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Date: date unknown
Description: Rowboat (Oriole) with four people in foreground near shoreline, with Sugarbowl in the background.
Date: 2004
Description: Jim and Donna Neitzel live on this farm at W2129 Hwy TW (Section 7). This barn has been in the Neitzel name for 100 years and will be deemed a Century Farm...
Date: 1949-02-17
Description: A teacher, probably Mrs. Regina Barnhart at Groves-Barnhart School for Secretaries, giving a shorthand test to two female students.
Date: 1950 ca.
Description: Portrait of a woman standing in a yard next to a maple tree, holding a child in her arms,
Date: 1939-12-02
Description: Group portrait of Phi Sigma Sigma fraternity formal party in the Loraine Hotel dining room.
Date: 1952-04-25
Description: Portrait of Fern (Mrs. D. L.) Kirkpatrick, trumpet soloist with the Madison Civic Symphony.
Date: 1982-05
Description: Alma Schmidt Petersen, left, walks arm and arm with her sister, Clara Theresa (Tessa) Reese on the grounds of Black Point Estate. Behind them are two of A...
Date: 1964
Description: Sandra Adickes (right), Freedom Summer volunteer teacher at Palmer's Crossing Freedom School. Students (left) are Rita Mae Crawford and Jimella Stokes.
Description: Studio portrait of a young Ho-Chunk man posed standing and wearing a suit and bow tie, flanked by two young Ho-Chunk women standing in front of a painted b...
Date: 1917 ca.
Description: Quarter-length portrait of a woman tying a scarf around her head and knotting it under her chin. The photo is one of a series demonstrating how to use a sc...
Date: 1954 ca.
Description: Group portrait of the Edgar Krueger Family standing in front of a line of trees. From left to right: Elna Krueger, Shirley Krueger Oestreich, Adelbert Oest...
Date: 1970
Description: A woman wearing a red dress, brown shoes, and a beehive or bouffant hairstyle is sitting on a rolling chair with an adjustable back at a desk. She is comp...
Description: Bride, groom, and attendants posed standing at a wedding.
Date: 1952-06-23
Description: New AFSCME union officers of Local 720 of the Dane County Courthouse and Lake View Santorium employees. Left to right: Doris Williams, secretary; Rodney S...
Description: View looking north down Ohio Street. On the left corner, Sedalia Trust Company (built in 1890) features a corner entrance with stone columns. Published b...
Date: 1938
Description: Female and male factory workers handle bags of twine in the McCormick Works Twine Mill.
Date: 1987-06
Description: The Wagner family and friends stage a stone picking bee on their farm.
Date: 1948
Description: A female factory worker creates balls of twine, possibly at the McCormick Twine Mill. Original caption reads: "This scene shows a battery of balling machin...
Date: 1915 ca.
Description: A group of students and a teacher sit at desks in a classroom while listening to a lesson on the dangers of house flies. Agricultural lecture charts about...
Date: 1946-08-10
Description: Helene Stratman-Thomas and Dr. Charles O. Lindberg, a Swedish singer.
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text: http://forum.xda-developers.com – Hello. I've noticed that, unlike my other droid based on the same SoC, the Sony Ericsson Live With Walkman, this X5 has a couple of problems when recording video, regardless of the ROM. First, when recording in low-light conditions, the framerate drops to an unusable level. Even in the photo camera, when moving around, I can see some "ghosting". The other issue concerns the very low bitrate of the audio recording, but it seems that it's improved in the ICS ROM's using the AAC codec. At least the bitrate is higher, but I haven't tried yet if that translates to a better sound recordin (HowTos)

subset: commoncrawl


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text: I chose this hotel to be close to family who were in from the north. It is a nice hotel. I don't have any real complaints. Although, when I booked online, I paid a small additional fee for a Premium Water View instead of the preserve/river. When I checked in I confirmed that this was what I was getting and she said only the 1 and 2 bedroom suites have the water view, not the standard room. I showed her my confirmation stating I was suppposed to get Premium Water. She then consulted with another employee and came back and said yes, you will have a water view on a high floor; it will be nice. We did get a nice, high view; it was still 2/3 preserve/river, but my guest actually preferred it. The private balcony is screeened in and I believe I heard her telling another guest that all the rooms are this way, so you can't take any photos from the balcony. (They probably have to be since there are no guard rails.) This hotel is not on the beach; it is near boats at a marina and there is not alot around, but it is nice and quiet, and I believe they have a free boat taxi to the near beach. They have a beautiful gazebo at which I believe weddings take place. We had breakfast at the Marker 92 restaurant, which is a bit limited in selection and pricey. In order to avoid a shortage of towels like other guests had reported, we asked for additional towels in the evening and we were accommodated quickly. One suggestion I have is a sign be put on the door of the fitness center, because I watched 4 other guests besides myself try to pull the door open, then try to use a guest key, when it actually pushed in.


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7 sports movies to be unveiled
The Tribeca Film Festival began as an idea in 2001 following the attacks on the World Trade Center in New York City. The trio of actor Robert De Niro, film producer Jane Rosenthal and real estate developer Craig Hatkoff wanted to spur the economic and cultural revitalization of lower Manhattan through an annual celebration of film, music and culture. The Festival's mission is to assist filmmakers to reach the broadest possible audience, enabling the international film community and general public ...
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For the fourth year, the Tribeca Film Festival teams with sports entertainment powerhouse ESPN for an exhilarating showcase of films celebrating the spirit of competition.
Robert De Niro, Jane Rosenthal and Craig Hatkoff founded the Tribeca Film Festival in 2001 following the attacks on the World Trade Center to spur the economic and cultural revitalization of lower Manhattan through an annual celebration of film, music and culture. Tribeca/ESPN sports films
- April 21: Festival opens
- May 2: Festival closes
KEY DATESFull schedule
PURCHASING TICKETSFor all ticketing information, including hours of operation, rules and regulations, pricing and package specifics, go to the Ticket Information page.
• The director, cast, and crew are often on hand for a Q & A after the screenings.
• The films, chosen from over 5,000 submissions, are from every corner of the globe and offer almost as many perspectives as New Yorkers have opinions.
• Almost every film is a North American, international, or even world premiere, so you could be among the first to see the next big hit.
THE BIRTH OF BIG AIR: In 1985, at the tender age of 13, Mat Hoffman entered into the BMX circuit as an amateur. By 16, he had soared to the top of the sport. Throughout his prodigious career, Hoffman has ignored conventional limitations, instead focusing his efforts on the purity of the sport and the pursuit of "what's next." Academy Award nominee Spike Jonze and extreme sport fanatic Johnny Knoxville, along with director Jeff Tremaine, showcase the inner workings and exploits of the man who gave birth to "Big Air."
FREETIME MACHOS: Matti and Mikko play for Finland's worst amateur rugby team. Overworked and domesticated, the two men long for a space to revel in their masculinity and bond with other men. Following the two friends and their teammates on a quest to end the season with just a single win, award-winning writer/director Mika Ronkainen ("Screaming Men") crafts a genuine and disarmingly funny love story about modern male friendship.
INTO THE COLD: The absolute top of the earth is a place few try to reach on foot. Even fewer succeed. With the vast arctic ice vanishing rapidly, photographer, extreme adventurer, and environmental advocate Sebastian Copeland sets out to reach the North Pole on the centennial of Admiral Peary's reach in 1909. This inspiring documentary follows their tumultuous two-month trek -- not just through piercing cold and merciless terrain, but straight into the depths of the soul.
KEEP SURFING: This kinetic and fast-paced documentary will put you right on the Eisbach in the heart of Munich, where river-surfing was invented 35 years ago. Stunningly shot with cameras literally on the surfboards, you can sense the exhilaration as they take to the water. With cameos by surfing legends like Nick Carroll and Kelly Slater, "Keep Surfing" will make you want to hit the waves!
THE LAST PLAY AT SHEA: The intersecting histories of a stadium, a team, and a music legend are examined in a documentary that charts the ups and downs of the New York Mets and the life and career of Long Island native Billy Joel, the last performer to play Shea Stadium. Set to the soundtrack of Joel's final Shea concerts, "Last Play" interweaves personal Joel interviews with exclusive concert footage -- featuring guests like Tony Bennett and Roger Daltrey.
STRAIGHT OUTTA L.A.: For rapper-turned-filmmaker Ice Cube, the emergence of gangster rap will be forever linked to the Oakland Raiders' move to Los Angeles in 1982. Ice Cube turns the camera on himself to tell how his genre-defining group NWA forged an unlikely relationship with the Raiders, a team whose swagger and style captivated LA during their troubled 13-season stay.
THE TWO ESCOBARS: Born in the same city in Colombia but not related, Andrés Escobar and Pablo Escobar shared a fanatical love of soccer. Andrés grew up to become one of Colombia's most beloved players, while Pablo became the most notorious drug baron of all time. While adeptly investigating the secret marriage of crime and sports, Michael Zimbalist and Jeff Zimbalist ("Favela Rising," TFF '05) reveal the surprising connections between the murders of Andrés and Pablo.


text:
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FeaturesDescribed as a 'from the city to the country' pack, the Berghaus Remote 20 has a laptop sleeve inside the zipped opening, walking pole attachments and a pair of mesh pockets on the side. The front zipped pocket is large enough for maps. The back has some stiffening and padding but there is no top tensioning strap and the waistbelt ...
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LULU Makes NYC Concert Debut at B.B. King's, 2/16
Britain's iconic singer LULU will make her NYC concert debut with a February 16th performance at B.B. King Blues Club & Grill, backed by an All-Star Band, featuring Will Lee, Jimmy Vivino and Rich Pagano from The Fab Faux, plus Paul Shaffer.
Lulu's name is synonymous with rock and a whole lot of soul. She first burst onto the scene in the sixties at the tender age of 15, with the enduring mega-hit Shout. Since then she has topped the charts in every decade (working with everyone from Jimi Hendrix, Johnny Cash and The Beatles to Elton John, Sting, David Bowie and Paul McCartney) and also become an accomplished actress, in such notable works as the cult classic 'To Sir With Love' and legendary guest appearances on the BBC comedy, 'Absolutely Fabulous'.
237 West 42nd Street, New York, NY 10036
Backed By An All-Star Band featuring JIMMY VIVINO, WILL LEE + Rich Pagano of The Fab Faux plus PAUL SHAFFER
February 16, 2013
Showtime @ 8:00PM
Doors Open @ 6:00PM
Tickets are $36.00 in advance, $40.00 day of show - 1-800-745-3000
General Admission Seating
First come, First seated
$80.00 VIP tickets include reserved premium seating, color show poster & a post-show meet & greet with Lulu.
About Author
- SAG-AFTRA Convention Continues, George Heller Memorial Awards Presented
- Hugh Jackman, Lady Gaga, and More to Appear on THE TONIGHT SHOW STARRING JIMMY FALLON
- David Leitch to Direct THE COLDEST CITY
- SAG-AFTRA Announces Gabrielle Carteris Re-Elected Executive Vice President at National Convention
- VH1's Primetime Ratings For Third Quarter Surge Ahead +34% Over Prior Quarter Among Adults 18-49
- Sara Bareilles, Idris Elba, Adam Lambert, and More to Visit LATE NIGHT WITH SETH MEYERS
- KEVIN HART PRESENTS PLASTIC CUP BOYZ to be Released on Comedy Dynamics 10/13
- The First Lady Joins Public Media Effort, American Graduate Day 2015
- Review Roundup: Matt Damon Stars in Action Adventure THE MARTIAN
- Original Motion Picture Score from Acclaimed Film THE MARTIAN Out Today
- Kaley Cuoco & More Attend Opening Day of MASTERS OF LA
- Mika Kaurismaki's THE GIRL KING Available 12/8
- Sony Classical Releases THE WALK Original Motion Picture Soundtrack Today
- Documentary PINK & BLUE: COLORS OF HEREDITARY CANCER Set for Worldwide Theatrical Premiere Tonight
- Warner Bros. Celebrates Release of PAN by Asking Audiences to 'Discover Your Neverland'
- THE DAILY SHOW's Trevor Noah to Participate In Politicon
- As World Preps or Release of SPECTRE, Gillette Celebrates 'Bond Moments' in Every Man's Life
- Innovid and Netflix Win 2015 IAB MIXX Gold Award
- Director Ridley Scott Incorporates GoPro into the Making of THE MARTIAN
- Special Screening of Documentary SIR IVAN: I AM PEACEMAN to Kick Off Transgender Awareness Campaign


text: I love this place. First off, you get free pickles that are outstanding! The sandwiches are usually pretty stuffed and very fresh. I would recommend any sandwich. I would also recommend asking for more pickles! Did I say that they were great. These folks are also pretty fast at service so you... read more


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I recently moved to SF from Buffalo, NY for a job that started September 1st in the Mission District. I do computer programming at a tech startup called Meraki ((website hidden)). I am 22 years old and recently graduated from Cornell University where I studied astrophysics. For fun I enjoy rock climbing, hiking and cooking. I also like to do yoga and go to farmers markets.


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Native Plants
Q. Who is Mr. Smarty Plants?
A: There are those who suspect Wildflower Center volunteers are the culpable and capable culprits. Yet, others think staff members play some, albeit small, role. You can torture us with your plant questions, but we will never reveal the Green Guru's secret identity.
Did you know you can access the Native Plant Information Network with your web-enabled smartphone?
Ask Mr. Smarty Plants is a free service provided by the staff and volunteers at the Lady Bird Johnson Wildflower Center.
Need help with plant identification, visit the plant identification page.
rate this answer
Thursday - August 19, 2010
From: Dallas, TX
Region: Southwest
Topic: Vines
Title: Propagation of trumpet vines from Dallas
Answered by: Barbara Medford
QUESTION:Can you tell me about trumpet vines, can they be rooted in water? I heard they reseed at the end of their growing season.
Campsis radicans (trumpet creeper) grows natively in the Dallas area, so they should do well where you are. We don't know if it can be rooted in water, but we do know it propagates itself to the point of madness. If you tried rooting it in water, it might take over your kitchen. Before you make any decisions about using this plant, we suggest you read this Dave's Garden Forum on Campsis radicans, especially the negative comments.
From our own Native Plant Database page on this plant, here are some more warnings:
"Native to eastern North America as far north as New York and Ontario, this vine is often cultivated for its attractive, reddish orange flowers and can escape cultivation, sometimes colonizing so densely it seems a nuisance, particularly in the southeast, where its invasive qualities have earned it the names Hellvine and Devils Shoestring. Its rapid colonization by suckers and layering makes it useful for erosion control, however, and its magnificent flowers never fail to attract Ruby-throated Hummingbirds within its range. Adapted to eastern forests, Trumpet creeper grows tall with support. It climbs by means of aerial rootlets, which, like English Ivy, can damage wood, stone, and brick. To keep it in check, plant it near concrete or an area that you can mow; mowing down the suckers will discourage them. Fairly drought tolerant within its range. Blooms most in full sun."
As an alternative, might we suggest Bignonia capreolata (crossvine)? It is more of an East Texas plant, but it grows in your area. The vines are related in that both belong to the Bignoniaceae Family, but while Crossvine also can grow rather vigorously, it is a little more controllable that the trumpet vine. Here are its propagation instructions:
"Propagation Material: Seeds , Softwood Cuttings , Root Cuttings
Seed Collection: Collect the large, woody capsules from late summer through fall when they are light brown and beginning to dry. Seeds remain viable one year in sealed, refrigerated containers.
Seed Treatment: Seed requires no pretreatment.
Commercially Avail: yes
Maintenance: Training to avoid crowding of stems will aid in the formation of flower shoots. Branches can be cut back in the spring to encourage flowering."
From our Native Plant Image Gallery:
More Vines Questions
Burned leaves on trumpet vine, Campsis radicans
August 10, 2009 - I have a trumpet vine, it is about 10 years old and I have never had any problems with it. This year I have noticed that the leaves on some of the branches are shriveling up, like it was burned with ...
view the full question and answer
Propagate a trumpet vine from a cutting in Toms River NJ
July 17, 2009 - Can you propagate a trumpet vine from a cutting?
view the full question and answer
Vine for yard with morning sun in California
May 24, 2014 - Hi, What is a good perennial vine to grow on the front of our house? We would like a fragrant flower too. It will be on a wood support beam, helping cover some pipes we have there. It will be an e...
view the full question and answer
Eliminating evasive Celastrus orbiculatus (Oriental bittersweet)
July 21, 2013 - I have Oriental Bittersweet growing pervasively in my shrub garden, strangling my shrubs and growing into my beautiful Victorian porch. I can't keep up with it! What can I do?
view the full question and answer
Are grape vines killing oaks in San Antonio
December 20, 2009 - I have some large live oaks growing in sand on some property I have south of San Antonio that are slowly dying one limb at a time. Some large mustang grape vines are growing in and around them and I ...
view the full question and answer
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Home Concert Tickets Jazz and Blues Joe Bonamassa
Joe Bonamassa Tickets
Find the Joe Bonamassa tickets you're looking for on viagogo, the world's largest ticket marketplace. Select an event to view the range of tickets available or view the Joe Bonamassa tour dates to find an upcoming event near you. You can also sell your spare Joe Bonamassa Concert Tickets on viagogo, and listing tickets is free. All Joe Bonamassa tickets are covered by the viagogo Ticket guarantee!
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In This Issue:
2012 Edition of Assessment Law; Countywide Reassessments; Annual Property Valuations Appeals; and Welcome Stranger.
Excerpt from Countywide Reassessments
Lehigh, Lebanon, Erie, Allegheny and Bedford County all are conducting countywide reassessments this year. That is, each parcel in those counties received a reassessed value and property owners either have received or will receive a reassessment notice in the mail with the new value. The deadline to file an appeal from a reassessment notice is 40 days from the date of the notice. If you are a taxpayer in one of those counties and have not already done so, you should immediately examine your reassessment notice to see if it reflects actual market value. It is important to note that the fact the notice indicates that your estimated taxes will not go up, does not mean that your property is properly assessed.
Please see full issue below for more information.
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DISCLAIMER: Because of the generality of this update, the information provided herein may not be applicable in all situations and should not be acted upon without specific legal advice based on particular situations.
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Install the base first, then add the crystals, right? (Embarrassing to ask!)
I love this accent for my bathroom. It sets a mood that relaxes me. Great buy for the price. It is not very bright so don't buy if you need adequate light....
Floating Crystal Bubble and Chrome Compact Flush Mount Chandelier
ITEM#: 14339930This uniquely designed chandelier features an iron and steel base with chrome-finished fixtures and clear crystal. The three-light indoor chandelier is chainless.
- Chain: N/A
- Setting: Indoor
- Fixture finish: Chrome
- Clear crystal
- Base: Iron, steel
- Number of lights: 3
- Requires three (3) 60-watt (GU10) bulbs (not included)
- Overall dimensions: 11 inches wide x 27 inches high
- Ceiling cap dimensions: 11 inches wide x 3 inches high
This fixture does need to be hard wired. Assembly is required. Professional installation is recommended.
|Lighting Type
|Flush Mounts
|See description
|See description
|Model Number
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Customer Reviews
- Disclosures & Guidelines
- Filter By:
- Most Helpful
- Most Recent
Love it!!
I love this accent for my bathroom. It sets a mood that relaxes me. Great buy for the price. It is not very bright so don't buy if you need adequate light.Read More
bits of sparkle
want to put some life in that ol' bathroom ceiling? Put an inexpensive chandelier with halogen lights, puts out great light and sparkles all over the room...looks great and puts a new twist to any decorRead More
The chandelier is just beautiful. The quality is great and it really changes my dining room. It was a little time consuming to string the crystals and then hang them on the mount but not hard and I did not mind. You really cannot complain at the price--I get so many compliments, people think I spent much more than I did. My sister has a similar one in her kitchen and hers is just as great! Definitely recommend.Read More
Rave reviews
It's very beautiful~ The electrician wasn't happy about installing it. But Everyone wants to know where I got it from. When it's on, the Chrystal sparkle.Read More
Modern looking chadelier
The light fixture took some time to install, just to put all the crystals together but once installed it looked great. It is a must-haveRead More
Floating Crystal Bubble Chandelier
Just a note to anyone considering purchasing this-every crystal globe has to be attached to a wire, and then you need to attach it to the light fixture. This ended up being a lot more time consuming than I had originally expected BUT the overall effect is great and I am very pleased with the light fixture. I have a very small dining room (8' x 9') and this fits the room just fine and looks very pretty. However the light is directed straight down so you may want to consider some other lighting options for the room (or just deal with a somewhat dark room). I opted to just use candles to add to the light in the dining room for now, but will need to figure out another option for a later date. I would not call this a high quality fixture, the globes are attached to wires that are similar to beading wires (used for necklaces) and you use a small key ring type thing to attach the globes on. Then on the other end of the wire is another key ring which you use to attach the wire & globe to the overall fixture. The small loops the wires attach through look like silver twist ties-so I have no idea how well everything will stay in place. Luckily we don't have children, because I am sure on hard pull and it will yank it out. Overall I am REALLY pleased with the effect and I think it looks quite nice. Even my friends have been asking me to help them put one of these together for their place so I am glad I have a new unique light fixture that was budget friendly.Read More
Questions & Answers
Thank you for your question. Chandeliers like these can be assembled either way. Other customers have suggested attaching the crystals first so your not trying to do it high up on a ladder or something of that nature. I really hope this helps you. Have a great day!
Can this be installed where I now have a recessed light?
Thank you for your inquiry. It is possible to depending on the size and wiring. We would recommend consulting a professional regarding the topic.
Are the bulbs halogen or LED? And where do you buy them?
Thank you for your interest. These bulbs for this fixture are LED. You can find them on our site, at a major hardware store, or a lighting store. Please feel free to contact us again with any further questions.
How bright is this light fixture?
This fixture is designed to take three 60-watt bulbs.
Can this product be on a dimmer switch?
Yes, it can be wired to a dimmer switch.
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Best Light Fixtures for Your Dining Room
Best Light Fixtures for Your Dining Room from Overstock.com. Our guides provide customers with information about the best light fixtures for dining rooms and advice about our many brand-name products.
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Summer by the Lake
You're invited to enjoy a weekend by the water. Paddle around in a canoe or settle into the hammock with a good book. Everything is better at the lake.
2015 Lighting Trends to love
If you want to refresh your home this spring, new pendants and lamps help create a chic, brightening effect.
Dipped in Gold
The glitter and glamour of gold is catching the eye as a 2015 home decor trend.
Wedding Bells At Pride Parade
"It's not just gay marriage, to us it is marriage. Whether it's gay, straight, lesbian, or anything in between, every person should have the right to marry whomever they want and to be happy."
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text:
The Saguaro Sabercats baseball team won Tuesday's away non-conference game against Queen Creek by a score of 6-4.
(March 27, 2012: Queen Creek, AZ 85242) The Saguaro Sabercats (Scottsdale, AZ) baseball the host Queen Creek Bulldogs (Queen Creek, AZ), 6-4 in Tuesday's non-league contest.
Coming up next, the Sabercats encounter the Coronado Dons (Scottsdale, AZ) in a non-league outing, on Wednesday, March 28. Saguaro will attempt to advance on its 15-3 season record. The Dons enter the outing with a 7-3 record after their 4-1 league win over Fountain Hills (Fountain Hills, AZ).
Next, the Bulldogs face the Perry Pumas (Gilbert, AZ) in a non-league outing, on Friday, March 30. Queen Creek will attempt to advance on its 11-7 season record. The Pumas enter the outing with a 9-10 record after their 8-4 league loss to Highland (Gilbert, AZ).
Saguaro's stats are presented by GameChanger: live mobile scorekeeping for baseballGameChanger Media, Inc (ai)


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Press Releases
- Federal Judge Blocks Legally Married Same-Sex Couples in Texas From Accessing FMLA Benefits
Mar 26, 2015
- Senator Inhofe Throws Red Meat At Anti-LGBT Base
Mar 24, 2015
- HRC Calls on Former Gov. Jeb Bush to Explain Support for Anti-LGBT "Religious Freedom" Legislation
Mar 23, 2015
- HRC Poll Shows Overwhelming Support for Federal LGBT Non-Discrimination Bill
Mar 17, 2015
- Vice President Biden Affirms Support for Federal Non-Discrimination Protections for LGBT People
Mar 06, 2015
- Ben Carson Faces Blowback After Human Rights Campaign Calls Him Out for His Anti-LGBT Rhetoric
Mar 05, 2015
- Hundreds of Nation's Top Companies Urge Supreme Court to Strike Down Same-Sex Marriage Bans
Mar 05, 2015
- When Did Ben Carson Choose to be Straight?
Mar 04, 2015
- HRC Commends Appointment of Benjamin Mizer as Interim Head of Civil Division at Justice Dept
Mar 02, 2015
- As Marco Rubio Gears up 2016 Presidential Campaign, HRC Highlights His Opposition to LGBT Equality
Feb 27, 2015
- As Jeb Bush Gears up 2016 Presidential Campaign, HRC Highlights His Opposition to LGBT Equality
Feb 27, 2015
- HRC on Jeb Bush: An "LGBT-friendly" Republican Should Support Marriage Equality
Feb 27, 2015
- As Ted Cruz Gears up 2016 Campaign, HRC Website Highlights His Opposition to LGBT Equality
Feb 26, 2015
- As Mike Huckabee Gears up 2016 Campaign, HRC Website Highlights His Opposition to LGBT Equality
Feb 26, 2015
- As Some Candidates Try to Duck, HRC Unveils Website Highlighting GOP Candidates' Record
Feb 25, 2015
- Department of Labor To Implement FMLA Rule Change Impacting Married Same-Sex Couples
Feb 23, 2015
- Defense Secretary Ashton Carter Addresses Transgender Service
Feb 22, 2015
- New Video Featuring Edie Windsor and Roberta Kaplan Highlights Importance of "The People's Brief"
Feb 19, 2015
- New EEOC Memo Says Title VII Protects Workers from Discrimination Based on Sexual Orientation
Feb 18, 2015
- HRC Praises Selection of Eric Fanning as Chief of Staff for the Secretary of Defense
Feb 18, 2015


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Now, the interesting question is why is Palestretz is so shy as to share details about what is going on in Lybia, Tunisia, and Egypt. At least its English speaking edition. Zaman in Russian edition sports 3 page editorial on the subject. JPost, hebrew edition, had a monthly from its well regarded reporter. And rest of Israeli media aflame about the troubles. Pelestretz, on the other hand, is all pinky pie. Sharia in Lybia? - ears plugged. Sharia in Tunisia? - ears plugged. Sharia in Egypt? - ears plugged. Now, they have a report from the constellation of Tau - Kita. About things they can't hear about because they have... technical difficulties.
Second rocket fired from Gaza explodes inside Strip (Haaretz)
from the article: Why did Israel attack Syria now, and why did the Syrians admit it?


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Image Unavailable
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Warning: Protective equipment should be worn. Not to be used in traffic.
|Manufacturer recommended age:
|10 months - 3 years
|Manufacturer reference
|Main Language(s)
|Best Sellers Rank
381,746 in Toys & Games (See top 100)
|Delivery Destinations:
|Visit the Delivery Destinations Help page to see where this item can be delivered.
|Date First Available
|24 April 2014
Smart Trike Spirit Smart Trike's new 4 in 1 tricycle designed for babies from 10 months that grows with the child!
With the lightest touch Spirit maneuvers like a stroller. The unique navigator button on the mudguard lets one switch from parent Touch SteeringTM to child ride-control.
Built-in suspension gives baby a 100% comfort ride.
Stage 1: At 10 months baby safely uses foot rest & has a padded high-back support.
Stage 2: At 18 months the seat converts while parents push and control the trike. Baby enjoys the ride while developing confidence, balance & motor skills.
Stage 3: At 24 months it becomes a training trike - kids learn to pedal.
Stage 4: At 30 months, remove parent handle, switch navigator button and the product becomes an independent trike that kids pedal by themselves.
The functional, washable, padded seat cover coordinated with trendy printed material canopy and bag gives a fun, fashionable look to the product!
Versatile and safe - it's the adorable convertible.
Lightweight parent steering handle equipped with a parent bag.
Patented Touch Steering® technology
Sturdy wide grip parent handle
Washable padded seat cover
Seat belts with shoulder pads
Bottle holder
From 10 Months to 36 Months


text:
Over seven million people in the UK have private medical insurance (PMI) to cover all or part of the cost of private medical treatment if they fall ill. With constant headlines about waiting lists and problems in the NHS, it's easy to see the appeal of private treatment, but is it worth the cost for you?
This article on private medical insurance (PMI) is written by Jackie Griffiths, a freelance journalist who writes health, medical, biological, and pharmaceutical articles for national and international journals, newsletters and web sites.
The advantages
People may choose PMI for a wide variety of reasons, but for most the biggest advantage is the peace of mind knowing that you can get the treatment you need, when you need it, without the anxiety of waiting. With limited funds prioritised towards urgent cases, you could wait months for a non-urgent operation on the NHS. With private medical insurance, you could have the same operation within a few days of making the decision to operate. Other advantages include:
- Convenience – with PMI you choose when and where you have your treatment, rather than fitting around a date given by the NHS.
- Choice – with PMI you can choose where you're seen and by whom.
- Quality – most private patients enjoy a single room with high-quality features, such as en-suite bathroom and satellite TV.
- Cleanliness – without the high turnover of patients, and with more staff, private hospitals have more time for cleaning, reducing the risks of super bugs.
- Service - without the high pressures under which NHS hospitals operate private consultants, doctors, and nursing staff also tend to have more time to spend with each patient.
Modern private medical insurance also has the advantage of being affordable, offering a choice of cover levels to suit a wide range of budgets.
The disadvantages
In an ideal world, your PMI policy would cover you for everything at a premium you could afford. In reality, the level of cover will vary according to the price you pay. As with anything, you get what you pay for. This means that you may find that your private medical insurance doesn't cover you for everything you presumed and you may be let down when you need it most. Generally, PMI policies will only cover curable, short-term illness or injury, with more complex conditions, such as cancer only available as an added extra.
Private medical insurance is unlikely to cover you for existing conditions, even if they have not been fully diagnosed when you take out your policy. This means that it may not provide the treatment you need most. It's important to be sure exactly what your PMI includes before you take out the policy.
Another disadvantage is that the cost of private medical insurance cover will increase with age, just as you're more likely to need it. This can leave you in a 'Catch 22' situation since changing policy providers will exclude any pre-existing conditions once again, so you could find yourself trapped with ever increasing premiums.
Another consideration is the size and speciality of most private hospitals. Although you're unlikely to be visited by a consultant with a group of student doctors in tow, you could miss out on the breadth of experience available in a large, multi-disciplinary teaching hospital. Furthermore, many private hospitals and clinics do not have intensive care support, so should anything go wrong you will have to be transferred to the local NHS unit.
How you can reduce your insurance costs
If, on balance, you decide to proceed with private medical insurance, there are many ways you can reduce your premium costs:
- The less conditions that are covered, the less you will pay, so you may decide to limit your PMI to the conditions that concern you most. For example, BUPA provide a heart and cancer only cover.
- Limit the range of hospitals available to you. By excluding the top hospitals, or those in places like London, you can cut the cost considerably.
- Limit the extras your PMI includes. For example, you can pay to cover just consultations and in-patient treatment, and exclude extras such as physiotherapy and convalescent care.
- Shop around. With more than a dozen leading PMI providers, and many more joining the market all the time, it's worth comparing prices to get the best deal. Make sure you compare like for like to get an accurate picture of a policy's value. As with all insurance, some of the best deals are available online.
- Include an excess on your policy. Paying the first £500 or £1000 will cut the cost but can also reduce the policy's effectiveness.
One of the best ways to cut the cost of private medical insurance is to get your employer to take out a group policy or to take a joint policy with other members of your family.
When considering the pros and cons of private medical insurance, it's worth remembering that everything that's covered by your policy is available to you free on the NHS. You need to decide how important the benefits are to you, and whether these benefits are worth the cost.


text:
Summer Class Schedule Available
Barbara Kennedy, Silversmithing Classes, Private Lessons, Lessons Available upon request
call for details 503.720.2925.
Certified PMC Instructor.
My Etsy Gallery
Checkout my wedding ring gallery
New Classes: Custom Classes -
Studio Time & Workshops Available for Beginners to Advanced Students.
Make & Take - Your Project is completed in a class..
Check class calendar for hours. *Master Card and Visa Accepted.
Open Lab Time Studio open for Experienced students - 3 hour minimum - work on your own pieces, materials available.
Metalsmithing Classes - hammered silver chain, stacked rings, bezel set stones,.Viking Knit,
Bead Workshops, and Silver Metal Clay.
To sign up for Metal Classes
contact Barbara at email@example.com
Phone 503.720.2925
Monday, Wednesday, Friday, Saturday
Studio location:
1500 NW 18th Ave. Suite 108
Portland, Oregon 97209


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- Home
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Sep 2015
Attorney Bruce Fein is interviewed after filing lawsuit at U.S. District Courthouse.
Sep 2015
Monday, Gallup published a new poll showing that 49% of Americans think that "the federal government poses an immediate threat to the rights and freedoms of ordinary citizens."
This is up from 30% in 2003.
Gallup also asked participants, "In what ways do you see the government posing an immediate threat to rights and freedoms of its citizens?"
Sep 2015
No hair jokes, just issues.
Nicholas Sarwark, Chair of the Libertarian National Committee issued the following statement:
Libertarianism is the idea that you have the right to pursue happiness in any way you wish as long as it doesn't hurt people or take their stuff. We support the fundamental rights of all people, all of the time. We do not denigrate or disparage any group. We respect the humanity of all people.
Sep 2015
The Libertarian Party applauds the efforts of Campaign Zero to go beyond merely protesting the injustices perpetrated by law enforcement on citizens of the United States to proposing specific, well thought-through changes in government policy. They include:
Jul 2015
Republican politicians and their allies are wailing over the recent U.S. Supreme Court ruling which affirmed provisions of Obamacare, blaming the president for the onerous, 17,000-page body of law that is driving up the cost of health insurance premiums and reducing health care access for Americans.
But Republicans are just as much to blame for Obamacare as the president.
Jun 2015
Libertarian Party four-decade advocacy for marriage equality pays off with US Supreme Court decision
In a 5-4 ruling today, the US Supreme Court held that the Fourteenth Amendment requires states to license same-sex marriage and recognize those marriages entered into in other states.
Libertarian candidates, including the party's 1976 presidential nominee, Roger MacBride, have been bravely calling for marriage equality since long before it was politically correct. In fact, they've been doing so when it was considered downright dangerous.
Jun 2015
The Libertarian Party joined Level the Playing Field, the successor group to Americans Elect, and the Green Party to file a lawsuit aimed at the Commission on Presidential Debates (CPD), a Democratic- and Republican-run agency that sets rules designed to exclude third parties and independents from presidential debates.
The lawsuit is filed against the Federal Election Commission (FEC), which is also run by Democrats and Republicans and which refuses to enforce laws that the CPD is blatantly violating.
Jun 2015
Secretive Trans-Pacific Partnership trade bill lets foreign governments and foreign special interests control American medical care, banking, the Internet, and even civil liberties
Republicans howled when Nancy Pelosi famously said, "We have to pass [Obamacare] so that you can find out what is in it." Now GOP lawmakers, who control the U.S. House, are following suit in their passage of a new Trans-Pacific Partnership (TPP) trade bill.
Sep 30
Sep 29
LP Video
View more videos here at LP.org
and on our YouTube channel!
Elected Libertarian
Robert Stephens
Elected to Springfield Mayor
Elizabeth Dahl
Elected to Crystal City Council Ward 1
Russ Monchil
Elected to Mirabile School Board
Joshua Katz
Elected to Planning Commission, Westbrook


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transmissive(adj.) A type of LCD screen in which the pixels are illuminated from behind the monitor��s screen (opposite of the user). Transmissive LCDs are commonly used in laptop computers because they offer high contrast and deep colors and are well-suited for indoor environments and low-light circumstances. Transmissive LCDs are at a disadvantage in very bright light, such as outdoors in full sunlight, as the screen can be hard to read.
Compare to reflective and transflective.
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From wacky alarm clocks to lecture hall tools and after class entertainment, these Android apps are a good fit for a student's life and budget. Read More »Sharing Threat Intelligence
A growing number of startups make the sharing of threat intelligence a key part of their solutions. Read More »Smiley Faces and Symbols
A text smiley face is used to convey a facial expression or emotion in texting and online chat conversations. This Webopedia guide shows you how... Read More »
The Open System Interconnection (OSI) model defines a networking framework to implement protocols in seven layers. Use this handy guide to compare... Read More »Network Fundamentals Study Guide
Networking fundamentals teaches the building blocks of modern network design. Learn different types of networks, concepts, architecture and... Read More »Computer Architecture Study Guide
This Webopedia study guide describes the different parts of a computer system and their relations. Read More »


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This hotel is perfectly located, walking distance from Laax lifts station and restaurants. The rooms are nice and clean and the service is good overall. The indoor parking is super convenient (for a fee). The quality of the breakfast is good too. All in all, good value for money in a great location. Highly recommended for a weekend stay!
If you own or manage Hotel Casa Selva, register now for free tools to enhance your listing, attract new reviews, and respond to reviewers.


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text: July 28, 2015 Postcode: L34 Ben and his crew did a very good job of installing a combi-boiler and extending the hot water supply to additional parts of my property. They disposed of the old boiler, hot water tank and other bits and pieces and cleaned up before they left, and were as courteous as they were efficient. Recommended! Service: Value: Get details »


text:
Motel 6 Sacramento
Today:Open 24 Hours
227 Jibboom St,
Sacramento, CA 95811
(916) 441-0733
Hotel Rooms
Secure online booking with Expedia Affliate Network Call 866-761-3335 to book by phone
The room rates listed are double occupancy per room unless otherwise stated and exclude tax recovery charges and service fees.
Business Details
- Hours:
Regular Hours
- General Info:
- Since its light first turned on in 1962, Motel 6 had been synonymous with great value travel. Our brand promise – providing a clean, comfortable room and great service for the lowest price of any national chain – remains, but much has changed in the last 50 years. A passion for growth and innovation, in addition to our unwavering commitment to a great guest experience, is how we have and will continue to leave the light on.
- Extra Phones:
Fax: (916) 446-5941
- Payment method:
- amex, discover, master card, visa, check, cash
- Location:
- With a stay at Motel 6 Sacramento-old Sacramento North in Sacramento, you'll be convenient to American River Bike Trail and California State Railroad Museum. This motel is within close proximity of Discovery Park and Sacramento History Museum.
- Neighborhood:
- Central Sacramento
- Other Link:
- Categories:
- Hotels, Lodging, Motels


text:
Bravo School of Art at Liberty Station
- 2690 Historic Decatur Road, Barracks 19, Studio 206, San Diego, CA 92106 | Directions
- 619-223-0058 | Website
Bravo School of Art offers over 50 different hands-on art, craft- and home-decorating classes, workshops, and summer camps for adults and children to explore their creativity in a safe, supportive, and fun environment. Taught by experienced artists, these workshops provide the creative outlet you've been seeking.
In our studio classroom, students are encouraged to color outside the box and to escape the daily grind by getting creative.
Workshops designed for creativity and self-improvement may include Mosaic, Book Arts, Digital Photography, Mixed Media, Drawing, Painting, Hat Making, Basketry, Jewelry Making, Printmaking, The Artist's Way, and more.
There is something for everyone. No previous art training is required.
Upcoming Events
Painted Furniture
Wednesday, October 7, 10:00 a.m.
Mosaic Address Sign
Sunday, October 11, 10:00 a.m.
Chalkboard Lettering
Monday, October 12, 10:00 a.m.
Paint Your Sneakers
Monday, October 12, 2:00 p.m.
Saturday, October 17, 2:00 p.m.
Creative Beginning Embroidery
Monday, October 19, 10:00 a.m.
Garden Art
Wednesday, October 21, 10:00 a.m.
Point & Shoot Digital Photography
Wednesday, October 21, 6:00 p.m.


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Not since his first book, Authentic Mexican, has there been such an accessible opportunity to learn about real Mexican cooking. Rick Bayless's Mexican Kitchen offers translations of authentic Mexican dishes that preserve their authenticity. The book opens with 14 salsas, sauces, and seasonings that Bayless calls "cornerstones of Mexican dishes." Other than some chile peppers essential to certain dishes, most ingredients are found in any supermarket. For any less common ingredients, a mail-order source or an easy substitution is provided. This brilliant book is engaging, informative, and inspiring. --This text refers to an alternate Hardcover edition.
This definitive collection from Chicago chef and James Beard Award winner Bayless, in collaboration with his wife (and fellow restaurateur) and food journalist Brownson, proves comprehensively that the best Mexican food requires?and amply rewards?dedication and, often, time. Bayless begins with 15 Essential Recipes for salsas and sauces that work as "building blocks." Substitutions are suggested for uncommon ingredients, and excellent descriptions identify fresh and dried peppers. Throughout the text, sidebars inform about such items as tortilla presses, cactus paddles, pumpkin seeds and the delicacy huitlacoche (black corn fungus). Bayless explains fat's importance in the Mexican diet and tells how to make good lard at home. The chapter on salads includes two versions of guacamole, one given a fresh twist with roasted tomatillos; the chapter on soups offers Chilied Tortilla Soup with Shredded Chard and Oaxacan Black Bean Soup. An array of authentic Mexican fare is explored in "Tacos, Enchiladas and Other Casual Fare" (Simple Red Mole Enchiladas with Shredded Chicken) and "Vegetable, Bean, Rice and Egg Dishes" (e.g, Green Poblano Rice). "Fiesta Food" includes recipes for moles and tamales. Gringo cooks can relax with simpler main dishes?Red Chile-Braised Chicken wreathed in ancho and garlic sauce, smoky Chipotle Shrimp or zesty Chile-Glazed Country Ribs. Desserts are as delectable as Modern Mexican Chocolate Flan and as unusual as Crunchy Amaranth Tart and Creamy Lime Pie. Mail-order sources and a bibliography are included. Photos not seen by PW.
Copyright 1996 Reed Business Information, Inc. --This text refers to an alternate Hardcover edition.
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Rick is very knowledgable and definitely the go to guy for Mexican cuisine .Published 6 months ago by linda k gates
great fast shipping, awesome item, better than described, would buy from again! Thanks, happy buyer.Published 6 months ago by dana dana
It is very evident when you first peruse this book that a lot of time and research went into creating an authentic experience with each dish. Read morePublished 7 months ago by D. White
Oh I do love this book! It is fun to explore authentic Mexican food, which does not resemble that which you receive at an Anglicized local cafe. Read morePublished 8 months ago by Katherine Howard


text: Wal-Mart Grand Re-Opening April 27, 2013 | 1:00 PM - 3:00 PM Where Wal-Mart Harmony and College, Fort Collins, CO 80525 United States Additional Information Join K99 and Pepsi for the grand re-opening of Wal-mart on Harmony and College in Ft. Collins! Share on Facebook Share on Twitter More Leave a comment Send to a friend! Print this page Pin it! Reddit This! Share on Tumblr! Discuss This Event


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Middle East recruiters expect new jobs in next 6 months
Naukrigulf survey reveals job creation and hiring much better in 2015 compared with 2014October 13, 2015 10:17
Register for our free newsletter
MIDEAST STOCKS-Markets to rise on stronger global environment
Abu Dhabi's ADWEA in talks with banks for $350 mln project bond
Emirates hints at no Dubai Airshow orders, pushes decision on A350/787 to next year
UAE minister says plans to boost oil output capacity on track
MIDEAST STOCKS-Saudi, Egypt markets barely move in early trade
Qatar Q2 GDP growth accelerates to 4.8 pct year/year
UAE says reforms will better protect foreign workers
United Arab Bank secures $125 mln three-year loan
MIDEAST STOCKS-Saudi hit by weak oil, commodities; Egypt down modestly
MIDEAST STOCKS-UAE markets narrowly mixed in thin, early trade
In case you missed these, here's the world's most expensive perfume; some gift ideas and a list of everything in 2011
December 9, 2011 12:12 by p.deleon
Smell good while doing some good: A million-dollar fragrance for a cause
Here's your chance to smell good and do good. DKNY Fragrances has partnered with celebrity jeweler Martin Katz to create the world's first million-dollar fragrance bottle. But don't go in a huff. They say the net proceed will go to Action Against Hunger.
Check out the video below to see how the pricey perfume was made:
The DKNY Golden Delicious Million Dollar Fragrance Bottle is carved from polished 14-karat yellow and white gold and features 2,700 Round Brilliant White Diamonds weighing 15.17 carats, 183 Golden Yellow Sapphires weighing 2.28 carats in a micro pave setting in 14kt. white gold making up the Manhattan skyline that is so iconic to the DKNY brand. The 2.43 carat Flawless Vivid Yellow Canary Diamond adorns the "cap" of the bottle alongside a DKNY Golden Delicious diamond oval logo. Whew.
Think the title above is total gibberish? Then you're out of touch. Apparently LMS is the top meme on Facebook. LMS as the silver generation among Kipp found out means "Like My Status". Well that's what Facebook is about isn't it really? This constant campaign and marketing of one's self to your peers? Oooh check me out with my cool hair, my duck face and love for funny internet quotes. As if. And if it wasn't bad enough, LMS was sometimes partnered with TBH "To Be Honest", as in LMS for a TBH. In this case, anyone who likes the status will be given an honest opinion from the person who posted the status. FML. Like so totally. Check out the title link above for other Facebook Memes.
Don't send us anything from this list of gift ideas
It'll take a special person to appreciate the items on this list. Have a look through the list. And if you find there's something here that one of your friends or family members would like having, then we'd seriously reconsider keeping a connection with them. This.is.just.weird. C'mon, you know you want to click that title link.
The Stories You Missed in 2011
We are inundated with news and stories this year. There were a lot of heart-wrenching and soul-searching stuff. But just in case you missed it, here's a slideshow of things that you may have missed in between your tweeting and Facebook preening. Oh yeah, just click the title link above already.
In the spirit of every goshdarnit list that you will ever see in the countdown to 2012, here is A LIST OF EVERYTHING 2011 (say with booming James Earl Jones voice) brought to you by Time.com. It's got pretty much everything. That's about a couple of hours of your time taken out. You're welcome, Kippers.
Dubai says no plans to restructure state firms' debt
December 8 2011
South Sudan: Land of opportunity, if you don't mind risk
December 10 2011


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Fisherman's Stew
Our recipe is a speedy version of cioppino -- a California specialty of fish simmered in a garlicky tomato broth. We cut back on the variety of seafood usually called for, helping us shave off a considerable amount of prep time.
Level: Easy
Advertisement - Continue Reading Below
- 1 tbsp. olive oil
- 1 medium red pepper
- 1 medium red onion
- 2 clove garlic
- .13 tsp. crushed red pepper
- ½ c. dry white wine
- 2 can diced tomatoes
- ½ tsp. salt
- 1 lb. cod fillet
- 1 lb. large mussels
- ½ lb. large shrimp
- ¼ c. fresh basil or parsley leaves
- In nonstick 5- to 6-quart Dutch oven, heat oil over medium-high heat until hot. Add chopped pepper and onion, and cook 6 to 8 minutes or until tender, stirring occasionally. Add garlic and crushed red pepper, and cook 1 minute, stirring. Add wine and heat to boiling; boil 2 minutes.
- Stir in tomatoes, salt, and 1/2 cup water; heat to boiling over medium-high heat. Add cod, mussels, and shrimp; heat to boiling. Reduce heat to medium-low and simmer, covered, 8 to 9 minutes or until cod and shrimp turn opaque throughout and mussel shells open. Remove Dutch oven from heat; sprinkle with basil.
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14 Genius Tricks to Keep Fresh Food From Spoiling Fast
Because you didn't pay $4.50 for a pint of moldy strawberries.
Prince Harry and William Ate These Mouse Cupcakes
And now you can, too!
How to Brew the Perfect Cup of Coffee
Everything you need to know — from the beans to the temperature.
McDonald's Will Soon Start Serving Organic Burgers
But there's only one place to get them.
More From Food & Recipes


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Page 1
Yeast 2009; 26: 507–521.
Published online 3 August 2009 in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/yea.1701
Yeast Functional Analysis Report
New selectable host–marker systems for
multiple genetic manipulations based on
TRP1, MET2 and ADE2 in the methylotrophic
yeast Hansenula polymorpha
Seon Ah Cheon1,2, Jinho Choo1, Vera M. Ubiyvovk3,4, Jeong-Nam Park1,2, Moo Woong Kim3,
Doo-Byoung Oh3, Ohsuk Kwon3, Andriy A. Sibirny4, Jeong-Yoon Kim5and Hyun Ah Kang1,2*
1Department of Life Science, College of Natural Science, Chung-Ang University, Seoul 156-756, Korea
2Research Centre for Biomolecules and Biosystems, Chung-Ang University, Seoul 156-756, Korea
3Integrative Omics Research Centre, KRIBB, Daejeon 305-806, Korea
4Institute of Cell Biology, NAS of Ukraine, Lviv, Ukraine
5Department of Microbiology, School of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, Korea
*Correspondence to:
Hyun Ah Kang, Department of
Life Science, Chung-Ang
University, 221 Heukseok-dong,
Dongjak-gu, Seoul 156-756,
South Korea.
E-mail: firstname.lastname@example.com
Received: 11 March 2009
Accepted: 23 June 2009
Interest has been increasing in the thermotolerant methylotrophic yeast Hansenula
polymorpha as a useful system for fundamental research and applied purposes. Only a
few genetic marker genes and auxotrophic hosts are yet available for this yeast. Here
we isolated and developed H. polymorpha TRP1, MET2 and ADE2 genes as selectable
markers for multiple genetic manipulations. The H. polymorpha TRP1 (HpTRP1),
MET2 (HpMET2) and ADE2 (HpADE2) genes were sequentially disrupted, using
an HpURA3 pop-out cassette in H. polymorpha to generate a series of new multiple
auxotrophic strains, including up to a quintuple auxotrophic strain. Unexpectedly,
the HpTRP1 deletion mutants required additional tryptophan supplementation for
their full growth, even on complex media such as YPD. Despite the clearly increased
resistance to 5-fluoroanthranilic acid of the HpTRP1 deletion mutants, the HpTRP1
blaster cassette does not appear to be usable as a counter-selection marker in H.
polymorpha. Expression vectors carrying HpADE2, HpTRP1 or HpMET2 with their
own promoters and terminators as selectable markers were constructed and used
to co-transform the quintuple auxotrophic strain for the targeted expression of a
heterologous gene, Aspergillus saitoi MsdS, at the ER, the Golgi and the cell surface,
respectively. The nucleotide sequences presented here were submitted to GenBank
under Accession Nos AY795576 (HpTRP1), FJ226453 (HpMET2) and FJ493241
(HpADE2), respectively. Copyright 2009 John Wiley & Sons, Ltd.
expression vector
Hansenula polymorpha; TRP1; MET2; ADE2; auxotrophic marker;
Hansenula polymorpha is a thermotolerant methy-
lotrophic yeast that serves as a useful system
for fundamental research and applied purposes.
H. polymorpha has been a favourable model in
which to study the genetic control mechanisms
of methanol metabolism, peroxisome biogenesis
and nitrate assimilation (Gellissen, 2002; Martin
et al., 2008; van der Klei et al., 2006). It is also
one of the promising microbial hosts for produc-
tion of recombinant proteins on an industrial scale
Copyright 2009 John Wiley & Sons, Ltd.
Page 2
508S. A. Cheon et al.
(de Bruin et al., 2005; Kang and Gellissen, 2005).
Moreover, peculiar physiological characteristics of
H. polymorpha, such as resistance to heavy met-
als, oxidative stress and heat, make this yeast
attractive for several biotechnological purposes
(Blazhenko et al., 2006). Recently, the capability
of this thermotolerant yeast in alcoholic fermenta-
tion of xylose at elevated temperatures has drawn
attention to the yeast as a good candidate for the
development of an efficient process for simulta-
neous saccharification and fermentation (Dmytruk
et al., 2008). With the completion of H. poly-
morpha genome sequencing (Ramezani-Rad et al.,
2003), post-genomic approaches such as transcrip-
tome (Park et al., 2007) and proteome analysis
(Kim et al., 2004b) are now feasible and enable
more systematic strategies for metabolic engineer-
ing and process improvement.
The availability of useful genetic selection mark-
ers for an organism is essential for its exploita-
tion as a cell factory, since the introduction of
a certain heterologous biosynthetic or metabolic
pathway might require expression of a large num-
ber of foreign genes, demanding versatile sets
of host strains and selection markers. Further-
more, multiple gene disruptions and subsequent
gene expression are required in investigations of
biological processes that include multiple func-
tionally redundant genes. Until now, only a few
selectable markers were available for the molecu-
lar manipulation of H. polymorpha. As biosynthetic
marker genes in combination with the correspond-
ing H. polymorpha auxotrophic strains, the ADE11
(PUR7), LEU2, MET6 and URA3 genes from H.
polymorpha were developed (Gidijala et al., 2007;
Haan et al., 2002; Kang et al., 2002). Heterologous
genes, such as Saccharomyces cerevisiae URA3 or
LEU2, are also used as selectable markers, espe-
cially for the selection of multi-copy integration,
based on their low level of expression in H. poly-
morpha (Agaphonov et al., 1999). Based on the
sensitivity of H. polymorpha to aminoglycoside
antibiotics, including G418 and hygromycin B, and
to Zeocin, selectable markers conferring resistance
to these antibiotics were developed, especially for
copy-number-controlled selection systems (Kang
et al., 2001; Sohn et al., 1999a; van Dijk et al.,
The most representative industrial application
of H. polymorpha has been the production of
recombinant therapeutic proteins (Kang and Gel-
lissen, 2005). Since more than 60% of secretory
proteins with therapeutic potential are glycopro-
teins, engineering of the yeast glycan biosynthetic
pathway to produce glycoproteins with human-
compatible oligosaccharides has been a major issue
in the development of H. polymorpha expres-
sion systems. Recently, we constructed glyco-
engineered H. polymorpha strains that were able
to produce human mannose-type N-glycans as a
major glycan (Kim et al., 2006; Oh et al., 2008).
To further modify the yeast glycosylation path-
way toward synthesis of human complex-type N-
glycans, we need more selective marker–host sys-
tems for subsequent introduction of several het-
erologous genes involved in glycan processing
into the glyco-engineered H. polymorpha strains.
Compared to auxotrophic mutant strains obtained
by random mutagenesis, defined mutants carrying
clear deletions of biosynthetic genes will be advan-
tageous for improving the vector/host systems,
avoiding possible complications derived from addi-
tional unknown mutations. Thus, here we report
the isolation and exploitation of three biosyn-
thetic genes, TRP1, MET2 and ADE2, to gener-
ate useful selective markers and new auxotrophic
strains to facilitate multiple genetic manipulations
in H. polymorpha.
Materials and Methods
Yeast strains and media
Strains and plasmids used and constructed in
this study are listed in Table 1. All H. polymor-
pha strains were derivatives of DL1-LdU (leu2
?ura3::lacZ) (Kang et al., 2002). Yeast strains,
except for the HpTRP1 deletion mutant (?Hptrp1)
strains, were routinely grown on YPD medium
(1% bacto-yeast extract, 2% bacto-peptone, 2%
glucose), synthetic complete (SC) medium (0.67%
yeast nitrogen without amino acids, 2% glucose,
all amino acid mixture supplemented with 30 mg/l
each of uracil and adenine) or synthetic mini-
mal (SD) medium (0.67% yeast nitrogen without
amino acids, 2% glucose), with appropriate nutri-
tional supplements at 37◦C. The ?Hptrp1 disrup-
tants were maintained in YPD supplemented with
90 mg/l tryptophan (YPD + 3 × T), YTD medium
(1% bacto-yeast extract, 2% bacto-tryptone, 2%
Copyright 2009 John Wiley & Sons, Ltd.
Yeast 2009; 26: 507–521.
DOI: 10.1002/yea
Page 3
New selectable host–marker systems for Hansenula polymorpha
Table 1. H. polymorpha strains and plasmids used in this study
Strain nameGenotype Reference
leu2 ?ura3::lacZ
leu2 ?ura3::lacZ ?trp1::lacZ-URA3-lacZ
leu2 ?ura3::lacZ ?trp1::lacZ
leu2 ?ura3::lacZ ?met2::lacZ-URA3-lacZ
leu2 ?ura3::lacZ ?met2::lacZ
leu2 ?ura3::lacZ ?trp1::lacZ ?met2::lacZ-URA3-lacZ
leu2 ?ura3::lacZ ?trp1::lacZ ?met2::lacZ
leu2 ?ura3::lacZ ?trp1::lacZ ?met2::lacZ ?ade2:: lacZ-URA3-lacZ
leu2 ?ura3::lacZ ?trp1::lacZ ?met2::lacZ ?ade2:: lacZ
leu2 ?ura3::lacZ ?trp1::lacZ-URA3-lacZ ?ade2::tc-TRP1-tc
leu2 ?ura3::lacZ ?trp1::lacZ? ade2::tc-TRP1-tc
leu2 ?ura3::lacZ ?och1::lacZ ?alg3::lacZ ?trp1::lacZ
leu2 ?ura3::lacZ ?trp1::lacZ-URA3-lacZ ?gsh2::lacZ-TRP-lacZ
(Kang et al., 2002)
(Kang et al., 2002)
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
Plasmid nameDescriptionReference
pDLMOX-GOD(H)Expression vector for A. niger GOD under MOX promoter with
the HpLEU2 marker
Vector for MsdS-HA-HDEL expression under MOX promoter
with the HpTRP1 marker
Vector for MsdS-HA-HDEL expression under MOX promoter
with the HpMET2 marker
Vector for MsDs-HA-HDEL expression under MOX promoter
with the HpADE2 marker
Vector for HpOch1(N-term 1-53aa)-MsdS-Flag) expression under
MOX promoter with the HpTRP1 marker
Vector for cmyc-MsdS-Flag-Tip1(C-term 40 aa) expression under
MOX promoter with the HpMET2 marker
Plasmid containing the Hptrp1::lacZ-HpURA3-lacZ cassette
Plasmid containing the Hpmet2::lacZ-HpURA3-lacZ cassette
Plasmid containing the Hpade2::lacZ-HpURA3-lacZ cassette
Plasmid containing the Hpade2::tc-HpTRP1-tc cassette
Plasmid containing the lacZ-HpURA3-lacZ pop-out cassette
Plasmid containing the tc-HpTRP1-tc cassette
Plasmid containing the lacZ-HpTRP1–lacZ cassette
Plasmid containing the HpTRP1 gene for sequencing
Plasmid containing the HpMET2 gene for sequencing
Plasmid containing the HpADE2 gene for sequencing
Plasmid containing the HpGSH2 gene
Plasmid containing the Hpgsh2::lacZ-HpTRP1–lacZ pop-out
(Kim et al., 2004a)
pDTMOX-msdSThis study
pDMMOX-msdS This study
pDAMOX-msdS This study
pDTMOX-HH1MSFThis study
pDMMOX-aaMSF-TIP1This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
glucose) or SC medium supplemented with 90 mg/l
tryptophan (SC + 3 × T). For recycling of a
selective marker gene, the HpURA3 pop-out cas-
sette (HpURA3 –lacZ blaster) was popped out on
YPD medium containing 0.5 g/l 5-fluoro-orotic
acid (5-FOA; Fermentas, EU), if necessary sup-
plemented with 90 mg/l tryptophan and 60 mg/l
uracil. For gene expression under the MOX pro-
moter, YPM (1% bacto-yeast extract, 2% bacto-
peptone, 2% methanol) was used as previously
described (Gellissen, 2002). For analysis of vector
stability, strains grown in 5 ml YPD medium
overnight were inoculated into 5 ml fresh YPD
medium by 100-fold dilution. After 24 h (ca.
20 generations), 50 µl culture was transferred to
5 ml fresh YPD medium and this procedure was
repeated to 120 generations, as described in a
previous study (Sohn et al., 1999b). Escherichia
coli DH5α for plasmid propagation was cul-
tured in LB medium (1% bacto-tryptone, 0.5%
bacto-yeast extract, 1% sodium chloride) with
ampicillin (100 µg/ml) at 37◦C.
Copyright 2009 John Wiley & Sons, Ltd.
Yeast 2009; 26: 507–521.
DOI: 10.1002/yea
Page 4
510S. A. Cheon et al.
Isolation and sequencing of HpTRP1, HpMET2
and HpADE2 genes
To determine the nucleotide sequences of the
HpTRP1, HpMET2 and HpADE2 genes, these
three genes were amplified from genomic DNA of
H. polymorpha DL1-L strain by polymerase chain
reaction (PCR), using the following primer sets:
TRP1-N NotI (5?-aaggaaaaaagcggccgccaaacctgaca
ccgcaactg-3?)/TRP1-C XbaI (5?-gctctagagaagaagct
gattgctggcg-3?), MET2 1F SalI (5?-agtgggtcgacatg
ctagccattcaagcgc-3?)/MET2 2B NdeI (5?-ctcccatat
gcgaaacaggcgttcaaacc-3?) and ADE2 1F XhoI (5?-
aactcgagcctcctgatgtgcactca-3?)/ADE2 2B NdeI
pectively (the sequences corresponding to the target
genes for disruption are in bold and the restriction
enzyme sites introduced for subcloning are under-
lined). The obtained HpTRP1 PCR product was
digested with NotI and XbaI and ligated into the
corresponding sites of the bacterial plasmid pBlue-
script II KS+(Stratagene, La Jolla, CA, USA)
to create the pBS-HpTRP1 vector. The HpMET2
and HpADE2 PCR products were subcloned into
the pGEM-T easy vector (Promega, WI, USA)
to generate pTHpMET2 and pTHpADE2 plasmids
(Table 1), respectively. Nucleotide sequences of
HpTRP1, HpMET2 and HpADE2 derived from
H. polymorpha DL1-L strain were determined
and submitted to GenBank under Accession Nos
AY795576, FJ226453 and FJ493241, respectively.
Disruption of HpTRP1 and HpMET2 by fusion
PCR and in vivo recombination
HpTRP1 and HpMET2 were disrupted with the
HpURA3 pop-out cassette (HpURA3 –lacZ blaster)
through modified fusion PCR and in vivo recombi-
nation, as previously described (Kim et al., 2006;
Oh et al., 2008). Two DNA fragments contain-
ing the promoter (TR1N) and terminator (TR1C)
regions of HpTRP1, respectively, were ampli-
fied with two PCR primer sets: TR1Nfor (5?-
cggaggatcgcaggagacgg-3?; target gene sequences
in bold)/TR1Nrev (5?-agctcggtacccggggatccgtctcg
cctgctcagggtcc-3?; complementary sequences of
a primer UNfor in italics) and TR1Cfor (5?-
complementary sequences of primer UCrev in ital-
ics)/TR1Crev (5?-cgccgaggaggtcattgctg-3?). The
N- and C-fragments (Z-UR3N and UR3C-Z) of
the HpURA3 –lacZ blaster were amplified from
the pLacUR3 plasmid with a 100 bp overlap at
the end, using two PCR primer sets: UNfor (5?-
ggatccccgggtaccgagct-3?)/UNrev (5?-caccggtagcta
atgatccc-3?) and UCfor (5?-cgaacatccaagtgggccga-
3?)/UCrev (5?-ctggcgaaagggggatgtgc-3?). Subse-
quently, the Z-UR3N and UR3C-Z fragments were
fused with the TR1N and TR1C fragments by
PCR, using two primer sets, TR1Nfor/UNrev and
UCfor/TR1Crev, to generate the fusion products
TR1N-Z-UR3N and UR3C-Z-TR1C, respectively.
The two fused PCR products with a 100 bp over-
lap in the HpURA3 region were introduced into
the H. polymorpha DL1-LdU strain for homolo-
gous recombination-mediated target gene replace-
ment in vivo with the HpURA3 –lacZ blaster to
generate the DL1-LdT strain (leu2 ?ura3::lacZ
?trp1::lacZ-URA3-lacZ). Additionally, the Hptrp1
amplified from genomic DNA of the DL1-LdT
strain by PCR with a primer set TR1Nfor/TR1Crev
and subcloned into the pDrive vector (Qiagen,
Hilden, Germany) to generate the pDHptrp1DU
HpMET2 was disrupted by the same procedure
as described above. Two DNA fragments con-
taining the N-terminal (MET2N) and C-terminal
(MET2C) regions of HpMET2 were amplified with
two PCR primer sets: MET2N 1F (5?-gaagcgtcaga
atgcctcg-3?)/MET2N 2B (5?-agctcggtacccggggatcc
catcatcacgcaccgtgca-3?) and MET2C 3F (5?-gcaca
tccccctttcgccagatcgaggactctccttc-3?)/MET2C 4B
(5?-caacgtcctcagcctca-3?), respectively. The result-
ing PCR fragments were fused with the UR3N
and the UR3C fragments using two primer sets
(MET2N 1F/UNrev and UCfor/MET2C 4B) to
make the fusion products MET2N-Z–UR3N and
UR3C–Z-MET2C, respectively. The two fusion
PCR products were introduced into H. polymorpha
DL1-LdU and the correct disruption of HpMET2
via in vivo recombination was confirmed by PCR
with primer set MET2 5F (5?-tgggcaacagaaaccatc-
3?)/MET2 6B (5?-atgacgacaaagacgagg-3?), gener-
ating DL1-LdM (leu2 ?ura3::lacZ ?met2::lacZ-
URA3-lacZ). Additionally, the Hpmet2::lacZ–
HpURA3–lacZ disruption cassette was amplified
from genomic DNA of the DL1-LdM strain by
PCR with primer set MET2N 1F/MET2C 4B and
subcloned into pDrive vector (Qiagen, Hilden, Ger-
many) to generate the pDHpmet2DU vector.
Copyright 2009 John Wiley & Sons, Ltd.
Yeast 2009; 26: 507–521.
DOI: 10.1002/yea
Page 5
New selectable host–marker systems for Hansenula polymorpha
Construction of HpTRP1–lacZ and HpTRP1-tc
pop-out cassettes
To construct a HpTRP1–lacZ blaster contain-
ing the lacZ repeat region upstream and down-
stream of HpTRP1, the 166 bp PvuII/AvaI C-
terminal fragment of LacZ, excised from pBlue-
script KS+, was subcloned into the SmaI and
SalI sites of pBS-HpTRP1 to generate pBS-
HpTRP1-LZ. To construct a pBHpTRP1-tc vec-
tor containing a HpTRP1-tc blaster with a 585
bp direct repeat of the tetracycline gene (tc), the
YlTRP1 region (XhoI–AflII fragment) of vector
pBTERP2 (Cheon et al., 2003) was replaced with
the HpTRP1 gene fragment amplified with primer
set TRP1 1F SalI/TRP1 4B AflII (5?-gactcacttaag
Construction of HpADE2 disruption cassettes
with the HpTRP1-tc and the HpURA3–lacZ
pop-out cassettes
Two parts of the HpADE2 ORF were amplified
by PCR with two primer sets: ADE2N 1F (5?-
ggcggatcccggctccttcaacgatga-3?)/ADE2N 2B (5?-
cacttcgcgatcactgctagctccttgg-3?) and ADE2C 3F
ADE2C 4B(5?-gacggatccgttaacgatgatgcaccgtatt
cc-3?), respectively. Two fragments of HpADE2
were fused by PCR with the primer set, ADE2N
1F/ADE2C 4B, and the fusion PCR product was
subcloned into pGEM-T easy vector (Promega, WI,
USA), resulting in vector pTHpade2D. Then the
HpTRP1-tc blaster cassette, which was obtained
by NheI–SpeI digestion of pBHpTRP1-tc, and the
HpURA3 –lacZ cassette, which was obtained by
NheI digestion of pTHpURA3LZ, were inserted
into the NheI site of pTHpADE2D to yield final
vectors pTHpade2DT and pTHpade2DU, respec-
tively. pTHpURA3LZ vector was constructed by
subcloning the HpURA3 –lacZ cassette, which
was amplified from pLacUR3 with primer set
HpURA3 1F NheI (5?-tatgctagcggatccccgggtaccga
gct-3?)/HpURA3 2B NheI (5?-gatgctagcctcgagcctc
ttcgctattacgccag-3?), into pGEM-T easy vector.
The HpADE2 disruption cassettes were generated
from pTHpade2DT and pTHpade2DU by NotI
digestion to transform strains DL1-LdTU and DL1-
LdTMU, respectively (Table 1), and the correct
disruption of HpADE2 was confirmed by PCR with
primers ADE2 5F (5?-cggccaagcagatcaattc-3?) and
Construction of HpGSH2 disruption cassette
using HpTRP1–lacZ
To generate the HpGSH2 disruption cassette, the
HpTRP1–lacZ blaster was amplified from pBS-
HpTRP1LZ by PCR, using a set of primers contain-
ing NcoI and AflII sites (1-F, 5-catgccatggtattacgcc
agctggcgaaa-3?; 2-R, 5?- agcggcttaagcgagggatccac
agttcta-3?). The HpTRP1–lacZ cassette digested
with NcoI and AflII was inserted into the cor-
responding sites of the pGLG61-GSH2 vector,
which contained the 4.3 kb HpaI–SmaI HpGSH2
gene fragment (Ubiyvovk et al., 2002) in the in
the backbone of pGLG61 (Sohn et al., 1999a);
this process produced the plasmid pgsh2-TRP1,
which contained the HpGSH2 disruption cassette
(Figure 3B). Plasmid pgsh2-TRP1 was digested by
AatII to generate the HpGSH2 disruption cassette
for transformation of DL1-LdT and DL1-LdTU
Construction of expression vectors
The H. polymorpha expression vectors pDTMOX-
msdS, pDMMOX-msdS
(Figure 4A), which contain the MsdS gene fused
with the ER retention signal, were constructed
by replacing the NdeI–SalI HpURA3 gene frag-
ment in pDUMOX-msdS (HA-HDEL) (Kim et al.,
2006) with the 1.45 kb HpTRP1, 2.4 kb HpMET2
and 2.4 kb HpADE2 gene fragments, respectively;
each contained its own promoter and terminator
sequences, which were amplified by PCR using
primers TRP1 1F SalI (5?-tagtgggtcgactcaaacctga
caccgcaac-3?)/TRP1 2B NdeI (5?-ctcccatatgaagaa
gctgattgctggc-3?), MET2 1F SalI/MET2 2B
NdeI, and ADE2 1F XhoI/ADE2 2B NdeI, respec-
tively. Vectors pDTMOX and pDMMOX were
obtained by self-ligation after deleting the HindIII
MsdS gene fragment from pDTMOX-msdS and
pDMMOX-msdS, while pDAMOX was generated
by self-ligation after removing the BamHI/NcoI
MsdS gene fragment and blunting the ends. For
the Golgi-targeted expression of msdS, the MsdS
gene was fused with the DNA fragment encod-
ing the N-terminal 53 amino acids of HpOch1p
(Kim et al., 2006) and then inserted into pDTMOX
vector, resulting in pDTMOX-HH1MSF. For the
surface-targeted expression, the MsdS gene was
fused with the DNA fragment coding for the C-
terminal 40 amino acids of HpTip1p (Kim et al.,
Copyright 2009 John Wiley & Sons, Ltd.
Yeast 2009; 26: 507–521.
DOI: 10.1002/yea


text:
LOS ANGELES, CA -The government shutdown has temporarily suspended the Small Business Administrations lending program and the process to obtain government contracts.
Yet the number of Latino-owned businesses is expected to double this year from the number that existed in 2002, according to a recent study released by the US Hispanic Chamber of Commerce and Geoscape. Another report from the Kaufman Foundation shows that immigrants were twice as likely to start businesses than native-born Americans in 2012.
Figuring out ways to help and bolster Latino businesses and entrepreneurship especially at this time is one of the key goals of The Latino Coalition's 2013 West Coast Economic Summit, taking place in Oakland, California.
The Latino Coalition is a non-profit national organization founded 18 years ago by a group of Hispanic business owners to research and develop policies relevant to Latino entrepreneurs. This free summit brings together business owners across California to network and share tools that support small business growth.
Hector Barreto, chairman of The Latino Coalition and the former administrator of the US Small Business Administration (SBA) during the George W. Bush administration, tells NBC Latino that the ongoing fiscal crises in Washington are hindering business expansion.
"The government shutdown has the US markets sliding and although government can shut down; small businesses need to keep moving forward," Barreto said. "Congress needs to come together with job creators that will free up corporate money and private sector investments in order to spur job growth and get small businesses expanding again."
Barreto's focus for the gathering was on what he refers to as the 3Cs: capital, contracts, and capacity. With three million Hispanic small businesses in the US, knowing how to get financing is key especially for new companies that do not have a credit history.
"The SBA is the largest guarantor of small business loans in the country. When I was there, we increased the number of loans going to Latinos, women, and other minority business owners," Barreto said.
Drawing on his experience at the SBA, Barreto explained that he has worked with banks to make smaller loans for the businesses that don't need large loans. At Wednesdays summit, Biz2Credit will be represented, which is an aggregator of loans that will find entities to make loans to small businesses based on size and the type of business. Barreto calls this process "match making for loans."
How to obtain government contracts will be addressed at the summit, along with capacity issues, which involve helping small businesses run more efficiently to keep more of their revenue. How to deal with regulation will also be addressed.
Coco Corona, a Latina business owner and President and CEO of By Design Multimedia, echoed the sentiment that Congress needs to come together to fund and re-open the government to keep businesses moving.
"Those of us who are in business are looking to see if our leaders in Congress can come to the table and negotiate. You have to be able to get along — you might not like who you are sitting in front of but these people are not sitting down and are not listening to what a majority of the US population wants," Corona said. "For Latino businesses to grow, they will need support. If nobody is working and funding loans, then businesses will be shut down and employees will be let go."
Adriana Maestas is a senior contributing editor at Politic365 and one of the co-founders of the DailyGrito.com. She resides in California.