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Software cracking

Modification of software, often to use it for free

Software cracking (known as "breaking" mostly in the 1980s[1]) is the modification of software to remove or disable features which are considered undesirable by the person cracking the software, especially copy protection features (including protection against the manipulation of software, serial number, hardware key, date checks and disc check) or software annoyances like nag screens and adware.

A crack refers to the means of achieving, for example a stolen serial number or a tool that performs that act of cracking.[2] Some of these tools are called keygen, patch, or loader. A keygen is a handmade product serial number generator that often offers the ability to generate working serial numbers in your own name. A patch is a small computer program that modifies the machine code of another program. This has the advantage for a cracker to not include a large executable in a release when only a few bytes are changed.[3] A loader modifies the startup flow of a program and does not remove the protection but circumvents it.[4][5] A well-known example of a loader is a trainer used to cheat in games.[6]Fairlight pointed out in one of their .nfo files that these type of cracks are not allowed for warez scene game releases.[7][4][8] A nukewar has shown that the protection may not kick in at any point for it to be a valid crack.[9]

The distribution of cracked copies is illegal in most countries. There have been lawsuits over cracking software.[10] It might be legal to use cracked software in certain circumstances.[11] Educational resources for reverse engineering and software cracking are, however, legal and available in the form of Crackme programs.

History[edit]

The first software copy protection was applied to software for the Apple II,[12]Atari 8-bit family, and Commodore 64 computers.[citation needed]. Software publishers have implemented increasingly complex methods in an effort to stop unauthorized copying of software.

On the Apple II, the operating system directly controls the step motor that moves the floppy drive head, and also directly interprets the raw data, called nibbles, read from each track to identify the data sectors. This allowed complex disk-based software copy protection, by storing data on half tracks (0, 1, 2.5, 3.5, 5, 6...), quarter tracks (0, 1, 2.25, 3.75, 5, 6...), and any combination thereof. In addition, tracks did not need to be perfect rings, but could be sectioned so that sectors could be staggered across overlapping offset tracks, the most extreme version being known as spiral tracking. It was also discovered that many floppy drives did not have a fixed upper limit to head movement, and it was sometimes possible to write an additional 36th track above the normal 35 tracks. The standard Apple II copy programs could not read such protected floppy disks, since the standard DOS assumed that all disks had a uniform 35-track, 13- or 16-sector layout. Special nibble-copy programs such as Locksmith and Copy II Plus could sometimes duplicate these disks by using a reference library of known protection methods; when protected programs were cracked they would be completely stripped of the copy protection system, and transferred onto a standard format disk that any normal Apple II copy program could read.

One of the primary routes to hacking these early copy protections was to run a program that simulates the normal CPU operation. The CPU simulator provides a number of extra features to the hacker, such as the ability to single-step through each processor instruction and to examine the CPU registers and modified memory spaces as the simulation runs (any modern disassembler/debugger can do this). The Apple II provided a built-in opcode disassembler, allowing raw memory to be decoded into CPU opcodes, and this would be utilized to examine what the copy-protection was about to do next. Generally there was little to no defense available to the copy protection system, since all its secrets are made visible through the simulation. However, because the simulation itself must run on the original CPU, in addition to the software being hacked, the simulation would often run extremely slowly even at maximum speed.

On Atari 8-bit computers, the most common protection method was via "bad sectors". These were sectors on the disk that were intentionally unreadable by the disk drive. The software would look for these sectors when the program was loading and would stop loading if an error code was not returned when accessing these sectors. Special copy programs were available that would copy the disk and remember any bad sectors. The user could then use an application to spin the drive by constantly reading a single sector and display the drive RPM. With the disk drive top removed a small screwdriver could be used to slow the drive RPM below a certain point. Once the drive was slowed down the application could then go and write "bad sectors" where needed. When done the drive RPM was sped up back to normal and an uncracked copy was made. Of course cracking the software to expect good sectors made for readily copied disks without the need to meddle with the disk drive. As time went on more sophisticated methods were developed, but almost all involved some form of malformed disk data, such as a sector that might return different data on separate accesses due to bad data alignment. Products became available (from companies such as Happy Computers) which replaced the controller BIOS in Atari's "smart" drives. These upgraded drives allowed the user to make exact copies of the original program with copy protections in place on the new disk.

On the Commodore 64, several methods were used to protect software. For software distributed on ROM cartridges, subroutines were included which attempted to write over the program code. If the software was on ROM, nothing would happen, but if the software had been moved to RAM, the software would be disabled. Because of the operation of Commodore floppy drives, one write protection scheme would cause the floppy drive head to bang against the end of its rail, which could cause the drive head to become misaligned. In some cases, cracked versions of software were desirable to avoid this result. A misaligned drive head was rare usually fixing itself by smashing against the rail stops. Another brutal protection scheme was grinding from track 1 to 40 and back a few times.

Most of the early software crackers were computer hobbyists who often formed groups that competed against each other in the cracking and spreading of software. Breaking a new copy protection scheme as quickly as possible was often regarded as an opportunity to demonstrate one's technical superiority rather than a possibility of money-making. Some low skilled hobbyists would take already cracked software and edit various unencrypted strings of text in it to change messages a game would tell a game player, often something considered vulgar. Uploading the altered copies on file sharing networks provided a source of laughs for adult users. The cracker groups of the 1980s started to advertise themselves and their skills by attaching animated screens known as crack intros in the software programs they cracked and released. Once the technical competition had expanded from the challenges of cracking to the challenges of creating visually stunning intros, the foundations for a new subculture known as demoscene were established. Demoscene started to separate itself from the illegal "warez scene" during the 1990s and is now regarded as a completely different subculture. Many software crackers have later grown into extremely capable software reverse engineers; the deep knowledge of assembly required in order to crack protections enables them to reverse engineerdrivers in order to port them from binary-only drivers for Windows to drivers with source code for Linux and other free operating systems. Also because music and game intro was such an integral part of gaming the music format and graphics became very popular when hardware became affordable for the home user.

With the rise of the Internet, software crackers developed secretive online organizations. In the latter half of the nineties, one of the most respected sources of information about "software protection reversing" was Fravia's website.

+HCU[edit]

The High Cracking University (+HCU) was founded by Old Red Cracker (+ORC), considered a genius of reverse engineering and a legendary figure in RCE, to advance research into Reverse Code Engineering (RCE). He had also taught and authored many papers on the subject, and his texts are considered classics in the field and are mandatory reading for students of RCE.[13]

The addition of the "+" sign in front of the nickname of a reverser signified membership in the +HCU. Amongst the students of +HCU were the top of the elite Windows reversers worldwide.[13] +HCU published a new reverse engineering problem annually and a small number of respondents with the best replies qualified for an undergraduate position at the university.[13]

+Fravia was a professor at +HCU. Fravia's website was known as "+Fravia's Pages of Reverse Engineering" and he used it to challenge programmers as well as the wider society to "reverse engineer" the "brainwashing of a corrupt and rampant materialism". In its heyday, his website received millions of visitors per year and its influence was "widespread".[13]

Nowadays most of the graduates of +HCU have migrated to Linux and few have remained as Windows reversers. The information at the university has been rediscovered by a new generation of researchers and practitioners of RCE who have started new research projects in the field.[13]

Methods[edit]

The most common software crack is the modification of an application's binary to cause or prevent a specific key branch in the program's execution. This is accomplished by reverse engineering the compiled program code using a debugger such as SoftICE,[14]x64dbg, OllyDbg,[15]GDB, or MacsBug until the software cracker reaches the subroutine that contains the primary method of protecting the software (or by disassembling an executable file with a program such as IDA). The binary is then modified using the debugger or a hex editor or monitor in a manner that replaces a prior branching opcode with its complement or a NOPopcode so the key branch will either always execute a specific subroutine or skip over it. Almost all common software cracks are a variation of this type. Proprietary software developers are constantly developing techniques such as code obfuscation, encryption, and self-modifying code to make this modification increasingly difficult. Even with these measures being taken, developers struggle to combat software cracking. This is because it is very common for a professional to publicly release a simple cracked EXE or Retrium Installer for public download, eliminating the need for inexperienced users to crack the software themselves.

A specific example of this technique is a crack that removes the expiration period from a time-limited trial of an application. These cracks are usually programs that alter the program executable and sometimes the .dll or .so linked to the application. Similar cracks are available for software that requires a hardware dongle. A company can also break the copy protection of programs that they have legally purchased but that are licensed to particular hardware, so that there is no risk of downtime due to hardware failure (and, of course, no need to restrict oneself to running the software on bought hardware only).

Another method is the use of special software such as CloneCD to scan for the use of a commercial copy protection application. After discovering the software used to protect the application, another tool may be used to remove the copy protection from the software on the CD or DVD. This may enable another program such as Alcohol 120%, CloneDVD, Game Jackal, or Daemon Tools to copy the protected software to a user's hard disk. Popular commercial copy protection applications which may be scanned for include SafeDisc and StarForce.[16]

In other cases, it might be possible to decompile a program in order to get access to the original source code or code on a level higher than machine code. This is often possible with scripting languages and languages utilizing JIT compilation. An example is cracking (or debugging) on the .NET platform where one might consider manipulating CIL to achieve one's needs. Java'sbytecode also works in a similar fashion in which there is an intermediate language before the program is compiled to run on the platform dependent machine code.

Advanced reverse engineering for protections such as SecuROM, SafeDisc, StarForce, or Denuvo requires a cracker, or many crackers to spend much more time studying the protection, eventually finding every flaw within the protection code, and then coding their own tools to "unwrap" the protection automatically from executable (.EXE) and library (.DLL) files.

There are a number of sites on the Internet that let users download cracks produced by warez groups for popular games and applications (although at the danger of acquiring malicious software that is sometimes distributed via such sites).[17] Although these cracks are used by legal buyers of software, they can also be used by people who have downloaded or otherwise obtained unauthorized copies (often through P2P networks).

See also[edit]

References[edit]

  1. ^Kevelson, Morton (October 1985). "Isepic". Ahoy!. pp. 71–73. Retrieved June 27, 2014.
  2. ^Tulloch, Mitch (2003). Microsoft Encyclopedia of Security(PDF). Redmond, Washington: Microsoft Press. p. 68. ISBN .
  3. ^Craig, Paul; Ron, Mark (April 2005). "Chapter 4: Crackers". In Burnett, Mark (ed.). Software Piracy Exposed - Secrets from the Dark Side Revealed. Publisher: Andrew Williams, Page Layout and Art: Patricia Lupien, Acquisitions Editor: Jaime Quigley, Copy Editor: Judy Eby, Technical Editor: Mark Burnett, Indexer: Nara Wood, Cover Designer: Michael Kavish. United States of America: Syngress Publishing. pp. 75–76. doi:10.1016/B978-193226698-6/50029-5. ISBN .
  4. ^ abFLT (January 22, 2013). "The_Sims_3_70s_80s_and_90s_Stuff-FLT".
  5. ^Shub-Nigurrath [ARTeam]; ThunderPwr [ARTeam] (January 2006). "Cracking with Loaders: Theory, General Approach, and a Framework". CodeBreakers Magazine. Universitas-Virtualis Research Project. 1 (1).
  6. ^Nigurrath, Shub (May 2006). "Guide on how to play with processes memory, writing loaders, and Oraculumns". CodeBreakers Magazine. Universitas-Virtualis Research Project. 1 (2).
  7. ^FLT (September 29, 2013). "Test_Drive_Ferrari_Legends_PROPER-FLT".
  8. ^SKIDROW (January 21, 2013). "Test.Drive.Ferrari.Racing.Legends.Read.Nfo-SKIDROW".
  9. ^"Batman.Arkham.City-FiGHTCLUB nukewar". December 2, 2011. Archived from the original on September 13, 2014.
  10. ^Cheng, Jacqui (September 27, 2006). "Microsoft files lawsuit over DRM crack". Ars Technica.
  11. ^Fravia (November 1998). "Is reverse engineering legal?".
  12. ^Pearson, Jordan (July 24, 2017). "Programmers Are Racing to Save Apple II Software Before It Goes Extinct". Motherboard. Archived from the original on September 27, 2017. Retrieved January 27, 2018.
  13. ^ abcdeCyrus Peikari; Anton Chuvakin (January 12, 2004). Security Warrior. "O'Reilly Media, Inc.". p. 31. ISBN .
  14. ^Ankit, Jain; Jason, Kuo; Jordan, Soet; Brian, Tse (April 2007). "Software Cracking (April 2007)"(PDF). The University of British Columbia - Electrical and Computer Engineering. Retrieved January 27, 2018.
  15. ^Wójcik, Bartosz. "Reverse engineering tools review". pelock.com. PELock. Archived from the original on September 13, 2017. Retrieved February 16, 2018.
  16. ^Gamecopyworld Howto
  17. ^McCandless, David (April 1, 1997). "Warez Wars". Wired. ISSN 1059-1028. Retrieved February 4, 2020.
Источник: https://en.wikipedia.org/wiki/Software_cracking

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Rons CSV Editor is the ultimate CSV editor, whether you need to edit a CSV file, clean some data, or merge and convert to another format, this is the ideal solution for anyone who regularly works with CSV files.

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THE LION KING: THE GIFT (2019)

THE LION KING: THE GIFT
  • Everything you see exist together in a delicate balance
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    PERFORMED BY JAMES EARL JONES / WRITTEN BY JEFF NATHANSON / SCORE BY BEYONCÉ, DEREK DIXIE / ARRANGEMENT AND ORCHESTRATION BY DEREK DIXIE / CO-ARRANGEMENT BY STEPHANIE MATTHEWS / STRINGS PERFORMED BY STEPHANIE MATTHEWS, STEPHANIE YU, CRYSTAL ALFORQUE, BIANCA MCCLURE, MARTA HONER, RHEA HOSANNY, TAHIRAH WHITTINGTON, ADRIENNE WOODS, CHELSEA STEVENS, JONATHAN RICHARDS / ADDITIONAL SOUNDS BY TYLER SCOTT / MIXED BY TYLER SCOTT, LESTER MENDOZA AT NRG RECODING STUDIOS NORTH HOLLYWOOD, CA / STRINGS RECORDED BY DANIEL PAMPURI AT NRG RECORDING STUDIOS NORTH HOLLYWOOD, CA / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY® ©2019 DISNEY ENTERPRISES, INC.

  • Verse 1: Beyoncé
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  • Let me tell you something my father told me
    Look at the stars
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    So whenever you feel alone
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    PERFORMED BY JAMES EARL JONES/WRITTEN BY JEFF NATHANSON/SCORE BY BEYONCÉ, DEREK DIXIE / MIXED BY ZACHARY ACOSTA, JOHN CRANFIELD / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY® / ©2019 DISNEY ENTERPRISES, INC.

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    Daddy used to take me walkin down the street
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    Daddy used to lead me back home all the time
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    Daddy used to teach me all my plays
    On a marathon told me run my race

    PERFORMED BY BEYONCÉ / WRITTEN BY BEYONCÉ, BRITTANY “STARRAH” HAZZARD, BUBELE BOOI, ROBERT MAGWENZI, ABISAGBOOLA “BANKULLI” OLUSEUN, OSARETIN OSABUOHIEN / PUBLISHED BY OAKLAND 13 MUSIC (ASCAP), ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, PEOPLE OVER PLANES (ASCAP)/THESE ARE SONGS OF PULSE, UNIVERSAL MUSIC PUBLISHING (SAMRO), ROBERT MAGWENZI PUB DESIGNEE (SAMRO), ABISAGBOOLA OLUSEUN PUB DESIGNEE, OSARETIN OSABOUHIEN PUB DESIGNEE / PRODUCED BY BEYONCÉ, BUBELE BOOI, ROBERT MAGWENZI /ADDITIONAL PRODUCTION BY GUILTYBEATZ, DEREK DIXIE / VOCAL PRODUCTION BY BANKULLI, DEREK DIXIE /ADDITIONAL VOCALS BY BANKULLI RECORDED AT PARKWOOD WEST LOS ANGELES, CA / BEYONCÉ VOCALS RECORDED BY STUART WHITE AT KINGSLANDING LOS ANGELES, CA / MIXED BY STUART WHITE AT NRG STUDIOS NORTH HOLLYWOOD, CA / RECORDING ENGINEER: DEREK DIXIE / ASSISTANT RECORDING ENGINEER: JENNA FELSENTHAL / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY®

    CONTAINS ELEMENTS OF “MARADONA” WRITTEN BY OSARETIN OSABUOHIEN AND PERFORMED BY NINIOLA

  • Go back to your den simba
    I dont babysit

    PERFORMED BY CHIWETEL EJIOFOR / WRITTEN BY JEFF NATHANSON / SCORE BY BEYONCÉ, DEREK DIXIE / MIXED BY ZACHARY ACOSTA, JOHN CRANFIELD / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY® / ©2019 DISNEY ENTERPRISES, INC.

  • Verse 1: Tekno
    Suzuki no be chang jiang
    (Suzuki is no chang jiang)
    Jet li no be liu kang
    (Jet li is no liu kang)
    You know me i no be pretend
    (You know me i dont pretend)
    From start of the week to the weekend
    I no de hide for corner
    (You won’t see me hiding in no corner)
    Foot work like pogba
    If you get my ton ton ton
    (If you get on my vibe)
    Me I want to get on your don don don
    (I can get on your vibe)

    Pre: Nana
    Sheep don’t run with lion Snake don’t swing monkey
    I can’t talk for too long
    got too much gold to try on

    Sheep don’t run with lion Snake don’t swing monkey
    I can’t talk for too long
    got too much gold to try on

    Hook: Nana
    Jealousy
    Don’t you jealous me
    That’s that jealousy
    Don’t you jealous me
    That’s that jealousy
    Don’t you jealous me
    That’s that jealousy
    Don’t you come for me

    Mr Eazi
    Baako – “one”
    Mienu – “two”
    Yenko – “let’s go”
    Aahbaaa

    Baako – “one”
    Mienu – “two”
    Yenko – “let’s go”
    Aahbaaa

    Baako – “one”
    Mienu – “two”
    Yenko – “let’s go”
    Aahbaaa

    Baako – “one”
    Mienu – “two”
    Yenko – “let’s go”
    Aahbaaa

    Verse 2: Yemi Alade
    O O Odogwu
    (Great Man)
    aBeg make them talk I go give them the action
    (please let them talk , I’ll give them action)
    I be the grand master
    Salute the commander
    Otunba
    Oga
    Saidi
    Chairman
    Mansa
    Oba
    (Titles of African Leaders)
    Yaro
    (Small boy/youngin)
    Clear Road
    (Make way)

    Pre: Nana
    Sheep don’t run lion
    Snake don’t swing with monkey
    I can’t talk for too long
    got too much gold to try on

    Sheep don’t run lion
    Snake don’t swing with monkey
    I can’t talk for too long
    got too much gold to try on

    Hook: Nana
    Jealousy
    Don’t you jealous me
    That’s that jealousy
    Don’t you jealous me
    That’s that jealousy
    Don’t you jealous me
    That’s that jealousy
    Don’t you come for me

    Mr Eazi
    Baako – “one”
    Mienu – “two”
    Yenko – “let’s go”
    Aahbaaa

    Baako – “one”
    Mienu – “two”
    Yenko – “let’s go”
    Aahbaaa

    Baako – “one”
    Mienu – “two”
    Yenko – “let’s go”
    Aahbaaa

    Baako – “one”
    Mienu – “two”
    Yenko – “let’s go”
    Aahbaaa

    PERFORMED BY TEKNO, LORD AFRIXANA, MR EAZI, YEMI ALADE/WRITTEN BY BEYONCÉ, AUGUSTINE MILES KELECHUKWU, NANA O. AFRIYIE, OLUWATOSIN OLUWOLE AJIBADE, YEMI ALADE, ARIOWA IROSOGIE, EARL PATRICK TAYLOR/PUBLISHED BY OAKLAND 13 MUSIC (ASCAP), ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, TEKNO PUB DESIGNEE, EPIPHANY S WRITING GROUP (BMI)/PRIMARY WAVE BEATS (BMI)/BMG PLATINUM SONGS (BMI), SONY/ATV UK (PRS), EFFYZZIE MUSIC LTD (PRS),WARNER/CHAPPELL MUSIC LTD (ASCAP), BELLA’S MONEY/THESE ARE SONGS OF PULSE (ASCAP)/ PRODUCED BY BEYONCÉ,P2J/ADDITIONAL PRODUCTION BY DEREK DIXIE/ADDITIONAL VOCALS BY JOZZY RECORDED AT PARKWOOD WEST LOS ANGELES, CA/MIXED BY STUART WHITE AT NRG RECORDING STUDIOS NORTH HOLLYWOOD,CA/RECORDED BY P2J, ARI PENSMITH AT PARKWOOD WEST STUDIOS WEST HOLLYWOOD, CA/MIX ASSISTANT: JOHN CRANFIELD/ASSISTANT RECORDING ENGINEER: JENNA FELSENTHAL/MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY®

    TEKNO APPEARS COURTESY OF ISLAND RECORDS UK UMG AFRICA
    LORD AFRIXANA APPEARS COURTESY OF BULLISH ENETERTAINMENT
    YEMI ALADE APPEARS COURTESY OF EFFYZZIE MUSIC LTD
    MR EAZI APPEARS COURTESY OF EMPAWA AFRICA LTD

  • This must be it
    C’mon
    Simba get down it could be dangerous
    Danger? Ha I laugh in the face of danger

    PERFORMED BY JD MCCRARY, SHAHADI WRIGHT JOSEPH / WRITTEN BY JEFF NATHANSON / SCORE BY BEYONCÉ, DEREK DIXIE / MIXED BY ZACHARY ACOSTA AT NRG RECORDING STUDIOS NORTH HOLLYWOOD, CA / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY® / ©2019 DISNEY ENTERPRISES, INC.

  • Verse 1: Burna Boy
    Your body go rest o
    (you will calm down)
    Papa tell piken
    (A father told child)
    Your body go resto
    Your body go resto

    Pre: Burna Boy
    Your tongue go confess o
    (you will speak the truth)
    Your head will correcto
    (Your mind will be focused)
    You no forgeto
    (You won’t forget)
    She you get the concept
    (Do you understand)

    Chorus: Burna Boy
    Dem no day tell person
    (Nobody will tell you)
    Ja Ara é Ja Ara é
    You go learn your own lesson
    (You will learn our own lessons)
    Ja Ara é Ja Ara é
    Everybody keeps on searching
    Ja Ara é Ja Ara é
    For miraculous blessings
    Ja Ara é Ja Ara é

    Verse 1: Burna Boy
    You go bow for Lagos
    (You will be confused Lagos) (back home)
    Ja Ara
    If you no know yourself you go lost
    (if you don’t know yourself you will get lost)
    Ja Ara
    If you get money you be bros
    (I you have money you will be respected)
    Ja Ara
    Omo nobody holy no apostles
    (Nobody is holy there are no apostles)
    Ja Ara

    Pre: Burna Boy
    Your tongue go confesso
    (you will speak the truth)
    Your head will correcto
    (Your mind will be focused)
    You no forget o
    (You won’t forget)
    Shey you get the concept
    (Do you understand)

    Chorus: Burna Boy
    Dem go tell you watch out when you go
    (They will tel you to watch out)
    Watch out pon de road
    Watch out for them hyenas
    Watch out when you go
    Watch out pon de road
    Watch out

    Bridge: Burna Boy
    Look around in all the places you go to
    Stand your ground and stay solid like ogun (God of iron (strength))
    Watch your back and just do what you’re supposed to
    Take care of family and people your close to

    Chorus: Burna Boy
    Dem no day tell person
    (Nobody will tell you)
    Ja Ara é Ja Ara é
    You go learn your own lesson
    (You will learn your own lessons) Ja Ara é Ja Ara é
    Everybody keeps on searching
    Ja Ara é Ja Ara é
    For miraculous blessings
    Ja Ara é Ja Ara é

    PERFORMED BY BURNA BOY / WRITTEN BY BEYONCÉ, DAMINI OGULU, RICHARD ISONG / PUBLISHED BY OAKLAND 13 MUSIC (ASCAP), ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, UNIVERSAL MUSIC PUBLISHING GROUP (ASCAP), P2J MUSIC (ASCAP)/BMG RIGHTS MANAGEMENT (UK)LTD (ASCAP) / PRODUCED BY BEYONCÉ, P2J / ADDITIONAL PRODUCTION BY DEREK DIXIE / MIXED BY STUART WHITE AT NRG RECORDING STUDIOS NORTH HOLLYWOOD, CA / RECORDED BY P2J AT RED ROOM, MILOCO STUDIOS LONDON, UK AND CECIL BERNARDY AT RECORD PLANT RECORDING STUDIOS LOS ANGELES, CA / MIX ASSISTANT: JOHN CRANFIELD / ASSISTANT RECORDING ENGINEER: JENNA FELSENTHAL / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY®

    BURNA BOY APPEARS COURTESY OF BAD HABIT/ON A SPACESHIP/ATLANTIC RECORDS

  • PERFORMED BY JD MCCRARY, CHIWETEL EJIOFOR / WRITTEN BY JEFF NATHANSON / SCORE BY BEYONCÉ, DEREK DIXIE / ADDITIONAL SOUNDS BY LESTER MENDOZA/MIXED BY ZACHARY ACOSTA, JOHN CRANFIELD AT NRG RECORDING STUDIOS NORTH HOLLYWOOD, CA / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY® / ©2019 DISNEY ENTERPRISES, INC.

  • Verse 1: Beyoncé and Kendrick
    One time I took a swim in the Nile
    I swam the whole way I didn’t turn around
    Man I swear
    It made me relax when I came down

    I felt liberated like free birds
    I’m stimulated now
    Plunging away unless my body on top

    All of these current might cost me my life
    Right now

    When danger finds me it follow with tides
    Many miles ahead of me still I’m in stride
    She said

    Bridge: Beyoncé
    Hey little buddy where you going
    I’m not sure but I know I’m still in motion
    This ain’t regular I seen regular
    These streams may take me out to the ocean

    Verse: Kendrick
    Told myself if I dive in it without precaution of a life jacket
    Then I’ll dive in until I’m exhausted and I’m type lackin’
    Waste line on 4 deep
    Senses on 4 deep
    Feel like there’s 4 me

    Chorus: Beyoncé and Kendrick
    One time I took a swim in the Nile
    (one time I took a swim)
    I swam the whole way I didn’t turn around
    Man I swear
    (I swear)
    It made me relax when I came down
    (when I came down)
    I felt liberated like free birds
    I’m stimulated now
    (stimulated) Plungin away unless my body on top
    (Stimulated now)
    All of these currents might cost me my life
    Right now
    (right now)
    When danger finds me it follows with tides
    (stimulated right now)
    Many miles ahead of me still I’m in stride

    That’s some good

    Verse: Beyoncé
    Got the Nile running through my body
    Look at my natural
    I’m so exotic
    Darker than berry
    Sweeter than fruit
    Deeper the wounded
    Deeper the roots
    Nubian doused in brown
    Boy I’m lounging in it
    Fountain of you, I said I’m drowning in it
    I’m in the Nile, deep in denial
    I’m in the Nile, deep in denial

    PERFORMED BY BEYONCÉ, KENDRICK LAMAR / WRITTEN BY BEYONCÉ, KENDRICK DUCKWORTH, MARK SPEARS, HYKEEM CARTER, KEANU DEAN TORRES, DENISIA “@BLU_JUNE” ANDREWS FOR @NOVWAV, BRITTANY “@CHI_CONEY” CONEY FOR @NOVAWAV / PUBLISHED BY OAKLAND 13 MUSIC (ASCAP), ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, HARD WORKING BLACK FOLKS INC (ASCAP)/TOP DAWG MUSIC (ASCAP)/WB MUSIC CORP (ASCAP), PUSHY PUBLISHING (ASCAP), TDE MUSIC LLC (BMI), HYKEEM CARTER PUB DESIGNEE (BMI) , KEANU DEAN TORRES PUB DESIGNEE (APRA), 114 SOUNDS/W.B.M MUSIC CORP (SESAC), CONEY COSMOS MUSIC PUBLISHING/WARNER-TAMERLANE PUBLISHING CORP. (BMI)/ PRODUCED BY BEYONCÉ, SOUNWAVE, HYKEEM CARTER, KEANU BEATS / BEYONCÉ VOCALS RECORDED BY STUART WHITE AT KINGSLANDING LOS ANGELES, CA/KENDRICK VOCALS RECORDED BY MATT SCHAFFER AT INTERSCOPE RECORDS LOS ANGELES, CA / MIXED BY STUART WHITE AT NRG RECORDING STUDIOS NORTH HOLLYWOOD, CA / MIX ASSISTANT: JOHN CRANFIELD / ASSISTANT ENGINEER: JOHNNY KOSICH, JENNA FELSENTHAL / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY®

    KENDRICK LAMAR APPEARS COURTESY OF TOP DAWG ENT/AFTERMATH RECORDS/INTERSCOPE RECORDS



    NEW LESSON (TIMON, PUMBAA, & YOUNG SIMBA INTERLUDE)

    PERFORMED BY BILLY EICHNER, SETH ROGEN, JD MCCRARY / WRITTEN BY JEFF NATHANSON / SCORE BY BEYONCÉ, DEREK DIXIE, LUDWIG GORANSSON / MIXED BY LESTER MENDOZA, JOHN CRANFIELD AT NRG RECORDING STUDIOS NORTH HOLLYWOOD, CA / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY® / ©2019 DISNEY ENTERPRISES, INC.

  • PERFORMED BY BILLY EICHNER, SETH ROGEN, JD MCCRARY / WRITTEN BY JEFF NATHANSON / SCORE BY BEYONCÉ, DEREK DIXIE, LUDWIG GORANSSON / MIXED BY LESTER MENDOZA, JOHN CRANFIELD AT NRG RECORDING STUDIOS NORTH HOLLYWOOD, CA / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY® / ©2019 DISNEY ENTERPRISES, INC.

  • Verse 1: Beyoncé
    I know my enemies prey on me
    So pray for me
    Tick tick wait on it
    I’m keeping down my body count
    I’m finessing like a trap bounce, trap bounce
    Cause everyday above ground is a blessing
    I done leveled up
    Now my view Panoramic
    None of my fears can’t go where I’m headed
    Had to cut em loose
    Now I’m loose break the levee
    I’m bout flood on em flood on a killa
    They ran it down, I’m ___, go Mutombo, no no killa
    You can’t dim my light
    Cause When we walk up in the club
    I need them sirens going off
    Then we look up in the sky
    The tears cry let us know that we’re alive, yeah yeah
    I get goosebumps every time
    I throw up my diamond
    Together we big timing
    Don’t make me have to remind ya

    Chorus: Beyoncé
    I got my cup to the heavens
    Another night I won’t remember
    Promise this my mood foreva
    Promise this my mood foreva
    Treasures dancing on the bezel
    The kinda feeling unforgettable
    Promise this my mood foreva
    Promise this my mood foreva
    Foreva and eva

    Verse 2: Jay-Z
    You heard that
    That’s the sound of the price going up
    Forever and eva eva
    And Eva Eva
    At the Saxon in the Madiba suite
    Like Mandela
    Bumping Fela on the puma jet
    Like we from Lagos man
    Mansa Musa reincarnated
    We on our levels that’s a Billi
    A thousand Milli
    First one to see a B
    Out these housing buildings
    I be feeling like Prince in 94
    Mike in 79
    Biggie in 97
    94 Nas
    Ali bumaye no Kumbaya
    Just give me the Somale
    I’m on lataj
    Helmet on the Jet Ski
    You know the vibes
    Hit my head, figured out me
    Oh my God without the God in the XY
    I’m afraid whole the game will be colonized
    The marathon will be televised for NIP
    Cause true kings don’t die, we multiply
    Peace

    Sub Verse (Verse 1.2): Beyoncé
    I’m so unbothered , I’m so unbothered
    Y’all be so pressed while I’m raising daughters
    Sons of empires y’all make me chuckle
    Stay in your struggle
    Crystal blue water
    Piña colada and you stay ramada inn
    My baby father blood line Rwanda
    Why would you try me
    Why would you bother
    I am Beyoncé
    Giselle Knowles-Carter
    I am the Nala
    Sister of Naruba
    Oshun Queen Sheeba
    I am the mother , ankh on my gold chain
    I sold my whole chain
    I be like soul food
    I am a whole mood

    When we walk up in the club
    I need the sirens going off
    Then we look up in the sky
    The tears cry let us know that we’re alive, yeah yeah
    I give goosebumps every time
    I throw up my diamond
    Together we big timing
    The children are our reminder

    Chorus: Beyoncé
    I got my cup to the heavens
    Another night I won’t remember
    Promise this my mood foreva
    Promise this my mood foreva
    Treasures dancing on the bezel
    The kinda feeling unforgettable
    Promise this my mood foreva
    Promise this my mood foreva
    Foreva and eva
    Eva , Eva
    Forever and eva

    Bridge: Beyoncé
    No shade no shade I got that lime light
    No trade no trade I wont’t give-up my pride
    I’m focused so get your mind right
    Cloud 9 cloud 9 I couldn’t ever tell you what’s going down

    Time to break it down now
    Woo..yeah
    Woo…yeah
    Woo…yeah

    Chorus: Beyoncé
    I got my cup to the heavens
    Another night I won’t remember
    Promise this my mood foreva
    Promise this my mood foreva
    Treasures dancing on the bezel
    The kinda feeling unforgettable
    Promise this my mood foreva
    Promise this my mood foreva
    Foreva and eva
    Forever and eva
    Eva eva eva eva eva
    Foreva and eva
    eva and
    eva and eva (reapet)

    PERFORMED BY BEYONCÉ, JAY-Z, CHILDISH GAMBINO, OUMOU SANGARÉ / WRITTEN BY BEYONCÉ, S. CARTER, DONALD GLOVER, KHALED KHALED, FLOYD NATHANIEL HILLS, DENISIA “@BLU_JUNE” ANDREWS FOR @NOVWAV, BRITTANY “@CHI_CONEY” CONEY FOR @NOVAWAV, ANATHI BHONGO MNYANGO,ANT CLEMONS, MICHAEL UZOWURU,TEO HALM, JEFF KLEINMAN, OUMOU SANGARE / PUBLISHED BY OAKLAND 13 MUSIC (ASCAP), ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, CARTER BOYS MUSIC, ADMINISTERED BY WARNER CHAPPELL (ASCAP), CHILDISH INDUSTRIES(BMI),BMG PLATINUM SONGS US (BMI)/GIVE THANKS PUBLISHING (BMI),DANJAHANDZ MUZIK (SESAC),114 SOUNDS/W.B.M MUSIC CORP (SESAC), CONEY COSMOS MUSIC PUBLISHING/WARNER-TAMERLANE PUBLISHING CORP. (BMI), GOLD TEA PRODUCTIONS (SAMRO), A. BLESSING PUBLISHING (BMI)/SONG OF UNIVERSAL (BMI), BEYOND CATEGORY LLC/UNIVERSAL MUISC PUBLISHING GROUP,SONGS OF UNIVERSAL, INC(BMI), UNIVERSAL MUSIC PUBLISHING (ASCAP),OUMU SANGARE PUB DESIGNEE / PRODUCED BY BEYONCÉ,DJ KHALED, DANJA/ADDITIONAL PRODUCTION BY MICHAEL UZOWURU, TEO HALM, JEFF KLEINMAN /BEYONCÉ VOCALS RECORDED BY STUART WHITE AND JOHN CRANFIELD AT KINGSLANDING LOS ANGELES, CA/ JAY-Z VOCALS RECORDED BYSTUART WHITE AND JOHN CRANFIELD AT KINGSLANDING LOS ANGELES, CA/CHILDISH GAMBINO VOCALSRECORDED BY RILEY MACKIN AT THE TEMPLE LOS ANGELES, CA/MIXED BY STUART WHITE AT NRG RECORDING STUDIOS NORTH HOLLYWOOD,CA / MIX ASSISTANT: JOHN CRANFIELD/ASSISTANT RECORDING ENGINEER: JENNA FELSENTHAL /MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY®

    CONTAINS ELEMENTS OF “DIARABY NENE” WRITTEN AND PERFORMED BY OUMOU SANGARE
    CONTAINS AN INTERPOLATION OF “SWEET GREEN FIELDS” WRITTEN BY JIMMY SEALS, PUBLISHED BY SONY/ATV ACUFF ROSE MUSIC (BMI).
    CONTAINS AN INTERPOLATION OF “(THINK) ABOUT IT” WRITTEN BY JAMES BROWN, PUBLISHED BY UNICHAPPELL MUSIC INC./DYNATONE PUBLISHING CO. (BMI).

    JAY-Z APPEARS COURTESY OF ROCNATION
    CHILDISH GAMBINO APPEARS COURTESY OF WOLF & ROTHSTEIN / RCA RECORDS

  • PERFORMED BY BEYONCÉ KNOWLES-CARTER, DONALD GLOVER / WRITTEN BY JEFF NATHANSON / SCORE BY BEYONCÉ, DEREK DIXIE/MIXED BY ZACHARY ACOSTA, JOHN CRANFIELD / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY®/©2019 DISNEY ENTERPRISES, INC.

  • Hook: Salatiel
    Baby Oh I’m not much of a talker
    Baby Oh can I drink from your water
    Baby Oh meet me down by the river
    We can dance to the rhythm

    Post: Pharrell
    Yes we can make it far
    Don’t need inflatables
    That’s what the waves are for
    What are you waiting for

    Yes we can make it far
    Don’t need inflatables
    That’s what the waves are for
    What are you waiting for

    Verse: Pharrell
    Oh darling if you leave
    I’ll go whereve you go
    I’ll where your heart on my sleeve
    So everybody here knows
    But if you tell me to stay
    You’ll never be on your own
    So baby , don’t wait too long

    Hook: Pharrell
    Baby oh
    I’m not much of a talker
    Baby oh
    Can I drink from your water
    Baby oh
    Meet me down by the river
    We can dance to the rhythm
    Till the sun is high and the water runs dry

    Post: Pharrell
    Yes we can make it far
    Don’t need inflatables
    That’s what the waves are for
    What are you waiting for

    Yes we can make it far
    Don’t need inflatables
    That’s what the waves are for
    What are you waiting for

    Verse 2: Beyoncé
    I’ll bring you back the moon
    Just so we got all night
    I’ll bring the sun down too
    So I can show you the light
    Trust me if you ever leave
    I’ll be right by your side oh
    For you, I’ll ride

    Hook: Pharrell and Beyoncé
    Baby oh
    I’m not much of a talker
    Baby oh
    Can I drink from your water
    Baby oh
    Meet me down by the river
    We can dance to the rhythm
    Till the sun is high and the water runs dry

    Post: Pharrell
    Yes we can make it far
    Don’t need inflatables
    That’s what the waves are for
    What are you waiting for

    Yes we can make it far
    Don’t need inflatables
    That’s what the waves are for
    What are you waiting for

    Hook: Pharrell and Beyoncé
    Baby oh
    I’m not much of a talker
    Baby oh
    Can I drink from your water
    Baby oh
    Meet me down by the river
    We can dance to the rhythm
    Till the sun is high and the water runs dry

    PERFORMED BY SALATIEL,PHARRELL, BEYONCÉ/WRITTEN BY BEYONCÉ, NANA O. AFRIYIE , NIJA CHARLES PKA NIJA, PHARRELL WILLIAMS, RICHARD ISONG, ALASTAIR O’DONNELL/PUBLISHED BY OAKLAND 13 MUSIC (ASCAP), ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, EPIPHANY S WRITING GROUP (BMI)/PRIMARY WAVE BEATS (BMI)/BMG PLATINUM SONGS (BMI), SONGS OF UNIVERSAL, INC. AND SONGS BY AMNIJA. ALL RIGHTS FOR SONGS BY AMNIJA CONTROLLED AND ADMINISTERED BY SONGS OF UNIVERSAL, INC, MORE WATER FROM NAZARETH (GMR), P2J MUSIC (ASCAP)/BMG RIGHTS MANAGEMENT (UK)LTD (ASCAP), NEO SONGS/BMG/PRODUCED BY BEYONCÉ, P2J/ADDITIONAL VOCALS BY MOONCHILD SANELLY / BEYONCÉ VOCALS RECORDEDBYSTUART WHITE AND JOHN CRANFIELD AT KINGSLANDING LOS ANGELES, CA/SALATIEL VOCALS SELF RECORDED/ PHARRELL VOCALS RECORDED BY MIKE CARSONBY RECORDING ENGINEER AT MOTORBASS RECORDING STUDIO PARIS, FR/MIXED BY LESLIE BRATHWAITE AT MEANS STREET STUDIOS IN ATLANTA, GA /ASSISTANT ENGINEER: JENNA FELSENTHAL, ANTOINE POYETON / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY®

    SALATIEL APPEARS COURTESY OF ALPHA BETTER RECORDS
    PHARRELL APPEARS COURTESY OF IIAMOTHER/COLUMBIA RECORDS

  • Intro (Chorus): Blue Ivy Carter, SAINt JHN
    Brown skin girl
    Ya skin just like pearls
    The best thing in the world
    I never trade you for anybody else baby
    Brown skin girl
    Ya skin just like pearls
    The best thing in the world
    I never trade you for anybody else, singin’

    Verse 1: WizKid
    She say she really grew up just like me
    Don’t believe in nothing but the almighty
    Just a little jeans and a pure white tee
    She never dream for ever be nobody wifey
    She Wilhemina pretty but she heart is a maze
    Play ya like a villain ‘cause she caught in a wave
    Tonight I am walking away
    9 to 5 mind on the grind ya ya

    Pre: WizKid
    Tonight I might fall in love
    Depending on how you hold me
    Iʼm glad that Iʼm calming down
    Can’t let no one come control me
    Keep dancing and call it love
    She fights it but falling slowly
    If ever you are in doubt
    Remember what Mama told me

    Chorus: WizKid
    Brown skin girl
    Ya skin just like pearls
    Your back against the world
    I never trade you for anybody else yeah

    Brown skin girl Ya skin just like pearls The best thing inna di world
    I never trade you for anybody else yeah

    Verse 2: Beyoncé
    Pose like a trophy when Naomi’s walking
    She need a Oscar for that pretty dark skin
    Pretty like Lupita when the cameras close in
    Drip broke the levee when my Kelly’s Rowland
    I think tonight she might braid her braids
    Melanin too dark to throw her shade
    She minds her business and whines her waist
    Gold like 24K, OK
    Pre: WizKid and Beyoncé
    Tonight I might fall in love
    Depending on how you hold me
    Iʼm glad that Iʼm calming down
    Can’t let no one come control me
    Keep dancing and call it love
    She fights it but falling slowly
    If ever you are in doubt
    Remember what Mama told me

    Brown skin girl
    Ya skin just like pearls
    Ya back against the world
    I never trade you for anybody else yeah
    Brown skin girl
    Ya skin just like pearls
    The best thing about di world
    I never trade you for anybody else yeah

    Bridge: Beyoncé
    Oh oh, have you looked in the mirror lately
    Wish you could trade eyes with me
    ‘Cause there’s complexities
    In complexion
    But your skin
    It glows like diamonds
    Pigment like the earth
    You be giving birth
    To everything alive
    Baby know your worth
    I love everything about you
    From your nappy curls
    To every single curve
    Your body natural
    Same skin that was broken
    Be the same skin taking over
    Most things out of focus view
    But when you’re in the room
    They notice you
    ‘Cause you’re beautiful
    Yeah you’re beautiful
    The men them gon’ fall in love
    With you and all of your glory
    Your skin is not only dark
    It shines and it tells your story
    Keep dancing
    They can’t control you
    They watching
    They all adore you
    If ever you are in doubt
    Remember what Mama told you

    Chorus: WizKid and Beyoncé
    Brown skin girl
    Ya skin just like pearls
    Your back against the world
    I never trade you for anybody else
    Brown skin girl
    Ya skin just like pearls
    The best thing in the world
    I never trade you for anybody else, say

    Chorus: Blue Ivy Carter
    Brown skin girl
    Ya skin just like pearls
    The best thing in the world
    I never trade you for anybody else, singin’

    PERFORMED BY BLUE IVY CARTER, SAINt JHN, BEYONCÉ, WIZKID/WRITTEN BY BEYONC&Ereg;, CARLOS ST. JOHN, ADIO MARCHANT, S. CARTER, STACY BARTHE, ANATHI BHONGO MNYANGO, MICHAEL UZOWURU, RICHARD ISONG/ PUBLISHED BY OAKLAND 13 MUSIC (ASCAP), ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, THE ST.

    SAINT JHN APPEARS COURTESY OF HITCO
    WIZKID APPEARS COURTESY OF RCA

  • PERFORMED BY BEYONCÉ KNOWLES-CARTER / WRITTEN BY JEFF NATHANSON / SCORE BY BEYONCÉ, DEREK DIXIE / ADDITIONAL SOUNDS BY ZACHARY ACOSTA / MIXED BY ZACHARY ACOSTA AT NRG RECORDING STUDIOS NORTH HOLLYWOOD, CA/MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY® / ©2019 DISNEY ENTERPRISES, INC.

  • Verse 1: Tiwa Savage
    See how the sun and the moon bow for you
    But you won’t open your eyes

    Omo oba ma gba gbe (Son of the king don’t you forget)
    Run ti oruko Baba re (Remember your father’s name)

    Pre: Tiwa Savage
    Tick tock pretty baby
    You can get it back don’t you waste it
    To grow from your past gotta face it
    Ahh, yeah

    Chorus: Tiwa Savage
    Ooh you are the remedy
    Don’t know what’s inside
    But you’re the key to the kingdom

    Ooh you are the remedy
    Don’t know what’s inside
    But you’re the key to the kingdom So believe
    Even if you can’t see I’ll never leave
    Cause You’re the key to the kingdom (2x)

    You‘re the key to the kingdom
    Oh you are the remedy
    Don’t know what’s inside
    But you’re the key to the kingdom
    You’re the key to the kingdom
    Hey, so believe even if you can’t see I’ll never leave
    You’re the key to the kingdom
    You’re the key to the kingdom

    Verse 2: Mr Eazi
    Zagadat
    Just like the tree, just like the Chinese bamboo tree
    See eyes might not see the greatness inside you, that lies within

    Oya come sit pon your throne,
    you know you no go stay down for long,
    whenever in doubt and alone
    Just remember, you’re the king inna the kingdom

    Bridge: Tiwa Savage
    You‘re the key to the kingdom
    Oh you are the remedy
    Don’t know what’s inside
    But you’re the key to the kingdom
    You’re the key to the kingdom
    Hey, so believe even if you can’t see I’ll never leave
    You’re the key to the kingdom
    You’re the key to the kingdom

    Post: Tiwa Savage
    Here’s some things you have to know it go rough for man to grow
    When you feel you had enough You gotta breathe
    Just remember who you are
    If you forget look to the stars
    Even the strong get weak
    But you’re the key , you’re the key

    Verse 2: Tiwa Savage
    Baby you’re the key
    It cost my life to pave the way
    You get it for free
    (Call on me when you need)
    It’s obvious to me
    Bloodline is royalty passed to you when I leave But
    (Call on me when you need)

    Pre Hook: Tiwa Savage
    Tick tock pretty baby
    You can’t get it back don’t you waste it
    To grow from your past

    PERFORMED BY TIWA SAVAGE, MR EAZI / WRITTEN BY BEYONCÉ, KIM KRYSIUK PKA “KAYDENCE”, RICKIE TICE PKA “CASO”, AKIL “FRESH” KING, TIWATOPE SAVAGE, GERALD “MIXEDBYGWHITE” WHITE, ARIOWA IROSOGIE, RICH KING, OLUWATOSIN OLUWOLE AJIBADE, RONALD BANFUL, DEREK JAMES DIXIE, RICHARD ISONG/ PUBLISHED BY OAKLAND 13 MUSIC (ASCAP), ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, SEVEN CORNERS/BMG (BMI), PEN PIPER PUBLISHING(BMI), 3M PUBLISHING/BMG RIGHTS, SONYATV/TIWASAVAGE PUBLISHING (ASCAP), GERALD WHITE PUB DESIGNEE (BMI), WARNER/CHAPPEL MUSIC LTD, HILLTOP AVENUE PUBLISHING (ASCAP), SONYATV UK (PRS), RONALD BANFUL COPYRIGHT CONTROL (PRS), PROKIZUM MUZIK PUBLISHING (SESAC), P2J MUSIC (ASCAP)/BMG RIGHTS MANAGEMENT (UK)LTD (ASCAP) / PRODUCED BY BEYONCÉ, NORTHBOI ORACLE / CO-PRODUCED BY GUILTYBEATZ, P2J, DEREK DIXIE / ADDITIONAL VOCALS BY ARI PENSMITH / BASS BY COURTNEY LEONARD / GUITAR BY ROD CASTRO / MIXED BY CHRIS GODBEY AT TIKI HILL STUDIOS DALLAS, TX AND STUART WHITE AT NRG RECORDING STUDIOS NORTH HOLLYWOOD,CA / RECORDING ENGINEERS: ARI PENSITH, DANIEL PAMPURI / MIX ASSISTANT: TYLER SCOTT, LESTER MENDOZA / ASSISTANT ENGINEER: JENNA FELSENTHAL, ZACHARY ACOSTA / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY®

    TIWA SAVAGE APPEARS COURTESY OF UNIVERSAL MUSIC GROUP
    MR EAZI APPEARS COURTESY OF EMPAWA AFRICA LTD

  • PERFORMED BY DONALD GLOVER, JOHN KANI / WRITTEN BY JEFF NATHANSON / SCORE BY BEYONCÉ, DEREK DIXIE / ADDITIONAL SOUNDS BY LESTER MENDOZA / MIXED BY LESTER MENDOZA AT NRG RECORDING STUDIOS NORTH HOLLYWOOD, CA / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY® / ©2019 DISNEY ENTERPRISES, INC.

  • Hook: Beyoncé
    Long live the king
    You a king you know it
    King already..
    Already you know it
    Top everything, everything you know it…
    King already
    Already you know it

    Verse 1: Beyoncé
    Mind body soul
    Got a king body…
    Body gon shine
    Bling bling body…
    Callin all the shots
    Ring ring body..
    Crown on ya head
    Got a king body….

    Hook: Beyoncé
    Long live the king
    You a king you know it
    King already…
    My baby you know it…
    Top everything
    Everything you know it…
    King already…
    Already you know it

    Post: Beyoncé
    Shine already…
    It’s time already
    In line already
    It’s time already
    Shine already…
    It’s time already
    In line already
    It’s time already

    Verse 2: Beyoncé
    Tried to stop it me say no, no, no
    Royalty say don’t you know
    Know, know, know
    Tried to stop it me say go, go, go
    Bubble up and watch it
    Grow, grow, grow

    Verse 3: Shatta Wale
    Every king Be Ruler,
    Be ruler yea
    Every warrior they conquer yea
    Every king be stronger yea
    King Dey rule them long yea.
    Osh Remember who you are ooh oh oh Osh Real king always win ooh oh.
    Osh You for be brave ooh oh oh.
    Osh And show your people more love ooh oh Osh
    It’s time already,
    I say it’s time already,
    In line already
    I say in line already.
    Only You got the remedy
    I say you got the remedy
    Shine your body
    Shine your body.
    Ha Ha HAahh......
    Be your own king Make nobody come rule your world.

    Yo Yo Yo......
    Be your own king Make nobody come rule your world.
    Ha Ha HAahh......
    Be your own king Make nobody come rule your world.
    Yo Yo Yo......
    Be your own king Make nobody come rule your world.
    Long Live the king
    You are a king and you know it
    Top everything, everything you know it Show them the way, you know it
    You know it, know it

    Bridge: Beyoncé
    Diamonds on my fist
    Fighting demons y’all
    Come and rest your head
    Take your crown off oh
    Woke up in a foreign
    Need to take it slow
    He said I’m moving too fast
    Said I need to take it too slow
    Take it slow take it slow
    Trying to take my baby home
    Take it slowww take it sloww

    Verse 4: Shatta Wale
    Osh remember who you are ooh oh oh
    Osh real king always win ooh oh
    Osh you for be brave ooh oh oh
    Osh and show your people more love
    Ooh oh Osh
    It’s time already
    I say it’s time already
    In line already
    I say in line already
    Only you got the remedy
    I say you got the remedy
    Shine your body
    Shine your body
    Ha Ha Haah… Be your own king
    Make nobody come rule your world
    Yo yo yo..
    Be your own king make nobody come

    Hook: Shatta Wale
    Long live the king
    You a king you know it
    Top everything, everything you know it…
    Show them the world
    You know it
    You know it
    You know it

    PERFORMED BY BEYONCÉ, SHATTA WALE, MAJOR LAZER / WRITTEN BY BEYONCÉ, BRITTANY “STARRAH” HAZZARD, TOUMANI DIABATE, CLÉMENT PICARD, MAXIME PICARD, THOMAS WESLEY PENTZ, CHARLES NII ARMAH MENSAH, RONALD BANFUL / PUBLISHED BY OAKLAND 13 MUSIC (ASCAP), ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, PEOPLE OVER PLANES (ASCAP)/THESE ARE SONGS OF PULSE, TOUMANI DIABATE PUB DESIGNEE (BMI), CLÉMENT PICARD PUB DESIGNEE (SACEM), MAXIME PICARD PUB DESIGNEE (SACEM), I LIKE TURTLES MUSIC (ASCAP), CHARLES NII ARMAH MENSAH PUB DESIGNEE (BMI), BANFUL COPYRIGHT CONTROL (PRS) / PRODUCED BY BEYONCÉ, DIPLO, PICARD BROTHERS / CO-PRODUCED BY GUILTYBEATZ / BEYONCÉ VOCALS RECORDED BY STUART WHITE AT KINGSLANDING LOS ANGELES, CA / MIXED BY CHRIS GODBEY AT TIKI HILL STUDIOS DALLAS, TX AND STUART WHITE AT NRG RECORDING STUDIOS NORTH HOLLYWOOD,CA / ASSISTANT RECORDING ENGINEER: JENNA FELSENTHAL / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY®

    SHATTA WALE APPEARS COURTESY OF FANTASY ENTERTAINMENT GROUP
    MAJOR LAZER APPEARS COURTESY OF MAD DECENT

  • As King I was most proud of one thing
    Having you as my son
    I never left you
    I never will
    Remember who you are
    Remember

    PERFORMED BY JAMES EARL JONES / WRITTEN BY JEFF NATHANSON / SCORE BY BEYONCÉ, DEREK DIXIE /ADDITIONAL VOCALS BY ERYN ALLEN KANE / MIXED BY LESTER MENDOZA, AND JOHN CRANFIELD AT NRG RECORDING STUDIOS NORTH HOLLYWOOD, CA / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY® / ©2019 DISNEY ENTERPRISES, INC.

  • If the storm comes
    If we burn up
    If the wells run dry
    You’re my reason
    To believe in
    Another life

    If it all ends
    And it’s over
    If the sky falls fire
    Best believe me
    You will see me
    On the other side

    If we wake up
    Lose our patience
    Or eben lose our lives
    I’ll feel lucky
    To say that you’ve been
    A friend of mine

    Best believe me
    You will see me
    On the other side

    Bankuli Swahili Chants
    B Swahili Chants

    PERFORMED BY BEYONCÉ / WRITTEN BY BEYONCÉ, SYDNEY BENNETT, DAVE ROSSER, NICK GREEN, ABISAGBOOLA “BANKULLI” OLUSEUN / PUBLISHED BY OAKLAND 13 MUSIC (ASCAP), ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, SONGS OF KOBALT MUSIC PUBLISHING (BMI), DAVE ROSSER MUSIC (BMI)/THESE ARE PULSE SONGS (BMI), SONGS FOR DON (BMI)/THESE ARE PULSE SONGS (BMI), ABISAGBOOLA OLUSEUN PUB DESIGNEE / PRODUCED BY BEYONCÉ, NICKY DAVEY / ADDITIONAL PRODUCTION BY DEREK DIXIE, SYDNEY BENNETT / ADDITIONAL VOCALS BY BANKULLI RECORDED AT PARKWOOD WEST STUDIOS LOS ANGELES, CA / BEYONCÉ VOCALS RECORDED BY STUART WHITE AT KINGSLASNDING LOS ANGELES, CA / MIXED BY TONY MASERATI AT MIRRORBALL STUDIOS NORTH HOLLYWOOD, CA AND STUART WHITE AT NRG RECORDING STUDIOS NORTH HOLLYWOOD, CA / RECORDING ENGINEER: DEREK DIXIE, LESTER MENDOZA, DANIEL PAMPURI / ASSISTANT RECORDING ENGINEER: JENNA FELSENTHAL / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY®



    WAR (NALA INTERLUDE)

    PERFORMED BY BEYONCÉ KNOWLES-CARTER / WRITTEN BY JEFF NATHANSON / SCORE BY BEYONCÉ, DEREK DIXIE/MIXED BY ZACHARY ACOSTA AND JOHN CRANFIELD AT NRG RECORDING STUDIOS NORTH HOLLYWOOD, CA / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY® / ©2019 DISNEY ENTERPRISES, INC.

  • Your reign is over Scar
    If you want to get him, you’ll have to get through us
    Are you with me lions?

    PERFORMED BY BEYONCÉ KNOWLES-CARTER / WRITTEN BY JEFF NATHANSON / SCORE BY BEYONCÉ, DEREK DIXIE/MIXED BY ZACHARY ACOSTA AND JOHN CRANFIELD AT NRG RECORDING STUDIOS NORTH HOLLYWOOD, CA / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY® / ©2019 DISNEY ENTERPRISES, INC.

  • Hook: Nija
    They’ll never take my power
    My power, my power
    They’ll never take my power
    My power, my power
    They feel a way Oh wow
    They feel a way Oh wow

    They’ll never take my power
    My power, my power
    They’ll never take my power
    My power, my power
    They feel a way Oh wow
    They feel a way Oh wow

    Verse 1: Tierra Whack
    I was always in the lead
    Who ya wanna be?
    I’m who they wanna be?
    B E A U T Y
    Never seen so much rage from a Queen
    Rage from a Queen
    Rage from a Queen
    Queen so strong
    Thought she was a machine
    Girl of ya dreams
    Synclaire Regine
    Turned to the max
    Can’t forget Maxine

    Refer to me as a Goddess!
    I’m tired of being modest
    100 degrees the hottest
    Ebony and Ebonics
    Black people win
    They say we being demonic
    Angel in disguise
    I hate I have to disguise it
    Why you gotta despise it
    Rich in the mind, That’s why I’m making deposits
    I carry all the power
    Time to realize it!

    Hook: Nija
    They’ll never take my power
    My power, my power
    They’ll never take my power
    My power, my power
    They feel a way Oh wow
    They feel a way Oh wow

    They’ll never take my power
    My power, my power
    They’ll never take my power
    My power, my power
    They feel a way Oh wow
    They feel a way Oh wow

    Verse 2: Beyoncé
    This that Rhythm
    This that lightening
    This that burn
    This ain’t no Perm
    This that Nappy
    This that herb
    This that Kinfolk
    This that Skinfolk
    This that war
    This that bloodline
    On the frontline
    Ready for war
    Where you gon run
    Ima get loose get loose
    Get low get low
    Ima get loose get loose
    Get low get low
    Gotta protect my grace
    Keep it locked in a safe
    Don’t make me get back to my ways
    My power they’ll never take

    Hook: Nija
    They’ll never take my power My power, my power They’ll never take my power My power, my power They feel a way Oh wow They feel a way Oh wow
    They’ll never take my power My power, my power They’ll never take my power My power, my power They feel a way Oh wow They feel a way
    Oh wow

    Vumani bo! (Do you agree/Are you with me?)
    Syavuma!!! (We agree/ We are with you)
    Selingenile iKumkani (The King has arrived)
    Ningangabazi amandi' ami (Do not doubt my power)
    Ngizo gobis' abanenkani (I will persuade those who are stubborn)

    Thulani bo! (Be Quiet)
    Senghleli!!! (We are still)
    Selivukil' iDimoni lami (My inner-demons have awakened)

    Nibabuzil' abaziyo ngam (Have you asked those who know of me)
    There will be peace when I'm done

    Jongani bo! (Look here)
    Syabona!!! (We See)
    Ndim neSkeem sami (It is me & my 'squad')
    Sizilethile Izikhali (We have prepared weapons)
    La amagwal awahlangani (Cowards have no place here)

    Hlalani bo (Sit! *plural*)
    Ibamb'Umthetho (be still & obedient)
    Niyabon' iyinkinga lento (can you see we have a challenge/predicament?)
    Soy'lungisa (We will resolve it)
    Themba Mina (Put your trust in me)
    Qoq amaqhawe! Soy'qeda manje! (Gather the Troops! We will finish it now!)

    Hook: Nija
    They’ll never take my power My power, my power They’ll never take my power My power, my power They feel a way Oh wow They feel a way Oh wow
    They’ll never take my power My power, my power They’ll never take my power My power, my power They feel a way Oh wow They feel a way
    Oh wow

    Bridge: Yemi Alade
    You no fit to touch am O
    You no fit to touch am O

    Verse 5: Moonchild
    Boom Boom check you later
    I roll with the danger
    Andoyiki n'inja
    (I’m not even scared of dogs)
    Mina ndili ninja
    (Because I’m a ninja)

    Boom Boom check you later
    I roll with the danger
    Andoyiki n'inja
    (I’m not even scared of dogs)
    Mina ndili ninja
    (Because I’m a ninja)

    PERFORMED BY NIJA, BEYONCÉ, BUSISWA, YEMI ALADE, TIERRA WHACK, MOONCHILD SANELLY, DJ LAG / WRITTEN BY BEYONCÉ, BUSISWA GQULU, YEMI ALADE, TIERRA WHACK, SANELISIWE TWISHA ,NIJA CHARLES PKA NIJA, DENISIA “@BLU_JUNE” ANDREWS FOR @NOVAWAV, BRITTANY “@CHI_CONEY” CONEY FOR @NOVAWAV, LWAZI ASANDA GWALA/PUBLISHED BY OAKLAND 13 MUSIC (ASCAP), ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, SON/ATV MUSIC PUBLISHING (SAMRO), EFFYZZIE MUSIC LTD (PRS), TIERRA WHACK PUB DESIGNEE UNIVERSAL MUSIC PUBLISHING GROUP (ASCAP), MOONCHILD SANELLY PUB DESIGNEE (SAMRO), SONGS OF UNIVERSAL, INC. AND SONGS BY AMNIJA. ALL RIGHTS FOR SONGS BY AMNIJA CONTROLLED AND ADMINISTERED BY SONGS OF UNIVERSAL, 114 SOUNDS/W.B.M MUSIC CORP (SESAC), CONEY COSMOS MUSIC PUBLISHING/WARNER-TAMERLANE PUBLISHING CORP. (BMI), DJ LAG PUB DESGINEE / PRODUCED BY BEYONCÉ, DJ LAG / ADDITIONAL PRODUCTION BY DEREK DIXIE / BEYONCÉ VOCALS RECORDED BY STUART WHITE AT KINGSLANDING LOS ANGELES, CA / BUSISWA VOCALS RECORDED BY GUILTYBEATZ AT EIGHT MUSIC STUDIOS IN JOBURG, SA / YEMI ALADE VOCALS RECORDED BY P2J AT PARKWOOD WEST STUDIOS LOS ANGELES, CA / TIERRA WHACK VOCALS RECORDED BY KENETE SIMMS AT NAKED PEOPLE STUDIOS PHILADELPHIA, PA / MOONHILD SANELLY VOCALS RECORDED BY XIVONAKI MANZINI AT THE COUSINS X AQUANOTE PRODUCTIONS SOUTH AFRICA AND ZAIN VALLY AT MAD COW STUDIO (ZA) / NIJA VOCALS RECORDED BY NIJA AT PARKWOOD WEST STUDIOS LOS ANGELES,CA / MIXED BY STUART WHITE AT NRG RECORDING STUDIOS NORTH HOLLYWOOD, CA / RECORDING ENGINEER: JENNA FELSENTHAL, DANIEL PAMPURI / MIX ASSISTANT: JOHN CRANGIELD / ASSISTANT RECORDING ENGINEER: JENNA FELSENTHAL, ZACHARY ACOSTA / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY®

    NIJA APPEARS COURTESY OF LEGION
    BUSISWA APPEARS COURTESY OF BUSISWA ENTERTAINMENT
    YEMI ALADE APPEARS COURTESY OF EFFYZZIE MUSIC LTD
    TIERRA WHACK APPEARS COURTESY INTERSCOPE RECORDS
    MOONCHILD SANELLY APPEARS COURTESY OF BAND OF WOLVES
    DJ LAG APPEARS COURTESY OF HYPERDUB

  • Give me one good reason why I shouldn’t rip you apart
    I can give you more than one

    PERFORMED BY DONALD GLOVER, CHIWETEL EJIOFOR / WRITTEN BY JEFF NATHANSON / SCORE BY BEYONCÉ, DEREK DIXIE, P2J / MIXED BY ZACHARY ACOSTA, AND JOHN CRANFIELD AT NRG STUDIOS IN NORTH HOLLYWOOD, CA/MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY® / ©2019 DISNEY ENTERPRISES, INC.

  • Verse 1: 070 Shake
    I wonder
    How and why it went so wrong
    It’s been so dark yea
    I wanna say my last goodbyes and Iʼll be gone For good left you guys behind oh
    Iʼm so lonely

    Verse 1: Jessie Reyez
    In the jungle
    Aint no bible
    Show time show time
    No recital
    Show no fear boy
    Where’s your spinal
    Where’s your back bone
    Where’s your pyro
    I had to be everything you couldn’t be for my survival
    My own idol
    Skipped my trial
    And took my rightful
    Rightful title

    No remorse
    The sin is final
    I found all my
    Own disciples
    I’m too far gone
    Down this spiral
    No white angels
    Just albinos
    Check my eyes man, they’re all white man, rolling back now
    where’d the light go
    Guess it died so
    Cold like kryo
    Crack my kneck back like in the chiro eeeeeee

    Hook: Jessie Reyez
    I’ve waited too long for this
    I need this like oxygen
    I’ve waited too long for this
    I need this like oxygen
    I’ve waited too long for this
    I need this like oxygen
    Dark side is fine by me

    Verse 2: 070 Shake
    Sorry
    Sorry
    My own family
    I slayed you
    Betrayed you
    Donʼt you grow to be like me Iʼm under
    Sinking to the darkest sea The darkest sea

    PERFORMED BY 070 SHAKE, JESSIE REYEZ / WRITTEN BY BEYONCÉ, DANIELLE BALBUENA, JESSIE REYEZ, RICHARD ISONG, TIM SUBY, DAVE HAMELIN, ARIOWA IROSOGIE / PUBLISHED BY OAKLAND 13 MUSIC (ASCAP), ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, DANIELLE BALBUENA MUSIC (BMI), FMLY USA/BMG GOLD SONGS (SOCAN), P2J MUSIC (ASCAP)/BMG RIGHTS MANAGEMENT (UK)LTD (ASCAP), NYIAN KING / EMI POP MUSIC PUBLISHING(GMR), ELECTRONIC BONGO PRODUCTIONS / SOUTHERN MUSIC PUBLISHING CO CANADA LTD., WARNER/CHAPPELL MUSIC LTD (ASCAP) / PRODUCED BY BEYONCÉ, P2J, TIM SUBY, DAVE HAMELIN / CO-PRODUCED BY ARI PENSMITH / ADDITIONAL PRODUCTION BY DEREK DIXIE, MIKE DEAN / ADDITIONAL KEYS BY ARI PENSMITH / RECORDING ENGINEER: DANIEL PAMPURI / 070 SHAKE VOCALS RECORDED BY JENNA FELSENTHAL AT PARKWOOD WEST STUDIOS LOS ANGELES, CA / JESSIE REYEZ VOCALS RECORDED BY SPENCER “MOOSE” MUSCIO AT LEFT FIELD STUDIOS TORONTO,CA / MIXED BY STUART WHITE AT NRG RECORDING STUDIOS NORTH HOLLYWOOD, CA / MIX ASSISTANT: JOHN CRANFIELD / ASSISTANT RECORDING ENGINEER: JENNA FELSENTHAL, ZACHARY ACOSTA / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY®

    070 SHAKE APPEARS COURTESY OF DEF JAM RECORDINGS
    JESSIE REYEZ APPEARS COURTESY OF ISLAND RECORDS

  • The kings time as ruler rises and falls like the sun
    One day Simba The sun will set on my time here and will rise with you as the new king
    Simba
    Im here mother. Im home.

    PERFORMED BY JAMES EARL JONES, ALFRE WOODARD, DONALD GLOVER / WRITTEN BY JEFF NATHANSON / SCORE BY BEYONCÉ, DEREK DIXIE / ADDITIONAL VOCALS BY MZANSI YOUTH CHOIR (SOUTH AFRICA) / MZANSI YOUTH CHOIR RECORDED BY JAMES BASSINGWAIGHTE / MIXED BY ZACHARY ACOSTA AT NRG STUDIOS NORTH HOLLYWOOD, CA / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY® / ©2019 DISNEY ENTERPRISES, INC.

  • Verse 1:
    Yeah and the wind is talking
    Yeah for the very first time
    Will the melody that pulls you towards it
    Paint in pictures of paradise
    Saying

    Pre:
    Rise up, to the light in the
    Sky yeah, watch the light lift your
    Heart up, burn your flame through the night
    Wooo

    Chorus:
    Spirit
    Watch the heavens open (open) yeah
    Spirit
    Can you hear it calling (calling) yeah

    Verse 2:
    Yeah and the waters crashing
    Trying to keep your head up high
    While you’re trembling
    That’s when the magic happens
    And the stars gather by
    By your side, side yeah

    Pre:
    Rise up, to the light in the
    Sky yeah, let the light lift your
    Heart up burn your flame through the night
    Woooo

    Chorus:
    Spirit
    Watch the heavens open (open) yeah
    Spirit
    Can you hear it calling (calling) yeah

    Post:
    Your destiny is coming close
    Stand up and fight
    Ohhhh

    Bridge:
    So go into that far off land
    And be one with the great I am
    I am boy becomes a man
    Wooo

    Chorus:
    Spirit
    Watch the heavens open (open) yeah
    Spirit
    Can you hear it calling (calling) yeah

    Chorus:
    Spirit
    Watch the heavens open (open) yeah
    Spirit
    Can you hear it calling (calling) yeah

    Your destiny is coming close
    Stand up and fight
    Ohhhh

    So go into that far off land
    And be one with the great I am

    PERFORMED BY BEYONCÉ / WRITTEN BY BEYONCÉ, TIMOTHY MCKENZIE, ILYA SALMANZADEH / PRODUCED BY BEYONCÉ, TIMOTHY MCKENZIE, ILYA FOR MXM PRODUCTIONS, LABRINTH / PUBLISHED BY OAKLAND 13 MUSIC (ASCAP), ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, MXM ADMINISTERED BY KOBALT (ASCAP)/WOLF COUSINS (STIM)/WARNER CHAPPELL MUSIC SAND (STIM) / BMG PLATINUM SONGS (BMI), ALL RIGHTS ADMINISTERED BY BMG RIGHTS MANAGEMENT (US) LLC., / PRODUCED BY BEYONCÉ, ILYA FOR MXM PRODUCTIONS, LABRINTH / VOCAL PRODUCTION BY BEYONCÉ / BEYONCÉ VOCALS RECORDED BY STUART WHITE AT KINGSLANDING LOS ANGELES, CA / DRUMS BY LABRINTH, ILYA, DEREK DIXIE / PROGRAMMING AND ARRANGEMENT BY LABRINTH, ILYA, BEYONCÉ / CHOIR PRODUCED BY DEREK DIXIE AT NRG RECORDING STUDIOS NORTH HOLLYWOOD, CA / CHOIR RECORDED BY DANIEL PAMPURI AND LESTER MENDOZA AT NRG RECORDING STUDIOS NORTH HOLLYWOOD, CA / MIXED BY STUART WHITE AT NRG STUDIOS NORTH HOLLYWOOD, CA / ASSISTANT ENGINEER CORY BICE, JEREMY LERTOLA, SAM HOLLAND, ANDREA ROBERTS, STEVEN XAI / MASTERED BY COLIN LEONARD AT SING MASTERING IN ATLANTA, GA USING SING TECHNOLOGY®

Homecoming (2019)

Homecoming
  • Slater Thorpe:
    Ladies and Gentleman welcome to Beyoncé Homecoming 2018

    PERFORMED BY BEYONCÉ / ANNOUNCER: SLATER THORPE / WRITTEN BY BEYONCÉ, QUINCY JONES, CHARLES EMANUEL SMALLS, MICHAEL COOPER, ASHETON TERRENCE O’NIEL HOGAN, KENDRICK LAMAR, ANTHONY TIFFITH, MICHAEL WILLIAMS, SHAWN C. CARTER, ELBERNITA CLARK, AND ERNEST DION WILSON / PUBLISHED BY OAKLAND 13 MUSIC (ASCAP) ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, YELLOWBRICK ROAD MUSIC (ASCAP), WB MUSIC CORP (ASCAP) KIDADA MUSIC (BMI), WARNER-TAMERLANE PUB (BMI), BRAVO AND ENCORE MUSIC (BMI), WARNER -TAMERLANE PUBLISHING CORP (BMI), HARD WORKING BLACK FOLKS INC (ASCAP), TOP DAWG MUSIC (ASCAP), WB MUSIC CORP (ASCAP), SONGS OF KOBALT MUSIC PUBLISHING (BMI), SOUNDS FROM EARDRUMMERS (ASCAP), CARTER BOYS MUSIC (ASCAP) ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. SONGS BY TWINKIE MUSIC PUBLISHING (BMI), OAKLAND 13 MUSIC (ASCAP) ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, LET THE STORY BEGIN PUBLISHING (ASCAP) AND BMG MONARCH (ASCAP) / PRODUCED BY BEYONCÉ AND DEREK DIXIE

  • Intro: Beyoncé Coachella.You ready? Let’s go get ‘em

    JAY-Z Background Vocals:
    Yes, so crazy right now
    It's so crazy right now
    Most incredibly, it's ya girl, B
    History in the making
    Part two, so crazy right now

    Verse 1: Beyoncé
    I look and stare so deep in your eyes
    I touch on you more and more every time
    When you leave, I'm begging you not to go
    Call your name two, three times in a row
    Such a funny thing for me to try to explain
    How I'm feeling and my pride is the one to blame
    I still don't understand
    Just how your love can do what no one else can

    Chorus: Beyoncé
    Got me lookin’ so crazy right now, your love’s
    Got me lookin’ so crazy right now your love
    Got me lookin’ so crazy right now, your touch
    Got me lookin’ so crazy right now your touc)
    Got me hopin’ you'll page me right now, your kiss
    Got me hopin’ you'll save me right now
    Lookin’ so crazy in love's
    Got me lookin’, got me lookin’
    Lookin’ so
    Lookin’ so
    Lookin’ so

    Big Sean: Drop it

    Bridge: Beyoncé
    Uh oh, uh oh, uh oh, oh, no, no
    Uh oh, uh oh, uh oh, oh, no, no
    Uh oh, uh oh, uh oh, oh, no, no
    Uh oh, uh oh, uh oh, oh, no, no

    Verse 2: Beyoncé
    When I talk to my friends so quietly
    Who he think he is? Look at what you did to me
    Tennis shoes, don't even need to buy a new dress
    If you ain't there, ain't nobody else to impress
    It's the way that you know what I thought I knew
    It's the beat in my heart skips when I'm with you
    I still don't understand
    Just how your love can do what no one else can

    Chorus: Beyoncé
    Got me lookin’ so crazy right now
    Got me lookin’ so crazy right now
    Got me lookin’ so crazy right now
    Crazy right now
    Crazy right now
    Got me lookin’ so crazy right now
    Got me lookin’ so crazy right now
    Crazy right now
    Crazy right now

    Beyoncé: Coachella, Y’all dance with me, Come on.

    JAY-Z Background Vocals:
    N---a ask
    N---a N---a ask about me
    N---a ask
    N---a N---a ask about me
    N---a ask
    N---a ask
    N---a N---a ask about me
    N---a ask

    PERFORMED BY BEYONCÉ / WRITTEN BY BEYONCÉ, SHAWN CARTER, RICHARD HARRISON, EUGENE RECORD, DWAYNE CARTER, TERIUS GRAY, BYRON O. THOMAS, TIM MOSLEY, SEAN MICHAEL ANDERSON, STANLEY KIRK BURRELL, ERNEST CLARK, JAMES JOHNSON, ALONZO MILLER, MARCOS PALACIOS / PUBLISHED BY BEYONCÉ PUBLISHING (ASCAP), WB MUSIC CORP (ASCAP), CARTER BOYS PUBLISHING (ASCAP), LIL LU LU PUBLISHING (ASCAP), EMI BLACKWOOD MUSIC INC (BMI), UNICHAPPEL MUSIC INC (BMI), MONEY MACK MUSIC (BMI), BREKA MUSIC (BMI) AND FRESH IS THE WORD (BMI), PUBLISHED BY SEAN MICHAEL ANDERSON MUSIC LLC (BMI), BUST IT PUBLISHING (BMI), TWO WORKS (ASCAP), SONY/ATV TUNES LLC (ASCAP), JOBETE MUSIC CO INC (ASCAP), FF TO DEF PUBLISHING LLC (BMI), SONGS OF UNIVERSAL INC (BMI), STONE DIAMOND MUSIC CORPORATION (BMI) AND VIVA PANAMA (ASCAP) / PRODUCED BY BEYONCÉ AND DEREK DIXIE

  • Verse 1: Beyoncé
    Trying to reign
    Trying to rain on the thunder
    Tell the storm I’m new
    I’ma walk
    I’ma march
    On the regular
    Painting white flags blue
    Lord forgive me I’ve been running
    Running blind in truth
    I’ma rain
    I’ma reign
    On this bitter love
    Tell the sweet I’m new

    I’m telling these tears gonna fall away fall away… oooh
    May the last one burn into flames

    Chorus: Beyoncé
    Freedom
    Freedom
    I can’t move
    Freedom cut me loose
    Freedom
    Freedom
    Where are you?
    Cause I need freedom too
    I break chains all by myself
    Won’t let my freedom rot in hell
    I’ma keep running cause a winner don’t quit on themselves

    Kendrick Lamar Background Vocals:
    Huh what you want from me is it true you see uh oh father can you hear me?
    Huh what you want from me is it true you see uh oh father can you hear me?
    Hear me?

    PERFORMED BY BEYONCÉ / WRITTEN BY JONATHAN COFFER, BEYONCÉ, CARLA WILLIAMS, ARROW BENJAMIN, KENDRICK DUCKWORTH, FRANK TIRADO, ALAN LOMAX, JOHN LOMAX SR, CALVIN BROADUS, AWOOD JOHNSON, CRAIG LAWSON, COREY MILLER / PUBLISHED BY B-UNIQUE MUSIC LIMITED/KOBALT PUBLISHING AMERICA INC (BMI), WB MUSIC CORP (ASCAP), OAKLAND 13 MUSIC (ASCAP), ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, EMI BLACKWOOD MUSIC INC (BMI), SONY/ATV SONGS (BMI) OWN CHIT PUBLISHING (BMI), ULTRA EMPIRE MUSIC (BMI), BLOCK OFF BROAD PUBLISHING (BMI) AND L P BOYZ MUSIC LLC (BMI) / PRODUCED BY BEYONCÉ AND DEREK DIXIE

  • Beyoncè:
    Lift every voice and sing
    Till earth and heaven ring
    Ring with the harmonies of Liberty
    Let our rejoicing rise
    High as the list'ning skies
    Let us march on till victory is won

    PERFORMED BY BEYONCÉ / WRITTEN BY JAMES WELDON JOHNSON

  • Intro: Big Freedia

    Baby, I'm back by popular demand
    B----, I’m back by popular demand
    I did not come to play with you h---
    I came to slay b----

    Refrain: Beyoncé
    Ya’ll haters corny with that illuminati mess
    Paparazzi catch my fly and my cocky fresh
    I’m so reckless when I rock my givenchy dress
    I’m so possessive so I rock his roc necklaces
    My daddy Alabama
    Mama Louisiana
    You mix that negro with that creole make a Texas-bama
    I like my baby hair with baby hair and afros
    I like my negro nose with Jackson 5 nostrils
    I earned all this money but they never take the country out me
    I got hot sauce in my bag… swag

    Chorus: Beyoncé
    I see it I want it
    I stunt yellow hornet
    I dream it I work hard I grind till own it
    I twirl on them haters
    Albino alligators
    El camino with the seat low sipping cuervo with no chaser
    Sometimes I go off
    I go hard
    Get what’s mine
    I’m a star
    Cause I slay… I slay I slay I slay
    I slay I slay I slay I slay
    I’m gon’ slay I slay I slay I slay
    All day I slay I slay okay okay

    Ok ladies now let’s get in formation
    Ok ladies now let’s get in formation
    Prove to me you got some co-ordination
    Slay trick or you get eliminated
    When he f--- me good i take his ass to red lobster
    when he f--- me good i take his ass to red lobster
    If he hit it right I might take him on a flight on my chopper
    Drop him off at the mall let him buy some j’s let him shoppa

    I might get your song played on the radio station
    I might get your song played on the radio station
    You just might be a black bill gates in the making
    I just might be a black bill gates in the making

    Chorus: Beyoncé

    I see it I want it
    I stunt yellow hornet
    I dream it I work hard I grind till I own it
    I twirl on my haters
    Albino Alligators
    El camino with the seat low sipping cuervo with no chaser
    Sometimes i go off
    I go hard
    Take what’s mine
    I’m a star
    Cause I slay… I slay I slay I slay
    I slay I slay I slay I slay
    We gon’ slay gon’ slay we slay i slay
    I slay okay I slay okay okay okay

    Ok ladies now let’s get in formation
    Ok ladies now let’s get in formation
    Prove to me you got some co-ordination
    Slay trick or you get eliminated
    Ok ladies now let’s get in formation
    Ok ladies now let’s get in formation
    You know you that b---- when you cause all this conversation
    Always stay gracious
    Best revenge is your paper

    Outro: Nina Simone
    I think what you are trying to ask is why am I so insistent upon giving out to them that blackness that black power that black…pushing them to identify with black culture.
    I think that’s what you’re asking. It’s…I have no choice over it in the first place.
    To me we are the most beautiful creatures in the whole world. black people.
    My job is to somehow make them curious enough or persuade them by hook or crook to get more aware of themselves and where they came from and what they are into and what is already there and just to bring it out. This is what compels me to compel them. And I will do it by whatever means necessary.

    PERFORMED BY BEYONCÉ / WRITTEN BY BEYONCÉ, MICHAEL L. WILLIAMS II, KHALIF BROWN, ASHETON HOGAN / PUBLISHED BY OAKLAND 13 MUSIC (ASCAP) ALL RIGHTS ADMINISTERED BY WB MUSIC CORP OBO ITSELF AND OAKLAND 13 MUSIC, SOUND FROM EARDRUMMERS LLC ASHETON HOGAN BMI PUB DESIGNEE, WARNER-TAMERLANE PUBLISHING CORP (BMI) OBO ITSELF EARDRUMMERS MUSIC PUBLISHING LLC (BMI) AND KHALIF BROWN BMI PUB DESIGNEE / PRODUCED BY BEYONCÉ AND DEREK DIXIE / CONTAINS AN EXCERPT OF NINA SIMONE’S “THAT BLACKNESS” SPEECH, “NINA SIMONE: THAT BLACKNESS”, FEBRUARY 2013. EXCERPT COPYRIGHT © 2013 BY NINA SIMONE

  • I grew up in Houston, Texas and visiting Prairie View
    We rehearsed at TSU for many years in Third Ward
    And I always dreamed of going to an HBCU
    My college was Destiny’s Child
    My college was traveling around the world and life was my teacher
    I wanted a black orchestra, I wanted the steppers, I needed the vocalists
    I wanted different characters, I didn’t want us all doing the same thing
    The amount of swag is just limitless, like the things that these young people can do with their bodies and the music they can play and the drumrolls, and the haircuts and the bodies.
    It’s just not right, it’s just so much d--- swag

    PERFORMED BY BEYONCÉ / WRITTEN BY BEYONCÉ / PUBLISHED BY OAKLAND 13 MUSIC (ASCAP) ALL RIGHTS ADMINISTERED BY WB MUSIC CORP OBO ITSELF AND OAKLAND 13 MUSIC

  • Beyoncé: Let me hear you sing

    Intro: Beyoncé
    I ain’t sorry
    I ain’t sorry
    I ain’t sorry
    N---- nah
    I ain’t sorry
    I ain’t sorry
    I ain’t sorry

    You trying to roll me up I ain’t sorry
    I ain’t picking up I ain’t sorry
    I’m headed to the club I ain’t sorry
    I ain’t thinking ‘bout you I ain’t sorry

    Me and my ladies sip my D'ussé cups
    I don’t give a f---- chucking my deuces up
    Suck on my b--- pause

    Beyoncé: Bug A Boos.
    I need a good laugh
    You Make me laugh
    Bug A Boo 1: Bug a Boos!
    Bug A Boos: ooooooooahhhhhhhh
    Beyoncé and Bug A Boos: laughing
    Beyoncé: Did that make us laugh?
    Audience: no!
    Beyoncé: Think about it.
    Bug A Boo 1: Bug a Boos!nBuuzzzzzzzzzz
    Beyoncé: Fall in line.
    Ladies. Ladies. Ladies.
    Are we smart?
    Audience: yes!
    Beyoncé: Are we strong?
    Audience: yes!
    Beyoncé: Have we had enough of the bullshit?
    Audience: yes!
    Beyoncé: Show em.
    Beyoncé + Performers:
    Suck on my b----
    Suck on my b----pause

    Suck on my b----
    Suck on my b----
    Suck on my b----
    Suck on my b----pause

    Hell na
    Hell na
    Middle Fingers up
    Middle Fingers up
    Put em hands high
    Put em hands high
    Wave it in his face
    Wave it in his face
    Tell him boy bye
    Tell him, tell him boy bye
    Tell him boy bye
    Tell him, tell him boy bye
    Tell him boy bye
    Tell him, tell him boy bye
    Tell him boy bye
    Tell him, tell him boy bye
    I ain’t think about you.
    Ugh

    Verse 2: Beyoncé
    Middle fingers up
    Put them hands high
    Wave it in his face
    Tell ‘em boy bye
    Tell ‘em boy bye
    Boy bye
    Middle fingers up
    Iain’t thinking 'bout you

    I ain’t sorry
    Iain’t sorry
    Iain’t sorry
    I ain’t thinking 'bout you
    I ain’t sorry
    I ain’t sorry
    I ain’t sorry
    No No , hell nah

    Now you want to say you're sorry
    Now you want to call me crying
    Now you gotta see me wilding
    Now I'm the one that's lying
    And I don't feel bad about it
    It's exactly what you get
    Stop interrupting my grinding

    Bridge: Beyoncé
    I can't believe I believed everything we had would last
    So young and naive for me to think she was from your past
    Silly of me to dream of one day havin' your kids
    Love is so blind it feels right when it's wrong
    I can't believe I fell for your schemes, I'm smarter than that
    So dumb and naive to believe that with me you're a changed man
    Foolish of me to compete when you cheat with loose women
    It took me some time, but now I am strong
    Because I realized I got
    Me, myself, and I, that's all I got in the end
    That's what I found out
    And it ain't no need to cry

    Beyoncé:I see you, how did you do that so fast?
    She has on my outfit y’all

    Sing it
    Sing it
    Sing it Ladies

    From now on I’ma be my own bestfriend
    Right Ladies?

    Verse 3: Beyoncé
    I left a note in the hallway
    By the time you read it, I'll be far away
    I'm far away
    But I ain't f------with nobody
    Let's have a toast to the good life
    Suicide before you see this tear fall down my eyes
    Me and my baby, we gon' be alright
    We gon' live a good life
    Big homie better grow up
    Me and my whoadies 'bout to stroll up
    I see them boppers in the corner
    They sneaking out the back door
    He only want me when I'm not there
    He better call Becky with the good hair
    You better call Becky with the good hair

    PERFORMED BY BEYONCÉ / WRITTEN BY DIANA GORDON, SEAN RHODEN P/K/A MELO-X, BEYONCÉ, SCOTT STORCH AND ROBERT WALLER / PUBLISHED BY WB MUSIC CORP. (ASCAP) OBO ITSELF AND LOTS OF LYRICS ENTERTAINMENT (ASCAP) ALL RIGHTS OBO ITSELF AND LOTS OF LYRICS ENTERTAINMENT ADMINISTERED BY WB MUSIC CORP., MELOXTRA PUBLISHING (BMI), WB MUSIC CORP. (ASCAP) AND OAKLAND 13M (ASCAP) ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, BEYONCE PUBLISHING (ASCAP), WB MUSIC CORP (ASCAP), NOTTING DALE SONGS INC (ASCAP), RESERVOIR MEDIA MUSIC (ASCAP) AND BLACK OWNED MUSIK (ASCAP) / PRODUCED BY BEYONCÉ AND DEREK DIXIE

  • Verse: Beyoncé
    Rock diamonds on my neck, got diamonds on my records
    Since fifteen coming down reppin’ Texas
    How you gon' neglect this? You is just a hot mess
    You can call Tyrone, you ain'tgots to lie Craig
    What about my body, body?
    You don’t want my body, body?
    Acting like I'm nobody
    You gon' make me call somebody

    Let’s go little Kitty Kat
    I think it’s time to go
    Let’s go little Kitty Kat
    He don’t want no more
    Let’s go little Kitty Kat

    Outro: Beyoncé
    He better call Becky with the good hair

    WRITTEN BY PHARRELL WILLIAMS, S. CARTER, BEYONCÉ / PUBLISHED BY EMI POP MUSIC PUBLISHING/MORE WATER FROM NAZARETH (GMR), B DAY PUBLISHING (ASCAP) AND WB MUSIC CORP (ASCAP) / PRODUCED BY BEYONCÉ AND DEREK DIXIE

  • Call Becky with the good hair

    Beyoncé: Coachella!
    Thank y’all so much for having us once again.
    I’m so honored to be on this stage tonight.
    This next song is for all of my queens.
    Do we have any beautiful queens in the house tonight?
    I want y’all to sing along if y’all know the lyrics.
    It’s called Bow Down.

    Verse 1: Beyoncé
    I know when you were little girls
    You dreamt of being in my world
    Don’t forget it, don’t forget it
    Respect that, bow down b----
    I took some time to live my life
    But don’t think I’m just his little wife
    Don’t get it twisted, get it twisted
    This my s---, bow down b----

    Bridge: Background track
    Bow down b----, bow bow down b----
    Bow down b----, bow bow down b----
    H-town vicious, H H-town vicious
    I’m so crown, bow bow down bitches

    I’m out that H-Town
    Coming, coming down
    I’m coming down dripping candy on the ground
    H, H-town, town, I’m coming down
    Coming down dripping candy on the ground

    PERFORMED BY BEYONCÉ / WRITTEN BY DIANA GORDON, SEAN RHODEN P/K/A MELO-X, BEYONCÉ , SHAWN C. CARTER, KIRK ANDRE BENNETT, MIGUEL COLLINS, MIKE DEAN, BOBBY DIXON, EBONY NAOMI OSHUNRINDE, JACQUES WEBSTER, DEMACIO CASTELLON, CHRIS GODBEY, LESLIE JEROME HARMON, GARLAND WAVERLY MOSLEY JR. AND TIMOTHY MOSLEY / PUBLISHED BY WB MUSIC CORP. (ASCAP) OBO ITSELF AND LOTS OF LYRICS ENTERTAINMENT (ASCAP) ALL RIGHTS OBO ITSELF AND LOTS OF LYRICS ENTERTAINMENT ADMINISTERED BY WB MUSIC CORP., MELOXTRA PUBLISHING (BMI), WB MUSIC CORP. (ASCAP), OAKLAND 13 MUSIC (ASCAP) ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, CARTER BOYS MUSIC (ASCAP), WB MUSIC CORP (BMI), DUB PLATE MUSIC PUBLISHERS LTD (ASCAP), ZABOGAUBHI MUSIC (ASCAP), PAPA GEORGE MUSIC (BMI), SONGS OF STB (BMI), WANER-TAMERLANE PUBLISHING CORP (BMI),BMG GOLD SONGS (ASCAP), TRAVIS SCOTT MUSIC (BMI), SONY/ATV BALLAD, DEMOLITION CREW PUBLISHING (ASCAP), SONY/ATV BALLAD (BMI), WARNER-TAMERLANE PUBLISHING CORP (BMI), 757 MUSIC (ASCAP) AND VB RISING PUBLISHING (ASCAP) / PRODUCED BY BEYONCÉ AND DEREK DIXIE

  • Intro:
    Coming coming down
    Dripping candy on the ground
    Coming coming down
    Dripping candy on the ground

    Beyoncé:Listen

    Verse: Beyoncé
    I been on, I been on, I been on
    Tell me who gon' take me off
    Take me off, take me off, take me off
    I been on, I been on, I been on
    Tell me who gon' take me off
    Take me off, take me off, take me off

    Rolling high, leather and wood
    Keep it trill, that's what good
    Kiss my momma, show that love
    Pop them bottles in that club
    I heard your boo was talking lip
    I told my crew to smack that trick
    Smack that trick, smack that trick
    Guess what they did, smack that trick
    Gold everything, gold a-- chains
    Gold a-- rings, gold a-- fangs
    You can see me stunt when you turn on ya screen
    You can see me stunt when you turn on ya screen
    I'm bigger than life, my name in the lights
    I'm the capital B, I don't need no hype
    The capital B means, I'm 'bout that life
    The capital B means, I'm 'bout that life b----

    PERFORMED BY BEYONCÉ / WRITTEN BY BEYONCÈ, POLOW DA DON, TIMOTHY MOSLEY, JONATAN "ANONYMOUS" SOLONNE-MYVETT, THERON THOMAS, TIMOTHY THOMAS, SONNY COREY UWAEZUOKE, CRAIG BAZILE, PERCY MILLER AND MIA YOUNG / PUBLISHED BY OAKLAND 13 MUSIC (ASCAP) ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, MY DIET STARTS TOMORROW INC/SONGS OF UNIVERSAL INC (BMI), VB RISING MUSIC PUBLISHING/OLE MEDIA MANAGEMENT LP II (ASCAP), I LOVE BELIZE PUB, U CAN’T TEACH BEIN THE SHH INC (BMI), T-N-T EXPLOSIVE MUSIC PUBLISHING/ UNIVERSAL MUSIC CORPORATION(ASCAP), SONY DIGITAL MUSIC GROUP/BANG VILLAGE 247 PUBLISHING LLC/IRVING MUSIC (BMI), BLOCK OFF ROAD PUBLISHING (BMI) AND LP BOYZ MUSIC LLC (BMI) / PRODUCED BY BEYONCÉ AND DEREK DIXIE

  • Verse 1: Beyoncé
    I've been drinkin’, I've been drinkin’
    I get filthy when that liquor get into me
    I've been thinkin’, I've been thinkin’
    Why can't I keep my fingers off it, baby I want you, nana
    Why can't I keep my fingers off it, baby I want you, nana

    Beyoncé: Coachella I wanna hear y’all sing

    Cigars on ice, cigars on ice
    Feeling like an animal with these cameras all in my grill
    Flashing lights, flashing lights
    You got me faded, faded, faded
    Baby, I want you, nana
    Can't keep your eyes off my fatty
    Daddy, I want you
    Drunk in love,

    Beyoncé: How y’all doing over here.

    I want you

    We woke up in the kitchen saying
    "How the hell did this shit happen?" Oh baby

    Beyoncé: I can see you all the way, all the way back there.
    Drunk in love
    We be all night
    Last thing I remember is our beautiful bodies grinding off in that club
    Drunk in love

    Beyoncé: Sing it y’all

    We be all night

    Sing!
    Come on sing!

    We be all night

    Sing!
    Hey!

    We be all night, and everything alright
    No complaints for my body, so fluorescent under these lights
    Boy, I'm drinking, park it in my lot, 7-11
    I'm rubbing on it, rub-rubbin’
    If you scared, call that reverend
    Boy I'm drinking, get my brain right
    I'm on the Armand de Brignac, yeah gangsta wife
    Louis sheets, he sweat it out, like washrags, he wet it up
    Boy, I'm drinking, I'm singin’ on the mic to my boy’s toys
    Then I fill the tub up halfway
    Then ride with my surfboard
    Surfboard, surfboard
    Grindin’ on that wood, grindin’, grindin’ on that wood
    I'm swervin’ on that, swerving-swervin’ on that
    Big body been swervin’ all this
    Surfs up
    Good good

    Bridge: Nina Simone
    I lost myself on a cool damp night
    Gave myself in that misty light
    Was hypnotized by a strange delight
    Under a lilac tree
    I made wine from the lilac tree
    Put my heart in its recipe
    It makes me see what I want to see
    And be what I want to be
    When I think more than I want to think
    Do things I never should do
    I drink much more that I ought to drink
    Because I brings me back you

    Last thing I remember is our beautiful bodies grinding off in that club
    Drunk in love
    We be all night

    Sing it!
    Hey!

    We be all night
    Riding with my surfboard
    Surfboard, surfboard

    Grindin’ on that wood, grindin’, grindin’ on that wood
    I'm swervin’ on that, swerve-swervin’ on his big body
    Ahhh! You!!

    Surfboard, surfboard, surfboard
    Grindin’ on that wood, grindin’, grindin’ on that wood
    Swervin’ on that, swerve-swervin’ on his big body
    Been Surfin on all his
    Surf Surfin on all his good good!

    Outro: Beyoncè
    To the left, to the left
    Everything you own in the box to the left
    Sing it!
    To the left, to the left
    Everything you own in the box to the left
    Say I’ma a Diva
    Sing it!
    I’ma a Diva
    Say I’ma a Diva
    Sing it!
    Louder
    I’ma a Diva
    Louder
    I’ma a Diva
    Can’t hear you
    I’ma a Diva
    Sing it!
    Come on!

    PERFORMED BY BEYONCÉ / WRITTEN BY SHAWN C. CARTER, RASOOL DIAZ, NOEL FISHER, JEROME HARMON, BEYONCÉ, TIMOTHY MOSLEY, ANDRE PROCTOR, BRIAN SOKO, JAMES EDWARD FAUNTLEROY II, CHRIS GODBEY, MIGUEL JONTEL PIMENTEL AND JUSTIN R. TIMBERLAKE, JAMES H. SHELTON, ALVITICUS BRYANT, KEVIN MICHAEL ERONDU, MICHAEL GORDON, JARED RICE, SEDARIUS SPEARMAN, KEISHAUN PATRICK WATTS, AMUND BJORKLUND, MIKKEL ERIKSEN, TOR HERMANSEN, ESPEN LIND AND SHAFFER SMITH

  • I’m-a diva
    i'm-a, i’m-a a diva
    I'm-a, i’m-a a diva
    I'm-a, i’m-a a diva
    I'm-a, i’m-a a diva
    I'm-a, i’m-a a diva
    I'm-a, i’m-a a diva
    i’m-a, i’m-a

    Na, na, na, diva is a female version of a hustla
    Of a hustla, of a, of a hustla

    Na, na, na, diva is a female version of a hustla
    Of a hustla, of a, of a hustla

    Stop the track
    Let me state facts

    I told you gimme a minute and i'll be right back
    Fifty million ‘round the world and they said that i couldn't get it
    I done got so sick and filthy with benji'si can't spend it
    Tell me somethin’ where ya boss at
    Where my ladies up in here that like to talk back
    I wanna see ha
    I'd like to meet ya
    What you said
    she ain't no diva
    No, no, no

    Beyoncé: Diva’s y’all help me sing this next verse

    Since 15 in my stilettos
    Been struttin’ in this game
    What's your age was the question they ask when i hit the stage
    I'm a diva best believe-a, you see her, she's gettin’ paid
    She ain't calling him a greeter
    Don't need him, her bed’s made

    This is a stick up, stick up
    I need them bags or that money
    I’m-a stick up, stick up, you see the mask, where that money

    All my ladies gettin’ up
    I see you, i do the same
    Take you to another level
    no passengers on my plane

    I need them bags or that money
    I’m-a stick up, stick up, you see the mask, where that money

    Outro: Everybody Mad
    I be getting to the money, everybody mad
    I think I'm getting too much money, everybody mad
    The same old nigga from the block
    The same old nigga with the pot
    The same old nigga from the trap
    Everybody hated on him, then he bounced right back

    I be getting to the money, everybody mad
    I think I'm getting too much money, everybody mad
    The same old nigga from the block
    The same old nigga with the pot
    The same old nigga from the trap
    Everybody hated on him, then he bounced right back

    I be getting to the money

    PERFORMED BY BEYONCÉ / WRITTEN BY SHONDRAE CRAWFORD, SEAN GARRET HAMLER, BEYONCÉ, AUBREY GRAHAM, ANTHONY PALMAN, MATTHEW SAMUELS, NOAH SHEBIB, JEREME ALERTA, ODIS FLORES, SHAWN C. CARTER AND TIM MOSLEY / PUBLISHED BY SONGS OF UNIVERSAL INC (BMI) TEAM S DOT PUBLISHING (BMI), B DAY PUBLISHING (ASCAP) EMI APRIL MUSIC INC (ASCAP), LEVEGAS PUBLISHING COMPANY INC (ASCAP), AUBREY DRAKE GRAHAM SOCAN PUB DESIGNEE, PARMA NINJA MUSIC (ASCAP), EMI APRIL MUSIC INC (ASCAP), RONCESVALLES MUSIC PUBLISHING (ASCAP), 1DAMENTIONAL PUBLISHING (ASCAP), SONY/ATV TUNES LLC (ASCAP), EMI BLACKWOOD MUSIC INC (BMI), LIVE WRITE LLC (BMI), NOAH SHEBIB SOCAN PUB DESIGNEE, JEREME BMI PUB DESIGNEE , SONY/ATV BALLAD (BMI), CARTER BOYS MUSIC (ASCAP), WB MUSIC CORP (ASCAP), ECAF MUSIC (BMI), SONY/ATV SONGS LLC (BMI) / PRODUCED BY BEYONCÉ AND DEREK DIXIE

  • It’s that Yoncé, your Yoncé
    In that lingerie, on that chardonnay
    Scoring touchdowns on your runway
    I’m Texas forever, like Bun B
    And I’m Redbonedyo
    I’m really rit’ like Donjae
    I’m camo in here yo
    These thots can’t clock me nowadays
    You wish I was your pound cake
    Boy, you know I look good as f---
    Wish I was your baby momma
    Want me to come around and give you good karma
    But no, we escalating, up in this b----, like elevators
    Of course sometime s--- goes down
    When it’s a billion dollars on an elevator

    Momma taught me good home training
    My Daddy taught me how to love my haters
    My sister taught me I should speak my mind
    My man made me feel so God damn fine
    I’m flawless

    Of course some s--- goes down when there’s a billion dollars on the elevator

    You wake up, flawless
    Post up, flawless
    Ride round in it, flawless
    Flossin on that, flawless
    This diamond, flawless
    My diamonds, flawless
    This rock, flawless
    My rock, flawless
    Sing!
    I woke up like this
    I woke up like this
    We flawless, ladies tell 'em
    I woke up like this
    I woke up like this
    We flawless, twins tell 'em
    Say I, look so good tonight
    G—d---, G—d---,
    Say I, look so good tonight
    G—d---, G—d---,G—d---

    How y’all feeling?
    Coachella how did you wake up this morning?

    Woke up to n---- looking like me
    Woke up to n----- talking like me

    Alight, I want y’all to repeat after me.

    I’m feelin myself.
    Say

    I'm feelin' myself, I'm feelin' myself
    I'm feelin' my, feelin' myself
    I'm feelin' myself, I'm feelin' my, feelin' my, feelin' myself
    I'm feelin' myself, I'm feelin' my, feelin' myself
    I'm feelin' myself, I'm feelin' my

    I'm feelin' myself, I'm feelin' myself
    I'm feelin' my, feelin' myself
    I'm feelin' myself, I'm feelin' my, feelin' my, feelin' myself
    I'm feelin' myself, I'm feelin' my, feelin' myself
    I'm feelin' myself, I'm feelin' my

    Changed the game with that digital drop
    Know where you was when that digital popped
    I stopped the world
    Male or female, it make no difference
    I stop the world, world, stop

    PERFORMED BY BEYONCÉ / WRITTEN BY BEYONCÉ, CHAUNCEY HOLLIS, BEYONCÉ, RAYMOND MARTIN, RASHAD MUHAMMAD, TERIUS NASH, ONIKA MARAJ, SOLÁNA ROWE SHAWN C. CARTER, KIRK ANDRE BENNETT, MIGUEL COLLINS, MIKE DEAN, BOBBY DIXON, EBONY NAOMI OSHUNRINDE, JACQUES WEBSTER, SHARON KAYE ABSHIRE, AUBREY GRAHAM, BERNARD JOSEPH GERARD, CHAUNCEY HOLLIS, RAYMOND MARTIN, NOAH SHEBIB, MARVIN THOMAS, JUSTIN GARNER, ANTOINE MCCOLISTER, WILLIAM ROBERTS, NAYVADIUS WILBURN, MICHAEL WILLIAMS, JORDAN CARTER, JORDAN JENKS, SYMERE WOODS, MARQUIS KING,MANUEL ALVARENGA, ANDRE BENJAMIN, PATRICK BROWN AND ANTWAN PATTON / PUBLISHED BY U CAN’T TEACH BEIN THE SHHH INC (BMI), SONY/ATV BALLAD (BMI), OAKLAND 13 MUSIC (ASCAP), WB MUSIC CORP (ASCAP); ALL RIGHTS ADMINISTERED BY WB MUSIC CORP.

  • DJ Khaled: Check this out
    After Beyoncé performs.
    After she done the dance.
    Coachella gotta rename Coachella to Beychella.

    New name alert!
    Beychella!

    Beyoncé:
    I’m the only lady here, still the realest n---- in the room
    I break the internet, top two and I ain’t number two
    My body, my ice, my cash, all real, I’m a triple threat
    F--- it up and then leave, come back, f--- it up and then leave again
    Top off the coupe and it look like Freaknik
    In the hood, hollerin’ “Freek Meek”
    Two deep, it’s just me and JAY
    Just posted in them courtside seats
    Woo! I’m like hol’up
    Woo! I might roll up
    If they tryna party with the queen
    They gon’ have to sign a non-disclosure, ayy

    I took the top off the Maybach, yeah
    I took the top off the Maybach, yeah
    I took the top off the Maybach, yeah
    Ayy, I took the top off of my Maybach b----
    I took the top off of my Maybach b----

    PERFORMED BY BEYONCÉ / WRITTEN BY S. CARTER, NAYVADIUS DEMUN, DENISIA ANDREWS, BRITTANY CONEY, KHALED KHALED, BEYONCÉ, JOE ZARILLO / PUBLISHED BY CARTER BOYS MUSIC (ASCAP), WARNER CHAPPEL NORTH AMERICA LIMITED (ASCAP), NAYVADIUS MAXIMUS MUSIC (BMI), 1114 SOUNDS (SESAC), WBM MUSIC (SESAC), CONEY COSMOS MUSIC PUBLISHING, WARNER-TAMERLANE PUBLISHING CORP (BMI), DJ KHALED PUBLISHING (BMI) , OAKLAND 13 MUSIC (ASCAP) WB MUSIC CORP (ASCAP) AND JOE ZARILLO PUB DESIGNEE / PRODUCED BY BEYONCÉ AND DEREK DIXIE

  • G—d---
    G—d---
    G—d---

    I know you care
    Smack it, smack it in the air

    Wave your hands side to side, put it in the air

    Clap, clap, clap like you don't care
    Smack that, clap, clap, clap like you don't care
    I know you care

    Clap, it clap, clap, clap it
    Foot up, my foot up
    Hold up now my foot up
    I'm spinnin' my foot up
    Foot up yeah my foot up
    I'm spinnin' my foot up
    Put my foot down
    Yeah my hands up

    Hold that cup like alcohol
    Hold that cup like alcohol
    Hold that cup like alcohol
    Don't you drop that alcohol
    Never drop that alcohol, never drop that alcohol
    I know I'm thinkin' bout that alcohol
    I know I'm thinkin' bout that alcohol

    Man this here like rollin' dice, d---this feel like rollin' dice
    Seven eleven, seven eleven, seven twice, man seven twice
    Man it feel like rollin' dice, d---this feel like rollin' dice
    Man it feel like rollin' dice
    Seven twice, seven twice

    Man I'm tryna kick it with you
    Damn I wanna kick it wit you
    Man I wanna kick it with you

    I spin' around and I kick it with you

    Shoulders sideways, smack it, smack it in the air
    Legs movin' side to side, smack it in the air
    Legs movin' side to side, smack it in in the air
    Shoulders move side to side smack it in the air
    Smack it in the air, smack it, smack it the in the air
    Smack it in the air, smack it, smack it in the air

    I know you care

    Wave your hands side to side
    Wave your hands side to side
    Wave your hands side to side
    Wave your hands side to side
    Ooh we Bey be freaky deaky
    Then you see she pink bikini
    Rock out that groovy dye dashiki
    Nefertiti edges kinky
    Sweatin' out my blow out
    Sweatin' on my press
    This trick about to go off
    Mad 'cause I'm so fresh
    Fresher than you
    I'm fresher than you
    Fresher than you
    Fresher than you, oh!

    PERFORMED BY BEYONCÉ / WRITTEN BY ADRIAN BRUESCH, NOEL C FISHER, BEYONCÉ, SWIFT SIDNEY, AUBREY GRAHAM, LELAND TYLER WAYNE AND NAYVADIUS WILBURN / PUBLISHED BY HIP HOPVILLE USA MUSIC (BMI), SONGS OF CROWD CONTROL PUBLISHING (BMI), IF YOU NEED ME DON’T LEAVE ME (BMI), EMI BLACKWOOD MUSIC INC (BMI), , IF YOU NEED ME DON’T LEAVE ME (BMI), OAKLAND 13 MUSIC (ASCAP), WB MUSIC CORP (ASCAP); ALL RIGHTS ADMINISTERED BY WB MUSIC CORP. OBO ITSELF AND OAKLAND 13 MUSIC, SONGS OF KOBALT MUSIC PUBLISHING (BMI), AND SONGS OF SWIFT SONGS (BMI), AUBREY DRAKE GRAHAM SOCAN PUB DESIGNEE, IRVING MUSIC (BMI), NAYVADIUS MAXIMUS MUSIC (BMI) AND PLUTO MARS MUSIC (BMI) / PRODUCED BY BEYONCÉ AND DEREK DIXIE

  • Bug-A-Boo 1: I’m Bug A Boo daddyAKA me, myself
    Yo let’s take a group pic bro– and I. Ha haha.

    Bug-A-Boo 2: I’m Bug A Boo Up–Grade–You. Also known as Shinin, ShininShinin Yea,

    Bug-A-Boo 3: I’m Bug A … I’m Bug A Boo sweet dreams also known as blow.

    Bug-A-Boo 4: I’m Bug A Boo Up disappear also known as *sings* Ave Maria

    Bug-A-Boo 5: I’m Bug A Boo Rocket. Also known as*sings* If I were a Boy.

    Bug-A-Boo 6: I’m Bug A Boo *sings* We gotta work it out hey.
    Also known as we like to party ay, ay, ay, ay.

    Bug-A-Boos: Lets go huh

    Bug-A-Boo 1: Bug A Boos

    Bug-A-Boos: All together now.

    PERFORMED BY BEYONCÉ / WRITTEN BY BEYONCÈ, BURT BACHARACH, JAHRON BRATHWAITE, INGRID BURLEY, SHAWN C. CARTER, HAL DAVID, FLOYD NATHANIEL HILLS, KHALED KHALED, IAN DENCH, MIKKEL ERIKSEN, AMANDA GHOST, TOR HERMANSEN, MAKEBA RIDDICK , CHAD HUGO, AND PHARRELL L. WILLIAMS / PUBLISHED BY OAKLAND 13 MUSIC (ASCAP), WB MUSIC CORP (ASCAP) BMG RIGHTS MANAGEMENT (UK) LTD (HAL DAVID) (ASCAP), CARTER BOYS MUSIC (ASCAP) , JA PUBLISHING GROUP INC (ASCAP), LOVE VIVIAN MUSIC (ASCAP), NEW HIDDEN VALLEY MUSIC CO (ASCAP), SONGS OF FUJIMUSIC (ASCAP),SONY/ATV ALLEGRO (ASCAP),BMG PLATINUM SONGS US (BMI), GIVE THANKS PUBLISHING (BMI), BC JEAN PUBLISHING (BMI), SONGS OF UNIVERSAL INC (BMI), BMG RUBY SONGS, GAD SONGS, LIEDELA MUSIC (ASCAP), B DAY PUBLISHING (ASCAP), EMI APRIL MUSIC INC (ASCAP), SONY/ATV MUSIC PUBLISHING (UK) LIMITED (ASCAP), EMI BLACKWOOD MUSIC INC (BMI), JANICE COMBS MUSIC (BMI), SONGS OF KOBALT MUSIC PUBLISHING (BMI), YOGA FLAMES PUBLISHING (BMI), EMI BLACKWOOD MUSIC INC (BMI) AND EMI POP MUSIC PUBLISHING/MORE WATER FROM NAZARETH (GMR)

  • I may be young, but I'm ready
    To give you all my love
    I told my girls you can get it
    Don't slow it down, just let it go

    So in love
    I'll give it all away
    Just don't tell nobody tomorrow
    So tonight,
    I'll do it every way
    Speakers knockin' til the morning light

    Cause we like to party hey, hey, hey, hey, hey, hey
    Cause we like to party hey, hey, hey, hey, hey, hey
    Cause we like to party

    hey, hey, hey, hey, hey, hey

    You a bad girl and your friends bad too, oh
    We got the swag sauce, she drippin' swagu
    You a bad girl and your friends bad too, oh
    We got the swag sauce, she drippin' swagu

    Outro: Beyoncé
    As a black woman
    I used to feel like the world wanted me to stay in my little box
    And black women often feel underestimated
    I wanted us to feel proud of not only the show but the process
    Proud of the struggle
    Thankful for the beauty that comes with a painful history and rejoice in the pain
    Rejoice in the imperfections and the wrongs that are so damn right
    And I wanted everyone to be grateful for their curves , their sas, their honesty
    Thankful for their freedom

Источник: https://www.beyonce.com/track/

Ron`s Editor 2021.01.26.1742 Crack With Serial Key Free Download Latest Version

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Muscle Cramping During Exercise: Causes, Solutions, and Questions Remaining

Abstract

Muscle cramp is a temporary but intense and painful involuntary contraction of skeletal muscle that can occur in many different situations. The causes of, and cures for, the cramps that occur during or soon after exercise remain uncertain, although there is evidence that some cases may be associated with disturbances of water and salt balance, while others appear to involve sustained abnormal spinal reflex activity secondary to fatigue of the affected muscles. Evidence in favour of a role for dyshydration comes largely from medical records obtained in large industrial settings, although it is supported by one large-scale intervention trial and by field trials involving small numbers of athletes. Cramp is notoriously unpredictable, making laboratory studies difficult, but experimental models involving electrical stimulation or intense voluntary contractions of small muscles held in a shortened position can induce cramp in many, although not all, individuals. These studies show that dehydration has no effect on the stimulation frequency required to initiate cramping and confirm a role for spinal pathways, but their relevance to the spontaneous cramps that occur during exercise is questionable. There is a long history of folk remedies for treatment or prevention of cramps; some may reduce the likelihood of some forms of cramping and reduce its intensity and duration, but none are consistently effective. It seems likely that there are different types of cramp that are initiated by different mechanisms; if this is the case, the search for a single strategy for prevention or treatment is unlikely to succeed.

FormalParaKey Points
Exercise-associated muscle cramp (EAMC) is a temporary but intense and painful involuntary contraction of skeletal muscle occurring during or soon after a period of physical activity. QuickBooks Enterprise Solutions License key
EAMC is highly unpredictable and it seems likely that different mechanisms may operate in different scenarios.
Proposed mechanisms include disturbances of water and electrolyte balance, and abnormal spinal reflex activity.
No prevention strategy or treatment is consistently effective.

Introduction

Few athletes escape the painful experience of muscle cramps at some stage during their sporting career. Cramps that occur during or soon after a bout of physical activity have been termed exercise-associated muscle cramps (EAMC), and these are commonly experienced as a “painful, spasmodic contraction of the skeletal muscle that occurs during or immediately after muscular exercise” [1].

This review is based in part on a review of the literature using the Web of Science database and the key words ‘cramp’, ‘muscle’ and ‘exercise’. Titles and abstracts of the 379 results returned were screened for relevance. The same search on PubMed returned 236 items. However, although the timespan for the Web of Science was set to 1900–2019, this search revealed no publications prior to 1966. No date was specified for the PubMed search, but the earliest relevant publication returned by the search appeared in 1960. These searches thus excluded all of the older literature, and this perhaps explains why most publications continue to ignore this. Earlier publications were identified from various sources.

Several surveys have attempted to identify the prevalence of EAMC in different sports populations, but comparing results across studies is hampered by different definitions and different measurement periods, and also by the use of different assessment tools. Nonetheless, EAMC has been reported to affect 67% of triathletes during or after training or racing [2], 18–70% of marathoners or endurance cyclists [3,4,5], and 30–53% of American football players [6, 7]. Although seemingly suggesting that cramp is common, these data are a mixture of incidence rates in single events and lifetime incidence. Most often, cramping is a relatively minor inconvenience: Schwabe et al. reported the incidence of serious muscle cramping to be less than one per thousand runners in a large cohort (65,865 runners) of participants in half-marathon and ultra-marathon events [8]. To put these data in perspective, Abdulla et al. reported that among an outpatient sample aged 65 years or older, 50% of outpatients experienced frequent muscle cramps [9], and that another survey of a similar population reported a similar prevalence of 56%, with half having cramps occurring at least once per week [10]. About 7–12% of patients with amyotrophic lateral sclerosis (ALS), a progressive, fatal neurodegenerative disorder, present with muscle cramping [11].

The statistics on EAMC from athlete populations do not reveal the fact that for some of those afflicted, it may be a rare occurrence—perhaps only one or two incidents over the course of a whole career, and therefore mostly of negligible impact—while others may be affected much more frequently and much more severely. The intensity and duration of cramps can vary greatly, from a minor spasm that resolves spontaneously within a few seconds, to the whole-body ‘lock up’ lasting several minutes that some athletes describe. In severe cases, the muscle pain may persist for hours or even days after the acute contraction has resolved, and may result in an inability to train or compete. At worst, repeated episodes can result in a premature end to an athlete’s career.

There are many different potential causes of muscle cramps, most of which are not associated with exercise but with a range of clinical conditions or the use of drugs for the treatment of those conditions [12,13,14]. Even within the narrow area of EAMC, the highly localised cramp in the calf that afflicts the football (soccer) player late in the game is very different from the whole-body cramps that some American football players and tennis players describe and that have been reported in some industrial settings. These in turn are different from the cramp that afflicts small muscles used in repetitive exercise, such as the hand in writers or typists [15]. Cramps typically occur spontaneously and may or may not occur predictably. Some cramps are associated with fasciculations or other prodromal symptoms, but there may be no warning in other cases [12]. Cramp in some small muscles can be induced in the laboratory, but not all cramps can be induced reliably and not all individuals are susceptible, making them difficult to study. Likewise, some cramps occur early on during exercise, while some occur only after prolonged periods of exercise; others still occur some minutes or even many hours after exercise. It is not clear that the mechanisms underpinning these different types of cramp are the same.

This uncertainty is reflected in the conclusion of several recent reviews that the causes of EAMC, and therefore the treatment options, remain uncertain [16, 17]. Two main hypotheses have been proposed and continue to be debated: a disturbance of water and salt balance, and a neurological cause resulting in sustained abnormal discharge of motor drive to the afflicted muscles [18]. Each of these has some support, but neither can fully explain the nature of EAMC.

Risk Factors for Exercise-Associated Muscle Cramp (EAMC)

Although EAMC has been observed in both training and competition in almost every type of sport, it does seem from the surveys cited above to be more associated with endurance-type activities and in team sports. An analysis of the evidence of cramp among American football players showed that the great majority (95%) occurred during periods of hot weather: EAMC occurred most often during the first 3 weeks of practice, when fitness and acclimation levels are likely to be lowest and when the training load is often the highest [19]. The incidence of heat cramps was 37% during the first week of the training camp, then 27%, 18% and 4% in the succeeding weeks. Notwithstanding these observations, the incidence of EAMC may also be high in endurance events taking place in cool or cold environments: Maughan found that 15 of 92 (18%) runners experienced cramp during a single marathon race taking place at 10–12 °C [3]. Most cases occurred in the later stages of the race, after an average of 35 km had been completed: no cases were reported to have occurred before 24 km, and 5 of the 15 instances occurred during the last 1.5 km.

Schwellnus and colleagues have made a number of attempts to characterise the primary risk factors predisposing to cramp in endurance events. Furthermore, Schwellnus suggested that EAMC in marathon runners is associated with high intensity, long duration, and hilly terrain, which can lead to ‘premature muscle fatigue’ in competitors with a history of cramping [20]. It is not immediately obvious what is meant by ’premature’ fatigue and how this might differ from the fatigue that is an inevitable consequence of participation in such events [20]. Schwellnus et al. reported that, in a prospective cohort study in 210 Ironman triathletes, independent risk factors for EAMC were a history of the condition and competing at a higher than usual exercise intensity, but that dehydration and serum sodium changes did not predict EAMC [21]. Manjra et al. analysed data from 1300 marathon runners and found that risk factors included those common to all participants in marathon races, including long distance (> 30 km) and the presence of fatigue, but also running at a faster pace than was normal in training [5]. Other risk factors included older age, a longer history of running, higher body mass index (BMI), shorter daily stretching time, irregular stretching habits, and a positive family history of cramping.

In a more recent analysis of cross-sectional data from almost 16,000 participants in two races over a distance of 21.1 km and 56 km, Schwellnus et al. identified a number of differences between runners who reported a history of EAMC (n = approximately 3000) and a control group with no such history (n = approximately 13,000) [22]. Factors associated with a history of EAMC included underlying chronic disease (including cardiovascular, respiratory, gastrointestinal, nervous system, kidney, bladder and haematological disease), as well as cancer, allergies, regular medication use, and a history of injury. More experienced runners were also at greater risk. Whether some underlying common factors underpin these associations is not at present clear.

In what seems to be the largest survey to date, but published only as an abstract, Swanevelder et al. analysed data from an online pre-race medical screening questionnaire completed by 41,698 distance runners who completed either a 21.1 km or 56 km run [23]. The investigators considered independent risk factors associated with EAMC (model 1: binary outcome), and risk factors associated with severe EAMC (model 2: defined as recurrent cramping history), and found some rather inconsistent outcomes. For model 1 (binary outcome), significant risk factors for EAMC were males, age > 40 years, increased BMI, history of any disease of the gastrointestinal tract Ron`s Editor 2019 Download - Crack Key For U kidney/bladder, chronic or regular medication use, history of a running injury in the last 12 months, running the 56 km race, recreational running for < 5 years, training/racing < 3 times/week, and slower runners (> 6 min/km). In model 2 (recurrent cramping history), the authors said that “EAMC was associated with all the risk factors for EAMC in model 1, but also included a history of any cardiovascular disease (CVD) symptoms. In model 2, a lower BMI and running in 21.1 km race were also specific risk factors for severe EAMC. Training volume and pace weren’t risk factors in model 2”. There seem to be some mutually contradictory statements here.

Possible Causes of EAMC

Two main causes for muscle cramps have been proposed and, depending on which an individual subscribes to, the choice of prevention and treatment strategies will be determined. This suggests an either/or dichotomy, and this is how the literature is often presented, with loud voices expressing strongly held views on either side [24, 25]. It should be recognised though that the picture is not at all clear, and the evidence on both sides of the debate is weak. It is unlikely that a single mechanism can account for all cramps in all situations, therefore the search for a single causal mechanism is probably futile. It follows from this that strategies for the prevention and treatment of the condition are also unlikely to be one-dimensional. However, whatever the primary cause, it is clear that cramp is accompanied by active contraction of the afflicted muscle, as evidenced by high levels of muscle electrical activity [26].

Disturbances of Hydration and Electrolyte Balance

The role of changes in hydration status and electrolyte balance as a factor in the aetiology of EAMC was dismissed by Schwellnus, who said that “Scientific evidence in support of the ‘‘electrolyte depletion’’ and ‘‘dehydration’’ hypotheses for the aetiology of EAMC comes mainly from anecdotal clinical observations, case series totalling 18 cases, and one small (n =10) case–control study” [25]. This assessment of the evidence has been repeated in many subsequent publications: for example, Qiu and Kang wrote that “its [i.e. the electrolyte imbalance-and-dehydration theory] supporting evidence comes mainly from anecdotal observations and case reports” [27]. There may however be more evidence than these authors admit.

The strongest evidence that sweat-related electrolyte imbalances are a factor in some muscle cramps is found in the large-scale observational and prospective studies of industrial workers—mainly studies on miners, ship’s stokers, construction workers and steel mill workers that were conducted in the 1920s and 1930s—where administration of saline drinks or salt tablets was able to greatly reduce the incidence of cramps [28,29,30,31,32]. These studies were inevitably limited by the methods available at the time, but they did have the advantage of access to large populations and the keeping of careful medical records related to productivity. It is easy to dismiss much of the older literature, but some of the observations were extensive and meticulous. They should also be read in the context of the normal publishing conventions of the time.

Although methodologies were limited, some of the observations were acute and sometimes remarkably prescient. For example, Moss published an extensive report in which he documented cases of cramp among coal miners and the factors that may have contributed to the development of these cramps [28]. He attributed the onset of cramps, which in some cases were seriously debilitating, to (1) high air temperatures; (2) excessive drinking of water caused by dryness of the mouth and throat; and (3) continued hard work.

He also observed that cramps tended to occur during the second half of a working shift and in men who were less physically fit, thus implicating not only sweat losses but also fatigue in the aetiology. It should be noted that cramp was not attributed to dehydration or increased serum electrolyte concentrations, but rather “to a form of water poisoning of the muscles brought about by the combination of great loss of chloride by sweating, excessive drinking of water, and temporary paralysis of renal excretion” [33]. Chloride was normally measured in body fluids as there was no good assay for sodium at the time, but there is a close relationship between sodium and chloride concentration in sweat [34]. This does not implicate dehydration, as most of the later writers say (e.g. Bergeron [24]), but rather inappropriate, and perhaps excessive, intake of plain water in combination with large losses of electrolytes in sweat. Schwellnus refers to ‘dehydration’ and ‘electrolyte depletion’ theories [25], while Qiu and Kang say that “this theory suggests that overly sweating and thus loss of electrolytes can cause muscles and nerves that innervate them to malfunction, thereby producing muscle cramps” [27]. This is not a true reflection of the theories proposed during the 1920s and 1930s.

It is also not correct to say that there have been no large-scale prospective studies to assess the role of water and salt balance in the aetiology of muscle cramps. Dill et al. reported the findings of intervention studies carried out at the site of construction of the Hoover Dam and in the steel mills of Youngstown, Ohio [32]. At both of these locations, large numbers of men undertook hard physical work in extremely hot environments on a daily basis. They found that those suffering from cramp displayed the following characteristics: (1) dehydration; (2) lowered concentration of sodium and chloride in blood plasma; (3) little or no sodium or chloride in urine; (4) increased serum protein concentration; (5) increased red cell count; and (6) normal osmotic pressure.

This presents a complex picture: some of these findings are typical of dehydration (1, 4 and 5), while others are consistent with overhydration (2, 3). However, they also reported that injection of isotonic saline normalised the blood profile and brought immediate relief from the symptoms. In the largest intervention study, reported in the same paper, they added saline to the water given to the 12,000 men employed in one of the mills, while those at neighbouring mills continued to be provided with plain water; this was effective in almost completely abolishing cases of muscle cramp, although in previous years, and at other mills in the same year where plain water was given, up to 12 cases of cramp required hospitalisation in a single day.

In a controlled environment, severe restriction of dietary sodium intake can result in hyponatraemia and may be associated with generalised skeletal muscle cramping in the absence of exercise [35]. Some more recent studies have assessed changes in hydration status and plasma electrolyte concentrations in athletes who have experienced muscle cramps; these studies have included marathon runners [3], participants in a 56 km road race [36], competitors in an Ironman triathlon [37], and participants in a 161 km ultramarathon [38]. None of these showed any association between cramp and serum electrolyte changes, but it is important to note that serum electrolyte concentrations may be of little relevance. Local intracellular and extracellular electrolyte concentrations may be relevant as they will affect the resting membrane potential of both muscle and nerve, but it is unlikely that changes in plasma concentrations can track these changes; there is good evidence that changes in the plasma concentration of these electrolytes do not reflect local intramuscular changes during either intense or prolonged exercise [39, 40]. It is also the case that blood samples have usually not been collected at the time of cramping, but only later, usually once the cramping has resolved; in some cases, this was several hours after resolution of the cramps, therefore the absence of any association is perhaps not surprising. Schwellnus et al. acknowledged that disturbances in electrolyte concentrations can lead to alterations in neuromuscular excitability, and this may have a role in the generalised skeletal muscle cramping reported in some industrial contexts, but argue that most EAMC affects only the muscles involved in the exercise task, suggesting that systemic disturbances must interact with local changes occurring within the active muscles [1].

There is some experimental evidence that individual athletes who lose large amounts of salt in their sweat may be more prone to muscle cramps. Unlike the earlier large-scale industrial records, this evidence does derive primarily from small studies, case reports and anecdotal reports, and is therefore inevitably rather weak [41, 42]. Stofan et al. found that sweat sodium losses during training sessions were larger in cramp-prone football players (n = 5) than in a group of players with no history of EAMC [41]. Subsequently, the same research group investigated a reference group of American football players (n = 8) without a cramping history, and a cramp-prone group (n = 6) [42]. Whole blood sodium concentration (as stated by the authors, but in reality this is plasma sodium concentration) remained unchanged after training in the control group (138.9 ± 1.8 to 139.0 ± 2.0 mmol/L), while it tended to decline (137.8 ± 2.3 to 135.7 ± 4.9 mmol/L) in the cramp-prone players. Additionally, three subjects in this group recorded values below 135 mmol/L. Those in the cramp-prone group consumed a greater percentage of their total fluid as plain water rather than electrolyte-containing sports drinks (although the difference in sodium intake was small) and had a higher sweat Ron`s Editor 2019 Download - Crack Key For U concentration (52.6 ± 29.2 mmol/L vs. 38.3 ± 18.3 mmol/L), thus incurring a greater sodium deficit over the course of the training session.

In support of a role for disturbances of water and salt balance as a causal factor, Ohno and Nosaka showed that a body fluid deficit of 3% of body mass induced by intermittent sauna exposure without exercise increased the number of subjects who developed EAMC during a muscle cramp test in the toe flexors, but not in the knee extensors [43]. Jung et al. had participants perform a fatiguing protocol in the calf muscles to induce EAMCs. In one trial, subjects consumed a carbohydrate electrolyte drink at a rate similar to sweat rate, while in the other trial, no fluid was consumed and mild (1% loss of body mass) hypohydration developed [44]. Nine participants experienced cramps in the carbohydrate–electrolyte trial, compared with seven in the hypohydration trial. Of the seven individuals who had EAMC in both trials, time to onset was more than doubled in the carbohydrate–electrolyte trial (36.8 ± 17.3 min) compared with the hypohydration trial (14.6 ± 5.0 min). Subjects who experienced cramps sweated more (2.0 ± 0.9 L/min) than those who did not (1.3 ± 0.6 L/min). It is not clear whether there was any treatment order effect in these studies that might have confounded the results (this is discussed further below).

Although numerous papers have disputed the findings above, two recent publications seem likely to reopen the debate on the role of disturbances of water and salt balance in the development of muscle cramps. Ohno et al. systematically investigated the susceptibility of voluntarily-induced EAMC in hamstrings after hypohydration of 1, 2 and 3% of body mass induced by sauna exposure without exercise [45]. No EAMC occurred in the nine participants in the control condition or after 1% dehydration; three subjects experienced EAMC in the 2% and six in the 3% condition. In the study by Lau et al., 10 men ran downhill in a hot environment until they lost 2% of their initial body mass [46]. Ten minutes after completing the run, they ingested either plain water or a commercially available oral rehydration solution (ORS) containing sodium (50 mEq/L), chloride (50 mEq/L), potassium (20 mEq/L), magnesium sulphate (2 mEq/L), lactate (31 mEq/L) and glucose (18 g/L) in a volume equal to the mass lost. Susceptibility of the calf muscles to electrically-induced cramp was assessed by a threshold frequency (TF) test applied at baseline before running, immediately after running, and 50 and 80 min after drink ingestion. Muscle cramp susceptibility assessed by TF did not change from baseline to immediately after running in either condition, but TF decreased after water intake by 4.3 Hz (at 30 min) and 5.1 Hz (at 60 min post-run), but increased after ORS intake by 3.7 and 5.4 Hz, respectively. The investigators reported that serum sodium and chloride concentrations decreased after water intake but were maintained after ingestion of the electrolyte-containing drink.

In accord with the mechanisms proposed by Moss, Haldane and others in the 1920s, these results suggest that the combination of sweat loss and water intake makes muscles more susceptible to electrical simulation-induced muscle cramp, but the susceptibility to muscle cramp decreases when a drink with a high electrolyte content is ingested. It is interesting to note that cramping is a recognised accompaniment of hyponatraemia (defined as a serum sodium concentration < 135 mmol/L) in clinical settings [47]. However, the extensive literature on exercise-associated hyponatraemia generally makes no mention of muscle cramping [48].

While cramp is often associated with large sweat losses during prolonged exercise in the heat, it also occurs in cool environments with little or no sweat loss, suggesting that sweat loss alone and the consequent disturbances of electrolyte balance cannot account for all cramps. Notwithstanding these observations, there is overwhelming evidence from large-scale industrial settings that cramping occurs more frequently in environments that are hot (although not necessarily humid) and where sweat losses are high [28, 31]. Supporting evidence that disturbances of electrolyte balance may be implicated in muscle cramps can be found in some non-exercise contexts. For example, the use of low-sodium dialysis fluids during maintenance dialysis may provoke cramping in renal patients [49], and normalisation of plasma osmolality and sodium concentration by use of the sodium profiling technique can significantly reduce the frequency of cramping during dialysis [50]. Whether this is relevant to the exercise situation though is uncertain.

Altered Neuromuscular Control

The idea that the cause of cramp is neurological rather than being related directly to events occurring within the muscle is not a new one. Telegraphists’ cramp, affecting the small muscles of the hand involved in the repetitive movements of those operating a Morse code instrument, was the subject of a UK Parliamentary enquiry that published its findings in 1911 [51]. The Committee wrote that “Some authorities have regarded it [i.e. telegraphists’ cramp] as a muscular disorder; others as a disease of the peripheral nervous system; others as a disease of the central nervous system”. They further wrote that “After careful consideration of these antagonistic theories of telegraphists’ cramp, and examination of a number of telegraphists affected with the disease, the Committee accept the last-named view; namely, that telegraphists’ cramp is a disease of the central nervous system, and is the result of a weakening or breakdown of the cerebral controlling mechanism in consequence of strain upon a given set of muscles”. As will be seen below, this is remarkably similar to the proposed mechanism in experimentally-induced muscle cramps. However, the findings of the Parliamentary enquiry seem to have been largely forgotten, along with much of the older literature.

As evidence accumulated in the 1980s and 1990s that cramp often occurred during exercise in the absence of substantial sweat losses or of gross disturbances in electrolyte balance, an alternative causation was sought. Schwellnus et al. hypothesised that cramp is caused by “sustained abnormal spinal reflex activity which appears to be secondary to muscle fatigue” [1]. In particular, EAMC was ascribed to an abnormality of sustained alpha motor neuron activity due to an abnormality of alpha motor neuron control at the spinal level, but this still does not identify the cause of this abnormality. Muscle fatigue was implicated through an excitatory effect on the muscle spindle afferent activity (type Ia and II) and an inhibitory effect on the type Ib Golgi tendon organ afferent activity (Fig. 1). Circumstantial evidence in support of this suggestion arose from the observation that passive stretching of the muscle during an episode of cramp may alleviate the symptoms as a result of an autogenic inhibition by the tendon organ reflex [52]. However, this still does not explain why cramp is not an inevitable consequence of exercise that causes fatigue, why it appears to occur more frequently in environments that impose high thermal stress, or why some individuals are affected while others are not.

Postulated abnormal spinal control of motor neuron function during exercise-associated muscle cramp. Based on a proposal by Schwellnus et al. [1]. CNS central nervous system

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The strongest evidence for an altered neuromuscular control is from laboratory studies of small muscles in humans and in animal models. In each of these two different scenarios, a story can be made, but in each case the story is incomplete. Because EAMC is notoriously unpredictable, laboratory models have been developed where cramp can be induced more reliably, whether by voluntary activation of muscles or by electrically-evoked contractions. It has been reported that cramping occurs more frequently when the muscle is activated while it is already shortened [1] (although no evidence to support this statement was presented). Various forms of this experimental model have been used in laboratory studies of cramping, even though this may not reflect the movement patterns of athletes. This is consistent with the proposal of Schwellnus et al. [1] as outlined above, as a reduction in the tension in the muscle tendon will reduce the inhibitory feedback from the Golgi tendon organ; this in turn has the potential to increase the motor drive to the alpha motor neuron. Consistent with this proposal, Khan and Burne [26] found that cramp induced by voluntary maximal activation of the gastrocnemius while it was held in a shortened position could be inhibited by electrical stimulation of tendon afferents in the cramped muscle. However, even under conditions that favoured cramping, 5 of their 13 subjects could not induce cramping, and in a further two it did not persist long enough for measurements to be made.

Athletes who are prone to muscle cramps are reported to demonstrate a lower threshold for muscle cramps evoked by electrical stimulation of motor nerves [53, 54]. Blocking of the motor nerves with anaesthetic does not abolish these electrically evoked cramps, but when the nerve is blocked, a greater stimulation frequency is required to induce cramping and cramp duration is reduced; altered motor unit discharge characteristics are consistent with the existence of a positive feedback loop involving afferent input from affected muscles and motor drive to those muscles [55].

Strong objections to the dehydration/electrolyte loss theory have been raised by studies that have provided fluids to prevent dehydration and found that this does not affect the onset of electrically-evoked cramps [56, 57]. However, these findings are contradicted by other studies referred to above [43,44,45,46]. It should be noted that marked hypernatraemia developed as a result of dehydration in the studies of Miller et al. [56] and Braulick et al. [57], Ron`s Editor 2019 Download - Crack Key For U this may be protective against the development of cramp [46]. Fatigue alone is also unlikely to be the cause, although it may be a contributing factor. In marathon runners, cramp tends to occur more frequently towards the end of races [3, 5]; however, everyone is fatigued in the later stages of endurance events such as a marathon race, but relatively few experience muscle cramps. The nature of the fatigue that occurs in sprinters is very different from that experienced towards the end of a marathon race, but cramp may occur in either situation.

Therefore, rather than focusing on an either/or approach, there are good reasons to suggest that different mechanisms may apply in different situations. We are all inevitably influenced by our own experiences and these may bias us towards one cause as being more likely or more common than another, but the key issue is how to treat or prevent an attack. With regard to treatment and prevention, it is important to note that a plausible mechanism can help to identify effective treatments, but it is not necessary to understand mechanisms to know if a treatment is effective or not.

Possible Preventive and Treatment Strategies

The early studies of muscle cramping that occurred in industrial settings identified large sweat losses and ingestion of large volumes of plain water as factors contributing to muscle cramping, therefore it is not surprising that ingestion of salt was proposed as a prevention strategy [33, 58]. The strongest evidence for the efficacy of this strategy is found in the work of Dill and colleagues [32], where large-scale prospective studies showed that the addition of salt to drinking water was effective in reducing the rate of cramping.

Schwellnus et al. said that “The treatment of acute muscle cramps is passive stretching” [1]. In support of this, they showed data from a single runner in whom stretching resulted in a dramatic decrease in the electromyographic activity of the affected muscle. The same group also suggested that ‘irregular stretching habits’ were associated with an increased risk of cramping [5]. They later hypothesised that variants in genes that code for connective tissue components of muscle may influence the susceptibility to EAMC [59]. To test this hypothesis, they recruited 116 ultraendurance athletes with a recent self-reported history of EAMC, and 150 participants who had never experienced EAMC. As per the hypothesis, the COL5A1 CC genotype was significantly overrepresented (p = 0.031) among the control group (21.8%) compared with the EAMC group (11.1%). However, none of the other related genes showed a differential distribution. A review by Nelson and Churilla stated that there is ‘strong evidence’ that passive stretching is the most effective treatment for muscle cramping, but did not identify this evidence [60]. More recently, Panza et al. tested the possible association between acute static stretching of the muscle and prevention of cramping, using an experimental model whereby cramp was induced in the flexor hallucis brevis muscle by electrical stimulation with the muscle held in a shortened position [61]. In a crossover study, static stretch was compared with a no-stretch condition; the cramp TF increased in both the control and stretching conditions, with no difference between conditions. Miller et al. subsequently reported similar findings [62].

There is a long history of the use of folk remedies for the prevention and treatment of muscle cramps, and many of these have included compounds that have a strong or bitter taste, including pickle juice, mustard, quinine, vinegar and various spices and herbs. Even homeopathic cures are reported to be effective, with anecdotal support from athletes often being used to promote these products, suggesting that both the placebo effect and athlete belief may play a powerful role [63]. As with other interventions, these have proved difficult to evaluate as muscle cramps generally resolve spontaneously before any intervention can be implemented. However, in the human model of electrically-invoked cramp, pickle juice (which has a high salt content and a sharp taste imparted by the acetic acid content) was reported to be effective in reducing the duration of cramps. Miller et al. found that cramp duration was reduced by about 37% on average when 1 mL of pickle juice was ingested 2 s after induction of cramping, compared with a trial where water was ingested (85 ± 19 s vs. 134 ± 16 s, respectively; p < 0.05); the intensity of cramping was not affected [64]. The same authors had previously shown that ingestion of small volumes of pickle juice had no measurable effect on plasma concentrations of sodium, potassium, magnesium or calcium concentration, or on plasma osmolality and plasma volume [65]. The authors proposed that, in the absence of any effect of the ingested pickle juice on circulating electrolyte concentrations, the mechanism by which pickle juice shortened cramp duration involved Ron`s Editor 2019 Download - Crack Key For U of receptors in the oropharyngeal region that resulted in a reduced firing rate of alpha motor neurons that innervate the affected muscle. However, it is important to note that this was not a study of EAMC, but of cramping induced by electrical stimulation during maximal voluntary contraction of a small muscle in the sole of the foot that was held in a shortened position; this cannot be taken as evidence of efficacy in the treatment of EAMC. However, this and the results of other similar studies, raise some interesting questions; crossover designs involve using the same subjects in treatment and placebo trials, usually in the case of a single treatment, with half receiving treatment before placebo and the order reversed in the other half. The statistical analysis applied in the study by Miller et al. [64] assumes that there was no treatment order effect, but we cannot be sure that this is true, with only 1 week for recovery between experimental trials [66]. The authors of this and other studies involving similar experimental designs should have reported whether the cramp intensity and cramp duration were different between the first and second exposures, and should perhaps also have habituated the subjects to the electrical stimulation process prior to the experimental trials. The importance of this is highlighted by a recent publication showing that repeated exposures to electrically-evoked cramps induce a long-lasting increase of the cramp TF in healthy subjects [67]. These authors induced EAMC in the gastrocnemius medialis of one leg twice a week, while the opposite leg served as the control leg; after four cramp training sessions, the cramp threshold frequency (CTF) increased in the intervention leg but not in the control leg. This same consideration of course applies to many other laboratory studies of electrically-evoked cramping, but becomes particularly acute when, as in the study of Miller et al. [64], a large difference between conditions occurs in the first trial, with possible consequences for the succeeding trial.

Quinine has been considered to have a possible role in the prevention of cramps. There is little research specific to EAMC, but a 2015 Cochrane review concluded that ingestion of quinine (200–500 mg daily) reduces cramp number and cramp days (low-quality evidence) and reduces cramp intensity (moderate-quality evidence), but has no effect on cramp duration [68]. They reported some evidence that ingestion of theophylline in combination with quinine improved Athentech Perfectly Clear Complete 3.11.2.1915 Crack serial key2021 - Free Activators more than quinine alone. They also drew attention to the risks of adverse events associated with quinine use. These conclusions are in general agreement with those of an earlier review [13].

A recently launched product has claimed that cramp can be prevented or treated by activation of transient receptor potential (TRP) in the mouth [69], although this has not been supported by other research [70]. TRP receptors form a family of 28 related ion channels that are thought to be important for mediating the sensations of taste and pain. The TRPV1 and TRPA1 channels are stimulated by the active components of spicy foods such as chilli peppers or wasabi. It may be that evidence will emerge to support the product, but there are some questions about the science. There is no doubt that unpleasant (or pleasant) sensations in the mouth will induce electrical activity in some regions of the brain, but there are some gaps in the chain of events between stimulation of oropharyngeal receptors and the inhibition of activity in motor nerves. TRPV1 is activated by capsaicin if the local pH is < 6 [71], but it is by no means certain that such a pH will be reached in the mouth after ingestion of this product. Having said that, it is clear that ingestion of foods containing chilli, ginger and many other foods has powerful effects on receptors in the mouth and elsewhere. Anyone who has put raw chilli in their mouth or near their eye will be aware that this causes not only pain and irritation but also a variety of physiological responses. Whether these signals can disrupt the electrical activity associated with spontaneous muscle cramps remains uncertain.

Conclusions

Exercise-associated muscle cramp is a relatively common occurrence in a range of sport and exercise activities. Onset is generally unpredictable, and the intensity and duration of muscle spasms are highly variable. Spontaneous muscle cramping in occupational settings involving hard physical effort suggests that high ambient temperature and large sweat losses accompanied by the ingestion of large volumes of plain water may be risk factors, and there is some evidence that the risk is reduced by the addition of salt to ingested fluids. Laboratory models of cramp involve either voluntary or electrically-evoked activation of muscle held in a shortened position. These studies have produced mixed results regarding the effects of disturbances of water and salt balance on the risk of cramping; however they do suggest that, at least in this model, sensory organs in the muscle invoke abnormal reflex activity that results in sustained motor drive to the afflicted muscles. There may be different mechanisms at work in different situations, and there is no conclusive support for any of the proposed mechanisms. Preventive and treatment strategies are not uniformly effective.

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Acknowledgements

This supplement was supported by the Gatorade Sports Science Institute (GSSI). The supplement was guest edited by Lawrence L. Spriet, who attended a meeting of the GSSI Expert Panel in March 2019 and received honoraria from the GSSI, a division of PepsiCo, Inc., for his participation in the meeting. Dr. Spriet received no honorarium for guest editing the supplement. Dr. Spriet suggested peer reviewers for each paper, which were sent to the Sports Medicine Editor-in-Chief for approval, prior to any reviewers being approached. Dr. Spriet provided comments on each paper and made an editorial decision based on comments from the peer reviewers and the Editor-in-Chief. Where decisions were uncertain, Dr. Spriet consulted with the Editor-in-Chief.

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    Ronald J. Maughan & Susan M. Shirreffs

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Correspondence to Ronald J. Maughan.

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This article is based on a presentation by Ronald Maughan to the GSSI Expert Panel in March 2019. Partial funding for attendance at that meeting was provided by the GSSI. Ronald Maughan received no honorarium for the preparation of this article or for preparation of the presentation to the GSSI Panel on which this article is based.

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Ronald J. Maughan and Susan M. Shirreffs have no conflicts of interest relevant to the content of this article.

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Maughan, R.J., Shirreffs, S.M. Muscle Cramping During Exercise: Causes, Solutions, and Questions Remaining. Sports Med49, 115–124 (2019). https://doi.org/10.1007/s40279-019-01162-1

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Dynamics of Singlet Oxygen-Triggered, RONS-Based Apoptosis Induction after Treatment of Tumor Cells with Cold Atmospheric Plasma or Plasma-Activated Medium

Abstract

Treatment of tumor cells with cold atmospheric plasma (CAP) or with plasma-activated medium (PAM) leads to a biochemical imprint on these cells. This imprint is mediated by primary singlet oxygen, which is mainly generated through the interaction between CAP-derived H2O2 and NO2. This imprint is induced with a low efficiency as local inactivation of a few membrane-associated catalase molecules. As sustained generation of secondary singlet oxygen by the tumor cells is activated at the site of the imprint, a rapid bystander effect-like spreading of secondary singlet oxygen generation and catalase inactivation within the cell population is thus induced. This highly dynamic process is essentially driven by NOX1 and NOS of the tumor cells, and finally leads to intercellular RONS-driven apoptosis induction. This dynamic process can be studied by kinetic analysis, combined with the use of specific inhibitors at defined time intervals. Alternatively, it can be demonstrated and quantified by transfer experiments, where pretreated cells are mixed with untreated cells and bystander signaling is determined. These studies allow to conclude that the specific response of tumor cells to generate secondary singlet oxygen is the essential motor for their self-destruction, after a singlet oxygen-mediated triggering process by CAP or PAM.

Introduction

Plasma medicine1,2 is coupling plasma physics and chemistry, biochemistry, and biology as rational basis for various fascinating medical applications of plasma3. Cold atmospheric plasma (CAP) and plasma activated medium (PAM) show impressive antitumor effects in vitro and in vivo4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24.

Most studies concluded that CAP- and PAM-mediated apoptosis induction acts selectively on tumor cells25,26,27,28,29,30,31,32,33,34,35,36. There is a general consent that RONS are centrally involved in CAP- and PAM-mediated antitumor effects5,37. The exact mechanism is the subject of an ongoing discussion.

The gaseous and liquid phase of CAP contains electrons, photons, superoxide anions (O2●−), hydroperoxyl radicals (HO2), hydrogen peroxide (H2O2), hydroxyl radicals (OH), atomic oxygen (O), singlet oxygen (1O2), ozone (O3), nitric oxide (NO), nitrogen dioxide (NO2), peroxynitrite (ONOO), nitrite (NO2), nitrate (NO3), dichloride radicals (Cl2●−) and hypochloride anions (OCl)3,16,37,38. Due to the high reactivity and short live time of most CAP-derived species, PAM (as well as other plasma-activated liquids) essentially contains only H2O2, NO2 and NO333,39,40,41. It is intriguing that a liquid with a RONS composition of such an apparently low complexity can trigger impressive antitumor effects in many tumor systems in vitro and in vivo, as shown by many groups5,14,30,33,34,35,36,40,42,43,44,45,46,47,48,49,50,51, and reviewed by Yan et al.52. Whereas Koensgen et al.50 and Canal et al.35 found equivalent antitumor effects of CAP and PAM in vitro, Saadati et al.53 reported on a stronger effect of CAP compared to PAM in vivo. Yan et al.54 reported that CAP, but not PAM, triggered H2O2 generation from tumor cells. Several groups reported on selective effects of PAM towards tumor cells30,33,34,35,36. Ikeda et al.51 presented evidence that PAM kills human cancer-initiating cells. PAM is not only effective in classical 2 D cell cultures, but also affects 3D multicellular tumor spheroids55in vitro and is effective in vivo36,42,46,53. There is very strong evidence that RONS contained in PAM are responsible for apoptosis induction in tumor cells, and that PAM treatment also triggers a RONS response of the target cells5,33,40,43,46,48. As H2O2 seems to play a central role for PAM-mediated antitumor effects, and as tumor cells express high concentrations of aquaporins in their membrane56, Yan et al.57,58 proposed that an increased influx of H2O2 through aquaporins determines the selective effect of PAM on tumor cells. Girard et al.40 and Kurake et al.33, concluded that a synergistic effect between PAM-contained H2O2 and NO2 is responsible for selective antitumor action of PAM. This view was substantiated by reconstitution experiments with defined concentrations of H2O2 and NO259,60. The interaction of these compounds was shown to result in the formation of primary 1O2 that caused local inactivation of membrane-associated catalase on tumor cells. This established autoamplificatory 1O2 generation by the tumor cells, catalase inactivation and reactivation of intercellular apoptosis-inducing RONS signaling. The apoptotic response required a preceding influx of H2O2 through aquaporins, which caused depletion of glutathione and sensitization of the cells for apoptosis induction through RONS signaling. This aquaporin-dependent step is analogous to the mechanism proposed by Yan et al.57,58, but required preceding inactivation of the gating catalase on the membrane. Freund et al.61 presented strong evidence that plasma-treated saline promotes an immunogenic phenotype of colon cancer cells, whereas Lin et al.62 concluded that PAM is not sufficient to induce immunogenic cell death, but that short-lived species in CAP are required. The discrepancy between these two studies might be explained by the low degree of induction of cell death by PAM in the study by Lin et al.62.

The preceding manuscript (Bauer et al.,63) has demonstrated that selective apoptosis induction in tumor cells by cold atmospheric plasma (CAP) generated by a portable air plasma ‘corona pen’ plasma source64 was essentially triggered by long-lived species from CAP-treated medium, i. e. by the components that are also found in plasma-activated medium (PAM). These are nitrite (NO2) and H2O2, whose synergistic interaction is required for their antitumor action33,40. Our preceding study has shown that the direct effect of short-lived singlet oxygen (1O2) from the gaseous phase of CAP on treated cells seemed to be neglectable compared to the effects that can be attributed to PAM-related compounds. This finding can be explained by the preferential reaction (i.e. quenching) of 1O2 with medium components above or around the cells. These interactions prevent 1O2 derived from the gaseous phase of CAP from reaching its target, i. e. the tumor cells. In contrast, NO2 and H2O2 are long-lived under the same conditions, but can lead to 1O2 generation in the vicinity of the target cells. Therefore, under our experimental conditions, the central effects of CAP and PAM treatment of tumor cells seem to be more or less the same, as they are both mainly based on long-lived species with the potential to generate 1O2 close to the target.

The preceding manuscript, as well as reconstitution experiments with defined sources of H2O2 and NO259,60, presented evidence that the long-lived species H2O2 and NO2 generated the “primary 1O2” through a complex cascade that started with the generation of peroxynitrite (ONOO) after H2O2/NO2 interaction65,66 (Fig. 1). This was followed by the reaction between OH  radicals, derived from peroxynitrous acid (ONOOH)67, with H2O268. The resultant hydroperoxyl radicals (HO2) then interacted with NO2 derived from ONOOH and thus enabled the formation of peroxynitric acid/peroxynitrate (O2NOOH/O2NOO)69 as source for the generation of 1O269,70. It was recognized that the generation of ONOOH from ONOO required the activity of membrane-associated proton pumps. This effect seemed to outcompete the consumption of ONOO through its favoured reaction with CO271,72,73. In addition, protonation of ONOO through membrane-associated proton pumps directed the subsequent generation of 1O2 to the site where it is required, i. e. close to its target catalase.

Apoptosis induction by CAP/PAM is mediated by the generation of primary and secondary singlet oxygen (1O2). NADPH oxidase 1 (NOX1) is expressed in the membrane of tumor cells and generates extracellular superoxide anions (O2●−) (#1). NO synthase (NOS) (#2) generates NO which can be either oxidated by NO dioxygenase (NOD) (#3) or pass through the cell membrane. Membrane-associated catalase (#4) protects tumor cells towards intercellular RONS-mediated signaling. Comodulatory SOD (#5) is required to prevent O2●−-mediated inhibition of catalase. Further important elements in the membrane are the FAS receptor (#6), Dual oxidase (DUOX) (#7), from which a peroxidase domain (POD) is split through matrix metalloprotease, proton pumps adobe dimensions models and aquaporins (#9). H2O2 and NO2 derived from CAP treatment and stable in PAM interact and generate peroxynitrite (ONOO) (#10). In the vicinity to membrane-associated proton pumps ONOO is protonated to peroxynitrous acid (ONOOH) (#11) and decomposes into NO2 and OH radicals (#12). OH radicals react with H2O2, resulting in the formation of hydroyperoxyl radicals (HO2) (#13). The subsequent generation of peroxynitric acid (O2NOOH) (#14) and peroxynitrate download idm full crack - Activators Patch (#15) allows for the generation of “primary singlet oxygen” (1O2) (#17). Primary 1O2 causes local inactivation of membrane-associated catalase (#18). Surviving H2O2 and ONOO at the site of inactivated catalase are the source for sustained generation of “secondary 1O2” through reactions #19- #24. Secondary 1O2 may either inactivate further catalase molecules (#25) and thus trigger autoamplification of 1O2 generation (#29), or activate the FAS receptor (#26) and in this way enhance the activities of NOX1 and NOS. This enhances the efficiency of secondary 1O2 generation. The site of action of specific inhibitors and scavengers are indicated. Please find details on the elements on the surface of tumor cells in references74,80,97,118,124,125,130, on singlet oxygen generation in references59,96,97,118, and on intercellular apoptosis-inducing signaling after catalase inactivation in references78,80,83,132.

Full size image

The generation and action of primary 1O2 derived from the interaction between H2O2 and NO2 in the presence of tumor cells seemed to be a relatively rare event59,60. This finding was not unexpected, as the generation of 1O2 in this biological system is limited by

  1. (a)

    the decomposition of PAM-derived H2O2 by membrane-associated catalase of tumor cells,

  2. (b)

    the relatively low reaction rate of ONOO generation through the interaction between NO2 and H2O266,

  3. (c)

    the decomposition of ONOO generated by PAM through membrane-associated catalase of tumor cells74,75 and

  4. (d)

    the reaction of ONOO with CO2 rather than with protons71,72,73, especially in locations realtively far from the proton pumps in the cell membranes.

Furthermore, the expected low concentration of primary 1O2 generated in the system only has a biological impact if it reaches its specific target on tumor cells, i. e. membrane-associated catalase, which is inactivated by 1O276,77. This step is limited by the high reactivity of 1O2 and the resultant small free diffusion path length, due to 1O2 reaction with other competing substrates.

Therefore, primary 1O2 seems to have no chance to directly damage tumor cells to a degree that induces cell death59,60. However, primary 1O2 can utilize tumor cells like a switchboard, on which it triggers the sustained generation of “secondary 1O2” [The term “secondary1O2” is used in this paper for1O2that is generated by tumor cells after they have been triggered through local catalase inactivation by primary1O2]. This is achieved through the interaction between tumor cell-derived ONOO and H2O2. Both molecular species are constantly generated by tumor cells, due to their active NOX1 and NOS, and are no longer decomposed at sites of catalase that has been inactivated by primary 1O2 (Fig. 1). This biochemical scenario seems to result in a strong autoamplificatory process that drives catalase inactivation to a point at which intercellular apoptosis-inducing RONS signaling gets activated60. In parallel, substantial inactivation of membrane-associated catalase allows an influx of H2O2 into the cells59. This leads to the depletion of intracellular glutathione and thus sensitizes the cells for the apoptosis-inducing effect of lipid peroxidation. This step explains the dominant role of aquaporins for plasma-mediated apoptosis induction, as reported by Yan et al.57,58 though the time frame of action is different from the model proposed by these authors. Intercellular apoptosis induction was mainly based on the HOCl signaling pathway, which depends on H2O2-dependent HOCl synthesis by the peroxidase domain of DUOX, and subsequent HOCl/O2●− interaction, leading to OH radicals that induce apoptosis through lipid peroxidation78,79,80.

In this way, even low concentrations of primary 1O2 trigger a massive, yet highly selective process directed towards tumor cells. The central overall process is summarized in the flowchart illustrated in Fig. 2, and further discussed in the light of this study and in relation to other models on CAP and PAM action, in Supplementary Figs 24 and 25. Nonmalignant cells remain unaffected by this process, as long as the H2O2 concentration in the system does not reach a level that leads to H2O2-mediated apoptosis induction in nonmalignant cells. Importantly, the apoptosis-inducing level of direct H2O2 application is lower for nonmalignant cells compared to tumor cells, due to the lack of membrane-associated catalase on nonmalignant cells59,74,81, (Bauer et al.,63. Therefore, selective apoptosis induction in tumor cells through the synergistic action of H2O2 and NO2 requires that the H2O2 level is below the damaging level for nonmalignant cells.

Flow chart of major steps in CAP leading to selective apoptosis of tumor cells. Step 1: CAP generates NO2 and H2O2 in cell containing medium for 1 minute. Alternatively, CAP is used to treat medium, creating PAM (step 1′). Defined concentrations of NO2 and H2O2 containing medium are used in reconstitution experiments (step 1”). Step 2: NO2- and H2O2 create primary 1O2 near cells following O2NOOH pathway, as described in Figure 1. Step 3: Few catalase molecules on a few cells are inactivated due to primary 1O2 near cells. Step 4: At the site of inactivated catalase, H2O2 and ONOO (generated through NOX1 and NOS) are no longer decomposed. Step 5: The reaction between H2O2 and ONOO is leading ultimately to secondary 1O2. Step 6: This additional 1O2 leads to further catalase inactivation and the process cycles back to step 4. Step 7: Increased H2O2 resulting from catalase loss from secondary 1O2 leads to H2O2 entering cells via aquaporins, leading to antioxidant glutathione depletion. Step 8: In parallel with step 7, increased H2O2 resulting from catalase loss from secondary 1O2 also leads to HOCl generation by peroxidase, in the presence of Cl. The interaction between NOX1-derived O2●− leads to OH formation near the cell membrane and lipid oxidation. Step 8′: If HOCl signaling is suppressed, an alternative NO/ONOO signaling can also lead to lipid peroxdiation. Step 9: If both lipid peroxidation and glutathione depletion occurs, then caspase-associated apoptosis can take place, finally leading to cell death. Steps 1–3 correspond to CAP triggering or activation of a few cells, thereby initiating propagating bystander signaling in steps 4–6. Steps 7–9 are the steps that lead to the final cell apoptosis. These steps are activated only if the repeated performance of steps 4–6 has caused a sufficiently high degree of catalase inactivation for reactivation of HOCl or NO/ONOO - mediated apoptosis-inducing signaling.

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Although Fig. 2 summarizes the key steps involved in selective tumor cell apoptosis following CAP treatment, it says nothing of the dynamics associated with each step. The present manuscript aims at the elucidation of the dynamics that drives the concerted action of primary and secondary 1O2 generation of CAP and PAM-treated tumor cells.

Materials and Methods

Materials

The NOX1 inhibitor 4-(2-Aminoethyl)benzenesulfonyl fluoride (AEBSF), the singlet oxygen (1O2) scavenger histidine, the NOS inhibitor N-omega-nitro-L-arginine methylester hydrochloride (L-NAME) and the HOCl scavenger taurine were obtained from Sigma-Aldrich (Schnelldorf, Germany).

The peroxynitrite decomposition catalyst 5- 10- 15- 20-Tetrakis(4-sulfonatophenyl)porphyrinato iron(III) chloride (FeTPPS), was obtained from Calbiochem (Merck Biosciences GmbH, Schwalbach/Ts, Germany).

The catalase mimetic EUK-134 [chloro([2,2′-[1,2-ethanediylbis[(nitrilo-κN)methylidyne]]bis[6-methoxyphenolato-κO]]]-manganese was a product of Cayman (Ann Arbor, Michigan, U.S.A.) and was obtained from Biomol (Hamburg, Germany).

Detailed information on inhibitors has been previously published74,82,83,84,85,86. The site of action of inhibitors and scavengers has been presented in detail in the Supplementary Material of references #81 and #85.

Cells and media for cell culture

The human gastric adenocarcinoma cell line MKN-45 (ACC 409) (established from the poorly differentiated adenocarcinoma of the stomach (medullary type) of a 62 year-old woman), was purchased from DSMZ, Braunschweig, Germany. MKN-45 were cultured in RPMI 1640 medium, containing 10% fetal bovine serum (FBS).

Fetal bovine serum (Biochrom, Berlin, Germany) was heated for 30 minutes at 56 °C prior to use. Medium was supplemented with penicillin (40 U/ml), streptomycin (50 µg/ml), neomycin (10 µg/ml), moronal (10 U/mll) and glutamine (280 µg/ml). Care was taken to avoid cell densities below 300 000/ml and above 106/ml.

Methods

The plasma sources

The portable air plasma ‘corona pen’ plasma source used here employs a neon-sign transformer with a rectifier and a high voltage multiplier and was developed in the framework of the frugal plasma biotech applications64. A high voltage needle electrode was inserted in a quartz tube. A DC-positive streamer corona discharge was generated on the needle electrode in ambient air, in a geometry similar to the discharge previously presented in87,88. The grounded electrode was a tin wire submerged in the cell culture medium at the bottom of the container. The distance of the needle tip to the medium surface was kept at 1 cm. The plasma discharge was directly hiting the liquid surface of the medium. The discharge voltage was kept at 10.7 kV and the maximum streamer pulse current was typically 17 mA with a repetitive frequency of 10 kHz. The streamer corona discharge generates RONS, such as O3, NOx, and OH radicals at very low deposited power (<0.1 W). All experiments described in this manuscript were performed with the streamer corona regime.

Treatment of cells with cold atmospheric plasma (CAP)

All treatments were performed in 24 well tissue culture clusters, 1 ml of medium and a grounded electrode. MKN-45 cells were used at a density of 125 000 cells/ml. The cells remain in suspension and only few cells attach firmly.

Standard treatment with CAP was by the streamer corona regime, with a distance of the plasma source from the top of the medium of 1 cm. Typical electrical parameters were voltage 10.7 kV, pulse amplitude 17 mA, and pulse frequency 10 kHz. The standard time of treatment was 1 min, unless otherwise indicated.

After treatment with CAP, the cells were either further incubated at 37 °C for the indicated times or subjected to washing steps and resuspension in fresh medium, depending on the protocol of the experiments. Whenever possible, culture was continued in 96 well plates with 100 µl medium after the washing step. These manipulations, which were essential for the analysis, are explained under “Strategy and design of our study”, as well as specified in the legends of the respective figures. The final step was always to determine the percentage of apoptotic cells induced by the treatment.

Generation and application of plasma-activated medium (PAM)

Complete medium without cells was treated with CAP for 1 min, unless otherwise specified. After 10 min, PAM was added to the cells that had been prepared at higher cell density, to reach a final concentration of PAM between 80–50%, as indicated. In some experiments, PAM was first serially diluted and then equal volumes of the dilution steps and cells of double standard density were mixed.

Determination of the percentage of apoptotic cells

After the indicated time of incubation at 37 °C and 5% CO2, the percentage of apoptotic cells was determined by inverted phase contrast microscopy based on the classical criteria for apoptosis, i.e., nuclear condensation/fragmentation or membrane blebbing74,82,89,90. The characteristic morphological features of intact and apoptotic cells, as determined by inverted phase contrast microscopy have been published74,82,91,92,93. At least 200 neighbouring cells from randomly selected areas were scored for the percentage of apoptotic cells at each point of measurement. Control assays ensured that the morphological features ‘nuclear condensation/fragmentation’ as determined by inverse phase contrast microscopy were correlated to intense staining with bisbenzimide and to DNA strand breaks, detectable by the TUNEL reaction83,91,92,93. A recent systematic comparison of methods for the quantitation of apoptotic cells has shown that there is a perfect coherence between the pattern of cells with condensed/fragmented nuclei (stained with bisbenzimide) and TUNEL-positive cells in assays with substantial apoptosis induction, whereas there was no significant nuclear condensation/fragmentation in control assays82,93. Further controls ensured that ROS-mediated apoptosis induction was mediated by the mitochondrial pathway of apoptosis, involving caspase-9 and caspase-393,94.

Statistics

In all experiments, assays were performed in duplicate. Quantitative data are presented as means ± standard deviations. The statistical analysis comprised the comparison of groups such as assay without apoptosis induction/assay with apoptosis inducer or assay without inhibitor/assay with inhibitor. Therefore, the differences between two groups were analyzed by Student’s t-test (two-tailed), with N = 500 in all tests, and double checked with the Yates continuity corrected chi-square test. The confidence interval used was 95%. P < 0.01 was defined as “significant”; P < 0.001 as “highly significant”. The modules for the calculation of the tests were taken from https://www.quantitativeskills.com/sisa/statistics/t-test.htm (t test) and from http://www.quantpsy.org/chisq/chisq.htm (Chi-square test).

Strategy and design of our analysis

In the first part of this study, tumor cells were treated with CAP or PAM, and the resultant kinetics of apoptosis induction were used to define central molecular species and their interactions, that are involved in this process. This approach was instrumentalized for the comparison between the unrestricted response of tumor cells to CAP and PAM, and responses in which distinct signaling events had been blocked. For example, secondary 1O2 generation was selectively prevented either by inhibition of NOX1 through AEBSF or NOS through L-NAME. The generation of primary 1O2 from long-lived species was prevented by decomposing ONOO and H2O2 through FeTPPS and EUK-134, respectively. Alternatively, the focus was put on the potential role of 1O2 derived from the gaseous phase of CAP. This was achieved through initial treatment of cells under the conditions where 1O2 generation by long-lived CAP-derived species as well as the generation of secondary 1O2 were blocked. To achieve a final read-out of apoptosis induction in all of these treatments, the initial treatment with its specific restrictions to defined signaling events needed to be followed by conditions that allow resumption of secondary 1O2 generation and subsequent catalase inactivation and apoptosis-inducing RONS signaling. In total, this approach gave insight into the dynamics and connection of the processes in our biological system of CAP and PAM-treated tumor cells.

Figure 3 explains the procedures that were used in the experiment described later in Fig. 8. This introductory experiment already defined some of the key concepts and findings that were essential for our study. Figure 3 A describes the control without CAP treatment. Under 3B, cells in medium were treated with CAP for 1 min and the cells remained in contact with the treated medium for another 25 min (at 37 °C), before a washing step (three cycles) was applied and the cells received fresh medium. The assays described under 3C was analogous to 3B, except for the presence of the NOX inhibitor AEBSF during CAP treatment and subsequent incubation. This prevented the generation of secondary 1O2, but allowed the generation of primary 1O2 through the interaction between long-lived species derived from CAP. The washing step stopped the generation of primary 1O2, but allowed the resumption of secondary 1O2 generation, as AEBSF is a reversible inhibitor. Therefore, tumor cells that had got an “imprint” (i. e. inactivation of some catalase molecules on their outside) by primary 1O2 were now able to generate cell-derived, secondary 1O2 and thus to induce a bystander effect in neighbouring cells. This finally resulted in a kinetics of apoptosis induction that was effective, but delayed compared to the assays without interference with signaling, such as in 3B. In the assays under 3D, E the medium was treated with CAP in the absence of cells. Cells were added to the treated medium afterwards. Under 3E, the contact between CAP-treated medium and cells was restricted to 25 min through a washing step. In the assays described by 3F, G, the cells were treated with CAP (in the absence or presence of AEBSF) and the treated medium was immediately removed after CAP treatment. This regime prevented the contact between the cells and primary 1O2 from long-lived species in the treated medium. Under G, also the generation of secondary 1O2 during the time of CAP treatment was prevented through AEBSF. 3F and G therefore allowed to focus on the imprint by primary 1O2 derived from the gaseous phase of CAP. In all assays, the percentage of apoptotic cells was determined at three different time points.

Scheme of the experimental procedures used in the experiment described in Fig. 8. (A) Untreated control cells (MKN-45, human gastric carcinoma cells) were cultivated in parallel to the other assays. (B,C) MKN-45 cells (125 000 cells/ml) in 24 well tissue culture clusters were treated with CAP for 1 min. After CAP treatment, the cells remained in contact with the same medium for 25 min. 100 µM of the NOX inhibitor AEBSF was present during CAP treatment and incubation in assay C. Assays were washed after the 25 min incubation step and were further cultivated in fresh medium. (D,E) Medium was treated with CAP for 1 min in the absence of cells and was then transferred to cells. The cells in assay (E) were washed after the incubation step and further cultivated in fresh medium. (F,G) Cells were treated with CAP for 1 min and then washed immediately. In assay (G), 100 µM AEBSF was present during CAP treatment. In all assays, the percentages of apoptotic cells were determined at 4, 6 and 7.5 h.

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The subsequent kinetic analysis, as described in Figs 9–12 all followed a protocol that is illustrated in Fig. 4. Treatment with CAP was for 1 min and was followed by an incubation for 25 min in the treated medium, before a washing step was performed (4B,C) or alternatively, the washing step was performed immediately after the CAP treatment (4D). Assays according to 4 C always contained AEBSF during CAP treatment and the subsequent incubation. Further inhibitors or scavengers, as specified in the respective figures were added either during treatment before the washing step or thereafter. The quantitation of the percentages of apoptotic cells was performed kinetically. The interpretation of the data requires to recapitulate that (i) histidine scavenges primary 1O2 as well as secondary 1O2, (ii) FeTPPS decomposes ONOO that is required for the generation of primary 1O2 through the interaction between H2O2 and NO2 as well as for the generation of secondary 1O2 and (iii) that AEBSF inhibits NOX1 and thus prevents the generation of cell-derived H2O2 and peroxynitrite that are required for the generation of secondary 1O2.

Basic scheme of the experiments described in Figs 9–13. (A) Untreated MKN-45 cells (control). (B,C) MKN-45 cells were treated with CAP for 1 min. The cells were further incubated in the same medium for additional 25 minutes and then washed (3 cycles) and resuspended in fresh medium. Inhibitors (INH) (as indicated in the respective figures) were present during CAP treatment and the incubation step. In the assays described under C, 100 µM AEBSF was present in all assays (for prevention of the generation of secondary 1O2), together without or with additional inhibitors, as indicated. Inhibitors were also added after the washing step, where indicated. (D) Cells were treated with CAP for 1 min, in the absence or presence of the indicated inhibitors and were then washed immediately and resuspended in fresh medium. Where indicated, inhibitors were added and cultivation was continued. The percentages of apoptotic cells were determined kinetically.

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The analysis shown in Fig. 13 used the same principle approach as described in Fig. 9, except that medium was treated with CAP in the absence of cells. The resultant plasma-activated medium (PAM) was then brought into contact to cells.

In the second part of the manuscript, tumor cells were pretreated with CAP or PAM under defined analytical conditions and were then added at increasing percentages to untreated tumor cells. This approach was based on a proof of concept experiment for “bystander effect inducing potential” of tumor cells with inactivated catalase60,95,96. The principle of this test system is shown in Figs 5 and 6.

The principle of bystander signaling between tumor cells with inactivated catalase and untreated tumor cells. (A) The principle. Left side: In a mixture between tumor cells with inactivated membrane-associated catalase and untreated tumor cells the final percentage of apoptotic cells would be strictly correlated to the percentage of pretreated cells in the population. Right side: Experimental evidence has been provided60,95,96 that tumor cells with inactivated catalase drive a singlet oxygen-mediated bystander signaling that causes inactivation of membrane-associated catalase, reactivation of intercellular RONS signaling and apoptosis induction. As a result, the percentages of apoptotic cells are much higher than to be expected from the percentage of pretreated cells in the mixedpopulation. (B) Quantitative analysis of bystander signaling. The percentage of apoptotic cells is much higher than the percentage of pretreated cells with inactivated catalase, when bystander signaling is effective. Inhibition of bystander signaling leads to percentages of apoptotic cells that strictly correlate with the percentages of pretreated cells. This system allows analysis of the underlying molecular mechanisms through application of defined inhibitors or scavengers at distince steps. It also allows to determine the percentage of bystander-inducing cells in a population through titration of cells from this population on untreated cells and determining the percentage of apoptotic cells. If the induction of bystander inducing potential is a rare effect, a large portion of a cell population will be needed to induce bystander signaling, whereas a population with a high percentage of bystander effect-inducing cells will allow bystander induction even with a low percentage of these cells within an untreated population.

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Interpretation of the results from bystander experiments. If a tested population of pretreated cells shows apoptotic potential, but no bystander effect induction, the dependency of apoptosis induction on the percentage of pretreated cells is a strict line. In the case of bystander signaling, the resultant curve is hyperbolic. It can be reduced to the linear relationship when inhibitors of bystander signaling (such as the singlet oxygen scavenger histidine or other relevant inhibitors) are present during the coculture phase. If pretreatment of tumor cells is a singlet oxygen-dependent pathway as well (as in the case of CAP and PAM), the presence of certain inhibitors will neither allow apoptosis induction nor bystander induction. If the number of bystander inducing cells in the pretreated population is low, the hyperbolic curve will be shifted more to the right, thus allowing quantitation of the bystander-inducing population.

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Combined with differential treatment and use of inhibitors/scavengers at defined steps, this approach allowed at least a semiquantitative estimation of the contribution of the individual signaling elements to the overall process and its underlying dynamics. The experimental procedures for the study of bystander signaling of tumor cells after treatment with CAP or PAM are summarized in Fig. 7.

Basic scheme of the bystander experiments performed in this study (Figs 14–19). Tumor cells were treated with CAP for 1 min and then either further cultivated in the same medium for additional 25 min, and then washed (A,B), or washed immediately after CAP treatment (C,D). Pretreated cells from assays A and C were added at increasing percentages to untreated tumor cells immediately after the washing step. Pretreated cells from assays B and D  were subjected to 25 min incubation after the first washing step, were then washed and added to untreated tumor cells at increasing percentages. As indicated in this figure and specified in the respective legends, defined inhibitors or scavengers could be applied at various steps. The titration of the pretreated cells, in combination with their potential for bystander signaling, Webcam Surveyor For Windows to quantitatively determine the number of cells that had obtained an “imprint” during a specific, experimentally defined step. This allows to conclude back on the dynamics of the underlying processes. The use of inhibitors and scavengers thereby allows to define the chemical biology involved in these dynamic interactions.

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The variability of the mode and time of CAP treatment, the placement of the washing steps, the length of the incubation steps and of the inhibitors/scavengers opens the chance to selectively pinpoint all intermediate steps and to obtain quantitative information on them.

The interpretation of the data obtained in this study was also based on and counter-controlled by results that had been obtained in reconstitution experiments, in which defined long-lived species from CAP and PAM, i. e. H2O2 and NO2 had been tested for their effects on tumor cells and nonmalignant cells59,60. Furthermore, experiments with the same plasma source as used in this study had allowed to define the essential steps in tumor cell/plasma interaction, such as generation of primary and secondary 1O2, catalase inactivation, glutathione depletion after H2O2 flux through aquaporins, intercellular apoptosis-inducing RONS signaling and activation of the mitochondrial pathway of apoptosis63.

Results

Dissection of the experimental system and kinetic analysis

Monitoring of apoptosis induction 4 h after treatment of tumor cells with CAP or PAM (Fig. 8 I) showed that direct treatment of tumor cells with CAP for 1 min, followed by 25 minutes of incubation in the same medium, and a subsequent washing step (condition B), caused a similar apoptosis-inducing effect as addition of PAM to untreated tumor cells (condition D,E). When the NOX inhibitor AEBSF had been present during CAP treatment plus the 25 min incubation step (condition C), no significant apoptosis induction was observed. Separation of the cells from the surrounding medium immediately after CAP treatment (condition F, G), also did not lead to detectable apoptosis induction within 4 h.

Kinetic and mechanistic aspects of different modes of CAP treatment of tumor cells. I. (A) MKN-45 cells remained untreated and were incubated for 4 h. (B) Medium and cells were treated with CAP for 1 min, followed by 25 min of incubation, a washing step and further incubation in fresh medium up to 4 h; (C) The NOX1 inhibitor AEBSF (100 µM) was present during the preatreatment of cells + medium with CAP for 1 min, followed by 25 min incubation, a washing step and further incubation in fresh medium. (D) Medium was treated with CAP for 1 min and then tumor cells were added to reach a final Ron`s Editor 2019 Download - Crack Key For U of PAM of 80%. The assays were further incubated up to 4 h. (E) Medium was treated CAP for 1 min and then tumor cells were added. The assays were further incubated for 25 min, washed, and further incubated in fresh medium up to 4 h; (F) Medium plus cells were treated with CAP for 1 min and then washed immediately, resuspended in fresh medium and further cultivated. (G) The assay was performed as described undeer F, with the modification that AEBSF was present during CAP treatment. II., III.: The conditions and assays are identical to those described under I, but the time of assessment of apoptosis was at 6 h or 7.5 h after CAP treatment, respectively. The results show that CAP treatment of tumor cells in medium, followed by 25 min incubation in the same medium causes apoptosis induction to the same degree as treatment of medium with CAP, followed by incubation of cells in this plasma-activated medium (PAM). This points to the dominant action of long-lived species for apoptosis induction. The presence of AEBSF during CAP treatment and 25 min incubation causes a kinetic delay, which is explained by initial prevention of secondary singlet oxygen (1O2) generation, followed by resumption of secondary 1O2 generation after the washing step and incubation in fresh medium. Separation of the CAP-treated cells from their medium immediately after 1 min CAP treatment results in a very long delay in apoptosis induction, as 1O2 from the gaseous phase of CAP is the only initial trigger under the conditions in assays F and G.

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When the quantitation of apoptotic cells was performed two hours later (Fig. 8 II), the inhibitory effect through the presence of the NOX inhibitor during CAP treatment and 25 min incubation phase (C) was no longer observable, whereas cells treated according to regime F and G still showed no apoptosis induction. At six hours, the assays that had already been positive at four hours seemed to have reached their plateau phase of apoptosis induction, as can be concluded from comparing Fig. 8 I with Fig. 8 II. However, Fig. 8 III (measurement at 7.5 hours) indicated that conditions F and G seemed to allow late occurence of apoptosis induction. These data point to a complex system of multiple CAP and PAM interactions with the tumor cells. These interactions seemed to occur in parallel, but with different requirements, kinetics and efficiencies. The data also show that long-lived species in plasma-activated medium trigger the major effect achieved with CAP treatment. The effect of the NOX inhibitor AEBSF points to the role of tumor cell-specific NOX1 in this process, conceivably through generation of secondary 1O2.

The next experiment allowed to differentiate between three kinetically distinct processes after CAP action. As shown in Fig. 9A, the overall process of apoptosis induction in CAP-treated tumor cells was completely inhibited when either scavenging of 1O2 by histidine, decomposition of ONOO by FeTPPS, or inhibition of NOX1 by AEBSF continued over the whole time of the experiment. This points to the dominating role of 1O2, ONOO and superoxide anions (O2●−) for this process, but does not yet allow to differentiate between the different phases of the process or between potentially existing multiple pathways.

Dissection of CAP-mediated apoptosis into three kinetically defined processes. (A) MKN-45 cells in medium (“M + C”) were not pretreated (control) or treated with CAP for 1 min in the absence or presence of either 2 mM of the singlet oxygen (1O2) scavenger histidine, 25 µM of the peroxynitrite (ONOO) decomposition catalyst FeTPPS or 100 µM of the NOX1 inhibitor AEBSF. The assays were incubated after CAP treatment for the indicated times (without any washing steps). (B) MKN-45 cells were not pretreated or treated for 1 min with CAP in the absence or presence of the indicated inhibitors. The assays were further incubated for 25 min and then subjected to three cycles of washing. The cells were resuspended in fresh medium and further incubated, as indicated. (C) The tumor cells were treated with CAP for 1 min in the absence or presence of the indicated inhibitors. Immediately after CAP treatment, the cells were subjected to three cycles of washing, resuspended in fresh medium and cultivated further, as indicated. In all assays, time zero in is the beginning of CAP treatment. The data show that Ron`s Editor 2019 Download - Crack Key For U modifications after CAP treatment allow to define three kinetically different processes.

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However, the use of specific inhibitors during the initial phase of CAP treatment and 25 min incubation before the washing step allowed a clear differentiation between three kinetically different processes (Fig. 9B). In the absence of inhibitors, an overall process (#1) started after a lag phase of 30 minutes and reached a plateau at about 5 hours of incubation. When NOX1 had been inhibited by AEBSF, and thus autoamplification of 1O2 generation had been prevented during the first 25 minutes of the experiment, the kinetics of apoptosis induction in this process (#2) was delayed for about 3 hours. It then approached the efficiency of the overall process. Process #2 seemed to depend on ONOO, as it was inhibited by FeTPPS. The curve obtained for process #2 inhibited by FeTPPS was indistinguishable from the kinetics of a further process (#3).

This kinetically different process (#3) started five hours after CAP treatment. It also was independent of NOX1 activity during CAP treatment and the first 25 minutes of incubation, but in addition did not require the presence of ONOO during this initial phase. Importantly, this third process was also demonstrated in full activity and unchanged kinetics in cells that had been removed from their surrounding medium immediately after CAP treatment (Fig. 9C), and therefore were not under the influence of the long-lived species in the medium for longer time. In contrast, process #1 and #2 were not observed in a cell population that had been separated from the medium containing long-lived CAP-derived species, immediately after the 1 min treatment with CAP. Process #3 was not inhibited by the presence of AEBSF or FeTPPS alone or in combination during CAP treatment, provided CAP treatment was followed by a washing step to remove the inhibitors.

Processes #1–#3 were completely prevented when the 1O2 scavenger histidine had been present during CAP treatment and the subsequent 25 min incubation step (Fig. 10). This demonstrates the central function of 1O2 for each single one of these processes.

The CAP-induced processes are dependent on the action of singlet oxygen (1O2). MKN-45 cells in medium were treated for 1 min with CAP in the absence of inhibitors or in the presence of the indicated inhibitors. All assays were further incubated after CAP treatment for 25 min, before they were subjected to three cycles of washing. Further incubation was performed in fresh medium without any inhibitors and the percentages of apoptotic cells were monitored kinetically. The results show three kinetically different processes which all were completely blocked when the 1O2 scavenger histidine (HIS) was present during CAP treatment and the 25 min incubation step following CAP treatment. Assays containing L-NAME showed no apoptosis induction, as L-NAME is an irreversible inhibitor of NOS. The inhibitors were applied at the following concentrations: HIS (2 mM); AEBSF (100 µM), FeTPPS (25 µM), L-NAME (2.4 mM).

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Addition of the 1O2 scavenger histidine or the ONOO decomposition catalyst FeTPPS after the 25 min incubation step had a differential impact on processes #1–#3 (Fig. 11). The overall process (#1) was no longer inhibited by histidine or FeTPPS under this condition of late addition of inhibitor (Fig. 11A) indicating that an early singlet oxygen- and ONOO-dependent process was completed at the time of addition of inhibitors. Process #2 was still partially inhibited by late addition of histidine and ONOO (Fig. 11B), indicating that a 1O2- and ONOO-dependent step was only partially completed at the time of addition of inhibitors. Importantly, process #3 was completely blocked by the late addition of histidine and FeTPPS (Fig. 11C). This indicates a remarkable switch of process #3 from an initial independence of ONOO to the dependence on ONOO later on, and the requirement for 1O2 during this late phase. In other words, process #3 seemed to be triggered by 1O2 that was not generated by a process that required ONOO as intermediate, whereas the propagation of its apoptosis-inducing effects required 1O2 generation with ONOO as an intermediate later daemon tools pro crack 2021 kinetically determined, CAP-induced processes show differential response to early and late addition of inhibitors. The experiments were performed in analogy to those described in Fig. 10. In addition, parallel assays also received either 2 mM of the singlet oxygen (1O2) scavenger histidine or 25 µM of the peroxynitrite (ONOO) decomposition catalyst FeTPPS after the washing step that followed CAP treatment and the first incubation step of 25 min. Assays without CAP treatment showed less than 5% apoptotic cells at all time points (not shown in the graph). (A) Process #1 was inhibited when histidine was present during CAP treatment and the first 25 min incubation step, but not when histidine had been added after the washing step. Likewise, FeTPPS added after the washing step had no inhibitory effect. (Note that addition of FeTPPS during CAP treatment would have shifted process #1 to process #3, shown below). Process #1 is the overall process, based on unlimited primary and secondary singlet oxygen generation. (B) Process #2 was induced when AEBSF was present during CAP treatment and the 25 min incubation step. Process #2 was completely inhibited by the presence of histidine during CAP treatment and the 25 min incubation step, and partially inhibited when histidine or FeTPPS had been added after the washing step. Process #2 reflects the regime in which secondary1O2generation is blocked during CAP treatment and the subsequent 25 min incubation step. After the washing step, secondary1O2generation resumes, driven by the imprinted signature established initially by primary1O2generated by long-lived species of CAP-treated medium. (C) Process #3 is independent of ONOO and NOX derived O2●− during CAP treatment and the 25 min incubation step, but completely dependent on 1O2 at this step. The process after the washing step is completely dependent on 1O2 and on ONOO. Process #3 depends on the imprinted signature by primary1O2from the gaseous phase of CAP. The imprinted signature drives secondary1O2generation after the washing step.

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When process #3 was induced specifically in cells that had been removed from their surrounding medium immediately after CAP treatment (in the absence or presence of AEBSF or FeTPPS or both of them), the dependency of this step on 1O2 as well as ONOO at the time following the washing step was clearly shown. Furthermore the dependence on 1O2, but independence on ONOO of this step during CAP treatment was confirmed (Fig. 12).

Further details of process #3. Process #3 was established either by treating tumor cells with CAP for 1 min, followed by an immediate washing step and further incubation in fresh medium. During CAP treatment, the tumor cells were not confronted with inhibitors (A), or with 100 µM of the NOX1 inhibitor AEBSF (B) or AEBSF plus 25 µM of the ONOO decomposition catalyst FeTPPS (C). As indicated, assays contained additional histidine or FeTPPS, either during CAP treatment, or during the incubation phase following the washing step. Control assays without CAP treatment did not show apoptosis induction above 4% (data not shown). The results show that process #3 is initiated by primary singlet oxygen from the gaseous phase of CAP, independent of H2O2/NO2-dependent primary 1O2 (as the effect was not inhibited by FeTPPS during treatment) and independent of secondary 1O2, as it was not inhibited by AEBSF. However, the imprinted signature induced by primary 1O2 from the gaseous phase of CAP allowed the generation of secondary 1O2 after washing, as seen by the inhibitory effects of histidine and FeTPPS.

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A kinetic analysis of PAM-treated tumor cells showed that PAM induced process #1 and #2 with equal efficiency and with identical kinetics and inhibition profiles as shown for direct CAP treatment (Fig. 13A,B). However, process #3 with its initial independence of both O2●− and ONOO, was not observed after treatment of cells with PAM (Fig. 13C). This finding indicates a relationship between processes #1 and #2 to the long-lived species contained in PAM. It also is in line with the conclusion that process #3 seems to be directly triggered by 1O2 from the gaseous phase of CAP. This process cannot be triggered by PAM, due to the short-lived nature of 1O2.

Plasma activated medium (PAM) triggers processes #1 and #2, but not #3. Medium was treated with CAP for 1 min and then an equal volume of cells of double standard density was added. Where indicated, inhibitors were added during the initial contact of PAM and cells. After 25 min of incubation, assays were subjected to three cycles of washing and the cells were resuspended in fresh medium. Where indicated, inhibitors were added at this step. Inhibitors were added at the following concentrations: AEBSF (100 µM); FeTPPS (25 µM), HIS (2 mM). The assays were further incubated and the percentages of apoptotic cells were determined kinetically. Time zero in all assays is the time of mixing PAM and cells. Control assays without PAM did not show apoptosis induction above 4% at all time points (data not shown). The results show that PAM induces the kinetic processes #1 and #2 with equal efficiency and characteristics as shown for direct CAP treatment in the preceding figures, whereas PAM has no potential to initiate process #3. This finding is in line with the short-lived nature of singlet oxygen derived from the gaseous phase of CAP that is responsible for process #3. These findings demonstrate the generation of primary 1O2 through the interaction of the long-lived species in PAM and to the role of secondary 1O2 that is generated by the tumor cells, dependent on their active NOX1.

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Supplementary Fig. 1 demonstrates the independence of process #3 from NOX-derived O2●−, and ONOO during the 1 min CAP treatment, but its dependence on the primary 1O2 during CAP treatment. It also shows the action of a 1O2- and ONOO-dependent step for more than one hour after the washing step that followed CAP treatment and separation of the cells from their treated medium. Finally, the strong inhibitory effect of the HOCl scavenger taurine added 4 h after the initial washing step showed that process #3 seems to be finalized by HOCl signaling.

Supplementary Fig. 2 shows that process #3 is also independent of the action of H2O2 during the 1 min of CAP treatment, as it was not inhibited in the presence of AEBSF and the catalase mimetic EUK-134. Also under these conditions, the process was dependent on 1O2 and ONOO after the washing step, in analogy to the results shown in Supplementary Fig. 1.

When CAP treatment of the cells was extended from 1 min to 5 minutes, the kinetics of apoptosis induction through process #3 started at 2 hours instead of 4 hours post treatment (Supplementary Fig. 3). The presence of the 1O2 scavenger histidine during CAP treatment, as well as after the washing step, confirmed that this process was initiated by 1O2 and required 1O2 for its kinetic propagation. The results are consistent with gas phase 1O2 triggering the effect. As expected, the onset of the kinetics is dependent on the time of CAP treatment. In contrast, 1O2 required for the propagation step must have been derived from the triggered tumor cells themselves.

Quantification of the dynamics of singlet oxygen-mediated catalase inactivation in the cell population

The kinetic analysis of CAP-and PAM-mediated effects on tumor cells, as well as the mechanistic studies in the preceding manuscript63, have indicated that 1O2 generated through the interaction between long-lived RONS seemed to trigger the tumor cells to generate secondary 1O2 to an extend that causes extensive inactivation of membrane-associated catalase. This then seems to allow the reactivation of RONS-dependent intercellular apoptosis-inducing signaling. We therefore endeavoured to obtain quantitative data on the relative contribution of processes #1–3 to this autoamplificatory mechanism.

Autoamplification requires cell-to-cell communication, sometimes referred to as the “bystander effect”. Cells that did not experience the initial exposure of primary 1O2 and the associated inactivation of catalase must somehow be induced to undergo apoptosis. Our postulate is that this cell-to-cell bystander effect communication occurs because a few cells with inactivated catalase will generate enough H2O2 and ONOO to generate secondary 1O2 that will inactivate catalase on its neighboring cells. Due to their inactivated catalase, these neighboring cells are now allowed to generate their own secondary 1O2 and the process can continue in a kind of apoptotic wave. This postulate can be tested by pre-treating a subset of cells (thereby inactivating catalase and rendering them capable of generating secondary 1O2), then mixing and co-culturing these pre-treated cells with untreated cells. The judicious use of the various chemical species scavengers and enzyme inhibitors at different periods of the pre-treatment and co-culturing stages allows more refined testing of which species are key to the process.

Based on previous approaches that utilized siRNA-mediated knockdown of catalase95, the effect of direct 1O2 generators96, or high concentrations of H2O297, as well as reconstitution experiments with defined concentrations of H2O2 and NO260 we utilized an experimental system that allows to define and characterize bystander signaling between CAP/PAM-pretreated cells and untreated tumor cells. It is based on the concept that apoptosis induction in mixtures of increasing concentrations of CAP/PAM-pretreated cells with untreated cells should merely be correlated to the percentage of pretreated cells if there was no bystander signaling. If bystander signaling occurred between pretreated and untreated cells, final apoptosis induction should be much higher than to be expected from the percentage of pretreated cells in the mixture. In other words, there should be an amplification effect: initially pre-treated cells co-cultured with untreated cells should induce the untreated cells to undergo apoptosis during co-culturing.

The validity and significance of this experimental system has been explicitely shown for mixtures of untreated tumor cells with increasing percentages of tumor cells that had been either pretreated by siRNA-mediated knockdown of their protective catalase95, inactivation of their protective catalase through 1O2 generated by an illuminated photosensitizer96, or by defined sources of H2O2 and NO260.

When MKN-45 cells were pretreated with CAP for 1 min, followed by an incubation of 25 min in the same medium, and were then washed and added to untreated cells at increasing concentrations, a remarkable bystander effect on apoptosis induction was observed (Fig. 14A). The first of five experiments illustrated in Fig. 14A is pre-treating and co-culturing with no inhibitors (open circles), with varying percentages of pre-treated cells used in the co-culture stage. With zero percent pretreated cells, i. e. the pure population of untreated cells, no apoptosis above background was observed. With 100 percent pretreated cells, i. e. the pure population of pretreated cells, the reference (maximum) value of approximately 40% apoptotic cells was observed. This case corresponds to the previous experiments. The addition of only 0.1% pretreated cells (i. e. 12 cells) to an excess of untreated cells caused significant apoptosis induction. The presence of 0.2% pretreated cells (25 cells) was sufficient to induce the maximal effect of apoptosis induction in the cell population. This is clear evidence that only a few cells that have been activated by CAP or PAM exposure will trigger the maximal level of apoptosis in the entire populations of cells.

Источник: https://www.nature.com/articles/s41598-019-50329-3