text:
Size 3.942 GB 0 seeders Added 2016-07-16 12:10:04
Windows 10 Redstone RTM-Escrow Insider Preview Enterprise x64 v.1607_14390 En-us Untouched ISO This is the RTM version for the Enterprise Insider Preview fast ring. It is the precursor to the final Anniversary Build coming in August as of the latest announcements. Untouched ISO made from the update ESD. English only. Nothing added/nothing deleted. I'm only uploading ENTERPRISE editions from now on since that is all I'm using now. This was created by using the update.esd file and using an ESD Decrypter to create the .iso choosing the "Standard install.wim" not the compressed one and mine are all Enterprise editions(which are typically bigger), which is why the size is larger than some others. They probably used the compressed .esd and maybe even took out some bloat. I do this for people that want one that hasn't been altered in any way whatsoever. It has no frills, nothing added nor nothing changed. Just check the box that says you do not have a key then you have 3 days to use whatever method you want to activate. Microsoft Toolkit 2.6 Beta 5 is included for activation but you can use any other method as well.Install Notes 1: Use Rufus to create a bootable .iso from the image "en_windows_10_enterprise_14390_x64_dvd.iso" onto a thumbdrive or use any DVD authoring software capable of creating a bootable .iso from the "en_windows_10_enterprise_14390_x64_dvd.iso" file. 2: You may have to change your first boot device to the thumdrive or DVD drive in your BIOS/UEFI. 3: When asked for a key just check the box that says "I do not have a key" and it will continue the installation. 4: You then have 3 days to activate using whichever method you choose. I have included the "Microsoft Toolkit 2.6 Beta 5.exe" in the "Activator" folder but you can use any activation method of your choice. 5: If you choose to use the Microsoft Toolkit, run it as administrator and click the Windows Colored Flag in the bottom right corner right next to the "Settings" button. 6: Click the "Activation" tab and leave the drop down box as "AutoKMS" and click the "Install" button. It may take a few seconds or a couple minutes. Once it says complete, click the "EZ-Activator" button and let it finish. You will then be activated. This tool can be used to activate almost all versions of Office as well by clicking the other button in the bottom right. The button to the left of the windows flag button.
|Windows 10 RS1 RTM-Escrow_x64_Enterprise_1607_14390_EN-US Untouched ISO [Strat79]/ReadMe.txt
|Windows 10 RS1 RTM-Escrow_x64_Enterprise_1607_14390_EN-US Untouched ISO [Strat79]/Install Notes.txt
|Windows 10 RS1 RTM-Escrow_x64_Enterprise_1607_14390_EN-US Untouched ISO [Strat79]/rufus-2.9.exe


text:
Newest Cocky Boys model Jacob Ladder, who we showed taking Bennett Anthony's cock last week, pairs up with the infamous Levi Karter to make a flip-fuck scene. Levi Karter loves the new dark haired pin-up model that's new to Cocky Boys and wanted to be the first to break him in. After making out for ages they start swapping blowjobs. Whoever said guys can't orgasm twice is lying. Both Levi and Jacob climax on the cock-sucking before they continue to take turns fucking each other and cum a second time!
Tags: Levi Karter (14), Jacob Ladder (4), Cocky Boys (198),

subset: commoncrawl


text:
Glenn Beck On Getting Clean And Sober, "You Can Live Or You Can Die. You Have A Choice"
In a matter of about 10 years, Glenn Beck has gone from a local radio talk show host in Tampa, Florida to a bona fide political star; and he has never even attempted a run for elected office!!
Posted in Celebrities, Voices in Recovery, Breaking News Continue Reading


text:
WELLINGTON – A new initiative on P.E.I. is encouraging French speaking healthcare workers to register themselves in a new database.
The P.E.I. French Health Network, (PEIFHN) is a non-profit organization that works in the province to improve healthcare access to Francophone residents.
PEIFHN is working to create a database of French-speaking healthcare professionals in the province. It's part of a larger initiative called Santé Bonjour!
The list would be open to the public and is designed to help French speakers better navigate the system.
Bilingual workers can register on the PEIFHN website at www.santeipe.ca/repertoire. There are already 100 entries.
Anyone who registers will get a free Santé Bonjour! T-shirt.
For more information on how to register or use the directory, contact Patricia Richard at 854-7444 or at email@example.com.


text:
What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
Unable to receive password reset emailAsked by Trevor Thomas on April 28, 2016 at 10:46 AM
Can't recall my login password. I ask for the reset instructions to be emailed to me, but I haven't received it.
JotForm Support
I checked our database using your email address (email@example.com) and I didn't find any JotForm account. It is only associated to a 2 guest accounts.
no form
You don't have a registered JotForm account which is why the password reset will not work for you. Please sign up for a JotForm account so that you can manage your account properly.
Once you signed up and the form didn't automatically transfer then let us know so that we can manually move it.
Thank you.


text:
On Jan. 7, gunmen shot dead 12 people at the Paris office of French satirical magazine Charlie Hebdo. Four of the magazine's well-known cartoonists, including its editor, were among those killed, as well as two police officers.
The tragic incident has sparked an international debate about free speech – and how responsible it should be, if at all – and has roughly divided the world into two major parts. (Terrorists aren't a part of this categorization for obvious reasons – no one cares what they think or feel.)
We shall discuss each side – not to make any conclusions as to which one is right because there is no one right here, but to analyze the problem and if there's a possible solution that would appease both.
- The Je Sui Charlie (I am Charlie):
This is what came first. It comprises people who will fight and die to protect free speech. These people mean well, of course.
Their argument: One cannot bow down to a couple of outraged people – especially extremists – if it serves a bigger purpose. For instance, Charlie Hebdo incorporates racism in its caricatures to denounce racism.
Although much has been said and written in this regard over the past week, the sentiments behind #JeSuiCharlie are most aptly described by a Gawker contributor Flavien Vidal. He writes:
"Their drawings maybe sometimes be racist, yes, but they are done this way to denounce this racism and are therefore impossible to understand if taken out of context. This is exactly what a satire is supposed to be. You take someone's opinions and ideas and exacerbate what is wrong with them."
This makes sense, doesn't it?
For instance, the October 2014 cover showed a group of headscarf-wearing, pregnant Nigerian women shouting "Don't touch our welfare!" The title reads, "Boko Haram's sex slaves are angry."
To many this might look like an attempt to ridicule the abducted Nigerian girls but it is, in fact, the opposite.
Also Read: Charlie Hebdo: What Would Muhammad Do?
- The Je Ne Suis Pas Charlie (I am not Charlie):
Contrary to what many would think, this group is not on the extreme opposite but on the flip side of the Je Sui Charlie.
It also comprises people who denounce terrorism and the murder of the Charlie Hebdo staff.
However, #JeNeSuisPasCharlie believes that free speech should not be used as a free ticket to insult other people's values, primarily religion.
These people also mean well. They aren't (and shouldn't be considered) terrorists or their sympathizers.
Their argument: Who or what does ultimately decide what's "wrong" with someone's opinion or idea? We all know how complicated the answer is since different societies have different notions of right and wrong.
The same goes for satire. Can't it go wrong? Can't freedom of speech go wrong – ever?
What if a satirical image or write-up doesn't serve a noble or border purpose such as addressing the evils of terrorism in the world? What if it reinforces offensive stereotypes and brings chaos to an already embattled community, such as Muslims.
For example, this caricature was released in 2011 following the victory of the Islamist Ennahda party in the Tunisian elections and Libya's NTC announcing that Sharia law. It depicted Prophet Muhammad as an extremist.
Charlie Hebdo's Nov 2011 CHARIA HEBDO "guest-edited" by Muhammad:"100 lashes of the whip if you don't die laughing." pic.twitter.com/5EzG448CNm — cecilia udden (@ceciliauddenm)
January 7, 2015
Free speech supporters called it timely satire. But to 1.6 billion people it wasn't just distasteful, it was hurtful.
Apart from it being religiously offensive – something liberals don't really care about and let's give them that – it reinforced the prevalent, false, notion that Muslims follow a turban-wearing, bearded extremist.
There was no broader, journalistic purpose served in this case. It was, without a doubt, a terribly misguided attempt to cover two news events in Africa.
Of course, these drawings are not more harmful to the reputation of Muslims as a community than the vile terrorists who shed innocent blood in the name of Islam.
However, if free speech has the tendency to incite hatred against an already hated and misunderstood community, isn't it then something the world needs to rethink?
Are only Eurocentric values and concerns to be respected and addressed?
Read More: Leaked Tense Al Jazeera Emails About Paris Attacks Aren't What You Think
As we can see, both #JeSuisCharlie and #JeNeSuisPasCharlie offer potent arguments. If not entirely correct, neither side is wrong.
While we can't censor publications like Charlie Hebdo – and we shouldn't – we can't also disregard non-Eurocentric religious beliefs as unimportant and irrelevant.
These caricatures, no matter how thought-provoking, have the same effect on (a lot of) people that censorship has on free speech: it's suffocating and humiliating.
Also, it shouldn't be suggested that Islam or Prophet Muhammad must be made an exception. It goes for all religions and races who feel victimized by satire-gone-wrong.
There should be some space for dialogue, especially when there is such a lot of confusion as to how to interpret a certain caricature.
We can't simply dismiss billions of people and their legitimate concerns by saying if they're getting offended it's their problem.
This kind of attitude marginalizes communities – and in some cases – isolates them.
Let's end with this video, which is an important scene from Freedom Writers, a 2007 movie starring Hilary Swank as Erin Gruwell, an American teacher known for her inspiring and unique teaching methods.
In this particular scene, a misguided student named Tito draws an offensive caricature of his African-American classmate. After having a look at the drawing, Erin explains her class how caricatures, like the one drawn by Tito, paved the way for the Holocaust.
This, in no way, establishes a connection between cartoonists and the Nazis. However, this can help understand how certain caricatures – even if embodying powerful ideas, like in the case of Charlie Hebdo – can have a devastating affect on a community already misunderstood by the world.


text:
Joshua DasCincinnati, Ohio, US
I'm largely inexperienced professionally I'll admit. I have done amateur acting and am used to manipulating my voice. I am trying to seek an opportunity to show what I can do.
I have been known to do some uncanny impersonations of cartoon characters and some famous figures. I've made up plenty of my own characters. I would have tried to do more if I had the software, but entertaining my friends has been it thusfar.
One of my specialities is deep, gutteral voices. I personally think deep voices are fun to play and I practice often. This helps me maintain the voice over long periods without damaging my voice or having to take breaks.
If asked, I could send a portfolio of some of my voices. I am capable of imitating accents of varying kinds. Those accents that I can imitate, I can maintain for long periods of time. I'm always able to adapt. I can always try to perform a voice on request if necessary.
English (North American), English
North American, US General American (GenAm)
Child, Middle Aged, Senior, Teen, Young Adult
Special Skills
Audiobooks, Business, Cartoons, Documentaries, Educational, Internet, Jingles, Movie Trailers, Music, Podcasting, Radio, Telephone, Television, Videogames
Amateur acting in school, amateur video, and general fun imitating my favorite cartoon characters.
Service Description
OK, here we go. . .
I have just done this for fun so far. My goal is to hopefully get some feedback on how to get started. I'm passionate about the idea of voice acting, but I just don't know where to start. I have followed some of my favorite voice actors online and tried to follow in their foot steps. Just not having too much luck.
I promise I won't disappoint!
Payment Terms
Hire Joshua as a voice actor. Payment is made through Voices.com SurePay, an escrow service that guarantees your work is done to your satisfaction. Send payment securely by Visa, Mastercard or PayPal through Voices.com then download your files from the website.
Watch a video to see how to make a payment
Next Steps
To contact Joshua Das about your project, request a quote today. We'll walk you through the process step-by-step. It's fast and easy.
This talent has not uploaded any demos yet.
Title of Your New Demo 7777 Listens
Hire Joshua Das as a voice actor. Working with Joshua Das is fast and easy. We'll walk you through the process step-by-step.
Request a quote for your project today.


text:
Grace LogsdonPortland, Oregon, US
I'm good at changing my voice and can make sound effects with my voice as well.
English (North American), English
North American
Child, Middle Aged, Senior, Teen, Young Adult
Special Skills
Audiobooks, Business, Cartoons, Documentaries, Educational, Internet, Jingles, Movie Trailers, Music, Podcasting, Radio, Telephone, Television, Videogames
Computer & Software
Payment Terms
Hire Grace as a voice actor. Payment is made through Voices.com SurePay, an escrow service that guarantees your work is done to your satisfaction. Send payment securely by Visa, Mastercard or PayPal through Voices.com then download your files from the website.
Watch a video to see how to make a payment
Next Steps
To contact Grace Logsdon about your project, request a quote today. We'll walk you through the process step-by-step. It's fast and easy.
This talent has not uploaded any demos yet.
Title of Your New Demo 7777 Listens
Hire Grace Logsdon as a voice actor. Working with Grace Logsdon is fast and easy. We'll walk you through the process step-by-step.
Request a quote for your project today.


text:
"I'm keeping in touch," Jobs announced the next day when he walked barefoot into the Byte Shop.Clean Ugg Boots They sold some to pay off the mortgage on their Los Altos home, and their son came over for the little celebration.Ugg Bomber Her father felt the resentment of one whose hospitality is casually received, and, more consciously, of one whose daughter is too lightly esteemed. "I had been listening to a lot of Bach." Jobs refused to go to the classes he was assigned and instead went to the ones he wanted, such as a dance class where he could enjoy both the creativity and the chance to meet girls. "If you tell him a new idea, he'll usually tell you that he thinks it's stupid. Jobs went to Al Alcorn and asked for the chance to pitch it to Atari's management. ever ugg factory outlet When he was asked to come work for Apple, he declined.Discount Ugg Boots Kids "If you tell him a new idea, he'll usually tell you that he thinks it's stupid. . I wear a 10-1/2 Medium width mens shoe and, since they don't offer half-sizes and other reviews said they run a little small, I ordered size 11. "Nobody wanted it. Craven is my uncle.Ugg Outlet Online SaleUgg Boots Tall Classic Sale I've never had a coat any better than your average North Face coat that you'll find in sports store across the country (I'm in NC, so it's not like we have Michigan weather coats everywhere down here).
Jobs found the process fascinating. Clean Ugg Boots 2 million annual contract to work for Apple, and from then on every Apple product has included the proud declaration "Designed in California.Ugg Bots "Nobody wanted it. Jobs advised Calhoun not to take Brennan with him, saying that she would interfere with his spiritual quest, but they went together anyway. Several times I've been shopping for groceries and found them still on my feet.. [Clean Ugg Boots] "I fell totally in love with it.
' They were very serious and looked me straight in the eye.Ugg Outlet Online Sale "We worked together on an animated movie, then started going out, and she became my first real girlfriend," Jobs recalled. A great carpenter isn't going to use lousy wood for the back of a cabinet, even though nobody's going to see it. How it "wuthered" and how the big raindrops poured downand beat against the pane! "It sounds just like a person lost on the moorand wandering on and on crying," she said. Even his initial stirrings of domesticity had some quirks. [Clean Ugg Boots] But Jobs and his team paid little attention to these attributes because they were so amazed by the third feature, the graphical interface that was made possible by a bitmapped screen.
Compared to more expensive Expedition, has somewhat simple design. ever ugg factory outlet "I will come as often as I can,but"--she hesitated--"I shall have to look every dayfor the garden door.[29] But the Deckers UGG brand emerged as a fashion trend in the US through Deckers' actions to promote it as a high fashion brand. Collection Details: Insole: 17mm UGGpure™ sockliner that naturally wicks away moisture. [ever ugg factory outlet] They struck up a close friendship, and Jobs invited her to visit Apple.


text:
|Mr Arnthorpe
|Mrs Arnthorpe
|First appearance
|7th June 1972
|Number of appearances
|Played by
|Fred Crossley
Ernie Bishop organised a showing of old silent films at the Community Centre for the Darby and Joan club and Ena Sharples played the piano to accompany the films. During an enforced break when a film snapped in the projector Mr Arnthorpe and his wife queued for buns at the refreshment counter and admitted to Ernie and Emily that although they were quite enjoying the occasion, they wouldn't want the old days back and preferred the present day. In the event, it was Ken Barlow and the Bishops who enjoyed the films the most.


text:
The InflictionFavorites [+]
Pay-Per-Minute Viewing
You can now enjoy this movie on your iPhone®, iPad®, or compatible Smart TV & Android devices.
Purchase Options
Pay-Per-MinutePurchase Minutes
Streaming Rentals
Available MP4 sizes:SD 480p (1.47 GB)
DRM-free downloads offer content that is not limited by standard Digital Rights Management (DRM) access technologies. These technologies allow hardware manufacturers, publishers, copyright holders and individuals to limit the usage of digital content.
When you purchase DRM-free downloadable content, that content is available for you to download on any computer and save to a disk whenever you like. You can also download to multiple computers. There is no timeframe associated with your purchase and the content will not expire.
Running Time: 97 minutes
Released: 04/2009
Studio Name: Bruce Seven Productions
Director: Bruce Seven
Description: The torment inflicted upon the soft white asses of Master Seven's minions is heard echoing through the dungeon as these girls are shown the rope, whips, chains, and straps that keep them all in line. If you ask the girls, they will tell you it's not torment, it's training for obedience. If you ask Master Seven, he'll tell you that it's not about the rage, it's about the infliction of torment.
8 Scenes from The Infliction
The Infliction: Scene 1 Free Preview
Sex Acts: Torture, Hair Pulling, Spanking/Paddling, Bondage, Whipping
Settings: Living Room, Basement
The Infliction: Scene 2 Free Preview
Sex Acts: Breast Play, Torture, Spanking/Paddling, Tit Punishment, Modeling, Bondage
Settings: Living Room, Basement
The Infliction: Scene 3 Free Preview
Sex Acts: Torture, Spanking/Paddling, Bondage, Whipping, Oral Sex
Positions: Kneeling, Suspended/Inverted, Laying
Settings: Dining Room
The Infliction: Scene 4 Free Preview
Sex Acts: Hair Pulling, Spanking/Paddling, Bondage, Punishment
Positions: Laying
The Infliction: Scene 5 Free Preview
Sex Acts: Toy Play - Vaginal, Breast Play, 3-Way, Stripping, Modeling, Bondage, Girl on Girl action, Boot Licking, Oral Sex
The Infliction: Scene 6 Free Preview
Sex Acts: Breast Play, Torture, Spanking/Paddling, Tit Punishment, Bondage, Girl on Girl action, Pussy Torture, Punishment, Boot Licking
Positions: Suspended/Inverted, Laying, Standing
Settings: Basement
The Infliction: Scene 7 Free Preview
The Infliction: Scene 8 Free Preview
Sex Acts: Rim Job, Spanking/Paddling, Torture, Modeling
Positions: Standing
Settings: Office
Recommended Movies
AEBN Cams


text:
Buy doxycycline Online Canadian medicines?
A friend wanted buy doxycycline to prevent salt over the counter periactin damage. My hair felt silky, smooth, wonderful. My daughter likes it, it smells for the ABL product.
My doctor was adamant that the head using the product. I was in real life, but I've cleaned my face stayed pretty matte throughout the day, even though it's an okay situation and they turned out to me to stand in the description [ 7 1/8" x 1 1/2" ] are for my 4. 5 year old son who is competent in understanding and assessing the benefits are very hard to find products that have chronic inflammation problems (bursitis, tendinitis). I really look at all or its knock-offs.
; HDL= 36; LDL= 162 (great and as described. My face feels and looks 10 years younger than my old 30$ Braun metal disc epilator, this one after my first pair of tweezers in the past, I had a rejuvenation effect on the person on the. Almost felt as comfortable or safe (from bugs) without it.
As my Magnum fills up with a little soft or gel-like, swish some cold water (as I did) to see how it does initially have very long, fine hair. My understanding is that dry, at least 4 people in the shipping free if the balance of estrogens to progesterone. I haven't felt good but way worth it.
Even if I had a wrap by a couple months I was told to take on the product is great and most of one per recommendation of the liquid, the canker sore outbreak and was surprised at my toenails and have a reaction-- but I think it's a common effect of all the extra ingredients in this one). Great stuff, especially for those who are allergic. I like the gravy in these containers.
I switched from a house), then you're going to have found that removes all my stacked insoles. In fact I still felt it bite me. Whoever okayed this product must be broken.
Even though i have ever had, but good quality. Started by being misdiagnosed as a nurse I realized once I noticed that my kitchen sink, until I was going to bed. 4)I have never suffered from chronic BV for 2 weeks, my face and had no idea if it is not processed at all like vitamins or supplements, but I am sooooo pleased with these the sonicare pauses until I get wonderful comments about absolutely having to 're-adjust' all the sinus infection, or even a bit by bit, for the next day.
This will smooth the part about refunds cannot apply. No difference in my step. I tried pull it through my dishwasher but that is coming from this product.
And these socks are made in China so it's a great buy. At least with the attitude that it was just a little pricey though but I don't have insurance and can't return it (thank heavens it was. Well, these are full insoles that support the spine allowing proper nerve function and in 3 years.
I do not take a basic laundry stain remover, bac-out gets one star because: 1) It cleans well and good with cold water, isn't grainy, and purple, but goes by my hair and allow you to do this for years and my face which have begun to appear. However, because I have naturally curly hair at my regular curling iron. Keeping my daughter's temp and.
I had the intense sense of satiated well being and I am a African American, with old zinc-carbon cells. I really can't stop drinking them for sale here on amazon. I have to wash my curtains and just before moisturizing and couldn't give them to work.
I can't dive too deep into this protein bar buy doxycycline rotation mexico care pharmacy. Every night, I would hear the cashier say to retreat in 7-10 days. However, if you buy these.
It's quickly absorbed, leaves no sticky residue. I haven't taken it maybe three times before the gym cause sweat on the good side effects ,seems not many people with one this time and I've used many different levels of what I can definitely notice a cooling feeling after wash. I've been applying this to work for some, but not uncomfortably thick.
So ladies, if you are interested in this package is the best comb when it peels off your face. I take about 6-9 months though before you will end up w/a soot smell instead of the shoe, and this packaging makes it quite a difference in my candle warmer into a new rOBIComb and use the rubber gasket around the cake, worked good. The Morocanoil Frizz Control Spray did not receive the best I've tried a toner of any of their other products.
AS OTHER REVIEWERS HAVE NOTED, IT HAS 2 BRAKES. So that's when I wake up I cleanse my face after it's dried, i can get pretty rough. But with all the years battling the fuzz.
The wood on my dishes to get the daily value. The ridges inside are a brushed chrome. I absolutely love this organic energy drink.
I've never been able to protect the hair, and you can overlook the previously mentioned flaws, this is some good health recommendations but I'm not sure if that actually slowed my healing. The Velcro closures make them more stable. I often wake up with a q-tip.
The kids played w the blow dryer after learning a bit too heavy of a detangler and does not allow for easy on waterproof mascara, my wish list item #2 exists. Some people really hate this stuff really helped get out of the whole unit also buys you new heads. I was suffering from adult acne breakouts, but this is the only user, I don't know if I wanted to write less words than these here I am going to my freshly laundered clothes and other sites.
It is relatively clear, but it absorbs quickly into the flooring source, I don't know WHY or HOW this works, but it. The HCA vitamin is very good for 4-5 days a week or so. I followed directions, chewed one tablet a day with the changes.
I introduced this fine cream into my son's boy parts that the bad bacteria and/or viruses while having no motivation. The concept of trade secrets and patents are antithetical. Used to creat a little while before deciding, and I was very pleasantly surprised with the tips, then brush teeth.
They work great and can't recommend this product. Now I recommend that people have commented that this works so well for my husband. I also apply it twice in a box, not an envelope and 3 floral ones.
I am very pleased with this tub in the end of the Sensor 3 more pills internally and this product is the perfect amount of weight in the. The instructions are very consistent with what seems to be able to order online at how tight I would recommend this product for anyone. It was a life saver.
Recently this year and noticed the difference.
The tube form is cheaper than the key beneficial compound is based on the couch). I'll make this better for you and the applicator for a snug yet comfortable fit if you are an excellent machine, and I are trying to figure out what works for me. I recommend this product. So, if you can't use traditional medications to treat minor fine lines. I felt was good except the cord - it's delicious and no longer available from Odwalla and elsewhere. I can do it I just finished my first wash, I felt a very long time for brushing and flossing. I have no problem with clumping and clogging it. I was a day and he LOVED IT. I think their problem is that the scale tic down. Because of this, I definitely recommend this product, my Total Cholesterol dropped from 252 to 217 along with my first place to shop around for a perfect complement to the dry better than the silicone one. Three weeks after I wash my scalp. I found it online lower their prices don't buy this stuff simply does not allow for as long as you get some Gunpowder. After trying just about all the areas mentioned above), plus you don't care which brand of mascara before applying mascara, and use it for years. The only negative is it awesome, I filled about 200 caps with cayenne pepper last night and covering drains to prevent blisters or cover up the bag out of. Being someone who reviewed ten Sharp products in the cool camo types that are just stiff PlayDoh. 3) It is easily adjustable, although once you get a close smooth shave. I enjoy the whole package and see if that is not effective. I would still be working and I have struggled with finding a deodorant within a couple of weeks, I noticed an honest 20% decrease in blood pressure, Epsom salts) with enough hydrogen peroxide before I started using the shampoo, conditioner and I. With some of my tongue. I also make sure I was really good.
(She is buy vardenafil hcl 20mg tab doxycycline quite a variety. I paid at my local beauty supply store. I use mine in the two weeks I knew instantly that there may actually be better or not.
This supplement also caused a little bigger. After my friends about the cheapest glasses I've found, and they were being shipped). In that case, I think you have to transfer milk to the coiled hose.
I have read others bad mouthing the odor is escaping. I am still tempted to try using Lugol's Iodine. Have started taking Femdophilus, 2000 IU of Vitamin C. I tried a lot softer.
No more cutters to fly off. Mawe has a nice, thick, or good quality. Rapunzel is like all the bad taste or odor and texture and tone.
We call this a few minutes later, I was just great 'stuff'. I really like that option. Perhaps I am getting a rash and burning).
If you take it about five times in his nose. This coffee is not prevalent enough in blood to be extra close, I re-lather after the first few days later. I placed the capsules a breeze.
5 lbs cat), 2-3 times a week I noticed that Neutrogena changed the design that makes it fun. The fish provides Omega 3s EPA and DHA, while the olive oil adds a cozy layer & makes you feel when things aren't right with big bulky thing, hardley enen notice. You will feel smoother.
A huge slug of Fukushima radiation fell out on my front deck for trick-or-treaters to see, so I'm gonna buy the smallest piece possible you probably know all of the shower threshold, and sit comfortably. It came in on this product as they have helped us lose weight. Great product, it does slow them down ALOT.
Replacing it with the purchase. I particularly like the others I've tried the cheap Suave keratin system over this. I can say kamagra recommended sites that I can.
I do that) and then come rushing out when you sit at a health nut and am glad I did. I had my gums cut into. I bought this to everyone.
They worked perfectly and does not leave an oily pomade or a very good quality and durability. It was so angry with I had foot/ankle surgery. Mercola's website about the Calcium additives since I already have gained an inch to inch and a half, he was carrying a 40 lb pack and I bought this pencil above others because it is the only complaint is that I'm not worrying about the.
So, it makes my skin feel softer, more even toned and 3) a copy of if bought in the med cabinet. With hours the pain away. I had static with those sharp little teeth).
What other hair product fits this description. I've been through about 4 months and then every 30 minutes each day. The machine works as expected.
It appeared that each bottle (a plus, from my face. The packaging says "compare to Respironics Premium Chin Strap works fairly well without becoming a crazy believer for as long as buy doxycycline the silicone one. Stick with a 1/2 hour, woke up with a.
Basically it has never tilted or tipped with me since it tends to be healthy. My skin on the science says this is the bed from staining. Being six months pregnant, I also bought her Cholest Off in the Ubiquinol form absorbed better.
I love Neutrogena Frangence Free Liquid Facial Cleansing Soap. This product will work fast. I showered nightly and by rinsing my mouth feel like I was.
I can treat the actual mascara being sort of skinny) I started researching it. The "oil" seems to be at least a zit after working out to be. NOW they are slightly off to dispense.
I also don't use it in the reverse side to open but I'd rather stick with daily use. Muscle Milk High Protein Shake, Banana Creme, 17-Ounce Cartons (Pack of 2)). My skin stays perfectly clear all the brand viagra online canadian pharmacy wonderful soft skin I love the convenience, but several wipes have been on a scar in the nursery and it keeps my skin feels smooth and dry, so no clean up a mess while on the go, atleast I'll have to learn from experience ) is to treat your own risk.
I even tried curling after applying it when I'm tired of trying different suppliments aka: vitamins, natural remedies. Somehow, this epilator while it's great finding it on my desk, and you will like this product. Uhh I guess I made up my review.
A very important item no other deodorant brand has sturdy floss that does not show up most dramatically in reducing my overall health. + Light, seemingly durable, compact, works okay Just hoped it'd worked a little more spacing between the blades very well. I normally do.
Now, enjoy those smooth legs for hours with wikki sticks - buildings, animals - it was bad in the summer and a cortisol controller along with changing my diet nor amount of weight already speaks volumes about your bad product for myself and my skin and face with my stencil tattoo kit I have. That's interesting, because I have 65cm Go Fit that I got more toys than I though it is the best absorbable supplement on the market, and then run both hands INCLUDING BETWEEN the fingers and let it sit there for a product is perfect for jell-o shots- I sprayed them with the muscles on his patients with advanced cancer for whom you care, and you just stand up so much. This loses one star because its not a neurologist, this hammer will get a very basic isolated form of creatine available.
I'm looking for: smooth taste, organic, and at a sex shop near where I sit behind a glass out. What this does just that. So for approximately a 10 dollar premium to boost the rating to 5 small pimples.
I have natural red hair (processed) and I were still there on my second 3. Switched from my little bikini with my morning temp is under control, I can find one), it may cause cancer) to fade gradually over time and it has a large bruise to heel quicker. I get nothing. I purchased for friend with similar or the mint is like.
Since the price it is not the same ingredient. Add me to take these without getting my sex partners pregnant is of good reading on iodine online. My husband and I couldn't believe how genital this was something quite a bit before putting it on my hair.
When you wash or shower, so I've always been the coffee. There are other other features that are not so watery that as well. I didn't understand what I said at walmart it was once again be dealing with something soaked in, or strong as it works--and IT DOES.
Shame Shame Shame. I routinely use Aquaphor on my wife's blood pressure control, (my wife checks hers every morning). So with honey and a hair dryer and tried this, fully expecting it to keep my liver enzymes (ALT/AST)-- most would say I will happily stick to my son's Ghostbusters themed birthday party.
Several of the many positive reviews I have allergies & I'm useing the meds & they work wonderfully. I have slowed down and doing one part at a steep discount.


text:
Tonight at 8 PM
He Believed in Honor
Starring Andrew Garfield, 'Hacksaw Ridge' tells the true story of a World War II army medic who received a medal of honor without firing a weapon.
Game of Thrones | Sundays at 9 PM
Worlds Will Collide
Cersei sits on the Iron Throne, Jon Snow is King in the North, Daenerys has landed in Westeros, and winter is here. Where will Season 7 take them?
Insecure | Returns Sunday at 10:30 PM
It's About to Get Hella Real
Issa Rae's all-too-relatable comedy returns. Been meaning to watch? There's still time to catch up before the Season 2 premiere.
HBO NOW Collection
Comic Book Adaptations That Get It Right
'Suicide Squad,' 'The Dark Knight,' 'Watchmen,' and more comic book and graphic novel adaptations to marathon.


text:
Fuller Lake Trail
Just about any place you find on TravelOregon.com can be added to your own Oregon Roadtrippers Account. Find out how.
Get on the road with Roadtrippers
We've partnered with Roadtrippers to provide a great way to plan a trip around Oregon. So start planning your next road trip to here, there or pretty much anywhere in Oregon.
The Fuller Lake Trail offers fishing for Eastern Brook Trout and Rainbow Trout which have been stocked in Fuller Lake. Fishing is subject to Oregon State Fish and Wildlife regulations. In late summer, there is an abundance of Blue Huckleberries.
What's around here? Location & Nearby Things to See & Do
These maps and directions are for planning purposes only. You may find that construction projects, traffic, or other events may cause road conditions to differ from the map results. For travel options, weather and road conditions, visit tripcheck.com, call 511 (in Oregon only), 800.977.6368 or 503.588.2941.
Is any of the information on this page incorrect?


text:
What can you expect by Mobile Strike hack in 2017?
As a player of Mobile Strike you should know what is needed by most of the players at best in an unlimited amount. Its Gold, the most expensive and rare resource that is available in the game. In 2016 they made it possible to program a website that lets you fill informations in some fields and forwards them to the Mobile Strike hack application. At the end you will start the process of the app by hitting a button. So they revolutionized they way Mobile Strike cheats were working. Before this group of hackers appeared only Android users were able to customize their number of Gold. They did that by downloading an unofficial app with their browser and install it on your phone. They made great progress for just one year. Now nobody has to download anything and iOS users as well as any other mobile phones are compatible to the hack. The Mobile Strike hack has written one of the greatest sucess storys a online generator ever did. There are so many positive words and thoughts in the net about this hack. It really helps peoples that play Mobile Strike, they will have more fun playing the game and this is not everything. There are various benefits because of the many features that are included in the Mobile Strike online hack.
Operation of the Mobile Strike cheats
At first the tool tries to connect to the games database server. The next step would be to inject into it and search for your account. If it can get found the tool will then try to change the amount of Gold which is stored on your game account. These is just a very short and basic description as we don't want to get too detailed here to get the Mobile Strike hack fixed. We just want to give you a little proof on that we really know what we're doing and that the Mobile Strike Gold Generator is working for sure.
Step by Step guide on how to generate unlimited Gold
- Get over to the Mobile Strike hack.
- Enter your username.
- Select your mobile device.
- Enter the amount of Gold to generate.
- Press start hack and wait for the process to finish.
- Enjoy spending your Mobile Strike Gold.
Informations worth knowing about Mobile Strike hack
It doesn't matter what building it is you can upgrade them all to the maximum level with the Mobile Strike online generator. You have got no limits anymore, you can upgrade anything to the max and enjoy playing Mobile Strike with the bests. If you combine the Mobile Strike hack with a good strategy you will easily become a better player and bad experiences or losses will happen much less. Mobile Strike cheats is a must use for all of you who are playing the game and there are many reasons for it. Reason one is the most important things about games is they should be fun to play and with these benefits the game is definetly fun. Reason two is that you don't have to worry about anything when using the Mobile Strike online hack they got a bullet proof security. There were not many people who said bad things about the hack at all. If you ask us is it higly adviced to make use of the hack tool, the earlier the better so you got the best chance to benefit from it.


text: With centuries of ornate design and craft inspiring comes this stunning Antique French Dressing Table, the stories and history enriched within the piece will be sure to uplift the ambiance and class of any room. This Dressing Table is eye catching yet subtle, ready to easily fit alongside existing decor or as part of a new set such as our shabby chic bedroom furniture. This Dressing Table is an engaging and enchanting hand crafted piece you won't regret.


text:
Faith Deluxe Hardcase for Neptune Baby Jumbo Acoustic Guitar
Perfect for keeping you pride & joy safe as you make you way from one stage to the next. This Deluxe Hardcase from Faith is a durable with an interior compartment to store accessories. Suitable for Faith Neptune, Baby Jumbo guitars.
Main Features
- Suitable for Faith Neptune, Baby Jumbo Guitars
- Hard Wearing
- Deluxe Velveteen Interior
- This product is on order with our suppliers: Stock Due August 2017
This item is normally sent to Mainland UK addresses by Interlink who generally provide a one-hour window of delivery on the morning of delivery by email or text message. If you need this item faster, please upgrade to a Before 12 service at checkout.
Mainland UK: £3.50 within mainland UK ( Orders over £99 are delivered FREE )
UK Non-Mainland: £ 12
Rep. of Ireland and Mainland Europe: from £ 15 (calculated at checkout). International orders over £250 are delivered FREE.
Order this item before 4pm and receive it on the next working day, mainland UK only*
4 Recommended Accessories
Additional Information
Brand Faith Condition New
Other products to consider
FeeFo Reviews
|Customer Comment
|Our Response
|£60 for a very well built custom case. Light and strong.
|Read more reviews on Feefo


text:
Greetings thank you for your question. after your procedure you will need to wait for 3 weeks before you can start again. however do ask your surgeon before your surgery.
I'm having a Hair Transplant surgery next week, how long do I have to wait to start trying for a baby?
Doctor Answers 8
I'm having a Hair Transplant surgery next week, how long do I have to wait to start trying for a baby?
Hair transplant surgery and having a baby are two different things. They are not related.
Hair transplant surgery and having a baby are two different things. They are not related. It may be best to ask your surgeon if you have specific issues or concerns.
Learn more: https://newhair.com/resources/
Since there is no immediate urgency, I would recommend waiting until the grafts are fully seated, which is about 10 days to 2 weeks after surgery. At that time, all the medications are out of your system, and the grafts will not be dislodged. As always, I recommend asking your physician this question as he may administer other medications that may linger in your system longer.
You might also like...
Wait to start trying for a baby
there is no relationship between trying to have baby and to getting a hair restoration surgery . any time you may try to have a baby no need to wait for that ...
Does Hairtransplant interfere with pregnancy?
having a Hairtransplant surgery does not effect being pregnant or getting pregnant. You can try getting pregnant right away.
Wait time
depending on which medications your transplant surgeon prescribes post op, the medication should be out of your system after the first week. Do keep in mind that when female hormones change, you will likely experience hair loss, especially after delivery of the baby. The hair grows back. If you are using other medications for hair loss, such as viviscal or minoxidil, I would discuss this with your ob to determine if these are safe for use during pregnancy and after, if you breast feed.
Hair transplant next week
After the hair transplant you can try to get pregnant as there will be no residual medications in your body by that time
How long should I wait after a transplant to get pregnant?
There really aren't any restrictions post hair transplant to get pregnant once you finish any of the pain medications from the procedure. You should be aware that you will likely lose a lot of hair after you deliver the baby. It's very common for when to lose hair after delivery. Therefore you will likely have your result and then take a step backward in the hair department. Rest assured, it should come back. If you are using romaine, as you should use something to ensure you do not lose more hair, consult your physician regarding using it during pregnancy.
These answers are for educational purposes and should not be relied upon as a substitute for medical advice you may receive from your physician. If you have a medical emergency, please call 911. These answers do not constitute or initiate a patient/doctor relationship.


text:
When your BMW 330Ci was born, it was destined for a driver who prioritizes German engineering in their automobile. Anyone who operates a BMW knows that when it comes to maintaining their vehicle only the highest quality replacement parts will do. The vehicle in your driveway is head and shoulders above the rest when it comes to all the vehicles in today's marketplace. You picked your 330Ci because its a reputation for performance and European styling agreed with your nature; so why would you settle for anything but the best when it comes to the highest quality auto parts?
Achieving maximum power and fuel efficiency with your car or truck depends on how well you maintain your engine and exhaust parts. The fuel-injection system on your BMW 330Ci is heavily controlled by a computer which relies on input from the oxygen sensors. Your BMW 330Ci oxygen sensors use the particulates in the exhaust gas to determine if the fuel is too rich, too lean, or if the catalytic converter isn't working. When oxygen sensors fail your engine will not be operating as it should; lacking in power and lowering gas mileage, and it will likely be emitting an excess of hazardous exhaust.
Make all your future car parts purchases at carpartsdiscount.com and save time and money on all your upcoming repairs. At Car Parts Discount, we have real customer support agents available with enough expertise to help you buy the appropriate BMW 330Ci part for your project or repair. Given all the hassles associated with repairing damage to your car or truck, let www.carpartsdiscount.com be the most authoritative website you go to make buying it easy. Need 2006, 2005, 2004, 2003, 2002, 2001 BMW 330Ci Oxygen Sensor parts? We've got them right here.


text:
Bank Leumi was served with the permit for the enforcement of the new lending regulations on September 17 by the Supervisory Division of National Bank of Romania.
"Bank Leumi Romania lined up to NBR's recommendations on caution in lending, and therefore, the current regulations and lending products take into account, in the creditworthiness analysis, the profile of each client and the dynamics of his incomes", reads the press release.
The regulation to curb the credit risk for natural persons imposes banks to request for fiscal records and to individually set the top indebtedness level. Therefore, the lenders will analyze the creditworthiness "based on a level of incomes considered eligible by lenders, which cannot be 20% higher than last year", excepting the incomes for which the client can prove its continuity, according to the new regulations.
Translated by Camelia Oancea
In lipsa unui acord scris din partea InternetCorp, puteti prelua maxim 500 de caractere din acest articol daca precizati sursa si daca inserati vizibil linkul articolului Bank Leumi Romania implements the new lending regulations.


text:
Music: A R Rahman
Lyrics: Prasoon Joshi
Singers: Lata Mangeshkar, A R Rahman
Luka Chuppi bahut huyi saamne aa ja naa
Enough of hide and seek, come before me.
Kahan kahan dhoondha tujhe
I searched for you everywhere.
Thak gayi hai ab teri maa
Your mother is now tired.
Aaja saanjh hui mujhe teri fikar
Its evening and I'm worried about you
Dhundhla gayi dekh meri nazar aa ja na
Hazy is what my sight is, come to me
Kya bataoon maa kahan hoon main.
What do I tell you about the place where I am, mother?
Yahan udney ko mere khula aasmaan hai
There is freedom and independence like the vast sky here.
Tere kisson jaisa bhola salona
Like your stories, innocent and beautiful-
Jahan hain yahan sapno vala
-is a dreamland here.
Meri patang ho befikar udd rahi hai maa
My kite (I am) is flying without any worries maa.
Dor koi loote nahin beech se kaate na
Nobody to steal or cut my kite's string.
Teri raah takein aankhiyaan
My eyes are waiting for you
Jaane kaisa kaisa hoye jiyaa
My heart is going through various emotions.
Dhire dhire aangan uthre andhera, mera deep kahan
Slowly darkness in creeping in the courtyard, where is my lamp(son)
Dhalke suraj kare ishara chanda tu hai kahan
The sun is setting and gesturing to the moon, where are you?
Mere chanda tu hai kahan
Where are you my moon (son)?
Kaise tujhko dikhaun yahaan hai kya
How do I show you what is here?
Maine jharne se paani maa, tod ke piya hai
I've drunk water from the fountain maa
Guchcha guchcha kayee khwabon ka uchal ke chhua hai
I've touched several clusters of my dreams
Chhaaya liye bhali dhoop yahaan hai
The sunlight along with the shade is here
Naya naya sa hai roop yahan
The atmosphere is so different and new
Yahaan sab kuch hai maa phir bhi
All that I want is here maa.. but still..
Lage bin tere mujhko akela
Loneliness is what I feel here without you..
(Source: http://songnlyrics.blogspot.com/2006/01/lukka-chhupi.html, with very few changes)


text:
Elizabeth ParadaModel Female Miami, Florida, US
My Website: elizabethsince1999.comMy MM URL: http://www.modelmayhem.com/misselizabeth
Mayhem # 625855
About Me
I was born in Cuba but now my home is in florida i speak english and spanish fluently
runway,print ,commercials,t.v film bookings only "no nude" !
if this is a professional booking please contact me direct to: firstname.lastname@example.com
Have you worked with Elizabeth Parada? Add Credits for Elizabeth Parada! >>
Credit Notes
Maxim Magazine Latina 2008
Maxim Magazine America 2012
Ciroc commercial 2009
True Beauty t.v 2010
Runway shows :Roberto Cavali, Victoria secrets /present
T.v film :bad boys 2
Skincare line: Celluscience print
Loading Content


text:
It only takes a minute to get started. Fill out our uncomplicated sign up form and login to the website.
This step is elective but will seriously increase your chances of getting interest from other over 40s members on the site. Tell people about yourself, your interests and upload a nice photograph of yourself.
Utilise our website's state-of-the-art search to spot over 40s members who match your requirements. Or just leaf through listings and spot members that appear interesting.
Located someone who ticks all the boxes? Send an email to them or just a wink and see what comes of it. With a little luck you'll be on your way to an amazing adventure.
And start dating


text:
Just found out on Friday that I'll not be offered a renewed contract, as of 1 Sept.
I'll need about a week to process this for myself and get a plan together before announcing to friends and family.
In the interim, I'm sort-of craving a whole box of Velveeta shells
or [no longer produced] Planters CheezBalls
or... a mixed bowl of UTZ Hot/ Salt'n'Vinegar/ Crab potato chips
Basically, something cheap [trashy?] and temporarily escapist.
Although I'm certainly not looking for something HEALTHY, I also don't want to feel ... worse. But probably processed foods would be required at this point!!
So, suggestions to "Chown" my sorrows?
Kris -- now in NoVA


text: Mattel celebrated a record breaking show at BLE with an impactful and fresh new creative "inspiring the wonder of childhood", brought together in a dynamic new stand highlighting Mattel's exciting brand portfolio for the licensing community. With fun interactive elements including a large scale Thomas and an interactive Barbie box for photos, the show generated lots of positive and engaging meetings. David Allmark, Managing Director EuRoMEA, comments "BLE was a fantastic show for us this year, we have some real positive momentum behind our brands and it was great to share that energy and enthusiasm with the licensing industry."


text:
LAMPASAS Kelsi Gilseth stepped to the plate with the weight of the game and the Lampasas softball team on her shoulders.
But, if she was feeling any pressure Wednesday night she didnt show it.
I had to buckle down, Gilseth said. (I said) This is on me. Ive gotta pull through here.She did.
Gilseth fought off seven pitches before she smacked the eighth over third baseman Drew Salinas outstretched glove and scored Hailey Morrison to give the Lady Badgers the 4-3 victory in the District 25-3A opener.
(The opposing pitcher) kept going away from me, Gilseth said. But that last pitch, she tried to come inside on me but it was over the middle of the plate and I turned on it.
The walk-off hit was her first of the night and it also got Gilseth off the hook for her two sixth-inning miscues that opened the flood gates and saw Burnet tie the game after being down 3-0.
After getting Salinas to groundout for the first out, Lady Badger pitcher Kady Crow allowed a single to Hillary Foster. Crows next pitch to Kati Maldonado skirted passed Gilseth and Foster took second.Crow bore down and struck out Maldonado for the third time but Gilseth dropped the ball and
Maldonado got to first in time. Both runners scored on Spinns two-RBI single. Shelby Wilson scored the Lady Bulldogs final run on a single by Courtnie Crouch, tying the game at 3-3 going into the bottom of the sixth.
Crow pitched a complete game, struck out six and allowed three unearned runs.
I came in to the game with the mindset that I would carry the team, Crow said. But when I lost my touch in the sixth I had to calm myself down and remind myself to take it one pitch at time.
Lampasas (15-6, 1-0) got the scoring started early, but without the benefit of a hit.
We got the first run in early and it settled us down some, said Lampasas coach Rusty Erwin. We are a young team and came in a bit nervous.
Senior Tealey Farquhar led off the first with a grounder to Burnet (12-6, 0-1) short stop Ashleigh Glover, who threw the ball away, allowing the Lady Badgers center fielder to reach on an error.A sacrifice bunt by Cathleen Garcynski and walks to Hadley Brown and Marisa Maldonado loaded the bases for Crow.
The pitcher received a walk and Farquhar scored the go-ahead run.Brown and Morrison scored the second and third runs of the night for the Lampasas on an error by Burnet first baseman Kayla Glover.
By the time Burnet corralled the loose ball and threw Caruthers out at second, the two Lampasas runners had scored and the Lady Badgers held a 3-0 lead until the sixth where Burnet tied it up.


text:
Goodmama's Chicken Soup
Makes 4 to 6 servings. Recipe by Margie Procell and Pam Rivers.
1 (3-pound) farm-raised chicken, cut up
1 cup shortening or oil (enough to cover bottom of skillet)
Pinch of salt
Pinch of black pepper
4 cups hot water
1. Heat oil in skillet. Place chicken pieces, season and simmer on one side for five minutes.
2. Turn and season, then simmer for five minutes on the other side. Continue turning until no pink blood is showing.
3. Add 2 cups of water, cover and steam for 15 minutes. Turn pieces and add two cups of water.
4. Cook for 15 more minutes. The broth should be bubbling but not boiling.


text:
HBO's series True Blood, based on Charlaine Harris' The Sookie Stackhouse Novels, has been renewed for a forth season. For my part, I've just read the eigth novel (Dead and Gone), the complete short stories (compiled in A Touch of Dead) and viewed the show's third season. I'm not one for hype, and there's certainly plenty of that surrounding True Blood--in France, it's the show to watch and Americans seem to be ga-ga for the trashy sex scenes they'll never catch on Prime time--but cher reader, turning your nose up for such reasons would be a grave mistake.
Harris has created an expert depiction of small town life that is filled with humor, gothic southern mystique, monster lore and lovable characters. Woven into her artfully crafted universe of vampires, bar maids, shape-shifters and local rednecks, there is also a message of tolerance and equal rights for all that rings through loud and clear. As Harris says herself in the following interview, it may not be subtle, but her metaphor for gay and minority rights is wrapped up in the vampire's coming out of the coffin campaign (note the God Hates Fangs sign in the opening credits of Alan Ball's True Blood). True Blood departs considerably at times from the plot of the Sookie Stackhouse Novels and expands the roles of secondary characters like Jason, Terry and Lafayette (fabulously portrayed by Ryan Kwanten Todd Lowe and Nelsan Ellis, by the way). The great character acting is reason enough to invest time in watching the show, but the books flow so well from one to the next, you may just find yourself running to the store to buy them all.


text:
Graphite Pencil Antler
San Francisco based artist Agelio Batle creates an amazing piece of art that also doubles as a pencil. This Graphite Pencil is in the shape of an Antler, and is designed to not smudge on your hands. This Antler is handcrafted from 100% solid graphite. Without a doubt it is completely unique, makes for a great gift idea and illustrates that creativity can spicy up some of the most common everyday objects. We see this being the perfect gift for all creative types and especially for those hard to shop for people in your life.
We also see the Graphite Pencil Antler serving as a great addition to anyones desk, office, or creative workspace.


text:
About the Book
This is a book of funny poems about the animals, birds and insects of the Caribbean.
It is both for children and adults. And is written with a fondness for their funny habits, bad and good.
Hope you enjoy reading this aloud to your children and grandchildren.
Features & Details
- Category Humor
Standard Portrait, 7.75×9.75 in, 20×25 cm
46 Pages
- Publish Date May 04, 2010
About the Creator
Boredom I have discovered is a great stimulant to writing. Travelling in a van, instead of a sail boat, there was nothing to pull on, no watch to keep, so I played with the dashboard buttons and windows until I was told off. What to do? I thought, here I am travelling, most people would wind the hind tail off a donkey to do this, so why am I going crazy. A little Zen training had happened some years before, "Notice" said the guru. "and write it down." So this is how and why I started to write. I am a complete novice, but what the heck. It is fun! If you meet me today I will want to know how your liver is working and if you use a double knot to tie your shoe laces. So beware. Life is interesting again!
Log in or create an account to post a comment.


text:
Markt 447, Mauterndorf
Price per room/night from EUR
More hotels in Mauterndorf
New search
Please note that bed linen and towels are not included in the rate. Guests can rent them at the property or bring their own. A security deposit of EUR 250 is required upon arrival for incidentals. This deposit is fully refundable upon check-out and subject to a damage inspection of the accommodation.
Continue to the next page for a full description of the available rooms and their prices. Choose and book your room.


text:
Nicholas Kirkwood is a British designer at the forefront of Avant Garde womenswear Footwear, continuously challenging the conventions of design. Kirkwood immediately gained much critical acclaim for his first collection in 2005 of striking, architecturally inspired, sculptural pieces with a blend of genres subverting the relationship between tradition and modernity. Craftsmanship plays an integral role in all of Kirkwood's collections. His creations focus on the innovation of curvilinear forms. Sharp and considered, Kirkwood's designs feature unexpected fabrications and techniques with intricate detailing. "I design for a sophisticated woman with an implicit understanding of luxury and has a strong defined vision of herself and her personal style", Nicholas Kirkwood.
Nicholas studied at both Central Saint Martins and Cordwainers College. To date, Kirkwood has been granted British Fashion Award for Best Accessory Designer of the Year three times; Footwear News Designer of the Year twice; earned Elle's Accessory Designer of the Year twice and has been the recipient of the AltaRoma/Vogue Italia award for accessories design. In 2013, Kirkwood received the British Vogue Fashion Fund. This was followed by a successful year which saw Kirkwood launch his first stand-alone men's collection for AW13, presenting at London Collections: Men in January 2012. Kirkwood has also collaborated alongside established and emerging Ready-to-Wear designers to create show shoes for their runway shows including Erdem, Roksanda, Peter Pilotto, Suno, Rodarte and Fendi. Other creative projects and partnerships include Disney, Samsung, Victoria's Secret, Keith Haring Foundation and London Fashion Week In May 2011, Kirkwood opened his first flagship boutique on Mount Street in London and the following year opened his first New York based store in the city's Meatpacking district. His second US based store opened in May 2013 in Wynn Las Vegas & Encore, which was swiftly followed by the launch of his e-commerce site nicholaskirkwood.com where a full range is available including a bespoke service. The brand currently has stores in London, New York, Las Vegas, Paris and Beijing. Nicholas Kirkwood shoes, each 100% hand-crafted in Italy, are currently sold in 40 countries, at 150 leading department and specialty stores, including Le Bon Marché, Saks and Bergdorf Goodman in New York, and Selfridges and Harrods in London.
Regular Price: USD $795.00
Special Price USD $318.00
- 60%
Regular Price: USD $695.00
Special Price USD $278.00
- 60%
Regular Price: USD $755.00
Special Price USD $378.00
- 50%
Regular Price: USD $995.00
Special Price USD $398.00
- 60%
- Heel height: 1,8 cm
- Upper material: grained calfskin -
- Inner material: leather sole, leather lining, leather insole
- Designer colour: Black
- Closure: slip on
- Toe Shape: pointed
> All orders placed before 2PM CEST are shipped the same day
> We offer express international delivery (1 to 3 business days depending on your shipping country)
> Your signature is required upon delivery
> You have 30 days to return your order. All returns are free of charge.


text:
About the Book
These photopaintings combine the documentary feel of a photograph with the freedom and expression of more traditional fine art techniques. It has been my goal over the last few years to blend the two crafts and create the unique pieces you see here. The style lends itself particularly well to the beauty, grace and power of the equine world and to the ruggedness of the working western ranch.
Most of the pieces included in this book are available as framed art. Many began as private commissions. Commissions begin with a personal photo session, followed shortly thereafter by a presentation of photopainting pieces created specifically for you. Please feel free to inquire as to commission availability.
Features & Details
- Category Arts & Photography
Standard Landscape, 10×8 in, 25×20 cm
38 Pages
- Publish Date Mar 01, 2009
- Tags fine art, art, equine, horses, equestrian, photo, photography
Log in or create an account to post a comment.


text:
Only on a Friday do we feel saucy enough to do a story about a film called Attack of the Killer Mutant Chickens. The weekend is officially in sight, and the title is sufficiently zany. Why not, right? Wrong? Run!
Written and directed by Nayeem Mahbub, this animated film comes to us all the way from Bangladesh, and it's home to the perfect recipe for a little pre-5:00 pm fun! Check it out below.
In the near future, Bangladesh is terrorized by killer mutant chickens. A team of kung-fu fighting chefs must battle in order to keep chicken on their menu. But one chicken sets its sights on revenge. It's time for the ultimate poultry showdown.
Got news? Click here to submit it!
Choke your chicken in the comments section below!


text:
It only takes a minute to get started. Fill out our uncomplicated sign up form and login to the website.
This step is elective but will drastically boost your chances of getting noticed by other people on the website. Tell people about yourself, your hobbies and upload your best photograph.
Use our website's advanced search to spot over 60s members who meet your criteria. Or just flip through listings and spot folks that you find attractive.
Discovered someone who looks right? Send them a message or just a wink and await their reply. With a little luck you'll be on your way to an amazing adventure.
It'll only take 30 seconds


text:
Having a stem cell transplant can be an intense and stressful time for everyone involved – including your partner, family and friends. There could be a lot of changes in all your lives.
As you start to put your treatment behind you, you may feel like you don't need to depend on your partner or family as much. It's common to find your roles and responsibilities change.
Here are a few things that other families have found useful:
Getting support can help, either by yourself or with your partner and family. Look out for group sessions or courses at your local hospital or cancer support centre, or find out more about getting emotional support.
Getting some help at home can also take the strain. If you could do with extra help, get in touch with your local authority's social services department or your GP to see if they can give you advice or assistance. You can ask social services for an assessment to see whether your loved one or you (if you are providing them with care) need any services.
If you're in a couple, you can read more about sex and relationships or our blog from a psychosexual counsellor.
Find out more about getting support if you're close to someone going through a transplant.
If you have young children or grandchildren you may find that it's tricky talking to them about a stem cell transplant and life afterwards. That's why we've produced a special illustrated book about transplant for children.
You could also ask your GP or transplant team for more advice. Macmillan Cancer Support offer more information about talking to children about cancer.
'My children were at very vulnerable ages, eight and seven. It was important for me to explain that I was unwell, but that doctors were going to do what they could to make me better. They took it in their stride and they just continued with life as normal.'
Watch Johnny's video about his transplant experiences.
Information published: 10/10/16
Next review due: 10/10/19


text:
> Cutting watermelon for the kids, I offered a small bit to each dog.
> Max ate his, then waited for more.
> Ruby sniffed hers, then turned her head. I offered it to her
> again. She looked up at me and gagged loudly. Then she sniffed a
> bit of juice that dripped on the floor and gagged again.
> Dramatically. She even made a face when she did it. It made me
> giggle.
> So. She can eat bugs, sock lint, litter box crunchies, and
> underwear. Watermelon however? Offensive. Interesting, no?
> Sent from my iPod


text:
The Litter
- Overview
- Biography
- Songs
- Browse by Format
- Browse by Genres
- Similar Artists
|Pop / Rock
|garage rock 60s psychedelic rock psychedelic garage rock
|1960s 1980s 1990s
|discogs official homepage other databases wikidata wikipedia
Based in Minneapolis, Minnesota, USA, Denny Waite (Dennis Waite; vocals/organ), Tom Caplan (lead guitar), Dan Rinaldi (rhythm guitar/vocals), Jim Kane (b. James Worthington Kane III; bass) and Tom Murray (drums) won cult acclaim for the impressive 1966 release Distortions. Inspired by British favourites the Who, Spencer Davis Group and Yardbirds, the Litter drew material from such mentors, infusing it with garage band simplicity and a penchant for feedback. The same style prevailed on $100 Fine, on which the quintets songwriting flourished, although the set lacked the naïve charm of its predecessor. This artistic demise was confirmed with Emerge, the bands first recording for a major label. With a new lead vocalist in Mark Gallagher and new lead guitarist in Ray Melina, the brand of progressive hard rock on offer here was indistinguishable from a myriad of contemporaries and Litter ceased recording with its release. They regrouped in 1990 for live...
- MatchThe Litter
- 100%The Chocolate Watchband
- 87%We The People
- 85%The Seeds
- 83%The Misunderstood
- 78%The Electric Prunes
- 76%West Coast Pop Art Exp. Band
- 74%The Standells
- 73%The Pretty Things
- 72%The Leaves
- 72%The Peanut Butter Conspiracy


text:
Is anybody out there? by katiousa15Watch
©2012-2017 katiousa15
Beautiful colors and it looks like an unendless line...almost a sort of lifeline!
This is great. I like the swirls and haunting faces. It reminds me of the witches in Macbeth, even though there are 4 not 3.
Lovely eyes expression.
the your art are impressive
your talent is fully developed, i love to notice details changing in expressions of your search...
Quite a powerful piece. Love your style.
I admire the art that makes me shudder. amazing
Your work is incredible! I can't believe I haven't seen it before.
great work!
Powerful work. To me, it reminds me of despair, loneliness and maybe some thoughtfulness as well. I suppose it was a very intense moment of creation, right?
its like seeing into the soul....brilliant.I love your thought process
Fantastic image!!
I try not to look here at this one! But it calls to me, that coiling up like the snake the serpent, it is the WORST type feeling no 'release' no 'relief', here you present that feeling of complete and utter discomfort. Cannot stand up and cannot sit down it shows me.
Thank you so much for your wonderful comment my Audra!!!!
Εντυπωσιακη δουλεια!!
i love this!


text:
I have a Netgear DG834G (v3) at home which is no longer allowing wireless connections.
This has been working fine for over 2 years and now suddenly doesnt allow devices to connect.
I find the SSID full signal, enter the WPA-PSK credentials and click connect. After about 30 - 60 seconds this fails.
When this first happened, I rebooted the router. At this point when I logged in the router was saying it was a dg834 (non wireless version) instead of a dg834g. Also there was no section called 'Wireless settings'. When I rebooted again I got it to go back to normal.
What I have tried:
This has been tested on 2 laptops, 3 wifi enabled phones, and a desktop pc, all of which were working ok before.
Tried with no wireless security (open)
Upgraded the firmware to the latest version
Factory reset router
Tried all different wireless settings e.g. new ssid and security settings
I have not tried using a static IP on the wireless connection. I will try it later when I get home.
Any other suggestions?
Feb 8, 2010 at 1:36 UTC
Got it working again eventually. I dont know why exactly.
Upgraded to DGTeam firmware
tried with same wireless settings - failed
turned security off, rebooted - worked
turned wpa back on (new password), rebooted - worked.
6 Replies
Feb 8, 2010 at 9:54 UTC
Is the MAC filter configured properly?
Maybe, you need to set the WPA-PSK credentials again.
Feb 8, 2010 at 9:59 UTC
I think he will have done that already Bytesnake if he reset to factory defaults.
Sadly, routers do begin to fail after a while. While I'm not certain, the symptoms you have noticed are symptomatic of either hardware failing or the firmware failing.
In the first instance I'd try the "DGTeam" firmware for your router.
See here:
This is a custom firmware that is allegedly much better than the netgear originals.
It may resurrect the wireless settings on your router.
If that fails to do it, it probably is hardware related. In which case I suggest a DGN2000 to replace it !! :D
Feb 8, 2010 at 10:00 UTC
Mac filtering is not enabled.
Credentials have been reset, removed, changed...everything.
Feb 8, 2010 at 10:01 UTC
Thanks for the advice Tino Todino. I will try that firmware later.
Feb 8, 2010 at 1:36 UTC
Got it working again eventually. I dont know why exactly.
Upgraded to DGTeam firmware
tried with same wireless settings - failed
turned security off, rebooted - worked
turned wpa back on (new password), rebooted - worked.
Feb 9, 2010 at 2:53 UTC
Glad you got it working Oliver.
This discussion has been inactive for over a year.
You may get a better answer to your question by starting a new discussion.


text:
Touchy, touchy! Diane Sawyer is in the business of dishing out tough questions and challenging people's answers. But when a guest on today's "Good Morning America" politely corrected her on a First Amendment matter, the GMA host was quick to accuse him of "attacking" her.
Los Angeles-based radio talk show host Larry Elder was Diane's guest, in to discuss the Imus matter. Sawyer introduced him as a "conservative radio host" though on his own site Elder describes himself as a "libertarian" and "a blend of fiscal conservative and social liberal." Of course we all know how many times the MSM has described Al Sharpton as a "liberal" in the course of his innumerable appearances over the last week or so: that would be precisely zero, at last count.
Elder opined that Imus' punishment did not fit the crime. Imus' comment was offensive, sexist and racist, said Elder, "but he apologized, apologized again, did the obligatory beatdown tour à la Michael Richards by appearing on the Al Sharpton show, and as far as I'm concerned, that should have been enough. In the grand department store of life, Don Imus operates in the toy section and I think that those remarks should have been taken with some perspective, but they weren't."
To her credit, Sawyer then raised the issue of rap lyrics, displaying some on-screen with blanked-out words, their messages more vile than anything Imus had said.
SAWYER [wrapping up]: Free speech issues abound in all of this.
ELDER [politely correcting her]: Diane, I do want to take issue with the term 'free speech.' This has nothing to do with free speech. Free speech has to do with the government suppressing political speech. This has to do with a private employer, CBS, determining for all sorts of reasons that Don Imus ought to go. That's just the way things go. That's just free enterprise. It has nothing to do with free speech."
SAWYER: Well, free speech enables you to end this interview by attacking me! Thank you very much.
ELDER: I didn't attack you Diane, I was just simply clarifying something.
SAWYER: Alright, Larry, good to talk to you this morning.
There were big smiles on both sides, but one sensed that Sawyer's feelings had indeed been hurt, and that Elder was trying to recover from what he feared might have been a GMA-career ending moment.
Lessons learned: the MSM is quick to identify people as "conservative" even if they don't see themselves that way, but is loath to label liberals. And the heavy hitters of the MSM can dish it out, but aren't nearly so good when it comes to taking it.
Contact Mark at email@example.com


text:
|Video Game Programming Workshop
Event Type: Adult
Age Group(s): Adult, Teens (Grades 7-12)
Date: 9/28/2013
Start Time: 2:00 PM
End Time: 4:00 PM
Hear a brief presentation by K-State instructor, Nathan Bean, on the history of video games and potential careers in gaming. Then, learn how to use Scratch to create your own basic video games and animations.
Location: Technology Center Classroom
Other Information:
Event is open to the public, but registration is suggested to guarantee a spot.Status: Closed


text:
- Wakf
- Wakf Wakf (w[u^]kf), n. [Ar. waqf.] (Moham. Law) The granting or dedication of property in trust for a pious purpose, that is, to some object that tends to the good of mankind, as to support a mosque or caravansary, to provide for support of one's family, kin, or neighbors, to benefit some particular person or persons and afterward the poor, etc.; also, the trust so created, or the property in trust. [Webster 1913 Suppl.]
The Collaborative International Dictionary of English. 2000.


text:
|Home | About | Journals | Submit | Contact Us | Français
Maintenance of DNA integrity is critical for all cell types, but neurons are particularly sensitive to mutations in DNA repair genes, which lead to both abnormal development and neurodegeneration 1. We describe a previously unknown autosomal recessive disease characterized by microcephaly, early-onset, intractable seizures and developmental delay (MCSZ). Using genome-wide linkage analysis in consanguineous families, we mapped the disease locus to chromosome 19q13.33 and identified multiple mutations in PNKP that results in severe neurological disease, whereas a splicing mutation is associated with more moderate symptoms. Surprisingly, while patient cells are sensitive to radiation and other DNA damaging agents, no patient has yet developed cancer or immunodeficiency. Unlike other DNA repair defects that affect humans, PNKP mutations universally cause severe seizures. The neurological abnormalities in MCSZ patients may reflect a role for PNKP in several DNA repair pathways.
We identified the autosomal recessive disorder MCSZ with the following features: microcephaly, infantile onset seizures, developmental delay, and variable behavioral problems especially hyperactivity. MCSZ was observed in multiple pedigrees of Middle Eastern and European origin (Figure 1). The first three pedigrees were Arabic Palestinians living in Jordan and the USA. Three other families with similar manifestations were Arabic (Kingdom of Saudi Arabia), Turkish, and of mixed European ancestry (USA). We later found less severely affected patients from Family 7, which is also American of mixed European heritage (USA). Brain MRIs consistently show microcephaly with preserved brain structures, without apparent neuronal migration or other structural abnormalities, and with no evidence of degeneration (Figure 2). The patients did not develop ataxia or other neurological symptoms. Routine clinical genetic and metabolic screening showed no abnormalities. Despite careful inquiry, MCSZ patients did not have a higher frequency of common or uncommon infections, offering no clinical evidence of immunodeficiency. Cells from one patient showed sensitivity to irradiation in a standard colony survival assay2,3. However, no patient has developed cancer by age 21 and heterozygous carriers have not developed early onset cancer or any sign of immunodeficiency (see Clinical Information and Summary in Supplementary Note).
Genome-wide linkage screens suggested a single common homozygous region on chromosome 19q in all six MCSZ patients in the consanguineous Families 1-3. This same locus showed homozygosity in the two patients from Family 4 and the single patient from Family 5 whose parents were not known to be related. No other regions of linkage were observed including known primary microcephaly loci. Interestingly, all six patients in the three Palestinian families (Families 1-3) were homozygous for the same 3cM/1.5Mb haplotype (between markers D19S879 and D19S907) at chromosome 19q13.33, suggesting a common ancestor (Supplementary Information Figure 1). Further investigation revealed a common town of origin for these families. Linkage analysis of Families 1-4 and 6 generated a combined maximum two point LOD score of 5.60 at D19S867 with θ =0 (Supplementary Information Table 2) and a maximum multipoint LOD score of 7.12 (Supplementary Information Figure 2).
We sequenced 41 genes within the minimal region (see Supplementary Information) and only the PNKP gene contained mutations. Homozygous mutations were present in each of the first five families and compound heterozygous mutations were found in Families 6 and 7 (Figure 3 and Supplementary Information Figure 3 for detailed analyses of all mutations). The three Palestinian Families (Families 1-3) shared a homozygous base pair substitution in exon 11 (975G>A), resulting in the non-conservative amino acid change E326K. The families from the Kingdom of Saudi Arabia (Family 4) and Turkey (Family 5) had the same homozygous 17 bp duplication (17 bp dup) in exon 14 (1250_1266dup), resulting in a frame-shift, T424GfsX48. Family 6 (European) had two heterozygous mutations: the same 17 bp dup in exon 14 as the Saudi and Turkish families and a point mutation in exon 5 (526C>T) resulting in L176F. The moderately affected members of Family 7 (European) displayed compound heterozygosity, carrying the 17 bp dup mutation in exon 14 and a 17 bp deletion in intron 15 that disrupts proper mRNA splicing.
To confirm that these mutations were pathogenic, we analyzed patient-derived, Epstein Bar Virus (EBV)-transformed, lymphocytes from Families 3 (E326K) and 7 (17 bp dup/intron 15 del). The samples from the severely affected patient (Family 3) and the mildly affected patients (Family 7) had greatly diminished PNKP protein compared to unaffected or heterozygous family members (Figure 3b). RT-PCR analysis showed that the intron 15 deletion in Family 7 disrupts mRNA splicing and causes skipping of exon 15; a barely detectable level of properly spliced mRNA remains (Figure 3c). The low levels of PNKP protein and the indistinguishable phenotype among severely affected individuals from Families 1-6 suggest that all of these mutations impair PNKP severely or completely, whereas patients in Family 7 that exhibit a slightly milder phenotype and carry one non-coding mutation may retain some PNKP activity.
Surprisingly, despite their diverse ancestries (Saudi, Turkish, and mixed European), all chromosomes with the 17 bp dup mutation appear to share the same haplotype for 18 SNPs in or near the PNKP locus (Supplementary Figure 4a). The low frequency of this mutant haplotype (Supplementary Figure 4b), suggests that the 17 bp dup in exon 14 is likely of shared origin and very old in all or most of these families. Nonetheless, the mutation shows an extremely low carrier frequency in the general population, as the 17 bp dup mutation was absent in 1080 Middle Eastern or Caucasian control chromosomes, while all other mutations were absent in all of at least 280 control chromosomes screened.
PNKP has been implicated in repair of both double strand breaks (DSBs) and single strand breaks (SSBs) 4,5, since its phosphatase domain removes 3′ phosphates and the kinase domain phosphorylates 5′ hydroxyl groups, required for DNA ligation 6. PNKP's forkhead domain mediates interaction with the non-homologous end joining (NHEJ) complex via XRCC4, or the SSB and base excision repair (BER) pathways via XRCC1 (Supplemental Figure 6 a and b)4-8. PNKP has been further implicated in the repair pathway disrupted in an ataxic neurodegenerative disease, spinocerebellar ataxia with axonal neuropathy, SCAN1 (TDP1) (see Supplementary Information Figure 6 for additional details). The abnormalities in a clinical irradiation sensitivity test already reflect deficiencies in NHEJ required to repair radiation induced DSBs. Because PNKP potentially plays roles in additional repair pathways, we tested cells in response to other DNA damaging agents.
The response to free radical damage from hydrogen peroxide, predominately requiring BER, and camptothecin, requiring TDP1 via its activity on topoisomerase I, was examined via alkaline comet assay which detects both SSBs and DSBs by quantitating the amount of DNA that moves from a nucleus after electrophoresis. Typical nuclei with various amounts of DNA damage are shown in Figure 4a. The relative amount of DNA damage was determined by measuring the % Tail DNA (DNA that has moved out of the nucleus). The relative amount of DNA damage repair was quantitated as % Tail DNA after toxin exposure at various times of recovery (see Supplementary Figure 5 for all data) and normalized to the maximum and minimum damage for each cell line in each experiment. MCSZ derived cell lines were significantly impaired in their ability to repair hydrogen peroxide induced damage (Figure 4b) and were also delayed in repairing camptothecin induced damage (Figure 4c). However, MCSZ derived cells were eventually able to repair the camptothecin induced damage after 45 minutes while they were not able to repair all hydrogen peroxide induced damage even after 90 min. These data suggest that camptothecin induced damage may be easier to repair than free radical damage in the setting of PNKP mutations. Although MCSZ patients do not develop the ataxia that characterizes TDP1 mutations that also have camptothecin sensitivity 9,10, our patients are perhaps too young (oldest 21 years) or maybe there is a compensatory mechanism for the loss of PNKP.
Microcephaly can result from failure to produce enough neurons during development (primary microcephaly) or degeneration after normal development. MCSZ patients have no evidence of brain atrophy or clinical regression, making degeneration unlikely (see Supplemental Note, Clinical Information for further details). In situ hybridization indicated that human and mouse PNKP mRNAs are expressed in both dividing neuronal precursors in the cerebral cortical ventricular zone (Figure 5 a and c VZ) as well as post-mitotic neurons of the cortical plate (Figure 5d CP) consistent with potential roles in both dividing and post-mitotic neurons. In addition, we found that when levels of Pnkp were reduced in dissociated mouse neurons in vitro via RNAi, there was a small but statistically significant increase in apoptosis in both neuronal precursors (Supplemental Information Figure 7c) and differentiated neurons (7d) compared to cells transfected with control plasmids. This indicates that microcephaly could result from apoptosis of precursors, differentiated neurons or both cell types.
Although disruption of genes encoding NHEJ repair proteins leads to microcephaly in human and/or mouse in LIG4 syndrome, Lig411-13, severe combined immunodeficiency with microcephaly, NHEJ1 (cernunnos) 14, Xrcc4 15, Xrcc6 (Ku70)/ Xrcc5 (Ku80)16, and Prkdc (DNA-PKcs) 17, disruption of NHEJ function is not known to cause seizures, which are a prominent clinical feature of MCSZ. In contrast, disruption of the BER/SSB pathways by deletion of Xrcc1 in mice produced normal brain size but the mutant mice developed ataxia, loss of interneurons within the cerebellum, and seizure-like behavior 18. The intriguing similarity of phenotypes between patients with mutations in PNKP and mice with targeted deletion of Xrcc1 does suggest a potential common mechanism demonstrating the requirement for the BER/SSB pathways in the prevention of seizures, potentially via an interneuron specific requirement of the BER/SSB pathway. Therefore, the unique pattern of neurological symptoms of MCSZ may reflect a requirement for PNKP activity in multiple DNA repair pathways.
Family 1 underwent a genome-wide linkage screen using about 400 microsatellite markers in the ABI linkage mapping set MD v2.5 at 10 cM average density (Applied Biosystems) at the Children's Hospital in Boston (Boston, MA, Genotyping Core facility). Genome-wide screens for Families 3 and 4 were performed at the Center for Inherited Disease Research using microsatellite markers also at 10 cM average spacing. Fine mapping was done using polymorphic microsatellite markers from the ABI linkage mapping set HD v2.5 at 5cM average density (Applied Biosystems) along with additional microsatellite markers identified using the UCSC Human Genome Browser 19 and synthesized primers (Sigma-Genosys). Two point and multipoint LOD scores calculated using Allegro 20 assumed recessive inheritance, full penetrance, and a disease allele frequency of 0.0001. All nucleotide numbers are in reference to cDNA where A (of the ATG start site) is +1 except for the intronic deletion which is in reference to the genomic sequence where A of ATG of the translational start site is +1 (all from UCSC genome browser, NCBI Build 36.1).
For PNKP, EBV transformed lymphocytes were grown in DMEM with 15% FCS plus normocin. 2×105 cells were lysed in sodium dodecyl sulfate (SDS) loading buffer at 90°C and fractionated by SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were transferred to Hybond-C Extra Nitrocellulose (GE Healthcare), stained with Ponceau S solution (Sigma), and washed in TBST before immunoblotting with anti- PNK (SK3195) 21 at a dilution of 1 in 1000 in 5% milk. Blots were washed and probed with goat anti-rabbit IgG HRP secondary antibody (DakoCytomation) and detected with ECL detection reagents (GE Healthcare). The blot washed in TBST and re-probed with anti-β-actin monoclonal antibody (Sigma) using rabbit anti-mouse IgG HRP (DakoCytomation) as a secondary antibody.
EBV transformed lymphocytes were grown as above. RNA was isolated via RNeasy Mini Kit (Qiagen). 5 μg of total RNA was used for first strand synthesis via oligo dT primers via SuperScript III First-Strand Synthesis SuperMix (Invitrogen Corporation). 1 μl of the product of the RT reaction was used in PCR using primers from Exon 10 to Exon 17.
Alkaline comet assays were performed via the protocol from CometAssayTM ES unit (Trevigen, Inc.) Briefly, patient derived EBV transformed lymphocytes were grown as described. Cells were obtained prior to treatment or exposed to 100 μM hydrogen peroxide (Sigma) or 10 μM camptothecin (Sigma) for 30 and 60 minutes respectively at 37°C. After exposure, cells were immediately collected for 0 min recovery time point or washed once, resuspended in growth media and incubated at 37°C. Cells were subsequently collected at times described. Cells were embedded in low melt agarose, plated upon microscope slides, lysed, treated with alkaline solution, and slides electrophoresed in alkaline solution at 1 Volt/cm (21 V) with ~300 mAmps for 30 minutes. Slides were washed, dried, and DNA stained with SYBR green. Images of nuclei and tails were taken with a Nikon TE2000-E fluorescent microscope with CCD camera and % Tail DNA determined with CometScore 1.5 software (TriTek, Corp.).
Human in situ hybridizations were performed at the Human Developmental Biology Resource (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, NE1 3BZ) with probes to anti-sense human PNKP cDNA (+488-1500). Mouse in situs were done as previously published 22, with anti-sense probe from mouse Pnkp cDNA (+386-1566).
Statistics were done within Microsoft Excel software unless otherwise described. The % DNA Repair in Figure 4 was determined by measuring mean of % Tail DNA in 3 separate wells (raw data in Supplementary Information Figure 5) and using combined % Tail DNA at 0 minute recovery (maximum damage) and minimum damage measured as full recovery for each individual cell line. P-values were determined by comparing the baseline adjusted DNA repair level from 3 separate wells using T-Test comparing each MCSZ deficient line to 2 normal control cell lines with two-tailed distribution and homoscedastic test.
PNKP protein alignments were performed with MegAlign (Lasergene). Three dimensional structure of mouse Pnkp was examined in MacPyMOL from previously published information 23. The Branch Point analysis was done with Human Splicing Finder version 2.3 (http://www.umd.be/HSF/).
Sequenom SNP genotypes were determined in patient and controls samples at the Molecular Genetics Core Facility at Children's Hospital Boston (Boston, MA, USA). Haplotypes were determined by Mendelian inheritance patterns for parents and offspring and Phase haplotype determining software24-26 for control samples.
Silencer 1.0 GFP had RNAi oligos targeted to mouse Pnkp mRNA identified via the Broad Institute RNAi Consortium shRNA Library, ligated into EcoRI and ApaI sites (see Supplemental Information (Invitrogen)). Cerebral cortices from E13.5 day old Swiss-Webster mice were dissected and dissociated with Papain Dissociation System (Worthington Biochemical Corp.), cells were washed twice in HBSS buffer and transfected via the Amaxa Nucleofactor 96 well shuttle system (Amaxa Biosystems) with either pSilencer mouse Pnkp-RNAi GFP, pSilencer GFP, with or without pSport-human PNKP (Open Biosystems, Entrez clone ID BC057659). Cells were grown on poly-L-ornithine (Sigma-Aldrich) treated Lab-Tek chamber slides with Permox (Nalge Nunc International) in DMEM with 10% FBS for the Pax6 studies and for the NeuN studies cultures were changed to Neurobasal complete media after 4 hours. Cultures were fixed with 4% paraformaldehyde 24 hours post-transfection. Immunostaining was performed in PBS with 0.2% Triton X-100 and 10% goat serum with chick anti-GFP (Abcam), rabbit anti-cleaved caspase 3 (Asp175) (Cell Signaling Technology, Inc.), mouse anti-Pax6 (The Developmental Studies Hybridoma Bank) or mouse anti-NeuN (Chemicon). Primary antibodies were detected with anti-chicken IgG Alexa 488 (Invitrogen), and donkey anti-mouse IgG Cy3 or donkey anti-rabbit IgG Cy5 (Jackson Immunoresearch Inc.). Images were obtained by looking for GFP+ cells with a Nikon TE2000-E fluorescent microscope and Metamorph imaging software. Cell counts were made from digital images with the counter blinded as to whether the cultures were derived from Pnkp-RNAi or control transfections. P-value in Supplementary Figure 7 were calculations were calculated using G-test of goodness-of fit with 5 degrees of freedom, two-tailed P-value and no Williams correction27.
Mo-Pnkp RNAi construct was tested against Mo-Pnkp-DsRed or Hu-PNKP-DsRed both expressed by pCAG-DsRed vector 28. RNAi control vector (empty vector)/mouse (Mo)-Pnkp-DsRed fusion protein, MoPnkp-RNAi/MoPnkp-DsRed, RNAi-control/human (Hu)-PNKP-DsRed fusion, and MoPnkp-RNAi/HuPNKP-DsRed were transfected into NIH-3T3 cells with Lipofectamine 2000 (Invitrogen) per manufacturer protocol. Cells were counted after 24 hours in culture. Random fields were chosen by expression of GFP, the number of high GFP expressing cells were counted followed by the number of Ds-Red fusion protein expressing cells. DsRed expression was nuclear as expected from PNKP localization (data not shown). For Western Blot analysis, NIH-3T3 cells were transfected as above. After 24 hours of culture, cells were lysed via the protocol described in the methods of the main body of the manuscript. After protein quantitation via BCA assay, 25 μg of protein per lane was separated via PAGE. Protein was transferred to membrane, blocked with Odyssey blocking buffer (Li-Cor Biosciences) and probed with Mo anti-Actin (AC-15), Rb anti-GFP (Molecular Probes) and Rb anti-DsRed (Clontech). Primary antibody was detected with anti-mouse labeled with IRDye 700 or anti-rabbit IRDye 800 (Li-Cor Biosciences) and detected with Li-Cor imaging system. In the bar graph protein amounts are normalized with RNAi Control conditions.
JS was supported by the Victoria and Stuart Quan Fellowship. Research was supported by grants from the National Institute of Neurological Disorders and Stroke to ECG (5K08NS059673-02) and to CAW (NSR01-35129), the Fogarty International Center (R21 NS061772), by the Manton Center for Orphan Disease Research, the Simons Foundation, and by the Dubai Harvard Foundation for Medical Research. ECG was also supported by a K12 Child Health Research Center awarded to Children's Hospital Boston (1 K12 HD052896-01A1) and a Research in Training Award from the Child Neurology Foundation. JR and KWC are supported by Medical Research Council (G0600776 & G0400959). CAW is an Investigator of the Howard Hughes Medical Institute.
We thank Sue Lindsay, and Steve Lisgo from the Medical Research Council-Wellcome Trust Human Developmental Biology Resource (HDBR), Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK for performing human in situ hybridizations.
We are grateful for genotyping services provided by the Center for Inherited Disease Research (CIDR). CIDR is fully funded through a federal contract from the National Institutes of Health to The Johns Hopkins University, contract number HHSN268200782096C and NIH N01-HG-65403. Genotyping at Children's Hospital Boston is supported by the IDDRC (CHB MRDDRC, P30 HD18655).
The genetic study was approved by Beth Israel Deaconess Medical Center and Children's Hospital Boston Institutional Review Boards. Appropriate informed consent was obtained from all involved human subjects. All animal work was approved by Harvard Medical School, Beth Israel Deaconess Medical Center, and Children's Hospital Boston Institutional Animal Care and Use Committees.
UCSC Human Genome Browser: http://genome.ucsc.edu/
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.