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Abadox: The Deadly Inner War Reviews (NES)
Published by
Developed by
Critic Score
100 point score based on reviews from various critics.
User Score
5 point score based on user ratings.
User Reviews
|It's gross!
|Adzuken (818)
|Entertaining Life Force rehash
|Multimedia Mike (18029)
|Odd Shooter into the Belly of a Beast
|Game22 (42)
Our Users Say
|User Score
|How well the game mechanics work (player controls, game action, interface, etc.)
|The quality of the art, or the quality/speed of the drawing routines
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|How much you personally like the game, regardless of other attributes
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|The quality of the sound effects and/or music composition
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|Overall User Score (15 votes)
Critic ReviewsMobyRanks are listed below. You can read here for more information about MobyRank.
Pixel-Heroes.de (Oct, 2003)
Abadox weiß von Anfang an richtig zu begeistern und ist für mich eines der besten NES-Spiele überhaupt. Auch wenn es nicht ganz leicht ist, ist es aufgrund der unendlich vielen Continues mit ein wenig Übung zu schaffen. Die vielen originellen Mittel- und Endgegner sowie die zahlreichen Waffen sorgen für eine extrem hohe Motivation.
Retro4Ever (May 23, 2012)
The graphics are superb as the living environments and grotesque enemies seemingly come from the minds of some true sick puppies. The sound effects are satisfying, and the music is catchy and varied. Just try to get the music from level 2 out of your head after an intense play-through, it won't be easy! Published by Milton Bradley, it was developed by Natsume, which these days is mostly known for Harvest Moon and Rune Factory spin-offs. Of all the games published by Milton Bradley on Nintendo, this one stands out as the best in my opinion, edging out even Captain Skyhawk. This kind of game features a difficulty not often seen in games of this generation, so be warned that it will only appeal to those with old-school sensibilities. If that's you, or if you are already a fan of games such as Life Force, then this is a must-play. If you can get a copy used, I urge you to add this to your collection.
Video Game Den (Apr 07, 2015)
All in all, Abadox is a great shooter with a fun premise and solid ideas throughout, with a focus on a very difficult gameplay that demands perfect maneuvering and careful attention to enemy attack patterns.
Electronic Gaming Monthly (EGM) (May, 1990)
It plays fine, and for most that's good enough - but I'd have like to see more.
All Game Guide (1998)
Despite these issues, Abadox is one of the better shooters available for the 8-bit system and fans of the genre should definitely check it out (if only for the interesting visuals and level design). While Abadox won't win any awards for innovation, it succeeds at being a fun game to play.
Game Freaks 365 (2006)
In conclusion, I must say that Abadox is generally an average game. It looks good sometimes and it plays good, but most of its features are generally the same as any other shooter when it comes down to it. Most of the variety is superficial, and when you have enough experience or have seen enough games like this and know about the history you see that it's more of a footnote than anything else. It has its moments and can be quite entertaining, but it's also somewhat marred by a poor difficulty curve and some pretty silly graphic and sound combinations. I definitely suggest it to NES fans nonetheless, but with a bit of caution.
HonestGamers (Apr 09, 2013)
If moments like that were representative of Abadox as a whole, I'd feel no chagrin about that 8/10 I once awarded it. Sadly, like I said, that moment of immersion represents the game's peak. Most of the time, I instead found myself playing a game that merely looked nice and was interesting almost solely because it possesses the sort of imagery that Nintendo usually was so good about preventing us poor Americans from seeing (especially this blatantly). I also found myself growing frustrated by the slew of deaths inflicted on my ship, particularly because each of those deaths might as well have been a "Game Over" screen thanks to the crippling loss of all my power-ups. Abadox has some merit, but a great game it is not…
The Video Game Critic (Nov 12, 2007)
The graphics are about average, but the multi-colored explosions look nice. There are two keys to beating Abadox: loading up on power-ups early, and memorizing the patterns. The game's uneven difficulty can lead to frustration, but even if that were fixed, Abadox would still be marginal at best.


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Stayed for 5 night with colleagues when visiting Dell. Not sure why somoen booked this place for us, as it is way below the standard of hotels we normally stay in.
However, if you are on very tight budget it isn't a bad option. Rooms are very large with two Queen double beds. They come well equipped with two Queen beds, fridge, coffee apparatus, iron and ironing board. Staff were fine and gave us good recommendations for restaurants within walking distance. There are quite a few of these, so I don't understand some of the comment below. I really recommend "Texas Land & Cattle Steakhouse", which we like so much we went there for two nights.
Location isn't very appealing (next to several main roads) and the place looks awful form the outside. Breakfast looked unappealing, so I didn't try it.
- Official Description (provided by the hotel):
- Americas Best Value Inn Austin, located in the state capitol of Texas and the live music capitol of the world. Visit the historic Governor's Mansion, which has been the official home of every Texas governor since 1856. Our hotel has two floors & 88 comfortable guest rooms. Each room is well appointed with free wifi, free local calls, microwaves & refrigerators, hair dryers, coffee makers and 25" TV with cable channels including HBO. Our property amenities include: Free Wifi, outdoor pool, exercise room & guest laundry. ... more less
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text:
Huawei sues T-Mobile over 4G patents
Consider it retaliation for T-Mobile's lawsuit from 2014.
Huawei confirms that smartphone cameras still aren't DSLRs
Even when they have Leica's name slapped on them.
Huawei's MateBook is beautiful, but fundamentally flawed
A terrible keyboard, trackpad and kickstand experience hold back its attractive hardware.
Huawei's MateBook heads to the US on July 11th for $699
Another Surface competitor enters the fray.
Huawei manager says it's making another Nexus device
Can we expect a successor to the Nexus 6P or even a tablet?
Huawei sues Samsung over cellphone patents
It claims that Samsung's phones are swiping its technology.
The smartphone market is peaking
Shipments have leveled off, and smaller phone makers are ousting the giants.
Huawei P9 review: New phone, familiar tricks
A fun camera doesn't save the P9 from being another iterative smartphone.
Of course Huawei is making a Gear VR rival
These are apparently the first VR headsets to offer 360-degree sound field on the go.


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Talk:Kolia Bhomora Setu
|WikiProject Assam
|WikiProject India / Assam
|(Rated Stub-class, Low-importance)
Bhomoraguri inscription facts?[edit]
The famous Bhomoraguri inscription where references to the intention of a bridge cross the Brahmaputra near Tezpur exists in the Chitralekha Udyan (formerly known as Cole Park). However, I have come across two different references as to who put the inscription.
- At the bottom of http://sonitpur.nic.in/archaeology.htm (Additional Information section) it states "The rock inscription lying on the Bhomoraguri hill near the Kolia Bhomora Bridge has not been recorded in the list of the protected monuments of Sonitpur district. The inscription states the construction of a rampart by Ahom King Pratap Sinha (1603-41 A.D.) after defeating the Mughals in the Saka year 1538. This is the first Assamese inscription (i.e. the language of the inscription is Assamese). So far discovered in Assam till now."
- At the bottom of http://www.assamonnet.org/index.php?article=siteseeing (click on Tezpur at the bottom to show the relevant section) it states "Bhomoraguri - The mammoth stone inscription made by the Ahom General Kalia Bhomora Borphukan, who planned to construct a bridge over Brahmaputra. The Kolia Bhomora Bridge is a 3.015 km bridge over the Brahmaputra River that connects Tezpur on the North bank with Nagaon on the south bank. This is one of the longest bridge in India and stands unrivalled in its grandeur."
Who actually made the inscription or plan the bridge - Ahom King Pratap Sinha or Ahom General Kalia Bhomora Borphukan? Can someone verify the fact please?-Deepraj | Talk 14:52, 14 June 2007 (UTC)
Image copyright problem with Image:Kbridge.jpg[edit]
The image Image:Kbridge.jpg is used in this article under a claim of fair use, but it does not have an adequate explanation for why it meets the requirements for such images when used here. In particular, for each page the image is used on, it must have an explanation linking to that page which explains why it needs to be used on that page. Please check
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This is an automated notice by FairuseBot. For assistance on the image use policy, see Wikipedia:Media copyright questions. --01:41, 18 September 2008 (UTC)
A giant Leap towards the development of the North-East India. The most striking feature of the picturesque North Eastern region, the hills and rivers, is the all-pervasive presence of the majestic Brahmaputra. This masculine river not only splits the state of Assam along its length but also cuts off the seven sister states of the North-East from the rest of the country.
What on earth does this bullshit actually mean? Skinsmoke (talk) 18:39, 3 December 2012 (UTC)


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Lyrics Depot is your source of lyrics to An Experiment In Homicide by Cannibal Corpse. Please check back for more Cannibal Corpse lyrics.
An Experiment In Homicide Lyrics
Artist: Cannibal Corpse
Album: The Bleeding
[Music by Webster, Barrett, Owen]
Walk away, into the dark
Come my way, terror
I am the evil within each soul
I take the spirits of those I kill
Decomposition, The body rots
Rotting, rotting body parts
I killed the first
To experiment
I don't want to hurt you
I just want to kill you
Torture, I start to cut
Carving, blood slowly clots
Horrors, mutilations
Homicidal experimentation
I set the dead on fire
To decay in flame
Homicidal desire
More are marked for murder
More victims to capture
Bodies hang dead
Drained of all their blood
Pieces sewn on backwards
Neck with no heads
Mutilated, dead
Homicidal desire
More are marked for murder
More victims to butcher
Cannibal Corpse Lyrics
Cannibal Corpse The Bleeding Lyrics
More Cannibal Corpse Music Lyrics:
Cannibal Corpse - Bloodlands Lyrics
Cannibal Corpse - Dormant Bodies Bursting Lyrics
Cannibal Corpse - I Will Kill You Lyrics
Cannibal Corpse - No Remorse Lyrics
Cannibal Corpse - Put Them To Death Lyrics
Cannibal Corpse - Sanded Faceless Lyrics
Cannibal Corpse - Severed Head Stoning Lyrics
Cannibal Corpse - Under The Rotted Flesh Lyrics


text:
Our panel looks at the big stories in the news.
The Short List: Mike Albano Enters Sheriff's Race, Lord Jeff No More, School Spending
Our panel looks at the big stories in the news.
With New Budget, A $10,000 Pay Hike For Mass. Governor's Councilors
The Boston Herald featured the pay raise on its front page Thursday with the headline, "Money for nothing!"
Springfield Courthouse Could Be Renamed For Former Justice Ireland
Legislation to rename the Hampden County Hall of Justice for Roderick Ireland, former chief justice of the Massachusetts Supreme Court, was filed on Thursday.
Albano To Question Supreme Judicial Court Pick On Work With FBI, U.S. Attorney
At a meeting last week, the nominee said nothing. The hours of testimony were devoted to speakers praising Associate Justice Ralph Gants, and members of the governor's council questioning those speakers.


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|Reviews for A Beautiful Mess
| DawnsWhimsy chapter 11 . 12/10/2011
| xtothey chapter 11 . 12/10/2011
**cries** NOOOOOOOOOOOOOOOO. Don't make him leave! HE'S EDWARD. lol
| parula8 chapter 11 . 12/10/2011
I saw this story recommended by iambeagle and I wanted to tell you how much I am enjoying it.. It's heartbreaking what Bella is going through, but so well written.. Thank you! :-)
| iambeagle chapter 11 . 12/10/2011
:( my heart hurts for Bella! I do want to slap her though to make her realize that she's not a charity case for Edward, but unfortunately it's not an easy fix. I know he won't give up though, and I just hope she allows him in. SHE HAS TO. Love this, love you, now off to review the other chapters that I skipped..
| deranged-dreamer chapter 11 . 12/10/2011
I was getting all jealous about Gianna.
Yeah i cant blame bella for her raction. I mean, she doesnt ally know him.
| twilly chapter 11 . 12/10/2011
Oh, that was rough. I hurt so much for both of them.
I totally get where Bella is coming from. Hopefully she'll process and open up a bit more rather than use it as an excuse. I guess that a lot easier said (by someone else) than done. Avoidance is my coping mechanism of choice. I rarely works out well.
Edward could probably use a little TLC after dealing with his friend for a week, and now this. And you know, he still doesn't have her number!
| MsHavisham79 chapter 11 . 12/10/2011
Somehow, despite the fact that I had already read this, it made me want to cry just now. This paragraph in particular just made me want to sit on the floor and cry - "Don't you dare do that," I shouted, my body vibrating with rage. "You have no idea, no fucking clue, what it's like to be in that kind of situation. To love someone so much and have them begging you to stay while completely crushing your spirit at the same time. To love someone and watch them slowly destroy themselves everyday and have them tell you that you are the reason that they go on, that you are the only reason they continue to live," I finished, gasping for air.
It is perfection, the whole thing and I totally get where Bella is coming from. As an outsider I can see that maybe Edward feels like the strength of what he feels for Bella means that he is where he is meant to be and that what he went through before with Gianna has made him able to deal with whatever he needs to deal with in order to be with Bella like his heart wants. But from Bella's perspective it does just look like he is trying to make up for past mistakes.
I hope Edward takes the time to prove he isn't going anywhere and in time that Bella can see it is HER he wants, not redemption.
And dude, making me all blush over calling me lovely. I love you and am honored you even ask me to read your stuff. I am not worthy but I do so love talking it all through with you.
| linz1628 chapter 11 . 12/10/2011
This Edward can't catch a break. I get where Bellas coming from, it looks really bad from her position. Let's hope some time and a clear head help her process everything. She has had a lot go on in the last few hours, she can have a little time.
| left1215 chapter 10 . 12/9/2011
Ok, seriously loved this chapter. Every single part of it.
At one point I was in my office with my hands over my mouth cause that is just what I do when I am FREAKING out about what is going happen.
Wasn't sure where you were going with the whole Sam thing. Glad I don't have to come in there in PP you :)
Looks can be decieving - even though Garrett looks better when he says "I just figured you'd finally gotten over whatever was wrong with you and we could start over again," he proves he is still an asshole.
'Edward's fist met Garrett's jaw with a deafening crack. I stood, stunned as Edward pummeled his fist into Garrett's face, blood splattering across the wall': Most satisfying moment of my week.
Alright hooker, you better have a good reason for Edward not being around for 10 days. Although I have a sneaking suspicion that I am not going to like it very much.
| jennlen522 chapter 10 . 12/9/2011
Yes, was about to beat the shit out of Edward for being absent for so long. What the hell? Okayyyy, way to addicted to characters and story Jenn. Anyway, I love this story. I love you and I am all up in this angsty place today so this is just what I needed. LOL Great as always. :)
| deranged-dreamer chapter 10 . 12/9/2011
he didnt have her phone?
seriously, cant they ban garret from the bar?
and there isnt ONE single person from her past that isnt an asshole?
| xtothey chapter 10 . 12/9/2011
I'm scared!
| jmcfall chapter 10 . 12/9/2011
Omfg I feel like I just got chocolate every time you update. So yea thanks so much .
| shehazi chapter 10 . 12/9/2011
Is it bad that I got a heck of a lot of satisfaction fromthe image of Edward beating the shit out of Garrett?
| bzwifenmom24-7 chapter 10 . 12/9/2011
I can't remember Garrett history with Bella. I thought Edward was her first.


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QR Code WordPress plugin to insert a QR code in your blog. The qr code contains the current URL or any other text you like.
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qrcode_wprhe.php to the directory
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Compatible up to: 4.2.9
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Active Installs: 1,000+
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Lonnie Brooks
Similar Artists
Son Seals - 100% match to Lonnie Brooks
Frank Seals Jnr., 14 August 1942, Osceola, Arkansas, USA, d. 20 December 2004, Chicago, Illinois, USA. Son Seals was one of 13 children of Jim Seals, an entertainer and club owner in rural Arkansas. Son began his musical education on the drums and worked with many of the later famous musicians who travelled through the area. Having taught himself to play the guitar, he forme Read more
Hubert Sumlin - 94% match to Lonnie Brooks
16 November 1931, Greenwood, Mississippi, USA. One of 11 children, Sumlins mother saved hard to buy him an $8 guitar when he was a child. Renowned for his guitar work, particularly in support of his mentor Howlin Wolf, Hubert Sumlin began his career in the Mississippi juke-joints. He joined Jimmy Cotton and first met Wolf when he was 19 in Memphis where he worke Read more
Carey Bell - 93% match to Lonnie Brooks
Carey Bell Harrington, 14 November 1936, Macon, Mississippi, USA, d. 6 June 2007, Chicago, Illinois, USA. Bell began to play harmonica after being inspired by the records of Muddy Waters, Little Walter and Sonny Boy Rice Miller Williams. He played with a white C&W band and with his stepfather, Lovie Lee. He moved to Chicago with Lee in the mid-50s Read more
Big Walter Horton - 90% match to Lonnie Brooks
6 April 1918, Horn Lake, Mississippi, USA, d. 8 December 1981, Chicago, Illinois, USA. Horton, also aka "Shakey", "Mumbles" and "Big Walter", claimed to have taught himself harmonica by the time he was five years old, and certainly the extraordinary skill he achieved speaks of a very special affinity with the instrument. By his teens, he was in Memphis and beginning to make Read more
Earl Hooker - 82% match to Lonnie Brooks
Earl Zebedee Hooker, 15 January 1930, Clarksdale, Mississippi, USA, d. 21 April 1970. Hookers interest in music was kindled at an early age. A self-taught guitarist, he began his itinerant career as a teenager, and having toured Americas southern states in the company of Robert Nighthawk, Ike Turner and many others, Earl made his first, rudimentary recordings in Read more
James Cotton - 77% match to Lonnie Brooks
Lowell Fulson - 68% match to Lonnie Brooks
31 March 1921, Tulsa, Oklahoma, USA, d. 6 March 1999, Los Angeles, California, USA. Blues guitarist Lowell Fulson (whose surname was often mistakenly misspelled Fulsom) recorded steadily from 1946 onwards, and performed regularly on the US and European club circuits into the 90s. One of the founding fathers of West Coast blues, Fulson blended the rural blues of his home stat Read more
Jimmy Rogers - 67% match to Lonnie Brooks
Otis Spann - 66% match to Lonnie Brooks
21 March 1930, Jackson, Mississippi, USA, d. 24 April 1970, Chicago, Illinois, USA. One of the finest pianists of post-war blues, Spann learned the instrument as a child. He initially played in his stepfathers church, but by the age of 14 was a member of a small local group. Having pursued careers in football and boxing, Spann moved to Chicago where he returned to musi Read more
Billy Boy Arnold - 65% match to Lonnie Brooks
16 September 1935, Chicago, Illinois, USA. Arnold first played blues harmonica with Bo Diddleys group in 1950 and became a well-known figure in Chicago blues throughout the following two decades. Among those he accompanied were Johnny Shines and Otis Rush. With a serviceable singing voice and a harmonica style influenced by John Lee Sonny Boy Williamson, Ar Read more
Little Milton - 60% match to Lonnie Brooks
Clarence "Gatemouth" Brown - 58% match to Lonnie Brooks
18 April 1924, Vinton, Louisiana, USA, some sources give Orange, Texas, where he was raised from the age of three weeks, d. 10 September 2005, Orange, Texas, USA. Browns father was a musician who taught him to play guitar and fiddle, and during his youth he heard the music of Tampa Red, Bob Wills, Count Basie, and others. He toured the south as a drummer with a travell Read more
Luther Allison - 58% match to Lonnie Brooks
17 August 1939, Mayflower, Arkansas, USA, d. 12 August 1997, Madison, Wisconsin, USA. Born into a family where he was the fourteenth child of 15, the young Allison worked with his siblings in the local cotton fields. In his youth, guitarist Allison sang with a family gospel group and moved to Chicago in 1951, where he attended school with one of Muddy Waters children. Read more
Magic Sam - 58% match to Lonnie Brooks
Samuel Gene Maghett, 14 February 1937, Grenada County, Mississippi, USA, d. 1 December 1969, Chicago, Illinois, USA. Although Maghetts immediate family were not musical, he received encouragement from his uncle, Shakey Jake, a popular blues singer on Chicagos west side. Maghett arrived in the city in 1950 and by the age of 20 had secured a recording contract with Read more
William Clarke - 58% match to Lonnie Brooks
29 March 1951, Inglewood, California, USA, d. 2 November 1996, Torrence, California, USA. Clarke began playing harmonica at the age of 16, inspired by the records of Junior Wells and Walter Horton. He turned professional two years later, and by the late 70s had become a well-respected harmonica player and singer on the Los Angeles blues scene. He associated with Smokey Wilso Read more
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Porn Bastards: Shaundi
New episode from Porn Bastards. Enjoy sexy babe Shaundi and her nice body. Click Next button to proceed the dialogs. At the bottom left corner you can change speed of sex and customize her looks.Wyświetlenia: 14779
The King of Porn City [June 2016]
This game is about the most outrageous pornstar ever, who love to destroy every partner he shoots with. Getting a whole city pregnant, by the most brutal porn and perverse lies possible, for his solely fame and deviant pleasure. Your task is to buy new locations and options, with money gathered by fucking women.Wyświetlenia: 261723
Umichan Maiko Classroom Havoc
In this 2D first person Hentai game you play as Maiko Umiya. Normally you play as her and enjoy regular life. But then you can turn all this into something unreal. Play this game with your mouse and keyboard. Be patient when multiple files preload during the game, there are some big files that may take a lot of time (guitar play mini game).Wyświetlenia: 98908
For all alien and monster sex lovers here's another game from AKATA. In this compilation you'll see 3 women from the Earth Defense Force and how they become totally helpless toys for monster aliens.Wyświetlenia: 37697
House of R'Thoth
Your task is to walk around with sexy succubus girl and go through 7 levels of hell. You'll meet a lot of different creatures on your way. Use W A S D to walk around and click your mouse (left and right) to attack. You must explore all area and defeat some of demons to unlock next level.Wyświetlenia: 42611
Two best friends William and Pierre had a bet about UK exit from EU like many of us did. Pierre put his wife on this deal and unfortunately he lost. Now all of them are going to satisfy each other in a threesome.Wyświetlenia: 23736
Resident Evil - Hounded
Another hardcore sex animation featuring Jill Valentine. She got captured once again and no one is going to save her from sex with evil monsters. Click on white arrow buttons at the bottom part in the game.Wyświetlenia: 41867
Big Top Bangeroo
Father of Victoria Vanderbilt has passed away, now she's a legal owner of International Circus legacy. She doesn't know how to run circus, because she was planning to sell it. But her father insisted that she must run it at least one year before selling it.Wyświetlenia: 24023
StationB 2 Alien Zoo Experiments
In this follow up of StationB mating experiments continue in a zoological laboratory. You'll meet 4 human babes with huge boobs and they all are going to get laid with those experimental resulting creatures.Wyświetlenia: 56005
Individual StationB
Scientists are going to survey a planet that is filled with strange and horny monsters. They all want to breed with sexy human ladies. Here you'll find 123 looping animations and 52 images. Censorship, Japanese .. as usual.Wyświetlenia: 47576
Velma Gets Spooked
Scooby Doo crew received a message from two girls from Amsterdam. They are trying to say that strange and creepy monsters have been seen around some abandoned castle. As always they will try to solve this mystery.Wyświetlenia: 63212
The Dirty Diary of Ms. Kaori
Enjoy this comedy porno about a sexy teacher with hairy armpits :) This set contains 56 looping animations and 37 pictures. Can't read a shit because everything's in Japanese, but still can enjoy artwork.Wyświetlenia: 38104
Mom, I will Make Your Face Expresses Ecstasy!
In this perverted game you'll check sexual adventures of Mio and Shou. They tell each other about their feelings and now they have felt too deep into forbidden world. Enjoy lots of sex styles like BDSM, Milking and many more.Wyświetlenia: 71446
Rogue Courier (Alpha Test)
This game still isn't complete, but now you can really check how it will look a like when finished - a mix of RPG, RTS, Arcade Space Shooter, Hentai and many more. Read dialogs and instructions to learn how to play. Basically move with W A S D and press Space to interact.Wyświetlenia: 86042
Individual Tentacle
Another strange story about a young women raped by tentacles and different monsters. Here you'll see 135 looping sex animations. As always all of this has a censorship and Japanese voice and text.Wyświetlenia: 39722
Amazon Island 3
As a boss of Amazons you can enjoy all benefits, but your tribe is being attacked by another group of warrior women. Your task is to protect your home and then you'll be able to fuck all invaders and turn them into slaves.Wyświetlenia: 122031
Tragic! Young Wife
In this story from Akata you'll see how hot young married woman gets laid with two guys in a clearing at the concrete park. She just wanted to pie :) Click on the next/back buttons to navigate through the game.Wyświetlenia: 79456
Gimmix Type90 - Ah Nanase-Sama vol.1
Another nice motion-comic game/movie from Gimmix. This set contains 5 episodes, 71 pages in total. You can simply press Next button or Jump into the part you like the most.Wyświetlenia: 29126
Dangerous Mask Spider Monster
This hardcore animation set is about a hot girl who's having sex with strange men and later with a giant spider monster. You can just click next button to move forward. Game is in Japanese and as usual - fucking censored.Wyświetlenia: 59100
Dangerous Mask 2
In this animated story you'll see how three masked girls protect their school from strangers using their naked bodies and red fucked pussies. Some views are not hot or beautiful, but some are, so don't judge too hard this submission.Wyświetlenia: 37532
Individual Outside
In this erotic 3D game you'll see adventures of the young and sexy brunette in an underground bunker with primates, lizards and horned dogs. Just click Next button at the bottom right corner to progress the game.Wyświetlenia: 51702
Train Fellow 3
In this full version of the game you'll meet our peeking friend Danny. He always finds some great situations to satisfy his addiction. This time you'll peek on sexy big breasted blonde at the train station.Wyświetlenia: 80406
Brad's Erotic Week [Updated Ep.5]
Your name is Brad. Early last week you saw an exotic woman walk into a nearby coffee shop and felt compelled to follow her in. Something about her intrigues you, causing you to come back every day to see her again and maybe ask her out. You time your arrival so it seems like a coincidental meeting, but you're a little early, and you need to kill some time.Wyświetlenia: 579357
Maid for You
What a mess! Someone must clean up your room. Guess who could that be? Of course, hot maid which will clean up not just your room, but also set free your load from your nuts.Wyświetlenia: 81596
Maddison - Virtual Date Girls
Meet Maddison - hot large breasted brunette. As always in such games - your task is to completely seduce her and fuck her in all possible locations and positions. This game has a huge story line, so find some free few hours to play this game.Wyświetlenia: 580304
Hot Candy Land
So you always wanted to be a farmer and chase magic dragons... Yeah right, what a load of crap. Now go grab your smart phone, start filming and get ready to fulfill your destiny as the King of the Adult Industry!Wyświetlenia: 2016
Slave Maker Revised v15.8
We already have this parody of the game Princess Maker. But this is more improved version. You are a slave trainer and you must turn a girl into the ideal slave. Improve their abilities, get new clothing, buy special potions and many more. You can save the game and continue to play anytime (I hope so).Wyświetlenia: 402119
Katy The Semen Witch Vol 01
Meet Katy, pretty schoolgirl who was attacked and left to die. But you saved her and made her into Semen Witch! Now you decide what she'll do - take it in her mouth or between her big boobs. Listen to all dirty talking and watch how your cock looks inside her.Wyświetlenia: 124797
- Obecna klasyfikacja 3.97/5
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- Obecna klasyfikacja 3.97/5
- Obecna klasyfikacja 3.97/5
- Obecna klasyfikacja 3.97/5


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How To Get Free WiFi Access Anywhere, Anytime
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Whether you live in the town or country, have a small or large garden, a Grillstream barbecue offers uncompromising quality in design, ease of use and the taste of your barbecue food.
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Knowledge + Good mood + Friendly atmosphere. We always do our best to make these tours as interactive and social as possible. You will get a decent amount of information about history but we don't want to overcrowd you with dry facts that you would forget very soon anyway, our goal is to make you enjoy your walk, meet new people and have a good time.
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Hox transcription factors specify numerous cell fates along the anterior-posterior axis by regulating the expression of downstream target genes. While expression analysis has uncovered large numbers of de-regulated genes in cells with altered Hox activity, determining which are direct versus indirect targets has remained a significant challenge. Here, we characterize the DNA binding activity of Hox transcription factor complexes on eight experimentally verified cis-regulatory elements. Hox factors regulate the activity of each element by forming protein complexes with two cofactor proteins, Extradenticle (Exd) and Homothorax (Hth). Using comparative DNA binding assays, we found that a number of flexible arrangements of Hox, Exd, and Hth binding sites mediate cooperative transcription factor complexes. Moreover, analysis of a Distal-less regulatory element (DMXR) that is repressed by abdominal Hox factors revealed that suboptimal binding sites can be combined to form high affinity transcription complexes. Lastly, we determined that the anterior Hox factors are more dependent upon Exd and Hth for complex formation than posterior Hox factors. Based upon these findings, we suggest a general set of guidelines to serve as a basis for designing bioinformatics algorithms aimed at identifying Hox regulatory elements using the wealth of recently sequenced genomes.
Hox genes encode a widely conserved family of transcription factors that control diverse cell fates by directly regulating the expression of downstream target genes (Mann et al., 2009; Pearson et al., 2005). Species across numerous phyla contain anywhere from five to thirty-nine Hox genes that are often clustered on chromosomes (Carroll et al., 2001; Duboule, 2007; Garcia-Fernandez, 2005; Lemons and McGinnis, 2006). Hox genes are differentially expressed along the developing anterior-posterior axis of individuals to specify diverse cell fates of specific organs and morphological structures. Between species, the modification of Hox gene numbers (duplication and deletion), Hox gene expression patterns, and/or Hox protein functions are thought to underlie many of the morphological changes observed in divergent body plans (Carroll et al., 2001; Galant and Carroll, 2002; Garcia-Fernandez, 2005; Ronshaugen et al., 2002). In addition to their role in development, Hox genes are widely expressed in adult life and play key roles in vertebrate hematopoiesis with altered Hox activity being directly linked to human leukemia (Lawrence et al., 2005; Lawrence et al., 1997; Lawrence et al., 1999; McGonigle et al., 2008). Thus, determining how Hox factors identify the appropriate set of target genes is critical for better understanding development, evolution, and disease.
Hox transcription factors have two defining molecular features: a highly conserved homeodomain that binds DNA, and a short pentapeptide motif (usually containing YPWM residues) N-terminal to the homeodomain (Chang et al., 1996; Chang et al., 1995; Lu and Kamps, 1996; Mann, 1995; Phelan et al., 1995). The homeodomain folds into three α-helices with the third helix contacting nucleotides in the major groove of DNA (Qian et al., 1989; Qian et al., 1993). Since each Hox factor performs highly specific functions in vivo, it was initially predicted that each would bind a distinct set of nucleotides. However, numerous studies over the past twenty years have established that Hox factors bind short stretches of AT-rich DNA (TNAT) with relatively low specificity in vitro (Affolter et al., 2008; Berger et al., 2008; Ekker et al., 1994; Noyes et al., 2008). Thus, a fundamental question remains: how do Hox transcription factors direct region specific cell fates in vivo when they bind similar DNA sequences in vitro?
One answer to the question of target specificity is that Hox factors form transcription factor complexes on DNA. The two best-characterized Hox co-factor proteins are Extradenticle (Exd, Drosophila) and Homothorax (Hth, Drosophila) and their vertebrate homologues Pbx and Meis, respectively (Burglin, 1997; Mann and Affolter, 1998; Moens and Selleri, 2006). Exd and Hth have C-terminal TALE homeodomains that contain a three amino acid loop extension between their first and second α-helices. Biochemical and crystallographic studies revealed that the Exd TALE motif forms a hydrophobic pocket that mediates interactions with the Hox pentapeptide to cooperatively bind adjacent DNA sites (Chan et al., 1994; Joshi et al., 2007; LaRonde-LeBlanc and Wolberger, 2003; Passner et al., 1999; Piper et al., 1999). Although the exact domains of interaction between Hox and Hth are not as clear, Hth also interacts with Hox factors on adjacent DNA binding sites (Shen et al., 1997). Since Exd binds a core sequence of TGAT and Hth binds a consensus sequence of TGACAG, the interactions between Hox proteins and these cofactors expand their DNA binding footprint and enhance DNA binding selectivity (Chang et al., 1997). In addition, Exd and Hth are obligate heterodimers that require each other for their in vivo activities (Abu-Shaar et al., 1999; Rieckhof et al., 1997). Exd-Hth interactions through conserved N-terminal domains thereby add further flexibility to the formation of transcription factor complexes with Hox proteins as Hox/Exd/Hth, Hox/Hth/Exd, and even Hox/Exd/Hth/Hox complexes form on DNA (Chan et al., 1997; Ebner et al., 2005; Ferretti et al., 2005; Gebelein et al., 2002; Gebelein et al., 2004; Jacobs et al., 1999; Li-Kroeger et al., 2008; Manzanares et al., 2001; Popperl et al., 1995; Ryoo and Mann, 1999; Ryoo et al., 1999; Samad et al., 2004; Tumpel et al., 2007). Thus, three direct protein-protein interactions (Exd-Hox, Hth-Hox, and Exd-Hth) contribute to the cooperative formation of higher-order Hox transcription factor complexes with enhanced target selectivity and affinity.
The formation of large Hox transcription factor complexes in which each protein binds specific DNA sequences suggests it may be possible to predict cis-regulatory elements from primary DNA sequences. With the large amount of genomic sequence data available from a broad range of organisms, the successful application of a bioinformatics approach has the potential to illuminate the Hox target genes required for animal development as well as many of the Hox-mediated changes underlying morphological evolution between species. However, to accurately predict target sites, one needs to first understand the DNA sequence requirements for the formation of active Hox complexes. In this study, we begin to address this problem by performing a comparative study on eight confirmed Hox target sites identified in vertebrates and Drosophila melanogaster. Through a combination of protein and DNA site mutations, we find that a great deal of flexibility exists in forming Hox complexes with Exd and Hth. However, we have uncovered differences between the formation of anterior and posterior Hox complexes and suggest several general rules that should aid future efforts aimed at identifying new Hox target sequences.
The following constructs were used to generate the Hox and Exd/Hth proteins for this study: His-Lab (Chan et al., 1996), His-Abd-A (Ryoo and Mann, 1999), His-Scr (Ryoo and Mann, 1999), His-Exd (Chan et al., 1997), His-Hth (Ryoo et al., 1999), Exd (untagged) (Gebelein et al., 2002), Hth (untagged) (Gebelein et al., 2002), His-Exd51A (Li-Kroeger et al., 2008), and His-Hth51A (Li-Kroeger et al., 2008). The Hox expression constructs were transformed in BL21 bacteria. Exd/Hth heterodimers were produced by co-transformation of a His-tagged Exd (or Hth) construct (pET14b, ampicillin) with an untagged Hth (or Exd) construct (pET9, kanamycin) placed under double antibiotic selection. Bacteria were grown in 100 mL of LB liquid culture to log phase, and induced using 0.2 mM IPTG for 1.5 hrs at 37°C. After centrifugation, bacteria were resuspended in 5 mLs lysis buffer (50 mM Tris, pH 7.5; 100 mM NaCl, 20 mM imidizole), and lysed on ice using sonication (4 × 20 seconds). Glycerol (final concentration of 10%) and Igepal (NP-40) detergent (final concentration of 0.5%) were added and samples incubated for five minutes with gentle rocking. Membranes and cellular debris were pelleted in eppendorf tubes (20 minutes at 13,000 rpm) at 4°C. Proteins were bound to 450 uLs of washed Ni-agarose beads with gentle rocking for 1.5 hrs at 4°C, washed three times with lysis buffer plus glycerol/Igepal, and eluted with the same buffer fortified with 250mM imidizole. Samples were dialyzed against 500 mL binding buffer (50 mM Tris, pH 7.5; 100 mM NaCl, 1 mM MgCl2, 10% glycerol) for 4 hrs at 4°C. Protein concentrations were measured by the Bradford assay and confirmed by SDS-PAGE and Coomassie blue analysis.
The forward and reverse complement oligonucleotides of the DMXR, RhoA, Lab48/95, EVIII, Hoxb1 R3-PM2, Hoxb2-PP2, Hoxa2 PM-PH2, and Hoxa3 PHP1 probes used in EMSA analysis are shown in Figure 1. DMXR1 and DMXR2 sequences are shown in Figure 2A; DMXR-Hox1m, -Hox2m, -Hox12m sequences are shown in Figure 2B; DMXR2-G>T and RhoA T>G sequences are shown in Figure 3A; DMXR2-Exdm, -Hthm, and -Hox2m sequences are shown in Figure 3B; DMXR1-Con, -ΔA, and −HoxC sequences are shown in Figure 4B; RhoA-Exdm, -Hthm, and -Hoxm sequences were previously described (Li-Kroeger et al., 2008). In each case, reverse complement oligonucleotides were synthesized and double-stranded probes generated by annealing oligonucleotides to a final concentration of 1 uM in STE buffer (100 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA pH 8.0). 1.75 uLs of each annealed oligonucleotide was end-labeled with T4 PNK and ATPγ-32P in a 5 uL reaction. The kinase reaction was terminated using 95 uLs of TE-stop solution (10 mM Tris pH 8.0, 7 mM EDTA pH 8.0).
EMSAs were performed using native PAGE essentially as previously described (Gebelein and Urrutia, 2001). In brief, 1 uL of labeled 32P probe was used in a 20 uL binding reaction (bringing the final concentration of probe to 8.75 × 10-10 M). The binding reaction buffer consisted of a final concentration of 10 mM Tris, pH 7.5; 50 mM NaCl; 1 mM MgCl2; 4% glycerol; 0.5 mM DTT; 0.5 mM EDTA; 50 ug/mL poly(dI-dC); and 200 ug/mL of BSA. For non-competition EMSAs, protein samples (see figure legends for amount of each protein used) were gently mixed with labeled probes, incubated at room temperature for 15 min, and run on a 4% polyacrylamide gel for 75 min at 150V. For competition assays, the appropriate amount of cold competitor was added with the 32P-labeled probe prior to the 15 min incubation. All experiments were performed at least three times. The dried acrylamide gels were exposed to phosphor-screens and densitometry was performed using ImageQuant 5.1 software.
To better understand the sequence requirements for Hox factors to bind DNA with Exd and Hth, we selected a set of Hox target sites from Drosophila and vertebrates for comparative analysis (Figure 1). The Drosophila target genes include: the Distal-less element (DMXR) that is repressed by the Abdominal-A (Abd-A) and Ultrabithorax (Ubx) Hox factors (A/U) in abdominal segments (Gebelein et al., 2002; Gebelein et al., 2004); a rhomboid (rho) activation element (RhoA) that is stimulated by Abd-A in the abdominal nervous system (Li-Kroeger et al., 2008); a labial (lab) auto-regulatory element (Lab48-95) and an element (EVIII) from CG11339 that are activated by Lab within the gut endoderm (Ryoo et al., 1999). The vertebrate target sites include a Hoxb1 auto-regulatory element (Hoxb1-R3-PM2) and elements within other anterior Hox genes (Hoxb2-PP2; Hoxa2-PM-PH2; and Hoxa3-PHP1), which are all regulated by HoxB1 (a lab homologue) and the Pbx and Meis co-factors within the hindbrain (Ferretti et al., 2005; Ferretti et al., 2000; Manzanares et al., 2001; Popperl et al., 1995; Tumpel et al., 2007). Importantly, each cis-element has been experimentally verified as a Hox target using a combination of genetic, DNA binding, and transgenic reporter assays.
Sequence comparisons between the eight cis-regulatory elements in Figure 1 reveals that while each contains an Exd(Pbx), Hth(Meis), and at least one Hox binding site, the order, orientation, and spacing among sites vary. For example, the two abdominal Hox targets contain neighboring Hth/Hox sites whereas the Lab and HoxB1 targets contain Exd(Pbx)/Hox sites with a distant Hth(Meis) site. Moreover, no specific order and/or orientation of the Hth(Meis) site relative the Exd(Pbx)/Hox site is favored as all four possibilities are represented in the six Lab/HoxB1 targets. These data indicate that a great deal of flexibility exists in forming Hox transcriptional complexes. However, as each site was studied independently, it is unclear how Hox complex formation compares between sites and if there are significant differences between the formation of anterior and posterior Hox complexes. In this study, we use a combination of protein and DNA binding site mutations to compare and contrast the ability of the Abd-A and the Lab Hox factors to form transcription factor complexes with Exd and Hth on their respective target sites.
Compared to other Hox-regulated elements, DMXR is unusual in that it contains Hox binding sites close to both an Exd site (Hox1) and a Hth site (Hox2). Previous studies revealed each independent Hox site is cooperatively bound by an Abd-A, Exd, and Hth complex, and that altogether, an abdominal Hox/Exd/Hth/Hox tetramer forms on DMXR to repress Dll and suppress leg development (Gebelein et al., 2004). However, it is unclear whether the two Hox sites synergize to further enhance binding cooperativity during Hox complex formation. To address this question, we compared the ability of Abd-A to form complexes with Exd and Hth on probes containing all four binding sites (DMXR), the Hox1/Exd/Hth sites (DMXR1), or the Exd/Hth/Hox2 sites (DMXR2, Figure 2A). Using a defined quantity of Exd and Hth with two concentrations of Abd-A, we found the amount of DMXR bound (68% and 81%) was approximately the sum of DMXR1 (47% and 62%) and DMXR2 (24% and 33%) (Figure 2C). Next, we analyzed the relative strength of Hox binding to each probe using unlabeled DMXR, DMXR1, and DMXR2 to compete with labeled DMXR. As shown in Figure 2D, the ability to compete for Hox complexes is in the following order: DMXR ≥ DMXR1 > DMXR2 with DMXR2 being significantly weaker than both DMXR and DMXR1. These findings suggest the Hox1 site mediates most of the cooperative binding to DMXR. Consistent with this idea, mutation of the Hox1 but not the Hox2 site significantly compromised binding to DMXR (Figure 2E). However, Hox1/Hox2 double mutations resulted in additional loss in competition, demonstrating both sites can mediate Hox complex formation (Figure 2E). Importantly, these DNA binding assays correlate well with transgenic reporter assays in Drosophila showing that Hox-mediated repression on DMXR is more sensitive to mutations in Hox1 than Hox2 but double mutations fully compromise gene repression (Gebelein et al., 2004). Nevertheless, these data indicate that the binding of either Abd-A/Exd/Hth on DMXR1 or Exd/Hth/Abd-A on DMXR2 is sufficient to mediate significant repression in vivo (Gebelein et al., 2004).
While Abd-A directly regulates the expression of several other genes, only one has a confirmed set of Exd, Hth, and Hox sites. The RhoA element within rhomboid contains Exd/Hth/Hox sites that are bound by an Abd-A complex to stimulate gene expression in abdominal sensory cells (Figure 1A) (Gebelein et al., 2004; Li-Kroeger et al., 2008). Like DMXR2, RhoA has adjacent Hth/Hox sites that closely match a consensus sequence bound by the vertebrate Meis/HoxA9 factors (Figure 3A, (Shen et al., 1997)). However, the RhoA site more closely matches this consensus, as DMXR2 contains a single nucleotide difference at a constrained position within the Hth site. RhoA and DMXR2 also differ in regards to the location of their Exd binding sites. The Exd site in RhoA is directly adjacent to the Hth site, a configuration found in an optimal Pbx/Meis binding site defined using selection assays in the absence of Hox factors (TGATTGACAG, Pbx site is italicized; Meis site in bold (Chang et al., 1997)). In contrast, DMXR2 has the same Exd site (TGAT), but in the opposite orientation and separated from the Hth site by seven nucleotides.
To compare transcription factor binding to RhoA and DMXR2, we performed competition assays with Exd/Hth in the absence and presence of Abd-A. As shown in Figure 3, RhoA has much higher affinity than DMXR2 for both Exd/Hth and Exd/Hth/Abd-A complexes (Figure 3C-D). To determine if the single nucleotide change within the DMXR2 Hth site is sufficient to alter binding, we performed gel shift assays using probes containing reciprocal nucleotide changes: DMXR2 G->T and RhoA T->G. As shown in Figure 3D, DMXR2 can be made into a significantly better Exd/Hth site by changing the G to T, but not to wild type RhoA levels. Changing the T to G in RhoA also significantly compromises Exd/Hth binding, but again the RhoA T>G change competes better than DMXR2. Surprisingly, however, neither RhoA T>G nor DMXR2 G>T results in significant changes in competition for the Exd/Hth/Abd-A complex (Figure 3C). Thus, enhanced Exd/Hth binding to RhoA is not sufficient to explain high affinity Hox complex formation relative to DMXR2.
Prior biochemical studies showed that the vertebrate Meis/HoxA9 factors cooperatively bind DNA in the absence of Pbx (Shen et al., 1997). To determine if Abd-A complex formation on DMXR2 and RhoA depends on Exd binding, we generated DNA binding compromised Exd and Hth proteins by mutating the highly conserved asparagine 51 residue of each of their homeodomains to alanine (N51A) (Gehring et al., 1994). Using equimolar amounts of wild type Exd/Hth, Exd51A/Hth, or Exd/Hth51A with Abd-A, we found that Exd binding was largely dispensable for Abd-A complex formation on RhoA and DMXR2 (Figure 3E-F). Moreover, point mutations in the Exd binding sites (Exdm) of RhoA and DMXR2 also have a negligible effect in competing Abd-A complexes compared to wild type probes (Figure 3H-I). In sharp contrast, the Hth protein mutation (Hth51A) abolished Hox complexes on DMXR2 and greatly diminished Hox complexes on RhoA (Figure 3E-F). To more easily visualize these results, we assigned the amount of wild type Exd/Hth/Abd-A bound to each probe to 100% and graphed the relative amount bound by each mutant protein (Figure 3G). For example, the Exd/Hth51A heterodimer results in a 90% decrease in Abd-A complexes on DMXR2 versus a 40% decrease on RhoA. We also found that mutation of either the Hth (Hthm) or Hox (Hoxm) sites in DMXR2 and RhoA strongly compromised Hox complex formation in competition assays (Figure 3H-I). However, the Hth mutations did so to varying degrees as Hthm within DMXR2 nearly eliminated competition, whereas the same Hth mutation in RhoA still competes over 50% of the Hox complex. Altogether, these data demonstrate that while most cooperative Hox binding on DMXR2 and RhoA is mediated by Abd-A and Hth, the presence of a nearby Exd site greatly enhances complex formation on RhoA.
Currently, no other Abd-A targets contain Exd/Hox and Hth sites like DMXR1 for comparative purposes. However, over twenty-five cis-regulatory elements for other Hox factors contain adjacent Exd(Pbx)/Hox sites (Mann et al., 2009), and sequence comparisons reveal DMXR1 is the only element that contains a nucleotide (Adenine) inserted between its Hox and Exd sites (Figure 4A-B). In fact, a previous study using vertebrate proteins found that inserting an extra nucleotide within a Pbx/Hox binding site abolished DNA binding (Chang et al., 1996). However, that study was performed in the absence of a Hth(Meis) protein and binding site. Hence, to test the dependence of Hox complex formation on Hth, we compared binding to wild type DMXR1 and a modified DMXR1 probe (DMXR1-Con) that removes the extra nucleotide and changes the Hox site from TAATTT to TTATGG (reverse complement is listed as most studies use this orientation) (Figure 4B, (Gebelein et al., 2002)). Using Exd/Hth, Exd51A/Hth, and Exd/Hth51A with Abd-A, we found that both Exd and Hth binding are required for forming Abd-A/Exd/Hth complexes on DMXR1 (Figure 4C). In addition, DMXR1 probes containing Exd, Hth, or Hox1 site mutations all significantly decreased competition for Hox complexes compared to wild type DMXR1 (Figure 4D). In contrast, the same concentrations of Exd/Hth and Abd-A proteins on DMXR1-Con revealed that Hth and Exd binding are largely dispensable for complex binding (Figure 4E). These data indicate that either Exd or Hth is sufficient to mediate complex formation on DMXR1-Con. To better test this idea, competition assays using Exd, Hth, and Hox DMXR1-Con binding site mutations revealed that: 1) Hthm competed as well as wild type DMXR1-Con; 2) Exdm did not compete as well as wild type but did compete significantly better than Hoxm; and 3) ExdmHthm double mutations resulted in a significant decrease in competition when compared to either mutation alone (Figure 4F). These findings indicate that a Hth binding site is more critical for Abd-A complex formation on a suboptimal Exd/Hox site (DMXR1) than on an optimal binding site (DMXR1-Con). Surprisingly, we also found a significant amount of complex forms on DMXR1-Con but not DMXR1 in the absence of Exd binding, suggesting additional differences in Hox complex formation between these two probes.
DMXR1-Con alters both the spacing between Exd and Hox sites as well as the Hox binding sequence. To determine which of these changes permits Abd-A complex formation independent of Exd and/or Hth binding, we used two additional probes: DMXR1-ΔA deletes the extra Adenine between the Hox1 and Exd sites, and DMXR1-HoxC changes TAATTT to TTATGG but leaves the extra nucleotide (Figure 4B). We tested the relative strength of each probe in competition assays with DMXR1 and found each behaves similarly in competition assays except DMXR1-Con, which is a significantly better competitor (Figure 4G). We next used wild type versus 51A mutant Exd/Hth proteins to determine the relative dependence of Hox complex formation on Exd and Hth binding to each probe. For comparative purposes, the amount of wild type Abd-A/Exd/Hth bound to each probe was assigned to 100% and the relative amount bound by each mutant was determined (Figure 4H). On DMXR1, Exd51A/Hth results in an 80% decrease and Exd/Hth51A results in over a 70% decrease in Abd-A complex formation. In contrast, the same amount of Exd and Hth mutant proteins decrease binding to DMXR1-Con by only 15% and 10%, respectively. DMXR1-ΔA, on the other hand is highly dependent upon Exd (60% decrease) but not Hth (25% decrease), while DMXR1-HoxC is highly dependent upon Hth (80% decrease) and relatively independent of Exd (20% decrease). Altogether these findings indicate: 1) If the spacing between Hox and Exd sites is optimal, then Hox complexes form relatively independent of Hth (DMXR1-Con and DMXR1-ΔA). 2) If the Hox binding site is TTATGG as opposed to TAATTT, then a distant Hth site can mediate Hox complexes independent of Exd binding (DMXR1-Con and DMXR1-HoxC). Further studies revealed that merely changing the Hox site from TAATTT to TTATTT is sufficient to make DNA binding dependent upon distant Hth sites (data not shown).
As mentioned above, previous studies using vertebrate proteins found that inserting an extra Cytosine nucleotide within a consensus Pbx/Hox binding site abolished DNA binding (Chang et al., 1996). To determine if the identity of the nucleotide inserted between the Hox1 and Exd sites makes a significant difference in DNA binding activity, we used three additional probes that change the Adenine to either Thymine (DMXR1-A>T), Cytosine (DMXR1-A>C), or Guanine (DMXR1-A>G) as cold competitors for binding Abd-A and Exd/Hth. We found that changing Adenine to any other nucleotide significantly decreased Hox complex formation revealing that Adenine is the preferred nucleotide, then Thymine, Cytosine, and lastly Guanine (Supplemental Figure 1).
We next tested if the behavior of the abdominal Hox complex on DMXR1 and DMXR1-Con can be extrapolated to other Hox factors and their binding sites. For this analysis, we selected a set of six cis-elements regulated by lab Hox genes. As opposed to the posterior Abd-A Hox factor, lab and its homologues are the most anterior Hox genes and regulate head structures in both vertebrates and invertebrates (Carpenter et al., 1993; Dolle et al., 1993; Mark et al., 1993; Merrill et al., 1989). Functional conservation between lab and vertebrate genes has been demonstrated by showing Hoxb1 rescues a lab null allele in Drosophila, and a vertebrate cis-regulatory element (Hoxb1 R3-PM2) drives gene expression in a lab-dependent pattern in transgenic Drosophila (Lutz et al., 1996; Popperl et al., 1995). Thus, we used purified Drosophila Exd, Hth, and Lab proteins to analyze complex formation on six Drosophila and vertebrate cis-regulatory elements that contain adjacent Exd(Pbx)/Hox sites with variably spaced/oriented Hth(Meis) sites (Figure 1B-C).
Lab complex formation on all six probes was first analyzed using direct competition analysis. As shown in Figure 5A, a constant amount of Lab and Exd/Hth was bound to the labeled Lab48/95 probe in the absence and presence of different concentrations of each probe as cold competitors. Importantly, while each probe significantly competes for Lab/Exd/Hth complexes, they did so to differing degrees and in the following order: Hoxb2-PP2 > Lab48/95 ≥ Hoxb1 R3-PM2 > Hoxa2 PM-PH2 ≥ EVIII = Hoxa3 PHP1. Similar results were found using comparative gel shift analysis and calculating the percent probe bound under identical Exd/Hth and Lab protein conditions (data not shown). In Table 1, we list these cis-elements from strongest to weakest in terms of Lab complex formation and compare strength of binding with cis-element architecture and sequence. Importantly, we find that no one orientation of binding sites is favored as both the strongest and weakest sites share the same configuration. We also compared the Exd/Hox and Hth binding site sequences using position weight based matrices (PWMs).
For this purpose, we generated PWMs from two data sets. First, we used the recently published binding sequences for the individual Exd, Hth, and Lab proteins identified using a bacterial one-hybrid assay (Noyes et al., 2008). Second, we used the published sequences identified using purified Meis1 and Pbx1/HoxB1 heterodimers bound to a random oligonucleotide library followed by reiterative purification/amplification (SELEX) (Chang et al., 1996; Shen et al., 1997). Sequences were imported into Target Explorer (http://luna.bioc.columbia.edu/Target_Explorer/) and the program assigned the best matrix for each binding site (Supplemental Data) (Sosinsky et al., 2003). Analysis of the six Lab/HoxB1 regulatory elements using each PWM revealed the following: 1) scores for the six Hth sites are uniformly positive using the bacterial one-hybrid PWMs, but the Exd and Lab scores vary greatly and when summated the total scores do not correlate well with strength of Lab complex formation. 2) In contrast, all six probes scored positively using the in vitro SELEX sites for Meis1 and Pbx1/HoxB1 and there is a strong correlation between strength of Lab complex formation and total PWM score. An additional interesting result that came from this analysis is that the reverse complement of the Exd/Lab site (TAATTGATCA; Exd in italics, Hox in bold) in the EVIII probe scored significantly higher than the suggested sequence (TGATCAATTA). This finding indicates that the orientation of these sites may differ from the original published report (Ebner et al., 2005). However, since our data cannot discriminate between these two possibilities and structural studies would be required to determine the correct orientation, we left the orientation of binding sites as previously published.
We next determined the dependence of Lab complex formation on Exd and Hth binding using the wild type and mutant Exd/Hth proteins. As shown in Figure 5B, all six Lab complexes are heavily dependent upon Exd binding as Exd51A abolishes nearly all complex formation to each probe. The one exception is that some Lab/Exd51A/Hth complex forms on HoxB2-PP2, but when normalized, even this binding is 75% less than wild type (Figure 5C). The Hth51A protein also significantly disrupted Lab complexes but to a variable degree. For example, Lab/Exd/Hth51A disrupted over 90% of binding to Hoxa2 PM-PH2 but only 55% of binding to Lab48/95 and Hoxb1 R3-PM2. Interestingly, the Hoxa2 element has a poor Hox binding site (AGACCG) compared to Lab48/95 (GGATTG) and Hoxb1 (GGATGG), suggesting that like abdominal Hox complexes on DMXR1, Lab complex formation is more highly dependent upon Hth binding if the Exd/Hox site is suboptimal.
Unlike our findings using Abd-A on DMXR1-Con, all six of the Lab complexes required wild type Exd and Hth proteins for strong binding. This finding could be due to inherent differences between the Abd-A and Lab Hox factors or due to differences between the cis-regulatory elements tested. To distinguish between these possibilities, we performed gel shift analysis using Lab with wild type Exd/Hth on DMXR1-Con and found Lab readily forms complexes on this probe (Figure 6A-B, note: slightly more Lab protein was used than Abd-A to achieve a similar level of Hox complex formation, 75.3% vs 79.4% respectively). To determine if Lab can bind DMXR1-Con independent of either Exd or Hth binding, we performed comparative gel shifts using wild type and Exd/Hth mutant proteins and found that both Exd51A and Hth51A formed significantly less complex with Lab than wild type proteins (an 80% decrease with Exd51A and a 60% decrease with Hth51A, Figure 6C). In contrast, the same amount of mutant Exd and Hth proteins disrupted less than 15% of binding with Abd-A (Figure 6B-C). As Lab is the most anterior Hox factor and Abd-A is one of the most posterior Hox factors, we also performed the same set of assays using the central Hox factor Sex Combs Reduced (Scr). As shown in Figure 6, Scr behaved much like Abd-A in that it is able to form a significant amount of Hox complex on DMXR1-Con with either Exd51A or Hth51A.
We next determined if Abd-A or Scr could form Hox complexes with Exd51A and Hth51A on two of the Lab-regulated cis-elements. For this purpose, we first selected the Hoxa3-PHP1 site because it closely resembles the Exd/Hox site of DMXR1-Con (Figure 6A). Using the same amount of each respective Hox factor as for the DMXR1-Con gel shifts, we found that Scr and Abd-A bind significantly more Hoxa3-PHP1 than Lab (Figure 6A). Moreover, when tested with equal amounts of Exd51A/Hth and Exd/Hth51A, the Abd-A Hox factor was able to significantly bind Hoxa3-PHP1 in the presence of either protein mutant (Figure 6E). Scr, on the other hand, was intermediate between Lab and Abd-A in terms of its ability to form complexes on Hoxa3-PHP1 in the absence of Exd binding (Exd51A). Lastly, we selected the Lab48/95 probe, which contains an Exd/Hox site that is significantly different from DMXR1-Con. This probe was similarly bound by all three Hox factors with wild type Exd/Hth (Figure 6A). However, unlike on DMXR1-Con and Hoxa3-PHP1, Lab, Scr and Abd-A mediated Hox complex formation is heavily dependent upon Exd binding (Exd51A). Thus, Abd-A and to a lesser extent Scr forms complexes on Exd/Hox and Hth sites in the absence of Exd binding, but only on specific Hox sites (TTAT). Moreover, the anterior and posterior Hox factors significantly differ in their ability to form complexes independent of Exd binding. As discussed below, these data have implications for the mechanisms underlying the general ability of posterior Hox factors to phenotypically suppress anterior Hox factors during development.
The Hox genes encode a family of conserved transcription factors that regulate numerous fundamental cell behaviors throughout the development and life of an organism (Carroll et al., 2001; Mann et al., 2009; Mann and Morata, 2000; McGinnis and Krumlauf, 1992). Expression experiments (mainly microarrays) from several organisms and/or tissues revealed that hundreds if not thousands of genes are affected by alterations in Hox gene activity (Chung et al., 2006; Ghannam et al., 2004; Hueber et al., 2007; Lei et al., 2006; Lei et al., 2005; Lu et al., 2008; Salsi et al., 2008; Takeda et al., 2006; Williams et al., 2005). Identifying which of these genes are direct targets is of primary importance to understand how Hox factors regulate cell growth and differentiation. Moreover, large-scale genomics have provided a wealth of sequence data that can be mined for the presence of potential Hox binding sites (as well as their co-factor proteins) near candidate target genes. However, our ability to use bioinformatics to predict Hox target sites is limited by our understanding of what Hox regulatory elements look like and the characteristics they share. In this study, we used protein and DNA site mutations to explore the binding characteristics of both anterior (Labial) and posterior (Abd-A) Hox complexes with the Exd and Hth transcription factors on cis-regulatory elements. Through this analysis, we determined that: 1) Hox factors interact with Exd/Hth on a variety of binding site combinations, and that suboptimal sites can be combined to yield functional Hox complexes; 2) in general, posterior Hox factors are less dependent on Exd/Hth binding than anterior Hox factors on the same DNA probes. Here, we discuss the implications of these findings and assess the relative accuracy of using position weight matrices (PWMs) to predict Hox complex binding sites.
The DMXR element provides a good example of the flexible nature of Hox complex formation on DNA. Unlike other previously characterized Hox regulatory elements, DMXR contains both Exd/Hox and Hth/Hox sites that mediate abdominal Hox tetramer formation. Importantly, each on its own is a relatively low-affinity binding site due to suboptimal spacing between sites. Together, however, they constitute a strong binding site that mediates abdominal Hox complex formation to repress Dll expression (Gebelein et al., 2004). Moreover, this combination of binding sites results in DMXR being a robust repression element. For example, mutating either the Exd or Hth sites in the context of the full DMXR element results in only a modest loss of both DNA binding (Supplemental Figure 2) and repression activity in vivo (Gebelein et al., 2004). In contrast, when the same mutations are introduced into the DMXR1 probe that lacks the Hox2 site, abdominal Hox complex formation is severely disrupted (Figure 4) and abdominal Hox-mediated repression is significantly more compromised than on the full DMXR element (Gebelein et al., 2002). Thus, suboptimal Hox binding sites can be combined to form robust regulatory elements, and as relatively few Hox cis-elements have been thoroughly characterized, we propose additional configurations of Exd, Hth, and Hox sites are likely to result in cooperative complexes on cis-regulatory elements.
By characterizing the dependence of Hox complex formation upon Exd and Hth DNA binding on several cis-regulatory elements, we made the unanticipated finding that in general posterior Hox factors (Abd-A) form more robust complexes than anterior Hox factors (Lab). For example, mutations within the Hth protein that disrupt DNA binding (Hth51A) consistently had a greater affect on Lab complexes than Abd-A complexes on the same DNA probes (Figure 6). More surprisingly, we found that a distant Hth site can mediate cooperative Hox complexes with posterior but not anterior Hox factors in the absence of Exd binding (Figure 6). On both Hoxa3-PHP1 and DMXR1-Con, for instance, Abd-A but not Lab, readily formed complexes with a DNA binding compromised Exd protein (Exd51A). Intriguingly, the ability of Abd-A to form these complexes was Hox site dependent, as DMXR1 and Lab48/95 were unable to mediate similar complexes in the presence of Exd51A. Further analysis revealed a single nucleotide change in DMXR1 (TAAT to TTAT) makes Hox complex formation relatively independent of Exd binding. Moreover, we found that a central Hox factor (Scr, Hox5 homologue), displays an intermediate ability to form Hox complexes independent of Exd binding on the same probes. Thus, at least for these three Hox proteins, the more posterior Hox factors are better able than anterior Hox factors to tolerate loss of either Exd or Hth binding and retain Hox complex formation.
The differential ability of anterior and posterior Hox factors to form complexes with Exd and Hth has two important implications. First, if anterior Hox complex formation is more dependent upon Exd and Hth binding, then anterior Hox cis-regulatory elements should be more likely to have binding sites for all three factors than posterior Hox regulatory elements. While the total number of characterized regulatory elements is still relatively small, it is interesting to note that of the eleven cis-elements regulated by Lab (or Lab homologues) seven contain characterized Hth/Meis binding sites (Mann et al., 2009). In contrast of the thirty-three cis-elements regulated by the central/posterior Hox factors, only two contain characterized Hth binding sites (Mann et al., 2009). This model also predicts that anterior Hox factors should be more sensitive to hypomorphic exd and hth gene mutations than posterior Hox factors. At least for cuticle formation in Drosophila, this prediction holds, as posterior segments have relatively normal cuticles in both exd and hth mutant embryos whereas anterior segments are transformed into posterior fates (Peifer and Wieschaus, 1990; Rieckhof et al., 1997).
A second implication of our work relates to the phenomenon of posterior dominance (also known as prevalence) of the Hox factors. Studies in Drosophila have shown that when an anterior and posterior Hox factor are co-expressed in the same segment, the fate of the segment is predominantly determined by the posterior Hox factor. Several explanations have been proposed regarding this phenomenon, including that posterior Hox factors make additional contacts with both DNA and Exd to raise their DNA binding affinity (LaRonde-LeBlanc and Wolberger, 2003; Merabet et al., 2007). Our studies are consistent with and add to this model by showing that posterior Hox factors form more robust complexes than anterior Hox factors. In particular, the ability of posterior Hox factors to bind DNA sequences relatively independent of Exd or Hth suggests the posterior Hox factors would bind additional target genes than anterior Hox factors. Congruent with this possibility, Hueber et al used microarray studies to show that Abd-A regulated many more target genes than the anterior Hox factors and that most of these targets were unique to Abd-A (i.e. not regulated by other Hox factors) (Hueber et al., 2007). Future studies focused on the identification and characterization of additional cis-regulatory elements will be required to determine how the Abd-A factor can specifically affect the regulation of so many downstream target genes.
Advances in DNA sequencing technologies have provided a wealth of genomic sequence data that can be searched for potential cis-regulatory elements. To successfully do so, we first need to accurately predict transcription factor binding sites. For the Hox factors, several biochemical and high throughput approaches have been used to identify sequences bound by individual Hox factors or in combination with Exd(Pbx) and/or Hth(Meis) proteins (Chang et al., 1996; Noyes et al., 2008; Shen et al., 1997). Here, we applied the results from these searches to generate PWMs and correlated their scores with the relative DNA binding activity of Lab Hox complexes. We found that using the bacterial one-hybrid PWMs did not result in a strong correlation with our DNA binding analysis. This result may be due to the Lab and Exd proteins being tested in isolation in the one-hybrid assays, whereas the cooperative binding of Exd/Lab heterodimers may alter/restrict the sequences that can be bound relative to monomer proteins. In contrast, the in vitro SELEX sites were identified using Pbx1/HoxB1 proteins together and the Meis1 protein alone, and the PWMs from these sites strongly correlated with Lab complex formation. However, there is an important caveat with this analysis. Since the SELEX sites were published prior to the identification of the cis-regulatory elements, the SELEX sequences are likely to have been used as a guide to identify the cis-elements in the first place. Nevertheless, the direct correlation between strength of Lab complex and total PWM score suggests using the SELEX matrices in a bioinformatics approach is likely a good strategy to identify additional Lab cis-regulatory elements.
What about using PWMs for the posterior Hox factors? Our biochemical data indicate that using a similarly strict search for both Exd/Hox and Hth sites for the posterior Hox cis-regulatory elements may not work as well due to posterior Hox complex formation being less dependent upon both Exd and Hth binding. However, we do believe some general guidelines can be useful in searching for Hox cis-regulatory elements. 1) As mentioned above, anterior Hox factors are likely to be more highly dependent upon Hth binding than posterior Hox factors. 2) Optimal Hth/Hox sites can mediate posterior Hox complex formation relatively independent of Exd binding. 3) A distant Hth binding site can permit posterior Hox complex formation on suboptimal Exd/Hox binding sites or on specific Hox binding sites independent of Exd binding. 4) Nearby low affinity (suboptimal) binding sites can be combined to mediate functional Hox regulatory complexes.
We would like to acknowledge the Gebelein and Cook laboratories for their helpful discussions of this project. This work was supported by the National Institutes of Health (GM079428 to BG).
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Journey with IONS to a Sustainable Future
Journey with IONS to a Sustainable Future
EarthRise is now certified as a green business!
Sept. 27, 2013 – IONS is thrilled to announce that EarthRise, our 194 acre campus at the border of Marin and Sonoma counties, is now a certified member of the Marin County Green Business Program—part of the Bay Area Green Business Program. This voluntary program recognizes and promotes businesses and organizations that implement environmentally responsible practices. The Green Business Program certifies that businesses meet higher standards of environmental performance such as using energy efficient processes, conserving water, recycling, using recycled products, reducing pollutants, and much more.
To receive this certification IONS EarthRise went through a detailed process to comply with more than 83 required, as well as additional, measures. This Green Business Certification is one component of the Institute of Noetic Sciences' wider commitment to sustainability and caring for the earth and its inhabitants, and to developing EarthRise into a world-class model of sustainability.
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A week of events dedicated to spirit
SAA hosts BEAT Week
SAA hosts BEAT Week
As Sanela Mrvoljak waited for her citizenship ceremony to begin, she couldn't help but wonder what her life could have been.
Bulldogs shock Shockers in triple-overtime
Living arrangements discussed
Friends, family and staff gather to share memories of first-year student
Stay caught up on what went down this past week.
Playing against a great opponent, a team that should have been nationally ranked by all means, the Bulldogs played their hearts out.
The Bulldogs' 77-62 victory over Bradley catapulted Drake into a three-way tie for third place in the Missouri Valley Conference.
Karys finishes 1st in long-jump
The Bulldogs quickly improved their season record to 3-1 with victories over Northern Illinois and Southern Illinois at Edwardsville.
Bulldogs top Depaul, Denver and fall to Harvard at Harvard Kick-Off Invitational
Tips, tricks and resources to find the best fit for you
Long lost relative found in an unexpected place
So, you're probably wondering why someone would devote an entire column to Hubbell Dining Hall. Valid question. Basically, we love it. There's no way around it.
Typically, Iowans spend the winter months bundled up in coats or staying inside to keep warm. Some people, however, are taking a plunge in sub-freezing temperatures for a good cause. Drake University Students in Free Enterprise is teaming up with the Des Moines winter games to host the first-ever Drake […]


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Crazy Substitute Teachers
Enjoy, NG4.03 / 5.00 2,382 Views
Vacuum Salesman
This dude and his Megazord vacuum cleaner4.00 / 5.00 2,694 Views
Markiplier Animated | Mark Of The Survey Corps
This took far too long to make...3.97 / 5.00 6,135 Views
The concept was genius, title has a great ring to it...
Your art was good, nice drawings but somewhat poorly animated... if you were to put alittle more time in it i'm sure you could make it alot smoother...
the story & humor was pretty ok, loved the super hero feel to it, i'm not a big fan of the carebear reference though...
The use of audio sucked...repetitive & anoying... could of used some music.
overal this had the potential to be great but turned out to be so-so.
I'd love to see a remake of this one day.
I liked the design of your robot it looked so tight, and i saw the little piece of sand falling in the preloader, that kept me somewhat entertained, lol. Make more and more!
You really need to add music or something along those lines. I found it really hard to pay attention when the only sound was the odd ringing of a bell.
This has been on newgrounds for too long..
Good idea.
What an excellent idea! Father time and the reaper teaming up to fight crime! Very cool. Art was great. and the pacing of the story was good..heres what you lose points for though... audio was boring and uninteresting.... also...i thought it was near perfect till the care bears showed up...you already had a great concept, why ruin by some dumb south parkish pop culture reference....lame.


text: Summer time - Stock Photo Golden sunset. Two boys riding a small bike and three girls on a bicycles Share this:PinterestGoogleFacebookTwitter Nikita Buida info Author Page Portfolio: 10624 images. Updates / RSS Network Image updated—July 28, 2013 Keywords bicycle, bike, calm, children, evening, fun, mist, road, scenics, summer, sun, sunset, vacations, warm, yellow Image Categories People Image #9180 Size Type File Info Price ( US Dollars $ ) Bloggeejpg3.47" × 2.21" @ 72 dpi250 × 159px3.00 Smalljpg8.33" × 5.31" @ 72 dpi600 × 382px6.00 Mediumjpg6.67" × 4.24" @ 300 dpi2000 × 1273px10.00 Largejpg11.18" × 7.12" @ 300 dpi3355 × 2135px20.00 End User License Agreement CHECKOUT (0.00) Symbiostock Community Symbiostock CommunityNetwork with illustrators, photographers, and designers. Symbiostock on TwitterSymbiostock news. Symbiostock on FacebookCommunity maintained Facebook hub. Symbiostock.infoSearch images on the Symbiostock network. Symbiostock SearchSearch Symbiostock images and network. Symbioguides.comSymbiostock knowledge base. Symbiostock.comSell your images and network with fellow microstock professionals. ClipArtof.comHigh Resolution Stock Illustrations & Clip Art ClipArtIllustration.comFirst Symbiostock Site, and home of the Orange Man Microstockgroup.comA meeting place for microstock professionals.