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A 42-year-old man in the southern province of Binh Phuoc has lost 40 kilograms, much of it shed skin, due to a strange condition that has also turned his face dark brown.
Doctors have been unable to diagnose the problem.
Relatives of Van Viet Dien, 42, told Tuoi Tre in a Friday report the man had pain in his ankle in July last year. He visited a private clinic and was given an injection of painkillers and medicine.
Ung Thi Ngoc Hanh, his 39-year-old wife, said the pain got worse and he started to shed skin, while his voice, vision, movement, and hearing deteriorated.
Dien, who weighed more than 70 kilograms last year, is now 30 kilograms.
Doctor Le Thanh Hung, who ran the clinic, denied giving Dien any shot.
Hung, who also works at the Binh Phuoc General Hospital, said he prescribed some anti-fever tablets and tonic.
Van Viet Dien, 42, (right), before his skin condition started to develop in July 2011. He has lost 40 kilograms.
But the condition did not improve in four days, so he advised the family to take Dien to a better hospital, the doctor told Tuoi Tre.
The family said they received various answers from doctors at leading hospitals in Ho Chi Minh City, including the Hospital for Tropical Diseases and Cho Ray, where he stayed for nearly two months.
They have spent more than VND300 million (US$14,400) and no treatments have helped.
A couple of doctors said he was allergic to some kind of medicine, some said he had septicemia or a blood infection, and others blamed pus in his lung, or Steven Johnson syndrome, a life-threatening skin condition.
Dien is being taken care at home as the family is confused and does not want to spend more money on uncertainties. | 2019-04-24T14:10:58 | http://www.thanhniennews.com/health/unidentified-skin-condition-causes-vietnamese-man-to-lose-40-kilos-5322.html |
Ken’s Tech Tips is a mobile technology blog. We focus on mobile phone networks in the UK, new mobile devices and mobile tariffs. We cover the latest mobile phone deals and share tips on how to get the most out of your mobile phone. Our core audience is consumers from the United Kingdom.
Logging in to Google Adwords and visiting the Adwords Display Planner.
In the “Your customers are interested in” box, search for kenstechtips.com.
Click on “Get placement ideas” (the grey box).
You should see our website listed at the top of the results.
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If you have any further questions about advertising on the website, please contact me via e-mail at ken at kenstechtips.com. I’d be happy to discuss a range of advertising formats and would be looking forward to receive your e-mail. | 2019-04-21T02:18:51 | https://kenstechtips.com/index.php/advertising |
Discussion in 'Basic4ppc Wishlist' started by JesseW, Mar 26, 2009.
Being a throwback from the VB6 days, I'm used to setting up variable names capitalized the way I like them in the DIM statements, then just typing along in lowercase and letting the IDE fix the other occurrences automatically. B4P does this with keywords... Can you add this functionality to variables, including library and control names, as well?
Are you using Ctl-SpaceBar? It auto-completes just about anything with the correct capitalisation.
No, I hadn't realized ctrl-spacebar. But even remembering ctrl-spacebar is too much for me. I type pretty fast, and just like to roll on without capitalizing or ctrl-spacing.
Erel, how bout it? If a variable is Dimmed and used later not in the same case, could the IDE correct the case? If a variable isn't dimmed, then it should be left as is?
I also favorably remember this VB6 behavior.
Will see how and when it will be implemented. | 2019-04-21T20:07:28 | https://www.b4x.com/android/forum/threads/auto-capitalized-variable-names.4069/ |
Montreal is a popular but so vastly different city than major cities around it, like Toronto and Ottawa. The city is built so beautifully with elevation changes, historic buildings and a particular focus on architecture and spacing.
What I really liked specifically was the ‘of-course’ mentality they have with architecture and design in the city. I feel like bad design dies on the drafting table before the design is even complete.
Of course this building should look different on the street than other buildings, why would we want boring buildings?
Feels like design is more of a way of life here, it’s refreshing. But getting to Montreal from Toronto, seems to take the enjoyment right out of the trip. Whether it’s getting there or getting back. With long, cumbersome commutes, you tend to forget the beauty of your trip.
But, I’m a businessman, I do business-y things and I can’t be sitting in traffic or waiting in line at the airport for a short trip, I need something different. I need to enjoy and make the best out of my commute.
This time we wanted to see what it would be like to take the train there instead. We still get to conduct business, sit back and relax, knowing we don’t have to drive and we can have a dinner that doesn’t come in a brown paper bag. It was a good decision.
We booked a business class trip through Via Rail from Toronto to Montreal, leaving at 6:00pm. First things first, we checked out the Via Rail business lounge, had soft drinks, free wifi, TVs and comfortable chairs. Worth waiting in.
Our train time was called and we headed straight to the train, no waiting line and got to our spot.
I had a great circle of people around me, heading back home to Kingston. We shared stories, wine and more, made the trip go much faster. The trip brought me into the station around 11:00pm, just the right amount of time to have a good night sleep ahead or drop my bag, freshen up and hit the town.
Everyone usually has issues with food during a commute when it comes to airline food, etc. However, with Via Rail, we were able to enjoy beverages while enjoying some great food (chicken or beef, fish, or pasta (vegetarian)). Tried the pasta on the way there, fish on the way back. Both great with it’s compartmentalized dishes with dessert, some cheese and wine on the side, delightful. It’s hard to have mass transit with the white tablecloths, the crystal wine glasses and silverware, but the food was great for the trip.
The staff were cheerful, talkative and provided a great social experience. I even had some of the same people from the train there for the train back to Toronto. The wifi was steady throughout the ride and the desks provided us enough space for our laptop to sit.
Overall, with the whole experience, if you aren’t in an absolute rush to get to Montreal and don’t want to worry about hitting rush hour traffic, having a consistent arrival and departure time, then maybe, just maybe, you should take the train.
For more information, be sure to visit Via Rail’s website for destinations, pricing and scheduling your next trip. | 2019-04-23T05:01:35 | https://lxry.ca/training-enjoy-weekend-montreal-via-rail/ |
From one painful surprise to another.
From one cheerful surprise to another.
From one fruitful surprise to another. | 2019-04-21T12:21:11 | https://www.srichinmoylibrary.com/gvp-44 |
For far too long, the only way to reverse the effects of aging has been a facelift. While this is an effective technique for many, it's also painful, invasive and the results fade over time.
A skin care regime that includes ingredients that tighten, firm, lift, sculpt, and contour the face can prove an excellent, noninvasive alternative.
When you use DMAE Serum, part of the Donna Bella Signature line of DMAE products, you can achieve dramatic effects. | 2019-04-20T19:09:30 | https://www.asseenontvlive.com/product/24k-gold-dmae-instant-lifting-serum?transfer= |
Jesus is the only good shepherd. The rest of us, no matter how steadfast or seemingly indispensable to the life of the community, are his lambs. And this is “the sum of the gospel.” We take comfort in the promise of his loving care, and rest on his shoulders.
The Sunday liturgy is rich with meaning. The gathering of sinner-saints, the order of service, the scriptural narrative, the sacraments, the music and lyrics, the liturgical art, the furniture – every detail has something to say about who God is for us, and who we are as people of God. The classic liturgical attire is no exception. If you’ve ever wondered to yourself, “What’s with the robe?” I’m sure you’re not alone. It may be that the leader’s robe, or alb, is such a longstanding symbol in churches like ours that we simply take it for granted. But, what does it mean?
One explanation I’ve heard is that the alb is reminiscent of a shepherd’s robe, signifying that the pastor is like a shepherd to his or her congregation, or “flock.” After all, the English word pastor comes from the Latin for “shepherd.” And, I imagine some pastors prefer to understand their role this way. There’s a certain privilege in being entrusted with the care and guidance of others. It feels good to be needed. Similarly, I imagine some congregants appreciate the idea that the pastor is their shepherd. It’s a comfort to have someone to look to for the fulfillment of your spiritual needs.
But, we should be cautious about the shepherd model for pastors. On the one hand, the pastor who sees herself as the shepherd of her flock may be tempted to believe that she is uniquely responsible for the congregation’s ministry, that she must become all things to all people, and that congregants themselves are not equipped to lead in faithful and meaningful ways. On the other hand, congregants who elevate their pastor to the role of shepherd may come to rely too heavily on her for leadership, to develop unrealistic expectations for her work, and to view ministry as a service the pastor provides rather than the work God calls us all to do.
The Fourth Sunday of Easter, traditionally known as Good Shepherd Sunday, is an occasion to remember who we all are in God’s sight, regardless of our various gifts and callings. “I am the good shepherd,” Jesus proclaims at the beginning of our Gospel from John. It’s a well-loved image for Jesus, and seemingly self-explanatory. But this short statement is brimming with significance, each word playing an important role.
“I am the good shepherd.” The first two words of Jesus’ pronouncement harken back to God’s mysterious self-designation early in the Book of Exodus. “I AM WHO I AM,” God responds when Moses asks for God’s name; “I AM WHO I AM,” because no proper name can capture the fullness of God’s identity. So, in Jesus’ famous “I am” statements in the Gospel of John – “I am the good shepherd,” “I am the bread of life,” “I am the vine,” and others – we hear the echo of God’s own name, and we recall the singular relationship between God the Father and the Beloved Son.
“I am the good shepherd.” Jesus is the true shepherd, the model shepherd. “I know my own and my own know me,” he insists, “just as the Father knows me and I know the Father. And I lay down my life for the sheep.” Unlike the hired hand, who is concerned only for his own well-being, Jesus knows and loves his sheep, and no sacrifice is too great for our sake. For this reason, we trust Jesus to be our guide in good times and bad. He is the one who makes us lie down in green pastures, who leads us beside still waters, who restores our souls. And, through him we come to know the heart of God, whose goodness and mercy follow us all the days of our lives.
So, what’s with the robe? If pastors aren’t shepherds, then what does the alb mean after all? In the first centuries of the church, the alb served as the basic symbol of baptism. Emerging from the baptismal pool early on Easter morning, the newly baptized were clothed with a bleached white robe, a sign that they had “put on Christ,” in the words of Paul, and joined all those who had “washed their robes and made them white in the blood of the Lamb,” in the words of Revelation. Although this practice has largely been lost, many parents still dress their children in white when they bring them to the font.
So, if it’s a symbol of baptism, then all the baptized might wear the alb, except that we might be mistaken for a cult. Instead, worship leaders wear the alb on behalf of everyone to signify that we are all held in the grace and mercy of God. Dear church, we are the beloved flock of the Good Shepherd. Joined to the death and resurrection of Christ in baptism, we are all precious in the Lord’s sight. So, take comfort in the promise of his loving care, and rest on his shoulders.
Barbara J. Essex, in Feasting on the Word, Year B, Vol. 2, 451.
Day by Day We Magnify You, 187.
Lorraine S. Brugh and Gordon Lathrop, The Sunday Assembly, 91. | 2019-04-25T14:38:20 | https://www.peacepuyallup.org/sermons/the-only-good-shepherd/ |
The Watertown Economic Development Authority (EDA) was created to serve as a resource to develop Watertown's local economy. The EDA is served by two City Council liaisons and three members from the general public. Members of the EDA are: Ken Grotbo (Chair), Corey MIttiness, Roxanne Wilems, Lindsay Guetzkow (City Council), and Deborah Everson (City Council). The City Administrator serves as the Executive Director of the EDA and as the primary contact.
The Watertown EDA is ready to work with existing businesses and new leads to ensure a robust and active local economy. The EDA has already partnered with downtown property owners for fascade improvements and new businesses in the Business Park.
The Watertown Economic Development Authority (EDA) has resources available to grow your business in Watertown! The local community has a long history of supporting local businesses, either welcoming those who are new to town or an established businesses seeking to expand. The EDA and City Council have a proven track record of using creative economic development tools at their disposale to help the local economy thrive. In addition to the various economic development tools available to the EDA and City like TIF, tax abatement, etc. The EDA administers two grant and loan programs for various business uses.
To learn more about Watertown and the unique and important statistics about our community please view our Community Profile and Welcome to Watertown video.
EDA Staff Contact: Shane Fineran, City Administrator sfineran@ci.watertown.mn.us or 952-955-2681.
101 Territorial Video Tour - Riverfront and Gateway parcel to the downtown area of Watertown. This parcel is eligible for TIF redevelopment incentive.
NE Watertown/Mill Ave Development Land - Approximately 118 acres of prime land available for residential development in the NE area of Watertown. Adjacent to the Forest Hills neighborhood and sweeping views of the Crow River valley.
609 Jefferson - Hwy 25 multi tenant space. Excellent visibility and access.
300 Lewis Ave S - Excellent location in the heart of downtown, ample private and public parking. Adjacent to the Luce Line regional trail.
676 Industrial Blvd - 13,600 sq ft industrial site with loading docks, office, and warehouse/manufacturing.
The Building Facade Improvement program aids businesses in commercial, industrial, and central business districts complete building envelope improvements. The program provides up to $5,000 in loan assistance and up to $2,500 in grant aid to local property owners who make improvements on the facade of their building.
The EDA makes available resources to help a business fund start up through the Revolving Loan Fund program. Loan proceeds of up to $25,000 can be used for capital acqusition, capital equipment, property acquisistion etc. to help a business locate or expand in Watertown. The loan offers competitive terms and finance rates.
A unique incentive program that provides grant funds for eligible projects that invest in the facade and interior of a home. Projects seek to increase the livability of the home for those aging in place or to meet the needs of contemporary home owners. Homes must be at least 35 years old. Up to $5,000 could be available in grant funds for eligible projects. | 2019-04-19T00:40:36 | https://www.ci.watertown.mn.us/government/economic-development-authority |
The DNA methyltransferase Dnmt3a suppresses tumorigenesis in models of leukemia and lung cancer. Conversely, deregulation of Dnmt3b is thought to generally promote tumorigenesis. However, the role of Dnmt3a and Dnmt3b in many types of cancer remains undefined. Here, we show that Dnmt3a and Dnmt3b are dispensable for homeostasis of the murine epidermis. However, loss of Dnmt3a-but not Dnmt3b-increases the number of carcinogen-induced squamous tumors, without affecting tumor progression. Only upon combined deletion of Dnmt3a and Dnmt3b, squamous carcinomas become more aggressive and metastatic. Mechanistically, Dnmt3a promotes the expression of epidermal differentiation genes by interacting with their enhancers and inhibits the expression of lipid metabolism genes, including PPAR-γ, by directly methylating their promoters. Importantly, inhibition of PPAR-γ partially prevents the increase in tumorigenesis upon deletion of Dnmt3a. Altogether, we demonstrate that Dnmt3a and Dnmt3b protect the epidermis from tumorigenesis and that squamous carcinomas are sensitive to inhibition of PPAR-γ.
Most of the cells in our body contain the same DNA. However, our bodies are made of many different types of cell, such as nerve cells or skin cells, which perform very different jobs. In each cell type only certain sets of genes encoded by the DNA are active. Proteins known as epigenetic regulators are responsible for producing the different patterns of gene activity. If epigenetic regulators are switched on or off at the wrong time, they can contribute to ageing and diseases such as cancer.
Enzymes known as DNA methyltransferases are one group of epigenetic regulators. DNA methyltransferases control the activity of genes by adding small chemical groups known as methyl groups to the DNA. Two of these enzymes – known as Dnmt3a and Dnmt3b – are important during development to help cells mature and specialize into different types. Mice that lack both of these enzymes either die as embryos or just after birth. Furthermore, these enzymes are mutated or less active in some skin cancers and various other human cancers.
Here, Rinaldi et al. investigated the role these enzymes play in adult mice. The experiments show that under ordinary laboratory conditions, mutant mice that lacked Dnmt3a and Dnmt3b were as healthy as normal mice. However, when the mice were exposed to chemicals that promote tumor growth, which mimics skin exposure to UV light, the mutant mice developed many more skin tumors than the normal mice. Furthermore, the tumors in the mutant mice were more likely to form secondary tumors in the lung. Rinaldi et al. found that Dnmt3a reduced the production of a protein called PPAR-γ, which helps to break down some types of fat molecules. Treating the mutant mice with a drug that inhibits PPAR-γ activity slowed the growth of the tumors.
Overall, these experiments show a new way in which DNA methyltransferases act in adult animals. Future research will investigate whether drugs that inhibit the breakdown of fats could help to treat cancers in which the Dnmt3a and Dnmt3b proteins are mutated or less active.
DNA methylation is an epigenetic mechanism that regulates several aspects of gene expression, such as long-term gene silencing, transcriptional elongation, and maintenance of genomic stability (Allis and Jenuwein, 2016; Avgustinova and Benitah, 2016; Rinaldi and Benitah, 2015). It is found throughout the vertebrate genome and is deposited by DNA methyltransferases on the fifth position of cytosine (5-mC), predominantly at CpG dinucleotides. The role of DNA methylation in establishing different cell fates during embryogenesis is fairly well understood. However, if and how DNA methylation is necessary to stably maintain the identity of adult stem cells, and how this process is disrupted during oncogenic transformation, is under intense investigation (Shen and Laird, 2013).
Three DNA methyltransferases are encoded in the vertebrate genome. Dnmt1 is predominantly associated with the maintenance of DNA methylation following cell division due to its high affinity for hemimethylated DNA. Consequently, depletion of Dnmt1 leads to a significant reduction of the global levels of 5-mC (Li et al., 1992; Lei et al., 1996). Dnmt3a and Dnmt3b are de novo DNA methyltransferases that establish genome-wide DNA methylation during mammalian embryogenesis and adult stem cell homeostasis (Okano et al., 1999). In mouse embryonic stem cells, the combined loss of Dnmt3a and Dnmt3b leads to the progressive loss of DNA methylation, suggesting that these enzymes are additionally involved in maintaining 5-mC levels (Chen et al., 2003).
Recently, progress has been made in identifying the molecular mechanisms underlying the biological functions of Dnmt3a and Dnmt3b by studying their genome-wide localization. For instance, Dnmt3b associates with and methylates the gene bodies of actively transcribed genes in murine embryonic SCs and human embryonic carcinoma cells (Baubec et al., 2015; Jin et al., 2012; Morselli et al., 2015). Likewise, it has been proposed that gene body methylation is responsible of most of the transcriptional changes underlying the ability of Dnmt3a to promote neural SCs differentiation, and in protecting the lung epithelium from tumor progression (Wu et al., 2010; Gao et al., 2011).
We have recently reported that Dnmt3a and Dnmt3b are required for the self-renewal of human keratinocyte progenitors, whereas Dnmt3a is also required for their proper differentiation (Rinaldi et al., 2016). Mechanistically, Dnmt3a and Dnmt3b bind to and promote the activity of enhancers in both human epidermal progenitors and differentiated keratinocytes (although Dnmt3a having a stronger affinity for enhancers in differentiated keratinocytes). Interestingly, both proteins preferentially associate to super-enhancers rather than typical enhancers. Nonetheless, they differ in their mechanism of action, since Dnmt3a (together with Tet2) is essential to maintain high levels of 5-hydroxymethylcytosine (5-hmC) at the center of its target enhancers, while Dnmt3b promotes 5-mC along the body of the enhancer. These regulatory regions dictate the transcription of essential genes necessary for epidermal stem cell identity and maintenance, such as FOS, ITGA6, TP63, and KRT5. Similar to its role in mouse ES cells, Dnmt3b also binds to and methylates the gene bodies of these genes to reinforce their expression (Rinaldi et al., 2016). Dnmt3a also associates to the enhancers regulating the expression of genes such as IVL, LOR, FLG2, and KRT1 which drive the differentiation of SCs into mature keratinocytes (Rinaldi et al., 2016). Interestingly, DNA methylation at active enhancers has also been recently reported in normal and cancer-derived human colon, mammary and prostate epithelial cells (Charlet et al., 2016).
However, to date, no in vivo studies have investigated the roles of Dnmt3a and Dnmt3b in adult epidermal function and malignant transformation. Using mouse models carrying an epidermis-specific ablation of either Dnmt3a or Dnmt3b, or both, we demonstrate that Dnmt3a and Dnmt3b are dispensable for skin homeostasis. However, Dnmt3a has a critical role in suppressing carcinogen-induced squamous tumor initiation, but not progression, while both Dnmt3a and Dnmt3b concertedly prevent tumor progression.
We first studied the pattern of expression of Dnmt3a and Dnmt3b during epidermal development, and in adulthood. At E14.5, Dnmt3a was expressed in the entire Keratin-14+ compartment comprising the basal layer of the embryonic epidermis and the hair placodes (Figure 1—figure supplement 1A). At P0, all Keratin-14+ basal cells were positive for Dnmt3a with the exception of the highly proliferative hair follicle bulb cells (Figure 1—figure supplement 1A–B). By the time animals reached adulthood, Dnmt3a levels remained high in the hair follicle bulge where most hair follicle stem cells reside (Solanas and Benitah, 2013), and decreased in the interfollicular epidermis, although some basal IFE cells expressed high levels (Figure 1—figure supplement 1A–D). On the other hand, we were not capable of detecting Dnmt3b by immunofluorescence staining in sections of developing or adult mouse epidermis (not shown), suggesting that Dnmt3b is expressed at low levels (Challen et al., 2014). In fact, RNA-seq data confirmed that Dnmt3a was enriched almost fivefold as compared to Dnmt3b in adult basal IFE keratinocytes (Figure 1—figure supplement 1C). However, Dnmt1, the main DNA methyltransferase, was the most abundant DNA methyltransferase, both in interfollicular epidermis and in hair follicle bulge cells (Figure 1—figure supplement 1C).
To gain insight to the roles of Dnmt3a and Dnmt3b in epidermal tissue function, we generated epidermis-specific conditional knockout (cKO) mice by crossing animals containing the Dnmt3a or Dnmt3b gene flanked by loxP sites with animals carrying the Keratin14-CRE-ROSA26-YFP-cassette (hereafter referred to as Dnmt3a/3b-cKO) (Gao et al., 2011). Surprisingly, neither Dnmt3a- nor Dnmt3b-cKO displayed noteworthy epidermal phenotypical differences as compared to their wild-type littermates at different postnatal ages (Figure 1—figure supplement 1D–E and Figure 2—figure supplement 1A). Likewise, despite its strong expression in hair follicle stem cells, the loss of Dnmt3a did not result in evident changes in hair follicle cycling and pelage growth (Figure 1—figure supplement 1E–F).
Deregulation of DNA methylation can alter gene expression, leading to tumor suppressor silencing or oncogene activation (Witte et al., 2014), and mutation/deregulation of Dnmt3a and Dnmt3b has been observed in several tumor types (Leppert and Matarazzo, 2014; Subramaniam et al., 2014). Recently, Dnmt3a has attracted much attention, as it is one of the most frequently mutated genes in cancer (Kim et al., 2013), especially in acute myeloid leukemia (Garg et al., 2015; Ley et al., 2010). In fact, a loss-of-function mutation of Dnmt3a is one of the earliest mutations that occurs in human acute myeloid leukemia (Shlush et al., 2014). Importantly, these mutations are functional since knock-in mice that model it develop a range of severe myeloid and lymphoid malignancies (Challen et al., 2011; Mayle et al., 2015). In addition, HSCs harboring inactivating mutations of Dnmt3a are clonally selected in ageing humans (Shlush et al., 2014). However, much less is known about how deregulation of Dnmt3a and Dnmt3b affect tumorigenesis in epithelial tissues.
To elucidate the roles of Dnmt3a and Dnm3b in skin tumorigenesis, we first generated tumors from the epidermis using the chemically induced carcinogenesis protocol based on DMBA/TPA (Ewing et al., 1988). The first epidermal squamous malignancies appeared significantly sooner in Dnmt3a-cKO than in their wild-type littermates, after only 2 months from the first DMBA treatment, indicating that Dnmt3a acts as a barrier against tumor initiation (Figure 1A,B). Dnmt3a-cKO animals also showed a significant increase in tumor burden, with an average of 17 tumors per animal compared to three tumors per wild-type animal after 6 months of initiating the experiment (Figure 1C and Figure 1—source data 1).
Dnmt3a loss shortens the onset of carcinogen-induced skin neoplasia, and increases tumor burden.
(A) Representative pictures of wild-type and Dnmt3a-cKO animals after 5 months of treatment with DMBA/TPA. Graph in panel A represents the percentage of animals WT (n = 6) or Dnmt3a-cKO (n = 6) that entered into anagen after 2 weeks of treatment of DMBA/TPA, p=0.02, Chi-Square test. (B) Time of appearance, expressed in percentages of skin tumors on wild-type or Dnmt3a-cKO animals, p=0.005. (C) Number of skin tumors after 3 or 6 months of DMBA/TPA treatment, p=0.001 and p=0.0007. (D) Representative images (hematoxylin/eosin staining) of different subtypes of skin tumors. (E) Histopathological analysis of the different subsets of skin tumors that appeared after DMBA/TPA treatment of wild-type or Dnmt3a-cKO animals.
Although Dnmt3a-cKO animals showed a strong increase in tumor initiation and burden, they developed the same percentage of squamous cell carcinomas than wild-type mice (Figure 1D,E). Indeed, a detailed histological analysis of the tumors collected from Dnmt3a-cKO and wild-type animals indicated that Dnmt3a-cKO mice developed the same percentage of benign tumors, such as keratoacanthomas and papillomas, as well as of malignant invasive papillomas and squamous cell carcinomas (SCCs) (Figure 1E). Dnmt3a-cKO mice only developed an increase in the percentage of sebaceous adenomas (Figure 1E). No metastases were scored in any of the animals, as expected using this protocol in mice with a C57/Bl6 genetic background (Sundberg et al., 1997). Altogether, these results indicate that loss of Dnmt3a dramatically increases initiation of epidermal squamous tumors without affecting their malignant progression, and slightly skews the histology of tumors towards the sebaceous lineage.
Dnmt3a suppresses K-Ras-driven lung tumor progression, whereas Dnmt3b is pro-tumorigenic in APC-deficient colorectal adenomas (Gao et al., 2011; Lin et al., 2006). Hence, we next tested whether Dnmt3a and Dnmt3b also exert opposing effects regarding tumorigenesis in the epidermis. Interestingly, there were no differences between wild-type and Dnmt3b-cKO mice with respect to either the timing of tumor initiation or tumor burden upon treatment with DMBA/TPA (Figure 2A-right panel). There were no significant changes in the histological appearance of the tumors, or the number of basal cells proliferating or undergoing apoptosis, between Dnmt3b-cKO and the wild-type controls (Figure 2—figure supplement 2 and Figure 2—figure supplement 3A–B).
Dnmt3a and Dnmt3b double cKO animals develop more aggressive tumors than wild-type, Dnmt3a-cKO and Dnmt3b-cKO mice.
(A) Left, representative images (hematoxylin/eosin staining) of skin tumors isolated from wild type and Dnmt3b-cKO littermates after 6 months of DMBA/TPA treatment. Right, time of appearance of tumors shown as percentages in wild-type and Dnmt3b-cKO animals, and number of skin tumors after 3 or 6 months of DMBA/TPA treatment. (B) Left, representative images (hematoxylin/eosin staining) of skin tumors isolated from wild type and Dnmt3a/Dnmt3b DcKO littermates after 6 months of DMBA/TPA treatment. Right, time of appearance of tumors represented as percentages in wild-type and Dnmt3a/Dnmt3b DcKO animals, and number of skin tumors after 3 or 6 months of treatment with DMBA/TPA. (C–D) Number of tumors (left) and time of appearance (right) expressed as percentages, in wild type, Dnmt3a-cKO and DcKO animals after 6 months of DMBA/TPA treatment. (E) Histopathological analysis of the different subsets of skin tumors that appeared after DMBA/TPA treatment of wild type or DcKO animals. (F) Representative images of metastatic nodules identified only in a percentage (33%) of the lungs of DcKO animals, scale bar = 100 μm.
To assess whether Dnmt3a and Dnmt3b potentially play redundant roles during tumorigenesis, we also induced tumors using the DMBA/TPA protocol in animals carrying an epidermis-specific deletion for both Dnmt3a and Dnmt3b in combination (DcKO). Strikingly, although DcKO animals had a severe depletion of DNA methylation in their epidermises, they formed a morphologically normal skin with all its appendages and did not develop any epidermal abnormality even up to 70 weeks of age (Figure 2—figure supplement 4A–B). This strongly suggests that de novo DNA methylation is dispensable for the long-term homeostasis of undamaged epidermis. However, when subjected to tumorigenesis, DcKO animals displayed a significantly shortened latency and significant higher tumor burden than wild-type mice (Figure 2B–F). Although these differences were similar to the ones observed in single Dnmt3a-cKO mice (Figure 2C–D), DcKO mice formed aggressive squamous cell carcinomas at a higher frequency as compared to the single cKOs of Dnmt3a or Dnmt3b (Figure 2E). In addition, metastatic nodules in the lungs were observed in 30% of DcKO animals, but in none of the wild-type, Dnmt3a-cKO, or Dnmt3b-cKO animals (Figure 2F). Recent reports show that epidermal squamous cell carcinomas that harbor cells undergoing epithelial to mesenchymal transitions are more metastatic than those that remain predominantly epithelial in nature (Latil et al., 2017; da Silva-Diz et al. 2016). Interestingly, DcKO tumors contained large areas with spindle-shaped cells that expressed lower levels of the epithelial markers E-Cadherin and Keratin14, compared to the wild type and to Dnmt3a-cKO tumors (Figure 2E, and Figure 2—figure supplement 5). These cells also expressed the mesenchymal marker Vimentin (Figure 2—figure supplement 6A–C). Importantly, these cells that had undergone a mesenchymal transition were still YFP+, thus deriving from the K14+ origin of the tumor (Figure 2—figure supplement 6).
Taken together, these results indicate that Dnmt3a and Dnmt3b are dispensable for epidermal homeostasis, and that Dnmt3a, but not Dnmt3b, suppresses skin squamous tumor initiation. However, both Dnmt3a and Dnmt3b repress the malignant transformation of epidermal cells into aggressive squamous cell carcinomas.
We next wanted to characterize the molecular mechanisms that might underlie the tumor-suppressive function of Dnmt3a in the epidermis. To this end, we isolated by FACS-based cell sorting the basal integrin α6bright tumor cells from four wild-type and eight Dnmt3a-cKO tumors, and performed whole-genome transcriptome profiling by RNA-seq (Figure 3A). It is important to note that our mouse pathologists scored these tumors as squamous cell carcinomas (SCCs), and that ADFP (Perilipin-2) expressing sebaceous adenomas were not included in this transcriptome study (Figure 3—figure supplement 1A–C).
Deletion of Dnmt3a results in increased tumor heterogeneity, and upregulation of genes related to lipid metabolism.
(A) Schematic representation of FACS sorting strategy to isolate both RNA and DNA from Itga6pos cells within the tumors. (B) Heatmaps representing gene expression (rlog transformed values) of the 391 differentially expressed genes between wild type and Dnmt3a-cKO sorted tumor cells. (C) Two-dimensional principal-component analysis (PCA) of RNA-seq samples from wild-type (n = 4) and Dnmt3a-cKO (n = 8) Itga6bright sorted tumor cells. (D) Gene ontology analysis using Genomatix Online Software of the 114 downregulated and 277 upregulated genes in Dnmt3a-cKO tumors, divided by biological processes and over-represented signal transduction pathways. (E) Immunofluorescence staining for Krt14 and PPAR-γ of skin tumors from wildtype and Dnmt3a-cKO animals.
The PCA analysis of the RNA-seq samples showed that the four wild-type tumors clustered together, indicating that overall their transcriptomes were defined by common genes (Figure 3B,C). In contrast, the transcriptomes of Dnmt3a-cKO tumors were substantially more heterogeneous, suggesting that the loss of Dnmt3a could result in the deregulation of numerous different pathways in cancer cells, or that in the context of Dnmt3a loss, different cell of origins (i.e. basal IFE cells, hair follicle stem cells, or Lrig+ stem cells) might be more prone to generate more transcriptionally divergent tumors. Nevertheless, 391 genes were consistently differentially expressed between wild-type and Dnmt3a-cKO tumors, of which 114 were downregulated and 277 were upregulated in the latter (Supplementary file 1). The downregulated genes were mainly associated with apoptosis, suggesting that loss of Dnmt3a promotes cell survival and protects against programmed cell death (Figure 3D); consequently, TUNEL and active Caspase3 staining confirmed that Dnmt3a-cKO tumors had fewer apoptotic cells as compared to wild-type tumors (Figure 3—figure supplement 2A–B and Figure 3—figure supplement 2—Source data 1). Dnmt3a-cKO tumors also expressed higher levels of several genes involved in cell proliferation (Figure 3D). Interestingly, proliferation was only significantly increased in pre-cancerous DMBA/TPA-treated Dnmt3a-cKO epidermis (Figure 3—figure supplement 3A–B), while no differences in proliferation were evident between the homeostatic epidermis and tumors of wild-type and Dnmt3a-cKO mice (Figure 3—figure supplement 3A–B). Altogether, this suggests that the loss of Dnmt3a endows pre-cancerous mutant basal cells with a survival and proliferative advantage, which could account for the increased number of tumors these mice develop. However, once tumors are formed, they progress with the same kinetics as wild-type tumors (Figure 3—figure supplement 3C and Figure 3—figure supplement 4—Source data 1).
Gene ontology (GO) analysis of the 277 genes that were upregulated in Dnmt3a-cKO basal tumor cells highlighted two principal pathways that were over-represented in all eight Dnmt3a-cKO tumors: Wnt signaling (including ligands and receptors), and more predominantly, lipid metabolism (Figure 3D). Interestingly, recent reports have associated an increase in lipid metabolism with increased tumorigenesis of chronic myeloid leukemia, as well as colorectal, liver, oral, and breast cancer (Beyaz et al., 2016; Ma et al., 2016; Camarda et al., 2016; Ye et al., 2016; Corbet et al., 2016; Schug et al., 2015; Pascual et al., 2017; Wahl et al., 2017; Bensaad et al., 2014; Luo and Puigserver, 2016). A number of genes associated with fatty acid and lipid metabolism were upregulated in Dnmt3a-cKO tumors (Figure 3D, Supplementary file 1). Among these, the most upregulated ones encoded the key pro-adipogenic transcription factors PPAR-α and PPAR-γ, which promote adipocyte differentiation and the expression of genes involved in fatty acid metabolism, and which are not expressed in homeostatic epidermal cells (Fajas et al., 2001). The role of these transcription factors in cancer is still poorly understood, although they tend to be upregulated in many types of human tumors (Fajas et al., 2001). Importantly, PPAR-γ was upregulated at the RNA and protein levels in all the Dnmt3a-cKO sequenced tumors (Figure 3E and Figure 3—figure supplement 4A–B). Interestingly, the expression of PPAR-γ has been extensively reported to be under epigenetic control by repressive mechanisms such as H3K9 methylation and DNA methylation (Wang et al., 2013; Zhao et al., 2013).
To further dissect the early molecular changes that might result in the tumor-suppressing role of Dnmt3a in the epidermis, we did a short (6-week long) DMBA/TPA carcinogenesis treatment (Figure 4A). We then FACS-isolated Itga6brightCD34neg cells, consisting mostly of epidermal basal cells (IFE), and hair follicle stem cells (Bulge; Itga6brightCD34pos) from pre-cancerous back skin of wild-type or Dnmt3a-cKO animals for RNA-seq analysis (Supplementary file 2). After this short DMBA/TPA treatment, most of the upregulated genes in epidermal cells (IFE) were already predominantly linked to lipid metabolism and cell proliferation, whereas they related mostly to cell proliferation, and Wnt signaling in bulge stem cells (Figure 4—figure supplement 1A–B). Interestingly, we did not observe a diminished expression of genes regulating apoptosis, as we did in tumor cells. Hence, these results suggest that most of the transcriptome changes observed in tumors upon deletion of Dnmt3a occur early, and that the transition from the pre-cancerous epithelium to tumor growth occurs subsequently by bypassing apoptosis.
Dnmt3a binds a subset of enhancers in tumor cells.
(A) Schematic representation of a short treatment of DMBA/TPA in wild-type and Dnmt3a-cKO animals. (B) Genomic localizations of Dnmt3a determined by ChIP-seq of Dnmt3a in epidermal cells isolated from wild-type animals after 6 weeks of DMBA/TPA treatment. (C) Gene ontology analysis of the 363 H3K27ac-enriched regions (located at least 4 kb away from the TSS) also bound by Dnmt3a in isolated epidermis from wild-type animals after 6 weeks of DMBA/TPA. (D) Screenshot of enhancers bound by Dnmt3a in DMBA/TPA-treated skin in the FOS locus. All tracks are normalized to the number of mapped reads.
Dnmt3a is responsible for establishing and maintaining the levels of both 5-mC and 5-hmC around enhancers and promoters (Colquitt et al., 2014; Yang et al., 2016). In addition, Dnmt3a directly methylates the center of its target enhancers resulting in their subsequent hydroxymethylation via Tet2 in human epidermal keratinocytes (Rinaldi et al., 2016). To study which targets are regulated directly by Dnmt3a during transformation of murine epidermis, we performed ChIP-Seq for Dnmt3a in DMBA/TPA-treated pre-cancerous back skin epidermises from wild-type or Dnmt3a-cKO animals (Figure 4A). We also compared the ChIP-seq data obtained with MeDIP-seq and hMeDIP-seq performed on FACS-sorted tumor cells. The profiles of MeDIP-seq and hMeDIP-seq around regulatory regions (transcription start sites (TSS) and enhancers) agreed with published data (Figure 5—figure supplement 1A), and the CG content in our MeDIP-seq/hMeDIP-seq was highly enriched as compared to the input, both of which are measures of good quality data (Figure 5—figure supplement 1B).
We detected 16,483 genomic locations bound by Dnmt3a in wild-type animals, but only 64 in Dnmt3a-cKO, confirming the specificity of the Dnmt3a antibody (Figure 4B and Supplementary file 3). Of the bound regions in the wild-type epidermis, more than 20% corresponded to intergenic regions (Figure 4B). ChIP-Seq for H3K27ac using the same samples allowed us to identify 3097 intergenic regions enriched for H3K27ac that corresponded to active enhancers, 10% of which were bound by Dnmt3a in wild-type cells (Figure 4A–C, Supplementary file 3). Interestingly, proximity-based analysis revealed that the active enhancers bound by Dnmt3a predominantly corresponded to genes essential for keratinocyte differentiation and transcriptional regulation, such as Evpl (encoding for Envoplakin), Ppl (encoding for Periplakin), Fos, Myc, Cebpa, and Fosl2 (Figure 4C–D), similarly to what we have previously reported in human epidermal keratinocytes (Rinaldi et al., 2016).
The active enhancers bound by Dnmt3a contained higher levels of DNA methylation and hydroxymethylation than those not bound by it (Figure 5A,C). Importantly, loss of Dnmt3a significantly reduced their DNA methylation and hydroxymethylation (Figure 5A,C). Intriguingly, a significant reduction in DNA methylation also occurred in enhancers not bound by Dnmt3a, albeit to a statistically significantly lesser extent than those directly targeted by Dnmt3a in wild-type cells (Figure 5A,B). Upon deletion of Dnmt3a, DNA hydroxymethylation was also significantly reduced in its target enhancers, and to a lesser extent in non-Dnmt3a-bound enhancers (Figure 5C). However, the ratio of 5-hmC levels at enhancers bound by Dnmt3a between wild-type and Dnmt3a-cKO epidermal cells is significantly higher as compared to the ratio of 5-hmC levels between the enhancers that are not normally bound by Dnmt3a (Figure 5D). This indicates that the presence of Dnmt3a correlates with significantly higher 5-hmC levels, likely because Dnmt3a provides 5-mC as a substrate for generating 5-hmC, as we have previously shown in human keratinocytes (Rinaldi et al., 2016).
Depletion of Dnmt3a leads to loss of DNA methylation and hydroxymethylation around its target enhancers.
(A) Relative methylation score (CpG count) measured around 363 enhancers bound by Dnmt3a (–5 kb, +5 kb) from independent biological replicates of FACS sorted tumor cells from wild type (n = 2) and Dnmt3a-cKO (n = 2) (p<2.2 × 10−16). (B) Relative methylation score (CpG count) measured around 2734 enhancers not bound by Dnmt3a (–5 kb, +5 kb) from independent biological replicates of FACS-sorted tumor cells from wild-type (n = 2) and Dnmt3a-cKO (n = 2) animals (p=2.374e−5). (C) Global levels of 5-hmC at enhancer center (–2Kb, + 2 Kb) were quantified using HOMER software in independent biological replicates of FACS sorted tumor cells from wild-type (n = 2) and Dnmt3a-cKO (n = 2) mice at enhancers bound or not by Dnmt3a. (D) Ratio between the 5-hmC levels at enhancers bound or not by Dnmt3a in wild-type and Dnmt3a-cKO tumor cells.
In addition to active enhancers, a significant proportion (19%) of the enriched regions for Dnmt3a corresponded to promoters/TSSs (Figure 4B and Supplement file 3). To understand if Dnmt3a was methylating these promoters, we overlaid the Dnmt3a ChIP-seq with the MeDIP-seq data. Notably, the promoters bound by Dnmt3a showed a strong and statistically significant loss of DNA methylation around the corresponding TSS (Figure 6A). The levels of DNA methylation were not significantly changed at promoters not bound by Dnmt3a (Figure 6B). Of note, Dnmt3a-target TSSs were not enriched for 5-hMC (not shown). The loss of DNA methylation at the promoters/TSSs bound by Dnmt3a was also accompanied by a general increase in the transcription of these genes, measured by RNA-seq in the tumors (Figure 6C). Altogether, these data suggest that Dnmt3a directly represses the expression of a specific subset of genes by methylating their promoters/TSSs.
Dnmt3a binds and methylates a subset of promoters of genes involved in lipid metabolism in DMBA/TPA-treated epidermal cells.
(A) Relative methylation score (CpG count) measured around active and silenced promoters bound by Dnmt3a (–5 kb, +5 kb) from independent biological replicates of FACS-sorted tumor cells from wild type (n = 2) and Dnmt3a-cKO (n = 2) animals. (B) Relative methylation score (CpG count) measured around promoters not bound by Dnmt3a (–5 kb, +5 kb) from independent biological replicates of FACS-sorted tumor cells from wild-type (n = 2) and Dnmt3a-cKO (n = 2) animals (p=0.104). (C) CPM (Counts por Million) values of genes bound at the TSS by Dnmt3a in DMBA skin tumors from wild-type or Dnmt3a-cKO animals. (D) Gene ontology analysis, using Enrichr online software, of the 3521 genes bound at their promoter by Dnmt3a. (E) Screenshot of PPAR-γ gene, with all tracks normalized. (F) Normalized methylation score measured around TSS of Ppar-γ (–1 kb to +1 kb) bound by Dnmt3a. (G) CPM (Counts por Million) values of PPAR-γ measured by RNA-seq in sorted Itga6bright cells from DMBA/TPA-treated IFE and from DMBA skin tumors in wild-type and Dnmt3a-cKO mice. (H) Immunofluorescence staining for Krt14 and PPAR-γ of DMBA/TPA-treated skin and skin tumors from wild-type and Dnmt3a-cKO animals.
Interestingly, a GO analysis of the promoters bound by Dnmt3a indicated that they regulated the expression of genes predominantly involved in cell proliferation and lipid metabolism, consistent with our RNA-seq results (Figure 6D and Supplement file 3). Interestingly, Dnmt3a bound the promoters of Ppar-α and Ppar-γ in wild-type but not Dnmt3a-cKO epidermis (Figure 6E and Supplement file 3). Furthermore, 5-mC levels were lower in the TSS of the PPAR-γ gene in Dnmt3a-cKO as compared to wild-type tumors, indicating a DNA methylation-dependent mechanism of transcriptional repression (Figure 6F).
Consistent with a transcriptional derepression of the locus following loss of DNA methylation, PPAR-γ mRNA and protein levels were upregulated both in pre-cancerous interfollicular epidermis and in tumors lacking Dnmt3a, suggesting that the upregulation of PPAR-γ is acquired at the pre-cancerous stage, even before overt tumors appear (Figure 6G,H and Figure 6—source data 1).
We next tested whether the increase in the expression of genes involved in lipid metabolism was required for the earlier onset of tumorigenesis and increased tumor burden in Dnmt3a-cKO mice. To this end, wild-type and Dnmt3a-cKO mice were subjected to the DMBA/TPA skin carcinogenesis protocol, but were separated into two cohorts, one treated topically with a PPAR-γ chemical inhibitor in combination with DMBA/TPA, and the other with the vehicle (Figure 7A-diagram) (Sahu et al., 2012; Grabacka et al., 2006). Interestingly, inhibition of PPAR-γ significantly delayed the onset of tumor appearance in Dnmt3a-cKO mice, and reduced the number of tumors developed by the Dnmt3a-cKO (Figure 7B–C and Figure 7—source data 1). However, the average size of the tumors was not affected by the inhibition of PPAR-γ (Figure 7D). Thus, inhibition of PPAR-γ could be a potential new therapy for cutaneous squamous cell carcinomas harboring low levels of Dnmt3a.
PPAR-γ inhibition revert the tumor initiation phenotype of the Dnmt3a-cKO.
(A) Schematic representation of the DMBA/TPA orthotopic treatment together PPAR-γ inhibitor (Sigma GW9662) treatment onto wild-type and Dnmt3a-cKO animals. (B) Time of appearance, expressed in percentages of skin tumors on wild-type or Dnmt3a-cKO animals (vehicle and GW9662 treated): p=0.008, Chi-Square Test. (C) Number of skin tumors after 3 months of DMBA/TPA treatment plus GW9662 treatment, p=0.007 (Unpaired T-Test). (D) Tumors sizes expressed in millimeters (mm) after 3 months of DMBA/TPA plus GW9662 treatment.
At last, using public available data from four different studies we determined that the expression of Dnmt3a is significantly reduced in squamous cell carcinomas and actinic keratosis, the premalignant stage of squamous tumors, compared to human healthy epidermis (Figure 7—figure supplement 1 and Figure 7—figure supplement 1—Source data 1).
Dnmt3a modifies cytosine at CpG dinucleotides and is responsible for the proper differentiation of murine hematopoietic stem cells and murine neural stem cells (Challen et al., 2011; Mayle et al., 2015; Shlush et al., 2014). Recently, we and others have shown in human epidermal keratinocytes and murine olfactory sensory neurons, respectively, that Dnmt3a regulates gene expression by cooperating with Tet to maintain high levels of 5-hmC at enhancers (Colquitt et al., 2014; Rinaldi et al., 2016). Importantly, Dnmt3a is not only frequently mutated in human tumors (Kim et al., 2013) but is perhaps one of the first mutations to occur during tumorigenesis (Shlush et al., 2014). Using knockout mouse models, we now have demonstrated that Dnmt3a is also tumor-suppressive toward carcinogen-induced epidermal squamous neoplasia. Its loss not only accelerated the onset of tumors, but also increased tumor burden. However, once formed, Dnmt3a-deficient tumors grew, and progressed to carcinomas with the same kinetics and proportions, respectively, as their wild-type counterparts. Recent works have shown that the absence of Dnmt3a or Tet2 in hematopoietic stem cells predisposes to leukemia formation (Yang et al., 2016; Rasmussen et al., 2015), but that restoring the expression of Dnmt3a after the leukemia had developed did not revert the phenotype (Yang et al., 2016). That said, the role of Dnmt3a in tumorigenesis is tissue specific, since in the lung it does not affect tumor initiation but rather tumor progression (Gao et al., 2011). Interestingly, in the work of Gao et al., most of the changes in gene expression in Dnmt3a-depleted cells were attributed to alterations in gene body methylation, rather than at promoters. Conversely, in our model, we see significant changes at regulatory elements (i.e. promoters and enhancers) that lead to changes in gene expression in Dnmt3a-depleted epidermal tumors.
Our results, together with accumulating evidence from other groups, demonstrates a clear relationship between the levels of Dnmt3a–Tet–5-hmC and tumorigenesis: the inactivation of this axis in adult stem cells predisposes them to tumor initiation. Interestingly, a global reduction of 5-hmC is a hallmark of several cancer types, including squamous cell carcinoma, and is often correlated with poor prognosis (Zhang et al., 2016; Liao et al., 2016; Shi et al., 2016; Ficz and Gribben, 2014; Lian et al., 2012). Our results indicate that Dnmt3a drives the DNA-methylation, and subsequent hydroxymethylation, of a subset of enhancers that regulate the expression of genes involved differentiation. Conversely, Dnmt3a binds to, and DNA methylates, the promoters of cell proliferation and lipid metabolism genes to repress their expression. Interestingly, however, deletion of Dnmt3a does not result in changes in the specification of the different keratinocyte lineages in the skin (epidermis, hair follicles, and sebaceous glands), nor their homeostasis in adulthood. What is more, even the combined deletion of Dnmt3a and Dnmt3b, albeit significantly reducing overall DNA methylation levels, did not result in any skin phenotype even in aged mice. On the other hand, we have recently shown that Dnmt3a and Dnmt3b are necessary for the self-renewal and differentiation of primary human keratinocytes (Rinaldi et al., 2016). This apparent contradiction in the phenotypes observed might be due to the fact that our work with human keratinocytes relied on culturing the cells, which was recently shown in murine skin keratinocytes to induce a wound healing damaged-like reversible state that affects their epigenome and transcriptome (Adam et al., 2015). Thus, in vivo deletion of Dnmt3a might not be sufficient to alter the homeostasis of undamaged skin, but renders the epidermis more susceptible to situations of damage. Accordingly, the epidermis of Dnmt3a-cKO mice responded to the treatment of DMBA/TPA in a much more pronounced manner. This effect might not be specific to Dnmt3a. For instance, Dnmt1 is the DNA methyltransferase most highly expressed in epidermal cells and is responsible for about 70% of DNA methylation levels (Li et al., 1992). Its depletion causes a strong loss of self-renewal of primary human basal keratinocytes (Sen et al., 2010). However, its loss in murine epidermis leads to a mild increase in proliferation, and to a partial alopecia, only in very aged mice (Li et al., 2012). Intriguingly, these results also suggest that Dnmt1 and Dnmt3a/3b exert different functions in epidermal homeostasis, although future work will be required to study these putative differences in depth.
Notwithstanding the differences between in vivo and ex vivo studies, our results show that the genomic localization of Dnmt3a is very similar between intact murine keratinocytes and cultured human keratinocytes. Besides its localization at active enhancers of genes involved in epidermal differentiation, Dnmt3a also bound to, and methylated, promoters of genes that regulate cell proliferation and lipid metabolism to repress their expression. Among these genes, were the master regulators of lipid metabolism and adipogenesis PPAR-α and PPAR-γ. Interestingly, a number of recent studies have highlighted the importance of a persistent lipid metabolism in promoting tumor transformation, and tumor metastasis in colorectal, liver, breast and oral squamous carcinomas, as well as for enhancing chemoresistance of leukemia stem cells (Pascual et al., 2017; Ma et al., 2016; Beyaz et al., 2016; Ye et al., 2016). The upregulation of these transcription factors upon deregulation of Dnmt3a might predispose the epidermis to develop more tumors, suggesting that an intriguing mechanistic link between lipid metabolism and the epigenetic regulation of tissue homeostasis through DNA methylation, might exist. A recent large clinical association study has already pointed to this by establishing a correlation between the expression of obesity-related genes and changes in the content of DNA methylation (Wahl et al., 2017). Importantly, our results show that PPAR-γ is partially responsible for promoting tumorigenesis in Dnmt3a-deficient epidermis, which considering that human skin tumors express lower levels of Dnmt3a, might provide us with a new therapeutic antitumor avenue against squamous cell carcinomas.
The work with mice was approved by the Ethical Committee for Animal Experimentation (CEEA) of the Scientific Park of Barcelona (PCB), and the Government of Catalunya. Inbred male or female Dnmt3a flox/flox (C57/Bl6) backcrossed to Krt14-CRE-YFP (C57/Bl6) for six to nine generations were used for all animal experiments. Chemically-induced skin carcinogenesis was performed as previously described (Nassar et al., 2015; Abel et al., 2009), with a slight modification to yield high-frequency SCCs in the C57/Bl6 genetic background. Briefly, the back skin of 8-week-old mice—at which time hair follicles are in their resting phase (telogen)—was shaved and treated with the mutagen 7,12-dimethylbenz[a]anthracene (DMBA; 200 µl of 0.25 mg/ml solution in acetone) and the pro-inflammatory and pro-proliferation agent 12-O-tetradecanoyl phorbol-13-acetate (TPA; 200 µl of 0.02 mg/ml solution in acetone) once weekly for 6 weeks. Specifically, DMBA was given on Monday and TPA always on the Friday of the same week. For short DMBA experiments, animals were sacrificed and back skins were processed 3 days after the sixth TPA application. For tumor formation studies, treatment continued twice weekly with TPA (200 µl of 20 µg/ml solution in acetone) for up to 20 weeks, or until the largest tumor of each mouse reached 1.5 mm diameter, at which point animals were sacrificed. In total, 12 wild type and 15 Dnmt3a-cKO tumors from 6 and 8 mice, respectively, were included for tumor analyses.
The chemically-induced skin carcinogenesis was performed as previously described above (Nassar et al., 2015; Abel et al., 2009). We used the chemical 2-Chloro-5-nitro-N-phenylbenzamide (Sigma Aldrich: GW9662) described as potent PPAR- γ inhibitor. Briefly, the shaved dorsal epidermis of wild-type and Dnmt3a-cKO mice was treated twice a week topically with 200 µl of 100 nmoles of GW9662 solubilized in acetone. We applied the PPAR-γ inhibitor together with the first DMBA treatment, and subsequently administered it at every DMBA or TPA treatment. The PPAR-γ inhibitor was applied always 2 min before every administration of DMBA or TPA (Sahu et al., 2012; Grabacka et al., 2006).
To isolate pre-cancerous epidermal cells following short DMBA/TPA treatment, back skins were dissected and processed to single-cell suspensions as previously described (Jensen et al., 2010). To purify tumor cells, DMBA/TPA-induced SCCs were mechanically dissociated using a McIlwain Tissue Chopper (The Mickle Laboratory Engineering Co. LTD). Minced tumor tissue was digested under agitation in serum-free EMEM medium without calcium containing 2.5 mg/ml Collagenase I (Sigma Aldrich, St. Louis, Missouri), and 0.75 mg/ml trypsin (Life Technologies) for 90 min at 37°C. Cells were pelleted, suspended in 1–2 ml of 0.25% pre-warmed trypsin/EDTA (Life Technologies) containing 100 µg/ml (Aldrich) per tumor, and incubated at 37°C for 2 min. Trypsin was inactivated by adding EMEM without calcium containing 10% chelated FBS. Cells were washed twice in PBS and filtered sequentially through 100 µm and 40 µm cell strainers.
For ChIP-seq, single-cell suspensions were cross-linked for 10 min at room temperature with 1% formaldehyde (methanol-free; Thermofisher, 28906) and quenched for 5 min to a final concentration of 0.125M of glycine. Cells were washed 2× with cold PBS and frozen at –80°C.
For flow cytometry analysis, epidermal or tumor cells were re-suspended at 1 × 107 cells/ml in PBS and labeled with CD49f-PE (clone NKI-GoH3, 1:200, AbD Serotec) and CD34-biotin (clone RAM34, 1:50, eBioscience) followed by streptavidin-APC (1:400, BD Biosciences). Tumor cell suspensions were additionally labeled with lineage-BV605 (CD31, clone 390; CD45, clone 30-F11; TER119, clone TER119; all 1:100) (Biolegend) to exclude stromal cell contamination. Both epidermal and tumor cells were positive for YFP due to the presence of the Rosa26-YFP allele in the mice.
Tumor cells (YFPbright/lineageneg cells), pre-cancerous epidermis of interfollicular epidermis (YFPpos/CD49fhigh/CD34neg cells), and bulge hair follicle stem cells (YFPbright/CD49fhigh/CD34pos cells) were FACS-sorted using a BD FACSAria Fusion flow cytometer (BD Biosciences). Approximately, 3–20 × 104 cells were sorted and lysed in 1 ml of TRIzol for RNA and DNA isolation. After adding 200 µl chloroform, samples were vortexed for 30 s and then centrifuged at 12,000 g to separate the RNA-containing supernatant from the organic phase. RNA was precipitated with 1× volume of isopropanol, washed twice with 70% ethanol, and then used for library preparation. The interphase of the TRIzol solution (after removal of the supernatant) was precipitated adding 1× volume of isopropanol, centrifuged for 1 hr at 4°C at 13,000 g, washed twice with ethanol, and digested overnight at 55°C with proteinase K (10 mg/ml) in TE 1× buffer. The following day, digested material was incubated 1 hr at 37°C with RNase A and purified using a conventional phenol/chloroform separation. The DNA pellet was quantified, and DNA was used for library preparation for MeDIP-seq and hMeDIP-seq experiments.
Purified genomic DNA (250 ng) from tumor cells was sonicated to obtain fragments of 300–700 bp. Adaptors from the NEBNext Ultra DNA Library Prep Kit for Illumina were added to the fragmented DNA. DNA was denatured for 10 min at 99°C and cooled to avoid re-annealing. Fragmented DNA was incubated overnight with 1 µg of antibodies (5-methylcytosine, Abcam cat. # ab10805; 5-hydroxymethylcytosine, Active Motif, cat. # 39769, RRID: AB_10013602) previously cross-linked with 15 µl of Dynabeads Protein A (Life Technologies). Immunocomplexes were recovered using 8 µl for 2 hr. The following morning, DNA was washed three times for 10 min each, and purified DNA was extracted using QIAquick MinElute (Qiagen). Amplified libraries were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina (E7370L) following the manufacturer's instructions.
The libraries of total RNA from wild type and Dnmt3a-cKO tumors was prepared using the TruSeqStranded Total Sample Preparation kit (Illumina Inc.) according to the manufacturer’s protocol. Each library was sequenced using TruSeq SBS Kit v3-HS, in paired end-mode with the read length 2 × 76 bp. A minimal of 137 million paired-end reads was generated for each sample run in one sequencing lane on HiSeq2000 (Illumina, Inc) following the manufacturer’s protocol. Images analysis, base calling, and quality scoring of the run were processed using the manufacturer’s software Real-Time Analysis (RTA 1.13.48) and followed by generation of FASTQ sequence files by CASAVA.
RNA-seq datasets were pre-processed by removing both low-quality bases from the 3′- ends of the reads and adapter sequences using Trimmomatic (version 0.33) (Bolger et al., 2014). The trimmed reads were aligned to the mouse genome (UCSC mm10) using TopHat (version 2.0.13) (Trapnell et al., 2009), with default parameters and –g 5. Gene and transcript expression levels were quantified with HTSeq (version 0.6.1p1) (Anders et al., 2015). From the raw counts, counts per million (cpm) and fragments per kilobase of transcript per million mapped reads (fpkm) values were calculated. Differential expression analysis was performed using DESeq2 (Love et al., 2014) using a q-value cutoff of 0.05 and a fold-change cutoff of 1.5 to identify differentially expressed genes.
ChIP was performed as previously described (Morey et al., 2012). Briefly, frozen pelleted were lysed in 1 ml ChIP buffer (150 mM NaCl, 10 mM Tris-HCl, 5 mM EDTA, 1% SDS, 0.5 mM DTT, and 1% Triton X-100) and sonicated for 30 min in a Bioruptor Pico (Diagenode). DNA fragments were de-crosslinked overnight at 65°C and checked with a bioanalyzer. After a DNA check, chromatin was diluted 1:5 with ChIP buffer with no SDS (150 mM NaCl, 10 mM Tris-HCl, 5 mM EDTA, 0.5 mM DTT, and 1% Triton X-100). Immunoprecipitation experiments for transcription factors used 30 µg of chromatin, and those for H3K27ac, 3 µg of chromatin. Antibodies (10 µg for Dnmt3a and 3 µg for H3K27ac) were incubated overnight with the chromatin in ChIP buffer. Immunocomplexes were recovered with 40 µl of protein A bead slurry (Healthcare, cat. # 17-5280-01). Immunoprecipitated material was washed three times with low-salt buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1% Triton) and 1× with high-salt buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 1% Triton). DNA complexes were de-crosslinked at 65°C overnight, and DNA was then eluted in 50 µl of water using the PCR purification kit (QIAGEN). Antibodies used for ChIP were Dnmt3a (SantaCruz H-295; RRID: AB_2093990) and H3K27ac (Merck Millipore, cat. # 07–360; RRID: AB_310550). Libraries for sequencing were prepared using NEBNext Ultra DNA Library Prep Kit from Illumina (E7370L) following the manufacturer's instructions.
ChIP-seq datasets were aligned to the mouse genome build mm10 using BowTie (version 1.0.1) (Langmead et al., 2009); the parameters used were –k 1, –m 1, and –n 2. UCSC browser tracks (Kent et al., 2002) were created from the mapped bam file after converting it to bedGraph (normalized to 10 million reads) and subsequently bigWig format. Peak calling of Dnmt3A to determine regions of ChIP-seq enrichment over the background was done with the MACS version 1.4.1. Peaks of the methylation and hydroxymethylation datasets were determined similarly. For histone marks, MACS version two was used with parameters –broad, -q 0.01, and –g mm. ChIP-seq peaks were annotated using the annotatePeaks.pl script of the HOMER suite (version 4.6) (Heinz et al., 2010) using the UCSC mm10 annotation. The coverage depths of different ChIP-seq experiments at specified regions were also calculated using the annotatePeaks.pl script. This generated a normalized coverage value of different sequencing experiments at equally spaced bins spanning the region of interest. Bin size was set to 1 bp.
For the differential regulation analysis of MeDIP-seq data with replicates, common peaks were first determined among the replicates of the wild type and KO samples separately. A consensus peakset was then created from the two common peaksets, and the read counts were calculated for all the peaks of the consensus peakset. DESeq2 (Love et al., 2014) was applied to calculate the differentially bound peaks using an adjusted p value of < 0.05.
RRID: AB_2093990), anti-PPAR-γ (1:100 Santa Cruz, sc-7196: RRID: AB_654710), and anti-keratin 14 (1:500, Biolegend SIG-3476: RRID:AB_10718041); secondary antibodies were anti-rabbit Alexa Fluor 488 (RRID: AB_141708) and anti-mouse Alexa Fluor 647 (1:500, Molecular Probes; RRID: AB_162542). For immunofluorescence staining anti-5-Methylcytosine (1:100, Abcam10805, clone 33D3: RRID: AB_442823), sections (after deparaffinization and before Triton incubation) were incubated for 15 min with 2N HCL to further denature DNA. Adipophilin (ADFP, ab37516, 1:100 dilution; RRID: AB_722641). Tunel Staining was performed using the Promega DeadEnd Tunel System following the manufacturer's instructions. Pictures were acquired using a Leica TCS SP5 confocal microscope.
To compare tumor burden between genotypes, we used a T-Test with 95% confidence. To compare free tumor survival differences and anagen entry differences, we used a Chi-Square test. To compare Relative Methylation Score (RMS) levels and to compare normalized 5-hmC levels between wild type and Dnmt3a-cKO sorted tumor cells we used a paired Wilcoxon Test. The same paired-Wilcoxon test was used to measure differences in RNA expression.
Skin and tumor sections were stained after deparaffinization with KI67 (Abcam ab15580; RRID: AB_443209) for 60 min. After two washes, section were incubated with Power Vision Rabbit (InmunoLogic) for 45 min. Positive staining was revealed using a chromogen DAB for 5 min (Dako). Counterstain for hematoxylin was incubated for 3 min (Dako).
Stained sections were scanned using a high-resolution NanoZoomer 2.0 HT (Hamamatsu). KI67-positive nuclei in the interfollicular epidermis were measured using the TMarker software (Schüffler et al., 2013). Positive and negative nuclei for the staining were trained using the color deconvolution plugin and quantified using the cancer nucleus classification plugin. The total number of positive nuclei was normalized to the total number of nuclei in the area considered. Unpaired parametric T-test was used to measure statistical difference among groups and genotypes.
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Thank you for submitting your article "Dnmt3a associates with promoters and enhancers to protect epidermal stem cells from cancer" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Fiona Watt as the Senior Editor. The reviewers have opted to remain anonymous.
The reviewers agreed that understanding the role of Dnmt3a/b in skin carcinogenesis is an interesting and unresolved question. In particular, the authors convincingly and thoroughly show that loss of Dnmt3a in keratinocytes promotes increased tumorigenesis after DMBA treatment, which is intriguing. Transcriptional profiling and ChIP-seq data reveal genes altered by Dnmt3a.
1) The reviewers all raised concerns regarding the functional link between Dnmt and gene regulation in tumorigenesis. While the authors link the function to the suppression of PPAR and a lipid-type program, there are no functional data in the manuscript in support of this claim and, as mentioned below, this might be linked to a sebaceous tumor phenotype. Functional analysis of Dnmt targets is needed to extend this manuscript to more fully understand how Dnmt impacts tumor progression.
2) In Figure 1A, they authors show that hairs regrow faster after DMBA treatment in Dnmt3a cKO mice. The authors suggested that "Dnmt3a might act as a break to maintain hair follicles in a dormant state". This potentially interesting phenotype has not been studied in detail and this information as it is might be a bit distracting.
3) It is clear that DNA methylation can affect non-transcriptional mechanisms such as genome stability and DNA repair mechanisms, and there are no studies presented to assess non-transcriptional effects of DNA methylation on tumorigenesis. Have the authors looked at mutation frequency and/or genomic stability in these tumors?
4) The authors argue that Dnmt3a selectively affects tumor initiation rather than tumor progression. These claims are questionable. Tumor initiation, which is difficult to study, refers to the first oncogenic conversion of a normal cell. In this study, the authors make these claims based on the macroscopic detection of tumors which could entirely depend on an effect on tumor progression. DMBA causes h-ras mutations and such tumors are highly sensitive to the presence of h-ras, indicating that this is the key initiation mechanism in the DMBA/TPA model. Did the authors observe an increase in h-ras mutations after DMBA application? Did the authors observe a difference in the repair of such mutations? Are skin tumors caused by activated k-ras also affected by Dnmt3a or is the effect specific to DMBA-induced tumors? In any case, to claim an effect on tumor initiation in epidermal stem cells, the authors would need to study earlier steps in tumorigenesis.
5) In the studies (Figure 3) where the authors isolated alpha6-high cells from WT and Dnmt3a-/- tumors for gene expression studies, it would be important to include images of the histology of each tumor as supplemental information. How did they ensure that all tumors were of the same type? Also, since the Dnmt3a tumors progress much faster, how can the authors rule out that the differences relate to different stages of tumorigenesis rather than different genotypes?
6) The findings of increased PPAR-γ and linked genes suggest the possibility that the authors may be detecting differentiation along the sebaceous lineage in these tumors. This is actually supported by the data indicating that a higher proportion of such tumors are found in the Dnmt3a ko mice. Again, having overview images of each of the tumors with staining for sebaceous markers would be helpful to rule this possibility out.
7) The characterization of apoptosis and link to Dnmt3a should be improved. Does Dnmt3a act after DNA damage and increase the number of cells undergoing apoptosis following DMBA? The authors should check for active caspase 3 after DMBA and show more convincing Tunel staining. The Tunel staining data (now as Figure 3—figure supplement 1) should be quantified with statistics, and also it would be important to make sure that the same types of tumors are compared across genotypes.
8) The initiation of hair wave domains after hair removal in second anagen is highly variable and 6 mice (as in Figure 1A) is probably not sufficient to conclude that there is a difference between genotypes. Perhaps smaller domains were initiated and missed. When did the WT mice exhibit anagen patches? Plotting the time of first anagen patch might be more convincing (also this is not a major point in the paper).
9) The differences in gene expression reported in Figure 6C may be significant because of the high number of genes included in the comparison, but these differences are very small and not likely to be biologically important. Also, the differences in DNA methylation reported in Figure 6A are very small.
10) In the title and throughout the manuscript the authors refer to the study of epidermal stem cells. Cultured human keratinocytes under low calcium conditions cannot be accurately referred to as "stem cells". Furthermore, the stem cells within epidermal tumors would need to be studied in more detail to substantiate the claim that epidermal stem cells are particularly affected by Dnmt function. In Figure 4 alpha6+/CD34 is used as marker of interfollicular epithelial cells, which likely includes non-stem hair follicle keratinocytes.
11) The characterization of epidermal differentiation should be performed more carefully. Are the suprabasal epidermal layers normal in these mouse mutants? If deletion of Dnmt3a and Dnmt3b singly or in combination has little effect on epidermal differentiation during development, and epidermal homeostasis, indicating that these enzymes are dispensable for adult epidermal stem cell function, this finding would be an important one to emphasize in the manuscript. The findings in the current study are much more plausible and in line with previous studies suggesting that reversible DNA methylation does not seem to have a prominent role in gene regulation in normal tissues (it has clear roles in irreversible silencing such as imprinting, X-inactivation, transposon silencing, irreversible promoter silencing, but a role in re gene regulation has been difficult to demonstrate outside cell culture; see for example Bestor et al., PNAS 2015). There is very little data supporting the idea that methylation and demethylation are important regulators of cellular differentiation during embryonic development; the current manuscript is consistent with this view and contradicts the authors' recently published study. The authors need to discuss the divergence in their two studies more clearly and mention this key finding in the Abstract.
We agree that providing functional analysis of Dnmt3a targets, in particular PPARg, would improve the mechanistic insight of our manuscript.
First, we would like to point out that we believe that the upregulation of lipid metabolism genes we observed is not a consequence of the higher incidence of sebaceous tumors in Dnmt3a-cKO mice. Although Dnmt3a-cKO mice develop more sebaceous adenomas than wild type mice, we made sure that this type of tumor was not used for any of the mechanistic studies included in our work. We apologize for not making this clear in the first version of our manuscript. To highlight this, we now have included a new figure (Figure 3—figure supplement 1) containing the histology of each tumor used of the RNA-sequencing studies, which was performed by our mouse pathologist to make sure that none was scored as a sebaceous adenoma. We have also performed immunostaining to detect the expression of ADFP to show that none of the tumors studied expressed this sebaceous marker (also included in Figure 3—figure supplement 1). In addition, we now include as Figure 3—figure supplement 4, the immunostaining to detect the expression of PPARg in each sample, showing that Dnmt3-cKO tumors (scored as squamous cell carcinomas, and not sebaceous adenomas) show high expression of PPARg. Furthermore, the RNA-seq data obtained from the short-term treatment with DMBA-TPA shows that basal IFE cells in Dnmt3a-cKO mice already start to express high levels of genes involved in lipid metabolism (including PPARg, as also shown in Figure 6H) without any sebocyte differentiation.
Regarding the mechanistic studies: since we do not have PPARg conditional knockout mice available in our lab, we decided to use a well-characterized inhibitor of PPARg to address this issue. We repeated the DMBA-TPA chemical carcinogenesis protocol in wild type and Dnmt3a-cKO mice, and divided the mice into two cohorts, one treated with the PPARg inhibitor, and the other one with the vehicle. As shown in Figure 7, inhibition of PPARg significantly delayed the time of tumor appearance, and reduced the number of tumors, in Dnmt3a-cKO mice. Interestingly, the size of the tumors was not affected by inhibition of PPARg, suggesting that this TF is necessary for the early onset of tumorigenesis, but not for the growth of tumors once they are formed. These results therefore suggest that inhibition of PPARg could be considered as a new possible therapy for skin squamous cell carcinomas.
We would like to thank you for making this interesting suggestion, which we believe has significantly enhanced the quality of our work.
We agree that this observation could distract the readers from the primary message of our work, which is to describe the role of Dnmt3a and Dnmt3b in skin tumorigenesis. We have therefore decided to remove panel A from Figure 1, and to study this interesting phenotype in more detail in the future.
We believe this is a very interesting question. There are several publications suggesting that the epigenetic landscape of chromatin can have a major influence in the mutational load of tumors (for instance, Schuster-Böckler B and Lehner B. Chromatin organization is a major influence on regional mutation rates in human cancer cells. Nature 2012). However, these studies are correlative and do not provide any functional data. In this sense, before we reply directly to the issue raised above, we would like to point out that we have been developing for the last 4 years a large project designed to specifically determine whether the chromatin landscape (i.e. presence or absence of different chromatin modifications, and whether the chromatin is more accessible or closed) affects the mutational frequency and types of mutations in tumors. We have performed whole exome sequencing, ATAC-seq, and ChIP-seq of several chromatin marks in different mouse models in which we have deleted a specific epigenetic regulator, to precisely determine whether altering different chromatin marks has any effect over the regional mutation rates, types of mutations, and number and type of driver mutations, in DMBA/TPA-induced cutaneous carcinoma. This ongoing project has resulted in a vast amount of results, with really interesting but also quite complex conclusions. We therefore believe this analysis is beyond the scope of this current study, as it constitutes a story by itself. Because of the complexity of the analyses and results, adding just some of the data to this manuscript would not be sufficient to explain if and how epigenetic modifications, including DNA methylation, shape the mutational landscape of tumors.
We have studied whether deletion of Dnmt3a results in increased genomic instability by looking at the DNA content in precancerous epidermis. To do so, we treated for six weeks three wild type and three Dnmt3a-cKO mice with DMBA/TPA. We then analyzed by FACS the DNA content in proliferative (Edu+) and non-proliferative (Edu-) Itga6brightCD34pos and Itga6brightCD34neg cells. Notably, we did not observe any significant change in the DNA content between Dnmt3a-cKO and wild type cells, suggesting that aneuploidy, or other signs of genomic instability, do not occur upon deletion of Dnmt3a in the epidermis (Author response images 1 and 2).
We also confirmed that the expression and localization of γ-H2AX, a marker of DNA double- strand breaks and general genomic damage, were the same in wild type and Dnmt3a-cKO tumors.
We took advantage of the high quality of our RNA-sequencing to start to understand if loss of Dnmt3a alters the mutational landscape in skin tumors. We obtained between 70-110 million reads per tumor; moreover, the reads were 125 nucleotides long, 2.5X more than the conventional 50 nucleotides; and also the sequencing was pair-ended. Importantly, we calculated that each nucleotide was covered in an average depth of 150-250x in each tumor (Author response image 3).
Although we do not know at the moment whether this change has any influence on the differences in the timing and number of tumors that Dnmt3a-cKO mice develop, we believe there might be a mechanistic explanation for it. Cytosine is one of the most-unstable bases, as it often spontaneously deaminates into Uracil (Gallinari et al., 1996). 5-methylcytosine also undergoes deamination, even more often than the native form, but instead it naturally mutates into Thymine instead of Uracil. In mammals Thymine-DNA glycosylase (TDG) is the enzyme primarily responsible for repairing the spontaneous deamination of 5-MethylCytosine, as part of the Base Excision Repair (BER) mechanism. TDG recognizes the 5mC>T mutation, excises the damaged base, and subsequently initiates the process to restore the native Cytosine (Jacobs et al., 2012). Interestingly, TDG interacts with many factors such as CBP/p300, ERα, but most importantly, with Dnmt3a (Li et al., 2006). In fact, the interaction with Dnmt3a positively regulates the glycosylase activity of TDG. Recent works have underscored the strong relationship between the DNA methylation machinery and the enzymatic activity of TDG (Cortellino et al., 2012; Dalton et al., 2013; Shen et al., 2013; Muller et al., 2014). Thus, altogether we believe that the depletion of Dnmt3a leads to an impairment of the activity of TDG glycosylase, resulting in the defective repair of 5mC>T mutations.
Although exome sequencing of Dnmt3a-cKO HSCs and leukemias has been published, the raw data is not available (or we have not been able to find it). Thus, unfortunately, we have not been able to compare our data with theirs to determine whether this difference in C-T mutations is generally associated to the loss of Dnmt3a (Mayle et al., 2015; Celik et al., 2015). In any case, and to conclude, we therefore believe that loss of Dnmt3a does affect the mutational landscape of DMBA/TPA-driven tumors. However, studying if and how these differences impact tumorigenesis is turning out to be very complex and is part of a large separate study in our lab (as mentioned above); hence, we believe that addressing this issue is beyond the scope of this current study.
4) The authors argue that Dnmt3a selectively affects tumor initiation rather than tumor progression. These claims are questionable. Tumor initiation, which is difficult to study, refers to the first oncogenic conversion of a normal cell. In this study, the authors make these claims based on the macroscopic detection of tumors which could entirely depend on an effect on tumor progression. DMBA causes h-ras mutations and such tumors are highly sensitive to the presence of h-ras, indicating that this is the key initiation mechanism in the DMBA/TPA model. Did the authors observe an increase in h-ras mutations after DMBA application? Did the authors observe a difference in the repair of such mutations?
These are very interesting questions and issues. Regarding the concept of tumor initiation versus tumor progression, we referred to differences in tumor initiation as the difference in the number of tumors that grow (or initiate) in Dnmt3a-cKO mice compared to wild type mice. On the other hand, we used the term progression to describe the transition from a benign lesion to a carcinoma. We believe these definitions are commonly accepted in the cancer field. According to these definitions, although Dnmt3a-cKO mice develop/initiate more tumors, they show the same proportion of benign and malignant lesions. Hence, loss of Dnmt3a does not favor this malignant conversion (progression) of squamous neoplasias.
However, as the reviewers point out, Dnmt3a-cKO mice might develop (or initiate) more tumors due to an increase in the percentage of cells mutated in Ras. In order to determine this, we would need to perform whole-exome sequencing of sorted epithelial tumor cells from a reasonably large cohort of wild type and KO tumors (for our other ongoing studies on the influence of chromatin on mutations we are sequencing at least 20 tumors per genotype). However, using the RNA-seq data included in the manuscript, and applying the GATK software described above, we could analyze mutations in HRAS and NRAS genes. As expected, we found mutations in HRAS and NRAS in wild type and Dnmt3a-cKO tumor cells. However, and being aware that the number of samples sequenced precludes reaching definitive conclusions, the percentage of mutations in H-RAS and N-RAS was not increased upon loss of Dnmt3a (Author response image 6). In addition, the sites of mutations were the same between wild type and KO tumors in HRAS (T > A mutation), and NRAS (A >G mutations). Importantly, the HRAS mutations spotted by our analysis in Dnmt3a-cKO mice are precisely the same T>A mutation resulting in the activating Q61L transition, shown in two recent articles from the Blanpain and Balmain laboratories (Nassar et al., 2015 and McCreery et al., 2015).
Are skin tumors caused by activated k-ras also affected by Dnmt3a or is the effect specific to DMBA-induced tumors?
In any case, to claim an effect on tumor initiation in epidermal stem cells, the authors would need to study earlier steps in tumorigenesis.
As mentioned above, we think that our results are compatible with the conclusion that Dnmt3a-cKO mice initiate more tumors earlier than wild type mice. We believe this accurately describes the phenotype. Hence, we are not sure why this conclusion is not considered valid. We are however open to discussing any suggestion to perhaps more accurately describe our results.
Regarding tumor initiation, we believe we have in fact tried to provide some insights into the early steps of tumorigenesis with the short-term treatments of DMBA/TPA (showed in Figure 4 and Figure 4—figure supplement 1). We have shown that the loss of Dnmt3a increases cell proliferation and the expression of lipid metabolism genes early after treatment with DMBA/TPA (Figure 3—figure supplement 2). However, we also show that, once formed, Dnmt3a- cKO tumors do not show increased cell proliferation compared to wild type tumors (Figure 1). Interestingly, a very similar conclusion has been reached by another work recently published by the laboratory of Margaret Goodell (Yang et al., 2016), where the authors state: “The inability to slow the leukemia by re-expression of DNMT3A suggests that DNMT3A loss is important for leukemia initiation but less so for maintenance”.
We have now included the histology for each tumor analyzed for gene expression as the new Figure 3—figure supplement 3. It is important to note that our mouse pathologist (Neus Prats, who is an author in the manuscript) performed the histological characterization of each tumor in a blind manner. In addition, we have now included the measurements of the time difference between tumor appearance and tumor collection for each mouse, showing the Dnmt3a-cKO tumors, although appearing before, do not progress faster than wild type tumors (new Figure 3—figure supplement 4C). The animals were sacrificed for humane reasons when the biggest tumor reached 1.5cm (as described into the methods section), but the average time between tumor appearance and this endpoint was the same between wild type and Dnmt3a-cKO tumors.
We agree this was an important issue that we should have clarified better in our first version of the manuscript. First, our mouse pathologist did not score any of the tumors used for RNA- seq (or any subsequent analysis) as a sebaceous adenoma. We have also now included in the new Figure 3—figure supplement 3, the results of the immunofluorescence staining for the sebaceous gland marker Adipophilin (ADFP), for each of the sequenced tumors. We did not observe any protein expression of ADFP in any of the 12 tumors sent for sequencing. Accordingly, ADFP was not differentially expressed at the mRNA level in Dnmt3a-cKO tumors compared to wild type lesions based on our RNA-seq analysis. As a positive control, we stained for ADFP in a sebaceous adenoma, which was not sent for sequencing. We would also like to note that sebaceous adenomas are generally small and quite different macroscopically from squamous cell carcinomas, so we could easily discriminate them when collecting the tumors for FACS sorting and subsequent analyses.
At last, we have also included in the new Figure 3—figure supplement 4 the expression analysis of PPAR-γ by immunofluorescence in all the 12 sequenced tumors that our pathologist scored as squamous cell carcinomas. We observed that PPAR-γ is consistently higher in all the 8 Dnmt3a KO tumors compared to the 4 wild type tumors, both at the RNA level (measured by RNA-seq) and also at the protein level. In addition, PPAR-γ is increased, upon Dnmt3a deletion, in the interfollicular epidermis after the short term DMBA/TPA treatment previous to any tumor formation, suggesting that the PPAR-γ is an early event not associated to a general sebocyte differentiation.
In conclusion, we believe we now provide new conclusive data indicating that deletion of Dnmt3a results in a stronger expression of lipid metabolism genes prior to tumor initiation, that persists in squamous cell carcinomas.
To properly quantify the changes in apoptosis in the Dnmt3a-cKO mice, we have stained and quantified, in all the sequenced tumors, for TUNEL and active Caspase-3. The results show a statistically significant decrease of apoptosis in Dnmt3a-cKO tumors compared to wild type lesions. These results are now included in the new Figure 3—figure supplement 2.
We agree with the reviewers that is not a major point of our work, and that perhaps we need to study this phenotype in more detail and with more mice. Therefore, we have decided to remove these results from the manuscript.
We respectfully disagree that the differences we show might not be biologically relevant. The changes in gene expression that occur upon deletion of Dnmt3a do in fact have a strong biological consequence regarding the timing and number of tumors that mice develop upon DMBA/TPA treatment. However, we would like to point out that the main reason we performed these analyses was to determine whether binding of Dnmt3a to promoters affected in any way the expression of the bound genes. In order to better show this, we have divided the Dnmt3a-target genes (as determined by our ChIP-seq data) based on binding strength; that is, we have separated them into the top 10%, 20%, 50% genes whose promoters are most strongly bound by Dnmt3a. Interestingly, the top 10-20% genes most strongly bound by Dnmt3a showed a stronger upregulation in their mRNA expression upon deletion of Dnmt3a compared to the top 50% of genes bound by Dnmt3a, or to all bound genes (Author response image 7). We believe these results therefore indicate that binding of Dnmt3a to promoters, correlates mostly with a downregulation of the expression of those target genes. Upon deletion of Dnmt3a, the average expression of those genes increases.
We also believe that the new data we have added showing that inhibition of PPAR-γ partially prevents the positive effects of the deletion of Dnmt3a over tumorigenesis in fact indicates that the differences in gene expression that we have observed are biologically relevant.
In order to verify this, we have performed a comparative clonogenic assay of cells isolated from the bulge and the IFE of wild type and Dnmt3a-cKO mice. As it can be observed in Author response image 8, Dnmt3a-cKO keratinocytes (irrespective of whether they come from the bulge or the basal IFE) show a significantly impaired clonogenic potential, just as we had previously reported in Rinaldi et al., for Dnmt3a-depleted primary human keratinocytes. Hence we believe that deletion of Dnmt3a renders primary basal keratinocytes more sensitive to situations of stress.
However, we would like to point out that previous work from the laboratory of Margaret Goodell has shown that deletion of Dnmt3a does result in changes in gene expression in adult murine hematopoietic cells. It is true that these differences result in mild differentiation phenotypes (that are mostly apparent when HSCs are serially transplanted into irradiated mice). But it is equally true that these mice end up developing leukemia, which is a clear alteration of tissue homeostasis (a phenotype that is exacerbated upon the combined deletion of Dnmt3a and Dnmt3b). Hence, we think that the statement: “…that reversible DNA methylation does not seem to have a prominent role in gene regulation in normal tissues (it has clear roles in irreversible silencing such as imprinting, X-inactivation, transposon silencing, irreversible promoter silencing, but a role in re gene regulation has been difficult to demonstrate outside cell culture”; is perhaps too strong, and that more in depth studies are needed to determine the exact role of these proteins, and importantly of DNA methylation, in the differentiation of different tissues. For instance we do not know if Dnmt3a and Dnmt3b compensate each other upon conditional deletion in tissues during embryonic development. Would their inducible deletion during adulthood result in the same phenotype? Perhaps the recently identified Dnmt3c, although specific to germ cells, could also compensate for the combined loss of Dnmt3a and Dnmt3b in somatic tissues. In addition, our results suggest that Dnmt1 is capable by itself to maintain some degree of DNA methylation in the long term in the absence of Dnmt3a and Dnmt3b. In fact, our manuscript contains some evidence supporting this last point. With our K14-Cre line, we have deleted Dnmt3a and Dnmt3b in the embryonic epidermis approximately at E13.5. However, when we looked at DNA methylation in old mice (over 70 weeks of age) with the combined deletion of Dnmt3a/Dnmt3b, we could still see some residual methylation present (Figure 2—figure supplement 4B). Hence, we would like to avoid concluding that DNA methylation/demethylation is completely dispensable for epidermal homeostasis, since with our KO models we have not completely eliminated DNA methylation from the epidermis.
The Spanish Ministry of Economy and Development (MINECO), and the Institute supported this project in the laboratory of SAB for Research in Biomedicine (IRB-Barcelona). IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain). We are very grateful to the laboratories of Rudolph Jaenisch and Rafii Ahmed for providing us the Dnmt3a and Dnmt3b flox/flox animals. LR was sponsored by La Caixa International PhD fellowship. We thank all the core facilities at the IRB-Barcelona for their assistance in our work, and Veronica Raker for editing the manuscript. The raw data for every dataset included in the manuscript can be found at GEO (GSE87412).
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the European Union. All of the animals were handled according to approved institutional animal care and use committee (CEEA) protocols (SAB-13-1522) of the Scientific Parc of Barcelona (PCB). The protocol was approved by the Committee on the Ethics of Animal Experiments of the Government of Catalunya.
© 2017, Rinaldi et al. | 2019-04-18T12:54:08 | https://elifesciences.org/articles/21697 |
Above methods save screenshot of entire screen. But, what if you want to save a part of your computer screen as an image? Here Windows built-in screenshot tool, Snipping Tool, comes into play that lets you save full or a part of screen as an image.... How to take a screenshot via the Game Bar in Windows 10 If you want another way to take screenshots, follow these steps. On your Windows 10 PC, press Windows key + G .
Above methods save screenshot of entire screen. But, what if you want to save a part of your computer screen as an image? Here Windows built-in screenshot tool, Snipping Tool, comes into play that lets you save full or a part of screen as an image.
How to take a screenshot via the Game Bar in Windows 10 If you want another way to take screenshots, follow these steps. On your Windows 10 PC, press Windows key + G . | 2019-04-25T14:51:25 | http://roaringbrookdairy.com/queensland/how-to-take-a-screenshot-on-your-computer-windows-10.php |
Artists include: Juan Bethancurth, Jessica Brouder, Elly Clarke, Julian Eicke, Liz Fletcher, Alexander Heaton, Sophia New and Dan Belasco / plan b, Terry Piercey, Daniel Sailsbury, Christian Sievers, Vajra Spook, Anna-Myga Kasten, E. Louise Wachler, Kym Ward and Andrea Winkler.
Curated by Elly Clarke / Clarke Gallery, 12 months on from its last presentation at Franklin Furnace in New York, Clarke Gallery was honoured to unpack the contents of WUNDERKAMMER once again, this time at TROVE in Birmingham, UK.
Translating as Cabinet of Curiosities, WUNDERKAMMER is a travelling show of sixteen small works by artists from Germany, the UK, Canada and the USA that grows and changes as it goes. Incorporating sculpture, photography, painting, (a new) ‘smart phone-dependent art’ (piece by Daniel Sailsbury) and a live presentation of “A Slide Show by… Andy Field” on the opening night, this is the forth stop WUNDERKAMMER has made. Starting out at Clarke Gallery in Berlin the exhibition then travelled to Eastern Edge in St. John’s, Newfoundland, Canada and Franklin Furnace in New York. For this presentation at TROVE, works by two additional artists are introduced: New York-based Columbian artist Juan Bethancurth (who I met during the last exhibition in NYC) and British Birmingham based artists Daniel Sailsbury and Sparrow+Castice.
WUNDERKAMMER is the first Clarke Gallery exhibition to have travelled beyond the confines of my Berlin apartment, where Clarke Gallery started up. After two years of hosting exhibitions in my space (involving, at different times, sculpture in my bed, performance in my bathroom, video installation in the kitchen, slide shows in my living room; art objects nestling amongst my personal things) – it felt right to branch out and bring the mobility that we as individuals today enjoy, to the gallery. Now, having lived in Birmingham for a year and with plans to find a more permanent setting for Clarke Gallery in the West Midlands, it is a great honour to bring this suitcase of work to TROVE. And also, for the first time, for the exhibition to have so much space to breathe. As well as to be able to remain there for a few weeks.
Artists include: Sophie Bancroft, Rachel Blake Butler, Michael Clulee, Benedict Davenport, Ryan Hughes, Hannah Humphries, Hannah Kershaw, Daniel Salisbury, Polly Saunders, Bronia Sawyer, Kate Spence, Matt Webb and Dane Worrallo.
An exhibition co-ordinated and curated by Sophie Bancroft, Daniel Salisbury and Kate Spence about the cosmos at TROVE, Birmingham.
Recent graduates and current MA students at BIAD came together for this exhibition based on Space, the Cosmos, the Universe. Highlighting in particular it’s never ending and unlimited source for ideas, thoughts and inspirations.
Non obsolescence opened at DownStairs with a preview reception on Saturday 29th October until February 2012.
Creative Machines, Minimalist Sculpture was an exhibition that developed from conversations between Birmingham curator, Charlie Levine, and London gallery director and artist, Minnie Weisz. Interested in forging and forming links with creative practices and artists outside their home cities, Levine and Weisz have formed a creative collaboration, which is linked by rail stations, both connecting north and south. So, very apt that this exhibition takes place at the very first station to link Birmingham directly to London; built in 1838, at the start of the Industrial Revolution.
For ‘The Event 2011’, Levine and Weisz curated an exhibition entitled Creative Machines, Minimalist Sculpture. It brought together artists from all over the UK who either create their own machine art works or have used machinery, or the idea of mechanics, to create the final Heath Robinson-esque whimsical, playful, scientific and experimental pieces.
The exhibition crossed sound, film and object/sculpture all based around the narrative of creative machines and minimalist sculpture. It was a look into pure machines meets pure minimalism, in a unique gallery setting.
August 2011 TROVE presented a weekend of performance and film based on the works of Dudley born film-maker, James Whale.
Best known for his 1930s Horror classics such as Frankenstein, The Invisible Man and Bride of Frankenstein, Whale worked in the theatre as an actor, set designer and stage manager before directing plays including ‘Journey’s End’, which toured to New York and attracted the attention of Hollywood film producers, landing Whale a contract with Paramount Pictures. He later signed to Universal where he made ‘Waterloo Bridge’ a film based on a Broadway play about a chorus girl who becomes a prostitute. Throughout his career Whale often chose the ‘outsider’ as the protagonist in his films and he himself was openly gay throughout his career, living with his partner, David Lewis, at a time where many chose to hide their true sexuality.
His films, often theatrical in style, also challenged the conventions of the time in many ways. He was a trailblazer who has inspired film-makers and artists alike still to this day.
Richard Peel presents a short theatrical remix of the Frankenstein myth, starring George Marchant as both the monster and the creator. Using the latest innovations in mime technology, the classic tale of alienation and revenge is blasted into the far future. And set on Mars.
TROVE invaded Fargo Space with a room full of sound in its latest exhibit presenting several artists working with sound and music.
By achieving the atmosphere of a void this project attempted to present its unique taste of meditative loud sound that envelopes the space within the confines of the mundane urban environment. This allows the audience to explore the space in a fleeting moment or stay a little longer to experience.
Artists include: Caroline Collinge, Tracey Eastham, Jo Gane, Caitlin Griffiths, Craig Marchington, Paul Newman and Edward Wakefield.
TROVE took to Manchester a plan chest. Three Birmingham based artists and three Manchester based artists, plus one trickster artist who hales from both locations, were invited to create a compact and moveable gallery space, each with their own drawer. Guests to the Club were invited to open the drawers and look inside, take the drawers fully out and sit in the TROVE space within the Burlington Fine Art’s Club.
As part of Birmingham Architectural Association’s Made in Birmingham 2011 series, SHOWCASE was an exhibition which celebrated the current work of local architectural practices and architecture students.
SHOWCASE was part of a number of architecture and built environment events held during June at TROVE in the heart of the Jewellery Quarter and at other venues across the city.
Create a more efficient way of doing things. This simple mantra has driven the advance of culture for millennia.
Better tools, smarter thinking, maximum output from smallest effort. With each new iteration of efficiency, a little more about the universe we live in is revealed. We move closer to a realization that a formula exists. There is a system. The system is perfect. The problem is not the system, the problem is purpose. The suspicion arises that the journey toward purpose will not be efficient. It will be messy. It will be the work not of the scientists, but of the combinatorialists.
Pattern will fail. Hypothesis will end. The Infinite Combinatorium has begun.
Mary Yacoob produced 3 new bodies of work for the Infinite Combinatorium; Part 0.
In her ‘Doodle’ drawings, the artist repeatedly draws a letter of the alphabet or a number whilst she listens to the radio, mapping the changes in sound, voice, and music with changes to direction, size and colour of the unit. A site specific drawing responds to the time of installation, the voices in the gallery space, and changes to music being played. The process altered a previous the artwork, a large Tipp-Ex rectangle created by Alastair Levy.
In ‘Research for Trove’, the artist has enlarged a photograph of the site as many times as it takes for the image to disappear. The largest white area on each photocopied image describing the magnitude of enlargement.
Her ‘Draft’ drawings were inspired by the technical diagrams in mechanical and electrical engineering textbooks, and relate to the site’s previous history as a science museum.
The ‘Proposition for Trove’ drawings are proposals for large scale installations for the site. Drawing on top of photographs sent to her by the Gallery Director, Yacoob generated ideas for 3d installations. For example, in one drawing colour lines are matched up to different tonal values in the photocopied image.
Donning metaphorical lab coats and overalls, Alastair Levy and collective-practice They Are Here will instigate a series of experiments and interventions inspired by the industrial, scientific and architectural legacy of the exhibition space: a former science and industry museum (1951 – 1997) and prior to that Elkington’s silver electroplating factory, the first of its kind in the world.
Inspired by notions of chemical reactions, manufacture and associative words: catalyst, conductive surface, the striking method, plating, gold, silver etc, the artists seek to explore how these ‘key words’ might become manifest in an artistic process that reacts to site and situation.
Significantly the opportunity to present work arose through a failed funding application that would have seen an alternative set of artists in-residence had it been successful. An attitude of expediency and rapid response has been pushed to the fore, necessitated by the development of ideas and work that began only thirteen days before the scheduled opening. This limited window of opportunity resonates with future of the site itself, scheduled to be converted into an office unit later this summer. This context has been embraced as a creative challenge to making work – hopefully encapsulating the spirit of efficiency and elegance of Victorian engineering.
The first event was Fierce Archive – the launch of Fierce’s research activity, for this project Fierce have delved into their archives to offer audiences a glimpse of their history. From the festival’s inception in 1998, then known as Queerfest, through to the current day, this selection of leaflets, programmes and pamphlets is there to start as a catalyst for conversations and memories about the festival’s past and the archive’s potential future display.
Acclaimed performance artist Dominic Johnson is premiering his new work Departure at TROVE on the 25th March, this piece is followed by a night of club performance entitled Human Salvage which has been co-curated by Fierce and Johnson. Departure sees Johnson being tattooed live in TROVE with an array of other performances, and live film feed of the event being projected around the space in this new sensitive durational piece.
Finally at TROVE is Sheila Ghelani’s Covet Me Care For Me. This extremely beautiful performance-installation hints at nostalgia, death and kitsch objects. Ghelani invites you to choose a glass heart before smashing it, then a ritual wrapping of what was inside; Ghelani then invites you to take home its precious contents.
Caroline Collinge’s A Picture Unfolds was the first in a new collaboration between galleries TROVE, Birmingham and Minnie Weisz Studio, London. Collinge, represented by Minnie Weisz Studio, made her first visit to Birmingham in February 2011. A Picture Unfolds opened in December 2010 at Minnie Weisz Studio, the show looks at the history of the A-Z and its maker as well as paper folding as a textile technique in her first solo show.
The London A-Z therefore sets the scene for this capriccio, an architectural fantasy of the city streets. Fact, mythology and fantastical combinations of origami, costume and architectural elements recreate the story of the London A–Z.
Fragments of the life of Phyllis Pearsall who created the A-Z are literally woven into Caroline’s exhibition. Born 1906 -1996 Phyllis Pearsall began to note down the names of the streets of London whilst lost one evening on her way to a party in Belgravia. Lost in the city and armed with the 1919 Ordnance Survey map, Pearsall could not find the address of the party and so begins her story of working for up to eighteen hours a day, walking a total of 3,000 miles while mapping London’s 23,000 streets.
Caroline Collinge is an artist working within film, installation and performance currently a PhD student at University of the Arts (London College of Fashion). Caroline studied Theatre Design BA at Wimbledon School of Art 2002 and MA in Scenography at LABAN Conservatoire of Dance 2004. Her artwork was shortlisted for the RIBA ‘Forgotten Spaces’ Competition 2010 and exhibited at The National Theatre, she has also been shortlisted for the Museummaker Contemporary Craft Commission 2010. Her Set Design work includes theatre productions at Liege Opera House, Shunt, The Arcola, Camberwell Arts Festival, Prague Quadrennial and The Roundhouse, London.
Paris Correspondence School is touring to Minnie Weisz Studio, London opening during February 2011.
See www.minnieweiszstudio.co.uk for further details. | 2019-04-19T18:52:30 | https://www.charlielevine.org/2011.html |
Yet another comic based on a real conversation with a coworker. He bought his girlfriend a bottle of perfume he selected because it was the largest bottle for the lowest price.
This was in Orlando. There are a lot of strip mall perfume stores in Orlando, most of them called “PERFUME.” They’re usually next to stores called “LUGGAGE,” “ELECTRONICS-SONY,” and “T-SHIRTS.” For some reason, tourist destinations seem to draw certain kinds of stores, even if they don’t fit the area’s primary draw. If you’re on a romantic trip to the Bahamas, perfume makes sense. Same goes for Hawaii or Cabo San Lucas, I guess. But if you’re going whale watching in Juneau, or spending a July day at Universal Studios Orlando, the last thing you want is something that will make you smell more. | 2019-04-24T02:56:47 | http://www.basicinstructions.net/basic-instructions/2018/9/6/how-to-avoid-a-false-bargain |
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A longtime friend and avid fly fisherman recently asked, as I was showing him pictures of some recent warm water fish, “With all of the trout fishing near you, why do you spend so much time on local ponds?” I paused for a moment, trying to give him an honest and thoughtful answer I figured he would understand. But, in the end, I shrugged with a smile and replied, “I like it.” In return I received a nodding head as any friend and fellow fisherman instantly recognizes as “I don’t get it, but if you like it, cool.” Yet, somehow, I felt I had missed a chance; I blew an opportunity to express myself adequately and it bugged me.
After that fish, I caught three or four “pounders” and then nothing else. Knowing the fish were there, I switched to a Gurgling Frog pattern. It is a foam Gurgler/frog pattern tied on a size 2 stinger hook as well. On my first cast I caught a lily pad, and after the disturbance of freeing my fly I kicked down another 20 feet and found another target. When the fly hit the water, a fish aggressively rolled over it almost instantly. I lifted the rod to a heavy fish, but knew at once by the circular jig it was dancing around the lily pad stalks that it was a panfish. Bringing it to hand, proved me right. It was a thick bull bluegill in splendid spawning colors and even eleven inches long. But again what caught my eye was the fact that he had completely swallowed the entire fly. Popping the size 2 hook out with my forceps, I was amazed. I had to use effort to pull the fly back through its mouth! What possessed this 11inch fish to attack something larger than the opening of its mouth? And obviously with zero inspection or hesitation! That fish knew the second my fly hit the water, it was dinner.
And then it hit me. That is what draws me to warm water so often. Being an addict of surface fishing through the summer, the aggressiveness is unmatched. The fish take a bass bug like it is the last meal on earth. Multiply that by the fact that you never know what exactly is going to take your fly, be it a bass, bluegill, crappie or chain pickerel…..and you have something unmatched by a trout stream. Don’t get me wrong, trout fishing has a hold on my fishing world, and that will never change, but a rise in the lily pads to a bass bug often has me stuck in the driveway deciding which way to point my truck. And each time I am finning my tube as the sun gets low, the frogs kick into a full concert and I see a boil large enough to move 10 feet of lily pads! Well…..that’s why. | 2019-04-24T20:27:21 | http://flyanglersonline.com/articles/whipfinish/2017/whipfinish20170816.php |
BlueBOLT®-enabled Panamax® power solutions help to protect the condos’ investment in top-of-the-line tech.
PETALUMA, CALIFORNIA — In luxury condo communities where high rents often afford fantastic views, superb locations and high-end accoutrements, residents expect the amenities and community areas to be easy-to-use and up-to-date. Scottsdale Waterfront Residences in Scottsdale, Arizona recognized this need and recently upgraded its common areas with the latest ELAN® control system to make the audio and video options accessible and easy to use for residents of all ages. According to the Residences’ Chief Engineer Patrick Bernardi, it was a welcome change that also resulted in residents having a better appreciation for control technology and how it can improve home living.
The Scottsdale Waterfront Residences is comprised of two identical 14-story towers, each featuring a stunning rooftop pool with multiple speakers for music, and a kitchen and bar area with two TVs, plus an indoor Club Room that offers a dining area, prep kitchen, wet bar, comfortable seating areas, an 80-inch TV and a surround sound stereo system. Catering to high-end residents and busy families, it was necessary to ensure the technology throughout the building was as simple to use as possible.
Jon Lunt, owner of JL Automation, the integration firm responsible for system design, installation and maintenance, sees the upgrades as a big step toward convincing people that home automation and control can make everyday living simpler and more enjoyable.
Each of the towers has a total of seven 7-inch touch panels for residents to control audio and video, while the club rooms each feature a larger 12-inch touch panel. While residents can only control the room they are currently in, staff have their own logins they can use to override or manage the system in any room in the building. This means if residents have pushed the music volume too loud at the pool and it needs to be lowered, a staff member doesn’t have to go to the roof to do so. Each rooftop pool has its own control system, including an ELAN touch panel, ELAN Controller, ELAN Multi-Room Audio Amplifier, multiple Gefen® 4K splitters and extenders to distribute the A/V signals, an Autonomic media server for Sirius/XM, Pandora, Spotify, Airplay and more, and two televisions.
With so much of the living experience riding on this interactive technology, JL Automation took the necessary precautions the protect the equipment and installed a Panamax® M4315 Power Conditioner with BlueBOLT® remote energy management functionality in each building’s main equipment rack and pool equipment rack to ensure smooth operation and protection in case of power surge or shortage. The club rooms also feature a BlueBOLT-enabled Panamax SM3-Pro system manager with surge protection. The BlueBOLT functionality in the Panamax units allows Bernardi to get instant text or email alerts of any power issues or problematic components, often solving problem remotely without rolling a truck.
This project was a welcome upgrade to the residents, who appreciate every effort to simplify their lives and make technology work for them, rather than against them. With rave reviews from residents, staff and Bernardi himself, nobody will be surprised if more residents begin to install ELAN systems in their condos and bring the pleasant, simple control experience to their homes.
ELAN, now part of Nortek Security & Control, develops an award-winning line of whole-house entertainment and control solutions distributed through a comprehensive channel of select dealers throughout the United States, Canada, and countries worldwide. The ELAN 8 update was honored with the “2017 Human Interface Product of the Year” award, and continues to expand its intuitive functionality with security, climate, surveillance and video distribution products and integrations. | 2019-04-20T06:32:18 | https://www.elanhomesystems.com/press-release/arizona-luxury-condo-complex-elan-control-community-areas-entices-residents-upgrade |
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Thank you for your interest. We are no longer accepting applications for this position.
Cleveland Leadership Center is looking for its next Director of (i)Cleveland and College Programs.
college students and recent college graduates. The director will forge relationships with local colleges and universities and with employers of college interns to advance these objectives. The Director will have the benefit of working collaboratively with a talented team of staff members that will provide strategic and programmatic guidance to ensure success. | 2019-04-18T16:55:41 | https://www.cleveleads.org/were-hiring/ |
The set of caddy spoons are now finished and avaliable at Payne & Son Oxford.
Commissioned by Payne & Son my brief was to design a set of spoons to celebrate the forthcoming Jubilee celebrations. On researching the coronation ceremony I discovered that the Queen wears more than one crown! She was invested in the St Edward crown (spoon on the right) and on the return journey back to Buckingham Palace she wore the Imperial State Crown (spoon on the left). The crown we are most likely to see her wear is the Diamond Diadem (centre spoon), which she wears on the occasions of travelling to the State Openings of Parliament.
I was also inspired by line in the poem written by the Poet Laureate, John Masefield which marked the Queen's Wedding, in 1947.
" Where a Crown shines, the courage cannot fail " | 2019-04-19T12:16:32 | https://ruthball.weebly.com/blog/category/payneson%20oxfordb881dd3a13 |
Should I Raise My Rental Rates?
When Should You Claim On A Vacation Rental Damage Deposit?
Is Your Vacation Rental Packaging Attractive? | 2019-04-24T02:22:11 | https://www.vacationrentalformula.com/category/managers/page/15/ |
"The issue here is the church will not allow anyone to examine it. When it does happen, it shows it to be a hoax."
So at least 5.5 billion against!
hahaha Should I write a letter?
Here is a random internet definition of morality: "principles concerning the distinction between right and wrong or good and bad behavior".
You think that a virgin gave birth. Your religion is a lie.
Yeah, that's really cool. Good job boo! I kinda feel proud for my bud ?????? | 2019-04-24T01:54:10 | http://tokoonlinedenature.com/tribbing/double-action-penetration-with-rita-hong-tube.php |
Department of Anatomy and Cell Biology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5.
Transcriptional activation of heat shock genes is a reversible and multistep process involving conversion of inactive heat shock factor 1 (HSF1) monomers into heat shock element (HSE)-binding homotrimers, hyperphosphorylation, and further modifications that induce full transcriptional competence. HSF1 is controlled by multiple regulatory mechanisms, including suppression by additional cellular factors, physical interactions with HSP70, and integration into different cellular signaling cascades. However, the signaling mechanisms by which cells respond to stress and control the HSF1 activation-deactivation pathway are not known. Here we demonstrate that HSP90, a cellular chaperone known to regulate several signal transduction molecules and transcription factors, functions in the regulation of HSF1. The existence of HSF1-HSP90 heterocomplexes was shown by coimmunoprecipitation of HSP90 with HSF1 from unshocked and heat-shocked nuclear extracts, recognition of HSF1-HSE complexes in vitro by using HSP90 antibodies (Abs), and recognition of HSF1 in vivo by HSP90 Abs microinjected directly into oocyte nuclei. The functional impact of HSP90-HSF1 interactions was analyzed by using two strategies: direct nuclear injection of HSP90 Abs and treatment of cells with geldanamycin (GA), an agent that specifically blocks the chaperoning activity of HSP90. Both HSP90 Abs and GA delayed the disassembly of HSF1 trimers during recovery from heat shock and specifically inhibited heat-induced transcription from a chloramphenicol acetyltransferase reporter construct under control of the hsp70 promoter. HSP90 Abs activated HSE binding in the absence of heat shock, an effect that could be reversed by subsequent injection of purified HSP90. GA did not activate HSE binding under nonshock conditions but increased the quantity of HSE binding induced by heat shock. On the basis of these findings and the known properties of HSP90, we propose a new regulatory model in which HSP90 participates in modulating HSF1 at different points along the activation-deactivation pathway, influencing the interconversion between monomeric and trimeric conformations as well as transcriptional activation. We also put forth the hypothesis that HSP90 links HSF1 to cellular signaling molecules coordinating the stress response.
Cells respond to heat and other forms of stress by upregulating expression of a family of highly conserved heat shock proteins (HSPs) which function under both normal and stressful conditions as molecular chaperones mediating the folding, assembly, translocation, and degradation of proteins (reviewed in references 25 and 34). In eukaryotes, stress-induced expression of HSPs is regulated primarily by heat shock transcription factor HSF1, which acts through heat shock regulatory elements (HSEs) found in the promoters of HSP genes (33, 73). Under nonstress conditions, HSF1 exists as a repressed non-DNA-binding monomer (16, 53, 75), and the activation-deactivation pathway of HSF1 in metazoan cells involves several independently regulated steps (reviewed in references40 and 73). Different forms of cellular stress trigger the rapid conversion of HSF1 from inert monomers to homotrimers, and this first step is accompanied by increased HSE-binding affinity (4, 53, 55, 66, 70). Detailed mutagenic analyses have suggested that HSF1 monomers are stabilized by intramolecular interactions between several hydrophobic heptad repeats and that activation during stress involves the disruption of these intramolecular interactions, leading to formation of intermolecular coiled coils with other HSF1 monomers (16, 55, 66, 75). Thus, the suppression of HSE-binding activity under normal conditions is regulated at least in part by hydrophobic sequences within HSF1, although other regions of the molecule have also been implicated in this regulation (48). Once it has trimerized, HSF1 undergoes additional modifications to upregulate transcriptional activity which appear to be regulated independently of trimerization and DNA binding (21, 59, 76). The final step of HSF1 regulation is deactivation or attenuation. Upon removal of stress, HSF1 dissociates into monomers and ceases to activate transcription (11, 44,52).
It has become clear that HSF1 is subject to complex regulation under both normal and stress conditions. First, a simple model in which HSF1 is regulated by temperature alone is contradicted by observations thathsp genes are switched on by multiple unrelated stresses and that HSF1 molecules expressed in heterologous systems are reprogrammed according to the appropriate physiological temperatures of the host cells (8, 11, 30, 53). It therefore appears that HSF1 is under negative regulation by unknown cellular factors. Second, HSF1 has been shown to be phosphorylated by several different signal transduction molecules; for example, heat-induced activation of HSF1 was enhanced by phorbol ester treatment of human erythroleukemia cells, and thus HSF1 is apparently targeted by protein kinase C (26). HSF1 has also been shown to be phosphorylated by ERK1, linking HSF1 to the Ras signaling pathway (29). The constitutive phosphorylation on serine and threonine residues and inducible hyperphosphorylation in response to stress also suggested that HSF1 is modulated by cellular kinases and/or phosphatases (4,30, 55). Although definitive correlations between different phosphorylation states and specific HSF1 activities have not been established (12, 31, 46, 72), recent evidence suggests that hyperphosphorylation could function both to increase transactivation potential and to delay the dissociation of trimers (74).
In addition to phosphorylation and suppression by cellular factors, it has been hypothesized that HSPs themselves, particularly HSP70, could function as part of an autoregulatory mechanism by which cells detect stress and regulate HSF1 (39). This idea is substantiated by a number of experimental observations with various cell types: activated expression of other HSPs after mutation of thehsp70 gene in yeast (9), continued transcription of hsp70 genes during recovery in cells blocked for HSP accumulation by treatment with translational inhibitors (13), and activation of hsp genes in response to artificially increased nuclear concentrations of denatured proteins (2, 37). It has been shown that physical interactions between HSP70 and HSF1 occur both in vitro and in vivo, but these appear to be unstable, and thus the stoichiometric relationships of these complexes have not been determined (1, 3, 5, 39, 47, 53,70). In addition, different studies have shown that direct overexpression of HSP70 in cultured cells either reduced the level of HSF1 activation during induction (5, 41) or increased the rate of HSF1 deactivation (41, 54), and so the precise influence of HSP70 on oligomerization steps of the HSF1 activation-deactivation have not been fully elucidated. More recently, HSP70 has been shown to repress HSF1 through direct interaction with the transactivation domain (61); therefore, the primary autoregulatory role of HSP70 appears to be downregulation of HSF1-mediated transcription.
Another well-characterized HSP, HSP90, functions as the core of several chaperone heterocomplexes that contain different assortments of noncovalently linked proteins, including HSP70 (18). Numerous studies have established that HSP90 is required for higher-order folding of certain signal transduction molecules and transcription factors (reviewed in references 7 and28) and that HSP90 functions in the later stages of protein folding after the formation of extensive secondary structure (35, 62, 63). HSP90 is thought not to be a general chaperone but rather to interact with and modulate the activity of various specific substrates, including hypoxia-inducible factor α (20), MyoD (58), steroid hormone receptors (50), the pp60v-src kinase (10), casein kinase II (15), and eIF-2α kinase (69).
Together, the known chaperone function and substrate specificity of HSP90, the conformational changes associated with HSF1 oligomerization, apparent integration of HSF1 into different signal transduction cascades, and a previous report describing an interaction between immobilized HSP90 and HSF from HeLa cell nuclear extracts (42) prompted us to consider HSP90 as a potential modulator of HSF1 activity. Further, HSF1 has recently been shown to assemble HSP90 and other chaperones common to the progesterone receptor assembly pathway in vitro (43). Here we investigated the potential autoregulatory role of HSP90 by using Xenopus oocytes. Results of coimmunoprecipitation and antibody recognition experiments provide clear evidence of a physical association in vivo between HSP90 and HSF1. Impairment of HSP90 function either by microinjected antibodies or by the specific binding agent geldanamycin (GA) resulted in a significant delay in HSF1 trimer disassembly during recovery and a marked reduction in heat-induced transcription from thehsp70 promoter. In addition, microinjected HSP90 antibodies (Abs) caused the activation of HSE-binding activity in the absence of heat shock, an effect which could be reversed by subsequent injection of exogenous HSP90. Together these observations support a new regulatory model in which formation of a heterocomplex between HSP90 and HSF1 plays a key role in modulating different steps of the HSF1 activation-deactivation pathway.
Oocyte manipulations, stress treatments, and microinjection. Xenopus laevis frogs were purchased from Xenopus I (Ann Arbor, Mich.). Ovary portions were surgically removed from adult female frogs, and follicle cells were removed from oocytes by treatment in calcium-free OR2 (45) buffer (82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 1 mM NaH2PO4, 5 mM HEPES [pH 7.8], 10 mg of streptomycin sulfate per liter, 10 mg of benzyl penicillin per liter) containing 2 mg of collagenase (type II; Sigma) per ml for 2 to 3 h at 18°C. Oocytes were washed extensively and allowed to recover overnight in OR2 containing 1 mM CaCl2at 18°C, and only stage VI oocytes were selected for experiments. Nuclei were obtained under OR2 by scoring animal hemispheres with a needle and gently squeezing the equatorial region with watchmaker’s forceps. In some experiments, oocytes were incubated either in OR2 containing 10 μg of GA (a gift of the Developmental Therapeutics Program, National Cancer Institute, Bethesda, Md.) per ml dissolved in dimethyl sulfoxide (DMSO) or in OR2 with 1:250 (vol/vol) DMSO for 2 h at 18°C, rinsed, and then exposed to heat shock treatments. In all experiments, a minimum of 20 oocytes was used for each sample.
Plasmid constructs used for microinjection experiments were the human cytomegalovirus (CMV)-chloramphenicol acetyltransferase (CAT) and theXenopus hsp70-CAT clones (kindly provided by Alan Wolffe, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md.), which were previously described (32). Following defolliculation, oocytes were incubated for several hours at 18°C, after which healthy oocytes were selected and injected into nuclei with 20 nl of a solution containing 2 ng of either CMV-CAT or hsp70-CAT plasmid per μl (40 pg) by using a Narashige IM 300 microinjector. After incubation overnight at 18°C, oocytes were subjected to heat shock or GA treatments and then incubated for an additional 12 h at 18°C, washed in OR2, and assayed for CAT activity.
For Ab microinjection experiments, Abs were diluted (1:1) in sterile H2O immediately prior to microinjection. In experiments in which the effects of HSP90 Abs on induced transcription were measured, Ab solutions (15 nl) were injected directly into the nuclei of oocytes containing reporter constructs (injected 12 h previously). Oocytes were then incubated for 30 min at 18°C, heat shocked, and then incubated for a further 12 h to allow for CAT expression. In experiments to measure the effects of HSP90, HSF1, or YY1 Abs on HSF1 binding, Abs were injected into (previously uninjected) oocyte nuclei. Injected oocytes were allowed to recover for 30 min at 18°C and then incubated at a control temperature (18°C) or heat shocked at 33°C. Protein extracts were then prepared for gel mobility shift analyses and immunoblotting. HSP90 polyclonal Abs (PAbs) and purified bovine HSP90 were gifts of R. Matts, Oklahoma State University, Stillwater; anti-HSP90 monoclonal Ab (MAb) (SPA 830) was purchased from StressGen, Victoria, British Columbia, Canada; HSF1 PAbs (55) were a gift of K. Sarge, University of Kentucky, Lexington; and anti-Ying Yang 1 (anti-YY1) transcription factor PAbs were purchased from Santa Cruz Biotechnology, Santa Cruz, Calif.
Protein extracts and gel mobility shift assays.Protein extracts were prepared by homogenizing oocytes in buffer C (50 mM Tris-Cl [pH 7.9], 20% glycerol, 50 mM KCl, 0.1 mM EDTA, 2 mM dithiothreitol, 10 μg of aprotinin per ml, and 10 μg of leupeptin per ml) (14) in a Dounce homogenizer (10 μl/oocyte). Homogenates were transferred to Eppendorf tubes and spun for 5 min at 15,000 × g (4°C). The resultant supernatants were placed in a fresh tube and then immediately frozen in liquid nitrogen and stored at −80°C.
DNA mobility shift assays were performed by using HSE oligonucleotide probes as previously described (19). DNA-binding reaction mixtures contained 10 μl of extract (one oocyte equivalent by volume, or 20 μg of soluble protein). The relative amounts of protein in all samples were confirmed by Bradford assays, and extract volumes were adjusted so that equal protein concentrations were added to each binding reaction mixture. Binding reactions were performed with 1 μg of poly(dI-dC)–10 mM Tris (pH 7.8)–50 mM NaCl–1 mM EDTA–0.5 mM dithiothreitol–5% glycerol in a final volume of 20 μl. Reaction mixtures were incubated on ice for 20 min and immediately loaded onto 5% nondenaturing polyacrylamide gels containing 6.7 mM Tris-Cl (pH 7.5), 1 mM EDTA, and 3.3 mM sodium acetate. Gels were electrophoresed for 2.5 h at 150 V, dried, and exposed to autoradiography with X-ray film (XAR; Kodak). In vitro Ab recognition experiments were performed by adding Abs directly in DNA-binding reaction mixtures or by mixing Abs with oocyte extracts for 20 min on ice prior to DNA-binding reactions.
Immunoblotting and immunoprecipitations.Protein extracts were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% acrylamide) and electroblotted onto polyvinylidene difluoride membranes, and the blots were blocked (for 2 h at room temperature) in TBST (20 mM Tris-Cl [pH 7.6], 137 mM NaCl, 0.1% [vol/vol] Tween 20) containing 5% milk powder. Abs were diluted in TBST with 2.5% milk (1:5,000 for anti-HSP90 PAb), and blots were incubated in primary antibody for 2 h at room temperature. Blots were washed in TBST and incubated with secondary Ab (horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G; Bio-Rad) diluted 1:10,000 in TBST and 2.5% milk for 2 h at room temperature. Blots were washed, and proteins were visualized by chemiluminescence (Renaissance system; Dupont NEN) and autoradiography with X-ray film (XAR; Kodak).
Immunoprecipitations were performed by the method described by Firestone and Winguth (17). Nuclei from nonshocked and heat-shocked (30 min, 33°C) oocytes were isolated as described above, and in each assay eight nuclei were subjected to immunoprecipitation with anti-mouse HSF1 PAb (55) or anti-YY1 transcription factor PAb (Santa Cruz Biotechnology).
CAT assays.CAT assays were performed with one oocyte equivalent of whole-cell extract from uninjected or microinjected oocytes as previously described (49). A pool of at least 20 microinjected oocytes was used for each experimental treatment. The acetylated products were separated by thin-layer chromatography and visualized by autoradiography.
Detection of HSP90 in immune complexes with HSF1.In this study we used the Xenopus oocyte model system to examine the potential role of the HSP90 chaperone in modulating the activities of HSF1. We have previously shown that endogenous HSF1 is a nuclear protein in Xenopus oocytes both before and after heat shock (36). Accordingly, we reasoned that endogenous HSP90 must be present in oocyte nuclei in order for HSP90-HSF1 interactions to occur in vivo. To test this, oocytes were manually enucleated and the subcellular distribution of HSP90 was determined by immunoblotting (Fig. 1A). In this assay, total protein from intact oocytes was compared directly to protein in a single large oocyte nucleus and in the cytoplasm of the same cell. Although the majority of HSP90 appeared to reside in the cytoplasm, HSP90 was clearly detectable in the nuclear compartments of both unshocked and heat shocked oocytes. Relatively equal quantities of HSP90 were detected in nuclei isolated from oocytes before and after heat shock, demonstrating that the nucleocytoplasmic distribution of HSP90 was not significantly affected by the heat stress conditions used in this experiment (33°C for 2 h). Heat shock did not result in a detectable increase in the total cellular levels of HSP90. This is in agreement with previous observations demonstrating that HSP synthesis is not significantly induced by heat shock in the single-cellXenopus oocyte (27).
Identification of HSP90-HSF1 heterocomplexes inXenopus oocyte nuclei. (A) Immunoblot showing subcellular distribution of HSP90. Oocytes were incubated at a nonshock (NS) temperature (18°C) or heat shocked (HS) at 33°C for 2 h, and proteins from single intact oocytes or from the nuclear and cytoplasmic fractions were separated by SDS-PAGE along with purified bovine HSP90 as a control. HSP90 was detected with an HSP90 PAb. The positions of molecular mass standards are shown on the left. (B) Coimmunoprecipitation of HSF1 and HSP90 from oocyte nuclei. HSF1 and YY1 were immunoprecipitated with rabbit anti-mouse HSF1 PAb (55) or YY1 PAb (Santa Cruz). Nuclear extracts were isolated from nonshocked or heat-shocked (33°C for 1 h) oocytes. Immunoprecipitated material was subjected to SDS-PAGE and transferred to nitrocellulose, and the blot was probed with HSP90 and HSF1 Abs as indicated.
In order to determine if in vivo HSP90-HSF1 interactions could be detected in vivo, HSF1 was immunoprecipitated from unshocked or heat-shocked oocyte nuclei by using HSF1 PAbs (anti-mouse HSF1 antiserum) (55), and the immunoprecipitated material was examined for the presence of HSP90 by immunoblotting (Fig. 1B). HSP90 was present in the HSF1-immunoprecipitated material from both nonshocked and heat-shocked nuclei, suggesting a physical association between HSP90 and HSF1. Identical quantities of HSP90 were detected before and after heat shock, indicating that HSP90 associates equally well with inactive HSF1 monomers or stress-activated HSF1 trimers. Control experiments show that the HSF1 PAbs used in these experiments do not cross-react with endogenous Xenopus or purified bovine HSP90 on immunoblots (data not shown). Also, as shown in Fig.1B, HSP90 was not detected in control immunoprecipitations with PAbs against YY1 (60), which is a relatively abundant transcription factor in Xenopus oocytes (36a).
HSP90 Abs recognize heat-activated HSF1 in vivo and in vitro.While coimmunoprecipitation of HSP90 and HSF1 suggested a physical interaction between these proteins, additional evidence was required to substantiate the existence of HSP90-HSF1 heterocomplexes. In the following series of experiments, we tested the ability of HSP90 Abs to recognize activated DNA-binding HSF1 trimers. HSP90 Abs were mixed with aliquots of unshocked or heat-shocked oocyte extracts prior to DNA-binding reactions with radiolabeled HSE probes, and the effect on the migration of HSF1-HSE complexes was analyzed in gel mobility supershift assays (Fig. 2). Heat-induced HSF1 complexes were clearly supershifted by an HSP90 MAb and were disrupted by HSP90 PAbs, although in comparison to the HSP90 MAb, a relatively large amount of antiserum was required for this effect. The results of these experiments were identical for each antibody when HSP90 MAbs or PAbs were mixed directly into DNA-binding reaction mixtures without preincubation (data not shown). We attributed the retardation or disruption of heat-induced HSE bandshifts observed in these assays to specific antibody recognition of HSP90 in direct association with HSF1 trimers. This interpretation is consistent with the coimmunoprecipitation of HSP90 and HSF1 (Fig. 1). The disruption of HSE binding by HSP90 PAbs, while differing somewhat from the clear supershift by the MAb, was similar to the results of supershift experiments with HSF1 PAbs (see Fig. 4B) (19). It is likely that recognition of multiple epitopes by PAbs present in HSP90 antiserum interfered with HSP90-dependent DNA-binding activities of HSF1.
Recognition of the heat shock-activated DNA-binding form of HSF1 in gel mobility shift assays by HSP90 Abs. Aliquots of nonshocked (NS) or heat-shocked (HS) oocyte extracts were mixed with HSP90 MAb or PAbs (antiserum) and incubated for 30 min prior to DNA-binding reactions with a 32P-labeled HSE probe, and the migration of HSF1-HSE complexes was analyzed in gel mobility shift assays. Lanes in which extracts were not mixed with antibodies are indicated (−), and the final antibody dilutions in the extract incubations are indicated above the panel. Positions of the nonspecific binding activity (ns) and heat-induced (HSF1) and supershifted (HSF1 + ab) complexes are indicated.
We next determined if the physical association between HSP90 and HSF1 implied by in vitro supershift experiments could be detected in vivo. A series of experiments in which HSP90 was targeted by direct introduction of HSP90 Abs into oocyte nuclei was performed. It was important, however, to first evaluate the efficacy of the Ab injection technique. Initial experiments were aimed at determining the stability of injected Abs during heat shock, as well as any quantitative effects that Ab injection might have on endogenous HSP90 levels. Following nuclear microinjection of HSP90 MAb, oocytes were allowed to recover for 1 h at 18°C and then were further incubated under nonshock or heat shock conditions (18 or 33°C for 2 h). Cell homogenates were then examined for the presence of anti-HSP90 MAbs by immunoblotting; in this experiment injected Abs were recognized by the secondary Ab used to detect endogenous HSP90 (Fig.3A). The results show that both the quantity and apparent molecular sizes of microinjected MAbs were similar before and after heat shock and also that HSP90 levels were not affected by the presence of MAbs. Thus, it appeared that this technique could effectively be used to target or sequester HSP90 into immune complexes in vivo without affecting total HSP90 concentrations.
HSP90 interacts with HSF1 in vivo. (A) Immunoblot of nonshocked (NS) and heat-shocked (33°C, 2 h) oocytes that were either uninjected or microinjected with HSP90 MAb. HSP90 was detected with HSP90 primary Ab, and injected HSP90 Ab was detected with the secondary antibody (as described in Materials and Methods). Endogenous oocyte HSP90 and injected HSP90 MAbs present in extracts are indicated on the right. The positions of molecular mass standards are indicated on the left. (B) The migration of HSF1-HSE complexes was analyzed by gel mobility shift assay of uninjected or HSP90 MAb-injected oocytes that were not shocked (NS) or heat shocked (HS) at 33°C for the times indicated. The heat-activated HSF-HSE (HSF1) and HSP90 Ab-supershifted (HSF1+ab) complexes are indicated on the right. ns, nonspecific binding activity. (C) An experiment similar to that for panel B was performed with HSP90 antiserum (pAb).
The effect of microinjected HSP90 Abs on the DNA-binding activity of HSF1 was analyzed both before and after heat shock. HSP90 MAbs significantly retarded the mobility of heat shock-induced HSF1-HSE complexes (Fig. 3B). The relative amounts of induced HSF1 were similar in both uninjected and HSP90 MAb-injected oocytes, indicating that these antibodies did not suppress trimerization or the DNA-binding activity of HSF1. Supershift of heat shock-induced HSF1-HSE complexes was not observed after injection of HSP90 PAbs (Fig. 3C), but this was not surprising, since the maximum volume of antiserum that could be injected into oocyte nuclei was much less than the amount required to disrupt HSF1-HSE bandshifts in vitro (Fig. 2). Despite the differences between the effects of HSP90 MAb and PAbs seen in these experiments, the overall results of Ab injections are consistent with our interpretations of immunoprecipitation and in vitro gel supershift experiments and provide further evidence that HSP90 associates with HSF1 in vivo.
HSP90 associates with HSF1 in vivo.In control experiments we also tested the effects of microinjected HSF1 Abs on HSF1 in vivo. We have previously shown that anti-mouse HSF1 PAbs (55) supershift or obliterate stress-activated HSF1 complexes in oocyte extracts in vitro (19). Similar to the results of in vitro gel supershifts, direct nuclear injection of HSF1 antiserum appeared to completely inhibit the formation of HSF1-HSE complexes in response to heat shock (Fig. 4B). We could not, however, distinguish between the possibility that HSF1 Abs actually bound to and inhibited the trimerization of inactive HSF1 monomers prior to heat shock and the possibility that HSF1 Ab-supershifted complexes were simply not detectable in these gel shift assays. The results of control experiments also showed that heat shock-induced HSF1-HSE complexes were not affected by sham nuclear injection of H2O or nuclear injection of Abs against the transcription factor YY1 (Fig. 4B), indicating that the supershift of heat-induced HSF1-HSE complexes by microinjected HSP90 Abs was attributable to specific effects on HSP90 in vivo.
HSP90 Abs activate the HSE-binding activity of HSF1 under nonstress conditions. (A) Left, gel mobility shift assay of uninjected oocytes (−) and HSP90 Ab-injected oocytes (MAb or PAb) that were incubated at nonshock temperatures (NS) or heat shocked for 1 h (HS). The positions of heat-activated or Ab-activated HSF1-HSE complexes (HSF1) and HSP90 Ab-supershifted complexes (HSF1 + ab) are indicated. Right, gel mobility shift assay showing the effects of coinjected bovine HSP90 on the formation of HSF1-HSE complexes. In the third lane (+HSP90), 50 ng of purified bovine HSP90 was injected into oocytes 2 h after injection of HSP90 PAbs, and extracts were made following incubation at a nonshock temperature for a further 1 h. ns, nonspecific binding. (B) Bottom, gel mobility shift assay of oocytes injected with PAbs against YY1 (Santa Cruz; see Materials and Methods). Top, comparison of the activation of HSF1 in uninjected (uninj) and sham-injected (H2O) or HSF1 antiserum-injected (HSF1 PAb) (55) oocytes. (C) Recognition of HSF1 by microinjected HSP90 Abs occurred in vivo. A gel mobility shift assay was performed with uninjected (−) or HSP90 MAb-injected oocytes. In some lanes, HSP90 MAb or purified bovine HSP90 was added to extracts as indicated below the panel. HSP90 (1 μg) was added to Ab-injected oocytes at the time of homogenization (fifth lane from left), and HSP90 MAb or protein (1 μg) was added to uninjected heat-shocked oocyte extracts just prior to DNA-binding reactions (seventh and eighth lanes from left).
HSP90 Abs induce HSE binding in the absence of heat shock.In the mobility gel shift assay with the results shown in Fig. 3B, HSF1-HSE complexes were not observed in unshocked oocytes after nuclear injection of an HSP90 MAb; however, in repeated experiments HSF1 appeared to be induced by the same MAb in the absence of heat shock (Fig. 4A). Similar heat shock-independent activation was also observed in experiments with microinjected PAbs in HSP90 antiserum (Fig. 3C and4A). Thus, it appeared that injection of HSP90 Abs mimicked the effects of heat shock in eliciting the DNA-binding activity of HSF1. The mobility of HSF1-HSE complexes induced by injected HSP90 Abs in unshocked oocytes did not appear to be supershifted but was similar to that of the heat-induced complexes in uninjected cells (Fig. 3C and4A). This suggested that Ab-induced activation may have been the result of Ab interaction with HSP90 not in direct contact with HSF1.
Several control experiments were performed to determine if the activation of HSF1 under nonshock conditions by microinjected HSP90 Abs was attributable to specific effects on HSP90 in vivo. First, induction of HSE binding by HSP90 Abs could be reversed by subsequent injection of 50 ng of purified HSP90 (Fig. 4A). Second, sham injection or the nuclear presence of YY1 or HSF1 Abs did not induce HSE binding in the absence of heat shock (Fig. 4B). Thus, HSF1 activation by HSP90 Abs was not due to stress caused by the injection procedure and/or the presence of nonspecific antibodies alone but rather was a result of specific Ab interactions with HSP90 in vivo. Since injected HSP90 Abs probably inhibit or disrupt HSP90 function in vivo, these results indicate that HSP90 participates in repression of trimerization, or maintaining the monomeric state of HSF1, under nonshock conditions. We also found that gel supershifts of activated HSF1-HSE complexes by HSP90 MAb added in vitro could be quenched or inhibited by the addition of excess purified HSP90 (1 μg/oocyte) to DNA-binding reaction mixtures (Fig. 4C). Addition of the same quantity of excess HSP90 to heat-shocked oocytes immediately upon homogenization did not suppress the supershift of HSF1-HSE complexes by injected HSP90 MAb (Fig. 4C). We therefore conclude that the effects on HSF1-HSE complexes observed after microinjection of HSP90 MAbs were the result of Ab interactions occurring in vivo.
Nuclear injection of HSP90 Abs delays attenuation of HSF1-binding activity.Taken together, the established role of HSP90 as a protein-folding chaperone targeting specific cellular substrates, the known structural changes associated with HSF1 oligomerization, and the results here of immunoprecipitation and antibody recognition experiments demonstrating association between HSP90 and HSF1 and activation of HSF1 by microinjected HSP90 Abs suggested that HSP90 could function as a modulator of HSF1 activity. To examine this further, we analyzed the effects of microinjected HSP90 Abs on the DNA-binding properties of HSF1 during the deactivation phase of the heat shock response. Since HSF1-HSE complexes persist indefinitely throughout prolonged heat shock treatments of oocytes (19), we could not monitor HSF1 deactivation during continuous stress. Instead, we compared attenuation of HSE binding in uninjected and HSP90 Ab-injected oocytes recovering at 18°C from a 33°C heat shock. As shown in Fig. 5, microinjected HSP90 MAbs caused a significant delay in the attenuation of HSE-binding activity relative to that for uninjected controls. The result of this experiment suggested that disruption of the chaperoning ability of HSP90 in vivo decreased the rate of HSF1 trimer disassembly. Thus, it appears that, in addition to suppressing trimerization, HSP90 also plays a role in the conformational changes required for the reverse step of HSF1 trimerization.
HSP90 Abs delay the deactivation of HSF1 in vivo. Uninjected or HSP90 MAb-injected oocytes were subjected to a heat shock at 33°C for 1 h (HS) and then allowed to recover at 18°C for the periods of time indicated, and the HSE-binding activity of HSF1 was analyzed by gel mobility shift assay. Both heat-induced and supershifted complexes are indicated as HSF1 at the left. ns, nonspecific binding activity.
HSP90 Abs inhibit HSF1-mediated transcription fromhsp70 promoter.The transcriptional activation domain of HSF1 is regulated independently of trimerization, and it has been hypothesized that full induction requires additional conformational changes (21, 59, 76). We postulated that HSP90-HSF1 interactions might also have a functional impact on heat-inducible transcription. To investigate this possibility, we measured the effect of HSP90 Abs on heat-inducible expression fromhsp70-CAT reporter constructs. Nuclear injection of HSP90 Abs resulted in a significant reduction of heat-induced CAT activity (Fig. 6A). The negative effect on HSF1-mediated CAT expression implied that binding of Abs to HSP90 in vivo could disrupt transactivation by HSF1 under heat shock conditions. In order to rule out the possibility that injected HSP90 Abs had a general inhibitory effect on transcription and/or CAT, parallel experiments were performed with CMV-CAT reporter constructs (Fig. 6A). The results show that expression from the CMV promoter was not affected by anti-HSP90. In additional control experiments, we assessed the effects of HSF1 Abs on heat shock-induced expression ofhsp70-CAT. The results show that CAT activity was downregulated by HSF1 antiserum (Fig. 6B), and, as was the case for HSP90 MAbs, expression from the CMV promoter was not affected by HSF1 antiserum. Thus, the negative effects of both HSP90 and HSF1 Abs appeared to be specific to HSF1. On the basis of these expression assays, we suggest that HSP90 Ab interactions with HSF1-HSP90 heterocomplexes in vivo hindered or inhibited physical changes to HSF1 required for the acquisition of full transcriptional competence in response to heat shock. We have not ruled out the possibility that the inhibitory effect of injected Abs was exerted through interaction with uncomplexed HSP90 molecules. It is also possible that HSP90 must dissociate from HSF1 trimers before transcriptional activation, and therefore Abs may have sterically inhibited conformational changes to the HSF1 activation domain.
Microinjected HSP90 Abs inhibit the heat-induced transcriptional activity of HSF1. (A) CAT assay of oocytes microinjected with hsp70-CAT (left) or CMV-CAT (right). Oocytes were injected with HSP90 MAb 1 h before heat shock (33°C for 1 h) treatment (HS), and the expression from each promoter was compared directly to that in similarly treated oocytes without injected Ab. Each experiment was repeated at least five times with different batches of oocytes, yielding similar results. The positions of unacetylated (Cm) and acetylated (Ac) forms of [14C,]chloramphenicol are indicated on the left of each panel. Background CAT activity of non-plasmid-injected oocytes is shown in the right panel (cont). NS, no heat shock. (B) CAT assay of oocytes microinjected with reporters as described above. HSF1 PAbs were injected into oocyte nuclei essentially as described above for HSP90 Ab.
Effect of GA on the DNA-binding and transcriptional activities of HSF1.In order to rule out potential artifacts of microinjected Abs, we examined the role of HSP90 in regulating HSF1 activities by an alternative method involving treatment of cells with GA. GA is a fungal benzoquinoid ansamycin known to block HSP90 function (65,71). GA binds specifically to HSP90 at the ATP-binding domain (22, 51, 67) and prevents HSP90-mediated renaturation of proteins (24, 56, 68). GA has been shown to inhibit the HSP90-dependent activities of numerous proteins, including the glucocorticoid receptor and pp60v-src(71), the Src family kinase p56lck(23), Raf kinase (57), and progesterone receptor (65).
Oocytes were incubated in medium supplemented with GA (10 μg/ml) for 2 h, and the DNA-binding activity of HSF1 was analyzed by gel shift assay with labeled HSE. As shown in Fig.7A, GA treatment alone did not activate HSF1. We did not observe HSF1 induction by GA at nonshock temperatures even after extended exposure of up to several hours or after acute exposure to GA concentrations as high as 100 μg/ml (data not shown). The failure of GA to induce HSE binding in the absence of heat shock indicated that GA inhibition of HSP90 in vivo was not sufficient to bring about HSF1 trimerization. In heat shock experiments, however, GA treatment (10 μg/ml) for 2 h prior to a heat shock of 30°C for 30 min caused a significant increase in the induced levels of HSF1-HSE complexes relative to those for identically stressed controls (Fig.7A). Elevated levels of HSE-binding activity were observed at several different points during the induction phase, from between 5 min and 1 h of heat shock.
(GA) enhances the heat-inducible HSE-binding activity of HSF1 and delays deactivation during recovery. (A) Oocytes were incubated for 2 h in the presence of 10 μg of GA (G) per ml or in DMSO (D) or left untreated (U) prior to heat shock at 30°C for the times indicated. The HSE-binding activities of HSF1 were then compared by gel mobility shift assay. (B) Gel mobility shift assay of oocytes that were treated with GA or DMSO as described above, heat shocked at 33°C for 1 h and then allowed to recover at 18°C for the periods of time indicated. The positions of activated HSF1 and nonspecific (ns) bands are indicated at the left of each panel.
Enhanced activation of HSF1 in GA-treated oocytes could have been attributed to a synergistic effect of heat shock in combination with GA or to a shift in the oligomerization equilibrium towards formation of HSF1 trimers caused by GA inhibition of HSP90-mediated dissociation. In an attempt to distinguish between these possibilities, we examined the effect of GA on the DNA-binding properties of HSF1 during the deactivation phase of the heat shock response. The results of recovery experiments consistently showed that deactivation of HSF1 was delayed in GA-treated oocytes relative to that in untreated controls (Fig. 7B). It is important to note that even after severe heat treatments, HSE binding of endogenous HSF1 in oocytes returns to control levels within 5 to 15 min of recovery (19). Thus, GA disruption of the chaperoning activity of HSP90 in vivo appears to decrease the rate of HSF1 trimer disassembly. The results of recovery experiments with GA were similar to those of HSP90 Ab injections, and together these data imply that HSP90 functions in controlling the conformational changes associated with conversion of HSF1 from trimers to monomers. From these experiments, however, we cannot be certain whether GA and HSP90 Abs exerted their effects directly by inhibiting the function of HSP90 molecules in complex with HSF1 or indirectly by inhibiting the function of HSP90 molecules not stably complexed with HSF1.
We next used GA to examine the functional significance of HSP90-HSF1 interactions for heat-inducible transcription. Expression from microinjected hsp70-CAT reporter constructs in untreated and GA-treated oocytes under nonshock and heat shock conditions was compared (Fig. 8). The results show that heat-induced CAT activity was significantly reduced in GA-treated oocytes relative to that in untreated controls. Thus, inhibition of HSF1-mediated CAT expression by GA mimicked the results with microinjected HSP90 Abs. Expression was unaffected by GA in parallel experiments with microinjected CMV-CAT reporter constructs (Fig. 8), thus ruling out the possibility that GA had a general inhibitory effect on transcription and/or CAT activity in oocytes. The results of expression experiments using both microinjected Abs and GA strongly suggest that HSP90 functions in regulating conformational changes to the transcriptional activation domain of HSF1.
GA inhibits the heat-induced transcriptional activity of HSF1. CAT activities of CMV-CAT- or hsp70-CAT-injected oocytes that were either untreated or treated with GA (10 μg/ml, 2 h) prior to incubation at 18°C (NS) or heat shock (HS) (33°C for 2 h) were compared. Each experiment was repeated at least five times with different batches of oocytes, yielding similar results. The positions of unacetylated (Cm) and acetylated (Ac) forms of [14C]chloramphenicol are indicated on the left.
HSF1 is one of the most intensely studied eukaryotic transcription factors. Numerous reports have described the functional domains and intrinsic properties of HSF1 molecules from different organisms, the changes in oligomerization and phosphorylation states, interactions with HSP70, and HSF1 activation by disparate stress signals. Despite knowledge of these events, however, the precise mechanisms by which cells regulate HSF1 are yet to be defined. This study is an important step towards understanding HSF1 regulation. It had previously been postulated, on the basis of HSP90-HSF1 heterocomplex formation in vitro, that HSP90 could modulate HSF1 activity (42, 43), an idea that has not been confirmed before this work. The results of this analysis, using novel experimental approaches made possible by theXenopus oocyte model system, clearly establish a physical association between HSP90 and endogenous HSF1 in vivo and provide evidence that HSP90 functions to modulate oligomerization and possibly the inducible transcriptional activity of HSF1.
The existence of HSP90-HSF1 heterocomplexes is corroborated by several independent lines of evidence, including the detection of HSP90 in HSF1 Ab-immunoprecipitated material from both heat-shocked and control cells, HSP90 Ab-directed induction of HSE-binding activity, HSP90 Ab-directed supershifts of heat-activated HSF1 both in vitro and in vivo, and the delayed deactivation of HSF1-binding activity after disruption of HSP90 with GA or microinjected Abs. Results showing HSP90 Ab induction of HSF1 in the absence of heat shock and delayed deactivation of HSF1 after targeting of HSP90 in vivo suggest that HSP90 contributes to the conformational changes required for suppression of trimerization as well as disassembly of HSF1 trimers. Furthermore, independent experiments with microinjected HSP90 Abs and cell treatments with GA show specific effects on heat-induced transcriptional activity of HSF1. Although we have not directly confirmed the universality of these findings, we assume that HSP90-HSF1 interactions and the apparent regulatory role of HSP90 are not unique to Xenopus oocytes; in fact, the likelihood that HSP90-HSF complexes must exist in other cells is strengthened by the evolutionary conservation of these molecules and previous reports of HSP90-HSF interactions in human cell extracts or reticulocyte lysates (42,43). Also of note is the recent observation by Farkas et al. (16) of a 94-kDa protein species copurifying with bacterially expressed HSF1; our results lead us to suggest that this could have been HSP90.
Based on the evidence presented here and the previously reported properties of both HSF1 and HSP90 molecules, we propose a regulatory model in which HSP90 functions as an HSF1-specific chaperone at different points along the activation-deactivation pathway: maintenance of the monomeric conformation under nonstress conditions, induction of transactivation potential, and disassembly of trimers to a non-DNA-binding state following stress (Fig.9). The model predicts that HSP90 is associated with HSF1 at different points along the activation-deactivation pathway and exerts its regulatory effects directly through interactions with HSF1. The model also predicts that, in addition to coordinating the dynamic changes in HSF1 oligomerization, HSP90 could function in the unmasking of the transcriptional activation domain. Previous studies have shown that the carboxy-terminal transcriptional activator domain is regulated independently of oligomerization (21, 31, 46, 59, 76). The effects on HSF1-dependent transcription of GA and microinjected HSP90 Abs suggest that this step could be regulated at least in part by complexed HSP90. It must be emphasized that the current model does not exclude the involvement of HSP70 in controlling HSF1 activities. Rather, it is more likely that HSP70 and HSP90 act coordinately in this regard. The idea of cooperative and/or coordinate regulation of HSF1 by both HSPs is congruent with the previous identification of HSP70 and HSP90 in large chaperone complexes (63, 64), with the ability of HSF1 to assemble HSP90 along with HSP70 and several components of the cytoplasmic molecular chaperone machinery, and with the recent demonstration that HSP70 binds to the activation domain and negatively regulates transcription (61).
Model of HSF1 regulation by HSP90. Our data indicate that HSP90 interacts with the inactive monomeric and active trimeric forms of HSF1 and could be involved in regulating the interconversion between these forms (A and C). Both HSP90 Abs and GA delay trimer disassembly and inhibit heat-induced transcription of HSF1 (D). These observations suggest a role for HSP90 in modulation of the transcriptional activation domain (B) or that HSP90 dissociation from trimers is required for transcriptional competence. We also hypothesize that HSP90 could link HSF1 to cellular signaling molecules (B). Details of HSF1 structure are not shown, although the shape change from a circle to an ellipse is intended to indicate conformational changes associated with trimerization. No data are presented for the one-to-one stoichiometric relationship between HSP90 and HSF1, which is shown only for simplicity. Although HSP70 is not represented here, we do not imply its absence of involvement in any of these processes.
Apart from providing a mechanistic framework in which the HSF1-regulatory function of HSP90 could be explained, the model shown in Fig. 9 describes a hypothetical mechanism by which HSF1 could be linked to disparate signaling pathways. We put forth the hypothesis that HSP90 functions as an integral part of the cellular stress detection machinery, tethering HSF1 to cell signaling molecules. We suggest that this could occur through transient or reiterative interactions between HSP90-HSF1 heterocomplexes and other HSP90-associated cellular kinases or other regulatory molecules. Alternatively, HSF1 could assemble with HSP90 and other molecules into large heteromeric complexes; however, to our knowledge these have not been detected in vivo through biochemical approaches. Our proposal of interactions between HSP90 molecules simultaneously in complex with HSF1 and cellular signaling molecules provides a plausible mechanism by which signaling molecules that participate in regulating the cellular stress activation-deactivation pathway could be specifically presented to HSF1. The formation of HSP90 homodimers (38) is consistent with this idea.
Specific recognition of HSF1-HSE complexes by using HSP90 Abs was demonstrated both in vivo (Abs injected into oocyte nuclei) and in vitro (Abs added to DNA-binding reactions mixtures). Also, both active and inactive forms of HSF1 coimmunoprecipitated with HSP90 from heat-shocked and unshocked nuclear extracts. These results are consistent with the previous findings showing an interaction between immobilized HSP90 and HSF in nuclear extracts of unshocked HeLa cells (42) and between HSP90 and bacterially expressed HSF1 in a cell-free reticulocyte lysate assembly system (43), but they are in contrast to results of other studies in which HSF1-HSP90 interactions were not detected by immunoprecipitation (53) or supershift analyses (5); it is possible that such discrepancies could be accounted for by the use of different Abs. The simplest interpretation of our results is that a subset of nuclear HSP90 molecules exist in heterocomplexes with inactive HSF1 monomers and remain associated with trimers. Coimmunoprecipitation of equal quantities of HSP90 under both nonshock and heat shock conditions implies that HSP90-HSF1 heterocomplex formation is independent of the oligomeric state of HSF1. What remains to be determined is the relative affinity between HSP90 and HSF1 monomers and trimers and the stoichiometric relationship between these molecules. Also of interest is whether HSP90 remains associated with HSF1 throughout activation and deactivation or whether HSP90 interacts reiteratively and transiently with HSF1 at each step in the pathway.
Induction of the HSE-binding activities of HSF1 under nonshock conditions after nuclear injection of HSP90 Abs, although not after GA treatment, leads us to hypothesize that HSP90 participates as a molecular chaperone in the folding of HSF1 monomers, either to establish the formation of intramolecular hydrophobic interactions of inactive HSF1 monomers, or to maintain the monomeric conformation. Interestingly, the HSP90 Ab-induced HSE-binding activity in unshocked oocytes did not appear to be supershifted, suggesting that this heat shock-independent activation could have been the result of Ab interaction with uncomplexed HSP90. Why did HSP90 Abs but not GA induce HSF1 in unstressed cells? Presumably, the binding of Abs to certain epitopes was sufficient to inhibit the apparent HSP90-mediated repression of HSF1, either directly through interactions with molecules in HSF1-HSP90 heterocomplexes or indirectly through interactions with HSP90 not complexed with HSF1. The fact that GA treatments failed to activate HSF1 does not necessarily contradict a potential role for HSP90 in the folding or repression of HSF1 monomers. GA binds the ATP-binding domain of HSP90 and is hypothesized to inhibit substrate release or lead to substrate degradation (22, 24, 51, 56, 67,68), effects that would not inexorably lead to trimerization.
Although the functional implications of an interaction between HSP90 and inactive monomers is still unclear, recovery experiments indicated a clear relationship between HSP90 and the disassembly of HSF1 trimers. Both GA treatments and nuclear injection of HSP90 Abs significantly delayed deactivation of HSF1 during recovery from heat shock. A number of potential mechanisms by which HSP90 might modulate reversion of HSF1 oligomerization could be considered. One is that HSP90 acts to stabilize the trimeric conformation of HSF1 and must be released prior to conversion to the monomeric form and deactivation of HSE binding. Alternatively, HSP90 could remain complexed with HSF1 throughout the disassembly process and act to mediate the conformational and folding changes associated with conversion of homotrimers to monomers. In either case, the delay in HSF1 deactivation by GA is probably due to inhibition of the ATPase activity of HSP90, but the mechanism by which HSP90 Abs inhibited HSP90 function in vivo is not known. It was interesting that HSP90 Ab-induced HSF1-HSE complexes detected in gel mobility shift assays were not supershifted. Consequently, we have not ruled out the possibility that GA or HSP90 Abs could have exerted their effects indirectly by affecting the chaperone activity of HSP90 molecules not in complex with HSF1.
Another key finding of this study is the repression of heat-inducible transcription after targeting HSP90 in vivo. It is very unlikely that this transcriptional repression of HSF1 was artifactual, since it was reproduced by two independent methods, microinjection of HSP90 Abs and cell treatments with GA. The results imply that physical binding of HSP90 by injected Abs or disruption of its ATPase domain by GA somehow blocked the conformational changes to the transactivation domain of HSF1. Therefore, we suggest, but have yet not confirmed, that the chaperoning function of HSP90 may be directly involved in this step of the activation pathway. This function would be entirely consistent with the known chaperoning role of HSP90 with other cellular proteins and transcription factors such as MyoD (58). Similar to the case for the delay of deactivation by HSP90 antibodies or GA, we have not yet determined whether these agents inhibited transcription directly through perturbing the function of HSP90 molecules complexed with HSF1 or indirectly through interactions with the uncomplexed pool of HSP90. Furthermore, additional factors such as HSP70 (61) or various signal transduction molecules could be involved in regulating the transcriptional activation domain.
There were two apparent contradictions in the results of expression experiments: (i) under heat shock conditions, GA treatment enhanced the HSE-binding activity of HSF1 while reducing transcriptional activity, and (ii) under nonshock conditions, HSP90 Ab injection induced HSE binding but not a corresponding increase in HSF1-mediated transcription from the HSP70 promoter. It could be argued that these results reflect the independent regulation of (i) trimerization and DNA binding and (ii) transactivation (21, 59, 76). In fact, the DNA-binding and transcriptional activities of HSF1 are also uncoupled inXenopus oocytes (6).
Rather than a direct role for HSP90 in regulating transcriptional competence of HSF1, the observations of repression by GA and HSP90 Abs could indicate that dissociation of HSP90 from trimers is a necessary precondition for transcriptional activation. In this case, reduced transcriptional activity caused by HSP90 Abs or GA could have been the result of inhibition of HSP90 release and steric blocking of modifications to the transcriptional activation domain. It is possible that in our experiments with oocytes, these agents acted to stabilize a transcriptionally incompetent intermediate in the activation pathway. Regardless of the interpretations of these experiments, however, it is clear that HSP90 is involved in the transcriptional activation of HSF1. The precise details of this role will probably require more knowledge of the stoichiometric relationships between these two proteins throughout the activation-deactivation pathway.
In conclusion, the data presented here provide the first clear evidence in support of a role for HSP90 in the regulation of HSF1. This study raises a number of key questions. For example, what is the molecular basis for HSP90-HSF1 interactions, and what are the dynamics of HSP90-HSF1 interactions throughout the stress response? Do ATP hydrolysis and HSF1 hyperphosphorylation affect the physical association between these molecules, or does HSP90 actually coordinate HSF1 phosphorylation through interactions with other factors? Do HSP70 and HSP90 cooperate in modulating HSF1 activities, and are other molecules involved? It will be interesting to unravel the precise roles of HSP90 in coordinating the physical changes associated with induction of HSF1 by stress and to determine if HSP90 plays a role in the signaling pathways used to sense stress and coordinate the appropriate changes in cellular activities.
This work was supported by a research grant from the Medical Research Council of Canada to N.O., a postdoctoral fellowship from the Health Services Utilization Research Council of Saskatchewan to A.A., and a PGSA scholarship from the Natural Sciences and Engineering Research Council of Canada to S.B.
We thank S. Hartson and R. Matts for purified HSP90 protein and antiserum; A. Wolffe for hsp70-CAT and CMV-CAT constructs; K. Sarge and R. Morimoto for HSF1 antiserum; A. Hnatov, J. Davies, and J. Xavier for technical assistance; and P. Mercier for critical reading of the manuscript.
A. Ali and S. Bharadwaj contributed equally to this paper.
Returned for modification 10 May 1998.
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Good morning world and thanks for tuning in to another Tasty Tuesday here on Planet Magnet and I hope you have brought your tastebuds with you because have we got a cracking dish for you here today….
The list goes on but trust me when I say this dish is most worthy of your consideration for a future feature and your table at least one meal time in the coming weeks….
So here is how to make my version of Nigel Slater’s wonderful dish.
Same bunch of Spring (Salad) Onions – roughly sliced.
2 Teaspoons Curry Powder (as hot as you prefer).
Measure out the 200gms of Pearl Barley.
Put the Pearl Barley into a pan.
Then add…. Start to boil and then leave to simmer.
Whilst this simmers, roughly chop the Spring Onions.
Then add the 20gms of Salted Butter to a pan and melt on a low heat.
Check on the Pearl Barley adding more water should it be required. Season With some Cracked Black Pepper and stir in well.
Now the butter has melted, add the Curry Powder.
Once the Pearl Barley is cooked with all of the liquid absorbed, remove rom the heat and then add the curried Peas and Onions and stir in well. You will note that this small amount of Curry Powder will spread easily through the Barley and colour it nicely. Place a lid on the pan to allow to continue cooking.
Remove the skins from the Mackerel and roughly shred into pieces before adding to the Barley. Mix in well and replace the lid to continue cooking.
Boil the three eggs, shell and cut into quarters.
Dish up the Kedgeree into a small bowl and add the eggs to one side.
Simple to make, healthy and wholesome, low in calories with a good amount of protein plus all of those lovely fish oils…. Yum!!
Well there you go guys, another Tasty Tuesday Recipe has been brought to you from Nigel Slater but as always with a Magnet twist on it. Hope you like it and I hope you try it very soon.
Thanks Lynn, I hope you like it….!
I have never tried pearl barley. I will have to search for this.
It’s very good for you. It is usually used in soups with lentils.
Love lentils. I will have to go to one of the large scale supermarkets that sell organic foods and a wide variety. Thanks for the suggestion.
LOL I will try it.
Well here is the chance you’ve been waiting for!!
Looking forward to catching up with everyone’s world and the progress they pursue. As the weather here warms and the sunshine remains longer each day, I think of your upcoming charity event. The time will pass quickly. Hope the training has gone well.
Hey Jonathan, welcome back. It’s good to hear from you.
I’m glad you like the recipe and have learnt something about another kind of pea….!
The walk is certainly coming along. We are moving closer each day although there is a ton of work to do. I need to continue with my training and things will be good of that I am sure.
Hope all is well with you and your father is on the mend.
Ha Ha Ha!!! Well only you know Kat but boy are you missing out…!
Well in that case I would expect a resounding success for you…. Do you usually use Smoked Haddock and rice? I really like that version, along with the Tuna and Basmati version but this one is quite excellent and I need to remember to thank Nigel Slater for thinking of it….!
I like it but I think I prefer the first one, creme fraiche and coriander. Yum…!
Mmmm. I wish I could hire you as my personal chef. Everything you make looks so good. And it’s stuff I NEVER see here in the states or would even think of making. It’s interesting!
I’m glad you approve Blair but believe me when I say if I can do it, ANYONE can do it!! | 2019-04-25T16:51:32 | https://worldsbiggestfridgemagnet.com/2016/03/08/tasty-tuesday-with-a-hint-of-spice/ |
Carey Mulligan, who’d rather work than stay at home, continued her efforts to promote her new film, ‘Wildlife’ at the premiere on Tuesday (November 6) in Paris, France.
The actress brought a touch of the emerald isle to Paris as her Erdem Fall 2018 Cordelia dress features a multicoloured floral print inspired by the gardens at Ireland’s Lismore Castle.
Embellished with bold hues and styled with lush velvet blue pumps, this graceful style perfectly reflects the British designer’s focus on femininity. | 2019-04-26T02:31:03 | https://www.redcarpet-fashionawards.com/2018/11/07/carey-mulligan-in-erdem-wildlife-paris-premiere/ |
More great photo coverage of the Daytona Turkey Rod Run put on by the Daytona Street Rod Group. Thanks to Bill Skirvin, known as Skirv on the HAMB traditional hotrod forum. Always one of the top shows in the country, the place was packed and as usual it had a very wide range of differenht astyle hotrods and customs. One of my old hnaunts as we were there year after year with the Old Farts Car Club. | 2019-04-23T20:58:13 | http://hotrodsonline.com/virtualrodrun/2012/12_Turkey/slides/img0254vw.html |
Having once vehemently ordered Russian hijackers to “Get off my plane!,” Harrison Ford can’t even get a few techie terrorists out of his living room in Firewall, suffering one indignity after another as the criminal squatters smack around his wife (Virginia Madsen), give his allergic son some deadly peanut butter cookies, and generally reduce him to a sniveling, submissive wimp. It’s an emasculating situation for an emasculating thriller, one that shrivels the former (and, hopefully, not future) Indiana Jones’s rugged everyman persona to a pathetic husk of his previously formidable self via a mundane heist-and-hostage narrative. Such a fate isn’t very surprising considering that Ford’s latest is helmed by Richard Loncraine, a director whose action pedigree doesn’t extend beyond the combat-free Wimbledon and HBO’s My House in Umbria. But it’s still depressing to see the star awkwardly shoehorn himself into such an embarrassingly ill-fitting role, in which he simultaneously comes off as too gruffly physical to be a cerebral, computer-savvy bank executive and security systems expert, and yet too geriatric to be a rock ‘em, sock ‘em villain-trouncing avenger.
Character suitability issues aside, Ford has nonetheless taken a significant step down the heroism ladder with Jack Stanfield, a dull, reactive protagonist who spends the bulk of this go-nowhere dud either lamely complying with, or feebly attempting to extricate his family from, his laptop and security camera-equipped captors. This malevolent crew is led by Bill Cox (Paul Bettany), a dapper Brit in the Alan Rickman-Die Hard mold, though any evocations of John McTiernan’s lone-ranger standard-bearer merely call attention to Firewall‘s meager scope and nonexistent swagger. Cox wants to rob the bank where Stanfield works, and sets about forcing the man—through threats to his continually cowering family, who soothe themselves by thinking about happy times on their private boat, The Lark—to carry out MacGyver-esque assignments with iPod hard drives and cellphone cameras. What ensues is lots of wrangling about infiltrating secure networks, cracking encrypted files, and subverting protocols, all hi-tech tasks far more briskly exploited by Fox’s 24, with which it also shares the emphatically weird Mary Lynn Rajskub as do-gooder Jack’s curt, bitchy, scowling sidekick.
Despite staging a promising early boardroom scene featuring Ford, Robert Patrick, Alan Arkin, and Robert Forster (a compelling collection of men’s men the film is wholly incapable of properly utilizing), Loncraine mainly deals in senselessly smeared slow-motion, grainy black-and-white surveillance footage, and disorienting close-ups for a climactic bout of fisticuffs. By the time Ford and Bettany square off at a lakeside cabin constructed solely out of break-away walls, windows, and tables, the filmmaker has long since squandered all opportunities to manufacture any intelligent suspense from his identity theft and homeland security-tinged scenario. But it’s hard to imagine any amount of directorial dexterity or pertinent subtextual commentary salvaging Firewall from the antiquated garbage heap for which it’s destined, since Joe Forte’s screenplay, when not regurgitating tired genre staples (women and children in peril, chase scenes in the rain), partakes in barking-mad plot developments—such as a third-act doozy involving curbside wireless internet service and a convenient GPS-enabled canine collar—that, like the rest of this information age-set claptrap, is for the dogs. | 2019-04-20T17:07:45 | https://www.slantmagazine.com/film/firewall/ |
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At BRM we ask a lot of questions regarding your preferences, because we take great pride in being able to provide the best service possible.
Thank you for your confidence !! | 2019-04-25T16:13:10 | https://www.bestrealtymexico.com/ |
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department 56 replacement light bulbs replacement light bulbs clear brite lites bulb factory north pole brite lites bulb factory north pole. | 2019-04-22T22:40:27 | http://tophat.info/department-56-replacement-light-bulbs/ |
February 17 2019, admin uploads String Letters. The String Letters has been created for your inspiration with ideas and combined by follow trend of printable Letter, so the String Letters will give you the real of certificate, template, letter you need. more over The String Letters.
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MultiWebQuiz 1.5 offers a complete On-Line Quiz Management System.
To use this Web application, your site must be hosted on a server that supports PHP (4.0+) + MySQL.
After following a few basic instructions to set up and configure the MySQL database and then adding password protection to your admin directory, all you need do is enter your questions, answers and explanations into the user-friendly quiz creation form. They can be easily copied and pasted from any Word Processor.
Unpack the Zip File !
First unpack the zipped file, ideally to a new folder on your hard drive. This operation will create two new subfolders, inc and admin. The first folder contains HTML code snippets such as the page header and footer dynamically included in the main quiz pages as well as configuration data. The admin folder contains the quiz management pages.
Set Up the Database !
The quiz management system will not actually create the database, because your server, administered in all likelihood by a remote Web host, may impose restrictions on the number and names of databases.
It's that simple. PhpMyAdmin is a good PHP and MySQL administration tool and may be preinstalled on your server. If not, download the latest version and then upload it to a special directory on your Web space. Remember to add password protection to all admin directories. Many Web hosting services will have an online control panel to let you do this effortlessly.
All this data is stored in one small file inc/db_config.inc.php .
Do not overwrite the inverted commas. If in doubt, contact your Web Host, who should furnish you with the correct details.
host: Type your host name here at the end in inverted commas. Localhost if it is on the same server. If it is on another server, place the port number after a colon at the end.
username: Type your database user name in inverted commas.
password: Type your database password here in inverted commas.
database: Type your database name here in inverted commas..
There are no links from the quiz in the main section to the admin zone, but nonethless remember to add password protection to the admin directory. Many Web hosting services will have an online control panel to let you do this effortlessly. If your site is hosted on an Apache server you can edit the .htaccess file.
Use an FTP program, which may be integrated in your HTML editor, to upload the files to your Website.
Navigate to main_quiz_directory/admin/index.php and click Create Tables. If this does not work, check your host name, user name, database password and database name. If the database is on a remote server, check the port number with your WebHost. Please note this command will overwrite any tables of the same name.
After creating the database, Questions and Subjects tables, just add at least one subject area. You can now add as many questions as you like.
Tick Use "Special Characters" to convert all special characters such as &, <, >, è or é to HTML entities for database storage. This will ensure such characters are viewable in HTML. This option will also convert line breaks to <br /> tags. Otherwise, all data will read as raw HTML so you can use tags within questions, answers and explanations.
MultiWebQuiz uses a stylesheet and some custom classes are essential to the Quiz, in particular p#bignav, span#subnav, table#qst and ol#numbered.
However, a little basic understanding of stylesheets should let you change much of formatting.
To add your own custom header, just edit inc/header.inc.php file and use standard HTML under the body tag. Remember the paths of all images you may include in your header are relative to the page and not to the header stored in the /inc/ folder.
As the quiz script relies on CSS2 formatting, it will not display correctly in Netscape 4.7. It has, however, been tested on IE 6, Opera 7, Mozilla 1.2 and Netscape 6.0.
Multiple choice with as many as 6 answer options (default), of which only one may be correct.
Multiple answer with as many as 6 answer options,of which more than one may be correct.
By Simply leaving the fourth, fifth or sixth answer fields blank in the question edit or update forms, MultiWebQuiz will add a code q#exc to this database field to exclude this option from the test.
If the Boolean checkbox is ticked [checked], answer option 1 becomes true and 2 false. MultiWebQuiz fills the other answer fields with xb##l and will shuffle only the true and false options.
If you see these codes in the question edit form, ignore them unless you wish to change the answer mode. | 2019-04-22T06:35:39 | http://multiwebvista.com/testzone/help.php |
Users of Iomega Zip drives who have experienced the so-called Click of Death outside Iomega's warranty period are still entitled to some recompense, according to the Consumers Institute. Assistant chief executive Peter Sutton says two different pieces of legislation cover business and personal users.
Users of Iomega Zip drives who have experienced the so-called Click of Death outside Iomega’s warranty period are still entitled to some recompense, according to the Consumers Institute.
Assistant chief executive Peter Sutton says two different pieces of legislation cover business and personal users.
Both acts provide more cover for users than just the manufacturer’s warranty. According to Sutton it’s the life expectancy of a product that is important, not whether it is still covered by a warranty or not.
“If you’d expect, say, a five-year life out of the drive, and it only lasted a year, then you’ve still got a claim.” Sutton says a product should work for a “reasonable” life span, something which varies from product to product. For a device which completely fails just outside warranty, Sutton says it is fairly straightforward.
One distributor is eager to sort out the Click of Death problem is Electronic Resources.
Director John Dunbar has known of the Click of Death for some time and has asked Iomega for an information pack to help “dispel the myths” surrounding the problem.
Dunbar says when the heads return to the outside of the track there is a clicking noise, hence the name. But calling it “Death” is misleading, he says.
“It’s quite normal. This sort of thing happens on floppies as well, but they have an infra-red sensor so you don’t hear it happen.” Any long-term problems with a disk are due to the oxidisation of the coating protecting the media itself.
“Once it’s got that outside layer on it’s very hard to get it off.” That can damage the heads of the drive itself, thus spreading the problem. Dunbar says this is most prevalent with Nomai disks.
Nomai and Iomega are currently engaged in litigation overseas regarding Nomai’s production of Zip-compatible disks. | 2019-04-19T22:47:45 | https://www.computerworld.co.nz/article/517263/zip_drive_users_entitled_recompense_says_consumers_institute/ |
This blog is about all the extra wonderful and unique happenings in the lives of my family members. Living abroad, pregnancy abroad, and especially about the newest addition to our family: our wonderful daughter, Anne Kathleen. Annie, as we call her, was born on April 1, 2011 in Rome. Annie was born with an extra chromosome – which we think is pretty darn special – and was diagnosed at birth with Down Syndrome, Trisomy 21. We’ve learned so much and experienced so much since then. She’s brought us so much joy, we thought we’d share some of it with all of you. Your thoughts, comments, and questions are extra welcome.
I love this, and I love all 3 of you. You are a beautiful, perfect little family, and I love any and all ways to know what you are up to over there….Love you more.
Colleen – I left my comment under your Sept 20th post…………but maybe I should have put it here! LOL – I’m not very up to date with all this “blogging” kind of stuff!!
Before now I’ve never seen the picture of her Rocky pose outside the Vatican after her Baptism…Hilarious.
What a wonderful writer you are, Col. Thank you so much for sharing. I am in tears while reading, but I LOVE it. Can’t wait to see you guys again. | 2019-04-20T10:42:43 | https://ourromababy.com/about/ |
DELIVERY AVAILABLE UNTIL 9PM ONLY.
-Sautéed fresh ginger, onions, bell peppers, mushrooms and scallions. Please select chicken,pork, beef or shrimp from popup menu.
-Sautéed with basil, bell pepper, bamboo shoots, hot chili and onions Please select chicken,pork, beef or shrimp from popup menu.
-Sautéed with garlic, black pepper, and sherry wine, Served on a bed of steamed vegetables. Please select chicken,pork, beef or shrimp from popup menu.
-Sautéed with cashew nuts, bell pepper, celery, mushrooms, carrots, water chestnuts and scallions. Please select chicken,pork, beef or shrimp from popup menu.
-Sautéed with mixed vegetable. Please select chicken,pork, beef or shrimp from popup menu.
-Simmered in red curry with coconut milk, bell peppers, bamboo shoots and basil. Please select chicken,pork, beef or shrimp from popup menu..
-Simmered in green curry with coconut milk, bell peppers, bamboo shoot and basil. Please select chicken,pork, beef or shrimp from popup menu.
-Jumbo Shrimps sautéed with yellow curry, onion, snow peas, carrots, red pepper, celery and egg.
–Grilled jumbo shrimp topped with chili sauce. Served on bed of mixed vegetables. | 2019-04-21T21:02:17 | http://orders.bluefinparkland.com/thai-menu |
Honey Dew Acres, LLC was founded out of our love of horses, children and horse sports. We started our lesson program on our three-acre farm at 635 Butternut Ridge Road in Canton in 1999 and have continued to grow ever since. In the Fall 2005, HDA moved to 169 Post Road in Canton. A much-anticipated expansion, the farm boasts 200+ acres providing us with much-needed turn-out, riding and driving space. Of course, with a bigger farm comes the much-needed bigger barn!
In 2010, we added a new (and 2nd) indoor arena with adjacent stalls and tackroom.
In 2011, we created an on-site tack store.
In 2012, we built the "New Addition" which holds 10 stalls, 2 sets of crossties, and it's very own boarders' tackroom.
In 2013, we started to build the decking for our upstairs viewing room that will overlook the indoor Dream Arena.
In 2014, we're planning to build a 2nd "new addition" off the parking lot side of the small indoor arena which will include more cross-tie space and stalls.
As for the name, “Honey Dew Acres,” it was really quite simple. Have you ever heard the phrase, “Honey do this, and honey do that”? And thus a farm name was born. | 2019-04-18T10:21:53 | http://www.honeydewacres.org/history |
PROBLEM TO BE SOLVED: To provide a novel amphiphilic polymer which has safety for living organisms and easily bonds or dissociates, and to provide a technology for applying the polymer to the delivery of various functional materials.
SOLUTION: Polysaccharide, a hydrophobic polymer and a hydrophobic material are premixed in an aprotic polar solvent or alkaline aqueous solution. By adding water thereto and mixing, the resultant mixture forms a self-assembled structure which is supposed to be micellar, and then a composite bonded with the hydrophobic material is formed in water. Various functional materials or organic solvents can be used as the hydrophobic material, and β-1,3-glucan is preferably used as the saccharide. | 2019-04-23T11:27:23 | https://jstore.jst.go.jp/nationalPatentDetail.html?pat_id=25478 |
West Elm starts us off with this gorgeous design. Hop on over and check out all of the fabulous selections that they have. You can literally build your very own piece with all of your wants and wishes.
If you’re looking for something that is extra roomy with a punch of style, then this tufted beauty from Overstock may be exactly what you need. Switch out the fabrics to your liking and choose and tone that coincides with your vision. We personally really love the beige linen and all of the styling possibilities it offers up.
Ashley Furniture has a piece that’s a bit smaller but still perfect for a big family. It’s more on the traditional side of interior design but one that will be versatile and fit into many homes. This creamy taupe is a wonderful way to start out your living room revamp.
Houzz showcases a lot of great inspiration and this peek was no exception. Going with an extra large sectional in a dark brown, leather will make for a great addition to a home with more masculine nuances. This fits inside a large home office or the formal living room as well.
Here’s another beautiful example of what an large, sectional sofa in a creamy neutral can do for your space. It fits nicely into this family room and is touched beautifully by all of the subtle, trendy accents. Thank you to Honey n Hydrangea for the idea!
Apartment Therapy featured this gorgeous living room on their site and we scooped it up too. You can even find sectionals that have a personalized style to them. Like this grey beauty for example, those studs give it a punch of industrial, modern vibes.
Kelley Nan gives us a beautiful view of this spacious living room. It has a crisp, clean vision and it fits a family nicely without any fuss. Sometimes all you need is the right amount of space and some comfy, throw pillows to get the job done.
If you’re really lucky you’ll find a sectional with both unique styling (as in its color) and size. We got this gorgeous look from Baker and Storehouse and found it to be quite inspiring. Even a family home – with need for all the seating space – can be trendy with some bohemian flair!
If you’re looking for something really simple, then this leather piece from Wolf Furniture may be what you’re shopping for. It’s traditional, it’s comfy, and you can really style and personalize it to your liking. If you find this scene too dull, just add pops of colors to its surroundings!
Even your ultra modern home can have some extra large seating arrangements. We’re loving this sharp and chic sectional sofa we found over at Home Designing. It fits in nicely with the rest of the room’s contemporary styling.
Over at Loaf, you’ll find this extra cozy and cushy blue sectional. It’s so lush that we, personally, want to dive right in. It also looks gorgeous in an open living room, especially contrasted with chic and creamy whites.
A smaller sectional design, this white piece from Architecture Art Design is another great choice for those looking to spruce up and get their modern home feeling a bit cozier. Just because you want posh lines doesn’t mean you have to skimp on comfort for the family. Go with a sleek piece like this to keep the vision and function intact.
Pinterest, as usual, brought us vast amounts of great ideas and inspiration. This plush, taupe sofa is one of the best ways to get a jumpstart on easy, family home decorating. Extra large pieces can really make or a break a room.
Modern Euro Design offered up this stunning, modern design. Dipped in a gorgeous shade of orange, this will fit right into a more family-oriented space by creating a unique focal point. Or, it can be slipped right into an ultra contemporary home easily as well.
And finally, for the most unique sectional sofa inspiration on the list then we ask you to visit Architectural Digest. This stunning, velvet masterpiece can be dressed up or dressed down while creating a gorgeous scene inside of your home. Contrast it with golds and creamy whites for the ultimate look. | 2019-04-24T08:19:42 | https://www.trendir.com/large-sectional-sofas/ |
By Scott Croft (As Amended By ISAF). Image, Janet BAXTER is presented with the Leadership in Women's Sailing Award:© BoatU.S.
With near-perfect weather and a capacity crowd, the sixth annual Women's Sailing Conference sponsored by BoatU.S. filled the Corinthian Yacht Club in scenic Marblehead, USA, with a lively day-long series of classes and workshops. A highlight of the day's events was the presentation of the Leadership in Women's Sailing Award to Janet BAXTER, a Chicago-based racer, sailing judge, ISAF Committee member and the first woman to be elected president of US SAILING.
BAXTER, a member of the ISAF Women's Sailing Committee and Women's Forum, was instrumental in preparing US SAILING for the 21st century with a comprehensive reorganization of the group. In accepting the award, BAXTER spoke graciously about how sailing has enriched her life and cemented friendships that last a lifetime. She has raced in 27 Chicago-to-Mackinac races and excelled in racing Lasers, Etchells, as well as offshore boats. She is a member of the Chicago Yacht Club.
The award is co-sponsored by BoatU.S. and the National Women's Sailing Association (NWSA) and honours a male or female who has a record of achievement in giving something back to the sport of sailing as well as inspiring and educating women. 'Janet BAXTER, in taking such a high-profile leadership position at US SAILING, shows that women can excel at every level - both on the water and in the board rooms,' said Elaine DICKINSON of BoatU.S. in presenting the award with NWSA President Valli COOK.
The 2 June Women's Sailing Conference was organized by the NWSA and gives women sailors, from novices to experts, an opportunity to network with other women while learning new skills or brushing up on old ones. The conference ran like clockwork as women took to the water for hands-on training sessions in man overboard, racing skills, spinnaker and up the mast. Land-based workshops included diesel engines, knots, anchoring, marine electronics and cruising preparation. With nearly 100% female instructors, many of them U.S. Coast Guard-licensed operators, this year's students showed the same enthusiasm as in years past.
Following a gala dinner, featured speaker Maureen MCKINNON-TUCKER described how she found a way to hold onto her passion for sailboat racing even after she suffered a paralyzing accident. She is the first woman named to the US Disabled Sailing Team and is vying for a berth in the 2008 Paralympic Games in China. Some 60 prizes were raffled off, including an inflatable boat donated by West Marine. The raffle raised $2,500 for the AdventureSail programme for at-risk girls and boys, which NWSA also sponsors.
The conference is one of several women's events sponsored by BoatU.S. (Boat Owners Association of The United States). To find out more visit www.boatus.com/women.
ISAF has a keen and long standing commitment to Women's Sailing, and both the ISAF Women's Sailing Committee and ISAF Women's Forum focus on the development of women's sailing. | 2019-04-21T10:46:36 | https://members.sailing.org/news/5000.php |
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Please use the drop down menu next to style to select the stamp of your choice!
High quality home decor stamps. Use with ink or paint. Add incredible detail to all your products. Stamp on painted surfaces, wood, furniture, fabric, paper and much more. Clean with soap and water. | 2019-04-19T11:10:04 | https://3monkeyshomeaccents.shop/collections/iron-orchird-designs-iod/products/stamps |
The material is good for this very business like skirt. Comes with a belt. The sizing is exactly like the site.
I like it. Good material. Have it for almost a year now and its holding up!
This skirt looked just as pictured, very well made, nice lining and stitching. It was a perfect fit in the hips and waist, a little shorter than I had hoped for but the measurements were exact. Good buy! | 2019-04-22T03:13:02 | https://www.sammydress.com/product271811.html |
After the Rise of the Planet of The Apes, came the Dawn… now its time for War. Quinny and Dion caught the final chapter of the new Planet of the Apes trilogy, thanks to 20th Century Fox, and now the real war has begun: The War for the Periodic Table of Awesome! Who will come out on top in this futile conflict; the peaceful, ape-like Quinny, or the rapidly-losing-his-humanity Dion?
Flawless CGI means you will forget you aren't watching real super-smart apes.A deep and involving plot that echoes other films whilst still being fresh and creativeFeatures some really Incredible performances.
Some of the human characters are a bit 2 dimensional. | 2019-04-26T05:38:26 | http://www.theperiodictableofawesome.com/war-for-the-planet-of-the-apes-spoilers-tptoa-podcast-35/ |
Gao Yanmin, a woman who was abducted but later came atop reports as a model teacher, has recently aroused heated discussion online, as was reported by Beijing News and also cited by major news websites in China on Tuesday.
Gao Yanmin was at a train station in 1994 when she was abducted by two female traffickers who promised to find her a job. She was then sold several rounds before she was finally bought by a local peasant as a wife in a village in Quyang of north China's Hebei Province.
Having failed to run away and being thrown back to tortures for several times, Gao finally settled down in the village and began working as a teacher at a local primary school.
Gao's plight brought public attention nine years ago when she was nominated for a "Touching Hebei" award. Her story received wide media coverage and was even adapted as a movie in 2009.
During the latest round of online discussion, however, many netizens believe that it is not appropriate for certain media to focus on Gao's contribution as a teacher in rural areas, but the first matter to be questioned should be her status as an abducted woman.Netizens say that the gang involved in the trafficking of Gao Yanmin should be identified and arrested.Online opinion was later echoed by Chen Shiqu, Director of the Anti-trafficking Office of China's Ministry of Public Security, who made a statement via his Sina microblogging account on Wednesday to say that Gao's case is already under police investigations.Chen also vowed steady efforts on human trafficking in an all-round way. | 2019-04-26T04:37:27 | http://zjnews.china.com.cn/redian/2015-07-31/48.html |
The quad port VSC8564 GbE PHY with Intellisec is ideal for securing cloud network applications such as e-commerce, databases, collaboration, smart grid, video, and enterprise or government communications. In addition, VSC8564 can be added into the design of FIPS 140-2 compliant products for use in a wide variety of markets including (but not limited to) medical, insurance, financial, banking, military, defense, security, retail, industrial, entertainment, and cloud computing.
Intellisec enables a realistic and affordable Layer 2 MACsec security solution. Intellisec is a patented technology enabling IEEE 802.1AE MACsec encryption end-to-end over any network (including multioperator and cloud-based networks), independent of the network's awareness of security protocols. Intellisec is not limited to traditional MACsec link-based box-to-box applications, and scales easily with the number of interfaces (delivering significant cost savings in network deployment), but is still capable of being configured to be 100% backward compatible to currently deployed 128-bit MACsec 802.1AE-2006 compliant solutions. The device also includes dual recovered clock outputs for timing references in Synchronous Ethernet solutions.
VSC8564 is pin compatible with the 1588v2 PTP-capable VSC8584 device. | 2019-04-23T16:32:35 | https://www.microsemi.com/product-directory/gigabit-ethernet-phys/3904-vsc8564 |
Originally established in 1921, the Arthur B. Sim Golf Course is the largest in Wichita. This 18-hole course features 33 sand traps, a pro shop, and food and drinks. They offer lessons here as well, if you're looking to improve your skills. Enjoy a day outdoors, playing a round with friends or business associates. | 2019-04-25T23:49:07 | https://cityseeker.com/wichita-ks/711934-arthur-b-sim-golf-course |
Our 53 Seater Coach is available for hire with Driver. We regularly collect within Worthing, Hove, Brighton, Chichester, Littlehampton, Lancing, Shoreham by Sea, Bognor Regis and surround areas and cover jobs internationally.
Bought From Woods Coaches. This is our newest vehicle to our fleet. We our very proud to have bought it from a company who looks after there vehicles as well as we do. Are first 53 Seater Coach. Many more to come. | 2019-04-20T10:50:25 | https://aandatravel.co.uk/53-seater-coach-hire/ |
The rarefied calm of Kettle's Yard, the much-loved, newly reopened modern art gallery in Cambridge, has enchanted numerous visitors and prompted many to wonder how they could recreate its subtle charm in their own homes. The good news? The founders of new west London gallery, 8 Holland Street, insist it's entirely possible to create a similarly rich mix in the vein of the Kettle's Yard founders, Jim and Helen Ede. "I studied History of Art at Cambridge and hung some of the less expensive works from the collection in my room at college, through the museum’s picture lending scheme," recalls Tobias Vernon, co-founder, with curator Rowena Morgan-Cox, of 8 Holland Street. "We regularly visit the collection to gaze at works by Ben and Winifred Nicholson, Christopher Wood and Barbara Hepworth, and take inspiration from the chalky white walls, and the artworks sitting happily next to furniture, glass, ceramics and natural objects." Here, they share their interiors tips.
Kettle’s Yard engenders an easy informality in the way that a Brancusi sculpture might sit alongside Edwardian glassware on a roughly hewn oak plinth. Ignore hierarchy and embrace the casual juxtaposition of an antique ladder-back chair with a vintage boucherouite rug and a contemporary hand-woven textile by Catarina Riccabona. Throw in a Clifford Ellis oil painting from the Fifties, joyful lithographs by John Hoyland and a round rope mirror attributed to Audoux-Minet.
Never underestimate the power of artistic tableware. For glassware we use carnival striped Murano glasses and carafes designed by Campbell Rey in collaboration with Laguna B, or multi-coloured, recycled Mexican shot glasses, tumblers and carafes. For crockery our favourites are Dora Good’s earthy pitchers (£170) and cups and Rachel Cocker's scalloped edged plates (from £68). Try these beside the vintage bon bon dishes, also on sale in the gallery, or Guido Gambone’s dessert set.
Using white, or another neutral, brings consistency and will allow you to move things around easily. Enliven the space with brightly coloured paintings and cushions. Here, a Clifford Ellis Fifties-era painting, an iconic Ivon Hitchens piece from the Seventies and coloured hand-blown glass lights by Venini contrast the natural tones of rattan and stripped back furniture.
Collecting doesn't have to be an expensive past time. Spring is also a good time to start trawling art markets and fairs for bargains. Whilst working at The Fine Art Society, Rowena bought inexpensive prints by artist husband-and-wife duo French-English artist Ethel Gabain and John Copley, as well as turn-of-the-century Whitefriars glassware from The John Scott collection. Tobias collects post-war print editions by Twentieth Century artists associated with the fishing town of St Ives in west Cornwall, such as Patrick Heron and Breon O’Casey, together with stoneware studio ceramics. Choose a period and begin researching: you'll be surprised what you can find if you trawl Etsy, Vinterior, Pamono and eBay.
Like the Edes, we enjoy working with our artist friends. Emily Buck is an exciting young sculptor and friend of 8 Holland Street. In our opening show we have on display both her textured black ceramics and carefully constructed gouaches. We are currently planning a talk with Emily during London Craft Week and a future exhibition of her work. Look our for local open house and studio events to meet the creatives behind artworks.
pieces from across the globe. Unattributed artefacts are a fun way to add diversity (and mystery) to your home on a smaller budget. Think of the Surrealists and get mystically drawn to an object in a charity shop à la André Breton. | 2019-04-23T10:50:42 | https://www.vogue.co.uk/gallery/kettles-yard-interiors-tips |
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For years our family has used the Playstation console to play video games.
The family has tons of games - most of the ones from the older console can be used on the newer machine.
Well, the current unit has begun to have a few problems.
Perhaps from too much use or just age itself, so the search began for a new console.
If we stayed with our current brand, the unit was going to cost between $350-$400.
We didn't see much use in picking up another used system like we have because it would be just as old as the one we already use.
My son decided that since he was buying it, he was going for the best deal he could find.
We now have an Xbox 360.
I really don't see much difference between it and the Playstation.
The graphics are a bit better.
Storage space on the hard drive is easier to use than the individual cards on the old system.
It was quite a bit less expensive for this system.
And I really like the wireless controllers. The rechargeable batteries last quite a while in them too.
Even with the hard drive, I don't see where this system is any faster than the old one though.
And the biggest problem we have - we don't have many games to chose from in our house at the moment.
But, as long as everyone is happy, I guess that is all that matters.
Perhaps it is the Ram? | 2019-04-19T22:50:40 | https://www.techtipsfromanongeek.com/2010/10/new-game-system.html |
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The Greens have been accused in the debate about particulate matter and nitrogen oxide limits of Ministers of transport, Andreas Scheuer (CSU) chumminess with the auto industry.
The Green-Chairman Anna Lena Baerbock called the debate more than irritating: “Will the Union actually, the scientific basis of the world health organization in question?” The Minister of transport seem to not just want to the facts to adjust, if the reality can fit into the stuff.
Scheuer had welcomed the Initiative of more than a hundred lung doctors, the applicable particulate matter and nitrogen oxide limits. On the Basis of these limits, it is Diesel in the next few months-driving bans in a number of cities. In Hamburg there is such a ban already.
the President of The employers Association of total metal, Rainer dulger pointed out, called for a halt in the nitrogen oxide limits: “If even lung specialists keep the limit in traffic for too low, needs to be promptly a new decision. The Federal government must insist on the in Brussels immediately,” said dulger pointed out. Previously, the economic Council of the CDU had demanded a Moratorium on driving bans. Scheuer wants to limit discussion, according to his Ministry in the EU. | 2019-04-24T09:12:13 | http://www.ccdiscovery.com/green-throw-scheuer-chumminess-with-the-auto-industry |
The turn of the year always sees people undertake new fitness regimes and diets.
Come on, admit it, how many of you are currently watching what you eat, or have joined a gym, in the hope by summertime you’ll be strutting about, feeling like a million dollars?
Well it’s not just us mere mortals who take on this mindset, numerous celebrities, including the likes of Chris Pratt and Chris Hemsworth are also getting into shape.
Hemsworth, who you might recognise better as Marvel’s very own Thor, recently took to Twitter to show his adoring fans – all 4.57m of them – how he’s getting into shape for 2019.
In the video, the Avengers star can be seen exercising on all fours across a basketball court, and I’ve got to admit – it looks pretty intense.
Meanwhile, over on Instagram, Hemsworth posted the same video, and some of the comments were hilarious.
It looked easy. I tried it. I almost died.
Uh…..based solely on this video my work out spirit animal is a panda…..
I completely relate to that one. Perhaps not even a panda though, just a giant sloth.
I realise I could be getting carried away with myself, but is Hemsworth getting into such great shape because he’s coming back as Thor, not only for Avengers: Endgame, but possibly another movie about everyone’s favourite Norse god?
Posting a video on Instagram in May last year, Hemsworth thanked fans for their continued support with everything MCU-related.
There was one sentence in particular which got fans – and myself – convinced there’ll be another Thor standalone film.
It was the biggest superhero film opening ever. The film continues to smash records left right and center and, I’ve said it before and I’ll say it again — it’s all thanks to you guys.
Thank you so much, everyone who continues to support these characters and the Marvel Universe.
We’re going to keep trying to crank them out for you, if you let us.
What’s that Chris? You’re going to keep cranking them out? Hmmmmmm. Obviously means there’s more coming, doesn’t it?
Whether he comes back for another Thor film, or not, I’m too excited to be worrying about that when we’ve got the next Avengers film to look forward to!
I literally cannot contain myself for the next instalment! | 2019-04-22T14:12:31 | https://www.unilad.co.uk/film-and-tv/chris-hemsworths-new-spirit-animal-workout-regime-looks-brutal/ |
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One of the major advantages of Cannabis is their Online procedure which basically allows you to purchase through the website and get it delivered straight to your home. They give a huge variety of premium flowers, centers, topicals, tinctures and tasty edibles.
This area is actually a one stop shop for most medical marijuana requirements. They appeal to over 40 unique strains and have a huge variety of products and accessories. Some of the best products include; Iced Grapefruit, Large Cheese, Bubba Kush and Pre 98 Dubba. Prices are cheap starting at about $10.
If you decide to use Cannabismo, you are going to understand what we’re talking about when we say”Top-Notch” service. They really understand client loyalty and perform their very best to provide you with the finest possible service. If they like you and you become a regular, you’ll see that they will begin to spoil you with a few nice freebies.
Cannabismo knows that not everyone wishes to return to their store and that is why they developed well-oiled online ordering procedure. (There is not any purpose missing out on a huge online market!) . With Cannabismo, you can order through their menu or simply phone them up and place an order. Deliveries are quick (1 to 3-day shipping throughout Canada). Also, it’s all very discreet.
Regrettably Cannabismo does not have a contact number, so in the event that you need something urgent you’ll have to email them. In their defense they’re pretty responsive through email.
By the time that I put my order (which Is before noon PST — order cutoff) it has never taken over 2 days for my doorstep, and is generally in 1 day. Follow-up with orders is practically instantaneous too — the processing time with these men is crazy.
Once I have placed my order, I get reassurance In a number of different ways. First I get a confirmation email, secondly I receive notification that my payment has been received, and after the order is sent I get a tracking number as well. No additional MoM is quite this thorough.
Cannabismo Delivers the best looking and user Friendly site of all the marijuana mail order solutions. The subject is simple and straight to the point. The site is easy to navigate and I need to spend barely any time whatsoever completing my purchase.
Although I’ve already mentioned speed, the Shipping and packaging this online dispensary utilizes is unmatched. There’s no possible way that your order could stink with the measures that have been taken to seal the package. Additionally the packaged includes postal tracking, which means you may always know where your order is. | 2019-04-19T21:06:37 | https://itechtrick.com/thc-for-sale-uk-cannabismo-mail-order-marijuana-review-2019/ |
Term entries in the full glossary matching "content"
The content associated with an element in the source document; not all elements have content in which case they are called empty. The content of an element may include text, and it may include a number of sub-elements, in which case the element is called the parent of those sub-elements.
In this specification, the noun "content" is used in three ways: It is used to mean the document object as a whole or in parts.It is used to mean the content of an HTML or XML element, in the sense employed by the XML 1.0 specification ([XML], section 3.1): "The text between the start-tag and end-tag is called the element's content." Context should indicate that the term content is being used in this sense.It is used in the terms non-text content and text content.Empty content (which may be conditional content) is either a null value or an empty string (i.e., one that is zero characters long). For instance, in HTML, alt="" sets the value of the alt attribute to the empty string. In some markup languages, an element may have empty content (e.g., the HR element in HTML).
The final part of a computed constructor is an expression enclosed in braces, called the content expression of the constructor, that generates the content of the node.
For the purpose of this specification, "content generation" refers to generating content appropriate to the user agent profile of the request by using the user agent profile as input to a dynamic content generation engine. The XSL and style sheets of the document are used to tailor the document to the user agent profile of the request.
The mechanism for selecting the appropriate representation when servicing a request. The representation of entities in any response can be negotiated (including error responses).
The mechanism for selecting the appropriate HTTP representation when servicing a request. The HTTP representation of entities in any response can be negotiated (including error responses).
This term was developed from that in Hypertext Transfer Protocol -- HTTP/1.1.
The practice of providing multiple representations available via the same URI. Which representation is served depends on negotiation between the requesting agent and the agent serving the representations.
A server that originates content in response to a request.
For the purpose of this specification, "content selection" refers to selecting an appropriate document from a list of possible choices or variants by matching the document profile with the user agent profile of the request.
Content element having only PCDATA, sep and presentation expressions as content. Represents either an identifier (ci) or a number (cn).
The content of a document refers to what it says to the user through natural language, images, sounds, movies, animations, etc. The structure of a document is how it is organized logically (e.g., by chapter, with an introduction and table of contents, etc.). An element (e.g., P, STRONG, BLOCKQUOTE in HTML) that specifies document structure is called a structural element. The presentation of a document is how the document is rendered (e.g., as print, as a two-dimensional graphical presentation, as an text-only presentation, as synthesized speech, as braille, etc.) An element that specifies document presentation (e.g., B, FONT, CENTER) is called a presentation element.Consider a document header, for example. The content of the header is what the header says (e.g., "Sailboats"). In HTML, the header is a structural element marked up with, for example, an H2 element. Finally, the presentation of the header might be a bold block text in the margin, a centered line of text, a title spoken with a certain voice style (like an aural font), etc. | 2019-04-25T18:35:06 | http://www.w3.org/2003/glossary/keyword/All/content.html |
Running errands on your bicycle is a great way to burn calories and get some exercise. When you need something small or you are just checking prices and availability, pedaling to the store can be a pleasant experience if you can find a bicycle-friendly route.
Several major shopping locations in the Rogue Valley can be accessed by bicycles without encountering excessive traffic. For example, both the Rogue Valley Mall and the Medford Shopping Center are easily accessible from the Bear Creek Greenway bicycle path.
It is somewhat more difficult to ride a bike to the big-box stores along Highway 62 (Crater Lake Highway). At these stores, the parking lots are full of cars, but I seldom see a bicycle. I have found three bicycle-friendly routes that will get you to the area of Costco, Lowes and Office Depot without having to negotiate through a lot of traffic.
1. From east Medford: get on Springbrook Road, which has bike lanes along most of its length, and head north. Turn left on Delta Waters, which also has bike lanes. Head west, crossing Crater Lake Highway at the traffic lights. Don't dally crossing at these lights as they don't stay green very long. Delta Waters turns into Lear Way which will take you to all the big-box stores.
2. From Poplar Drive, anywhere north of McAndrews Road: Poplar Drive has bike lanes. Head north and cross Highway 62 at the traffic signals. These lights also change quickly from a bicyclist's perspective. After crossing Highway 62, Poplar becomes Bullock Road. Look to your right for a paved bike lane, just before Elite Motorsports on the right. If you pass Lithia Body and Paint and the batting cages on the left, you have gone too far. Turn right on the paved Medco Haul Road bike path. It climbs a short hill and then proceeds along the edge of the airport for 1.3 miles. Turn right on Commerce Drive and right on Lear Way, which passes behind Costco and the other stores.
3. From the Bear Creek Greenway: travel northward. Be cautious riding on the Greenway bike path as it has unmarked bumps and cracks hiding in the shadows which could dislodge you from your bike if you hit them unexpectedly.
From where the bike path goes under McAndrews Road, it's about 1.3 miles to where you turn right onto the path that leaves the Greenway to get to Hilton Court. Look for a green sign on the greenway that says "Biddle Road" with a picture of a bicycle. Upon entering Hilton Court, cross the street to get into the eastbound turn lane.
Turn left at the traffic light at the intersection with Biddle Road (which has bike lanes). Then turn right into the Bureau of Land Management entrance and follow their access road until you reach Bullock Road. Take a right on Bullock and proceed about four-tenths of a mile. (If you miss the BLM turn, go to the next traffic signal and turn right on Lawndale Road, then right onto Bullock Road.) Be alert on Bullock Road as there are no shoulders, vehicles seem to travel fast and drivers are often reluctant to share the road.
Before approaching the raised median divider on Bullock (as you approach Lithia Body and Paint), cross the oncoming traffic lane and get on the sidewalk in front of Elite Motorsports. Proceed about 50 yards and turn left onto the Medco Haul Road bike path. From here follow directions as described for route 2.
Either of these routes provides bicycle-friendly access to a lot of shopping opportunities. Be careful in the big parking lots as drivers are not used to seeing bicycles. Stay in the traffic routes and don't cut through the parking areas.
Take a good lock to secure your bike while you do your shopping.
Carry a small backpack, pull a trailer or have some method of carrying your goods that leaves your hands free to steer your bike.
Enjoy your shopping; I hope that using your bicycle is less stressful than you anticipate. Have a great ride. | 2019-04-21T05:08:11 | https://mailtribune.com/oregon-outdoors/biking/exercise-by-biking-to-shops |
Building engineering is the foundation of every safe structure. Architectural and structural Engineers guarantee that buildings are sound by being included in every step from the blueprint to the finished home or building. The men and women at Structural Engineer Pros in Eros, LA in this field are responsible for the safety of all who walk into the structure after them, and do have a solid knowledge of building regulations and codes along with the creative spark.
Our building Engineers at Structural Engineer Pros good Engineers that are able to creatively solve any problems that may arise due to local circumstances, altering priorities and altered blueprints. As professionals we can customize on the fly and still keep everything up to code in accordance with Eros, LA. Our experts are able to see a project through from start to finish, and brings a knowledge and professionalism to any site.
For Structural Engineer Pros in Eros, LA every structure should be safe for the people living or working in it depend on it. Additionally, rising environmental awareness has put the spotlight on higher efficiency and lower energy costs. As building engineering seal on official blueprints raises the credibility of the architect, and promises the best build for any major structure.
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Photos by Maryann Harlow Chatham second doubles player Sydney Berkson hits a backhand during the 2018 MCT Tennis finals. Berkson teamed with Emma Sheldon and prevailed at second doubles for the Cougars, who won their sixth consecutive title.
RANDOLPH _ Chatham's Sydney Berkson and Emma Sheldon were sweating through a first set tie-breaker at second doubles when players began finishing their matches during the Morris County Girls Tennis Tournament finals.
Berkson and Sheldon consider every point, every match, important but had no idea how critical their match was until they prevailed, 7-6 (7-3), 6-0, over Morristown-Beard's Sophia Maney and Emma Duffy and their coach, James Cai, approached them.
"High five," a smiling Cai said, putting his right hand up. "You just won the county title."
The Cougars' sixth consecutive championship, attained on Sunday, Sept. 23 at County College of Morris, was extremely satisfying because it was so hard fought. Berkson and Sheldon, along with second doubles player Libby White, were the only ones from Chatham to advance to the finals. Berkson and Sheldon's triumph allowed the Cougars to edge out second-place Morristown by one point, 17-16.
Kinnelon and Morristown-Beard tied for third with 15 points apiece. Mountain Lakes was next with 14 so it was actually a good thing that Berkson and Sheldon didn't know what was going on with the team scores.
"We came here so nervous," said Berkson, a sophomore. "After warmups, we were confident and got more focused. The first set was difficult. I thought, 'Whoa, what's going on?' during the tie-breaker. I didn't think it would be easy... It was getting frustrating. In the second set, they were wearing out. I couldn't believe it was 6-0."
"It was so close," Sheldon, a senior, noted. "We know that every single point counts. We had to miss as little as possible. One by one, everyone was leaving (the courts). There have been times when we've been the last ones on the court but... We had to power through. We had tunnelvision. It was better if we didn't know if the team was winning or losing. We just kept a positive attitude and played as hard as we could."
Berkson, who has been striving to be more consistent and to improve her mindset, exhibited strong backhand strokes. First serves were Sheldon's forte. The duo was very pleased by their second doubles crown because this is their first season on varsity.
"We play well together," Sheldon said. "We bring each other up. We heard the cheering and kept a positive attitude."
Kinnelon senior Britany Lau garnered her third straight first singles crown, dispatching Hannah Blake of Morristown-Beard, 6-1, 6-0. Lau, who went up 4-0 in the first set, relied on solid serves and solid play at the net.
"I can't think of exact words to describe this," Lau said. "I'm very lucky and very excited. I got to represent my school in this tournament for another year. I focused on every point and came to the net often. I put away a lot there. I moved through the court more."
Lau, of course, was in a familiar environment, having played many of her matches at CCM over the last three years. She was delighted for freshman teammate Claire Zhang, who topped Chatham's Libby White, 6-0, 6-0, to finish atop the field at second singles.
"I've played challenge matches with Claire and hit with her," Lau said. "Having her win at second singles is big for our school. She's similar to me - very focused on her game, consistent, fast. Claire is crafty with her hands."
Zhang, introduced to tennis at the age of 7 by a neighbor, planned to be aggressive and "put a little more on my serve." She was anxious at first during the final but quickly settled in.
"It feels really good," Zhang said. "I've never been in a tournament this big."
Kinnelon coach John Cataldi, naturally, was proud of Lau and Zhang.
"What they did is awesome," Cataldi said. "Both of them were on their games for the entire tournament. They are consistent and dedicated and have a passion for the sport. They have great personalities and are leaders on and off the court. Both girls needed mininal coaching. I spoke to each of them once (in the finals). They were calm, cool and collected the entire time."
Maddie Siegel of Morristown defeated Montville's Amy Zheng, 6-2, 6-2, to take the third singles title. At first doubles, Mountain Lakes' Megan Matiwsky and Alexis Buchanan beat Isabella Galinkin and Alex Fisher, 6-4, 6-1. | 2019-04-25T17:43:12 | https://soar-nj.com/sports/tennis/item/1537-mct-tennis |
DISCLAIMER: I have a limited knowledge of baking, and no business doing so. Now that we’ve got that out of the way, let’s make a pie!
Pumpkins are in season, you may have noticed. Pumpkin spice and flavoring can be found in every food and beverage, while corpses of Jack’o’laterns rot on every door step. I’m not big on pumpkin flavoring, but I do love the culinary delights that highlight it’s deliciousness, like pie, or soup. Pumpkin pie was my favorite growing up, so I've dabled in a few recipes. What follows is a hybrid of what I liked best from each of them.
First, let's focus on the crust which will make or break any pie. I've been experimenting with whole wheat crust. Not a mix with white flour, but entirely whole wheat. It’s a bit tricky, and takes some patience and care. Here's how I do it.
It’s a basic crust recipe you’ll run across everywhere in regards to ingredients, I just do a straight flour substitution.
Cube up the butter, and let it sit in the freezer for 15 minutes. Meanwhile, whisk the dry ingredients together in a large bowl.
Cut the cubes of butter in to the flour mixture with a dough blender, or a couple of butter knives. It’s important that the butter is cold, and you do this in a timely manner. When the butter is cut down to pea size pieces you’re done.
Stir in just enough of the ice water so that you can work the dough into a ball, but don’t over do it. Divide the ball in two, and shape the halves in to disks. Since this pie doesn’t call for a top layer of crust, I used about 2/3 of the dough for a thicker crust, and kept the rest aside to make cookies.
Wrap the disks in plastic, and let them sit in the refrigerator for at least an hour. Now lets look at what I put into the filling.
I’m not convinced that the sugar pie pumpkins make better pies. Growing up we always used carving pumpkins and never complained. But they’re available, so why not.
First you need to cook the pumpkin. There are several ways, but I prefer to bake it. Simply cut it in half, scrape out the inside, and set face down in a pan with about 1/4 in of water.
Bake at 350 until you can easily pass a skewer through. I over bake it to the point where the skin separates, making it easy to get at the filling.
If you want to be fancy, you can process the pumpkin through fancy kitchen appliances. I don’t have any thing like that, so I just stir it well and call it good. The result is a bit rustic, but no less delicious.
In a mixing bowl, beat the two eggs. First the whites, then work in the yokes. Mix in the cream and sugar, and finally the spices. Lastly, stir in the pumpkin and set the whole mixture aside for a bit at room temperature. The ingridients need some time to mingle.
Now pull that crust disk out of the fridge and let it sit for 10 or 15 minutes before trying to roll it out.
While you’re rolling it out, think of pushing and pulling the crust into shape, rather than flattening it with all your pressure downward. Because it’s 100% whole wheat, you’ll have to flip it over a few times so that it doesn’t crumble apart. It won’t have the same elasticity as it would with white flour. And the end result might not be pretty, or a perfect circle, but you can fix it in the pan.
Once you have the crust shaped out in the pan, put it back into the fridge for at least 30 minutes. Stir your filling a bit in the meantime, and preheat the oven to 400.
Now we’re going to give the crust some extra attention so it comes out crispy and flaky. If you fill it at this point, you’ll end up with a soggy crust. If you’re cool with that it’s no problem, and a lot less work. But a good pie really is all about the crust, so let’s talk about how to do that.
Pull the crust out of the fridge, and line it with foil. Fill the pan to the rim with uncooked rice. This will keep the crust from shrinking away from the edges of the pan while we pre cook it.
Place the rice loaded crust into the lower third of the oven for 20 minutes. Remove the foil lining with the rice and set it aside. Poke some holes in the crust with a fork, and return to the oven for about 5 minutes. You can store the rice and cook it normally any time. It might even have a nice toasted flavor.
Whisk 2 egg yolks in a bowl with a pinch of salt, and brush this mixture over the entire crust. Return it to the oven just long enough for the eggs to set. This will provide a moisture barrier between the filling and crust, preventing a soggy end result.
If you take your time and do all this right, you’ll end up with a flaky, crispy crust when everything has cooked and cooled.
While the crust is still warm, pour in the filling, set the oven temp to 375, and drop the pie onto a rack in the middle of the oven. | 2019-04-20T04:51:04 | https://1077theend.radio.com/zach-van-lue/tis-season-pie-pumpkin-pie |
What is the difference between an Account and a Company?
How do I disconnect from QuickBooks?
How do I switch between companies? | 2019-04-25T16:31:25 | https://bookvalu.zendesk.com/hc/en-us/sections/201847886-General-Administration |
These days radio and television are filled with stories of the downturn in the United States Real Estate Markets and many think that this crisis will tumble across the border into Canada. There may be market corrections in some pockets of Canada, particularly in the East and in higher end, over priced homes but what will happen in a community like Maple Ridge?
With two major Bridge construction projects curently being undertaken, The New High Level Pitt River Bridge linking Maple Ridge & Pitt Meadows to Port Coquitlam and the Golden Ears Bridge linking Maple Ridge & Pitt Meadows to Langley, the future looks brighter here than almost anywhere else in North America. With quick access to the South towards the US Border and allowing residents to access the southern communities without having to cross two bridges, and needless to say the Pitt River bottleneck being eliminated to the West Maple Ridge is poised to grow in all areas, Residential, Commercial & Industrial. With all this construction completing in the Fall of 2009 and the the continuation of low interest rates to prop up the United States and Eastern Canadian Economies there is unlikely to be any loss of value in the Maple Ridge Pitt Meadows region any time soon, because as you see Real Estate is a local phenomenon.
So if you are sitting on the shelf waiting for lower prices in this region, you may be sitting a long time. | 2019-04-25T14:36:11 | http://mapleridge-realestate.ca/?page=24 |
Sarah King is a Cardiff based writer and art critic. She has a background in History and travel writing, and currently contributes to Wales Arts Review. (http://www.walesartsreview.org) where this article first appeared.
Y Gwyll/ Hinterland is beautiful. There is no doubt about that. With record ratings, a second series confirmed and rave reviews, the S4C/BBC crime drama can only be described as a roaring success. Having been sold to DR, the Danish network behind The Killing, before it was even filmed, this should be Welsh TV jumping out of its corner fists flying, punches thrown left, right and centre. This should be it.
All the ingredients are there. A brilliant idea, a strong script, excellent directors and actors, money and backing from two big TV networks. The result is a visual tour de force from vast, imposing landscapes to soulless, sepia interiors through mists of rain, frost and windswept mountains. A promotion campaign of epic proportions was staged and the viewer was promised raw, scary, gritty drama in the style of Scandinavian noir.
But does it live up to the hype? Aside from the usual clichés of the genre, and the fact that, actually, it is not that scary, it almost does.
Shot back to back in both Welsh and English, fraternal twin versions of the series were produced. The first, Y Gwyll, was shown on S4C at the end of 2013. The second Hinterland, a ‘bi-lingual’ version, was recently shown on BBC Wales.
Taking the viewer through the four stand-alone stories that make up the first series is DCI Tom Mathias and his trio of dedicated detectives. Richard Harrington makes a handsome and brooding DCI Mathias. He is newly transferred to sunny Aberystwyth from the big smoke of London following (surprise, surprise) a personal tragedy. Having risen through the ranks at a young age through sheer determination and hard work, he is an intense and committed protagonist with a good beard and very tight trousers.
His team is made up of 33-year-old DI Mared Rhys who lives at home with her parents and her emotionally distant teenage daughter. A local girl who rose through the ranks at a young age through sheer determination and hard work. Then there is the blonde one, DS Sian Owens, who gets shouted at by DI Rhys and hit on by the lonely men of Aberystwyth. She is young, pretty and rising through the ranks through sheer determination and hard work.
Finally there is the guy with the glasses, DC Lloyd Ellis. We do not really know much about him, except that he is young, has glasses and will probably be rising through the ranks quickly through sheer determination and hard work. Four hard-working, determined and serious characters. Not a lot of drinking games and party hats with this bunch, but despite the obvious clichés this team really does work. Superbly acted, it slugs its way through rain and mud in search of that ever-elusive holy grail of policing: the truth.
The natural environment plays an important role in Y Gwyll/Hinterland. Almost to the point of being a character in itself, the vast, open landscapes mirror the loneliness and isolation felt by the characters, a theme that runs throughout the series. The wide sweeping shots of long yellow grass swaying in the wind, barren trees and the coiling waves of the ocean, though enigmatic, sometimes overshadow the narrative.
It is in the interiors that we get a real feel for the characters, especially DCI Mathias. In the interrogation rooms, the blood-soaked bathrooms, the caravans and the dilapidated farm-houses, the dialogue seems more natural and the acting subtle and restrained.
The series has created a stylised version of Aberystwyth. Any semblance of Gregg’s or Tesco has been removed and a picture is painted of a lonely, timeless town. Nobody seems to laugh, least of all DCI Mathias. The series revolves around him, and his growing obsession with the cases he works on. Like any good TV detective, this is a character long overdue for a sit-down with the police psychologist.
The cases, unfortunately, are what let the series down. Yes, the series is set in Wales. Yes, it is set in the backwaters of Aberystwyth. But that does not mean sheep, land deeds and philandering university lecturers are very interesting or very scary stories. Escaped Nazis, stalkers and a suspicious, isolated community had potential, but never really took off. In an attempt to make the crimes relevant to the location the writers have ended up pastiching Wales rather than pushing it forward.
The one story with real meat on the bones is the murder of a former children’s home manager. The sadistic abuse she subjected the children to slowly unravells, and in one of the final scenes it is hinted at that the story that will continue into the second series. Fingers crossed!
I cannot write about Y Gwyll/ Hinterland without addressing the role of the Welsh language. Having watched both versions of the series, the fact is they are almost identical. They are not set in a different country or with different actors. No, it is the same actors saying exactly the same thing in exactly the same locations – but in two different languages. ‘Why?’ I hear you ask. Exactly. Why? Perhaps the 80,000 people who watched Y Gwyll on S4C, and the 350,000 viewers of Hinterland on BBC One Wales cannot read subtitles? Of course they can!
Viewers tune en masse to watch subtitled shows like The Killing, The Bridge and The Returned. Why not Y Gwyll? Making the two versions just adds to the view that Welsh language TV is only for Welsh speakers, and Hinterland unfortunately ends up undermining Y Gwyll whichever way you look at it.
But do not worry Y Gwyll. You are still a contender. It appears it is the Welsh version that has interest abroad, and by far the majority of people who watched it on S4C had the subtitles on.
The European audience are used to subtitles, and if they like it in America they will probably just make their own. So here’s to having a little more faith in Welsh language TV, hitting a little harder, playing a little less safe and looking forward to a second series a lot.
This series may not be as satisfying as others in the crime genre, but surely it’s a cut above Midsomer Murders! The important thing about this is that it gives S4C/BBC and Wales a profile in the genre. Scotland has Rebus, Yorkshire has Banks, Norway and Sweden have a slew of detectives. Wales has………..? It’s a start.
I watched it and thought it was average. Perhaps I’d have thought it was a lot better were it not for the barage of hype pre release. Like many things in Wales currently, this seems to be a case of ‘excellence’ being measured in terms of the amount of Welsh language content, rather than the true quality….. education would be another classic example.
I don’t think there is a Gregg’s in Aberystwyth. Or a Tesco!
By any standard the series was very good. Wales has had other well regarded detective series in the past as well including Yr Heliwr [ A Mind to Kill] with Philip Madoc. That ran for 5 series. So not unique at all.
To say they were poor storylines is very harsh and simply not true. How we Welsh often hate success in others – it is beyond me why and it finds it’s way to this website more than others.
Recently I watched (for the first time ) the original series of the acclaimed Nordic noir “The Killing”.
On a Box set which and I got through all 20 one hour episodes in a week.
Enjoyed it, but watching it over such a short time span meant that the introduction of extra plot lines of variable relevance and holes in the main plot become evident. Both of which if watched over a twenty week period would I expect be far less noticeable.
I think the Y Gwyll, although there’s scope for criticism stands up well in comparision. And the quality and appeal should have been evident to BBC executives who have chosen basically to trial it twice, on S4C then on BBC2 Wales before allowing it out of Wales and onto a BBC UK wide channel. It’s not something they do with a series produced in Scandinavia.
Nordic Noir actually varies a lot in quality – the final series of ‘The Killing’ was ultimately a bit disappointing but ‘The Bridge’ is better than any contemporary drama produced in the UK since the 1980s – and there is no reason why something just as good could not be made in Wales.
Indeed, ‘Yr Heliwr’/’A Mind to Kill’ was a classy production that deserved a higher profile – I remember Philip Madoc telling me how he had to telephone Channel 5 to boost the signal. It was in many ways ahead of its time. It may also be cited as proof of the main point of this article, that we need to make more positive use of our natural environment in Welsh productions. The lesson of ‘Midsomer Murders’ is that scenery sells.
The more basic point is that the recent boom in production in Wales – with the Pinewood Studios development in Cardiff the latest example – remains essentially a back office operation. The serious decisions are still made in London or LA. It is as difficult as ever for Welsh-based writers or producers outside the ‘magic circle’ to get Welsh-themed work on to the big or little screens.
How much of that thirty million allocated to and then by the Welsh Assembly as part of the Pinewood deal will end up in the hands of Welsh talent rather than English or American businessmen? | 2019-04-18T12:42:51 | https://www.iwa.wales/click/2014/02/welsh-landscape-key-character-in-crime-drama/ |
The overstuffed roster for X-Men: Days of Future Past just got a little bit smaller. Although Anna Paquin‘s Rogue was reported as being part of the sequel all the way back in January, director Bryan Singer now reveals that the character has sadly been cut out of the film altogether. Hit the jump to find out why.
According to EW, Paquin was set for just one scene in the movie — a big rescue sequence featuring Rogue, Magneto (Ian McKellen), Professor X (Patrick Stewart), and Iceman (Shawn Ashmore). The footage was shot early in the production process, but as the film moved into post-production, Singer realized he didn’t have room for it after all.
The gigantic cast for X-Men: Days of Future Past has had fans wondering how Singer could possibly find time to service them all in a two-hour film. The fact that Rogue was cut out so easily confirms what we suspected — that some of the stars’ roles will amount to little more than cameos. | 2019-04-23T22:45:18 | https://www.slashfilm.com/rogue-cut-out-of-x-men-days-of-future-past/ |
Take a non-hormonal holiday from hot flashes!
Many women are searching for a natural, non-hormonal treatment for their hot flashes associated with menopause.
The good news is that there is Relizen: A non-estrogenic supplement that is endorsed by gynecologists, and has clinically shown efficacy. This safe and natural option helps women ease into menopause comfortably and naturally.
Relizen is a patented nutritional supplement for the relief of hot flashes associated with menopause.* Already one of France’s top natural menopause products, and used by over one million women in Europe over the last 15 years, Relizen is now available in the U.S. for the first time ever.
We know how annoying and uncomfortable those hot flashes can be, which is why we want to give away a three month supply of Relizen to one lucky reader.
Visit us on Facebook and leave a comment on the page telling us why you want to win Relizen!
One winner will be selected on December 16, 2014 and announced December 17, 2014.
To learn more about Relizen, visit www.Relizen.com.
This entry was posted in happiness, Contests. Bookmark the permalink. | 2019-04-23T12:06:55 | http://www.menopausemakeover.com/2014/12/01/relizen-for-hot-flashes-giveaway/ |
Property Management at it’s BEST..!!
Proptension was founded with a vision to provide the best property management solutions to the homeowners, minimizing hassles in asset management and maintenance. Since its rise in 2014, we have focused on ensuring the best services and experience for all real estate property owners.
Our dedication and drive keep us poised which is why we have adopted the measures of transparency and timely reporting to make sure that our clients real reasonable returns from their real estate and assets. One of its kind property management firms in Bangalore, Proptension brings together world class service with the traditional values of trust and transparency.
The founders Menaka Ramesh and Ramesh Mariyappan have propelled the company towards its success and within its short span of inception, the company has been hailed as one of the best customer focused estate management firms.
With a goal to establish ourselves all over India, we are working towards a scalable progressive network that will bring together home owners and tenants and minimize the hassle filled processes. | 2019-04-25T06:15:44 | https://www.proptension.com/Home/AboutUs |
Hermeneutics is the art and science of interpretation; biblical hermeneutics is the art and science of interpreting the Bible.
D.A. Carson from "Must I Learn How To Interpret The Bible?"
We ought to read the Scriptures with the express design of finding Christ in them. Whoever shall turn aside from this object, though he may weary himself throughout his whole life in learning, will never attain the knowledge of the truth; for what wisdom can we have without wisdom of God?
The history of Jesus is thus the hermeneutical key to the biblical canon as a whole. Jesus Christ is the hermeneutical key not only to the history of Israel but to the history of the world, and hence to the meaning of life, for he is the Logos through whom all things were made.Jesus Christ is the content of the Scriptural witness, the one who interprets the Old Testament witness, and the one who commissions the New Testament witness. Accordingly, Jesus is both the material and the formal principle of the canon: its substance and its hermeneutic. | 2019-04-25T23:54:07 | https://www.monergism.com/topics/hermeneutics |
Fire is among the oldest representations of divinity and sacredness. In mystical tradition the hearth maintains the divine essence of fire and connects it with household, Clan, and the ancestral spirit. Our beautifully detailed hearth honors this ancient tradition, and can be used as an altar piece or a shrine adornment. Place offerings on its mantle and light a candle within the hearth to embrace this spiritual and magical lineage. The cauldron inside can be used as an offering dish, charging bowl, or a tealight holder. This Altar hearth is truly unique and is quite large! Decorate your altar hearth as the sabbats come and go, or use it a beautiful decor! Our altar hearths are extremely detailed with a fiery cauldron in the middle, a besom on the left side, a staff poker on the other and detailed wood and bricks are found throughout. There is even a mantle and a stone etched base as well. A truly magickal piece indeed!
The finish on this item is done by hand, so please allow for minor variations of color. | 2019-04-18T21:30:13 | https://www.eartisans.net/collections/other-statuary/products/sacred-magical-altar-hearth |
There are many things that earn me the name Super Standard Sherlock – Defender of Poodle-kind. Take my tongue for example. As you can see from the photo above, it is epic. My human thinks I have it out so much because it is hot here in Houston, but I am really just showing the world what a fine body part it is. In fact, it is too glorious to be kept hidden in my mouth. So, I share it with my admiring public.
I have heard of this creature called an Ant Eater that is suppose to have a better tongue than I. Let me first say, eating ants is silly. I have eaten a cockroach – was not good – tasted like the rubbish bin. I have eaten June bugs – not bad – be careful not to get a leg caught in your throat. I have eaten moths – very tasty – wings are a little dry but the flavor is a little “taste like chicken.” But an ant? Why bother. I won’t even waste my time on a cookie crumb that small. So this critter might have a longer tongue than I, but he has questionable taste as to what he does with it.
I will let you judge for yourself as to whether the tongue pictured above is not the best darn licker you have ever seen. And if you want proof, make an appointment with my secretary and bring a jar of peanut butter. I will be happy to prove it in person.
Canine Conversations – New Year Revolutions. | 2019-04-23T20:34:00 | https://2spoos.com/2014/12/30/tongue-out-tuesday-the-stuff-of-legends/ |
Facilitate cross-department coordination of release management functions.
Track and maintain products-wide release calendar.
Measure release cadence and effectiveness using common metrics across the entire software portfolio.
Create common standards and templates for release rollout processes and maintain the production deployment runbook.
8+ years of expertise and leadership in Release Management.
2+ years of working in the financial enterprise applications.
Knowledge of ITIL standards and best practices.
Bachelor’s degree or higher in Computer Science, Computer Engineering, Management Information Systems and/or equivalent work experience.
The RM should possess the below skills.
Must be well versed in Visual Studio/TFS Continuous Integration & Delivery process.
Very strong analytical and problem-solving skills are desired. | 2019-04-26T06:16:40 | https://wuzzuf.net/jobs/p/158121-Release-Manager-Veripark-Cairo-Egypt |
YPR-PVR WEEKLY EXP (16541) departs from यशवंतपुर जं. Railway Station at 18:00.
YPR-PVR WEEKLY EXP reach on day 2 to पंढरपुर Railway Station. The arrival time of YPR-PVR WEEKLY EXP at पंढरपुर Railway Station is 11:35.
Distance covered by YPR-PVR WEEKLY EXP?
YPR-PVR WEEKLY EXP covers 890 km to reach पंढरपुर Railway Station at average speed of 51 km/hr. YPR-PVR WEEKLY EXP passes through 13 stations. | 2019-04-19T02:19:14 | https://www.ndtv.com/indian-railway/ypr-pvr-exp-16541-train-in-hindi |
If students learn these poems by heart and recite them to each other, they will never repeat those "silly" mistakes with "make" vs. "do" anymore. Make an effort and you will see that you will feel far more comfortable speaking English.
Additional poems with worksheets and keys are to be found in DOC in QualityTime-ESL: The Digital Resource Book Version 2.0. | 2019-04-22T15:01:39 | https://qualitytime-esl.com/spip.php?article95 |
Free players aka free agents play a mayor role in Football Manager 2017. Top 10 Free Defenders Left are without a club and are contract free which means that you can get them without any transfer fee, this is great news for any team without transfer budget, especially lower league clubs.
We'll take a look at top 10 free defenders left in Football Manager 2017. Free players aka free agents are a great addition to your squad. | 2019-04-22T11:05:22 | https://www.footballmanagerblog.org/2016/11/top-10-free-defenders-left-fm2017.html |
Home / News / Britain's oldest protester has won a court battle to have his name removed from a police "extremism" database after he was added despite never committing a crime.
Britain's oldest protester has won a court battle to have his name removed from a police "extremism" database after he was added despite never committing a crime.
John Catt, 94, a peace and human rights campaigner, was mentioned in 66 entries in the National Domestic Extremism Database after he took part in demonstrations between 2005 and 2009.
Details included what he was wearing at particular rallies and the fact he was making sketches of the protests.
Mr Catt, from Brighton, has been fighting the decision to retain his details on the database, even though he was never convicted of any offences, for the past eight years.
He first took legal action in 2010 when the Association of Chief Police Officers (ACPO) refused to permanently delete information about him in the database, which is maintained by the National Public Order Intelligence Unit.
The Court of Appeal ruled in his favour in March 2013 but police chiefs successfully challenged the decision at the Supreme Court.
Mr Catt's legal battle with police came to an end today after the European Court of Human Rights overturned the Supreme Court's decision and ruled that his Article 8 right to privacy had been violated.
His lawyer Shamik Dutta tweeted: "This ruling sets an important precedent that it is unlawful for governments across Europe to label citizens engaged in peaceful protest domestic extremists and put them on a database for a potentially indefinite period."
Mr Catt has been attending public demonstrations since 1948 and began participating in protests organised by Smash EDO in 2005.
The group's objective was to close down the activities of EDO MBM Technology Ltd, an American-owned company which manufactured weapons and weapon components and had a factory in Brighton.
Mr Catt was arrested twice for obstructing a public highway at two demonstrations organised by Smash EDO but was never convicted of any offence.
He also participated in rallies at the Labour Party Conference in Bournemouth in 2007 and a pro-Gaza demonstration in Brighton in 2009. | 2019-04-25T21:59:58 | http://www.madnesshub.com/2019/01/britains-oldest-protester-has-won-court.html |
ALEX DIY Friends Forever jewelry kit lets young ones make colorful friendship bracelets the easy way. Designed for crafty kids ages eight and up, this kit includes the friendship wheel, a handy tool that simplifies the weaving process so you can make as many bracelets as you have friends. The kit contains 22 different colors of string, beads, needle, and color-coded looms that can be used again and again with other string. Everything comes in a take-me-anywhere suitcase so you can make bracelets on the bus, in the car, at your friend’s, and more.
Includes 4 color coded looms, 22 colors of embroidery floss (26ft / 8m), beads (0.3oz / 8g), beading needle, instruction book, and carrying case. Recommended for children 8 years of age and older. | 2019-04-23T12:34:05 | https://www.alexbrands.com/product/arts-crafts/friends-4-ever-bracelet-kit/ |
“So what do you do?” “What’s next?” “What are your plans after school/ uni/ this job…?” We have all been there. When the prospect of a social event fills you with dread, anticipating the same tired questions. As if you have to justify why you have not mapped out your entire lifespan already. That tight feeling starts to grip your stomach as you see yourself falling behind the ranks of other bright young things marching confidently into the future with a swing in their step. Resist the pressure these well-meaning social interrogators are pushing on to you. They are usually thinking more about their own life than yours or just trying to making polite conversation. To my disgust, I even caught myself doing it recently (and choked into my wine glass).
The truth is, decision-making fills many of us with dread. I remember lying awake for long hours at night thinking of how I was going to fill my gap-year; of what it would look like on my C.V.; of what friends, family, future employers would think of me; of what I could be missing out on. Yes, it is true, the decisions you make shape your life. Who your friends are, what you do, who you know, where you live, whether you go to that late event on a work night… There is huge pressure on each of us to make decisions and to make them earlier and earlier in childhood, apparently with lasting implications. The opportunities are seemingly limitless and therefore the likelihood of making the wrong decision all the greater.
If you, like me, are you in your twenties, then right now you are probably facing an obstacle course of crossroads, opportunities and decisions that you need to take. Probably even several at the same time. Various aunts and uncles have cheerfully told me how much they hated being in their twenties. A decade of head pummelling whilst you ride white-knuckled on rollercoaster after rollercoaster. You get hurt, hurt people, get bitterly disappointed, have doors slammed in your face whilst others fly open and it is all a bit intense (and still somehow fun!). Before you curl up in the foetal position, clutching a tub of Ben and Jerries’, take a deep breath, exhale and realise that not all is lost! The good news, so they tell me, is that this stage only lasts until you find your particular groove in life, after which you get on with it, stick with it and life is much simpler. Do-able? In the meantime, we can give ourselves a hand with all this decision-making by making sure we keep in good shape for it.
That is because good decision-making is more about an attitude, a way of being rather than a TV quiz show with right or wrong answers. The focus should be on you, the decision maker rather than on the decisions themselves. I urge you not to spend hours at night over-analysing your options from every angle and playing out possible outcomes. I have been there so many times. You wake up exhausted and even coffee cannot save you. If you are in good shape yourself, then you will be in the right place, with the capacity for making good decisions as and when opportunities inevitably come along. It is a bit like keeping fit so that when the marathon (or dance off) comes up, you can go for it.
Decision-making flows from you, from where you are at when making the decision. It is not separate from the rest of your life. This means that your support network, your physical, emotional and mental health will all have an impact, so ensure you are maintaining good health and balance in these areas. At the same time, beware the trap of holding off all decision-making until you are feeling ‘ready’. You never are to be honest; you just get on and make them when they come up. As the old saying goes, moderation in all things – including asking others for advice. So please do not lose your individual opinion and capability by involving the whole sorority in your decision-making. Whilst fond of sharing, I avoid telling too many people about big decisions coming up, and only have about five key people whose opinion I really take on-board. Make that three people….At the end of the day you have to decide for yourself and to be at peace with your decision. If you decide based on the opinion of others, then it will come back to bite you.
This leads to the next decision-making 101 – and this is uncomfortable. Ask yourself, am I deciding from a place of freedom? As recently as this summer, I decided to give up my job, friends, family and country, essentially life as I knew it, to go abroad for a year with a friend. My friend had a concrete reason to go. I thought I did, but then realised it was her path, not mine. I was caught up in the excitement, in wanting to go with her, in a desire to escape my current life. Sometimes we can choose someone else’s pathway, especially when you care deeply about them. One of the things I found helpful in making my decision was a YouTube video by Fr Mike Schmitz who recommended asking yourself, ‘What would the freest version of me do?” You can tell this by looking at the fruits. Does this give me peace? Because that is the giveaway sign of living in freedom. I had been feeling restless and uncomfortable as the proposed departure date crept closer for us to leave England. This was when I realised that my decision-making was operating on shaky foundations, like the desire to please others and therefore was not free. These kind of questions are not nice but sometimes we have to grit our teeth and face ourselves and this takes time.
Time is one of the greatest luxuries of our so-called ‘developed world’ and we deal it out like misers, especially when it comes to spending it on ourselves without a concrete result. Rebel! Create the space for decision-making and allow yourself the time. I took a week off in the autumn and restrained myself from filling it with activities and people. Instead, I went for long walks in the countryside, spent the afternoons writing and then spent time with my grandma and one of my oldest friends who both know my weaknesses and what my life should look like. Thanks to this week off, I had time to recover my balance away from the whirl of work, life and London, to perk up thanks to a potent combo of fresh air, good food, laughter and daylight and spend time with people who really know and love me. The big decision I had to make then naturally rolled itself out like a red carpet over the course of the week, and by Saturday evening, I had made a complete turn around and arrived at a place of peace with my decision.
Even if you have taken the time out and ensured you are in good health for decision-making, it is not always clear and in this case you have to take a step forward, even if turns out to be in the wrong direction! This is scary but you will learn something positive from the experience. Nothing is wasted. Even unsuccessful relationships are not all bad, if you step back and reflect on why it did not work out, what your non-negotiables and priorities are and what should trigger the warning lights. Making any decision will also start the ball rolling. Things start to happen. The worst position to be in is when we freeze. I saw this on the London Catholic dating scene (or lack thereof). People would want to find the right person so badly, but they would rarely take the risk of rejection or give that sweet guy/girl a chance by going on a date. As if going on a date was as binding as an engagement ring! Risk is good. Step out of your comfort zone because it can grow stagnant and we need to keep open and growing.
Practise decision-making in the small things to keep you in shape for when the big decisions come along. Do not underestimate the small decisions – they can have greater impact than you realise. It is a bit like the maxim that if you look after the pennies, the pounds take care of themselves. Another benefit is this saves you from living in limbo, waiting on your ‘big break’. This is a very real danger of fixating on what we are going to do, what we want and of living in the future. I remember actually saying to one guy when we broke up (over the phone, nasty), “But I had such a beautiful future mapped out for us!” My vision for our future was so beautiful that I was willing to overlook warning signs in how the relationship was in the present, in order to get that dream. You will be delighted to hear that this is unhealthy and unhelpful. If you do live in the future, you will most likely grow discontented and bitter, prone to navel-gaze, become boring to be around and ironically start to miss out. That is because life is being lived right here and now, in the present. We are making our futures right here and now. You are not in limbo, this time is full of potential. Time to root yourself, to find your character, your core values, who your true friends are, what your weaknesses are, how you react in given situations and practise key inter-personal skills. Building up who you are, so that what you do then comes about naturally from that. Are we making the most of this time? | 2019-04-19T01:01:44 | https://handmaidmag.co.uk/know-thyself-1/2018/1/15/hpozt3ogtmkvjems37vbsfjhixcl70 |
RH) You live in an apartment, in a building known collectively by its residents as the Pigeon Palace. In 2015 you were all able to stave off eviction by securing a loan to purchase the building as a cooperative. As you know I am fascinated by what is going on in Spain, with En Comu, and in in Los Angeles, by the emergent constellation of citizen led organizations challenging the city for the wealthy and the developers. In this time of economic and social crisis, I’m wondering how you as an urbanist relate to the possibility of the contemporary grassroots urban politics?
Another gorgeous sunset view from Bernal Heights, a short distance from my home.
Getting up the hill looks like this at sunset.
Great show of Victor Arnautoff was held at the Labor Archives at SF State… this is his rendition of Joe McCarthy in the early 1950s.
I’m so rarely in a car, or on the bridge, so this photo I took while driving across the Bay Bridge made me happy for some inexplicable reason.
Embarcadero palms and the Vaillancourt Fountain at dusk… made for a weird view. | 2019-04-21T15:09:07 | http://www.nowtopians.com/work-and-the-economy/from-nowtopia-to-rebel-city |
Will you be celebrating Valentine’s of Gal-entine’s this year? Either way, you can’t beat a delicious array of tempting cocktails in various pink-hues to 'cheers' with your loved ones. The lovely team at Roast have shared three of their tantalising cocktail recipes that are inspired by Valentine’s Day. They’re bound to shake and stir up some warm and fuzzy feelings.
Mix on all the ingredients (except the sparkling wine) in a cocktail shaker.
Give everything a good shake.
Strain in a cup glass and top up with sparkling wine and garnish with 2 little prunes and a lime twist.
Put the four strawberries in a blender and process the berries until they are pureed.
Mix the strawberry purée with on all the ingredients.
Strain in a cup glass and garnish the glass with heart-shaped strawberry and pink sugar rim.
Crush 1/3 apple in a blender and add the Seedlip Grove 42, coconut water, lychee juice and lemon juice.
Strain the mix into a little bottle.
Serve the bottle on the side of a cocktail glass with heart-shaped candy floss in the glass.
Pour the preparation and the candy floss will dissolve in the mix. | 2019-04-19T03:00:44 | https://www.arloandjacob.com/trio-cocktails-valentines-day/ |
High waist shaping brief. Firm compression providing a high level of support. Shapes the waist, supports the bottom, smooth the tummy and supports the back. Natural cotton gusset. | 2019-04-19T03:15:16 | https://minaebazaar.co.uk/products/311064 |
Maximum utility of your office space is necessary to enhance not just the internal productivity but also to incorporate a lasting impression on your international clients. Refurbishing and rearrangement of furniture and office accessories definitely impart a new look to your already existing office surrounding. Office fitouts can also be done by professional interior designers if you want an envious look to your office. For the primary step, you need to go for the careful planning technicality. These are used in order to make the fit out project a huge success among all. There are certain tips available, which can help you to plan out a program, accordingly.
This part of the article is going to take help of the right kind of office fitouts steps, which can help in making the right plan as per the needs and demands of the office environment. These are solely designed for the official environment, and not suitable for other interior panning areas.
Set up the goals: You are asked to determine the results, which you want to achieve the fitness goals. Are you looking for fit out structure, to reinforce the branding service of the business? Is your main aim to increase the motivation power of the employees and to provide them with a good working environment? Make sure to keep a list of all these services beforehand, and set a goal accordingly.
Keep a proper checklist: For this step, you need to list down the present requirement along with the most inevitable priorities, which can match up the dreadful timelines. In case you are planning to purchase a new office fixture, or want to take help of architect for designing the layout of office, you are asked to list the tasks, in order to keep the office fitouts programs in proper and organized manner.
Install the fixtures: You are asked to deal with some of the various fixtures, as installed after the official hours. These ensure that the employees, who can work comes in term with the undisturbed services, mainly under the official hours. These are likely to include the schedule of the installation procedures or any other jobs, associated with the present checklist. These will help you to deal with the aspect of the project, and those need to be done, on time.
Do not compromise on quality: There are different types of fixtures and furniture, which can be availed at this present moment. It is not at all wrong to keep the costing down, associated with the project downing trend. You have to search for the branded accessories and fit-out elements in the market, which come within the lowest price range, and start looking for the most durable ones. Make sure to check the quality of the product first, before considering the price rate.
Including special items available: There are certain major items, which are to be included, when the main area relates with office fitouts. Some of those are storage cabinets, IT infrastructure, power and phone outlets, and the list is practically endless. These can be defined as the most proficient cost effective segment, which include various other updates. These are related to the official fit out. Make sure that the workstations along with furniture appear likely on the presented layout.
Focus towards your research point while dealing with office fitouts is quite good and an important point. This point can be defined as a right option while dealing with the fit out points. There are various trends and innovations, which are available nowadays due to the varied nature of interior planning. Thus, make sure to get in touch with the right research platform, just like you have wanted. These points are for your benefit. | 2019-04-21T08:38:33 | http://mcn2.com/ways-to-choose-the-best-office-fitouts-without-negativity/ |
What if I cannot find the exact device that I am looking for on MedWOW?
If you have not found what you are looking for on MedWOW, you may post a Wanted Item request, which will be available for sellers to view, post items for sale/auction that match your Wanted Item`s criteria in response.
Another option is to Request an Alert, that will notify you if an item for sale/auction matching your Wanted Item`s criteria is posted on MedWOW.
I need to purchase equipment individually or in bulk. Can MedWOW handle the process for me?
MedWOW’s Professional Purchasing Service is specifically geared at assisting buyers in finding comprehensive solutions for purchasing used medical equipment individually or in bulk. MedWOW`s highly qualified team of medical equipment experts handle the entire process of searching, finding and comparing numerous options that match the buyers` requests. MedWOW also manages the negotiations on behalf of the buyer, and formulates a purchasing package for the buyer. | 2019-04-22T00:32:56 | http://www.medwow.com/helpnew.php?itemid=127&pid=6&action=topicsL&tp=Buying |
International friendlies – Follow the Football match between Argentina and Mexico live with Eurosport. The match starts at 00:00 on 21 November 2018. Our live coverage lets you follow all the key moments as they happen.
Who will come out on top in the battle of the managers Lionel Scaloni or Gerardo Martino? Find out by following our live matchcast.
Have your say by voting on who will win between Argentina and Mexico? Enjoy some pre-match reading with related articles about these two Enjoy some pre-match reading with related articles about these two Football teams.
Head-to-head: see historical stats and visit our detailed profiles for Argentina vs Mexico. Get all the latest on Football: fixtures, results and tables. | 2019-04-23T20:05:37 | https://www.eurosport.co.uk/football/international-friendlies/2018/live-argentina-mexico_mtc1083220/live.shtml |
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Clears tables, using proper method to stack dishes, glassware and silverware. | 2019-04-21T08:21:10 | http://allindiajobbank.com/9/Hotels-Restaurant-Hospitality-jobs.html |
One-in-five voters now regularly get news and political updates on their phones or other portable electronic devices.
Voters under 30 are more likely to get routine news updates this way than those who are older. Democrats are more likely to get these regular news feeds than Republicans or voters not affiliated with either party.
Use of the Internet to find political and other news also remains generally unchanged. A plurality (44%) go online to read political news at least once a week, including 20% who seek such news online every day or nearly every day. However, 33% say they rarely or never go online to read about politics, elections and government.
The survey of 1,000 Likely Voters was conducted December 26, 2010 by Rasmussen Reports. The margin of sampling error is +/- 3 percentage points with a 95% level of confidence. Field work for all Rasmussen Reports surveys is conducted by Pulse Opinion Research, LLC. See methodology. | 2019-04-21T08:51:09 | http://www.rasmussenreports.com/public_content/politics/general_politics/december_2010/19_get_regular_news_updates_via_phone_or_other_devices |
At a Stewart City Council meeting on Monday night, councilors approved the sale of a fire truck and heard from a freelance writer working with the McLeod County Historical Partnership.
Councilors — with Council Member Kurt Glaeser absent and with the new addition of Council Member James (Jim) Eitel — were heartily surprised to receive a bid on the sale of the city’s old fire truck at $2,500, with the conditions that the truck be picked up within 30 days, weather permitting. Other bids were as high as $1,200 and as low as $500.
In what was at least his second meeting in as many weeks, Brian Leehan, a freelance writer working with the McLeod County Historical Partnership, approached the council in hopes of adding to his list of names and numbers to document the history of McLeod County. He was also at the most recent Brownton City Council meeting. | 2019-04-24T06:05:29 | http://glencoenews.com/content/stewart-city-council-accepts-bid-hears-freelance-writer |
England’s hopes in Auckland were over as soon as they were dismissed for 58 on the first day. Kane Williamson’s faultless hundred then ensured that England would have to bat exceptionally to make New Zealand bat again, a task thy fell just short of achieving despite a more concerted effort in the second innings.
The match saw a record equalling 12th match away from home that England have failed to win. The previous bar was set between 1939-1948.
New Zealland have won 3 of their last four tests at the venue and a year ago in beating Bangladesh Boult and Southee combined to take 15 wickets.
The most likely outcome here is another New Zealand win, though much as ever depends on Williamson and Taylor with the bat but at odds against 11/10 with Bet365 New Zealand are certainly a backable proposition.
March 26, 2018 - Offer valid as of date published. T&Cs apply. | 2019-04-23T04:30:26 | http://www.betpal.com/new-zealand-v-england-2nd-test-betting-preview |
State health officials are urging Texans to protect themselves from mosquito bites — but they aren't helping counties coordinate or pay for those protective measures.
More rain is coming, and so are more mosquitoes.
As parts of South and Southeast Texas enter peak season for Aedes aegypti, the species of mosquito apparently posing the greatest risk for transmitting the Zika virus, state health officials are ramping up a public awareness campaign urging people to protect themselves from insect bites.
What the state is not doing, public health experts say, is paying for protective measures — such as mosquito eradication and handing out insect repellants — that could reduce the risk of Zika transmission. By and large, making prevention happen — and finding the money for it — is left to local governments, which experts say are often ill-equipped to handle the job.
So far, state officials are declining to pay for certain Zika-preventive services through Medicaid, the federal-state insurance program for the poor and disabled. The federal government announced on Wednesday that states could use Medicaid funds to pay for mosquito repellants and family planning services to reduce pregnant women’s risk for contracting Zika, which has been linked to microcephaly, a birth defect that causes babies to have abnormally small heads and improper brain development.
While Texas health officials are stressing the importance of prevention in legislative hearings, public service announcements and on a central website, on Thursday a government spokesman said Texas has not yet decided whether it will allow Medicaid to pay for the additional preventive services, such as mosquito repellant.
Bryan Black, a spokesman for the Texas Health and Human Services Commission, said the agency was still “reviewing” the federal government's proposal.
“For several months, the agencies that make up the Texas Health and Human Services System have been collaborating to pursue every avenue to protect our citizens from this potential health crisis,” Black said in a prepared statement.
Texas has a decentralized system of public health programs that largely makes counties responsible for disease prevention services, such as mosquito control and monitoring. Experts say that arrangement means there is little uniformity in the state’s preparedness for preventing infectious diseases — and will almost certainly leave Texans in the poorest parts of the state more vulnerable to Zika.
The Aedes aegypti mosquito is prevalent in some of the state’s most impoverished regions, such as the Rio Grande Valley in South Texas.
“Where you are going to see the problems is in the high poverty areas,” said Catherine Troisi, an infectious disease epidemiologist at the University of Texas Health Science Center at Houston School of Public Health.
Local health departments in Zika-vulnerable areas say they are struggling to provide preventive services because they don't have the money — and in some cases the jurisdiction — to address problems.
Lack of funding has hamstrung Hidalgo County's ability to eradicate mosquitos, Eduardo Olivarez, chief administrative officer of Hidalgo County Health and Human Services, recently told the Senate Health and Human Services Committee. He said poor housing conditions left many of his county’s residents vulnerable to mosquito bites, and stray tires proved fertile breeding grounds for the mosquitoes that carry the virus.
“I have hundreds of thousands of tires in my county, and there’s no place to take them,” Olivarez said.
In one example, Olivarez said a local tire dumping ground had been abandoned after federal officials deemed it environmentally unsuitable, yet tires there remain full of mosquito larvae today.
“It’s halfway filled and sitting there, collecting water and bringing mosquitoes,” he said.
In Houston, health care providers say they fear a lack of preventive services, and city leaders have said they are $7 million short for mosquito control and debris cleanup after widespread flooding in recent weeks.
At the Senate hearing, lawmakers appeared supportive of efforts to wipe out Aedes aegypti and other species.
But Peter Hotez, dean of the National School of Tropical Medicine at Baylor College of Medicine, said in a recent interview it would take significant government involvement to wipe out the pests.
John Hellerstedt, commissioner of the Texas Department of State Health Services, told lawmakers this month that while the state had taken a lead on a public awareness campaign to prevent Zika transmission, most of the on-the-ground preventive work would take place at the county level — and would “vary from community to community” — because Texas is a home-rule state.
In addition to the public service announcements, Hellerstedt noted Gov. Greg Abbott had assembled a task force on infectious disease preparedness in February. | 2019-04-22T02:31:22 | https://www.texastribune.org/2016/06/03/texas-struggling-zika-prevention/ |
The AKP, the latest of several Turkish Islamist political reincarnations, rose to power in November 2002 on a wave of popular dissatisfaction with economic malaise and corruption scandals within establishment parties. Although the AKP captured barely a third of the vote, this translated into a two-thirds parliamentary majority because much of the popular vote went to parties that failed to meet the 10% electoral threshold for winning seats.
When the AKP came to power, Erdogan disavowed any intention to implement the Islamist agenda he had embraced in the past. Nevertheless, his government worked to weaken or disable all of the inherent checks that would prevent the establishment of an Islamic state in the longer run.
The Erdogan government has tried to undermine Turkey's secular educational tradition, most notably by lifting a long-standing ban on religious attire in universities. According to Egitim-Sen, a left-of-center teachers' union, Islamic influences are creeping into textbooks. Only fierce public opposition stalled more sweeping educational initiatives.
The AKP has had more success exerting influence over the lower courts. In December 2007, the government enacted a new law that requires all judicial candidates to take an oral exam administered by the AKP-controlled Ministry of Justice (codifying a practice already in place). The Union of Turkish Bar Associations organized a demonstration by thousands of lawyers, arguing that this law would allow the ministry to screen candidates based on their political and religious views. According to the US State Department's annual report on human rights practices in Turkey, the Erdogan government has "launched formal investigations against judges who had spoken critically of the government."
Wherever the AKP has managed to penetrate the judiciary, the results have been worrisome. Pro-AKP judges have placed liens against the property of political opponents, seized media outlets, and overturned earlier decisions levied against Islamists.
One of the most egregious abuses of power in the criminal justice system involved Yucel Askin, rector of Yuzuncu Yil University in Van. Askin had staunchly opposed Erdogan's efforts to reduce barriers to college admission for students educated in exclusively religious seminaries and also had enforced the ban on Islamic headscarves on campus. In 2005, police raided his house in search of illicit artifacts (Askin was a known collector of antiquities) and hauled him off to jail. However, they were forced to release him after it was discovered that he had government licenses for every artifact in his possession. Three months later, police arrested him again, this time on charges of accepting kickbacks from the university's purchase of medical equipment. Again, however, he was released when a judge determined that the university bought the medical equipment in question a year before Askin became rector. While Askin got his life back, the university's general secretary was not as lucky. Enver Arpali committed suicide after being held for months in prison without trial in the same case.
Rather than bridge the gap between Turkey's religious and secular constituents, Erdogan has widened it. Although the AKP won 47% of the popular vote in the latest parliamentary elections last year, millions of Turks took part in the waves of anti-government demonstrations that erupted the preceding May. In one recent public opinion poll, only 30% of respondents said they would vote for the AKP if elections were held today.
The timing of these explosive revelations raised suspicions, occurring just weeks before parliament was scheduled to elect a new president, amid widespread speculation that the AKP would attempt to put a dedicated Islamist in the post. While Gul (like Erdogan) has moderated his public pronouncements over time, he was once very direct. As Islamists rose in political power in the mid-1990s, Gul said, "This is the end of the republican period . . . the secular system has failed and we definitely want to change it."
Kuddusi Okkir was arrested in June 2007 on suspicion of financing the alleged Ergenekon plot and held for over a year without charge. For the first eight months he was held solitary confinement, with the authorities refusing even to allow his wife to visit. When he was diagnosed with lung cancer while in prison, officials rejected numerous petitions to enable him to receive outside medical treatment. They finally relented when he fell into a coma in early July 2008, but by then it was too late - he died four days later without ever regaining consciousness. Another detainee held without charge, Ayse Asuman Ozdemir, developed liver disease while in captivity and was also denied critical medical treatment. She finally received furlough after the death of Okkir caused an embarrassing uproar for the government, but it may also be too late to save her.
Especially troubling is that, despite being a couple thousand pages long, the indictment lacks specificity as to which suspects are charged with what crimes. Indeed, many of the charges center on incitement and interfering in government work, the type of language more common in dictatorships like Syria and Saudi Arabia than in Turkey. Selcuk, for example, is accused of "providing guidance, with his writings, to the suspects engaged in a coup effort," a charge that an Islamist newspaper has also leveled against this writer.
Another concern is the fact that Zekeriya Oz, the lead prosecutor in the case, is a virtual unknown, in his early thirties, with previous experience only as a public prosecutor in two small towns. This has raised questions as to his competence and whether he has the stature to resist political interference.
Throughout this saga, pundits close to the ruling party have repeatedly drawn equivalence between the Constitutional Court case and the Ergenekon investigation. "Circles who invited everyone to have respect for the judicial process in the [AKP] closure case raised hell the other day during the Ergenekon arrests and made accusations that Turkey has become a 'police state,'" columnist Cengiz Candar wrote, "But these same groups regarded the closure case as the judiciary's business." Ali Aslan, a columnist for the Islamist daily Zaman, expressed similar logic. The obvious subtext of such columns, many of which reference private conversations with the prime minister, is that those who defend Turkey's secular tradition have no right to demand rule of law and or complain about prosecutorial misconduct. They also indicate that the ruling party may be more interested in headlines than in actually seeing the Ergenekon prosecution through. | 2019-04-25T05:51:46 | https://www.meforum.org/1968/erdogan-ergenekon-and-the-struggle-for-turkey |
United Medical Solutions was founded in 1996 by Renae C. Cardinal, RCC, and Karen L. Clark, CPC, with the goal of providing outstanding client service and patient support while offering competitive rates. Delivering that quality of service now includes a staff of more than 30 highly trained staff, analysts, and certified coders.
In association since 1989, Renae and Karen have worked with a broad spectrum of providers, from those just starting a new practice with concerns about beginning cash flow, to large practices with established revenue cycles and annual expectations. Specialties include anesthesia, ambulatory surgical centers, interventional and diagnostic radiology, orthopaedic surgery, general surgery, and ambulance, among others.
United Medical Solutions is a unique full service partner with medical providers. They have been successful because they have elevated the billing function to encompass the whole practice, providing cost effective and quality service. Their purpose is to help clients grow by removing the complexities of the medical billing function so medical providers can focus on the practice of medicine.
They have developed a well-trained, dedicated, dependable staff with knowledge of coding intricacies and industry regulations. United Medical Solutions, LLC, has firsthand practice management experience and possesses the knowledge, experience, resources, and tenacity to get the job done. UMS is dedicated to providing services which are second to none for individual physicians and group practices. | 2019-04-23T02:32:38 | http://unitedmedicalsolution.com/company_history.php |
For more than 45 years, Jarlette Health Services has focused on elevating resident comfort and care. When you care for more than 2,000 residents across 20 retirement lodges and long-term care homes throughout Ontario, a reliable and efficient digital platform is paramount in keeping your workforce productive, connected and engaged. To enhance existing infrastructure and amplify its exceptional standard of care, Jarlette has introduced Google Chrome into its operations.
Our frontline staff has benefited tremendously from the migration to the Chromebook infrastructure. They are thrilled with the improved ergonomics, increased mobility of their cart-mounted devices, and that the user-friendly interface has streamlined the many data entry elements of their respective roles.
Going Google: How Google Cloud Transforms Any Enterprise. Register here.
Onix Achieves Google Cloud Location-Based Services Partner Specialization. Read More.
Strong Data Accessibility & Recovery Mark New Onix Alliance. Meet Actifio.
Cloud Computing 101: A Crash Course for the New Year. Get the facts.
Workplace Cloud Collaboration Simplified. Learn how.
The Onix Top 10: Hot 2018 Cloud News and Trends. Read the most popular stories.
The start of a new year is a perfect time to reassess where your cloud journey is taking you. Maybe it needs a new direction...in the form of maps, that is. There’s no better time to explore what location-based services have to offer to your organization. View the infographic. | 2019-04-19T09:21:09 | https://www.onixnet.com/newsletter-january-2019 |
The following samples a range of art production, from Ideal X’s own 2D and 3D works to public art design for other artists. Always present is the dynamics of perception related to a work’s material and spatial construction. The design work for other artists is under their authorship, not Ideal X. Highlighted here are some of the means, methods, and material details of our design contributions. Links to an artist’s work accompany their specific work.
Wave Echo, Catherine Wagner, Artist.
Hands Across…, Kota Ezawa, Artist.
Ghost Grove, Catherine Wagner, Artist.
Atmospheric Flurry, Catherine Wagner, Artist.
Bridges + Connections, Falliers with F. Domeyko.
Steel and concrete installation for exhibition, Thinking the City, Cambridge MA. | 2019-04-21T01:03:30 | https://www.idealxdesign.com/art-public-art-design/ |
This stunning abandoned power station was part of a large industrial site in Corbehem which consisted of a number of paper mills.
So I came to the site for a control room that had been a rather popular location for urban explorers. We arrive to a demolition zone with a number of buildings in the distance. The pin we had was this building so we wondered through rubble. We didn't have long and I was concerned we may not be able to find the control room in time. We easily found a way inside and I couldn't believe my eyes! I was welcomed with the second shot of this report. A stunning control panel and a big turbine. How can this be a tourbus location where lots of urban explorers go and I haven't seen this beauty before? Anyway we crack on with the little time we had and grabbed our shots.
The power plant was built in 1936 a smaller Alstom unit and a much larger Rateau Schneider, this was flanked by an impressive control panel which nearly spanned the length of the turbine hall. Who needs a stupid control room anyway. Well it turns out Kat did.
While exploring the building I discover there is even more, this isn't just a power plant, its a paper mill that is amidst demolition. Even though a lot of it had been demolished there was still a fair bit to see, which will be featured in my next report.
It turns out the popular control room is across the river. At this point I realised I had to return, there was so much more to see here. When I got home I managed to find someone who had been here before me and I had certainly missed some parts of the power plant. I returned two weeks later with Behind Closed Doors to finish up the power station and see what else was here.
Thanks for looking feel free to leave your views on this article below. | 2019-04-19T23:12:55 | http://www.darbiansphotography.com/beghin-stora-enso-centrale-one-corbehem |
The Green Party is made up of a wide range of individuals who volunteer their time to support various projects. These projects span campaigning against fracking to promoting cycling, campaigning for nuclear disarmament to lobbying for a memorial to the Peterloo Massacre. But what they all have in common is a vision for a better world.
We are immensely proud of the effort our members make within their local communities and the support they offer campaigns that have a global reach and impact. Here are just a few of the causes that our local Manchester Green Party Members are supporting now.
The Green Party has made its stance on fracking very clear: it makes no climate sense. While fracking continues, it keeps us hooked on fossil fuels that are fast running out and diverts our attention from other sustainable energy resources.
Our members have actively supported anti-fracking campaigners around the UK, going from site to site and protesting regularly. With more locations now under threat, our members are making a concerted effort to defend their local areas by raising money, helping to rally protesters and keeping morale high.
Some members of the Green Party are also members of the CND (the Campaign for Nuclear Disarmament). This group campaigns for the elimination of nuclear weapons, an end to illegal, interventionist wars and the closure of the nuclear power industry.
Again, the Green Party and its member are clear that other sustainable energy sources should be promoted well above nuclear power stations.
Remembering the Peterloo Massacre through a respectful, informative and permanent memorial has been a key issue for many Green Party Members this year. The Peterloo Memorial Campaign has worked hard to raise awareness by hosting commemoration events.
Manchester City Council have now committed to a permanent memorial which will be displayed in St Peter’s Square. Council Leader Richard Leese announced that Jeremy Deller, Turner Prize winner, has been invited to work on a Peterloo Memorial.
Amnesty International is a champion of human rights and many of our members support the campaigns of the Manchester branch with a range of fundraising and awareness events. Our members have been involved in a diverse range of activities including: concerts, comedy nights, greeting cards campaigns, information stalls, local events, and a yearly street collection.
The case work at the Manchester branch currently includes the human rights activist and blogger, Ahmed Mansoor, who has recently been sentenced to 10 years in prison and the human rights lawyer Mohamed al-Roken, both of whom are in the UAE.
Human rights is a central notion in the policy of the Green Party and it is great to see our members being so active in this particular area. | 2019-04-21T07:07:48 | https://manchestergreenparty.org.uk/campaigns.html |
As more and more Internet-connected devices find their way into our homes and businesses, it’s important to remember that they represent a security risk. The Internet of Things (IoT) is growing rapidly, and in the rush for convenience, our privacy and safety is often an afterthought. Leaving them unsecured is the digital equivalent of leaving the back door unlocked.
There are 5.5 million new things getting connected every day in 2016, as we head toward more than 20 billion by 2020, according to Gartner. That’s an awful lot of devices. They might bring all sorts of handy new features, but, whether it’s the latest cutting-edge baby monitor or a wireless doorbell camera that links to your phone, it’s also a network-connected computer and should be treated as such. Here are eight tips to help you secure those IoT devices.
The first step is to consider what functionality you need from the device. Just because your TV or fridge can connect to the internet, doesn’t mean you definitely want to hook it up. Take a good look at the features it offers and learn exactly what internet connectivity brings before you connect.
If you want to make sure you have the latest security patches and reduce the chances of a successful attack, then you need to keep your firmware fully updated. Vulnerabilities and exploits will be fixed as they emerge, so your IoT devices and your router need to be regularly updated. Automate this wherever possible or set a schedule to check for updates every three months or so. | 2019-04-23T08:46:47 | https://www.csoonline.com/article/3085607/8-tips-to-secure-those-iot-devices.html?token=%23tk.NWWNLE_nlt_networkworld_security_alert_2016-06-22 |
Wick's Tavern was founded in 2000. Wick's Tavern specializes in Retail - Tavern (drinking Places). Wick's Tavern has 3 employees and estimated revenues of $150,000.00. | 2019-04-25T12:47:05 | https://www.dandb.com/businessdirectory/wickstavern-phoenix-az-3312.html |
China is a vast country with a rich culinary culture. Over a long period of time, different styles of regional cuisine took shape due to factors such as available resources climate, geography, history, tradition and customs. The best-known and most influential among them are the cuisines of Shandong, Sichuan, Jiangsu and Guangdong. They are quite different in taste; for example, Jiangsu cuisine( Huaiyang cuisine in particular) is known for its light and delicate flavors, while Sichuan cuisine is known for its hot and spicy flavors.
Chinese people are very particular about food. The three essential factors by which Chinese cooking is judged are color, aroma and taste. The Quanjude Restaurant chain is the most popular of the traditional Peking roast duck restaurants. It uses an open oven. with fires of fruit tree wood, After roasting, the ducks become plump and claret-colored, with a shiny gloss. Slicing cooked duck Is quite demanding, as each duck should be carved into 100 to 120 pieces of almost the same size.
The real charm of Chinese food comes from its taste, which lies in proper seasoning. The original tastes of the food, the post-cooking taste, plus the taste of other ingredients and seasoning should be all integrated. In contrast to taste-oriented Chinese food, Western food is more focused on a rational diet. Whatever the color, aroma and taste of the food, its nutrition must be guaranteed with proper calories, vitamins and protein.
Tea comes from the buds of the tea bush, an evergreen perennial woody plant. China is the original tea-growing region, and was the first country to produce and drink tea.
Tea is a daily necessity for the Chinese people. Tea also has an indissoluble bond with art. Most ancient Chinese scholars enjoyed drinking tea, and pursuing spiritual purity and tranquility by tasting tea.
Modern science has proved that tea contains elements beneficial for human health. Tea can refresh the mind, relieve internal heat, assist digestion, eliminate phlegm, reduce fat, improve eyesight and relieve diarrhea.
Longjing (dragon well), one of China’s most renowned teas, is produced in the Xihu District of Hangzhou, Zhejiang Province. The Longjing leaves are long and narrow. The buds are tender and of similar sizes, with one bud plus one or two stemless leaves. They are green-yellow in color, smooth and faintly scented. Biluochun produced in Dongshan Town, Suzhou, is another kind of excellent green tea. Together with Longjing, it is on the list of China’s Ten Famous Teas. | 2019-04-25T21:46:57 | https://www.china-silkroad-travel.com/our-blog/chinese-culinary-culture.html |
Two SRS (supplemental restraint system) front airbags. The driver’s airbag is stored in the center of the steering wheel; the front passenger’s airbag is stored in the dashboard. Both are marked ‘‘SRS AIRBAG’’.
Two side airbags, one for the driver and one for a front passenger. The airbags are stored in the outer edges of the seatbacks.
Both are marked ‘‘SIDE AIRBAG’’.
Two side curtain airbags, one for each side of the vehicle. The airbags are stored in the ceiling, above the side windows. The front and rear pillars are marked ‘‘SIDE CURTAIN AIRBAG’’.
Automatic front seat belt tensioners.
Sensors that can detect a moderate to severe front impact or side impact.
Sensors that can detect whether a child is in the passenger’s side airbag path and signal the control unit to turn the airbag off.
Sensors that can detect whether the driver’s seat belt and the front passenger’s seat belt are latched or unlatched.
A driver’s seat position sensor that monitors the distance of the seat from the front airbag. If the seat is too far forward, the airbag will inflate with less force.
Weight sensors that monitor the weight on the front passenger’s seat. If the weight is about 65 lbs (29 kg) or less (the weight of an infant or small child), the passenger’s front airbag will be turned off.
A sophisticated electronic system that continually monitors and records information about the sensors, the control unit, the airbag activators, the seat belt tensioners, and driver and front passenger seat belt use when the ignition switch is in the ON (II) position.
An indicator on the instrument panel that alerts you to a possible problem with your airbag system components.
An indicator on the instrument panel that alerts you that the passenger’s side airbag has been turned off.
An indicator on the dashboard that alerts you that the passenger’s front airbag has been turned off.
Emergency backup power in case your vehicle’s electrical system is disconnected in a crash. | 2019-04-26T05:52:02 | http://www.haccord.org/airbag_system_components-312.html |
NGINX [Engine-X] is an HTTP(S) server, HTTP(S) reverse proxy and IMAP/POP3 proxy server written by Igor Sysoev. It has been running on many heavily loaded sites, including Facebook, Zappos, Groupon, LivingSocial, Hulu, TechCrunch, Dropbox, Tumblr and WordPress.
Evolution provides integrated mail, addressbook and calendaring functionality to users of freedesktop.org compliant desktop environments.
Dovecot is an open source IMAP and POP3 server for Linux/UNIX-like systems, written with security primarily in mind.
Exim is a message transfer agent (MTA) developed for Unix systems connected to the Internet. It is similar to Smail 3, but expands on Smail features.
Sendmail is a remarkably flexible program, supporting many kinds of mail transfer and delivery including the overwhelmingly popular SMTP.
Gnus is a mail- and news-reader that is incorporated into GNU Emacs. Gnus is independently developed, and is frequently merged into GNU Emacs. | 2019-04-21T20:26:28 | https://www.openhub.net/tags?names=mail |
NVR is a highly regarded practice amongst clinicians and parents.
NVR is a highly regarded practice amongst clinicians and parents as per the high number of referrals we receive and the positive feedback from parents. The highest percentage of referrals is for C&YP with a combination of neurodevelopmental, behavioural and attachment conditions who live in multi-stressed environments; definitely this is the therapeutic option when the C&YP do not wish to engage with the service. Positive outcomes include: improvement in the relationship between parents and C&YP, reduction of aggression between siblings, reduction and prevention of escalation, improvement of family relationships, reduction of risk behaviours and violence in the family; more engagement of extended family, parents and teachers as well as parents feeling more hopeful and confident of their own abilities.
Clinicians describe how the approach has had the effect of restoring the strengths, resources and abilities not only within families but also within and between clinicians and has mobilised difficult and challenging situations into productive direct actions. | 2019-04-22T22:39:41 | https://www.partnershipprojectsuk.com/testimonial/nvr-is-a-highly-regarded-practice-amongst-clinicians-and-parents/ |
This is the Hunger Heroes 5K. This race will help the Weekend Food Program here in Navarre. They will use all of the proceeds to feed our school children including Navarre HS students. The race will use the usual course on Navarre Beach. | 2019-04-25T15:02:21 | http://navarrecert.org/index.php/cert-calendar/25-hunger-heroes-5k |
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