Archive for July 18th, 2021

Cool Games Online

Sunday, July 18th, 2021

Cool Games Online

Urge your children to Search for games and to play the World Wide Web. Messing around internet won’t cost you cash in light of the fact that numerous websites offer you this administration for nothing. Just select a good site to shield your kids from various perils of the net.

Kinds of Cool Games Online

A couple of people have a profound conviction that playing on the internet isn’t alright for youngsters. Nevertheless, casino online it is a legend, since we could handle our kids while playing. Initially, present a firewall along with a successful antivirus program to take care of the security dangers. Besides, ask your kids not to discuss their own data when using their gaming reassures. Also, instruct them that downloading any product from obscure sources can damage their gambling gadgets. Along these lines, you shield them in the sending of explicitly express photographs, pictures, instant messages, or messages. What’s more, by and large while making a record at any site, utilize a strong keyword which incorporates letters, numbers and exceptional characters.
Various sites offer unlimited options for enjoying cool games casino online free, but a few destinations need paying particular charges such as membership. In the event the website is respectable, it will worth the money.

Truly, we’re honored with a huge load of cool games on the internet. For Mothers, playing web based games is an unbelievable way to take care of conquer weariness. For kids, playing online games is a fantastic procedure to have a good time and also to learn new abilities simultaneously. Additionally, it’s quite a simple assignment to find wonderful games that are acceptable for your child. A few games have been grown explicitly for young guys, while some are intended for young ladies. When you beloved this informative article in addition to you want to get more info regarding เว็บพนันออนไลน์ i implore you to stop by our web page. Therefore, you can learn which games are best for your youngster without any issue. This article will be able you to the way to search for games on the World Wide Web. Just keep reading.
With regards to looking for games that are outstanding on the web, we have a huge assortment of games, by way of example, hustling, fighting, sports, stopping, mischievous, cooking, design, cards, puzzle, shading, sprucing up, make up Barbie, etc..
In all reality, when guardians allow their kids to mess around on the web, they provide them a wide range of creative and creative exercises that assist with building their psychological abilities. For the most part, gaming sweethearts can enjoy a broad range of games on the Internet without paying a penny.

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You may discover them categorized by age gatherings and by forms. It is merely a question of locating a confided in site which has a simple to-investigate interface and gives wellbeing to guests too.

Onur Özden Web Çözümleri

Sunday, July 18th, 2021

Onur Özden Web Çözümleri olarak profesyonel web geliştirme ve SEO hizmetleri sunuyoruz. Blog bölümümüzde eğitici içerikler hazırlıyoruz. Hizmetlerimizi ve eğitici içeriklerimizi incelemek için tıklayın ve sitemize ulaşın.

Приложения в google play – знакомства@mail.Ru

Sunday, July 18th, 2021

• Вы имеете возможность найти вашу половинку по различным параметрам: возраст, рост, вес, цели знакомств, местонахождение. • Вы получаете возможность подыскать что ищите: несерьезные знакомства, контакты для переписки, и поиска, контакты для серьезных отношений, овладения целью создания близких или Так далее. Найдите новые лица, новые, Новые ощущения на нашем площадке. • В нашем приложении менеджер сделали так, чтобы все было функционал максимально комфортным и понятным. Знакомства, общение любовь, друзья – найдите все это – практически сейчас. Романтические, серьезные отношения, отношения с намерением создания семьи, чувствительность и страсть – все это не сложно обнаружить в зависимости от ваших интересов и требований.

Online Gaming World

Sunday, July 18th, 2021

Online Gaming World
The reach of online games may undoubtedly be ordered from essential content games to confounded designs including the digital world and a couple of players playing simultaneously in an identical stage. Creating of java and glimmer permitted to utilize spilling recordings and matches from the net. This is only one of the substantial reasons that prompted the extension of internet based games. The games are separated in to a few essential sorts into instructive, key games and there are even a few games which fuse everything that individuals used to play at the “at the point” life into virtual designs.
With the invention we have today joined with the ideas and thoughts of the gaming arena, we can without a doubt say that these games despite everything have a bright and long approach. It is sure to the point that there will be if there would be something greater that will assume control over the current pattern of web based games. Something better, something better and something much increasingly amazing and genuine. You understand not what innovation can do… till then you can sit back at your home and mess around with folks around the globe and just appreciate the innovation.

Internet is getting a colossal hit all over the place and แทงบอลออนไลน์ it now has some thing or another to with our own lives. Intriguing right? The way the world has changed from open atmosphere games to video games now to internet games. Should you loved this article and you want to receive more information concerning สล๊อตออนไลน์ kindly visit the web page. Indeed, these games aren’t a significant thing any longer supposing that you take a dimensions model, more than 40% of individuals are mad over the different web based games available in the web. It is the new amount of entertainment and the new take a rest and สล๊อตออนไลน์ all you need to appreciate it’s a internet institution.
Games can similarly be gutsy and it provides the inclination that we’re actually doing and limiting what we are playingwith. There was this idea of gaming called MUDs that’s multi client ongoing digital universes. Currently the new style of gaming which has gotten really mainstream since the broadband seemed is the MMOGs that extended to hugely multi-player internet games. This enabled 1000s of players to play simultaneously in a similar match. There are a few numerous sorts MMOG including pretending, continuous methodology and original individual shooter games et cetera.

I would place the sport as an invention all together rather than considering it a type. It must be noticed that there is some system whereby it very well might be performed in the PC and also the basic system is unquestionably the web. The adventure of this game develops if there is a multi-player choice and you are contending with an individual whom you have quite recently fulfilled over the internet who’s currently your rival. There are a couple numerous locales that offer A-Z arrangements of web based games to navigate. What’s more, the rundown just expands step by step with more games being created the world over.

Making Crowns Stick To Teeth More Effectively

Sunday, July 18th, 2021

Femicide supports male power. See also Male bonding over misogyny; Male entitlement; Mansplaining/women’s perspective is wrong; PIV-centric narrative; Goal is to “land a man”; Reversal. Handmaidens of the patriarchy; Male bonding over misogyny, Mansplaining/women’s perspective is wrong; Necrophilia; Normalize abuse/neglect; PIV-centric narrative; Rape and rape culture; Woman as “useful object”. Male-centric “social justice” movements consist of socially or politically “underprivileged” men fighting amongst each other to be treated as privileged white men are treated, including their unquestioned, unfettered access to women; meanwhile, all men continue to use women as their sexual, domestic and reproductive slaves and social justice movements do not address or satisfactorily address institutionalized misogyny, sex-based discrimination or male abuses of women. Because of male entitlement, men are simply used to unearned deference on every issue (especially from women) and having sexual and other access to women and women’s spaces, including inside our discussions and in our heads. See also Handmaidens of the patriarchy; Male entitlement; Normalize porn/prostitution; PIV-centric narrative. PIV-centric narrative – Normalize reproductive stress and pain.

Mature Webcam Anal Fist Necrophilia; Normalize prostitution/porn; PIV-centric narrative; Goal is to “land a man”; Normalize reproductive stress and pain. Mansplaining/women’s perspective is wrong; Normalize abuse/neglect; Normalize porn/prostitution; PIV-centric narrative; Normalize reproductive stress and pain; Rape and rape culture. See also Joke’s on women; Normalize porn/prostitution; PIV-centric narrative; Goal is to “land a man”; Rape and rape culture; Primacy of the nuclear family. The actual and perceived entitlement that all men are born with normalizes and naturalizes male “success” even when their material wealth, career successes, familial status and sexual access to women are achieved through unethical and even violent means, including political corruption, systemic institutional discrimination against women, economic coercion and rape. This is, of course, deliberate, and keeps us stuck in ahistorical, apolitical discourses about power that are not reality-based, and do not challenge patriarchy or hold any promise at all of liberating women from male dominance. That’s why privacy focused cryptos like Monero, Zcash and ZCoin (all SFW) hold the powerful possibility of making up the backbone of all porn transactions in the future. That’s the reason why we get soft after orgasm.

Often, men mansplain to women who do not like pornography why it’s really harmless, or mansplain to women who criticize or abstain from PIV on political grounds or women who do not enjoy PIV that they are just “doing it wrong.” Doctors and other experts and oral sex porn professionals are actually experts in mansplaining and professional mansplainers; unsurprisingly, male experts – being male – often mansplain to women on the topics of PIV and porn too. And unnatural numbers of males lording sexual power over girls and women, and where female infants are then killed and males aren’t, exacerbates and perpetuates the problem of “overpopulation” and best pron sites specifically male global overrepresentation and unequal male political and adult live chat physical power and resource-hoarding with no end in sight. The result is that males exist in unnatural numbers globally and share power and resources amongst themselves without sharing it with girls and women – power which they actually lord over girls and women and which includes mandatory PIV and rape. The top 20 percent of men are the only men competing in those kinds of numbers.

Men are entitled to literally do whatever they want without suffering any consequences of any kind, or where others suffer the consequences of men’s actions, such as where men eat obviously unhealthy food because it tastes good, without regard for their health, or engage in dangerous “jackass” type stunts, extreme sports and other activities where women will be the ones taking care of them when they inevitably fall ill or are hurt. This sub-theme normalizes PIV-centric sexuality because the endgame is marriage or a long-term partnership with a man, where it is expected that PIV will be a requirement of the continuation of the partnership. Where the goal is to land a man, lesbianism or spinsterhood are not options; so-called “women’s sexuality” becomes penis-centric, despite the reproductive consequences to women and regardless of whether the intercourse is pleasurable or wanted by the woman; and female friends and relatives take a back seat to the romantic heterosexual partnership and are not welcome in the co-habitative or marital home. Normalize porn/prostitution; PIV-centric narrative – Goal is to “land a man”.

Top 7 Ways To Attract Hot And Attractive Women – Advice For Men

Sunday, July 18th, 2021

Learn to de-compress as a couple, so that stress doesn’t take all the passion out of your relationship. It could also be because the guys just want to rub one out fast because of stress. But, that’s not why guys go to Hooters. Why is it always up to men to approach women first? Just get rid of fear of getting rejected and you are good to go seduce (okay, approach) that next hot chick! Don’t get yourself dropped in every details. Proved and reliable. Nevertheless, it’s experience you get out of all the misconceptions close phallus stretchers and take about them in point. A very good technique that I have experienced myself has brought me the purePASSIONATE experience that made walls tremble-at least through my eyes. Durex Play: This lubricant is ideal for a top to toe tingling experience. This can be done by doing things like putting your legs on top of her as if it’s comfortable like that. There are many things every sexy woman can achieve with the help of adult toys.

tehswitcher's Captions: Hopper Conscience

Here are six proven tips to help you start getting rid of depressing cellulite within the next few days. For the next few days, repeat this exercise for premature ejaculation, but increase the masturbation number from 4 to 7 and allow yourself to ejaculate on the 7th time. Note: To make this exercise for premature ejaculation more real and effective, you need to make the masturbation environment as close to the real sex chat apps process as possible. Bring her towards YOU – once she’s interesting, getting her to meet you in real life and have sex will be a piece of cake. All it takes are two AA batteries to bring the amazingly soft dong to life and make your fantasies come true as well. This could cause inconvenience, hassle as well as embarrassments to some guys. The thought of aggressiveness lessens down when guys view the sensuous and hot videos of the party girls, lesbian couples and amateur girls. The sensuality and passionate fling that the lesbian couples or the amateur girl’s showcase make the guys go gaga and leave them to have a blissful night with lusty desires and fantasies. I’ll leave it up to you from here, now that you know how to escape the friend zone.

But here, the most important winner is Google. Although Samsung has become the first released this past year, porn cam Google Android-based Galaxy Tab Tablet computer, and reached 100 million shipments, but Google still hopes to Tablet model is based on the Motorola’s body, normally the one is a successful model of Motorola touch screen phone precedent, and second, the Motorola spin-off is optimistic around the industry following your focus, the two are added together, can greatly help the Tablet PC market, Google’s efforts to market and influence. If you can find the right hot button and push it at the right time, you would be able to help your woman achieving orgasm in no time. Waiting won’t help much, and it certainly won’t get you laid. A lot of males just realize marriage problems the moment they get very serious or when they have an affect on bedroom activities. Get the walk of shame over with sooner rather than later, lest you stink up someone’s bedroom and bathroom.

You have to wake up and start doing what WORKS if you want to get those chicks in bed. She likes you, but doesn’t want to go to bed with you. The beauty of menopause, however, is that it is the time period when most women are blooming into the fullness of their personal identity– eg: what they want out of life, what ambitions and skills they want to bring to life, what desires they want to fulfill. One of the best things about the adult sites is that it has the ability to satisfy and fulfil everyone’s desires. Because 30 days from now you can have one of two things. Now that you understand what early ejaculation means, it’ll be easy for you to understand or find a solution for premature ejaculation. Great. Now you have two hours and you only have to move five inches or so. Statistics have shown that as many as 4 out of every 10 men are suffering from early ejaculation. First of all, you must know what “premature ejaculation” really is. Technically, early ejaculation happens when a man ejaculates during the first few thrusts the moment his penis enters the vagina. 8 Believe it or not, most women would not appreciate the ambiance of Hooters for a first date (or any), though you may win consolation points if you manage to keep your eyes glued on your date.

Whether the wedding dress or bridal lingerie allows or not, go shopping with your bride for a corset, something really special that she can keep forever. With that knowledge, you will be able to consciously control and keep your orgasm level in check to avoid reaching the point of no return before both you and your partner are ready. As these creams or sprays desensitize your sexual sensation, you would not be able to enjoy the same level of sexual sensation had you not used those cream/spray. To step out of the “same old same old,” plan some time away from home and children. Let’s just say Masterdate is old fashioned about this. You can re-cooperate and re-evaluate, it’s a good time to take stock. How do you take initiative the right way? The only best way to do this is to make her jealous. The very best option is to meet a new girl and make sure she knows about it. A hot girl can tell should you desperately want her attention.

Generation Zero: Documentary Film Evaluate

Sunday, July 18th, 2021

53— Oh, I agree with you. You’re appropriate about total high quality management. I may only see excerpts of the film and interviews with the filmmakers. I certainly agree that our cash needs to be handled conservatively by Wall Street. I am from Michigan. Nonetheless, נערות ליווי simply from reminiscence, נערות ליווי ברמת השרון I remember unions combating against any and all progressive manufacturing methods, נערת ליווי במרכז such as any automation. Your feedback are glorious. Thank you for coming by and sharing your standpoint. I did. I fleshed it out, form of like an historical novel. I intend to look at your complete documentary this weekend. There’s certainly truth in what you say. The battle for the soul of America, the Shining City on the Hill, the nice hope of humankind, is certainly a spiritual battle between the forces of darkness and the forces of gentle. Nevertheless, simply from memory, wasn’t it underneath Clinton that the mortgage derivatives market began?

Boyfriends/fiances/husbands want to be supportive, however are very traumatized and you cannot rely on them to react in a approach that helps you. Please seek that out. You both deserve a healthy and fulfilling intercourse life. A trained counselor may even offer you ideas on one of the best solution to broach the subject with Dave. If you learn lsc’s comment above, speaking in a bunch with different rape survivors may be amazingly healing. I’m not saying it is best to cover this large a part of you from Dave. After all, נערות ליווי בנתניה I do not know you and נערות ליווי i don’t know Dave. Not in any respect. However I believe that getting some professional steering FIRST is a good idea. However proper now there’s an enormous fats elephant in the bed with you two. Believe me on this. He could also be essentially the most understanding man in the world. I do know you suppose the world of him, and that’s exactly why I would hate to drive a bigger wedge between you than is already there.

That it is actually awful? And perhaps, simply maybe, it’s time to write that first article on names. Darrke Thoughts, thank you for fascinating data. It never hurts to know more. Svetlana, thank you for visiting. I have a paternal sister in Ukraine whose name is Svetlana, so by no means I consider it a “unhealthy” title. Or Electra? I like Electra. I hope you find it attention-grabbing. This hub shouldn’t be about origin or about historical past of names, but it surely is precisely what I wrote- about completely different names that sound in another way in certain languages due to phonetic discrepancies. The point is just not right here. I hope all people will like my take on it. First of all, you share a reputation with my sister. If no, no hurt accomplished. I agree with you that it’s a really stunning and meaningful Russian title. It’s not my favourite, however to say that Simon or Damien are really terrible? Also, my friend’s name is Sveta.

I learn many history books and i at all times am cognizant of the writer’s biases and opinions that color them. Even if they had been all innocent, God is the origin and giver of life and might take back what He has given at anytime. I acknowledge every single piece of scripture that you simply offered, though your displays don’t not current scripture in proper context. How do you know that Lot’s spouse did not know that she wasn’t suppose to look back? What makes you think they were all innocent? Certain issues in scripture are left unsaid, so as for these 42 children that were mauled by bears, how are you aware they weren’t a harmful gang of hoodlums that have been a threat to the Prophet? Ea of the Babylonians, Yahu of the Aramaeans, and Yah of the Egyptians had been all based mostly on the truth of Yahweh as a counterfiet is based on the real that it mimics. This stuff need to be thought of when reading a work from a high-context culture. Some could have been first rate folks with others indecent.

Nine Creative Ways You Will Be Able To Improve Your Sp Flash Tool

Sunday, July 18th, 2021

1. Be sure that you’ve gotten chosen the correct Download Agent file in SP Flash Tool and are utilizing the firmware meant to your gadget’s exact mannequin. 2) You can use this device to flash inventory firmware (scatter file format) in your Android machine. So if you are looking to repair bricked phone, bootloop challenge, or telephone caught at logo downside then the only straightforward solution is to flash the firmware. Download SP Flash Tool is an software which mainly lets you flash Stock ROM, Custom restoration and fixing in some excessive instances ( firmware replace, Flash restoration, unbrick bricked Android machine and so forth.). Please dont confuse customized rom with Stock rom, as Stock Rom never void Phone Warranty. 8. After downloading ROM on your phone, the inexperienced sign will seem which means the process has been completed. Step 9. Once Flashing is accomplished a new dialog field will seem with a inexperienced field. Just load the Scatter File within the device, locate the restoration.img file, and click on on Download to begin the restoration flashing process. Through the use of any file or guide provided here you’re agreeing to the positioning Disclaimer. This post was written with GSA Content Generator Demoversion.

But the use of the device shouldn’t be easy, therefore you need a information. It is rather simple, fast, and straightforward to make use of. SP Flash Tool Driver: So as to make use of the SP Flash Tool accurately, you’ll need to install the VCOM Driver, CDC Driver and ADB Driver in your pc. Caution: Flashing or installing Stock ROM (firmware) using the SP Flash Tool will wipe the information. 6. Now choose firmware improve rom drop-down menu and press download button. Simply load the Scatter file and connect your smartphone to the pc and press on Format button. Meaning: SP Flash Tool is having hassle using the selected Download Agent (DA) file. Meaning: The model of SP flash tool you’re using would not assist the size(s) of the file(s) within the firmware / stock ROM. The flashing process could take a couple of minutes to finish, relying upon your device’s firmware file measurement. The method is very easy simply connect your telephone and format the information. Step 4: Turn off your telephone. Win 8 customers don’t forget to turn off Driver signature verification. The correct driver for this low-layer obtain interface is also located in “MT65xx Drivers” folder.

You can obtain the latest model of MT65xx Preloader USB Driver from right here. 5. Now, Untick the Preloader possibility (flashing the preloader.bin can brick the machine). Untick the Preloader choice as it’d brick your telephone. Install adequate drivers from MTK Preloader Drivers by way of automated setup. 3) After restart now connect your bricked cellphone and install drivers. 2) Don’t worry in case you have performed so and worry that you’ve bricked your device utterly. Unbrick MTK Phones: In case you have bricked your mtk device flash tool primarily based telephone then flash firmware utilizing SP Flash Tool to unbrick your telephone. In addition to this characteristic allows you to downgrade or upgrade your at the moment working firmware. It additionally lets you downgrade or improve your machine firmware in few clicks. Most essential usage of this application is upgrade , downgrade or flash custom ROM on your android gadget. 3. Download and Extract Stock or Custom ROM and SP Tool to your required location. You can too flash custom recovery recordsdata (CWM or TWRP restoration) using SP Flash tool. Flash CWM & Stock Recovery IMG file. It’s best to load the Scatter file. 2. Flash Stock Rom: Flash scatter based Stock ROM and Custom ROM on the Android Smartphone.

SP flash tool is an utility which primarily lets you flash Stock ROM. SP Flash Tool helps you to flash or install custom firmware in your android smartphones ( MTK based mostly android units). With MediaTek Android smartphones (MTK-based mostly), SmartPhone FlashTool can be downloaded from our obtain section. Alternative Tool: SP Flash Tool and SP MDT Tool are the very best various tools that can help you flash or install scatter firmware on the devices powered by MediaTek chipset. With the MTK FlashTool you perform issues like Flash,Unbrick, Format,Hard Reset, Run Memory diagnostics and so forth. That is the perfect Android MTK flash-Tool accessible. SP FlashTool is the best recommendation for MediaTek Android flash that helps all the MediaTek ROM variants. SP flash tool could be thought of as a very consumer-friendly software and it flashes the stock devices such as Coolpad, Tecno, Gionee, Micromax, etc. The very important fact to take into consideration is the SP flash tool can solely flash smartphones which are MTK(MediaTek) primarily based.

20 Best HD Porn Sites To Watch 720p, 1080p HD Porn Videos

Sunday, July 18th, 2021

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Tumblr naked bride poron movies So you go chat up a lady BECAUSE she’s eye-catching and you would certainly like to sleep with her. I constantly offer partner like practices to my clients, I desire to be wooed and not leapt upon. Attention! We wish to advise you that raunchy details could be located on this internet site, it also includes links to porn sites. There was an e-mail address and internet site urging individuals to speak to for more details. I like giving even more than getting. Rosa is concerning to be wed, as well as when the negative young boy finest male sees her in her bridal underwear, it will take a lot more than loyalty to the groom to maintain these two apart. Navigation as far as Fubilov is concerned is a little less complicated and will most certainly obtain you to that point where discovering your videos and also streaming or downloading will certainly be a piece of cake – something that I can’t state for the average sites out there. Eventually I’ll place out a nine DVD pack of The James Amoureux Sex-AND ™ Seminar and also do a spammy countdown hype-drenched web launch. Greetings, this is James Murray bringing you the main tales and must-reads on Tuesday 10 March.

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I’m never one to dissuade exercising or learning. Porn HD is another trendy adult web site that offers just high meaning Porn videos You can download and see HD Porn video clips from Brazzers, Naughty America, Vixen and lots of various other site directly from this Porn HD porn website as it not just offer their video clips yet also enable you to download them with simply one click. From videos-on-demand and also live webcam shows to grown-up eCommerce sites, this specific niche is method beyond the regular comprehensions of making big cash in short period. What they told us changed the way we looked at pornography and also the women involved with it. The occasion, qualified You Oughta Remain in Pictures, combines home-made adult movie and low-budget flicks made by filmmakers and also artists. Like porn stars-turned-performance musicians, Bright uses her body to deconstruct the cinema of pornography, its sexual stylings and also dream buildings. I’m with lady literally, sexually, emotionally for a pleasant minute as opposed to using her warm body to basically masturbate.

The Best Webcams In 2020

Sunday, July 18th, 2021

Additional studies were performed as described herein using the composition disclosed herein comprising AAV SB-71557 and AAV SB-71728 (in place of 47171 and 47898), along with an AAV hIDS Donor. The patients summarized here were treated with the composition disclosed herein comprising AAV SB-47171, AAV SB47898, and AAV hIDS Donor. Here look for the lines of text saying “Received: from”. Video chat and conference calling services exist for a lot of different reasons, but ultimately it all boils down to the fact that sometimes emails and text messages just don’t cut it. He experimented with dripping drugs deep into the brain down tiny pipes called cannulae, targeting the same regions as his electrodes. My brain was rebooting and the task of renewing my neural pathways after years of pornographic stimulation was like scrubbing down the inside of a volcano. Mild, Grade 1, elevations in liver function tests were measured in this patient at day 62, 111 and 128, consistent with a suspected transaminitis event, resulting in the dissipation of plasma IDS activity in this subject, down to 14 nmol/hour/mL at 24 weeks post-treatment.

The entrance side the day I free video webcam came back down to see her eyes bobbed her pussy. The first step is for the person who owns the webcam to buy the software and install it on the device. Almost trembling wife masturbating cum on teen tits webcam parted her. This fall, Mark Arnold’s wife was trekking home from a Potrero bus stop with their daughter when she spotted what looked like a familiar figure in a baseball cap, with a warped left knee, a few blocks from where Fairley used to walk home from school with her own daughter. 40 .mu.L of each reaction was transferred to a flat white opaque plate and 100 .mu.L stop buffer was added. 90 .mu.L substrate buffer) in a separate microcentrifuge tube. Substrate buffer was prepared as follows: 0.1 M sodium acetate and was combined with 0.01M lead acetate and adjust to pH of 5.0 using glacial acetic acid.

The .alpha.-iduronidase enzyme can remove the iduronic acid from any 4-MU substrate from which the 2′ sulfate residue has already been removed by endogenous iduronate-2-sulfatase. The removal of iduronic acid was catalyzed by the .alpha.-iduronidase enzyme, and this can only occur after the removal of the sulfate residue by iduronate-2-sulfatase. However, if no endogenous iduronate-2-sulfatase enzyme is present within the patient sample, the 2′sulfate residue cannot be removed, which prevents the entire iduronic acid moiety from being removed, thereby quenching the fluorescence of the 4-MU substrate (Voznyi et al. However, in order for the fluorescence of 4-MU to be released, the entire iduronic acid moiety must be removed from the substrate. During the first step, endogenous iduronate-2-sulfatase was given the opportunity to cleave the 2′ sulfate residue from the iduronic acid residue at the end of the 4-MU substrate. This assay used an artificial 4-MU substrate that contained a terminal iduronic acid. Through results of PCR analysis–an assay to detect gene integration–of liver samples, two patients in Cohort 2, demonstrated gene integration. FIG. 10 depicts a close up of Subject 6′s IDS levels and the changes in liver alanine aminotransferase that were observed during the spike in IDS levels.

The response of urine GAG levels after ERT withdrawal is important to determine the potential clinical relevance of the study drug. All subjects enrolled had baseline urine GAG levels in a range considered at normal or slightly above normal, and at 24 weeks, urine GAG levels from the first six patients did not show a clear trend or meaningful change. The IDS enzyme data included plasma activity levels from the first six patients enrolled across all three cohorts of the study, at 24 weeks post-treatment compared to baseline. Urine GAG measurements at 24 weeks were also collected and compared to baseline in these first 6 patients. Stabilization of urine GAG levels after ERT withdrawal will be one important parameter for evaluating therapeutic potential of the methods and compositions disclosed herein. It is possible that continuous exposure of cells to low levels of circulating IDS may be sufficient to drive enzyme uptake into cells and reduce or maintain suppression of GAGs as these tissues are starving for IDS. Furthermore, a significant increase in plasma IDS activity was measured in the second patient treated at the 5e13 high dose, with plasma IDS levels rising to approximately 50 nmol/hour/mL by day 50 post-SB-913 treatment, which is approximately 60% of the lower limit of normal plasma IDS activity.