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10x10 Biltmore
10x10 Biltmore
An outdoor room goes a long way in finishing your yard and creating a dedicated space for entertaining, relaxing and privacy. The Biltmore pergola by New England Arbors is an easy solution to personalizing your yard; from the distinctive trellis style posts that easily support climbing plants and create an attractive oasis, to the easy installation with included brackets. The Biltmore pergola is designed to be an attractive and practical addition to any yard.
Because the Biltmore is manufactured with modern techniques using premium materials, you get all the beauty and none of the maintenance. An occasional rinse with a garden hose will keep your pergola looking great for years. Even better – we'll guarantee it for a lifetime!
The Biltmore Pergola requires a drill, level, ladder, measuring tape, and two people to install, rest assured our detailed instructions give you all the information you need to install and mount your pergola.
The Biltmore pergola has a 10'x10' footprint. Inside height measures 88 1/4" to the bottom beams and outside height 95 7/8".
|Item Code: VA42081
Approx. Time Commitment: ?2 people, 6-8 hours
Posts: 19 x 19
Color: White
Number of Boxes: 5


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Dear NIAE NEC, Fellows and Members,
Members are enjoined to prepare adequately, participate actively and plan to stay throughout.
The opening ceremony shall be personally graced by the Hon Minister of Agriculture, and shall witness special Fellowship Award session for Dr Ahmed Adekunle, Dr Yomi Kasali and Engr Adebambo Ademiluyi.
The Dinner shall also incorporate another Award Session for more Fellows and likely Branches/ Regions.
The Plenary and Technical Sessions shall be fantastic and the AGM shall be concluded with election. Members should please zero their account to be eligible to participate in the election.
Engr. Prof. BA. Adewumi, fnse, fniae
NIAE National Chairman

subset: commoncrawl


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Brochure Design
Design Concepts Ready In Less Than 1 Business Day
Sancassano.com is the one-stop resource for all your brochure design needs! Here you will find the solutions for all your requirements – from the outline of concepts and messages of your brochure, to the final printing.
Brochures are the spearhead of marketing efforts: their purpose is not only to inform the customer about the products or services you offer, but also to emphasize the fact that you offer better solutions than your competitors.
Our approach to brochure design revolves around these concepts and around one key element: your own input.
The creation of such a delicate and important tool for your business must be left in the hands of professional designers! Our experience and skill ensure that the brochure we create for you will be of the same caliber of marketing materials used by large corporations, but you will pay only a fraction of the price these companies spend on their designs.
Contact Us
You need a quote? You are looking for a smart strategy? Communicate with us
The advantages of Brochure Design Service over our competition
Whether you need a mailer or a flyer, a tri-fold brochure, sales collateral, a training manual, a booklet, a folder or a catalog we have the expertise to deliver.
- Design concepts ready in less than 1 business day. Same-day rush service available.
- We guarantee 100% satisfaction and ensure positive results by providing unlimited revisions. You can request as many revisions as you like till final design approval.
- A professional multi-disciplinary staff working on your brochure: graphic designers, marketing experts, illustrators, copywriters.
- We have been serving thousands of clients all around the world for more than 10 years.
- Affordable and high-quality end products. 100% custom design, no templates.
- Custom designs – including embossing, die cuts, and unique folds. We can also create e-mailable or downloadable PDF brochures.
- Internet-based design process that is faster, more reliable and cost-effective than a traditional brochure design agency.


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Tourist places in Kolkata offer an interesting mix of sightseeing attractions, historical monuments, street shopping places and some of the country's best museums, food and drink nightlife places. Victoria Memorial Hall, as it is called is full of surprises and the list of places to visit in Kolkata is bursting at the seams.
Some of the most talked about tourist attractions in Kolkata include the Howrah Bridge, which is among the best and most visited in the country. Shopping at New Market and praying at the religious site Dakshineswar Kali Temple are among the most popular activities to experience the best the city has to offer.
Sightseeing in Kolkata would include water them parks to the celebrated Hooghly river and wildlife sanctuaries, Kolkata offers a range of options for all types of travellers, be it families, couples or solo travellers. Eclectic and vibrant, the city is both cosmopolitan and yet full of traditional flavours and nuance. There's never a shortage of places to visit in Kolkata.
Kolkata is well connected to all major cities in India by air and all modes of public transportation. The best way to get around the city is on public transportation and taxis.
The best time to visit most places in Kolkata the months between October to March, when the weather is cool and perfect to experience all the top tourist attractions in Kolkata.
Frequent search terms leading to this page:
Tourist Places in Kolkata, Tourist Attractions in Kolkata, Places to see in Kolkata, Sightseeing in Kolkata


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I don't have a quote tonight because, frankly, I'm being lazy and don't want to go downstairs to get my folder that has the quotes in it. I have 10 days until Ironman Florida and I am in taper mode. I still have workouts on the calendar, but they are shorter and the body is saying "take it easy".
I had a swim and a bike on the schedule today, but we also had haircuts, so my time got cut short. I went to do the swim and about 30 minutes in decided I was done. I came home, grabbed something to eat, made myself a cup of hot chocolate and am now relaxing in the recliner. The bike will not get done tonight. I may move it to another day. Or I may not. At this point I'm not going to gain any additional fitness. I can just maintain.
I have short workouts this weekend and will spend my "free" time packing. I will be heading out early next week. I am getting excited and can't wait to be around other triathletes doing the race. We got bib numbers assigned this week, so that made things more real. I am #2484. At least it's fairly easy to remember.
Stay tuned in the coming days for more updates. I will try to do some updating from the road and once I get to Panama City Beach. I'm sure I will have lots of thoughts going through my head.
The time I wanted to take down a nurse.
2 days ago


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Considering that there are 26 days remaining from the end of July, it will be no suspense to finally break millions of dollars.
Design,Atom equipped with a 2.45-inch TFT display, 8-core 2.0GHz processor, 4GB RAM, 64GB ROM, front 8 million + rear 16 million lenses, supports 4G dual card dual standby, and even fingerprint, NFC, IP68 waterproof, FM Radio and other functions, battery capacity 2000mAh.
Of course, Atom's shape is also very tough, a little outdoor three anti-cell phone feel, measurements 96x45x18mm, weight 108g.
According to the official introduction, after the successful completion of Atom crowdfunding, shipments will begin in October.
Pictured is Jelly
It is worth mentioning that the Unihertz team from Shanghai, former Jelly and Posh Mobile Micro XS240 are all the main small-size Android phones, which have caused a wave in the United States and Europe.
Recommended use China IT News APP
Download flyfish app to read news


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culture puncture
From March 1, 2014 to November 1, 2014
CulturePuncture: a Toolkit for cultural operators to interact with their neighbourhood and identify public spaces for cultural interventions.
The toolkit aims to connect cultural spaces and entities to their social, cultural and geographic environment through a participatory mapping process. CulturePuncture combines two strong independent definitions: Culture and Acupuncture. The analogy with acupuncture is fascinating, as this traditional Chinese understanding defines the body as a balanced system with nodes, flows and pressure points. This is an attempt to apply this concept to the effects that cultural and artistic interventions might have on a defined territory and is fundamental to understanding the framework of a participatory exploration.
The purpose of this toolkit is to give inspiration as to how to identify physical locations in a neighbourhood as potential CulturePuncture spots as strategic and "neuralgic" points on which "to press" with a cultural intervention – just like in acupuncture – to have a positive effect on the whole territory through a simple and easily manageable 2-day exploration by members of a cultural organisation & more. The CulturePuncture project was created in March 2014 and carried out by MCE Productions and Mahatat for contemporary art throughout a year within the context of the Tandem/Shaml network, a cultural managers exchange program. The tools and tips emerged from the participatory explorations of MCE Productions' Noailles neighbourhood in Marseille (France) in October 2014 and Mahatat for contemporary art's Manial/ El Rohda neighbourhood in Cairo (Egypt) in November 2014.
Check out the toolkit here: https://issuu.com/mahatat/docs/cp_web
Project Partners
MCE Productions, European Cultural Foundation, Robert Bosch Stiftung, DOEN, Mimeta, Al Mawred Al Thaqafy, Mitost, Mimeta, Anadolu Kültür, Romain Quesada


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Following the company's impressive Q2 earnings report, Bank of America has upgraded U.S. regional casino operator Boyd Gaming Corporation BYD 0.36% from Underperform to Neutral. In a new report, analyst Shaun Kelley explained why the firm now sees stability returning to Boyd's business.
Fundamental Recovery
According to Kelley, Boyd's 3.5 percent gross gaming revenue (GGR) growth in Q2, along with the company's effective cost-cutting measures, was the driving force behind its Q2 EBITDA beat. Bank of America had predicted that Q2 would be a strong quarter for U.S. regional operators Boyd, Gaming and Leisure Properties Inc GLPI 0.6%,Pinnacle Entertainment, Inc PNK 0.49% and Penn National Gaming, Inc PENN 1.61%, and so far, the results have confirmed that prediction.
In addition to the company's top-line growth, Kelley also pointed out Boyd's 6.2x net debt/EBITDA ratio, financial leverage that can magnify equity returns.
Despite the improving environment, Bank of America still sees…
Click here to continue reading
Want to learn more about how to profit off the stock market? Or maybe you just want to be able to look sophisticated in front of your coworkers when they ask you what you are reading on your Kindle, and you'd prefer to tell them "Oh, I'm just reading a book about stock market analysis," rather than the usual "Oh, I'm just looking at pics of my ex-girlfriend on Facebook." For these reasons and more, check out my book, Beating Wall Street with Common Sense. I don't have a degree in finance; I have a degree in neuroscience. You don't have to predict what stocks will do if you can predict what traders will do and be one step ahead of them. I made a 400% return in the stock market over five years using only basic principles of psychology and common sense. Beating Wall Street with Common Sense is now available on Amazon, and tradingcommonsense.com is always available on your local internet!


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Youth Engaged in Philanthropy Grants
YEP Grants
Through the Youth Engaged in Philanthropy (YEP) Grants program, the Community Foundation supports organizations and projects that improve the quality of life for youth in DeKalb County. Projects considered for funding must be dedicated to the community, directly involve youth, and promote youth empowerment and leadership.
As a youth led program of the DeKalb County Community Foundation, Youth Engaged in Philanthropy provides opportunities for youth from all across DeKalb County. Members attend monthly youth led meetings to build leadership skills, learn about nonprofits in DeKalb County, and become life-long philanthropists. Each year, the pinnacle of YEP is the grant allocation meeting in April. During this meeting, members are responsible for allocating approximately $11,000 to nonprofits with youth serving programs and projects in DeKalb County. Once recommendations have been made, the decisions are presented to the DeKalb County Community Foundation Board of Directors for final approval.
Applying for a Grant
Applications for YEP Grants are due each year on February 1. Applicants may request a maximum of $2,500 and are required to present their proposal to the YEP committee through a site visit or presentation. Nonprofits with youth initiatives in DeKalb County are encouraged to apply. Grant guidelines should be reviewed prior to applying online. For more information or to inquire about an application, please contact YEP Staff Advisor Noah Nordbrock at 815-748-5383 or email@example.com.
Past YEP Grants Awarded
- 2018 YEP Grants - $12,562 in YEP Grants Benefit Youth Programs and Organizations in DeKalb County
- 2017 YEP Grants - $11,588 in YEP Grants Benefit Youth Programs and Organizations in DeKalb County
- 2016 YEP Grants - $11,370 in YEP Grants Benefit Youth Programs and Organizations in DeKalb County
Funding for YEP Grants is made possible through the DeKalb County Youth Grantmakers Fund at the Community Foundation. As this Fund continues to grow, so does the impact for youth in DeKalb County!If you have a desire to make a positive impact on youth in our community, consider making a gift to the DeKalb County Youth Grantmakers Fund. For questions or additional information, please contact Executive Director Dan Templin at 815-748-5383 or email@example.com.


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Report Bullying
If you or someone you know is experiencing a bullying, or other discipline-related concerns at JISD, submit your concern to us with the report below. You do not need to leave your name, unless you want the principal to follow up with you. Specific dates, times, locations, and other information related to the incident is critical to resolving your concern.
Your concern will be taken seriously and will be worked on promptly – as the information is sent directly to the Campus Administrator. If your concern needs immediate attention, you are urged to tell a teacher, a counselor, school administrator or other trustworthy adult as soon as possible.
JISD believes all students should feel safe to learn and grow. It is the goal of the Jourdanton Independent School District to change the norms regarding bullying behavior and to restructure the school setting itself so that bullying is less likely to occur or be rewarded.
To report bullying, click on the appropriate campus link and provide as much information as possible:
High School Anonymous Reporting
Elementary Anonymous Reporting


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Sheriff's Message, Week of November 20th
Community Friends,
An old adage wrongfully attributed to Winston Churchill states a pessimist sees the difficulty in every opportunity; an optimist sees the opportunity in every difficulty. What he did say was "For myself I am an optimist—it does not seem to be much use being anything else…." A life filled with optimism produces less life stresses, which has shown to prevent chronic disease, and helped people live longer, happier, and more productive lives. The recent election results, regardless of the chosen candidate or ballot question has produced many pessimists on both sides; I however, have chosen to be an optimist.
In a civil case that originated in Moundhouse over a denied gun sale to a medical marijuana card holder, the 9th Circuit Court of Appeals ruled in September of this year that the marijuana card applicant was not allowed to purchase the gun from the dealer, and the dealer was right in denying the sale. This is a very liberal court; however, they have established case law in that no firearm transfers can be made to marijuana users.
The ATF is updating their background check form to include a warning that medicinal and recreational use of marijuana is illegal for gun transfers. Sometime in January 2017, a new ATF form will be used with the new warning. Our new state law mandates transfer background checks, in which firearm buyers have to answer questions, including the marijuana question. Answer truthfully and the transfer is denied; lie on the question and they could be prosecuted for a federal crime; transfer the gun without a background check conducted by a licensed FFL dealer, it is a gross misdemeanor under the new state law.
We are working with our District Attorney's Office in the development of a county ordinance for vehicle marijuana prohibitions, similar to the open alcohol container laws. If approved by our commissioners, the ordinance can be enacted long before our state legislature can pass a law to help improve road safety.
The provision in the new law prohibiting marijuana cultivation within a 25 mile radius around retail marijuana establishments is causing concern in group of marijuana growers. In fact, they are already talking about passing another petition to eliminate the 25 mile rule. We believe county zoned retail marijuana establishments can stabilize crime rates by minimizing home invasions and burglaries associated with home grows and stored cash from illegal sales through regulated and controlled cultivation facilities and retail establishments.
There has been some citizens asking us to retire our K-9, Deputy Bo because of the new marijuana law. Deputy Bo is trained for the detection of marijuana, and we still need him in our schools detecting marijuana products to help deter the drug from coming on campuses. Marijuana is still illegal for person under 21 to possess, he can assist us in other criminal investigations, and he is more than just a drug do; he is a dog that can apprehend criminals and help protect our deputies on the street. We will not be retiring him.
And finally, one of our local manufacturing companies, Trex, recently donated $5,000 to the Lyon Sheriff Advisory Council (LSAC) in support of our canine program. We wanted to thank them for their support in helping to make our community stronger and safer.
As always, keep the faith.
Archive for the 'rifle ranges in Nevada' Category
I word from the Lyon County sheriff in Nevada on the new gun background checks and recreational marijuana. Be aware!November 27, 2016
Sheriff's Message, Week of November 20th


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Microsoft has outlined the future of their patch releases for Windows 10 and Office 365 in a blog post on their website.
Here are more details.
- Windows is committing to a predictable twice-per-year feature release schedule, targeting September and March of each year, aligning with Office 365 ProPlus. The next Windows 10 feature update will be targeted for September 2017.
- Each Windows 10 feature release will be serviced and supported for 18 months. This is consistent with our current Windows 10 approach, but adds further clarity and predictability to organizations by aligning with Office 365 ProPlus.
- In addition, System Center Configuration Manager will support this new aligned update model for Office 365 ProPlus and Windows 10, making both easier to deploy and keep up to date.
Basically, we can now expect to see the major feature releases either in March or September from now on in both Windows and Office. Of more note it that Microsoft will only be conducting extended support for these updates for 18-months or about 3 release versions. This should help out on the deployment of updates in the enterprise environment, however, I would be concerned about the 18-month service & support window for releases. It will be nice to know that I should be looking for a major feature set update on about a 6 month cycle now.
Bleeping Computer Article: https://www.bleepingcomputer.com/news/microsoft/microsoft-sets-new-release-schedule-for-windows-10-with-two-major-updates-per-year/


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|From: J. Ian Johnson (ianj at ccs.neu.edu)
Date: Wed Oct 31 08:26:45 EDT 2012
I have a 64-bit machine and OS with 16GB RAM. I have some benchmarks that I want to time out after an hour or die if they use 4GB memory. Unfortunately, the entire VM dies with OOM instead of carrying on. I'm running from the terminal. Is there some other option I need to pass to not limit the VM's overall memory? Xubuntu has as default that processes can use as much memory as they want, last time I checked. Thanks, -Ian
|Posted on the users mailing list.


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The receptor‐type protein tyrosine phosphatases (RPTPs) have been linked to signal transduction, cell adhesion, and neurite extension. PTPRT/RPTPρ is exclusively expressed in the central nervous system and regulates synapse formation by interacting with cell adhesion molecules and Fyn protein tyrosine kinase. Overexpression of PTPRT in cultured neurons increased the number of excitatory and inhibitory synapses by recruiting neuroligins that interact with PTPRT through their ecto‐domains. In contrast, knockdown of PTPRT inhibited synapse formation and withered dendrites. Incubation of cultured neurons with recombinant proteins containing the extracellular region of PTPRT reduced the number of synapses by inhibiting the interaction between ecto‐domains. Synapse formation by PTPRT was inhibited by phosphorylation of tyrosine 912 within the membrane–proximal catalytic domain of PTPRT by Fyn. This tyrosine phosphorylation reduced phosphatase activity of PTPRT and reinforced homophilic interactions of PTPRT, thereby preventing the heterophilic interaction between PTPRT and neuroligins. These results suggest that brain‐specific PTPRT regulates synapse formation through interaction with cell adhesion molecules, and this function and the phosphatase activity are attenuated through tyrosine phosphorylation by the synaptic tyrosine kinase Fyn.
The functions of PTPRT/RPTPρ, a protein tyrosine phosphatase (PTP) expressed only in the brain, have not been defined, although several potential substrates have been suggested (Besco et al, 2006; Zhang et al, 2007). PTPRT is a classical PTP that has a core cysteine residue in the active site and is localized in the plasma membrane by its trans‐membrane domain (McAndrew et al, 1998; Paul and Lombroso, 2003; Besco et al, 2004; Sallee et al, 2006). PTPRT is classified in the same subfamily as PTPRK, PTPRM, and PTPRU (PCP‐2), all of which have a common MAM domain in the extracellular N‐terminus followed by one Ig and several FN domains. PTPRT has two intracellular catalytic domains; the membrane–proximal catalytic domain is active, whereas its membrane distal catalytic domain is inactive similar to many other PTPs (Alonso et al, 2004). Several PTPRT gene mutations have been found in colon cancer, and transformation could be inhibited by overexpression of wild‐type (WT) PTPRT (Wang et al, 2004). However, based on in situ hybridization data, PTPRT has a brain‐specific expression pattern in contrast with PTPRK, PTPRM, and PTPRU (Paul and Lombroso, 2003; Besco et al, 2004).
Several PTPs are reported to control axonal outgrowth, guidance, and synapse formation in the central nervous system (CNS), but many PTP‐knockout mice have not shown significant phenotypes because PTP has many subfamilies in the brain that can compensate for the knockdown activity (Burden‐Gulley and Brady‐Kalnay, 1999; Harroch et al, 2000; Xie et al, 2001; Johnson and Van Vactor, 2003; Petrone et al, 2003; Dunah et al, 2005; Uetani et al, 2006). It has been questioned whether PTP has substrate specificity because structural analyses have not shown large differences among the catalytic domains of PTPs (Hoffmann et al, 1997; Nam et al, 1999, 2005; Johnson and Van Vactor, 2003; van Montfort et al, 2003). PTP is thought to have limited access to the phospho‐tyrosine residues of substrates, and fine regulation of its activity may be accomplished by interaction with partners. PTP activity can be regulated by alternative splicing, modulation of protein levels, posttranslational modifications, dimerization, and subcellular localization (Paul and Lombroso, 2003; van Montfort et al, 2003). Especially, dimerization and active‐site blockage was proposed for downregulating the catalytic activity of receptor‐type PTP (RPTPα) and other RPTPs (Bilwes et al, 1996). The wedge formed by amino‐terminal helix‐turn‐helix segment of RPTPα D1 inserts into the active site of a dyad‐related D1 monomer, which would block the access of the substrates. Some PTPs communicate with protein tyrosine kinase (PTK) directly or indirectly, but it is rare for PTP activity to be controlled by PTK; PTK is activated by PTP in many cases (Zeng et al, 1999; Tsujikawa et al, 2002; Berman‐Golan and Elson, 2007). Despite the likelihood that tyrosine phosphorylation occurs in PTP, auto‐dephosphorylation would make it difficult to detect.
Homophilic or heterophilic interactions among the ecto‐domains of cell adhesion molecules are well characterized for both cis‐ and trans‐interactions (Boggon et al, 2002; Dean et al, 2003; Boucard et al, 2005; Kim et al, 2006). Trans‐synpatic interaction between neuroligin and neurexin is known to regulate synapse formation and synaptic functions at excitatory and inhibitory synapses (Graf et al, 2004; Chih et al, 2005; Chubykin et al, 2007; Sudhof, 2008). However, signal transduction mechanisms that regulate the functions of neuroligins and neurexins have not been identified (Dean and Dresbach, 2006; Craig and Kang, 2007). Some RPTPs have ecto‐domains similar to those of cell adhesion molecules, and likely interact with other RPTPs or cell adhesion molecules through these ecto‐domains. The extracellular ecto‐domains of RPTP are highly divergent and consist of a wide variety of different structural motifs, whereas the cytoplasmic catalytic domains are conserved. However, heterophilic interactions through the ecto‐domain of RPTP have not been reported except in PTPσ, PTPζ/β, and LAR (Brady‐Kalnay et al, 1995; Gebbink et al, 1995; Krasnoperov et al, 2002; Johnson and Van Vactor, 2003; Anders et al, 2006; Woo et al, 2009). The ecto‐domains of PTPRK and PTPRM failed to interact in a heterophilic manner despite their structural similarity (Zondag et al, 1995). PTPRM is known to interact with E‐cadherin through intracellular domains, not ecto‐domains (Brady‐Kalnay et al, 1995). When RPTPs interact with cell adhesion molecules through ecto‐domains, many cytoplasmic substrates could be made accessible to RPTPs for further signal transduction.
In this study, the functions of PTPRT in the brain were investigated. When overexpressed, PTPRT increased the density of dendritic spines and excitatory and inhibitory synapses in cultured neurons. Knockdown of PTPRT resulted in a decrease in the number of dendritic spines and excitatory synaptic transmission. The soluble PTPRT ecto‐Fc protein reduced the number of dendritic spines and excitatory synapses by inhibiting the interaction between ecto‐domains. Endogenous interaction partners of PTPRT were examined in the rat brain synaptosome by immunoprecipitation using a PTPRT‐specific monoclonal antibody and tandem mass spectrometry for amino acid sequencing. As a result, neuroligin was identified as a candidate interaction partner of PTPRT. Both neuroligin and neurexin interacted with PTPRT through the extracellular ecto‐domains. Neuroligin was recruited by overexpressed PTPRT and synapse formation by neuroligin was not induced when PTPRT was knocked down in cultured neurons. A specific tyrosine residue (Y912) in the wedge formed by helix‐turn‐helix segment of PTPRT catalytic domain was found to be phosphorylated by Fyn. The clashes of phosphorylated tyrosine with the surrounding residues would help the wedge into the catalytic pocket of other monomer and strengthen the dimeric interaction. An in vitro PTP assay showed that the catalytic domain of a PTPRT phosphorylation‐mimic mutant (Y912E) had a severely reduced activity. Fyn interfered with synapse formation by increasing homophilic interactions of PTPRT and by inhibiting interaction between PTPRT and neuroligin. PTPRT‐induced synapse formation was attenuated by co‐expression with Fyn and the augmentation of synapses did not occur when neurons were transfected with PTPRT mutant mimicking phosphorylation. Thus, brain‐specific PTPRT/RPTPρ regulates synapse formation by gaining access to synaptic substrates connected with cell adhesion molecules, and its activity seems to be regulated through tyrosine phosphorylation by Fyn PTK.
PTPRT is localized in the brain and neuronal synapses
According to in situ hybridization data, PTPRT is expressed only in the CNS in contrast to PTPRM, PTPRK, and PTPRU (PCP‐2), which are ubiquitously expressed in many organs. A PTPRT‐specific monoclonal antibody was produced against the catalytic domain, and was shown to recognize only recombinant PTPRT but not PTPRM, PTPRK, or other PTPs (Supplementary Figures S1A and B). When overexpressed in heterologous cells, only PTPRT reacted with the monoclonal antibody, but PTPRM, PTPRTK, or PTPRU did not (Figure 1A) (Jiang et al, 1993). In mouse samples, PTPRT proteins appeared as two major bands, similar to other members of this protein subfamily (Brady‐Kalnay et al, 1995): a full‐length 210 kDa form and a cleaved 110 kDa form. These forms were detected only in the brain, similar to the brain‐specific scaffolding protein post‐synaptic density‐95 (PSD‐95) (Figure 1B). To understand the nature of short form, a predicted cleavage site for furin in PTPRT was changed (Mut‐PTPRT; K635N, R637L) (Figure 1C). The short form (indicated by an arrowhead) disappeared by the mutation of the furin‐cleavage site (KSRR → NSLR). In the early development stages of the rat brain, another 90 kDa cleavage fragment or possible alternative splicing form of PTPRT was evident, but the expression level of full‐length (210 kDa) and 110 kDa proteins gradually increased up to the 3 weeks of age, whereas the expression of the 90 kDa fragment became undetectable from 3 weeks (Figure 1D) (Besco et al, 2004). In subcellular fractions of the brain, the majority of PTPRT proteins were localized in the synaptosomal (P2), crude microsome (P3), synaptic plasma membrane (LP1), and synaptic vesicle (LP2) fractions (Figure 1E). In PSD fractions, PTPRT was retained in the PSD III, although it was not enriched to the same extent as PSD‐95.
In brain slices, PTPRT monoclonal antibodies revealed PTPRT expression in specific brain regions (F2), whereas signals could not be detectable in the kidney (a negative control tissue) (F4) (Figure 1F). PTPRT signals were eliminated when PTPRT monoclonal antibodies were pre‐incubated with cognate antigen proteins (G4) (Figure 1G). In cultured hippocampal neurons, PTPRT was present in both neurons and astrocytes, marked by staining for MAP2 and GFAP, respectively (Figure 1H and I). PTPRT was localized to dendritic spines, marked by PSD‐95 (Figure 1J) and soluble RFP (Supplementary Figure S1D) (arrowhead). PTPRT co‐stained with the representative synaptic markers synaptophysin and PSD‐95 and appeared to be localized to neuronal synapses (Figure 1K and L, arrowhead). When EGFP‐tagged PTPRT was expressed in cultured neurons, PTPRT was shown to co‐localize with PSD‐95 and vGLUT, which are markers for excitatory synapses (Figure 1M, arrowhead). PTPRT was also localized to inhibitory synapses stained with gephyrin and vGAT (Figure 1N, arrowhead).
PTPRT induces synapse formation in hippocampal‐cultured neurons
We next tested whether overexpression of PTPRT has any effects on neuronal synapses (Figure 2). The density of dendritic spines resulting from the overexpression of WT PTPRT (WT‐RT) significantly increased compared with control neurons (Figure 2A; Supplementary Figure S2A). In the case of the activity‐dead mutant (2cs‐RT), the spine density was less than that of WT‐RT, but was not reduced to the control level, suggesting that some interaction partner recruitment occurred through the ecto‐domain or the endogenous substrate was trapped to 2cs‐RT (Barford et al, 1995). In some cases WT and the activity‐dead mutant PTP show similar phenotypes because both of them could block signal transductions by dephosphorylating (WT) or trapping (the activity‐dead mutant) substrates. However, the spine length of dendrites in these mutants was greater, indicating immature synapse formation because of the elimination of PTP activity (below, Supplementary Figure S2B). The distributions of the representative synaptic markers were examined to obtain more evidence to support the role of PTPRT in synapse formation (Figure 2B–D). The density of PSD‐95 increased with transfection of WT‐RT but not with transfection of 2cs‐RT compared with the control (Figure 2B). Many areas in which increased spines were induced by 2cs‐RT expression did not appear to be real synapses; therefore, PTP activity is necessary for mature synapse formation (Supplementary Figure S2B). The density of vGLUT was increased by WT‐RT and considerably increased by 2cs‐RT (Figure 2C). The density of vGAT was increased by WT‐RT and not by 2cs‐RT (Figure 2D). To verify the excitatory synapse formation vGLUT‐positive PSD‐95 clusters were examined (Figure 2E). When PTPRT was overexpressed, the excitatory synapses were increased. When vGAT‐positive gephyrin clusters were examined, the inhibitory synapses were also increased by overexpression of PTPRT (Figure 2F).
Synapse formation is inhibited by knockdown of PTPRT
To inhibit PTPRT expression in cultured neurons, specific short‐interfering RNA (siRNA) (RTsiRNA#1) was produced in the pSuper.gfp/neo vector and the effects of PTPRT knockdown on synapse formation were examined (Supplementary Figure S2C). Spine density was dramatically decreased by PTPRT knockdown and somewhat rescued by the overexpression of PTPRT (Resc RT) (Figure 3A). However, the rescued spine morphology did not appear as mature as the control, which suggests that the effects of PTPRT knockdown by siRNA are irreversible. Knockdown of PTPRT caused PSD‐95 puncta to significantly decrease (Figure 3B), and density of vGLUT and vGAT was attenuated (Figure 3C and D). To verify the effect of PTPRT knockdown new siRNA (RTsiRNA#2) was produced and synapse formation was examined (Supplementary Figure S2D). Spine density was decreased by PTPRT knockdown and rescued by the overexpression of PTPRT (Figure 3E). Contrary to former siRNA the rescued spine morphology was as mature as the control. The frequency of mEPSC was reduced dramatically, but not the amplitude by PTPRT knockdown in cultured neurons (Figure 3F). These results confirm that PTPRT regulates synapse formation in hippocampal neurons.
PTPRT interacts with neuroligin and neurexin through its extracellular ecto‐domain
Rat brain synaptosomal fractions extracted with sodium deoxycholate were immunoprecipitated with anti‐PTPRT monoclonal antibody to determine the identity of endogenous PTPRT interaction partners. Co‐precipitants were resolved by SDS–PAGE, then the proteins in ∼50 gel pieces were digested with trypsin and identified by tandem mass spectrometry (Figure 4A) (Husi et al, 2000). Among the candidates of interaction partners, neuroligin 3 was identified by two peptides (Figure 4B). The amino acid sequences of these peptides were conserved in all neuroligins (NLG1,2,&3) except for one amino acid. PTPRT associates with neuroligin and neurexin in the brain synaptosome (Figure 4C). When immunoprecipitation was performed using an anti‐PTPRT monoclonal antibody, neuroligin and neurexin co‐precipitated with PTPRT. As neuroligin and neurexin are receptors of each other in the synapse, the co‐precipitated neurexin was considered a reasonable result. Conversely, PTPRT and neuroligin were co‐precipitated using anti‐neurexin‐specific antibody (Figure 4C; Supplementary Figure S1C). The interactions between PTPRT and neuroligin or neurexin were investigated through co‐immunoprecipitation in heterologous cells (Supplementary Figures S3 and S4). PTPRT seems to associate with members of the neuroligin and neurexin families with varying intensity.
To define the domains of PTPRT that interact with neuroligin and neurexin, several PTPRT truncation mutants were tested in heterologous cells (Figure 4D; Supplementary Figure S5). Only full‐length PTPRT co‐precipitated with neuroligin and neurexin, whereas truncated PTPRT without the extracellular ecto‐domain did not (Figure 4E). Only ecto‐domain of PTPRT without intracellular domains (ΔJDD&ΔDD) was sufficient for co‐immunoprecipitation with neuroligin and neurexin even though neurexin seems to interact with PTPRT in somewhat different ways from neuroligin (Figure 4F; Supplementary Figure S6). PTPRT seemed to interact with neuroligin and neurexin through extracellular ecto‐domains. In cultured neurons, PTPRT and neuroligin were co‐localized as much as neuroligin and PSD‐95 (Figure 4G, arrowhead). On the basis of these results, PTPRT seems to associate with neuroligin and neurexin in the pre‐ and post synapse.
PTPRT interacts with neuroligin and neurexin functionally
The expression level of neuroligin was examined in cultured neurons when PTPRT was overexpressed. The numbers of neuroligin1 and neuroligin2 puncta was increased by overexpression of WT PTPRT, which suggests that PTPRT recruited synaptic neuroligin (Figure 5A and B). To analyse whether the synapse‐inducing activity of neuroligin is regulated by PTPRT, the density of dendritic spine was examined when neuroligin was overexpressed in the same neurons that the expression of PTPRT was inhibited by siRNA (Figure 5C). The synaptic formation induced by neuroligin seemed to be attenuated by PTPRT knockdown whereas neuroligin functioned normally in cultured neurons that still expressed PTPRT as reported earlier (Chih et al, 2005). Soluble PTPRT ecto‐Fc was produced in heterologous cells, and purified PTPRT ecto‐Fc was incubated with the hippocampal primary cultured neurons. After several days, the density of dendritic spines and the synapsin puncta was significantly decreased compared with control cells (Figure 5D).
To define the main cause of inhibition, a pull‐down assay using purified PTPRT ecto‐Fc 'pre‐bound' on protein A‐beads was performed (Figure 5E). It was hypothesized that the trans‐interactions could be shown mainly in this scheme because the streric hindrances from pre‐bound protein A‐bead inhibit the cis‐interaction. Interestingly, the interactions between the ecto‐domains of PTPRT and neuroligins/neurexins were not as strong as the PTPRT homophilic interactions. Next purified PTPRT ecto‐Fc was mixed with neuroligin before addition of protein A‐beads and significant neuroligins were pulled down (Figure 5F). These results suggest that the interactions between PTPRT and the cell adhesion molecules could be a cis‐interaction rather than a trans‐interaction. To confirm cis‐interaction between PTPRT and cell adhesion molecules, PTPRT ecto‐Fc was co‐expressed with cell adhesion molecules in heterologous cells and then a pull‐down assay was performed in the cell lysate (Figure 5G). This time, neuroligin and neurexin were pulled down by PTPRT ecto‐Fc proteins suggesting cis‐interaction between PTPRT and cell adhesion molecules.
Tyrosine phosphorylation of PTPRT by Fyn attenuates synapse formation
Phospho‐tyrosine levels are regulated through PTP–PTK interactions in the cell, and PTP could be a substrate for PTK, and hence the functions of PTP could potentially be regulated by tyrosine phosphorylation. The phospho‐tyrosine level of PTPRT was examined in cultured neurons treated with pervanadate, a potent phospho‐tyrosine phosphatase inhibitor, and PTPRT was shown to be phosphorylated at tyrosine residues (Figure 6A). Of several possible PTK candidates responsible for tyrosine phosphorylation of PTPRT, Fyn PTK was examined because Src‐related PTKs were discovered as interactive factor from IP‐proteomics (data not shown). When the constitutively active form of Fyn PTK (caFyn) (Liu et al, 2006) was co‐expressed in heterologous cells with PTPRT, Fyn was recruited to PTPRT and tyrosine residues of PTPRT were phosphorylated (Figure 6B). On the other hand, PTPRT was not phosphorylated when co‐transfected with kinase‐dead mutant of Fyn (kdFyn). WT‐RT seems to be auto‐dephosphorylated, and the phospho‐tyrosine level of WT‐RT was lower than that of the activity‐dead mutants (csRT and 2csRT). Then two tyrosine residues (Y912, Y1027) in the first catalytic domain of PTPRT, predicted to be candidates for phosphorylation by Fyn PTK, were changed to phenylalanine for phospho‐tyrosine immunoblotting (Figure 6C). As the phospho‐tyrosine residues of WT‐RT underwent auto‐dephosphorylation, YF mutants were constructed in the activity‐dead PTPRT context (2csRT). The level of tyrosine phosphorylation was significantly decreased in the Y912F mutant compared with single 2cs and the Y1027F mutant although not decreased to the 2YF mutant, therefore the 912 tyrosine residue seems to be the major target of Fyn PTK.
To determine the effects of Fyn PTK on the synapse formation activity of PTPRT, caFyn PTK was co‐expressed with PTPRT in cultured neurons (Figure 6D). PTPRT‐mediated synapse formation was attenuated by caFyn PTK, which suggests that Fyn PTK regulates the function of PTPRT through tyrosine phosphorylation during synapse formation, but expression of Fyn alone had no effect on synapse formation in this system (Supplementary Figure S7A). To confirm the 912 tyrosine residue is the major target of Fyn; the density of dendritic spine was examined when YF mutants were co‐expressed with Fyn (Supplementary Figure S7B). Y912F mutant still increased the number of dendritic spine, but WT and Y1027F mutant did not when Fyn was co‐expressed. The Fyn‐targeted tyrosine residue was changed to a glutamate residue to mimic a phosphate group, and then the effect of this mutation on synapse formation was examined by overexpressing Y912E‐RT in cultured neurons (Figure 6E). As expected, synapse formation by overexpression of PTPRT was attenuated when tyrosine 912, but not tyrosine 1027, was mutated to glutamate (Supplementary Figure S7C). When tyrosine residues were changed to phenylalanine to block phosphorylation, synapse formation was induced as much as in WT (Supplementary Figure S7C). From these results, we suggest that tyrosine residue 912 is a major target of Fyn and synapse formation can be regulated by phosphorylation of this residue.
PTPRT activity and the PTPRT–neuroligin interaction are regulated by Fyn
To elucidate the regulatory effect of tyrosine phosphorylation on PTPRT‐induced synapse formation, an in vitro PTP assay was first carried out using recombinant proteins. PTP activity was remarkably reduced when the Y912E mutation was introduced, but not when the Y1027E mutation was introduced (Figure 7A). Therefore, Fyn seems to regulate PTPRT activity by phosphorylating specific tyrosine residue in PTPRT's catalytic domain.
The effects of tyrosine phosphorylation on the homophilic interactions of PTPRT were then examined, because cis‐homophilic interactions of PTPRT were suggested in the PTPRT ecto‐Fc experiment (Figure 5F and G) and it is possible that an inactive cis‐dimer of PTPRT may exist, as seen for RPTPα (Blanchetot et al, 2002). Although considerable PTPRT seemed to compose dimer already, Fyn was shown to reinforce the homophilic interaction of PTPRT in experiments using differentially tagged PTPRT (Figure 7B). The effects of tyrosine phosphoryaltion on homophilic interaction of PTPRT were examined in cultured neurons (Figure 7C). The clustering of PTPRT could be reflected by puncta of PTPRT in cultured neurons even though the possible difference between molecular and cellular phenomena cannot be ruled out. The area and intensity of PTPRT‐EGFP puncta were increased by overexpression of Fyn whereas the number of PTPRT‐EGFP puncta was decreased significantly. To know effects of PTPRT clustering on synaptic localization, co‐localizations of PTPRT and neuroligin were evaluated (Figure 7D). When Fyn was co‐expressed, less neuroligin puncta were found to be with PTPRT and it seems to be related with attenuated synapse formation.
Homophilic multimerization—possibly cis‐interaction seems to decrease PTPRT activity and as a result synapse formations could become attenuated by uncontrollable phosphorylation of synaptic substrates (Supplementary Figure S9). Dimerization of PTPRT could be enhanced by PTPRT ecto‐Fc in reduced synapse formation because Fc‐fusion proteins compose dimer (Figure 5D). Inactivated PTPRT clusters might deform neuronal synapses and appear to be far from neuroligin. Thus, Fyn PTK induces multimerization and inactivation of PTPRT by phosphorylating tyrosine residue 912 in the catalytic domain resulting in the attenuation of synapse formation.
PTPRT regulates synapse formation by gaining access to synaptic substrates connected with cell adhesion molecules, and its activity seems to be regulated through tyrosine phosphorylation by Fyn PTK. Inactivated PTPRT could not control synaptic substrates nor induce synapse formation.
The modelling of PTPRT catalytic domain
The 912 tyrosine residue seems to be the major target of Fyn and the homophilic interaction of PTPRT was induced by Fyn (Figures 6C and 7B). On the basis of these results and a model for dimerization of RPTPα, one hypothesis in the structural context of PTPRT was developed (Bilwes et al, 1996). In the model of RPTPα, a wedge formed by helix(α1′)‐turn‐helix(α2′) segment at the N‐terminus of one monomer occludes the catalytic face of the other monomer within dimer, which would completely block the access of the substrates (Supplementary Figure S8). Tyrosine 912 of PTPRT belongs to helix α2′ of wedge and is situated in the interface between wedge and the body of the catalytic domain. In the crystal structure of PTPRK/RPTPκ (pdb code: 2c7s), tyrosine 892, equivalent to tyrosine 912 in PTPRT, participates in extensive hydrogen‐bonding interaction (Figure 7E) (Eswaran et al, 2006). The phosphorylation of 892 would result in the clashes with the surrounding residues and the detachment of the wedge from the body of the catalytic domain. The resulting flexible wedge may enter the catalytic pocket of other monomer deeply, strengthen the dimeric interaction, and eventually inhibit catalytic activity.
Thus, Fyn PTK might induce cis‐multimerization and inactivation of PTPRT by phosphorylating tyrosine residue 912 in the wedge of the catalytic domain resulting in the attenuation of synapse formation.
The regulation of synapse formation by PTPRT and Fyn PTK
There have been many reports that PTK activity is regulated through dephosphorylation by PTP. CD45 PTP is thought to dephosphorylate the non‐receptor Lck PTK, thereby enhancing PTK signal transduction activity (Mustelin and Altman, 1990). RPTPα regulates Fyn activity through interaction with NCAM and the elongation of neurites (Bodrikov et al, 2005). RPTP LAR was also suggested to regulate Lck and Fyn PTK activity, although no linker between LAR and PTK has been discovered to date (Tsujikawa et al, 2002). PTPRO functions in negative regulation of the Eph receptor tyrosine kinase in retinal axon growth (Shintani et al, 2006). Conversely, examples of PTK controlling the activity of PTP through tyrosine phosphorylation have been suggested in several cases such as PTP D1 and RPTPα (Vogel et al, 1993; Zheng et al, 2000).
Here, PTPRT seems to be regulated by Fyn PTK through tyrosine phosphorylation. The catalytic activity of PTPRT was decreased to basal levels through phosphorylation of a specific tyrosine (Y912) by Fyn. PTPRT seems to be connected to its substrates through neuroligin and neurexin, well‐known synaptic molecules inducing synapse formation. When PTPRT activity is attenuated by phosphorylation by Fyn, the functions of synaptic molecules may be impaired by hyper‐phosphorylation. In our system, overexpression of Fyn alone did not induce a significant increase in synapse formation (Supplementary Figure S7A). Although hyper‐phosphorylation of synaptic molecules by Fyn itself could not be completely ruled out, inactivation of PTPRT through tyrosine phosphorylation by Fyn could account for the attenuation of synapse formation by Fyn.
As PTPRT seems to be auto‐dephosphorylated, the level of phosphorylation in the YF mutant was not significantly different from that of non‐YF mutant in a WT background (WT‐RT, data not shown). To visualize contrasts of phosphorylations, we constructed a YF mutant in the activity‐dead mutant (2csRT, Figure 6C). It is possible that Fyn PTK could be a substrate of PTPRT, because the phospho‐tyrosine level of WT Fyn was somewhat decreased by WT‐PTPRT (data not shown). PTP and PTK could therefore be substrates of one another, and their activities could be fine regulated by phospho‐tyrosine levels in the neurons. Neuroligin and neurexin do not seem to be substrates of Fyn or PTPRT, because no phospho‐tyrosine forms of these proteins were detected in neuronal samples. However, it is possible that a rapid reaction results in no detectable neuroligin or neurexin phospho‐tyrosine residues.
Roles of the interaction between PTPRT and cell adhesion molecules
The ecto‐domains of full‐length proteins seem to facilitate trans‐homophilic interactions although PTPRT, PTPRM, and PTPRK are partially processed. PTPRM has been demonstrated to interact with E‐cadherin through its intracellular domain (Brady‐Kalnay et al, 1995). PTPRT has also been reported to interact with cadherins and catenins through intracellular domains (Besco et al, 2006). However, STAT3, a substrate candidate of PTPRT, was found by using only the extracellular portion of PTPRT expressed in HEK293T cells (Zhang et al, 2007). It is therefore necessary to examine the function of RPTPs through ecto‐domain interactions. In this study, neuroligin was identified as a protein that interacts with PTPRT through immunoprecipitation and mass spectrometry. Experiments using various truncations showed that the ecto‐domain of PTPRT is responsible for its interaction with neuroligin. PTPRT also showed trans‐homophilic interactions, similar to PTPRM and PTPRK, in experiments using ecto‐Fc recombinant proteins (Figure 5E) (Aricescu et al, 2007). This raises the question as to how PTPRT interacts with cell adhesion molecules. Cell adhesion molecules are generally considered to act by clustering and are thought not to function properly as single molecules. However, X‐ray scattering and neutron contrast variation data revealed that two neurexin monomers bind at symmetric locations on opposite sides of the long axis of the neuroligin dimer (Arac et al, 2007; Comoletti et al, 2007). PTPRT could interact with neuroligin and neurexin in a cis‐manner and consolidate the interaction between neuroligin and neurexin by adding its homophilic trans‐interaction. If PTPRT is a component of the cell adhesion molecule cluster, the homophilic interaction of PTPRT could have a key function in cell adhesion molecule cluster function. The number of neuroligin puncta was increased by overexpression of PTPRT and synapse formation induced by neuroligin was attenuated by knockdown of PTPRT (Figure 5A–C). These results suggested PTPRT and neuroligin are functionally connected with each other for synapse formation in the neuronal synapses. Interestingly, PTPRT seems to make cis‐homophilic interactions in addition to trans‐homophilic interactions, and these cis‐homophilic interactions seem to be reinforced by Fyn (Figures 5G and 7B). Sequentially, decreased PTP activity could result in failure in dephosphorylation of various neuronal substrates and could not consolidate neuronal synapses, and cis‐multimerized PTPRT seems to collapse neuronal synapses and be dissociated from neuroligin (Figure 7D). The cis‐interaction between PTPRT and neuroligin or neurexin could make substrates linked to cell adhesion molecules accessible to PTPRT for consolidation of synapse formation. When PTPRT was knocked down in cultured neurons, dephosphorylation might be not regulated, resulting in severe synapse depletion.
Neuronal disease related to PTPRT
Mutations in two X‐linked genes encoding neuroligins 3 and 4 were found in the brains of siblings with autism‐spectrum disorders (Jamain et al, 2003). As synapse formation is regulated by the interaction of PTPRT with neuroligin, and because many PTPRT mutations have been reported in colon cancer (Wang et al, 2004), defining the relationship between neuronal diseases and the PTPRT gene would be of significant interest. PTPRT and Fyn kinase could be a possible drug targets for neuronal disease treatment.
Materials and methods
DNA constructs
Full‐length human PTPRT (NM_133170, aa 1–1460; from a human brain cDNA library; Clontech) was subcloned into GW1‐CMV. For FLAG tagging the N‐terminus, a 3xFLAG epitope (MDYKDHDGDYKDHDIDYKDDDDK; p3xFLAG‐CMV‐7.1, Sigma) was inserted between amino acid residues 26 and 27 of PTPRT. For the construction of Fc‐fusion proteins, the ecto‐domain of PTPRT was fused with human Fc domain at the C‐terminus. For siRNA knockdown, nt 3188–3206 and nt 2685–2703 of rat PTPRT (CGGAACCATGACAAGAACC, RTsiRNA#1; GAAAGGCTACCATGAGATC, RTsiRNA#2; NM_001108603) were subcloned into pSuper.gfp/neo (OligoEngine). The C1103S (cs), C1103,1397S (2cs), Y912E, Y1027E, Y912,1027E, Y912F, Y1027F, Y912,1027F, and K635N, R637L PTPRT constructs were generated by site‐directed mutagenesis using the Quick‐Change system (Stratagene). The mouse RPTPκ construct was kindly provided by Dr Sap (Universite Paris Diderot Paris 7, France). The human PTPRM and PTPRU (PCP‐2) constructs were generously donated by Dr Brady‐Kalnay and Dr Wang (Case Western Reserve University, USA). The Fyn constructs (Fyn Y531F, constitutively active and Fyn K299M, kinase dead) were generously provided by Dr Yamamoto (University of Tokyo, Japan). The YFP‐neuroligins and CFP‐neurexins were kindly donated by Dr Craig (University of British Columbia, Canada).
The PTPRT monoclonal antibody was raised against the catalytic domain (aa 908–1164) as an antigen. A PTPRT‐specific clone was selected by ELISA using purified antigen, and the ascites was produced in mice. The PTPRT polyclonal antibody was produced against the juxta‐membrane domain (aa 804–966) as an antigen in the guinea‐pig. Two peptide epitopes of neurexin 2α (RGRSPTMRDSTTQN and KAPAAPKTPSKAKK) were used for the production of a polyclonal antibody in rabbit. The SK‐7 antibody was generously provided by Dr Brady‐Kalnay (Case Western Reserve University, USA). Other antibodies were purchased from commercial sources: synapsin1, vGLUT, vGAT, neuroligin1, neuroligin2, and neurexin (Synaptic Systems GmbH); 4G10 (anti‐phosphotyrosine, Upstate); Fyn and β‐catenin (Santa Cruz); MAP2 and FLAG (Sigma); GFAP (Abcam).
Cell culture and transfection
HEK293T cells, maintained in DMEM containing 10% foetal bovine serum, were transfected by the calcium phosphate method. Primary hippocampal neuronal cultures were as described earlier and transfected by the calcium phosphate method (Lee et al, 2004).
Subcellular fractionation
Rat brain homogenates (H in Figure 1C) were centrifuged at 900 g to remove nuclei and other large debris (P1). The supernatant was centrifuged at 12 000 g to obtain a crude synaptosomal fraction (P2). The supernatant (S2) was centrifuged at 250 000 g to obtain light membrane (P3) and cytosolic fraction (S3). In parallel, the P2 fraction was subjected to hypotonic lysis and centrifuged at 25 000 g to precipitate synaptosomal membrane (LP1). The supernatant (LS1) was further centrifuged at 250 000 g to obtain a crude synaptic vesicle‐enriched fraction (LP2) and soluble fraction (LS2). To obtain PSD fractions, the synaptosomal fraction was extracted with detergents, once with Triton X‐100 (PSD I), twice with Triton X‐100 (PSD II), once with Triton X‐100, and once with sarcosyl (PSD III).
Immunoprecipitation and mass spectrometry
PTPRT‐specific monoclonal antibody was used for immunoprecipitation in rat brain synaptosomal fraction. A total of 8 mg sodium deoxychorate‐extracted synaptosome (P2‐DOC) was incubated with 80 μl mouse ascites for immunoprecipitation. The resulting complex was pulled down by protein G‐agarose beads, and proteins were separated from the resin by boiling with SDS‐sampling buffer. Samples were resolved with 10% SDS–PAGE and stained by Coomassie blue staining. Approximately 50 SDS‐gel bands were excised, destained, and digested with trypsin. The resultant peptide mixtures were analysed by online liquid chromatography/tandem mass spectrometry (Waters nanoACQUITY/Q‐Tof Premier, Milford, USA) to generate peptide sequence information. Two intense ions, which met the predetermined MS survey scan criteria, were selected for collision‐induced fragmentation. The collision energy of the selected ion was automatically determined according to its charge and mass. The acquisition software collected MS/MS spectra for 7 s for each selected ion, and then acquisition was returned to MS survey mode. The mass spectral data were processed into peak lists containing m/z values, charge states of the parent ion, fragment ion masses, and intensities, and these were then correlated with protein sequence databases using Mascot software (Matrix Science, London, UK). Proteins were identified on the basis of matching the MS/MS data with mass values calculated for selected ion series of a peptide.
Competition with soluble PTPRT ecto‐Fc
The ecto‐domains of PTPRT (aa 1–749 or 1–763) were fused to the Fc region of human immunoglobulin (RT‐ecto‐Fc) and Fc‐fusion proteins were purified in HEK293T cell maintained in serum‐free culture medium. Cultured neurons transfected with pSuper.gfp/neo (for visualization of dendritic protrusions; days in vitro (DIV) 9–20) were treated with RT‐ecto‐Fc for 4 days (4 μg/ml).
Image acquisition and quantification
Images captured by confocal microscopy (LSM Meta, Zeiss) were analysed blindly using MetaMorph software (Universal Imaging). The density of dendritic protrusions (0.4–2.5 μm) and synaptic protein clusters was measured from 40–50 dendrites of 8–10 neurons; the total dendritic length of ∼50 μm was measured from the first dendritic branching points. Means from multiple individual dendrites were averaged to obtain a population mean and s.e.m.
In vitro tyrosine phosphatase assay
Recombinant proteins containing the PTPRT catalytic domain (aa 886–1164 into pET28a) were produced and purified by affinity and size‐exclusion chromatography. PTP activity was assayed with 6,8‐difluoro‐4‐methylumbelliferyl phosphate (DiFMUP, Molecular Probe) as a substrate. The reaction was performed for 20 min at room temperature and stopped by adding 1 mM sodium orthovanadate. Fluorescence excitation of hydrolysed DiFMUP was measured at 355 nm, and emission was detected at 460 nm in a fluorescence plate reader.
Supplementary data
Supplementary data are available at The EMBO Journal Online (http://www.embojournal.org).
Conflict of Interest
The authors declare that they have no conflict of interest.
Supplementary Information
Supplementary Data [emboj2009289-sup-0001.pdf]
Review Process File [emboj2009289-sup-0002.pdf]
We thank Dr Ann Marie Craig for the kind gifts of neuroligin and neurexin expression constructs, Dr Tadashi Yamamoto for providing Fyn expression constructs, Dr Jan Sap for generous gifts of RPTPκ plasmids, and Dr Brady‐Kalnay for kind donation of RPTPmu, PCP‐2 plasmids, and antibodies. This work was supported by the Korea Science and Engineering Foundation (KOSEF), a grant funded by the Korea government (MOST) (R01‐2007‐000‐11499‐0 to J‐RL), the KRIBB research initiative programme to J‐RL, and the joint research collaboration programme for Chinese–Korean–Japanese research cooperation to D‐YY.
- Copyright © 2009 European Molecular Biology Organization


text:
Niklaus Troxler (b.1947)
A Tribute to the Music of Theolonius Monk
Silkscreen, printed by Graffik-Studio Niklaus Troxler, Luzern, Switzerland 1986. Condition: Near Mint (A) , backed on linen.
Sheet size: 50 H. X 36 W.
In this award winning design for a tribute concert to jazz pianist and composer Thelonious Monk, Troxler created "Round Midnight" typography which outlines the very recognizable goateed profile of Monk. "Round Midnight" is Monks most recorded composition. The random changing of the color of the text plays tribute to Monk's legacy of improvisation, new chords, syncopations and runs.
In the collection of National Portrait Gallery, Smithsonian Institution
Reference: Troxler pg 167; Rotzler Scharer Wobmann pg 86
Inventory #11181
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text:
With the election just around the corner, last week's was never going to be a punishing Budget and the Chancellor delivered a range of carefully measured giveaways as usual.
Twelve months ago saw the most revolutionary Budget for pensions that I had ever seen and this time around it was savers who benefited. George Osborne announced that basic rate tax payers will now have a £1000 tax-free savings income band on top of their normal tax-free personal allowance. This means, for example, that cautious savers could have £20,000 in the Santander 123 account earning £600 and £10,000 in the National Savings 3 year pensioner bond, earning £400, all without being liable to pay tax.
Higher rate taxpayers will however only receive £500 of interest before paying any tax. There will still be some paperwork required to claim back any tax paid on the pensioner bond and to receive the interest tax-free on any other savings accounts.
ISA savers will now benefit from the flexibility of being able to make withdrawals from an ISA and pay money back in during the same tax year. Previously if an ISA was fully funded and money was taken out, it could not be put back in until the next tax year. This welcome change is on top of the recent announcement that on death, an ISA can pass to a spouse without losing its tax-free status.
Probably the most dramatic ISA change is the announcement of a new first time buyer ISA. Savers can put £1,000 initially and up to £200 per month into a first time buyer ISA and the Government will add 25 per cent to the amount saved when it is use to buy a property. The maximum that can be saved in this way is £12,000 and the Government will add £3,000. A couple could each benefit from the £3,000 incentive, resulting in an extra £6,000 towards a deposit.
The scheme will apply to houses valued up to £250,000 outside of London.
One disappointing aspect of the Budget was the reduction of the lifetime pension allowance to £1m. Anyone approaching retirement with funds close to or above this amount will need to take action to protect their pension from a potential 55 per cent tax charge.
David Hill is a Chartered Financial Planner and Independent Financial Adviser at Hills Financial Planning, 15 Agnew Street, Larne. He can be contacted on 028 28276814 or by email: firstname.lastname@example.com


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'When I saw this picture of Jessica Biel I felt so violated by her outfit it compelled me to write you and plea for you to smear her on your fashion blog. HAHA. Ok no, seriously though take a look. It's so wrong all over - from the earrings to the blazer with that t-shirt, awkward skirt and absolutely HORRIFIC shoes to match the whole look. How is she dating JT and dressing like this? No stylist? FAIL. No surprise, Biel always dresses to disappoint, but as JT's girlfriend she should know better."
I've got to agree with Sam. This look is beyond tragic. It's disturbing that someone with all of the connections (seriously JT is one of the most stylish guys out there) can always look so wrong. I don't think she's ever worn an outfit that I've liked. I actually like some of the pieces that she's wearing here but put together it's a horrible combination. I have a soft spot for Roger Vivier flats but not with this outfit. Good thing she's got such a great body! It's the only thing going for her.
So what do you think? Do you agree or disagree with our thoughts on Jessica's outfit?
Source: Just Jared


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R. J. Campbell was an English Congregational Minister, who, at the turn of the 20th century, occupied the pulpit of one of the most prestigious churches in England – the City Temple in London. From
this pulpit, Campbell began to articulate a doctrinal position which he termed "The New Theology". Campbell invited 'Abdu'l-Bahá to give His first ever public address at the City Temple in 10th September 1911.
(The Bahá'í movement) "is one of the most remarkable religious movements of this or any age, a movement which includes, I understand, at least three million souls… The Bahá'í movement, as it is called, in Hither Asia rose on that soil just as spontaneously as Christianity rose in the middle territories adjoining, and that faith – which, by the way, is very closely akin to, I think I might say identical with, the spiritual purpose of Christianity – that movement stands for the spiritual unity of mankind; it stands for universal peace among the nations. These are good things, and the man ('Abdu'l-Bahá) who teaches them and commends them to three millions of followers must be a good man as well as a great."


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Sigma Systems is the global leader in catalog-driven software solutions for communications, media, and high-tech companies. The company's portfolio spans enterprise-wide Catalog, Configure Price Quote (CPQ), Order Management and Provisioning products. Sigma utilizes an agile approach to implementing its B/OSS products for its customers.
Etiya provides the MOST COMPLETE catalog-driven B/OSS offer in the market. Etiya is the ONLY VENDOR bridging rule-based systems and autonomous learning algorithm-driven applications. It incorporates powerful ARTIFICIAL INTELLIGENCE in its preintegrated products and customized solutions to help you transform your business, reduce costs, and delight your customers with in-context business insights and predictions.
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text: Veteran's Administration (VA) Loan - Home Buying process provided by Helene Cass and Team specializing in McDowell Mountain Ranch HOME CONTACT US RE/MAX Fine Properties 21000 N. Pima Road, Suite 100 Scottsdale, AZ 85255 (480) 563-3333 888 471 9500 View our listings at The Ranch Search MLS The Purchase Process Financing Your New Home Mortgage Calculators Frequently Asked Questions An Introduction to the Community Our MMR Neighborhoods A Community Map Our Homeowner's Association Our Communication Tools Our Community Facilities An Historical Perspective Our Neighboring Communities The Scottsdale School System Recreational Opportunities Our Other Community Assets An Area Map Arts & Entertainment Environmental Impacts Government & Politics Healthcare Resources Jobs & Education Real Estate Resources Sports & Recreation Local Media Weather Travel & Tourism Utilities Current Issue Archives Regional Map Community Map Directions to My Office Directions to Anywhere My History My Past Performance My Awards My Education My Sales Team Testimonials Directions to My Office About RE/MAX My Extra Efforts My Newsletter Contact Helene Site Map Financing Your Purchase > Types of Mortgages > Veteran's Administration (VA) Loan 30 Year Fixed Rate 15 Year Fixed Rate Adjustable Rate Loans Interest Only Loans FHA Loans VA Loans "God, please just give us one more real estate boom." - Steve Hewson Veteran's Administration (VA) Loan: Loans for qualified veterans are backed by the Department of Veterans Affairs and permit the veteran to make a purchase with either a low down payment or none at all. These mortgages are subject to the VA mortgage funding fee, a premium of up to 2.15% of the loan amount, depending on the size of the down payment. VA loans can be assumable to qualified buyers. Hypothetical Home Cost: $400,000 Down payment: None Mortgage Amount: $417,000 (Maximum Available) Term of Loan: 30 years Interest Rate: 5.25% Monthly Payment: $2,302.70 «Back Home | About Helene | Homes for Sale | The Ranch | Local Businesses | Site Map Each Office is independently owned and operated. Privacy | Terms © 2001 - 2009 Helene CassHosted and Powered by Pier 1 Solutions


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Raise The Titanic! was the 3rd novel by Clive Cussler and was published in 1976.
It was originally the 3rd of the Dirk Pitt books but later became the 4th installment as a later release – Pacific Vortex! – was set earlier chronologically.
This novel was also adapted for the big screen as a movie starring Richard Jordon as Dirk Pitt.
Raise The Titanic! book description
The President's secret task force has developed an unprecedented defensive weapon that relies on an extremely rare radioactive element – and Dirk Pitt has followed a twisted trail to a secret cache of the substance.
Now, racing against brutal storms, Soviet spies, and a ticking clock, Pitt begins his most thrilling mission – to raise from its watery grave the shipwreck of the century…


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Little Red Bunny is taking a breather outside the Eden Roc in Miami Beach when a soon-to-be fan approaches her. A middle-aged man wearing cargo shorts, an orange Polo and a visor waddles up as the famous cam girl takes in a pool-side view.
"Your look is cute," the dweeb says. "Where are you from?"
It's a question that Little Red Bunny is asked a lot on her site, but one she never answers outside of private chat. So typically, her honest response costs $4.99 per minute. In this case, the woman with a vaguely Eastern-European accent lies and says her distinct manner of speech is derived from her parents who are "Irish-French."
The random interloper doesn't seem to catch on to Little Red Bunny's sarcasm. He invites her to a party and tries to exchange contact information. When the stunning red-head hands over a card with her URL listed, the dorkus maximus tries to get further, saying he prefers telephone numbers.
"I don't do that," she replies coolly. "My life is very public, so I keep that information private."
Although the Eden Roc guest might not know who he's talking to, his conquest this past Friday afternoon was one of the most popular webcam models in the world. He's far from being the only dude who wants to know more about her. Every day, Little Red Bunny (real name Ophelia) broadcasts shows from her apartment in New York and attracts hundreds of customers. A trained ballet dancer, this model was named "Favorite Cam Girl" by the Adult Video Network this year. The distinction, along with her particular brand of burlesque, caused the Daily Beast to call her "The Queen of Cam Girls" in March.
Little Red Bunny spent the weekend in Miami Beach as part of Camming Con, the first such industry event in North America. She was the headliner, too, being the prime example of how someone can go from making $1.99 an hour (and giving 35 percent back to her hosting site) to charging more than double as an entrepreneur. She has an affiliate site off of Jasmin, which is pretty much the biggest cam site in the world. Hours before she was pestered by this random admirer, she sat down with New Times to discuss her business model and unusual calling.
How did you get started, and what was your background before camming? I saw a pop up of a girl sitting on the computer typing. Before that, I'd done a lot of artistic stuff. I've done years of classical ballet. I've done concert piano -- just years worth of stuff in New York City. So my relationship to my body has always been very close. I thought the idea of stripping could be nice, but it's not what it used to be. It's no longer glamorous or burlesque. I was curious how I could be sexy while dancing, but I didn't want to be a stripper.
What was attracting to me was that I wanted to work from home. I didn't want to go out and have a boss, so I started working for Jasmin instead. Now I work all night for them.
Some sites, like Playboy Live, provide training to their cam models. What were some of the challenges you faced in learning how it works on your own? I started from nowhere, and I was shy. It was trial and error. That's probably why I have my own style in a way -- I did not study other girls to see what they do in free chat or how they handle things.
I'm not the kind to show myself too much or go out with a super miniature skirt or all that. I thought I would see if i was comfortable and then keep doing it a little longer, so I did it for a week or two.
The first few days are really weird. I've never done stripping or porn or anything. There's the whole technological aspect of it, too. Learning how to position yourself with the cam to give people what they want. You have to understand the people and the dynamic, too.
So what's your 9-5? People ask me how much I work at the gym, because I'm so muscular and strong, but [camming] is what I do. I kind of apply belly moves in an erotic way in my bed. I can work the hours i want, but it's become like 6 to 6 or 4 to 4.
Twelve hours? Really? Sometimes I do 10. Sometimes 8. But there are nights where I pull up to 16 to 18 hours a night because you think you're going to go to bed and then there's that person from Belgium or France that just woke up and says "I've gotta see you." So I stay. People wonder how I get the energy, and I have no idea. But I think it's like a bubble where you lose sense of time and space.
What's the scene like at your site? There's that whole atmosphere at my free chat that's like having a party. People say to each other, "Where were you? I haven't seen you the past two weeks." They really interact between themselves. And then there's some people who see me and have a drink before they go to bed or who have had a bad day and know it's always a party in my room, really. It's a social event for most people. But in private chat there are people who come a few times a week, or a few times a month. It varies a lot.
How does camming interfere with your personal life? Do you have to keep up an air of mystery with your regulars? There are some people I've known for five years. Some people gain the kind of trust to gain something personal back. What's private, I keep private. Especially in free chat -- I keep that very separate. I think for your own health you have to keep some things to yourself. You have to have your own little world.
You're pretty much at the top tier of cam girls right now. Where do you hope to go? Sometimes there are bad nights. To make sure you're busy all the time is a big thing. Beyond that, as a person, I have much more to me than just the camming. The sexuality is kind of on the side, and I am good at what I do. But I have an artistic side. Maybe someday even the dance side is going to merge with that. But life is like a puzzle that gets created as you grow up, and you never know how it's gonna be until the end. I didn't expect to even be here today.
Does a site like Jasmin tell you what to do? There are a lot of restrictions, which is why a lot of models don't like to work on that site. But that's why I like to work there. You cannot show your breasts on free chat, for instance. But I would never do that. You cannot lick toys. You cannot have your cat walk on the bed. When you start, you question a lot of them, but when you think about it, it makes sense. They have their morality, too. They wanna make sure it's OK for children to come on their site, so that in free chat the kid wont come and see crazy stuff.
What does your family think of your career? People who know me would say I have always had an old-fashioned clothing style. That's followed me since I was a teenager. I was kind of on the hippie side. I had friends that were punks with the studs and friends that were totally nerds. There was a lot of variety-- my best friend was goth. I was always artistic and a good student, but not the kind that was always good at everything just like that, I had to work for it and study. [As for the AVN Award], nobody knows. I don't talk with them all the time. I say, "I do performance and work on some projects." But one thing that might make it better is if they knew that I did win the award, so even if they were like, "What the hell," I could say I was at least good at what I was doing. They do want to know that I'm financially secure and like what I do.
People say that camming is the new porn, but do you consider yourself a porn star? Hell no. But people say, "This a jerk off site, what are you doing, ballet?" There's more substance to what I do. If I was doing porn, people would be touching me and people would end up in my bed that would never be there in real life. What also makes me never want to do porn in real life is that i need a bubble. Sex is something that should only happen with the person you're with. Online I touch myself, but these guys don't touch me, they don't smell me, they don't taste me. This keeps something sacred for your real relationship. I still have a real sexuality.
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Regime Security Theory: Why Do States With No Clear Strategic Security Concerns Obtain Nuclear Weapons?
Beasley, Matthew
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Beasley, Matthew
Current realist explanations of why states decide to develop nuclear weapons cannot account for the behavior of states that lack a clear strategic threat. An alternative explanation is necessary to explain such behavior. I argue that domestic regimes in states with no clear strategic threat may develop nuclear weapons in order to ensure their survival. Such regimes are internationally isolated, under pressure from major powers in the international system and possess some preexisting nuclear capacity. Under these conditions, increasing domestic instability causes regimes to pursue a course of nuclear development. Nuclear weapons allow a regime to change the preferences of the great powers that would otherwise prefer to see the regime overthrown. If the regime possesses nuclear weapons, because of the costs and risks associated with those weapons, the great power will favor maintenance of the status quo and may even prop up a regime it intensely dislikes.


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TLC Home is here to create a customized approach to help you meet your organizing goals.
Whether you have committed to an organized lifestyle or just considering organizational changes in your home, we begin with a 15 minute phone consultation.
At this time, we can learn more about your needs and goals and you will learn how we will work together to achieve them.
(You may request a self-assessment by email, to determine if we are a good match for your needs.)
Next we schedule an in-home assessment, if needed. Or if you know you are ready to begin, we can schedule your first organizing session. Either way, we will go over our Agreement and payment schedule, if you select a package from the pricing options, at our first visit.
When we are ready to organize together, I will send you a Welcome Letter describing how to be prepared and how we can make most of our session.
Then we organize together! We get you started on the organized life you've been dreaming of. In the first session we will pick the area to begin. You will learn my 5 C method of organization and you will be blown away by how much we accomplish.
You won't need any bins or containers at this time. We'll make do with what we find around the house. Once we've selected the best system for you, I'll give you a list of items to purchase or you can hire us to purchase them for you.
Love your home; feel great about your spaces!
Stay in touch, ask questions, and be sure to take advantage of the free follow-up consultation, if you purchased a package.
Spread the Word!
If you loved working with us (and I know you will,) please Like us on Facebook and tell all of your friends and family.
We'd love a brief testimonial too!


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Dupuytren's Contracture Symptoms and Signs
The presence of Dupuytren's Disease may not be known to an individual until it is discovered that they experience difficulty placing their hand flat on an even surface, such as a table top (see Figure 4). Additionally, some people notice increasing difficulty with activities such as washing, wearing gloves, shaking hands, and putting their hands into their pockets.
Figure 4: Dr. Nathan's picture of a tabletop test
Initial symptoms of Dupuytren's Contracture usually include the development of a small nodule, or lump, on the palmar surface of the hand or fingers. Some individuals may only develop small lumps or cords, while others will develop severely bent, curved fingers. Often, these nodules, or lumps, are mistaken for calluses because they are generally firm and adhere to the skin.
As Dupuytren's progresses, the nodules appear to grow together to form a line in the palm or digit. This line is referred to as a "cord." These cords, which commonly are mistaken for tendons, actually lie between the skin layer and the tendons. In many cases, cords occur most commonly in line with the Ring and Little fingers; however, contractures can occur in any of the digits in both hands. Similarly to Dupuytren's Disease, nodules, lumps, and/or cords can occur in the soles of the feet (Ledderhose's Disease), and in male genitalia (Peyronie's Disease).
Dupuytren's Disease is not typically painful, although when hand nodules initially develop they may be tender. Over time, several nodules may occur, progress and/or seize in development. Unfortunately, the degree of involvement and progression may vary and is mostly always unpredictable.
To learn more about Dupuytren's Contracture and the various treatments to correct it, request a consultation, or contact The Hand & Wrist Center at (562) 424-9000.


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This headache relief salve is a light, aromatic mix of peppermint and lavender oils. I can honestly say that this is the most used salve I make in my home. Everyone benefits from it. It works wonders for tension headaches, head colds, migraines and more.
Peppermint is known to relax muscles and spasms, making it ideal for releasing headache tension. Peppermint helps relieve pain and the added benefits of soothing Lavender magnifies the healing properties. Lavender acts as a mild anti-spasmodic, alleviating inflamed blood vessels causing the head pain.
The aroma is magnificent. I find this blend helpful as an aromatherapy "inhaler" also. It's light scent is relaxing, good for relieving stress, nervousness and even depression.
I've included an extension to this post. Please see migraine compress and soak for extra relief.
What You'll Need:
Recipe 1 (Solid Salve):
This one can be kept out of the fridge without melting. For a softer base, see Recipe 2.
- 5 drops Peppermint Oil
- 5 drops Lavender Oil
- 1 tbsp Beeswax or 1/2 tbsp Carnauba Wax
- 6 tbsp Coconut Oil
Depending on your preferences, you can add a few more drops of each essential oil.
Recipe 2 (Softer Base & Simplest Recipe):
This one will liquefy to an oil out of the fridge but solidify when kept cool. It will melt easily when introduced to your skin.
- 5 drops Peppermint Oil
- 5 drops Lavender Oil
- 5 tbsp of Coconut Oil
In my first batch I used peppermint oil I had made from Infusing my own oil. If you're going to infuse your own oil, you can use it as your base oil and leave out the coconut oil. See recipe below if you want to use your own infused oil.
Recipe 3 – if you're infusing your own oil:
- 1/2 cup Infused Peppermint Oil
- 5 drops Lavender Oil
- 1/2 ounce Beeswax or 1/4 ounce Carnauba Wax
*These are basic measurements. Depending on the size of container you are using to store your finished product will depend how much you need. (In tbsp amounts, use 1 tbsp of beeswax to 6 tbsp of carrier oil)
Preparation Items You Will Need:
- Melting Pot / Double Boiler or a pan on the stove (low heat)
- Mixing Stick (I use a chopstick)
- Storage Jar(s)
Preparation for Recipe 1:
- Melt your Beeswax or Carnauba Wax first.
- Stir in coconut oil and allow for that to melt. (low heat)
- Reduce Heat & add in your Essential Oils. Stir well until everything is combined.
- Remove from heat and pour into your storage container immediately. (It doesn't take long for this to harden)
Preparation for Recipe 2:
- Melt coconut oil in your pot (low heat)
- Reduce Heat (or remove from heat) & add in your Essential Oils. Stir well until everything is evenly combined.
- Remove from heat and pour into your storage container immediately. (It doesn't take long for this to harden)
Preparation for Recipe 3:
- Melt your Beeswax or Carnauba Wax first.
- Next, add in Infused Oil and stir ingredients together until everything is combined uniformly.
- Reduce Heat & add in your Essential Oils.
- Remove from heat and pour into your storage container immediately. (It doesn't take long for this to harden)
Depending on the firmness you prefer, you can add more beeswax or add more carrier oil to make a softer base. I like mine to be soft, plus I keep it in the fridge so it stays hard, but melts easily on the skin.
If you aren't happy with your finished salve, you can always melt it back down and add in more of what you need.It's best to keep your mixture in the fridge. (At it's best if used within 3 – 6 months)
Apply to temples, base of neck, (right under hairline) the pain site (across forehead) and bottom of the feet.
*You can also use these oils in a migraine compress & hand soak.
Please Also See our post on Earache & Headache Relief for Children. ❤
Happy Healing! ❤
I recommend Mountain Rose Herbs & Starwest Botanicals for all of your herbal healing needs! For my Canadian Friends, you can purchase your oils at Harmonious Mind. You can purchase any extra supplies over at Amazon.ca. Images Edited using PicMonkey. Some of the links here are affiliate sites, meaning if you make a purchase using my link, I earn a small commission! Thank You for supporting The Herbal Healing Mama. & Please also see my disclaimer. Thanks Friends.


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St. Demetrios Spring Greek Fest
This event occurred in the past.
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The aromas of Greek cuisine waft over Lake Country on May 2 and 3 as the first Spring version of its hugely successful Summer Greek Festival is offered. Feast on full meals of Athenian chicken, souvlakia (kabobs) and gyros, finishing with the signature baklava and other delicious Greek pastries. Onsite dining and take-away boxes for both meals and pastries. Greek beer and homemade Greek yogurt. The St. Demetrios Bookstore offers spiritual books, icons and an assortment of fun Greek import items. Fr. Cosmas Halekakis answers questions about Orthodoxy and shows the church's stunning architecture with its gold dome and larger-than-life icons.


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We shift gears in this episode and not only talk about a single band but a single album from a single band. Our shift in gears is analogous to said single band's shift in gears, musically. Angry sludgesters Baroness turn into deft indie rockers on their new Yellow & Green. Spacey '60s narcotics in one hand, sledgehammer in the other, the band lobotomizes longtime listeners and will undoubtedly acquire many new ones as well.
This new double LP reaches across multiple genres in tapping a well-spring of influences and will appeal to a broad range of music fans. Comparisons to Mastodon, Queens of the Stone Age, and Cave In will be peppered with critics' invocations of Pink Floyd, Killing Joke, the Beach Boys' Pet Sounds, and perhaps Radiohead. Dig a little deeper and you might hear buzzing insects or a dash of alt-country. Turned off already, metal purist? Well, you never gave Baroness the time of day anyway. Yellow & Green will surely recruit as well as alienate, but go back to even the early EPs and you'll hear that Baroness have been on said trajectory all along. They are obviously interested in pushing themselves. We're glad they have the guts, and we're hoping that it'll turn out net positive. A major-label masterpiece may be within the next two or three records.


text: About Kitchens by Regal – Toronto Renovation Contractor Kitchens by Regal is a family owned business, where quality and satisfaction is our number one priority. We have over 12 years of experience in kitchens and custom cabinetry. Through the years we have acquired a team of remarkable craftsmen and designers that, with hundreds of possible colour and finish combinations, they can bring any unique idea or design to life. Your thoughts are discussed with a member of our highly skilled and experienced team, and following this consultation, a set of drawings will be created to guide our craftsmen in building your dream. Call Kitchens by Regal company for any quote related renovations, remodelling, floors or stairs refacing or other home improvement issues.


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Jennifer Ann Holt released her second novel, "Discovering Peace," last month. The book is a story about mistakes, healing, and moving forward.
Book synopsis:
Placing her baby girl for adoption isn't the mistake Ally is running away from, but everyone else seems to think it is. As Ally tries to move forward in her life, her past keeps pulling her back, and she wonders if she will ever find true love again. Only holding onto hope can help Ally find the peace she's seeking.
Holt is no stranger to the adoption process, having adopted two children while battling infertility and a difficult pregnancy that resulted in her youngest son being born three months prematurely.
We're grateful to Jennifer for taking a few minutes to field questions in the Fiction Fest hot seat.
Is "Discovering Peace" a sequel to "Delivering Hope"?
Yes, although people are telling me that both can be read as stand alone titles, as well.
What role has your personal experience with adoption and infertility played in these books?
I think my personal experience has made the emotions much more realistic. I wanted the story to be very true to life, so I dug into many of my own feelings to help the story mirror reality very closely.
What is your current work in progress?
I have a couple of things I'm working on…a Christmas novella and a general fiction novel for the national market.
What is your writing routine like?
I squeeze in writing whenever I can. I have three children who are in elementary school, and they are my first priority. I usually make time to write while they are at school.
What do you like most about being a published author?
I like that my stories are able to be enjoyed by many people. It's great to get a note from a reader telling how one of my books helped them through some of their own struggles.
What's the most joy writing has brought you?
For me, the most joy has come through getting my words down on paper. I had a story in my mind that I was very passionate about telling, and it was great watching it come to life on the page.
Which authors had the most influence on your writing style?
I love the Bronte sisters and Jane Austen. They really influence my writing, and I have to work hard to make my style more modern.
Who would play the main characters in the motion picture version of "Discovering Peace"?
Hmm…for Olivia, probably Keri Russell, and for Allison…maybe Emma Watson.
What's your sure-fire method for battling writer's block?
Take a break and read something fun.
What advice would you like to offer to aspiring novelists?
Write about something you know and are passionate about. Even if it's complete fantasy, you should know it inside and out and understand the characters (think J.K. Rowling with Harry Potter).


text: From a central geographic point between the Greater Toronto Area (GTA) and the US/Niagara border, to being consistently ranked one of the top places to invest in Canada, to having the most diversified economy in the nation, investing in Hamilton puts your business at an advantage in so many ways. Known as the Canadian hub for advanced manufacturing, Hamilton is also steadily being recognized as a food and beverage manufacturing leader, a pioneer in life science discoveries, and burgeoning tech scene. Explore this site to see how Hamilton is home for your next investment.


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A study that surveyed more than 500 dairy farmers found that naming cows had a significant impact on milk yields. Those farmers who called their cows by name got around 2,105 gallons (7,968 liters) of milk over the cows' 10-month lactation cycle, compared to around 2,029 gallons (7,680 liters) reported by those who didn't name their cows. Scientists believe that addressing the cows by name might have a calming effect on the animals, reducing the production of the stress hormone cortisol, which can impede milk production.
Key Facts In This Video
The average cow can produce about 100 glasses of milk each day. 00:41
Cows don't have four stomachs—they have one stomach with four compartments. 01:06
Holstein cows produce more milk per day than any other type of cow. 01:51
Written by Curiosity Staff December 7, 2015
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