I still believe side stories didn't get much reviews compare to main stories, which we all understand your workload that main stories are the focus for time priorities/constraints despite a few gaidens stood out to me.
There are some parts I see such as that Kuiper flashback you wrote for chaos. Neat stuff to see their kingdom having their enjoyment and training. That's how it goes.
Ken and Richard duel, but the latter uses a remaining Seal of Orichalcos card and turns the tide. I see Dark Rebellion XYZ, and that Neo Galaxy Eyes makes Kaiba have a dragon fetish in obsessed with another powerful card. Well the duel is over.
Now I get why the Paradais Knights are freed due to not Psyren (I recall that woman in Season 1), but surprisingly Cain. Okay, so I predict the Paradais Knights will become Duel Monsters (the same ones of Shinnen) and remained themselves the Defiant Knights?
That means another difference here to examine two versions: We already did the last part of the Paradais Knights/Paradox on email for Shinnen, but I don't know your take on them for Defiants.
| Generic Reviewer chapter 24 . 11/4/2015
Cain Bearer is still a problematic loose end. He doesn't give up.
Its also real nice Kensuke lives in a dimension where most of the anime bosses are just regular cards you can buy in a store. And I'd be scared too if someone went full Anna at you naked. Poor guy.
Not much else to say. Later days!
| JNaegi chapter 23 . 10/15/2015
Duels non-withstanding there is one battle that was something to make a note about; Rainbow Kuriboh vs. Kuriboh/Wing Kuriboh!
New butterball on the turf and it's understandable why the duo would feel threatened by some puffball that could be a pet to Rainbow Brite.
Puffs in the hood is a go! XD
Lance and the British duelist, eh? Need to some development but I'm intrigued. Yay for a buxom blonde lady tempting Richard to use a new card against Ken (it's good character development).
Wow Takato pretty lax with his dueling, Rika going to crack the figurative whip on his butt.
Till next time.
| Generic Reviewer chapter 23 . 10/14/2015
I'm amused Kensuke didn't show the weird manga Signer dragons to Team 5Ds.
I also wonder if Takato was using Evols with Darwino. He's still going to get dragged by his ear while complaining when this is over though.
Later days!
| Chaosblazer chapter 23 . 10/14/2015
all the edits and additions you made fit pretty dang well dude, this is awesome.
Oh yes the fun is coming, and so is the pain_ lol.
| Ford1114 chapter 23 . 10/14/2015
Didn't see that one coming, well Chaos did mention this since he's taking over Ninetalesuk's position in writing duels. Knowing that Zexal and Arc V will be separate dimensions like Xros Wars (though Data Squad remind you did exist in YYGDM).
It's interesting to see Zexal cards onward exist in the YYGDM dimension. Yugi got Legendary Magician of Dark and plans to give Joey his new ace. Jeri has Lionheart. Seeing Takato having Volcasaurus gets something to be interested, and good to see Rika is the first to have an XYZ card. Several versions of BEWD, a hint that Kaiba will get his soon.
It seems that our protagonists are facing the Grandra siblings, lets see how these card play. No way, the Paradais Knights are back? (To coincide in Shinnen that they appear as the Da'ath Knights alongside Mandark)
| Ford1114 chapter 13 . 10/6/2015
It has been 5 years since this chapter, way before Gaiden is revived.
I wonder what that antimatter force is that attack the AU senshi's world? Well I ask it on email to know.
Yeah so that's how these Senshi's became Amazons and fight alongside the main senshi.
Ah, the first concept ideas of the multiverse as some of the concepts to utilized throughout this decade.
| Guest chapter 1 . 8/13/2015
I'm not really interested in the anime of your fanfic but i respect you o much.
| JNaegi chapter 22 . 8/12/2015
Good overall side story with Daiki-kun! Along with GJ for Lazerwulf coming up with the premises!
So that's how Dai got his arm taken? The scenario was spot on along with the interactions between them. Always nice to see Jami and she's a respected doctor in her own right next to her senpai, Ami!
I also liked Helbot's own development, she reminds me of KOS-MOS in that respect. It would be something if Dai placed some black box in her body that made her act human just like the synthetic droid, ya know "Blue-eyed KOS-MOS" with feelings? But that's just a random thought! XD
Mechanics, Legendary Warriors seal their spirits; damn Usagi cleaned up the world quick if they can do all that advancement? Of course with the other various stories of the eventual demon battle and Ken's own involvement in his main story, peace does get broken for a minute.
Anyway I'm rambling on, again good chapter!
One more thing; it's a twist that in this axis Daiki get's his arm taken and given that YYGDM axis where the revamped story takes place, wouldn't that mean Dai (when he's born and grows up) will have the exact thing happen to him? Paradox?
| Belletiger BT chapter 22 . 8/11/2015
I really loved Daiki's flashback and how he did loose the arm. And I loved how you added the friendship he has with her. Kinda reminds me the relationship of Tony and JARVIS. Anyway, I can't wait to see more!


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Latest Human Rights NewsFollow us on facebook or on twitter Statement Civil Society Condemns Conviction of Human Rights Defender Ny Chakrya
September 22, 2016
We, the undersigned civil society organisations, condemn in the strongest terms today's unjustified conviction of former Head of the Human Rights and Monitoring Section of the Cambodian Human Rights and Development Association ADHOC) and deputy secretary-general of the National Election Committee (NEC), Mr. Ny Chakrya, by the Phnom Penh Municipal Court. After a four and a half hour trial, Judge Khy Chhay deliberated for a mere 15 minutes before deciding to convict Mr. Ny Chakrya on charges of defamation, malicious denunciation, and publication of commentaries intended to unlawfully coerce judicial authorities, under Articles 305, 311 and 522 respectively of the Cambodian Criminal Code. Mr. Ny Chakrya was sentenced to six months in prison and given a fine of six million Riel (approximately $1,500).
September 19, 2016 This morning, Boeung Kak Lake representatives Tep Vanny, Bo Chhorvy, Kong Chantha and Heng Mom were found guilty of obstruction of a public official with aggravating circumstances and insult relating to a November 2011 protest.
Judge Ly Sokleng sentenced all four women to six months' imprisonment after a three-hour hearing at the Phnom Penh Municipal Court. Tep Vanny was sent back to pre-trial detention in CC2 prison – where she has now been for nearly five weeks – for separate charges relating to a protest held by Boeung Kak Lake community in 2013 in front of the Prime Minister's house. No special enforcement order was issued so Bo Chhorvy and Kong Chantha – as well as Heng Mom, who was tried in absentia – were not arrested as their sentence will not be enforced until all appeals are exhausted. None of the evidence presented at the trial sufficiently proved the guilt of the four women.
About 30 community members and supporters from other land communities and civil society groups gathered outside the court during the trial.
Related Material:
August 22, 2016
Equitable Cambodia (EC), Cambodian League for the Promotion and Defense of Human Rights (LICADHO), and Center for Alliance of Labor and Human Rights (CENTRAL) are extremely disheartened by today's verdict against Eang Vuthy, Chheang Phea and Phen Kimsong. From the outset, it has been our position that there was no legal basis for this case. This was made clear during the trial hearing, when the prosecutor informed the trial judge during his closing remarks that there was no evidence to support the charge of defamation.
August 18, 2016
We, the undersigned civil society groups, demand the immediate release of two Boeung Kak Lake activists – Ms. Tep Vanny and Ms. Bov Sophea – who were charged and sent to provisional detention in Prey Sar's CC2 prison on 17 August 2016, in relation to their involvement in the "Black Monday" campaign. We also call on authorities to drop the spurious incitement charges against them, and allow for peaceful assemblies, such as those in support of the Black Monday campaign, as protected under Cambodian and international law.
August 18, 2016
We, the undersigned international, regional, and Cambodian non-governmental organizations, urge your delegation to support the adoption of a resolution addressing Cambodia's gravely deteriorating human rights situation at the 33rd session of the Human Rights Council.
This resolution should highlight and condemn the ongoing and systematic human rights violations in the country and impunity for their perpetrators; build on the concerns expressed by a number of States at the Council's 32nd session; and urge the Cambodian Government to urgently take corrective action to preserve the legacy of the Paris Peace Agreements, ahead of their 25th anniversary and of key municipal and general elections.
July 24, 2016
At 7 o'clock this morning, mourners will begin a procession carrying the body of Dr. Kem Ley from Wat Chas, Phnom Penh, to his home village of Ang Ta Kok, Takeo Province. It is expected that thousands of mourners will take to the streets to join the procession. On 10 July, Kem Ley was shot dead in a busy Caltex petrol station cafe in central Phnom Penh. One suspect, a former soldier, was arrested shortly after the shooting and remains in custody.
July 11, 2016
We, the undersigned civil society groups, are outraged at the brutal murder of political analyst and grassroots organiser Kem Ley yesterday morning, and call for a prompt, thorough and independent inquiry into his death.
June 8, 2016
Prey Speu "Social Affairs" center has made the headlines again recently following the decision by the Phnom Penh Municipality to keep the detention facility open despite Prime Minister Hun Sen's support for its closure (Cambodia Daily, "Officials Keep Prey Speu Open After PM's Rebuke", June 3, 2016).
May 10, 2016
Yesterday's detention of eight human rights workers and activists was an egregious violation of the right to freedom of expression in Cambodia, civil society groups said today. The detentions, which followed the pre-trial detention of four other rights workers and an election official last week, highlight an alarming surge in the Cambodian government's latest campaign of intimidation against civil society.
May 2, 2016
We, the undersigned human rights and other civil society organisations, condemn in the strongest terms the politically-motivated charging of six human rights defenders from a Cambodian human rights group, the country's National Election Committee (NEC) and the United Nation's Office of the High Commissioner for Human Rights (UNOHCHR). The targeting of these individuals, five of whom were sent to pre-trial detention today, is the latest escalation in a far-reaching government assault on civil society ahead of upcoming local and national elections, and is a clear reprisal for support provided by rights workers in a politically-sensitive case.
April 29, 2016
We, the undersigned civil society organisations, strongly condemn the summonsing and interrogation of civil society members for conducting vital and legitimate activities to protect human rights. This amounts to an outrageous misuse of the Anti-Corruption Unit (ACU) as a political tool to attack and intimidate civil society.
April 29, 2016
The European Union (EU) must use the upcoming human rights dialogue with Cambodia to demand that authorities stop the harassment, arbitrary arrest, and imprisonment of opposition MPs, members, and their supporters, FIDH and its member organizations, the Cambodian Human Rights and Development Association (ADHOC) and the Cambodian League for the Promotion and Defense of Human Rights (LICADHO), said today. This year's EU-Cambodia human rights dialogue is scheduled to take place from 2 to 4 May in Phnom Penh.
March 31, 2016
Behind a façade of "technical" intent, the new Law on Telecommunications (Telecoms Law) poses a severe threat to freedom of expression in Cambodia, targeting not only online public expression but also any private communications made using telecommunications devices. In a briefing paper released today, LICADHO analyses the law's most egregious provisions – which, among others, allow the government to secretly intrude into the private lives of individuals, destroy evidence before criminal trials, and seize control of the entire telecoms industry if arbitrarily deemed warranted. Its excessive measures, particularly those creating new criminal offenses, reveal the true intent of the law: to intimidate individuals, punish the exercise of fundamental rights and freedoms and quash individual and group dissent.
March 28, 2016
In this video, LICADHO's medical team pays tribute to Volker Jantzen, of Swiss NGO Dynamic Share, who died in January 2016 after a long illness.
Since 2013, support from Dynamic Share has been invaluable for LICADHO's work providing medical care to victims of human rights abuses in prisons, at relocation sites, to human rights defenders and on-the ground during protests and demonstrations. From all of us at LICADHO, we want to express our gratitude to the generous support Mr. Jantzen, through Dynamic Share, has provided to Cambodia.
March 7, 2016
Tomorrow is International Women's Day. To mark the event LICADHO is releasing a new audio book and summary audio clip titled Getting Away With It: The Treatment of Rape in Cambodia's Justice System. Both the book and the summary present evidence of the immense failure of the Cambodian justice system to properly investigate and prosecute cases of rape involving women and children. They provide details of multiple systemic flaws – corruption, discriminatory attitudes towards women and girls, misinterpretation of the law, and lack of resources – which, together, mean that many perpetrators of rape receive only very lenient punishment or go completely unpunished.
February 23, 2016
In response to the brutal attack on protesting workers on February 6, 2016, we, the undersigned trade unions, community representatives and civil society organizations (CSOs), declare our strong support for the ongoing boycott of Capitol Co. Ltd, also known as Capitol Tours, and call upon the general public to join the boycott in solidarity with the wrongfully dismissed Capitol employees, and the wider Cambodian trade union movement. Capitol Tours has relentlessly violated the labor rights and fundamental freedoms of its employees and we wish to unreservedly condemn the company's actions.
February 15, 2016
In response to the latest unjust charges brought against three further members of the environmental NGO Mother Nature, we, the undersigned civil society organizations (CSOs), declare our support for Mother Nature, and condemn the Royal Government of Cambodia (RGC)'s crackdown on the organization and Cambodian civil society in general.
February 6, 2016
We, the undersigned civil society groups, strongly condemn this morning's violence by members of the Cambodia for Confederation Development Association (CCDA) against protesting bus drivers and their supporters, as well as the woefully inadequate police response, call for the release of two men arbitrarily arrested and demand an investigation into the violence.


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Nutanix exceeds expectations in upsized IPO
Sept 29 Nutanix Inc said on Thursday it sold a more than expected 14.87 million shares priced at $16, valuing the company at more than $2 billion.
* GMAC's halt comes day after Massachusetts lawsuit
* GMAC parent is limiting correspondent lending
* Other U.S. banks have shuttered correspondent units
By Joe Rauch and Brenton Cordeiro
Dec 2 GMAC Mortgage, the mortgage arm of Ally Financial Inc, said on Friday it will stop buying new mortgage loans in Massachusetts that were made by other correspondent lenders and wholesale brokers.
GMAC said it will honor all commitments made with such lenders as of Dec. 5, 2011, and will continue to service its existing customers and honor its contractual obligations as a servicer.
The move comes a day after the Massachusetts attorney general filed a lawsuit against five large U.S. banks -- Bank of America Corp , JPMorgan Chase & Co , Citigroup , Wells Fargo & Co and GMAC -- accusing them of deceptive foreclosure practices, such as using robo-signers and false documents.
"GMAC Mortgage has taken this action because recent developments have led mortgage lending in Massachusetts to no longer be viable," the company said in a statement.
GMAC said it will continue to make loans to Massachusetts customers through its direct lending business.
A spokesperson for Ally Financial said in an e-mailed statement that "GMAC Mortgage is currently operating in full compliance with the law. Any suggestion related to past activity will be heard before the court, and we are confident in our ability to prevail."
Analysts said the move is unlikely to be followed by other lenders named in the Massachusetts lawsuit.
"There's so much posturing in this," said Marty Mosby, bank analyst with Guggenheim Securities. "It's almost like someone shoots across someone's bow, and they just shoot back."
Massachusetts Attorney General Martha Coakley criticized the move and in a prepared statement said that GMAC must "follow the law before foreclosure on homeowners."
"With today's action, it appears GMAC has acknowledged it has a problem following those laws and being held accountable for doing so," she said.
Ally Financial was the state's fourth-largest mortgage lender, when ranked by volume of loans sold to Fannie Mae and Freddie Mac, through the first six months of 2011, said Guy Cecala, publisher of Inside Mortgage Finance, an industry trade publication.
Ally Financial sold $1.5 billion loans to the U.S. government-backed mortgage investment companies, totaling 8,000 loans so far this year, Cecala said.
Overall, Massachusetts is the seventh-largest U.S. state for loans sold to Fannie Mae and Freddie Mac, Cecala said.
In November, Ally Financial announced plans to scale back its correspondent lending operations company-wide, following other U.S. banks in limiting those types of mortgage loans.
In correspondent lending, banks buy loans originated by outside banks.
An Ally Financial spokeswoman said the cutback is due to the rising costs of mortgage lending in Massachusetts, and is separate from the company's broader rollback.
Other large banks have also curbed their correspondent lending efforts.
Bank of America, the second-largest U.S. bank by assets, said earlier this year it planned to shutter its correspondent lending unit by the end of 2011.
Sept 29 Nutanix Inc said on Thursday it sold a more than expected 14.87 million shares priced at $16, valuing the company at more than $2 billion.
SAN FRANCISCO, Sept 29 In the run-up to its initial public offering, cloud computing company Nutanix scrambled to avoid a situation that has increasingly marred technology IPOs: a price that is below the most recent private funding round.
Sept 29 Oregon State Treasury said on Thursday that it will press Wells Fargo & Co to make reforms in its management structure and executive compensation in light of a sales scandal that has become a major issue in Washington and on Wall Street.


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Top Definition
Being up to your elbow in something (ie. mud, money, a twat...)
Guy #1: Holy shit bro, I swear, last night I got twat-deep in Amy's pussy!
Guy #2: WTF!!
Guy #2: WTF!!
Grant Martz tarafından 10 Eylül 2007, Pazartesi
Trending Now
Can you define these popular missing words?


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February 14, 2013
I'm from the South.
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