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text:
New University Act: Statement by the NRW Rectors' Conference
On January 14, 2012, the Rectors' Conference of North Rhine-Westphalia (LRK NRW) published a statement on the draft version of the new NRW University Act. According to the universities in NRW, the new Act poses a threat to university autonomy and the quality of teaching and learning.
Excerpts of the Press Release by the NRW Rectors' Conference
The development of the universities in North Rhine-Westphalia over the last years is a success story. They successfully managed the double cohort of secondary school leavers and increased their research performance.
The NRW universities are convinced that in order to be able to maintain this level of performance, the new University Act needs to be fundamentally revised before coming into effect. The critique of the current draft version of the Act focuses on five central points:
The Quality of Teaching and Learning is Under Threat
The improved competitiveness of the NRW universities was made possible by increased flexibility and room for autonomous action of the University Autonomy Act currently in force. The planned limitation of responsibility and autonomy on the part of the university will result in a substantial, sustainable impairment of the universities' research performance and their development potential in teaching and learning.
The Act Creates Opaque Structures and Undermines Democratic Participation
The new University Act aims to establish a system which gives inordinate power to the Ministry of Innovation, Science and Research. The ministerial bureaucracy will have the capacity to bypass the university and its bodies in decision making processes and act largely without parliamentary control. In this way, structures and decisions are made much less transparent.
Severe Limitation of Academic Freedom
The new Act makes it possible for the NRW Ministry of Innovation, Science and Research comprehensively to control the universities, possibly even trespassing the constitutionally guaranteed academic freedom. The Act stipulates, for example, that the ministry may withdraw a faculty's right to award doctoral degrees. Such a grave encroachment on academic freedom unprecedented and questionable with regard to the ethics of science.
Massive Bureaucratization at the Expense of Students and Research
The regulations stipulated in the draft version of the Act and the unbalanced increase in bureaucratic powers of the Ministry will result in a significant growth in bureaucracy in general. None of the newly introduced regulations helps the universities in fulfilling their roles. The increase in administrative expenditure is expected to generate costs in the millions, and this at a time of limited public funds.
2500 Jobs under Threat – Weakening of NRW as a Location of Science and Business
Constraining the universities' scope for action means to weaken their innovative power; in this way, North Rhine-Westphalia as a site of business will lose important economic impulses. Under the new Act, it will no longer be possible to guarantee partners from business and industry the necessary confidentiality for research collaborations. Thus, in research alone, about 2500 positions are under threat.
Full Text of the Statement of the NRW Rectors' Conference (de)
You Are Here:New University Act: Statement by the NRW Rectors' Conference

redpajama_set_name: RedPajamaCommonCrawl


Electric 94. 9s Acoustic Christmas 17 at Freedom Hall on Wednesday, December 13, 2017 - Event Page - TriCitiesNights Our 2018 Almost Acoustic Christmas Tickets Can Be Sorted By Price, Or Section To Assist You In Narrowing Down The Particular Almost Acoustic Christmas 2018 Tickets You're Wanting And For Any Future Almost Acoustic Christmas Lineup 2018 That You Would Need To Attend When It Travels To Your City.
Top 40 hit music for Johnson City, Kingsport and Bristol with Jinger& Izzy in the morning. Listen live, get the latest music news and more. Contact us. Freedom Hall Civic Center 1320 Pactolas Road Johnson City, TN (423) 461-4884 Box Office (423) 461-4855 Administration Lower Prices On 2018 Acoustic Christmas Tickets Online. We Offer 2018 Acoustic Christmas Tour Tickets To All The Major 2018 Acoustic Christmas Concert Nationwide, And Information On The 2018 Acoustic Christmas Tour Schedule.
Latest Stories. Hailey Baldwin Debuts Massive Engagement Ring From Justin Bieber; Top 9: Thursday, August 23; Most Wanted @ Noon; WATCH: Pink Stops a Show to Hug a Girl Who's Mother Died Monday morning, Electric 94. 9 announced this year's lineup for Acoustic Christmas – the Tri-Cities' annual holiday concert. Monday morning, Electric 94. 9 announced this year's lineup for. To buy Acoustic Christmas tickets for sale at discounted prices, choose from the Acoustic Christmas schedule and dates below.
Stub offers cheap Acoustic Christmas tickets for 2018 Acoustic Christmas events along with Acoustic Christmas cost information. For questions on purchasing Acoustic Christmas tickets or general. See Who's Going to KROQ Almost Acoustic Christmas 2018 in Inglewood, CA! The legendary KROQ Almost Acoustic Christmas in Los Angeles, California, at the Shrine Auditorium features an acoustic performance to kick off two nights of incredible live music by nationally acclaimed rock and alternative artists.
The one-of-a-kind lineup. Organizers from the City of Johnson City announced on Friday the performers for the upcoming Acoustic Christmas at Freedom Hall. The concert is schedule for Friday, December 16 and will start. UPCOMING EVENTS.
Senior Services offers Trail Mix and Mingle. The Johnson City Senior Foundation Inaugural Indoor Pickleball Tournament will be held Nov. 2-3 at Memorial Park Community Center, 510 Bert St. Sponsorship revenue from the event will make this the Foundation's signature fundraiser for 2018 and help provide outreach.
1320 Pactolas Rd, Johnson City, Tennessee. Show Map. Hide Map 1320 Pactolas Road, Johnson City, Tennessee. Show Map.
Hide Map List of all concerts taking place in 2018 at Freedom Hall Civic Center in Johnson City. This event has been added to. Electric 94. 9's Acoustic Christmas 2017 Fall Out Boy. Freedom Hall Civic Center, Johnson City, TN, US 1320 Pactolas Rd. I was there. Saturday 08 April 2017; Moon Taxi. Freedom Hall Civic Center, Johnson City, TN.
(Tickets for the originally scheduled March 1, 2018 show will be honored at the November 11, 2018 concert) Find cheap Acoustic Christmas at Johnson City Tickets at Mark's Tickets. Mark's Tickets specialize in sold out Acoustic Christmas at Johnson City Tickets and last minute Acoustic Christmas at Johnson City tickets. Acoustic Christmas Schedule.
No upcoming events found. Click here to see Acoustic Christmas events at all. Almost Acoustic Christmas is an annual concert run by the Los Angeles radio station KROQ-FM. From 1989 through 2012 the show took place at the Universal Amphitheatre in Universal City, except for 1998 where it was held at the Shrine. Date, Lineup. This page was last edited on 19 May 2018, at 07: 44 (UTC). Acoustic Christmas '17 · Summer Hangout '17 · Acoustic Christmas '16 · Acoustic Christmas.
Schedule is as follows: April 29 at 7: 30 p. m. June 28 at 8: 30 p. m. July 27 at Acoustic christmas lineup 2018 johnson city 30 p. m. September 01, 2018 Sunday, 10am until 8pm. 15th starting at 6pm at the Johnson City Country Club, 1901 E. Unaka Ave, Johnson City, TN. WAEZ Public Inspection File Public File Assistance EEO Public File. © 2018 Bristol Broadcasting Company, Inc. All Rights Reserved.
We're already planning the Biggest Hit Music Show of the Year for the Tri-Cities. Electric 94. 9's Acoustic Christmas will be here before you know it and we want.


text:
More South Florida Flights Canceled Due to Nor'easter
Nor'easter in northeast leads to flight delays, cancellations
Published at 11:38 am on November 8, 2012
The nor'easter that moved into the northeast Wednesday continued to lead to flight cancellations and delays in South Florida Thursday, officials said.
As of 11 a.m., six fights were canceled and another nine were delayed at Fort Lauderdale-Hollywood International Airport, Broward County Aviation Department spokesman Greg Meyer said.
Nor'easter Brings New Outages for Sandy-Weary Northeast
The cancellations were impacting Newark and JFK Airport in the New York City area, Meyer said. The delays are impacting flights to Newark, JFK, Long Island, Washington, D.C., Boston and Newburg.
At Miami International Airport, a total of 16 flights were canceled Thursday, Miami-Dade Aviation Department spokesman Marc T. Henderson said.
On Wednesday, the nor'easter led to 18 canceled and 6 delayed flights at FLL. Another 33 flights were canceled at MIA Wednesday.
Nor'easter Slams Sandy-Battered States


text:
Hong Kong drama bags nominations at Golden Horse Awards
Posted: Nov 27, 2021 / 08:02 AM EST
Updated: Nov 27, 2021 / 08:17 AM EST
TAIPEI, Taiwan (AP) — Taiwan's annual Golden Horse Awards kicked off Saturday with a Hong Kong drama receiving the most nominations for the Asian equivalent of the Academy Awards.
The Golden Horse Awards, which began in 1962, is considered one of the most prestigious awards for Chinese-language films.
This year, the Hong Kong film "Drifting", which is based on a 2012 court case involving homeless people in the working neighborhood of Sham Shui Po, received 12 nominations including for Best Director, Best Leading Actor and Best Cinematography.
Two Taiwanese movies, titled "The Soul," "The Falls" and "Till We Meet Again" received 11 nominations each.
"The Falls" won Best Original Screenplay, while "Drifting" won Best Adapted Screenplay.
"Revolution of Our Times," a documentary about the Hong Kong political unrest in 2019 by Hong Kong director Kiwi Chow, won Best Documentary Feature.
The film was a controversial topic in Hong Kong and Chow was accused of flouting coronavirus rules after authorities raided a private screening of the documentary in Hong Kong. Under Hong Kong laws, private gatherings are not limited by coronavirus restrictions.
Chow has since sold the copyright of the documentary to a European distributor and disposed of all his footage, in light of risks from a sweeping national security law implemented in the city following the political unrest.
The Golden Horse Awards took place at the National Dr. Sun Yat-sen Memorial Hall in Taipei, with Singaporean actor Mark Lee and Chinese-American actress Bai Ling presenting awards.
This year, a total of 573 entries were submitted to the Golden Horse Awards, up from 465 last year.
Taiwanese actress Chen Shiang-chyi arrives at the 58th Golden Horse Awards in Taipei, Taiwan, Saturday, Nov. 27, 2021. Chen is nominated for Best Leading Actress…
Taiwanese actress Chen Shiang-chyi arrives at the 58th Golden Horse Awards in Taipei, Taiwan, Saturday, Nov. 27, 2021. Chen is nominated for Best Leading Actress for the film "Increasing Echo" at this year's Golden Horse Awards, one of the Chinese-language film industry's biggest annual events. (AP Photo/ Billy Dai)
Aspirus Medical Monday: Blood Donor Month
DNR seeking public comment on wolf management plan
Heikki Lunta Winter Festival back for 2022
Stephenson man sentenced for stealing lumber
Bay Mills President Whitney Gravelle shares the impact …
Sisu: Defining the U.P.
The best embroidery machine


text:
Joseph Gannascoli Booking
Joseph Gannascoli (Vito Spatafore) Booking Information
Joseph Gannascoli
Best known for the role of Vito Spatafore on the HBO Hit "The Sopranos". Joe also lost 160lbs on VH1's Celebrity Fit Club.
Specialities: Veteran Character Actor, Narration and voice-over, Personal Appearances and self taught Chef. Joe has his own book (A Meal to Die For) and line of pasta sauces (A Sauce to Die For).
Joseph Gannascoli Booking Form
Joseph Gannascoli Bio
Joseph Gannascoli IMDB
Joseph R. Gannascoli, "Vito" from "The Sopranos," joined Steve to discuss the life and death of cast mate, James Gandolfini, remembering him as a "humble, down to earth, regular guy."


Small in size, but mighty in power, the ECO HEPA Air Purifier has a four-stage filtration process to remove allergens, odors and other airborne contaminants. It features advanced true HEPA filtration to capture 99.97% of dust, allergens, smoke, pet dander and pollen as small as 0.3 microns. A carbon filter removes odors, while the pre-filter removes large particles, such as dust, hair and fur. Finally, the ECO HEPA Air Filter produces negative ions to freshen the air. Automatic fan adjusts speed or shuts off depending on air quality. Recommended for rooms up to 326 sq. ft.
Only 7 remaining. Expected to sell out in 1 day!
The ECO HEPA Air Purifier features four stages of filtration to remove airborne contaminants, and freshens the air with negative ions. It's small in size, but mighty in power.
• Recommended for rooms up to 326 sq. ft.


Today the olive tree has moved back out to the veranda steps, a sure sign that it's spring at last. There's tea in my mug, newly wound yarn in my yarn bowl, Bamse is sitting on my feet and I have new sunglasses. What more could I ask for?
There's no nicer way to spend a Sunday morning! We filled our pockets with all kinds of wonderful treasure.


Allo Mates! My name is Kydee Kristina and I just want to welcome you to my blog. I've been blogging since I was 19 and I LOVE IT! Writing has become who I am and I hope my quirky stories, tips and experiences resonate with you guys.
Are you ready for Life & Style according to Ky?


text:
The Shelby County School system has canceled all sporting events until April 3 due to the threat of COVID-19. (For the Reporter/Dawn Harrison)
Shelby County Schools cancels all sporting events until April 3
By ALEC ETHEREDGE | Managing Editor
With the spread of COVID-19 (Coronavirus) precautions have been taken across the country, specifically in the sports world, and on Friday, March 13, sports will also come to a halt for Shelby County Schools.
It was announced by the Shelby County School system that all sports activities were suspended for not just the day, but until at least April 3.
"At the end of the day, the decision we made was what we felt was the best for the community," said Shelby County Schools Supervisor of Athletics David Hogue. "We felt it was in our communities best interest after talking with the Alabama Department of Education."
Hogue said there were meetings earlier in the day with all District 5 superintendents and the decision was easy for Shelby County from there.
"After that, it was a decision that was made very quickly on our end because of the best interest of everyone involved," he said.
This came shortly after the Alabama Athletic Association announced that events with 500 or more people in attendance from outside the school's student body should be cancelled or limited to the recommended number.
Coming a week before the school system is expected to be on spring break, that means the cancelation of trips to several popular spring break tournaments as well.
It also means the earliest games would start back would put teams less than three weeks from the playoffs beginning for spring sports, making the entire season outlook bleak at this point depending on if other school systems follow suit.
Hogue said he hopes the season will continue after April 3, but wants to make sure everybody is safe.
"At the end of the day, when the president declares a National Emergency, we're talking about a pandemic," he said. "We take that into consideration as well as the hard work these student athletes have put in when making these decisions."
Hogue said the main reason they set the April 3 date was to be able to come back and reassess the matter then, in case there is a possibility of not losing the season completely.
"We have the opportunity to reassess things and put a pause on things," Hogue said. "This is a rapidly developing situation. As it moves forward, we'll reassess. I'd love to play again, but we'll look at that later."
Schools had been on high alert throughout the day with the AHSAA sending updates early in the morning, which included limiting the Alabama-Mississippi All-Star basketball game in Mississippi to no fans, while the Alabama High School Hall of Fame Ceremony scheduled for Monday, March 16, has been moved until this summer on June 22.
"We are saddened to have to reschedule this prestigious event," Denise Ainsworth of the AHSAA said. "We apologize for the inconvenience this might cause. However, the health and safety of our inductees, their families, and supporters must be paramount. Tickets purchased will be honored. We appreciate the cooperation of the Renaissance Hotel and everyone's understanding and flexibility."
The AHSAA also released a statement through social media earlier in the day that said they are closely monitoring statewide conditions regarding COVID-19 with the Alabama Department of Education, Alabama Department of Public Health and Center of Disease Control.
Right now, the AHSAA is uncertain of what this means for spring sports championships, but said a Spring Sports Contingency Committee has been formed to analyze current information to develop alternative options for championship play if necessary.
"Decisions will be made in the best interest of coaches, administrators, fans and most importantly, student-athletes.
Right now, Alabaster City, Pelham City and Briarwood Christian School have yet to suspend play. This story will be updated as more schools cancel sports or other activities.
Vincent's JROTC competes in Raider Ruck Race
By KATHY COPELAND / Community Columnist Vincent/Harpersville On Friday, Feb. 21, two teams and two individuals from the Vincent Middle... read more


text:
require 'spec_helper'

describe RainForest::S3 do
  let(:credentials){ double("AWS Crendetials") }
  let(:client){ double("AWS Client") }

  before do
    expect(::Aws::Credentials).to receive(:new).with(ENV["RAIN_FOREST_AWS_AKID"], ENV["RAIN_FOREST_AWS_SECRET"]).and_return(credentials).at_least(:once)
    expect(::Aws::S3::Client).to receive(:new).with(region: ENV["RAIN_FOREST_AWS_REGION"], credentials: credentials).and_return(client).at_least(:once)
  end

  describe "#write" do
    it "works with default public-read permissions" do
      expect(client).to receive(:put_object).with(bucket: ENV["RAIN_FOREST_AWS_BUCKET"], key: "STORAGE-KEY", body: "Some test data...", acl: "public-read").and_return(true)

      success, message = RainForest::S3.write("STORAGE-KEY", "Some test data...")

      expect(success).to eq(true)
      expect(message).to eq(nil)
    end

    it "accepts other options" do
      expect(client).to receive(:put_object).with(bucket: ENV["RAIN_FOREST_AWS_BUCKET"], key: "STORAGE-KEY", body: "Some test data...", acl: "public-read", content_encoding: 'utf8').and_return(true)

      success, message = RainForest::S3.write("STORAGE-KEY", "Some test data...", content_encoding: 'utf8')

      expect(success).to eq(true)
      expect(message).to eq(nil)
    end

    it "handles errors" do
      expect(client).to receive(:put_object).with(bucket: ENV["RAIN_FOREST_AWS_BUCKET"], key: "STORAGE-KEY", body: "Some test data...", acl: "public-read").and_raise(Exception.new("KABOOM!"))

      success, message = RainForest::S3.write("STORAGE-KEY", "Some test data...")

      expect(success).to eq(false)
      expect(message).to eq("KABOOM!")
    end
  end

  describe "#read" do
    let(:response_body){ double("Response Body", read: "Some test data...") }
    let(:response_object){ double("Response Object", body: response_body) }

    it "works" do
      expect(client).to receive(:get_object).with(bucket: ENV["RAIN_FOREST_AWS_BUCKET"], key: "STORAGE-KEY").and_return(response_object)

      success, data = RainForest::S3.read("STORAGE-KEY")

      expect(success).to eq(true)
      expect(data).to eq("Some test data...")
    end

    it "handles errors" do
      expect(client).to receive(:get_object).with(bucket: ENV["RAIN_FOREST_AWS_BUCKET"], key: "STORAGE-KEY").and_raise(Exception.new("KABOOM!"))

      success, data = RainForest::S3.read("STORAGE-KEY")

      expect(success).to eq(false)
      expect(data).to eq("KABOOM!")
    end
  end

  describe "#content_length" do
    let(:response_metadata){ double("Response Metadata", content_length: 12345) }

    it "works" do
      expect(client).to receive(:head_object).with(bucket: ENV["RAIN_FOREST_AWS_BUCKET"], key: "STORAGE-KEY").and_return(response_metadata)

      response = RainForest::S3.content_length("STORAGE-KEY")

      expect(response).to eq(12345)
    end

    it "returns -1 for errors" do
      expect(client).to receive(:head_object).with(bucket: ENV["RAIN_FOREST_AWS_BUCKET"], key: "STORAGE-KEY").and_raise(Exception.new("KABOOM!"))

      response = RainForest::S3.content_length("STORAGE-KEY")

      expect(response).to eq(-1)
    end
  end

  describe "#delete_objects" do
    let(:content_1){ OpenStruct.new(key: "abc") }
    let(:content_2){ OpenStruct.new(key: "123") }
    let(:contents){ [content_1, content_2] }
    let(:objects_response){ double("Objects", contents: contents) }
    let(:deleted_response){ double("Deleted Objects", deleted: contents) }

    it "works and returns the number of delete objects" do
      expect(client).to receive(:list_objects).with(bucket: ENV["RAIN_FOREST_AWS_BUCKET"], prefix: "SOME-PREFIX").and_return(objects_response)
      expect(client).to receive(:delete_objects).with(bucket: ENV["RAIN_FOREST_AWS_BUCKET"], delete: {objects: [{key: "abc"}, {key: "123"}]}).and_return(deleted_response)

      result = RainForest::S3.delete_objects("SOME-PREFIX")

      expect(result).to eq(2)
    end

    it "returns -1 for errors" do
      expect(client).to receive(:list_objects).with(bucket: ENV["RAIN_FOREST_AWS_BUCKET"], prefix: "SOME-PREFIX").and_raise(Exception.new("KABOOM!"))

      result = RainForest::S3.delete_objects("SOME-PREFIX")

      expect(result).to eq(-1)
    end
  end

  describe "#move" do
    it "works" do
      expect(client).to receive(:copy_object).with(bucket: ENV["RAIN_FOREST_AWS_BUCKET"], copy_source: "#{ENV["RAIN_FOREST_AWS_BUCKET"]}/SOURCE-KEY", key: "DEST-KEY")
      expect(client).to receive(:delete_object).with(bucket: ENV["RAIN_FOREST_AWS_BUCKET"], key: "SOURCE-KEY")

      success, message = RainForest::S3.move("SOURCE-KEY", "DEST-KEY")

      expect(success).to eq(true)
      expect(message).to eq(nil)
    end

    it "handles errors" do
      expect(client).to receive(:copy_object).and_raise(Exception.new("KABOOM!"))

      success, message = RainForest::S3.move("SOURCE-KEY", "DEST-KEY")

      expect(success).to eq(false)
      expect(message).to eq("KABOOM!")
    end
  end

  describe "#copy" do
    it "works" do
      expect(client).to receive(:copy_object).with(bucket: ENV["RAIN_FOREST_AWS_BUCKET"], copy_source: "#{ENV["RAIN_FOREST_AWS_BUCKET"]}/SOURCE-KEY", key: "DEST-KEY")

      success, message = RainForest::S3.copy("SOURCE-KEY", "DEST-KEY")

      expect(success).to eq(true)
      expect(message).to eq(nil)
    end

    it "handles errors" do
      expect(client).to receive(:copy_object).and_raise(Exception.new("KABOOM!"))

      success, message = RainForest::S3.copy("SOURCE-KEY", "DEST-KEY")

      expect(success).to eq(false)
      expect(message).to eq("KABOOM!")
    end
  end

  describe "#set_index_document" do
    it "works" do
      expect(client).to receive(:put_bucket_website).with(
        bucket: ENV["RAIN_FOREST_AWS_BUCKET"],
        website_configuration: {
          index_document: {
            suffix: "INDEX-DOCUMENT"
          }
        }
      )

      success, message = RainForest::S3.set_index_document("INDEX-DOCUMENT")

      expect(success).to eq(true)
      expect(message).to eq(nil)
    end

    it "handles errors" do
      expect(client).to receive(:put_bucket_website).and_raise(Exception.new("KABOOM!"))

      success, message = RainForest::S3.set_index_document("INDEX-DOCUMENT")

      expect(success).to eq(false)
      expect(message).to eq("KABOOM!")
    end
  end
end

redpajama_set_name: RedPajamaGithub


Mezza9 Located midst the IT hub of Hinjewadi in Pune . The first thing that hits you is the space that is available. Its very nice place for couple and also comfortable tent like enclosures for the large crowd, from a semi covered to the open air this place has quite a few options.
The menu is mainly Indian, but has a great choice, The best part is of course the food in terms of presentation and taste. Really liked it.
The Tandoori dishes were great. Must ... must ... try surmai tandoori.
Food: Food quality is very good and most of dishes we have were very tasty.
Service: Service is bit poor and sometimes you have to ping them so they can serve to little bit fast. If time is not constraint then you will be fine with this kind of slow service.


text:
DOTr forms transition team to handle MRT maintenance ops
By Aerol John Pateña November 6, 2017, 7:48 pm
MANILA -- The Department of Transportation (DOTr) has created a transition team that will handle the maintenance operations of the Metro Rail Transit Line 3 (MRT-3) after it terminated the maintenance contract with Busan Universal Rail Inc. (BURI).
The DOTr said the transition team consisting of the DOTr-MRT 3, Philippine National Railways (PNR) and the Light Rail Transit Authority (LRTA) will temporarily take over the maintenance works of MRT-3 while the procurement of a new maintenance provider is being processed.
"The PNR and LRTA have committed to provide adequate technical support and expertise to ensure the smooth transition of the MRT maintenance operations," the DOTr said in a statement Monday.
The technical personnel from BURI which are vital in maintenance works will likewise be absorbed by the MRT-3 to avoid service disruptions.
The DOTr, in an order signed by Secretary Arthur Tugade dated November 3, cited BURI's failure to address various issues such as poor performance, failure to ensure availability of required number of trains, failure to implement a feasible procurement plan for spare parts and noncompliance with the requirements of a complete and up-to-date Computerized Management System.
"The safety and welfare of the riding public is a priority of the DOTr," Reiner Paul Yebra, Undersecretary for Legal Affairs and Procurement, said in a press briefing.
Some of the companies that expressed interest to bid for the new MRT maintenance provider are Sumitomo Corporation, French public transport operator RATP Group and Singapore's SMRT Corporation, according to DOTr Undersecretary for Railways Cesar Chavez.
For its part, BURI wanted the DOTr to undergo arbitration proceedings to settle the issue on the MRT maintenance contract.
This comes as the firm asked the Quezon City Regional Trial Court (QC RTC) to resolve its petition for a protection order that will stop the termination process, compel the DOTr to submit to arbitration and settle their unpaid maintenance fees.
"The maintenance contract requires for an arbitration process," BURI legal counsel Maricris Pahate said.
BURI reiterated that the glitches currently being experienced by the MRT system are due to the design flaws and not maintenance issues.
"Why are the glitches of the MRT being blamed on us? We have sent letters to the DOTr stating that unless the rails are fixed, the MRT will always experience train derailments and other breakdowns," Pahate said.
"There is no basis for the termination. Why are they not addressing our concern that the rails should be replaced?" she added.
In its statement, BURI said that since it assumed maintenance operations in January 2016, it wrote the MRT-3 regarding the defective tracks that cause severe vibration affecting the rolling stock, signaling power and related system, and its track systems.
The letter stated the likely effect is detachment of component parts in the train underbody and interrelated systems.
BURI said it has consistently delivered the required number of train availability under the contract from averages of 18.75 to highs of 20.35 and 20.48 for November and December 2016 which are above the minimum of 18 trains required for peak hours.
The firm said its operational fleet availability for MRT-3 is 91.67 percent (66 out of 72 cars) which is higher than LRT 1's 74.82 percent (102 of 139 cars) and LRT 2's 66.67 percent (48 of 72 cars).
"The reputation of our company was damaged and we need to be vindicated," Pahate said. (PNA)
Marcos wants to fast-track upgrade of aviation safety system
DOTr to prevent more delays in PNR Clark Phase 2 project
Workers' group welcomes return of free rides program
DOTr to continue 'comprehensive' modernization of PCG
CAAP apologizes over fiasco; to procure CNS/ATM backup system
'Libreng Sakay' to continue with P1.3-B budget: DBM


text:
GLEE 2×02: Britney/Brittany
Like some sort of truth in advertising campaign, this week's GLEE focused on the homonyms Britney and Brittany – showcasing the music of one and the dancing of the other. The story focused on the power of fantasy, and several of the production numbers were like a dream come true.
While Will (Matthew Morrison) wanted to focus on easy-listening icon Christopher Cross, the kids – especially Kurt (Chris Colfer) – wanted to perform a Britney Spears number for the homecoming pep rally. But Will was completely against it, because he did not consider her a solid role model. And, strangely enough, Brittany (Heather Morris) was against drawing from the pop princess' catalog. That's because Brittany's full name is Brittany Susan Pearce, making her Brittany S. Pearce, or "Brittany Spearce."
HAWAII FIVE-O 1.2: 'Ohana
Turns out that Steve McGarrett's state police task force is staffed by some interesting characters – and this week's HAWAII FIVE-O gave those people some room to breathe.
In the case of the week, Roland Lowry (Scott Cohen), an NSA expert in cyberterrorism, was kidnapped because he invented a software "skeleton key" that can be used to enter any computer system in the world, even those employing advanced asymmetric encryption. McGarrett (Alex O'Loughlin) and posse track down the cyber bad guys through the liberal application of hard work, sideways thinking, gunplay, explosions, and a (literal) dollop of blood.
MAD MEN 4.10: Hands and Knees
This episode was about the past catching up with you; chickens coming home to roost; reaping what ye sow – and any other clichés you care to mention about consequences and responsibilities. In this case, the causes were Don't secret identity, Joan and Roger's recent tryst, Roger coasting at work, and Lane's marriage collapsing. They were facing the prospects of being suitably humbled; getting on their knees or holding out their hands to beg forgiveness. Actions have consequences, even for the great Don Draper. Perhaps especially for the great Don Draper Dick Whitman.
Joan (Christina Hendricks) told Roger (John Slattery) she's late, and Greg has been gone for seven weeks, so "it" has to be Roger's. And there's no way she can go to her regular OB-GYN. Luckily, Roger knew a doctor, who was (slightly) less judgmental but still accused him of "ruining her") and begrudgingly referred them to a clinic doctor who does abortions. Joan said she'll "take care of it." At the clinic, she encountered a mom whose 17-year-old daughter was having a procedure. The woman was devastated, and Joan (perhaps out of sympathy more than mere embarrassment) pretended to be there with a 15-year-old daughter. I suppose that was a sort of atonement, accepting social blame, but it seemed like Joan was dodging admitting she was the one who "got in trouble."
THE AMAZING RACE 17.1: They Don't Call It the Amazing Race for Nothin'
And THE AMAZING RACE has not won all those Emmys for nuthin', either! This is my favorite of the so-called "reality series," an unscripted race around the world that plays like an edge-of-your-seat horse race crossed with an extended slo-mo trainwreck.
Now that I have officially given up on SURVIVOR: NICARAGUA because the cast is just too boring, I will be concentrating my unscripted attention on this show and UNDERCOVER BOSS.
NIKITA 1.3: Kill Jill
NIKITA struck a blow for bloggers in the latest episode. Well, not really, but it was nice to see the-powers-that-be keeping up with the times for their reimagining. Newspapers and TV are so last decade.
Percy (Xander Berkeley) ordered muck-raking blogger Jill Morelli (Julie Gonzalo) silenced before her latest discovery — clandestine video of Division agents retrieving smuggled cocaine from an AirMerica crash site — can be published, ruining and its CEO Sampson (Christopher Cousins), a crony of Percy's. Agent Donnegan killed newspaper editor Jeremy and framed Jill, but Nikita (Maggie Q) was able to stop Jill from being kidnapped.
HELLCATS 1.3: Beale St. After Dark
It got a little harder to root for HELLCATS this week, considering there was little actual cheerleading, and the plotting was even more unfocused than last week's episode. I did appreciate the continued emphasis on characterization, though.
Just like those feisty kids on GLEE, the Lancer Hellcats were dismayed to discover they had to foot the bill for their own road trip, because their funding was reallocated to the ladies volleyball team. These tortured attempts to make the cheerleaders look like some kind of oppressed, underdog minority are not ringing true, and are really starting to annoy me. And is everyone on the squad a scholarship student (except bitchy Alice?) It's like we're supposed to feel sorry for the beautiful people, because being attractive, talented and popular is such a burden.
GLEE 2.1: Audition
GLEE faced the monumental challenge of following up last year's breakout season as the critical darling or, if you prefer, the teacher's pet of television. So what could GLEE do you do for an encore? Why, more of the same – only different, of course! And, for the most part, the sophomore season's debut succeeded by quickly establishing a new status quo that was actually the old status quo with a fresh coat of paint.
Fresh off losing at Regionals (McKinley High finished third out of…three schools.); the members of New Directions find themselves back for another fall semester at the bottom of the social ladder. (They are the "plankton" of the food chain, Kurt (Chris Colfer) notes, wryly. Will (Matthew Morrison) decided the answer lay in recruiting new members. Two promising newbies presented themselves: Sam Evans (newcomer Chord Overstreet) and Sunshine Corazon (pop star Charice). The problem was, Sunshine was a little too good. Rachel (Lea Michelle) feared that Sunshine might hog the spotlight from her, so Rachel set about preemptively eliminating Sunshine before she got a chance to shine.
HAWAII FIVE-O 1.1: Pilot
The reboot of HAWAII FIVE-O was a triumph of form over substance. Which is not necessarily a bad think; I don't mind a TV show set in Hawaii that looks really great. And who needs another procedural that gets bogged down in the intricacies of police procedure?
By now, I have seen so many procedurals I feel like I could not only investigate a murder, but follow it to court and prosecute it to conviction. So I really wasn't looking forward to seeing an iconic 1970s series reworked for the meticulous modern age. And, luckily, that's not what we got. In fact, HAWAII FIVE-O seemed remarkably uninterested in the nitty-gritty details of Steve McGarrett's (Alex O'Loughlin) investigation into a terrorist family human-smuggling ring guy who killed his father. Or something like that.
LONE STAR 1.1: Pilot
The premise of LONE STAR seems interesting yet limiting at the same time. Interesting because of how it amplifies the idea of a con man running a long con and escalates it all the way to faking his entire life. Twice over. And limiting because…where does one go with this high-concept in the future?
When I first heard about this concept, it sounded to me like an extended version of that horrible sitcom cliché, the dummy who accidentally makes two dates for Saturday night and decides to take both women to the same restaurant and try to juggle them. Hilarity is sure to ensue.
Well… not so much.
CHASE 1.1: Pilot
If you're going to call your TV show CHASE, it's probably a requirement that it contain an actual chase sequence. NBC's new CHASE gets that out of the way early by hitting the ground literally running, continuing the theme throughout the hour, and wrapping up with a chase by land, air and sea (well, river, at least).
The episode hits the ground literally running, with U.S. Marshal Annie Frost (Kelli Giddish) chasing a fugitive on foot through Fort Worth, Texas. She is joined in this pursuit by her partner, Jimmy Godfrey (Cole Hauser). Eventually Annie runs down the fugitive and after a vigorous bout of hand-to-hand combat, she subdues him.


text:
Juventus – Inter
Derby d'Italia – Juventus against Inter. It's time once again to enjoy one of the most iconic matches in Italian football between two clubs who don't love each other very much. A true football rivalry.
It won't be much of a surprise to note that Juventus are once again leading the table in the Serie A. Max Allegri's team is four points ahead of Roma after 22 rounds played, but Juve also have a game in hand. Teams like Roma and Napoli still have a theoretical chance to beat The Old Lady for the title, but there are very few people who actually believe that it could happen.
Inter on the other hand are enjoying a terrific run of results under the management of Stefano Pioli. The 51-years old tactician was appointed as manager in November and he quickly turned the Nerrazzuri into a real contender for a Champions League football next season. Inter are currently in a run of a seven consecutive wins in the Serie A, which allowed them to occupy the 4th spot in the table and move just three points behind Napoli. The Champions League dream is once again alive for the San Siro giants.
But are Inter good enough to take something from Juventus in Turin? After winning seven games in a row, most certainly they will travel to Juventus Stadium with high confidence and morale. But on the other side of the pitch we have arguably the best team in Italy at the moment.
Max Allegri can use all of his first team players for the Derby d'Italia, so we should expect to see the best possible Juve squad on the pitch. Last week The Old Lady didn't face any problems against Sassuolo away from home, winning comfortably thanks to the goals of Gonzalo Higuain and Sami Khedira.
There aren't any major injuries or suspensions in Inter's squad as well. In their last game the Nerrazzuri scored three times against Pescara, winning with 3-0 at the Stadio San Siro in Milan.


Sally is derived from a frozen, imported embryo and is full sister to Hope. She kidded triplets (Stanley, Simon, Sophie) this year and she could easily feed a fourth one due to her large, soft udder. She is friendly, patient and a very good mother.


Teaching a friend to use ethereal software (aka Egregores, a matrix, The Universe) without relying on rituals.
Two beginner-friendly techniques for better communication with magickal forces and spirits.
This week, I made two improvements to my magick communication.
What I learned about communicating with spirits and moving energy in the last 7 days.


In heavy clay and bricks industry, reduction of fuel consumption is the absolute priority of refractory linings. Special materials are used which aim at the reduction of firing circles with exceptional results in saving energy. Depending on the operating conditions, individualized solutions are proposed with advantages like low weight and big thermal output in combination with high tolerances in all temperature range and thermal shock resistance. Prefabricated solutions are also available for kilns of each type and kiln cars of all sizes.


The main ingredient in coffee, tea and some sodas is caffeine. This is the substance that is responsible for the energizing effect that such beverages give to us when we take them. This effect, however, is not true to all people. There are those who complain about the tremor attacks and nervousness that they feel when they drink anything with caffeine in it.
If you are among the people who get the positive kick of coffee, then better take coffee before you go to the gym. It will surely be of great help in energizing you and motivating you to stay longer in your workout sessions. Be wary though because caffeine has this diuretic effect which means that you might be urinating all the time. To compensate, drink lots of fluids.
Before you sip coffee from your cup, make sure that you know its side effects. Other than diuresis, coffee can cause an increase peristalsis which means that you might suffer bowel problems if you are not used to drinking it. Make sure that you have not eaten too much prior to going to the gym or else you will be increasing your risk for an embarrassment.
While coffee increases alertness, there are those who experience the opposite of this – they rather become irritable and have lack of concentration. Those who love drinking coffee and have nervous conditions should be extra careful with this because they are usually the kind of people who overdose their coffee intake.
Coffee increases your heart rate thereby increasing the amount of blood flow to the different tissues and organs of the body. Because of this, oxygen and nutrients are better delivered to the body cells making you more alert and awake. This is the reason why you experience a different high with coffee. It is also the same reason why people with heart conditions should contact their physicians first before coffee intake.
There is also a belief that coffee decreases the intake of energy by the muscles and increases the flow of adrenaline which makes it a good stimulant. Whenever you feel stressed, fatigued and tired, better drink a cup of Joe.
Whenever we exercise, our body muscles make use of glycogen for energy. A by-product of this chemical process is lactic acid. Lactic acid is the one that makes us feel pain while we workout and this is the main culprit why we tend to stop exercising because we already are feeling uncomfortable. What coffee does is make the muscles of the body utilize fat rather than glycogen for energy. As a result, lesser lactic acid is produced and thereby lesser pain. Lesser pain means longer workout periods and furthermore a greater amount of fats burned. This is the reason why you can lose weight more when you drink coffee.
Apart from the above effect, coffee also increases the release of endorphins which is a hormone in the body known to increase pleasure. Not only do we feel more energized while we exercise but we also feel good about it.
However, coffee is not all good. There is only a specific amount by which coffee can bring about positive effects. If you drink more than the advised amount, you feel the opposite – the adverse effect. So the next time you drink coffee, make sure that you have been cautious enough with the amount you are taking in. For better effect, the advised amount is just two to three cups (8 oz.) of coffee an hour before a workout and this is also the recommended amount of daily intake of coffee.


hd00:27Closeup shot of blackberry growing on the bush. Unknown person is picking berries off.
4k00:18Red flowers on a summer day on a background of green grass and trees, a panorama of beautiful flowers on a windy day, flowers swaying beautifully in the wind.


If you're looking for car parts to replace damaged or worn parts on your car then you've come to the right place. Euro Car Parts in Hastings supply a range of new car parts for all makes and models at low prices, and our team of experts are available to provide assistance and advice. We've got an extensive range of standard and performance car parts available, ranging from car lighting parts to lubricants and fluids, even workshop tools and accessories. Visit us in store today to browse the full range.


text:
St. Philip's may refer to:

 St. Philip's, Newfoundland and Labrador, Canada
 Rural Municipality of St. Philips No. 301, Saskatchewan, Canada
 St. Philip's Church, Brunswick Town, former church in North Carolina; listed on the National Register of Historic Places
 St. Philip's Castle, a fortress
 St. Philip's Cathedral, Birmingham, a Church of England cathedral
 St Philip's Church, Sydney
 St. Philip's School, a grammar school
 Bristol St Philip's railway station
 St Philip's Footbridge, a bridge in Bristol

See also
 Saint Philip (disambiguation)
 St. Philip's College (disambiguation)
 St. Phillip's, Kingston, Tortola, British Virgin Islands

redpajama_set_name: RedPajamaWikipedia


text:
PS Now Streaming service gets 25 new games
Game of the Year 2016: Best Story
Warhammer: End Times - Vermintide Review: Fight against the rats
Tagged Deus Ex: Mankind Divided
Deus Ex: Mankind Divided next story DLC announced
One of the most unique video game franchises to have a new beginning in the past few years has been Deus Ex. Set in a dystopian future, players take control of Adam Jensen, an ex-SWAT member who's body was augm... Read More...
Deus Ex soundtracks become available December 2nd
There are a lot of pieces that go into making a great game. Some may be more obvious than others, but each piece does its part to make sure the player is completely immersed into the virtual world they are step... Read More...
Canadian Video Game Awards: Winners announced!
While The Game Awards aren't until December 1st, the Canadian Video Game Awards winners have been announced-- with big winner Deus Ex: Mankind Divided. On November 15th, in Montreal's Palais de Congres, more than 50 games developed in Canada were up for 20 di... Read More...
Deus Ex: Mankind Divided gets PS4 Pro update
At this year's E3, Microsoft showed off their enhanced console, the Xbox Scorpio. It had been talked about for some time and Sony eventually answered with the official announcement of their upgraded PS4 Pro. Wi... Read More...
Details for Deus Ex: Mankind Divided's first DLC pack revealed
Details for the first piece of story DLC for Deus Ex: Mankind Divided, entitled System Rift, have been released, as well as a release date for the DLC pack. "In Deus Ex: Mankind Divided -System Rift, you wil... Read More...
Deus Ex: Mankind Divided Review
Even today, the original Deus Ex is spoken of with reverence and enduring love from those that played the game when it came out in 2000. The 2011 reboot/prequel Human Revolution had the monumental task of bringing the franchise into the modern era of gaming. I... Read More...
Deus Ex: Mankind Divided's microtransactions angering players
It seems that a number of players are upset over Deus Ex: Mankind Divided's use of microtransactions. Despite Mankind Divided's positive reception, the pre-order DLC has sparked an angry reaction on the titl... Read More...
Deus Ex: Mankind Divided gets its first PC patch
Deus Ex: Mankind Divided has received its first patch on PC. The patch, v1.0 build 524.7, focuses on critical issues that PC players encountered while playing the latest Deus Ex title. The patch introduce... Read More...
Deus Ex: Mankind Divided launch trailer delivers what fans want
Deus Ex: Mankind Divided's launch trailer has just been released, and it looks to give the game's themes the weight they deserve. Deus Ex is a series known for its options. There's plenty of side content and characters, and nearly any situation can be approac... Read More...
Deus Ex: Mankind Divided Dubai gameplay shown at E3
Deus Ex: Mankind Divided's first map was shown off at E3, featuring the beautiful city of Dubai. The first map bridges the gap between Human Revolution and Mankind Divided, providing a setup for the newest game... Read More...


text:
Local DPP Chair saddened at Glengormley blaze
Chair of Newtownabbey District Policing Partnership, Cllr Tom Campbell, has expressed sadness at news of a fire in a taxi office on Carnmoney Road in Glengormley. The fire was reported to authorities at around 3.30am this morning, and police have yet to reveal the circumstances surrounding the blaze.
Newtownabbey Alliance Councillor Tom Campbell said: "It is extremely sad that there has been a fire in this local business. With fires of this nature, the public need to know the circumstances behind them. I await the police releasing further detail, when they have concluded they work at the site.
"I want to pass on my best wishes to the owners and staff of this taxi company who may be facing disruption due to the fire. I hope that this fire does not impact negatively on the business."


text:
Justia Patents 15 To 23 Amino Acid Residues In Defined SequenceUS Patent for Cell-penetrating peptides Patent (Patent # 9,644,001)
Cell-penetrating peptides
May 26, 2016 - Universite Pierre Et Marie Curie (Paris 6)
A vector that includes a cell-penetrating peptide linked to a therapeutic agent. The therapeutic agent can be a cytotoxic agent, an anti-viral agent, an anti-bacterial agent, or an anti-parasitic agent.
Latest Universite Pierre Et Marie Curie (Paris 6) Patents:
HYCANTHONE DERIVATIVES AND PRIMAQUINE DERIVATIVES FOR USE IN THE PREVENTION AND/OR THE TREATMENT OF DISORDERS ASSOCIATED TO GAMMAHERPESVIRUS
PROCESS FOR MANUFACTURING AN IMPLANT FOR FOCAL ELECTRICAL STIMULATION OF A NERVOUS STRUCTURE
High MAST2-affinity polypeptides and uses thereof
General technical field and state of the art
Method for producing polyphenol compounds
This application is a continuation of U.S. application Ser. No. 14/366,199, filed on Jun. 17, 2014, which is the National Stage of International Application No. PCT/EP2012/076968, filed on Dec. 27, 2012, which claims the benefit of European Application No. 11306784.7, filed on Dec. 27, 2011. The content of each prior application is hereby incorporated by reference in its entirety.
The invention relates to shuttle peptides that penetrate the cell membrane.
Apoptosis is a genetically programmed cell death and its deregulation is associated among other pathologies, with cancer. While apoptosis is known to rely on the Bcl-2 family members and caspases, recent data suggested that two major families of serine/threonine phosphatases, PP1 and PP2A, are key actors involved in cell life or cell death decision. The Ser/Thre phosphatase PP2A has been implicated in both, induction and prevention of apoptosis, pointing to a complex interplay of phosphatase actions. Several phosphatases have recently become attractive targets for the treatment of a variety of diseases, including cancers. However, the only clinical drugs targeting a phosphatase are the immunosuppressive cyclosporine A and FK506.
Cell penetrating peptides (CPP) are molecules which can translocate into cells without causing membrane damage, leading to their proposed use as vectors for delivering therapeutic cargo. Several CPP have been identified such as Tat, antennapedia, or SHV1 VP22. These peptides can cross the cell membrane and reach the cytoplasm and/or the nucleus. Penetrating peptides interacting with PP1/PP2A proteins were designed. This approach, named "Drug Phosphatase Technology" (DPT), was described in Guergnon et al, 2006 and International patent applications WO2003/011898 and WO2004/011595. A pro-apoptotic peptide, called DPT-C9h, that specifically deregulates the interaction between caspase-9 and PP2A, used this penetrating sequence (international patent application WO2010/112471).
However this peptide shows a short half-life, which is a real draw-back for clinical uses.
The present mutated peptides overcome this problem since they are not digested by human serum proteases. This new property makes it possible to reduce the dose of peptide injected as well as the schedule of administration.
The invention provides a peptide comprising the following amino acid sequence (I):
X1-KKKIK-Ψ-EI-X2-X3(I) (SEQ ID NO:1)
wherein X1 is vacant, is a lysine residue, or valine-lysine;
X2 is vacant, is a lysine residue, or lysine-isoleucine;
X3 is vacant or is an amino acid sequence of 1 to 4 amino acids;
and Ψ is an amino acid residue that is different from arginine,
or a proteolysis-resistant peptide deriving from sequence (I) by one or more chemical modifications, or a substantially homologous peptide deriving from sequence (I) by one or more conservative substitutions.
The invention further provides a vector comprising said peptide, as a cell penetrating peptide, coupled to a molecule of interest.
The invention further provides a chimeric peptide construct, comprising said peptide, as a cell penetrating peptide, fused to a pro-apoptotic peptide, wherein the penetrating peptide is preferably fused at the N-terminus of the pro-apoptotic peptide.
Another aspect of the invention is a nucleic acid comprising a sequence coding for the cell penetrating peptide or for the chimeric peptide construct.
Still another aspect of the invention is a vector comprising a nucleic acid comprising (i) a nucleotide sequence coding for the cell penetrating peptide coupled to (ii) a nucleotide sequence of interest, for use in gene therapy or gene transfer in vivo or ex vivo.
Using the chimeric peptide construct, or of a nucleic acid encoding said chimeric peptide construct, for inhibition of cell proliferation in vitro, is further encompassed.
A further subject of the invention is a pharmaceutical composition comprising said vector or a chimeric peptide as herein described, in association with a pharmaceutically acceptable carrier.
The invention further relates to the use of the chimeric peptides or the pharmaceutical composition according to the invention for treating hyperproliferative diseases or parasitic diseases.
The inventors have worked to improve stability of the peptides disclosed in WO2010/112471, in particular peptide DPT-C9h that is subjected to degradation by proteases. This peptide corresponds to a penetrating peptide associated to the sequence of the binding site of caspase-9 to PP2A. This peptide induces apoptosis in human cell lines. In addition, it has a specific apoptotic effect only in tumoral B cells isolated from chronic lymphocityc leukemia patients without effect on healthy cells. In addition, the peptide induces important reduction in the size of tumor when injected in mice bearing human breast cancer xenograft.
DPT-C9 consists of sequence VKKKKIKREIKI-YVETLDDIFEQWAHSEDL (SEQ ID NO:6), where VKKKKIKREIKI (SEQ ID NO:7) is the penetrating peptide.
The inventors have shown that a mutation in the penetrating peptide dramatically increases the stability of the whole peptide, while maintaining its properties, in particular its ability to induce apoptosis.
According to the invention, a mutation of the arginine residue in the penetrating peptide prevents cleavage from proteases.
The inventors have thus designed peptides comprising the following amino acid sequence (1):
X1-KKKIK-Ψ-EI-X2-X3 (I) (SEQ ID NO:1)
wherein X1 is vacant, is a lysine residue, or valine-lysine:
X2 is vacant, is a lysine residue, or lysine-isoleucine:
and Ψ is an amino acid residue that is different from arginine.
In a preferred embodiment, Ψ is A, K or N. Still preferably Ψ is non-conservative with respect to arginine. In a preferred embodiment, Ψ is thus an amino acid residue different from lysine, asparagine, or glutamine. Preferably Ψ is alanine.
In a preferred embodiment.
X1 is valine-lysine;
X2 is lysine-isoleucine:
and X3 is vacant.
The preferred peptide is VKKKKIKAEIKI (SEQ ID NO:2).
Another peptide is VKKKKIKKEIKI (SEQ ID NO:10).
Still another peptide is VKKKKIKNEIKI (SEQ ID NO: 11).
The term "patient" refers to a human or non human animal, preferably a mammal, including male, female, adult and children in need of a treatment wherein a pro-apoptotic effect is desired.
As used herein, the term "treatment" or "therapy" includes curative and/or prophylactic treatment. More particularly, curative treatment refers to any of the alleviation, amelioration and/or elimination, reduction and/or stabilization (e.g., failure to progress to more advanced stages) of a symptom, as well as delay in progression of a symptom of a particular disorder.
Prophylactic treatment refers to any of: halting the onset, reducing the risk of development, reducing the incidence, delaying the onset, reducing the development, as well as increasing the time to onset of symptoms of a particular disorder.
The term "penetrating peptide" or "cell-penetrating peptide" (or "CPP") or "shuttle peptide", as used interchangeably, means that the peptide is able to translocate into cells without causing substantial membrane damage, and can be used as a vector of other molecules when linked to them. The terms refer to cationic cell penetrating peptides, also called transport peptides, carrier peptides, or peptide transduction domains. The CPP, as shown herein, have the capability of inducing cell penetration of a peptide fused to the CPP within 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of cells of a given cell culture population, including all integers in between, and allow macromolecular translocation within multiple tissues in vivo upon systemic administration. A cell-penetrating peptide may also refers to a peptide which, when brought into contact with a cell under appropriate conditions, passes from the external environment in the intracellular environment, including the cytoplasm, organelles such as mitochondria, or the nucleus of the cell, in conditions significantly greater than passive diffusion. This property may be assessed by various methods known by the skilled person.
Two amino acid sequences are "homologous", "substantially homologous" or "substantially similar" when one or more amino acid residues are replaced by a biologically similar residue or when greater than 80% of the amino acids are identical, or greater than about 90%, preferably greater than about 95%, are similar (functionally identical). Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program, or any of the programs known in the art (BLAST, FASTA, etc.). Preferably, these homologous peptides do not include two cysteine residues, so that cyclization is prevented.
The term "conservative substitution" as used herein denotes the replacement of an amino acid residue by another, without altering the overall conformation and function of the peptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, shape, hydrophobic, aromatic, and the like). Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Neutral hydrophilic amino acids, which can be substituted for one another, include asparagine, glutamine, serine and threonine.
By "substituted" or "modified" the present invention includes those amino acids that have been altered or modified from naturally occurring amino acids.
As such, it should be understood that in the context of the present invention, a conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Examples of conservative substitutions are set out in the Table 1 below:
Conservative Substitutions I
SIDE CHAIN CHARACTERISTIC AMINO ACID Non-polar G A P I L V Polar-uncharged C S T M N Q Polar-charged D E K R Aromatic H F W Y Other N Q D E
Alternatively, conservative amino acids can be grouped as described in Lehninger, 1975, as set out in Table 2, immediately below.
Conservative Substitutions II
SIDE CHAIN CHARACTERISTIC AMINO ACID Non-polar (hydrophobic) A. Aliphatic: A L I V P B. Aromatic: F W C. Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl: S T Y B. Amides: N Q C. Sulfhydryl: C D. Borderline: G Positively Charged (Basic): K R H Negatively Charged (Acidic): D E
As still another alternative, exemplary conservative substitutions are set out in Table 3, immediately below.
Conservative Substitutions III
Original Residue Exemplary Substitution Ala (A) Val (V), Leu (L), Ile (I) Arg (R) Lys (K), Gln (Q), Asn (N) Asn (N) Gln (Q), His (H), Lys (K), Arg (R) Asp (D) Glu (E) Cys (C) Ser (S) Gln (Q) Asn (N) Glu (E) Asp (D) His (H) Asn (N), Gln (Q), Lys (K), Arg (R) Ile (I) Leu (L), Val (V), Met (M), Ala (A), Phe (F) Leu (L) Ile (I), Val (V), Met (M), Ala (A), Phe (F) Lys (K) Arg (R), Gln (Q), Asn (N) Met (M) Leu (L), Phe (F), Ile (I) Phe (F) Leu (L), Val (V), Ile (I), Ala (A) Pro (P) Gly (G) Ser (S) Thr (T) Thr (T) Ser (S) Trp (W) Tyr (T) Tyr (Y) Trp (W), Phe (F), Thr (T), Ser (S) Val (V) Ile (I), Leu (L), Met (M), Phe (F), Ala (A)
Peptide Preparation:
Peptides described herein can be synthesized using standard synthetic methods known to those skilled in the art, for example chemical synthesis or genetic recombination. In a preferred embodiment, peptides are obtained by stepwise condensation of amino acid residues, either by condensation of a preformed fragment already containing an amino acid sequence in appropriate order, or by condensation of several fragments previously prepared, while protecting the amino acid functional groups except those involved in peptide bond during condensation. In particular, the peptides can be synthesized according to the method originally described by Merrifield.
Examples of chemical synthesis technologies are solid phase synthesis and liquid phase synthesis. As a solid phase synthesis, for example, the amino acid corresponding to the C-terminus of the peptide to be synthesized is bound to a support which is insoluble in organic solvents, and by alternate repetition of reactions, one wherein amino acids with their amino groups and side chain functional groups protected with appropriate protective groups are condensed one by one in order from the C-terminus to the N-terminus, and one where the amino acids bound to the resin or the protective group of the amino groups of the peptides are released, the peptide chain is thus extended in this manner. Solid phase synthesis methods are largely classified by the tBoc method and the Fmoc method, depending on the type of protective group used. Typically used protective groups include tBoc (t-butoxycarbonyl), Cl—Z (2-chlorobenzyloxycarbonyl), Br—Z (2-bromobenzyloyycarbonyl), Bzl (benzyl), Fmoc (9-fluorenylmcthoxycarbonyl), Mbh (4, 4′-dimethoxydibenzhydryl), Mtr (4-methoxy-2,3,6-trimethylbenzenesulphonyl), Trt (trityl), Tos (tosyl), Z (benzyloxycarbonyl) and Clz-Bzl (2,6-dichlorobenzyl) for the amino groups; NO2 (nitro) and Pmc (2,2,5,7,8-pentamethylchromane-6-sulphonyl) for the guanidino groups); and tBu (t-butyl) for the hydroxyl groups). After synthesis of the desired peptide, it is subjected to the de-protection reaction and cut out from the solid support. Such peptide cutting reaction may be carried with hydrogen fluoride or tri-fluoromethane sulfonic acid for the Boc method, and with TFA for the Fmoc method.
Alternatively, the peptide may be synthesized using recombinant techniques. In this case, a nucleic acid and/or a genetic construct, comprising or consisting of a nucleotidic sequence encoding a peptide according to the invention, polynucleotides with nucleotidic sequences complementary to one of the above sequences and sequences hybridizing to said polynucleotides under stringent conditions.
The invention further relates to a genetic construct consisting of or comprising a polynucleotide as defined herein, and regulatory sequences (such as a suitable promoter(s), enhancer(s), terminator(s), etc.) allowing the expression (e.g. transcription and translation) of a peptide according to the invention in a host cell.
Thus, in another aspect, the invention relates to a host or host cell that expresses (or that under suitable circumstances is capable of expressing) a peptide of the invention; and/or that contains a polynucleotide of the invention or genetic construct of the invention.
The method of producing the peptide may optionally comprise the steps of purifying said peptide, chemically modifying said peptide, and/or formulating said peptide into a pharmaceutical composition.
Chimeric Constructs:
The peptide X1-KKKIK-Ψ-EI-X2-X3(1) (SEQ ID NO:1) is useful in the invention as cell penetrating peptide (CPP).
The invention thus provides vectors, comprising said peptide, as a cell penetrating peptide, coupled to a molecule of interest.
The molecule of interest may be coupled to one or several such CPP.
The molecule of interest may be any therapeutic agent, including a cytotoxic agent (preferably a pro-apoptotic peptide), an anti-viral agent, or anti-bacterial, or anti-parasitic agent.
In a preferred embodiment, chimeric peptide constructs, comprising said peptide, as a penetrating peptide, fused to a pro-apoptotic peptide, can be prepared.
Preferably the pro-apoptotic peptide is fused at the C-term of the penetrating peptide.
The pro-apoptotic peptide may be of any pro-apoptotic peptide of interest.
The chimeric peptide construct may preferably have a length comprised between 23 to 70 amino acids, preferably between 23 to 40 amino acids.
In a preferred embodiment, the pro-apoptotic peptide is a fragment of caspase-9 protein.
According to one embodiment, chimeric peptide constructs useful in the invention comprise, or consist in the following amino acid sequence:
Y-X4a-ETLD-X4b-I-X5-EQWA-X6-S-X7 (SEQ ID NO:3)
X4a is valine or isoleucine;
X4b is aspartic acid or glycine;
X5 is phenylalanine or leucine:
X6 is arginine or histidine;
X7 is vacant or is glutamate, or glutamate-aspartate, or glutamate-aspartate-leucine; or
a proteolysis-resistant peptide deriving from said pro-apoptotic peptide by one or more chemical modifications, or a substantially homologous peptide deriving from SEQ ID NO:3 by one or more conservative substitutions.
Such proteolysis-resistant or homologous peptides induce cell apoptosis, in vitro and/or in vivo. Assays for determining if a molecule, for instance a peptide, induces cell apoptosis are well-known in the art and include, for instance, incubating cells with the candidate peptide and determining if apoptosis is induced by said candidate peptide, e.g. by Annexin V and PI labelling of cells and identifying as apoptotic cells, those being Annexin V+ and PI−.
X4a is valine;
X4b is aspartic acid;
X5 is phenylalanine;
and X6 is histidine.
In a particular embodiment, the chimeric peptide construct is
(SEQ ID NO: 4)
VKKKKIKAEIKI-YVETLDDIFEQWAHSEDL
also herein designated Mut3-DPT-C9h.
In another particular embodiment, the chimeric peptide construct is
VKKKKIKAEIKI-YIETLDDILEQWARSEDL
(SEQ ID NO: 12)
VKKKKIKKEIKI-YVETLDDIFEQWAHSEDL
VKKKKIKKEIKI-YIETLDDILEQWARSEDL
VKKKKIKNEIKI-YVETLDDIFEQWAHSEDL
In still another particular embodiment, the chimeric peptide construct is
VKKKKIKNEIKI-YIETLDDILEQWARSEDL
In still another embodiment, the pro-apoptotic peptide is a PP2Ah peptide that comprises or consists of:
a) the amino acid sequence DTLDHIRALDRLQEVPHEGP (SEQ ID NO:8);
b) an amino acid sequence substantially homologous to SEQ ID NO:8, preferably at least 80% identical to SEQ ID NO:8, which induces cell apoptosis; or
c) a proteolysis-resistant peptide which induces cell apoptosis and which derives from the peptide defined in a) or b) by one or more chemical modifications.
In a preferred embodiment, the pro-apoptotic peptide comprises or consists of the sequence DTLDHIRALDRLQEVPHEGP (SEQ ID NO: 9).
VKKKKIKAEIKI-DTLDHIRALDRLQEVPHEGP
VKKKKIKKEIKI-DTLDHIRALDRLQEVPHEGP
VKKKKIKNEIKI-DTLDHIRALDRLQEVPHEGP
Further Protection Against Proteolysis:
The N- and C-termini of the peptides described herein may be optionally protected against proteolysis. For instance, the N-terminus may be in the form of an acetyl group, and/or the C-terminus may be in the form of an amide group. Internal modifications of the peptides to be resistant to proteolysis are also envisioned, e.g. wherein at least a —CONH— peptide bond is modified and replaced by a (CH2NH) reduced bond, a (NHCO) retro-inverso bond, a (CH2-O) methylene-oxy bond, a (CH2-S) thiomethylene bond, a (CH2CH2) carba bond, a (CO—CH2) cetomethylene bond, a (CHOH—CH2) hydroxyethylene bond), a (N—N) bound, a E-alcene bond or also a —CH═CH-bond.
For instance the peptide may be modified by acetylation, acylation, amidation, cross-linking, cyclization, disulfide bond formation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, phosphorylation, and the like.
The peptides of the invention may be composed of amino acid(s) in D configuration, which render the peptides resistant to proteolysis. They may also be stabilized by intramolecular crosslinking, e.g. by modifying at least two amino acid residues with olefinic side chains, preferably C3-C8 alkenyl chains, preferably penten-2-yl chains, followed by chemical crosslinking of the chains, according to the so-called "staple" technology described in Walensky et al, 2004. For instance, amino acids at position i and i+4 to i+7 can be substituted by non-natural aminoacids that show reactive olefinic residues. All these proteolysis-resistant chemically-modified peptides are encompassed in the present invention.
In another aspect of the invention, peptides are covalently bound to a polyethylene glycol (PEG) molecule by their C-terminal terminus or a lysine residue, notably a PEG of 1500 or 4000 MW, for a decrease in urinary clearance and in therapeutic doses used and for an increase of the half-life in blood plasma. In yet another embodiment, peptide half-life is increased by including the peptide in a biodegradable and biocompatible polymer material for drug delivery system forming microspheres. Polymers and copolymers are, for instance, poly(D,L-lactide-co-glycolide) (PLGA) (as illustrated in US2007/0184015, SoonKap Hahn et al).
Nucleic Acids
The invention also relates to a polynucleotide comprising or consisting of a nucleotide sequence encoding a peptide according to the invention.
The genetic constructs of the invention may be DNA or RNA, and are preferably double-stranded DNA. The genetic constructs of the invention may also be in a form suitable for transformation of the intended host cell or host organism, in a form suitable for integration into the genomic DNA of the intended host cell or in a form suitable for independent replication, maintenance and/or inheritance in the intended host organism. For instance, the genetic constructs of the invention may be in the form of a vector, such as for example a plasmid, cosmid, YAC, a viral vector or transposon. In particular, the vector may be an expression vector, i.e. a vector that can provide for expression in vitro and/or in vivo (e.g. in a suitable host cell, host organism and/or expression system).
In a preferred but non-limiting aspect, a genetic construct of the invention comprises i) at least one nucleic acid of the invention; operably connected to ii) one or more regulatory elements, such as a promoter and optionally a suitable terminator; and optionally also iii) one or more further elements of genetic constructs such as 3′- or 5′-UTR sequences, leader sequences, selection markers, expression markers/reporter genes, and/or elements that may facilitate or increase (the efficiency of) transformation or integration.
In a particular embodiment, the nucleic acid encoding the cell-penetrating peptide of the invention is coupled or fused to a nucleic acid that encodes a peptide or protein of interest. The peptide of interest may be a pro-apoptotic peptide as described herein. More generally it may the peptide or protein of interest may be any peptide or protein to express, such as therapeutic peptide or polypeptide, as well as any antigenic or immunogenic peptide if desired.
The nucleic acid may especially be carried by a viral vector, such as an adenovirus or a lentivirus, for ex vivo or in vivo infection and expression of the peptide or protein of interest coupled to the cell-penetrating peptide.
Pro-Apoptotic Activity:
The chimeric peptides as defined herein, or nucleic acids that encode said peptides, are useful for inhibition of cell proliferation in vitro or in vivo.
They are useful therapeutic agents, in particular for treating hyperproliferative diseases.
It is thus described a method of treatment of a hyperproliferative disease in a patient in need thereof, which method comprises administering said patient with the chimeric peptide construct, or a nucleic acid encoding said construct.
The peptides (or nucleic acids that encode said peptides) are useful for the treatment of a tumor, in particular a cancer tumor, preferably in a human patient.
The hyperproliferative disorder may be cancer, such as a haematologic cancer, in particular acute myelogenous leukaemia (AML), chronic lymphocytic leukaemia (CLL), multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma, B cell, cutaneous T cell lymphoma, or a non-haematologic cancer, for instance brain, epidermoid (in particular lung, breast, ovarian), head and neck (squamous cell), bladder, gastric, pancreatic, head, neck, renal, prostate, colorectal, oesophageal or thyroid cancer, and melanoma.
Different types of cancers may include, but are not limited to fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelio-sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, lymphoma, leukemia, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma, uveal melanoma and breast cancer.
More particularly the peptides described herein (or nucleic acids that encode said peptides) are useful in the treatment of cancers which exhibit a deregulation of PP1 and/or PP2A or which exhibit an over-expression of the anti-apoptotic protein Bcl-2, an apoptotic regulator that interacts with and is controlled by PP1 and PP2A.
High levels of expression of the human bcl-2 gene have been found in all lymphomas with t (14; 18) chromosomal translocations including most follicular B cell lymphomas and many large cell non-Hodgkin's lymphomas. High levels of expression of the bcl-2 gene have also been found in leukemias that do not have a t(14; 18) chromosomal translocation, including lymphocytic leukemias of the pre-B cell type, neuroblastomas, nasophryngeal carcinomas, and many adenocarcinomas of the prostate, breast, and colon. Especially overexpression of bcl-2 was found in chronic lymphocytic leukemia (CLL) (Deng et al, 2009; Prickett et al, 2004).
In a preferred embodiment, the cancer tumor is thus a lymphoma, especially a leukemia, such as chronic lymphocytic leukemia (CLL).
Furthermore, the chimeric peptides (or nucleic acids that encode said peptides) may be used for the treatment of metastases.
According to another embodiment, the hyperproliferative disorder may be a non-cancerous hyperproliferative disorder such as benign hyperplasia of the skin (e.g., psoriasis) or prostate (e.g., benign prostatic hypertrophy (BPH)), rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas, vascular occlusion, restenosis, atherosclerosis, or oral hairy leukoplakia.
The chimeric peptides (or nucleic acids that encode said peptides) as described herein may also be used for treating parasitic diseases.
In particular, the chimeric peptides (or nucleic acids that encode said peptides) may have the ability to decrease the parasite load in a subject of at least 50%, 60%, 70%, 80%, 90% or 100%.
The invention also provides a method of treatment of a parasitic disease in a patient in need thereof, which method comprises administering said patient with a chimeric peptide or a nucleic acid that encode said peptide.
Preferably, the parasitic disease is due to a parasite that belongs to the species Trypasonoma, Theileria or Plasmodium.
The parasitic disease caused by the Trypanosoma may be sleeping sickness disease in humans, Chagas disease in humans, Nagana disease in ruminant livestock, horses and pigs, Trypanosomiasis in birds, dourine or covering sickness in horses and other Eauidae.
The parasitic disease caused by Theileria may be the tropical theleriosis, the Mediterranean Coast Fever, the East Coast Fever or the equine or ovine piroplasmosis.
The parasitic disease caused by Plasmodium may be malaria.
Pharmaceutical Compositions:
The vectors of the invention, in particular chimeric peptides (or nucleic acid that encode said peptide) may be administered by any convenient route including intravenous, oral, transdermal, subcutaneous, mucosal, intramuscular, intrapulmonary, intranasal, parenteral, rectal, vaginal and topical. Intranasal route is of particular interest.
Advantageously, intra-tumoral administration is also contemplated.
The therapeutic agent is formulated in association with a pharmaceutically acceptable carrier.
The pharmaceutical composition may also include, or be combined with any other active principle, such as in particular an anti-cancer agents, e.g. conventional cytotoxic chemotherapies with inhibitors of DNA replication such as DNA binding agents in particular alkylating or intercalating drugs, antimetabolite agents such as DNA polymerase inhibitors, or topoisomerase I or II inhibitors, or with anti-mitogenic agents such as alkaloids. In a further embodiment, the vectors of the invention, in particular chimeric peptides (or nucleic acid that encode said peptide), may be combined with protease (kinase, aromatase, ATPase) inhibitors, monoclonal antibodies or hormones or hormone analogs.
In a preferred embodiment, the therapeutic agent may be administered by electroporation. Electroporation, also known as electropermeabilization or electroinjection, is the permeabilization of cell membranes as a consequence of the application of certain short and intense electric fields across the cell membrane, the cells or the tissues. Typically, electroporation consists of injecting compounds, preferably via intramuscular or intradermal route, followed by applying a series of electric pulses by means of electrodes connected to a generator. The conditions for applying an electric field in the injection zone are now well known to those persons skilled in the art, and are in particular described in the U.S. Pat. No. 5,468,223. Those persons skilled in the art will be able to adapt these conditions according to each case. The electric field may be 50-200 microseconds pulses of high-strength electric fields in the range of 1-5000 V/cm and with a frequency between 0.1 and 1,000 hertz. Typically, a sequence of eight 100 microseconds pulses of 1000-1500 V/cm with a frequency of 1 hertz is applied.
The therapeutic agent, such as the chimeric peptide, is formulated in association with a pharmaceutically acceptable carrier.
The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically such compositions are prepared as injectables either as liquid solutions or suspensions; however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified. In particular, the pharmaceutical compositions may be formulated in solid dosage form, for example capsules, tablets, pills, powders, dragees or granules.
The choice of vehicle and the content of active substance in the vehicle are generally determined in accordance with the solubility and chemical properties of the active compound, the particular mode of administration and the provisions to be observed in pharmaceutical practice. For example, excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate and disintegrating agents such as starch, alginic acids and certain complex silicates combined with lubricants such as magnesium stearate, sodium lauryl sulfate and talc may be used for preparing tablets. To prepare a capsule, it is advantageous to use lactose and high molecular weight polyethylene glycols. When aqueous suspensions are used they can contain emulsifying agents or agents which facilitate suspension. Diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol and chloroform or mixtures thereof may also be used.
Preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that may provide controlled or sustained release of the product.
The dosing is selected by the skilled person so that a pro-apoptotic effect is achieved, and depends on the route of administration and the dosage form that is used. Total daily dose of the chimeric peptide administered to a subject in single or divided doses may be in amounts, for example, of from about 0.001 to about 100 mg/kg body weight daily and preferably 0.01 to 10 mg/kg/day. Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated.
Further aspects and advantages of the present invention will be disclosed in the following experimental section, which should be regarded as illustrative and not limiting the scope of the present application.
FIG. 1 is a graph that shows stability of the mutated peptides analyzed by Proteominer fractionation and Maldi-Tof. The ratio of intensity of each pick is represented, relative to its own control. The R residue was mutated to K (Mut1-DPT-C9h), N (Mut2-DPT-C9h) or A (Mut3-DPT-C9h). Round: control DPT-C9h peptide (SEQ ID NO:6), Square: Mut1-DPT-C9h, Triangle: Mut2-DPT-C9h, Diamond: Mut3-DPT-C9h.
FIG. 2 shows an analysis of apoptosis by annexin-V-FITC staining of breast cancer cell line HBCx-12A was treated for 24h with 100 μM of the control and mutated peptides.
FIG. 3 shows biodistribution of Cy5DPT-C9h and Cy5Mut3DPT-C9h. Mice were grafted in interscapular by luminal breast cancer xenograft HBCx-3. They received one intraperitoneal injection of DPT-C9h or Mut3DPT-C9h labelled with Cy5 at 5 mg/kg. Control mouse received the control excipient (glucose 5%). Mice were imaged between 0 (prior to injection) and 168h after injection. Fluorescence of the tumors was calculated and normalized by using Living Image software.
Design and Characterization of Mutated Non-Degradable DPT-C9h Penetrating Peptides
1.1. Materials and Methods
Peptide Synthesis and Sequence
Peptides were synthesized in an automated multiple peptide synthesizer with solid phase procedure and standard Fmoc chemistry. The purity and composition of the peptides were confirmed by reverse phase HPLC and by amino acid analysis.
Analysis Peptide Stability in Human Serum
Analysis of peptides degradation was done by Proteominer and Maldi-Tof as previously described.
1.2. Results
DPT-C9h is VKKKKIKREIKI-YVETLDDIFEQWAHSEDL,
The R residue was mutated to K (Mut1-DPT-C9h), N (Mut2-DPT-C9h) or A (Mut3-DPT-C9h).
FIG. 1 shows that Mut3-DPC-C9h peptide is not degraded upon 24h of contact with the human serum. In addition, the other mutants showed a higher stability compared to control peptide (DPT-C9h).
Effect of Mutated DPT-C9h on Apoptosis
Human breast cancer HBCx-12A, cell line has been isolated from primary human cancer xenografts and was cultured in RPMI medium supplemented with 10% of FCS.
Detection of Apoptosis by Annexin-V-FITC Staining
Apoptotic cells were detected using Annexin-V (-FITC from BD biosciences) as described by the manufacturer. Briefly, the cells were washed in 1× binding buffer, centrifugated and then resuspended in 200 μl of 1× binding buffer containing Annexin V-FITC (0.1 μg/ml) and PI (0.5 μg/ml). After incubation at room temperature in the dark for 10 min, cells were analyzed by flow cytometry. Data acquired by FACSCalibur (BD biosciences) were analyzed with Cellquest Pro software.
The inventors have then analyzed whether the mutated peptides retain the capacity to induce apoptosis. The breast cancer cell line HBCx-12A was treated for 24h with 100 μM of the control and mutated peptides. Apoptosis was analyzed by annexin-V-FITC staining. As shown in FIG. 2, the analyzed peptides induce similar levels of apoptosis. The same result was observed when using the cell lines HBC-x3 and HBCx-17.
Biodistribution of Mut3DPT-C9h in Tumors
Peptides (DPT-C9h and Mut3DPT-C9h) were synthesized as described above. The fluorochrome Cy5 was added during the synthesis of the peptide.
Fluorescence Assays
Mice were IP (intraperitonally)-injected with the peptide Cy5DPT-C9h or Mut3DPT-C9h (5 mg/kg) and then analyzed at different times after injection.
Fluorescence imaging was performed with the IVIS imaging system (IVIS 100, Caliper Life Sciences, USA). Mice were anesthetized upon analysis. Imaging acquisition time was from 1 s to 10 s, depending on the fluorescence signal. Analysis was performed using software Living Image V. 2.50 (Caliper Life Sciences).
Biodistribution of Mut3DPT-C9h and DPT-C9h in the breast cancer xenograft models.
The inventors were interested in analyzing and comparing the biodistribution of both peptides. FIG. 3 shows the biodistribution of Cy5DPT-C9h and Cy5Mut3DPT-C9h in a breast cancer xenograft model. Mice were intraperitonally (IP) injected and biodistribution analyzed at different times upon injection.
FIG. 3 shows that 6 h after IP injection, the inventors were able to detect both peptides in the tumor. The maximal peak of detection of Cy5 Mut3DPT-C9h is detected 23h after injection, slightly decreasing the intensity of the fluorescence after this time. Finally, considerable level of Cy5Mut3DPT-C9h was detected 168h after injection. The maximum level of Cy5DPT-C9h fluorescence was detected 6h after injection, slightly decreasing after this period of time. Low level of Cy5DPT-C9h was detected upon 168h of treatment.
Taken together, these results show that Cy5-labelled DPT-C9h and Cy5-labelled Mut3DPT-C9h reach the tumor. More importantly, Cy5-labelled Mut3DPT-C9h showed to be more stable that the original peptide, DPT-C9h.
The mutated peptide Mut3DPT-C9h shows a biodistribution in the tumor more sustained than the original peptide (DPT-C9h) since we are able to detect the fluorescence of the Cy5 fluorochrome longer that the fluorescence of DPT-C9h.
This new property will allow to reduce the dose of peptide injected as well as the schedule of administration. In summary, the new mutants have a clear new advantage compared to control peptide and have a new characteristic since they are not degradable by serum proteases.
Deng X, Gao F, and W. Stratford May. Dephosphorylation and up-regulation of Bcl2-p53 binding Protein phosphatase 2A inactivates Bcl-2's antiapoptotic function by dephosphorylation and up-regulation of Bcl2-p53 binding. Blood. 2009 Jan. 8; 113(2):422-8.
Guergnon, F. Dessauge, V. Dominguez, J. Viallet, X. Cayla, A. Rebollo, V. Yuste, S. Susin, P E. Bost and A. Garcia Use of penetrating peptides interacting with PP1/PP2A proteins as a basis for a new Drug Phosphatase Technology. Mol. Pharmacol. (2006) 69:1115-1124.
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Pitton, C., Rebollo, A., Van Snick, J., Theze, J. and Garcia, A. (1993) High affinity and intermediate affinity forms of the human IL-2 receptor expressed in an IL-9-dependent murine T cell line deliver proliferative signals via differences in their transduction pathways. Cytokine, 5, 362-371.
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1. A vector comprising a penetrating peptide consisting of the amino acid sequence VKKKKIKAEIKI (SEQ ID NO:2) coupled to a therapeutic agent.
2. The vector of claim 1, wherein the vector is a chimeric peptide construct.
3. The vector of claim 1, wherein the therapeutic agent is selected from the group consisting of a cytotoxic agent, an anti-viral agent, an anti-bacterial agent, and an anti-parasitic agent.
4. The vector of claim 3, wherein the cytotoxic agent is a pro-apoptotic peptide.
5. The vector of claim 4, wherein the penetrating peptide is fused to the pro-apoptotic peptide, and the vector is 23 to 70 amino acids in length.
6. The vector of claim 4, wherein the penetrating peptide is fused to the pro-apoptotic peptide at the C-terminus of the penetrating peptide.
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Date of Patent: May 9, 2017
Assignees: Universite Pierre Et Marie Curie (Paris 6) (Paris), Institut Curie (Paris)
Inventors: Angelita Rebollo Garcia (Paris), Fariba Nemati (Paris), Didier Decaudin (Verrieres le Buisson), Jeronimo Bravo Sicilia (Valencia), Jesus Maria Fominaya Gutierrez (Madrid)
Primary Examiner: Julie Ha
Current U.S. Class: 15 To 23 Amino Acid Residues In Defined Sequence (530/326)
International Classification: A61K 38/00 (20060101); A61K 38/10 (20060101); A61K 38/04 (20060101); A61K 38/16 (20060101); C07K 7/08 (20060101); C07K 14/47 (20060101); C07K 7/06 (20060101); C12N 9/64 (20060101); C12N 7/00 (20060101); A61K 48/00 (20060101);


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