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Last night's theme was country music, with Martina McBride mentoring the silly seven. Hard to say who's getting heaved this evening. Phil Stacey has been hovering around the bottom for the past three weeks, so I thought he'd get the boot, but the dude really nailed his Keith Urban cover. Of course, Americans were raised to distrust Lex Luther. And many of us are probably offended by walking penises. Phil looks like both, and that might be hurting his chances for remaining another week. Hasn't he learned anything from Sanjaya? Hair is important!
And then there's Sanj himself. You've got to hand it to him for being self-aware with his song choice, Bonnie Raitt's "Something to Talk About." But you also have to hand it to Simon for calling him out on not having any talent, rather than just politely smiling and saying stuff like, "You do you, dawg."
The situation actually got a little heated as Simon was verbally bitch-slapping Sanjaya, and Seacrest, ever the chivalrous one, stepped in to defend the honor of his lady, er, singing competition contestant. Simon's response was to wave Seacrest aside with a mini hand motion, which sort of enraged Seacrest. The whole time that this was happening, Paula was trying to nervously laugh off the whole thing, like a peace-keeping aunt at an extremely tense family dinner. And Sanj hung tough: his barndance bandana didn't fall off at all.
Tune in tonight to see our hero's creepy smile when Seacrest tells him he's safe.


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Mobility Summit – Washington, D.C.
Date: Tuesday, July 22, 2014
Time: 8:00 am - 2:00 pm
Location: Marriott Marquis Washington, D.C.
Is secure mobility an oxymoron? Which apps do industry leaders use to securely transform the way they do business? How are BAE Systems deploying a secure mobility strategy?
Join us at our Mobility Summit in Washington, D.C. to find out! This is a must-attend event for CIOs, CTOs, CSOs and those with responsibility for secure mobility in Government and Enterprise. Share best practices and lessons learned with peers; see demos of the industry's most innovative mobile apps from companies like Accellion, Acronis, Aternity, BoardPad, CellTrust and EyeVerify; connect with mobility thought leaders during a networking lunch!
By attending the Washington, D.C. Mobility Summit you'll get the insight and tools you need to unleash the potential of secure mobility in your organization, including how to:
- Build a mobility roadmap from MDM to business transformation
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- Develop a strategy to create, manage and deploy enterprise mobile apps
Plus, you'll see demos of some of the industry's hottest mobile apps! Space is limited so RSVP today!
Thank You To Our Platinum Sponsor:
|8:00 - 8:45 am
|Registration, Breakfast & Networking
|8:45 - 9:00 am
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|9:30 - 10:00 am
|Technology Vision & Demo: Hemant Sahani, Product Management, Good Technology
|10:00 - 10:30 am
|Samsung KNOX™: Enterprise-Grade Security for Android™: Phil Fernandez, Director Alliances, Samsung
|10:30 - 10:45 am
|Coffee Break Sponsored by Samsung
|10:45 - 11:30 am
|Panel Discussion: Accellion, Acronis, CellTrust, EyeVerify, moderated by Brian Reed, Chief Mobility Evangelist, Good Technology
|11:30 - 12:00 pm
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subset: commoncrawl


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|9 posts found
Greetings, programs!
I'm a FFXI vet; started playing at launch and played on and off (mostly on) for over 7 years before they started making changes I didn't agree with and slowed down my playing. Abyssea, and the way it totally changed the way people play the game (hence, making it harder for me to enjoy it as I had been and wished to continue), turned me off completely to it.
One thing I noticed my last time back, is that the general attitude of many (most?) players hasn't changed in XI - one of the ugly parts of the game as I remember...
Unless there's something in it for them, most people are completely unwilling to help others out - even those who'd helped them in the past (that happened to me plenty). It used to be that they'd simply make excuses to avoid it. Now, though, it seems to be that, when asked to help with different content, people are just told "hit 99 and then solo it". Thing is, I don't want to race to 99 (you can see my view on leveling in my sig). I don't want to *solo* the content when it's become so easy that it can be. I'd like to experience the content at the level it was intended, as the challenge it was intended to be. Unless there are people out there who enjoy going back to do that earlier level content at the intended job level(s), then it looks like I'm SOL on that.
So, I guess at this point what I'm wondering is - for someone who played the game almost religiously for over 7 years and continue to consider it the single best MMO they've personally ever played (when it was at its best, IMO), but felt that SE had begun to lose the plot sometime after WoTG, and then lost it completely with Abyssea - is there anything that SE has done since that might enable me to at least experience some of the awesome stuff I'd done back then? Now that the "rush to 99" thing is (presumably) not front and center in people's attention, are people a bit more willing to go back and do older content at its intended challenge level, just for the fun of it - or to help someone out?
Or is it time I put a bullet in the head of this ailing old dog and just move on completely?
This is an honest post and I'm asking a sincere question. No attempt to bash or troll the game at all. I'd love to come back to XI and "live" in Vana'diel again.. I just want to know how much of it is left for me to come back to.
Edit: I really need to let the coffee kick in before typing up a post lol.
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Spotlight Poster
Joined: 3/28/11
I have never played FFXI, mind you, but after reading your post, I could identify with some of what you are saying. What I have learned, and what you probably already know, is that you can never go back.
You can never go back home. You can never go back in time. The way it was is over. It's time to take up your bag and wander. It is the pathless way. When you find your game again, you will know it.
Advanced Member
Joined: 11/10/06
PWN_FACE is dead on right.
And awesome link too!
So, I should take that as "no, there's really nothing in FFXI anymore for you at this point"?
I mean, that's kind of the conclusion I'd come to on my own in my times attempting to go back.
But, sometimes something like even a partial shift in the focus of players, or something like that, could make all the difference.
After all, it wasn't the content of Abyssea itself that caused XI to change so drastically. All that other content was/is still there. It was more the players' reaction to Abyssea and how they changed their playstyle completely to take advantage of it (PL'ing from 30, etc).
Even the presence of a small sub-community within the game that enjoys playing it more the old-school way could make all the difference.
So, one could also say, "not likely, but never say never".
So, after thinking it over, I'm going back to XI.
Some friends of mine are still playing, and seem to have basically the same approach as me - much more into the story and exploring and enjoying the journey; not in a hurry to get to the end.
I have a list of things I want/need to complete, and so I'll have plenty to keep me busy before I even have to begin to think about Adoulin, etc.
Novice Member
Joined: 6/28/10
which server do you play on?
Originally posted by Standadeezy
My server is Asura. Though, I'm not playing at the moment. Real Life's been trolling me a lot lately, and it's been difficult to spend any amount of time on anything, really.
Not sure about the changes upcoming in the game either. I'm not a fan of SE's 'cross-marketing' efforts lately (promoting DQ and FFXIV in XI, promoting XIII in XIV, etc, etc). Vana'diel is vast, deep and interesting self-contained world. Using it as a marketing platform for their other games only cheapens it, IMO. But... that's what they're doing. So... I dunno.
I think SE's given up any hope of trying to pull it back together with XI, after the damage done to the game overall with Abyssea (which they admitted themselves). Ya can't put a cat that large back in the bag because you realize it was a mistake letting it out in the first place. Seems they've decided to just go in a completely different direction with the game altogether.
Will update when ever something new comes up.
Apprentice Member
Joined: 11/24/05
And the science gets done, so you make a neat gun, for the people who are still alive.
I'm on Asura and was thinking of coming back to see how the game has changed since I left in 08.
If you get on let me know, I think I'm going to piss away 15 dollars for a one day look around lol.
Novice Member
Joined: 4/18/12
Now is probably the best time to come back and play. No real need for most abyssea gear outside of macro pieces now. All the new armor and weapons are pretty easy to get. They have introduced new upgraded AF and new hard mode Ark Angel fights. There is also supposed to be new relic gear upgrades coming and even Empy upgrades down the road. The new FATE system is pretty cool too, though Tenzen won't do Cosmic Elucidation :(


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Why Do Mexis Like Wrought-Iron Fences?
By Gustavo Arellano
By R. Scott Moxley
By Alfonso Delgado
Women For: OC are Stitching and Bitching
By Courtney Hamilton
Santa Ana River Ready for Its Renaissance
By Joel Beers
By Peter Maguire
Can Tech Ruin the Kim Pham Murder Trial?
By Charles Lam
MLG Anaheim: This eSporting Life
By Charles Lam
Ross issued his ruling on Aug. 23, after four days of testimony from the two sides: The Bernals could stay in their house, and the plaintiffs had to pay their legal fees. Ross wrote in an opinion that housing covenants "have a tendency to be . . . injurious to the public good and society; violative of the fundamental form and concepts of democratic principles" and that he agreed with Marcus' contention that such discrimination violated the Fifth and 14th amendments. Taking note of the ethnic stock of most of Sunnyside's residents, Ross gave them a dose of their own venom. "I do feel there is a paranoia among the German people," he remarked in court. "I don't think Hitler is the only one. I would rather have people of the type of the Bernals living next door to me than Germans of the paranoiac type now living in Germany."
The local press didn't pay attention to Bernal's victory; the Fullerton News-Tribune devoted just a 200-word story to the ruling. But the Bernal case received international attention. The Mexican consul, noting the Good Neighbor Policy between Mexico and the United States at the time, said the decision "proved that the politics of inter-American rapprochement isn't an utopia, but a reality." United Press put the story on its wire, and Time wrote a brief report complete with pictures of a suited Alex, his wife, and daughters Maria Theresa and Irene, everyone smiling—the all-American family that had "moved across the tracks to stay." Time also dramatized the Bernal story in its nationally syndicated radio program, March of Time. And the case even received mention in American Me, a 1949 book that was one of the first positive portrayals of Mexicans in American publishing.
Meanwhile, a Los Angeles-based African-American weekly, the California Eagle, splashed the Bernal decision across its front page, with headlines proclaiming, "California Judge Jolts Nation," "Race Property Bars Held Illegal!" and "Race Housing Bars are Falling!" The Eagle, along with other African-American organizations, had long tried unsuccessfully to fight housing covenants in Los Angeles against blacks; the newspaper saw the Bernal victory as crucial to its cause, contrasting it to the "stolid silence of city and county officials concerning race restrictions on the city's housing."
"This decision," the paper concluded, "may be an important instrument in helping to solve the serious wartime-housing situation in the Los Angeles area as it applies to Mexicans and especially to Negro people."
But the case wasn't finished. On Sept. 20, Hagenstein filed a motion taking exception to Ross' ruling. He argued that the 14th Amendment didn't apply to the Bernals, as they were Mexicans, and mentioned again that Mexicans would bring down housing prices. Two weeks later, he filed another motion, now seeking to vacate Ross' decision; Hagenstein cited case precedents in arguing for the legality of housing covenants.
The judge was not swayed. In his final ruling on Nov. 15, Ross wrote that he felt that if he enforced housing covenants, a higher court would eventually find them unconstitutional. His bigger concern, however, was that it was against the philosophy "of the United States and of the state of California to enforce a restriction on occupancy based solely on nationality of the persons against whom the restrictions are sought. This is especially true when the nationality affected is that of a friendly neighbor and when one particular nationality is named."
* * *
In April 1967, Los Angeles County Superior Court Judge Loren Miller sat down for an interview with Lawrence de Graaf that is now part of the collection at Cal State Fullerton's Center for Oral and Public History. Miller would pass away a couple of months later, but he was already a legendary figure in the civil-rights movement for attacking the very sort of housing covenants that Bernal and Marcus had defeated 25 years earlier. It was Miller who, along with future Supreme Court Justice Thurgood Marshall, in 1948 successfully argued Shelley v. Kraemer before the nation's highest court, a decision that ended housing covenants nationwide. It was Miller who brought national attention to the issue in 1945 with what's commonly called the Sugar Hill case; like Doss v. Bernal, it involved white neighbors suing to remove African-Americans from their neighborhood, in this instance African-American movie stars such as Hattie McDaniel and Ethel Waters who lived in the West Adams district of Los Angeles. And it was Miller who had argued the successful California Supreme Court appeal of Fairchild v. Raines, a 1944 case involving a Pasadena African-American family that historians usually cite as the first legal finding against housing covenants.
Miller was also a historian of civil-rights lawsuits—his 1966 book, The Petitioners: The Story of the Supreme Court of the United States and the Negro, is a beautifully written chronology of the African-American legal experience. The judge knew the importance of the cases he had tried as a lawyer. But ever the humble judicial officer, he told de Graaf that it was the judge in the Sugar Hill case, Thurmond Clarke, who deserved the most credit for ending housing covenants, as he claimed that the decision was the first time someone had successfully cited the 14th Amendment in fighting segregation in the courts. "Unfortunately for him and for his place in history," said Miller, the case didn't go further than Los Angeles Superior Court. "[Clarke is] entitled to a great deal of credit for his courage and his foresight in looking ahead and correctly gauging the trend of constitutional decisions."
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I received my first IPL nearly 18 months ago, and did it right before summer. I didn't know to get numbing cream, and I plowed ahead despite the seasons heading into high sun. Big mistake. My spots faded off, but came right back because of sun exposure (even with SPF). This time, I did it right: October time period and I got numbing cream before. I'm that much older now, too, and can see... more
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I have had two IPL treatments with Dr. Linell, and other IPL treatments elsewhere, and I am always happiest with my results from her. I'm not sure why, but I am always more comfortable during the procedure at her office, and the results are beyond my expectation each time. I literally feel that the more I see her, the healthier my skin looks. She is conservative but effectve with all of her... more
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Starting A New Church
When starting a new church, ministry or even carrying forth an existing ministry, the most important aspect that many overlook is the simple issue of prayer.
7. What Paul and Barnabus teaches us in Acts 14, in the the seventh step is praying.
(Acts 14:23 "they commended them to the Lord"). Prayer was a reality in their lives. Prayer is what makes it happen. You can't teach it in a classroom. Jesus' disciples said Lord teach us to pray, not how to pray. We don't need to learn how to pray, we need to learn to pray. If you learn to pray, you'll know all the how to you need to know. Yet, how many local churches, and believers take it for granted and simply don't do it.
It's not about saying the right magic words, he sorts all that out.
Matthew 9:38 tells us missionaries are selected through prayer. That's how they come, not anything else! We must pray for missionaries. We must labor in prayer. Pray for people individually. Pray as we send out missionaries and to support them.
William Carey left for India in 1792. In 1746 there was a prayer campaign that lasted for 7 years in America and England. In 1786, John Sutcliff organized Baptist churches in England to pray the first Monday of each month. That made it back across the ocean again after this prayer movement started in 1746. Finally in 1792 – 1793, William Carey was leaving for England. This was the key to new church planting as missionaries blanketed the world in the golden age of missions.
We get discouraged that our prayers aren't answered as fast as we want but it's the power for the mission. It is necessary and required when starting a new church.
8. The eighth step is they commended them to the Lord (Acts 14:23). It means to turn them over to God. That's the same word that Jesus used when he was dying on the cross when he said Father into your hands I commend my spirit.
Father it's all yours. The hardest thing is to commend your missionary work to God when you leave. To say I'm not in charge anymore and to see God put leaders in a position that you may not have chosen. To see the church or church ministry suffer and go in directions that you may not want it to go.
The same thing happens when you give your daughter in marriage. You commend her to somebody else. You may not like your son-in-law but you can't go back in 5 years and say I'm taking her back now. One of the hardest things for missionaries to do, and sometimes God has to do it for them, through sickness, war etc. is to get them to leave.
One of the greatest causes of failure in the work of Christ are missionaries (or the church planter) who stay to long. That doesn't mean that some missionaries don't invest an entire lifetime, sometimes that's necessary. Sometimes that missionary must say it's time for me to commend this work to the Lord. Same thing that parents must do when they commend their children to go as missionaries.
Prayer and trusting the Lord are essential elements when starting a new church and carrying forth ministries.
Read the previous steps from Acts 14 to church planting:
All church planting topics:


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It might be out of the frying pan and into the minimum security women's prison for Lindsay Lohan, due to probation violations connected to the slap-fight she instigated with a rehab technician last month. Less than a day after Lindsay was released from rehab, investigators allegedly want Lilo charged with battery for her late-night shoving match with Betty Ford Center employee Dawn Holland on December 12. The altercation constituted a violation of Lohan's probation for her 2007 DUI conviction, which in part requires that she "obey all laws." All of them? Every single one? Well that was setting the bar pretty high for Lohan, now wasn't it?
Sheriff Joe Borja released a statement Monday indicating that authorities probing the incident would be sending their report to the Los Angeles County Probation Department this week…and it does not look good for Lindsay. "The investigation determined Ms. Lohan violated several aspects of her probation, including the battery [charge]," the Palm Desert Police Department said. Depending on the whims of L.A. judge/Lohan's archnemisis Elden Fox, the violation could land Lohan up to six months in jail. So here's hoping Lindsay really enjoyed herself last night as she peered through the blinds at Sam Ronson's house. It might be the last evening she spends out of a regulation orange jumpsuit for a long time.


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Direct gene transfer of genetic material into an external or internal target cell site ("microseeding"), in optional combination with a wound treatment chamber, are particularly effective as a way of obtaining long term expression of native or non-native polypeptides in a host. A wide variety of proteins and materials can be expressed, either for secretion into the general blood and lymphatic system, or to alter the properties of the protein, for example, to not express proteins eliciting an immune response. The use of the optional wound chamber system for gene transfer to skin target sites also allows non-invasive assessment of the success of transfer by assaying for the presence of the expressed protein in wound fluid, in contrast to the prior art use of invasive techniques, such as biopsies, in order to achieve the same assessment of early expression.
1. A method for introducing genetic material into a cell the method comprising the step of
repeatedly injecting the genetic material into a plurality of cells of a target tissue at an in situ site on a human or non-human animal with a microneedle, whereby a change in the animal attributable to the delivery of the genetic material is detectable.
2. The method of claim 1 wherein the cell is selected from the group consisting of a superficial keratinocyte, a stem cell keratinocyte, a dermal fibroblast, a smooth muscle, a striated muscle, a connective tissue, an epithelial tissue, a wall of an abdominal passage, a liver, a kidney, a stomach, an appendix, an intestine, a pancreas, a lung, a heart, a bladder, a gall bladder, a brain, an endocrine gland, a reproductive gland, and a lymphatic gland.
3. The method of claim 1 wherein the genetic material is attached to microparticles.
4. The method of claim 3 wherein the microparticles are iron oxide particles ranging in size from 0.5 to 1 micron in diameter.
5. A method for delivering genetic material into a human or non-human subject, the method comprising the steps of:
contacting a tip of a microneedle with a solution comprising the genetic material;
penetrating the tip repeatedly into a site on a surface of a target tissue at an in situ site on the subject, to a depth in the tissue selected to allow contact between the tip and a plurality of target cells that can take up the genetic material, until a sufficient amount of the genetic material is delivered to the target cells such that a change in the subject attributable to the delivery of the genetic material is detectable.
6. A method as claimed in claim 5 wherein the target tissue is selected from the group consisting of skin cells and a wound.
7. A method as claimed in claim 5 wherein the target tissue is internal to the subject.
8. A method as claimed in claim 7 wherein the target tissue is selected from the group consisting of a smooth muscle, a striated muscle, a connective tissue, an epithelial tissue, a wall of an abdominal passage, a liver, a kidney, a stomach, an appendix, an intestine, a pancreas, a lung, a heart, a bladder, a gall bladder, a brain, an endocrine gland, a reproductive gland, and a lymphatic gland.
9. A method as claimed in claim 5 wherein less than about 500 micrograms of the genetic construct are delivered to the target cells.
10. A method as claimed in claim 5 wherein between about 20 and 200 micrograms of the genetic construct are delivered to the target cells.
11. A method as claimed in claim 5 wherein the penetrating step is repeated between about 500 and 5000 times per cm2 of the surface of the target tissue.
12. A method as claimed in claim 5 wherein the penetrating step is repeated between 3000 and 4000 times per cm2 of the surface of the target tissue.
13. A method as claimed in claim 5 wherein the tip penetrates the target tissue to a depth of less than 5 mm.
14. A method as claimed in claim 5 wherein the tip penetrates the target tissue to a depth of less than 3 mm.
15. A method as claimed in claim 5 wherein the genetic material encodes a non-native growth factor.
16. A method as claimed in claim 15 wherein the non-native growth factor is epidermal growth factor.
17. A method as claimed in claim 5 wherein the subject is non-human.
18. A method as claimed in claim 17 wherein the subject is selected from a group consisting of a dog, a cat, cattle, swine, a goat, and a sheep.
19. A method as claimed in claim 5 wherein the subject is a human.
20. A method as claimed in claim 5 further comprising the step of:
placing the microneedle in a concentrated solution of hydrochloric acid before the penetrating step.
21. A method for delivering genetic material into a target tissue of a subject, the method comprising the steps of:
contacting a tip of a microneedle with a solution comprising the genetic material;
penetrating the tip repeatedly into a site on a surface of the target tissue, to a depth selected to allow contact between the tip and a plurality of target cells that can take up the genetic material, until a sufficient amount of the genetic material is delivered to the target cells such that a change in the subject attributable to the delivery of the genetic material is detectable,
wherein the target tissue is selected from the group consisting of skin cells and a wound, the method further comprising the step of covering the target cells with a protective chamber.
This is a continuation-in-part of U.S. Ser. No. 08/076,550, filed Jun. 11, 1993, now abandoned, which is a continuation-in-part of U.S. Ser. No. 07/897,357, filed Jun. 11, 1992, for "System and Method for Transplantation of Cells", by Elof Eriksson and Peter M. Vogt, now U.S. Pat. No. 5,423,778, which is a continuation-in-part of U.S. Ser. No. 07/707,248, filed May 22, 1991, by Elof Eriksson, now U.S. Pat. No. 5,152,757, which is a continuation of U.S. Ser. No. 07/451,957 filed Dec. 14, 1989, now abandoned.
The invention relates to a system for delivering genetic material into cells in situ in a patient.
Various methods for introducing genetic material into an internal or external target site on an animal exist. Most prominent are methods of accelerated particle mediated gene transfer, such as are described in U.S. Pat. Nos. 4,945,050 (Sanford, et al.), 5,204,253 (Sanford, et al.) and 5,015,580 (Christou, et al.).
Other methods have focussed on introducing genetic material into skin cells, particularly keratinocytes. Keratinocytes are the principle cells which cover the surface of the body. They are capable of producing proteins, particularly keratin, which constitute the main surface barriers of the body. For several different reasons, keratinocytes are attractive potential targets for gene transfer. Since they are located on the surface of the body, they are easily accessed both for gene manipulation and monitoring. If complications from gene transfer would occur, for instance, the development of local tumors or local infections, these could more easily be treated in the skin than elsewhere.
To date, genetic manipulation of keratinocytes has been done in one principal way. Skin has been harvested, the keratinocytes have been separated from the fibroblasts, and then the keratinocytes individually isolated and brought into suspension. These suspensions of keratinocytes have then been cultured to confluence using tissue culture techniques, as reported by Rheinwald, J. G., Green, H. Serial Cultivation of Human Epidermal Keratinocytes: The Formation of Keratinizing Colonies From Cells. Cell G, 331-343, 1975.
The new genetic material has been introduced into the keratinocyte while being grown in vitro using either a viral vector or plasmid, as reported by Morgan, J. R., Barrandon, Y., Green, H., Mulligan, R. C. Expression of an Exogenous Growth Hormone Glue by Transplantable Human Epidermal Cells. Science, Vol. 237, 1476-1479 (1987) and Tenmer, J., Lindahl, A., Green, H. Human Growth Hormone in the Blood of Athymic Mice Grafted With Cultures of Hormone-Secreting Human Keratinocytes. FASEB J., 4:3245-3250 (1990). The transfected cells are then usually resuspended and grown on selective media in order to increase the yield of transfection. Sheets of keratinocytes are then transplanted back to the mammal from which the keratinocytes were harvested.
Even though the in vitro yield has been acceptable, the in vivo yield has been unacceptably low, both short and long term. It has been very difficult to document any significant long term (more than thirty days) expression with these techniques, for example, as reported by Garlick, J. A., Katz, A. B., Fenvjes Esitaichman, L. B. Retrovirus Mediated Transduction of Cultured Epidermal Keratinocytes. J. Invest. Dermatol., 97:824-829, 1991.
Direct gene transfer of genetic material into internal or external target sites in optional combination with the use of an "in vivo" culture chamber is particularly effective for long term expression of polypeptides.
Direct delivery of genetic material is accomplished by repetitive microneedle injection into intact skin cells, open skin wounds, and internal tissues or organs. In a 1 cm2 target area, microneedles make between 500 and 5000 separate injections of an aqueous solution comprising genetic material at a desired concentration.
By employing the optional culture chamber system, direct in vivo gene transfer to exposed cells in an open wound can be performed. If these cells were not covered by the chamber, they would desiccate and die. The chamber also completely seals the wound from the outside, eliminating the spread of genetic material and vectors to places outside of the wound. At the same time, the chamber prevents the accidental introduction of undesired contamination, including viruses and other microorganisms and chemical contaminants into the wound.
The use of the chamber system for gene transfer also allows non-invasive assessment of the success of transfer by assaying for the presence of the expressed protein in wound fluid, in contrast to the prior art use of invasive techniques, such as biopsies, in order to achieve the same assessment of early expression.
A wide variety of proteins and materials can be expressed, either for secretion into the general blood and lymphatic system, or to alter the properties of the protein, for example, to not express proteins eliciting an immune response against the transplanted cell.
It is an object of the present invention to provide a method for delivery of genetic material which is economical, and practical, and can be customized to the patient with minimal effort and expense.
It is another object of the present invention to provide an apparatus suitable for direct delivery of genetic material to internal target sites.
FIG. 1 is a schematic representation of a microseeding device for delivering genetic material into cells of a patient.
FIG. 2 is a cross-sectional view of a microneedle 10 shown in FIG. 1.
FIG. 3 (a and b) are representations of microseeding devices for delivering genetic material into cells of a patient.
FIG. 4 is a schematic of the exposed undersurface of a partial thickness skin flap used to expose hair follicles to obtain epidermal stem cells.
FIG. 5 shows the EGF concentration in target sites at various times after microseeding with an EGF-expressing genetic construct.
FIG. 6 shows the levels of EGF in fluid and in tissue biopsies taken 3 days after delivery of pWRG1630 into intact skin and into partial thickness wounds.
The method described herein is a method for delivering genetic material into cells at an internal or external target site on an human or non-human patient using a microneedle delivery apparatus. This process has been termed "microseeding" by the inventors. Examples of disorders that can be treated include wounds such as burns, pain, tumors, and infections.
Also described herein is a microneedle delivery apparatus for delivering DNA according to the method of the present invention.
Also described herein is use of a treatment system for wounds and other skin disorders that can optionally be employed in the method of the present invention when delivering DNA to an external target site. The treatment system has previously been described in U.S. Pat. No. 5,152,757, which is incorporated herein by reference.
The method of the present invention delivers genetic material into any accessible target cell type in any human or non-human patient. Among non-human patients, most preferred are mammalian animals, especially domesticated animals such as dogs, cats, cattle, swine, goats, sheep and the like, in which the method is envisioned as a desired veterinary therapy.
The preferred target cells in all target animals are skin cells, which are most readily transduced with genetic material because of their proximity to the exterior of the patient. "Skin" is intended to encompass cells in all skin layers including epidermal, dermal, and subdermal layers. More specifically, skin includes superficial keratinocytes, stem cell keratinocytes and dermal fibroblasts. For purposes of this patent application, "skin" also encompasses the muscular tissue beneath the skin that can be accessed from the exterior by the microneedles described herein. The target skin can be intact or can be prepared for treatment by wounding. In addition, internal tissues and organs within a patient are also desirable target sites into which exogenous genetic material can be introduced in keeping with the method. Suitable internal sites include any soft tissue that can be pierced by microneedles as described herein. For purposes of this patent application, "internal sites" are those target sites which are not accessible from the exterior of the patient but which are accessible using the microneedle delivery device for internal use, as disclosed herein. Without intending to limit application of the method to particular internal sites, specifically envisioned as targets are smooth and striated muscle, connective and epithelial tissues, walls of abdominal passages, and internal organs including, but not limited to, liver, kidney, stomach, appendix, intestines, pancreas, lungs, heart, bladder, gall bladder, brain and other nervous system cells, as well as reproductive, endocrine, lymphatic and other glandular tissues.
In any particular target tissue, it is envisioned that a subpopulation of cells (e.g., keratinocytes in skin cells) may be most preferred target cells, insofar as they exhibit a superior ability to take up or express introduced genetic material. The particular preferred subpopulations can readily be determined empirically in an experimental system designed to measure a particular response to introduction of a particular genetic molecule. If a particular subpopulation of cells is found to be a preferred target, one of ordinary skill will understand how to direct the genetic material into that subpopulation either by orienting the microneedle delivery apparatus toward a portion of a target tissue, or by selecting a preferred delivery depth to which the genetic material is delivered, or both.
The genetic material that is introduced into the target cells using the method can be any native or non-native genetic molecule that can provide a desirable activity to a target cells. The genetic material can encode any native or non-native protein or polypeptide having such a desirable activity in a target cell. Alternatively, the introduction of the genetic material itself can alter the target in a desirable way, such as by interference with the transcription or translation of a gene normally present in the target. "Non-native" means that neither the genetic material, nor any protein or polypeptide product encoded by the genetic material is detectable in the untreated target cells or tissue. The nature of the introduced genetic material itself forms no part of the present invention.
The genetic material, can be RNA, but is preferably DNA and is most preferably supercoiled plasmid DNA. The genetic material is prepared according to any standard preparation or purification method among the many known to the art and is provided in an aqueous solution, buffered or unbuffered. The amount of genetic material delivered is not absolutely critical, though better results have been observed for DNA molecules tested when the amount of DNA is below 500 μg, and preferably below 200 μg. Suitable concentrations can readily be determined by routine experimentation. Delivery to the target site of approximately 100 μl of an aqueous solution containing genetic material in the preferred concentration range is sufficient to function in the method. The genetic material can be attached to microparticles such as iron oxide particles in the range of 0.5 to 1 micron in size. Alternatively, unsupported genetic material in solution is also suitable for delivery according to the method.
The genetic material typically includes an expressible DNA sequence that encodes a native or non-native polypeptide. The DNA can be of any length and can include genomic DNA fragments, engineered DNA produced in a microbial host, or synthetic DNA produced according to known chemical synthetic methods, including, but not limited to, the Polymerase Chain Reaction. The art is cognizant of the various required and preferred elements (including promoters, terminators, transcription- and translation-regulating sequences, and the like) that one of ordinary skill would provide on an expressible genetic construct. One of ordinary skill is also able to select appropriate elements from the many known elements to facilitate or optimize expression in a particular target animal. The native or non-native polypeptide produced in the method can be maintained intracellularly or secreted to the extracellular space. One of ordinary skill in the art is familiar with the genetic elements necessary to direct a sequence to a particular cellular or extracellular microenvironment and with methods for constructing a genetic construct to facilitate such direction. For example, a polypeptide expressed after gene transfer can be directed to cross a cell membrane by adding an appropriate signal peptide to the gene that encodes the polypeptide.
Desirable proteins that can be expressed after gene transfer include, without limitation, growth factors, hormones, and other therapeutic proteins. These would speed the healing of wounds and correct certain deficiencies, such as parathyroid, growth hormone, and other hormone deficiencies, as well as deficiencies of certain clotting factors such as factor VIII. For instance, if the gene encoding the growth factor is introduced into the genome or cytoplasm of target skin cells in a wound, these cells can be made to produce a desirable growth factor. The expressed growth factor will then not only speed the healing of the wound, but may also help to heal wounds that would not heal otherwise.
Cells can also be engineered to not express a protein, such as a protein involved in an immune response, for example, a human leukocyte antigen (HLA). This can be accomplished in a variety of understood ways, the most common being the introduction of "antisense" genetic material that hybridizes with mRNA present in a cell to prevent translation of the mRNA.
This approach can be used to insert other genetic material of interest into target cells in order to eliminate, or restore, missing or defective functions of patients having any of a variety of skin diseases. Also, the mode in which cytokines act can be changed in that when a factor is expressed in a cell that normally does not produce that factor, a paracrine pathway can be changed into an autocrine pathway. In the same fashion, target cells can be genetically engineered to repair or compensate for inherent genetic defects, such as epidermolysis bullosa. The target cells in a superficial excisional wound can be microseeded with appropriate genetic material to generate a new epidermis with the desired features. Problematic wounds may require coverage of the defect and fast healing.
Genetic material can be delivered directly into target cells of a patient using a microneedle delivery apparatus to repeatedly puncture a target area, and to deliver, with each puncture, a small amount of the genetic material provided in an aqueous solution. A microneedle delivery device 10, suitable for use in the method of the present invention, such as the preferred devices shown in FIGS. 1-3 (a and b), include microneedles 12 having beveled tips 14, mounted in a row on a support 16, such as a handle. The microneedle diameter is about 300 microns. The beveled tip 14 tapers to a zero diameter along the 2 mm closest to the tip; at 100 microns from the tip, the diameter is about 60 microns, while at 50 microns from the tip, the diameter is about 35 microns. The beveled tip 14 facilitates puncturing of the selected target site by the microneedles 12 and opening of cells along the needle path, which is thought to facilitate high expression levels after delivery. The microneedles 12 can be solid (FIG. 3) or can have hollow centers (as in FIG. 2), although preferred microneedles are solid, since no genetic material is lost to the interior of the needle during delivery. If hollow needles are used, the hollow center should terminate to the side, rather than the bottom, of the tip 14, as is shown in FIG. 2. Moreover, the solid interior applies significantly higher force than hollow needles to the solution containing the genetic material, to help direct the material into the target cells.
The microneedles 12 are reciprocally driven by a power source 18, secured to the support, which can be an electric motor or any other suitable power source. The microneedles 12 have a proximal end and a distal end, relative to the power source 18. If the motor is electric, as in the preferred devices, the motor is attached to a switch that controls whether electric power is applied to the motor. The switch can provide variable or constant power to the device. The motor is connected by a cam to a reciprocating piston 20, which includes an attachment slot thereupon. The microneedles are joined to the piston attachment slot through a bar connected to the microneedles and inserted into the piston attachment slot. When energized, the piston can oscillate the microneedles at a wide range of speeds between 8 and 50 oscillations per second. The speed of the needles is not thought to be critical to their use in the invention. The amplitude of the oscillating microneedles can vary up to approximately 5 mm. The amplitude is also not believed to be critical, the effect of a greater amplitude simply being a deeper penetration depth. A 3 mm amplitude is suitable. Both the speed and amplitude can be predetermined by, for example, varying the power applied to the motor.
Surrounding the microneedles is a needle tube 22, shaped to accommodate the microneedles 12, which supports the microneedles 12 in all four directions and urges the microneedles 12 into a very small delivery area. The needle tube 22 itself is rigid or semi-rigid and is secured to the support 16 with a fastener, such as a wing nut, in a narrow opening.
If hollow microneedles 12 are used, as in FIG. 2 (schematic), the hollow centers of the microneedles are in fluid communication with controllable port 24 for connecting a fluid receptacle that holds the solution comprising the genetic material. The fluid receptacle can be a syringe. The solution can flow from the fluid receptacle through the hollow centers to the distal tips 14 of the microneedles 12.
If solid microneedles are used, delivery of the solution comprising genetic material through the microneedles to the tips is not practical. No fluid delivery structure is needed if the device is to be used for delivery only to an external target site, such as an intact skin or wound target site because fluid delivery can be readily accomplished by manual delivery of the solution to the site before applying the microneedle device. However, tubing for delivering the genetic material, as described elsewhere herein, is preferably also used for delivery to an external target site because less DNA is consumed when delivery is performed through delivery tubing.
When the genetic material is to be injected into internal sites within the patient, the genetic material needs to be delivered to the distal tips 14 of the microneedles 12. In a preferred apparatus 25 that facilitates direct gene transfer to an internal or external target site, separate structures are provided as part of the device, to bring the genetic material in solution to the distal tips of the solid microneedles. As shown in both embodiments of FIGS. 3a and 3b, the tube that surrounds the needle can be provided with one or more additional channels 26 on its inner or outer surface to serve as fluid conduits. The channels 26 can be separate from and attached to the needle tube, or can be formed directly thereto or therein. A first channel terminates at a first end at the motor end of the device at a controllable port 28 connectable to a fluid receptacle such as a syringe. A second end of the channel is at the distal end of the microneedles 12. A second, optional, larger channel, connectable by a controllable port 30 at one end to an external suction device, also opens at the opposite end near the tips, and provides suction of fluid from the delivery site. The second channel can alternatively be provided with a second fluid receptacle and used as a second fluid delivery channel to deliver a second fluid, such as a sealant, a biological glue or a hemostatic agent, to the distal ends of the solid microneedle tips.
The overall size of the apparatus 25, and, in particular the length of the microneedle 12, is such that that power source remains user-operable and external to the patient when the distal end of the microneedle 12 is positioned at the internal target site. Thus, the length will depend upon the distance of the internal site from the incision through which the apparatus 25 is introduced into the patient. A suitable length of the device for internal use is 10-15 inches, preferably 13 inches, which allows adequate manipulation to numerous internal target sites.
The inventors have determined that optionally placing the microneedles 12 in a 35% (12.1M) solution of hydrochloric acid for 12 hours before use in the method etches the needles slightly. Another way to modify the needles is to bombard the needles with aluminum oxide microbeads. It is not yet known whether needle surface modification provides additional surface area onto which the genetic material can adhere during delivery, or whether the modified needle surface advantageously affects the needle's ability to puncture the cells. Although not essential, dipping the microneedle tips 14 into hot paraffin can also prevent DNA from sticking irreversibly to the microneedles during microseeding.
Microseeding can be used to insert genetic material directly into target cells in situ in a human or non-human patient. One or more microneedles are ideal for this purpose. In the method, a solution containing genetic material is placed on the target surface. The microneedles are then moved as a group across the target site to "inject" and thereby deliver the genetic material through the surface of the target to the underlying tissue along several parallel lines. The microneedle or microneedles repeatedly penetrate a site on a surface of the target tissue to a depth within the tissue at which a plurality of preferred or most preferred target cells are found. The penetrating and delivering steps are repeated in the vicinity of the target cells until sufficient genetic material has been delivered that a change in the patient attributable to the delivery of the genetic material is detectable, preferably within twenty-four hours. The change can be physiological, biochemical, histological, genetic, or immunological, or otherwise. Detection of an added or eliminated characteristic of the patient, genetic or phenotypic, is sufficient.
The method can be used to deliver unsupported genetic material in solution, or genetic material attached to carriers, such as iron oxide microparticles. This technique is useful for delivery of a suspension of genetic material alone, without delivery of infectious viral material or attachment to microparticles. This is desirable because of the minimal sample preparation time required. In addition to persistent expression, high transient expression levels are observed.
It is preferred that a large number of independent microneedle penetrations be performed to ensure delivery of an adequate amount of genetic material in the method. The number of penetrations should range from between about 500 to about 5,000 per cm2 of surface at the target site. Preferably, between 3,000 and 4,000 independent penetrations should be performed at each 1 cm2 site. To reduce the total delivery time, it is highly preferred that more than one oscillating microneedle, preferably six or more oscillating microneedles, be used to deliver the genetic material. Because of the large number of microinjections, it is also highly preferred that the oscillation be accomplished using an apparatus designed for repeated penetration of oscillating microneedles, such as either of the microneedle delivery devices described herein. Such a microneedle delivery device, placed under control of a scanner 27, can systematically and accurately cover a predefined area of skin or other tissue, and can deposit the genetic material very evenly into a large number of target cells in the predefined area.
For delivery into a wound or intact skin, the microneedles are placed over a desired treatment site that has previously been prepared, as needed. No particular preparation is necessary for delivery into intact skin. The site for delivery of genetic material into a wound is prepared by removing infected or burned skin, if necessary, or by creating an appropriate wound for the purpose of in situ delivery of genetic material. An artificially created wound is shown in FIG. 4. Stem cells located in the hair follicles 30 deep to the epidermal dermal junction can be exposed by creating a flap 32 of epidermis where the deep portion of the flap contains the basal layer of the epidermis. The exposed wound surface 34 contains the portion of the hair follicles with the stem cell keratinocytes. After delivery of the genetic material, the epidermal flap 32 of an intentional wound is sutured back in place and the wound is sealed with a dressing or in a chamber, as described below.
When internal delivery is desired, surgical access to a desired delivery site is provided for a microseeding apparatus such as that described herein. Surgical access to the interior of the patient can be provided through laparoscopic ports made in a manner known to the art. The laparoscopic ports can also accommodate a viewing scope and an assisting instrument for accurate placement of the microseeding apparatus. In such cases, no wound chamber is needed after treatment, since the treated cells are not exposed to the air and will not dry out.
Regardless of location of the delivery site, the beveled tips of a plurality of microneedles are surrounded with an aqueous solution comprising the genetic material. As noted above, the genetic material is provided to the microneedle tips manually or via a channel provided on the microneedle delivery device. Oscillation of the microneedles placed on the target site at a rate of between 8 and 50 oscillations per second is initiated by energizing the power source. The power source reciprocates the piston at a predetermined rate so that the microneedles repeatedly enter the treatment site to a desired depth. The desired depth, which can range from 0 to about 5 mm, is selected to deliver the solution containing genetic material to a depth at which a large number of cells competent to take up and express the genetic material are found. For instance, it has been determined by the inventors that, when the target is skin, the preferred target cells are keratinocytes, generally located at about 2 mm beneath the skin surface. The optimal penetration depth for other delivery sites can readily be determined empirically by looking under controlled experimental conditions for a desired change in an art-accepted model system, for example, a pig, using a physiological, biochemical, chemical, histological, genetic, immunological or like detection method.
After treatment, no particular method steps need to be followed. However, should one desire to either regulate the fluid in which the treatment site is bathed, or to analyze the products produced at the treatment site, isolation of the site in a vinyl adhesive chamber, or other such isolating chamber is recommended.
The technique can also be enhanced by practicing it in combination with other biological agents, such as liposomes.
This method of introducing genetic material into cells may be superior to gene transduction using plasmids or retroviral vectors, because the latter have an unacceptably low yield. Transfer of genetic material with accelerated particles, employing "gene guns," have a higher yield than plasmid or retrovital transduction, but can, in some embodiments, have other disadvantages, such as a very loud blast and a risk of accidental discharge. Moreover, this method does not leave particles inside patients after treatment. For internal delivery, the microneedles are advantageous because it is not necessary to fully expose internal organs before delivery; rather, only an few incisions large enough to receive a manipulable microneedle apparatus, assisting tool, and viewing scope are made.
Thus, microseeding of genetic material into cells with oscillating microneedles provides a practical, affordable, and a predictable method of insertion of genetic material for localized or systemic expression. The invention finds particular utility as a method for immunization against a non-native polypeptide, and as a method for introducing a foreign genetic material as a therapeutic agent.
If a polypeptide is secreted from treated intact skin or wound cells, it may be desirable to localize the secreted polypeptide at or near the target site. Such is the case, for example, when the secreted polypeptide accelerates wound healing. In such cases, the target site can be isolated after treatment by enclosing the treated wound site in a sealed chamber, in the manner of U.S. Pat. No. 5,152,757, which is incorporated herein by reference. The structure of the chamber is described in the referenced patent, and is not described in detail herein.
Isolation of the treatment site has an additional benefit of protecting the treatment site from external pathogens such as bacteria and viruses. Moreover, one has the ability to surround the treated target site with fluid having a desired composition, and the ability to analyze samples of the fluid to evaluate the concentrations of desired or undesired compounds in the fluid. However, if delivery is made into a non-wound skin site, use of the sealed chamber may not be necessary, as the skin provides sufficient protection to transduced cells.
In a preferred embodiment, treated skin and wound cells are protected by a system including a chamber, treatment fluid (which may be nutrient media, physiological saline, or some other compatible solution), treatment additives (such as antibiotics and buffering agents), means for controlling treatment variables and means for monitoring cell growth.
The treatment system includes a chamber securable about the periphery of a wound, having portal means for introduction into and removal of treatment fluids from the chamber, treatment fluid, at least one treatment additive, control means for treatment variables, and monitoring means for monitoring wound conditions. The chamber is secured about the periphery of a wound, a treatment fluid and at least one treatment additive are introduced into the chamber, and the treatment variables are controlled according to wound conditions.
The wound chamber, which is made of vinyl or other flexible transparent material, such as polyurethane, has a bellows shape and an opening which corresponds to the size of the wound, either the chronic wound or a superficial wound created specifically for the purpose of gene transfer. The chamber contains a small amount of normal saline with antimicrobial agents. When the microneedle is used, the DNA is delivered into the cell and the chamber with normal saline and antibiotics is then attached to the perimeter of the wound. Wound fluid is sampled in order to assay expression of the secretable gene product (e.g., growth factor) at 24 and 48 hours. The wound is treated in the chamber until healed.
The chamber encloses a predetermined surface area about the treatment site. The chamber provides protection for the wound from the surrounding non-sterile environment, control of treatment variables, containment for continuous fluid treatment, an effective delivery system for additives, direct monitoring of cell growth. Monitoring can be accomplished visually if the chamber is formed of a transparent material, or by extraction and analysis of fluid from the chamber. Fluid extracted from the system can be analyzed for factors which provide an indication of the status of healing, as well as the presence of undesirable components such as microorganisms, low oxygen, high carbon dioxide and adverse pH. The fluid may be tested for the number and type of bacteria and other microorganisms, the number and type of cells, the amount and type of proteins secreted by the patient and the cells within the chamber, and other factors such as drug levels, oxygen, carbon dioxide and pH.
The treatment system provides control over variables including temperature, specific ion concentration, colloid osmotic pressure, glucose concentration, amino acid content, fat concentration, oxygen concentration and carbon dioxide concentration and pH.
Portal means provide access for the introduction of treatment fluids and treatment additives into the chamber and extraction of fluid from the chamber. In some embodiments, treatment fluid is introduced into the chamber by injection with a conventional hypodermic syringe through the wall of the chamber, preferably made of a flexible, self-repairing plastic. In other embodiments, the chamber has an inlet and outlet port or separate inlet and outlet ports. Valve mechanisms are necessary where the apparatus is not to be connected to a treatment fluid reservoir and a drain or connected to a continuous perfusion system. The seals of the ports would be broken at an appropriate time for connection to other apparatus, such as a continuous perfusion system, at a hospital for example.
A preferred embodiment of the treatment system incorporates continuous perfusion of treatment fluid through inlet and outlet ports. A pump or gravity may be used to move the treatment fluid. The treatment fluid may be recirculated after filtering and other appropriate action (eg. heating or cooling). Alternately, fresh treatment fluid may be introduced and contaminated fluid disposed of. In an embodiment described in U.S. Pat. No. 5,152,757, the chamber contains a reservoir or more than one chamber, with the additional chamber serving as a source of fresh culture media, oxygen, and treatment additives.
As those skilled in the art will readily recognize, a removable sheet for protecting the adhesive and maintaining the sterility of the interior of the chamber is desirable. The chambers may be stored in a sterile pack for years. This chamber can take many shapes in order to fit wounds from the size of one square centimeter up to the size of a whole extremity. It is important that the adhesive surface be sufficient to secure the bandage to the skin surface to ensure a leak-proof seal.
As previously mentioned, treatment fluid and treatment additive introduction and subsequent extraction may be accomplished directly through the chamber walls by a needle and syringe. A self-repairing material to construct chamber 10 is contemplated. An alternative method would be to use inlet and outlet ports allowing the introduction and extraction of various substances into the chamber.
If the chamber is to be used to cover a wound or intact skin site, the skin adjacent to the site is cleaned so that there will be good adhesion between the chamber and the skin. The open portion of the chamber is then placed over the site, with the adhesive edges securing the chamber to the skin, then an appropriate culture medium and cells are introduced into the sealed chamber. The treatment fluid may be introduced and then extracted in favor of fresh culture media in a continuous or batch process. Selected treatment additives may be introduced into the chamber continuously or at a predetermined time or at periodic intervals. Appropriate control of treatment variables is also effected. Monitoring is accomplished by examination of the patient and visual examination of the fluid within the chamber and the wound itself. In addition, samples of fluid are extracted from the chamber for analysis and diagnosis. The chamber is removed once sufficient healing of the wound has occurred.
For example, to determine whether or not the wound is healed, the protein content of the extracted fluid is analyzed. When the protein content of the extracted fluid decreases to the level present in chambers containing fluid that are placed over normal skin, the wound is healed. Methods for determining protein content are well known in the art and are inexpensive and fast. The types of protein and the relative amounts of the types of protein can also be determined to further evaluate healing and expression of exogenous genetic material.
Treatment of each patient is specific for the conditions within the chamber. Control over treatment variables can include continuous cooling to 34° C. for the first 24 hours. Monitoring can include analyzing extracted fluid for protein and microorganisms, with samples extracted every 24 hours. For example, when the number of microorganisms is less than 104 per milliliter or per cc, infection has been resolved. Protein levels checked every day should be less than 24 mg/dl/cm2.
As noted above, there are a number of treatment variables which may be controlled by the system. One such treatment variable which may be controlled is temperature. It has been found that heating the wound from a temperature of approximately 27° C. (a common temperature of a lower extremity wound) to 37° C. accelerates wound healing. Experimental data has shown that at a wound temperature of approximately 37° C., the rate of wound healing is more than twice as fast as at a temperature of 27° C. The temperature of the wound area can be achieved by heating the treatment fluid. Cooling has also been proven beneficial in the case of acute burn and other traumatic wounds. Cooling reduces pain, swelling and destruction of tissue. In general terms, acute wounds benefit from cooling during the first hours after occurrence of the wound and later, wounds benefit from a temperature of approximately 7° C. Cooling can similarly be effected by cooling the treatment fluid.
Other treatment variables may also be optimized. For example, ion concentrations should be kept close to extracellular ion levels. Glucose, amino acid and fat concentrations should be kept close to the concentrations present in plasma or corresponding to a skin tissue culture medium. Oxygen and carbon dioxide concentrations should also be maintained at their normal tissue levels. Oxygen is an important treatment additive, and is essential for cell growth.
Normal or physiological buffered saline is the basic culture media. Buffering agents, anesthetics such as lidocaine, antibiotics such as penicillin or streptomycin, chemotherapeutic agents, and growth factors including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), and cholera toxin (CT) can be added to the culture/treatment media. Tissue culture mediums and fluids which increase osmotic pressure and oxygen accessibility may also be introduced to the chamber as treatment additives.
Selection of treatment additives is wound specific. For example, if an infection has been diagnosed, antibiotics are added in the amount of one single parenteral dose per 1,000 cc of fluid. Furthermore, a treatment additive of gentamicin, tobramycin or carbenicillin is appropriate for a wound infection with Pseudomonas, detected by analyzing extracted fluid. When hypoxia has been diagnosed, the liquid is passed through an oxygenating chamber before entering the chamber. If a tumor has been diagnosed, chemotherapy is given in an amount of one single parenteral dose per 1,000 cc of fluid. In situations involving a wound containing necrotic tissue and debris, proteolytic enzyme is added to the liquid. Immune modulators are added to the treatment fluid if an inflammatory reaction is exhibited. Epidermal growth factor is added in a concentration of 10 nanograms per cc when required.
The present invention will be further understood by reference to the following nonlimiting examples.
Iron oxide particles ranging in size from 0.05 microns to 1 micron in diameter were mixed with DNA plasmids in Tris-EDTA buffer. Drops of this material were placed on intact human skin. A microseeding was placed on the material on the skin to insert, or inject, the DNA into superficial keratinocytes as well as stem cell keratinocytes in the deep epidermis, or dermal fibroblasts.
The method of the present invention was tested in a laboratory model system for wound healing. Dorsal skin sites on domestic female Yorkshire pigs (3-4 months old, 40-45 kg) were outlined and were randomly assigned for partial-thickness wound or intact skin treatment. Wounds (15×15×1.2 mm) were created using a dermatome. Animals were maintained in accordance with the Harvard Medical Area Standing Committee on Animals. Surgical procedures were performed under Halothane (1-1.5%) anesthesia in a 3:5 mixture of oxygen and nitrous oxide.
Supercoiled plasmid DNA was introduced into the intact skin or into the wound bed using an oscillating microneedle apparatus driven by an electric motor and a piston. The oscillating microneedle apparatus for external (skin and wound) use is commercially available from Spaulding and Rogers Manufacturing, Inc. (Voorheesville, N.Y.). Controls included skin and wound sites untreated with DNA. 100 μl of solution containing supercoiled plasmid DNA in water was introduced into the intact skin or into the wound bed. After delivery, the skin and wound site were covered with sealed vinyl adhesive chambers, of the type described in U.S. Pat. No. 5,152,757, containing 1.2 ml of isotonic saline with 100 units/ml penicillin and 100 mg/ml streptomycin.
a. Histochemical Determination of Transduced Cells
Plasmid pCMVβ-gal, described by MacGregor, G. R. and C. T. Caskey, Nucl. Acids Res., 17:2365 (1989), was delivered to wound sites. Plasmid pCMVβ-gal encodes a β-galactosidase enzyme which is histochemically detectable upon the addition of 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal chromogen), which forms a blue precipitate in transduced cells. The treated tissues and control tissues were frozen and thin sections prepared from the frozen samples were histochemically stained for β-galactosidase activity. Positive staining was observed only in the epidermal keratinocytes and in the hair follicles of sites treated with pCMVβ-gal DNA. No stained cells were observed in control sections.
Skin biopsies were flash-frozen, embedded in O.C.T. and cut into 8 micron cross-sections. The sections were then fixed in 1.5% glutaraldehyde and were stained for β-galactosidase activity. See MacGregor, G. R. et al., Somat. Cell Molec. Genet. 13:253 (1987). The tissues were counterstained with hematoxylin. Blue spots were observed in portions of the tissues having β-galactosidase activity.
b. Delivery of Epidermal Growth Factor-Encoding Plasmid DNA
Plasmid pWRG1630, an epidermal growth factor-encoding expression plasmid, contains an in-frame fusion of the hGH secretory signal peptide to the mature EGF polypeptide. Plasmid pWRG1630 includes, upstream of the chimeric hGH-EGF gene, the cytomegalovirus (CMV) immediate early transcriptional promoter. Downstream of the mature human EGF coding region is the 3' untranslated sequence and polyadenylation signal from the bovine growth hormone gene (obtained from pRc/CMV, commercially available from Invitrogen, Inc.).
The complete nucleotide sequence of plasmid pWRG1630 is attached hereto as SEQ ID NO: 1. The 93 amino acid long polypeptide, encoded in two separate exons by pWRG1630, is shown in SEQ ID NO: 2. Referring now to SEQ ID NO: 2, the hGH secretory signal peptide is the first 26 amino acids. These amino acids are cleaved during signal-peptide processing at a cleavage site between amino acids 26 and 27. The next 14 amino acids are encoded in part by the hGH DNA and in part by the plasmid polylinker. Following the 14 amino acid long portion, is a 53 amino acid long mature EGF portion that corresponds to the 53 amino acids of the naturally occurring mature EGF peptide. The ability of this plasmid to produce an EGF polypeptide has been demonstrated by showing that after transduction of the plasmid into cultured fibroblast KB-3-1 cells, a polypeptide secreted into the culture medium reacted with hEGF monoclonal antibodies in ELISA and Western Blot assays. The culture medium containing the secreted polypeptide was biologically active in an 3 H!-thymidine incorporation assay using primary human foreskin fibroblasts and Madine-Darvy Canine Kidney (MDCK) cells.
c. Expression After Delivery
Initial experiments were performed to determine DNA transfer conditions to obtain high specific transfer (expression level per μg of input DNA). Parameters tested included the amount of DNA delivered per wound, the depth of needle penetration, and the density of penetrations. These parameters were adjusted to yield the greatest concentration of hEGF polypeptide in wound fluid after treatment. The preferred conditions for this plasmid were determined to be 20-200 μg of pWRG1630 DNA per wound delivered to a penetration depth of 2 mm at a density of 3,330 penetrations per cm2 surface area. In the experiment, 7500 penetrations were performed using the oscillating microneedle apparatus in a 2.25 cm2 surface area over a 25 second duration. Microneedles used for microseeding DNA were routinely pre-treated by dipping the tips of the microneedles into hot paraffin and then placing the microneedles into a 35% (12.5M) solution of hydrochloric acid for 12 hours. This pre-treatment scarifies the needles and provides increased surface area for trapping DNA during delivery.
Plasmid pWRG1630 was delivered into wounds at 20 μg, 200 μg or 2,000 μg per wound. At 72 hours post delivery, the wound tissue and the chamber fluid bathing the wound were tested for EGF polypeptide. EGF gene expression was monitored daily after gene transfer by measuring EGF concentrations in wound tissue, intact skin, and wound fluid for at least 7 sites for each group. Wound fluid was withdrawn from the wound chambers every 24 hours. The fluid was immediately chilled on ice, filtered and centrifuged. Samples from each group were then pooled, flash frozen and stored at -70° C. Skin biopsies were homogenized and protein was extracted from the homogenate before analysis. A commercially available EGF ELISA assay (Quantikine, R & D Systems, Minneapolis, Minn.) was used to determine EGF concentrations in samples. The minimum detection limit was 0.2 pg EGF per ml.
When 20 μg of DNA were delivered, between about 25 and 75 (average about 50) pg/ml of EGF were observed.
When 10-fold more EGF-encoding DNA (200 μg) was delivered, both the chamber fluid and the skin extracts contained about 150-175 pg/ml on average. The range of concentrations in the chamber fluid samples was between about 75-250 pg/ml, while the range in the skin extract samples was between about 50-300 pg/ml and was, on average, slightly higher than in the chamber fluid samples.
When yet another 10 fold increase in EGF-encoding DNA (2000 μg) was delivered into wounds, only about a 2 fold increase in EGF concentration was observed over the previous level. In this case, the EGF concentration in the chamber fluid samples ranged from about 250 to about 400 with an average of about 325 pg/ml while the concentration in skin extract samples was, on average, about 400 pg/ml with a range of between about 250 and 575 pg/ml.
From these data, it was determined that a maximally efficient response was observed in the range of 20-200 μg of DNA per wound. In future examples, 20 μg of DNA were delivered per wound or skin site, unless otherwise indicated.
Temporal variation in EGF level after gene delivery was monitored in chamber fluid over 5 days. Maximal EGF concentrations in fluid from wound sites (276±149 pg/ml) were observed 48 hours after microseeding. In fluid from intact skin treatment sites, a maximal EGF level of 165±113 pg/ml was observed 72 hours after microseeding. Detectable EGF concentrations were maintained over the entire 5 day monitoring period, as is shown in FIG. 5. The figure shows the EGF concentration in intact skin sites (Stippled) and in wound sites (Filled). Controls from wound sites seeded with 20 μg of pCMVβ-gal, and wound sites treated topically with 20 μg of EGF DNA are not shown. However, no EGF was detected in these controls using the ELISA assay. No evidence of abnormal cell growth, dysplasia tissue disorganization or the like has been observed in cells microseeded with pWRG1630.
d. EGF Yield in Fluid and Tissue after Delivery
Three days after delivery of the pWRG1630 EGF-encoding plasmid DNA into partial thickness wounds and into intact skin, analysis of the fluid surrounding the delivery site and of biopsies of the tissue itself were performed to determine the yield of EGF in such tissues. The results shown in FIG. 6 demonstrate that EGF levels in biopsied tissue (filled bars) are much higher than EGF levels in the fluid (stippled bars), and can reach nanogram levels. In FIG. 6, EGF in both wound and intact skin was above 2000 pg (2 ng), on average, and in some samples was above 4000 pg (4 ng), three days after treatment.
e. Persistence of Transferred DNA
Wound biopsy specimens were evaluated using the polymerase chain reaction (PCR) on days 1 through 30 to analyze the persistence of the transgenes. DNA was prepared from wound biopsies on days 6, 9, 12, 15, and 30 after delivery using a Puregene kit, commercially available from Gentra. To monitor for the presence of the introduced DNA, 400 ng of DNA prepared from the wound biopsy was mixed in a PCR reaction with 0.2 μg of each primer and 2.5 units of AmpliTAQ® DNA polymerase (from Perkin Elmer) in 100 μl of 10 mM Tris-HCl, pH 9.0 (25° C.), 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl2, and 0.2 mM of each dNTP. The sequences of the primers used are attached hereto as SEQ ID NO: 3 and SEQ ID NO: 4. After 3 minutes at 96° C., the reactions were subjected to 30 cycles of 60° C. for 75 seconds, 72° C. for 60 seconds, 96° C. for 60 seconds. See Mullis, K. B. and F. A. Faloona, Meth. Enzymol. 155:335 (1987). A 10 μl aliquot of each reaction mix was analyzed by agarose gel electrophoresis. The PCR products were visualized by staining with an ethidium bromide and their identities were confirmed by Southern Blot (Southern, E. M., J. Mol. Biol. 98:503 (1975) using internal hybridization probes. The PCR results demonstrated that plasmid DNA persisted in the wound site for at least 30 days. It is as yet unclear whether the persistent DNA resides within cells or is present in the extracellular matrix. These results contrast with the observation that both EGF and β-galactosidase protein expression diminish after 5-6 days.
An anesthetized Yorkshire pig was placed on its back. Laparoscopic ports were placed into the abdomen to accommodate a viewing scope, an assisting instrument, and a microseeding instrument adapted for use inside the patient as is shown in FIG. 3. A DNA solution containing the pCMVβ-gal plasmid and a small amount of a black iron oxide pigment was delivered to four sites in the liver, two sites in the stomach, and two sites in the abdominal wall. The black iron oxide pigment was added to the DNA solution to facilitate visualization of the treated area after the experiment. The DNA in solution was not coated onto the iron oxide.
Three days later, the tissues were harvested from the pig for processing to visualize expression of the inserted lac-Z gene. Strong expression was observed in the stomach. No expression was found in the liver. Expression in the abdominal wall was questionable. A reference site on the skin that was microseeded as a control showed strong expression. It is possible that the rapid healing of the liver tissue may have obscured the target sites and that the sites were missed during harvesting. In any event, the utility of the microseeding instrument for internal use was demonstrated by the strong lac-Z expression levels in the stomach.
The invention is not intended to be limited to the preferred embodiments nor to the specific examples, but is intended to include all such modifications and variations of the invention as fall within the scope of the appended claims.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 4(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4283 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: circular(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc ="Plasmid DNA"(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: join(713..721, 981..1250)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:[encoded data, 4872 characters](2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 93 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:MetAlaThrGlySerArgThrSerLeuLeuLeuAlaPheGlyLeuLeu151015CysLeuProTrpLeuGlnGluGlySerAlaPheProThrIleProLeu202530TyrGlnAlaSerIleSerArgValAsnSerAspSerGluCysProLeu354045SerHisAspGlyTyrCysLeuHisAspGlyValCysMetTyrIleGlu505560AlaLeuAspLysTyrAlaCysAsnCysValValGlyTyrIleGlyGlu65707580ArgCysGlnTyrArgAspLeuLysTrpTrpGluLeuArg8590(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc ="PCR Primer"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:TCAATAGTGACTCTGAATGTCCCC24(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc ="PCR Primer"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:GGCTGATCAGCGAGCTCTAG20__________________________________________________________________________
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|Best Western Keller hotels are listed below. Search for cheap and discount Best Western hotel prices in Keller, TX for your upcoming meeting or individual travels. We list the best Best Western Keller hotels/lodging so you can review the Keller Best Western hotel list below to find the perfect place.
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We apologize but we are unable to process online orders at this time as we are building a better PetSupermarket.com. To purchase any of our products, please visit one of our retail locations or visit our Amazon store here. You can find your nearest Pet Supermarket by using our Store Locator
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text:
The South's Best Fall Color
Hop in the car and road-trip through the South to see some of the country's most stunning fall colors.
Ellijay, GA
About 80 miles north of Atlanta, the small town of Ellijay sits on the edge of the Chattahoochee National Forest. Renowned as the "Apple Capitol of Georgia," Ellijay and surrounding Gilmer County are home to 10 pick-your-own apple orchards and the annual Georgia Apple Festival.
gilmerchamber.com or 706/635-7400.
Bernheim Arboretum and Research Forest, Clermont, KY
The Bernheim Arboretum in Clermont, (about 30 miles south of Louisville) includes 14,000 acres of fields and forests, as well as 35 miles of hiking trails and a bike route that winds along the fall-color-filled Long Lick Creek.
bernheim.org or 502/955-8512.
Caddo Lake, Karnack, TX
Located about 25 miles from Shreveport (and spreading into Texas), Caddo Lake is one of the South's largest natural lakes. Its 26,800 acres of freshwaters and cypress swamp are home to largemouth bass, crappie, and catfish. The best place to drop a line, and enjoy the fall colors, is from Caddo State Park in Karnack.
tpwd.state.tx.us or 903/679-3351.
Hanging Rock State Park, Danbury, NC
With mountaintops rising more than 2,500 feet, this state park about 30 miles north of Winston-Salem boasts some of North Carolina's brightest fall colors. Cascading waterfalls and more than 18 miles of trails lined with oak trees make the 7,024-acre park a popular place for an autumn outdoor adventure.
ncparks.gov/Visit/parks/haro/main.php or 336/593-8480
Lover's Leap Loop Trail, Hot Springs, NC
A 1.6-mile section of the Appalachian Trail brings hikers to this scenic view of the French Broad River and town of Hot Springs. The Lover's Leap Loop Trail overlooks 500 feet above the river, offering unobstructed panoramic views of the French Broad River Bridge and Blue Ridge Mountain area.
visitmadisoncounty.com or 828/680-9031
Harpers Ferry, WV
This historic town about an hour northwest of Washington D.C. was the site of abolitionist John Brown's infamous raid in 1859. Now it's better known as the "psychological half-way point" of the Appalachian Trail, where weary hikers can spend a day exploring the mountain town's shops, restaurants, and inns.
historicharpersferry.com or 304/535-6955.
Mount Cheaha, AL
The highest point in Alabama, the top of Mount Cheaha sits 2,407 feet above sea level. Called "Chaha" (or high place) by the Creek Indians, the mountaintop is now home to Cheaha State Park, a resort park complete with a hotel, restaurant, cabins, and campgrounds.
alapark.com/cheaharesort.com or 256/488-5115.
Baltimore County, MD
The rolling fields and hardwood forests north of Baltimore are filled with fall color. One of the best places to check out autumn leaves is Oregon Ridge Park. Located about two miles west of I-83, the 1,034-acre Baltimore County park features the Oregon Ridge Nature Center and hiking trails lined with flame-colored trees.
oregonridge.org or 410/887-1815.
Smoky Mountain National Park, Gatlinburg, TN
Spreading across 800 square miles of southern Appalachian Mountains in Tennessee and North Carolina, the nation's most visited national park offers acres of fall color. One of the most popular places to see the leaves and wildlife (including white-tailed deer, wild turkeys, and black bears) is Cades Cove, a broad valley at the northwestern corner of the park near Townsend, Tennessee.
nps.gov/grsm or 865/436-1200.
Natchez Trace Parkway, TN
Running through Tennessee, Alabama, and Mississippi, the scenic Natchez Trace Parkway connects the cities of Nashville, Tennessee and Natchez, Mississippi. The Tennessee section of the Parkway, which stretches for about 100 miles in the state, passes small towns such as Leipers Fork, and historic spots such as the Meriwether Lewis Site (where Lewis of the "Lewis and Clark Expedition" is buried).
nps.gov/natr or 800/305-7417.


text:
The University of Charleston will slash tuition rates by 22 percent next year in an effort to bolster enrollment.
The tuition decrease is one of several changes that UC President Ed Welch and a committee of stakeholders at the private school have been considering.
In an economic climate that caused public colleges and universities across the state to increase tuition last summer, Welch said he realized that the move to decrease UC's $25,000 yearly tuition to $19,500 was a bold move.
"It's a gutsy thing to do, but we know it's the right thing to do," Welch said.


text:
News and Information Staff
University media relations
Susan Griffith
senior media relations specialist
Susan is the veteran of the media relations team. Currently, she focuses on health sciences and humanities but has covered nearly every subject in her 15 years with the university. An award-winning photographer, Susan came to Case Western Reserve following two decades of experience as a newspaper reporter and editor. She can be reached at 216.368.1004 and email@example.com.
Marv Kropko
senior media relations specialist
Marv recently completed 30 years of service with the Associated Press, where he focused on business journalism. In the course of his Cleveland-based AP career, he has helped cover eight presidential elections, reported on an array of trials and rulings in state and federal courts and interviewed prominent corporate chief executive officers. He covers business and administrative news at Case Western Reserve University. He can be reached at 216.368.6890 and email@example.com.
Kevin Mayhood
senior media relations specialist
Kevin covers engineering, sciences and technology. He was an award-winning newspaper reporter for 25 years, most recently covering science for the Columbus Dispatch. He can be reached at 216.368.4442 and firstname.lastname@example.com.


text:
MiMI/Metabolomics API
The service provides a data repository supporting analysis in systems biology of genetic and metabolomic effects and molecular interactions. Available data include the MiMI database of protein interaction characteristics, which gathers input from multiple databases and sources within the field of biology. The repository also draws on daily updates of PubMed resources and reports from biomedical research. This single point of access allows queries and analysis against multiple interrelated data sources. API methods support search by free text or by a specific gene identifier. Returned data can include known compounds, interactions, and reactions. Methods can also return a list of interactions discussed in biological literature, discovered via natural language processing.
Java, Perl
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Oasis Hong Kong Airlines is Hong Kong's first low fare, long-haul passenger airline. Starting in October 2006, Oasis Hong Kong Airlines will offer non-stop service to London-Gatwick for the same price as many travelers presently pay for a 1- or 2-stop flight. Flights to Milan, Berlin, Cologne/Bonn (Germany), Oakland and Chicago will be successively phased in during the months after we launch service to London-Gatwick.
Oasis Direct to Europe as low as HK$1,000!!!!
so come to HK to shop and have fun at HKDL!!
website for Oasis Hong Kong
Source: Times Online
Budget route to Hong Kong opens
By Tom Chesshyre
IT'S Hong Kong for a song: the former British colony is about to get its first budget flights from Europe.
Tickets to Hong Kong costing £150 return, plus an estimated £106 in taxes, will be available from mid-October on a new airline called Oasis Hong Kong Airlines.
The airline, which says it is "opening up the Orient" to "budget-conscious travellers", is accepting registrations from passengers on its website before tickets go on sale in September.
Oasis, which is owned by Hong Kong investors, has two Boeing 747-400s with 359 seats, including 81 "luxury business" seats. On each 12-hour non-stop flight from Gatwick, 10 per cent of seats will be available at £150.
Traditionally, airlines have struggled to make the budget airline business model work on long-haul routes. However, Oasis hopes to overcome this with the extra revenue it hopes to make from its business cabin.
A spokeswoman said: "Oasis will offer low-fare travel with the same comfort and service you would expect from any airline." The airline believes that people will fly to Hong Kong to go Christmas shopping now that fares have dropped.


text:
By Nigel M Smith | iW NOW July 16, 2010 at 4:37AM
Average criticWIRE rating: A-
Arguably one of the year's most anticipated films, Christopher's Nolan's follow-up to "The Dark Knight," "Inception," finally hits theaters this week, and the positive buzz surrounding this movie couldn't be stronger, as indieWIRE reported earlier this week. The film boasts a starry ensemble that includes Leonardo DiCaprio, Michael Caine, Cilian Murphy, Ellen Page, Joseph Gordon-Levitt and Marion Cotillard. DiCaprio plays Dom Cobb, a 'dream extractor,' who goes in and steals valuable secrets from the subconscious of big corporate players while they dream. "Inception" tracks Cobb as he embarks on his most challenging mission yet; to actually plant an idea into the subconscious, an 'inception' if you will.
The first review to hit the press was from Rolling Stone's Peter Travers whose early praise caught on like wildfire. "The mind-blowing movie event of the summer arrives just in time to hold back the flow of Hollywood sputum that's been sliming the multiplex," Travers writes. ""Inception," written and directed by the visionary Christopher Nolan, will be called many things, starting with James Bond Meets "The Matrix." You can feel the vibe of Ridley Scott's "Blade Runner" in it, and Nolan's own "Memento" and "The Dark Knight." But "Inception" glows with a blue-flame intensity all its own."
The Hollywood Reporter's Kirk Honeycutt offered his take on the film's box office appeal: "Following up on such ingenious and intriguing films as "The Dark Knight" and "Memento," Nolan has outdone himself. "Inception" puts him not only at the top of the heap of sci-fi all-stars, but it also should put this Warner Bros. release near or at the top of the summer movies. It's very hard to see how a film that plays so winningly to so many demographics would not be a worldwide hit."
Similarily, Time's Richard Corliss forsees the film doing great business, mainly due to the labaryth plot which might require multiple viewings to fully uncode. He writes: "... seeing "Inception" - or seeing it twice, which we suggest - does not answer all the riddles. This is a film more to admire than to cherish, one that aims to fascinate rather than to satisfy familiar impulses. It's a beautiful object, like a perpetually spinning top, not a living organism."


text: Leif Pedersen wrote: >but seriously, it seems like overkill to have a whole package for (usually >tiny) patches No, you have it backwards. It's overkill to recompile the whole system just for (usually tiny) patches. A package is the small simple solution; I don't know what you mean about a "whole" package; it's just a tarball of the changed binary files. Remember, not everyone compiles the whole system at all, and I'd guess most never compile more of OpenBSD than the kernel. It's simply impractical in many cases (again, my IPX, or an installation with limited disk space). >...and if it's something big, you'll do more work than >installing current. Especially when considering: what about a patch on a >patch (esp in the kernel)? So ya distribute the latest kernel...but then >what about the required userland support? Pretty soon you might as well >upgrade to current. Nobody is suggesting packages for changes to -current. We're talking about tracking -stable here. -current is irrelevant here until it becomes the next release. This would be silly for -current. As for a "patch on a patch", you could simply overwrite the old files with the new ones. Yes, this might take some tracking at the packager end (patch version numbers would be useful), but anyone with experience with other binary patch systems (such as Sun's) should be able to put this together. A first pass would just overwrite files as needed, and expect people to apply all patches in the correct order. If I had the hardware, I'd put it together, but I don't. -- ============= R o b F u n k =============|======> funk+_(_at_)_osu_(_dot_)_edu <======= "A microscope locked in on one point | rfunk_(_at_)_wks_(_dot_)_uts_(_dot_)_ohio-state_(_dot_)_edu Never sees what kind of room that it's in"| rfunk_(_at_)_funknet_(_dot_)_net -- Chris Mars, "Stuck in Rewind" | http://www.funknet.net/rfunk