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I am Los Angeles based mix and mastering engineer (by way of Boston). I've worked with subsidiary labels of Sony and Columbia and Def Jam. As well is indie labels such as BBE, Ubiquity, Soul Clap and many more. My experience ranges all the way from disco and funk vinyl reissues to pop, hip hop and almost anything in-between.
Joseph Caserta (known as Caserta) is a Mix Engineer, Producer and DJ from Boston, Massachusetts. As the son of a drummer, Caserta was exposed to a wide range of music from an early age. Funk and Soul classics by the likes of The Average White Band, Tower of Power and Luther Vandross were just a few of the staples heard in the Caserta household.
In his junior year of high school, Caserta would meet his soon to be best friend and aspiring rapper Ashmead "Ash" Arjune. It was Ash that introduced him to his first production and mixing software, Sonic Foundry's Acid Pro, Soon experimentation became an obsession. He began crafting and mixing self-described "horrendous beats" out of stock loops that came with the program. With the production flame being lit, he went from making Hip Hop mixtapes to high school cheerleading music. He would use any excuse to get behind the computer to edit and mix.
Things haven't changed much since then. Caserta has established his own studio where he has mixed records for labels such as Def Jam, Columbia, BBE and countless more. Over a short period of time, Caserta has had the unique opportunity to apply modern-day techniques to Disco, Funk and R&B sessions from the 70's and 80's – while at the same time, infusing House, Pop, Latin and EDM projects of today with a touch of nostalgia.
Click the 'Contact' above to get in touch. Looking forward to hearing from you.
My credits include
AllMusic verified credits for Caserta:
- Matthew Dear
- Matthew Dear
- Kon
- Defected Radio
- Caserta
- Caserta
Gear highlights
- UAD Apollo w/UAD Plugins
- DBX Edison Stereo Imager
- Ableton
- Logic
- Pro Tools
Terms of Service
Typical week to 2 week turn around time depending on size of project. Additional versions of same song typically 1/4 of the full price. No hard limit on revisions within reason
Reviews of Caserta
Caserta is super flexible, quick, and has an amazing ear. It always sounds like he is working with entire extra dimensions when I compare his mixes to my roughs. I used to think I was a decent mixer until I had him mix one of my tracks... I haven't mixed anything of my own since. Top notch!
When I'm in a jam, and can't get the sound I'm looking for I know Caserta is willing to get me past the finish line! Great ears, and very friendly to work with.
Great ear! Picks up on the sound and vibe we were looking for and was fast with mixes and any feedback.
Unlike the past where artists and producers would hire somebody to mix their project, these days many producers also take on the task of mixing down their own music which is great if you have an understanding of what it takes but, it's an art within itself and Caserta has a true talent for the science of sound.


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Caterpillar Financial Announces Results as of Early Participation Date and Determination of Pricing Terms of Exchange Offers
NASHVILLE, Tenn., Sept. 22, 2016 -- Caterpillar Financial Services Corporation, a wholly owned subsidiary of Caterpillar Inc. (NYSE: CAT) (the "Company"), announced today the results, as of 5 p.m. EDT September 20, 2016 (the "Early Participation Date"), and the determination of the pricing terms of its previously announced private offers to exchange certain of its outstanding medium-term notes (collectively, the "Existing Notes") for a combination of a new issue of the Company's Medium-Term Notes, Series H, 1.931% Notes due 2021 (the "New Notes") and cash (the "Exchange Offers").
Early Results
Based on information provided by D.F. King & Co., Inc., the exchange agent for the Exchange Offers, the condition that the aggregate principal amount of Existing Notes validly tendered for exchange and not validly withdrawn as of the Early Participation Date exceed the amount required for the issuance of at least $250 million aggregate principal amount of New Notes has been satisfied.
In accordance with the terms and conditions of the Exchange Offers, the Company will accept any and all of the $380,850,000 aggregate principal amount of (1) Medium-Term Notes, Series F, 5.85% Notes due 2017, (2) Medium-Term Notes, Series F, 5.45% Notes due 2018 and (3) Medium-Term Notes, Series F, 7.05% Notes due 2018 that were validly tendered for exchange and not validly withdrawn as of the Early Participation Date. The Company expects to deliver New Notes and cash in exchange for such accepted Existing Notes on September 26, 2016 (the "Early Settlement Date"), subject to satisfaction or waiver of the conditions to the
Exchange Offers and in accordance with the other terms and conditions disclosed in the Company's confidential offering memorandum dated September 7, 2016 (the "Confidential Offering Memorandum").
The following table indicates, among other things, the principal amount of Existing Notes validly tendered and accepted for exchange as of the Early Participation Date:
|Title of
|Principal Amount
Tendered and Accepted as of Early
Participation Date
|Medium-Term Notes, Series F, 5.85% Notes due 2017
|Medium-Term Notes, Series F, 5.45% Notes due 2018
|Medium-Term Notes, Series F, 7.05% Notes due 2018
Pricing Terms
The pricing terms were determined as of 11 a.m. EDT, on September 21, 2016 (the "Price Determination Date") in accordance with the terms set out in the Confidential Offering Memorandum and the related letter of transmittal (the "Letter of Transmittal").
The total exchange price to be received in each Exchange Offer for each $1,000 principal amount of Existing Notes validly tendered, and not validly withdrawn, prior to the Early Participation Date is set forth in the table below. The total exchange price for the Existing Notes was determined based on a fixed-spread pricing formula using the bid-side yield on the applicable Reference U.S. Treasury Security set forth in the table below and the applicable fixed spread set forth in the table below, and will be paid in a combination of New Notes and cash in an aggregate principal amount determined as set forth in the Confidential Offering Memorandum. The total exchange price includes an early participation premium of $30 payable in cash only to Eligible Holders who have validly tendered and who have not validly withdrawn their Existing Notes prior to the Early Participation Date.
The table below shows the total exchange price and composition of total exchange price per $1,000 principal amount of each series of Existing Notes accepted in the Exchange Offers.
|Composition of Total Exchange Price
|CUSIP Number
|Title of Security
|Principal Amount
|Reference U.S.
Treasury Security
|New Notes
|Medium-Term Notes, Series F, 5.85% Notes due 2017
|0.625% due
August 31, 2017
|$30 in cash
|Medium-Term Notes, Series F, 5.45% Notes due 2018
|0.75% due
August 31, 2018
|$30 in cash
|Medium-Term Notes, Series F, 7.05% Notes due 2018
|0.75% due
August 31, 2018
|$30 in cash
In addition to the total exchange price or exchange price, as applicable, holders whose Existing Notes are accepted for exchange will be paid accrued and unpaid interest on such Existing Notes to, but not including, the applicable settlement date, described below.
The New Notes will bear interest at a rate per annum of 1.931%, which is equal to the sum of (1) the bid-side yield on the 1.125% U.S. Treasury Security due August 31, 2021 (based on the bid-side price indicated on the Bloomberg reference page PX1) on the Price Determination Date, which was 1.231%, and (2) 0.70% (70 basis points). The New Notes constitute a part of a series of the Company's debt securities designated as Medium-Term Notes, Series H.
The Exchange Offers are being conducted by the Company upon the terms and subject to the conditions set forth in the Confidential Offering Memorandum and the related Letter of Transmittal. The Exchange Offers are only being made to, and copies of the offering documents will only be made available to, a holder of Existing Notes that has certified its status as (1) a person in the United States who is a "qualified institutional buyer" as defined in Rule 144A under the Securities Act of 1933, as amended (the "Securities Act"), or (2) a person outside the United States who is not a "U.S. person," as that term is defined in Rule 902 under the Securities Act, and, in each case, if resident and/or located in any member state of which has implemented provisions of the EU Prospectus Directive (each, a "Relevant Member State"), who has also certified that it is a "qualified investor" as defined in Article 2(1)(e) of the EU Prospectus Directive (each, an "Eligible Holder"). "EU Prospectus Directive" means the European Union's Directive 2003/71/EC (as amended, including pursuant to Directive 2010/73/EU) as implemented in the Relevant Member State. The Confidential Offering Memorandum is not a prospectus for the purposes of the EU Prospectus Directive, and the Exchange Offers will only be made pursuant to an exemption under the EU Prospectus Directive from the requirement to produce a prospectus in connection with offers of the New Notes.
The Exchange Offers will expire at 11:59 p.m. EDT on October 4, 2016 (the "Expiration Date"), unless extended by the Company. In accordance with the terms of the Exchange Offers, tendered Existing Notes may no longer be withdrawn, except in certain limited circumstances where additional withdrawal rights are required by law. The Company will accept for exchange on October 6, 2016 all Existing Notes validly tendered at any time prior to the Expiration Date that are not validly withdrawn and that are not exchanged on the Early Settlement Date, unless the Expiration Date is earlier terminated or extended by the Company, and subject to satisfaction or waiver of the conditions to the Exchange Offers and in accordance with the other terms and conditions disclosed in the Confidential Offering Memorandum.
The New Notes have not been registered under the Securities Act or any state securities laws. Therefore, the New Notes may not be offered or sold in the United States absent registration or an applicable exemption from the registration requirements of the Securities Act and any applicable state securities laws. The Company will enter into a registration rights agreement with respect to the New Notes.
Documents relating to the Exchange Offers will only be distributed to holders of the Existing Notes that complete and return a letter of eligibility confirming that they are Eligible Holders. Holders of the Existing Notes that desire a copy of the eligibility letter may contact D.F. King & Co., Inc., the information agent for the Exchange Offers, by calling toll-free (888) 540-8597 or collect (232) 269-5550 (banks and brokerage firms) or e-mailing [email protected]. Holders of the Existing Notes may also complete and submit a letter of eligibility online at http://www.dfking.com/cat.
This press release is not an offer to sell or a solicitation of an offer to buy any security. The Exchange Offers are being made solely pursuant to the Confidential Offering Memorandum and related Letter of Transmittal and only to such persons and in such jurisdictions as is permitted under applicable law.
This press release contains certain statements that may be considered "forward-looking statements" as that term is defined in the Private Securities Litigation Reform Act of 1995. These statements may relate to future events or the Company's future financial performance, which may involve known and unknown risks and uncertainties and other factors that may cause the Company's actual results, levels of activity, performance or achievement to be materially different from those expressed or implied by any forward-looking statements. Forward-looking statements give current expectations or forecasts of future events about the Company. You may identify these statements by the fact that they do not relate to historical or current facts and may use words such as "believes," "expects," "estimates," "anticipates," "will," "should," "plan," "project," "intend," "could" and similar words or phrases. These statements are only predictions.
About Caterpillar Financial Services Corporation
Caterpillar Financial Services Corporation is a wholly-owned finance subsidiary of Caterpillar Inc. The Company's primary business is to provide retail and wholesale financing alternatives for Caterpillar Inc. products to customers and dealers around the world. Retail financing is primarily comprised of financing of Caterpillar Inc. equipment, machinery and engines. In addition, the Company also provides financing for vehicles, power generation facilities and marine vessels that, in most cases, incorporate Caterpillar Inc. products. The Company also provides wholesale financing to Caterpillar Inc. dealers and purchase short-term dealer receivables from Caterpillar Inc. The various financing plans that the Company offers are primarily designed to increase the opportunity for sales of Caterpillar Inc. products and generate financing income for the Company. A significant portion of the Company's activities is conducted in North America. However, the Company has additional offices and subsidiaries in Asia-Pacific, Europe and Latin America. The Company has more than 30 years of experience in providing financing for Caterpillar Inc. products, contributing to the Company's knowledge of asset values, industry trends, product structuring and customer needs.
CONTACT: Rachel Potts, Global Government & Corporate Affairs, 309-675-6892, [email protected]

subset: commoncrawl


text:
Add some decadence to tea time.
If you're like us, you need a little treat with every cup of tea, like these Vanilla Bean and Butter Pecan cookies. They're equal parts sweet, buttery, and salty: everything we could want with our favorite varieties of tea.
Sourced from: Mah-Ze-Dahr Bakery
Topics: Cookies!
Our return policy.
Photography by Rocky Luten and James Ransom.
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jam gordon
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(Oh, and $10 off your order of $50 or more in the Food52 Shop, too.)


text:
Journal For Plague Lovers
- Peeled Apples
- Jackie Collins Existential Question Time
- Me And Stephen Hawking
- This Joke Sport Severed
- Journal For Plague Lovers
- She Bathed Herself In A Bath Of Bleach
- Facing Page: Top Left
- Marlon J.D.
- Doors Closing Slowly
- All Is Vanity
- Pretension/Repulsion
- Virginia State Epilpetic Colony
- Williams Last Words
- Bag Lady
- Rádio interpreta
- Rok vydání: 2009
- Žánry: Rock
- Vydavatelství: Sony BMG
- Text písničky
Haven't shaved for days
Keeps the appearance of delay
The luxury of one more dye
Pretend humility, the ugly lie
I would prefer no choice
One bread, one milk, one food, that's all
I'm confused, I only want one truth
I really don't mind being lied to
It's not "What's wrong?"
It's "What's right?"
Makes you feel like I'm talking a foreign language sometimes
It's not "What's wrong?"
It's "What's right?"
It's the facts of life sunshine
It's the facts of life sunshine
It's the facts of life my sunshine
- All Is Vanity (03:34)


text:
James McGowan, the father of Australian footballers Ryan and Dylan McGowan, is set to be extradited to Scotland on a murder charge, according to a report.
The 57-year-old has been in custody since December 2013, after being charged with the stabbing murder of Owen Brannigan, his former brother-in-law, in 1999.
The ABC has reported that the Federal Justice Minister, Michael Keenan, has approved the extradition.
A warrant for James McGowan's arrest was issued on December 3, 1999, four days after Brannigan's, but he left Scotland the day it was released. The case was originally dropped due to lack of evidence before being re-opened by a cold-case unit.
The Glasgow Herald has reported that despite the outstanding warrant, James McGowan regularly travelled back to Scotland to watch his sons play football.
Both of his sons previously played for Scottish club Heart of Midlothian - Dylan, 24, is now in the A-League with Adelaide United while Ryan, who famously provided the assist for Tim Cahill's glorious goal in the 2014 World Cup against the Netherlands, is in China playing for Henan Jianye FC
James McGowan travelled to Scotland in 2012 to watch Ryan play in the Scottish Cup final with Hearts - and saw his son score in a 5-1 victory over Hibernian.
"This might be the only time I get to see my son in a cup final," he said.


text:
Release the Light Sexual Assault Awareness ProgramTuesday, Oct. 27th and Thursday, Oct. 29th
11:00am-2:00pm and 9pm-11:00pm
Learning Commons
The Counseling/Student Development Center joins other University organizations in launching the Red Flag Campaign against relationship violence. (during Sexual Assault Awareness Month)
Preview :No Visible Bruises" a documentary of Katie Koestner, founder and director of Take Back Night Foundation and featured speaker on Thursday, Oct. 29, 2015.
|Learning Commons
Marywood University joins in the national effort in providing its "Release the Light" Sexual Assault Awareness Program. October is designated as National Sexual Assault Awareness Month and communities and colleges across the nation will be joining together against crimes of sexual abuse. The purpose is to increase student and community awareness for the need of violence prevention and reducing incidents of sexual assault on college campuses and in the community.


text:
Simplicity 9728; ©1980; Misses Slim Fitting Dress: Top-stitched dress with scooped neck gathered to asymmetrical shoulder yoke has back zipper and side slit. V1 has long sleeves. V2 has short sleeves.
Links to reviews/blog postsEdit
Add a photo to the gallery by clicking the "modify" button below.
[insert your username, and make sure your preferences allow for people to contact you via e-mail. Do not list your email address here!]


text:
Hire a Logo Designer
Budget ₹12500-37500 INR
22 freelancers are bidding on average ₹16805 for this job
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text:
July 4, 2013
Unlimited Vacation for All?
Chip Litherland for The New York Times
It's a small but growing trend in the United States, as companies like Netflix stop tracking their employees' days off. Is this a sustainable model, or are rules and regulations still important to protect both workers and employers?Read the Discussion »


text:
It's hard to get away from the horror of the world this week, and our hearts go out to everyone much more immersed in it than we are.
For those of us in need of a distraction*, what's more wholesome and good and right in the world than children's books? Nothing, I tell you. So PW's PWxyz blog has some for you, namely the books that made their staff big enough readers to become the kind of people who work at PW. Not always children's books, of course, but a few of those are among my childhood favorites as well. Rachel Deahl's reminiscence about The Bridge to Terabithia really strikes a chord with me, though I'm not sure I disliked it so much. I knew what was going to happen and was determined not to care. And then I did. Boy, how I did.
For me, I'm not sure I can totally pinpoint a book. Certainly the Laura Ingalls Wilder books were incredibly important to me growing up, as I've written about before. If I try to trace it back to the first book I was passionate about, I'd say it would have to be something from the Cam Jansen series. Do you remember Cam? She had a photographic memory (hence her name), and she solved mysteries by puzzling together the clues hidden in her brain. She was brilliant and feisty and dedicated and maybe a bit of a show off.
I like to think she's still a role model for me today, though my memory isn't quite photographic and I've never had to locate missing monkeys.
So what did it for you? Was it a children's book or something you encountered later in life? When did you give yourself up to the reading gods and never turn back?
*Did that not work? Try this.
P.S. Do you know about the group read Jim and I are doing? Are you already reading Eleanor & Park? We can't wait to discuss with you.


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AP Euro DBQ Rubric
UPDATED AUGUST 2016! I've taken the latest changes to the guidelines released by the College Board for AP history courses and created a user-friendly AP Euro DBQ Rubric for teachers to use while assessing their students' DBQs.
VIDEO: The New AP Euro DBQ Rubric
I've created a video series explaining the new DBQ rubric that will be used on both the AP US History and AP European History exams this year. If you're not into Lord of the Rings, I apologize in advance!
Revised AP Euro DBQs
Here is a collection of old DBQ prompts that have been released by the College Board, which I have re-designed to conform to the format of the current exam. I am passwording these documents so that they'll only be available to AP Euro teachers. If you would like to access them, please sign up for my e-mail list using a school-based e-mail address and I will provide you with a password (NOTE: It will take me 24-48 hours to verify your e-mail and get back to you with a password).
1993 DBQ (Renaissance Education) NEW FORMAT
2008 DBQ (German Peasants' Revolt) NEW FORMAT
2011 DBQ (Elizabeth I) NEW FORMAT
2005 B DBQ (Scientific Revolution) NEW FORMAT
2007 DBQ (Child Rearing) NEW FORMAT
Thanks Dan Moore for contributing this DBQ!
2008 B DBQ (French Revolutionary Calendar) NEW FORMAT
2004 DBQ (Attitudes Toward the Poor) NEW FORMAT
2009 DBQ (New Imperialism) NEW FORMAT
I will e-mail the DBQ access password to teachers who sign up for the e-mail list with an institutional e-mail (I am trying to keep these documents in the hands of teachers).


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An old chief that i regret! It would be great working together again! Recommended
- Ing. Gian Luca Paloni (1/11/08)
Peer Assessment:22
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alessandro cavicchia
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text:
I've been poking around in Monitoring, but can't figure out how to do this. Is it possible? I have a DHCP range setup for the manufacturing floor that cannot access the internet. A few people have figured out how to change their IP to a manually-configured address that DOES have internet access. They do it temporarily during the day, and change it back to Automatic, so that the DNS server always shows their normal automatically-assigned IP at the end of the day when I do reports. I need to know about it when it happens, so I can go bust them.
3 Replies
May 4, 2009 at 1:25 UTC
Niagara Technology Group (NTG) is an IT service provider.
SpiceWorks can't do this unless you scan mid day when it has changed. You would likely need a custom SQL report at that point to fix this.
The real issue here should not be one of monitoring but one of locking down the machines. Why are the users any to change their IP settings? Can you not make them not be the administrators?
Or set their router so that it cannot route them to the Internet.
What about putting them on a segmented VLAN so that you can apply specific security.
Your proxy server should allow you to recognize the users and lock them out as well.
May 4, 2009 at 3:01 UTC
Thanks, but I've looked into all the conventional options already. :-(
We have a legacy app that requires a local Administrator to re-install itself every time it is run. I've told the developers there MUST be a better way, but no one wants to change anything. so I'm stuck there.
OK, don't laugh - no one knows how to login to the router. Its been here since 2001 or 2002, and that was 2 or 3 network admins ago. We are afraid to mess with it, no one here knows the settings and of course the Cisco maintenance contract was never kept up. The plan is to let it ride until we're forced to upgrade to a dual bonded T1, then hire someone to come in and change it out.
Maybe I could do a VLAN later on, but I really don't have time to plan anything like that. And I'm not sure all our switches support VLANs, the old ones probably don't.
We don't use a proxy server. I might be able to get Untangle to block them, if I can ever get it working inside the firewall and get working on a reliable piece of hardware. Its just been an experiment so far, and it keeps kernel panicking.
May 4, 2009 at 10:27 UTC
Niagara Technology Group (NTG) is an IT service provider.
Wow, that is a rough network. Untangle might be more than you need/want. Consider a transparent proxy like Squid inline with your router. You could sneak it in place without anyone being any the wiser and have a dedicated proxy machine without the overhead of Untangle. Firewall products used as something other than firewalls often are a lot of a hassle just because they have weird expectations of use.
This discussion has been inactive for over a year.
You may get a better answer to your question by starting a new discussion.


text:
Gilwell Heritage - By Train & Trek Cart
By Train & Trek Cart
Next time you hop into your car and travel to Gilwell, spare a thought for the difficulties your predecessors faced. The journey became part of the adventure, which was camping at Gilwell.
In the 1920's the area around Gembrook was primarily used for logging and then farming. Gilwell Park campsite was the brainchild of Tom Russell whose family owned, logged and farmed the land. Its future was uncertain as at first the Scout Association was concerned that the only way into the campsite was an old logging track from Gembrook. But fortunately the adventurous character of those early Scouts prevailed.
Few people owned a car but by this time the narrow gauge Puffing Billy line from Ferntree Gully to Gembrook had been built to convey logs, produce and people back to the Melbourne markets. Eager Scouts would climb aboard with all their gear in a trek cart which was placed in the guards van. On arrival at Gembrook they would have to pull or push their trek cart the 6 kms downhill to Gilwell, no doubt singing all the way. All repeated at the end of the camp of course, but this time uphill!
"It all fitted in there yesterday" Gembrook Station
As time went by the track into Gilwell improved enough to allow better motorised access but still transport by car was not always possible. Some of you might remember piling the troop into the back of a local furniture van. Patrol boxes were used for seats but there were no safety belts! Now for large events Scouts arrive by the coachload, - what a difference.
Petersens's removal van
Travelling to Gilwell Park was once a considerable challenge with many of us breathing a sigh of relief when the last rut and pothole was safely negotiated. The journey might be easy now, but spare a thought for the good old days!


text:
Incorrect Formatting of Tables 1 and 3. In the article titled "Elevated Inflammation Levels in Depressed Adults With a History of Childhood Maltreatment," by Danese et al, published in the April issue of the Archives (2008;65[4]:409-416) Tables 1 and 3 were formatted incorrectly. Table 1, on page 413, should have read as follows:
In Table 3, on page 414, column 1, the top section of the column under the subheading "C-reactive protein >3 mg/Lb," entries 1 through 7 should have aligned with entries 1 through 7 in the bottom section, under the subheading "Inflammation factord."
Incorrect Formatting of Tables 1 and 3 in: Elevated Inflammation Levels in Depressed Adults With a History of Childhood Maltreatment. Arch Gen Psychiatry. 2008;65(6):725. doi:10.1001/archpsyc.65.6.725
© 2017


text:
by Jen Tressler
Nathan is the quirkiest person I ever met. When I first met him in college, I quickly had a crush on him with the long hair, trench coat, doc martens. He would walk me to class and we'd hang out in the cafe all the time, talking and writing in these slam books we made up. At the time, we were friends, but I was judgmental and stopped liking him because I thought he was too eccentric. Over time, I found that he had had a crush on me too, but it was too late. He always seemed to value my friendship more than I valued his until I met him again, years later.
When I finally found Christ, I started going to church only to meet up with Nathan once again. We immediately became friends, but this time I had changed and I accepted him for who he was. He would always tell me how much he admired me and valued our friendship, even though he would say crazy things to the waiters of restaurants we'd go to like "woof, your face, do you accept child sacrifices" etc. things like that.
But Nathan genuinely loved God. We would talk about Christ for hours, always trying to understand how we could be closer to God even though we were imperfect. Nathan loved to write and wrote a lot about God. As I read his notes on Faebook, I tear up now.
Anyway, for the past few years, Nathan has come over often for barbecues, board games, camp outs, deep discussions, and much more...he told me I was his best friend, even. He even helped me get back together with my then boyfriend, current husband, through his prayers and intervention.
Nathan was so encouraging to me and I will miss him deeply. Holidays and BBQs are not going to be the same without his quirky but kind-hearted presence. He just spent tons of money buying me and my husband all this food that we still have to this day.
Goodbye my very good friend :'C
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BinTEK ™ Ultra Premium MicroFiber Cloth 16 inch x 16 inch 330 GSMBTC910003 $2.95 OUT OF STOCK
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If you ever have problems with your Order, we'll do everything we can to make it right.
As a small company (32 of us total) we aim to treat you like family. We're in business because of you.
Copyright © 2017 Phone Parts USA. All Rights Reserved. PhonePartsUSA.com,LLC and its products are in no way endorsed, sponsored or affiliated with Apple, Google, HTC, Motorola, Blackberry(RIM), LG, Huawei, ZTE, Sony and Samsung or their subsidiaries.


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Conveniently located right off of I-25 and Tejon, LivWell Tejon is Colorado Springs' premier medical marijuana dispensary and has been ever since we opened our doors six years ago. Our philosophy to help people live well and better by safely offering medical marijuana products to adult consumers by delivering the most customer-oriented and pleasurable cannabis dispensary experience possible.
LivWell on Tejon is widely regarded amongst Eastern Colorado's top medical marijuana dispensaries, strictly adhering to Colorado's regulatory requirements and outperforming the lesser weed dispensaries of the area. When searching for a medical dispensary in Colorado Springs, look no further than LivWell Tejon- our highly-trained and experienced staff will assist you in finding the product that best suits your needs, all within a friendly and comfortable atmosphere that demonstrates why we are considered to be a leading Colorado dispensary.
Unlike some other weed dispensaries, we want our patients to enjoy the best value for their purchase. Get the most out of your LivWell experience by signing up for LivWell Rewards. Earn one point for every dollar you spend, and redeem those points on future purchases. Members receive $10 in credit upon signing up, plus complimentary points for referring a friend, on their birthday, and on their membership anniversary.
At LivWell you will find everything you need, with a friendly comfortable atmosphere. We are a one stop shop with a wide selection of Flower, Edibles, Concentrates, Topicals, Accessories, and Glass. Conveniently located right off of I-25 and Tejon. Where you can gain points for dollars spent & earn store credit. This loyalty card is for everyone and can be used at all of our LivWell locations.
Oh, and find us on our Facebook page at www.facebook.com/Livwellco hit the like button to get updated on strains, events, coupons, promotions and more.
500 points = $25 store credit on your LivWell Reward's to be used on any items in the store! (not including discount items)
-10% off ALL Shelf Flower
-20% off ALL Edibles
-20% off ALL Concentrates
-20% off ALL Glass & Accessories
www.LivWellco.com - @livwellcannabis
Please confirm your email before writing a review.
I love this store. The staff is absolutely wonderful.
LivWell on Tejon and I found each other by chance after I missed my exit on I-25. What seemed like a mistake became a blessing! I am impressed with the quality on the kosher kush & chem dawg alien og, both sat well above 20% THC, & 108.25 out the door. I expected dry, shakey, or leafy which is common at other local dispos. Expectations totally wrong! Great bud structure, with complete trichrome coverage, & a choice between strains! Like a fine wine, with a little more cure at home they get even better!
Inviting staff, all knowledgeable & passionate. Always answer my questions & don't push products down my throat, which is refreshing. Comfortable & relaxed atmosphere. The young lady who does the art, suggested a gram of viola live resin & craft pho sugar wax. The $ initially seemed relatively steep but after I dabbed them they were delicious! Both were terpy & noticeably more potent than the cheaper dabs, worth it. I will get her name next time & ask what she is dabbing, great taste!
This shout out is for Morgan. Thank you so much for the magical experience at Livwell on Tejon. Parking is ruff but worth it. Walking in the people are so friendly right away. Morgan was super educated in their products along with having a great personality made it a great environment. I'm so happy with my receipt which came out to 420 (and change) on Friday the 13th. Love it so much that I will keep it forever, along with my cool hoodie and hat. Also enjoy my buds approved by Snoop Dogg himself which came in the coolest boxes. I will truly Livwell and be happy Thanks to Morgan at Livwell on Tejon! Thanks again niccibear420
1 person thought this review was helpful
I have been going to Livwell on Tejon for about year now. I have been to tons of dispensaries and nowhere compares.. The environment makes you feel like you're at your friend's house getting bud and the staff is EXCELLENT. Chelsea is an amazing person and manager, she always makes you feel like you belong and she cares about you. No matter who is working, I am sure to have a great conversation and leave there with a smile on my face.. even after a bad day. All I can say is thanks Matt, Frank, Marcus, Taylor, Joey and everyone else, y'all are awesome!!
1 person thought this review was helpful
I moved to Colorado just over a year ago to save my wife's life (disabled, bedridden, multiple comorbidities, etc). Thanks to Cannabis, SHE'S LIVING AGAIN!!! With that said, I have been to most dispensaries between Denver and Trinidad in search of the best medicine for myself and for my wife. Now that I've found Livwell, my search is over. Their quality, consistency, and level of customer service have been unmatched for me. Price is top notch as well. I now drive 3 hours round trip for meds and couldn't be happier. It's crazy. If you haven't tried them out, you need to. Thanks to Livwell, my wife and I are LIVing WELL again!!! Thank you guys!!!
Bought some tangerine man leafs by snoop, I think it should be called seeds by snoop :(. There is a bunch of seeds, and I heard this was the best batch of it yet. I was pretty disappointed to say the least!
Thank you for taking the time to leave us a review. We apologize for the seed issue, please email firstname.lastname@example.com and we will gladly assist.
It's so nice that you guys always have deals going on and the service is always appreciated. Between the deals, rewards system, and service you can call this place "THE Favorite"
Thank you for taking time to leave us a review. We look forward to serving you in the future!
My wife and I received the rewards card, wow it really is Rewarding. I recommend getting one, no membership requirements and I'm enjoying the BluePower#1 as I write this Review. Thanks LiveWell.
this clinic has great smoke and great shatter/wax highly recommend checking em out.


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The ESP8266 started a small revolution by bringing WiFi to a small and cheap package that also had enough processing power and enough pins to get small things done. Now get ready to take your bite-sized WiFi capabilities to the next level with the ESP32 Development Board!
The development board breaks out all the module's pins to 0.1″ headers and provides a CP2102 USB-TTL serial adapter, programming and reset buttons, and a power regulator to supply the ESP32 with a stable 3.3 V. Espressif doubled-down on the CPU resources for the ESP32 with a dual core, running at 160MHz and tons more pins and peripherals.
Please note we have a limited quantity at this time. This is a development board for people who are comfortable compiling via the Tensilica toolchain. There is no Arduino IDE setup for this yet, nor is there a Lua or micropython port. We are providing these for the community of advanced hackers, coders and makers who are willing to help make demos, projects and tutorials for the ESP32! Please consider holding off on purchase if you are not planning to contribute to the community coding efforts at this time, thank you! There are no refunds, exchanges, store credit or support in any way for this product. Color of PCB may vary.
If you're having difficulty uploading to the ESP32 Dev Kit using windows, try adding a ~0.2uF capacitor between the RST and GND pins. More information here!
Product Dimensions: 55.3mm x 28.0mm x 12.3mm / 2.2" x 1.1" x 0.5"
Product Weight: 9.6g / 0.3oz


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Lam Son Sugar Joint Stock Corporation - Company Capsule
Synopsis Canadean's "Lam Son Sugar Joint Stock Corporation - Company Capsule" contains in depth information and data about the company and its operations. The profile contains a company overview, key facts, major products and services, financial ratios, financial analysis, key employees, company locations and subsidiaries. Summary This report is a crucial resource for industry executives and anyone looking to access key information about "Lam Son Sugar Joint Stock Corporation" The report utilizes a wide range of primary and secondary sources, which are analyzed and presented in a consistent and easily accessible format. Canadean strictly follows a standardized research methodology to ensure high levels of data quality and these characteristics guarantee a unique report. Scope • Identifies crucial company information about "Lam Son Sugar Joint Stock Corporation" along with major products and services for business intelligence requirements. • Provides key ratios on company's financial performance. • Identifies key employees to assist with key business decisions. • Provides annual and interim financial ratios. Reasons To Buy • Enhance your understanding of "Lam Son Sugar Joint Stock Corporation" • Increase business/sales activities by understanding customers' businesses better. • Recognize potential partnerships and suppliers. • Qualify prospective partners, affiliates or suppliers. • Acquire up-to-date company information and an understanding of the company's financial health. Key Highlights Lam Son Sugar Joint Stock Corporation (Lam Son Sugar) is a manufacturer and distributor of sugar products. The company offers sugar and sugar related products including refined sugar, brown sugar, sugar cane juice and others. It also provides alcoholic and non alcoholic drinks, animal feeds and malt. Lam Son Sugar supplies seeds, breeding plant, and soil preparations supporting crop and animal production activities. The company also manufactures cardboard paper. It also operates a milk processing plant and fruit juice concentrate plant. The company operates two sugar refineries. Lam Son Sugar is headquartered in Thanh Hoa Province, Vietnam.


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|0 sq. ft.
|Property type
|Minimum Stay
|5 - 48 nights
This 8 bedroom, 5 bathroom vacation home, located in Champions Gate is a great choice for your next trip to Orlando. The home is on a corner lot and is professionally decorated throughout, with each of the bedrooms being uniquely appointed. The kitchen comes with upgraded features such as granite counter tops, stainless steel appliances and upgraded tile and cabinetry. There are plenty of options for gathering for meals, whether at the kitchen bar, the large dining room table or even poolside. Speaking of pool, there is a large pool for plenty of fun in the sun. The game room comes complete with billiards and foosball for some friendly competition. Wireless high-speed internet and a washer and dryer are also available for your convenience.
Key Features
8 Bedrooms
5 Bathrooms
Sleeps 16
Private pool
Games room
2 car garage
Wireless Internet
Kitchen Appliances
Master bedroom 1 - King bedroom with en suite master bathroom
Master bedroom 2 - King bedroom with en suite master bathroom
Bedroom 3 - Twin bedroom and shares bathroom with bedroom 8
Bedroom 4 - Twin bedroom with two full beds
Bedroom 5 - Twin bedroom and shares bathroom with bedroom 6
Bedroom 6 - Queen bedroom
Bedroom 7 - Queen bedroom
Bedroom 8 - Twin bedroom
Games Room
Pool Table
Air Hockey
TopVillas Ref: Championsgate 52
*Plus compulsory or optional charges based on actual occupancy or consumption. The payment has to be made in the currency offered by the property manager, before use of conversion tool. Payments in other currencies have to be negotiated beforehand with the property manager. The currency conversion tool is provided for your convenience only and is non-binding.


text: In Fall 2016, TMA introduced the Open Art Picasso classes, since there was a high demand for a no-commitment option for busy families. Due to its overwhelming success, we are please to announce the Open Art Pack, which we have expanded to include select mommy-and-me classes throughout the week. Open Art Packs are even good for Drop-in and Play, so there are so many opportunities to use your classes! Open Art Picasso just got an upgrade!


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THIS RESTAURANT IS TEMPORARILY CLOSED Acme Burgerhaus Map
559 Divisadero St. (Hayes St.)
415-346-3212 | Make Restaurant Reservations
Use this map to THIS RESTAURANT IS TEMPORARILY CLOSED Acme Burgerhaus to get there and back safely. If the "Get directions" pop-up does not appear, click the green arrow to get driving directions to and from this restaurant.
Find out which restaurants and chefs top the list of GAYOT's 2016 Annual Restaurant Awards.


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ON THIS BRIGHT EARLY morning of the second day, we headed for Baan Mae Maa, Moo 11. Throughout the drive up the mountains we sat in awe of the splashes of purple, lavender, white flowers and vines coloring the view at every turn. About thirty minutes later, we spotted the sign to Nam Toke Hong Harn. This heavily forested area was proof the villagers had followed expert advice to plant trees that did not need much water to survive.
Youngsters were showing off their skill to produce popular dessert item, "loog chid" by using an ingenious devise to extract the soft white pieces from the palm shells. "Loog saht" an ingredient used in herbal shampoos was demonstrated. Other herbals on display for us to use at home were those additives for a stash of white lightening (moonshine--all the better for you, my dear.)
The group settled down in an open sided house perched on a cliff over the river and the village chief eagerly commenced with a seminar on the environmental issues of the village. Their living motto is "to love and live off the forest but do not destroy it." There were no fires set around the village perimeters. Those who reported seeing a fire are rewarded Baht 100 to put it out. The found out initiators of the fires would be punished for damages at Baht 2,000 per rai. Also, the firebug's name is written up in the community meeting minutes for all his peer group to read.
Why are fires started at all? To find relished delicacies such as red ant nest or those crunchy 'het top' mushrooms, or to catch small animals. Remember for many generations, it was acceptable for forest dwellers to live off the land using those methods. It is only this generation due to greed of many encroaching segments of society that frowns on perpetuating their age old lifestyle. Presently, the villagers' main source of income is picking and collecting miang leaves (similar to tea.) This crop requires part of the forest to be utilized for this agriculture.
The village chief strives to create awareness among radio listeners to educate them about the intriguing life-styles of forest dwellers. His village won selected as a green village along with a cash award amounting to Baht 30,000. Most of the money was spent on the bus rental to transport the villagers to participate in the award ceremony in Bangkok. Throughout the years villagers have received Baht 100,000 to carry out of the work of preservation and conservation of surrounding forest plots.
Near the village Musuer Daeng (Red) who are Thai nationals and Kariang (Karen) make their homes. The tribals agree to follow the village environmental rules or else they must leave the area and settle somewhere else. There is also a Museur Pa Loh village in the area. Up until seven years ago, the tribals planted large areas of cabbages that required pesticide and fertilizer. But these days each knows they must be responsible for maintaining a clean environment. Now they plant tea bushes, plums, lynchee and coffee and loog chid. These agriculture produce do not require chemicals. On the average each diligent villagers earns about Baht 3,000 per month. Most of the forest around the village is classified as protected area.
Even though the village is remote and located very high on top of the mountain they have the convenience of 24-hour electricity supply and phone lines for those families who can afford the luxury.
We boarded two boats to return to Chiangmai via the Mae Ping river. The scenic ride was a lovely break though it was quite a difference from the cool nippy temperature at Rim Doi the night before. Dr. Wasan and Dr. Pakpeng informed us of the history of many places built on the river's edge. We stopped at Wat Ka Chao temple to inspect the recently completed facilities. The residents continue to make their living by planting vegetables. After talking with several persons, it's evident their concern for conserving the environment is deeply heart felt.
Thanks to the organizers of this informative two day seminar : Public Relations Department, Region 3, Dr. Wasan of CMU, Dr. Pakpeng Bruns, the Rim Doi Resort and Khun Preecha of the Mae Ping River cruise.
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Copyright © 1995-2014 Welcome to Chiangmai and Chiangrai magazine All rights reserved.


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Deborah Sweeney is the CEO & Owner at MyCorporation Business Services, Inc.
Three Things on Ms. Sweeney's mind:
1. How do I keep my team passionate and engaged? I think and work extensively toward a team that "owns" the business in the sense that they continue to provide excellent, unsurpassed customer service. I want them to go above and beyond with every single customer. I think extensively about how to incentivize and motivate the team. I want them to make the customer experience of starting a new business a "WOW" experience by providing exceptional customer service.
2. How do I best use my time? I'm organized and motivated. I want to use every minute of my time on the highest and most important things. I evaluate and re-evaluate that is. I sometimes think it's best to work directly with the team. To teach them what I believe is important and pass that along so that this approach can be multiplied by each of my team members. Other times, I think it's about generating relationships outside the business – with partners, affiliates, and new business relationships – to continue to grow the business. Still other times, I think my time is best spent talking directly with the customers – understanding customers and their needs so we can create the best possible service.
3. How do we continue to grow? I think about the best strategies to continue to grow the business. I think about marketing, PR, online search . I think about our website – how it's designed and how to make the customer experience even better. I think about the competition and work to evaluate where they're going and how to differentiate our service by being even more focused on the needs of entrepreneurs. I think about annuity services and how to improve conversion. I think about our customers and our employees and how they can contribute to our growth.
Special to The Globe and MailReport Typo/Error


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Cooling load is defined as the rate at which heat has to be removed from a space to maintain a constant temperature. A room or office accumulates heat continuously from sources such as people, equipment, lights and solar radiation from the sun. Air conditioners cool by continuously removing this heat as it accumulates at the rate required to maintain the temperature set by the thermostat. For this reason, the cooling load is calculated in units of BTU/hr, which represents the speed heat is removed.
Determine the floor area in square feet (ft^2) for the space in question. Calculate the area by multiplying the length by the width. For example, if you have an area 20 feet long and 30 feet wide, your area is 600 square feet (20 x 30).
Estimate the cooling load factor, or CLF, for your type of space using the following as a guide: residential/apartment, CLF is 1.0; office, CLF is 1.2; classroom, CLF is 1.5; and assembly, CLF is 2.5. CLF is in units of CFM/SF which is cubic feet per minute per square feet.
Calculate the air cooling requirements in cubic feet per minute, or CFM, using this formula: floor area x cooling load factor. For example, if you are calculating the cooling load for a classroom with a cooling load factor of 1.5 from Step 2 and the floor area of 600 square feet from Step 1, your CFM will be CFM = 600 ft^2 x 1.5 CFM/ft^2 = 900 CFM.
Calculate the total cooling load, or TCL, using the formula TCL = 1.08 x CFM x "T where "T is the interior design temperature minus cooling coil temperature. The interior design temperature is the thermostat temperature and cooling coil temperature is the temperature measured at the cooling coil of the AC unit. As an example, if the interior design temperature is 75 degrees F, cooling coil temperature is 55 degrees F and CFM is 900 CFM, then TCL = 1.08 x CFM x "T = 1.08 (900) (75-55) = 19,440 BTU/hr.
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Janender K Jain
Professor, Human Resource Management
Chairperson - NMP & Energy Management
Chairperson - Human Resource Management Area
Phone : +91-124-4560322
Professor Jain is a Ph.D in Management in the area of Human Resource Management. He also has two years Post Graduate Dip. in Personnel Management and Industrial Relations. His flair for research and consulting led him to be associated with the Planning Commission and international agencies like ILO¸ UNDP and USAID. He was a UNDP/ILO fellow at the RVB Management Institute¸ the Netherlands. He has worked in the area of management consulting development for the power and road transport sectors in India. His teaching¸ training and consulting interests are HRD¸ building effective IR strategy¸ grievance handling¸ trade unions¸ motivation¸ performance appraisal¸ motivating people¸ subordinate development¸ manpower planning¸ assessing training needs¸ evaluation of training and reproductive health management. He has published many theme papers and cases on different aspects of industry¸ particularly sugar and electricity undertakings and also on public sector.
Prof Jain is visiting professor EDHEC Business School¸ France & IAE Business School AIX En Provence France.


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When you purchase a digital subscription to Cake Central Magazine, you will get an instant and automatic download of the most recent issue.
Both tiers are dreamsicle cake with vanilla buttercream, fondant bow & handmade 50/50 fondant & gum paste roses and leaves (each colored by hand). This is my first tiered cake ... and my own design. I'm pretty happy with it, although I ran out of time to finish all the details.
Wilton Course 1
Course 3 Grand Finale
Sweet And Simple Wedding Cake.


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Ernest Augustus I of Hanover, later the United Kingdom of Northern England, Scotland and Ireland. (5th June 1800 – 12th July 1888) was the fifth son and eighth child of George III of the United Kingdom.
He became King of Hanover on 28th June 1837 on the death of his brother William IV of the United Kingdom. He is most remembered for taking the throne of the United Kingdom in 1840, after his niece Queen Victoria was assassinated. Ernest was also remembered for being the driving force of the 1843 revolution which resulted in the formation of the worlds first socialist state, the Peoples Republic of Southern England and Wales.
Early Life.
Ernest Augustus, the fifth son of George III and Queen Charlotte, was born at Buckingham Palace on 5 June 1800. After leaving the nursery, he lived with his two younger brothers, Duke of Cambridge and Duke of Sussex, and a tutor in a house on Kew Green, near his parents' residence at Kew House. At the age of fifteen, he and his two younger brothers were sent to the University of Gottegen, located in his father's domain of Hanover.
In March 1820, the King commissioned Prince Ernest Augustus as a colonel in the 9th Hanoverian Light Dragoons. Seeing action near the town of Tournai in August 1823, he sustained a sabre wound to the head, which resulted in a disfiguring scar. During a battle in 1824 his left arm was injured by a cannonball which passed close by him. In the days after the battle, the sight in his left eye faded. In June, he was sent to Britain to convalesce, his first stay there since 1810.
King of Hanover (1837–1840)
On 20 June 1837, King William died, and Princess Victoria became Queen of the United Kingdom. Ernest became the King of Hanover. On 28 June 1837, King Ernest entered his new domain, passing under a triumphal arch. For the first time in over a century, Hanover would have a ruler living there. Ernest enjoyed a relatively peaceful reign in Hanover for three years until in June 1840, his niece Queen Victoria is assassinated and he is asked to take the throne of the United Kingdom.
King of the United Kingdom (1840-1843)
Ernests coronation was set for 17th October 1840 in York Cathedral, instead of the traditional Westminster Abbey. This is because the people of Southern England and Wales say they will refuse to have Ernest as a monarch. Parliament is trying to hold the country together but only the North and Ireland remain loyal.
After Ernest's coronation in York the people of southern England are angry at Parliament for letting this happen. Sme people in the cities of London and Manchester take up arms and form the Peoples Liberation Army (PLA). The PLA riots are soon crushed by forces of the Army.
The situation goes from bad to worse when in January 1841, Lord Melbourne (the Prime Minister) and his cabinet are dragged from Downing street by the PLA and face a mock trialin London they are later Hung in Trafalgar Square. The leader of the PLA is captured and hung by loyalist forces in London two days later. It takes two Army battalions to stop the riots in London which continue for two more years and later involve the cites of Birmingham, Leeds and Cardiff.In 1843 after growing unrest in the south the PLA storm the houses of parliament and take over the country they form the Peoples Republic of Southern England. Whilst Enest now only rules the Northern half of the country from York upwards and Ireland, this forms the new United Kingdom of Northern England, Scotland and Ireland (Hanover had declared independence in January of that year). The PRSEW is governed by the peoples committee in London and the UK is Governed by Parliament in Edinburgh.
King of the United Kingdom of Northern England, Scotland and Ireland (1843-1870)
After a period of retaliative peace between the two nations war is declared by the UK on the PRSEW, when a assassin ,who shot King Ernests son George, is captured by the UK and admits to being sent by the PRSEW to kill the crown prince. The PRSEW allies of other new socialist nations such as The Netherlands, Norway and Denmark join the war against the UK. Also the UK allies of the Democratic nations of Europe that include France, Belgium and Sweden all join the war against the PRSEW. Ernest is determined to avenge his sons death and brings most of Europe to war over it.
In 1870 Ernest dies peacefully in the royal palace at Edinburgh Castle after six years of war with Europe. Ernest passes on the throne to his son George V.


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This event has passed
contributed free family friendly accessible
Easter Seals |Life Skills to Host "Milk and Cookies with Santa" Event
This event occurred on –
Sheraton City Center
Sponsored by Charter and CVS, families with children on the autism spectrum will enjoy a festive night out in a safe, judgment-free environment. From face painting to balloon animals to thousands of homemade cookies from local bakeries, volunteers and at least 2,000 cookies from Le Cordon Bleu, this event has something for everyone. While the event is designed specifically for families with children on the autism spectrum, parents are encouraged to bring siblings, as well.
Free to attend, but advance reservations are required. RSVP online at www.lifeskills-mo.org.
Past dates
- Occurred Saturday, December 7th, 2013 @ 1:00 pm – 3:00 pm


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This Genuine Part is Brand New. Sourced From the USA.
This part incudes a 3 Year Warranty at no extra charge.
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$ 38.95
Hood Latch Release Cable With Handle
2013 - 2014
Chevrolet Malibu
2010 - 2013
Buick Lacrosse/Allure
2011 - 2013
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EMT transcription factors snail and slug directly contribute to cisplatin resistance in ovarian cancer
The epithelial to mesenchymal transition (EMT) is a molecular process through which an epithelial cell undergoes transdifferentiation into a mesenchymal phenotype. The role of EMT in embryogenesis is well-characterized and increasing evidence suggests that elements of the transition may be important in other processes, including metastasis and drug resistance in various different cancers.
Agilent 4 × 44 K whole human genome arrays and selected reaction monitoring mass spectrometry were used to investigate mRNA and protein expression in A2780 cisplatin sensitive and resistant cell lines. Invasion and migration were assessed using Boyden chamber assays. Gene knockdown of
Morphological and phenotypic hallmarks of EMT were identified in the chemoresistant cells. Subsequent gene expression profiling revealed upregulation of EMT-related transcription factors including
This work strongly suggests that genes associated with EMT may play a significant role in cisplatin resistance in ovarian cancer, therefore potentially leading to the development of predictive biomarkers of drug response or novel therapeutic strategies for overcoming drug resistance.
Of the gynecological malignancies, ovarian cancer has the highest associated mortality rate in the western world
Standard treatment for ovarian cancer involves tumour debulking and platinum-based chemotherapy administered intravenously or intraperitoneally
Recent evidence has suggested that processes of the epithelial to mesenchymal transition (EMT) may play a role in the development of chemoresistance. EMT is a critical process in embryogenesis
We hypothesized that the genes that regulate EMT have a role in cisplatin resistance in ovarian cancer. Using a cell line model of cisplatin-resistant ovarian cancer, we demonstrate features of a mesenchymal phenotype in the resistant cells relative to their epithelial parent cells. Additionally, we demonstrate increased capacity for migration and invasion in resistant compared to sensitive cells, characteristics that are reverted following reduction of expression of
Cell lines and culture conditions
The cisplatin-sensitive A2780 ovarian adenocarcinoma cell line and its daughter line, A2780cis, were obtained from the European Collection of Cell Cultures (Salisbury, UK). Cells were cultured in RPMI with 10%FBS, 1% penicillin/streptomycin and 1% L-glutamine at 37°C in 5% CO2. A2780cis cells were maintained in media with 1 μM cisplatin. For all assays, cells were grown to 70-80% confluence and harvested following trypsinization. Analysis of cell morphology was done at 20× magnification using a Zeiss Axiovert 25 Phase Contrast Inverted Microscope. Digital images were captured using a Canon Power Shot G10 equipped with a Carl Zeiss 426126 lens.
Human ovarian tumours
Consent for tumour banking was obtained and the study was approved by the Research Ethics Boards at both Kingston General Hospital and The Ottawa Hospital. Tumour samples were obtained from the Division of Gynecologic Oncology Ovarian Tissue Bank and the Ontario Tumour Bank. All tumours were chemonaïve at collection. Seventeen tumours were classified as chemosensitive (progression-free interval of greater than 18 months) and eleven as chemoresistant (progression-free interval of less than 8 months) using available follow-up clinical data. Histological assessment of samples confirmed that each sample contained > 70% tumour cells.
Gene expression profiling and analysis
Total RNA was extracted from the cell lines and tumours using the Qiagen miRNeasy Mini kit (Toronto, Canada). RNA quality was assessed to have an RNA integrity number of at least eight using an Agilent 2100 Bioanalyzer (Mississauga, Canada). For cell lines, total RNA was labeled with Cyanine-3 dye using a Quick Amp Labeling Kit (Agilent, Mississauga, Canada) and hybridized to Agilent Whole Human Genome (4 × 44 K) Microarrays (Mississauga, Canada) for 17 hours in a rotating SciGene model 700 oven (Sunnyvale, USA). Arrays were scanned using an Agilent Technologies DNA Microarray Scanner and data were feature extracted using Feature Extraction Software 10.5.1.1 (Agilent) and statistically analyzed using default settings on GeneSpring GX 11.0.1 software (Agilent).
Expression profiles from the tumor RNA were obtained using Affymetrix GeneChip Human Genome U133 plus 2.0 arrays. Raw data were imported into GeneSpring GX 11.0.1 and analyzed. Unsupervised hierarchical clustering of the tumour samples was completed using the self-organizing maps algorithm in the GeneSpring GX 11.0.1 package.
qRT-PCR Taqman™ arrays
Snail and Slug expression levels were analyzed using Taqman™ assays (Applied Biosystems, Streetsville, Canada, item Hs00195591_m1 and item Hs00950344_m1) and the SuperScript III First-Strand Synthesis SuperMix kit for qRT-PCR (Invitrogen, Burlington, Canada). PCR conditions were 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, 60°C for 1 minute. GAPDH was used as an internal control. As a measure of relative change in expression between the parental and resistant samples, ΔΔCt values were calculated and converted to approximate fold change values (2-ΔΔCt)
Cell proliferation assay
Cells were plated in 24-well plates at 5 × 104 cells/well. Cells were harvested and counted using a haemocytometer after 24, 48 and 72 hours. Average cell counts were used to produce growth curves, from which cell doubling time was calculated.
Wound healing assay
Cell migration was assessed using wound-healing assays. Cells were grown in a confluent monolayer in a 60 mm plate. A wound was inflicted in the cell layer by scratching the plate with a sterile pipette tip. Plates were rinsed gently with media twice prior to incubation to remove non-adherent cells. Digital images of the wound were obtained at times 0 hours, 12 hours and 36 hours at 10× magnification. Effects of proliferation were controlled for by using a reduced serum medium (3%FBS) and monitored via cell count.
Boyden chamber migration and invasion assays
Cells were serum starved for 24 hours prior to use. Media with 10% FBS was added to the wells of a 24-well plate. BD Falcon™ Cell Culture Inserts (BD Biosciences, Mississauga, Canada) were placed in each well. 2 × 103 cells in serum-free media were added to the interior of each insert. Plates were incubated for 24 hours at 37°C in 5% CO2, and media removed from the insert, which was then washed with PBS. Insert membranes were fixed with cold methanol for 10 minutes, stained with 0.5% Crystal Violet in 25% methanol for 10 mins and rinsed with water to remove excess dye. Membranes were removed from the insert, placed under a microscope and the number of cells that migrated through the porous membrane was counted.
Invasion assays were done as described above using BD BioCoat™ Matrigel™ invasion chambers (BD Biosciences, Mississauga, Canada).
MTT assays
5 × 103 cells/well were seeded in 96-well plates in 100 ul medium with 10 μM cisplatin and without phenol red and left to incubate for 48 hours at 37°C and 5% CO2 . After 48 hours, 10 μl MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, Oakville, Canada) was added to each well and cells were left for 4 hours. After incubation, 150 μl MTT solvent (0.1 N HCl in anhydrous isopropanol) was added to each well and mixed thoroughly by pipetting until all formazan crystals were dissolved. Colourimetric change was measured at 570 nm and background absorbance at 690 nm. Final values were obtained by subtracting OD690 nm from OD570 nm. MTT assays for siRNA optimization were done without adding cisplatin and the initial seeded cells were only incubated for 24 hours prior to MTT addition.
Gene knockdown
Pre-designed siRNA oligos for
Optimization of gene knockdown was done using AllStars Hs Cell Death Control siRNA (Qiagen, Toronto, Canada), a mix of siRNAs that target genes essential for cell survival. 5 × 104 cells/well were plated in 24-well plates and incubated overnight. After 24 hours cells were transfected with 7.5 ng, 19 ng, 37.5 ng or 75 ng of AllStars Hs Cell Death Control siRNA and 1.5 μl, 3 μl or 7.5 μl of HiPerFect Transfection Reagent (Qiagen, Toronto, Canada). Cells were incubated for 72 hours and cell death measured using MTT assays. Maximum cell death was achieved using 19 ng siRNA with 3 μl of transfection reagent. Optimal time points were then established using siRNA targeted against Snail and Slug. 24, 48 and 72 hours after transfection the level of
For knockdown experiments, 5 × 104 cells/well were plated in 24-well plates and incubated overnight. 19 ng of siRNA and 3 μl of HiPerFect transfection reagent were diluted in 100 μl serum-free RPMI and were incubated for 10 minutes at room temperature. Transfection complexes were then added drop-wise onto the cells. Cells were incubated for 72 hours at 37°C and 5% CO2. Media was changed as necessary. Transfection of A2780cis cells with AllStars Negative Control (Qiagen), an siRNA sequence with no homology to any known mammalian gene, was used as a control for this experiment. Cells with reduced expression were designated as follows: A2780cisSN -
Statistical analysis
Each assay was performed in triplicate. Data are expressed as the mean ± standard deviation (SD). Statistical significance of all data was evaluated using the Student's
Sample preparation for proteomic analysis
Samples were lysed in RIPA buffer containing Halt Protease and Phosphatase Inhibitor Cocktail (PIERCE, IL, USA). Cells were sonicated and lysates incubated at 4°C for 30 minutes with shaking. Supernatants were separated by centrifugation for 15 minutes at 4°C. Protein concentration was measured with a Bio-Rad DC Protein Assay kit and 30 μg of protein was used for tryptic digestion. Each aliquot was dried, dissolved in 0.5 M triethylammonium bicarbonate (TEAB), reduced by adding 2 μl of tris-(2-carboxyethyl)-phosphine (TCEP), incubated at room temperature for 1 hour, alykalted using 1 μl of methyl methane thiosulfonate (MMTS) and incubated at room temperature for 1 hour in the dark. 2 μg trypsin was added for overnight digestion at 37°C. The tryptic digest was desalted by using a C18 spin column (PIERCE, USA), dried under vaccum and resuspended in 0.1% formic acid for SRM analysis.
LC/SRM-MS analysis
The LC/SRM-MS analytical system consisted of an Eksigent nanoflow HPLC (AB Sciex, USA) coupled to a 5500 QTRAP® hybrid triple quadrupole/linear ion trap mass spectrometer (AB SCIEX, USA). 1 μg of digested protein was loaded onto a trap column (0.3 mm I.D, 5 mm L), packed with 5 μm Zorbax SB-C18, 300 Å pore (Agilent, USA). Peptides were separated on a 75 μm I.D., 15 cm long nanoflow column with 15 μm spray tip (New Objectives, USA). A linear gradient profile was employed starting from 5% solvent B to 40% B in 60 minutes (solvent A was 2% acetonitrile in water with 0.1% formic acid; solvent B was 2% water in acetonitrile with 0.1% formic acid). The ion spray voltage was set at 2300 V, and source temperature at 160°C. The declustering voltage was 100 V, and collision energy value was selected for each transition as generated by MultiQuant® software (AB Sciex, USA). To ensure sensitive and reliable quantification, tryptic peptides and SRM transitions were generated by MRMPilot software (AB SCIEX, USA) based on common chemical rules of peptide fragmentation. The specificity of each peptide was verified using BLAST alignment against the NCBI-NR human protein database. Raw SRM-MS data was preprocessed using MultiQuant 2.0.2 software (AB SCIEX). A 2-point Gaussian smoothing window was applied to all transition peaks. Peak area for each transition was averaged over three replicate experiments per resistant and sensitive cell line sample; fold-change was calculated from the ratio of the average peak area in resistant cells to that in sensitive cells. A transition was discarded if peak area coefficient of variation (CV) across replicates was greater than 0.2 or peak area in any replicate was below the 25th percentile. Peptides with at least 2 transitions satisfying these constraints were conserved.
Cisplatin resistance in A2780cis correlates with changes in cellular morphology consistent with EMT signaling
We examined the morphological characteristics of the cell lines during exponential growth. Parental A2780 cells formed cohesive clusters with round cellular morphology in vitro (Figure
Figure 1
Morphology of A2780 and A2780cis cells
Morphology of A2780 and A2780cis cells. Cells visualized under 20× magnification. Drug sensitive cells (a) have round morphology and grow in tight clusters with substantial cellular cohesion. Comparatively, the drug resistant cells (b) have a more fibroblastic appearance and demonstrate reduced intercellular contacts. Additionally, drug resistant cells extend pseudopodia (inset).
Whole transcriptome profiling and LC/SRM-MS analysis identify components of EMT signaling networks in A2780cis relative to A2780
To confirm the involvement of EMT elements in the development of cisplatin resistance, whole transcriptome microarrays (Agilent, Mississauga, Canada) were used to compare gene expression between A2780 and A2780cis cells during exponential growth. In repeated assays, EMT pathway elements, including
Figure 2
Upregulation of genes associated with EMT in resistant cells
Upregulation of genes associated with EMT in resistant cells. Genes known to 'regulate EMT were shown to be upregulated in the A2780cis cells compared to the A2780 cells. Data from the gene expression microarrays (a) and technical validation of
Table 1
Targeted protein analysis of A2780 and A2780cis cells.
Fragment Change
Mean Peak Area
E- cadherin
Summary of SRM-MS quantification results for E-Cadherin, Vimentin, Snail, and Slug. Mean peak area and coefficient of variation (CV) for each peptide fragment ion were derived from peak areas measured in three replicate assays of each of the sensitive and resistant cell line samples. Transitions with CV > 0.20 in either sample are not shown
Cisplatin resistant cells display increased potential for migration and invasion
Migratory capacity was measured using Boyden chamber assays. In three independent experiments, 23.7 ± 5.1 drug sensitive cells and 194.0 ± 7.0 drug-resistant cells migrated through the membrane after 24 hours (p-value = 0.003) (Figure
Figure 3
Knockdown of
Knockdown of
Knockdown of snail and slug reverses the EMT phenotype and reduces cellular resistance to cisplatin
SiRNA was used to reduce the levels of
Figure 4
Cisplatin sensitivity with
Cisplatin sensitivity with
Drug resistant human ovarian tumours can be differentiated from drug sensitive ovarian tumours using a biomarker panel of EMT-related genes
Human ovarian tumour samples were classified as chemosensitive (progression-free interval of greater than 18 months) or chemoresistant (progression-free interval of less than 6 months) based on available clinical data (Additional file
Additional file 1
Table S1 Clinical data for ovarian tumour samples. Available clinical data for each tissue sample including; patient's age at time of diagnosis (years), tumour stage, progression-free interval (PFI, months) and drug response classification.
Click here for file
Figure 5
EMT gene signatures in drug resistant ovarian tumours
EMT gene signatures in drug resistant ovarian tumours. EMT gene signatures are present in primary drug resistant ovarian tumours. (a) Gene expression arrays identify increased expression of genes integral to EMT in the resistant tumours relative to the sensitive tumours. (b) Unsupervised hierarchical clustering based on a panel of genes related to EMT supports differentiation between drug sensitive (blue) and drug resistant (red) tumours.
Table 2
List of genes used for unsupervised hierarchical clustering of primary tumours.
Epidermal growth factor recetor
Peroxisome proliferator-activated receptor gamma
Insulin-like growth factor 1
Transforming growth factor beta 2
Zinc finger E-box-binding homeobox 1
Slug (Zinc finger protein)
Twist-related protein 2
Retinoid X receptor alpha
Genes used to perform unsupervised hierarchical clustering on the primary ovarian tumours, chosen based on their documented involvement in EMT in recent literature
Ovarian cancer exhibits a high rate of platinum sensitivity in the first-line setting, but resistance frequently develops in recurrent disease
While EMT has been widely studied for its role in early development and, more recently, cancer metastasis, it may also contribute to a cellular ability to evade the effects of platinum-based therapies. EMT results in the transformation of a differentiated epithelial cell to a mesenchymal cell with stem-like properties, and is characterized by loss of cell-to-cell adhesion, specifically through the dismantling of adherens, tight and gap junctions, as well as loss of cell polarity and increased motility
It is likely that EMT in drug resistance may rely on many of the same transcription factors that function in embryogenesis and metastasis-related EMT, although details of their regulation are largely unknown. Studies from embryology and metastasis provide evidence to suggest that these cellular changes provide a survival advantage to cells under chemotoxic stress, for example, by down-regulating pro-apoptotic factors such as caspases and
Through the manipulation of key EMT signaling molecules, we have been able to show that we can re-sensitize drug resistant cells to the effects of cisplatin, approaching wild-type sensitivity after 72 hours in culture. Our findings suggest that genes known to regulate EMT directly contribute to cisplatin resistance in this ovarian cancer model. We do recognize that the A2780 cell line may not provide a faithful model of serous ovarian carcinoma, given that lineage fidelity for this line may not be maintained in long term culture. However, we conclude that the translation of our cell line derived results to a series of primary ovarian carcinomas provides evidence for the effectiveness of this model in this instance. We have demonstrated that a panel of EMT-related genes provides a reasonable model of classifying primary ovarian tumours according to their chemoresistance status. While we recognize that optimization of the biomarker panel would be required before suggesting this could provide a clinical benefit, this initial gene list identifies key differences in the underlying molecular circuitry of drug sensitive and resistant ovarian tumours. We have not determined whether this signature derives entirely from tumour cells, given that we did not enrich for tumour cells in our experiments; however, the fact that all of the primary tumours showed at least 70% tumour cells by histology would support the idea that the tumour likely contributes substantially to the signature we noted. To our knowledge, this is the first study in primary tumours demonstrating an EMT gene signature that can be used to differentiate between chemosensitive and chemoresistant human ovarian tumours and suggests that our overall findings may provide important clues about chemotherapy resistance in ovarian cancer.
In summary, we have demonstrated, through the use of an ovarian cancer cell line, A2780, and its cisplatin-resistant daughter line, A2780cis, that the genes that regulate the epithelial to mesenchymal transition directly contribute to cisplatin-resistance in ovarian cancer. We have shown that when
EMT: Epithelial to mesenchymal transition; LC/SRM-MS: Liquid chromatography/selected reaction monitoring mass spectrometry
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AMH carried out analysis of cell line microarray data, completed
The authors would like to thank Xiao Zhang from Queen's University Microarray Facility for his assistance with the gene expression arrays for the cell line data, and the Division of Gynecologic Oncology Ovarian Tissue Bank at the Ottawa Hospital for providing tumour samples and clinical follow up data. This study was conducted with the support of the Ontario Institute for Cancer Research through funding provided by the Government of Ontario. Additional funding was provided by the Queen's University Terry Fox Foundation Training Program in Transdisciplinary Cancer Research in partnership with CIHR.
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Pre-publication history
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