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Worldwide / United States / Los Angeles, CA / Rugs & Carpets
Los Angeles, CA Rugs & Carpets
Jose Flooring
8311 S. Main Street, Los Angeles, 90003323-422-3844
Affordable quality carpet sales and installation, including hardwood flooring.
View detail page - Jose Flooring
Alex Carpet Cleaning
Serving around, Panorama City, 91402818-384-2638
With Alex Carpet Cleaning, you will experience a new level of carpet cleaning in Panorama City. Our attention to detail and unsurpassed quality has made us the first and only choice for businesses and homes throughout Panorama City and surrounding ...
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In Your Place Furniture
Special Offer: Add Some Flair, With Outdoor Furniture!
5943 Sepulveda Blvd, Van Nuys, 91411818-902-1114
View detail page - In Your Place FurnitureProducts and Services
So White Carpet Cleaning LLC
4821 Lankershim Blvd Ste F, North Hollywood, 91601855-769-4483
We STEAM CLEAN, deodorize, sanitize & rejuvenate: carpet, rugs (all types), furniture, mattresses, chairs.. and other type of fabrics. We are also specialized in PET stains as well as pet odor control. We also offer Teflon protection for ...
View detail page - So White Carpet Cleaning LLC
Water Damage & Restoration Woodland Hills
24115 Friar st. , Woodland Hills, 91367818-330-1083
**24/7 Emergency Service Water damage restoration: We offer water damage repair & restoration services. Emergency water damage service by certified techs Best and fast Water Damage Woodland Hills is ready and available to handle all things ...
View detail page - Water Damage & Restoration Woodland Hills
Magic Carpet Sales
152 E Martin Luther King Jr Blvd., Los Angeles, 90011323-877-6941
View detail page - Magic Carpet Sales
Water Damage Restoration Canoga Park
7210 Jordan Ave #c53, Canoga Park, 91303818-337-5262
**24/7 Emergency Service Water damage restoration: We offer water damage repair & restoration services. Emergency water damage service by certified techs Water Damage Restoration Canoga Park is ready and available to handle all things ...
View detail page - Water Damage Restoration Canoga Park
DLB Antique & Vintage Rugs
4444 N La Jolla Ave, Los Angeles, 90048323-443-8787
DLB Vintage & Antique Rugs is one of the leading manufacturers and suppliers antique rugs and carpets carpets in the world. We have been a leader in this category for the last five decades and continue to be the center of attraction in the ...
View detail page - DLB Antique & Vintage Rugs
Water Damage & Rewstoration West Hills
23673 Gerrad Way, West Hills, 91307818-337-5288
**24/7 Emergency Service Water damage restoration: We offer water damage repair & restoration services. Emergency water damage service by certified techs Water Damage & Restoration West Hills is ready and available to handle all things ...
View detail page - Water Damage & Rewstoration West Hills
American Flooring Experts Inc
8612 Vine Valley Drive, Sun Valley, 91352818-335-2931
Flooring Contractor
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Livin' Right Floors
Special Offer: Senior and Military Discounts Available
North Hollywood, 91601818-209-0340
View detail page - Livin' Right FloorsBios Products and Services
Alas Hardwood Floors
735 E. 98th St., Los Angeles, 90002323-893-4182
Alas Hardwood Floors Incorporated has been serving residential and commercial customers in the greater Los Angeles for six years. Alas provides top quality flooring including floor covering with affordable pricing. Our goal is to restore your ...
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Klein Carpets and Tile Cleaning
1935 Rosalia Rd, Los Angeles, 90027323-475-9114
Carpets are an extremely popular option for flooring as they make the whole ambiance quite inviting. Surely, you might also have some beautiful carpets at your home or office space, and if you are living in Los Angeles County, there is nothing to ...
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Amazing Care Chem-Dry
1533 W. 111 Place, Los Angeles, 90047562-244-2975
At Amazing Care Chem-Dry we delight our customers with the cleanest and fastest drying carpets & upholstery in the areas of Long Beach, Lakewood, Greater Los Angeles County. We pride ourselves with excellent customer service over the phone and ...
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Valley Carpet One Floor & Home
5836 Sepulveda Boulevard, Van Nuys, 91411818-922-8569
Valley Carpet One Floor & Home is your local Van Nuys, CA flooring store for great service and quality carpets, wood flooring, floor tiles, vinyl and more in a wide range of colors and prices. Visit us in store or online to learn more.
View detail page - Valley Carpet One Floor & Home
Finmark Carpet One Floor & Home
19601 Parthenia Street, Northridge, 91324818-671-0026
Finmark Carpet One Floor & Home is your local Northridge, CA flooring store. We carry quality carpets, hardwood, vinyl, tile and more in a wide range of styles and prices. We also offer Shop at Home, Healthier Living Installations, remnants and ...
View detail page - Finmark Carpet One Floor & Home
Anderco Carpet Co Inc.
10467 Roscoe Blvd, Sun Valley, 91352818-551-4242
At Anderco Carpet Co Inc., we offer a large variety of carpet styles for commercial (high traffic), and residential customers. Stop in today!
View detail page - Anderco Carpet Co Inc.
Anderco Carpet Co Inc.
10467 Roscoe Blvd,, Sun Valley, 91352818-551-4242
We offer numerous styles of carpet both for high traffic known as commercial, and residential. All prices vary, but we can guarantee you will be satisfied and you will leave our warehouse with the cheapest price in town. Anderco Carpet offers great ...
View detail page - Anderco Carpet Co Inc.
Master Oriental Rugs
11918 Santa Monica Blvd., Los Angeles, 90025310-279-3994
Master Oriental Rugs specialized in repair and restoration of antique oriental rugs and carpets.
View detail page - Master Oriental Rugs
Renteria Flooring Specialist
523 North Alvarado Street, Los Angeles, 90026213-422-2060
Have you been on the search for the best flooring contractor in the Los Angeles, CA area that also offers amazing prices? Are you building or remodeling a home and need a professional hardwood floor installer to get the flooring done just the way ...
View detail page - Renteria Flooring Specialist
Sutherland Carpet Cleaning
773 W 24th St, San Pedro, 90731310-704-4558
Sutherland Carpet Cleaning is the best residential and commercial carpet cleaning company in San Pedro, CA. We are an organic carpet cleaning company that provides services such as tile cleaning, grout cleaning, upholstery cleaning, pet odor ...
View detail page - Sutherland Carpet Cleaning
I.D.N. Hardwood & Carpet Superstore Inc.
7947 Canoga Ave., Canoga Park, 91304818-888-8051
I.D.N Hardwood & Carpet Superstore started as an interior designer company back in 1991, a year after in 1992 Rina Interior Design Consulting Services established in Santa Monica. We provided our customers flooring need from different sources ...
View detail page - I.D.N. Hardwood & Carpet Superstore Inc.
Elite Floorcovering
2255 S Sepulveda Blvd, Los Angeles, 90064310-231-5111
Offering a wide range of flooring options, our full service commercial and residential hardwood, carpet and laminate floor covering options are sure to fit any style. Renovations can be overwhelming but our professional staff will help your floor ...
View detail page - Elite Floorcovering
ALESSCO INC. 13161 Sherman Way Building C North Hollywood, CA, Los Angeles, 91605818-821-8278
Find great deals on softfloor for foam floor mats, martial arts mats, foam tiles and gym mats. All the products are available at a reasonable price and excellent in quality. Interlocking rubber mats and trade show flooring are well-known in the ...
View detail page - Softfloor
K & A Foam Carpet Cushion and Cristal Materials
6825 McKinley Ave, Los Angeles, 90001323-244-2464
Your one-stop site for all your foam needs! Here at CRISTAL MATERIALS, we carry a large selection of foam products for a wide range of applications, all at competitive prices! Products range from bedding, packaging and acoustical foam to cushions, ...
View detail page - K & A Foam Carpet Cushion and Cristal Materials
Certified Carpet Care
22569 Enadia Way, West Hills, 91307323-877-4372
Everyone at Certified Carpet Care is passionate about giving you an excellent cleaning service. We focus on consumer education and are dedicated to providing you with the latest information, so you can make informed decisions when choosing a ...
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The Home Depot
6345 Variel Ave, Woodland Hills, 91367818-716-9141 Calendar
The Home Depot
22855 Victory Blvd, West Hills, 91307818-887-7083 Calendar
The Home Depot
21218 Roscoe Blvd, Canoga Park, 91304818-348-9400 Calendar
Discount Carpet For You
23045 Ventura Blvd, Woodland Hills, 91364818-224-3838
View detail page - Discount Carpet For You
Sherman Oaks Family Carpet Cleaning
14226 Greenleaf St, Los Angeles, 91423818-869-6383
If you are in the market for a carpet cleaning professional in Sherman Oaks, CA that knows the best way to clean your carpeting to perfection, then you need to make the call to our Sherman Oaks carpet cleaning experts today so we can provide you ...
View detail page - Sherman Oaks Family Carpet Cleaning
Carpet Depot Inc
13451 Sherman Way, North Hollywood, 91605818-732-8235
Carpet Depot Inc is located at 13451 Sherman Way, North Hollywood, CA, 91605-4416. USA. We provide all major brands of carpeting, hardwood flooring, vinyl tiles, warehouse equipment, wood products and many more. We offer top quality service at ...
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Tom Duffy Company
14736 Keswick St, Van Nuys, 91405818-901-2629
The region's leader in flooring store products available to all flooring contractors. We specialize in a diverse item selection that will help you finish any floor covering project. Whether its ceramic tile flooring, hardwood flooring, or even ...
View detail page - Tom Duffy Company
C.R.C. Carpet Care
3521 W 27th Street Suite 17, Los Angeles, 90018323-602-4642
View detail page - C.R.C. Carpet Care
J & S Custom Hardwood Floors Inc
6402 Maryland Dr., Los Angeles, 90048323-459-8710
Jacob and Shaul 15+ Years Experience Serving The Greater Los Angeles Area
View detail page - J & S Custom Hardwood Floors Inc
Regal Restoration
6829 Canoga Ave #2, Canoga Park, 91303800-707-3425
We at Regal Restoration are committed to a 24hr, 365 days of the year water damage emergency service that will include an immediate response and arrival to your home in order to help you salvage your precious belongings and dwelling. We can arrive ...
View detail page - Regal Restoration
The LA Carpet Cleaners
11924 Montana Ave #10, Los Angeles, 90049213-810-5737
The LA Carpet Cleaners is the premier carpet cleaning service in the area because we are constantly committed to providing the highest quality experience for our customers... yes, that means more fun & more memories and fantastic ...
View detail page - The LA Carpet Cleaners
AZ Imports LLC
1933 S Broadway 9th Floor Unit 922, Los Angeles, 90007213-741-1155
The Largest Closing Clearance Sale in Rug History! SAVE 70 to 80 % off First Come, First Serve BUY NOW! Don't Miss It All Inventories must be SOLD. It's so hard for us to say GOODBYE after 35 years in business, but NOW the time has come ...
View detail page - AZ Imports LLC
Expert Carpet Cleaning
246 South Mansfield Ave, Los Angeles, 90036800-997-5161
Expert Carpet Cleaning Los Angeles is a leading carpet and rug cleaning company in Los Angeles. We have been working in Los Angeles for more than 20 years. We do organic carpet cleaning which is safe for your health and effective than traditional ...
View detail page - Expert Carpet Cleaning
Discount Carpet Center
4109 Chevy Chase Dr, Los Angeles, 90039818-500-9946
At the Discount Carpet Center we are ready to serve your flooring needs. Whether you are building, remodeling, or your 2 year old just has a whoopsie that made your carpet instantly 'outdated', we are here to help.Discount Carpet Center is offering ...
View detail page - Discount Carpet Center
ENC Bail Bonds
16847 Ventura Boulevard, Encino, 91436818-334-5116
We Make Getting Out of Jail Easy! Call Us 24 hours a day for a quick quote and friendly bail bonds service. We have 1000's of satisfied clients sinceread more
View detail page - ENC Bail Bonds
Area Rugs Online Shop
7277 Hayvenhurst Ave #B12, Van Nuys, 91406844-800-0050
Area Rugs Online Shop is a leading source of japer area rugs in the US.
View detail page - Area Rugs Online Shop
Lowe's Home Improvement
8383 Topanga Canyon Blvd, West Hills, 91304818-610-1960 Calendar
Lowe's Home Improvement
13500 Paxton Street, Pacoima, 91331818-686-4300 Calendar
Lowe's Home Improvement
4550 West Pico Boulevard Unit D-101, Los Angeles, 90019323-617-9570 Calendar
Lowe's Home Improvement
19601 Nordhoff St, Northridge, 91324818-477-9022 Calendar
At Home In the Valley
16531 Saticoy St, Van Nuys, 91406818-780-4663
Marisela and our friendly staff will help you decorate your rooms. Bring in your measurements, fabrics, samples, photos.... and we will help you put your rooms together with the perfect rug. We are very passionate in being able to bring your colors ...
View detail page - At Home In the Valley
Prius Carpet Cleaning
19234 Hamlin st #2, Los Angeles, 91335818-388-9955
If you are looking for first-class 100% Organic service, you have come to the right place! We aim to be Environmental friendly and approachable. Our Products are 100% Organic. We Will Protect Your Family Health! prius carpet cleaning 91335
View detail page - Prius Carpet Cleaning
Carpet Fresh
300 South Spring Street #1700 Suite G, Los Angeles, 90013310-853-8847
Patented hot-water extraction system for cleaning carpet. We also clean rugs, mattresses, sofas and assorted upholstery.
View detail page - Carpet Fresh
Rug Trader International
11151 W Pico Blvd., Los Angeles, 90064310-575-3200
View detail page - Rug Trader International


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Standing position rehabilitation exoskeletons
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HANK is a lower limb exoskeleton designed for rehabilitation of adults between 1.50 and 1.95 m in height, with a maximum body weight of 100 kg, such as stroke patients following neurological insults. ...
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3 technology disruptions to emulate natural walkFirst usage of dynamic robotics algorithms in an exoskeleton. Successful technological bet to mimic human walkFirst exo capable of replicating the motions of the human body. ...
... community. It is the most customizable exoskeleton and is configured specifically for you. This precise fit optimizes safety, function and joint alignment. The battery-powered system features a light, wearable exoskeleton ...
The Phoenix exoskeleton is the world's lightest and most advanced exoskeleton designed to help people with mobility disorders to be upright and mobile. In the clinic, at home, and in ...
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Features Indego's® unique features make it a valuable tool for use with mobility impaired patients in the clinic or at home. Lightweight At just 26 pounds, Indego's elegant design has no exposed cables or upper-body apparatus and does ...
EXOATLET - Russian exoskeleton for rehabilitation The mission of ExoAtlet project is to design and develop the first Russian exoskeleton for medical rehabilitation ...
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Hi Everyone,
Thanks for taking the time to read this. Really appreciate any and all help.
My fiance and I will be heading to Tokyo on September 16th after our wedding and will spend three nights there and three nights in Kyoto. Here is what I have so far, and need help on:
Staying at the Ritz Carlton (in Minato-ku)
Arrive at about 12:30pm
Lunch: We'll be tired, but will walk around to try and beat the jet-lag. Something easy and relatively quick. Any suggestion on a ramen/udon/soba place nearby? Please note: Not ippudo, we have one in NYC and while I'm sure Tokyo is better, I'd like to branch out.
Dinner: Again, something easy, fun, preferably close to the hotel. I've heard a lot of talk (good and bad... mostly good) about Birdland, Kushimura and Gonpachi - not sure which is considered "best" and which is closest to hotel.
Following Day:
Fish Market, Sushi Breakfast (probably Sushi Dai depending on the line)
During the day we'll probably do the following:
Meji Shrine -> walk to Harajuku -> walk to Omotesando -> walk around and get lost.
If anyone knows any good lunch spots along that route, that would be great. Again, noodles sound great - but we're totally open, would eat anything. Your recommendations are valued.
Dinner: Tempura Kondo (is this place stuffy? Or cool?)
Following Day:
Breakfast: No idea, probably hotel
Lunch: Maybe Sushi Saito? Heard it's the "best lunch deal in town" or I read an article about doing a food crawl through Asakusa stopping at places like: Raishuken, Koyanagi, Asakisa Namiki Yabu Soba, Sansada, Kamiya Bar, Suzuyo
We have sumo tickets for that day, but I understand that this starts around 3-4pm
During the day I've heard Naka-Meguro and Daikanyama are great neighborhoods to walk around and get lost. Maybe we go to the major anime area (forget the name).
Dinner: No idea really - so many options and I have been reading until my eyes bleed.
I think Park Hyatt for drinks (a bit cliche but heard it's still great)
then for dinner:
Sushi Mizutani
Sushi Kyubey
Ramen: Gogyo, Yatai Nagahamya #1, Maisan for tonkotsu. Menya Kissou for Ramen (any of these near where we will be in previous days? or by the hotel?)
This place apparently called Yakitori Alley by Westerners only: http://blog.wandr.me/2011/07/dining-u...
Obviously if we went to Mizutani or Kyubey (or any other sushi place for dinner we wouldn't do Saito for lunch)
The next day we travel to Kyoto and are staying at Ryoken Tawaraya that night. So dinner that night and breakfast the next morning are done. Then we check into the Ritz.
I've been focusing all my efforts on Tokyo, so Kyoto is totally up in the air. I've heard of the following:
Shoraian: Tofu place, right? Could be cool
Yamashita: Is all over chowhound as an awesome spot
Samboa Bar (and some steak place that the bartender will take you to if you ask)
A friend of mine said the following:
There is a duck ramen joint which was hard to find but delicious. There is a long strip that used to be the red light district which is now all converted to restaurants and bars. we found an amazing counter only sushi spot there - marble topped and sushi put straight on it bit by bit.
For both Tokyo and Kyoto - we like cool spots, great food, great drinks - I don't want to go to the Robot Restaurant, I don't want to go where there are all ex-pats and Westerners. I really want to experience Japan and blow my mind.
Well, that's all I got. Really appreciate you reading all the way through and helping me figure out the important parts of my honeymoon.
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The nomination of Deputy Energy Secretary Dan Brouillette to become energy secretary easily cleared the Senate Energy and Natural Resources Committee on Tuesday by a 16-4 vote. The full Senate has not yet scheduled a vote on the nomination, a source said. The previously…
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The San Francisco Presidio is the only park – and only national park – in the U.S. to win the prestigious Green Flag award — Photo courtesy of Presidio Trust In the middle of the urban paradise that is San Francisco, there's an oasis of green that makes you feel like you're far away from
Napa Valley may get the glory, but Missouri has a longer history of growing grapes and winning awards, especially in and around the small river town of Hermann. Founded in 1837 by German settlers, Hermann has the perfect micro-climate for growing grapes and was once the nation's second largest producer and exporter of wine and
Photo courtesy of The Creole Creamery Fun riffs on the Mardi Gras treat As soon as early January hits, a special treat starts popping up at parties, offices and homes all over Louisiana. "King cake is a New Orleans staple," says Adrienne Rousse, store manager at 71-year-old Gambino's Bakery which sells 100,000 of these baked goods annually.
Photo courtesy of Omni Bedford Springs Resort Iconic inn revitalized The Omni Bedford Springs Resort has come a long way since its humble 1804 opening as a 24-room inn for travelers drawn to the area's healing spring water. The property enjoyed great success in the early 1900s, but struggled during the Great Depression and World
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Wishlisted by Matt Mayer
There's no menu, just tell the bartender what flavors you like. Check the map because this is down a very anonymous looking soi.
Cocktail · Special Occasion · Flexible · Recommended - Good Food · Recommended - Great Service · For Two · Small Groups · Solo
16, 125/13 Sukhumvit 55, Thonglor, Klongton-Nue, Watthana, Bangkok, Thailand
ellisliu posted a wishlist
2 months ago
lemi posted a recommendation
2 years ago
Step into this tunnel-like bar hidden behind a non-descript door to disappear from Bangkok's busy streets.
From the beetle-covered steel walls to the sparkling golden lights, the details are small but loom large in J. Boroski. After all, it's the concept of famed Australian interior designer Ashley Sutton.
Don't be surprised at the lack of a menu, baristas create personalized drinks just for you and your preferences.
snacklord posted a wishlist
3 months ago
This literal (rabbit) hole in the wall is yet to be discovered! 🐰 The door to the bar is a huge wooden door with no signages, located in between a japanese bar and ramen place. I've read it's tricky to find but totally worth it! Look at that interior. 😍 Can't wait to have drinks in this ambience. 🍸
dustflyfly posted a recommendation
a year ago
I adore the neon-tinted atmosphere of Bound by Hillywood. It is a bright beacon in the night, drawing out Hong Kong's creatives from the underground. They offer some creative concoctions that use local rice liquor. My favorite: a strawberry, Wing Lee Wai Rice Wine, and Yakult combo called New Romance.
yanni posted a recommendation
In the back streets of central Athens, you will find this popular local hangout. This place is full of locals drinking their coffee by morning and Negroni's by night.
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Arizona is a great state to hike. Even without the magnificent hikes in and around the Grand Canyon, there are plenty of other trails to explore and enjoy. This book does it all! It is an exceptionally well-designed book for hikers across the spectrum. Whether you're a neophyte or have climbed Denali, this book contains all the essential information you need to tackle the hikes listed. The photos are all in color and are breathtaking! You really get a sense of what each hike will look like before you undertake it. Every hike also has a color map to accompany the text description. The maps are easy to follow and instructive.
Equally pleasing is that the authors take the time to describe each hike in extensive detail, though they are never wordy. They list the elevation gains, give succinct but necessary directions to each trailhead and provide ample analysis of the strengths/weaknesses of each trek. The book is small and light enough to carry in your backpack, if you feel the need to consult it while on the trail.
I have over 50 hiking books in my library and it would be hard to imagine a more complete, more photographically stunning or better written guide. I enthusiastically recommend this gem!
- Paperback: 238 pages
- Publisher: Mountaineers Books; 2 edition (1 June 2000)
- Language: English
- Product Dimensions: 17.1 x 1.9 x 22.9 cm
- Boxed-product Weight: 771 g
- Customer Reviews: Be the first to review this item


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Roger Federer was dealt a challenge after landing in the same group as on-form Novak Djokovic after the Tuesday draw for the ATP Finals.
Should Rafael Nadal – who had to quit with an abdominal muscle injury before his Paris Masters weekend semi-final – end up not playing, then the balance of power at the eight-man showpiece will rest in the Federer-Djokovic group.
Nadal is due to test his serving at the end of the week for Sunday's London start and make a decision on whether to play.
Along for the ride in the top-heavy Borg group are two-time Roland Garros finalist Dominic Thiem and Italy's first-timer Matteo Berrettini.
World No. 1 Nadal's group also features US Open finalist Daniil Medvedev, Stefanos Tsitsipas and London holder Alexander Zverev.
Nadal has a patchy attendance record at the season wrapup, withdrawing injured four times since 2009. he had to quit two years ago after playing one group match.
His best showings at the event have been a pair of runner-up finishes. he and Djokovic will be duelling for year-end top ranking status.
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Cablegram Winch 14 LONDON, 25 August 1943, 2.07 a.m.
Received 26 August 1943
Having agreed upon the setting up of the combined South East Asia
Command [1] I have arranged with General Marshall and Admiral
King, with the approval of the President, that I can have an
officer of General's rank as Liaison Officer at MacArthur's
headquarters. This will enable MacArthur to correspond freely with
me and will enable me to follow with closer attention than
heretofore the developments in the Pacific theatre. I talked this
over with Dr. Evatt when he was in London and understood from him
that you would like some such arrangement as this. [2] 2. While I
should never wish to intrude upon domestic party issues in
Australia, I hope I may send my private and personal
congratulations on the return of your Government with so large and
stable a working majority. [3] This should favour continuity in
the war effort and the taking of increasingly important decisions
which will be required during this and future campaigns in the
3. Will you kindly show this to Dr. Evatt to whom I am also
sending a personal telegram [4] in reply to his very kind personal
message to me. [5]
1 The establishment of a South-East Asia Command was agreed on by
the U.K. Govt in June and endorsed by the Combined Chiefs of Staff
at the Quebec Conference on 24 August. The Command covered Burma,
Thailand, Malaya, Sumatra and Ceylon and its initial objective was
to facilitate a more effective offensive against the Japanese in
Burma. Lord Louis Mountbatten was appointed Allied Supreme
Commander and Lt Gen Joseph W. Stilwell as Deputy Supreme
Commander. See John Ehrman, Grand Strategy, HMSO, London, 1956,
vol. V, pp. 135-48.
2 Curtin replied in cablegram Johcu 70 of 31 August (FA:A3196,
1943, 0.23908) that he fully agreed with the proposal, which would
be 'of great mutual advantage'. On 8 October Churchill advised in
cablegram Winch 17 (FA:A3195, 1943, 1.41474) that his Liaison
Officer with MacArthur would be Lt Gen Herbert Lumsden.
3 At the elections on 21 August Curtin's Australian Labor Party
gained forty-nine seats, the United Australia Party-Country Party
opposition twenty-three seats and independents two seats. The
A.L.P. also gained control of the Senate where it had previously
been in a minority. On 22 September R. G. Menzies replaced W. M.
Hughes as leader of the U.A.P. and, as the U.A.P.-Country Party
coalition was terminated, Menzies, as leader of the larger
opposition party, also replaced A. W. Fadden as Leader of the
4 Document 265.
5 See Document 265, note 3.
Category: International relations
Topic: History


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GLCF Announces Completion of Silver Star Phase II Supportive Housing Community
Great Lakes Capital Fund, along with other collaborative partners, announced the completion of Silver Star Apartment Homes' Phase II, which will bring an additional 100 units to the Battle Creek, Mich. housing market.Midwest 12 November 2013 17:18
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September 2, 2017
WELL, IT'S NOT LIKE THEY'RE A GATEWAY DRUG TO PATRIOTISM, CHIVALRY, AND CLASSIC NOVELS, OR ANYTHING: Millennials don't really care about classic movies.
That explains why landmark films are being banned because of their problematics, which is why the reels that contain Gone with the Wind are now currently smoldering in the Memory Hole. In Casablanca, Ingrid Bergman's character once refers Dooley Wilson's Sam as being Rick's "boy." Time to ban that one as well, despite the counterculture falling in love with Rick's coolness in the 1960s?
Found via Kathy Shaidle, who writes, "This is literally the cause of all our problems…I'm not being sarcastic. You can learn almost everything about life from movies," adding, "You will learn, for example, that you are not the first generation to have Problem X or 'Solution' Y."
Creationists believe that life on earth began 5,000 years ago. Millenials seem to think it started with the Beatles. Or maybe Star Wars.


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SoulCycle is a 45-minute indoor cycling class that features high-intensity cardio, muscle-sculpting strength training, and rhythm-based choreography. But it's so much more than just a workout it's a powerful mind-body experience. We ride together as a pack in candlelit studios to the rhythm of one-...
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If you're anything like me and you're a lover of healthy, delicious drinks, you know there's nothing like freshly squeezed orange juice! Especially since industrial fruit juices are becoming more and more controversial!
The cause: most of them contain more sugar than sodas. What's more, they are much lower in vitamins, minerals, fibre and antioxidants than natural fruit juices. What's worse, they could significantly increase the risk of obesity, diabetes, and cardiovascular disease.
Not to mention over-packaging and heavy juice packs to carry, which are bad for the environment… and your back. But also for your wallet, because these juices are not cheap!
So the alternative of making your own fruit juices quickly becomes indispensable. It is also the best way to get fresh and healthy juices, and to choose quality citrus fruits! The days of squeezing fruit by hand for many minutes are well and truly over! Thanks to electric juicers, making fresh juice has become a child's play and a pleasure in everyday life!
The electric version has many advantages over the manual version and is also more functional. It is equipped with a powerful motor, which gives it the ability to generate a high centrifugal force. This makes juice extraction easier and more efficient. The quantity of juice obtained is, therefore, more consequent.
And you can vary the taste by using all kinds of citrus fruits: lemons, tangerines, oranges, grapefruits, pomelos and so on!
But it is true that there are tons of different references and it is very difficult to make a choice. Luckily, I've had the opportunity to test a large number of electric juicers. This is simply because we served fresh orange juice with the pastries in most of the shops where I worked. So I have gained some experience in the field over the years.
Here's a list of my 4 favorite juicers! Keep in mind that I only recommend products that I've tried and loved – I either own them or tried them at work or in a cooking class.
|KRUPS ZX7000 Stainless Steel...
|Check availability
|Philips Citrus Press 85 W
|Check availability
|Mueller Austria Juicer Ultra 1100W...
|Check availability
|Cuisinart CCJ-500 Pulp Control...
|Check availability
Before presenting you with the best models on the market, here are first some important criteria to check:
The type of device
There are two types of electric juicers on the market.
1. Domestic juicers – These appliances are intended for domestic and light use. They are less powerful than professional models but also cost much less. They are suitable for occasional users.
2. Professional juicers – These are the models we use in bakeries. These machines are very powerful and required for intensive and regular use. They are more expensive than the models of "Mr. Everyman" but generally have a better life span (better materials for manufacturing, more powerful motors…).
The power
Let's come to another rather important criterion: the power of your future juicer. For a domestic model, I recommend choosing a power greater than 20 watts. This will be sufficient for the type of use of the machine. On the other hand, if you intend to use it regularly, go for a professional model with a minimum power of 100 watts.
The capacity
There are two solutions for the juicer: either it has a juice tray to collect the extraction, or it can be poured into the container of your choice.
The second solution remains, of course, the best because you can directly place a glass or a small bottle. For the juice container, I advise you to choose a model with a minimum capacity of 0.5 liters. This will avoid unnecessary handling while squeezing your fruit.
The features
An electric juicer is still a fairly simple device to use. But some of them are equipped with interesting features. I'm thinking in particular of the pulp selector which will allow you to make fruit juices with or without pulp, by simply changing the filter. But also the anti-drip system so that you don't end up with fruit juice dripping after using the machine.
The material
The ideal is undoubtedly to choose a stainless steel appliance. It is a healthy, durable and robust material that is also easier to maintain and is not afraid of oxidation. You can also leave on a plastic appliance if it is of good workmanship. But the most important thing is to make sure that the plastic is Bisphenol A-free. This is a controversial substance that is found in too many appliances designed with plastic. All the models presented in this article will, therefore, be 100% BPA-free.
Ease of cleaning
An electric juicer is clearly there to save you time and enjoy fresh produce. But if you spend your time cleaning it afterward, this type of device quickly loses interest and may discourage you from using it. Ease of maintenance becomes a determining factor in the choice of this type of appliance.
First of all, it is preferable that the various parts of the juicer be removable (juice tray, different filters, lid, etc…). And if possible, compatible with your dishwasher, so you will save a lot of time. The fact that the appliance is made of stainless steel is also useful for cleaning.
The noise
If you plan to prepare fresh fruit juices for the morning (and not wake the whole neighborhood up), it is also important to pay special attention to the noise emitted by your future juicer. Preferably, I advise you to choose a model that does not exceed 60-65 dB. This is equivalent to the noise level of a normal conversation between two people or a television set in operation. This is still reasonable and you won't wake up the whole house by preparing breakfast. In this article, I have only selected juicers that do not exceed this sound level.
The brand and the warranty
Forget about juicers from unknown brands or from Asia. These machines are very noisy, inefficient and break too quickly to be interesting. Conversely, a brand name juicer will offer you much better guarantees.
Personally, I find that the best professional quality machines are from Krups and H.Koenig. For entry-level domestic models, you can find good things at Moulinex or Philips, for example. The ideal is also to have a 2-year guarantee and a 5-year parts availability. That way, if you lose or break a filter, you can always replace it without having to buy the whole juicer again.
The price
This remains an important buying argument for many people. But as we have seen above, it is still very inadvisable to go for a first-rate juicer. You can find good machines from $50 to almost $200 for the most advanced models.
I advise you to buy your future juicer online. The prices are generally much cheaper on commercial sites such as Amazon. In shops, references are rare and prices are often higher, especially during certain periods of the year.
Customer reviews
As with all appliances, I advise you to take a brief look at the evaluations of previous buyers. It is often an interesting indicator and a very good reflex to have. It would be quite inadvisable to advise you to buy a product with a rating of fewer than 3 stars.
Advantages of an electric juicer
Some people may wonder what's the point of having an electric juicer in the home. Here are all the advantages that quickly convinced me:
✅ First of all, because with an electric juicer you can squeeze your fresh fruit juices with less effort. Indeed, the very powerful motor of these small devices will allow you to obtain juices quickly and efficiently!
✅ Thus, you will be able to make fresh fruit juices at a lower cost and limit the environmental impact by no longer buying juice cartons that are not always recyclable.
✅ Also, it's a small device that doesn't cost very much. You can find a very good electric juicer for less than $50!
✅ But you should also know that a juicer can squeeze other fruits and not only oranges. Some models have an interchangeable cone in order to adapt to the food from which the juice is to be extracted.
✅ Finally, it should be noted that the juicer is easily dismantled and therefore easy to maintain. But it is also a device that is fairly compact and therefore not very bulky


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3 Bedroom Plus Den, 3.5 Baths, 3-car Garage. Impact Glass Windows, Engineered wood floors, crown molding, California-style cabinets, magnificent kitchen and bath upgrades, R-30 Batts insulation, Electric blinds, Plantation shutters, solid core doors and so much more. Please call for a full list of upgrades. Valencia Reserve is a top 55+ community with tons of amenities. HOA Fee includes exterior painting of house, lawn care, alarm monitoring, cable and internet. Virtual Tour is Valencia Reserve Clubhouse.
Basketball, Bike - Jog, Billiards, Cabana, Cafe/Restaurant, Clubhouse, Community Room, Exercise Room, Game Room, Manager on Site, Pickleball, Picnic Area, Pool, Sidewalks, Spa-Hot Tub, Tennis, Workshop
Cable, Public Sewer, Public Water, Underground
2+ Spaces, Driveway, Garage - Attached, Vehicle Restrictions
Built-in Shelves, Foyer, Laundry Tub, Pantry, Roman Tub, Split Bedroom, Volume Ceiling, Walk-in Closet
Auto Garage Open, Cooktop, Dishwasher, Disposal, Dryer, Microwave, Range - Electric, Refrigerator, Smoke Detector, Storm Shutters, Washer, Water Heater - Elec
Auto Sprinkler, Covered Patio, Lake/Canal Sprinkler, Open Patio, Screened Patio, Zoned Sprinkler
|Status Changed from New to Active
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Listing information last updated on February 24th, 2020 at 9:00am EST.


text:
The genomic sequence of Trypanosoma cruzi, the protozoan causative of Chagas disease was published more than a decade ago. However, due to their complexity, its complete haploid predicted sequence and therefore its genetic repertoire remains unconfirmed. In this work, we have used RNAseq data to improve the previous genome assembly of Sylvio X10 strain and to define the complete transcriptome at trypomastigote stage (mammalian stage). A total of 22,977 transcripts were identified, of which more than half could be considered novel as they did not match previously annotated genes. Moreover, for the first time in T. cruzi, we are providing their relative abundance levels. We have identified that Sylvio X10 trypomastigotes exhibit a predominance of surface protein genes, specifically those encoding trans-sialidase and mucin-like proteins. On the other hand, detailed analysis of the pre-mRNA processing sites revealed some similarities but also some differences in the spliced leader and different polyadenylation addition sites compared to close related kinetoplastid parasites. Our results also confirm that transcription is bidirectional as occur in other kinetoplastids and the proportion of forward-sense and reverse-sense transcripts is almost equivalent, demonstrating that a strand-specificity does not exist.
Trypanosoma cruzi is a protozoan parasite causative of Chagas disease, bearing the name of its discoverer the Brazilian scientist Carlos Chagas. This parasite has a complex life cycle that includes an insect vector and a broad range of mammalian hosts, including domestic animals and sylvatic reservoirs1,2 and therefore, depends on several key points of cellular reprogramming through its entire life cycle to adapt to the different biological enviroments3. The insect vector is infected by non-replicative trypomastigotes present in the blood of mammalian reservoirs and differentiate into replicative epimastigotes in the midgut of the vector. Next are eliminated with the faeces as metacyclic trypomastigotes and once inside the host, infect and penetrate cells and differentiate into intracellular replicative amastigotes until the cell is completely swollen and then transform back to trypomastigotes, lyse the cell and release them into the bloodstream completing the cycle4,5,6. These adaptations have been determined mainly as morphological changes but very little is known about the molecular and metabolic processes necessary through the life cycle to produce these changes7.
Contrary to most eukaryotes, T. cruzi, as other kinetoplastids, does not regulate their gene expression by the differential recruitment of polymerase II and just a few potential transcription factors have been described8,9,10,11. Instead, they transcribe long RNAs containing up to hundreds of genes also called polycistronic transcription units (PTUs)12,13. A second step of maturation consisting in the addition of a capped 39-nt spliced leader (SL) in the 5′ start and polyadenylation on the 3′ end, produces the individual and mature mRNAs14. In other species, such as Leishmania, has been demonstrated that transcription initiation preferentially occurs at divergent strand-switch regions (SSR), where PTUs raise in opposite directions on opposing DNA strands15,16.
While other kinetoplastids that cause human diseases such as Leishmania (Leishmaniasis) and T. brucei (sleeping sickness) have been and are still extensively studied regarding genomics and transcriptomics, allowing the description of most of its molecular regulatory mechanisms through its life cycle and host interaction, T. cruzi remains poorly understood7.
To date, some attempts to describe the global genome of T. cruzi have been performed but his high intra-species variability has made of this a complex challenge17,18,19,20,21. In recent years T. cruzi has gained some attention at transcriptomic and proteomic level3,22,23,24,25 but contrary to other kinetoplastids, little is known about the parasite molecular switches. In this work, we performed RNAseq analysis of Sylvio X10 strain to describe the trypomastigote transcriptome, but importantly it helped to improve significantly the previous genome sequence of this strain and also allow describing for the first time some of its main regulatory molecular mechanisms of T. cruzi mRNA processing.
Results and Discussion
Genome correction, transcriptome assembly and quantification
RNA from trypomastigotes of T. cruzi (strain Sylvio X10) was sequenced after polyA+ selection on Illumina MiSeq sequencing platform, generating 17,332,912 of paired-end reads (length: 75 nucleotides). Raw data was deposited to the SRA database under accession number PRJNA546488. Raw reads trimmed and filtered (maximum and minimum length: 100 and 50 respectively, minimum mean quality 25, phred score based) were mapped to SylvioX10 genome deposited on Tritryp (http://tritrypdb.org/, "TcruziSylvioX10-1") allowing up to three mismatches with bowtie2. Considering that about 81.75% of the total reads were successfully mapped to the reference genome, we use it as bona fide genome reference.
The Sylvio X10 genome is composed by 47 "chromosome-like sequences" (C-LS) that are long scaffolds, no complete chromosomes. Thus, it is important to notice that non-aligned reads may correspond to undefined genomic regions (gaps) or missing chromosomes. Consequently, RNAseq reads were used to improve genomic assembly, such as the correction of deletions, insertions, SNPs etc. For this, we performed an assembly correction using Pilon, a bioinformatic tool for correcting drafted haploid and diploid microbial assemblies using paired end reads. Also, a previous paired DNAseq reads under the NCBI bioproject number PRJNA395140 were included to this genomic correction.
Results of Pilon improvement are summarized in Fig. 1. About 68% of the total genome bases were confirmed, corresponding to about 28 Mb of information (including gaps). Sequence correction affected mainly coding regions, 3,415 SNPs and 39 ambiguous bases were corrected, 72,887 insertions removed and 1,810 previous deletions were also corrected. Eighteen of the forty-seven C-LS decrease in total length up to 23 Kb as C-LS 4; the longest C-LS (1) decrease 7.2 Kb and the second longest C-LS decrease 12.8 Kb, while the shortest (C-LS 47) decreased 7.2 Kb. In total, 133.35 Kb of nucleotide insertions were trimmed from the previous assembly, while 43.66 Kb of new information was added to the new genome. In total, 177.02 Kb were corrected. In addition, it is important to notice that C-LS 17 and 47 had the lowest DNA-RNA sequencing coverage and the biggest ratio gaps per assembled base. This may be indicative that these sequences correspond to structural non-coding chromosomes, ambiguous assemblies or spurious sequences. But it clearly needs to be confirmed by further deeper genomic studies (Annotation in Supplementary File 1).
After genome correction 82.35% of the trimmed RNAseq reads were successfully mapped and were used in further analysis.
In trypanosomatids a discriminatory mechanism for the initiation of transcription at individual loci is absent in most genes. Thus, long RNAs containing more than one gene are transcribed at the same time by the polymerase II into long polycistronic transcription units (PTUs), requiring further mRNA maturation process that had been described before and named as trans-splicing and polyadenylation26,27,28. In this sense, the total transcripts were obtained in two steps: firstly, primary assembly (potential PTUs) was performed using Stringtie identifying 9,108 transcripts, corresponding to previous transcriptomic analysis24,29, but this is less than 50% of the recently predicted ORF's (20,058) that constitutes the T. cruzi genome21, or even the total genes annotated in Sylvio X10 (20,619).
We cannot discard the presence of unprocessed and stable PTUs in T. cruzi in our primary assembly as it has been describe in some instances in tryponosomatids30. However, since translation regulation relays mainly on mRNA stability, we found potential mature messengers from the same PTUs showing clear different levels of abundance (Fig. 2A), some of them match to more than one predicted gene. Surprisingly, most of PTUs did not match to any predicted genes.
In order to define the mature transcripts, we took advantage of the 39-nucleotide mini-exon sequence (SL) and polyA tail present in the 5′ and 3′-UTRs respectively of all mRNAs-31,32,33, as described by Rastrojo et al.34. 1,086,343 reads containing the SL sequence were trimmed and aligned back to the reference genome identifying 42,700 total SL-insertion sites (Fig. 2B), whereas 85,452 reads containing polyA-reads and 14,512 poly-Adenylation addition sites were identified. SL-containing sequences aligning in contrary orientation in any single PTU, confirming that in T. cruzi trans-splicing occurs only in the same orientation of the PTU similar to what has been described in Leishmania major Friedlin transcriptome34.
Considering that the previous annotated genome sequence was modified by our RNA analysis, the new Sylvio X10 genome was re-annotated using Companion and our transcriptomic evidence.
Using the SL and polyA insertion sites to define the 5′ start and 3′ end, respectively, most of the primary assembled PTUs were finally divided into 18,666 mature mRNAs. Of these, 1,172 were annotated as polycistronic, corresponding to sequences that contain one or more annotated ORFs, but that were not divided into mature transcripts by the presence of SL or poly-Adenylation-containing reads. Eight hundred seventy-two were annotated as truncated transcripts, corresponding to transcripts that do not cover its entire predicted ORF length (5′ or 3′ sequence). Although polycistronic and truncated mRNAs needs to be confirmed by future genomic and transcriptomic analysis, most cases correspond to low coverage transcripts. This may simply reflect that they are low-abundant transcripts at trypomastigote stage.
The remaining reads (about 3 million paired-end) that did not map to the reference genome or to kinetoplastid known sequences, were assembled de novo into potential transcripts using rnaSPAdes. Total potential assembled transcripts were filtered by coverage (RDC >= 10), length (>1 kb), sequence redundancy (≥90%) and contained ORFs (just contigs containing one ORF were considered). Finally, we obtained a total of 4,311 extra-transcripts (About 9.62 Mb). The abundance of these transcripts were also calculated using Stringtie and the sequences were added to the final transcriptome obtaining a total of 22,977 potential mRNAs. Those transcripts were named using the same nomenclature in other trypanosomatids: Trypanosoma cruzi "TC", Strain; "SX10", Polycistron Number, Monocistron number and Sense; "F", "R" or "X" (no sense identified) e.g TCSX10.1.1.F. De novo assembled transcripts were named using the contig number instead of polycistron and monocistron numbers, by default sorted by length (increasing order) and adding "ND" (non-defined) e.g TCSX10_ND.1.F, the sense was defined by the encoded ORF.
Taken together our results indicate that about the 79.95% of the T. cruzi genome (strain Sylvio X10) corresponds to coding sequence and therefore about 20% correspond to non-conding structural sequences. This is in sharply contrast with previous genomic analysis were only 37.73% of the total genome were defined as corresponding to coding sequences. Thus, our results have more than duplicated this estimation, largely improving the previous genomic annotation (Fig. 3). Besides, taking into account the length of ND transcripts, the haploid genome for Sylvio X10 may be higher than previously reported (at least 51 Mb).
In related species such as Leishmania, transcription initiation preferentially occurs at divergent strand-switch regions (SSR), where PTUs raise in opposite directions on opposing DNA strands15,16. In the Sylvio trypomastigote stage, we have identified a total of 226 SSR transcription initiation sites across the entire genome and up to 15 in a single C-LS (TcX10_chr24). All C-LSs contains at least one SSR (except TcX10_chr17) and 4.8 on average per chromosome. There is a medium positive correlation (0.54) between the number of SSR and C-LS length. On the other hand, there is a strong positive correlation between the total transcripts per chromosome and the truncated/polycistronic (0.86 and 0.84, respectively) transcripts found (as expected), whilst, there is no significant correlation between the number of forward, reverse and undefined transcripts (Fig. S1).
Seventeen thousand three hundred thirty-four transcripts have a FPKM (fragments per kilobase per million mapped reads) greater than or equal to 10 and quartile 3 includes values up to 66, whereas 59 transcripts have the maximum values (outliers, FPKM greater than or equal to 1,000) as shown in Fig. S2. These 59 top abundant transcripts (with FPKM greater or equal to 1,000) and its predicted function are listed in Table 1. Surprisingly, 83% (49) of the 59 more abundant transcripts correspond to de novo assembled transcripts and therefore were not annotated on the actual available genome for this strain.
The top abundant transcript corresponds to a ND transcript (TCSX10_ND.1840.F) containing an ORF coding for a highly conserved (in T. cruzi) elongation factor 1-alpha (EF-1-alpha). Different roles have been assigned to EF-1-alpha in various cellular processes, including metabolism, cytoskeletal organization, oncogenic transformation, apoptosis and anti-apoptosis and in T. cruzi has been described as potential regulator of gene expression35. Interestingly a second elongation factor (EF-2, TCSX10_ND.7486.F) was found in this top abundant list. The second and fifth top abundant transcripts correspond to mucin-like glycoproteins (TCSX10_ND.7941.F and and TCSX10_ND.5081.F) that are likely associated with parasite cell invasion and survival in Tc I strains36. Furthermore, 4 of these top abundant mRNAs in Sylvio X10 corresponds to heat shock proteins, as occurs in Leishmania (promastigotes) where they make up to 2.1% of the total protein in unstressed conditions37.
RNA binding proteins (RBPs), due their role in regulation of gene expression may represent one of the most important gene family for which parasite stage differential expression/transcript abundance information is of special interest. Unexpectedly, we found just one transcript (TCSX10_ND.7601.F, FPKM = 2301.46829) showing RNA recognition motif 3 (RRM3) of type I (polyA-binding proteins: PABPs) which is conserved in proteins that bind to the poly(A) tail of most eukaryotic mRNAs38. It may constitute a potential drug target for a translational inhibitor at trypomastigote stage that deserve deeper analysis across the entire parasite's life cycle. Additionally, we found being part of this list 17 mRNAs coding for ribosomal proteins (9 for 60S, 5 for 40S and one for S25, L24 and L37). It has been demonstrated that striking differences in ribosomal composition of trypanosomatids comparing to other eukaryotes (mainly the mammal host) may lead also to specific drug targets in Chagas disease39,40,41. The remaining abundant transcripts encode canonical histones H2B and H2A (TCSX10_ND.4352.F, TCSX10_ND.4559.F) and some others corresponding to proteins involved in cell metabolism.
The minimum transcript length was of 200 nucleotides and the maximum about 20.5 Kb (TCSX10.4085.2.F, a polycistronic transcript not divided by SL-containing or polyA-containing reads but containing 2 ORFs) and TCSX10_ND.1.F (a de novo assembled) transcript of about 20.2 Kb containing an ORF of 6,635 residues encoding a conserved transferase. The predominant length for Sylvio X10 transcripts is from 800 nucleotides to 2.2 Kb.
Finally, 12,146 of the C-LS transcripts (65%) and 4,197 (97%) of the ND transcripts were successfully identified by blastx hit to nr protein database, non-blasted transcripts were annotated as "Not Found".
Functional enrichment analysis
In order to define and visualize their functions, mature transcripts obtained in this study were ascribed gene ontology IDs (GO terms) with the Blast2GO tool. Level 3 of the gene ontology (GO terms) families mapped (at least 10 transcripts to be considered significant) were used to visualize the complete classification and functional enrichment as show in Fig. 4. In agreement to previous results, we found a set of transcripts ubiquitously abundant on epimastigotes, amastigotes and trypomastigotes24: 149 related to microtubule movement/processes (biological process/molecular function), 125 for chromosome organization/DNA packing (cellular component) and 17 for stress response (biological process) among others.
In Sylvio X10 trypomastigotes, the most abundant transcripts corresponding to cellular components belongs to membrane products (up to 200), cytoplasm and ribosomes. A total of 1,198 transcripts were classified into this level. GO terms associated to biological process corresponds to specific objectives that the organism is genetically programmed to achieve, including mRNAs that are implicated along the complete process or in the final outcome. A total 2,812 transcripts correspond to this group. Since the sequenced RNA was extracted from trypomastigotes (non-replicative infective form), is not surprising that the most represented biological process corresponds to pathogenesis (316), proteolysis (185), protein phosphorylation (142), cell adhesion (90) necessary in infection steps. These findings are also in agreement to Berná et al.24, which describes the enrichment of processes related to movement, adhesion, invasion and signalling in trypomastigotes (undefined strain). GO terms of Molecular function include transcripts involved on specific activities, and we found that in the Sylvio X10 transcriptome this is the GO family that includes most of the transcripts mapped (4,624). ATP-binding (480), protein-binding also known as glycoprotein-binding (398), exo-alpha-sialidase activity (334), protein kinases (134) and nucleic acid binding constitutes the top 5 most enriched functions.
Additionally, to the transcriptomic classification by GO terms, a second functional-based classification allow us the identification of the most abundant gene families over-represented across the entire transcriptome (Fig. S3). A total of 1,632 functionally related gene families were identified (17,929 transcripts), comprising 239 families (containing 10 or more genes) and corresponding to about the 52% (11,960) of the total transcriptome. Interestingly, 928 transcripts matching to Interpro GO ids did not show similar functions to other transcripts (single family) and 5,048 were not identified. This grouping of transcripts into functional families confirms that the trypomastigote physiological needs are covered by the transcription of multi-copy genes. As expected, some of these families correspond to the most abundant genes annotated along the T. cruzi genome21 such as; 1,329 transcripts coding for sialidase activity, 235 mucin-like proteins, 284 RHS (retrotransposon hot-spot) and 338 kinase proteins. Interestingly, about 428 transcripts corresponded to WD40-repeat-containing domain superfamily, proteins which are found in all eukaryotes and implicated in a variety of functions ranging from signal transduction and transcription regulation to cell cycle and apoptosis42. Specifically, WD40 motifs may act as a site for protein-protein interaction, and proteins containing WD40 repeats are known to serve as platforms for the assembly of protein complexes or mediators of transient interplay among other proteins. Other interesting abundant functionally related families are the P-loop containing nucleoside triphosphate hydrolase (695 transcripts), which is composed by proteins containing the most frequent domain of nucleotide-binding proteins43. This superfamily is characterized by the concanavalin A-like lectin/glucanase domain which includes proteins like glycosyl hydrolases, lectins, lyases, Beta-D-xylosidases, vp4 sialic acid binding protein among others44.
Trans-splicing and poly-Adenylation sites
The addition of about 39-nt mini-exon (SL) to the 5′ start of all mRNAs is an essential part of the maturation process in trypanosomatids45,46. As a result, an AG dinucleotide has been described as the consensus sequence for SL trans-splicing, but unfortunately, no specific signal for polyadenylation had been defined in Trypanosomatids, just a global polypyrimidine tracts of variable length is supposed to compose these regions34,47,48,49,50. Small differences between Leishmania and T. brucei in nucleotide composition surrounding the AG nucleotides and polyadenylation sites have been described before, suggesting that slightly different specific mechanisms may control the mRNA maturation process across trypanosomatid species34,48. In contrast, in-depth analysis of these sites in T. cruzi has not been performed to date. Thus, in this work, analysis of SL-containing and polyA-containing reads allowed us to differentiate between mature transcripts and long RNAs or PTUs, but also to analyse trans-splicing and polyadenylation sites to single nucleotide resolution.
We search for sequence enrichment/motifs associated to SL-addition sites by the calculation of the weblogo sequence on the ±30 nucleotide region from the trans-splicing and polyadenylation sites (Fig. 5). A total of 18,589 SL-addition sites were found. In agreement to previous results34,48, the AG dinucleotide was determined as the conserved insertion signature with 97.39% and 97.19% of probability for each base, respectively (Fig. 5A). Surprisingly, in contrast to Leishmania34 (having a Cytosine) but concordantly with T. brucei48, an Adenine nucleotide was the most probable (31.84%) residue before the dinucleotide AG (position -3), followed by Cytosine (29.18%), Thymine (26.53%) and Guanine (12.42%), interestingly at position -4 Guanine is the most probable nucleotide (31.20%) as occurs in L. major but contrary to T. brucei where a polyT tract starts and continue as the most frequent composition up to 50 nucleotides upstream.
The most marked differences between T. cruzi with L. major and T. brucei in the DNA regions surrounding the SL addition sites are in the pyrimidine nucleotide enrichment. Whereas in Leishmania C-T dinucleotides clearly consist about the 70% of nucleotides present upstream and downstream the AG dinucleotide, in T. cruzi, this pattern is different. T. cruzi just conserved the C-T pattern in the upstream 5′ region while downstream region is composed of purine nucleotides (A-G) up to 60% (Fig. 5A). T. brucei, also only had the C-T pattern in the upstream 5′ region but downstream A-T dinucleotides are the most frequent bases, indicating that in general, T. cruzi transcripts show more proportional nucleotide composition and G + C profiles (as ocurr in L. major) than T. brucei.
Regarding the polyadenylation signals, trypanosomatids, contrary to other eukaryotes, do not share the conserved AAUAA polyadenylation motif51,52. In contrast, an A-T, A-T, A-T in T. brucei and A-G, A-T, A-T for L. major have been described as the most probable dinucleotides preceding the polyadenylation site. In addition, sequence heterogeneity surrounding ±40 bases of these motifs between these T. cruzi related species have been determined. L. major displays a more variable sequence composition upstream and downstream polyadenylation site (specially the 5′)34,47 whilst, in T. brucei is possible to visualize a uniform pattern in both extremes composed by T-A nucleotides. In the T. cruzi, we found and analysed 9,789 polyadenylation sites that allow us to identify and visualize the surrounding genomic region (Fig. 5B).
Interestingly and contrary to other trypanosomatids where a AA dinucleotide has been found on the polyadenylation site34,53, we found that a single nucleotide is the most probable signal of polyadenylation start. Cytosine is the most frequent signalling nucleotide (45.32%), Adenine and Guanine are almost equally frequent bases (26.34% and 21.52%, respectively) and Thymine is the less frequent nucleotide with 6.79%. Likewise, our analysis allowed us to identify abundant Thymine composition (up to 95.45% at position −1) in upstream region, and a higher T + A composition (up to 89%) on the downstream sites, starting from +2 position. Together, our results indicate that mRNA maturation processes in T. cruzi may differ significantly from its closely related kinetoplastid counterparts.
In this work and for first time in T. cruzi, we have described some of the most important transcriptomic features in the tripomastigote stage of Sylvio X10 strain, as well as to contribute to the improvement of its genome sequence. Firstly, we corrected about 177 Kb of ambiguous genomic regions using RNAseq data and secondly we identified, classified, quantified and annotated 22,977 mature mRNAs. Their identification and quantification has allowed us to identify the main probable regulatory elements, and to significantly improve the entire genomic annotation.
We have also identified the transcription initiation SSRs across the genome, and determined that are positively correlated to the "chromosomal" length and therefore there is no strand-specificity as it occurs in Leishmania. Slighter abundance of Forward transcripts, also suggest that PTUs in T. cruzi are equally transcribed and that according to the genomic annotation the packaged genes may be or not functionally related. Besides, our results have identified that Sylvio X10 trypomastigotes exhibit a predominance of surface protein mRNAS, mainly those encoding trans-sialidase and mucin-like proteins which also constitutes the most expanded gene families, confirming that the gene copy number act as secondary regulator mechanism of protein expression.
Finally, but equally important, we have identified and described the trans-splicing and polyadenylation sites in T. cruzi which are different to other closely related kinetoplastids such as Leishmania and T. brucei, mainly on the polyadenylation signal and surrounding sequences.
Material and Methods
Parasite cultures and RNA extraction
The Sylvio X10 strain was obtained from Dr. M. Miles (London School of Hygiene and Tropical Medicine, London, UK) through the European program ChagasEpiNet. Vero cells were grown in RPMI medium supplemented with 5% fetal bovine serum (FBS), 100 UI/mL of antibiotics mixture, 10 µg/mL streptomycin and 2 mM glutamine at 37 °C in an atmosphere of 5% CO2 until the cells reached 80% confluence. The cell monolayer was subsequently infected with metacyclic trypomastigotes of T. cruzi Sylvio X10 strain. After 4 days, the supernatant medium was collected, Vero cells and amastigotes were removed by centrifugation at 1000 g by 5 minutes. Trypomastigotes were collected by centrifugation at 1600 g for 10 minutes. Three biological replicates where mixed before RNA isolation.
RNA was isolated using the RNeasy Mini Kit (Qiagen) and treated with RNAse-free DNAse I. RNA samples were quantified by absorbance at 260 nm using the Nanodrop ND-1000 (Thermo Scientific), all samples showed an A260/A280 ratio higher than 2.0. In addition, RNA integrity was checked in a bioanalyzer (Agilent 2100) resulting on RIN value higher than 8.
RNAseq and transcriptome analysis
RNA-seq was performed at the Massive Sequencing Platform of Cantoblanco (CSIC-PCM, Madrid, Spain). Standard libraries for massive sequencing were generated using the TruSeq RNA Sample Prep Kit (Illumina). Briefly, poly-A + RNA was selected by oligo-dT chromatography, and RNA fragmentation was achieved using divalent cations under elevated temperature. Afterwards, these fragments were used to generate a cDNA library, and cDNA fragments corresponding in size to about 450–550 bp were isolated from an agarose gel. Paired-end reads of 100 nucleotides were obtained, and raw reads were subject to quality-filtered using the standard Illumina process and analysed using FASTQC tool54.
Reads were mapped to the reference genome using Bowtie255, transcript assembly and abundance was calculated with Stringtie56 and assembly corrections were performed using Pilon57. Transcript identification/classification was performed by Blas2GO suite58, Blastx using the protozoan non-redundant (nr) proteome downloaded from the NCBI and in-house python scripts. Trans-splicing and polyadenylation sites were identified as described before by Rastrojo et al.34 and the local version of the WebLogo tool59. All figures and statistical analysis were performed using R and Rstudio60.
Additional bioinformatics tools were used to handle, parse, analyse or visualize sequencing data such as samtools61, CD-HIT62 and IGV63.
Data availability
Raw data has been submitted to the NCBI repository (SRA) under the accession number PRJNA546488. The new genome version, transcript sequences and annotation file have been also submitted to the TritrypDB for its public access. Both data will be immediately available after the publication of our manuscript.
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This work was supported by the "Consejo Nacional de Ciencia y Tecnología" (CONACYT, México) through the FC-H Ph.D. studentship number 411595 and the "Consejo de Ciencia, Tecnología e Innovación de Hidalgo (CITNOVA), México. By grants from "Ministerio de Economía y competitividad" (MINECO/FEDER) SAF2015-63868-R, and "Ministerio de Ciencia, Innovación y Universidades" (MICINN-FEDER) PGC2018-096132-B-I00, to N.G., SAF2016-75988-R (MINECO/FEDER) to M.F.; "Red de Investigación de Centros de Enfermedades Tropicales" (RICET RD12/0018/0004) to M.F.; European Union (HEALTH-FE-2008-22303, ChagasEpiNet) to M.F.; Comunidad de Madrid (S-2010/BMD-2332) to M.F.; and Institutional grants from "Fundación Ramón Areces" and "Banco de Santander". The authors would like to thank Dr. Ricardo Ramos responsible of the "Unidad de Genómica del Parque Científico de Madrid" and to Dr. Begoña Aguado and Fernando Carrasco, responsibles of the "Servicio de Genómica del CBMSO (CSIC/UAM)" for their valuable support on sequencing project.
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Callejas-Hernández, F., Gutierrez-Nogues, Á., Rastrojo, A. et al. Analysis of mRNA processing at whole transcriptome level, transcriptomic profile and genome sequence refinement of Trypanosoma cruzi. Sci Rep 9, 17376 (2019). https://doi.org/10.1038/s41598-019-53924-6
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