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Crib/Toddler Bed Transition Question
FTM to Zoey - 10/28/2011
Maybe one of those dealies that can transition? I think our IKEA cribs were $119. They transition to toddler beds when the time comes.
ETA: Looks like all IKEA cribs convert. That is, if you have a store nearby and if you have someone willing to put things together.
Unable to breastfeed, too lazy to circ.
Heather (38)
FTM to Michael born 10-31-2011 and Charlotte born 11-10-2014
Mama to three little boys...Cullen 12.09, Bryce 10.11, and Declan 1.14
***Other Kyla Jet***
Definitely crib.
(And I would just like to say that having your crib stolen would SUCK! Nothing like getting into a good routine with a LO to have it shaken up like that!)
My son hated his crib from day 1.. luckily it was free. He has always slept in the pack n play.. and we plan on keeping him there until its time for a big bed.
Sarah Anne *Wife to an amazing husband*
March 7th-Westley&October 7th-Nolen&May 7th-Braxton
Love all my little men <33
reba, mom to isabel, 5/14/09
...and annie 10/30/11
I agree with a crib
Wife to D. 8/11/05~ FTM to my baby boy C. 01/2010 shortly followed by M. 10/2012


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What all is need to properly set up a 100-150 shot on an 03 Ram
I have a 03 ram with a 214/218 cam, inertia performance heads and an edge pro series stall. I'm wanting a little more now until I can get my PT7675 on. As of right now I'm just tuning with cmr but my new engine will be retrofitted with an 05 harness and then ill be using the hp tuners new software. What all is needed to run a 100-150 shot properly on these trucks? I have the opportunity to buy a bnib nos sniper kit for 150, any luck with these?
as long as it's a wet kit you really only need a colder plug (ngk 4306) and premium gas... no need to adjust the tune unless you really want to...I'm almost identically setup the same way and have been runnin a zex box 150 shot for years...
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subset: commoncrawl


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David Birenbaumc. October 24, 1998 - Present Chairman, Coalition U.S. Fin. Support of U.N.
- Videos: 1 c. September 17, 2004 - Present Senior Analyst, Wilson (Woodrow) International Center for Scholars
- Videos: 0
Mr. Birenbaum discussed headline news and U.S. debts to the United Nations. He responded to audience telephone…


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Tonight's WWE RAW – Rock and Cena Face to Face, Undertaker, More
by Amish Patel - March 25, 2013
– Tonight's WWE RAW takes place from the Wells Fargo Center in Philadelphia, Pennsylvania with WWE Champion The Rock, John Cena, Chris Jericho and others.
There will be no Brock Lesnar or Triple H on tonight's show unless last minute changes were made. The Undertaker is being advertised by the arena but not by WWE. No matches have been announced for tonight but The Shield vs. Cena, Sheamus and Ryback is advertised locally.
WWE is focusing on the following points for tonight's show:
* What will happen when Cena and Rock come face to face?
* Can AJ Lee disrupt Kane and Daniel Bryan?
* Does CM Punk have an unprecedented advantage over The Streak?
* What will Chris Jericho have to say to Fandango?
* Will Jack Swagger feel the wrath of Alberto Del Rio?
The Hottest WWE news, photos and videos delivered to your inbox in one convenient newsletter. Sign up now and give it a try!
|Doink The Clown's Mother Suing WWE For Wrongful Death, Fraud, And Negligence
|WWE Censors Cesaro After He Flipped Off John Cena During Their Match On RAW
|What Main Events Is WWE Planning To Do At WrestleMania 32?
|The WWE 2K16 Video Game Cover To Be Revealed During This Monday's Episode Of RAW
|Vince Russo Posts A New Blog About The Original Plans For Chris Jericho's WWF Debut


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Contemporary Theories of Learning
Learning Theorists … In Their Own WordsIn this definitive collection of today's most influential learning theorists, sixteen world-renowned experts present their understanding of what learning is and how human learning takes place. Professor Knud Illeris has collected chapters that explain both the complex frameworks in which learning takes place and the specific facets of learning, such as the acquisition of learning content,
International Perspectives on Competence Development
Developing Skills and CapabilitiesIn today's complex and ever-changing world it has become obvious that even highly developed knowledge and skills are no longer sufficient to meet new challenges, situations and problems facing individuals, organisations and nations. This raises an enormous and potentially confusing issue for educators and trainers: how is it possible to generate and assess abilities to deal with challenges and
Learning and Non-Learning in School and BeyondHow We Learn deals with the fundamental issues of the processes of learning, critically assessing different types of learning and obstacles to learning. It also considers a broad range of other important questions in relation to learning such as: modern research into learning and brain functions self-perception, motivation and competence development teaching, intelligence and learning style
The Handbook of Transformative Learning
Theory, Research, and PracticeThe Handbook of Transformative Learning provides a comprehensive and critical review of more than three decades of theory development, research, and practice in Transformative Learning (TL). It will help adult educators understand what transformative learning is, distinguish it from other forms of learning, and foster it in their practice. The book covers five broad areas: historical, theoretical,
Understanding the Potential for TransformationThis new book from the award-winning author of Psychology and Adult Learning puts the spotlight on the kind of learning that brings about significant personal change. Tennant explores the techniques, processes, and practices educators can use to promote learning that leads to change and examines assumptions about self and identity, how we are formed, and our capacity for change. Throughout the
Collaborative Practice in Psychology and TherapyCollaborative Practice in Psychology and Therapy provides mainstream academics and practitioners with easy access to cutting-edge thinking in social constructionist psychology and therapy. This unique book is geared to readers who may not be familiar with narrative, social constructionist, or critical psychology and therapy, presenting contemporary theory and practice with a minimum of jargon. The
$50.39This book provides readers with an overview of the implications for research of the theoretical work which acknowledges a debt to the writings of L.S. Vygotsky. A concise introduction to Vygotsky's original thesis and discussions on his approach to research methods is given; this is followed by an exploration of the research practices which have arisen in fields developed on the basis of his
The Future of Educational ChangeThis timely book provides a systematic overview and critique of contemporary approaches to educational change from some of the best-known writers and scholars in the field, including Andy Hargreaves, Larry Cuban, Ivor Goodson, Jeannie Oakes, Milbrey McLaughlin, Judyth Sachs and Ann Liebermann. Divided into four sections, the book addresses the key themes: What has been the impact of
Creating Sustainable Work Systems
Developing Social SustainabilityCurrent trends reveal that increasing intensity at work has major consequences at individual, organizational and societal levels. New organizational approaches to work are needed so the balance between intensive and sustainable work can be achieved, yet there are no guiding models, theories or examples on how this can be done. In exploring the development of sustainable work systems, this book


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Date/Time: Thursday, April 16th - Saturday, April 18th, 12:00 pm – 6:00 pm daily
Place: The Skyline Room at the Tapp's Arts Center, 1644 Main Street, Columbia, SC 29201
General Admission: Free
Indie Bits is a celebration of independent gaming and interactive media designed to foster collaboration between developers and enthusiasts, academics and artists interested in games and gaming technologies. The showcase of games from across the Southeast will be located in the Skyline Room at the TAPP's Art Center – with additional video game installations sprinkled amongst your favorite Indie Grits events as well as various opportunities to get involved on the pixelated front. Additionally, there are plans for a life-sized pacman inspired game called GRITman at our opening party. You really couldn't ask for a more immersive experience (just short of being TRON'd into a computer). The Indie Grits Festival Pass will gain you access to all events at Indie Bits. Stay tuned for more programs and announcements to come!
Why Submit? Indie Bits is the wild, intelligent step-cousin the world renowned Indie Grits Festival has penciled into its family tree. Benefits of acceptance into the Indie Grits family include:
- 2 festival passes
- Access to the private filmmaker lounge (with complimentary food and drink)
- Free transportation to and from the Columbia Metropolitan Airport
- Free access to musical events, late-night parties, and other special programs
- $2,000 worth of awards and prizes
- Guidelines – Desktop, mobile, console, experimental, single-player, multi-player… you name it, we want it. Indie Bits accepts all genres on all platforms, provided the game-maker is from or the work concerns itself with the southeastern United States (South Carolina, North Carolina, Virginia, Georgia, Florida, Alabama, Mississippi, Tennessee, Louisiana, Kentucky, Arkansas, and Texas). If you feel you qualify, download and complete our submission form, found here. March 6th, 2015 is the deadline for submissions–don't delay!


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LOL are you kidding me?
So that's how to get her to keep her kids!
She's a mess and he's a douche!! I'm sure y'all are not shocked by her reaction. Damn girl, have that man of yours buy a damn suite...its time to go to court! Those boys will be asking...shit, I see begging to stay with y'all soon enough.
I understand you have that new baby (should be working on a girl but, oh well lol). Ya better entertain that idea....its a coming.....
You've got to be kidding! My husband probably would have told him that if he intended him to feel something it wouldn't be via a stroller! Lmao- I commend you for keeping your sanity dealing with these grown children.
I especially liked how you stated that that is how you get bm to keep her own damn kids. Ha Ha!!! She's such a jerk off it seems. N
Quoting faerie75:
Party boy is such a god damn pussy.
Seriously? Wtf
I call mine "fruit loop" I tried to think of one for you but I've got nothing :-(
Quoting sunshinedaisy75:
LMAO at the name party boy....hey can you make up a name for the BM I deal with? She is a control freak who needs to control every aspect of everyones life, thinks she is never wrong and is a class A bitch on top of it


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Originally written on Fox Sports | Last updated 4/4/13
Richard Pitino did everything Florida International wanted for the last year. That is, except stick around a while longer. The Panthers' search for their next basketball coach is already under way, with Pitino now at Minnesota after going 18-14 - far better than most anyone expected - in his one season at FIU. Pitino, the son of Louisville coach Rick Pitino, accepted the job offer from the Gophers on Wednesday, and on Thursday, FIU was already in the process of finding his replacement. ''Richard delivered on everything he promised he would,'' FIU director of sports and entertainment Pete Garcia said Thursday. ''He delivered quicker than I think even he himself thought he would. The job he did here was amazing. It has proven to myself, to President (Mark) Rosenberg, to the trustees and the administration, that FIU could be relevant in basketball. And he got us on the map.'' Garcia could not have been more effusive in his praise for Pitino on Thursday, not in any way sounding upse...
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I have Transactions that belong to a Category and can have one Fuelup. The Fuelup belongs to a Car and a Transaction:
class Transaction extends AppModel {
public $belongsTo = array('Category');
public $hasOne = array('Fuelup');
class Fuelup extends AppModel {
public $belongsTo = array('Transaction','Car');
class Car extends AppModel {
public $hasMany = array('FuelUp');
class Category extends AppModel {
public $hasMany = "Transaction";
My tables look as follows:
transactions id|category_id|description
categories id|name
cars id|name|numberplate
fuelups transaction_id|car_id|amount|mileage
In the index-function of the Transactions, I want to index all Transactions and also show, if existent, the Fuelup:
public function index() {
$this->set('transactions', $this->Transaction->find('all'));
How can I access the car's name in this view? I can use $transaction['Fuelup']['car_id'] without problems, but cannot access $transaction['Fuelup']['Car'] or such.
I am running the latest version of CakePHP 2.3.1.


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Leisure Products
Company Overview of Precious Moments Company, Inc.
Company Overview
Precious Moments Company, Inc. engages in the production and distribution of dolls in the United States. The company offers museum and chapel exclusives, figurines, baby and wedding items, doll bulletin boards, and doll tags. It serves customers through dealers, as well as online. The company was formerly known as Precious Moments Country, Inc. and changed its name to Precious Moments Company, Inc. in 1992. Precious Moments Company, Inc. was incorporated in 1984 and is based in Carthage, Missouri.
4105 Chapel Road
Carthage, MO 64836
United States
Founded in 1984
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Sponsored Financial Commentaries
Sponsored Links
To contact Precious Moments Company, Inc., please visit www.pmcdolls.com. Company data is provided by Capital IQ. Please use this form to report any data issues.
Our data partners will research the update request and update the information on this page if necessary. Research and follow-up could take several weeks. If you have questions, you can contact them at firstname.lastname@example.com.


text:
- Home
- Racing & Wagering
- News / Videos / Photos
- Calendar
- Plan Your Visit
- Parking / Maps / Directions
- Entering Churchill Downs
- General Information
- Guest Services
- Hotel Partner
- Group Sales
- Tours
Dubai Majesty Repeats in the Winning Colors at Churchill Downs
The Martin Racing Stable and Dan Morgan's Dubai Majesty swept past favored Hot Dixie Chick on the far turn and cruised to an authoritative 4 ½-length victory over Secret Gypsy in the seventh running of the $110,500 Winning Colors (Grade III) for fillies and mares on Monday at Churchill Downs.
Favored Ailalea Holds Off Tap Tap Tapping to Win 36th Dogwood
LOUISVILLE, KY (Saturday, May 29, 2010) - Starlight Partners' Ailalea assumed command at the head of the stretch and then held off a late bid from Tap Tap Tapping by a length to win the 36th running of the $110,200 Dogwood (GIII) for 3-year-old fillies on Saturday afternoon at Churchill Downs.
Riley Tucker Rallies to Win 22nd Aristides at Churchill Downs
LOUISVILLE, KY (Saturday, May 29, 2010) - Zayat Stables' Riley Tucker, under a ground-saving trip from jockey Shaun Bridgmohan captured the 22nd running of the $107,900 Aristides (GIII) by two lengths over Atta Boy Roy on Saturday at Churchill Downs.
Defending Champion Dubai Majesty Faces 3-Year-Old Hot Dixie Chick, Six Others In Winning Colors
The Martin Racing Stable and Dan Morgan's Dubai Majesty will try to become the first repeat winner of the $100,000-added Winning Colors (GIII) when she faces seven rivals in the featured event of a special holiday racing program on Monday, May 31.
Baffert's Spun Silver wins Ingredient Resource Corporation Classic
John Sikura and Stetson Land and Cattle's Spun Silver surged to the lead in the upper stretch and drew off to a 1 ½-length victory over Beyondallboundarys to take Thursday's featured $49,600 Ingredient Resource Corporation Classic at Churchill Downs.
Churchill Downs Winner Atta Boy Roy Heads Aristides; Eight 3-Year-Old Fillies in Saturday's Dogwood
R.E.V. Racing's Atta Boy Roy, who scored a wire-to-wire victory in the seven-furlong Churchill Downs (GII) on Kentucky Derby Day, will face five rivals Saturday in the 22nd running of the $100,000 Aristides (GIII) for older sprinters.
Free Fighter Upsets Louisville Handicap Field
Thomas Fedro Sr. and Team Block's Free Fighter, ridden by Francisco Torres, surged past dueling leaders Eagle Poise and Telling on the far turn and went on to win the 73rd running of the $113,800 Louisville Handicap (GIII) at Churchill Downs by two lengths over Bearpath.
Veteran Brass Hat Bids for Repeat Victory in $100,000 Louisville Handicap
Fred Bradley's veteran Brass Hat will face 10 rivals Saturday as he attempts to become the seventh horse to score multiple victories in the $100,000-added Louisville Handicap (GIII).
Thiskyhasnolimit Returns to Win Matt Winn
Thiskyhasnolimit, returning from six months on the sidelines because of injury, rallied in the stretch under jockey Shaun Bridgmohan to wear down favored Cool Bullet to win the ninth running of the $108,000 Matt Winn Stakes for 3-year-olds at seven furlongs at Churchill Downs.
Thiskyhasnolimit, Cool Bullet Duel For Hot Stables In Churchill Downs' $100,000-added Matt Winn
The stables that have been the hottest of the opening two of weeks of Churchill Downs' 42-day Spring Meet clash head-on Saturday when 2009 Iroquois Stakes (Grade III) winner Thiskyhasnolimit and Hansel Stakes winner Cool Bullet meet in Saturday's ninth running of the $100,000-added Matt Winn Stakes for 3-year-olds at seven furlongs.


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If you are interested in purchasing Blue Dungeon Tiles or Expansion Sets, you can now do so online at this site on the BLUE DUNGEON TILES page in the header or sidebar.
Petty Gods: Revised and Expanded Edition
Expanded Petty Gods is now out due to all the help of the old-school roleplaying community and the herculean effort by Richard LeBlanc of New Big Dragon Games. If you are not familiar with his stuff, you should definitely check out his website which is full of high quality old-school adventures and supplements.
You can download the Petty Gods pdf for free or get softcover or hardback books via print on demand at Lulu. See more at this link:
I contributed two illustrations to the project: Berenedril and Gilthigoet.
Prepare Encounters with Blue Dungeon Tiles
Click through the gallery below at the bottom to see the following steps:
(if you hover over the gallery image, the text shown below will appear)
B – Use drafting tape, drafting dots, or clear tape to temporarily connect and hinge the tiles.
C – This shows the first and second tile hinged.
D – To make the tavern fold up to a nice stack, hinge the second and third tile on the opposite side. (tiles shown are flipped)
E – Now, flipped back to the original side, you can see that all three tiles hinge to fold up.
F – The new Tavern element can be labelled or other information added and prepared before play.
G – The tiles hinge and can form into a "Z".
H – The "Z" hinged tiles stack and store nicely until needed.
Thundarr the Barbarian – Princess Ariel for 5e Dungeons and Dragons
Ariel, Human, 3rd level Sorceress
Armor Class: 13 Hit Points: 17
Speed: 30′ Proficiency Bonus +2
Saving Throws: Constitution +3 and Charisma +5
Alignment: Lawful Good
Str 10(+0) Dex 14(+2) Con 12(+1) Int 16(+3) Wis 15(+2) Cha 17(+3)
Cantrips (1/turn, 4 known):
• Fire Bolt (1d10)
• Mage Hand
• Ray of Frost (1d8, target's speed -10′ until next turn)
• Shocking Grasp (1d8, target loses action until next turn)
1st Level Spells (4 slots, 3 known ):
• Color Spray
• Sleep (90', 5d8 hit points of creatures for 1 minute)
• Shield (+5 AC until next turn)
Quarterstaff (1d6 bludgeoning or 1d8 with both hands, versatile)
Dagger (1d4 +2 piercing, finesse, thrown range 20/60)
Bracers of Defense +1 to Armor Class
Sorcerer Features:
• Spell Attack Roll +5 (Proficiency +2 + Charisma +3)
• Spell DC 13 (8+ Proficiency +2 + Charisma +3)
• Wild Magic (Random effect on a roll of 1 from a d20 after casting a spell or using Tide of Chaos)
• Font of Magic – 3 Sorcerer Points to create more Spell Slots or power Metamagic features
• Metamagic – Distant Spell for 1 Sorcerer Point will double a spells range or increase a Touch spell to 30'
• Metamagic – Twinned Spell for 1 Sorcerer Point for a Cantrip or 1 per the spell level will enable Ariel to use a spell that has a single target be used on a second target in range.
• Tide of Chaos grants Ariel Advantage on one attack, Ability Check or Saving Throw once until restored after a long rest.
Noble Background:
• Personality Trait: Commoners love Ariel for her kindness and generosity and she feels that despite her noble birth, she is not above others.
• Ideal: Ariel feels that it is her duty to protect and care for people.
• Bond: Ariel wants to be seen as a hero of the people.
• Flaw: Ariel often wears unsuitable footwear which causes her to trip.
• Feature: Because of her noble birth, people are inclined to think the best of Ariel and accommodate her. She can obtain an audience with local nobles if she needs to meet.
Skill Proficiencies:
+2 Acrobatics (Dex)
+2Animal Handling (Wis)
+5 Arcana (Int) – Sorcerer Skill
+0 Athletics (Str)
+3 Deception (Cha)
+5 History (Int) – Noble Skill
+4 Insight (Wis) – Sorcerer Skill
+3 Intimidation (Cha)
+3 Investigation (Int)
+2 Medicine (Wis)
+3 Nature (Int)
+2 Perception (Wis)
+3 Performance (Cha)
+5 Persuasion (Cha) – Noble Skill
+3 Religion (Int)
+2 Sleight of Hand (Dex)
+2 Stealth (Dex)
+2 Survival (Wis)


text:
1971 Cadillac Fleetwood 60 Special Station WagonTo be OFFERED AT AUCTION at Auctions America's California event, July 17-18, 2015. Click here to view this... More
This 1993 Cadillac Fleetwood Brougham Sedan features a 5.7L 8 CYLINDER 8cyl Gasoline engine. It is equipped with a 4 Speed Automatic transmission. The... More
38K Actual Miles / Fully Loaded... More
1954 Cadillac Fleetwood ID# 546048876 This Car was purchased new by the late Maurice Guerry of Castleford,ID in 1954. One year later it was... More
CADILLAC: 1931 V16 all-weather phaeton Fleetwood, 2-tone blue/grey wool/tan top, dsm, wire wheels, exceptionally well restored car in excellent running and driving condition. 800-837-9902,... More


text:
pink and brown knit hood by ArchaicalWatch
©2012-2015 Archaical
knit hood, my first attempt!
Is there a pattern for this online or did you make it yourself? It looks so amazing and I would love to make one or buy one online if you still sell any!
I understand your etsy store is closed, but if you sold a pattern for this on Ravelry, that would be amazing. (: I'm amazed at how simple yet gorgeous these hoods are!
I love the design do you think you could send me a copy of the pattern?
Beautiful! I absolutely love the tied back and the ribbed front. Where can I get this pattern?
can u please send me the pattern u used for this!
Would it be possible to have you send me the pattern/instructions for this? It is beautiful.
Looks great! Would make a House Elf very happy!
Absolutely beautiful. Can I get copy of the pattern please?
Amazing job. I love the design. would it be ok if I use your pattern to make one for myself?
Love these colours! /Download.
You're pregnant in the pic? Congrats on the shorty, hope it's going/went well.
You're pregnant in the pic? Congrats on the shorty, hope it's going/went well.
might i me able to get some instructions on how to make this?? it is amazing! excellent work! <3
Love it! Thank you so so much for the knitting instructions
I Love
May I ask how many yards of yarn you used to make this hood?
roughly 200-250
So amazing, love ur creativity
Nice! Do you sell it?
What is the brand of yarn you use?
loops and threads sometimes, but various ones
Which Loops and Threads? Charisma or Impeccables? (Sorry if I seem nosy, I work in a Michales, that's our brand, and I just was wondering~!)
Very cool~!
Very cool~!
i think impeccabllesss... sometimes i used a ball of chrism
I Love it...Great Job!
so cool! I gotta find a tutorial for this!
I could definitely use one of these for when I shave my head for Cops for Cancer xD It's so rad!
I love it! Did you do any sort of ribbing(may or may not be the wrong term for the 1x1 pattern) or was it just knit
not sure what you mean... stockinette stitch?
Where can I comission this!?!? I would love one before it snowed here!
nice work!


text:
Oil and gas will drive future control of the South China Sea
Kai Ryssdal
Mar 25, 2014 A new book explores the possible disputes over who owns the South China Sea, and its oil reserves.
Outsourcing the NSA's phone-call database
Dan Weissmann
Mar 25, 2014 Under proposals leaked this week, the National Security Agency would stop its blanket collection of phone-call data. Instead, phone companies would keep records, to be provided on court-sanctioned demand.
Diabetes' new frontier: China
Rob Schmitz
Mar 24, 2014 Hundreds of millions of Chinese have moved to the city, leaving behind farmer's diets.
Where to sell Alibaba's treasure?
Sabri Ben-Achour
Mar 17, 2014 China's Alibaba Group says it will list in New York instead of Hong Kong.
A raft of weak economic data out of China
Rob Schmitz
Mar 14, 2014 Industrial output, fixed asset investment, retail sales, you name it: all slipped the first two months of the year, defying expectations. But we've seen this before.
Troubling economic data from China indicates slowdown
Rob Schmitz
Mar 13, 2014 Troubling signs indicate the world's second largest economy isn't recovering
Hot Pot: The meal of China's consumer class
Rob Schmitz
Mar 10, 2014 American fast food chains are doing well catering to China's new consumer class.
Why China is lowering the bar on its growth
Rob Schmitz
Interview with
Mar 5, 2014 China chases 7.5 percent economic growth, less pollution, and Internet banking.
House of Cards is a huge hit in China
Rob Schmitz
Interview with
Feb 28, 2014 China allows a show about U.S. corruption to breach the "Great Firewall."
China's take on Twitter, Sina Weibo, plans Wall Street IPO
Rob Schmitz
Feb 25, 2014 Chinese Internet company Sina Corporation plans to raise $500 million for its initial public offering. But analysts say Sina's timing could be way off.


text:
Indian Actress Prerna Wanvari, who was seen in shows like 'Bandini' and 'Parichay' is excited to have signed a project on the Indian Television with Bollywood filmmaker Anurag Basu's 'Khoka Babu Paribartan' which will be based on an adaptation of Tagore stories. 74 more words
Tags » Bandini
Review: GRT - The Jungle Book: Fall Of The Wild
GFT – The Jungle Book: Fall Of The Wild #4
Writer: Mark L Miller
Art: Michele Bandini
Colors: Grostieta
Letters: Matt Krotzer
Editor: Nicole Glade… 360 more words
A Journey...
I woke up in the wee hours of the morning very distressed. I thought about my ongoing dilemma on how to help my mother be free of her present circumstances, and the flood gates opened! 275 more words
Arya : Ultra Modern Bathroom Faucet from Bandini
Born on the signal of an ideal stability between innovation, form and functinality, the Arya trendy taps symbolize the highest of the manufacturing vary of Bandini. 40 more words
Of the power of a woman
One of the better movies of recent times, Mardaani, that marked the comeback of Rani Mukerji, or should I say Rani Chopra, released recently and managed to do fairly well in terms of box office collection. 434 more words
Methods Hanes Womens 2 Pack Bandini Bra
cheap Nike Air Max 118 sell buy supply Heels Online Sale For Women sell Womens Cheap Nike High Heels Shoes On Sales Black Purple White.Mar 11, 2011 Cheap Womens Nike free run orange gray white for sale, free shipping fee. 308 more words


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12 things to do before going on maternity leave
You've told your manager and work mates that you're pregnant and now you're on a countdown to... more
9 Everyday foods to boost your immune system
Sian Breslin, co-founder of Sian's Plan, has outlined a week's worth of meals that the whole... more
Top 5 Family Staycations
Couldn't face lugging the whole family and the entire contents of your house abroad for a family holiday this year? There are some... more
Love your skin! New Thérapie Clinic opens in Dublin
Is your skin looking for a bit of TLC? Well, Thérapie Clinic, Ireland's leading advanced skin care specialists, have launched a new... more
8 Things that seem crazy until you have a baby
1. The Butt Sniff We've all been there. One minute you are having a nice conversation with your life-long friend. The next minute she... more
5 Top Tips for Relieving Dry Skin
1. Reduce time spent in the bath Lengthy baths can dry out your child's skin. By spending too much time in the bath, you are not only... more
15 Tips to make work travel less stressful
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Protect your family from household chemicals
Whether we realise it or not, most of us will use chemicals at home every single day. They're just an everyday part of life. Cleaning... more
8 beauty products you need to have this summer
Summer make up is all about minimalism, but achieving the 'no make up look' isn't as easy as it looks. And of course we all want to spend... more
Let's get cooking
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Don Walker offers news, notes and perspective on the sports business landscape
Favre's Pack, Jets jerseys still selling
National Football League officials reported Monday that NFLShop.com, the online merchandising arm of the league, sold 12,000 Brett Favre Jets jerseys from last Thursday through Sunday. That's a record for a four-day period.
Favre's jersey is now the No. 1 seller on NFLshop.com since April 1.
But Favre's Packers jersey is still selling, too, a league official said. Favre's Packers jersey, which was No. 1 since April 1 until Thursday, is now the second most sold jersey.
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Our Programs
Adam's Camp offers a variety of therapeutic and adventure programs for special needs infants through young adults and their families. All activities and types of therapies focus on each child's specific strengths and assets, providing coordinated, outcome-based treatment.
Register for Camp
Are you ready to have the time of your life while watching your child grow, thrive, gain independence, and succeed? Register for our Therapy and Adventure camps.
Work/Volunteer at Camp
Working at a camp provides a unique opportunity for volunteers, counselors, therapists and other professionals interested in meaningful, challenging work.
Adam's Camp Blog Snapshot
- Appreciating the moments – June 25, 2015 - "Woah-ho, come take my hand, we're riding out tonight to case the promise land." – "Thunder Road" by Bruce Springsteen There are things in this life you just do not understand until you experience them first hand. You can read about them, talk about them, dream about them, but until you live them, you just
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- Standing a little taller – June 24, 2015 - "What doesn't kill you makes you stronger, stand a little taller." – "Stronger" by Kelly Clarkson It is not exactly a secret that it is a challenge two work with the Adam's Camp population and that it takes incredible skill, enthusiasm, experience, patience and empathy to manage situations that can arise in the blink of an
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Adam's Camp Highlights
Male Volunteers Needed
Guys, are you ready to work hard AND have the best time of your lives? Due to cancellations, we have 2 openings for male volunteers, ages 16 and up for Adventure Camp, July 5-10 and a few later in the summer too.
Contact Helga today! email@example.com


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TBJ poll: NCSU does more than Duke, UNC for Triangle economy
North Carolina State University contributes more to the Triangle economy than UNC-Chapel Hill, Duke or any of the area's other institutions of higher learning, voters in TBJ latest online poll say.
NCSU captured 50 percent of the 1,424 ballots cast in the unscientific survey, which was available on trianglebusinessjournal.com from July 14 through July 20. Visitors to the Web site and recipients of TBJ's Daily E-mail Update were asked: "Which Triangle university do you think contributes the most to the Triangle economy?"
UNC-Chapel Hill, rated as one of the top public universities in the United States, finished a distant second to NCSU with 28 percent of the vote. Duke, rated among the top 10 U.S. universities, took third place in the Triangle with 15 percent of the vote.
Bizspace Spotlight
Wake Technical Community College followed at 4 percent, while N.C. Central, Meredith College and other each tallied about 1 percent of the ballots cast.
• Click here to vote in this week's poll: "Is the Wake County Schools diversity-policy controversy hurting the area's reputation?
Reporter e-mail: email@example.com


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Company Description
Founded in 1984, Enersol is a non-profit charitable organization developing and introducing sustainable energy solutions for rural communities. By supporting the use of clean, renewable solar energy for health and education applications in Latin America, we are committed to demonstrating that it's possible to improve the quality of life in rural areas of developing nations around the world.
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Top 10 Houses on Dwell This Week
As the weekend slowly draws to a near, we've gathered up ten of our most clicked on homes on Dwell this week for your perusal.
November 14, 2013
The Dwell Guide to Kitchen Design
Is your kitchen too drab? Feeling the pinch of cramped cooking quarters? Appliances dating from the stone age? We've got you covered. To coincide with our Rooms We Love special issue now on newsstands, we polled a handful of our favorite interior designers for expert tips and advice on how to solve common kitchen dilemmas and whip your space into tip-top shape. (Perfecting that Chicken au Poivre recipe, well that's up to you.)
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Ahhh the living room, or should we say the everything room. It's your respite from the world, the place you kick your shoes off and curl up on the couch after a long day, sometimes dinging room, often entertainment room, and even guest room in a pinch. We all want our living rooms to reflect the best of our personal style, after all they are the rooms we really live in, but sometimes we need a little inspiration from others. Here, we've gathered seven of our current favorites to give you some great ideas for your own space.
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Confronted with an historic loft in New York's SoHo district in need of an update, architects Bronwyn Breitner and Luigi Ciaccia executed a gut renovation to introduce more natural light throughout the interior while honoring the original character of the former industrial space. Though the loft was close to 2,300 square feet, the long and narrow footprint—23 feet by 100 feet—was a challenge. "By punching holes in walls and aligning site lines with the windows, we were able to create privacy to meet the clients' needs while also providing a sense of light and expansion from the common areas," says Breitner, co-founder of the firm 590BC. We detail the design moves in the following slideshow.
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Kansas City, Missouri, is the home of Make It Right's latest community revitalization project. Though the neighborhood of Manheim Park hasn't been affected by a natural disaster, it's been afflicted with decades worth of decline. Bancroft Elementary, a formerly abandoned school dating from 1904, is the projects locus. Make It Right and local firm BNIM transformed the vacant building into low-income housing for families, veterans, senior citizens, and youths transitioning out of foster care. Architect Tim Duggan, director of Make It Right's Innovations department, spearheaded the effort. We chatted with him about the project, "urban acupuncture," and how this redevelopment plan serves as a model for other cities. "We see this as a model both locally and nationally to identify a catalytic project and revitalize it," Duggan says. "So often conventional development says, it won't pencil out or it won't work unless there are inferior quality materials, or it'll pencil out with less community space or less attention to quality. We didn't want to do that." See what else he has to say in the following slideshow.
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Winter Inspirations: Icy Blues and Snowy Whites
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November 12, 2013
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January 5, 2014
| Article
The sixth Nail Tech Event of the Smokies took place August 25-26 in Gatlinburg, Tenn., with 225 nail techs in attendance and 30 nail company booths.
Quarter Dinh's friendly personality, singing voice, and unique business model has created quite a stir in the Detroit area where he is known as Quarter The Singing Manicurist.
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In a special guest column, salon and school owner Tina Caton talks about training nail techs to be the best they can be.
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Building a Bond: "I grew up with horses. When I was young I'd ride and work with them whenever I got the chance. I'd watch them for hours, studying their body language and their interactions with each other and with me," says nail tech and horse trainer Ruthy Carter.
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You Might Also Like: Gelish NailStyle App Available for iPhone and iPad
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Have you been using Twitter as a way to promote your business? Whether you use 140 characters or less to share appointment openings, Instagram photos, or retweet your favorite clients, this social network is a must for the modern day nail artist.
It's hard to ignore the presence of bloggers in the nail industry. They have taken the world of beauty by storm and some blogs are even a part of a daily diet of reading. These women post reviews, paint swatches, and create exquisite art that keeps their followers coming back for more. But I bet you didn't know that a good number of these nail enthusiasts are actually professionals, and that many of them made the decision to go to nail school after establishing their blogs. Learn how a tiny corner of the Internet encouraged these nail lovers to become licensed nail techs.
November 30, 2013
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A former corporate professional, VikkiAnn Albanese imagined a tranquil lifestyle as the owner of a more natural nail salon in New Jersey. Though she opened the salon without a single client in a seasonal area during a rough economy, Albanese thrived, eventually relocating and expanding the salon.
November 21, 2013
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Gelish U.S. Dean of Education MaeLing Parrish utilized her background in art to establish herself as a nail educator in the U.S. Find out what makes her tick in the one-on-one interview below.
November 21, 2013
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Coty Inc. announced today that OPI founder and CEO George Schaeffer is retiring, but he will continue to work with OPI's management team through a newly created role as OPI's strategic board advisor. He'll support long-standing artistic director Suzi Weiss-Fischmann and newly appointed GM Mary van Praag.
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November 1, 2013
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The present invention is directed to a liposomal preparation which is based on specific lipid components. The liposomal compounds are also combined with nucleic acids, forming nucleic acid-liposomal compounds. These compounds are useful in drug delivery, where specific therapeutic nucleic acids are used in the liposomes. The specific lipid components of the present invention provide a highly efficient and stable delivery system for nucleic acids. Consequently, the liposomal preparations are suitable for use in gene therapy.
1. A liposome composition comprising a cationic lipopolyamine and a neutral lipid, wherein the cationic lipopolyamine comprises spermine-5-carboxy-glycinedioctadecylamide.
2. A liposome composition according to claim 1 wherein the neutral lipid comprises dioleylphosphatidyl ethanolamine or phosphatidylethanolamine.
3. A liposome composition of claim 1 wherein the neutral lipid is dioleylphosphatidyl ethanolamine.
4. A liposome composition of claim 1 wherein the neutral lipid is phosphatidyl ethanolamine.
5. A nucleic acid-liposome composition comprising the liposome according to claim 1 and a nucleic acid.
6. A nucleic acid-liposome composition comprising the liposome according to claim 2 and a nucleic acid.
7. A nucleic acid-liposome composition comprising the liposome according to claim 3 and a nucleic acid.
8. A nucleic acid-liposome composition comprising the liposome according to claim 4 and a nucleic acid.
9. A nucleic acid-liposome composition according to claim 5 wherein the nucleic acid comprises an antisense oligonucleotide.
10. A nucleic acid-liposome composition according to claim 5 wherein the nucleic acid comprises a plasmid DNA.
11. A liposome composition according to claim 1 prepared by a method comprising:
a-mixing a cationic lipopolyamine and a neutral lipid forming a mixture;
b-evaporating the mixture to dryness, forming a dried lipid film;
c-rehydrating the dried lipid film forming the liposome.
12. A liposome composition according to claim 11 wherein the method of preparation further comprises: adding a nucleic acid, after rehydrating the dried lipid film.
13. A liposome composition according to claim 11 wherein the method of preparation further comprises: adding a nucleic acid, upon rehydration.
14. A pharmaceutical composition comprising a nucleic acid-liposome composition according to claim 5 and a suitable carrier.
15. A pharmaceutical composition comprising a nucleic acid-liposome composition according to claim 6 and a suitable carrier.
16. A pharmaceutical composition comprising a nucleic acid-liposome composition according to claim 7 and a suitable carrier.
17. A pharmaceutical composition comprising a nucleic acid-liposome composition according to claim 8 and a suitable carrier.
18. A method of introducing a nucleic acid into cells of a subject comprising administrating to the subject an effective amount of a nucleic acid liposome composition according to claim 5.
19. A nucleic acid-liposome composition comprising the nucleic acid-liposome of claim 5 and adenovirus particles.
20. A nucleic acid-liposome composition comprising the nucleic acid-liposome of claim 8 and adenovirus particles.
21. A liposome composition according to claim 12, the method of preparation further comprising the step of:
mixing the liposome with adenovirus particles.
22. A liposome composition according to claim 13, the method of preparation further comprising the step of:
mixing the liposome with adenovirus particles prior to the addition of the nucleic acid.
The present invention is directed to a liposomal preparation which is based on a composition of nucleic acids or analogues and specific lipids which form liposomes. It is also an object of the present invention to provide a method for preparing a liposomal nucleic acid composition which is simple and very efficient.
As advances continue to be made in the molecular biology of inherited or acquired diseases, modulation or modification of the genetic program of living cells is looked upon with growing interest as a new therapeutic approach. Two different strategies have emerged: Gene therapy and oligonucleotide-based therapeutics. To be successful these two approaches must be mediated by an efficient "in vivo" transfer of the nucleic acid material to the target cells and there is a need to provide an efficient and safe delivery system of nucleic materials.
Gene therapy involves the transfer of normal, functional genetic material into cells to correct an abnormality due to a defective or deficient gene product. Typically, the genetic material to be transferred should at least contain the gene to be transferred together with a promoter to control the expression of the new gene. In general, two gene transfer strategies are considered: Viral (retrovirus or adenovirus) vectors and synthetic gene-transfer vectors.
Viral agents have been demonstrated to be highly efficient vectors for the transfection of somatic cells. Retroviruses in particular have received a great deal of attention because they not only enter cells efficiently, but also provide a mechanism for stable integration into the host genome through the provirus. However, clinical use of retroviral vector is hampered by safety issues. A first concern is the possibility of generating an infectious wild type virus following a recombination event. A second concern is the consequences of the random integration of the viral sequence into the genome of the target cell which may lead to tumorigenic event. In addition, as retroviruses would only complete their life cycle in dividing cells, a retroviral vector would be inefficient in targeting cells which are not dividing. DNA viruses such as adenoviruses are potential gene carriers but this strategy is limited in the size of the foreign DNA adenoviruses can carry and because of the restricted host range. However, the advantage of adenoviruses over retroviral vectors is their ability to infect post-mitotic cells.
Synthetic gene-transfer vectors have been subject to intense investigation since this strategy appears to be clinically safe. Potential methods of gene delivery that could be employed include DNA/protein complexes (1) or liposomes (2-7). The genetic material to be delivered to target cells by these methods are plasmids. Plasmids are recombinant DNA which are circular forms of double stranded DNA. They are constructed so that they have, at the minimum, a promoter and the gene coding for the protein of interest. Plasmids can be expressed in the nucleus of the transfected cells in a transient manner. In rare events, the plasmids may be integrated or partly integrated in the cell host genome and might therefore be stably expressed. Episomal plasmid vectors are plasmids able to replicate in the nucleus of the transfected cells and may therefore be expressed in a total growing cell population. Plasmids may have a promising potential considering the fact that they may be applied in combination with a synthetic vector as carrier and that gene therapy by this means may be safe, durable, and used as drug-like therapy.
Plasmid preparation is simple, quick, safe, and inexpensive representing important advantages over retroviral vector strategy. The successful use of this genetic tool for "in vivo" approaches to gene therapy will rely on the development of an efficient cell delivery system.
Liposomes have been shown to be efficient vehicles for many in vitro and in vivo applications. Liposome encapsulated DNA have been used in vitro (3,5) and in vivo (2,4,6,7) for the expression of a given gene through the use of plasmid vectors. The term "liposomes" describes a closed structure composed of lipid bilayers surrounding an internal aqueous space. Liposomes may be used to package DNA for delivery to cells, even in the case of plasmids of large size which could potentially be maintained in a soluble form that would allow direct application to in vivo systems by a simple intravenous injection. Formation of complexes of DNA with cationic liposomes has recently been the focus of research of many laboratories. In particular, lipofectin (Gibco BRL, Gaithersburg, Md.) has been successfully used for the transfection of various cell lines in vitro (3) and for systemic gene expression after intravenous delivery into adult mice (6).
Lipofectin is formed with the cationic lipid DOTMA, N 1-(2,3-dioleyloxy) propyl!-N,N,N-trimethyl-ammonium chloride, and DOPE, dioleylphosphatidyl ethanolamine at a 1:1 molar ratio. The liposomes prepared with this formulation spontaneously interact with DNA through the electrostatic interaction of the negative charges of the nucleic acids and the positive charges at the surface of the cationic liposomes. This DNA/liposomal complex fuses with tissue culture cells and facilitates the delivery of functional DNA into the cells (3). New cationic liposomes have been developed: Lipofectamine (Gibco BRL), composed of DOSPA, 2,3-dioleyloxy-N 2(sperminecarboxamido)ethyl!-N,N-dimethyl-1-propanaminium trifluoracetate and DOPE at a 1:1 molar ratio. Lipofectace (GIBCO BRL) composed of DDAB, dimethyidioctadecylammonium chloride and DOPE at a 1:1 molar ratio. DOTAP (Boehringer Mannheim, Ind.) 1-2-dioleoyloxy-3 (trimethyl ammonia) propane.
Behr et al. (9,10) have recently reported the use of a lipopolyamine (DOGS, Spermine-5-carboxy-glycinediotadecylamide) to transfer DNA to cultured cells. Lipopolyamines are synthesized from a natural polyamine spermine chemically linked to a lipid. For example, DOGS is made from spermine and dioctadecylamidoglycine (9). DOGS spontaneously condence DNA on a cationic lipid layer and result in the formation of nucleolipidic particles. This lipospermine-coated DNA shows high transfection efficiency (10).
It is an object of the present invention to provide an efficient and safe delivery system for nucleic acid materials.
The present invention relates to a nucleic acid-liposome preparation, useful in delivery of nucleic acid-based materials to cells. The present invention further relates to a method of preparing the nucleic acid-liposome preparations. In addition, the present invention relates to the administration of the nucleic acid-liposome preparations to cells. The invention further relates to administration of the nucleic acid liposome preparations to patients as a therapeutic agent.
The liposome compositions of the present invention provide highly efficient delivery of nucleic acids to cells. Liposome vesicles are prepared from a mixture of a cationic lipopolyamine and a neutral lipid. Nucleic acids are associated with the liposomes in two ways: (1)complex formation between the cationic liposome vesicle and negatively charged nucleic acid or (2) partial encapsulation and partial complex formation in and with the cationic liposome vesicle. A preferred embodiment of the present invention uses a spermine-5-carboxy-glycinedioctadecylamide (referred to herein as "DOGS") as the cationic lipopolyamine and dioleylphosphatidyl ethanolamine (referred to herein as "DOPE") as the neutral lipid.
Another embodiment of the present invention is directed to a combination of a DOGS/DOPE liposome preparation (referred to herein as "DLS-liposomes") anchored through hydrophobic interactions with an adenovirus particle. Since adenoviruses enter cells via receptor-mediated endocytosis, the combination of adenovirus particles and the DLS-liposomes produces an enhanced transduction efficiency.
One embodiment of the present invention is directed to a liposomal preparation which is based on a composition of nucleic acids or analogues and DOGS containing liposomes. The liposomes of the present invention efficaciously deliver nucleic acids into the cytoplasmic compartment of human cells. Use of such liposomal vehicles, and use of plasmid DNA as a carrier of a gene of interest, make possible high expression of such gene in cells or in a patient.
The present invention also encompasses a method of preparing such a composition. The presence of neutral lipid, such as DOPE or phosphatidylethanolamine in combination with a lipopolyamine such as DOGS makes possible the formation of liposomes after hydration, whereas use of lipopolyamine alone merely leads to the formation of lipid particles.
The liposomal preparations are further embodied in the present invention as a method for drug delivery suitable for use in gene therapy. Therefore the present invention is further directed to a method of treating a subject with a suitable formulation of nucleic acid-liposomes in order to deliver specific nucleic acids to target cells of the subject. Such a method of treating subjects provides effective delivery of oligonucleotides or gene-expressing plasmids into cells. Therefore, such a method of drug delivery is useful for the transport of nucleic acid based therapeutics.
The present invention provides a pharmaceutical liposomal formulation for the delivery of nucleic acids useful systemic administration and provides long-term expression of a given gene in dividing cells.
FIG. 1: β-Galactosidase expression in HeLa cells transfected with DOGS/DOPE liposomes (referred to herein as "DLS" liposomes).
FIG. 2: Comparative study of the transfection efficiency of DLS and other liposomal delivery systems. Luciferase activity was assayed in HeLa cells transfected with pRSV luc plasmid in presence or in absence of serum (15%) T: DOGS or Transfectam (Promega, Madison, Wis.); LF: Lipofectin (Gibco BRL, Gaithersburg, Md.); LFA: Lipofectamine (Gibco BRL).
FIG. 3: Effect of preincubation in culture medium on DLS transfection efficiency in HeLa cells. Luciferase activity was ascertained following transfection of liposomal DNA pre-incubated in serum containing medium (15%).
FIG. 4: Transfection efficiency in HeLa cells with liposomal DNA. Luciferase activity was determined.
FIG. 5: Relative transfection efficiency of DLS-1 and DLS-2 in HeLa cells using pRSV luc.
FIG. 6: Intracellular localization of FITC-end labeled oligodeoxynucleotides (20 mers)) in HeLa cells following DLS treatment. Cells were exposed to 2 μM FITC-labeled oligodeoxynucleotides for 24 hr (A, B) and the post-incubated in drug-free medium for 24 hr (C)--Cells were treated with 2 μM free FITC-labeled oligodeoxynucleotides for 24 hr (D). Photographs represent computer-enhanced images from laser-assisted confocal microscopy. Magnification, �320.
The present invention relates to the discovery that nucleic acids may be associated with liposomes of a specific composition in a very effective manner. The liposome-associated nucleic acids may then be injected into a mammalian host to effectively express a given gene through the use of plasmid DNA, or to inhibit expression of a cellular gene through the use of antisense oligonucleotides. The efficiency of a liposome-mediated nucleic acid drug delivery system is directly dependent upon the liposome composition and its resulting association with cellular membranes.
Nucleic acids-based therapeutics may be of a broad use in therapy of a wide variety of diseases and disorders, such as, inherited or acquired genetic disease or viral infections.
The present invention may utilize one or more nucleic acids in conjunction with the liposomal carrier.
The term "nucleic acids" means any double strand or single strand deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) of variable length. Nucleic acid analogs such as phosphorothioates, phosphoramidates, phosphonates analogs may be associated to the liposome composition in accordance with this invention. In one embodiment of the present invention, modified phosphorothioate oligonucleotides (alternatively referred to as "oligo(dN)") are used and demonstrate increased nuclease protection and increased cellular transport.
The liposomes of the present invention are prepared by drying a lipid mixture containing a lipopolyamine and then by hydrating the subsequent dry lipid film. Two methods of associating nucleic acids with liposomes are described in this invention. The first method comprises hydrating a dried lipid film by introducing an aqueous solution, and completely dispersing it by strongly homogenizing the mixture with a vortex, magnetic stirrer and/or sonication. Subsequent liposomes are mixed with a nucleic acid solution allowing complex formation between positive charges of the lipopolyamine-containing liposomes and the negative charges of the nucleic acids. The second method comprises hydrating the dried lipid film using a low and defined (5-10 μl/μg lipids) volume of aqueous solution containing concentrated nucleic acids. Dispersion is completed by strongly homogenizing the mixture using a vortex or magnetic stirrer. Nucleic acids are partly encapsulated in the liposomes during the formation and also are partly complexed through electrostatic interaction between the nucleic acid and the cationic liposomes.
The lipid mixture of the present invention must contain a lipopolyamine compound. Lipopolyamine, such as DOGS, condenses plasmid DNA and coats it with a cationic layer following simple mixing. Other lipopolyamines useful in the present invention include polyamines, spermidine and spermine. In order to form stable liposomes, the lipopolyamine is added to phosphatidylethanolamine (PE) or a derivative of PE such as DOPE (dioleoyl-phosphatidylethanolamine). For optimal transfection efficiency a DOGS/DOPE molar ratio of 0.5:1 is used. However, liposomes may be formed using a 0.1 to 5 molar ratio and showing significant nucleic acids delivery.
Such liposomes are mainly endocytosed by the cell and are consequently concentrated in intra-cytoplasmic vesicles. These endocytic vesicles release liposome content depending on the membrane fusogenic property of the liposomes and the nature of the liposome content. As drug therapy principally relies on the drug's ability to access the biological target, cytoplasmic delivery of nucleic acids transported through liposomes is critical. Enhancement of cytoplasmic delivery of liposome-associated compounds may be achieved using PE and especially the derivative DOPE in the liposome's composition.
Nucleic acids delivered using the carrier system of the present invention are efficiently released from endocytic vesicles and as a result a high cytoplasmic and nuclear distribution of nucleic acids is achieved.
The presence of DOPE or PE in combination with a lipopolyamine such as DOGS makes possible the formation of liposomes after hydration, whereas use of lipopolyamine only leads to the formation of lipid particles. Formation of phospholipidic bilayer membrane (liposomes) may allow for a enhanced blood circulation, stability and effectiveness of transduction. In addition, the liposomal membrane facilitates anchorage to its surface of other substituents, which can increase gene transfer and allow cell targeting, such as viral particles, virus fusogenic peptides or antibodies.
Liposomal delivery in accordance with the present invention may be used for increasing recombinant retrovirus infection. Retrovirus entry into cells is mediated via ligand-receptor recognition, and consequently their uptake is very low in certain cells which do not present those receptors. Associating a retrovirus or other recombinant virus to be used for gene therapy to liposomes may enhance penetration and/or expression of the viral agents.
The liposomal delivery system of the present invention makes possible high transduction efficiency in numerous cells such as human adenocarcinoma, HeLa, murine carcinoma, NIH3T3, human embryonic kidney 293, human leukemia MOLT-3 cell lines, and primary cultures of human macrophages and human vascular endothelial cells.
The two methods of forming the liposomes of the present invention lead to liposome-complexed and liposome-encapsulated nucleic acids, respectively. Liposome-encapsulated nucleic acids have been shown to be more efficient in transducing cells in cell cultures. However, the ability to sonicate the lipid vesicles in the liposome-complexed nucleic acids allow for more homogenized and smaller liposome particles, and consequently for the ability to circulate for longer periods in blood following systemic injection.
The liposomal composition of the present invention has shown to be stable in a biological environment, demonstrating that nucleic acids associated with the liposomal carrier are completely protected from enzymatic attack such as nucleases, and that stability in circulating blood after administration may be achieved.
The liposomal composition of the present invention may be systematically administered into patients parenterally in order to achieve gene therapy or oligonucleotide delivery. Moreover, this technique may be used for "ex vivo" gene therapy where tissue or cells are removed from patients, then treated and finally reimplanted in the patient.
Many diseases can be treated via the drug delivery system of the present invention. Diseases such as diabetes, atherosclerosis, and chemotherapy-induced multi-drug resistance can be treated using the present drug delivery system. One particular condition which can be treated via the system of the present invention relates to HIV and HIV-related diseases, such as anemia, leukopenia and thrombocytopenia. These clinical conditions are significantly related to a decrease or disappearance of hematopoietic progenitor cells in bone marrow of HIV-1 patients. Transfection of bone marrow stem cells, bone marrow stroma cells and embryonic stem with gene coding for immuno-restoring compounds might enhance the differentiation and proliferation capacity of such cells.
This delivery system can be useful for correcting the ion transport defect in cystic fibrosis patients by inserting the human CFTR (cystic fibrosis transmembrane conductance regulator) gene. Oral administration such as nebulization could particularly suitable. In addition, DLS-liposomes can be used for the inhibition of tumor cells by administering in tumor cells a gene coding for a molecule inhibiting tumorigenesis such as antisense oligonucleotides directed to angiogenic factors. Intra-lesional or intravenous administration appear suitable for this case.
The DLS-liposomes containing the nucleic acid drug can be administered by intravenous, intramuscular, intraperitoneal, subcutaneous intra-lesional and oral means.
A proposed daily dosage of active compound for the treatment of man is 0.5 mg DNA/kg to 4 mg DNA/kg, which may be conveniently administered in one or two doses. The precise dose employed will of course depend on the age and conditions of the patient and on the route of administration. Thus a suitable dose for administration by inhalation is 0.5 mg DNA/kg to 2 mg DNA/kg, for oral administration is 2 mg DNA/kg to 5 mg DNA/kg, for parenteral administration is 2 mg DNA/kg to 4 mg DNA/kg.
The compound of the invention may be formulated for parenteral administration by bolus injection or continuous infusion. Formulation for injection may be presented in unit dosage form in ampoules, or in multi-dose containers with an added preservative. The compositions may take such forms as suspension, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.
The compounds according to the invention may be formulated for administration in any convenient way. The invention therefore includes within its scope pharmaceutical compositions comprising at least one liposomal compound formulated for use in human or veterinary medicine. Such compositions may be presented for use with physiologically acceptable carriers or excipients, optionally with supplementary medicinal agents. Conventional carriers can also be used with the present invention.
For oral administration, the pharmaceutical composition may take the form of, for example, tablets, capsules, powders, solutions, syrups or suspensions prepared by conventional means with acceptable excipients.
The following examples serve to illustrate further the present invention and are not to be construed as limiting its scope in any way.
Liposomes are formed by mixing 1 mg DOGS and 1 mg DOPE (0.5:1 molar ratio). After thorough stirring, the mixture is evaporated to dryness in a round bottomed borosilicate tube using a rotary evaporator. The subsequent dried lipid film is resuspended in a low volume of ethanol (10 to 40 pl/mg lipid). Formation of liposomes is carried out by adding an excess of distillated water (at least 200 μl/mg lipid). After homogenization by slight vortexing, the mixture is incubated for at least 15 min. If needed, the resulting suspension may be sonicated in a fixed temperature bath at 25 C for 15 min.
Complex formation of nucleic acids to the liposome bilayer membrane is achieved by simply mixing the preformed DOGS/DOPE liposomes (DLS-liposomes) to a solution of nucleic acids. In an Eppendorf tube, DLS-liposomes are mixed in a 150 mM NaCl solution to nucleic acids at a concentration of 12.5 μg total lipids (liposomes)/μg nucleic acid for double strand DNA, and a concentration of 6 μg liposomes/1 μg nucleic acid for oligonucleotides. The mixture is slightly mixed and incubated for at least 30 min at room temperature. Complex formation is very effective and nearly complete since at least 80% of nucleic acids were assimilated into the liposomes.
Liposomes were formed by mixing 1 mg DOGS and 1 mg DOPE (0.5:1, molar ratio). After thorough stirring, the mixture is evaporated to dryness in a round bottomed borosilicate tube using a rotary evaporator. The subsequent dried lipid film is resuspended in a minimal volume (7 μl/mg lipid) of water solution containing nucleic acids (1400 μg/ml). Formation of liposomes is carried out by thorough stirring. The subsequent liposome preparation may be diluted in 150 mM NaCl. Entrapment and/or assimilation of nucleic acid by the liposomes is very efficient and nearly complete since at least 80% of nucleic acids are assimilated by the liposomes.
Gene transfection efficacy was ascertained in vitro using reporter genes such as genes coding for p-galactosidase or luciferase. Two plasmid constructs containing the CMV (Clonetech) and RSV (Promega) promoters were used as genetic vectors for the β-galactosidase and luciferase genes, respectively. These plasmid vectors were delivered to the human carcinoma HeLa cells via the liposome carrier system in accordance with this invention.
One microgram liposomal DNA was added to the medium of a HeLa cell culture at a 50-70% confluency (500,000-700,000 cells/ml culture medium/7 cm2 culture plate surface area). Cells were incubated at 37� C. with the liposomal DNA for at least 4 hr. Determination of gene expression was carried out for both types of plasmids following an incubation of 2-3 days at 37� C.
β-galactosidase activity was observed using a staining procedure after cell fixation on the culture plate using a conventional method. Cells expressing the β-galactosidase were readily identified by their intense blue staining. As shown in FIG. 1, more than 60% of the HeLa cells treated by DOGS/DOPE liposome-associated DNA, actively expressed β-galactosidase.
Luciferase activity was detected in HeLa cells using a standard method (Promega, Madison, Wis.). Luciferase gene containing plasmid was used for a comparative study of the transduction efficiency of the liposomal delivery in accordance with this invention and liposomal vectors commercially available. This study was carried out using the optimal experimental conditions for each tested method.
In serum-containing cell culture medium transduction efficiency in HeLa cells treated with the liposomal system in accordance with this invention appears to be 11-fold, 10-fold and 37-fold higher than that of DOGS or Transfectam (Promega, Madison, Wis.), Lipofectin and Lipofectamine (GIBCO BRL, Gaithersburg). (FIG. 2)
In serum-free medium which is not corresponding to a biological environment, transduction efficiency using DOGS/DOPE liposomes appears equivalent to that determined when cells are incubated in serum-containing medium. In contract, use of Lipofectamine in serum-free conditions make possible a high transfection efficiency. The dramatic decrease in transduction efficiency (186-fold) using Lipofectamine in a medium containing Fetal Bovine Serum (10%) underlines the high instability of DNA when exposed to nucleases, and the need for complete DNA protection toward enzymatic attack in a biological environment. DOGS/DOPE liposomes prepared in accordance with this invention exhibit an effective cell delivery and an efficient DNA protection during transport.
As illustrated in FIG. 3, preincubation of liposomal DNA in serum containing medium up to 48 hours does not decrease transfection efficiency. In contrast, a significant increase was observed, suggesting the presence in the serum of a factor helping gene transfer.
FIG. 4 shows optimal transfection efficiency by using a 12:1 liposome/DNA weight ratio for the preparation of the liposomal DNA.
As illustrated in FIG. 5, use of plasmid DNA delivered by DLS-2 showed better transduction efficiency in HeLa cells when compared to the DLS-1 delivery. This may be due to increase in DNA protection toward nuclease attack and/or better release from endocytic vesicles when encapsulated in liposomes.
Oligonucleotides may be complexed or encapsulated in DLS-liposomes using the methodological procedure described in examples 2 and 3, respectively. The only modification is that a two-fold higher nucleic acid/liposome ratio may be used for DLS-1 (20 μg/60 μg, weight ratio) at equivalent encapsulation efficiency. This leads to a higher concentration of oligonucleotides in the liposome preparation and consequently a higher efficiency of delivery in terms of quantity of nucleic acids delivered per cells.
Nucleic acid cell penetration and its intracellular distribution following delivery using DLS-liposomes were observed using laser-assisted confocal microscopy and FITC-labeled oligodeoxynucleotides (20 mers). The former technique allows for the high resolution of optical sections of suspension cell preparations and can readily specify the intracellular distribution of a fluorescent compound. FIG. 6 presents images of hepatocyte HepG2 cells treated with DLS-1 encapsulated oligodeoxynucleotides for 24 hrs.
We observed a high penetration of the labeled oligodeoxynucleotides in all intracellular compartments (FIG. 6A). In order to investigate where oligodeoxynucleotides are highly concentrated, we significantly reduce the gain of the laser beam used for confocal microscopy observation of the same cell, and we observed a punctuated intra-cytoplasmic distribution of the oligonucleotides (FIG. 6B). This suggests that DLS transports oligonucleotides into cells via endocytosis and then oligonucleotides quickly escape from endocytic vesicles leading to a release of free oligonucleotides in the cytoplasm. Oligonucleotides are immediately transported from the cytoplasm to the nucleus (FIG. 6C). An extremely weak fluorescence intensity was observed in cells incubated with free labeled oligonucleotides, suggesting poor penetration and/or degradation by nucleases present in the serum-containing culture medium (FIG. 6D).
This observation is of great interest since it shows efficient delivery of nucleic acids to cells, total and immediate escape from endocytic vesicles where active degradation could take place, and nuclear localization after cell treatment. Theoretically, cell delivery of plasmid-DNA via DLS may use the same pathway of cell internalization.
Formation of liposomes make possible anchorage to the membrane bilayer of products which may increase transduction efficiency. Viruses, in general, are inherently excellent gene transfer vectors. Viral capsids or envelopes exhibit specific structure and contain molecules leading to efficient delivery of their genetic content to the infected cells. In order to exploit this property, adenovirus particles (without DNA or denatured by irradiation) have been attached to the liposomal membrane of the liposomes prepared according to the present invention. It is established that adenoviruses enter cells via receptor-mediated endocytosis. A specific fusogenic mechanism makes possible the release of the viral genetic content from the cellular endocytic vesicles after internalization. Use of adenovirus to facilitate gene transfer has been reported (1). Although DLS-liposomes clearly show a significant escape from intracellular vesicles, presence of adenovirus capsids at the liposome surface may enhance transduction efficiency by facilitating intracellular vesicle disruption.
The adenovirus strain used in the present invention is the dl 312 strain received as a gift from T. Shenk (Princeton Univ., Princeton, N.J.). Any adenovirus strain can be used in the present invention. Preparation of adenovirus capsids (no DNA) using cesium chloride gradient method may be performed following adenovirus collection and preparation. Whole adenovirus particles (with DNA) but inactivated by UV irradiation (10 J.m-2 8-1) may also be used. Hydrophobic binding of adenovirus to the liposomes is carried out by simply mixing the liposome suspension with the adenovirus concentrate. For instance, particles equivalent to 108 PFU (Plating Forming Unit) are added to 12.5 μl of a DLS-liposome-associated DNA preparation corresponding to 1 μg DNA and 12.5 μg lipids. The mixture is slightly homogenized and then incubated at 37 C for 1 hr with gentle shaking. Immediately after incubation adenovirus-DNA liposome preparation is added to cell culture. Transfection procedure is the same as that used for DLS liposomes.
FIG. 7 illustrates that adenovirus associated liposomes greatly enhance transfection efficiency in HeLa cells by a factor 4.5. Transfection efficiency obtained after simultaneous addition in cell culture medium of liposomal DNA and adenovirus particles at equivalent concentration was significantly lower (2.7-fold). This demonstrates the specific additional effect of the adenovirus particles attachment to the liposomes on gene expression and particularly on the plasmid DNA escape from endocytic vesicles.
DLS-liposomes and Transfectam TM (Promega, Wis.) reagent (DOGS) samples were submitted to negative staining and Transmission Electron Microscopy (TEM) analysis. The following is a part of the observations independently made by ABI Inc (Columbia, Md.).
DLS-liposomes: "Lipidic particles were observed throughout this preparation and were found in large quantities". "Each different particle appeared to display a heavily stained core region, which was surrounded by many different layers of membranes or envelopes. The particles contained so many different layers of membranes, it was difficult to establish the size of one lipidic particle to the next. Although it was difficult to measure the overall size of the particles, due to their pleomorphic shape and varied number of layers, it appeared the particles ranged from 200 to 3000 nm in diameter. The grid areas showed a high concentration of smaller lipidic particles throughout the background of the sample".
Transfectam reagent (DOGS): "Possible lipidic particles were found in this sample, in small quantities. The particles found in this sample were very different from those observed in the previous sample. The lipidic particles observed appeared to be either in the process of breaking down or they had never properly been formed. Large areas of lipid-like material were observed, however, they did not display any ultrastructural detail, such as different layers of membranes. The only similarity between this sample and the previous sample was that the lipidic particles were heavily stained. Very little debris was found in the background of the sample."
Thus, TEM analysis demonstrated that DLS-liposomes are formed of bilayer membranes vehicles. This specific ultrastructure differentiate them to Transfectam reagent or other cationic liposomes so far commercialized such as Lipofectin (BRL Co., N.Dak.). These later when complexed with DNA is not constituted of membrane bilayer-containing vesicles but is rather a lipid coating particle that presumably contain nucleic acids. Thus they may not be liposomes in the true sense of the term. DLS-liposomes better efficacy in transferring DNA may be explained by their liposomal structure. Furthermore, we may expect improved pharmacokinetic properties such as increased plasma half life. In addition, the presence of membrane bilayer in DLS-liposomes makes possible the anchorage of antibody to their surface which may result in cell targeting.
The mdr-1 gene expresses the P-glycoprotein ("P-gp"), a plasma membrane protein involved in the emergence of the Multi-drug Resistance phenotype which may occur after chemotherapy. The mdr-1 gene was used in this example as a marker of gene delivery in order to assess the efficacy of bone marrow transplant of mdr-1 gene transfected bone marrow cells by DLS-liposomes.
In order to assess the efficacy of bone marrow transplantation for "ex vivo" gene therapy, murine bone marrow cells were transfected with this plasmid and the DLS-liposomes and transplanted into Balb-C mice. The proliferation and differentiation of transduced hematopoeitic progenitor cells were detected up to 21 days after transplantation in the spleen and the bone marrow, suggesting that the bone marrow transplant had taken place.
Murine bone marrow cells were harvested and quickly transfected with the pHaMDR GA plasmid encapsulated in DLS-liposomes. Seven different experiments have confirmed that the mdr-1 gene was expressed in bone marrow cells since cells continue to grow under selective pressures (vincristine). In addition, lymphocyte, macrophage and fibroblast populations have been shown to exhibit the MDR phenotype after selection (using the rhodamine drug efflux method).
Plasmid vector containing the luciferase gene as a marker gene were delivered by DLS-liposomes in Balb-C mice. Various formulations of liposomes encapsulated plasmid at various DNA/lipid ratios were assayed. In these experiments transgene expression has been assayed in liver, lung and spleen. Luciferase activity was determined by bioluminescence measurement (2-3 mice/point). More than 100 mice have been studied. PCR analysis showed the long lasting expression of the lucriferase gene in all tissues tested (lung, liver, heart, spleen, skeletal muscle, blood cells, bone marrow, and ovary) up to at least 2 months post-injection. Only episomal replicating DNA vectors showed positive results.
Luciferase gene expression was detected in lungs liver and heart after 3, 10, 30 days post-injection of DLS-liposomes containing plasmid DNA at a dose of 50 and 100 ug DNA/mouse. The relative expression in these organs largely depends on the DNA/lipid ratio used. The optimal liposome formulation was determined to be 10 mg DNA/50 mg lipid. The toxicity was very limited for doses lower than 100 ug/mouse. The toxicity in humans is approximately 5 mg DNA/kg.
Luciferase gene expression was detected in spleen 3 days post-injection. The dose injected per mouse was 100 ug. No toxicity was detected.
Intravenous injection of mice with DLS-liposomes containing pHaMDR GA plasmid resulted in expression of the mdr-1 gene in circulating lymphocytes and granulocytes 10-15 days post-injection. Mice were pretreated with 5-FU before administration of DLS-liposomes.
The present invention can be used in the therapy of Kaposi's Sarcoma ("KS"). Two KS cell lines, showing tumorigenic properties in vitro and in vivo, have recently been established. One cell line, KS Y-1, was derived from a lesion of an HIV-infected individual. The second cell line, KS N1506, was derived from a lesion of a non-HIV associated immunodepressed individual. High amounts of IL-6, IL-8, and VEGF are produced in these cell lines. Correspondingly, high levels of these cytokines have also been found in the serum of AIDS-KS patients. In this example, antisense oligo(dN) was used as a specific molecular tool to inhibit KS cell production of these factors.
0.1 uM VEGF antisense phosphodiester oligodeoxynucleotides encapsulated in DLS-liposomes completely blocked KS Y-1 cell colony formation in semisolid culture. Lipofectin liposomes required 7-10 fold higher concentration to achieve the same inhibitory effect.
The hereinbelow list of references provides a complete citation of each of the references cited hereinabove. All of the references mentioned in the present application are incorporated in toto into this application by reference thereto.
1. CRISTIANO R. J., SMITH L., and WOO S. L. Proc. Natl. Acad. Sci. USA 90, 2122-2126 (1993)
2. NABEL E. G., PLAUTZ G., and NABEL G. J. Science 249, 1285-1288 (1990)
3. FELGNER P. L., GADEK T., HOLM M. et al. Proc. Natl. Acad. Sci. USA 84, 7413-7417 (1987)
4. WANG C. and HUANG L. Proc. Nat. Acad. Sci. USA 84, 7851-7855 (1987)
5. GAO X. and HUANG L. Biochem. Biophys. Res. Commun. 179, 280-285 (1991)
6. ZHU N., LIGITT D., LIU Y., and DEBS R. Science 261, 209-211 (1993)
7. SORIANO P., DIJUSTRA J., LEGRAND A. et al. Proc. Natl. Acad. Sci. USA 80, 7128-7131 (1983)
8. OSTRO M. J. and CULLIS P. R. Amer. J. Hosp. Pharm. 46, 1576-1587 (1989)
9. BEHR J. P., DEMENEIX B., LOEFFLER J. P. and PEREZ-MOTUL J. Proc. Natl. Acad. Sci. USA 86, 6982-6986 (1989)
10. BARTHEL F., REMY J. S., LOEFFLER J. P. and BEHR J. P. DNA Cell Biol. 12, 6, 553-560 (1993)
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Isabel Lucas to Star in Elizabeth Allen's CAREFUL WHAT YOU WISH FOR
Hyde Park-Image Nation, Troika Pictures and Merced Media Partners have announced that they will finance and produce the thriller CAREFUL WHAT YOU WISH FOR. The film, to be directed by Elizabeth Allen ("Ramona and Beezus," "Aquamarine"), was written by Chris Frisina and will star Isabel Lucas ("Transformer 2," "Immortals").
Producing are Hyde Park's Ashok Amritraj along with Troika's Robert Stein, Michael A. Helfant and Bradley Gallo, and Myriad Pictures' Kirk D'Amico. Executive Producers are William Gallo, Chris Frisina and Merced Media Partners' Raj Singh, Stuart Brown and Kevin Frakes. Philip Cohen is Co-Executive Producer. Hyde Park International will represent worldwide rights at the Toronto International Film Festival.
Self-conscious high school student Doug Martin gets more than he bargained for when he starts a torrid affair with Lena-the young, lonely wife of a wealthy businessman renting the lake house next door. But when a serious crime rocks their sleepy vacation town, Doug is let in on a devastating secret and suddenly finds himself a suspect in this sexy thriller reminiscent of "Basic Instinct" and "Body Heat."
Lucas is in high demand after completing roles that include the upcoming Terrence Malick film KNIGHT OF CUPS, as well as starring in the new RED DAWN and The Loft. In addition to Lucas, two other A-list stars will be cast in other roles.
The film will commence principal photography in the first quarter of 2013.
"I look forward to bringing the fascinating characters to life in this provocative and sexy coming of age thriller. The lead character of Lena is a uniquely complex femme fatale -- a compelling archetype that hasn't been brought to the screen in some time and is due a revival. I am thrilled to be collaborating with the talented Isabel Lucas on this project and am excited by the depth and mystery she will infuse into the film," said Allen.
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art direction // experiential design // hand-lettering // mysteries solved.
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1/28/2015 - I've announced the 10 winners of my 5k Giveaway on my instagram feed . Unfortunately, I'm sold out of prints for "I woke up like this" but will be making some more buyable stuff soon!
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Welcome to Saint George Parish Website
St. George Serbian Orthodox Church is a parish of The Western American Diocese of the Serbian Orthodox Church in North and South America. The parish was established on May 4, 1952 when the thirty founders met with their spiritual father, Fr. Nikolaj Dazgich. Today, our growing community welcomes all to worship with us and to inquire about our Orthodox Christian Faith. We eagerly look forward to your visit. God bless you!
Address: 3025 Denver Street, San Diego, CA, 92117; Tel. 619-276-5827
St. Petersburg Men's Ensemble Concert, Sunday, June 21st, 2015 @ 1:15pm
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Communique of the Holy Assembly of Bishops of the Serbian Orthodox Church
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Communique of the Holy Assembly of Bishops of the Serbian Orthodox Church held in Belgrade May 14-29, 2015
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King's College London Welcomes "The Christian Heritage of Kosovo and Metohija"
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On May 10, at King's College London, one of the top 20 universities in the world (2013-4 QS international world rankings), a rare history and fine art book, published by Sebastian Press, was presented to a wider public interested to study or expand their knowledge about the cultural heritage of Kosovo and Metohija (Serbia).
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Mon December 23, 2013
Most Books At N.Y. High School Are Digital
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Originally published on Mon December 23, 2013 6:00 am
Good morning. I'm Steve Inskeep with a milestone in White Plains, New York. Many schools are adding computer tablets and cutting back on books. Archbishop Stepinac High School is doing without books almost entirely. From biology to computer programming, texts will be on a digital bookshelf on laptops and tablets. The only exception is books on religion. Administrators at this Catholic boys school did not have quite enough faith in the digital future to throw out their bibles. You're listening to MORNING EDITION. Transcript provided by NPR, Copyright NPR.


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1964 Studebaker Hawk GT, 1 of 12 Non-Package R2 Supercharged Hawk with automatic transmission. The 1964 is the rarest and most refined of the... More
This beautiful Hawk is a fantastic original type of car that is both easy on the eyes and a blast to drive. Mostly fun... More