text:
Aen Seidhe
Mikolai Stroinski
The Witcher III: Wild Hunt - Aen Seidhe
The Witcher III: Wild Hunt - Aen Seidhe
Also I promised myself that today I would get so much writing done for already existing fics, except all I can actually think about is some vague random idea inspired by Sir, You Are Being Hunted, basically some survival thing with Noct on his own somewhere being hunted down by robots, but instead of Victorian gentlemen robots they're MTs or other Niflheim things or some shit.
i was playing around with values and filter effects for a while and ended up with this weird little thing and i thought it would be fun to turn into a fake snap hahah
When baby Jones is born, DO NOT comment about how much you dislike the name that has been picked. Or how *insert name* would have been better. Or I totally knew they were going to pick it because (Michael/Lindsay/anyone else) once mentioned it here in *insert video*
Congratulate the couple and comment on how cute the baby is. How the name is adorable. Literally anything positive.
But don't you dare belittle Austin's best couple for their name choice.
me: i don't really like country
kesha: hunt_you_down.mp3
We lost this wonderful man two years ago today, I think it's safe to say the world still misses you. ❤
(Gun use, alcohol mention, amazingly- no animal death)
So you may remember Uncle Popeye from A Holiday Story, when he and grandpa tried to shoot a pheasant and fucked it up real bad. I called the Ohio Relatives. They have no idea how the family knew Popeye either, but that his given name was Richard, but got tired of being called "Dick" and after losing an eye in WW2, went by Popeye.
Look man, Ohio DOES things to people.
Popeye fancied himself the Great Outdoors-man, despite a long list of evidence to the contrary- besides the shooting incident, there was the time he got lost in the woods behind his house for a week despite being less than a mile from his house and six major roads, the time he almost poisoned the whole family after mushrooming in the hills only to be stopped by GG, and the time he got in a fight with a Woodcock and Lost.
The worst though, was Snowflake.
Near where my Ohio relatives lived, and continue to live, there is a Military Armory. (You know that joke about "If all your relatives all live in the same postcode, you might be a redneck?" Yeah, check that. Mom was the first to leave the state, and keeps urging the others that they are free to leave, they can't keep you there. But I digress). The armory is actually kind of a large campus, several hundred acres in size, where they take lots of old munitions and aircraft and whatnot, and figure out how to take apart and dispose of them without blowing everything up to fuck. The whole area is fenced off to keep the locals from helping themselves to the munitions (A serious issue in redneck country), which trapped the deer in the forest inside.
The deer, no longer having to worry about hunters, but cut off from the outside population, basically went full Deliverance, and the resulting mutants are… rather pretty.
The mutation is Luecistism, not albinism, but it makes for pretty, pretty very stupid deer. Like, even dumber than white-tail already are, and whitetail are DUMB. But the deer on the armory could afford to be easy to spot and have no natural fear of anything, because there were no predators or hunters, and the soldiers stationed there had better things to do
The prettiest of them all was Snowflake, the large white buck named Snowflake, because soldiers are great at naming things. He was, by all accounts, a truly splendid creature- snow-white and shapely, with a well-developed rack. Not unlike a porn star, apparently. And many a man Lusted after snowflake, desperate for his head.
Or other things. Ohio's a pretty fucked up place.
But unlike other men, who would only stare wistfully from afar, Popeye was absolutely determined to have Snowflake. The issue was, the military, having a few moments of sense, had decreed that having people wandering around a munitions decommissioning plant with firearms was likely to result in fire and death, declared that there was to be no hunting on their grounds. The only way Popeye could feasibly shoot Snowflake would be if he were somehow able to get him on the other side of the fence. But he couldn't just cut a hole in the fence- it was fairly regularly checked, and he'd be caught. Nope. Somehow, Popeye had to get Snowflake on the other side of the fence without damaging it or the Military noticing.
It was during an afternoon of boozing and watching western documentaries, Popeye hit upon a solution. He was watching a tourism promotion for all the great outdoor activities in Colorado, when he saw the solution to his problem.
He could FISH for deer.
Specifically, he fly-fish. In his mind, he could clearly see how it would play out. he'd simply find a heavy-duty line, cast it over the fence, tangling it in Snowflake's antlers, and then reel him over the fence, where it would be perfectly legal to shoot him and then he'd be the envy of all the men down at the elks lodge. Hah! Genius!
So that spring, Popeye began tossing corn over the fence to lure deer to that particular secluded corner, and was immensely pleased when Snowflake started turning up regularly. He'd get his trophy AND some fat venison! All summer and into fall, he continued this, with the deer getting entirely too casual about his presence. he also got his hands on some deep-sea fishing line and practiced ensnaring the antlers of his dummy deer in the backyard. Just to make sure he had the leverage to haul Snowflake in, he got the harness that attaches the pole to your hip. All was going according to plan.
So the first day of hunting season, Popeye goes to his corner where he's been feeding the deer, and Snowflake is there, waiting for breakfast. Great. Popeye backs his pickup truck up to the fence, and stands on the bed so he can cast over the fence. The deer, being imbeciles, fail to notice anything amiss. He casts, and miracle of miracles, he gets the loop over Snowflake's antlers on the first try! Popeye whips the line around some more, making sure Snowflake is good and tangled, before reeling him in.
Apparently snowflake just stood there for this part, presumably looking confused. Then the line began to pull on him.
As Popeye would later recount from the hospital: "That's when I realized. Deer ain't Mackinaw."
Popeye had, in all his planning, not taken into consideration that a 200-pound buck at the height of his testosterone-riddled rut might be somewhat disinclined to be pulled over a fence. Furthermore, Popeye had failed to account that at 5′5″, he was of similar size to the deer, and in nowhere near as good of shape.
He recalled ALMOST flying over the fence as Snowlfake turned and ran for the safety of the base. He did not quite make it, and cracked both knees as they slammed into the fence, jeans and harness shredding on the barbed wire. it was not enough to separate him from the harness, only enough to slide it down his legs and tangle around his ankles, so that once he hit the ground, Popeye was dragged for half a goddamn mile by his feet as Snowflake frantically tried to get away.
Once at the base, and all manner of bruised, cut up and abused, Popeye was relieved when they finally came to a halt. he regretted it half a second later when he realized that Snowflake had only turned around, and was now bearing down on his sorry ass full-tilt. Several puncture and kick wounds later, Popeye managed to kick off the harness, freeing himself from Snowflake, and had to run back to where he thought he'd left the truck. In the middle of the night, in the woods, with cracked patellas and without pants.
It took him all night to find the fence and truck, but managed to get back over the fence and to the hospital without being spotted. In a fit of paranoia that almost pased for good sense, he drove to three counties away to be treated, so the police wouldn't find him, bleeding all the way. He neglected beforehand, to tell any of his friends or family where he was going, except that he was deer-hunting.
He was very disappointed when he turned up a week later and found out nobody had gone looking for him.
Snowflake was found tangled up in a tree, and was cut loose by the soldiers, apparently upset but unharmed. Concerned that the poachers were getting too creative for their own good, the base petitioned the state legislature to maybe make a law that you aren't allowed to fish for deer, Christ, we only found the poor man's pants.
The state legislature, in a fit of rabid libertarianism, declared that such a law would be too restrictive upon the freedom of Ohioans, so the Army tried the country. The county, which had to actually deal with this kind of bullshit on a semi-regular basis, agreed, and it is now illegal to Hunt any bird, fish or quadruped with devices and equipment not intended for such purpose.
Popeye never went deer-hunting after that, and Snowflake went on to sire many many more pretty inbred deer.
Gem Hunt aired one year ago today! ♡♡♡
normal is a myth
she's dead
"no thanks monty, you're not man enough."
tape 2 side a / tape 5 side b
me: I'll always respect gaga, but i just can't get behind this country shtick she's got going on for joanne
kesha: I wanna be your favorite and always by your side, I wanna talk forever with babies down the line, Yodel-odel-oh, oh oh, Yodel-odel-oh, oh oh oh
this is a sloppy-ish comic about a strong headcanon of mine which is basically:
hanzo, due to years of coping badly with life, sees genji in a very idealised, 'always-a-child' way, and can't quite connect that to 35-yrs old actual genji(this is turn, makes genji feel like hanzo thinks he's inferior). essentially: it's hard work patching things together, but they try.
2009-2017 was just one big slow burn lovers to enemies to friends to lovers fic and we're getting to the happy ending pretty soon i can feel it in my bones
If you all don't have a cover letter template written up so you can just fill in the blanks during a job hunt, you're doing yourself a major disservice bc man does it look professional and it can be SO fast and easy if you do it beforehand.
This is what mine looks like:
Your Name
Company Name
Company Street Address
City, State, Zip
Dear Hiring Manager/Hiring Department (pick 1 depending on how big the company sounds)
I am interested in applying for the (Insert job position here) that was (insert how you found the job here). My resume is included for your review.
My background includes extensive customer service experience in fast-paced environments (If you were in a lot of fast food/retail). Through those experiences I've developed a large focus on customer experience and satisfaction, that would easily fit within your company's dedication to positive customer relations. Insert something specific to the company's creed here. My skills include (relevant program/being eager to learn new skills), (a thing you can do), and (probably something like "reliable work pace").
I would sincerely appreciate getting to meet with you to talk about this opportunity further. Please feel free to email me or call to get in contact with me. Thank you very much for your consideration.
Your name again
Enclosure (it means a resume is attached too)
Short, sweet, to the point, and totally valid in p much any workplace. I wrote this up as a homework assignment for a business class and oh man it's helped me out so much.
You can do this, cover letters don't have to be intimidating!
Martha stans.


text:
Giordano Glorious Special Offer
Ready for you now to get its shocking special offer at Giordano on Fashion like no other shocking special offer. What are you waiting
Summer bundle surprise on selected items
Summer bundle surprise on selected items
Special Offer
- Ibn Battuta Mall
- Dubai Marina Mall
- Mirdif City Centre
- Deira City Centre
- Mall of the Emirates
- Lamcy Plaza
- Al Reef Mall
- Outlet Mall
- Al Madina Mall
- Al Ghurair Centre
- Dubai Festival City
- Dubai mall
- Safeer Mall Sharjah
- Sharjah City Centre
- Sharjah Mega Mall
- Sahara Centre
Abu Dhabi
- Abu Dhabi Mall
- Marina Mall
- Khalidiyah Mall
- Al Wahda Mall
- Mushrif Mall
- Ajman City Centre
- Fujairah City Centre
Ras Al Khaimah
- Al Manar Mall
- Al Hamra Mall
Al Ain
- Al Jimi Mall
- Bawadi Mall
- Al Ain Mall
Similar Offers
Deal Is Now On Buy 2 And Get 2 Free At THE CHILDREN'S PLACE
Search for deal ? We've got you covered. Shop at THE CHILDREN'S PLACE and buy Fashion for prices you never imagined
Up To 50% Off Sale At Ted Baker
Look no further for Sale . Avail yourself of this Sale at Ted Baker
Brioni Sale Up To 30% Off
Forget about any other Sale at Brioni and get your best chance and buy
Sale Up To 30% Off Currently At Joseph
Most generous Sale you could come across at Joseph. Why paying more when you can pay less

subset: commoncrawl


text:
Director Dee Rees On Making Southern Culture Come To Life in 'Mudbound'
The film hits Netflix on Friday.
Bow Wow is at it again with the controversial tweets. This time, though, it wasn't his comments about one of his exes that had people raising eyebrows, but about his own racial heritage.
It all started when someone asked Bow who he planned to vote for in the upcoming election, Hillary Clinton or Donald Trump.
Bow responded by saying neither of them. When a fan pushed him to acknowledge that it's especially important for Black folks to vote because of what ancestors sacrificed for the privilege, he pretty much said that's not his problem:
To prove his point, Shad shared a rare throwback photo of his estranged father, who appears to be light-skinned, on Instagram:
The debate continued from there, with the actor/host continuing to anger fans by saying that he doesn't think voting matters because he voted eight years ago and isn't sure what difference it made. Ultimately, he did endorse one candidate for president:
Kanye West https://t.co/00tiUbIBIg— Shad Moss(Bow Wow) (@smoss) July 28, 2016
Have a #TBT moment with some of Shad's most infamous Twitter drama with BET Breaks above.
(Photo: @Parisa / Splash News)
Presented by


text:
Reebok i run treadmill
1 year old used only 5 times so as new. Cost me £349
ProShox™ Lite impact absorbing running surface 1–20 kph (14.5mph) speed range plus 0–12% Incline.
24 Goals-based workout programs ( 8 Calories, 8 Distance and 8 Time) and iFit Live™ Compatible iPod® Compatible.
ProShox™ Lite Cushioning
Compressing with each step, this cushioning system responds evenly to your stride and offers a truly comfortable workout, reducing the impact on your joints by up to 21 % compared an outdoor run.
SpaceSaver® Design
Saving on space, the deck folds vertically, reducing the storage space of your treadmill when it is not used.
Dual-Grip EKG™ Heart Rate Monitor and Polar Chest
Monitor your heart rate with these convenient built-in handlebar sensors or use the Polar Chest Strap included.
iFit Live™ Compatible
iFit Live™* is the most advanced, interactive and result-specic tness experience available. With workouts featuring Google Maps™, automatic speed and incline controls, and audible coaching, you get the training you want in the comfort of your living room.
Backlit Display
This backlit display panel makes it easy to track your speed, time, distance and calories burned so you can monitor your progress throughout your workout.
CoolAire™ Workout Fan
Stay cool during your workout with the innovative CoolAire™ Workout Fan. The fan is built right into the console, just like the AC in your car. You'll stay comfortable longer, so you can reach your goals
Calorie App with 8 Workouts
Designed by a Certied Personal Trainer, these workouts will help you burn calories and lose weight! Input your weight for a more accurate calorie count. Choose between 200-700 calorie-burning workouts.
Timed App with 8 Workouts
Select between 20 - 60 minute workout times. Each workout is designed by a Certied Personal Trainer and automatically adjusts your speed and incline to match your goals.
Distance App with 8 Workouts
These workouts oer training programs for specic distance races. Each is designed by a Certied Personal Trainer and automatically adjusts your speed and incline to match the workout. Choose from 1-mile, 2-miles, 3K, 5K, 5-miles, half marathon or marathon distances.
Compatible Music Port for iPod®
Pump up the volume with this built-in sound system. Plug your iPod®*, or MP3 Player, into the Interplay™ Music Port for unbeatable sound quality. With the iPod resting on the console shelf, it's within easy reach and leaves the controls right at your fingertips.
Intermix Acoustics™ 2.0
Enjoy your favorite media with this built-in sound system that produces a broad range of frequencies and rich, equalized sound.
Ad ID: 1275153436
Instant delivery quotes powered by AnyVan
Find out if you'll be accepted before you apply.
Intelligent Lending ltd (Credit Broker). Capital One is the exclusive lender.
1 year old used only 5 times so as new. Cost me £349
Selling this treadmill as I have hardly used it! Great condition, used a handful of times. All parts included. Folds up easily when not in use to save space. Collection Cawston NR10
Nearly new , used approximately a dozen times, still has protective film on display. Z8 model selling at argos for £479.99. This is a really professional machine with various programmes, speeds, incline levels. Very heavy and will need
I have opened it and used it once its like brand new
I have a brand new York Treadmill that I want to sell! Its a compact treadmill for the use in the home or in the garage!! It has not been used and still is in the box. If anyone is interested I would be happy to answer any questions!
Heavy duty foldable treadmill 1km/h to 12km/h speed - with auto speed dialing - start building your fitness by walking, and progress up to a run at your own pace Auto incline with auto incline dialing up to 12% Multi-function large LED Display showi
Excellent Condition Allows you to select programmes for personalised fitness Heart and Pulse monitor Collection only
Hardly used ProFitness motorised treadmill. Folds up easily for storage. Good quality. Collection only.
Motorised roger black treadmill. Good working order. Buyer will need to collect. Any questions plz don't hesitate to ask.
Reebok Z9 treadmill. Mains Powered Multi programmes. Excellent condition. Folds up to create extra space if needed. Rarely used. Heart rate monitor and calorie counter. All functions work. This is an excellent piece of fitness equipment bought


text:
|Numéro de publication
|Type de publication
|Numéro de demande
|Numéro PCT
|Date de publication
|20 avr. 2004
|Date de dépôt
|30 avr. 1998
|Date de priorité
|5 mai 1997
|État de paiement des frais
|Autre référence de publication
|Numéro de publication
|Eugen Uhlmann, Anuschirwan Peyman, David William Will, Esther Chang, Kathleen Pirollo, Antonina Rait
|Cessionnaire d'origine
|Aventis Pharma Deutschland Gmbh
|Exporter la citation
|BiBTeX, EndNote, RefMan
|Citations de brevets (8), Citations hors brevets (8), Référencé par (12), Classifications (38), Événements juridiques (8)
|Liens externes: USPTO, Cession USPTO, Espacenet
This invention relates to a specific modified oligonucleotide complementary to a section of the human Ha-ras gene and mRNA, and its use to specifically regulate, modulate or inhibit expression of the Ha-ras gene, and its use as a pharmaceutical for the treatment of conditions arising from abnormal expression of the Ha-ras gene.
Antisense oligonucleotides (AO) have proven to be specific inhibitors of gene expression in a large number of systems, both in vivo and in vitro. (Uhlmann and Peyman, Chem. Rev. 1990, 90, 543).
One of the major problems encountered when using unmodified oligonucleotides containing only phosphodiester internucleoside linkages (PO-oligonucleotides) is the rapid degradation of this type of oligonucleotide in cells and biological fluids, such as, for example, serum and cerebrospinal fluid, by a range of nucleolytic activities. A wide range of chemical modifications to oligonucleotides have been carried out in order to improve their nucleolytic stability (Uhlmann and Peyman, Chem. Res. 1990, 90, 543). These modifications include the modification or replacement of the phosphodiester internucleoside linkage, the sugar unit, the nucleobase; or the sugar-phosphate backbone of the oligonucleotides. The most thoroughly investigated type of modification is alteration of the internucleoside linkage, including phosphorothioate (PS), methylphosphonate (MeP) and phosphorodithioate (PSS) linkages. It should be stressed that modification of the oligonucleotide alters not only its nuclease stability but also other characteristics of the oligonucleotide, such as, for example, their cellular uptake, RNaseH activation, and the strength and specificity of binding to their target nucleic acid, and the like. It should be borne in mind that the stability of the modified oligonucleotide in serum, frequently used to determine the nuclease stability of the oligonucleotide, is not the sole determinant of intracellular activity (P. D. Cook in "Antisense Research and Applications", Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993, Chap.9, p149 et seq.).
The phosphorothioate (PS) modified oligonucleotides are the most widely used type of modified oligonucleotide. The following strategies have been developed for the positioning of PS linkages in antisense oligonucleotides:
(1) Replacement of all phosphodiester internucleoside linkages with phosphorothioate linkages.
The resulting all-phosphorothioate oligonucleotides are much more stable to nucleases than PO-oligonucleotides (Monia et al. J. Biol. Chem. 1996, 271, 14533). For example, degradation of all-PS oligonucleotides by endonucleases is slowed down by a factor of 2-45 relative to a PO oligonucleotide (Stein et al. Nucleic Acids Res. 1988, 16, 3209). In Xenopus oocytes or embryos, the degradation of microinjected PO oligonucleotides proceeds with a half-life of 30 minutes, while all-PS oligonucleotides have a half-life of over 3 hours under the same conditions (Woolf et al. Nucleic Acids Res. 1990, 18, 1763). All-PS oligonucleotides retain their ability to activate RNaseH. The major disadvantages of all-PS oligonucleotides are that their ability to form stable hybrids with their target nucleic acid is reduced, and that they frequently give rise to unspecific "non-antisense" effects (Monia et al., J. Biol. Chem. 1996, 271, 14533).
(2) Oligonucleotides containing both phosphorothioate and phosphodiester internucleoside linkages.
In an effort to overcome the non-antisense effects observed with all-PS oligonucleotides, oligonucleotides containing both phosphorothioate and phosphodiester internucleoside linkages have been synthesized and tested for stability and biological activity.
Ghosh et al. (Anticancer Drug Design 1993, 8, 15) describe a PS—PO oligonucleotide containing various percentages of PS linkages. Their construction follows, for example, the pattern (PS—PO—PO—PO)n, (PO—PO—PS)n, (PS—PO)n, [(PO)2—(PS)2]n, [PO—PS—PS]n. They teach that a PS linkage content of at least 50% is required for selective translation inhibition in vitro and that activity drops drastically when the PS content is less than 50%. More recently it has been demonstrated that an oligonucleotide containing 50% PS-linkages arranged in the pattern (PS—PO)n showed no biological activity in an assay system where an all-PS oligonucleotide and "end-capped" PO—PS oligonucleotides (see below) of the same sequence were highly active (Monia et al., J. Biol. Chem. 1996, 271, 14533).
(3) "End-capped" Oligonucleotides, where one, two or three internucleoside bridges on the 5′ and/or the 3′ end of the oligonucleotide are phosphorothioate modified. (also known as the "gap technique")
This type of modification is designed primarily to protect the oligonucleotide from degradation by exonucleases. In particular modifications at the 3′-end of the oligonucleotide are desirable as they offer protection from 3′-exonucleases, which are the most abundant nucleases in serum (Uhlmann and Peyman, Chem. Rev. 1990, 90, 543).
An interesting comparison of strategies is found in Hoke et al. (Nucleic Acids Res. 1991, 19, 5743). The authors compare the activity of a range of antisense PS-oligonucleotides against HSV-1 in cell culture. Their findings confirm that 3′, or 3′+5′, end-capped oligonucleotides (the first three internucleoside linkages being modified in each case), similarly to all-PS oligonucleotides are sufficiently protected against degradation by nucleases in serum. In contrast internally modified (three PS bridges) oligonucleotides and oligonucleotides in which only the 5′-end has been capped (again, the first three internucleoside linkages being modified) are degraded rapidly. In contrast, the authors found that neither 5′ nor 3′ end capping nor both are sufficient for activity within the cell, and they drew the conclusion that a uniform modification (all-PS) is required to achieve sufficient stability to nucleases in cells.
More recently it has been discovered that pyrimidine nucleosides are the most nuclease susceptible points in oligonucleotides (Peyman, A. and Uhlmann, E., Biol. Chem. Hoppe-Seyler 1996, 377, 67; EP 0 653 439 A2). It was found that a combination of end-capping and PS protection of the pyrimidine positions of oligonucleotides (the so called "minimal modification" approach) is sufficient to make them highly resistant to nuclease degradation. The biological activity (against Herpes simplex virus) of an oligonucleotide with this type of PS modification pattern was comparible to that of an all-PS oligonucleotide.
One of the major problems encountered when using AOs, whether or not they are stabilized against degradation, is their poor cellular uptake. Many approaches have been tried to attempt to ameliorate this problem. Most of these approaches involve the attachment of a variety of substances to the oligonucleotide. Modifications include: the attachment of peptides to oligonucleotides (Lemaitre, M. et al. Proc. Natl. Acad. Sci. USA 1987, 84, 648) and the attachment of lipophilic residues, such as alkyl chains or cholesterol, to oligonucleotides (Saison-Behmoaras, T. et al. EMBO J. 1991, 10, 1111-1118; Will, D. W. and Brown, T. Tetrahedron Lett. 1992, 33, 2729). It has been found, however, that in many cases the introduction of a lipophilic group causes biological effects which are independent of the sequence of the oligonucleotide. Non-specific effects have been reported for cholesterol-oligonucleotide conjugates (Henderson, G. B. and Stein, C. A. Nucleic Acids Res. 1995, 23, 3726.), and for oligonucleotides attached to alkyl chains (Shea, R. G. et al. Nucleic Acids Res. 1990, 18, 3777). Saison-Behmoaras et al (EMBO J. 1991, 10, 1111-1118; WO 96/34008) have reported that a 9mer all-PO oligonucleotide derivatized with a 3′-dodecanol moiety and a 5′-acridine crosslinking agent, and antisense to mutated Ha-ras inhibited T24 human bladder carcinoma cell proliferation. No comparison of the antiproliferative activity of this oligonucleotide with that of the corresponding oligonucleotide without a dodecanol conjugate was made. The cellular uptake of the dodecanol oligonucleotide was reported to be 4 times that of the unmodified oligonucleotide in T24 cells. The acridine-dodecanol modified oligonucleotide had no effect on the proliferation of a human mammary cell line carrying an unmutated Ha-ras gene. This effect can either be attributed to the sequence specificity of the antisense oligonucleotide or to insufficient cellular uptake. Since the uptake of the acridine-dodecanol oligonucleotide was not determined in the human mammary cell line, the effect may be entirely, or at least partly due to the fact that the oligonucleotide was not taken-up by the human mammary cell line, and thus did not have an opportunity to exhibit non-sequence-specific effects on cell proliferation.
About 20% of human tumors have a mutation in one of the three ras genes (Ha-ras, Ki-ras, and N-ras) leading to over-expression of p21 protein which plays an important role in the transformed phenotype (Bos, T. L. 1988 Mutation Res. 1988, 195, 255). It has been reported that inhibition of different ras genes in different cell lines can either have no effect, or inhibit cell proliferation depending on which of the ras genes is controlling the cell proliferation (Chen et al. J. Biol. Chem., 1996, 271, 28259-65). It was suggested that differential modulation of individual ras genes may be an approach to inhibit tumor growth while minimising effects on normal cell growth. One approach to devising successful therapy of Ha-ras-induced tumors is to regulate, modulate or inhibit expression of the Ha-ras gene. Antisense oligodeoxynucleotides (oligonucleotides) have been shown to act as specific inhibitors of ras mRNA expression in cell-free systems, in transformed cells in culture (T. Saison-Behmoaras et al. EMBO J. 1991, 10, 1111-1118; Monia et al. J. Biol. Chem. 1996, 271, 14533), and in ras-activated tumors in vivo (Gray, G. D. et al. Cancer Research, 1973, 53, 577).
In order to modulate the expression of the ras-gene in wild type or in mutated form Brown et al. (Oncogene Res. 1989, 4, 243-252) propose the use of anti-ras oligodeoxy ribonucleoside methylphosphonates which are complementary to the initiation codon region of Balb-ras P21 mRNA (a mouse version of the human Ha-ras Gene). However, it is well known that methyl phosphonates show some major disadvantages compared to phosphorothioates, among other their poor cellular uptake.
Monia et al. (J. Biol. Chem. 267, 19954-19962, 1992; WO 92122651) disclose all-PS antisense oligonucleotides and antisense oligonucleotides containing various percentages of PS linkages (WO 94/08003) directed to the translation initiation site or to codon 12 of the human Ha-ras gene, which however, have the above-mentioned disadvantages concerning the cellular uptake and their poor stability.
Pirollo et al. (Biochem. Biophys. Research Comm. 230, 196-201, 1997) propose the use of anti-ras all-PS oligodeoxynucleotides in order to reverse radio resistance of cancer cells. However, these antisense oligonucleotides show the disadvantages (poor cellular uptake, unstability) as mentioned above.
Therefore, this invention aims to provide an oligonucleotide, modified to improve its stability and cell uptake, complementary to Ha-ras mRNA which specifically regulates, modulates or inhibits expression of the Ha-ras gene in the form of its wild type as well as in its mutated forms, and which can be used to inhibit the proliferation of cancer cells, to reverse radio-resistance in cancer cells, and to treat conditions arising from abnormal expression of the Ha-ras gene.
According to the invention, this problem is solved by providing a modified oligodeoxynucleotide of the sequence SEQ ID NO: 1 5′-TxAxTxTxCxCxGxTxCxAxT-3′-O—PO2—O—R
x is o or s
A is 2′-deoxyadenosine,
G is 2′-deoxyguanosine,
C is 2′-deoxycytidine and
T is thymidine.
The modified oligodeoxynucleotide according to the invention is particularly
characterized in that SEQ ID NO:1 has one of the following nucleotide linkage
(a) 5′-TsAsToToCsCoGoTsCsAsT-3′-O—PO2—O—R,
(b) 5′-TsAsToToCsCoGsToCsAsT-3′-O—PO2—O—R,
(c) 5′-TsAoTsToCsCoGoTsCsAsT-3′-O—PO2—O—R,
(d) 5′-TsAoTsTsCsCoGoTsCsAsT-3′-O—PO2—O—R,
(e) 5′-TsAoTsToCsCsGoTsCsAsT-3′-O—PO2—O—R,
(f) 5′-TsAoTsTsCsCsGoTsCsAsT-3′-O—PO2—O—R,
(g) 5′-TsAsToToCsCsGsToCsAsT-3′-O—PO2—O—R,
(h) 5′-TsAoTsTsCsCsGoTsCsAsT-3′-O—PO2—O—R,
(i) 5′-TsAsTsTsCsCsGoTsCsAsT-3′-O—PO2—O—R,
(j) 5′-TsAsTsToCoCsGoTsCsAsT-3′-O—PO2—O—R,
(k) 5′-TsAsTsToCsCsGoTsCsAsT-3′-O—PO2—O—R,
(l) 5′-TsAsTsToCsCoGoTsCsAsT-3′-O—PO2—O—R,
(m) 5′-TsAsToTsCsCoGoTsCsAsT-3′-O—PO2—O—R,
(n) 5′-TsAsToToCsCsGoTsCoAsT-3′-O—PO2—O—R,
(o) 5′-TsAsToTsCsCoGoTsCoAsT-3′-O—PO2—O—R,
(p) 5′-TsAoTsTsCoCsGoTsCoAsT-3′-O—PO2—O—R,
(q) 5′-TsAsTsToCsCsGoToCsAsT-3′-O—PO2—O—R,
(r) 5′-TsAsTsToCoCsGoToCsAsT-3′-O—PO2—O—R,
(s) 5′-ToAoTsToCsCoGoToCsAsT-3′-O—PO2—O—R or
(t) 5′-TsAsTsTsCsCsGoTsCsAsT-3′-O—PO2—O—R,
wherein all the variables o, s, R, m, n and q and A, G, C and T have the above-mentioned meanings.
The modified oligonucleotide according to the present invention is complementary to the DNA or RNA deriving from the human Ha-ras gene. The oligonucleotide is complementary to the translation initiation region of Ha-ras mRNA. Therefore it inhibits both wild-type and mutant ras expression resulting in inhibition of tumor cell proliferation. The oligonucleotide is modified to improve its stability and cell uptake characteristics. The oligonucleotide contains four to nine phosphorothioate linkages at certain positions which are especially vulnerable to attack by nucleases. The oligonucleotide is modified at the 3′-end with a C8-C21-alkyl or with a C1-C21-alkyl, preferably C16-alkyl chain, covalently attached through either a phosphodiester bridge, an oligoethyleneglycol phosphodiester linkage, or a glyceryl ether phosphodiester linkage.
A particularly preferred embodiment of the present invention is a modified oligodeoxynucleotide in which SEQ ID NO:1 has the linkage variation of 5′-TsAsToToCsCoGoTsCsAsT-3′-O—PO2—O—R (SEQ ID NO: 1), containing six phosphorothioate linkages, wherein the variables o, s, R, n, m, and q and A, G, C and T have the above-mentioned meanings.
R is particularly preferably —CH2CH(OH)CH2O(CH2)15CH3, —(CH2)15CH3 or —(CH2CH2O)n—(CH2)15CH3, wherein n is an integer from 1 to 6; preferably, n is 1, 2 or 3.
The invention further relates to the preparation of a modified oligonucleotide. Preferably the oligonucleotide is synthesized using standard phosphoramidite chamistry as outlind in examples 1 and 2. Phosphorothioate linkages are for example introduced by sulfurization using the Beaucage reagent.
Surprisingly it was found that particularly the glycerol alkyl ether, i.e. the group —CH2—CH(OH)CH2O—(CH2)q—CH3, q being an integer from 7 to 20, in particular the group —CH2CH(OH)CH2O(CH2)15CH3, linked by a phosphodiester linkage to the 3′-end of the oligonucleotide renders an oligonucleotide which no longer depends on the mixing with uptake enhancers for optimal biological activity. This is the first time that an oligonucleotide has been found to exhibit the same biological activity with and without the addition of an uptake enhancer.
The invention further relates to the preparation of a modified oligonucleotide. Preferably the oligonucleotide is synthesized using standard phosphoramidite chemistry as outlined in examples 1 and 2. Phosphorsthioate linkages are for example introduced sulfuization using the Beaucage reagent.
The modified oligodeoxynucleotide according to the present invention as described above is particularly suitable for use as a pharmaceutical. Therefore, a further object of the present invention is the modified olidodeoxynucleotide as described above for use as a pharmaceutical, in particular a pharmaceutical formulation which contains an effective amount of at least one modified oligodeoxynucleotide according to the invention. More particularly, the oligodeoxynucleotide according to the present invention is suitable for preparing a pharmaceutical for the treatment of a disease arising from the overexpression and/or mutation of the Ha-ras gene or diseases arising from a hyperproliferative disorder. Such diseases are in particular cancer, restenosis, or psoriasis.
Transforming activation of ras occurs in approximately 10-20% of human tumors by amplification of the ras gene at the DNA level or by overexpression of the ras protein at the mRNA level. Ras genes can be activated by point mutation at codon 12, 13, or 61 (Barbacid, M. Ann. Rev. Biochem. 1987, 56, 779-827; Bos, supra; Lowy, supra). Point mutation of the Ha-ras gene at codon 12 converts the normally regulated protein to one that is continually active. It is thought that the loss of regulation of normal ras protein function is responsible for the transformation from normal to malignant cell growth.
The modified oligodeoxynucleotide according to the invention is particularly suitable for the regulation, modulation, or inhibition of Ha-ras gene expression. The oligodeoxynucleotide is complementary to the translation initiation region of Ha-ras mRNA. Therefore, it inhibits both wild-type and mutant ras expression resulting in inhibition of tumor cell proliferation. The oligodeoxynucleotide according to the invention is further modified to improve its stability and cell uptake characteristics. The oligodeoxynucleotide contains four to nine, preferably six, phosphorothioate linkages at certain positions which are especially vulnerable to attack by nucleases. The cellular uptake of the oligodeoxynucleotide is further improved by a C8-C21-alkyl group or by an C1-C21-alkyl group covalently attached through either a phosphodiester bridge, an oligoethyleneglycol phosphodiester linkage, or a glyceryl ether phosphodiester linkage. Due to the improved stability and to the improved cellular uptake the oligodeoxynucleotide according to the present invention depends no longer on the mixing with uptake enhancers and is rendered for optimal biological activity.
Therefore, the oligodeoxynucleotide according to the present invention is particularly suitable for preparing a pharmaceutical which is directed to the regulation modulation or inhibition of Ha-ras gene expression of both, of the Ha-ras wild type gene and its mutants.
In case that the disease resulting from mutation of the Ha-ras gene is cancer, the oligonucleotide according to the invention is particularly suitable for preparing a pharmaceutical which inhibits the proliferation of cancer cells.
However, on the other hand the oligodeoxy nucleotide is advantageously used for preparing a pharmaceutical against cancer which is used in combination with chemotherapy, in particular in cases where the cancer cells have become resistant to chemotherapy, since the oligodeoxynucleotide according to the invention reverses chemoresistance in cancer cells. Therefore, a further object of the present invention is the use of the modified oligodeoxynucleotide as described above for preparing a pharmaceutical which reverses the chemoresistance in cancer cells.
In particular due to this particular effect the oligonucleotide according to the present invention is suitable for preparing a pharmaceutical formulation which contains an effective amount of at least one further chemotherapeutically effective agent. Such chemotherapeutically effective agents are for example cis-platinum and its derivatives, preferably cis-platinum, N-lost derivatives, preferably cyclophosphamide, trofosfamide and ifosfamide, aziridine derivatives, preferably thiothepa, N-nitrosourea derivatives, folic acid antagonists, preferably methotrexate, analogues of purine and pyrimidine bases, preferably 5-fluoro-uracil, cytostatically effective antibiotics, preferably adriamycine, mitomycine and daunorubicine, estrogene antagonists, preferably tamoxifene, and nucleoside derivatives, preferably MDL 101, 731 ((E)-2′-deoxy-2′-(fluoromethylene)cytidine; Cancer Research 54, 1485-1490, 1994).
Ras genes are known to be involved in signal-transduction of various factors for growth, differentiation and oncogenesis. Recent studies have implicated the raf-1 oncogene, which is downstream from ras in the signal transduction pathway, in the expression of the radiation resistance phenotype. It can be shown that the antisense oligonucleotide according to the present invention which is directed to the initiation codon of Ha-ras reverses the radiation resistance level of cancer cells.
Therefore, a further object of the present invention is the use of the modified oligodeoxynucleotide according to the invention for preparing a pharmaceutical for the treatment of cancer in combination with radiotherapy. Particularly, the modified oligodeoxynucleotide according to the present invention is suitable for preparing a pharmaceutical which reverses the radioresistance in cancer cells.
FIG. 1 shows the antitumor effects of PPS-C16 in vivo.
FIG. 2 shows the reversion of radioresistance in cell culture by treatment with PPS-C16.
The modified oligonucleotide according to the invention is prepared using an 1-alkanol and oligoethyleneglycol monoalkyl ether, respectively, in conjunction with the 3′-derivatization method described in EP 0 552 767 A2 and EP 0 552 766 A2; or a glyceryl ether phosphodiester linkage, using the solid-support described in Example 1.
List of Oligonucleotides used in Examples.
Sense + 3′-C16-
Antisense + 3′-C16-
Synthesis of solid support for the synthesis of oligonucleotides functionalised at the 3′-end with a glycerol hexadecyl ether linked by a phosphodiester linkage.
DL-a-Hexadecylglycerol (1 mmol) was dried by coevaporation with 3×5 ml anhydrous pyridine, then dissolved in anhydrous pyridine (3 ml). The solution was cooled to 0° C. and 4,4′-dimethoxytrityl chloride (1.15 mmol) was added. The reaction was stirred overnight. The reaction was quenched by the addition of water (100 ml) and evaporated to dryness. The residue was taken-up in dichloromethane (20 ml) and extracted 3 times with 10 ml 0.1 M phosphate buffer pH7. The organic phase was dried over sodium sulfate, filtered and evaporated. The residue was dried by coevaporation with 2×5 ml anhydrous pyridine, then dissolved in anhydrous pyridine (2 ml). 4,4-Dimethylaminopyridine (1.4 mmol), and succinic anhydride (2.1 mmol) were added. The reaction was stirred overnight. The reaction solution was evaporated to dryness and coevaporated twice with toluene:dichloromethane/1:1. The residue was taken-up in dichloromethane (20 ml), extracted with 10% citric acid (11 ml) and washed 3 times with water (11 ml). Two drops of triethylamine were added to the organic phase which was then dried over sodium sulfate, filtered and evaporated. The product was purified by silica gel chromatography using a gradient of 1-4% methanol, 1% triethylamine in dichloromethane. The product was obtained as a clear oil in 510 mg (62%) yield. The product (0.056 mmol), N-ethylmorpholine (0.07 mmol) and TBTU (0.056 mmol) were dissolved in DMF (2 ml) and added to aminopropyl controlled-pore glass (CPG; 500 mg; 500Å, Fluka). The mixture was shaken for 4 h and the CPG was filtered-off and washed with methanol and dichloromethane. The CPG was capped for 1 h using acetic anhydride/N-methyl imidazole in THF, filtered and washed with methanol, dichloromethane, THF and diethyl ether. After drying in vacuo the loading of dimethoxytrityl groups was determined to be 80 mmolg−1.
Oligonucleotide Synthesis
The oligonucleotides were synthesized using an Applied Biosystems 394 DNA synthesizer (Applied Biosystems, Inc., Foster City, USA) and standard phosphoramidite chemistry. After coupling, phosphorothioate linkages were introduced where required by sulfurization using the Beaucage reagent (lyer et al., 1990) followed by capping with acetic anhydride and N-methylimidazole in tetrahydrofuran. After cleavage from the solid support and final deprotection by treatment with conc. ammonia, the oligonucleotides were purified by gel electrophoresis. For the synthesis of the hexadecyl-modified oligonucleotides the solid support described in Example 1 above was employed. All oligonucleotides were analyzed by negative ion electrospray mass spectroscopy (Fisons Bio-Q) which in all cases confirmed the calculated mass.
Inhibition of in vitro Translation of Ha-ras mRNA
0.06 mg RNA was annealed with the appropriate oligonucleotide by heating them together in 10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 25 mM KCl and 1 mM DTT at 90° C. for 2 minutes and cooling slowly to room temperature. The translation reaction mixture (10 μl) consisted of 0.06 mg of RNA, varying amounts (1, 5, 10 or 20 μM) of oligonucleotide, 8 U of RNasin Ribonuclease Inhibitors (40 U/μl) (Promega Biotec), 20 μM of amino acid mixture minus methionine (Promega Biotec), 0.8 mCi of 35S-methionine (>1000 mCi/μM, Amersham), 3-6 μl of nuclease-treated Rabbit Reticulocyte Lysate (Promega Biotec), and 1 μM dithiothreitol (DTT). Translation proceeded for 1 hour at 30° C.
After translation, 10 μl of RIPA buffer (PBS, 1% NP40, 0.5% sodium deoxycholeate, 0.1% SDS) with fresh inhibitors (0.1 mg/μl PMSF, 1 mM sodium orthovanadate, 30 ml/μl aprotinin, Sigma, Cat. #A6279) was added to each sample, and the mixture incubated on ice for 20 minutes. 7 μl of 4×SDS sample buffer containing 1.25 M Tris-HCl pH 6.8, 6% SDS, 40% glycerol, 12% 2-mercaptoethanol and 0.001% bromphenol blue was added, and the sample was boiled for 5 minutes. 10 μl of each sample was electrophoresed in a 5-12.5% polyacrylamide gel (PAGE). The completed gel was fixed, dryed, and exposed to Kodak XAR-5 film with an intensifying screen at −80° C.
The claimed antisense oligonucleotide 5′-TsAsTTCsCGTsCsAsT-(PO2—O—CH2CH(OH)CH2O(CH2)15CH3)-3′ (PPS-C16) (SEQ ID NO:1) inhibited translation of ras p21 protein by approximately 10, 40, 50 and 80% at concentrations of 1, 5, 10, and 20 μM respectively. Controls using the sense oligonucleotide 5′-AsTsGACsGGAsAsTsA-(PO2—O—CH2CH(OH)CH2O(CH2)15CH3)-3′ (PPS-C16-S) (SEQ ID NO:2) showed no inhibition of translation until 20 μM at which point an 11% inhibition was observed. This strongly suggests that the C16 alkyl chain does not give rise to non-sequence specific effects in this system.
Inhibition of p21 Ras Protein Expression in Cell Culture
Cell Lines
NIH3T3 mouse fibroblasts (Chang et al. Biochemistry 30 (1991) 8283-86) transformed by the human Ha-ras (RS485) were used as an experimental cell line to evaluate the sequence-specific inhibition of cell growth and p21 ras protein expression by oligonucleotides directed to the first eleven nucleotides of the initiation codon region of Ha-ras mRNA.
RS485 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM, Flow Labs) supplemented with 10% heat inactivated (30 min. at 65□ C) fetal calf serum (FCS), antibiotics (50 U/μl each of penicillin, streptomycin, and neomycin), and 4 mM of L-glutamine.
Oligonucleotide Treatment of RS485 Cells in Culture
RS485 cells were seeded in 6-well plates (105 cells per well) and, after 20-24 hours (40-60% confluency), were treated with oligonucleotides. Medium was removed from the wells, 1 ml of fresh medium with oligonucleotides at various concentrations was added to each well, and the plates were incubated at 37° C. in an humidified atmosphere containing 5% CO2/95% air for 2448 hours.
Oligonucleotide Lipofection of RS485 Cells in Culture
RS485 cells (40-60% confluency) were transfected with oligonucleotides using Lipofectin™ Reagent and the supplier's protocol (from Life Technologies). Briefly, varying concentrations of oligonucleotides were diluted in 100 μl of serum-free medium. In a separate tube Lipofectin was added to serum-free medium at a concentration of 7 μl/100 μl. After 30 minutes at room temperature, 100 μl of Lipofectin solution was mixed with 100 μl of oligonucleotide solution, incubated for an additional 15 minutes at room temperature, and mixed with 800 μl of serum-free medium. Cells were washed two times with serum-free medium, and the total solution (1 ml) containing the oligonucleotide-Lipofectin complex was overlayed onto the cells. After 6 hours, 1 ml of fresh medium containing 8 mM L-glutamine and 20% calf serum was added, and the cells were incubated for an additional 38 hours. Treated cells were washed with PBS, harvested with trypsin, and cellular proteins were extracted.
The effect of PPS and PPS—C16 oligonucleotides on p21 expression in RS485 cells was compared in the absence or presence of Lipofectin (supra). After treatment total protein was extracted and p21 levels were determined by Western Blot analysis (infra). Lipofectin alone had no effect on p21 expression. Both the PPS— and PPS—C16-Lipofectin complexes almost completely inhibited p21 expression in RS485 cells at 1 μM concentration. Surprisingly the PPS—C16 oligonucleotide without Lipofectin completely inhibited p21 expression at 5 μM concentration. The PPS oligonucleotide without a C16 modification inhibited p21 expression by only 50% at the same concentration.
A comparison was then made of the effect of PPS-Lipofectin complex and PPS—C16 alone on p21 expression in RS485 cells. The p21 level was decreased by 30% and 43% at 0.25 μM and 0.5 μM PPS-Lipofectin concentrations respectively. Remarkably, the PPS—C16 oligonucleotide without any added Lipofectin showed approximately the same level of inhibition, namely 33% at 0.25 μM and 40% at 0.5 μM. Complete (100%) inhibition of p21 expression was observed at 1 M concentration, indicating that Lipofectin delivery does not augment PPS—C16-mediated inhibition.
Finally, a comparison of the inhibition of p21 expression in RS485 cell by PPS and PPS—C16 in the absence of Lipofectin was made. PPS alone gave an inhibition of approximately 50% at 5 μM. However, at a concentration of only 0.75 μM the PPS—C16 oligonucleotide exhibited nearly 60% inhibition of p21 expression in RS485 cells.
Inhibition of Cell Growth and p21 Ras Protein Synthesis in Cell Culture
Cell Growth Inhibition Studies
Cells treated with antisense oligonucleotides, or cells transfected with the oligonucleotide-Lipofectin complexes were harvested and lysed in RIPA buffer. Total protein levels in each well were determined spectrophotometrically. Inhibition of cell growth was estimated as the ratio of protein (mg) in the oligonucleotide-treated cells compared with that in the control. The control was either protein from cells treated with Lipofectin only, or protein from untreated cells.
Western Blot Analysis of Ras Protein
Total cellular protein was prepared by lysing cells in RIPA buffer with freshly added inhibitors (PMSF, aprotinin, sodium orthovanadate) and homogenizing through 21-gauge needles according to the protocol provided by Santa Cruz Biotechnology, Inc. The extracts were cleaned by centrifugation at 15,000×g for 20 min at 4° C. and the protein concentration of the supernatant was determined. 40 μg of protein was size fractionated in 5-12.5% PAGE and electroblotted onto a nitrocellulose membrane. The membrane was incubated with primary antibody against Ha-Ras (C-20) (Santa Cruz Biotechnology, Inc.) and then with Horseradish Peroxidase-conjugated goat anti-rabbit immunoglobulin G. The ECL chemo-luminescent Western system (Amersham, Arlington Heights, Ill.) was used to detect secondary probes.
A series of experiments were performed to study the sequence—sequence toxic effect of the PPS and PPS—C16 oligonucleotides on RS485 cells shows that treatment of RS485 cells with 1-5 μM of PPS and PPS—C16 in the presence of Lipofectin (supra) reduces cell growth by approximately 35-50% over a 2 day period. Lipofectin alone has a slight toxic effect (ca. 15%) on cells. Comparison of the Western blot analysis of p21 expression inside RS485 cells (supra) with the cell growth inhibition by PPS and PPS—C16 demonstrates that a 1 μM oligonucleotide/Lipofectin complex concentration totally inhibits p21 expression, but inhibits cell growth by only 35%.
PPS—C16 without Lipofectin inhibits cell growth by 11% at 0.75 μM while inhibiting p21 expression by 60% (supra). PPS inhibits cell growth by 22% at a concentration of 5 μM, a concentration at which it inhibits p21 expression by 50%. Lipofectin exerts a toxic effect when used with PPS at concentrations higher than 1 μM and that the PPS—C16 oligonucleotide without Lipofectin is, in the concentration range 0.25-1 μM, just as effective as the PPS oligonucleotide with Lipofectin.
Reversal of Radioresistance—Animal Study
Antitumor Effects of Antisense Oligonucleotides in vivo
RS504 cells are NIH 3T3 cells transformed by the Ha-ras oncogene isolated from EJ/T24, a human bladder carcinoma. Tumor were induced by injecting 5×106 RS504 cells subcutaneous in female athymic NCr-nu mice. Forty-eight hours later, when tumors were evident (−6 mm3), partially phosphorothioated C16-modified [PPS—C16] antisense [AS] or sense [S] oligonucleotides were injected directly into the tumors at a concentration of 50 μl of 5 μM solution (250 pmol) per tumor. After 24 hours, tumors were irradiated with a 2.0 Gy dose (thin arrows, see FIG. 1). Irradiation was repeated every 48 hours until an accumulated dose of 20 Gy had been delivered. Oligonucleotide injections were repeated at 48 hours [50 μl], 192 hours [50 μl), and 240 hours [100 μl] following the initial injection (bold arrows, see FIG. 1). Tumor volume was recorded in mm3. Controls consisted of tumors that were not injected with oligonucleotides and not irradiated, tumors that were not injected with oligonucleotides but were irradiated; and tumors injected with antisense PPS—C16 oligonucleotides but not irradiated.
Tumor Growth
Ras genes are known to be involved in signal-transduction of various factors for growth, differentiation and oncogenesis. Recent studies have implicated the raf-1 oncogene, which is downstream from ras in the signal transduction pathway, in the expression of the radiation resistence phenotype. It can be shown that the antisense oligonucleotide according to the invention reverses the radiation resistance level of the cells. To determine if there were differences in the effects of radiation on ras-induced tumors following treatment with oligonucleotides, RS 504-induced tumors in nude mice were treated with antisense and sense PPS—C16 oligonucleotides. FIG. 1 shows that antisense PPS—C16 oligonucleotide treatment in conjunction with radiation was most effective in inhibiting tumor growth during the 17 days of observation and treatment.
Reversion of Radioresistance in Cell Culture
Reversion of radioresistance phenotype in cell culture by treatment with PPS—C16 Bladder (T24) carcinoma cell lines (obtained from ATCC, Rockville, Md.) were maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum 50 μg/ml each of penicillin, streptomycin anfd neomycin and 2 mM L-glutamine.
For oligonucleotide treatment, the cells were plated at 1×105 cells/well in 6-well tissue culture plates. Twenty-four hours later, at approximately 40-60% confluency, the cells were transfected with oligonucleotides, facilitated by Lipofectin Reagent, using essentially the protocol supplied by the manufacturer, Life Technologies, Inc. After 6 hours, the lipofection solution was removed and the monolayer washed with fresh medium containing 8 mM L-glutamine and 20% serum. The cells were then incubated for an additional 16-18 hours in 1 ml of this medium. Cellular response to radiation was evaluated by the colony survival assay. Exponentially growing monolayer cultures of each cell line were treated with the oligonucleotides as described above the cells were harvested 24-48 hours later, suspended in fresh medium and irradiated at room temperature with graded doses of 137Cs γ rays at a dose of approximately 36 Gylminute in a J. L. Shephard and Associates Mark I irradiator. Afterward, the cells were diluted and plated at a concentration of 300 to 5000 cells per well in a 6-well tissue culture plate. Two to three days after plating, the cells were supplemented with 0,5 ml of serum plus 5 μg/ml hydrocortisone. Approximately 7-14 days later, the cells were stained with 1% crystal violet and colonies (comprising 50 or more cells of normal appearance) were scored. The D10-value (radiation dose required to reduce survival of cells to 10%) for antisense-treated (PPS—C16) T24 cells drops from the highly resistant level of 5.5 (untreated or sense-treated PPS—C16—S) to 4.5 Gy, a value dose to what is considered to be radiosensitive. These results are shown in FIG. 2.
<213> ORGANISM: Artificial Sequence
<223> OTHER INFORMATION: Description of Artificial
#Sequence: synthetic
tattccgtca t
# 11
<213> ORGANISM: Artificial Sequence
<223> OTHER INFORMATION: Description of Artificial
#Sequence: synthetic
tattccgtca t
# 11
<213> ORGANISM: Artificial Sequence
<223> OTHER INFORMATION: Description of Artificial
#Sequence: synthetic
atgacggaat a
# 11
<213> ORGANISM: Artificial Sequence
<223> OTHER INFORMATION: Description of Artificial
#Sequence: synthetic
atgacggaat a
# 11
<213> ORGANISM: Artificial Sequence
<223> OTHER INFORMATION: Description of Artificial
#Sequence: synthetic
tattccgtca t
# 11
|Brevet cité
|Date de dépôt
|Date de publication
|26 aoűt 1994
|19 nov. 1996
|Isis Pharmaceuticals Inc.
|Antisense oligonucleotide inhibition of the RAS gene
|14 déc. 1992
|10 déc. 1996
|Isis Pharmaceuticals, Inc.
|Antisense oligonucleotides to the RAS gene
|19 aoűt 1994
|10 déc. 1996
|Isis Pharmaceuticals, Inc.
|Antisense oligonucleotide inhibition of the ras gene
|5 juin 1995
|9 déc. 1997
|Hoechst Aktiengesellschaft
|3'-modified oligonucleotide derivatives
|21 janv. 1993
|23 juil. 1993
|Eugen Uhlmann
|Oligonucleotide analogs, their preparation and use
|10 nov. 1994
|13 mai 1995
|Anuschirwan Peyman
|Stabilized oligonucleotides and their use
|1 oct. 1993
|14 avr. 1994
|Isis Pharmaceuticals, Inc.
|ANTISENSE OLIGONUCLEOTIDE INHIBITION OF THE ras GENE
|10 aoűt 1993
|28 avr. 1994
|Temple University - Of The Commonwealth System Of Higher Education
|Combination of antineoplastic agent and antisense oligonucleotides for treatment of cancer
|Bitonti, A.J., et al., "Regression of Human Breast Tumor Xenografts in Response to (E)-2'-Deoxy-2'-(fluoromethylene)cytidine, an Inhibitor of Ribonucleoside Diphosphate Reductase," Cancer Research, vol. 54, pp. 1485-1490 (1994).
|Bitonti, A.J., et al., "Regression of Human Breast Tumor Xenografts in Response to (E)-2′-Deoxy-2′-(fluoromethylene)cytidine, an Inhibitor of Ribonucleoside Diphosphate Reductase," Cancer Research, vol. 54, pp. 1485-1490 (1994).
|Brown, D., et al., "Modulation of ras Expression by Anti-sense, Nonionic Deoxyoligonucleotide Analogs," Oncogene Research, vol. 4, pp. 243-252 (1989).
|Peyman, A., et al., "Facile Preparation of 3'-derivatized oligodeoxynucleotides," Bioorganic & Medic. Chemistry Letters, vol. 5, No. 21, pp. 2469-2472 (1995).
|Peyman, A., et al., "Minimally Modified Oligonucleotides of End-Capping and Pyrimidine-Protection," Biol. Chem. Hoppe-Seyler, vol. 377, pp. 67-70 (1996).
|Peyman, A., et al., "Facile Preparation of 3′-derivatized oligodeoxynucleotides," Bioorganic & Medic. Chemistry Letters, vol. 5, No. 21, pp. 2469-2472 (1995).
|Pirollo, K.F., et al., "Evidence Supporting a Signal Transduction pathway Leading to the Radiation-Resistant Phenotype in Human Tumor Cells," Bioch. and Biophys. Res. Comms., vol. 230, pp. 196-201 (1997).
|Zon, G., et al., "Phosphorothioate oligonucleotides," Eckstein, F., Ed., Oligonucleotides and Analogues, Ch. 4, pp. 87-108 (Oxford Univ. Press, 1991).
|Brevet citant
|Date de dépôt
|Date de publication
|22 avr. 2010
|12 juil. 2011
|Leonid Beigelman
|RNA interference mediated inhibition of myostatin gene expression using short interfering nucleic acid (siNA)
|28 déc. 2006
|3 janv. 2012
|Whiteland Institute for Biomedical Research
|Use of FOXC2 in diagnosing, preventing and treating cancer metastasis
|1 déc. 2011
|29 oct. 2013
|Whitehead Institute For Biomedical Research
|Methods of diagnosing, preventing and treating cancer metastasis
|14 avr. 2010
|29 oct. 2013
|Postech Academy-Industry Foundation
|Nucleolin specific aptamer and use thereof
|24 sept. 2013
|28 avr. 2015
|Postech Academy-Industry Foundation
|Method of diagnosing cancer using a nucleolin specific aptamer
|24 sept. 2013
|28 avr. 2015
|Postech Academy-Industry Foundation
|Method of treating hyperproliferation of cells using a nucleolin specific aptamer
|4 aoűt 2006
|4 janv. 2007
|Sirna Therapeutics, Inc.
|RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
|22 avr. 2010
|9 sept. 2010
|Mcswiggen James
|RNA INTERFERENCE MEDIATED INHIBITION OF MYOSTATIN GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
|14 avr. 2010
|16 déc. 2010
|Postech Academy-Industry Foudation
|Nucleolin Specific Aptamer and Use thereof
|24 sept. 2013
|3 avr. 2014
|Postech Academy-Industry Foundation
|Method of diagnosing cancer using a nucleolin specific aptamer
|24 sept. 2013
|31 juil. 2014
|Postech Academy-Industry Foundation
|Method of treating hyperproliferation of cells using a nucleolin specific aptamer
|2 aoűt 2007
|17 oct. 2012
|Medimmune Limited
|Human antibodies to ERBB2
|Classification aux États-Unis
|Classification internationale
|Classification coopérative
|Classification européenne
|17 févr. 2000
Owner name: HOECHST MARION ROUSSEL DEUTSCHLAND GMBH, GERMANY
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:UHLMANN, EUGEN;PEYMAN, ANUSCHIRWAN;WILL, DAVID WILLIAM;AND OTHERS;REEL/FRAME:010612/0918;SIGNING DATES FROM 19991018 TO 20000204
|18 aoűt 2000
Owner name: AVENTIS PHARMA DEUTSCHLAND GMBH, GERMANY
Free format text: CHANGE OF NAME;ASSIGNOR:HOECHST MARION ROUSSEL DEUTSCHLAND GMBH;REEL/FRAME:011019/0234
Effective date: 20000204
|18 nov. 2005
Owner name: SANOFI-AVENTIS DEUTSCHLAND GMBH, GERMANY
Free format text: CHANGE OF NAME;ASSIGNOR:AVENTIS PHARMA DEUTSCHLAND GMBH;REEL/FRAME:016793/0789
Effective date: 20050901
Owner name: SANOFI-AVENTIS DEUTSCHLAND GMBH,GERMANY
Free format text: CHANGE OF NAME;ASSIGNOR:AVENTIS PHARMA DEUTSCHLAND GMBH;REEL/FRAME:016793/0789
Effective date: 20050901
|17 sept. 2007
|Fee payment
Year of fee payment: 4
|11 oct. 2007
Owner name: THE BOARD OF TRUSTEES OF THE STANFORD JUNIOR UNIVE
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:SANOFI-AVENTIS DEUTSCHLAND GMBH;REEL/FRAME:019943/0341
Effective date: 20071010
|5 déc. 2011
|Maintenance fee reminder mailed
|20 avr. 2012
|Lapse for failure to pay maintenance fees
|12 juin 2012
|Expired due to failure to pay maintenance fee
Effective date: 20120420


text:
Brand Label: Rachel Zoe
Available for Purchase Online at Farfetch US for $395.00Pin It
Black frill detail dress from Rachel Zoe. Size: 2. Gender: Female. Material: Polyester/Acetate.
Shop other brand label items from Rachel Zoe on FashionStylist.com.
This item was published for sale online by Farfetch US on October 23, 2017 10:15 am and last updated by us November 15, 2017 10:49 am.
Editor Picks: Dress Yourself
Proenza Schouler Women's Patch Embellished Bomber
Loewe Women's Tie Waist Cotton Poplin Shirtdress
Yigal Azrouel Women's Crocodile Fil Coupe Midi
Lela Rose Women's Metallic Floral Fil Coupe Top
Etro Women's Floral Paisley Jersey Dress 6 Us
Editor Picks: So 70's Clothing Inspiration
Alice Olivia Lyn Paneled Suede Fringe Cape Tan
Isabel Marant Open Front Embroidered Long Coat
Adam Lippes Pencil Skirt W Fringe Hem Black
Burberry Prorsum Patchwork Blanket Fringe Poncho
Valentino Crochet Mitered Stripe Poncho Sorbet
Editor Picks: Holiday Fashion Trends for Women
Jason Wu Point Desprit Long Sleeve Gown
Chloe Chunky Cashmere Crewneck Sweater
Piazza Sempione Deco Dot Tie Dye Cardigan Yellow
Victoria Beckham Hexagon Fitted Pencil Skirt Black
Atm Leather Stretch Moto Pants Black
Editor Picks: Black to Basics
Helmut Lang Women's Cotton Angora Rib Knit Top
T By Alexander Wang Women's Argyle Stitch Cardigan
Rodarte Women's Rohearte Heart Graphic Sweatshirt
Vince Women's Colorblock Tshirt X Black T Shirt
Enza Costa Women's Loose V Neck Cotton Cashmere
Editor Picks: Relax in Black with Sweaty Betty
Perfect Moment High Neck Half Zip Base Layer Top
Sweaty Betty Double Time Tank
Sweaty Betty Superfine Slv Tshirt T Shirt
Sweaty Betty Assemble Knit Hoody Hoodie
Sweaty Betty Connection Dance Capri
Browse More Fashion Apparel by Rachel Zoe (view more)
- $395.00
Rachel Zoe Maida Sequin Pants
- $395.00
Rachel Zoe Monner Ruffle Trimmed Shift Dress
- $525.00
Rachel Zoe Isla Fringe Jacket
- $99.99
Rachel Zoe Stagedoor Studio Bag
- $49.99
Rachel Zoe Amalia Leather Wallet
- $124.99
Rachel Zoe Hallah Open Toe Leather Platform Sandals
- $99.99
Rachel Zoe Florence Leather Open Toe Flats
- $49.98
Rachel Zoe Raina Slip On Open Toe Sandals
- $104.99
Rachel Zoe Grechen Leather Block Heel Sandals
- $49.98
Rachel Zoe Belcaro Embroidered Slides Shoes
New promo codes and links offering limited time promotions, sales coupons, and special offers on Rachel Zoe designer fashions and accessories will be posted on each designer label apparel page. For the latest up to date information on this Rachel Zoe Frill Detail Dress Women Polyester Acetate 2 Black Polyester Acetate | Clothing fashion item, including product specifications, ordering options, pricing, shipping details, high resolution pictures, shopper comments, and other sales media, visit the Farfetch US Online Store.


text:
Fighting An Consuming Disorder IF IT IS 'Hard To NEED TO GET Better'
Enlarge this photo toggle caption Image Courtesy of Samantha Hackett; Elizabeth Birnbaum Photograph Courtesy of Samantha Hackett; Elizabeth Birnbaum
When you're facing a significant life change, it can help to talk to someone who has recently been through it. WITH THAT SAID is connecting persons on either part of a shared encounter, and they're letting us eavesdrop on the conversations in our series Been There.
All of a sudden, Maddy Rich found herself in the center of a crisis. It sneaked up on her, in February 2016, during her sophomore season at Cornell University.
When Maddy got on campus, she started working out a whole lot. "Oh, my gosh, there are these sportsmen around me, that's seriously inspiring," she remembers thinking. She started eating less, too: "Oh, I must go the lab, I don't possess time to eat anything."
Share Your Encounter Are you going to undergo a significant life change, like start your have business or deploy overseas found in the military? Or perhaps you have been through one already? WITH THAT SAID invites you to talk about your encounter, either to ask problems or pass on your own lessons discovered. Email us at firstname.lastname@example.com, with "Been There" found in the subject line.
Maddy had developed an taking disorder: anorexia nervosa – and it had been buying worse. She was obsessing, she says, thinking about food all the time. She began to get physically unhealthy.
Gradually, she hit a turning point, during a telephone call with her mother. "She was just thus upset, like 'Why will be you falling aside? I don't understand what's happening,'" Maddy says, "and I was certainly sitting down there with a glass of watered-down soup that would come to be my main lunch time. And for reasons uknown I couldn't eat it, and I simply started crying."
Maddy, now 21, got help. She eventually spent three weeks in a home treatment program. But recovery from anorexia can be an ongoing challenge. "It had been hard to admit that I needed that many help," Maddy says, "and also hard to need to get better that much."
Maddy uses a metaphor when she foretells persons about anorexia. "Even though I'm doing better, it's like there's chained to my ankle, a dead human body that I'm merely dragging around with me."
Julia Sinn knows what that's like. She halted wanting to eat when she was about 16, she says, and struggled with her ingesting disorder – which experienced symptoms of both bulimia and anorexia – for years before getting help. And she says there's still part of her mind that thinks destructive thoughts.
"There was a whole lot of fuel on that fire," 29-year-old Julia says, "but every day, I put a bit more fuel on the additional fire, which is, being individual with myself and being compassionate, and hearing what's heading on in my body."
This has been lightly edited for clarity.
Advice from Julia Sinn
On attempting to get better
We was struggling for seven or eight years. And I didn't need to get better. Naturally I really was unhappy, but there's this defiance, you know like, "I like it," or, "I'm better this way," or, "This is exactly what I wish to be doing." I would say 80 percent of the struggle of getting into recovery had been like, "I don't want to do this anymore."
On keeping destructive thoughts in check
Every day, I must be like, "Oh, I've been scrolling through Instagram feeds of really skinny models for 20 minutes, maybe this isn't great." Or, "Oh, I in fact did skip breakfast deliberately. It wasn't 'cause I was too active or blah, blah, blah." Like, checking atlanta divorce attorneys day, to see what I'm doing and where it's heading.
On learning to forgive herself for mistakes
My life will go on if one day is different from how I thought it had been supposed to be. And this helped me heal from disordered patterns: I had to recognize that it had been OK easily had relapsed, just like it's OK easily can't make it to the fitness center tonight.
On learning to live with taking disorder thought patterns
It probably is generally going to be there, but it doesn't mean that it has to be a good cloud over your head or a chain you every day. It's such as a little yappy dog, but eventually it becomes portion that you're just like, "that's there." And some days it's seriously loud, and some days and weeks pass where it's not, and it's simply sleeping in the part. But it will likely be there. And I don't prefer you to receive disheartened or scared of this. And, it's nearly empowering to recognize that. Because, when you're in the depths of ingesting disorder patterns and thoughts, you are feeling defined because of it. It's all you can consider. So there's merely something empowering to owning your ingesting disorder without letting it own you.
Former NPR Story Lab intern and freelance journalist and producer Mette Lützhøft contributed to this report.


text:
Some girls intrestet for 19m for date? Add me on facebook stefan mladenovski (teddybear) or on instagram s.mladenovski or snapchat steffamily
Stefan Mladenovski
![alt text](![image url])
By using TalkWithStranger, you are accepting our privacy and usage terms . You must be 18+ or 13+ with parental permission to use our online chatting site.


text:
FilterAlbany, NY
Coupons worked with the employee discount (in 1332 reviews)
It allowed for remote working which was great for work-life balance (in 252 reviews)
Work life Balance cab be a challenge at times (in 355 reviews)
The stress of opening credit cards (in 829 reviews)
I worked at Macy's part-time (More than a year)
Great people to work with and friendly people. Fun job and love helping customers. Busy faster paced retail environment.
Not enough hours given on schedule though you can pick up extra shifts on line
Advice to Management
Give more hours in schedule
I worked at Macy's full-time (Less than a year)
The company is so well recognized around the country as one of the premier places to shop for the holidays.
Management does not keep up with the quality of service towards customers which is making it more difficult for associates and store managers to keep relationships.
Advice to Management
Focus on ups and downs of individual stores quality of service, the appearance, more rewards for lower level jobs.
I worked at Macy's (Less than a year)
Good discount for employees when you purchase items
Only when you work with associates that don't really want to do their job
Advice to Management
I worked at Macy's part-time (More than a year)
Friendly and good customers and good environment to work in. I enjoyed putting a smile.in customer I wa the truck. I love you. gdn the
Poor management and not employees appreciation tr the truck. I love you. gdn the truck. I love you. gdn the truck. the 20 in the bathroom 2nd St Paul McCartney I was
Advice to Management
U know what I'm 6th Ave and I am applying the money to pay a little while, so I'm sure it was great and we are not an option for me,
I have been working at Macy's full-time (More than 3 years)
Managers and Associates are easy to work with.
Depending on the hours, sometime it is difficult to find a manager.
Helpful (2)
I have been working at Macy's part-time (More than 8 years)
Flexible hours, great suport and training, many opportunities to earn extra dollars. Great discounts.
Too many promos sales and discounts that over lap or are subject to qualifying items/ departments. Confusing to the customer.
Advice to Management
Commission sales staff pay back hours worked from commission earnings however; when non commission hourly sales staff is limited unavailable then commission sales staff spends many hours of their commission sales time ringing returns and sales that they dont get commission for due to the fact the items and customers are from other departments that are under staffed but, those hours still come out of commission check. Macys says its part of "Making Macys Magic" and yes it is but I feel solution could be to at least give commission sales staff some percentage of items they ring that are from outside departments.
I worked at Macy's (Less than a year)
good place to start out for college kids
low pay..next to no raises no matter how much you bust your butt
Helpful (1)
I worked at Macy's part-time (Less than a year)
Free samples from the counter and friendly co-workers. I worked during the holiday season so everyone was pretty cherry and delightful. Macy's also provided adequate training which I appreciated.
Long hours at times and competitive sales goals when working at a counter. The holiday season is fun to work but it also get pretty hectic, be prepared to work long hours.
Advice to Management
I just wish that those who work in the beauty department could be a little more professional.
I worked at Macy's part-time (Less than a year)
Great Experience alot of appreation Im Currently the Mens Polo Specialist
Poor Managament, alot of pressure on the employees
Advice to Management
Looking to Transfer to Bloomindales for a stronger clintel
Helpful (2)
I worked at Macy's part-time (More than a year)
Gave me knowledge about bedding, learned how to use a cash register pretty well.
Horrible scheduling system, incompetent management.
Advice to Management
I don't even feel like bothering because no one listened to me when I was there or even after I left, but this was probably the worst decision for a job I could have ever made. I thought because of how Macy's portrayed themselves it would be an okay job with some decent people, I was completely wrong. All of the staff was miserable, the people who did nothing or simply disobeyed the rules got rewarded the most. If you're a hard worker, obey the rules, actually put forth an effort for your position, and make sure a customer is happy after the emotional and mental abuse for coupons, don't expect to get rewarded or even told you're doing a good job by your higher up. You barely see your managers unless you're ready to play hide and seek or phone tag for about 24 - 48 hours. Don't expect to get days off in advanced, it's not happening. The credit system is a complete joke. The hours are god awful, and forget it if you're in college. You can give every manager in the store a copy of your schedule, but they still give you hours that conflict with your school schedule. When you work here, you don't have a life. You live and breathe Macy's. Keep letting the magic happen, the magic that's horrible.
This will replace the current featured review for targeted profile. Are you sure you want to replace it?
Are you sure you want to remove this review from being featured for targeted profile?


text:
20/05/2016 Clothing
Wonderful White Embroidery Work Net Brocade Gown.This beautiful gown is showing Embroidery work done on neck with Diamond Work and patch work. The suit is designed with different fabrics, like; net and brocade top,santoon inner and bottom,Net sleeves with work and chiffon dupatta.
Work from the comfort of your home, or as you travel, on your computer, tablet, or smartphone. ➡Start FREE, no obligati...
Category : Internet Posted : 21/11/2017 Price : 1.00 US$ Location : Brooklyn(New York)
We're a team of professionals working around the clock to fulfills the client requirement and make the Discount movers t...
Category : Moving - Storage Posted : 21/11/2017 Price : 120.00 US$ Location : Dubai(Dubai)
Moving - Storage
✌✌Hi ..friends.......join with DIGITAL INDIA concept......and earn daily 200 - 500 rs....by doing part time work.....It ...
Category : Marketing Posted : 21/11/2017 Price : Check with seller Location : Kolkata(West Bengal)
CJM Enterprises Pre-Owned Harley Davidsons, Motorcycles, ATV's and UTV's, Trucks and SUV's. Over 30 years' experience wi...
Category : Motorcycles Posted : 21/11/2017 Price : Check with seller Location : Loveland(Colorado)


text:
Needing exciting and sugary man (Rancho Santa Margarita, California )
I am just searching for an individual who would like to gather have some enjoyable. I am curvy and sexy and I love to celebration and get exposed ! married women looking for men to find guys who like to get together and acquire naked ! ! I'm trying to find a alluring connection with a hot gentleman who, like me personally, is wonderful to check out. i desire a man that may be not affraid to show his experiencing toward a girl in public areas. If you want to know more, then just ask. If you are anything but sincere.., Dont respond.
I could simply be me (92688, Rancho Santa Margarita, California)
I would desire to just fulfill an exciting gentleman to hang by helping cover their and see where it is going. Willing to try to see what it gets me... hopefully I get what I'm looking for, although im not too sure about this! You have to have outstanding nice and clean-ness, that's usually significant, and learn how to be serious having a NSA arrangement. My position married women looking for men available but that is perfectly up to you.... I don't mind in either case. Ought to be DnD cost-free. Any images will almost always be beneficial
married women looking for descrete relationships
Dare to join your neighbours gangbang tonight? Register and find out!
Regardless if I'm in bed I'm ready for sex (92688, Rancho Santa Margarita, CA, Orange County)
We have just come to be individual now and again married women looking for lovers is time for me personally to get out and get together difficult. Foreplay is extremely important to I and me require a man who believes that exact same way too. I devote hrs in the fitness center each week keeping my entire body seeking this divine. I possess a wilderness creative thinking within the room and desire a person having a single way too. I am just painful for many warm passion during my lifestyle. Need to see how exciting my ceiling is?
Ladies who adores in the open air.... (92688, Rancho Santa Margarita, CA)
Hey Effectively internet marketing just one females who performs times.... so its hard for me personally to satisfy men and women... I have got a job, my and car individual condominium. I really like getting out side, camping, playing, hunting and married senior women looking for sex swimming pool area amongst other things. If you are considering chatting with me... Be more than 23 and also a pic for me to respond and put your best shade from the subject range therefore i know your real.... Well hope to hear from ya and good luck.
Relaxed date this weekend with heavy/curvy girl? (Rancho Santa Margarita, 92688, CA )
Internet marketing simply looking to just go and enjoy yourself this saturday and sunday having a fairly sweet fine man. Cocktails, dancing, 420. dinner, whichever. Just want to have fun! I'm 5'4, latina/white-colored, simple short head of hair, generally curly. I'm fun, always, sweet and sarcastic downward for a great time.Please be 25-45. I enjoy white colored or hispanic but struck me together with your greatest married women looking for men
Yes... but would you feel the same if your wife was the one being shared? I utilized to enjoy once i was younger... I needed standard lovers who liked in my opinion ahead above per week...
Not looking for the love of my well being (Rancho Santa Margarita, 92688 , Orange County)
.. nor a true love , not wanting to get married or have your young ones not seeking to fulfill on top of you together with invest each and every waking next in your life along with you. Searching for sexual intercourse , trying to find Great gender , seeking someone thoroughly clean having a filthy dirty thoughts , searching for somebody black married women looking for men dosnt have nasty intimate ailments , looking for someone who is aware wherein a womans clit is without needing directions. Appear to be you? hit me up.
Close friends first then far more (92688, CA, Orange County)
I would desire to just satisfy an entertaining man to hang out with to see in which it is. Im not too sure about this, but willing to try to see what it gets me... hopefully I get what I'm looking for! black women looking for married men should have outstanding clear-ness, that's always crucial, and realize how to be intent on a NSA set up. My spot is accessible but that depends on you... I really do not brain in either case. Must be DnD free of charge. Any images are always useful
Yeah, I found myself THAT young lady (Rancho Santa Margarita, California )
In high school graduation. Stuff has transformed, I'm not the crazy youngster I had been. If I don't miss it, but damn! Would like to perform 'back inside married women looking for men day' with me? I have my cheerleading outfit and I'd gladly strut my stuff to suit your needs! I truly do overlook that old time. Funny issue is? The folks I wouldn't offer the time of day to back then are those I get eye-catching now. Geeks. Go figure. Write me!


text:
Home - Church Sewing Items - Cross sets - Machine-embroidered crosses - Donets cross vestment set
We are very happy and everything measured fit great. I am glad you questioned our measurements because we brought in a woman we knew could do the measuring correctly and we did end up changing the measurements we began with.Stephen Steineck Abbey Spring
I placed some vestment orders as gifts for a fellow priest. He opened his gifts yesterday and was immensely pleased with your vestments. Thank you for your continuing fine work.Fr. Alexis
You are doing a wonderful job!William Barnes
Dear Istok. Thank you, I have received my fabrics yesterday. Everything is perfect. Thank you again. Very much appreciated.Nadejda Vladimiroff
Many thanks for the undercassock which arrived at a most auspicious time. Your work is the highest quality and the fit is perfect. Many Blessings to You!Thank you so very much for the kind extension- I have never met such beautiful people in this...Br. Gregory Dean
Thank you so much! We have our complete order and everything was beautiful. I can not thank you enough and we got the large Icon and necklace in time for the Chrismation. What a wonderful Holy Saturday and Pascha we had.Jeannie Elizabeth
We love you fast service and wonderful products! The special offers are always amazing. We will buy many more products from here to outfit our new church.Linnea Kaminsky
My vestments have been received. Nice job. Thank you!Roy Salthouse
Thank you for the amazing work you do.John Collins
Glory to Jesus Christ! I received the beautiful cassock yesterday. Thank you very much! I truly appreciate your support and efforts and look forward to doing business in the future.Rev. Frank Avant
Dear friend,
We've added new products to our store:
You can conveniently see them on the What's New page.
We're also pleased to inform you about the following special offers:
These offers expire on Oct. 27/Nov.10 at 24:00 CST or until supplies last.
Tip: One of the best tools for caring about your candle-stands and other metal ware is German Metal polishing paste.


text:
Berlin, MD 21811MLS Number: 506291
Originally $749,900
Beds: 4
Baths: 2 Full / 2 Half
SqFt: 2804
View the Virtual Tour
Beds: 4
Baths: 2 Full / 2 Half
SqFt: 2804
View the Virtual Tour
Request Information
DescriptionOver 5 1/2 acres of meticulously landscaped ground in a private country setting. This beautiful 4 bedroom custom built Piney Island home, with an oversized bonus room that could easily be converted to another bedroom must be seen to be appreciated! Situated just minutes from Ocean City, Berlin and Delaware, this gorgeous property has many possibilities! Wood floors and tile throughout the home, multiple fireplaces, tons of natural light. Custom paver patio with an outdoor kitchen boasting Viking appliances. A five bay contractor shop offers even more versatility to this already very desirable property. Make an appointment to see this home today!
General DetailsBedrooms: 4
Bathrooms, Full: 2
Bathrooms, Half: 2
Living Area: 2804
Lot Area: 5.59
Property Type: Single Family Home
County: Worcester
Year Built: 2005
Sub-Division: N/A
Historical District: No
Public Maintained Road: Yes
Area: Worcester East of 113
Disclosures: Seller Disclaimer
Stories: 2.0
Acres: 5.59
Exterior FeaturesFoundation: Concrete Block
Garage(s): None, 0.00 spaces
Roof: Architectural Shngle
Exterior Features: Off Street Parking,Satellite Dish,Screens,Storage Shd/Out Bldg,Storm Doors,Storm Windows
Exterior Type: Vinyl Siding
Waterview: No
Interior FeaturesAppliances: Dishwasher,Dryer,Microwave,Range - Gas,Refrig. Ice Maker,Refrigerator/Freezer,Washer, whole house generato
Attic: Walk-In
Basement: Crawl Space
Heating: 2 gas furnaces
Cooling: Central Air
Interior Features: 1st Floor Bed & Bath,Cathedral Ceiling,Ceiling Fan(s),Fireplace-Gas,Skylight(s),Walk-in Closet(s)
Lot InformationNumber: 4
SqFt: 5.59
Description: Cleared
School InformationElementary School: Showell
Middle School: Stephen Decatur
High School: Stephen Decatur
Fees & TaxesCity Taxes: $0
Condo Fee: $0
County Taxes: $3,315


text:
Oyster Rock Featured Listings
15080 Oyster Shell, Milton, DE
This Stunning Home Pairs Convenience And Elegance Into A One Of A Kind Home Owning Experience. Entertain In Style In Front Of The Living Room's Double-sided Fireplace Or Curl Up With A Book In The Gorgeous Sun Room. The Top Of The Line Kitchen Includes Gas Cook Top, Stainless Appliances, Cherry Cabinets And Granite Counter Tops. Huge Second Floor Bedroom Could Be A Home Office, Gym Or Bunk Room And Includes A Full Bath. Other Highlights Include Bamboo Flooring; Vaulted Ceilings; Tankless Hot Water Heater; Trex Decking; Weather Treated Aluminum Fencing And Professional Landscaping. Insulated Concrete Form (icf) Construction With 1 Ft. Thick Walls For Energy Efficiency, Safety And Noise Reduction. Home Is Pre-wired For Cable, Wifi And Security System. Community Allows On-site Storage Of Boat Or Rv. Located On The East Side Of Route 1. The Address Says Milton But Home Is Close To Beaches, Shopping, Historic Downtown Milton And The Lewes And Rehoboth Beaches. Home Warranty Provided!
30425 Pearl, Milton, DE
Perfect Cleared Lot To Build On In The Community Of Oyster Rock. Minutes To The Broadkill River, Beaches And Shopping.


text:
Dear Sir,
Are you having one or two difficulties from other financial instrument lender? I want you to take a chance with us you will never regret your partnership with our firm.We have direct and efficient providers.
I am the sole (Direct) mandate to several genuine efficient providers for lease/sales BG/ SBLC and other financial instruments, at reasonable prices, Issuance by top AAA rated Bank in Europe.Presently, we focus on BG/SBLC for Lease and Sale transactions, However, our Lease BG/SBLC/MTN is 6+2% and Sale at 32+2%.
Should you find this interesting and acceptable? Kindly, contact us and we shall review and respond with DOA within 48hrs maximum.
Please request for full procedure details if interested.(WE MOVE FIRST)
For further inquiry contact:
Robert Francis,
Skype: robfrancis7
Similar Listings in Business Services classifieds
Angel Fundraise, Angel Funding made easy in Sweden.
Country: Sweden
The Angel Investment Network platform helps entrepreneurs with great�business id..
Country: United Kingdom
Dear Sir/Ma I am a direct mandate to a Financial Institution who is also known a..
Get All-in-one Online Arbitrage Solution to Make Huge Profits Online!
Country: United States
Login with SaleFreaks to get all-in-one arbitrage solution that makes drop shipp..
Background Screening Partner - Avvanz Pte Ltd
Country: Singapore
Avvanz, the leading background check company in Singapore offers a wide range of..
Best proposal for investment in China.
Country: Singapore
The Angel Investment Network platform helps entrepreneurs with great business id..
Country: United Kingdom
Cooperative Energy is a venture of UKs leading Midcounties cooperative Company s..


text:
Sweet, contrib gets closer!
My personal taste is unacceptable, agreed.
Album Rating: 4.5yea
But delete that rating you're not fooling anyone, not to mention it's blasphemy.
Album Rating: 4.0album kicks all kinds of culos
Lol soilwork.
6 has like best taste in the site so I dunno what pleby is talking about. :]
Must be that time of da month. Dun worry, jax. Just be patient y'all. :]
damn that was a dark joke hahaha
love this
Album Rating: 4.5so good
Fag central.
Album Rating: 4.5what
Nothing crazyy redd
Album Rating: 4.0http://www.sputnikmusic.com/forums/image.php?u=474208&dateline=1378903337
Thanks for reminding me dude.
sxc pic
Gotta rep Death m/ m/ m/
Bands: A B C D E F G H I J K L M N O P Q R S T U V W X Y Z


text:
Arkhangel'skoye Destination Guide
Discover Arkhangel'skoye in Kazakhstan!
Arkhangel'skoye in the region of Soltüstik Qazaqstan is a place located in Kazakhstan - some 270 mi (or 434 km) North-West of Astana, the country's capital.
Local time in Arkhangel'skoye is now 08:03 AM (Monday). The local timezone is named "Asia / Almaty" with an UTC offset of 6 hours. Depending on your mobility, these larger cities might be interesting for you: Tyumen', Kurgan, Petropavl, Peterfel'd, Nadezhka or Ivanovka. When in this area, you might want to check out Novokamenka, Arkhangel'skoye and Zaton as well. We found some clip posted online. Scroll down to see the most favourite one or select the video collection in the navigation.
Weather Conditions Today & Next Days Forecast
|Morning Temperature
|Evening Temperature
|Night Temperature
|Chance of rainfall
|Air Humidity
|Air Pressure
|1028.2 hPa
|Wind Speed
|with 9 km/h (5 mph) from North-East
|Cloud Conditions
|Overcast clouds, covering 88% of sky
|General Conditions
|Light snow
Tuesday, 21st of November 2017Sky is clear, gentle breeze, clear sky.
Day: -2°C (28 °F)
Night: -6°C (22 °F)
Wednesday, 22nd of November 2017Sky is clear, gentle breeze, broken clouds.
Day: -1°C (30 °F)
Night: -2°C (28 °F)
Thursday, 23rd of November 2017Sky is clear, light breeze, scattered clouds.
Day: -1°C (30 °F)
Night: -5°C (22 °F)
Camp Dostik
Svetopolisart.wordpress.com In the summer of 2008, I spent 2 weeks working at Camp Dostik (Friendship) near Petropavlovsk, Kazakhstan. At this camp I worked with 4 other Peace Corps volunteers (PCVs) ...
Videos provided by Youtube are under the copyright of their owners.
Interesting facts about this location
Avangard Stadium
Karasai (last name is Avangard) Stadium is a multi-use stadium in Petropavl, Kazakhstan. It is currently used mostly for football matches and is the home stadium of FC Kyzylzhar.
Located at 54.78, 68.95 (Lat. / Lng.), less than 10 mi away.
Related Locations
Information of geographic nature is based on public data provided by geonames.org, CIA world facts book, Unesco, DBpedia and wikipedia.


text:
You are here
The University Celebrates the Outstanding Students Who Passed the Enrichment and Summer Training Programs and Honors the Supporting and Participating Entities
Under the patronage of His Excellency Prof. Dr. Badran Bin Abdulrahman Al-Omar, the University's Rector, a ceremony was held to honor the outstanding students who passed the enrichment and summer training programs in the program of distinguished...
Cybersecurity Workshop Organized at KSU
Center of Excellence in Information Assurance (CoEIA) at King Saud University and King Abdullah University of Science & Technology (KAUST) co-organized a workshop on 15 March 2017 on Cybersecurity Capacity and Capability Building: Research,...
Workshop on "Forefront of Scientific Publishing: Impact on Achieving Saudi Vision 2030"
A workshop entitled, "Forefront of Scientific Publishing: Impact on Achieving Saudi Vision 2030" was organized on 8 November 2016 at the auditorium of College of Dentistry, King Saud University. The KSU Journals Unit hosted this workshop with...
KSU Professor Delivers Invited Lecture at Information Security Forum
The 2nd Annual forum on 'Information Security: Challenges, Insights, and Trends' was held from 4-5 October 2016 at Naif Arab University for Security Sciences, Saudi Arabia. The purpose of this forum was to bring together local and international...
Development and transfer of knowledge and technology in the areas of food science and human nutrition, and provide sources of knowledge and research and training...more
Be a section based global center for education, research and consultancy, and a pioneer in the field of food science and human nutrition in the Kingdom and...more
Objectives: Rehabilitation specialists in the areas of food science and human nutrition appropriate knowledge and skills for the labor market, by providing...more
About section
About the department: When it established the Faculty of Agriculture in 1385 with the beginning of the academic year 1402 - 1403 she joined the students......more


text:
Best Grammy Gifts Mug
per mug
Select an option:
Classic Mug
- $2.50
Two-Tone Mug
- $1.25
Combo Mug
Frosted Glass Mug
+ $3.75
Morphing Mug
+ $5.00
+ $7.50
Travel/Commuter Mug
+ $10.00
About This Product
- Sold by
Style: Combo Mug
Funny, unique, pretty, or personal, it's your choice for the perfect coffee mug. The outside of the mug features a bright white base for your photo, logo, pattern, or saying, while the rim & handle are vividly glazed in rich color. Match or complement the color of your existing dinnerware set, or gift your friend a mug in his or her favorite color.
- Available in 11-ounce or 15-ounce
- Dimensions:
- 11-ounce: 3.8" h x 3.2" diameter
- 15-ounce:4.5" high x 3.4" diameter
- Microwave and dishwasher safe
- Strong, ceramic construction
- Meets or exceeds FDA requirements for food and beverage safety
- Printed on demand in San Jose, California
Will be made by
Zazzle Home
Reno, NV
About This Design
Best Grammy Gifts Mug
Celebrate the World's Greatest Grammy and the special bond you share with these great t-shirts, mugs, buttons, tote bags and more. With designs ranging from classic to funky to sweet and cute, we have a great design that will appeal to your favorite Grandmother.
available on 16 products
Artwork designed by
Amazing Family Birthday Party Gifts
NC, United States
There are no reviews for this product yet.
Showing 1-0 of 0 reviews
Page 1 of 1
Showing 1-0 of 0 reviews
Page 1 of 1
Have you purchased this product? Write a review!
Other Info
Product ID: 168498530556066124
Created on:
Rating: G Report this product


text:
Your cart is empty.
How do I get started?
ORDER NOWfor Christmas delivery
Already own it?
DaisyJewel Tarantula Love Skull Dangle Earrings. Nickel Free/Lead Free Silvertone Alloy with Gunmetal and Black Accents, These 3-Chain Dangles Measure Just About 7.6cm . Long (7cm . from top of bail to bottom of heart charm). Lightweight and Great for Day or Night, These Dangles Feature 3 Different Chains of Varying Lengths and Links that End with 3 Unique Charms: A Smiling Silvertone Skull & Crossbones, a Black Heart, and an Iridescent Rhinestone Bellied Tarantula Spider - Arachna-Awesome! Each Super Spidey Pair of Earrings Arrives in Complementary Organza Sachet Gift Bag.
Their Email:
Already own this item?
Sell Yours and earn some cash.
It's fast and free to list! (Learn More.)
You can earn a 5% commission by selling DaisyJewel Tarantula Love Skull Dangle Earrings on your website.
It's easy to get started - we will give you example code. After you're set-up, your website can earn you money while you work, play or even sleep!
Are you the Author/Publisher? Improve sales by submitting additional information on this title.
This item ships from and is sold by Fishpond.com, Inc.
Fishpond's Best Deals Delivered to You Every Day
Thanks, your email was successfully added
You will receive special offers to your inbox daily.
© 2004-2017 Fishpond Ltd. All prices are in Australian dollars.


text:
John Betts - Fine Minerals > Home Page > Minerals For Sale > Minerals from African Countries For Sale > Apatites & Phosphates For Sale
This mineral specimen of Vanadinite with Calcite on Barite from Mibladen, Atlas Mountains, Khénifra Province, Morocco is available for sale from John Betts - Fine Minerals, mineral dealer located in New York City, New York.Follow on Facebook
To purchase mineral specimens from this site go to:
Detailed information on mineral for sale No. 71113:
|Mineral Species: Vanadinite with Calcite on Barite.
Location of mineral deposit: Mibladen, Atlas Mountains, Khénifra Province, Morocco.
Description: Lustrous orange-red transparent-to-translucent vanadinite crystals to 5 mm with translucent white calcite crystals to 1 mm coating bladed white barite crystals to 18 mm. The vanadinite crystals are tabular hexagonal form. Though the crystals are clean and in good condition, a few show damage upon close inspection, but they are minor compared to the relative size of the specimen. Collected in 2001.
Overall size of mineral specimen: 31x18x17 mm.
Size of individual crystals: micro to 18 mm
Price: $34 (plus shipping).
Click here to see more Vanadinite For Sale
Thumbnail-sized Specimen
See similar minerals for sale:
See more minerals for sale from:
© 29 October 2017 John Betts
- Fine Minerals, New York , NY - All
Rights Reserved.
The minerals illustrated on this site were purchased from collectors or wholesale suppliers for resale purposes. The owner of this site did not collect the minerals and collectors should not assume access is permitted to the property based on inclusion in this site. Please contact the property owner to obtain legal permission before entering private property.