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Dates: 3rd & 4th July, 2015
Venue: Hotel Residency, Coimbatore
Pediatric, Neonatal & Fetal Neuroimaging Course
Date: 21st December, 2014
The event was a grand success...
Prof. S. Suresh, Director, Mediscan Systems addressed a gathering of around 25 gynecologists and consultants of PGI and Govt. Medical College, ...
Dr. Sujatha Jagadeesh has been recognised as a "Guide" for candidates in research work leading to the award of Ph.D. Degree, under "The Tamil Nadu Dr. MGR. Medical University"
MediScan donates 6 computers to Sri Ramakrishna Vidyalaya, a school educating 350 children from rural areas through Computer Kindness Foundation (CKF).


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Learn how to use Typekit to choose from thousands of fonts, and deploy them on your websites quickly and gracefully.
Explore the creative possibilities of CSS typographic effects, including text shadows, animation, transforms, and 3D depth.
"My biggest problem is figuring out what I want to watch next. Just when I think I'm all set, I find something ELSE that is fascinating." —John O.
more from our members »

subset: commoncrawl


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We call this "cabbage tetherball". That's 'Orange Chicken' ready to take her next hit.
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what kind of plant is this?
supaflyzSeptember 12, 2009
My mom got this plant from her friend. My aunt told us that she saw them in hawaii before. Her friend told her that it will have red flowers, but for 2 years nor it hasn't even bloom yet. The leaves are very big. It has these little plants with roots coming at the tips of it leaves.


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- Home >
- Our Editors >
- Suzanne Denbow
Suzanne DenbowHuntington Beach, CA
I currently drive a Subaru Impreza that is neither glamorous nor particularly attractive, but it's pretty handy for hauling my Huskies around in. I often dream of upgrading to the Impreza WRX STi, and sometimes if I close my eyes I can almost hear the muffled-whump of airborne dog bodies as I scream sideways through a corner.
Naturally, I jest.
Outside of the automotive space I also enjoy things like restoring salvaged furniture (more fun than it sounds), bike rides by the beach, and reading a good book outside in the sunshine. I also like weird webcomics.


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It's starting to get cold outside with winter approaching….but it's only warming...Read Article
Hello everyone! As of March 2015 we have brought back the room-only rates. So you now have the...Read Article
Hi Everyone, We've just come through a busy January, and we were lucky...Read Article
As of the 1/2/15 the Jasper Kitchen will no longer be serving our dinner menu in the restaurant,...Read Article


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Suzi's Blog
Boston Lettuce Salad with Cream Dressing from Jacques Pepin
If you mention the name "Jacques Pepin" to someone at random, you stand a pretty good chance hearing, "That's somebody I've heard of, although …" Foodies, of course, can pinpoint this name of one of the world's top chefs. But non-foodies galore will look at you and struggle to answer. They have heard the name,…
Read More »


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"World on Edge details the vice closing around us: a quadruple squeeze of global warming and shortages in food, water and energy. Then it explains the path out—and how little time we have left to take that path. Got anything more important to read than that?" —Peter Goldmark, former head of the Port authority of New York and New Jersey, President of the Rockefeller Foundation, and CEO of the International Herald Tribune
Earth Policy Institute
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Washington, DC 20036
Phone: (202) 496-9290
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Email: [email protected]
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Please contact: Reah Janise Kauffman, Vice President (202) 496.9290 x 12 [email protected]
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Please send requests to Reah Janise Kauffman. Include the subject of the conference, when and where it is being held, the type of presentation, the type of audience, whether a speaking fee is provided and what that might be, and if you cover transportation and expenses. The Institute relies on speaking fees to support its work, thus an honorarium is an important consideration. All honoraria go directly to support the Institute.
Please note that the Institute receives a large number of speaking requests of which its staff is able to accept only a fraction.
Translation Inquiries:
If you are interested in publishing one of our books or translating our other publications, please contact Reah Janise Kauffman, [email protected].


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Outsider Threat Protection: Building a Kill Chain Defense [ Source: Digital Guardian ]
November 2014- The Kill Chain Defense exploits a critical weakness in the outsider attack model; for an attack to be successful, all steps must be completed and the target data exfiltrated from the organization. This white paper explains the Kill Chain Defense model and how it can be used to pragmatically prevent outsider attackers from succeeding.
Advanced Threat Defense Tactics [ Source: GENERAL DYNAMICS Fidelis Cybersecurity Solutions ]
March 2015- Advanced Persistent Threats (APTs) in the online realm are a painful reality for companies of all sizes, from the largest enterprises down to small and medium-sized businesses (SMBs). Business leaders might be tempted to think that their own organizations are exempt from APTs, or that the security measures they already have in place are adequate to defend against these persistent, methodical attacks. Yet those temptations must be avoided. If your business has something of value ...
Who are you trying to protect? [ Source: Gemalto ]
August 2014- If we are to progress towards more secure and convenient authentication solutions, we have to start thinking beyond passwords. Multi-factor solutions that operate independent of passwords, are the future. But not all users are created equal. Some need more stringent forms of authentication than others. So who are you trying to protect?
The Right Way to Protect Against DDoS Attacks [ Source: F5 ]
October 2014- CIOs want harmony. Security directors loathe point products. Network operations won't buy into anything new. CIOs can get the harmony they need around DDoS mitigation by extending the F5 Application Delivery Controller into a hybrid solution: on premises with a new cloud component. Read the white paper to learn more.
The Expectation of SSL Everywhere [ Source: F5 ]
March 2015- SSL is the set of cryptographic protocols that secure data in transit. Today SSL is often the only tool standing between your organization and the bad actors. The stakes around SSL have been upleveled to the limit. Whether or not it's convenient to admit, it's time for organizations to uplevel their overall security posture to protect this last line of defense. Read this white paper to learn how your organization can properly embrace a higher ...
Next-Generation Network Security: A Buyers' Guide [ Source: Cisco ]
March 2015- Read this buyers' guide for an in-depth explanation of the factors that impel organizations to look at next-generation security solutions.
Achieve Deeper Network Security and Application Control [ Source: Dell SonicWALL ]
March 2015- Discover a next-generation firewall solution that combats emerging threats effectively and without compromise while reducing the total cost of ownership (TCO). Read this paper and learn how NGFWs help you achieve a deeper level of security and safeguard your organization against today's evolving threats.
G2000 Firm Secures Critical Financial Applications and Generates 192% ROI (Forrester) [ Source: Veracode ]
March 2015- Learn how a global firm secured 400 critical applications and generated an ROI of 192% with cloud-based automation and centralization. The firm previously relied on a traditional on-premises scanning tool, but success was limited because specialized expertise was required to tune and interpret results. As a result, the firm's AppSec program only covered a small fraction of the firm's applications. This Forrester case study includes a detailed financial model showing how the firm leveraged centralized ...
How a Global Manufacturer Secures Its Software Supply Chain [ Source: Veracode ]
March 2015- A global manufacturer found that over 90% of their vendor applications had critical OWASP Top 10 vulnerabilities. The company now leverages Veracode's automated cloud-based service to audit hundreds of third-party applications per year without requiring access to proprietary vendor source code. It also tracks vendor progress with supplier scorecards from the Veracode platform, and has modified its procurement process to contractually require suppliers to meet its security policies. The company has also dramatically scaled its program ...
Hacking Exposed 7 - Chapter 10: Web and Database Hacking (McGraw-Hill) [ Source: Veracode ]
March 2015- Read this classic reference text to get into the cyberattacker's mind and understand the latest attack vectors and web application threats. The prerequisite for dealing with cybersecurity is knowledge: download this critical chapter to learn about web application vulnerabilities and hacking techniques; freely-available crawling tools; and countermeasures to protect your web application infrastructure.
Secure Agile Development (Securosis) [ Source: Veracode ]
March 2015- This white paper is for security professionals who want to understand how to embed security into the Agile SDLC. It describes Agile development and the issues developers face, so both teams can work together better. Agile frameworks have become the new foundation for code development, and secure development practices, just like every other facet of development, must fit within the Agile framework not the other way around.
The CISO's Handbook - Presenting to The Board (Forrester) [ Source: Veracode ]
March 2015- This report describes strategies for articulating your risk posture and security strategy to executives so you can position yourself as a key influencer in the boardroom. Written by a CISO, it offers guidance on: answering common questions such as "How secure are we"; describing how your strategy aligns with the goals of the business; and helping the board understand complex security issues.
Four Reasons Not to Nuke an Infected Machine: Reimaging Isnt The Only Option [ Source: ThreatTrack Security ]
March 2015- Many organizations still cling to the rigid practice of reimaging every infected machine. However, the costs of reimaging often outweighs the benefit. Learn why you should adopt an alternative approach to reimaging to remediate threats.
Is it Phishing or an APT? [ Source: ThreatTrack Security ]
March 2015- Spear phishing is a primary means by which APTs target and infiltrate networks. This paper describes the element of a phishing attack and how to keep your organization safe from advanced threats.
Losing the Battle - The Need for a New Approach to Advanced Protection [ Source: ThreatTrack Security ]
March 2015- This paper outlines the challenges of fighting APTs and outlines a solution purpose-built to find and stop attacks in progress allowing for mitigation before it's too late.
SaaS Security: Mind the Gap [ Source: Adallom ]
November 2013- This joint paper from EMC and Adallom provides a brief overview of emerging gaps and trust issues common to SaaS providers.
IDC Analyst Connection: The Evolving Threat Landscape [ Source: ThreatTrack Security ]
February 2015- Today's cybercrime environment has evolved from quick smash-and-grab tactics to persistent campaigns involving specialized malware. In response, a new category of security technology aimed at detecting, analyzing and preventing such threats is emerging. ThreatTrack Security discussed this trend with Charles Kolodgy, IDC's Research Vice President for IDC's Security Products Service.
How to Stop Social Media Hacks [ Source: Proofpoint ]
February 2015- A how to guide describing the key steps that organizations should take to prevent their social media accounts from being hacked.
Securing Enterprise Applications [ Source: Onapsis ]
November 2014- Business-critical platforms such as SAP and Oracle have been in place for more than a decade, however a majority of firms using these applications currently have gaps in their security program. There are many reasons for these security gaps ranging from a reliance on generic security tools, to IT teams lacking complete understanding of how application platforms work.
Implementing SAP Security Solutions [ Source: Onapsis ]
January 2015- Gaps in security practices of business-critical applications running on SAP are causing organizations to rethink their current approach and embrace a new strategy.
The SAP Security Survival Guide [ Source: Onapsis ]
January 2015- As a CISO, learn which questions to ask in order to uncover security challenges facing your SAP systems.
Analysis of Cybercrime Infrastructure [ Source: Proofpoint ]
January 2015-
Proofpoint security researchers have published an analysis that exposes the inner workings of a cybercrime operation targeting online banking credentials for banks in the United States and Europe. This Proofpoint research report provides a detailed and rarely seen inside view of the infrastructure, tools and techniques that enabled this cybercrime group to infect over 500,000 PCs.
Key facts from the Proofpoint analysis:
• Qbot (aka Qakbot) botnet of 500,000 infected systems sniffed "conversations&...
Open Source Software -Security Risks and Best Practices [ Source: Rogue Wave Software ]
January 2015-
Third-party applications, including open source software (OSS), make up an increasing proportion
of enterprise applications. By some estimates, up to 80 percent of the source code in many new
commercial applications is open source. It is easy to understand why. OSS allows developers to
build applications faster, adding functionality without writing source code from scratch. Open source
communities provide new features, shortening time to market and helping organizations gain
competitive advantage.
Open source can ...
The Business Case for Earlier Software Defect Detection and Compliance [ Source: Rogue Wave Software ]
January 2015- By providing developers with the right tools to detect, understand, and fix problems early, your business can simplify software development, shorten development lifecycles, and improve the quality of software code. The end result is increased innovation, secure applications, and a faster time to market - all at a lower cost. Read this whitepaper to learn more.
Protect Your Applications - and Reputation - with Symantec EV Code Signing [ Source: Symantec ]
January 2015-
For years, developers have known that one of the best ways to reassure users is by signing code using a digital signature accessed via a private key issued by a respected certificate authority. But signed code is not invulnerable. Due to lax key security and vetting processes, malware has managed to infiltrate applications with signed code.
Extended Validation (EV) Code Signing Certificates help to halt malware infiltration by requiring a rigorous vetting process ...


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Comfort Suites near NASA - Clear Lake
16931 N. Texas Ave
Webster, TX 77598
Phone: (281) 554-5400
Fax: (281) 554-5441
Beauty & Spas
Bikram Hot Yoga Houston
2438 South Blvd
Houston, TX 77098
+1 713 664 5333
Mo to Th from 06:00 AM to 08:30 PM,Fr from 06:00 AM to 06:30 PM,Sa to Su from 08:00 AM to 06:30 PMYoga is rather daunting to those unfamiliar with its benefits. Now imagine physically challenging yoga poses in a controlled environment of 105 degrees Fahrenheit with 65% humidity. Houston's premiere spot for this "Hot Yoga" is located near the happening Rice Village. Drink PLENTY of water well ahead of time and come in for a body nurturing 90 minutes of sweaty breathing concentration. Be prepared to sweat buckets next to a refreshing range of first-timers and super experts, and everyone in between. They say your challenge for your first class is merely staying in the room for the duration of the class. The addiction will be your chase for improvement. -Adam Rosen
Earn valuable points toward free nights.


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My Freight Train
A little boy, living out his fantasy of driving his own freight train, describes the different cars, what each carries, how they are connected, and more.
Publisher: New York : Henry Holt, 2002.
Edition: 1st ed.
Characteristics: [25] p. :,col. ill. ;,21 x 27 cm.
From the critics
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Billabong Carter Hybrid Shorts - Men's
Platinum X Quad stretch walkshort made with H2 Repel. This quick-dry walkshort is fully submersible and very versatile.
Key Features of the Billabong Carter Hybrid Shorts:
- 22
Core Fit Zero Gravity
- Workwear chino styling
- Welt back pockets
- Sleek cell phone pocket at the side seam
- Woven work wear patch at the back hip
- 89% polyester/11% spandex
This product can only be shipped within the United States. Please don't hate us.


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Date: 1902-05
Description: Group portrait, with Ernst Goetsch standing on a dirt road next to a team of oxen hitched up to a drag scraper on the left, and a team of oxen on the right...
Date: 1962
Description: A trucker uses a pay phone in a phone booth inside the Mt. Victory service plaza while taking a break from the road.
Date: 1938-05-01
Description: Side view of a car parked at curb decorated to participate in the May Day parade. The car has two large megaphones mounted on top and a banner that reads,...
Date: 1957
Description: Advertising postcard featuring a color illustration of a man driving an International A-120 4x4 pickup truck with a winch (tow truck). The side of the truc...
Description: Canal in Binh Hung Vietnam with numerous small boats and a crowd of people walking alongside. In the background is a large cross and a number of large that...
Date: 1997-09-22
Description: "In Lomira, a Kondex Corp. employee was killed when three derailed railcars plowed into the concrete block building in the background at 5:30 AM. Eleven of...
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Date: 1962 ca.
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Date: 1917
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Missions in GTA 2Talk0
10,551pages on
this wiki
this wiki
Category page
See Missions in GTA 2 also.
Pages in category "Missions in GTA 2"
" Taxi For Traitors!, also known as RC Taxi!, is a mission in Grand Theft Auto 2 given to...
Taxi For Traitors!
" The Final Job is the final mission in Grand Theft Auto II taking place in the Industrial...
The Final Job! (Industrial District)
" The Final Job! in the Residential District, like other districts in Anywhere City, involves the...
The Final Job! (Residential District)
" Valdez Alert! is a mission in Grand Theft Auto 2 given to protagonist Claude Speed by The...
Valdez Alert!
" Water Carry On!, also known as The Dam!, is a mission in Grand Theft Auto 2 given to protagonist...
Water Carry On!


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Chimney hood venting issues (soffit?)
We started demo on Tuesday for our DIY kitchen. I was curious to see what was there when the ceiling came down to see how easy it would be to vent our planned chimney hood. The plan was to run the vent along the joists and out the side of the house. Unfortunately, the joist that is above where the range will be is the one joist with pipes running the length of the kitchen. So there's not room for the vent there. So it seems like our options are to use another joist, which would require building a soffit to hide the ductwork to get it to the next joist, or to scrap the whole chimney hood plan and get an under cabinet. (We already have the chimney hood, which I like, so I'm not loving that option either).
Any pictures I find of chimney hoods with soffits are in kitchen with a soffit all around, so they are hidden with nice crown molding. But we would only need a soffit in between the cabinets over the range, which I think might look weird. Otherwise, our cabinets will go to the ceiling with a small crown molding at the top. Am I missing any other options I haven't thought of?
We already have our cabinets, so moving the range isn't ideal- we have a very small kitchen, so we're not working with a lot of room to spare- I think it would look funny.
Since your range placement is set, you have three options: move plumbing pipes ($$), hide with soffit as you mention, or change to under cabinet vent hood.
Of the three, I like the under cabinet vent hood the best as you can then order the trim from Ikea to align with the other to the ceiling trim. That's provided your HVAC team can deal with either the pipes (10 x 3 inch ducts) or go lateral a bit.
I know it's frustrating to be in your position. For a small kitchen, having cabinets over the range (30" above burner surface) offers space for cooking and baking items and helps to keep you from moving all around the kitchen. I found that handy in fact.
Your kitchen designs look nice btw. Enjoy.
The HVAC team is my husband, so we have to figure it out ourselves... I was thinking if we did the under the cabinet then we could just use the cabinets to hide the ductwork needed to go to the next joist. If we can deal with the pipes then couldn't we just do a chimney hood as originally planned?


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Navigation Lights
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Courtney the Dancer
Join Date: Nov 2000
Location: San Juan Islands., WA, USA
Posts: 3,868
Thanks: 4
Thanked 21 Times in 20 Posts
Rep Power:
Sailingdog- What I was saying was that when you see just red and green deck lights there is no reason to suspect that it is a sailboat, is there? So why wouldn't you be mistaken for a power vessel, exact same lighting. That makes the argument for the red over green. If you also have a masthead tricolor on, which according to the rules I don't think is legit(not positive on that, anyone have a copy of the rules handy? ) I would hope most people would conclude "sailboat" though. What would you think if you saw deck lights and red over green vertical lights? Even if you did not know what it was you would know it was something "different". As far as the meter seperation, that's about the same height of most VHF antennas so overall height wouldn't increase, and with LED lights and aluminum tubing I'm sure I could put something together that was very light weight and low wind resistance, so I don't see the big problem there. Different strokes for different folks I guess, I'd rather be identified as a sailboat, and if more of us start using red over green, it will be commonplace and well known.
Boasun- Oopsie #1. You are absolutely right, what was I thinking? Thanks for the wakeup call. Oopsie #2. Not sure what you mean, I just said that masthead lights are a pain to replace and add windage, etc., didn't say they were required/not required.
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|Reviews for A Light in the Darkness
| ilovedana53 chapter 23 . 1/12/2014
Sam don't play with the big boys if you can't handle it
| ilovedana53 chapter 22 . 1/12/2014
wonder who is coming back to life. sam is stupid. glad that georgie has liz
| ilovedana53 chapter 18 . 1/12/2014
well it seems jase has seen the light where sonny is concerned now he needs to see the light where his skank of a wife is concerned. hope carly is being sincere.
| ilovedana53 chapter 17 . 1/12/2014
Sam wants liz dead why I don't know since she has Jason she should let Liz be. Carly is right Jase needs to let Liz go especially since he walked away from her. Sonny has snapped
| ilovedana53 chapter 16 . 1/12/2014
yea they got married and why is lucky allowed to investiage when he is always yelling and verbally abusing liz
| ilovedana53 chapter 15 . 1/12/2014
Jason has to know how it looked to liz. hope anthony got shot.
| ilovedana53 chapter 13 . 1/12/2014
lucky is an abusive controlling ass. sonny needs to leave liz alone and stop obsessing over brenda. hope they kill anthony soon
| ilovedana53 chapter 11 . 1/11/2014
Carly should learn how to nind her own business.
| ilovedana53 chapter 10 . 1/11/2014
Sam is up to something and like I said before jason is being a hypriciote. hoping that ric dies
| ilovedana53 chapter 7 . 1/11/2014
Jase is being a hypriciote.
| ilovedana53 chapter 6 . 1/11/2014
I'm a liason fan but I can't stand who jason became in this time period. jase and sonny have no right to tell her what to do
| ilovedana53 chapter 2 . 1/11/2014
so happt that she is letting JZ help her. wonder who he called
| ilovedana53 chapter 24 . 10/11/2013
who would have thought Johnny O and Liz. hope they catch and the skank soon and hoping that we find out that the baby sam is carrying is not jason's but maybe sonny's. love that zander is alive, always wished that he, Johnny O and Francis were still on the show. can't wait for more
| ilovedana53 chapter 20 . 10/11/2013
OMG Diego is alive and raped Georgie and Lucky is paying him to go after Liz. Lucky is sicker than I realized. both Luckless and the skank want liz to miscarry and for her life to be ruined
| ilovedana53 chapter 19 . 10/11/2013
beginning to wonder if Sam baby is even Jason's. Jason needs to wake up to Sam's true colors. Lucky is an abusive prick


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Posts from People
Design Q&A with Jiun Ho
Designer Q&A with Tyler Dawson
Designer Q&A with Alison Damonte
Design Q&A with Chad DeWitt
From the episodes of This Old House that he watched with his dad as a kid, to his appearances on HGTV's Designer Finals, television has had a significant impact on interior designer Chad DeWitt's career. Catch a glimpse of his work during the American Craft Council Show which begins August 8 in San Francisco. Here's a look at his room and how it evolved from the piece that he chose as his inspiration:
Designer Q&A with April Powers
Designer Q&A with John Anderson
Welcome to "Naturally, Danny Seo" - Danny Launches a New Mag!
Yesterday, my old friend, Danny Seo, came by the offices and showed off his stunning new magazine, Naturally, Danny Seo. A return to his first glory in magazines, but with a great new edge, he takes his optimistic, can do and intelligent sensibility to subjects as far and wide as cooking, recycling, interior design, travel and beauty. Print is back, and it's eco and techy too (there are videos embedded in the magazine). I urge you to check out the first issue on newstands this week. → Check out the website with articles and subscribe right here.
Sophie Blackall Tells Kids Where Babies Come From (So You Don't Have To)
Book Review: The Baby Tree
As a mother, I relish those rare, heart-to-heart conversations I have with my son. And I always tell him the truth. ("Can I have another cookie?" "No...they're all gone." "Did you just say a bad word?" "Definitely not, I said SHIP! I thought I saw a ship.") But I am rather dreading the "birds and bees" convo and am hoping my husband is the lucky one to field this particular query. Or, better yet, I think I'll just let author/illustrator Sophie Blackall handle this one. Her new book The Baby Tree tackles this tricky topic in a way that is factual, age-appropriate and charming.
Meet Our Small Cool Judges: Patrick J. Hamilton
We're happy to announce that two super talented design pros have lent their expertise to our judges panel for Small Cool 2014. They are both New Yorkers which mean they know all about the challenges (and rewards!) of living Small and Cool. We can't think of anyone better suited to help us choose the finalists in this, our tenth year of Small Cool, than our very first Small Cool winner, Patrick J. Hamilton!
Meet Our Small Cool Judges:
Amy Preiser
We're happy to announce that two super talented design pros have lent their expertise to our judges panel for Small Cool 2014. They are both New Yorkers which mean they know all about the challenges (and rewards!) of living Small and Cool. First up, say hello to the digital editor of Hearst Design Group, Amy Preiser, who helps to make House Beautiful, Elle Decor and Veranda some of our favorite online destinations...


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over my head genetics
Hi everyone, I found this page while looking up how to cause mutations or inbreeding in daylilies. It is over my head for the most part, but maybe someone could brake it down for some of us here :)
In the last part it discusses that a blue daylily will come from crossing a blue eye with a white. Interesting stuff here...Enjoy :)
Here is a link that might be useful: genetics of daylily color
The suggestion that blue could be produced by selecting using crosses of 'bluish' eyed plants with 'near-whites' was made quite some time ago. I think hybridizers who tried that strategy were not successful.
A reasonable strategy is to select as parents plants with the most 'bluish' flower colours and cross them together. If one has only one such plant then crossing it to a near-white is reasonable, but then so would be crossing it to a lavender or perhaps even a pink or dark bluish-purple. There is no easy wasy to get bluish daylilies except to select generation after generation for more blue in the flowers.
There is an idea tha if one can produce a 'bluish' eyed plant then one could select parents so that the eye came to cover the whole petal. I think that will be difficult as the petal in most plants, including daylilies, has two slightly different parts (lower half versus upper half - not top versus bottom of the petal). The different parts are important for developing the flower and for what happens when the flower bud opens and the petals expand. Eyes typically do not extend much past the half-way point on petals and may be linked to the differences in the two petal parts.
If one was to breed for petals that were all eyes one could do that by keeping the size of the petal large and selecting for wider eyes and smaller petal remainder while maintaining the petal length. Or one could reduce the size of the petal so that it was only eye. In that case one would select for smaller petal remainder while maintaining the eye width (selecting for shorter petals that were all eye). The two strategies are not necessarily genetically equivalent.


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The Chamber Office is located in the old city hall building on Highway 78. Our physical and mailing address is:
Sachse Chamber of Commerce
5560 Highway 78 | Sachse, TX 75048
Join us for our Monthly Networking Luncheon on the 2nd Tuesday of each month.
Next Luncheon:
Tuesday, April 14, 2015
11:30am - 1:00pm
Woodbridge Golf Club, 7400 Country Club Drive, Wylie, Texas
Speaker: Lieutenant Marty Cassidy, Sachse Police Department
Sponsor: Jeanie Marten Real Estate
$15 for Chamber Members with an RSVP by noon the day before
$20 for non-members and walk-ins
Click Here to RSVP
Join us every Tuesday morning from 8:00 to 9:00am. Bring a friend! Open to members and non-members.
First meeting of 2015 will be on January 13th at our new location.
Photos by Jack Palmer Photography
Ambassador of the Month
Anna Morris
Business of the Month
North Pointe Church of Christ
Pleasant Valley United Methodist Church


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I am currently a member of Lexington Law Firm, and they have done wonders for my credit reports. I do have a few negative items remaining. Namely, a credit card and car repo I got stuck with from a messy divorce. I have tried PFD letters which I have sent out twice but I never get a response. Can anyone give me guidance on what I should do next? The credit card is handled by SST, the car was with American Honda Finance and I also sent one for a Dish Network collection through AFNI. Any help is much appreciated.
In my 3 years of studying repair, I've sent hundreds (no joke) of PFDs between CapOne, Verizon (landline and wireless), AFNI, and a couple others. I probably got a response to 10% of my letters, even when offering a payment in full each time. My only success was with Verizon Wireless.
Keep trying.
If AFNI is reporting, try sending the PFD via e-mail (firstname.lastname@example.com).... trust but verify the address.
Thanks for the info. I'll keep trying.
11-19-2010 09:06 PM - edited 11-19-2010 09:08 PM
What have you been saying in your PFD letters as an incentive for them to respond?
As you know, they dont have to ever respond to a PFD request.
In fact, their credit reporting guidelines with the CRAs tell them never to delete prior reporting based on payment. So the burden is on you.
So to just request a PFD deletion requries a knowledge of the facts, and some forethought in your letter of how to spark their capitalistic interest.
Are you sending it to an OC or a debt collector? If an OC, then maybe future promises of dealings with them might spur some corporate profit incentive. The same is hard to offer to a debt collector, for they breed on lack of payment, now and in the future.
How close is the debt from expiration of your state statute of limitations? Their inability to take future legal action may be a factor.
How much is the total debt, and how much, as the percentage of the total debt owed, are you offering as terms in your PFD offer?
Those are business terms they might listen to. My 'dawg just died, I lost my job, the roof is a'leaking. my wife left/divorced me, may work more to your detriment than bendfit unless you show how those prior woes have been overcome.
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Side entry garage
The new build will have a 2 car side garage, with windows. That's new to me, we've had a front garage with no windows.
Did you put blinds on the windows, or leave them open?
I put blinds on the windows for light/heat control as well as privacy. That's more important in a hot climate with south or west facing windows, but it can be a concern for some east facing windows as well in a super hot climate. They aren't always shut, but they are tilted most of the time. I shut them when I do a garage strip of filthy gardening clothes that are too dirty to even walk into the mudroom. Also, with the blinds always there and used occasionally, it doesn't look so "we're not home" if I close them so you can't see the absent vehicle when we travel. I'd be really uncomfortable if there were no window coverings in a subdivision that had a small front yard where you could see directly into the garage. If you're off of the street a good distance, and you aren't in a hot climate, I think it's much less of a concern.
Mine is open (I have 1 big decorative window). Mom & Dad simply put up valances so the windows looked pretty from the inside & they match up with all the other windows from the outside.
The bigger questions that need to be asked are: What will you be putting in the garage? Other than a vehicle, is there anything in there that an unsavory character might want to steal that they can see plainly from the window? Is your garage always neat and organized or do things tend to get out of control in there? What direction do the windows face? If you need to conceal either a messy garage or items of value that are in plain view or the windows face south or west, I would recommend some type of window covering fro privacy or to decrease sunlight & heat.
Just my $0.02. Hope it helps!
Thanks for your helpful replies!
We are the first house entering the development of 21 houses. In the current garage we keep one car, the lawn tractor, snow blower, bikes, trash/recycle cans, misc stuff. Baiscally everything you would have in a shed.
It's funny how you never really think about something like garage windows, when you don't have any.
Thanks again! I think we will put in some sort of blinds.
We tried to match the appearance of our garage windows to the other front-facing windows in the house. We have about 1" vertical blinds in the house, so did the same in the garage. However, we were able to get a much cheaper blind, since function isn't as important.
I plan on blacking out our garage windows. Where we live, flies are a problem. In a garage with windows, the flies beat themselves against the window trying to get out. In a garage without windows, flies will find the tiny pinpoint of light where they actually can escape. I've never had flies in my garage (no windows) and my parents' garage (windows) is like a fly version of Woodstock.
I agree with mydreamhome.... windows that aren't covered are an invite for thieves to see what is inside.
No one here leaves windows like that uncovered.
With that aside, I haven't ever seen a neat garage.... so I prefer to obscure the view anyway. Because of dirt, bugs and such, I just have replaceable, temporary blinds in the same shade as other blinds in my house. I use the kind that creates privacy but filters as little light as possible... It might be our windy climate, but I wouldn't be able to keep anything else clean for long.
I agree, cover them. I recommend window film that you can find at any big box store. I prefer Home Depot's brand. When you put up blinds - if not cleaned regularly (as is often the case in a garage) the blinds get dusty, spider webs,and bugs often find themselves a home... the window film helps to eliminate a hiding spot for the nastiness. The window film comes in a few different styles, IMO the frosted version looks good from the road.
I think the dirt thing may be overblown but it depends on your tolerance. I just cleaned ours for the first time after 3 years installed. They are the 2 inch faux wood white type.
I guess each situation is different but ours are high enough that only a 7 footer could see in and then he would have to be pretty close since the garage isn't lit up. He also has to get by the rose bushes and wouldn't that be suspicious looking. The blinds are left partly open depending on the season.
Our neighborhood of 55 side entry garages has over 50 garages with blinds in the windows - there might be a few that don't but I haven't noticed any bare or window films.
Opaque or semi-opaque interior window film can cause the inner pane to become warmer than the outer pane and put unexpected stress on the perimeter seal therefore it often voids the glass seal warranty. Professional film installers sometimes provide a warranty for the glass they modify.


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Systems, methods and kits that utilize uniquely coded microparticles for performing protein assays are provided. The uniquely coded microparticles are used as a substrate for reverse protein delivery into cells. The microparticles and methods offer the possibility of studying the biological functions of either a single protein of interest in multiple cell types per assay or multiple proteins in a single cell type.
1. A method for multiplexed functional screening of proteins in cells, comprising:
contacting a first microparticle having a unique identification code with a protein-containing mixture under conditions to allow the protein to attach to the microparticle; and
contacting the microparticle having a protein attached thereto with cells under conditions sufficient to deliver protein into the cells.
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[0001] The present invention relates to biological assays. More particularly, the invention pertains to methods and articles for reverse protein delivery into living cells using coded microparticles.
[0002] Systematic- and proteomic-wide studies of the biological functions of proteins have drawn increasing interest during the past several years as the realization that mRNA expression levels may not accurately reflect the activity/expression level of their corresponding protein. And proteins, not genes, are the true targets of modem medicines. The prevailing approach for analyzing protein function in vivo is use of cell-based assays. These types of assays are used to study the function of one particular gene in a cellular context by gene transfection and protein delivery.
[0003] Protein delivery, which is known in the art as protein transduction, is the process by which a peptide or protein motif is delivered across the plasma membrane into the cell. Traditionally, methods to introduce antibodies, peptides or other membrane-impermeable molecules into cells include micro-injection and electroporation. The obvious disadvantages of these techniques are that they tend to be toxic to the recipient cells, they are non-specific (i.e., anything can enter or exit the cell once the membrane is disrupted), and they exhibit low transduction efficiency and substantial variability. To overcome the disadvantage associated with these techniques, researchers have developed a number of protein-transduction domains (PTDs) that mediate protein delivery into cells. These PTDs or signal peptide sequences are naturally occurring polypeptides of 15 to 30 amino acids, which normally mediate protein secretion in cells. They are composed of a positively charged amino terminus, a central hydrophobic core and a carboxyl-terminal cleavage site recognized by a signal peptidase. Recently, researchers have shown that a number of membrane-translocating peptides can successfully mediate delivery of polypeptides, protein domains, and full-length protein, including antibodies into cells using solution-based protein transduction protocols. Recently, researchers have also demonstrated the use of lipid liposomes or the like for protein delivery. Traditionally, however, these approaches have been limited since they are solution-based formats. Only one gene or protein may be studied per assay. As there are more than 35,000 genes present in the human genome, and approximately 10,000 of these genes are expressed as proteins in any given cell type, a high-throughput method for studying gene function is needed. It would be desirable to provide systems, including, but not limited to, methods, microparticles, and kits for multiplexed analysis protein and cell interaction within a single experiment.
[0004] The invention relates to systems, methods and kits that utilize uniquely coded microparticles for delivering protein into cells. According to certain embodiments of the invention, uniquely coded microparticles are used as a substrate for reverse protein delivery into cells. The microparticles and methods offer the possibility of studying the biological functions of either a single protein of interest in multiple cell types per assay or multiple proteins in a single cell type.
[0005] Advantages of the invention will be apparent from the following detailed description. It is to be understood that both the foregoing general description and the following detailed description are exemplary and are intended to provide further explanation of the invention as claimed.
[0006]FIG. 1 is a schematic view of microparticles contacted with a protein that can be delivered into cells according to one embodiment of the invention; and
[0007]FIG. 2 is a schematic view of a microplate and a plurality of coded microparticles for multiplexed screening according to one embodiment of the invention.
[0008] Before describing several exemplary embodiments of the invention, it is to be understood that the invention is not limited to the details of construction or process steps set forth in the following description. The invention is capable of other embodiments and of being practiced or carried out in various ways.
[0009] The invention relates to systems, methods and kits including uniquely coded particles for cell analysis. Certain embodiments of the invention provide uniquely coded microparticles used as a substrate for reverse protein delivery into cells. Other embodiments relate to methods that use uniquely coded microparticles in which proteins are reverse delivered to cells. Delivery of proteins to cells can occur by contacting microparticles with a protein-containing mixture that includes a protein of interest, and then contacting the microparticles with cells under conditions sufficient to deliver the proteins to the cells. The microparticles can be used in high-throughput analysis of protein functions in prokaryotic or eukaryotic cells (e.g., bacterial, mammalian, human, insect, or plant cells). The microparticles may be used for rapid and direct functional screening of protein or enzymatic functions or any given intracellular protein interaction in the natural environment of a living cell. Moreover, the microparticles also are useful for high-throughput screening of other biological and chemical analytes, such as drugs, which affect the functions of these proteins.
[0010] According some embodiments of the invention, proteins can be pre-complexed with membrane-disrupting pore-forming reagents and placed in contact with microparticles, or can be pre-complexed with specific receptor-binding reagents and placed in contact with microparticles. Cells are then placed in contact with the microparticles including the proteins bound thereto, which then uptake the proteins. Unlike reverse transfection methods that are limited to genes that encode proteins, the reverse protein delivery can be used to deliver a broad range of protein-like biomolecules, including functional peptides, protein domains, proteins, protein mutants, engineered proteins with specific modifications, antibodies, and particle-protein conjugates.
[0011] As used herein, the term �contact� or �contacted� means attaching a protein or a cell to a microparticle directly or indirectly by a linking group through a binding mechanism. Binding mechanisms for proteins include, but are not limited to ionic bonding, hydrogen bonding, electrostatic interaction, van der Waals interactions, and/or hydrophilic-hydrophobic interaction. Binding mechanisms for cells include, but are not limited to, biospecific binding, for example, cells adhere onto a surface through the interaction between RGD peptides presented on a surface and cell surface receptors.
[0012] As used herein, �code,� �coded,� and �encoded� refers to association of identifying information with a microparticle to identify the microparticle. The identifying information could be information about cells and/or biomolecules in contact with the microparticles. For example, if microparticles are individually in contact with a single, different protein, the code could contain information to identify each protein in contact with each microparticle. The code could be based on the size, shape, or other physical property of the microparticle such as density, weight, color, plurality of colors, fluorescence, or other detectable property. In certain preferred embodiments, the code is based on a detectable optical property associated with the microparticles. In other preferred embodiments, the microparticles are encoded with at least one rare earth element. In embodiments in which rare earth elements are used to encode the particles, the coding could be based on the quantity, size, or type of rare earth element associated with a particle. The particles could be encoded with two or more rare earth elements to provide a barcode. The rare earth elements can be separated spatially to provide a pattern or array similar to a bar code. Each of the different rare earth elements, or same elements doped at a different concentration or location on the particle provides a discrete optical signal upon illumination capable of detection by conventional optical equipment and can be used to identify the particle. Rare earth elements are preferred because certain rare earth elements, do not spectrally interfere with traditional fluorescent channels such as Cy3, Cy5, Texas Red, FITC, and other fluorescent reporter dyes. For example, a particle coded with Eu2O3 produces red fluorescence, a particle coded with Tb2O3 fluoresces green, and a particle code with Tm2O3 or CeO2 fluoresces blue. These rare earth elements are exemplary only, and a wide variety of other rare earth elements could be used in accordance with the present invention. In addition, mixtures of rare earth could be used to produce a wider variety of fluorescent colors. The particles containing rare earth elements can be excited with a mercury lamp at either 254 or 365 nm. At these wavelengths, fluorescent tags such as the commonly used Cy-3 and Cy-5 are not excited, and therefore, crosstalk and interference between these common fluorescent tags and the particles encoded with rare earth elements does not occur.
[0013] As used herein, the term microparticle means a small piece of material that can be used as a host or a carrier for conducting reverse protein delivery experiments on a surface of the microparticle. The microparticle can be in shape of sphere, cylinder, circular disk, square plate, or any irregular-shaped disk. Commercially available microparticles range in size tens of microns to thousands of microns in diameter. Examples of suitable microparticles include glass, metal, ceramic, or polymeric beads. The uniquely coded microparticle, according to one specific embodiment, is a small piece of glass uniquely coded with rare earth metals or other state-of-the-art microbarcode methods. The uniquely coded microparticle, in another embodiment, is a macroporous and/or microporous microparticle with a code embedded inside the particle, such as cytodex�, cytoline�, cytopilot� mini�, cytopore�, and the like, which are available from Amersham. These commercially available cyto carriers do not have a code embedded inside, however, these carriers can be further modified with state-of-the-art methods to embed a code inside them. Cytodex� microcarriers are based on cross-linked dextran beads. The microporous beads are transparent, spherical and hydrated, and are substituted with positively charged groups. Cytodex� microcarriers have a mean diameter of 200 μm and a density of 1.04 g/ml. Their small size allows them to be easily transported through tubing. Cytoline� microcarriers are based on a matrix of polyethylene and silica. The polyethylene makes the microcarrier hydrophobic whilst the silica gives it a slightly negatively charge. Cytoline microcarriers are lentil-shaped with a length of 2 to 2.5 mm. This size makes their transfer through tubing more difficult, but possible. Cytopore� microcarriers are hydrophilic and have a mean diameter of 230 μm and a density of 1.03 g/ml. They are based on a cross-linked cotton cellulose matrix and have an average pore size of 30 μm. Cytopore� microcarriers are both macroporous and microporous. Cytopore� microcarriers are easily transported through tubing.
[0014] The unique coding information on the microparticles can be embedded inside the. microparticle using a variety of state-of-the-art methods. For example, a nanobarcoded glass bead can be encapsulated inside the host microparticle. In another embodiment, a DNA or protein delivered into the cells can serve as the barcode. A non-uniquely coded microparticle coated with a protein of interest is directly used to deliver protein to cells, and the resulting cells themselves can be indirectly or directly used as a barcode due to the expressed protein. For example, different proteins can be labeled with different fluorescent dyes, quantum dots, gold nanoparticles, paramagnetic nanoparticles, silica nanoparticles or other nanoparticles. In another embodiment, a uniquely coded molecule is mixed with the protein to be delivered into cells and used as a code. For example, purified green fluorescent protein or yellow fluorescent protein can be mixed with a protein of interest and co-delivered onto a microparticle host. Alternatively, different cell types can be co-transducted with DNAs encoded with different fluorescent proteins, such as green fluorescent protein (GFP) or yellow fluorescent protein (YFP). In another alternative, the protein delivered into the cell could be fused to a protein that serves as an identifier. Examples of such fusion proteins include a GFP fusion protein. The fluorescence of these proteins can be used as a barcode.
[0015]FIG. 1 is a schematic view according to one embodiment of the present invention. FIG. 1 shows a first microparticle 20 and a second microparticle 40. The first microparticle includes a unique identification code 22, and second microparticle includes a unique identification code 42. As shown in FIG. 1, the unique identification codes 22, 42 include a colored barcode pattern comprising at least two different colors. The embodiment shown in FIG. 1 illustrates one of many ways that particles can be marked with a unique identification code. According to certain embodiments, the unique identification code is used to identify a characteristic or the identity of a protein associated with the microparticles 20, 40. For example, if two different proteins of interest were being assayed, code 22 would identify a first protein associated with microparticle 20, and code 42 would identify a second protein associated with the microparticle 40. In certain embodiments, the proteins may be functionally related. In other embodiments, the proteins may the same type of protein but have different point mutations. In another embodiment, the microparticles 20, 40 could be associated with the same protein, and the microparticles 20, 40 bound to the proteins could be used to study interaction with two different cell types. According to this embodiment, the codes 22, 42 would represent first and second cell types.
[0016] Still referring to FIG. 1, the microparticles 20, 40 are placed in contact with protein-containing mixtures such that a protein 24 is bound to the first microparticle 20, and a protein 44 is bound to the second microparticle 40. In the embodiment shown in FIG. 1, different proteins are bound to the first and second microparticles. After the proteins have been bound to the microparticles 20 and 40, cells 26, 46 are then placed in contact with the microparticles 20 and 40 in a manner such that protein is delivered to the cells. For example, mammalian cells are cultured on the mixture-coated surface. The cells take-up the protein(s), which may affect cellular functions or observable events.
[0017] According to certain embodiments, the microparticles of the present invention can be used to provide a system or kit for multiplexed screening of proteins that could include reagents, microparticles and other equipment to perform multiplexed screening, for example, a microplate. Referring to FIG. 2, a multiwell plate 60 have a plurality of wells 62 can be used for contacting a plurality of groups of different microparticles 64 a-h, 66 a-h, 68 a-h, 70 a-h, etc., each microparticle attached to a different protein of interest. After each microparticle has been placed in its respective well, cells can be cultured in each of the multiwell plates under conditions such that the proteins in each well are delivered to the cells. Thereafter, the microparticles can be removed from the wells for functional screening. In certain embodiments, the same type of cell is cultured in each well of the multiwell plate. In other embodiments, the microparticles in each well are contacted with the same protein of interest, and different cell types are cultured in each of the different wells.
[0018] Proteins can be delivered to cells according to a �reverse delivery� technique in which a mixture containing: 1) a protein-of-interest, or 2) a protein-of-interest complexed with a carrier reagent, or 3) a protein-of-interest-conjugate with a carrier reagent, or 4) a combination thereof, is contacted with the microparticle and so that the proteins are bound to the surface of the microparticle. Cells are placed in contact with the microparticles including proteins, wherein the microparticle bearing the protein is used to culture the cells. Under appropriate conditions, the proteins enter the cells.
[0019] Proteins may include intracellular proteins, cell surface proteins, toxin proteins, antibodies, synthetic peptides, bioactive peptides, and protein domains; also protein-nucleic acid conjugates, and protein-nanoparticle conjugates, or multi-protein complexes. Additionally, conjugates between a protein-organic chemical entity or protein-inorganic chemical entity (e.g., Biotin, fluorescent dyes, silage derivatives, mass spectrometry tags, or low-molecular weight chemical moiety, etc.) are included.
[0020] Examples of intracellular proteins include, but are not limited to: oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, kinases, phosphoproteines, and mutator transposons, DNA or RNA associated proteins (for example, homeobox, HMG, PAX, histones, DNA repair, p53, RecA, robosomal proteins, etc.), electron transport proteins (for example, flavodoxins); adaptor proteins; initiator caspases, effector caspases, inflammatory caspases, cyclins, cyclin-dependent kinases, cytokeletal proteins, G-protein regulators, small G proteins, mitochondria-associated proteins, PDZ adaptor proteins, PI-4-kinases, etc. Recombinant proteins of unknown functions may also be used.
[0021] Applicable cell surface proteins include, but are not limited to: G-protein coupled receptors (e.g. the aderenergic receptor, angiotensin receptor, cholecystokinin receptor, muscarinic acetylcholine receptor, neurotensin receptor, galanin receptor, dopamine receptor, opioid receptor, erotonin receptor, somatostatin receptor, etc), G proteins, ion-channels (nicotinic acetylcholine receptor, sodium and potassium channels, etc), receptor tyrosine kinases (e.g. epidermal growth factor (EGF) receptor), immune receptors, integrins, and other membrane-bound proteins. Mutants or modifications of such proteins or protein functional domains or any recombinant forms of such proteins may also be used.
[0022] Toxin proteins include, but are not limited to, cholera toxin, tetanus toxin, shiga toxin, heat-labile toxin, botulinum toxin A & E, delta toxin, pertussis toxin, etc. Toxin domains or subunits may also be used. Antibodies include, but are not limited to, organism-specific antibodies such as mouse and human antibodies, monoclonal and polyclonal antibodies, intact antibodies or single-chain antibodies.
[0023] Synthetic and bioactive peptides and protein domains also can be delivered to cells using the method of the present invention. For example, a synthetic peptide comprising a sequence of AAYANAAVE may be delivered to cells and monitor the protein tyrosine kinase (PTK) activity in cells. This peptide has recently been used as a universal PTK substrate for rapid detection of PTK activity in recombinant yeast (Clark, D. D. & Peterson, B. R., J. Am. Chem. Soc., 1, 207-209 (2002)). Foreign proteins such as streptavidin and lectins and polymers may also be used.
[0024] Nanoparticle-protein conjugates may be delivered to proteins and used to deliver proteins to and visualize cells. The particles can include fluorescent tags, quantum dots, gold nanoparticles, paramagnetic nanoparticles, silica nanoparticles, or beads of silica glass or polymer material, or the like. In other embodiments, protein-DNA conjugates are used. The DNA can be directly used as a code or be used to encode a unique protein.
[0025] In another embodiment, proteins immobilized on the surface of the microparticle are preferably originated (produced) by using in vitro translation of nucleic acids previously deposited on the surface. In another embodiment, the proteins are preferably synthesized on the surface in situ. After the protein is synthesized on a surface of a microparticle, it can then be mixed with a carrier material to allow the protein to be delivered into cells after the cells become contact with the surface.
[0026] A library of fusion proteins can be deposited on a plurality of microparticles, and then adherent cell lines can be placed in contact with the microparticles. By this method, it is possible to assess the function of the protein in vivo in eukaryotic cells. Interactions between the delivered protein and other native cellular proteins may be studied in vivo. Fusing the delivered protein with an auto-fluorescent marker, such as the green fluorescence protein (GFP), may monitor intercellular localization of the delivered protein. Reverse delivery into cells could be accomplished, for instance, using VP22 fusion protein, herpes simplex protein, or any other protein with the similar properties. The fusion protein can be made using state-of-the-art methods. Some commercially available vectors for the production of VP22 fusion protein in mammalian cells and Escherichia coli (for example, pVP22/myc-His-2 from Invitrogen, Carlsbad, Calif.) are applicable. The fusion protein can be deposited directly and affixed onto microparticle, or the fusion protein can be pre-mixed with a helper reagent and then deposited and affixed onto the microparticle for delivery to cells.
[0027] According to certain embodiments of the invention, particular vehicle or carrier reagents are used to facilitate reverse protein delivery or reverse transduction. Carrier reagents may comprise a variety of reagents. In one embodiment, the carrier reagent is a bioactive cell membrane-permeable reagent, or other peptides containing protein-transduction domains (PTDs) (i.e., single peptide sequences comprising about 15 to about 30 residues). Protein-transduction domains (PTDs) mediate protein secretion, and are composed of a positively charged amino terminus, a central hydrophobic core and a carboxyl-terminal cleavage site recognized by a single peptidase. Examples of such membrane-transducing peptides include Trojan peptides, human immuodeficiency virus (HIV)-1 transcriptional activator (TAT) protein or its functional domain peptides and other peptides containing protein-transduction domains (PTDs) derived from translocation proteins such as Drosophilia homeotic transcription factor Antennapedia (Antp) and herpes simplex virus DNA-binding protein, VP22, and the like. Some commerically available peptides, for example, penetratin 1, Pep-1 (Chariot reagent, Active Motif Inc., Calif.) and HIV GP41 fragment (519-541), can be used.
[0028] Other carrier reagents include signal sequences, which have been used efficiently to target proteins to specific locations in both prokaryotic and eukaryotic cells, and a number of membrane-translocating peptides. Membrane-translocating peptides have been applied successfully to mediate membrane-translocation and the importation of a polypeptide, protein domain, full-length protein, or antibody into a cell using standard solution-based delivery protocols. The carrier reagent is a bioactive peptide or ligand that can specifically bind to and activate cell surface receptors. After binding to the cell surface receptors, the receptor and bound carrier-protein complex will undergo internalization, resulting in the delivery of ligand-protein complexes into cells. The proteins may be complexed with the ligand beforehand or in situ. The ligand can be complexed with the protein of interest or the protein to be introduced into cells by means of non-covalent interaction such as hydrophobic interaction or electrostatic interaction or both, or coupled covalently to the protein, or by means of a ligand-receptor binding interaction. For example, a carrier reagent can be modified with a ligand that can bind specifically to the protein of interest. To illustrate, a synthetic ligand termed �Streptaphage� has efficiently delivered streptavidin to mammalian cells by promoting non-covalent interactions with cholesterol and sphingolipid-rich lipid raft subdomains of cell plasma membranes (Hussey, S. L. & Peterson, B. R., J. Am. Chem. Soc., 124, 6265-6273 (2002)).
[0029] In another embodiment, the carrier reagent is a lipid liposome or any other reagent that can complex with a protein of interest and promote the delivery of the protein into the cell. The carrier reagent could be a naturally occurring or synthetic lipid . For example, the protein encapsulated in the formulation binds to the negatively vehicle lipids for delivery (O. Zelphati et al., J. Bio. Chem., 276, 35103-19 (2001)). Products available commercially can be used, such as BioPORTER (Gene Therapy Systems), or ProVectin (Imgenex, San Diego, Calif.).
[0030] Protein delivery reagents (e.g., Chariot� by Active Motif, or BioPORTER� by Gene Therapy Systems) can help save time by bypassing the traditional DNA transfection, transcription and protein translation processes associated with gene expression. Depending on the nature of the particular reagent employed, fusion proteins or chemical coupling in some embodiments would not be needed. The reagent forms a complex with the protein, stabilizes the macromolecule and protects it from degradation during delivery. Once internalized in a cell, the complex can dissociate, leaving the macromolecule biologically active and free to carry out its biological function.
[0031] Certain embodiments involve the use of at least one helper reagent. In one embodiment, the helper reagent is a polymer such as DEAE-dextran, dextran, polylysine, and polyethylamine. In another embodiment, a helper reagent can also be a cell adherent-enhancing protein, such as fibronectin and gelatin. The helper reagent can be a sugar-based compound (e.g., polyethylene glycol) or a synthetic or chemical-based gelatin, such as acrylamide. In a further embodiment, the helper reagent can be a RGD peptide, such as Arg-Gly-Asp-Ser, Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro, and the like. Alternatively, the helper reagent can be a mixture of a hydrogel and a RGD peptide, and combination of any the aforementioned molecules. The use of helper reagents enhances the efficiency of protein delivery into the cells.
[0032] In one embodiment involving a reverse protein delivery method, the protein-containing mixture comprises protein and a vehicle reagent that is present in an appropriate solvent, such as phosphate silane buffer. The mixture is incubated with a microparticle for certain time under appropriate conditions, and washed to remove excess mixture in solution. This results in a microcparticle bearing (having affixed or bound thereto) a protein-containing mixture.
[0033] In one embodiment, uniquely coded microparticles having a set of different proteins of interest bound to the microparticles are independently used to transduct or deliver protein to a single cell type. After delivery of the proteins to the cells, the microparticles carrying the cells containing the proteins are mixed together for rapid and direct screening the functions of the multiple proteins or any given intracellular proteins interacting with these proteins, as well as for high-throughput screening of drugs on these proteins in a given single cell type. In another embodiment, uniquely coded microparticles are preferably contacted with a single protein of interest, and used to deliver protein to multiple cell types independently. After delivery of the protein to the cells, the microparticles carrying the protein-bearing cells are mixed together for rapid and direct screening the functions of the single protein or any given intracellular proteins interacting with this protein, as well as for high-throughput screening of drugs on this protein in multiple cell types.
[0034] Reverse protein delivery on uniquely coded microparticles allows for screening of multiple protein targets within a single experiment. For example, by placing a mixture of 25 unique uniquely coded microparticles with 25 different proteins within the wells of a 96-well microplate and carrying out surface-mediated delivery, these 25 different targets can be screened simultaneously within each well in a high throughput fashion for drug interactions, ligand binding, etc. Another advantage of using uniquely coded microparticles is that the same protein can be studied in multiple cell types. This is accomplished by separately contacting multiple cell types on uniquely coded microparticles coated with the same protein followed by mixing the microparticles together in a common well.
[0035] The use of uniquely coded microparticles as a platform for carrying out reverse protein delivery affords flexibility compared to the conventional array format. Using uniquely coded microparticles allows the assembly of arrays within the well of a microplate by adding any desired combination of coated microparticle-protein elements. This allows flexibility in the content of the arrays to be manufactured, depending on the need and desire of the end-user/customer.
[0036] In addition, this flexibility also means a greatly simplified manufacturing process. Instead of having to contact print the protein in a predefined array pattern onto a solid support (e.g., a slide or microplate well), the protein can be applied to uniquely coded microparticles in bulk lots and then mixed together in the appropriate variety and ratios to form an array in the bottoms of a microplate wells.
[0037] Without intending to limit the invention in any manner, certain embodiments of the present invention will be more fully described by the following example.
[0038] In this example, three G protein-coupled receptors are used as model proteins to be delivered into cells using reverse protein delivery approach on coded microparticles. They are neurotensin receptor subtype I (NTR1), opioid receptor subtype mu and delta2. Rare earth metal doped glass beads obtained from the Coming Incorporated are used as the coded microparticles on which cells attach, grow, and uptake the pre-coated proteins to be delivered. The resulting protein-transducted cells are used for screening compounds that can effect cell signaling pathways.
[0039] Materials and Methods
[0040] Purified or unpurified recombinant human NTR1, mu and delta2 receptors are individually solubilized in 1% dodecyl maltoside using state-of-the-art methods (for example, discussed in Sklar, L. A. Vilven, J. Lynam, E. Neldon, D. Bennett, T. A., Prossnitz, E. �Solubilization and Display of G Protein-Coupled Receptors on Beads for Real-Time Fluorescence and Flow Cytometric Analysis� BioTechniques 2000, 28,976-985). Each solubilized receptor is pre-complexed with BioPORTER (Gene Therapy Systems). In some cases, helper reagent such as DEXE-dextran or RGD peptides is added into the mixture to enhance protein-delivery efficacy. The mixture is added to a well of 96-well microplate that contains 100 rare earth doped glass beads. These beads have a dimension of 20 micron in height, 50 micron in width and 200 micron in length, and have a unique code. After a 2 hour incubation and then wash, the beads are coated with the receptor-containing complexes. Three types of beads coated with different receptor-containing complexes are added into a same well in 24-well microplate. 250000 cells in serum-free medium are added into the cells. After 2 hours, serum media is used to replace serum-free media. Within a relative short of time (generally 1-3 hours) these cells become attached, and uptake the proteins pre-coated on these coded beads. The protein-transducted cells are ready for further studies. For example, these protein-transducted cells can be used for screening compounds that can selectively activate one of these three receptors in living cells by using state-of-the-art methods, such as Ca2+ flux fluorescence assays, fluorescence cAMP assays or beta-arrestin-GFP translocation assays.
[0041] The present invention provides several advantageous and unique aspects, which differentiate it from other processes, including the reverse transfection method. A surface-mediated protein delivery to living cells is able to transduct multiple proteins in a single assay using the array format over traditional, solution-based cell protein delivery techniques. This virtue provides for simultaneous, parallel analysis of many different proteins for a desired cellular readout (e.g., apoptosis, changes in cell morphology, effects on signaling pathway, etc.). Such a high-throughput capability signifies that many more proteins can be screened per assay. The ability to screen more proteins per assay also reduces the amount of reagents consumed per assay, which can greatly reduce assay costs.
[0042] Delivery of proteins into cells has certain advantages over delivering DNA into cells by transfection. Since the protein molecule itself is being delivering into the cell and not the precursor gene, the present technique bypasses the transcription-translation process associated with gene expression. Hence, the protein will begin performing its biological functions immediately after entry into the cell, greatly shortening the duration of time until cells can be assayed for protein function. The shorter time period (<24 hrs.) required to manifest the effects of the delivered protein in the cell is due in part to an ability to bypass the transcription and translation process associated with gene expression. This feature is another advantage of the present method. Typically, one can see changes within 12 hours. In some cases, greater than 95% of proteins can be delivered in as little as 3-6 hours, or even as short as within 1 or 2 hours.
[0043] Another advantage of the method is that it can be used to assess the role of post-translational modification (PTM) on protein functions. Previously in DNA transfection methods, a protein would be modified after translation according to specific signals on the protein (e.g., glycosylation sites), which would be dependent on the availability of a correct set of enzymes in the target cell line. The present method circumvents this need to find a cell line that will perform these PTMs. Using an array of the present device, one can test the effect that different PTMs in a protein (e.g., various sugar groups), which have been engineered in vitro, have on the biological function of the protein in a cell, without need to mutate the DNA sequence, or alter the signals and/or transfect the DNA into an appropriate cell line.
[0044] Moreover, unlike gene transfection that is limited to only the expression of gene products, the present protein delivery approach can be used to transfect cells with a much broader range of biologicals, for instance, including bioactive peptides, proteins domains, proteins, antibodies, protein-nucleic acid conjugates, antibody-nucleic acid conjugates, nanoparticle-protein conjugates, multi-protein complexes, and any amino-acid containing moiety.
[0045] Furthermore, the method can make amenable studies of the same protein in different cells (e.g., a protein in differentiated and undifferentiated stem cells). For instance, even though certain mammalian cells are notoriously difficult to transfect, of the mammalian cell types tested to date according to the present invention, all were receptive to protein transduction (delivery). Another advantage of the present method is the ability to better control the biological effect by varying the dosage or quantity of protein per cell.
[0046] It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention covers modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.
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(2 of 2 customers found this review helpful)
Works like a champ!
By Greg
from Rochester, NY
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Comments about Powerstands Racing Powerstands Max Rear Lift Stand:
I love this stand, very sturdy, heavy duty metal, disassembles into U, handle, and arms so it's pretty compact when disassembled, easy pins slide in/out to assemble/disassemble. Liked it so much I bought the front head/fork stand combo for $175.
Bottom Line Yes, I would recommend this to a friend
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I would buy again!
By Mike
from Houston
I've been looking for front and rear stands for my Honda CBR for a couple of months and finally decided against going with the "cheaper" options offered on some of the other popular sites and bite the bullet for the PSR set. I am glad I did because the construction of these is top notch. I am an engineer and can tell you that the pipe that these guys use will hold up for years under the common stress of supporting the bike. My only concern is with the rod connecting to the v-notch for the spools. The rods seem to be somewhat thin and will have to watch for bending. Be careful if you're not savvy enough to assemble items without instructions because there are none (and I could not find a website that might have any either). This bothers me considering that they tell you that they are not responsible for any damage if the stands fail due to improper assembly.
As good as expected
By Bikeman
from Hemet CA
The swingarm spool v-notches had an extra-tall rear leg that actually blocked access to the axle nut. Didn't work when I switched L for R, either. A few minutes with my bench grinder fixed this problem. Comes with fast-take down pins or more permanent bolts for main frame. Handle removes with pin. Love the four solid wheels. Seems very strong, no worries with my loaded Suzuki DL1000.
(1 of 1 customers found this review helpful)
Great product
By Dr. Speed
from Connecticut
The stand works great on my CBR 1000RR. I can pick up my bike without any help; very stable.
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Bamboo Hair and Scalp Stimulator for Women
We know you want the goods as soon as possible, which is why orders received by 3pm ET leave our warehouse the very same day. We'll send you an email that day to confirm shipment and the email will include your tracking information.
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Spectral RS is the most advanced minoxidil-free formula that delivers astonishing results. It is clinically proven to increase hair growth, arrest fall out, and increase diameter of hair strands. Spectral RS uses nanosome technology to maximize delivery of compounds into the scalp and is an ideal treatment for thinning hair and diffuse hair loss. Contains research grade ingredients including Aminexil.New Price:


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From The Collaborative International Dictionary of English v.0.48 [gcide]:
Voodooism \Voo"doo*ism\, noun [Probably (through Creole French vaudoux a negro sorcerer) fr. F. Vaudois Waldensian, because the Waldenses were accused of sorcery.] A degraded form of superstition and sorcery, said to include human sacrifices and cannibalism in some of its rites. It is prevalent among the negroes of Haiti, and to some extent in the United States, and is regarded as a relic of African barbarism.
From WordNet (r) 3.0 (2006) [wn]:
1: a religious cult practiced chiefly in Caribbean countries (especially Haiti); involves witchcraft and animistic deities [syn: {voodoo}, {vodoun}, {voodooism}, {hoodooism}]
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Mission Statement
As a Catholic school we inspire our pupils with faith, hope, justice and love, enabling them to grow and live in peace with each other. We hope to follow in the footsteps of Mother St Mary, our foundress, by creating in the heart and mind of each child a strong desire to learn and achieve fulfilment and success.
So how lovely to receive this email from Christine Hayward at CAFOD:-
Dear Mrs Baines: Just a quick email to thank you, Miriam, the staff and children for the warm welcome we received at Virgo Fidelis this morning. It is such a pleasure to visit your school - the children are a credit to you. Please pass on my thanks to the children for listening so well (and patiently!) and to the pupil who assisted with the technology. The prayers and generosity of Virgo Fidelis Prep School are so very greatly appreciated - many, many lives have been saved and touched by your school's steady and thoughtful support. Thank you so much. With very best wishes, Chris Hayward
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World Book Day at Virgo Prep
Fluent reading is central to a good education and a full life. We set great store by it here at Virgo Prep as a character-building activity that underpins access to the wider learning programme. And it's fun! Our Librarian, Mrs Coates, puts a great deal of effort and imagination into encouraging the love of reading. So, of course, when World Book Day comes along, we enter into the spirit and this year is no exception. As you can see, our pupils and staff were encouraged to dress as their favourite character from literature. Judge for yourself - we all had a great literary day!


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Currently about 700 people work in the BioPark in a range of positions including PhD level scientists, business development and marketing, legal and regulatory, lab technicians, clinical healthcare workers and administrative support positions. If you are interested in job opportunities in the BioPark, please visit our tenant page for the list of potential employers and their contact information or see the list below for current opportunities.
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Our energetic and inquisitive African blackfooted penguins are visitor favorites.
Watch carefully and you might even see them communicating with each other. When penguins flap their wings or bow their heads, they're telling each other how they feel. Throwing their heads back and wings out means "I'm happy." Leaning forward and opening their beaks means "Go away."
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In a battle of mantis shrimp versus disco clam, who wins? The answer may surprise you. The peacock mantis shrimp is well known as a tiny but deadly predator, but the disco clam dishes out more than just a flashy light show. Both live side by side—but apart—in our Splash Zone exhibit!
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Come and See the Works of the Lord
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Come and See the Works of the Lord composed by Patti Drennan. For Choral, Children's Choir (SAB). Fred Bock Publications. 10 pages. Fred Bock Music Company #BG2520. Published by Fred Bock Music Company (HL.8753125).
Selected for the Steve Roddy Houston Children's Chorus Series, this joyful number dances to a 3/4 meter. Paul's words speak to children and adults alike, and teaming with composer Patti Drennan has proved rewarding. Perfect for almost any season.
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Sewing or Football???
It's not quilted, but I'm very happy to have completed the top (40"x40") for the donation quilt. I did the center mariners' compass last weekend, then added the borders this weekend. The sun face images are from a Laurel Burch panel called Celestial Dreams. I was actually done in time to join the neighbor's for their superbowl party this evening.
Searching through my stash, I found a pretty multicolor batik that will work for the backing. There won't be much room to spare but there's enough. I'll use up every scrap of the yellow fabric for the binding too, but again, there's just enough to do it.
If I'm really diligent I might even get it done before Friday when the ALS group has their next fundraising. We're working out the details on how to do the quilt raffle and aren't ready to start it yet, but it would be fun to bring it to the Walk to show everyone.
The colors in the picture came out a little dark. The two inner borders and the first two dark stripes in the outer triangles are blue and purple. In the picture it just looks like one black stripe. I'll post a better picture when it's all done.
Good for you to get this far before the Game - nothing like a deadline to motivate!
Gorgeous! You are so creative using panels-something I want to get better at. This was a too busy Panel, even for LB; you successfully broke it down and highlighted each element beautifully.
Can you do FM bubbles or pebbles? They are easy, once you get going. They would look great in the white - bigger circles in the borders or a ribbon candy. The compass only needs something simple - I'm not good at SID.
Thank you for the nice comments. This project is close to my heart. My ALS friend loves bold colors and even tho the quilt isn't for her, I wanted her to be pleased with it. I have a few of the sun face panel pieces left and am planning to make a similar quilt for her before she moves back to Puerto Rico this summer. Sadly, the ALS has started to progress more rapidly than we all hoped and she won't be able to work much longer. What a terrible disease this is! Did any of you see the segment that was broadcast on CBS Sunday Morning this past weekend? It was about an ALS physician who was diagnosed with the disease himself a few years ago. DH and I were both in tears by the end of the piece.
I agree that ALS is a wicked condition. One of the worst in MHO. I am so sorry for your friend.
You have to be proud of you quilt. It is great. I love the fabric and I am not a big LB fan.
My suggestion for quilting a mariners compass is in the ditch. I just did a small one in SBQ and I feel that it makes the points very expressive. I have also done a pillow that way. Not sure about the rest but I know I would leave the suns plain. Really like what you have done with the fabric.
Kate, I didn't see the segment, but I'm very aware of the disease and what it does to a person as one of my uncle's had it. I'm sure advances have been made since 1970 when he passed and it's donations like yours are what helps make it happen. Thank you.
A couple weeks ago Rosajoe posted that she had just bought a new book. First I laughed, then I found one too! It was the inspiration for this quilt.
Here is a link that might be useful: Rosa's post on books
Thanks Kate - I thought I had lost my mine completely! I remember now ~ all the dust bunnies from that post got in my way! :~)
Rosa is right ~ great use of that book!
I also wanted to say how wonderful what you are doing to help educate and fight ALS ~ ~ sorry for your friend. Start thinking about a very special Inspirational label for her quilt. Some labels are more meaningful than others.


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ROSE TATTOO'S GUITARIST SET TO RELEASE DEBUT ALBUM – July 2014 "Big Man Movin" the first single release of debut album "Killin' Time". Download "Big Man Movin" On the back of what has been a big year for the Dai Pritchard Band they are set to kick it up a notch with the release of […]
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For those eagerly awaiting the launch of Apple Watch, reports of a Companion app in the new iOS 8.2 Beta indicates a release is getting closer.
With iPhone 6 rumors heating up and an official announcement expected soon, it's worth taking a look back at the evolution of the Apple iPhone.
A small percentage of consumers may have experienced an issue with faulty iPhone 5 sleep/wake buttons over the past two years.
With iPhones prices already
Buyer beware the dangers of faulty device chargers, as these iPad charger users discovered.
With the launch of the iPhone 5S and 5C, Apple is covering the both high-end and mid-range cell phone
Last week marked the first time ever Apple unveiled two new iPhones at the same time at their September iPhone event. Analysts have been weighing in on...
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