text:
Culture, Food & Drink, Storytelling
The Cheapside Way of Life
The Cincinnati food scene has long maintained no shortage of wonderful dinner options, but when it came to a late weekend morning, feet propped up with the New York Times, there was nothing. There was a deafening weekend silence as this town ached for a solid brunch destination. A late breakfast has long been a staple to getting my weekend started. I can take my time because the day is mine. I may first head to Modo Yoga to sweat out the previous night's toxins but I'll always find my way to the tranquility of a good cup of coffee and eggs.
The routine of the weekend often resembles that of my week. Every Tuesday, though I've since switched it up, you could find me at The Rookwood noshing on the Barnesdale Burger and drinking a glass of pinot grigio. I know, it sounds weird to have wine and a burger but it somehow makes sense to my palette! As I used to sit at The Rookwood, then head bartender and cocktail mixologist extraordinaire, Rom Wells told me their team was opening a breakfast and lunch outpost downtown. I immediately pulled out my phone and put their opening date in my calendar.
Cheapside Café earned its name for the alley it sits adjacent to, which runs parallel to Broadway St. and was once used for a canal-boat turnaround in the 19th century. How's that for a Throw-Back-Thursday? The Miami Canal used to be 274 miles of narrow waterways, much of which is now I-75. It connected Toledo with Cincinnati and featured short pit stops along the way in small towns like Piqua, Troy, Sharonville, and Lockland.
The most compelling elements of an establishment's food and bar culture often has little to do with the actual food on the plate or libation in the glass. It's the culture that happens behind the bar and in the seats that shapes its personality and appeal. Cheapside has accomplished what most organizations try to do but are not as successful. They've managed to create an authentic culture without forcing a false story upon the patrons. We had the lovely opportunity to chat with co-founder Rom Wells a few months ago, where he talked about building their concept and the path they took to get there.
You've been crafting cocktails before it was a popular trend. What interested you most in making the beverage & restaurant industry a career?
The short, not very exciting answer is that I needed to make money my senior year in college, so I started bartending as a means to do so. As it turned out, though, I was a fairly talented barkeep. When I moved to Cincinnati after graduating, I kept bartending and, even though I didn't really realize it initially, there was plenty of space here for someone who took the job seriously and treated it like a multifaceted craft with plenty of room for attention to detail. I made a good amount of connections in what was a rapidly-growing, creative food and beverage scene, including my current partners at Cheapside Café, and that helped lead me to where I am right now.
Cheapside has been very well received - what's the key to the brand's success?
I believe a lot of factors make Cheapside work. Primarily, I think that it's such a straightforward, no frills concept. When we were conceptualizing the project, one of the first images that we had for reference was an image of, like, a club sandwich on a white background that said "simple ass lunch." It's more than that, of course, but it's still pretty much "simple ass coffee, breakfast and lunch." I think that makes people comfortable - the options aren't overwhelming, we don't really try to promise too many things, we just try to do a few things as well as we can and really service the area we're in. I think the simplicity of the design mirrors the philosophy behind what we offer; we obviously put a lot of thought into the design, but it's pretty minimal, really. We kept staring at this image of a very plain room where only the textures of the materials and negative space were really important. The more nuanced details in the space--the food, the beverages, the plants - really stand out in that environment. The experience of my partners and myself has a lot to do with it. Joe has experience founding and designing other restaurants, and he is familiar with the difficulties and pitfalls inherent in the endeavor. Jon is a very talented chef. His techniques, consistency, and culinary experience are invaluable to the success of Cheapside. My beverage training has helped me approach coffee with the same attention to detail and consistency as someone running a bar program. We had a really great team of people working on the concept as well; it would take a good amount of space here to thank everyone who helped personally, but if you did any work in or on the space, got erratic calls and emails, attended meetings that may or may not have seemed fruitful at the time, painted, sanded, or caulked, you know who you are and we thank you. Finally, the staff helps make the place work; never underestimate the staff.
There's something to the attitude of Cheapside that says "this is where the cool kids hang," how do you create that vibe that is almost undefinable?
Ha beats me. We knew we'd be spending a lot of time in the space, so that has a lot to do with the vibe. Nothing lasts forever, though, and hopefully it's still a solid place even if the cool kids don't wanna hang there anymore.
Where did you get your inspiration for the Cheapside aesthetic? The name?
I mentioned a couple images that we dwelled on during the design process above. There was one image in particular that Joe found that helped inspire the layout. Personally, I was in some imaginary Nordic landscape with a bunch of hand carved Swedish woodenware and ancient monoliths on snow white deserts, highly caffeinated, with Gorgoroth on repeat. As for the name, it's just the cross street we're located on; East Eighth Street Café sounded too vanilla.
Tagged: Cheapside Cincinnati, Cheapside, Storytelling, Micah Paldino, Micah Paldino Cincinnati, Cincinnati Public Relations, PR Cincinnati, Coffee, Cincinnati Storytelling


text:
require 'spec_helper'

describe "should be_predicate" do
  it "passes when actual returns true for :predicate?" do
    actual = stub("actual", :happy? => true)
    actual.should be_happy
  end

  it "passes when actual returns true for :predicates? (present tense)" do
    actual = stub("actual", :exists? => true, :exist? => true)
    actual.should be_exist
  end

  it "fails when actual returns false for :predicate?" do
    actual = stub("actual", :happy? => false)
    lambda {
      actual.should be_happy
    }.should fail_with("expected happy? to return true, got false")
  end

  it "fails when actual returns false for :predicate?" do
    actual = stub("actual", :happy? => nil)
    lambda {
      actual.should be_happy
    }.should fail_with("expected happy? to return true, got nil")
  end

  it "fails when actual does not respond to :predicate?" do
    lambda {
      Object.new.should be_happy
    }.should raise_error(NameError, /happy\?/)
  end

  it "fails on error other than NameError" do
    actual = stub("actual")
    actual.should_receive(:foo?).and_raise("aaaah")
    lambda {
      actual.should be_foo
    }.should raise_error(/aaaah/)
  end

  it "fails on error other than NameError (with the present tense predicate)" do
    actual = Object.new
    actual.should_receive(:foos?).and_raise("aaaah")
    lambda {
      actual.should be_foo
    }.should raise_error(/aaaah/)
  end
end

describe "should_not be_predicate" do
  it "passes when actual returns false for :sym?" do
    actual = stub("actual", :happy? => false)
    actual.should_not be_happy
  end

  it "passes when actual returns nil for :sym?" do
    actual = stub("actual", :happy? => nil)
    actual.should_not be_happy
  end

  it "fails when actual returns true for :sym?" do
    actual = stub("actual", :happy? => true)
    lambda {
      actual.should_not be_happy
    }.should fail_with("expected happy? to return false, got true")
  end

  it "fails when actual does not respond to :sym?" do
    lambda {
      Object.new.should_not be_happy
    }.should raise_error(NameError)
  end
end

describe "should be_predicate(*args)" do
  it "passes when actual returns true for :predicate?(*args)" do
    actual = mock("actual")
    actual.should_receive(:older_than?).with(3).and_return(true)
    actual.should be_older_than(3)
  end

  it "fails when actual returns false for :predicate?(*args)" do
    actual = mock("actual")
    actual.should_receive(:older_than?).with(3).and_return(false)
    lambda {
      actual.should be_older_than(3)
    }.should fail_with("expected older_than?(3) to return true, got false")
  end

  it "fails when actual does not respond to :predicate?" do
    lambda {
      Object.new.should be_older_than(3)
    }.should raise_error(NameError)
  end
end

describe "should_not be_predicate(*args)" do
  it "passes when actual returns false for :predicate?(*args)" do
    actual = mock("actual")
    actual.should_receive(:older_than?).with(3).and_return(false)
    actual.should_not be_older_than(3)
  end

  it "fails when actual returns true for :predicate?(*args)" do
    actual = mock("actual")
    actual.should_receive(:older_than?).with(3).and_return(true)
    lambda {
      actual.should_not be_older_than(3)
    }.should fail_with("expected older_than?(3) to return false, got true")
  end

  it "fails when actual does not respond to :predicate?" do
    lambda {
      Object.new.should_not be_older_than(3)
    }.should raise_error(NameError)
  end
end

describe "should be_predicate(&block)" do
  it "passes when actual returns true for :predicate?(&block)" do
    actual = mock("actual")
    delegate = mock("delegate")
    actual.should_receive(:happy?).and_yield
    delegate.should_receive(:check_happy).and_return(true)
    actual.should be_happy { delegate.check_happy }
  end

  it "fails when actual returns false for :predicate?(&block)" do
    actual = mock("actual")
    delegate = mock("delegate")
    actual.should_receive(:happy?).and_yield
    delegate.should_receive(:check_happy).and_return(false)
    lambda {
      actual.should be_happy { delegate.check_happy }
    }.should fail_with("expected happy? to return true, got false")
  end

  it "fails when actual does not respond to :predicate?" do
    delegate = mock("delegate", :check_happy => true)
    lambda {
      Object.new.should be_happy { delegate.check_happy }
    }.should raise_error(NameError)
  end
end

describe "should_not be_predicate(&block)" do
  it "passes when actual returns false for :predicate?(&block)" do
    actual = mock("actual")
    delegate = mock("delegate")
    actual.should_receive(:happy?).and_yield
    delegate.should_receive(:check_happy).and_return(false)
    actual.should_not be_happy { delegate.check_happy }
  end

  it "fails when actual returns true for :predicate?(&block)" do
    actual = mock("actual")
    delegate = mock("delegate")
    actual.should_receive(:happy?).and_yield
    delegate.should_receive(:check_happy).and_return(true)
    lambda {
      actual.should_not be_happy { delegate.check_happy }
    }.should fail_with("expected happy? to return false, got true")
  end

  it "fails when actual does not respond to :predicate?" do
    delegate = mock("delegate", :check_happy => true)
    lambda {
      Object.new.should_not be_happy { delegate.check_happy }
    }.should raise_error(NameError)
  end
end

describe "should be_predicate(*args, &block)" do
  it "passes when actual returns true for :predicate?(*args, &block)" do
    actual = mock("actual")
    delegate = mock("delegate")
    actual.should_receive(:older_than?).with(3).and_yield(3)
    delegate.should_receive(:check_older_than).with(3).and_return(true)
    actual.should be_older_than(3) { |age| delegate.check_older_than(age) }
  end

  it "fails when actual returns false for :predicate?(*args, &block)" do
    actual = mock("actual")
    delegate = mock("delegate")
    actual.should_receive(:older_than?).with(3).and_yield(3)
    delegate.should_receive(:check_older_than).with(3).and_return(false)
    lambda {
      actual.should be_older_than(3) { |age| delegate.check_older_than(age) }
    }.should fail_with("expected older_than?(3) to return true, got false")
  end

  it "fails when actual does not respond to :predicate?" do
    delegate = mock("delegate", :check_older_than => true)
    lambda {
      Object.new.should be_older_than(3) { |age| delegate.check_older_than(age) }
    }.should raise_error(NameError)
  end
end

describe "should_not be_predicate(*args, &block)" do
  it "passes when actual returns false for :predicate?(*args, &block)" do
    actual = mock("actual")
    delegate = mock("delegate")
    actual.should_receive(:older_than?).with(3).and_yield(3)
    delegate.should_receive(:check_older_than).with(3).and_return(false)
    actual.should_not be_older_than(3) { |age| delegate.check_older_than(age) }
  end

  it "fails when actual returns true for :predicate?(*args, &block)" do
    actual = mock("actual")
    delegate = mock("delegate")
    actual.should_receive(:older_than?).with(3).and_yield(3)
    delegate.should_receive(:check_older_than).with(3).and_return(true)
    lambda {
      actual.should_not be_older_than(3) { |age| delegate.check_older_than(age) }
    }.should fail_with("expected older_than?(3) to return false, got true")
  end

  it "fails when actual does not respond to :predicate?" do
    delegate = mock("delegate", :check_older_than => true)
    lambda {
      Object.new.should_not be_older_than(3) { |age| delegate.check_older_than(age) }
    }.should raise_error(NameError)
  end
end

describe "should be_true" do
  it "passes when actual equal?(true)" do
    true.should be_true
  end

  it "passes when actual is 1" do
    1.should be_true
  end

  it "fails when actual equal?(false)" do
    lambda {
      false.should be_true
    }.should fail_with("expected false to be true")
  end
end

describe "should be_false" do
  it "passes when actual equal?(false)" do
    false.should be_false
  end

  it "passes when actual equal?(nil)" do
    nil.should be_false
  end

  it "fails when actual equal?(true)" do
    lambda {
      true.should be_false
    }.should fail_with("expected true to be false")
  end
end

describe "should be_nil" do
  it "passes when actual is nil" do
    nil.should be_nil
  end

  it "fails when actual is not nil" do
    lambda {
      :not_nil.should be_nil
    }.should fail_with(/^expected: nil/)
  end
end

describe "should_not be_nil" do
  it "passes when actual is not nil" do
    :not_nil.should_not be_nil
  end

  it "fails when actual is nil" do
    lambda {
      nil.should_not be_nil
    }.should fail_with(/^expected: not nil/)
  end
end

describe "should be <" do
  it "passes when < operator returns true" do
    3.should be < 4
  end

  it "fails when < operator returns false" do
    lambda { 3.should be < 3 }.should fail_with("expected: < 3\n     got:   3")
  end

  it "describes itself" do
    be.<(4).description.should == "be < 4"
  end
end

describe "should be <=" do
  it "passes when <= operator returns true" do
    3.should be <= 4
    4.should be <= 4
  end

  it "fails when <= operator returns false" do
    lambda { 3.should be <= 2 }.should fail_with("expected: <= 2\n     got:    3")
  end
end

describe "should be >=" do
  it "passes when >= operator returns true" do
    4.should be >= 4
    5.should be >= 4
  end

  it "fails when >= operator returns false" do
    lambda { 3.should be >= 4 }.should fail_with("expected: >= 4\n     got:    3")
  end
end

describe "should be >" do
  it "passes when > operator returns true" do
    5.should be > 4
  end

  it "fails when > operator returns false" do
    lambda { 3.should be > 4 }.should fail_with("expected: > 4\n     got:   3")
  end
end

describe "should be ==" do
  it "passes when == operator returns true" do
    5.should be == 5
  end

  it "fails when == operator returns false" do
    lambda { 3.should be == 4 }.should fail_with("expected: == 4\n     got:    3")
  end
end

describe "should be ===" do
  it "passes when === operator returns true" do
    Hash.should be === Hash.new
  end

  it "fails when === operator returns false" do
    lambda { Hash.should be === "not a hash" }.should fail_with(%[expected: === "not a hash"\n     got:     Hash])
  end
end

describe "should_not with operators" do
  it "coaches user to stop using operators with should_not" do
    lambda {
      5.should_not be < 6
    }.should raise_error(/not only FAILED,\nit is a bit confusing./m)
  end
end

describe "should be" do
  it "passes if actual is truthy" do
    true.should be
    1.should be
  end

  it "fails if actual is false" do
    lambda {false.should be}.should fail_with("expected false to evaluate to true")
  end

  it "fails if actual is nil" do
    lambda {nil.should be}.should fail_with("expected nil to evaluate to true")
  end

  it "describes itself" do
    be.description.should == "be"
  end
end

describe "should_not be" do
  it "passes if actual is falsy" do
    false.should_not be
    nil.should_not be
  end

  it "fails on true" do
    lambda {true.should_not be}.should fail_with("expected true to evaluate to false")
  end
end

describe "should be(value)" do
  it "delegates to equal" do
    self.should_receive(:equal).with(5)
    5.should be(5)
  end
end

describe "should_not be(value)" do
  it "delegates to equal" do
    self.should_receive(:equal).with(4)
    5.should_not be(4)
  end
end

describe "'should be' with operator" do
  it "includes 'be' in the description" do
    (be > 6).description.should =~ /be > 6/
    (be >= 6).description.should =~ /be >= 6/
    (be <= 6).description.should =~ /be <= 6/
    (be < 6).description.should =~ /be < 6/
  end
end

describe "arbitrary predicate with DelegateClass" do
  it "accesses methods defined in the delegating class (LH[#48])" do
    require 'delegate'
    class ArrayDelegate < DelegateClass(Array)
      def initialize(array)
        @internal_array = array
        super(@internal_array)
      end

      def large?
        @internal_array.size >= 5
      end
    end

    delegate = ArrayDelegate.new([1,2,3,4,5,6])
    delegate.should be_large
  end
end

describe "be_a, be_an" do
  it "passes when class matches" do
    "foobar".should be_a(String)
    [1,2,3].should be_an(Array)
  end

  it "fails when class does not match" do
    "foobar".should_not be_a(Hash)
    [1,2,3].should_not be_an(Integer)
  end
end

describe "be_an_instance_of" do
  it "passes when direct class matches" do
    5.should be_an_instance_of(Fixnum)
  end

  it "fails when class is higher up hierarchy" do
    5.should_not be_an_instance_of(Numeric)
  end
end

redpajama_set_name: RedPajamaGithub


Skipper Jason Holder and Kemar Roach took 4 wickets each as England collapsed to lose the second Test by 10 wickets as West Indies sealed the series win 2-0.
England went from 35-0 to 132 all out, with Roach and Holder holding sway over the tourists.
With West Indies chasing only 14, John Campbell wrapped up victory with a six.
West Indies 306 (Bravo 50, Brathwaite 49, Broad 3-53) and 17 for 0 (Campbell 11*, Brathwaite 5*) beat England 187 and 132 (Buttler 24, Holder 4-43, Roach 4-52) by 10 wickets.


text:
Texila American University (TAU)  is an offshore private medical school, owned by the Texila American University Ltd – Hong Kong (TAU-HK). TAU-HK is a project of the ALLTERE Education Management Company. Texila's U.S. offices are in New Jersey. The University is located in Guyana, South America and offers undergraduate and postgraduate degrees in medicine, nursing, public health and allied sciences.

The school is registered with the National Accreditation Council of Guyana and received accreditation from the Caribbean Accreditation Authority for Education in Medicine and other Health Professions in 2023. It is listed in both FAIMERs and the World Directory of Medical Schools.

Management and Organization
TAU was established in Guyana in September 2010 offering courses in medicine and dentistry. The initial classes ere offered at the Critchlow Labour College on Woolford Avenue in Georgetown, and in 2011, they moved to a campus in Goedverwagting, Sparendaam. In 2012, the university expanded its programs to offer nursing certifications. TAU graduated its first set doctors in 2014, followed by hundreds of graduate physicians in 2015, 2016 and 2017 out of a total enrollment of 422 students from 35 countries. In 2014, the school began an expansion hoping to accommodate up to 1000 students at a new campus located in Providence, East Bank Demerara.

Texila American University has a campus in Zambia and Fujairah in the United Arab Emirates.

Programs Offered

College of Medicine
Students also have the option of completing their training at independent facilities in Europe or Asia.

Completion of the full program will result in students receiving an MBBS for the pre-medical courses and the Doctor of Medicine degree for completion of the entire course. Options are available for students who have completed the pre-med elsewhere or have attained the equivalent of a BS and should contact the university for a determination.

College of Nursing
Nursing programs encompass a four-year program which is completed in 3 semesters per year. Subjects include biology and chemistry, as well as basic nursing and training in patient restorative care. The school utilizes both laboratory and technology-based education programs with students completing two years study on site at the Guyana campus and then completing two years of practicum in Canada, the United States, the United Kingdom, or other affiliated universities. Completion of the course will result in a Bachelor of Science in Nursing degree. Advanced classes for Registered nurse can be the undertaken in a 2-year RN-BSN program completed at the campus in Guyana.

Postgraduate Programs
Several postgraduate programs are also offered for students who have earned their bachelor's or other higher degrees. Fellowship programs vary and completion time will range from few months to one year. Masters and postgraduate programs are rendered in partnership with the Central University of Nicaragua.

Accreditation
Texila is registered with,  the National Accreditations Council of Guyana, Higher Education Authority, Ministry of Education, Guyana. It is listed in the FAIMER International Medical Education Directory (IMED) effective in 2011 as School #F0002428 and is listed in the World Health Organization's World Directory of Medical Schools. By virtue of its listing in IMED, students graduating from Texila are authorized to take part in the United States Medical Licensing Examination three-part examinations. Those who pass the examinations are eligible according to the Educational Commission for Foreign Medical Graduates to register for and participate in the National Resident Matching Program (NRMP). TAU received accreditation from the Caribbean Accreditation Authority for Education in Medicine and other Health Professions in 2023. Texila is also accredited by the Medical Council of India (MCI).

References

External links
School website

Medical schools in Guyana
Universities in Guyana
Buildings and structures in Georgetown, Guyana
2010 establishments in Guyana
Educational institutions established in 2010

redpajama_set_name: RedPajamaWikipedia


text:
COACHELLA 2015: Diners get an upscale treat…
COACHELLA 2015: Diners get an upscale treat from L.A. chefs
By Vanessa Franko | vfranko@scng.com | The Press-Enterprise
Imagine six tasting courses from one of Los Angeles' hottest chefs, served in an exclusive enclave where succulents sit as centerpieces on long white communal tables.
In the distance, you can hear the soulful sounds of Charles Bradley and His Extraordinaires.
The scene isn't an industry party in Hollywood, but rather the Coachella Valley Music and Arts Festival. The event, which is in its 16th year at the Empire Polo Club in Indio, has continued to ramp up its food offerings with three new full service pop-up restaurants. In 2014, Coachella followed the path of other big festivals by adding more menu items to the swath of shawarma, pad thai and pizza. Popular restaurants set up a miniature version, booth style, on the grounds.
This year, for $50 you could get a full sit-down service experience, but still with biodegradable paper plates and plastic utensils
In a VIP area of the grounds, the three Los Angeles chefs created desert outposts: Pot, Roy Choi's Korean hot pot restaurant; Terrine, Kris Morningstar's miniature version of the French favorite; and a mash-up of Chef Phillip Frankland Lee's two restaurants–the meat-eater's heaven Scratch | Bar and the vegetarian-friendly The Gadarene Swine.
After construction of the restaurants delayed their opening on Friday of weekend one, the foodie oasis was in full swing by the end of the weekend and continued to build buzz in weekend two.
At the Scratch | Bar/The Gadarene Swine combo, staff from the full-service locations were on hand to explain the dishes, serving the set menu of courses that opened with Scratch | Bar's crispy piece of pork belly topped by a raw oyster and covered in a coconut milk and chamomile tea emulsion.
The menu, while adapted from the two restaurants, encapsulated the Coachella experience in each dish. The dry-aged hanger steak, served medium rare with roasted mushrooms and onions atop a creamy potato, picked up the earthy notes, not unlike the polo field itself. A sugar-blistered tomato and a grilled asparagus with lemon added the bright, acidic notes of the desert sunshine.
The affable Lee popped around to each of the tables during the opening of Friday's service. He explained that he had a food truck at the film, music and interactive conference South By Southwest in Austin, Texas. Lee said Nic Adler, who curates the food for the festival, had heard about what they had done in Austin and approached them about doing something at Coachella.
Back in the kitchen, a team of chefs that included Vince Fearing, Julian Tham, Sage Avina and Jonathan Portela, moved deftly in a makeshift space that worked as a full kitchen and is larger than their usual digs, with a flat top grill, ovens, burners and a deep fryer. As orders were called out, they moved in their spaces, adding cauliflower puree to plates, or slicing the hanger steak for orders.
Another favorite was the crispy yucca on an avocado creme, served with both a chimichurri and a roasted red pepper sauce and topped with sweet and sour red onions.
For a $25 upgrade, diners could get two extra courses and dine inside the kitchen, much like the opportunity at the full-size Scratch | Bar.
Like Coachella's special guest stars and new music, the pop-up's menu ended with a new sweet creation at the festival only–a coconut pudding with mojito granita from pastry chef Margarita Lee, a refreshing ending to a one-of-a-kind meal.
Contact the writer: vfranko@pe.com or 951-368-9575
Vanessa Franko
Vanessa Franko is the Digital Director of Entertainment for the Southern California News Group. The lure of palm trees and covering pop culture brought her to The Press-Enterprise in Riverside in 2006. Vanessa has reported on everything from the Palm Springs International Film Festival to the MLB All-Star Game as a reporter, photographer, videographer and on-camera personality. She's won awards for her coverage of the Coachella Valley Music and Arts Festival and for crime reporting in her home state of Maryland. Vanessa studied multimedia storytelling as a Knight Digital Media Center fellow in Dec. 2011 and has taught college courses in digital journalism. She's seen shows at every major concert venue in Southern California, but most special was when Paul McCartney played the high-desert roadhouse Pappy & Harriet's in Pioneertown for a couple hundred fans in Oct. 2016. Her album collection numbers in the thousands (including a couple hundred on vinyl) and when she isn't hunting for records, she and her husband like to check out the best in Southern California craft beer and watch sports. She also had a cameo in the 1992 Atlanta Braves highlight film, Lightning Strikes Twice!
Follow Vanessa Franko	@vanessafranko

redpajama_set_name: RedPajamaCommonCrawl


text:
https://www.barrons.com/articles/federal-reserve-corporate-debt-buying-power-51590590141
The Fed Might Not Use All of Its Corporate Debt Buying Power. Why the Program Could Be a Success Anyway.
Alexandra Scaggs
May 27, 2020 10:36 am ET
The Federal Reserve building.
Photograph by Olivier Douliery/AFP/Getty Images
The Federal Reserve may only use about one-fifth of its capacity to buy corporate bonds over the next year, says UBS. That would be a sign of the program's success.
Strategists at UBS said in a recent note that the takeup of the Fed's two bond-buying facilities—one created for newly issued bonds and one created for outstanding bonds and exchange-traded funds—may be limited.
They expect the Fed to buy between $140 billion and $165 billion of bonds and ETFs over the next year, only around one-fifth of its maximum $750 billion of potential purchases.
That is because the program has already started to achieve the Fed's goals, even though the central bank has only been buying ETFs; it hasn't started buying bonds just yet.
Every weekday evening we highlight the consequential market news of the day and explain what's likely to matter tomorrow.
Corporate borrowing costs have already declined substantially from their March highs. Investment-grade corporate bonds now yield 2.8%, or 1.9 percentage points more than Treasuries. That is down from 4.7%, or 4 percentage points above Treasuries, when the Fed introduced its programs on March 23.
Yields are a good measure of how much compensation bond investors are demanding for the risk of lending to companies—especially companies' yield spreads over ultrasafe Treasuries, which face little to no risk of default.
In other words, the Fed's pledge to backstop corporate debt markets has made it easier for companies to borrow from investors. (And to do other things like sell equity, for that matter.)
Strategists say companies should prefer to tap the market instead of the Fed's facilities anyway. The central bank's purchases are limited to bonds issued by investment-grade borrowers or "fallen angels," companies that have been downgraded from investment grade to junk since the introduction of the facilities. And it imposes a 1-percentage-point fee on companies that want to sell new bonds directly to the central bank.
Of course, the investor confidence engendered by the Fed has now made bond markets expensive, UBS says. They recommend investors stick within the market for higher-quality bonds, because the central bank's backstop doesn't extend far into the market for lower-quality debt.
"And the next marginal buyer of U.S. [investment-grade corporate bonds] may come from overseas, particularly Asia," as currency-hedging costs decline, the strategists write.
Write to Alexandra Scaggs at alexandra.scaggs@barrons.com
The Federal Reserve may only use about one-fifth of its capacity to buy corporate bonds over the next year, says UBS.


The 2% current value growth seen in grocery retailers in 2016 can be considered a good performance, especially compared with the 1% decline seen in 2015. Grocery retailers managed to transform the negative performance in 2015 to a positive performance in 2016 through different strategies. S Group, which actively reduced the prices of its grocery products throughout 2015-2016, achieved growth by increasing its volume sales. This boost was much needed in the poor economic conditions. Lidl Suomi also continued to use pricing as its main promotional tool, but it also offers a better selection of more premium products. Kesko's response to the price-based competition was to hold back from joining in, and it offered local outlet-owner-led stores that were tailored to consumers' needs. In addition, it invested heavily in expanding its outlet numbers by acquiring Suomen Lähikauppa's grocery retail brands. Such different strategies benefited these retailers, which all saw growth in 2016.
S Group led grocery retailers with a 42% value share in 2016, seeing a slight decline from 2015. Kesko ranked second with a 36% value share. This was six percentage points up from 2015, as the company acquired Suomen Lähikauppa's Siwa and Valintatalo chains in convenience stores. Lidl Suomi was third with a 9% value share; half a percentage point higher than in 2015.
The transformation of grocery retailing in Finland is expected to continue in the forecast period. All the main retailers are likely to focus on distinguishing their profiles from those of competitors. If successful, in the medium term Finnish grocery retailers will have clearer target consumer groups. Until recently, the leading companies Kesko and S Group had similar profiles for their several chains. Another factor which will have a substantial impact on value sales in the forecast period is whether the current price competition continues.
Discover the latest market trends and uncover sources of future market growth in the Grocery Retailers industry in Finland with research from Euromonitor's team of in-country analysts.
If you're in the Grocery Retailers industry in Finland, our research will save you time and money while empowering you to make informed, profitable decisions.
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The role of chamaejasmine in cellular apoptosis and autophagy in MG-63 cells
Dawei Yang, Hao Zhang, Jianjun Wu, Ruishuang Ma, Zongyu Li, Kunzheng Wang, Fan Yang
Bioscience Reports Jan 15, 2019, 39 (1) BSR20181707; DOI: 10.1042/BSR20181707
Dawei Yang
Department of Orthopedics, The Fourth Affiliated Hospital of Harbin Medical University, Harbin, China
Hao Zhang
Department of Orthopedic Surgery, The Fifth Hospital of Harbin, Harbin, China
Jianjun Wu
Department of Cardiology, The Second Affiliated Hospital of Harbin Medical University, Harbin, China
Ruishuang Ma
The Department of Internal Medical Oncology, Harbin Medical University Cancer Hospital, Harbin, China
Zongyu Li
Kunzheng Wang
Department of Orthopedics, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China
Department of Cardiology, The Second Affiliated Hospital of Harbin Medical University, Harbin, ChinaKey Laboratories of Education Ministry for Myocardial Ischemia Mechanism and Treatment, Harbin, Province Heilongjiang, China
For correspondence: yangfana@sina.com
Background: Osteosarcoma (OS) is the most common malignant neoplasm in children and adolescents with a very high propensity for local invasion and poor response to current therapy. Anti-cancer effect of chamaejasmine is newly discovered from Stellera chamaejasmine L. Our study focuses on investigating the effect of chamaejasmine on the cellular apoptosis, proliferation, autophagy, and the underlying mechanisms in MG-63.
Methods: Our study investigated the concentration of chamaejasmine in MG-63 cells by MTT and verified that chamaejasmine inhibited cell invasion by transwell. We also used Hoechst staining as well as apoptotic associated-proteins in MG-63 cells. Meanwhile, we also detected the lysophagesome and autophagsome by Lysotracker. Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) knockdown was performed with siRNA.
Results: Our results show that chamaejasmine exerts cellular growth inhibition, pro-apoptotic and pro-autophagic effect via activating AMPK in MG-63 cells. Furthermore, chamaejasmine significantly increases autophagic cell via the inhibition of mammalian target of rapamycin (mTOR) and activation of AMPK signaling pathways. Administrated with chamaejasmine also induces reactive oxygen species (ROS) generation, indicating cross-talking between these two primary modes of programmed cell death.
Conclusion: Our results show that chamaejasmine promotes apoptosis and autophagy by activating AMPK/mTOR signaling pathways with involvement of ROS in MG-63 cells. Chamaejasmine is a promising anti-cancer agent in OS treatment, and further studies are needed to confirm its efficacy and safety in vivo or other cancer cells.
AMPK/mTOR
chamaejasmine
Osteosarcoma (OS) is the most common type of primary malignant bone tumor, ordinarily manifesting as malignant mesenchymal cells producing osteoid or immature bone [1]. The improvement of medical techniques has led to an obvious increase of 5-year survival rate of OS [2]. Although a large number of chemotherapeutic agents are currently in use, most of patients need limb amputation and undergo lung metastasis [3]. Until now, the pathogenesis and molecular mechanisms of osteosarcoma are still unknown. Therefore, novel strategies are essential to develop more effective therapies for OS treatment.
Many plants have anti-tumor pharmacological effects, and about two-thirds of current anti-cancer drugs such as vinblastine, topotecan, etoposide, and paclitaxel are originally plant-derived compounds [4,5]. 'Lang-Du'(Chamaejasmine), as a traditional Chinese herbal, has been reported that chamaejasmine has an anti-tumor activity [6]. It is one of the major bioactive ingredients of Stellera chamaejasmine L [7]. However, to the best of knowledge, there has been little research on chamaejasmine and/or elucidating the anti-tumor molecular mechanisms of actions in osteosarcoma cells.
Apoptosis is an important self-stabilizing mechanism for multicellular organisms [8], which is characterized by particular morphological changes, including plasma membrane bleb, cell shrinkage and DNA fragmentation [9]. It is reported that complex interaction of molecular events results in the activation of the apoptotic cascade [10]. Studies reveal that reactive oxygen species (ROS) generation and disruption of the mitochondrial membrane potential contribute to drug-induced apoptosis [11,12]. In normal cells, autophagy is essential for the maintenance of cellular energy and is responsible for the regulation of organelle turnover [13]. In response to various cellular stresses, autophagy is up-regulated in order to prevent the accumulation of misfolded or damaged proteins and organelles [14–17]. As immortal cells, the occurrence of tumor cells is not only with the abnormal cell biogenesis, but also with the abnormalities of apoptosis. It has been demonstrated that spontaneous apoptosis in malignant tumors may have therapeutic effects on tumors [18]. Accumulating evidence has linked the apoptosis to autophagy [19]. Thus, apoptosis or/and autophagy are interesting mechanisms to induce the death of cancer cells. Until now, the role of apoptosis in cancer therapy is still controversial.
Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) is an evolutionarily conserved sensor of cellular energy status that is activated by pathological stresses such as oxidative and cellular stresses that deplete ATP and increase AMP [20–25]. Cell growth, proliferation, differentiation, and cell cycle regulation could be controlled by mammalian Target of Rapamycin (mTOR). It is reported that activation of mTOR suppresses autophagy, and the inhibition of mTOR activity promotes it [26–29]. As the mechanism of action, AMPK activation regulates tumor cell growth through the inhibition of mTOR pathway, which is recognized to be associated with translation initiation and protein synthesis [30,31].
Our study aimed to demonstrate the anti-cancer effects of chamaejasmine against MG-63 cells by investigating its effects on apoptosis increasing, inhibition of cell invasion, and autophagy induction. To the best of our knowledge, the present constitutes the first report on this plant species in OS as its pro-autophagic effect.
Methods and materials
Cell lines and chemicals
Osteosarcoma cell lines MG-63/U2Os/Saos-2/KHOs were obtained from the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM complemented with 10% FBS and antibiotics in 37°C humidified atmosphere with 95% O2 and 5% CO2. Chamaejasmine (purity ≥98%) was obtained from the Zhongbiao Biochemistry Inc. (Wuhan, China). Compound C (SML2017-5mg), Bafilomycin A1 (02911643), NAC (A9165-5g) and 3-MA (M9281-100MG) were purchased from Sigma Chemical (St. Louis, MO).
Cell viability assay
Inhibition of cell proliferation by chamaejasmine was measured by using MTT. Briefly, cells were subcultured in a 96-well plate with a density of 2 × 104 cells/ml and incubated overnight at 37°C under an atmosphere of 95% O2 and 5% CO2. After 24 h, the medium was replaced with various concentration of chamaejasmine (0, 40, 80, and 160 μM). After 24- or 48-h incubation, culture media were modified for serum-free culture media. Then, the serum-free culture media containing MTT were discarded and DMSO was added to each well to dissolve the precipitate. The optical densities were measured at 490 nm spectral wavelength using Easy Reader 340 AT (SLT-Lab Instruments).
Cell invasion assay
The effects of chamaejasmine on the MG-63 invasion were used in modified Boyden chambers consisting of transwell (Corning Costar Corp., U.S.A.). The upper surfaces of the transwell membranes were coated with 1 mg/ml Matrigel matrix (Collaborative Biomedical Products, U.S.A.) overnight. Cells were cultured in DMEM containing 40, 80, and 160 μM chamaejasmine and then plated into the top chambers. After incubation for 24 h at 37°C, cells on the upper surface of the membrane were removed with a cotton swab. The cells on the lower surface were stained with Crystal Violet for 20 min. Invading cells from three random fields were counted under a light microscope using a magnification of 200×.
Cell apoptosis analysis by flow cytometry
The effect of chamaejasmine on cell apoptosis was analyzed by flow cytometer with Annexin V/PI kit (BD, Biosciences). Briefly, the medium was removed and the cells were treated with serum-reduced medium (0.5% FBS) with vehicle or different concentrations of chamaejasmine for 48 h. Cells were suspended in 300 μl Binding buffer, and then stained with Annexin-FITC and/or propium iodide. Positive controls for apoptosis were stained with only Annexin-FITC. Positive controls for necrosis were stained with only propium iodide. At least 104 cells were analyzed by flow cytometer (BD, Biosciences).
Caspase activity assays
Caspase activity assays in multi-well plate formats represent powerful tools for understanding experimental modulation of the apoptotic response. Caspase 3, Caspase 8, and Caspase 9 Multiplex Activity Assay Kit (Fluorometric) (ab219915) were purchased by Abcam. It a fluorescent assay that detects the activity of caspase 3/ 8/ 9 in cell lysates. We followed the protocol in https://www.abcam.com/ps/products/219/ab219915/documents/. Subtracting blank readings from all measurements (control and treated), using fluorescent intensity, determines fold change between control and treated cells. The fluorescence intensity was measured with FlexStation fluorescence microplate reader at the indicated wavelength. Caspase 3 (Ex/Em = 535/620 nm), Caspase-8 (Ex/Em = 490/525 nm), and caspase-9 (Ex/Em = 370/450 nm), activities can be detected in a single assay without interferences from other caspases.
Morphological apoptosis
Hoechst 33258 staining was carried out to observe morphological characteristics of apoptosis. In brief, cells were plated on the coverslips in 12-well plates and then treated with 0–160 μM chamaejasmine for 48 h. Then, the cells were washed twice with PBS, and then stained with Hoechst 33258 solution (5 μg/ml) for 10 min in the dark. Cells were observed using a fluorescence microscope (Olympus Optical Co., Japan) for morphological changes in the nucleus.
MG-63 cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. The protein concentrations were measured using a BCA protein assay kit (Beyotime, China). Proteins (40 mg) were separated by SDS-PAGE and transferred to a NC membrane (Bio-Rad, U.S.A.). After blocked with 5% non-fat milk for 1 h at room temperature, the membrane was incubated overnight at 4°C with antibodies against p-AMPK (1:1000), AMPK (1:1000), cleaved caspase 3 (1:1000), cleaved caspase 9 (1:1000), p-mTOR (1:1000), mTOR (1:1000), Bcl-2 (1:1000), Bax (1:1000), and β-actin (1:1000). After three washes, the membrane was incubated with secondary antibodies for 1 h at room temperature. The optical density of these protein bands was quantified using the Quantity One software after the separated proteins were visualized using ECL kits (Beyotime, China).
Measurement of intracellular reactive oxygen species (ROS)
Intracellular ROS production was measured using a DCFH-DA fluorescent dye (Beyotime, China). MG-63 cells were cultured in six-well plates at a density of 1 × 105/well. After different treatment, cells were incubated with 10 μM of DCFH-DA at 37°C for 15 min and then washed twice with ice PBS. Cells were observed using the fluorescence microscope mentioned above. Experiments were repeated at least three times to calculate the mean fluorescence intensity of DCFH-DA and data analyzed by Image Pro Plus.
Lysotracker view
LysoTracker Green (Cell signaling technology, U.S.A.) were diluted by 50 μl DMSO and added into normal growth media for a working concentration of 50 nM. After specific treatment, cells were imaged live without fix by 4% paraformaldehyde. The labeled cells were observed at 200× magnifications under a fluorescence microscope.
Transfection using siRNAs for gene silencing
Silence interfering (si) RNA oligonucleotides for AMPK was purchased from Cell signaling technology and transfected to suppress gene expression. A scrambled siRNA was used as a control. Transfection was performed with Lipofectamine 2000 (Invitrogen). According to the manufacturer's directions, siRNA was mixed with Lipofectamine 2000 in serum-free media. The cells mixed with siRNA were diluted in complete medium and transfected for 6 h until changed the normal DMEM. Cells were cultured for another 48 h after transfection.
All the experiments were performed in triplicate. Measurement data are expressed as mean ± SEM. Statistical analysis was performed with Graphpad Prism5. Statistical analyses were performed using the Student's t test for comparisons of two groups and using one-way analysis of variance for multi-group comparisons. Significance was set at P<0.05.
Chamaejasmine inhibits osteosarcoma cells viability
We assessed the effects of various doses of chamaejasmine on the viability of OS cells (MG-63, Saos-2, KHOS, and U2OS) using the MTT assay. As shown in Figure 1A, chamaejasmine inhibited the cell growth of cell lines effectively in a time- and dose-dependent manner (n=3, P<0.05). The growth inhibition caused by chamaejasmine is partially attributable to apoptosis, as evidenced by the activity of caspase; however, there are some differences among different cell line (Figure 1B). In MG-63 and KHOS cell lines, caspase-3 activity is significantly increased compared with control group. Whereas caspase-9 activity is much more sensitive and increasing in Saos-2 and U2OS cell lines. The invasion of the cells treated with chamaejasmine was significantly inhibited compared with that in the control cells by transwell invasion assay (Figure 1C,D). Our results revealed that the cytotoxic effect of the chamaejasmine showed a dose- and time-dependence.
Figure 1 Chamaejasmine inhibits growth and induces apoptosis in OS cells
(A) The viability of chamaejasmine in OS cells at three different time intervals and three different concentrations. Data are expressed as the mean ± S.E.M of at least three independent experiments. *P<0.05 vs 0 μM chamaejasmine. (B) Caspase activity of 3, 8 and 9 in MG-63, Saos-2, U2Os, and KHOs. The results were expressed as the mean ±S.E.M (*P<0.05 vs control group, n=3). (C and D) Cells with 0–160 µM chamaejasmine showed decreased invasive capacity compared with the control as documented by transwell invasion assay.
Chamaejasmine induces apoptosis in MG-63 cells
We used Hoechst 33258 staining to investigate the apoptosis induced by chamaejasmine. As shown in Figure 2A, MG-63 cells were caused condensed and fragmented nuclei, as characteristic of apoptosis after treatment with different concentration of chamaejasmine for 48 h (Figure 2B). The protein expression of cleaved caspase 3 (c-caspase 3), cleaved caspase 9 (c-caspase 9), and bcl-2/bax were all remarkably increased after treatment with 160 μM chamaejasmine in MG-63 cells (Figure 2C). All the results indicate that chamaejasmine provokes cellular apoptosis by activating both the extrinsic and intrinsic pathways in dose-dependent manner.
Figure 2 Effects of chamaejasmine on MG-63 cells apoptosis in concentration-dependent treatment groups
(A) The number of apoptotic MG-63 cells from 0 to 160 µM chamaejasmine was assessed by Hoechst/PI staining and visualized at 200× magnification. (B) Data are the mean ± SEM (n=3). *P<0.05 compared with control group. (C) Proteins level of caspase 3, Bcl-2, Bax, and caspase- 3 determined by Western blotting assay. The upper trace of each group shows representative blots of the respective proteins, and the lower panels show the bar graphs summarizing the immunoblot data. (D–F) Densitometric results are expressed as a fold increase. Data are the mean ± SEM (n=3). *P<0.05 compared with control group; #P<0.05 with 40 µM group.
Chamaejasmine induces autophagy in MG-63 cells
We then determined whether chamaejasmine could induce autophagy in MG-63 cells. The increased acidic vesicular organelles (AVOs) are related to autophagosomes, which are the hallmark of autophagy [32]. We used LysoTracker Green to label lysosomes and autolysosomes. Our results showed that cells treated with chamaejasmine exhibited more AVOs in the cytoplasm (Figure 3A). Next we investigated the expression of autophagy-related proteins in cytoplasm by Western blotting analysis in chamaejasmine-treated and control cells. A significant conversion of LC3-I to LC3-II was detected after exposing MG-63 cells at 0 to 160 μM chamaejasmine for 48 h (Figure 3B). The accumulation of LC3-II positively correlated with the treatment dose, suggesting that chamaejasmine induced autophagy in a dose-dependent manner (Figure 3B). Our results demonstrated that chamaejasmine increased the level of ATG-7, LC3B-II and beclin-1 on MG-63 cells in concentration-dependent manner (Figure 3C–E).
Figure 3 Protective autophagic effect of chamaejasmine on MG-63 cells
(A) The punctate dots of a green fluorescent protein tagged of Lysotracker View in MG-63 cells after treatment with chamaejasmine 0–160 μM, magnification: 200×. (B–E) The ratio of LC3-II/I and the relative protein levels of ATG-7 and Becllin-1 was determined by densitometric analysis, and data were measured by Western Blot. β-Actin was used as an equal loading of proteins. Data are the mean ± SEM (n=3). *P<0.05 compared with control group; #P<0.05, with 40 µM group.
Chamaejasmine activates the activity of the AMPK/mTOR signaling pathway
We investigated the effect of chamaejasmine on AMPK/mTOR signaling pathway to explore the molecular mechanism of autophagy activity. Our results showed that chamaejasmine increased the phosphorylation of AMPK and decreased the phosphorylation of mTOR in concentration-dependent manner (Figure 4).
Figure 4 Chamaejasmine activates the AMPK/mTOR signaling pathway
Total cell lysates were isolated 48 h after treatment with chamaejasmine of MG-63 cells and were subjected to immunoblot analysis with anti-pAMPK, -AMPK, p-mTOR, or –mTOR antibodies. β-Actin was used as a loading control. Results from three independent experiments are presented as means ± S.E.M. *P<0.05 compared with control group; #P<0.05 with 40 µM group.
Chamaejasmine induced ROS production in MG-63 cells
To examine how chamaejasmine affects the production of ROS in MG-63 cells; DCFH-DA staining was performed. The production of ROS was increased in chamaejasmine-treated MG-63 cells compared with control group and NAC group (Figure 5A). Next, we investigated the underlying mechanism of AMPK activation by chamaejasmine in MG-63 cells. Significantly, the antioxidant NAC decreased the production of ROS and inhibited chamaejasmine-induced AMPK activation (Figure 5B). Meanwhile, hydrogen peroxide (H2O2) increased the ROS level and induced AMPK activation in MG-63 cells, prevented by NAC pre-administration. We conclude that chamaejasmine-induced AMPK activation requires ROS production.
Figure 5 Effects of chamaejasmine on the ROS and p-AMPK expression in MG-63 cells
(A) Random micrographs of dichlorofluorescein (DCF)-derived fluorescence in MG-63 cells from different treatment groups. The histogram showed the quantitative analysis of the mean fluorescence intensity in the indicated groups. Data are the mean ± SEM (n=3). DMEM only vs NAC treatment, *P<0.05. (B) Western blot of p-AMPK and AMPK proteins in MG-63 cells from different treatment groups. Data are the mean ± SEM (n=3). DMEM only vs NAC treatment, *P<0.05. DCF, dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species; S.E.M, standard error of the mean.
Inhibition of AMPK facilities chamaejasmine-induced apoptosis in MG-63cells
To investigate whether chamaejasmine-induced apoptosis and autophagy have a connection with AMPK/mTOR signaling pathway, we used bafilomycin-A1 (Baf) (autophagy inhibitor, 100 nM) and Compound C (AMPK inhibitor, 2.5 μM) for further exploration. When the cells were treated with Compound C for 48 h, the proportion of Annexin V-staining positive cells (total apoptotic cells) markedly increased from 4.43% in control to 5.38%, respectively. Thus, flow cytometric analysis demonstrated that Compound C could reverse the cell apoptosis induced by chamaejasmine (Figure 6A). Chamaejasmine obviously increased the activity of p-AMPK and ATG-7 in MG-63 cells, while Baf (100 nM) decreased the level of p-ampk/ampk and the expression of ATG-7. In addition, the Baf markedly decreased the effects of chamaejasmine in the chamaejasmine +Baf co-treatment group. It proved that the AMPK pathway signaling was involved in chamaejasmine-mediated autophagy role in MG-63 cells (Figure 6D–F). In order to demonstrate the effect of chamaejasmine on the relationship between apoptosis and autophagy, MG-63 cells were treated with 3-MA, an autophagy inhibitor. Our data showed that cell apoptosis ratio was increased in MG-63 cells after treatment with chamaejasmine or 3-MA (Figure 6G,H) compared with chamaejasmine group. Whereas there was no significant changes between chamaejasmine and 3-MA groups. Our result revealed that apoptosis was possible partly dependent on autophagy.
Figure 6 Activation of AMPK mediates chamaejasmine-induced autophagy in osteoblastoma cells
(A–C) The cells were treated with 0 µM (control), 160 µM chamaejasmine, CC and CC+160 µM chamaejasmine for 48 h and analyzed using fluorescence-activated cell sorting. R1, R2, R3, and R4 quadrants show percentage of necrotic, early apoptotic, normal healthy, and late apoptotic cell populations, respectively. CC, Compound C; 160 µM, chamaejasmine; PI, propidium iodide. (D–F) MG-63 cells were divided into control, or 160 μM of chamaejasmine, and/or with Baf treatment group. Phosphor (p)- and total AMPK and ATG-7 were examined by Western blotting and quantified by Image Lab. Data were summarized from at least three different experiments (*P < 0.05 vs control). (G–H) MG-63 cells were treated by chamaejasmine and NAC with 3-MA. Representative photographs of double staining of PI and Hoechst 33258. The apoptotic cells were observed as nuclei pyknosis by Hoechst 33258. PI positive cells (red/pink) are regarded as the necrotic cells. The results were expressed as the mean ± S.E.M (*P<0.05 vs control group, n=3).
AMPK silencing impairs chamaejasmine-induced cell apoptosis and autophagy
To assess the effect of AMPK inhibition on signal transduction rather than a modulation of other cellular activities, accounting for the chamaejasmine-induced cell apoptosis and autophagy in MG-63 cells, we analyzed cleaved caspase 3 and Beclin-1 after silence interfering RNA (siRNA)-mediated silencing of AMPK. As shown in Figure 7A,B, the target-specific siRNA of AMPK significantly suppressed the cellular AMPK level by about 70% in chamaejasmine-treated MG-63 cells. Cells with AMPK knockdown led to a large decrease of cleaved caspase 3. AMPK knockdown also inhibited Beclin-1 after chamaejasmine treatment (Figure 7C,D). These results indicate that activation of AMPK is partly responsible for the increased cellular apoptosis and autophagy. All above findings suggest that apoptosis and autophagy induced by chamaejasmine in MG-63 cells could be mediated by the activation of AMPK/mTOR signaling pathways.
Figure 7 Effects of AMPK-siRNA on chamaejasmine-elicited apoptosis
(A and B) Suppression of autophagy in MG-63 cells were transfected with AMPK siRNA at 150 nM for 72 h. Protein levels of AMPK were measured by Western blotting with anti-AMPK antibody. (C and D) MG-63 cells were transfected with control siRNA or with AMPK siRNAs. Cells with AMPK siRNA transfection were co-incubated with chamaejasmine for 48 h. The upper trace of each group shows representative blots of the respective proteins, and the lower panels show the bar graphs summarizing the immunoblot data. Densitometry results are expressed as a fold increase. *P<0.05 vs control siRNA group; #P<0.05 vs chamaejasmine group. (E) Schematic diagram of the mechanisms of chamaejasmine-dependent apoptosis and autophagy in regulation of ROS and AMPK in osteosarcoma cells. Chamaejasmine-induced autophagy elicited cytotoxic effects through activated-AMPK-mTOR pathway and suggested a novel mechanism of chamaejasmine-induced MG-63 cells inhibited effect.
OS, the most common malignant bone tumor, occurs predominantly in children and adolescents. Until recently, a 5-year survival rate has been improved through surgery combined with adjuvant and neoadjuvant chemotherapy and radiotherapy [33,34]. However, recurrence, metastasis, and drug resistance are responsible for the poor effectiveness of cytotoxic drugs [35]. Therefore, it was urgent to develop novel treatments with potent anti-tumor effects to target the malignant behavior of OS cells and improving the treatments for patients.
In the past few years, researchers have reported that chamaejasmine can induce apoptosis in multiple cancer cell lines [36–38]. However, the exactly mechanism of the cause for apoptosis or autophagy of tumor cells remains unclear. We have demonstrated that chamaejasmine could induce apoptosis via p53 pathway in osteosarcoma cells [38]. ROS at a low concentration contribute to the improvement of cellular function and cell survival whereas excessive ROS causes the capability to promote programmed cell death [39]. We hypothesized that caspase and AMPK/mTOR signaling pathway may take part in this process. Our study first revealed that chamaejasmine-induced apoptosis and inhibited growth of MG-63 cells via AMPK activation. Here we report that chamaejasmine-induced early autophagy is mainly dependent on the induction of autophagosomes, conversion of LC3-I to LC3-II, expression of Atg7 and Beclin-1 and inhibition of Bcl-2/Bax, caspase 3, and caspase 9.
In the present study, after treatment with different chamaejasmine concentrations (0–160 μM) for 24 and 48 h, MTT assays showed that the cell viability was significantly decreased in a concentration- and time-dependent manner in MG-63 cells (Figure 1A). The effect of chamaejasmine on cell invasion, hallmark of malignancy, was examined by transwell assay. Our study demonstrated that chamaejasmine reduced cell invasion in a concentration-dependent manner (Figure 1C).
Apoptosis is a key strategy for protective mechanism against tumor biogenesis. Many anti-cancer drugs play roles in apoptosis induction and cell arrest eradicating the cancerous cells [40]. It has been identified that there are two distinct pathways of apoptosis in mammalian cells: the extrinsic and intrinsic pathways [41]. The extrinsic pathway is activated by pro-apoptotic receptor signals at the cellular surface; the intrinsic pathway is governed by Bcl-2-family proteins members, which affect the release of cytochrome C into the cytosol, resulting in caspase 9 and 3 activation [42,43]. The apoptosis induced by chamaejasmine was further confirmed in a concentration-dependent manner by Hoechst staining fluorescence imaging (Figure 2A). Our study demonstrated a decrease in the ratio of Bcl-2/Bax in MG-63 cells after treatment with different concentrations of chamaejasmine. Meanwhile, chamaejasmine-induced apoptosis was mediated by caspase 9 and caspase 3 in MG-63 cells (Figure 2C-F).
It has been mentioned that AMPK activation is involved in cell growth and reprogramming metabolism and autophagy through regulating its many downstream kinases [44,45]. Because AMPK plays a critical role in response to autophagy [27], we assessed the effect of chamaejasmine on AMPK pathway in osteosarcoma. It remains controversial about how autophagy modulates the balance between cytoprotection and cell death through AMPK pathway. Existing research demonstrated that activation of AMPK might inhibit cell growth and induce cancer cell apoptosis under stress condition [20,45]. While other studies indicate that AMPK is pro-survival and anti-apoptotic [46]. In addition, previous reports have established p-AMPK/mTOR serving as a key signaling pathway, which negatively regulates apoptosis and autophagy [47] in glucose/glycogen metabolism. ROS is well-known as the activator of AMPK [48,49] and directly induces autophagy by up-regulating autophagy-associated gene (ATG) expression [50]. The mechanism of chamaejasmine-mediated induction of oxidative stress is not clear. Here, we have provided evidence to support that ROS production and cancer cell apoptosis are involved in AMPK activation by chamaejasmine. In our study, ROS and AMPK activation significantly increased after chamaejasmine treatment (Figure 5). The AMPK inhibitor, Compound C, significantly inhibited the induction of apoptosis by chamaejasmine (Figure 6A). Indeed, while an increase in LC3B-II level in steady state conditions corresponds to an increase in the amount of autophagosomes in cells (Figure 3B), this may be due to activation or late inhibition of the autophagic process. Therefore, in order to distinguish between these opposite circumstances, it is necessary to compare autophagic-related proteins with those of the corresponding samples treated with lysosomal protease inhibitors (such as Bafilomycin A1 and Chloroquine): if autophagic flux is increased, the amount of LC3B-II or ATG-7 or Beclin-1 will be higher in presence of inhibitors (the autophagic process is active) while, if the autophagic process is inhibited, the amount of LC3B-II or ATG-7 or Beclin-1 will not increase in presence of inhibitors (the flux is blocked). Through exploring the further mechanism signaling of AMPK, NAC also decreased chamaejasmine-induced AMPK activation, suggesting that ROS production might be required for AMPK activation and cell autophagy by chamaejasmine. As a matter of fact, AMPK activation by chamaejasmine could activate oxidative stress and to increase the apoptotic cells.
Therefore, we investigated the relationships between chamaejasmine-induced apoptosis and autophagy. Autophagy and apoptosis control the turnover of organelles and proteins within cells, and of cells within organisms, respectively, and many stress pathways sequentially elicit autophagy, and apoptosis within the same cell. Generally, autophagy blocks the induction of apoptosis, and apoptosis-associated caspase activation shuts off the autophagic process. However, in some cases, autophagy or autophagy-relevant proteins may help to induce apoptosis or necrosis. In our study, co-incubation chamaejasmine with Baf also increased the expressions of p-AMPK/AMPK and ATG-7 (Figure 6D), indicating that the autophagy induced by chamaejasmine via the activation of AMPK. In order to demonstrate the effect of chamaejasmine on the relationship between apoptosis and autophagy, the administration of 3-MA, an autophagy inhibitor, treated with MG-63 cells. Our data showed that the increasing cell apoptosis ratio in MG-63 cells treated with chamaejasmine (Supplement 1), NAC, and 3-MA decreased compared with chamaejasmine group in MG-63 cells. Our result revealed that apoptosis was possible partly dependent on autophagy.
The important role in regulation apoptosis and autophagy is the cellular redox state. Activation of AMPK could influence the balance between the formation and degradation of intracellular ROS, and further influence the redox state of cell [51]. Therefore, the relationship between autophagy and apoptosis induced by chamaejasmine might help regulate these processes and improve the anti-tumor effects of chamaejasmine. AMPK inhibition by Compound C or AMPKsiRNA was added for the mechanism study. Compound C as an AMPK inhibitor decreases AMPK expression. However, Compound C may also regulate the expression of other proteins, such as AKTT308, AKTS473 or CK1, CAMMK according to previous studies. Therefore, we used AMPKsiRNA to demonstrate the phenotypic alteration by biological inhibition of AMPK. As shown in Figure 7C, AMPK inhibition decreased the expression of cleaved-caspase 3 in MG-63 cells; however, we could not figure out the exactly mechanism of chamaejasmin in the relationship between apoptosis and autophagy. More interestingly, as designed in our experiments, there were not any physical contact between chamaejasmine and MG-63 cells, which means there must be one or some secretory protein(s) that bridged the communication between cells and triggered autophagy. Identification of such soluble mediators is needed and will be essential for depicting the detailed molecular behaviors of chamaejasmine in the cross-talk between autophagy and apoptosis. As depicted in Figure 7E, chamaejasmine appears to regulate process and program that might affect the growth and/or survival of cancer cells.
Our findings indicated that chamaejasmine exerted its cytotoxic effect against MG-63 cells through the activation of AMPK/mTOR pathway, which was responsible for autophagy. However, further study is needed to explore whether chamaejasmine is suitable for providing an attractive target for cancer therapy. For example, further analysis is required to investigate its cytotoxic potential in vivo models as a promising anti-cancer medicine for osteosarcoma therapy. In addition to assessing the possibility of chamaejasmine, more information is needed to determine whether AMPK pathway contributes to the association with apoptosis and autophagy during osteosarcoma.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Supporting Data Availability
We declared that materials described in the manuscript, including all relevant raw data, will be freely available to any scientist wishing to use them for non-commercial purposes, without breaching participant confidentiality.
The authors declare that there are no competing interests associated with the manuscript.
This study was supported by a grant from the Key Project of Harbin Municipal Science and Technology Bureau [2016RAXYJ068, 2013RFXYJ041, 2006AA9BS146]. This study was supported by Graduate innovation foundation of Harbin medical university. This study was also supported by Dean foundation of the Fourth Affiliated Hospital of Harbin Medical University, Postdoctoral Science Foundation of Heilongjiang and Postdoctoral Science Foundation of China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Author Contribution
Y.D.W. contributed to the conception of the study. Z.H. and W.J.J. contributed significantly to analysis and wrote the manuscript. M.R.S. and L.Z.Y. performed the analysis with constructive discussions. K.Z.W. and Y.F. conceived and designed the experiments. All authors read and approved the final manuscript.
This work was also supported by the Key Laboratory of Myocardial Ischemia Mechanism and Treatment (Harbin Medical University), Ministry of Education. The authors thank the graduate students in Prof. Yubo 's laboratory in Harbin Medical University, Heilongjiang, China.
Abbreviations: 3-MA, 3-methyladenine; AMPK, adenosine 5′-monophosphate (AMP)-activated protein kinase; Baf, bafilomycin-A1; CC, Compound C; mTOR, mammalian target of rapamycin; NAC, N-acetyl-L-cysteine; ROS, reactive oxygen species
© 2019 The Author(s).
This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY).
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Friends, I had a marvelous day with this lovely group in Armagh on Saturday at the Marketplace Theatre and Arts Centre.
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