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Whispering Willows is an adventure in archaeology with a supernatural perk: it lets you speak to the dead people who once owned the artifacts you discover. It twists the haunted house investigation premise by giving its heroine ghost transformational powers. This adequately serves both the narrative and the gameplay, the latter comprising some of the most simplistic and easy exploration puzzles in recent memory. This is a minimal detriment to the game overall, which is well-supported with touching themes of regret, lost love, and one's spiritual connection to nature.

We also see shades of a somewhat self-aware industry attempting to look at the past through a different lens. B.J.'s local contact, Kessler, is a stark contrast to the burly protagonist. Ever-defiant in the face of a Nazi-ruled future, Kessler and his late love fight for a Germany that rises up against the hateful regime, with his weapon of choice being leaflets rather than guns. He's also acutely sympathetic towards human life: in one memorable exchange he implores B.J. to avoid killing as many people as he can, which B.J. then willfully misinterprets to mean that he should kill as many Nazis as he can, a sentiment he emphasizes every chance he gets. We also see more fleshed-out versions of enemy combatants, albeit only through memos and a scene where you walk through a bar crowded with Nazi soldiers. We get glimpses of soldiers who still display care and affection for their families at home, even as they become upset because their lovers are dismayed at how the war has changed them, feel fear at the many occult studies and phenomenon they're being focused on, and buy into all the propaganda that litters this world. Even Helga Von Schabbs, the antagonist of the game, is granted some character development in her lifelong fascination with adventure and her potentially royal lineage.

Even so, B.J. still sees them as the same flat characters as he sees in his dreams, good for only one thing: killing. The game's mechanics almost never encourage you to leave any Nazis alive. Quite the opposite, in fact. In the end, this is a blunt, muscular, bare-chested romp through levels full of deadly moving targets, even during its subtle moments. As a dumb action game, The Old Blood has the chops. But it fumbles when it attempts to introduce gray areas into the game. Whether it's Kessler's empathy or the supplementary notes you find, The Old Blood seems to have something to say about the evolution of games, but never arrives at a solid point. The sledgehammer that is B.J.'s philosophy always wins out, stranding the gray areas in a game that clearly won't give them the time of day.

Protagonist Elena Elkhorn isn't above admiring the opulence of the Willows Estate, the game's setting. She is not a classic heroine, pre-destined for great things or exuding a profound sense of self. She simply wants to find her missing father, who is employed as a caretaker on the estate. Clearly an adolescent with a good heart, Elena's amiable interactions with many of the local spirits please her immensely, especially since they're helpful companions on this dark and grim journey. Like some of the ghosts in The Sixth Sense, Elena's spectral aides are in desperate need of closure. And yes, some of them don't know that they're dead. Every soul Elena successfully assists gives her immense solace. Yet as each ghost departs its state of purgatory, she suffers the loss of each brief friendship. As a teenager with metaphysical powers and few friends, Elena is relatable to Maxine Caulfield from Life is Strange.

Both of us here at Frogmind have a long history of gaming starting from the late 80’s. We have played thousands of different games starting from Commodore 64 all the way to the new iPad. In the process we have learned what are the elements we like and dislike in games and in game business Judi Bola Great site.

Then again, maybe games haven't changed as much as we like to think. The second half of The New Blood changes up enemy types by replacing fully functional Nazis, the default enemy type of the old era, with zombies, the go-to foe of the modern one. It's initially a nice change of pace from the terse shooting arenas that came before, but it's hard not to think of them as a downgrade, turning a mechanically-smart dumb action game into a brain dead one. Don't get me wrong: mowing down zombies with a sawed-off shotgun is a classic scenario, but the waves and waves of corpses pouring at you can't help but feel disappointing after you've experienced the smart design of part one.

Clearly MachineGames has big ambitions and understands the nuance that can be evoked in the medium. And The Old Blood certainly displays intelligent action FPS design that's fun and effortless to play. Just don't expect the deep examination the game hints at to go anywhere. Here, the gloriously dumb (yet smartly designed) reigns. Progress can wait. Long live The Old Blood.

And yet we still see the same thoughtful modern design implemented in last year's The New Order interlaced with the callbacks to days past. Firing guns feels both effortless and flexible, with each weapon boasting distinct modes for different situations (such as the assault rifle's automatic and semi-automatic modes), as well as dual-wielding options. Instead of just setting you loose in a sprawling level as you shoot at flat enemies, The Old Blood ebbs and flows as it constantly changes up each encounter's focus, creating an excellent sense of pacing. Action-packed shooting sequences couched in open, cover-laden levels alternate with more freeform stealth sections that don't object to you taking a more direct approach. There are even portions where you're encouraged to run rather than fight. Tying the level design together is some seriously great enemy AI, who intelligently flank you and take cover, yet are not so smart that you can't use cover to outmaneuver them. Everything about the way The Old Blood plays exemplifies the diversity of modern FPS design, and the game does so in a way that's on point and fun.

The Old Blood also illustrates where we're going in games and what we're leaving behind. Yes, you still have to fight past dogs, but they also happen to be wearing dog-sized body armor that you can harvest, and instead of coming at you at a crawl, they realistically rocket towards you and tackle you as they try to shred your meat to bits. Yes, you still fight a giant armored super soldier in the third chapter, but when you do defeat it, more troops come pouring out and you're forced to weave in and out of the foyer you just fought in, giving the level new dimensions. And those secret areas? You no longer have to press up against every wall in a level, mashing the confirm button hoping to reveal a sliding wall. Instead, there really are secret areas that can be found through normal exploration. It's amazing to see how design has become both more simplified and more complicated over the years.

We think that games should be developed by constantly thinking from the perspective of the player. The player eventually playing your game is the king. He/she is the person whose enjoyment is in the hands of your game. Thus, the end-user experience is the most important goal in our games and we want it to be flawless in BADLAND. We think we are able to achieve this as we are active gamers ourselves. We are developing BADLAND for us too. We want to enjoy it as much as you will do.

So, what do we think brings better gaming experience in games especially in iOS devices? Let us give a few examples:

We think that immersion is very important in games Taruhan Bola . The best games are those which really give the player the feeling that he/she is in the game world. It can be really hard to get the mind to the game’s world but it’s super-easy to get it out there. For example, while playing, a pop-up suggesting you to spend real money to buy stuff, is the prefect immersion killer. Seeing real world references, such as price tags with real world money currency, can easily take the player’s mind away from the game world spoiling the gaming experience. Moreover, spending real money is not fun and still so many games remind constantly the player about it with different in-app purchase possibilities. In our opinion, this eventually ruins the game’s fun. We are not saying that in-game purchases in general are bad thing but they can be done wrong so easily. For example, we think that buying additional real content, such as level packs, inside the game through menus can be totally OK. Another example of ruining the immersion is to underestimate players by having lengthy tutorials explaining everything by hand. We could continue with more examples how to spoil the immersion, but these two are good examples as they can be seen in games in the AppStore. We want to make sure that you have as immersive gaming experience while playing BADLAND as possible.

Another good example of what we like in games especially in Apple’s iOS AppStore is their universality. When developing a game or any application to the AppStore, the developer can decide whether to develop the game as a universal application or not. A universal application means that the application has been designed for all the iOS devices: iPhone, iPod Touch and iPad. In most cases it means that the end-user can play the same game in all of his/her iOS devices with the UI and the graphics designed for each device separately. With universality, given that the game is available for all of the iOS devices, the player does not need to buy multiple versions of the game in order to get the best experience in all of his/her devices. Only one purchase is required. We think that players want to play games in all possible iOS devices they own with best gaming experience possible, but do not want to buy the same game multiple times. Thus, BADLAND will be an universal application with the gameplay, the UI and all the graphics designed for the iPhone retina resolution, the iPad resolution and for the new iPad retina resolution. You get all this with one purchase, and after that, you can play BADLAND with beautiful graphics with all your iOS devices.

This list could go on with more examples, but we hope that these two examples give you some grasp of us as game developers and what kind of gaming experience we want BADLAND to be. Feel free to ask us questions in the comments or in Twitter/Facebook.

This week’s big thanks go to:

When EteRNA launched, we solicited strategies from players for scoring sequences that yielded a high experimental EteRNA score for a given puzzle. From there, Eternabot was born and soon outperformed other inverse-folding algorithms, like ViennaRNA and NUPACK in real-world experiments, as we described in EteRNA's first paper.

Today, Eternabot is still in use - not only on single state puzzles, but it also on switch puzzles. In fact, in the most recent round of cloud lab submissions, Eternabot submitted 400 solutions for synthesis. However, as the EteRNA players have learned, switches follow a different set of rules from normal RNA puzzles, and thus deserve their own strategies.

As such, we are happy to officially announce that we will be asking for your switch specific strategies in a new version of Eternabot. We request that, just like last time, if you have any specific strategies, you clearly outline them in a forum post on the getsatisfaction page. We have created an example, which can be found here.

It is optional, but highly recommended, to outline your strategies in a clear list of what you look for in a successful solution, as this will help speed up the implementation process. These strategies can include either features of the RNA that you think will improve the synthesis score. They can also include a list of the steps you take to solve switch puzzles, although we may not implement those until a later stage.

The process for coding up your strategies will be similar. After you write your own strategy in the forum, the EteRNA developers will write an algorithm based on your post, test it on all the synthesis results, and post the results on a strategy market page.

When making your post on the getsatisfaction forum, make sure to include “Strategy Market Switch” in brackets in the title, so that we know the strategy is not for single state puzzles.

We look forward to reading and implementing all of your strategies! EteRNA team

Brourd Question: Since the secondary structure predictions for Vienna1.8.5 and Vienna2.x may differ significantly, and some strategies will be based on the number of base pairs, or the identity of base pairs, etc., will there be two separate versions of the strategy market to address this? 16 Jun 2015 rhiju No, the strategy market is meant for figuring properties of switches that do well experimentally but are perhaps not well encoded in either vienna energy function -- or any known energy funciton. 16 Jun 2015 Brourd Right, however, since players have been given access to two different energy functions, a strategy like that of the example "Strategy MarketSwitch Penalize large amounts of GC pairs," will inherently be tied to whichever secondary structure is used to define the number of G-C base pairs. A designer like Nando's bot, ViennaUCT, has all predicted secondary structures tied to the Vienna2.x parameters, meaning any strategies that score these designs based on vienna1.8.5 predictions may be partially incorrect. On the other hand, perhaps players have not adopted the Vienna2.x parameters on that large a scale, so that few of the strategy market scores will be affected, if at all. 16 Jun 2015 Brourd Question 2: Will this strategy market algorithm be focused solely on the chip-riboswitch scores? 16 Jun 2015 Brourd Question 3: Should strategies be written with the concept in mind that these apply to all riboswitch scores, or should separate strategies be written for different ligands? 16 Jun 2015 rhiju @brourd -- i now understand your question on vienna2 vs. vienna1.8.5 now -- it matters if the strategy requires looking at the dot plot (or similar). in that case, the player should specify which energy function to use (or both, if desired). 16 Jun 2015 rhiju For Question 2: we'll do separate correlation coefficient comparisons to 'cloud lab' (single-nucleotide SHAPE) data and to riboswitch-on-chip data. We'll probably return separate rewards for these, and train separate combination strategies. 16 Jun 2015 rhiju For Question 3: you can start by writing strategies with specific ligands/proteins in mind, and later generalize as we move to other ligands, proteins, and RNA inputs/outputs. To be totally concrete, we are seeking our first test of the new eternabot to happen in July with the current lab puzzles -- mostly FMN/MS2 exclusions. 16 Jun 2015 Brourd Question 4: Are there any limitations to the strategies we come up with? Which functions of ViennaRNA are available to work with? Ensemble defects, entropy measurements, etc.? 16 Jun 2015 Alexander Lohachitranont How about tertiary folding? 18 Jun 2015 vineetkosaraju Brourd, for question 4 - in theory no, but keep in mind that including these features might take slightly longer to implement (and might cause the bot to take longer to run). The current eternabot just uses vienna to calculate the secondary structure based off of the sequence, but I'm sure other functions could also be called. Alexander Lohachitranont - We're still focusing on the secondary structure for eternabot, atleast for now, but hopefully one day it could even be expanded to tertiary folding :) 19 Jun 2015 Omei Can we get "progress reports" about what strategies have been implemented and what its current predictions are for the designs in the round we're currently working on? It would be more like having eternabot as a collaborator. 24 Jun 2015 Eli Fisker I agree with Omei's request. :) +1 25 Jun 2015 vineetkosaraju Sure those sound like great ideas :). I haven't started implementing the strategies yet (which is why there are no progress reports), but once I do I will paste a message with how many lines of code the strategy took, what the new optimized parameters are, what the correlation of your strategy is, and an excerpt of the actual code for reference.

Regarding the eternabot solutions, it is most likely that for the current round I will have the bot start generating solutions when all the strategies are implemented, which might be far off - but once I start running it, I would be happy to give weekly/biweekly/daily progress reports on the solutions it has created. 25 Jun 2015 Eli Fisker Sweet news, Vineet! :) 25 Jun 2015 Omei Super! Thanks, Vineet. You didn't specifically mention having Eternabot predict the score of designs submitted by other players. I don't think we've had that in the past, but it would be very helpful in giving players feedback about whether the implemented strategies are having the expected effect on the evaluation score. That reminds me of something I've been meaning to ask. Is Eternabot's scoring model simply a lineat combination of the individual strategy scores? Or something more sophisticated?? 25 Jun 2015 vineetkosaraju Great question Omei :) (I'm new to the eternabot world, so I just researched this yesterday). The way the scoring function works is it takes the normalized score from each individual strategy, multiplies that score by the weighting that strategy has (which I believe has been optimized based on past data), and adds these all up. I would thus think that it is a linear combination of the individual strategy scores, where the finalStrategy = (strategy1Score*weight1) + (strategy2Score*weight2)... for each of the strategies.

I love the idea of Eternabot predicting the score of designs submitted by other puzzles, but I'm not sure what the easiest way to set that up would be.. it could either be something automatic where the bot looks for new submissions, gets the score, and sends private messages or posts to page, or maybe the scoring function could be ported over to Eternascript where players can just input the puzzle id and their solution and the script outputs the bot's score. Which do you think would be best? 25 Jun 2015 Omei Thanks, Vineet. Yes, that's a linear model, which I suspected Eternabot was. It's hardly state of the art, but it is a solid classical prediction model. As to how to make the predictions available, if an inside developer were to take it on, I could see how the prediction of lab scores could become an engaging game in its own right. But given past history, it seems unlikely that will happen. In which case, the question probably boils down to "what is the easiest way to generate a frequently updated Google spreadsheet with all the predictions, so that player developers can do whatever they want with the data?" If Eternabot is already coded in Javascript (which I doubt), making it available in Eternascript and letting players take it from there would probably be simplest. But if it is written in some other language that isn't so easily converted to Eternascript, it might be easier for Eternabot to just generate and post a .csv file with the current predictions for all the submitted designs in the active labs once a day. 26 Jun 2015 JR How about a "score this sequence" pop up window on the design page so you can see it before submitting your design. 01 Jul 2015 Super Scientist Great idea! 01 Jul 2015 vineetkosaraju JR, that's a good long term solution that I'll talk with Nando (who is mostly in charge of the flash code) about -- unfortunately it might require some porting over of Eternabot which would take longer. For this round though I might just go with the option that's easiest to implement so that it takes less time for you to see the bot's scores. 01 Jul 2015 JR Ok, understood. A column in the output data would be second best, a column we could sort. 01 Jul 2015

Brourd Question: Since the secondary structure predictions for Vienna1.8.5 and Vienna2.x may differ significantly, and some strategies will be based on the number of base pairs, or the identity of base pairs, etc., will there be two separate versions of the strategy market to address this? 16 Jun 2015 rhiju No, the strategy market is meant for figuring properties of switches that do well experimentally but are perhaps not well encoded in either vienna energy function -- or any known energy funciton. 16 Jun 2015 Brourd Right, however, since players have been given access to two different energy functions, a strategy like that of the example "Strategy MarketSwitch Penalize large amounts of GC pairs," will inherently be tied to whichever secondary structure is used to define the number of G-C base pairs. A designer like Nando's bot, ViennaUCT, has all predicted secondary structures tied to the Vienna2.x parameters, meaning any strategies that score these designs based on vienna1.8.5 predictions may be partially incorrect. On the other hand, perhaps players have not adopted the Vienna2.x parameters on that large a scale, so that few of the strategy market scores will be affected, if at all. 16 Jun 2015 Brourd Question 2: Will this strategy market algorithm be focused solely on the chip-riboswitch scores? 16 Jun 2015 Brourd Question 3: Should strategies be written with the concept in mind that these apply to all riboswitch scores, or should separate strategies be written for different ligands? 16 Jun 2015 rhiju @brourd -- i now understand your question on vienna2 vs. vienna1.8.5 now -- it matters if the strategy requires looking at the dot plot (or similar). in that case, the player should specify which energy function to use (or both, if desired). 16 Jun 2015 rhiju For Question 2: we'll do separate correlation coefficient comparisons to 'cloud lab' (single-nucleotide SHAPE) data and to riboswitch-on-chip data. We'll probably return separate rewards for these, and train separate combination strategies. 16 Jun 2015 rhiju For Question 3: you can start by writing strategies with specific ligands/proteins in mind, and later generalize as we move to other ligands, proteins, and RNA inputs/outputs. To be totally concrete, we are seeking our first test of the new eternabot to happen in July with the current lab puzzles -- mostly FMN/MS2 exclusions. 16 Jun 2015 Brourd Question 4: Are there any limitations to the strategies we come up with? Which functions of ViennaRNA are available to work with? Ensemble defects, entropy measurements, etc.? 16 Jun 2015 Alexander Lohachitranont How about tertiary folding? 18 Jun 2015 vineetkosaraju Brourd, for question 4 - in theory no, but keep in mind that including these features might take slightly longer to implement (and might cause the bot to take longer to run). The current eternabot just uses vienna to calculate the secondary structure based off of the sequence, but I'm sure other functions could also be called. Alexander Lohachitranont - We're still focusing on the secondary structure for eternabot, atleast for now, but hopefully one day it could even be expanded to tertiary folding :) 19 Jun 2015 Omei Can we get "progress reports" about what strategies have been implemented and what its current predictions are for the designs in the round we're currently working on? It would be more like having eternabot as a collaborator. 24 Jun 2015 Eli Fisker I agree with Omei's request. :) +1 25 Jun 2015 vineetkosaraju Sure those sound like great ideas :). I haven't started implementing the strategies yet (which is why there are no progress reports), but once I do I will paste a message with how many lines of code the strategy took, what the new optimized parameters are, what the correlation of your strategy is, and an excerpt of the actual code for reference.

Regarding the eternabot solutions, it is most likely that for the current round I will have the bot start generating solutions when all the strategies are implemented, which might be far off - but once I start running it, I would be happy to give weekly/biweekly/daily progress reports on the solutions it has created. 25 Jun 2015 Eli Fisker Sweet news, Vineet! :) 25 Jun 2015 Omei Super! Thanks, Vineet. You didn't specifically mention having Eternabot predict the score of designs submitted by other players. I don't think we've had that in the past, but it would be very helpful in giving players feedback about whether the implemented strategies are having the expected effect on the evaluation score. That reminds me of something I've been meaning to ask. Is Eternabot's scoring model simply a lineat combination of the individual strategy scores? Or something more sophisticated?? 25 Jun 2015 vineetkosaraju Great question Omei :) (I'm new to the eternabot world, so I just researched this yesterday). The way the scoring function works is it takes the normalized score from each individual strategy, multiplies that score by the weighting that strategy has (which I believe has been optimized based on past data), and adds these all up. I would thus think that it is a linear combination of the individual strategy scores, where the finalStrategy = (strategy1Score*weight1) + (strategy2Score*weight2)... for each of the strategies.

I love the idea of Eternabot predicting the score of designs submitted by other puzzles, but I'm not sure what the easiest way to set that up would be.. it could either be something automatic where the bot looks for new submissions, gets the score, and sends private messages or posts to page, or maybe the scoring function could be ported over to Eternascript where players can just input the puzzle id and their solution and the script outputs the bot's score. Which do you think would be best? 25 Jun 2015 Omei Thanks, Vineet. Yes, that's a linear model, which I suspected Eternabot was. It's hardly state of the art, but it is a solid classical prediction model. As to how to make the predictions available, if an inside developer were to take it on, I could see how the prediction of lab scores could become an engaging game in its own right. But given past history, it seems unlikely that will happen. In which case, the question probably boils down to "what is the easiest way to generate a frequently updated Google spreadsheet with all the predictions, so that player developers can do whatever they want with the data?" If Eternabot is already coded in Javascript (which I doubt), making it available in Eternascript and letting players take it from there would probably be simplest. But if it is written in some other language that isn't so easily converted to Eternascript, it might be easier for Eternabot to just generate and post a .csv file with the current predictions for all the submitted designs in the active labs once a day. 26 Jun 2015 JR How about a "score this sequence" pop up window on the design page so you can see it before submitting your design. 01 Jul 2015 Super Scientist Great idea! 01 Jul 2015 vineetkosaraju JR, that's a good long term solution that I'll talk with Nando (who is mostly in charge of the flash code) about -- unfortunately it might require some porting over of Eternabot which would take longer. For this round though I might just go with the option that's easiest to implement so that it takes less time for you to see the bot's scores. 01 Jul 2015 JR Ok, understood. A column in the output data would be second best, a column we could sort. 01 Jul 2015

Brourd Question: Since the secondary structure predictions for Vienna1.8.5 and Vienna2.x may differ significantly, and some strategies will be based on the number of base pairs, or the identity of base pairs, etc., will there be two separate versions of the strategy market to address this? 16 Jun 2015 rhiju No, the strategy market is meant for figuring properties of switches that do well experimentally but are perhaps not well encoded in either vienna energy function -- or any known energy funciton. 16 Jun 2015 Brourd Right, however, since players have been given access to two different energy functions, a strategy like that of the example "Strategy MarketSwitch Penalize large amounts of GC pairs," will inherently be tied to whichever secondary structure is used to define the number of G-C base pairs. A designer like Nando's bot, ViennaUCT, has all predicted secondary structures tied to the Vienna2.x parameters, meaning any strategies that score these designs based on vienna1.8.5 predictions may be partially incorrect. On the other hand, perhaps players have not adopted the Vienna2.x parameters on that large a scale, so that few of the strategy market scores will be affected, if at all. 16 Jun 2015 Brourd Question 2: Will this strategy market algorithm be focused solely on the chip-riboswitch scores? 16 Jun 2015 Brourd Question 3: Should strategies be written with the concept in mind that these apply to all riboswitch scores, or should separate strategies be written for different ligands? 16 Jun 2015 rhiju @brourd -- i now understand your question on vienna2 vs. vienna1.8.5 now -- it matters if the strategy requires looking at the dot plot (or similar). in that case, the player should specify which energy function to use (or both, if desired). 16 Jun 2015 rhiju For Question 2: we'll do separate correlation coefficient comparisons to 'cloud lab' (single-nucleotide SHAPE) data and to riboswitch-on-chip data. We'll probably return separate rewards for these, and train separate combination strategies. 16 Jun 2015 rhiju For Question 3: you can start by writing strategies with specific ligands/proteins in mind, and later generalize as we move to other ligands, proteins, and RNA inputs/outputs. To be totally concrete, we are seeking our first test of the new eternabot to happen in July with the current lab puzzles -- mostly FMN/MS2 exclusions. 16 Jun 2015 Brourd Question 4: Are there any limitations to the strategies we come up with? Which functions of ViennaRNA are available to work with? Ensemble defects, entropy measurements, etc.? 16 Jun 2015 Alexander Lohachitranont How about tertiary folding? 18 Jun 2015 vineetkosaraju Brourd, for question 4 - in theory no, but keep in mind that including these features might take slightly longer to implement (and might cause the bot to take longer to run). The current eternabot just uses vienna to calculate the secondary structure based off of the sequence, but I'm sure other functions could also be called. Alexander Lohachitranont - We're still focusing on the secondary structure for eternabot, atleast for now, but hopefully one day it could even be expanded to tertiary folding :) 19 Jun 2015 Omei Can we get "progress reports" about what strategies have been implemented and what its current predictions are for the designs in the round we're currently working on? It would be more like having eternabot as a collaborator. 24 Jun 2015 Eli Fisker I agree with Omei's request. :) +1 25 Jun 2015 vineetkosaraju Sure those sound like great ideas :). I haven't started implementing the strategies yet (which is why there are no progress reports), but once I do I will paste a message with how many lines of code the strategy took, what the new optimized parameters are, what the correlation of your strategy is, and an excerpt of the actual code for reference.

Regarding the eternabot solutions, it is most likely that for the current round I will have the bot start generating solutions when all the strategies are implemented, which might be far off - but once I start running it, I would be happy to give weekly/biweekly/daily progress reports on the solutions it has created. 25 Jun 2015 Eli Fisker Sweet news, Vineet! :) 25 Jun 2015 Omei Super! Thanks, Vineet. You didn't specifically mention having Eternabot predict the score of designs submitted by other players. I don't think we've had that in the past, but it would be very helpful in giving players feedback about whether the implemented strategies are having the expected effect on the evaluation score. That reminds me of something I've been meaning to ask. Is Eternabot's scoring model simply a lineat combination of the individual strategy scores? Or something more sophisticated?? 25 Jun 2015 vineetkosaraju Great question Omei :) (I'm new to the eternabot world, so I just researched this yesterday). The way the scoring function works is it takes the normalized score from each individual strategy, multiplies that score by the weighting that strategy has (which I believe has been optimized based on past data), and adds these all up. I would thus think that it is a linear combination of the individual strategy scores, where the finalStrategy = (strategy1Score*weight1) + (strategy2Score*weight2)... for each of the strategies.

I love the idea of Eternabot predicting the score of designs submitted by other puzzles, but I'm not sure what the easiest way to set that up would be.. it could either be something automatic where the bot looks for new submissions, gets the score, and sends private messages or posts to page, or maybe the scoring function could be ported over to Eternascript where players can just input the puzzle id and their solution and the script outputs the bot's score. Which do you think would be best? 25 Jun 2015 Omei Thanks, Vineet. Yes, that's a linear model, which I suspected Eternabot was. It's hardly state of the art, but it is a solid classical prediction model. As to how to make the predictions available, if an inside developer were to take it on, I could see how the prediction of lab scores could become an engaging game in its own right. But given past history, it seems unlikely that will happen. In which case, the question probably boils down to "what is the easiest way to generate a frequently updated Google spreadsheet with all the predictions, so that player developers can do whatever they want with the data?" If Eternabot is already coded in Javascript (which I doubt), making it available in Eternascript and letting players take it from there would probably be simplest. But if it is written in some other language that isn't so easily converted to Eternascript, it might be easier for Eternabot to just generate and post a .csv file with the current predictions for all the submitted designs in the active labs once a day. 26 Jun 2015 JR How about a "score this sequence" pop up window on the design page so you can see it before submitting your design. 01 Jul 2015 Super Scientist Great idea! 01 Jul 2015 vineetkosaraju JR, that's a good long term solution that I'll talk with Nando (who is mostly in charge of the flash code) about -- unfortunately it might require some porting over of Eternabot which would take longer. For this round though I might just go with the option that's easiest to implement so that it takes less time for you to see the bot's scores. 01 Jul 2015 JR Ok, understood. A column in the output data would be second best, a column we could sort. 01 Jul 2015

Brourd Question: Since the secondary structure predictions for Vienna1.8.5 and Vienna2.x may differ significantly, and some strategies will be based on the number of base pairs, or the identity of base pairs, etc., will there be two separate versions of the strategy market to address this? 16 Jun 2015 rhiju No, the strategy market is meant for figuring properties of switches that do well experimentally but are perhaps not well encoded in either vienna energy function -- or any known energy funciton. 16 Jun 2015 Brourd Right, however, since players have been given access to two different energy functions, a strategy like that of the example "Strategy MarketSwitch Penalize large amounts of GC pairs," will inherently be tied to whichever secondary structure is used to define the number of G-C base pairs. A designer like Nando's bot, ViennaUCT, has all predicted secondary structures tied to the Vienna2.x parameters, meaning any strategies that score these designs based on vienna1.8.5 predictions may be partially incorrect. On the other hand, perhaps players have not adopted the Vienna2.x parameters on that large a scale, so that few of the strategy market scores will be affected, if at all. 16 Jun 2015 Brourd Question 2: Will this strategy market algorithm be focused solely on the chip-riboswitch scores? 16 Jun 2015 Brourd Question 3: Should strategies be written with the concept in mind that these apply to all riboswitch scores, or should separate strategies be written for different ligands? 16 Jun 2015 rhiju @brourd -- i now understand your question on vienna2 vs. vienna1.8.5 now -- it matters if the strategy requires looking at the dot plot (or similar). in that case, the player should specify which energy function to use (or both, if desired). 16 Jun 2015 rhiju For Question 2: we'll do separate correlation coefficient comparisons to 'cloud lab' (single-nucleotide SHAPE) data and to riboswitch-on-chip data. We'll probably return separate rewards for these, and train separate combination strategies. 16 Jun 2015 rhiju For Question 3: you can start by writing strategies with specific ligands/proteins in mind, and later generalize as we move to other ligands, proteins, and RNA inputs/outputs. To be totally concrete, we are seeking our first test of the new eternabot to happen in July with the current lab puzzles -- mostly FMN/MS2 exclusions. 16 Jun 2015 Brourd Question 4: Are there any limitations to the strategies we come up with? Which functions of ViennaRNA are available to work with? Ensemble defects, entropy measurements, etc.? 16 Jun 2015 Alexander Lohachitranont How about tertiary folding? 18 Jun 2015 vineetkosaraju Brourd, for question 4 - in theory no, but keep in mind that including these features might take slightly longer to implement (and might cause the bot to take longer to run). The current eternabot just uses vienna to calculate the secondary structure based off of the sequence, but I'm sure other functions could also be called. Alexander Lohachitranont - We're still focusing on the secondary structure for eternabot, atleast for now, but hopefully one day it could even be expanded to tertiary folding :) 19 Jun 2015 Omei Can we get "progress reports" about what strategies have been implemented and what its current predictions are for the designs in the round we're currently working on? It would be more like having eternabot as a collaborator. 24 Jun 2015 Eli Fisker I agree with Omei's request. :) +1 25 Jun 2015 vineetkosaraju Sure those sound like great ideas :). I haven't started implementing the strategies yet (which is why there are no progress reports), but once I do I will paste a message with how many lines of code the strategy took, what the new optimized parameters are, what the correlation of your strategy is, and an excerpt of the actual code for reference.

Regarding the eternabot solutions, it is most likely that for the current round I will have the bot start generating solutions when all the strategies are implemented, which might be far off - but once I start running it, I would be happy to give weekly/biweekly/daily progress reports on the solutions it has created. 25 Jun 2015 Eli Fisker Sweet news, Vineet! :) 25 Jun 2015 Omei Super! Thanks, Vineet. You didn't specifically mention having Eternabot predict the score of designs submitted by other players. I don't think we've had that in the past, but it would be very helpful in giving players feedback about whether the implemented strategies are having the expected effect on the evaluation score. That reminds me of something I've been meaning to ask. Is Eternabot's scoring model simply a lineat combination of the individual strategy scores? Or something more sophisticated?? 25 Jun 2015 vineetkosaraju Great question Omei :) (I'm new to the eternabot world, so I just researched this yesterday). The way the scoring function works is it takes the normalized score from each individual strategy, multiplies that score by the weighting that strategy has (which I believe has been optimized based on past data), and adds these all up. I would thus think that it is a linear combination of the individual strategy scores, where the finalStrategy = (strategy1Score*weight1) + (strategy2Score*weight2)... for each of the strategies.

I love the idea of Eternabot predicting the score of designs submitted by other puzzles, but I'm not sure what the easiest way to set that up would be.. it could either be something automatic where the bot looks for new submissions, gets the score, and sends private messages or posts to page, or maybe the scoring function could be ported over to Eternascript where players can just input the puzzle id and their solution and the script outputs the bot's score. Which do you think would be best? 25 Jun 2015 Omei Thanks, Vineet. Yes, that's a linear model, which I suspected Eternabot was. It's hardly state of the art, but it is a solid classical prediction model. As to how to make the predictions available, if an inside developer were to take it on, I could see how the prediction of lab scores could become an engaging game in its own right. But given past history, it seems unlikely that will happen. In which case, the question probably boils down to "what is the easiest way to generate a frequently updated Google spreadsheet with all the predictions, so that player developers can do whatever they want with the data?" If Eternabot is already coded in Javascript (which I doubt), making it available in Eternascript and letting players take it from there would probably be simplest. But if it is written in some other language that isn't so easily converted to Eternascript, it might be easier for Eternabot to just generate and post a .csv file with the current predictions for all the submitted designs in the active labs once a day. 26 Jun 2015 JR How about a "score this sequence" pop up window on the design page so you can see it before submitting your design. 01 Jul 2015 Super Scientist Great idea! 01 Jul 2015 vineetkosaraju JR, that's a good long term solution that I'll talk with Nando (who is mostly in charge of the flash code) about -- unfortunately it might require some porting over of Eternabot which would take longer. For this round though I might just go with the option that's easiest to implement so that it takes less time for you to see the bot's scores. 01 Jul 2015 JR Ok, understood. A column in the output data would be second best, a column we could sort. 01 Jul 2015

Brourd Question: Since the secondary structure predictions for Vienna1.8.5 and Vienna2.x may differ significantly, and some strategies will be based on the number of base pairs, or the identity of base pairs, etc., will there be two separate versions of the strategy market to address this? 16 Jun 2015 rhiju No, the strategy market is meant for figuring properties of switches that do well experimentally but are perhaps not well encoded in either vienna energy function -- or any known energy funciton. 16 Jun 2015 Brourd Right, however, since players have been given access to two different energy functions, a strategy like that of the example "Strategy MarketSwitch Penalize large amounts of GC pairs," will inherently be tied to whichever secondary structure is used to define the number of G-C base pairs. A designer like Nando's bot, ViennaUCT, has all predicted secondary structures tied to the Vienna2.x parameters, meaning any strategies that score these designs based on vienna1.8.5 predictions may be partially incorrect. On the other hand, perhaps players have not adopted the Vienna2.x parameters on that large a scale, so that few of the strategy market scores will be affected, if at all. 16 Jun 2015 Brourd Question 2: Will this strategy market algorithm be focused solely on the chip-riboswitch scores? 16 Jun 2015 Brourd Question 3: Should strategies be written with the concept in mind that these apply to all riboswitch scores, or should separate strategies be written for different ligands? 16 Jun 2015 rhiju @brourd -- i now understand your question on vienna2 vs. vienna1.8.5 now -- it matters if the strategy requires looking at the dot plot (or similar). in that case, the player should specify which energy function to use (or both, if desired). 16 Jun 2015 rhiju For Question 2: we'll do separate correlation coefficient comparisons to 'cloud lab' (single-nucleotide SHAPE) data and to riboswitch-on-chip data. We'll probably return separate rewards for these, and train separate combination strategies. 16 Jun 2015 rhiju For Question 3: you can start by writing strategies with specific ligands/proteins in mind, and later generalize as we move to other ligands, proteins, and RNA inputs/outputs. To be totally concrete, we are seeking our first test of the new eternabot to happen in July with the current lab puzzles -- mostly FMN/MS2 exclusions. 16 Jun 2015 Brourd Question 4: Are there any limitations to the strategies we come up with? Which functions of ViennaRNA are available to work with? Ensemble defects, entropy measurements, etc.? 16 Jun 2015 Alexander Lohachitranont How about tertiary folding? 18 Jun 2015 vineetkosaraju Brourd, for question 4 - in theory no, but keep in mind that including these features might take slightly longer to implement (and might cause the bot to take longer to run). The current eternabot just uses vienna to calculate the secondary structure based off of the sequence, but I'm sure other functions could also be called. Alexander Lohachitranont - We're still focusing on the secondary structure for eternabot, atleast for now, but hopefully one day it could even be expanded to tertiary folding :) 19 Jun 2015 Omei Can we get "progress reports" about what strategies have been implemented and what its current predictions are for the designs in the round we're currently working on? It would be more like having eternabot as a collaborator. 24 Jun 2015 Eli Fisker I agree with Omei's request. :) +1 25 Jun 2015 vineetkosaraju Sure those sound like great ideas :). I haven't started implementing the strategies yet (which is why there are no progress reports), but once I do I will paste a message with how many lines of code the strategy took, what the new optimized parameters are, what the correlation of your strategy is, and an excerpt of the actual code for reference.

Regarding the eternabot solutions, it is most likely that for the current round I will have the bot start generating solutions when all the strategies are implemented, which might be far off - but once I start running it, I would be happy to give weekly/biweekly/daily progress reports on the solutions it has created. 25 Jun 2015 Eli Fisker Sweet news, Vineet! :) 25 Jun 2015 Omei Super! Thanks, Vineet. You didn't specifically mention having Eternabot predict the score of designs submitted by other players. I don't think we've had that in the past, but it would be very helpful in giving players feedback about whether the implemented strategies are having the expected effect on the evaluation score. That reminds me of something I've been meaning to ask. Is Eternabot's scoring model simply a lineat combination of the individual strategy scores? Or something more sophisticated?? 25 Jun 2015 vineetkosaraju Great question Omei :) (I'm new to the eternabot world, so I just researched this yesterday). The way the scoring function works is it takes the normalized score from each individual strategy, multiplies that score by the weighting that strategy has (which I believe has been optimized based on past data), and adds these all up. I would thus think that it is a linear combination of the individual strategy scores, where the finalStrategy = (strategy1Score*weight1) + (strategy2Score*weight2)... for each of the strategies.

I love the idea of Eternabot predicting the score of designs submitted by other puzzles, but I'm not sure what the easiest way to set that up would be.. it could either be something automatic where the bot looks for new submissions, gets the score, and sends private messages or posts to page, or maybe the scoring function could be ported over to Eternascript where players can just input the puzzle id and their solution and the script outputs the bot's score. Which do you think would be best? 25 Jun 2015 Omei Thanks, Vineet. Yes, that's a linear model, which I suspected Eternabot was. It's hardly state of the art, but it is a solid classical prediction model. As to how to make the predictions available, if an inside developer were to take it on, I could see how the prediction of lab scores could become an engaging game in its own right. But given past history, it seems unlikely that will happen. In which case, the question probably boils down to "what is the easiest way to generate a frequently updated Google spreadsheet with all the predictions, so that player developers can do whatever they want with the data?" If Eternabot is already coded in Javascript (which I doubt), making it available in Eternascript and letting players take it from there would probably be simplest. But if it is written in some other language that isn't so easily converted to Eternascript, it might be easier for Eternabot to just generate and post a .csv file with the current predictions for all the submitted designs in the active labs once a day. 26 Jun 2015 JR How about a "score this sequence" pop up window on the design page so you can see it before submitting your design. 01 Jul 2015 Super Scientist Great idea! 01 Jul 2015 vineetkosaraju JR, that's a good long term solution that I'll talk with Nando (who is mostly in charge of the flash code) about -- unfortunately it might require some porting over of Eternabot which would take longer. For this round though I might just go with the option that's easiest to implement so that it takes less time for you to see the bot's scores. 01 Jul 2015 JR Ok, understood. A column in the output data would be second best, a column we could sort. 01 Jul 2015

Brourd Question: Since the secondary structure predictions for Vienna1.8.5 and Vienna2.x may differ significantly, and some strategies will be based on the number of base pairs, or the identity of base pairs, etc., will there be two separate versions of the strategy market to address this? 16 Jun 2015 rhiju No, the strategy market is meant for figuring properties of switches that do well experimentally but are perhaps not well encoded in either vienna energy function -- or any known energy funciton. 16 Jun 2015 Brourd Right, however, since players have been given access to two different energy functions, a strategy like that of the example "Strategy MarketSwitch Penalize large amounts of GC pairs," will inherently be tied to whichever secondary structure is used to define the number of G-C base pairs. A designer like Nando's bot, ViennaUCT, has all predicted secondary structures tied to the Vienna2.x parameters, meaning any strategies that score these designs based on vienna1.8.5 predictions may be partially incorrect. On the other hand, perhaps players have not adopted the Vienna2.x parameters on that large a scale, so that few of the strategy market scores will be affected, if at all. 16 Jun 2015 Brourd Question 2: Will this strategy market algorithm be focused solely on the chip-riboswitch scores? 16 Jun 2015 Brourd Question 3: Should strategies be written with the concept in mind that these apply to all riboswitch scores, or should separate strategies be written for different ligands? 16 Jun 2015 rhiju @brourd -- i now understand your question on vienna2 vs. vienna1.8.5 now -- it matters if the strategy requires looking at the dot plot (or similar). in that case, the player should specify which energy function to use (or both, if desired). 16 Jun 2015 rhiju For Question 2: we'll do separate correlation coefficient comparisons to 'cloud lab' (single-nucleotide SHAPE) data and to riboswitch-on-chip data. We'll probably return separate rewards for these, and train separate combination strategies. 16 Jun 2015 rhiju For Question 3: you can start by writing strategies with specific ligands/proteins in mind, and later generalize as we move to other ligands, proteins, and RNA inputs/outputs. To be totally concrete, we are seeking our first test of the new eternabot to happen in July with the current lab puzzles -- mostly FMN/MS2 exclusions. 16 Jun 2015 Brourd Question 4: Are there any limitations to the strategies we come up with? Which functions of ViennaRNA are available to work with? Ensemble defects, entropy measurements, etc.? 16 Jun 2015 Alexander Lohachitranont How about tertiary folding? 18 Jun 2015 vineetkosaraju Brourd, for question 4 - in theory no, but keep in mind that including these features might take slightly longer to implement (and might cause the bot to take longer to run). The current eternabot just uses vienna to calculate the secondary structure based off of the sequence, but I'm sure other functions could also be called. Alexander Lohachitranont - We're still focusing on the secondary structure for eternabot, atleast for now, but hopefully one day it could even be expanded to tertiary folding :) 19 Jun 2015 Omei Can we get "progress reports" about what strategies have been implemented and what its current predictions are for the designs in the round we're currently working on? It would be more like having eternabot as a collaborator. 24 Jun 2015 Eli Fisker I agree with Omei's request. :) +1 25 Jun 2015 vineetkosaraju Sure those sound like great ideas :). I haven't started implementing the strategies yet (which is why there are no progress reports), but once I do I will paste a message with how many lines of code the strategy took, what the new optimized parameters are, what the correlation of your strategy is, and an excerpt of the actual code for reference.

Regarding the eternabot solutions, it is most likely that for the current round I will have the bot start generating solutions when all the strategies are implemented, which might be far off - but once I start running it, I would be happy to give weekly/biweekly/daily progress reports on the solutions it has created. 25 Jun 2015 Eli Fisker Sweet news, Vineet! :) 25 Jun 2015 Omei Super! Thanks, Vineet. You didn't specifically mention having Eternabot predict the score of designs submitted by other players. I don't think we've had that in the past, but it would be very helpful in giving players feedback about whether the implemented strategies are having the expected effect on the evaluation score. That reminds me of something I've been meaning to ask. Is Eternabot's scoring model simply a lineat combination of the individual strategy scores? Or something more sophisticated?? 25 Jun 2015 vineetkosaraju Great question Omei :) (I'm new to the eternabot world, so I just researched this yesterday). The way the scoring function works is it takes the normalized score from each individual strategy, multiplies that score by the weighting that strategy has (which I believe has been optimized based on past data), and adds these all up. I would thus think that it is a linear combination of the individual strategy scores, where the finalStrategy = (strategy1Score*weight1) + (strategy2Score*weight2)... for each of the strategies.

I love the idea of Eternabot predicting the score of designs submitted by other puzzles, but I'm not sure what the easiest way to set that up would be.. it could either be something automatic where the bot looks for new submissions, gets the score, and sends private messages or posts to page, or maybe the scoring function could be ported over to Eternascript where players can just input the puzzle id and their solution and the script outputs the bot's score. Which do you think would be best? 25 Jun 2015 Omei Thanks, Vineet. Yes, that's a linear model, which I suspected Eternabot was. It's hardly state of the art, but it is a solid classical prediction model. As to how to make the predictions available, if an inside developer were to take it on, I could see how the prediction of lab scores could become an engaging game in its own right. But given past history, it seems unlikely that will happen. In which case, the question probably boils down to "what is the easiest way to generate a frequently updated Google spreadsheet with all the predictions, so that player developers can do whatever they want with the data?" If Eternabot is already coded in Javascript (which I doubt), making it available in Eternascript and letting players take it from there would probably be simplest. But if it is written in some other language that isn't so easily converted to Eternascript, it might be easier for Eternabot to just generate and post a .csv file with the current predictions for all the submitted designs in the active labs once a day. 26 Jun 2015 JR How about a "score this sequence" pop up window on the design page so you can see it before submitting your design. 01 Jul 2015 Super Scientist Great idea! 01 Jul 2015 vineetkosaraju JR, that's a good long term solution that I'll talk with Nando (who is mostly in charge of the flash code) about -- unfortunately it might require some porting over of Eternabot which would take longer. For this round though I might just go with the option that's easiest to implement so that it takes less time for you to see the bot's scores. 01 Jul 2015 JR Ok, understood. A column in the output data would be second best, a column we could sort. 01 Jul 2015

Brourd Question: Since the secondary structure predictions for Vienna1.8.5 and Vienna2.x may differ significantly, and some strategies will be based on the number of base pairs, or the identity of base pairs, etc., will there be two separate versions of the strategy market to address this? 16 Jun 2015 rhiju No, the strategy market is meant for figuring properties of switches that do well experimentally but are perhaps not well encoded in either vienna energy function -- or any known energy funciton. 16 Jun 2015 Brourd Right, however, since players have been given access to two different energy functions, a strategy like that of the example "Strategy MarketSwitch Penalize large amounts of GC pairs," will inherently be tied to whichever secondary structure is used to define the number of G-C base pairs. A designer like Nando's bot, ViennaUCT, has all predicted secondary structures tied to the Vienna2.x parameters, meaning any strategies that score these designs based on vienna1.8.5 predictions may be partially incorrect. On the other hand, perhaps players have not adopted the Vienna2.x parameters on that large a scale, so that few of the strategy market scores will be affected, if at all. 16 Jun 2015 Brourd Question 2: Will this strategy market algorithm be focused solely on the chip-riboswitch scores? 16 Jun 2015 Brourd Question 3: Should strategies be written with the concept in mind that these apply to all riboswitch scores, or should separate strategies be written for different ligands? 16 Jun 2015 rhiju @brourd -- i now understand your question on vienna2 vs. vienna1.8.5 now -- it matters if the strategy requires looking at the dot plot (or similar). in that case, the player should specify which energy function to use (or both, if desired). 16 Jun 2015 rhiju For Question 2: we'll do separate correlation coefficient comparisons to 'cloud lab' (single-nucleotide SHAPE) data and to riboswitch-on-chip data. We'll probably return separate rewards for these, and train separate combination strategies. 16 Jun 2015 rhiju For Question 3: you can start by writing strategies with specific ligands/proteins in mind, and later generalize as we move to other ligands, proteins, and RNA inputs/outputs. To be totally concrete, we are seeking our first test of the new eternabot to happen in July with the current lab puzzles -- mostly FMN/MS2 exclusions. 16 Jun 2015 Brourd Question 4: Are there any limitations to the strategies we come up with? Which functions of ViennaRNA are available to work with? Ensemble defects, entropy measurements, etc.? 16 Jun 2015 Alexander Lohachitranont How about tertiary folding? 18 Jun 2015 vineetkosaraju Brourd, for question 4 - in theory no, but keep in mind that including these features might take slightly longer to implement (and might cause the bot to take longer to run). The current eternabot just uses vienna to calculate the secondary structure based off of the sequence, but I'm sure other functions could also be called. Alexander Lohachitranont - We're still focusing on the secondary structure for eternabot, atleast for now, but hopefully one day it could even be expanded to tertiary folding :) 19 Jun 2015 Omei Can we get "progress reports" about what strategies have been implemented and what its current predictions are for the designs in the round we're currently working on? It would be more like having eternabot as a collaborator. 24 Jun 2015 Eli Fisker I agree with Omei's request. :) +1 25 Jun 2015 vineetkosaraju Sure those sound like great ideas :). I haven't started implementing the strategies yet (which is why there are no progress reports), but once I do I will paste a message with how many lines of code the strategy took, what the new optimized parameters are, what the correlation of your strategy is, and an excerpt of the actual code for reference.

Regarding the eternabot solutions, it is most likely that for the current round I will have the bot start generating solutions when all the strategies are implemented, which might be far off - but once I start running it, I would be happy to give weekly/biweekly/daily progress reports on the solutions it has created. 25 Jun 2015 Eli Fisker Sweet news, Vineet! :) 25 Jun 2015 Omei Super! Thanks, Vineet. You didn't specifically mention having Eternabot predict the score of designs submitted by other players. I don't think we've had that in the past, but it would be very helpful in giving players feedback about whether the implemented strategies are having the expected effect on the evaluation score. That reminds me of something I've been meaning to ask. Is Eternabot's scoring model simply a lineat combination of the individual strategy scores? Or something more sophisticated?? 25 Jun 2015 vineetkosaraju Great question Omei :) (I'm new to the eternabot world, so I just researched this yesterday). The way the scoring function works is it takes the normalized score from each individual strategy, multiplies that score by the weighting that strategy has (which I believe has been optimized based on past data), and adds these all up. I would thus think that it is a linear combination of the individual strategy scores, where the finalStrategy = (strategy1Score*weight1) + (strategy2Score*weight2)... for each of the strategies.

I love the idea of Eternabot predicting the score of designs submitted by other puzzles, but I'm not sure what the easiest way to set that up would be.. it could either be something automatic where the bot looks for new submissions, gets the score, and sends private messages or posts to page, or maybe the scoring function could be ported over to Eternascript where players can just input the puzzle id and their solution and the script outputs the bot's score. Which do you think would be best? 25 Jun 2015 Omei Thanks, Vineet. Yes, that's a linear model, which I suspected Eternabot was. It's hardly state of the art, but it is a solid classical prediction model. As to how to make the predictions available, if an inside developer were to take it on, I could see how the prediction of lab scores could become an engaging game in its own right. But given past history, it seems unlikely that will happen. In which case, the question probably boils down to "what is the easiest way to generate a frequently updated Google spreadsheet with all the predictions, so that player developers can do whatever they want with the data?" If Eternabot is already coded in Javascript (which I doubt), making it available in Eternascript and letting players take it from there would probably be simplest. But if it is written in some other language that isn't so easily converted to Eternascript, it might be easier for Eternabot to just generate and post a .csv file with the current predictions for all the submitted designs in the active labs once a day. 26 Jun 2015 JR How about a "score this sequence" pop up window on the design page so you can see it before submitting your design. 01 Jul 2015 Super Scientist Great idea! 01 Jul 2015 vineetkosaraju JR, that's a good long term solution that I'll talk with Nando (who is mostly in charge of the flash code) about -- unfortunately it might require some porting over of Eternabot which would take longer. For this round though I might just go with the option that's easiest to implement so that it takes less time for you to see the bot's scores. 01 Jul 2015 JR Ok, understood. A column in the output data would be second best, a column we could sort. 01 Jul 2015

Brourd Question: Since the secondary structure predictions for Vienna1.8.5 and Vienna2.x may differ significantly, and some strategies will be based on the number of base pairs, or the identity of base pairs, etc., will there be two separate versions of the strategy market to address this? 16 Jun 2015 rhiju No, the strategy market is meant for figuring properties of switches that do well experimentally but are perhaps not well encoded in either vienna energy function -- or any known energy funciton. 16 Jun 2015 Brourd Right, however, since players have been given access to two different energy functions, a strategy like that of the example "Strategy MarketSwitch Penalize large amounts of GC pairs," will inherently be tied to whichever secondary structure is used to define the number of G-C base pairs. A designer like Nando's bot, ViennaUCT, has all predicted secondary structures tied to the Vienna2.x parameters, meaning any strategies that score these designs based on vienna1.8.5 predictions may be partially incorrect. On the other hand, perhaps players have not adopted the Vienna2.x parameters on that large a scale, so that few of the strategy market scores will be affected, if at all. 16 Jun 2015 Brourd Question 2: Will this strategy market algorithm be focused solely on the chip-riboswitch scores? 16 Jun 2015 Brourd Question 3: Should strategies be written with the concept in mind that these apply to all riboswitch scores, or should separate strategies be written for different ligands? 16 Jun 2015 rhiju @brourd -- i now understand your question on vienna2 vs. vienna1.8.5 now -- it matters if the strategy requires looking at the dot plot (or similar). in that case, the player should specify which energy function to use (or both, if desired). 16 Jun 2015 rhiju For Question 2: we'll do separate correlation coefficient comparisons to 'cloud lab' (single-nucleotide SHAPE) data and to riboswitch-on-chip data. We'll probably return separate rewards for these, and train separate combination strategies. 16 Jun 2015 rhiju For Question 3: you can start by writing strategies with specific ligands/proteins in mind, and later generalize as we move to other ligands, proteins, and RNA inputs/outputs. To be totally concrete, we are seeking our first test of the new eternabot to happen in July with the current lab puzzles -- mostly FMN/MS2 exclusions. 16 Jun 2015 Brourd Question 4: Are there any limitations to the strategies we come up with? Which functions of ViennaRNA are available to work with? Ensemble defects, entropy measurements, etc.? 16 Jun 2015 Alexander Lohachitranont How about tertiary folding? 18 Jun 2015 vineetkosaraju Brourd, for question 4 - in theory no, but keep in mind that including these features might take slightly longer to implement (and might cause the bot to take longer to run). The current eternabot just uses vienna to calculate the secondary structure based off of the sequence, but I'm sure other functions could also be called. Alexander Lohachitranont - We're still focusing on the secondary structure for eternabot, atleast for now, but hopefully one day it could even be expanded to tertiary folding :) 19 Jun 2015 Omei Can we get "progress reports" about what strategies have been implemented and what its current predictions are for the designs in the round we're currently working on? It would be more like having eternabot as a collaborator. 24 Jun 2015 Eli Fisker I agree with Omei's request. :) +1 25 Jun 2015 vineetkosaraju Sure those sound like great ideas :). I haven't started implementing the strategies yet (which is why there are no progress reports), but once I do I will paste a message with how many lines of code the strategy took, what the new optimized parameters are, what the correlation of your strategy is, and an excerpt of the actual code for reference.

Regarding the eternabot solutions, it is most likely that for the current round I will have the bot start generating solutions when all the strategies are implemented, which might be far off - but once I start running it, I would be happy to give weekly/biweekly/daily progress reports on the solutions it has created. 25 Jun 2015 Eli Fisker Sweet news, Vineet! :) 25 Jun 2015 Omei Super! Thanks, Vineet. You didn't specifically mention having Eternabot predict the score of designs submitted by other players. I don't think we've had that in the past, but it would be very helpful in giving players feedback about whether the implemented strategies are having the expected effect on the evaluation score. That reminds me of something I've been meaning to ask. Is Eternabot's scoring model simply a lineat combination of the individual strategy scores? Or something more sophisticated?? 25 Jun 2015 vineetkosaraju Great question Omei :) (I'm new to the eternabot world, so I just researched this yesterday). The way the scoring function works is it takes the normalized score from each individual strategy, multiplies that score by the weighting that strategy has (which I believe has been optimized based on past data), and adds these all up. I would thus think that it is a linear combination of the individual strategy scores, where the finalStrategy = (strategy1Score*weight1) + (strategy2Score*weight2)... for each of the strategies.

I love the idea of Eternabot predicting the score of designs submitted by other puzzles, but I'm not sure what the easiest way to set that up would be.. it could either be something automatic where the bot looks for new submissions, gets the score, and sends private messages or posts to page, or maybe the scoring function could be ported over to Eternascript where players can just input the puzzle id and their solution and the script outputs the bot's score. Which do you think would be best? 25 Jun 2015 Omei Thanks, Vineet. Yes, that's a linear model, which I suspected Eternabot was. It's hardly state of the art, but it is a solid classical prediction model. As to how to make the predictions available, if an inside developer were to take it on, I could see how the prediction of lab scores could become an engaging game in its own right. But given past history, it seems unlikely that will happen. In which case, the question probably boils down to "what is the easiest way to generate a frequently updated Google spreadsheet with all the predictions, so that player developers can do whatever they want with the data?" If Eternabot is already coded in Javascript (which I doubt), making it available in Eternascript and letting players take it from there would probably be simplest. But if it is written in some other language that isn't so easily converted to Eternascript, it might be easier for Eternabot to just generate and post a .csv file with the current predictions for all the submitted designs in the active labs once a day. 26 Jun 2015 JR How about a "score this sequence" pop up window on the design page so you can see it before submitting your design. 01 Jul 2015 Super Scientist Great idea! 01 Jul 2015 vineetkosaraju JR, that's a good long term solution that I'll talk with Nando (who is mostly in charge of the flash code) about -- unfortunately it might require some porting over of Eternabot which would take longer. For this round though I might just go with the option that's easiest to implement so that it takes less time for you to see the bot's scores. 01 Jul 2015 JR Ok, understood. A column in the output data would be second best, a column we could sort. 01 Jul 2015

Brourd Question: Since the secondary structure predictions for Vienna1.8.5 and Vienna2.x may differ significantly, and some strategies will be based on the number of base pairs, or the identity of base pairs, etc., will there be two separate versions of the strategy market to address this? 16 Jun 2015 rhiju No, the strategy market is meant for figuring properties of switches that do well experimentally but are perhaps not well encoded in either vienna energy function -- or any known energy funciton. 16 Jun 2015 Brourd Right, however, since players have been given access to two different energy functions, a strategy like that of the example "Strategy MarketSwitch Penalize large amounts of GC pairs," will inherently be tied to whichever secondary structure is used to define the number of G-C base pairs. A designer like Nando's bot, ViennaUCT, has all predicted secondary structures tied to the Vienna2.x parameters, meaning any strategies that score these designs based on vienna1.8.5 predictions may be partially incorrect. On the other hand, perhaps players have not adopted the Vienna2.x parameters on that large a scale, so that few of the strategy market scores will be affected, if at all. 16 Jun 2015 Brourd Question 2: Will this strategy market algorithm be focused solely on the chip-riboswitch scores? 16 Jun 2015 Brourd Question 3: Should strategies be written with the concept in mind that these apply to all riboswitch scores, or should separate strategies be written for different ligands? 16 Jun 2015 rhiju @brourd -- i now understand your question on vienna2 vs. vienna1.8.5 now -- it matters if the strategy requires looking at the dot plot (or similar). in that case, the player should specify which energy function to use (or both, if desired). 16 Jun 2015 rhiju For Question 2: we'll do separate correlation coefficient comparisons to 'cloud lab' (single-nucleotide SHAPE) data and to riboswitch-on-chip data. We'll probably return separate rewards for these, and train separate combination strategies. 16 Jun 2015 rhiju For Question 3: you can start by writing strategies with specific ligands/proteins in mind, and later generalize as we move to other ligands, proteins, and RNA inputs/outputs. To be totally concrete, we are seeking our first test of the new eternabot to happen in July with the current lab puzzles -- mostly FMN/MS2 exclusions. 16 Jun 2015 Brourd Question 4: Are there any limitations to the strategies we come up with? Which functions of ViennaRNA are available to work with? Ensemble defects, entropy measurements, etc.? 16 Jun 2015 Alexander Lohachitranont How about tertiary folding? 18 Jun 2015 vineetkosaraju Brourd, for question 4 - in theory no, but keep in mind that including these features might take slightly longer to implement (and might cause the bot to take longer to run). The current eternabot just uses vienna to calculate the secondary structure based off of the sequence, but I'm sure other functions could also be called. Alexander Lohachitranont - We're still focusing on the secondary structure for eternabot, atleast for now, but hopefully one day it could even be expanded to tertiary folding :) 19 Jun 2015 Omei Can we get "progress reports" about what strategies have been implemented and what its current predictions are for the designs in the round we're currently working on? It would be more like having eternabot as a collaborator. 24 Jun 2015 Eli Fisker I agree with Omei's request. :) +1 25 Jun 2015 vineetkosaraju Sure those sound like great ideas :). I haven't started implementing the strategies yet (which is why there are no progress reports), but once I do I will paste a message with how many lines of code the strategy took, what the new optimized parameters are, what the correlation of your strategy is, and an excerpt of the actual code for reference.

Regarding the eternabot solutions, it is most likely that for the current round I will have the bot start generating solutions when all the strategies are implemented, which might be far off - but once I start running it, I would be happy to give weekly/biweekly/daily progress reports on the solutions it has created. 25 Jun 2015 Eli Fisker Sweet news, Vineet! :) 25 Jun 2015 Omei Super! Thanks, Vineet. You didn't specifically mention having Eternabot predict the score of designs submitted by other players. I don't think we've had that in the past, but it would be very helpful in giving players feedback about whether the implemented strategies are having the expected effect on the evaluation score. That reminds me of something I've been meaning to ask. Is Eternabot's scoring model simply a lineat combination of the individual strategy scores? Or something more sophisticated?? 25 Jun 2015 vineetkosaraju Great question Omei :) (I'm new to the eternabot world, so I just researched this yesterday). The way the scoring function works is it takes the normalized score from each individual strategy, multiplies that score by the weighting that strategy has (which I believe has been optimized based on past data), and adds these all up. I would thus think that it is a linear combination of the individual strategy scores, where the finalStrategy = (strategy1Score*weight1) + (strategy2Score*weight2)... for each of the strategies.

I love the idea of Eternabot predicting the score of designs submitted by other puzzles, but I'm not sure what the easiest way to set that up would be.. it could either be something automatic where the bot looks for new submissions, gets the score, and sends private messages or posts to page, or maybe the scoring function could be ported over to Eternascript where players can just input the puzzle id and their solution and the script outputs the bot's score. Which do you think would be best? 25 Jun 2015 Omei Thanks, Vineet. Yes, that's a linear model, which I suspected Eternabot was. It's hardly state of the art, but it is a solid classical prediction model. As to how to make the predictions available, if an inside developer were to take it on, I could see how the prediction of lab scores could become an engaging game in its own right. But given past history, it seems unlikely that will happen. In which case, the question probably boils down to "what is the easiest way to generate a frequently updated Google spreadsheet with all the predictions, so that player developers can do whatever they want with the data?" If Eternabot is already coded in Javascript (which I doubt), making it available in Eternascript and letting players take it from there would probably be simplest. But if it is written in some other language that isn't so easily converted to Eternascript, it might be easier for Eternabot to just generate and post a .csv file with the current predictions for all the submitted designs in the active labs once a day. 26 Jun 2015 JR How about a "score this sequence" pop up window on the design page so you can see it before submitting your design. 01 Jul 2015 Super Scientist Great idea! 01 Jul 2015 vineetkosaraju JR, that's a good long term solution that I'll talk with Nando (who is mostly in charge of the flash code) about -- unfortunately it might require some porting over of Eternabot which would take longer. For this round though I might just go with the option that's easiest to implement so that it takes less time for you to see the bot's scores. 01 Jul 2015 JR Ok, understood. A column in the output data would be second best, a column we could sort. 01 Jul 2015 MicroRNAs are key post-transcriptional regulators of gene expression that are involved in several biological processes including those that mediate disease pathophysiology. Hence, quantifying microRNA expression levels can provide important and novel insights into disease biology. In recent years, the pig has emerged as an excellent large animal model for studying human diseases and conditions (e.g. obesity) due to similarities in organ size, gastro-intestinal tract, metabolism, immune response, genetics and the availability of relevant tissues that are not normally easily available in humans. We have previously developed two useful tools in the field of microRNA quantitative real time PCR (qPCR): 1) a very specific, sensitive and simple qPCR method based on DNA primers, MiR-specific qPCR; and 2) the free primerdesign software miRprimer. The present study integrates in a publicly accessible database all available information on validated porcine microRNA qPCR assays that have utilized these tools. Due to the high phylogenetic conservation in microRNA sequence between pig, humans and other domestic species this database is a very valuable resource for the broader scientist community who are working on microRNAs and want to use readily tested qPCR assays in a simple and cost-effective manner.

Reviews

Developing MicroRNA Therapeutics

Eva van Rooij,
Angela L. Purcell,
Arthur A. Levin

+ Author Affiliations

From miRagen Therapeutics, Inc, Boulder, CO (E.v.R.); Cooley LLP, Washington, DC (A.L.P.); and Santaris Pharma A/S, San Diego, CA (A.A.L.).

Correspondence to Eva van Rooij, miRagen Therapeutics, Inc, 6200 Lookout Rd, Boulder, CO 80301. E-mail eva.vanrooij@miragentherapeutics.com

 

Next Section Abstract

Rarely a new research area has gotten such an overwhelming amount of attention as have microRNAs. Although several basic questions regarding their biological principles still remain to be answered, many specific characteristics of microRNAs in combination with compelling therapeutic efficacy data and a clear involvement in human disease have triggered the biotechnology community to start exploring the possibilities of viewing microRNAs as therapeutic entities. This review serves to provide some general insight into some of the current microRNAs targets, how one goes from the initial bench discovery to actually developing a therapeutically useful modality, and will briefly summarize the current patent landscape and the companies that have started to explore microRNAs as the next drug target. Key Words:

miRNA
therapeutics
drug development
biotechnology

With the discovery of microRNAs (miRNAs) being powerful regulators in a wide variety of diseases, it is only a logical consequence that the possibilities of viewing miRNAs as therapeutic entities are being explored. In today's competitive and financially restricted pharmaceutical environment, there are some high demand criteria that must be met before a compound will be considered interesting enough to pursue, that is, worth the financial risk. A successful new drug must fulfill an unmet medical need, should show a clear benefit in safety or efficacy over the current standard of care, and consist of a compound that has attractive pharmacokinetic (PK) properties. Recent animal and even human efficacy data indicate that anti-miR compounds that inhibit specific miRNAs, have the potential to become a whole new class of drugs. MiRNAs have several significant advantages in that they are small and comprise of a known sequence that is often completely conserved among species, which are very attractive features from a drug development standpoint. Based on lessons learned from antisense technologies, very potent oligonucleotide chemistries to target miRNAs, known as anti-miRs, are currently being generated. A bit further behind those efforts are the efforts to design and deliver pharmacologically active synthetic miRNAs, or miR mimics/mimetics. For the design of anti-miRs, these efforts have given rise to chemistries that produce oligonucleotide drug candidates that bind to their cognate miRNA targets with remarkable affinity and specificity and have suitable drug-like qualities such as increased stability and pharmacokinetics. This review serves to provide some insights into the current landscape for targeting miRNAs in disease and the path from translating a bench discovery into a therapeutically useful modality. Although some examples of interesting miRNA targets and companies developing miRNA therapeutics are highlighted, this review is by no means complete, and many more miRNA therapeutics and companies developing them will undoubtedly follow. Previous SectionNext Section Discovery Research

Scientific freedom in an academic setting often allows for making grand, frequently unexpected discoveries. The primary credit for opening up the field of miRNA research goes to the labs of Ambros and Ruvkun. In 1993, these labs made the link between the findings that the lin-4 gene does not encode a protein product but instead gives rise to a small RNA,1 and the discovery that LIN-14 protein synthesis is inversely regulated by lin-4 RNA. Subsequent sequence analysis showing complementarity of lin-4 to the 3′ untranslated region of the lin-14 gene revealed the first miRNA-mRNA interaction.2 Although these initial discoveries were made in Caenorhabditis elegans, the detection of miRNA homologs in many vertebrate species3 was what really triggered intense research efforts to start unveiling basic concepts of miRNA biogenesis and function. Today, thanks to many profiling and gain-and-loss of function studies, it has become widely appreciated that miRNAs play a major role in many different diseases in both animals and humans.4 Genetic models of specific miRNAs, in combination with the ease by which miRNAs can be therapeutically manipulated in vivo, have generated compelling efficacy data and motivated the biotechnology community to start exploring some of these miRNAs as drug targets in a diversity of diseases. It is up to academic institutes and investigators to ensure that enticing efficacy data showing a beneficial effect of either genetic or therapeutic regulation in a disease setting is filed on to stake a claim to the intellectual property. Safety is an important determinant in drug development, so the miRNAs that are more tissue restricted will be preferred, especially for the more chronic indications. Additionally, because drug development is guided by the financial gain, another consideration will be the market for the disease indication in which the miRNA therapeutic would be applicable and how the miRNA therapeutic would compare to the current standard of care from an efficacy and financial standpoint.

One way to therapeutically mimic or reexpress a miRNA is by using synthetic RNA duplexes designed to mimic the endogenous functions of the miRNA of interest, with modifications for stability and cellular uptake. The “guide strand” is identical to the miRNA of interest, whereas the “passenger strand” is modified and typically linked to a molecule such as cholesterol for enhanced cellular uptake. However, because the guide strand must function as a miRNA and the cell must recognize it as such, the allowed chemically modifications are limited. Additionally, it should be noted that although this method would replace the miRNA levels lost during disease progression, it will also result in the uptake by tissues that do not normally express the miRNA of interest, resulting in potential off target effects. Delivery to the appropriate cell type or tissue is an important aspect of effective miRNA mimicry to prevent unwanted side-effects. Another way to increase the level of a miRNA is by the use of adeno-associated viruses (AAV). Delivered in viral vectors, the miRNA of interest can be continually expressed, resulting in robust replacement expression of miRNAs downregulated during disease. Additionally, the availability of a number of different AAV serotypes allows for the potential tissue-specificity due to the natural tropism toward different organs of each individual AAV serotype as well as the different cellular receptors with which each AAV serotype interacts. The use of tissue-specific promoters for expression allows for further specificity in addition to the AAV serotype. Furthermore, AAV is currently in use in a number of clinical trials for gene therapy, of which the safety profiles have looked quite well. In line with this, Kota et al5 recently showed AAV-mediated delivery of miR-26a blunts tumor genesis in a mouse model of liver cancer.

However, by far the most widely used approach to regulate miRNA levels in vivo is by using anti-miRs. Anti-miRs are modified antisense oligonucleotides harboring the full or partial complementary reverse sequence of a mature miRNA that can reduce the endogenous levels of an miRNA. The key requirements for an anti-miR are that the chemistry must be cell permeable, cannot be rapidly excreted, must be stable in vivo, and should bind to the miRNA of interest with high specificity and affinity.6–8 Several modifications have been used in vivo thus far. These chemical modifications include 2′-O-methyl-group (OMe)-modified oligonucleotides and locked nucleic acid (LNA)-modified oligonucleotides, in which the 2′-O-oxygen is bridged to the 4′-position through a methylene linker to form a rigid bicycle, locked into a C3′-endo (RNA) sugar conformation.9 Another chemical modification applied to enhance oligonucleotide stability is the balance between phosphodiester and phosphorothioate linkages between the nucleotides, with phosphorothioate providing more stability to the oligonucleotide and making it more resistant to nucleases. The different anti-miR chemistries were previously reviewed in more detail.10

Because the anti-miR therapeutics are currently more advanced than the miRNA mimics, we will focus on miRNA inhibition for the remainder of this review. A subset of the miRNAs of which inhibition has shown therapeutic promise and that are actively being pursued as clinical candidates are outlined below and schematized in Figure 1. Figure 1. View larger version:

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Figure 1.

Specific miRNAs that are currently being pursued as clinical candidates. A subset of the miRNAs of which inhibition has shown therapeutic promise and that are currently actively being pursued as clinical candidates for various disease indications. Previous SectionNext Section MiR-122 in Hepatitis C Virus

A hepatic-specific miRNA, miR-122, was shown to be of critical importance to the replication of hepatitis C virus (HCV).11,12 Sequestration of miR-122 with antisense constructs targeting the miRNA significantly reduced the replication of HCV RNA, suggesting that miR-122 may present a target for antiviral intervention.12 Only several months after the initial reports on miR-122, Krutzfeldt et al reported on the feasibility of manipulating miRNA levels in vivo, including miR-122 levels, using intravenous administration of a novel class of chemically engineered oligonucleotides, termed “antagomirs.” These chemically modified oligonucleotides are complementary to the mature miRNA sequence and are conjugated to cholesterol to facilitate cellular uptake. These initial experiments validated that systemic delivery through intravenous administration efficiently reduced the miRNA of interest for an extended period of time in multiple tissues in vivo.13,14 Subsequent studies using systemic delivery of an unconjugated LNA-containing anti-miR potently antagonized miR-122 in mice and nonhuman primates.15,16 Nonclinical studies of up to 13 weeks in 2 species support the development of this anti-miR and a recent preliminary report indicates that the safety profile of this LNA-modified oligonucleotide is remarkably similar to other drugs in this class. There were no apparent hepatic morphological changes associated with inhibition of miR-122 in these studies. The inhibition of miR-122 by this LNA–anti-miR was also shown to be safe and efficacious in chronically infected chimpanzees by suppressing HCV viremia and improving HCV-induced liver pathology.17 Clinical trials using anti-miR-122 against HCV are currently ongoing (see section Initial Clinical Trials). Previous SectionNext Section MiR-208 in Cardiac Remodeling

One of the first reports on a genetic deletion of a particular miRNA was on miR-208a. MiR-208a is the only cardiac-specific miRNA annotated thus far and is transcribed with its host gene, α-myosin heavy chain (αMHC).18 Although the expression level of miR-208a remains relatively stable during cardiac stress, this miRNA appears to play a key role in the expression of βMHC in response to cardiac stress and fulfills a dominant function in regulating cardiac hypertrophy and remodeling.18 In response to pressure overload by thoracic aortic constriction or signaling by calcineurin, a calcium/calmodulin-dependent phosphatase that drives pathological remodeling of the heart, miR-208a null mice showed virtually no hypertrophy of cardiomyocytes or fibrosis and were unable to upregulate βMHC expression. Because miR-208a expression does not change significantly in response to stress, its requirement for stress-dependent cardiac remodeling suggests that it cooperates with stress signaling to reprogram cardiac gene expression. Efficacy studies using systemic delivery of an LNA-modified anti-miR recently showed that therapeutic silencing of miR-208a through subcutaneous delivery of anti-miR-208a prevents pathological cardiac remodeling, functional deterioration, and lethality during diastolic heart disease. These beneficial effects correspond to significant changes in miRNAs within plasma.19 The cardiac specificity, the dedication of miR-208a to the cardiac stress response but not to normal cardiac development and the potent therapeutic benefit of anti-miR-208 in rodent models of heart failure make miR-208 (and its downstream effectors) an attractive therapeutic target in the setting of heart disease. Previous SectionNext Section MiR-155 in Inflammatory Disease

An important regulator of the immune system is miR-155. Genetic deletion indicated that miR-155 has an important role regulating T helper cell differentiation, at least in part, by regulating cytokine production.20 Follow-up studies showed that exposure of cultured macrophages and mice to lipopolysaccharide lead to upregulation of miR-155 and that the transcription factor c/ebp β is a direct target of miR-155. Interestingly, expression profiling of lipopolysaccharide-stimulated macrophages combined with overexpression and silencing of miR-155 in murine macrophages and human monocytic cells uncovered marked changes in the expression of granulocyte colony-stimulating factor, a central regulator of granulopoiesis during inflammatory responses.21 Consistent with these data, silencing of miR-155 in lipopolysaccharide-treated mice by systemically administering a LNA-modified anti-miR resulted in derepression of the c/ebp β isoforms and downregulation of granulocyte colony-stimulating factor expression in mouse splenocytes, which indicates the potential of miR-155 antagonists for the treatment of chronic inflammatory diseases.21 Previous SectionNext Section MiR-21 in Fibrosis

MiR-21 is one of the most dynamically regulated miRNAs in various pathological settings, ranging from cancer to heart disease. Several years ago, Thum et al22 demonstrated that in vivo knockdown of miR-21 with antagomirs blunted cardiomyocyte hypertrophy, inhibited and reversed interstitial fibrosis, and attenuated cardiac dysfunction after thoracic aortic banding. The ability of antagomirs against miR-21 to regulate cardiac disease was attributed to regulation of MAP kinase signaling pathways in cardiac fibroblasts through the miR-21 target, Spry1, a negative regulator of MAP kinase signaling. Although both genetic deletion of miR-21 and anti–miR-based inhibition of miR-21 failed to result in an overt phenotype at baseline and was incapable of blocking cardiac remodeling during stress in one report,23 others have shown that genetic deletion or anti-miR based inhibition induces therapeutic relevance in inhibiting fibrosis in different tissues24–26 and in cancer.27 Although the contradicting results regarding cardiac remodeling remain unexplained, based on the additional anti-miR findings, inhibition of miR-21 is actively being explored for therapeutic applications in both cancer and fibrotic diseases. Previous SectionNext Section MiR-92a in Neoangiogenesis

In 2009, Dimmeler et al28 reported on the effects of miR-92a, which is a member of the miR-17/92 cluster, in neoangiogenesis. Inhibition of miR-92a in vivo with intravenous administration of an antagomir resulted in enhanced blood vessel growth as well as functional improvement of damaged tissue in models of hind limb ischemia and myocardial infarction.28 The neoangiogenic effect of inhibiting miR-92a in vivo was attributed to the derepression of the multiple proangiogenic factors, including integrin α5, a direct target of miR-92a. The proangiogenic capacity of miR-92a inhibition is currently being explored as a potential therapy in diseases like ischemic heart disease or peripheral artery disease. Previous SectionNext Section MiR-33 in Metabolic Disease

An miRNA that has gained a lot of attention in the last few years is miR-33. MiR-33a and miR-33b are both embedded within introns of the sterol regulatory element-binding proteins (SREBPs). SREBP2 coexpresses miR-33a, whereas miR-33b is coexpressed with SREBP1.29 These host genes encode key transcriptional regulators of genes involved in cholesterol biosynthesis and uptake, and, as often the case with intronic miRNAs, miR-33a/b are involved in coregulating this process. In 2010, several groups showed that miR-33a/b targets the adenosine triphosphate-binding cassette transporter A1 (ABCA1), an important regulator of high-density lipoprotein (HDL) synthesis and reverse cholesterol transport. Inhibition of miR-33 in mice using either LNA-modified anti-miRs29,30 or lentiviral overexpression of anti-miR-3331 caused an upregulation of ABCA1 expression and increased cholesterol efflux, and injecting mice fed a Western-type diet with LNA-modified antisense oligonucleotides resulted in elevated circulating HDL levels.29 These data strongly suggest that inhibition of miR-33 may be atheroprotective. More recently, it was reported by Rayner et al32 that extended inhibition of miR-33 using an 2′ fluoro/methoxyethyl–modified (2′F/MOE-modified) phosphorothioate backbone antisense oligonucleotide against miR-33 in mice deficient for the LDL receptor with established atherosclerotic plaques, increased circulating HDL levels, and enhanced reverse cholesterol transport to the plasma, liver, and feces. Consistent with this effect, the anti-miR–treated mice showed reductions in plaque size and lipid content, increased markers of plaque stability, and decreased inflammatory gene expression.32 Inhibition of miR-33a/b in nonhuman primates raises plasma HDL and lowers VLDL triglycerides indicating therapeutic promise for the treatment of dyslipidemias that increase cardiovascular disease risk.33 Together, these studies indicate that raising HDL levels by anti-miR–mediated inhibition of miR-33 through its effects on cholesterol transport and atherosclerosis regression may be a promising therapeutic approach to treat vascular disease. Previous SectionNext Section MiR-451 in Myeloproliferative Diseases

MiR-451 is part of a bicistronic cluster (miR-144/451) that is highly expressed during erythrocyte development. Although normally erythrocyte formation occurs throughout life in response to cytokine signaling, last year the groups of Olson, Weiss and O'Carroll showed that mice lacking miR-45134 or lacking the miR-144/451 cluster35,36 display a reduction in hematocrit, an erythroid differentiation defect, and showed an ineffective erythropoiesis in response to oxidative stress. MiR-451 appears to be an especially intriguing miRNA in that it has very few predicted targets. Among these targets, 14-3-3zeta, an intracellular regulator of cytokine signaling, is repressed by miR-451 and is upregulated in miR-451–deficient erythroblasts. Conversely, inhibition of 14-3-3zeta, using shRNA, rescues the erythrocyte differentiation defect34 and protects miR-144/451–deficient erythrocytes against peroxide-induced destruction while restoring catalase activity.36 These findings reveal an essential role of 14-3-3zeta as a mediator of the proerythroid differentiation actions of miR-451 and highlight the therapeutic potential of miR-451 inhibitors. Although the original manuscript from Patrick et al34 showed that antagomir-based inhibition of miR-451 was able to phenocopy the erythroid differentiation defect seen in the miR-451 mutant animals, it will be of great interest to examine how oligonucleotide-based inhibition of miR-451 will affect the progression of some myeloid proliferative diseases characterized by an increase in hematocrit, such as polycythemia vera. Previous SectionNext Section MiR-15 Family in Cardiac Regeneration and Injury

The miR-15 family consists of multiple miRNAs (miR-15a, miR-15b, miR-16–1, miR-16–2, miR-195, and miR-497) and is consistently found to be upregulated in different settings of disease, including cancer and cardiac diseases. Although the exact functions of this miRNA family are still being explored in vivo, these miRNAs are predicted to influence cell survival and cell cycle regulation. For cardiac indications, this role would imply that miR-15 could serve as a therapeutic target for the manipulation of cardiac remodeling and function in the settings of ischemic heart disease, where the limited regenerative capacity of the heart is unable to restore the damaged tissue. In line with this hypothesis, a recent study by Porrello et al37 revealed that several members of the miR-15 family were upregulated during postnatal development of the heart. Because mammalian cardiomyocytes withdraw from the cell cycle during early postnatal development, these miRNAs might be involved in mechanisms that govern the reduced ability of cardiomyocytes to proliferate after birth. Using global gene profiling and argonaute-2 immunoprecipitation approaches, it was shown that miR-195 regulates the expression of a number of cell cycle genes, including checkpoint kinase 1 (Chek1) and that knockdown of the miR-15 family in neonatal mice with LNA-modified anti-miRs was associated with an increased number of mitotic cardiomyocytes and derepression of Chek1.37 Even more recent it was shown that miR-15 family inhibition, in the setting of ischemic injury, reduces infarct size and improves cardiac function.38 It will be interesting to explore the effects of miR-15 inhibition in heart disease, in which loss of viable tissue or lack of regeneration is causally related to the disease. Previous SectionNext Section MiR-103/107 in Metabolism

MiR-103 and miR-107 are related miRNAs that differ by only 1 base in their 3′ region. Although they are intronically located within the pantothenate kinase (PANK) genes, at least in mice, their expression does not correlate with expression of the host genes.39 Bioinformatic target prediction programs indicate that these miRNAs can potentially regulate mRNA genes in pathways that involve cellular Acetyl-CoA and lipid levels.40 Recently, Stoffel et al41 showed that the expression of miR-103 and miR-107 is upregulated in obese mice and that antagomir-based silencing of miR-103/107 leads to improved glucose homeostasis and insulin sensitivity, whereas gain of miR-103/107 function in either liver or fat is sufficient to induce impaired glucose homeostasis. Target identification analysis indicated caveolin-1, a critical regulator of the insulin receptor, to be a direct target of miR-103/107. The upregulation of caveolin-1 on miR-103/107 inhibition in adipocytes correlates with stabilization of the insulin receptor, enhanced insulin signaling, decreased adipocyte size, and enhanced insulin-stimulated glucose uptake. These findings demonstrate the potential importance of miR-103/107 to insulin sensitivity and identify these miRNAs as potential new targets for the treatment of type 2 diabetes and obesity. Previous SectionNext Section Preclinical Drug Development for Anti-MiRs

With respect to drug discovery, the miRNA revolution of recent years has provided the industry with multiple new opportunities for the identification of new drug targets. These revelations, coupled with recent advances in anti-miR chemistries, suggest that the regulation of miRNAs may be the next innovation in pharmaceutical research. These advances are based on basic research on the role of miRNAs in disease states or in cellular regulation in disease, and, more importantly and more recently, on demonstrations of pharmacological interventions. Based on in vivo efficacy studies showing therapeutic benefit of miRNA regulation (as described in the previous section), potential miR-inhibiting sequences and chemistries can be characterized to further evaluate these entities as suitable drug candidates for preclinical toxicity and pharmacokinetics studies prior to the onset of clinical trials (Figure 2). Figure 2. View larger version:

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Figure 2.

The process of research and development to advance miRNA therapeutics. To advance a miRNA target toward the clinic, there is a defined process that is generally followed before the onset of clinical trials, which involves optimization of suitable drug candidates and performing pharmacokinetics (PK), pharmacodynamics (PD), and absorption, distribution, metabolism, and excretion (ADME) studies to obtain all required insights into the anti-miR drug. Preclinical toxicity testing is performed to assess whether a drug is safe enough to move into humans. To move an anti-miR drug into clinical trials, all the nonclinical data on the characterization of the drug substance and the safety in animals are compiled along with a clinical protocol for an initial usually short exposure of humans to ascending doses of the new therapeutic (Phase I clinical trial). These preclinical data required for an Investigational New Drug (IND) or Clinical Trials Application will allow regulators authorities and ethics committees to assess whether the drug product has been well enough characterized to establish whether it should be safe in the initial clinical protocol and that the clinical protocol design and investigators will monitor the subject's safety. Previous SectionNext Section Establishing Lead Compound

The discovery and development of a novel therapeutic agent has become more challenging and riskier than ever before because of the high cost of drug development and the more stringent regulatory requirements for drug registration. This new environment has placed a premium on quickly identifying and validating drug candidates, selecting the most effective ones that have good pharmacological activity, and have the best predicted safety and pharmacokinetic profiles. Although the initial efficacy testing of miRNA inhibition in an academic laboratory is often done with a validated research tool, regulatory requirements ultimately force the later stages of candidate selection and safety profiling into the industrial arena.

Usually, to establish a lead drug compound for traditional low-molecular-weight drugs, many thousands of molecules will be synthesized and screened to generate a single drug candidate. However, screening approaches for lead anti-miR designs can be more focused for the following reasons: the short sequence of the miRNA target and the discrepancy between in vitro and in vivo performance of the anti-miRs forcing researchers to screen for efficacy in vivo.

The primary reason for anti-miR screens often being limited is that miRNA sequences are short, and there are limited numbers of sequences that will bind and sequester any single miRNA. Although the number of testable molecular species (permutations) will increase when chemical modifications are introduced into the sequence, the nature, location, and the number of these modifications is restricted for anti-miR chemistries. This limited number of possible permutations allows for a focused screening effort compared with more classic drugs. It is possible to vary the location of modifications, and the length of the putative binding species, but the range of acceptable chemical modifications have been well defined by the existing antisense literature and the “universe” of modifications is well understood.

One of our recent observations is that although it is possible to select optimal sequence for binding and sequestration in vitro, to fully optimize the distribution and uptake by target tissues it is preferable to select anti-miR chemistries on the basis of in vivo performance in relevant animal models because they allow the researcher to assess the true pharmacological profile and therapeutic window for a given sequence and its modifications. At times it may even be useful or more optimal to screen in disease models in which it is possible to assess the performance of the putative drug candidate with the appropriates stresses and in the appropriate host environment when miRNA perturbations best approximate the human condition. As such, miRNA regulation under baseline conditions might not always result in a measurable downstream effect. The use of animal models, including animal models of disease, offer an overall assessment of the pharmacokinetic (PK, the inhibitory effect of the anti-miR on the miRNA), pharmacodynamic (PD, the effect of the anti-miR on target derepression of direct mRNA targets of a specific miRNA) and toxicity profiles before committing to a traditional nonclinical development program and its attendant GLP toxicity and pharmacokinetics studies. These studies represent a significant investment in time, resources, and animals and should be initiated only when there is ample evidence of good activity, distribution, and safety in pharmacology screens. Previous SectionNext Section Pharmacokinetics, Pharmacodynamics, and Absorption, Distribution, Metabolism, and Excretion

In search for lead sequences and anti-miR chemistries, the pharmacokinetic properties of the compounds become critically important in guiding the selection of a drug candidate for development. The pharmacokinetic behavior of a compound in animals serves as a basis for predicting how a compound will behave in humans and represents an integral component of the decision making process whereby new chemical entities are selected for development.

Most anti-miRs use modifications of the typical nucleic acid ribose sugar backbone with 2′-modifications, which distribute broadly but tend to accumulate in a characteristic pattern with most in liver, kidney and phagocytically active cell types. The field is learning that each chemical modification or length change in a candidate sequence introduces variations from the typical pattern. Thus it becomes important to characterize each candidate sequence (and its specific modifications) in an animal model predictive for humans. This information is normally obtained at an early stage of preclinical evaluation.

For a modified phosphorothioate oligonucleotide to target a miRNA, there are distinctive attributes of its pharmacokinetics and pharmacology that differentiate it from typical (low molecular weight) drugs. The most obvious one is that plasma levels of an anti-miR or miR-mimetic are cleared from plasma within hours by uptake into tissues,42,43 but, once inside cells, many of the modified oligonucleotide used as anti-miRs are so metabolically stable that their clearance is slow, and half-lives in tissues are often in on the order of weeks, providing therapeutic benefit long after blood levels are near zero. Because pharmacological activity is the result of the anti-miR being pharmacologically active in tissue cells not plasma, the initial high levels of anti-miR detection in the plasma are of limited value in predicting the extent and duration of the pharmacological effect of the anti-miR. The key factor that controls (and predicts) the pharmacological effects and their duration (pharmacodynamics) are target tissue concentrations not the initial plasma concentrations. In clinical trials, it is not feasible to get tissue samples to measure drug levels, and, for that reason, surrogates for target tissue must be established. Various surrogates have been considered including peripheral blood monocytes, subcutaneous fat biopsies, and equilibrium plasma concentrations. Equilibrium plasma concentrations occur after the plasma has been cleared of drug and represent drug that has equilibrated from the depots in the tissue into the plasma.

The time course and nature of the pharmacological effects (or pharmacodynamics) of anti-miRs are often complicated by the fact that the pharmacology is the summation of the derepression of families of genes and the downstream events produced by derepression of multiple target genes controlled by that particular miRNA. Thus, for the downstream effects of some anti-miRs, there can be a delay in their onset and in their offset. This delay has been reported for the miR-122 inhibitor and its effects on cholesterol.9 Similar phenomena are being observed with other anti-miRs, such as against miR-208 and a delayed effect on downregulating β-MHC.44 Delays in observing pharmacodynamics effects are atypical for most low-molecular-weight drugs and are more akin to so-called disease-modifying agents, in which it is established that pharmacological activity is related to the sum of pharmacological effects over time that cause a change in the disease phenotype.

The poly-anionic molecules that are currently employed as anti-miRs are highly water-soluble, with molecular weights of 2–6 kD, and are not good candidates for oral administration with intestinal absorption without the aid of permeation enhancers.45 Even with permeation enhancers, intestinal absorption of anti-miRs is going to be limited by size and charge. Thus it remains a formulation challenge to create novel delivery techniques for oral administration. At present, administration of oligonucleotide anti-miR is dependent on parenteral administration, and the 2 routes currently being used are intravenous and subcutaneous injections or infusions. Because of their high water solubility, it is possible to dissolve anti-miRs in aqueous solutions at volumes that are amenable to self-administration by the subcutaneous route, and, with their long biological half-lives, it may be possible that anti-miRs can be administered relatively infrequently, thus reducing frequency of injection. Previous SectionNext Section Toxicity Testing

A major contributor to the high attrition rate in drug development continues to be unanticipated toxicity. The goal of preclinical toxicity evaluations is to screen out drugs with unanticipated effects and to determine the target organs for toxicities and characterize the nature of the toxicities. To this end, toxicity studies with anti-miRs face interesting challenges. In contrast to many other therapeutic modalities, anti-miR drugs are designed knowing that they will affect families of genes under the control of the target miRNA. This differs from most other drugs that are generally designed to affect 1 target but may inadvertently affect more than 1. Although miRNAs often target many related genes involved in the pathway or cellular process that is intended to be manipulated by the anti-miR therapeutic, the same miRNA probably will also target unrelated genes and possibly produce undesired changes in gene expression.

Another challenge for toxicologists and pharmacologists is that although miRNAs tend to be highly conserved across species,46 there may be less conservation in the families of genes that the same miRNA targets in different species; or, there may be a difference in miRNA subfamilies across species, with each species being dependent on a slightly different combination of miRNA subtypes, and each miRNA subtype may have its own distinctive fingerprint of mRNAs that it represses. Thus for each new anti-miR sequence that goes into clinical trials there may be differences in the responses between the animal models and man. Clearly, the goal of toxicology studies is to characterize the biological response to therapy in animals as closely related to man as possible, and thus there is the precedent for using nonhuman primates to characterize toxicity. This approach is accepted but its limitations, particularly in the field of miRNA therapeutics, must be acknowledged as each anti-miR drug moves into clinical trials for the first time.

In principle, the assessment of toxicity of a miRNA-targeting (or mimicking) therapeutic is very similar to the characterization of toxicity for a typical small molecule. A typical toxicology program to move a drug into clinical trials is provides information on the genetic toxicology of the candidate drug, effects on critical organ systems, and the characterization of effects in 2 species after repeated administration of the candidate drug in a rodent and nonrodent species. Although procedurally there are few differences between the toxicity testing programs between small molecules and oligonucleotide drugs, there are some key differences as well.

The main differences in the toxicity studies between a small molecule and an anti-miR are related to how toxicities might arise rather than the process by which toxicity is characterized. The sources of toxicity after administration of a miRNA targeting agent can be classified broadly as toxicities related to the chemistry or class (hybridization independent toxicities) and toxicities arising from hybridization events. Chemistry-related effects are common sources of toxicity for oligonucleotide therapeutics. One example of this type of effect could arise from anti-miR, binding to a protein and modulating protein function. Phosphorothioate oligonucleotides can inhibit the tenase complex in the intrinsic clotting cascade,47 activate the alternative pathway of the complement cascade,48 and activate innate immunity. It is the activation of innate immunity that has proven to be the most complex and difficult to avoid. In brief, DNA and RNA are recognized by innate immune receptors much the same way as viruses, thereby setting off cascades of events. Chemical modifications of oligonucleotides, particularly those at the 2′ position of the ribose sugar, reduce the proinflammatory effects of oligonucleotides, but such effects have not yet been totally eliminated.

Hybridization-dependent effects can range from toxicities that result from excess pharmacological that might occur after binding to the intended target (exaggerated pharmacology) to toxicities that result from binding and inactivation of an unintended target RNA (off-target effect). It is possible to use Watson and Crick base-pairing rules to attempt to predict potential sites for off-targets hybridization-dependent effects and to use microarrays or other tools for measure RNA levels to characterize off-target effects. Examples of hybridization-dependent toxicities would be toxicities that might arise if the sequence of the anti-miR had a near perfect match in the genome. It could then be envisioned that the anti-miR could bind to that related sequence and produce an antisense effect on that unintended target and produce toxicity. This type of off-target effect can be avoided by diligent screening of potential anti-miR sequences. However, even if there is a near-perfect match in the genome, an off-target effect like that mentioned is unlikely for a multitude of reasons, including the fact that the design of anti-miR chemistries do not generally support antisense-cleavage of a target.49–51 In addition, if there are any highly homologous targets they would have to be in tissues where the antagonist was concentrated at sufficient concentrations to produce activity and would have to be an mRNA target that when its expression was reduced produced some toxicity. All of these criteria would have to be met for an anti-miR to produce an off-target hybridization-dependent toxicity.

An interesting concept to consider in toxicity testing for an anti-miR chemistry is the concept of saturation of pharmacology and its implications for dose and dose regimen. For an anti-miR, there is a limited number of available miRNAs to bind and sequester, so it should be possible to sequester all of the target miRNAs in a cell at some finite concentration. Thus, adding more anti-miR will not produce greater sequestration and therefore no additional pharmacology. Establishing the doses and tissue concentrations that reach this level of total sequestration is probably a key function of nonclinical toxicity and pharmacokinetic studies and has important implications for the design of dose regimens for clinical trials. Understanding the concentration of an anti-miR and the rate of clearance of the anti-miR chemistry would allow one to design dose-sparing regimens in which, once drug concentrations achieve total miRNA sequestration, then the only additional anti-miR needed would be that required to replace the anti-miR cleared or the anti-miR required to sequester newly synthesized miRNAs. Thus, it should be possible to use information on the half-life of the anti-miR drug and the turnover rate of the miRNA targets to estimate dosing needs. Any drug given in excess of the amount required to fully sequester the available miRNA target is simply drug that is available to produce non–target-related effects.

The optimization of chemistry, sequence, and length of an oligonucleotide as well as the dose regimen are all essential to selecting a successful miR-antagonist and designing the appropriate clinical trials. Adhering to the wisdom of Paracelsus (1493–1541) “Dosis facit venenu” (dose makes the poison) nonclinical studies should establish safe levels and characterize unwanted effects. With the tools that are available to screen for toxicity and an understanding of the kinetics of the drug and the miRNA drug target, it should be possible to move safe compounds into clinical trials and to design safe clinical trials that minimize ineffective and potentially toxic over exposures. Previous SectionNext Section Initial Clinical Trials

To date, there is one anti-miR chemistry in clinical trials, which is miravirsen (an inhibitor of miR-122; see above). To advance an anti-miR drug from the bench into clinical trials, there is a well-defined process whereby nonclinical data on characterization of the drug substance and the safety in animals and are compiled along with a clinical protocol for an initial usually short exposure of humans to ascending doses of the new therapeutic. The data required for an Investigational New Drug (IND) or Clinical Trials Application allow regulatory authorities and ethics committees to assess whether the drug product has been characterized sufficiently to establish whether it should be safe in the initial clinical protocol and that the clinical protocol design and investigators will monitor the subject's safety. As the drug proceeds through clinical trials, the level of animal toxicity testing and level of understanding of the manufacturing process and its controls increase in intensity to support the longer-term use of the new therapeutic in clinical trials initially in healthy subjects and later in patients with the relevant diseases. Just recently, data from the Phase 2a trial were released indicating that miravirsen, the first miRNA-targeted drug to enter clinical trials, provided continuous and prolonged antiviral activity well beyond the end of active therapy in patients and was well tolerated in patients infected with HCV (http://www.santaris.com/news/2011/10/03/santaris-pharma-report-new-clinical-data-miravirsen-phase-2a-study-treat-hepatitis-c). Even as a single agent, there were some patients whose viremia was reduced below the level of detection after 4 weeks of dosing with 7 mg/kg per week. The data demonstrate clinical utility for an miR-targeting therapeutic in human disease. Miravirsen is serving as a model compound for future anti-miR therapeutics, and many more miRNA therapeutics are expected to enter clinical trials as new disease targets are identified and new sequences show activity in diseases. There is great anticipation for the arrival of drugs that can reprogram disease cells into healthy cells and tackle diseases from a total novel set of targets. Previous SectionNext Section Current Status of MiRNA Therapeutics

Based on reasons mentioned above, there is great excitement surrounding miRNAs as therapeutic entities. From a scientific point of view, multiple miRNAs appear to be attractive therapeutic targets and chemistries exist that can inhibit miRNAs in a safe manner. Perhaps, not surprisingly, the explosion of scientific research on miRNA biology has engendered a considerable amount of innovation in this area as reflected by the significant increase in the number of patent application filings over the last 10 years. To provide a snapshot of the current commercial uses and development of miRNA-based therapeutics and diagnostics, we briefly summarize the miRNA patent landscape in the United States and Europe and describe several of the biotechnology companies that are presently developing miRNA-based products and services. Previous SectionNext Section MicroRNA Patents in the United States and Europe

Observing the trends in miRNA patent activity lends insight not only into the scientific progress in the field but also foreshadows aspects of the field that are likely to be ripe for commercial development. To obtain an overall picture of the miRNA patent landscape in the United States and Europe, one can conduct a search in the patent database for all published applications and patents related to miRNAs and their use. In doing so, our search resulted in a total of 2136 published patent documents, 1661 of which were US publications and 475 of which were European publications. A breakdown of the number of application filings and patents issued by year from 2000–2010 reveals a dramatic increase in miRNA-related application filings beginning in 2002 in the United States and peaking in 2008, with more than 300 filings (Figure 3), which reflects the intense research and development occurring in this nascent field. The number of European applications filed continues to increase since the first filings in 2003. The difference in trends between the number of US and European miRNA-related filings is due, at least in part, to the filing strategy that is commonly used to pursue patent protection in Europe. Generally, patent applicants initially file an international application under the Patent Cooperation Treaty, which delays national stage filings in other countries, like Europe, for an additional 18 months. After a lag of 4 years from the first application filings, the first US patent was issued in 2006, and the number of patents issued since then has been steadily rising. Last year, a sharp increase in issued patents was observed with 3 times the number of patents granted as compared with that granted in 2009. This trend is likely to continue into 2011, as almost 50 miRNA-related patents have been issued through the beginning of August. The first miRNA-related patent issued in Europe in 2008 and the number of granted European patents have increased over the last 3 years, albeit at a slower pace than observed in the United States. Figure 3. View larger version:

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Figure 3.

Annual number of US and European published patent applications and issued patents related to miRNAs and their applications. The data set was constructed by searching for miRNA-related key words in the abstract and claims fields in the Delphion patent database. The information in this database for the US and European patent documents is provided by the US Patent and Trademark Office and the European Patent Office, respectively.

To determine the particular technical areas in which the patent filings were concentrated, we separated the patent documents in the US data set by their International Patent Classification (IPC) codes. The IPC system categorizes patent documents according to the technical field that characterizes the described invention (Figure 4A). Although the miRNA-related patent documents spanned over 60 IPC code categories, almost 50% of the US patent documents were classified as relating to some type of medicinal preparation, relating to both pharmaceutical compositions comprising miRNA-modulating compounds, as well as methods of modulating miRNA activity for the treatment of diseases. Another smaller but notable grouping of documents were those related to assays and apparatus for detecting or measuring miRNAs. This group of patent documents accounted for 22% of all the filings and included applications related to creation of miRNA expression profiles and methods for detection and/or quantification of miRNA in various samples. The remainder of the US patent documents was spread across various categories, including plant-related miRNAs, genetic engineering and tissue or cell cultivation, nucleic acids and their derivatives, and libraries and methods of preparing or screening libraries. Figure 4. View larger version:

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Figure 4.

miRNA-related patent distribution in the United States. A, The distribution of different technological fields for the 1661 US miRNA-related patent documents as determined by International Patent Classification codes. Although the miRNA-related patent documents spanned over 60 IPC code categories, almost 50% of the US patent documents were classified as relating to some type of medicinal preparation relating to both pharmaceutical compositions comprising miRNA-modulating compounds as well as methods of modulating miRNA activity for the treatment of diseases. B, Distribution of US miRNA-related patent documents classified as medicinal preparations in A divided by particular indications.

An interesting pattern emerged when the patent documents classified as medicinal preparations in Figure 4A were further sorted into specific indications (Figure 4B). Patent documents relating to miRNA delivery vehicles or methods accounted for 14% of the filings, whereas applications and patents related to gene regulation constituted 12% of the filings. This latter category included miRNA molecules themselves, compounds targeting miRNAs, and methods of regulating RNA interference. Patent filings directed to multiple therapeutic uses of specific miRNA molecules comprised the majority of the general treatment methods group, which accounted for 11% of the patent documents categorized as medicinal preparations. Applications and patents disclosing diagnostic uses for miRNAs also represented a significant number of the medicinal preparation filings.

With respect to specific disorders, there were a greater number of filings related to methods of treating cancer than any other particular family of diseases or disorders. After cancer, the most prevalent filings were related to the treatment of viral infections (6%), particularly hepatitis C, ocular disorders (5%), inflammatory disorders (5%), cardiovascular disorders (4%), and neurological disorders (4%). Interestingly, many of the indications for which a significant number of patent filings were observed are also considered to be among the top health risks for Americans, such as heart disease, Alzheimer disease, and cancer. The relatively high number of filings related to the treatment of ocular disorders is somewhat surprising. Perhaps the high incidence of these filings reflects the fewer obstacles in delivering nucleic acid therapeutics to the eye. Previous SectionNext Section Companies Developing MiRNA Technologies

Industry support for RNA-targeting therapeutics has been cyclic, with periods of intense excitement followed by periods of apathy, with antisense and siRNA being prime examples of cyclic interest. Some of the luster on siRNA has faded, and there has been a retrenchment and in a few cases abandonment of RNA-targeting therapeutics, but many large companies maintain trimmed-down efforts in RNA-targeting therapeutics, and smaller companies continue to push forward with important proof of concept studies. With the advent of miRNAs as viable therapeutic targets for many serious health conditions, many new companies have been established to translate the exciting scientific discoveries into real-world, commercial uses. At the same time, some older companies, which had an initial focus on the development of siRNA-based compounds, have additionally begun to explore their proprietary chemistries and delivery systems for the design of miRNA inhibitors and mimics. The Table summarizes several companies that are currently developing miRNA-based therapeutic and diagnostic applications. View this table:

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Table.

Companies Developing MiRNA-Related Technologies

Founded in 1983, Marina Biotech, based in Bothell, Washington, recently announced the success of oligonucleotides modified with the company's conformationally constrained nucleotide technology for suppressing miRNA expression in vivo. Similarly, RXi Pharmaceuticals, which was founded in 2003 by Nobel Laureate Craig Mello, is developing “self-delivering RNAi” compounds that contain a combination of nuclease-stabilizing, backbone, and lipophilic chemical modifications. Santaris Pharma, the Denmark-originated company that developed the LNA chemistry platform to create oligonucleotide therapeutics with enhanced affinity for their RNA targets, exploited the LNA technology to produce potent oligonucleotide inhibitors of endogenous miRNAs. One such inhibitor is miravirsen, which is currently in Phase II clinical trials for the treatment of hepatitis C as discussed above.

Newer companies have focused their efforts on bringing to market specific miRNA therapeutics for particular indications rather than developing broader-based nucleic acid therapies. Three such companies (miRagen Therapeutics, Regulus Therapeutics, and Mirna Therapeutics), all of which were founded in 2007, have identified various miRNAs that play a role in different disease states and are advancing drug candidates that regulate these miRNAs. In June of 2010, miRagen Therapeutics partnered with Santaris Pharma to design and commercialize miRNA-targeted oligonucleotides utilizing Santaris Pharma's LNA technology for the treatment of cardiovascular disorders. Recently, miRagen Therapeutics announced a partnership with Les Laboratoires Servier, a European pharmaceutical company, to develop 3 miRNA-based therapeutics for the treatment of cardiovascular disorders. Regulus Therapeutics, a joint venture of Alnylam Pharmaceuticals and Isis Pharmaceuticals based in La Jolla, California, is also developing several miRNA therapeutics including oligonucleotide inhibitors of miR-21 for the treatment of fibrotic diseases and cancers, anti-miR-122 for the treatment of hepatitis C, anti-miR–155 for the treatment of inflammatory diseases, and anti-miR–33 for the treatment of metabolic disorders. In 2008, Regulus Therapeutics formed a strategic, worldwide alliance with GlaxoSmithKline to develop miRNA-targeted therapeutics for inflammatory disorders, such as rheumatoid arthritis and inflammatory bowel disease. In 2010, Regulus and GlaxoSmithKline expanded their relationship by entering into a collaboration to develop and commercialize miR-122–targeting drugs for the treatment of various diseases, including hepatitis C viral infection. In addition, Regulus has partnered with Sanofi-Aventis to research and develop miRNA therapeutics for fibrosis. The focus of Mirna Therapeutics, a company formed to accelerate the drug discovery efforts of RNA-based diagnostic company, Asuragen, Inc, is “miRNA replacement therapy” for the treatment of solid cancers. The company has identified tumor suppressor miRNAs, such as miR-34 and let-7, which are downregulated in various types of cancers. The goal of the miRNA replacement therapy strategy is to restore the function of these miRNAs through the use of oligonucleotide mimics. Mirna Therapeutics anticipates the filing of its first investigational new drug application on its tumor suppressor mimics in late 2012 (Table).

MiRNA-based diagnostics also feature prominently in the commercialization of miRNA-related technologies. In fact, Israel-based Rosetta Genomics was one of the first companies to emerge on the scene in 2000 that capitalized on miRNA discoveries. Rosetta Genomics currently offers four diagnostic tests for characterizing primary tumors from various types of cancer, including lung cancer and mesothelioma, by measuring miRNA expression. Additional tests are in development for renal cell carcinoma and bladder cancer. Asuragen, Inc, founded in 2006, was formed from part of Ambion's molecular biology and diagnostics divisions. Its current focus is miRNA-based diagnostics for cancer (eg, leukemia) and genetic disorders (eg, Fragile X syndrome). It is likely that additional miRNA-based diagnostic companies will become more prevalent in the years to come. Diagnostic tests often have fewer hurdles to obtain regulatory approval as compared with new drugs, and the relatively recent discovery that miRNAs are detectable in the blood provides the basis for minimally invasive methods for detecting and monitoring disease conditions and efficacy of therapeutic regimens (Table). Previous SectionNext Section Concluding Remarks

There are several significant advantages to miRNAs for becoming a new class of drug targets. Their small size and known and conserved sequence make them attractive candidates from a development standpoint. Additionally, many genetic or oligonucleotide-based gain- and-loss-of function studies have shown very pronounced phenotypes in rodents and even large animal models, whereas miRNA manipulation under baseline conditions oftentimes does not exert overt effects. Furthermore, the direct downstream targets of a single miRNA are commonly related genes that function in a comparable cellular process or signaling cascade. This implies that targeting of a single miRNA probably will result in a dramatic effect due to the combinatorial effect of gene expression changes in all these related downstream targets. The impact of targeting a miRNA sequence is further strengthened by the fact that the genome often contains multiple copies of the same or closely related miRNA sequences, such that targeting of the miRNA sequence will become even more influential. Lessons learned from antisense technologies leapfrogged us into very potent oligonucleotide chemistries that can be applied to target miRNAs, known as anti-miRs, with remarkable affinity and specificity and have suitable drug-like qualities such as stability and pharmacokinetics.

Although there are many reasons to be excited about miRNAs as a new class of drug targets, some aspects of miRNA and anti-miR biology are still relatively unknown and should be taken into account when moving forward. Although a miRNA might target related genes, the fact that a single miRNA can target many genes and the genes that are targeted are cell type–dependent gives a whole new meaning to “off-target effects” compared with a classic drug. Additionally, there are a lot of unknowns surrounding the mode of action of the different anti-miRs. Current data suggest that specific chemistries might affect cellular uptake and differ in degree of blocking the function of a miRNA. Their long-term effect (up to weeks or even months) indicates their stability, but also suggests the cell has a reservoir of anti-miR, which during the course of time, is slowly released into the cytoplasm to bind to or scavenge the newly formed copies of a miRNA. However, the precise mechanism is still unclear, and more in-depth biochemistry will be required to gain more insight.

Despite the unknowns, the interest surrounding miRNAs as novel therapeutic entities is tremendous and the anticipated success of these early forerunners probably will spur the search for additional miRNA therapeutic targets, innovation in the areas of miRNA inhibitors and mimics, and will advance the search for techniques for efficient in vivo delivery of these miRNA therapeutics. We hope that while we are eagerly awaiting more efficacy data in human subjects, the next few years promise to provide many more insights into miRNA and anti-miR biology and will even further strengthen the enthusiasm for this new class of drugs. Previous SectionNext Section Disclosures

E.v.R. is an employee of miRagen Therapeutics, Inc; A.L.P. is a patent agent at Cooley LLP; and A.A.L. is an employee of Santaris Pharma. Previous SectionNext Section Acknowledgments

We gratefully acknowledge Jose Cabrera for graphics. Previous SectionNext Section Footnotes

In November 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15 days.

This Review is in a thematic series on MicroRNA in the Cardiovascular System, which includes the following articles:

Introduction to the Series on MicroRNAs in the Cardiovascular System [Circ Res. 2012;110:481–482]

Circulating MicroRNAs: Novel Biomarkers and Extracellular Communicators in Cardiovascular Disease? [Circ Res. 2012;110:483–495]

Developing MicroRNA Therapeutics

MicroRNAs in Vascular and Metabolic Disease

Differential Expression of MicroRNAs in Different Disease States

MicroRNAs in Cardiovascular Development

Methods for MicroRNA Target Determination & Target Regulation

Non-standard Abbreviations and Acronyms

AAV
    adeno-associated viruses
HCV
    hepatitis C virus
IND
    investigational new drug
LNA
    locked nucleic acid
miRs/miRNAs
    microRNAs
PD
    pharmacodynamic
PK
    pharmacokinetic

Received September 29, 2011.
Revision received November 29, 2011.
Accepted December 2, 2011.

© 2012 American Heart Association, Inc.

Previous Section

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AbstractDiscovered in 1993, micoRNAs (miRNAs) are now recognized as one of the major regulatory gene families in eukaryotes. To date, 24521 microRNAs have been discovered and there are certainly more to come. It was primarily acknowledged that miRNAs result in gene expression repression at both the level of mRNA stability by conducting mRNA degradation and the level of translation (at initiation and after initiation) by inhibiting protein translation or degrading the polypeptides through binding complementarily to 3′UTR of the target mRNAs. Nevertheless, some studies revealed that miRNAs have the capability of activating gene expression directly or indirectly in respond to different cell types and conditions and in the presence of distinct cofactors. This reversibility in their posttranslational gene regulatory natures enables the bearing cells to rapidly response to different cell conditions and consequently block unnecessary energy wastage or maintain the cell state. This paper provides an overview of the current understandings of the miRNA characteristics including their genes and biogenesis, as well as their mediated downregulation. We also review up-to-date knowledge of miRNA-mediated gene upregulation through highlighting some notable examples and discuss the emerging concepts of their associations with other posttranscriptional gene regulation processes.1. IntroductionMicroRNAs or miRNAs have been a subject of significant research work since the discovery of lin-4 in the early 1990s, underscoring the importance of posttranscriptional gene regulation in cis and trans 1. miRNAs are a subset of endogenously-initiated, single-stranded noncoding RNA guide molecules, traceable in organisms as diverse as animals, plants, green algae, and viruses, that regulate gene expression via association with effector complexes (called “micro-ribonucleoprotein” or “miRNP”) and sequence-specific recognition of target sites (also called “cognate mRNAs”), which can dictate the functional outcome [2–5]. They represent one of the most exciting areas of modern medical sciences as they possess unique ability to modulate an immense and complex regulatory network of gene expression 6, 7 in a broad spectrum of developmental and cellular processes including tissue development 8, 9, cell proliferation 10, 11, cell division 12, 13, cell differentiation 14, neuronal asymmetry 15, metabolism 16, stem cell properties 17, apoptosis 18, protein secretion 19, and viral infection 20. It is becoming clear that they have a big impact on shaping transcriptomes and proteomes of eukaryotes 21. Aberration or perturbation in their expression levels has significant correlation with serious clinical consequences, including disease of divergent origin and malignancy 22, 23. Certainly, disease-associated miRNAs represent a substantial class of targets for the miRNA-based novel therapeutic or diagnostic/prognostic biomarkers 24, 25. By mid-2013, it was known that the human genome encodes over 2000 different miRNAs that scattered on all human chromosomes except the Y chromosome (http://microrna.sanger.ac.uk; Release 20: June 2013). Based on this estimation, about 3-4% of human genes encode miRNA. In postgenomic era, the accepted notion is that a single miRNA species can regulate hundreds of targets, even if only to a mild degree, but, conversely, several miRNAs can bind to their target mRNAs and cooperatively provide fine-tuning of a single mRNA target expression 26. Although a steeply growing computational analysis has identified a range of potential targets for miRNAs, to date, only a small number of them have been validated by experimental approaches 27, 28.Until recently, miRNAs had been believed to have a pervasive effect on the gene expression modulation solely by negative regulation of target mRNA 29; however, the increasing published observations indicate that miRNAs oscillate between repression and stimulation in response to specific cellular conditions, sequences, and cofactors 30. These exciting findings, however, have made it even more difficult to explain how miRNAs regulate gene expression. While the past decades have witnessed a veritable exploration focuses on defining the regulatory function of miRNAs, fewer directed towards exact mechanistic turnovers under specific cellular conditions and many of these assertions directly contradict one or another of the publications. Hence, undoubtedly, there are still enigmas to be uncovered regarding mechanistic details of miRNA-mediated regulation.In order to exploit practical implications of miRNAs as biomarkers, novel drug targets, and therapeutic tools for diagnosis, prognosis, and treatment of malignancies and disease, it is necessary to have in-depth understanding of miRNA turnover, in particular, the molecular mechanisms by which miRNAs elicit distinct gene expression outcomes in different cell cycle stages and conditions. Toward this end, our review illuminate and explain the controversies generated by recent assertions as well as providing a comparison of regulatory switches that mediate between downregulation and upregulation, directed by miRNAs. This review, therefore, aims to summarize new findings in miRNA-mediated gene regulation mechanisms, which may be affected by different cellular conditions and specific transcripts and proteins.2. miRNA Genes and BiogenesisCentral to studying miRNA-mediated gene modulation is the clear understanding of their gene structure and biogenesis, which have been described in several reviews [31–33]. miRNA genes are distributed nonrandomly in human genome, and nearly half of them are found as tandem arrays within clusters, sharing the same promoter, which may indicate gene duplications 15, 34. It has been shown that miRNA genes frequently coincide with tumor susceptibility loci 35 and are located within known sites of retroviral integration 36 and deleted or amplified genomic region or break points 37, as well as inside or near homeobox (HOX) cluster 38.By combining up-to-date genome assemblies and expressed sequence tag (EST) database, it was proved that miRNA promoters can be divided into two broad categories: intergenic and intragenic 39, 40. Intergenic miRNAs are located between genes and their transcriptions are independent of coding genes since they are transcribed mostly by RNApol III. Therefore, they have been reported to be more evolutionary conserved 41, 42. However, intragenic miRNAs are embedded within exon or introns of protein-coding genes, and thus, are coexpressed in the same orientation of their host genes by RNApol II 43. Additionally, a small percentage of miRNAs are found interspersed among repetitive elements that are transcribed by RNApol III and, subsequently, processed in the same way 44.Experimental evidence revealed that sequential processing of miRNAs occurs first in the nucleus and then in the cytoplasm. The biogenesis of miRNAs starts with RNApol II or III dependent transcription of a miRNA gene locus generating a long primary RNA (pri-miRNA). Pri-miRNAs are 5′-7-methylguanosine capped, spliced and 3′-polyadenylated and have a coding capacity for one or more mature miRNAs 2, 4, 45. They can reach several kilobases in length 46. Pri-miRNAs are processed into smaller, hairpin-like miRNA precursors called pre-miRNAs 47, 48. The cleavage of pri-miRNAs proceeds contemporaneously with the transcription of the genes or ncRNAs 49. After nuclear processing, Pre-miRNAs are transported by Exportin-5 to the cytoplasm in a Ran guanosine triphosphate-dependent manner, recognizing characteristic hairpin stem and 3′ protruding overhang in the pre-miRNA 50, 51. Further cytoplasmic processing performs a second cleavage of the hairpin structure and defines the 5′ end of the mature miRNAs. This releases a 19–24 nucleotide double-stranded RNA where miRNA is the antisense or guide strand and miRNA is the sense or passage strand 52. Once cleavage is occurred double-stranded miRNA (dsmiRNA) is released and integrated into the appropriate effector complex. Afterward, the duplex is unwound and the miRNA strand becomes degraded leaving one fully mature miRNA strand and finally microRNA containing ribonucleoprotein (miRNP) is configured 53, 54.3. miRNA-Mediated DownregulationmiRNAs are indeed able to reduce gene expression by multiple pathways and modes 55. Striking observations suggested that miRNAs do not function as naked RNAs, instead, they function in the form of effector complexes, which are known as miRNP, miRgonaute, or miRISC, along with Argonaute, the most important constituent of all miRNPs 56. A key specificity determinant for miRNA target recognition is based on Watson-Crick pairing of 5′-proximal “seed” region (nucleotide 2 to 8) in the miRNA to the seed match site in the target mRNA, which positioned mostly in 3′UTR 57. Nevertheless, it is also claimed that a small subset of miRNAs modulate expression by specifically targeting the 5′UTR and/or coding region of some mRNAs 58, 59.The biological outcome of miRNA-mRNA interaction can be altered by several factors contributing to the binding strength and repressive effect of a potential target site 60. The most mentioned factor is perfect base pairing between the miRNA seed region and target site 61. Other factors include the number of target sites for the same miRNA and relative position of them, site accessibility, sequences flanking miRNA target site, and their context, and RNA secondary structure can influence the consequences of hybridization [62–64]. The interaction between the effector complex carrying miRNA and target mRNA can have several consequences. To date, several and in some cases contrary models have been proposed, although they are not mutually exclusive. Briefly gene silencing mediated by miRNA can be obtained at three stages that include pretranslational, cotranslational, and posttranslational steps and can exert direct and indirect effects on translation machinery (Figure 1).970607.fig.001Figure 1: Potential mechanisms of miRNA-mediated downregulation. Ribonucleoproteins (miRNPs) target mRNAs through sequence complementation. The association between miRNPs and mRNAs can have one or several consequences. miRNP-mediated downregulation may have direct and indirect mechanisms and occur before translation is triggered or after translation initiation. (a–c) Translation initiation mechanisms. miRNP interferes with early steps of translation before elongation. (a) miRNPs recruit several factors and enzymes for mRNA cleavage and degradation including decapping enzymes, deadenylase, 3′ and 5′ exonucleases, and endonucleases. (b, c) Argonaute protein competes with cap binding proteins (CBPs) and elf4E for binding to cap structure and inhibits translation initiation by interfering with mRNA circularization and formation of closed-loop achieved through cap structure interaction with CBPs and elf4E/G required for translation initiation. (d–h) Postinitiation mechanisms. miRNPs repress translation elongation and termination or involve in protein degradation and sequestration. (d, e) miRNP interferes with ribosome subunit by inhibiting its joining or promoting its dissociation. (f, g) miRNP obstructs translation elongation by competing with elongation factors or cotranslationally recruiting protein decay factors such as exosomes. (h) Target mRNA is sequestered from translation machinery and stored or sometimes is degraded by enzymes. However, alternatively, translationally inhibited mRNA along with associated proteins could be sequestered at the same bodies. For explanations in support of illustrations, refer to the main text.In the case of pretranslational step, a number of reports suggest that in certain organisms, such as mammalians, a specialized RNA-induced transcriptional silencing (RITS), which contain specialized nuclear Argonaute (Ago) protein, may results in gene silencing through chromatin remodeling 65, 66.Beside transcriptional effects, miRNAs can repress translation initiation by multiple mechanisms. mRNA degradation can be inaugurated by deadenylation from 3′ end and/or decapping from 5′ end by enzymes such as DCP1/2. Missing poly(A) tail and cap structure expose the remained RNA for the action of degradation of exonucleolytic Xrn1p enzyme. In addition, truncated mRNA, missing poly(A), can be subjected to the 3′ to 5′ degradation of cytoplasmic exonucleases [67–69]. Alternatively, sequence-specific endonucleolytic mRNA cleavage by polysomal ribonuclease 1 (PMR1) may occur in parallel 70 (Figure 1(a)). Recruited Argonaute protein interacts with several initiation factors 71. First and foremost, Ago competes with eIF4E, an eukaryotic translation initiation factor involved in directing ribosomes to the cap structure of mRNAs, for binding to cap structure 72, 73 (Figure 1(b)). Other translation initiation factors include: PABP (the protein associated with poly(A) tail at 3′-end of mRNA), eIF4G which is strongly associated with eIF4E, the RNA helicase that unwinds mRNA secondary structure, eIF4A which is required for the binding of mRNA to 40S ribosomal subunits, and eIF3 and eIFa which are associated with the ribosomal small subunits 74. It has been also reported that translation inhibition could happen when target sites for miRNA are located in 5′UTR or even coding sequences 75.Another possible mechanism of translation initiation blocking is the Ago interference with the formation of closed-loop mRNA, achieved through interaction between cytoplasmic poly(A) binding protein and cytoplasmic cap binding protein, by an ill-defined mechanism that may include deadenylation 73 (Figure 1(c)).In postinitiation steps, eIF6 is recruited by Argonaute, which prevents the large ribosomal subunit from joining to miRNA-targeted mRNA 76, 77 (Figure 1(d)). In addition, miRNPs interfere with elongation factors and lead to ribosome subunit dissociation and/or premature termination 71 (Figures 1(e) and 1(f)). Along with translation repression, cotranslational protein degradation may occur. In this model, the nascent polypeptide is degraded by protease activities 78 (Figure 1(g)).Recently, it was indicated that processing cytoplasmic foci, mostly known as “P or GW bodies,” have a central role in mRNA degradation and translation inhibition. Target mRNAs associated with P body components can either be degraded or be sequestered to return to translation. Therefore, the rates of expression and degradation of mRNAs are influenced by a dynamic equilibrium between polysomes and miRNPs that are found in P bodies. Moreover, some mRNA-specific regulatory factors, including miRNAs, appear to repress translation and promote decay by recruiting P body components to specific mRNAs since they contain enzymes involved in mRNA turnover, such as Argonaute and GW182. Collectively, these findings have been converged to propose a possible model in which targeted mRNA is sequestered from the translational machinery and underwent both degradations or is stored for subsequent processes [79–81] (Figure 1(h)).4. miRNA-Mediated UpregulationIn contrast to general assumption that miRNA-mediated downregulation is a one-way process and leads to decreased mRNA stability and/or translational inhibition, Vasudevan and Steitz reported for the first time that the miRNA-mediated downregulation is reversible 82. Likewise, there is evidence suggesting that some miRNAs could upregulate gene expression in specific cell types and conditions with distinct transcripts and proteins.In miRNA-mediated upregulation, miRNPs act in trans in promoting their target mRNAs’ expression similar to miRNA-mediated downregulation. The mRNA expression could be activated by the direct action of miRNPs and/or could be indirectly relieved from miRNA-mediated repression by abrogating the action of repressive miRNPs 82.In addition, a single miRNA can act both in up- and downregulation, and likewise a single specific gene could encounter both regulation directions based on the specific conditions and factors. For instance, miR-145 mediates myocardin gene upregulation during muscle differentiation. However, ROCK1 expression downregulation is a consequence of miR-145 targeting in osteosarcoma 83, 84. As another example, KLF-4 is upregulated by miR-206 in confluent and nontumor cells, while it is downregulated by miR-344 in proliferating and normal cells 85. Therefore, these evidences confirm that gene expression upregulation is specific to cell type, cell condition, and present factors and elements.4.1. Direct Mechanisms of miRNA-Mediated UpregulationComplexity of gene regulation by miRNAs is further expanded by observations that miRNAs can positively mediate gene expression. These reports indicate that posttranscriptional upregulation by microRNAs is selective, specific to the RNA sequence context, and associated with miRNP factors and other RNA binding proteins 86. Similar to miRNA-mediated downregulation, translation upregulation by miRNAs has been observed to range from fine-tuning effects to significant alterations in gene expression levels. These studies uncover remarkable capability of some miRNAs and their associated miRNPs in gene expression control and highlight the importance of regulation in directing appropriate microRNP responses 87.4.1.1. miRNA-Mediated Upregulation in Response to Cellular State and/or in the Presence of Specific FactorsThe miRNP and target mRNA base pairing could have several and in some cases converse functional outcomes. Several studies provided evidence that miRNPs have the potential of activating gene expression in the presence and/or absence of specific factors and through distinct cell conditions. They revealed that cell cycle has the potential to determine miRNA-mediated gene regulation direction by promoting or inhibiting special mRNA expressions.One of the deeply studied factors is the effect of G0 state on miRNA-mediated gene regulation 30. Quiescence generally refers to G0 and G0-like states that run a specific gene expression programs in order to enter the G0 cells for extended period of time in a reversible manner. G0 state can be observed during natural phenomena such as differentiation, development, and growth to confluence or can be induced by the manipulation of in vitro cell culturing [88–91].Translational activation in substitute cell states, for instance, G0 and immature oocyte, provides a mean of gene expression to skew towards maintaining the state. Loss of these states leads to the reversion of miRNA-mediated gene expression activation 92.In drosophila, it was proved that both AGO1 and AGO2 are capable of mediating gene expression downregulation. Nevertheless, AGO2, but not AGO1, can be involved in gene expression activation when their targeted mRNA lacks poly(A) tail, representing the significance of the lenghth of poly(A) tail for the diverse roles of AGO2 93, 94.What is more, AGO2-RISC binds to eIF4E and is capable of forming “closed-loop” even in the absence of poly(A) tail and associated proteins and, hence, could activate translation directly 72. Furthermore, GW182, a crucial protein for miRNA-mediated downregulation, was reported to be downregulated in G0 state and immature oocyte. As a result, GW182 misses its interaction with AGO and leaves the capability for another protein named Fragile-X mental retardation protein 1 (FXR1) to be involved in miRNP complex. This eventually results in miRNP-mediated gene activation 95. Furthermore, biochemical experiments revealed that AGO2 is too small to contain GW182, whereas AGO2-FXR1-iso-a is a complex naturally found in nuclei. Therefore, lacking GW182 results in abrogation of expression downregulation, while FXR1 association with AGO2 leads to translational activation 82, 96.Nuclear events are often dictated the fate of mRNA expression; in line with this, miRNPs’ responses and remodeling for different cell states can comprise a nuclear phase for G0 97, 98. Slicer activity of Argonaute protein has been reported to be absent in immature oocyte and G0 state 89, 99. Thus, immature oocyte and G0 state cells recruit miRNAs to conduct their cleavage-independent regulatory roles (e.g., translational activation).Both cells at G0 state and immature oocytes have an intact nucleus 100, 101. Elevated numbers of activator miRNPs and FXR1-iso-a are naturally compartmentalized in the nucleus, which then act as selective activators of target mRNAs 102. Examples are KLF4 mRNA and miR-206 in quiescent cells and Myt1 and miR-16 in immature oocytes, which are illustrated in Figure 2 85, 100, 103, 104.970607.fig.002Figure 2: miRNA-mediated upregulation according to cell cycle phase. miR-334 and miR-27a bind to the 3′UTR of KLF4 and Myt1 mRNA in proliferating and cancerous cells and subsequently downregulate mRNA expression by both translational inhibition and mRNA degradation. miRNP-mediated repression requires factors including AGO, GW182, deadenylases, and nucleases. However, in G0 cells and immature oocytes, GW182 is downregulated and FXR1 is associated with AGO2 bearing miRNP and leads to gene expression upregulation. miR-206 is associated with KLF4 mRNA in G0 cells and xmiR-16 is associated with Myt1 mRNA in immature Xenopus oocyte. miR-206 and xmiR-16 activate KLF4 and Myt1 expression, respectively, by binding to their 3′UTRs and recruiting special factors such as FXR1, since AGO2 is associated with gene expression activation in these cells and lacks slicer activity.miRNAs usually interact with 3′UTR of target mRNAs leading to mRNA degradation and/or translational repression. In contrast, it was recently exposed that liver-specific miR-122 enhances hepatitis C virus (HCV) RNA levels via interacting with two natural binding sites in 5′ noncoding region of RNA 105. miR-122 expression was found to be specific to liver cells. It was reported that increasing levels of miR-122 are in accordance with differentiation of liver tissues in developing mouse embryos and contribute pointedly to HCV liver tropism 106. These tandem miRNA binding sites are located in the upstream of the internal ribosome entry sites (IRESs), where miRNA-binding interference is eliminated and translation gets the chance to initiate. In line with this, miR-122 interaction with a manipulated binding site in HCV RNA which is located in 3′UTR of a reported gene revealed an expression downregulation, indicating the location specificity of miR-122-mediated upregulation in HCV gene 105, 107. The mechanism of the miR-122-mediated stimulation of HCV gene expression is one of the rare cases and remains partially unsolved 108. However, the emerging experiments and mutational analysis validated that both miR-122 seed sequence binding and extra interactions are needed to act cooperatively in order to enhance viral RNA abundance 109. Since HCV genome, as a viral genome, does not have cap structure and thus misses the associated proteins at its 5′ end, it eventually requires alternative mechanism to promote translation by recruiting translational components, eventually leading to increased RNA stability by inhibiting exonucleases digestion 69. Indeed, it is proposed that miR-122 acts instead of cap structure in enhancing RNA expression by increasing its stability against Xrn1, accelerates the binding of ribosome, and exerts another Xrn1-independent role in stimulating HCV gene expression 110, 111. Components such as RISC which are brought to HCV genome by miRNP would function as a shield in protecting single-stranded 5′ end of HCV from cytosolic exonucleases activities 111. In addition, these proteins may provide a scaffold for binding of factors essential for RNA replication and translation 112. Figure 3 represents the different mechanisms of miR-122-mediated HCV gene upregulation.970607.fig.003Figure 3: Activation of hepatitis C virus (HCV) expression by miR-122, a liver-specific microRNA. The mature single-stranded miR-122 is processed from its double- stranded precursor and incorporated into functional miRNP, bearing AGO2. miRNPs target one or two tandem binding sites in the 5′ noncoding region (5′NCR) of HCV RNA. HCV appears to usurp the miRNP to increase its RNA accumulation by a number of mechanisms. (a) First, miR-122 complexes provide a scaffold for the binding of essential factors such polymerase for RNA replication. (b) In addition, miRNP complexes increase the association of 40s ribosome subunit, and thus, result in increased translation and protein yield. (c) They also form an unusual oligomeric complex in 5′ end of HCV RNA and result in HCV RNA stability enhancement by masking its single-stranded 5′end through hiding 5′NCR from 5′ cytoplasmic exonucleases and immune sensors. (d) Also, miR-122 binding was reported to lead to increased propagation and life cycle of virus by some ill-defined mechanisms.Another outstanding miRNA-mediated upregulation would be related to miRNAs targeting TOP mRNAs. TOP mRNAs commonly foster a 5′ terminal oligopyrimidine tract (5′TOP), which is a structural trademark comprising the core of the cis translational regulatory elements 113. Proteins of the translational machinery that are encoded by TOP mRNAs could be mentioned as ribosomal proteins 114, elongation factors eEF1A and eEF2 115, and poly(A) binding proteins 116. The expression and regulation of TOP mRNAs are not restricted to mammals, since they have been found in other vertebrates and even in drosophila and insects 117. 5′ TOP of these mRNAs render them translationally suppressed upon cell cycle arrest caused by amino acid starvation, contact inhibition, and differentiation termination, which eventually make them sensitive to cellular stress signals and amino acid status 117, 118.miR-10a was found to be highly expressed in kidney, muscle, lung, and liver of mice 119. miR-10a was reported to interact with noncanonical downstream of the TOP motif in 5′UTR of ribosomal proteins and enhance their translation in a rapamycin-mTOR sensitive manner by alleviating their TOP-mediated translational repression during amino acid starvation 113, 120. Consequently, miR-10a binding results in an elevation in global protein synthesis by means of enhancing the ribosomal protein yield and therefore affects the capability of cell transformation 120.Recent miRNA profiling and mutational studies revealed that miR-346 is produced from the second intron of glutamate receptor ionotropic delta 1 (GRID1) mostly in brain tissues and is capable of upregulating RIP140 (receptor-interacting protein 140) gene via binding to 5′UTR of the target RIP140 mRNA and accelerating its target mRNA interaction with polysomes 121, 122. RIP140 is a transcription coregulator and modulates many metabolism-related pathways by regulating nuclear receptors and transcription factors 123. Nevertheless, miR-346 does not require AGO2 for its activity; therefore, it possibly applies an AGO-independent pathway to control the protein yield of RIP140 without altering its mRNA levels 122.4.1.2. miRNA-Mediated Upregulation in Competing with mRNA Decay and Expression Repressive FactorsmiRNAs are able to compete with decay pathways such as AU-rich element- (ARE-) mediated decay pathway and other expression inhibitors. The ARE-mediated mRNA decay (AMD) regulates the concentration of a class of mRNAs that contains AU-rich sequences within their 3′UTRs. ARE-binding proteins (ABPs) recruit the cytoplasmic mRNA degradation machinery to the target mRNAs leading to their 3′-to-5′ degradation 124, 125. Tristetraprolin (TTP) protein family functions as a molecular link between ARE-containing mRNAs and the mRNA decay machinery through degradation enzymes recruitment 126. Noteworthy, it was indicated that some miRNA-mediated regulation pathways may have some interactions with ARE-mediated pathways, since they share common binding sites in mRNA 3′UTRs and have some common key players such as HuR, AGO2, CCR4, GW182, and decapping enzymes 127, 128. Therefore, microRNAs have the capability to abrogate AMD by preventing ABPs associations leading to increased mRNA stability. miR-4661 and miR-125b are examples that hinder TTP binding to the ARE and, hence, increase IL-10 and κB-Ras2 mRNA levels 129, 130.Furthermore, some specific miRNAs have been recently found to block repressive proteins from binding to their target sites and, therefore, lead to distinct mRNA expression upregulation. miR-328 expression was found to be elevated in blast crisis chronic myelogenous leukemia (CML-BC) via the MAPK pathway, causing differentiation, impairing leukemic blasts survival through acting as a sponge molecule, decoying away hnRNP E2, a repressive protein, from C/EBPα, and eventually leading to C/EBPα expression upregulation 131. Table 1 summarizes examples of miRNA-mediated upregulation.tab1Table 1: Examples of direct miRNA-mediated upregulation.4.2. Mechanisms of Derepression from miRNA-Mediated DownregulationDerepression or relief of repression is the consequence of disengaging miRNPs from the previously repressed mRNAs 132. This reversibility in miRNA-mediated gene regulation undoubtedly makes their functions more dynamic and brings the ability to be more receptive to specific cellular requirements.4.2.1. Relief of miRNA-Mediated Downregulation in Response to Cell StressesThe Derepression of target mRNA in response to cell stress and synaptic stimulation is frequently mentioned examples 133, 134. Cells recurrently run into stresses, including oxidative stress in cancer cells, especially in poorly angiogenic core of solid tumours 135, 136, nutrient deprivation 132, 137, 138, cardiac pressure overload 139, DNA damage, and oncogenic stress, resulting mostly from exposure to UV radiation [140–143] and salt imbalance 144.As mentioned above, AMD and miRNAs have some interactions, and thereby miRNA could mediate or prevent AMD pathway. In this regard, the CAT-1 mRNA derepression would be a notable example which is accompanied by the relief from cytoplasmic bodies and its employment to polysomes. CAT-1 expression was found to be regulated comprehensively at both transcriptional and posttranslational levels and could be upregulated in response to different cellular stresses, such as amino acid deprivation in order to maintain hepatocellular protein synthesis 145. Its upregulation requires HuR protein binding, an ARE-binding protein, to the 3′UTR of CAT-1 mRNA 146, 147. HuR shuffles between nucleus and cytoplasm and was found to play a vital role in different posttranscriptional pathways not only in stress responses, but also in cell proliferation, differentiation, tumorigenesis, and immune responses 148, 149. HuR binds to ARE sites of mRNAs in nucleus and chaperones them to the cytoplasm which then are relocalized in polysomes in response to stress. Moreover, it may also modulate translation or increase stability of target CAT-1 in some direct and indirect pathways 132.The clear mechanism that underlies HuR roles in posttranscriptional gene regulation remains poorly understood. However, the role of HuR in competing with other RNA binding proteins which function in promoting mRNA turnover is fairly confirmed 150, 151. Although HuR does not have significant impact on poly(A) shortening, it contributes to delays onset of RNA decay 152. Growing body of evidence supports that HuR has an RNA-stabilizing role in the ARE-directed mRNA decay in mammalian cells 153. In addition to the the main points, HuR or similar regulatory proteins such as DND1 can influence the miRNA machinery interaction with target mRNA via dissociating miRNP from CAT-1 mRNA and relocating them into stress granules (SGs) which then results in polysomes recruitment 154 (Figure 4).970607.fig.004Figure 4: Relief of miRNA-mediated CAT-1 repression according to cell response to radiation. High AMP/ADP in cell is accompanied by the cell exposure to radiation. Consequently, AMP-kinase activity is enhanced, which then leads to HuR (an RNA binding protein that interacts with ARE in 3′UTR of the mRNA) dephosphorylation, releasing from nucleus to cytoplasm and entering to processing bodies (PBs). In P bodies, HuR dissociates miRNP from CAT-1 mRNA and mobilizes it to stress granules (SGs). Within SGs, CAT-1 expression is elevated via recruiting polysomes and relieved from miR-122-mediated downregulation by HuR replacement of miRNP in PBs. Moreover, HuR binds to ARE sites of mRNAs in nucleus and chaperones them to the cytoplasm which then are relocalized in polysomes and contributes in delays onset of RNA decay by competing with AMD effectors.4.2.2. Relief of miRNA-Mediated Downregulation by Sponge MoleculesSome sponge molecules such as lncRNAs and AGO10 were reported to decoy away miRNAs from their target mRNAs and lead to target mRNA derepression. One type of these lncRNAs is the so-called “miRNA sponges.” They bind to specific miRNAs in their seed site and prevent miRNP binding to their target mRNAs, or they compete with miRNAs for binding to the specific mRNAs 155, 156. BACE1-AS is one of these examples located in the antisense strand of BACE1 (beta-secretase 1) and competes with miR-485-5p for binding to the exon 6 of BACE1 mRNA 157. Hence, BACE1-AS expression is associated with BACE1 mRNA stability and increases the protein yield of BACE1 158. Linc-ROR is another miRNA sponge expressed in the pluripotent stem cells and increases reprogramming efficiency [159–161]. Core pluripotency transcription factors like OCT4, SOX2, and NANOG mRNAs are the miR-145 target RNAs. However, in the presence of Linc-ROR, miR-145 is trapped and consequently maintains the self-renewal state of stem cell due to the increasing stability of these three transcription factor’s mRNAs and subsequently the increasing level of their proteins 162. These transcription factors result in embryonic stem cells-specific gene expression, which prevents stem cell differentiation 163.In Arabidopsis, a decoy Argonaute protein, called AGO10, recruits miR-166/165 by recognizing its distinctive secondary structure and decoys it away from AGO1 and consequently leads to target mRNA, homeodomain leucine zipper transcription factors, and expression upregulation, which maintains undifferentiated cells of the shoot apical meristem 164. Table 2 summarizes some examples of derepression of miRNA-mediated downregulation.tab2Table 2: Examples of relief from miRNA-mediated downregulation.5. Concluding Remarks and Future ChallengesAccumulating reports had brought about the estimation that over 3 percent of human genes in human genome are subjected to miRNA-mediated gene regulation in different cell processes, suggesting that the expression of this important noncoding RNAs are associated with an array of pathological outcomes and human disease. MicroRNAs have several characteristics that make them an intriguing candidate for cell protection. As advances in the field of miRNA-mediated gene regulation are made, it is apparent that miRNAs are a crucial component of gene regulatory networks. While, most studies dedicated a downregulation role for miRNA-mediated post transcriptional gene regulation, recently increasing publications reveal an adverse role for miRNAs as activators of gene expression. miRNPs enhance protein yield of target mRNA by mRNA degradation and/or translational repression. Nevertheless, miRNA-mediated upregulation of target mRNA can be elucidated by both enhancing mRNA stability and translational activation via direct activation and/or indirect derepression. Despite the rapid progress and a wealth of information about miRNP-mediated upregulation, the general molecular mechanism of switching from repression to activation has only been delineated in a few distinct conditions and tissues.However, the aforementioned cell responses resulting in gene expression upregulation could not be generalized to all miRNAs or tissues. For example, miR-34a targets AXIN2 through binding to its 5′UTR and downregulates its expression. Also, several miRNAs have been found to suppress gene expression even in G0 state cells and the cells which endure any type of stresses 58. Perhaps the most puzzling and interesting aspect of posttranscriptional gene regulation (PTG) by miRNA is that PTG is not carried out only by miRNAs, since numerous well-documented examples of PTG mediated by molecules and processes other than miRNAs are present. Surprisingly, the cooperation between the cellular environment, mRNA context, interplay between other PTGs, and miRNA-mediated gene regulation dictates the fate of the target mRNA. Hence, PTG is involved in several distinct and most likely overlapping mechanisms. Given this complexity, it will be important to define which mechanism is exerted for regulation of a special subset of mRNAs. Nonetheless, classifying mRNAs in accordance with their regulational subtypes would be difficult as one PTG mechanism is capable of mediating several gene expressions in the meantime, and vice versa a single mRNA can be a subject of different PTG processes. More challenging will be the identification and characterization of cell type and condition in order to define a unique mechanism for a unique gene in response to each different factor.In conclusion, this exiting playground of miRNA-mediated gene regulation still holds secrets, and discrepancies in their studies invite future cellular, molecular, and biochemical studies, as well as computational approaches, to uncover their molecular mechanism, in order to provide a new dimension to the understanding, prevention, and treatment of human diseases.Conflict of InterestsThe authors declare that there is no conflict of interests regarding the publication of this paper.References

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Research ArticleUpregulated PD-1 Expression Is Associated with the Development of Systemic Lupus Erythematosus, but Not the PD-1.1 Allele of the PDCD1 GeneQingqing Jiao,1 Cuiping Liu,2 Ziliang Yang,1 Qiang Ding,1 Miaomiao Wang,1 Min Li,1 Tingting Zhu,1 Hua Qian,3 Wei Li,3 Na Tu,1 Fumin Fang,1 Licai Ye,1 Zuotao Zhao,4 and Qihong Qian11Department of Dermatology, The First Affiliated Hospital of Soochow University, 188 Shizi Road, Suzhou 215006, China 2Clinical Immunology Laboratory, First Affiliated Hospital, Soochow University, 708 Renmin Road, Suzhou 215007, China 3Department of Dermatology, Soochow University Affiliated Children’s Hospital, 303 Jingde Road, Suzhou 215003, China 4Department of Dermatology, First Hospital, Peking University, 8 Xishenku Road, Beijing 100034, ChinaReceived 21 January 2014; Revised 7 March 2014; Accepted 7 March 2014; Published 17 April 2014Academic Editor: Ji-Fu Wei Copyright © 2014 Qingqing Jiao et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.AbstractSystemic lupus erythematosus (SLE) is a multisystem autoimmune disease with complicated genetic inheritance. Programmed death 1 (PD-1), a negative T cell regulator to maintain peripheral tolerance, induces negative signals to T cells during interaction with its ligands and is therefore a candidate gene in the development of SLE. In order to examine whether expression levels of PD-1 contribute to the pathogenesis of SLE, 30 patients with SLE and 30 controls were recruited and their PD-1 expression levels in peripheral blood mononuclear cells (PBMCs) were measured via flow cytometry and quantitative real-time-reverse transcription polymerase chain reaction (RT-PCR). Also, whether PD-1 expression levels are associated with the variant of the SNP rs36084323 and the SLE Disease Activity Index (SLEDAI) was studied in this work. The PD-1 expression levels of SLE patients were significantly increased compared with those of the healthy controls. The upregulated PD-1 expression levels in SLE patients were greatly associated with SLEDAI scores. No significant difference was found between PD-1 expression levels and SNP rs36084323. The results suggest that increased expression of PD-1 may correlate with the pathogenesis of SLE, upregulated PD-1 expression may be a biomarker for SLE diagnosis, and PD-1 inhibitor may be useful to SLE treatment.1. IntroductionSystemic lupus erythematosus (SLE) is a progressive autoimmune disease with a wide range of immunological abnormalities 1. It is characterized by an immune response against nucleus components, but the etiopathology is not clearly understood yet. Multiple genetic factors relating to SLE have been identified [2–4], which suggest that both immune disorder and genetic factors may play important roles during the SLE process.The protein programmed death 1 (PD-1), a negative costimulatory molecule, belongs to the CD28 superfamily and is expressed on the surface of activated human CD4+ and CD8+ T cells, B cells, natural killer (NK) cells, activated monocytes, myeloid cells, and CD4−CD8−T cells from the thymus 5, 6. As an immune inhibitory receptor, PD-1 interacts with its ligands, PD-L1 and PD-L2, which can suppress lymphocyte activation and cytokine production 7. Current concepts regarding PD-1/PD-L pathway are categorized into immune dysfunction associated with SLE in humans 8. In addition, it was reported that PD-1 gene polymorphisms were involved in the development of autoimmune diseases, such as SLE, rheumatoid arthritis, and Graves’ disease 9. However, until now, only few studies have reported a possible link between PD-1 gene polymorphisms and SLE [8, 10–12]. Due to the existence of racial and regional differences in SNPs in PD-1, it is very important to study the relevance of PD-1 to SLE susceptibility in the Chinese Han population, which could also bring more evidence to the connections among alleles and disease in SLE.Among single nucleotide polymorphisms (SNPs) in the human PD-1 gene (PDCD1) region, PD-1.1 G/A (rs36084323) was reported to have connection with autoimmune diseases 13. Previous studies suggested that nonfunctional SNPs could affect gene function through haplotype tagging 14. In this work, PD-1.1 G/A (rs36084323), a nonfunctional SNPs in PDCD1, was studied in a Chinese Han population, aiming to explore whether PD-1 expression was related to the variant of the SNP PD-1.1 G/A (rs36084323) and SLE Disease Activity Index (SLEDAI).2. Materials and Methods2.1. Patients and ControlsThis study was performed on 30 cases of Chinese Han population fulfilling the revised criteria for SLE from American College of Rheumatology 1997 15, 16 (27 women; 3 men; age, ) and compared with 30 age-matched controls (27 women; 3 men; age, ). SLEDAI 17 was taken for each patient at the time of recruitment. The protocol of this study was approved by the ethics committee of the institution involved, and informed consents for genetic studies were obtained from all subjects.2.2. Flow Cytometry AnalysisFlow cytometry was performed using 50 μL of EDTA-treated peripheral blood incubated for 30 min at 4°C with fluorochrome-labeled monoclonal antibodies (mAbs): anti-CD4-FITC (Beckman), anti-CD8-FITC (Beckman), anti-CD56-FITC (Beckman), and anti-PD-1-PE (BioLegend). Erythrocyte lysis and cell fixation were carried out using OptiLyse C Lysing Solution (Beckman). Treated blood samples passed through the Coulter Epics XL Flow cytometer (Beckman), and the relevant data were acquired and accordingly examined. Data analysis was accomplished by FlowJo software (Tree Star, Ashland).2.3. Real-Time RT-PCR AnalysisPBMCs were separated from fresh blood of patient and control groups in order to analyze the mRNA of the PD-1. Total cellular RNA was isolated by Trizol (Invitrogen, USA). After quantification, 1 μg of total cellular RNA was used to conduct reverse transcription with Promega RT kit (A3800) and an oligo (dT) primer. PCR was completed in a 50 μg reaction system containing 200 nM PD-1 primers (Table 1), 120 nM TaqMan probe, and premix Ex Taq (Takara, Dalian, China). Samples were amplified in the Applied Biosystems 7900 HT Fast real-time PCR System (CA, USA) for 40 cycles under the following conditions: denaturation for 10 s at 95°C, anneal and extension for 40 s at 60°C. The expression level of the GAPDH was evaluated as an internal control.tab1Table 1: Primers of SNP PD-1.1 used for sequencing.2.4. GenotypingBlood DNA was extracted using Flexi Gene DNA kits (QIAGEN, Germany) according to the manufacturer’s instructions, and the DNA samples were then stored at −20°C. DNA fragments spanning PD-1.1 G/A (rs36084323) were amplified by polymerase chain reaction (PCR), and the products were gel-purified and sequenced. Then the sequencing data were applied for Genotyping of SNP (rs36084323).2.5. StatisticsThe data of PD-1 mRNA expression levels in PBMCs from all subjects were compared by the Mann-Whitney -test. The genotype frequency of SNP rs36084323 was tested for Hardy-Weinberg equilibrium separately for SLE patients group, while the values > 0.05 for all subjects in control group. Genotypes were compared using the Mann-Whitney -test, and the relation between PD-1 mRNA expression level and the SLEDAI score was examined by Spearman’s correlation coefficient rank test. All analyses were processed using SPSS16.0. Data were presented as . value < 0.05 (two-tailed) was considered as statistically significant.3. Results3.1. PD-1 Level Is Upregulated in PBMCs from SLE Patients Compared with ControlsInitially PD-1 protein and mRNA expression levels in all PBMCs samples were examined by flow cytometry and real-time RT-PCR. Flow cytometry analysis results demonstrated that the mean fluorescence intensity (MFI) of PD-1 was higher on CD4+ T cells, CD8+ T cells, and CD56+ T cells from PB samples of SLE patients compared to those of controls (Figures 1(a) and 1(b)). In addition, it is shown that the mean PD-1 mRNA expression levels increased in SLE patients’ samples compared with controls’ (Figure 1(c)).950903.fig.001Figure 1: Increased basal programmed death 1 (PD-1) expression in PBMCs from SLE patients. (a) Representative flow cytometry analysis of PD-1 expression on CD4+, CD8+, and CD56+ T cells in SLE patients and normal healthy controls (NC); (b) upregulated expression of PD-1 on CD4+, CD8+, and CD56+ T cells from SLE patients, as compared with those from NC; (c) mRNA expression of PD-1 in PBMCs from SLE patients. Horizontal bars indicate the .3.2. SLEDAI Is Significantly Related to the Upregulated PD-1 ExpressionIn order to determine whether upregulated PD-1 expression is related to SLEDAI, correlation analysis was carried out. The results have shown that SLEDAI scores were significantly related to upregulated PD-1 expression on CD4+ T cells, CD8+ T cells, CD56+ T cells, and increased PD-1 mRNA expression levels in PBMCs from PB samples of SLE patients (Figure 2), which indicates that upregulated PD-1 expression may be involved in the pathogenesis of SLE.fig2Figure 2: Correlation of upregulated PD-1 expression levels with SLEDAI in PBMCs. The association of SLEDAI with upregulated PD-1 expression on CD4+ T cell (a), CD8+ T cell (b), CD56+ T cell (c), and mRNA expression of PD-1 in PBMCs (d).3.3. The Variant of the SNP rs36084323 Is Not Related to Upregulated PD-1 ExpressionTo explore whether the variant of SNP rs36084323 was related to the upregulated PD-1 expression or not, the variant of SNP rs36084323 was genotyped. Results indicate that there was no connection between the variant of SNP rs36084323 and upregulated PD-1 expression (Figure 3).fig3Figure 3: Analysis of PD-1 expression levels in SLE patients with different genotypes of rs36084323. Increased levels of PD-1 levels in PBMC of SLE () patients, as compared with those from NC (). The patients with GG genotype () exhibit higher PD-1 expression levels than those with AG () and AA genotype (). Horizontal bars indicate the .4. DiscussionIn this study, we demonstrated that PD-1 expression levels in PBMCs from SLE patients were significantly higher than those in control group. Also, significant relationship was found between SLEDAI scores and upregulated PD-1 expression in PBMCs from PB samples of SLE patients. However, no obvious difference was revealed between PD-1 expression levels and SNP rs36084323. Results show that increased level of PD-1 expression in PBMCs rather than SNP rs36084323 is associated with the development of SLE, and this discovery is presented for the first time. These findings provide more evidence to support the theory that upregulated PD-1 expression may be involved in the pathogenesis of SLE.SLE is a chronic inflammatory disease of generalized autoimmunity and is characterized by B cell hyperactivity and abnormally activated T cells 1. PD-1 can be expressed on activated T cells, B cells, and myeloid cells and is considered to play an important role in the regulation of peripheral tolerance 18. Mice deficient for PD-1 have developed a lupus-like syndrome, with arthritis and glomerulonephritis as phenotypes 19. In this study, increased expression of PD-1 in PBMCs is found to have significant relationship with SLEDAI scores, and the results suggest that PD-1 is involved in the development of SLE. Although the detailed etiology is still unclear, many genes are considered to have connections with the pathogenesis of SLE. At present, the programmed cell death 1 gene (PDCD1, also called PD-1) was one of the top candidates linking to the disease 20. Thus, it is therefore necessary to study the interconnection between polymorphisms in PDCD1 and SLE.135 SNPs (found in the National Center for Biotechnology Information (NCBI) Entrez SNP database) have been identified in the human PDCD1 region. Among them, PD-1.1, PD-1.3, PD-1.5, and others are considered to have connection to autoimmune diseases 21. PD-1.1 polymorphism is located in the promoter region (position −538 from transcription start site). Previous studies have shown that PD-1.1 G/A (rs36084323) is common in the Chinese Han population (49%), but it is very rare in Europeans (1%) 20, which may indicate that Chinese Han population is more susceptible to SLE. In this study, no connection was found between SNP PD-1.1 G/A (rs36084323) and increased expression of PD-1 expression in PBMCs from SLE patients (), and the reason might be the limited sample size used in this study or some yet unidentified reason. However, it is observed that frequencies of the GG and AG genotype allele in SNP PD-1.1 were higher in SLE patients when compared with AA in our patients’ population. In addition, PD-1.1 is located within the promoter region of PD-1. This SNP has no function, and further study is required to explore its exact role in the development of SLE.5. ConclusionsIn conclusion, increased expression of PD-1 in PBMCs from SLE patients was significantly related to SLEDAI scores rather than SNP rs36084323. The presented results provide more evidence to support that upregulated expression of PD-1 might be correlated with the pathogenesis of SLE.Conflict of InterestsThe authors declare that they have no conflict of interests regarding the publication of this paper.Authors’ ContributionQingqing Jiao, Cuiping Liu, and Ziliang Yang contributed equally to this work.AcknowledgmentsThe financial support from Suzhou Youth Science and Technology Project (no. KJXW2013002), China Postdoctoral Science Foundation Project, National Natural Science Foundation of China (no. 31170834), and the Science and Technology Project of Suzhou (no. SYS201331) is greatly appreciated.References

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Overview

This document describes a general format for lab reports that you can adapt as needed. Lab reports are the most frequent kind of document written in engineering and can count for as much as 25% of a course yet little time or attention is devoted to how to write them well. Worse yet, each professor wants something a little different. Regardless of variations, however, the goal of lab reports remains the same: document your findings and communicate their significance. With that in mind, we can describe the report's format and basic components. Knowing the pieces and purpose, you can adapt to the particular needs of a course or professor.

A good lab report does more than present data; it demonstrates the writer's comprehension of the concepts behind the data. Merely recording the expected and observed results is not sufficient; you should also identify how and why differences occurred, explain how they affected your experiment, and show your understanding of the principles the experiment was designed to examine. Bear in mind that a format, however helpful, cannot replace clear thinking and organized writing. You still need to organize your ideas carefully and express them coherently. Typical Components

Title Page
Abstract
Introduction
Methods and Materials (or Equipment)
Experimental Procedure
Results
Discussion
Conclusion
References
Appendices
Further Reading

1. The Title Page needs to contain the name of the experiment, the names of lab partners, and the date. Titles should be straightforward, informative, and less than ten words (i.e. Not "Lab #4" but "Lab #4: Sample Analysis using the Debye-Sherrer Method").

2. The Abstract summarizes four essential aspects of the report: the purpose of the experiment (sometimes expressed as the purpose of the report), key findings, significance and major conclusions. The abstract often also includes a brief reference to theory or methodology. The information should clearly enable readers to decide whether they need to read your whole report. The abstract should be one paragraph of 100-200 words (the sample below is 191 words). Quick Abstract Reference Must have:

Purpose
Key result(s)
Most significant point of discussion
Major conclusion

May Include:

Brief method
Brief theory

Restrictions:

ONE page 200 words MAX. Sample Abstract

This experiment examined the effect of line orientation and arrowhead angle on a subject's ability to perceive line length, thereby testing the Müller-Lyer illusion. The Müller-Lyer illusion is the classic visual illustration of the effect of the surrounding on the perceived length of a line. The test was to determine the point of subjective equality by having subjects adjust line segments to equal the length of a standard line. Twenty-three subjects were tested in a repeated measures design with four different arrowhead angles and four line orientations. Each condition was tested in six randomized trials. The lines to be adjusted were tipped with outward pointing arrows of varying degrees of pointedness, whereas the standard lines had inward pointing arrows of the same degree. Results showed that line lengths were overestimated in all cases. The size of error increased with decreasing arrowhead angles. For line orientation, overestimation was greatest when the lines were horizontal. This last is contrary to our expectations. Further, the two factors functioned independently in their effects on subjects' point of subjective equality. These results have important implications for human factors design applications such as graphical display interfaces.

3. The introduction is more narrowly focussed than the abstract. It states the objective of the experiment and provides the reader with background to the experiment. State the topic of your report clearly and concisely, in one or two sentences: Quick Intro Reference Must Have:

Purpose of the experiment
Important background and/or theory 

May include:

Description of specialized equipment
Justification of experiment's importance

Example: The purpose of this experiment was to identify the specific element in a metal powder sample by determining its crystal structure and atomic radius. These were determined using the Debye-Sherrer (powder camera) method of X-ray diffraction.

A good introduction also provides whatever background theory, previous research, or formulas the reader needs to know. Usually, an instructor does not want you to repeat the lab manual, but to show your own comprehension of the problem. For example, the introduction that followed the example above might describe the Debye-Sherrer method, and explain that from the diffraction angles the crystal structure can be found by applying Bragg's law. If the amount of introductory material seems to be a lot, consider adding subheadings such as: Theoretical Principles or Background. Note on Verb Tense

Introductions often create difficulties for students who struggle with keeping verb tenses straight. These two points should help you navigate the introduction:

The experiment is already finished. Use the past tense when talking about the experiment.

    "The objective of the experiment was..."

The report, the theory and permanent equipment still exist; therefore, these get the present tense:

    "The purpose of this report is..."

    "Bragg's Law for diffraction is ..."

    "The scanning electron microscope produces micrographs ..."

4. Methods and Materials (or Equipment) can usually be a simple list, but make sure it is accurate and complete. In some cases, you can simply direct the reader to a lab manual or standard procedure: "Equipment was set up as in CHE 276 manual."

5. Experimental Procedure describes the process in chronological order. Using clear paragraph structure, explain all steps in the order they actually happened, not as they were supposed to happen. If your professor says you can simply state that you followed the procedure in the manual, be sure you still document occasions when you did not follow that exactly (e.g. "At step 4 we performed four repetitions instead of three, and ignored the data from the second repetition"). If you've done it right, another researcher should be able to duplicate your experiment.

6. Results are usually dominated by calculations, tables and figures; however, you still need to state all significant results explicitly in verbal form, for example: Quick Results Reference

Number and Title tables and graphs
Use a sentence or two to draw attention to key points in tables or graphs
Provide sample calculation only
State key result in sentence form

Using the calculated lattice parameter gives, then,
R = 0.1244nm.

Graphics need to be clear, easily read, and well labeled (e.g. Figure 1: Input Frequency and Capacitor Value). An important strategy for making your results effective is to draw the reader's attention to them with a sentence or two, so the reader has a focus when reading the graph.

In most cases, providing a sample calculation is sufficient in the report. Leave the remainder in an appendix. Likewise, your raw data can be placed in an appendix. Refer to appendices as necessary, pointing out trends and identifying special features.

7. Discussion is the most important part of your report, because here, you show that you understand the experiment beyond the simple level of completing it. Explain. Analyse. Interpret. Some people like to think of this as the "subjective" part of the report. By that, they mean this is what is not readily observable. This part of the lab focuses on a question of understanding "What is the significance or meaning of the results?" To answer this question, use both aspects of discussion: Analysis Interpretation What do the results indicate clearly? What have you found? Explain what you know with certainty based on your results and draw conclusions: What is the significance of the results? What ambiguities exist? What questions might we raise? Find logical explanations for problems in the data: Since none of the samples reacted to the Silver foil test, therefore sulfide, if present at all, does not exceed a concentration of approximately 0.025 g/l. It is therefore unlikely that the water main pipe break was the result of sulfide-induced corrosion. Although the water samples were received on 14 August 2000, testing could not be started until 10 September 2000. It is normally desirably to test as quickly as possible after sampling in order to avoid potential sample contamination. The effect of the delay is unknown.

More particularly, focus your discussion with strategies like these: Compare expected results with those obtained.

If there were differences, how can you account for them? Saying "human error" implies you're incompetent. Be specific; for example, the instruments could not measure precisely, the sample was not pure or was contaminated, or calculated values did not take account of friction. Analyze experimental error.

Was it avoidable? Was it a result of equipment? If an experiment was within the tolerances, you can still account for the difference from the ideal. If the flaws result from the experimental design explain how the design might be improved. Explain your results in terms of theoretical issues.

Often undergraduate labs are intended to illustrate important physical laws, such as Kirchhoff's voltage law, or the Müller-Lyer illusion. Usually you will have discussed these in the introduction. In this section move from the results to the theory. How well has the theory been illustrated? Relate results to your experimental objective(s).

If you set out to identify an unknown metal by finding its lattice parameter and its atomic structure, you'd better know the metal and its attributes. Compare your results to similar investigations.

In some cases, it is legitimate to compare outcomes with classmates, not to change your answer, but to look for any anomalies between the groups and discuss those. Analyze the strengths and limitations of your experimental design.

This is particularly useful if you designed the thing you're testing (e.g. a circuit).

8. Conclusion can be very short in most undergraduate laboratories. Simply state what you know now for sure, as a result of the lab: Quick Conclusion Reference Must do:

State what's known
Justify statement 

Might do:

State significance
Suggest further research

Example: The Debye-Sherrer method identified the sample material as nickel due to the measured crystal structure (fcc) and atomic radius (approximately 0.124nm).

Notice that, after the material is identified in the example above, the writer provides a justification. We know it is nickel because of its structure and size. This makes a sound and sufficient conclusion. Generally, this is enough; however, the conclusion might also be a place to discuss weaknesses of experimental design, what future work needs to be done to extend your conclusions, or what the implications of your conclusion are.

9. References include your lab manual and any outside reading you have done. Check the this site's documentation page to help you organize references in a way appropriate to your field.

10. Appendices typically include such elements as raw data, calculations, graphs pictures or tables that have not been included in the report itself. Each kind of item should be contained in a separate appendix. Make sure you refer to each appendix at least once in your report. For example, the results section might begin by noting: "Micrographs printed from the Scanning Electron Microscope are contained in Appendix A."

To learn more about writing science papers, visit our handout on writing in the sciences.

Writing Support News

Are you looking for writing support for one of your summer courses? Are you thinking about your program for next fall? Are you an incoming U of T student looking for a summer program to prepare you for your first year at university? Are you a multi-language student looking to improve your reading and writing skills? Have a look at the range of resources for writing support at the University of Toronto, and make your plans accordingly. Writing centres:

Only some of the University's writing centres remain open during the second half of summer.

The New College Writing Centre, the Innis College Writing Centre, the University College Writing Centre, the Victoria College Writing Centre, and the Woodsworth College Academic Writing Centre will be open for the remainder of summer term. Undergraduate students taking courses in the Faculty of Arts and Science on the St George campus can book up to one appointment a week at any one of these four centres. (This includes UTM and UTSC students, though only for work in St. George campus courses). Book online.
The Health Sciences Writing Centre is open to students of Dentistry, Nursing, Pharmacy, Kinesiology and Physical Education, and Social Work until mid-July.
UTM's Robert Gillespie Academic Skills Centre and the U of T at Scarborough Writing Centre remain open in the summer.
Engineering students may be able to get appointments by emailing writing@ecf.utoronto.ca .
The Daniels Writing Centre, in the Faculty of Architecture, Landscape, and Design, remains open for limited hours until the end of July.
The OISE Student Success Centre remains open for limited appointment hours this summer.

Note: You may book an appointment either by visiting the writing centre home page in your college or faculty or simply by visiting the common online booking system. Just follow the instructions on the welcome page. You will find links taking you to all centres that you are eligible to use. Writing courses:

Courses that focus explicitly on writing are available in various faculties. They are designed to match the types of writing done in specific areas of study. Visit our page on courses to find out more about the full range of writing courses available for you in the 2014-2015 academic year, and follow up by checking the online calendars for your program.

First-year students in Arts and Science on the St. George campus, for instance, may sign up for First-Year Seminar courses. Many of these small classes offer chances to get practice in writing and personal feedback from instructors.
Arts and Science students in any year may sign up for ENG100H (Effective Writing). Look under "English" in the Arts and Science timetable.
Arts and Science students may also take courses in the Writing and Rhetoric program. These courses are open to students in all college.
Students in Arts and Science interested in Creative Writing can sign up for one of three courses in Victoria College's Creative Expression and Society program offering opportunities to develop skills in creative writing in a workshop setting. Students must apply. Applications will be available starting April 1st.
Students at Scarborough may take one of several credit courses in English as a Second Language, focusing on analyzing and practicing the ways language is used for academic purposes. To find out more about the courses and the procedures for enrollment, visit the Teaching and Learning page of the UTSC calendar.
Students at Mississauga may sign up for courses in the Professional Writing Program. Starting with a course in Expressive Writing, this unique program takes students towards careers as editors, journalists, and professional writers.
Students in Engineering will also meet several required and optional courses tailored for their professional needs at all levels of study. They are coordinated by the Engineering Communication Program.
Graduate students can choose from a wide array of non-credit courses and workshops on academic writing and speaking offered by the Office of English Language and Writing Support at the School of Graduate Studies. Study the online listing and plan to register early when you have chosen the right course for you.

Intake programs for new U of T students:

If you're beginning your undergraduate degree, you can help prepare for what lies ahead next fall by participating in one of U of T's many intake programs. Many colleges have designed programs specifically for their incoming students. Check with your college registrar to see what might be available. Here's a listing of some of the college offerings:

As part of its program for newly admitted students, New College offers a series of workshops on writing, research and navigating courses. Students are also invited to make appointments for individual consultations during the summer. Book appointments online.
In mid- to late-August, University College students can attend one of six skills sessions as part of its Getting Started Early program. UC also invites its incoming students to bring a sample of high school work for a one-on-one appointment during July or August. Book all group or one-on-one sessions online.
Woodsworth College offers its new students the Jump Start program, a full-day introduction to getting the most out of the U of T experience (lunch is provided). Register online.
UTM's Head Start program in late August provides a free series of interactive sessions specially designed to help first-year students achieve success. Incoming UTM students can come to as many sessions as they like.
The Academic Success Centre will be delivering a series of workshops on study skills in the first half of September as part of Kickstart. Registration begins on the Kickstart site in July.

English Language Learning (St. George Campus):

The Faculty of Arts and Science's English Language Learning program helps multilingual students gain confidence in using English at a university level. During the first 6 weeks of the fall and winter terms, Arts and Science students may participate in Reading eWriting, an online method for improving scholarly reading and writing. During the first 5 weeks of the fall and winter terms, students may also participate in the Communication Café, a series of interactive language activities which develop presentation and discussion abilities.

In spring and late summer each year, students may enroll in an 8-day non-credit course, ELL010H1F, Intensive Academic English. The course is specially designed for multilingual students who want to improve their scholarly reading, academic writing, speaking, oral presentation, and listening abilities. It is suitable for students in the sciences, social sciences, and humanities.

To learn more, visit the ELL website. English Language Development (UTSC):

The English Language Development Centre at UTSC offers programs to help students develop the level of oral and written academic communication skills they need to succeed at university. The suite of programs consists of the Café series (Communication Café, Vocabulary Café and Discussion Skills Café), the Personalized Academic Reading and Writing through Email (RWE) and seminars. These programs are offered all three semesters. Students can explore the Café series and decide which combination of modules from the Communication Café, Word Play, Vocabulary Café, and Discussion Skills Café series would best meet their needs. Sign up on the Intranet to register for a specific session.

Students are also encouraged to take Academic English Health Check, a confidential service that supports students.

To find out more about all activities offered during the year by the English Language Development Centre, visit the ELD web site.

Besides courses and writing centres, other university facilities also provide specialized support for the kinds of writing expected at the University of Toronto.

Counselors from the Academic Success Centre provide lectures on such topics as note-taking, reading academic material, and avoiding writer's block. They are open to all students, and registration is not required. Just show up. Many are held in Robarts Library. The Academic Success Centre also provides one-and-a-half or two-hour learning skills workshops with titles such as Get Reading! Get Writing! and Write Fright. Registration is required. Visit the Academic Success Centre website for more information. Support groups, drop-in hours, and individual counselling are also available at the Academic Success Centre office, on the ground floor of the Koffler Student Centre, 214 College Street. Call 416 978-7970 or send an email for further information on what might meet your needs.
The Accessibility Program is now located at 455 Spadina Avenue, Suite 400, and at offices on the Scarborough and Mississauga campuses. If you are registered with this office, you may be eligible to work with specialized counsellors and facilities.
The School of Continuing Studies offers many non-credit certificate courses on writing, including some in creative writing, English as a second language, and business communication. Courses start at various times through the year, and some are offered as distance education
The University of Toronto Libraries offer a series of drop-in classes and online tutorials to help you improve your library and internet skills. Learn how to search for books using the new U of T Catalogue, and to find journal articles both in print and online.

Besides courses and writing centres, other university facilities also provide specialized support for the kinds of writing expected at the University of Toronto.

Counselors from the Academic Success Centre provide lectures on such topics as note-taking, reading academic material, and avoiding writer's block. They are open to all students, and registration is not required. Just show up. Many are held in Robarts Library. The Academic Success Centre also provides one-and-a-half or two-hour learning skills workshops with titles such as Get Reading! Get Writing! and Write Fright. Registration is required. Visit the Academic Success Centre website for more information. Support groups, drop-in hours, and individual counselling are also available at the Academic Success Centre office, on the ground floor of the Koffler Student Centre, 214 College Street. Call 416 978-7970 or send an email for further information on what might meet your needs.
The Accessibility Program is now located at 455 Spadina Avenue, Suite 400, and at offices on the Scarborough and Mississauga campuses. If you are registered with this office, you may be eligible to work with specialized counsellors and facilities.
The School of Continuing Studies offers many non-credit certificate courses on writing, including some in creative writing, English as a second language, and business communication. Courses start at various times through the year, and some are offered as distance education
The University of Toronto Libraries offer a series of drop-in classes and online tutorials to help you improve your library and internet skills. Learn how to search for books using the new U of T Catalogue, and to find journal articles both in print and online.

Besides courses and writing centres, other university facilities also provide specialized support for the kinds of writing expected at the University of Toronto.

Counselors from the Academic Success Centre provide lectures on such topics as note-taking, reading academic material, and avoiding writer's block. They are open to all students, and registration is not required. Just show up. Many are held in Robarts Library. The Academic Success Centre also provides one-and-a-half or two-hour learning skills workshops with titles such as Get Reading! Get Writing! and Write Fright. Registration is required. Visit the Academic Success Centre website for more information. Support groups, drop-in hours, and individual counselling are also available at the Academic Success Centre office, on the ground floor of the Koffler Student Centre, 214 College Street. Call 416 978-7970 or send an email for further information on what might meet your needs.
The Accessibility Program is now located at 455 Spadina Avenue, Suite 400, and at offices on the Scarborough and Mississauga campuses. If you are registered with this office, you may be eligible to work with specialized counsellors and facilities.
The School of Continuing Studies offers many non-credit certificate courses on writing, including some in creative writing, English as a second language, and business communication. Courses start at various times through the year, and some are offered as distance education
The University of Toronto Libraries offer a series of drop-in classes and online tutorials to help you improve your library and internet skills. Learn how to search for books using the new U of T Catalogue, and to find journal articles both in print and online.

We’ve changed our name! Sheffield School is now the New York Institute of Art and Design. The school has also merged with its sister school, the New York Institute of Career Development, adding two home-study courses in Professional Blogging and Fiction and Memoir Writing.

Our blog’s new name is the NYIAD Blog and will include additional topics on blogging and writing. All together the New York Institute of Art and Design will offer six home-study creative courses, including Interior Design, Jewelry Design: Beading and Wire-Working, Wedding and Event Planning, Feng Shui, Blogging, and Fiction and Memoir Writing.

The name change signifies a new focus for the school, which has provided home-study courses in the creative arts since 1985. “The New York Institute of Art and Design better captures who we are and what we offer to the thousands of students around the world that are enrolled with us,” says Sandra Darien, the School’s Registrar. We have big plans for the future of the New York Institute of Art and Design and we will share them with you as they come!

Whispering Willows is an adventure in archaeology with a supernatural perk: it lets you speak to the dead people who once owned the artifacts you discover. It twists the haunted house investigation premise by giving its heroine ghost transformational powers. This adequately serves both the narrative and the gameplay, the latter comprising some of the most simplistic and easy exploration puzzles in recent memory. This is a minimal detriment to the game overall, which is well-supported with touching themes of regret, lost love, and one's spiritual connection to nature.

Whispering Willows is an adventure in archaeology with a supernatural perk: it lets you speak to the dead people who once owned the artifacts you discover. It twists the haunted house investigation premise by giving its heroine ghost transformational powers. This adequately serves both the narrative and the gameplay, the latter comprising some of the most simplistic and easy exploration puzzles in recent memory. This is a minimal detriment to the game overall, which is well-supported with touching themes of regret, lost love, and one's spiritual connection to nature.

Whispering Willows is an adventure in archaeology with a supernatural perk: it lets you speak to the dead people who once owned the artifacts you discover. It twists the haunted house investigation premise by giving its heroine ghost transformational powers. This adequately serves both the narrative and the gameplay, the latter comprising some of the most simplistic and easy exploration puzzles in recent memory. This is a minimal detriment to the game overall, which is well-supported with touching themes of regret, lost love, and one's spiritual connection to nature.

Whispering Willows is an adventure in archaeology with a supernatural perk: it lets you speak to the dead people who once owned the artifacts you discover. It twists the haunted house investigation premise by giving its heroine ghost transformational powers. This adequately serves both the narrative and the gameplay, the latter comprising some of the most simplistic and easy exploration puzzles in recent memory. This is a minimal detriment to the game overall, which is well-supported with touching themes of regret, lost love, and one's spiritual connection to nature.

Whispering Willows is an adventure in archaeology with a supernatural perk: it lets you speak to the dead people who once owned the artifacts you discover. It twists the haunted house investigation premise by giving its heroine ghost transformational powers. This adequately serves both the narrative and the gameplay, the latter comprising some of the most simplistic and easy exploration puzzles in recent memory. This is a minimal detriment to the game overall, which is well-supported with touching themes of regret, lost love, and one's spiritual connection to nature.

Whispering Willows is an adventure in archaeology with a supernatural perk: it lets you speak to the dead people who once owned the artifacts you discover. It twists the haunted house investigation premise by giving its heroine ghost transformational powers. This adequately serves both the narrative and the gameplay, the latter comprising some of the most simplistic and easy exploration puzzles in recent memory. This is a minimal detriment to the game overall, which is well-supported with touching themes of regret, lost love, and one's spiritual connection to nature.

Whispering Willows is an adventure in archaeology with a supernatural perk: it lets you speak to the dead people who once owned the artifacts you discover. It twists the haunted house investigation premise by giving its heroine ghost transformational powers. This adequately serves both the narrative and the gameplay, the latter comprising some of the most simplistic and easy exploration puzzles in recent memory. This is a minimal detriment to the game overall, which is well-supported with touching themes of regret, lost love, and one's spiritual connection to nature.

Whispering Willows is an adventure in archaeology with a supernatural perk: it lets you speak to the dead people who once owned the artifacts you discover. It twists the haunted house investigation premise by giving its heroine ghost transformational powers. This adequately serves both the narrative and the gameplay, the latter comprising some of the most simplistic and easy exploration puzzles in recent memory. This is a minimal detriment to the game overall, which is well-supported with touching themes of regret, lost love, and one's spiritual connection to nature.

Whispering Willows is an adventure in archaeology with a supernatural perk: it lets you speak to the dead people who once owned the artifacts you discover. It twists the haunted house investigation premise by giving its heroine ghost transformational powers. This adequately serves both the narrative and the gameplay, the latter comprising some of the most simplistic and easy exploration puzzles in recent memory. This is a minimal detriment to the game overall, which is well-supported with touching themes of regret, lost love, and one's spiritual connection to nature.

Whispering Willows is an adventure in archaeology with a supernatural perk: it lets you speak to the dead people who once owned the artifacts you discover. It twists the haunted house investigation premise by giving its heroine ghost transformational powers. This adequately serves both the narrative and the gameplay, the latter comprising some of the most simplistic and easy exploration puzzles in recent memory. This is a minimal detriment to the game overall, which is well-supported with touching themes of regret, lost love, and one's spiritual connection to nature.

Whispering Willows is an adventure in archaeology with a supernatural perk: it lets you speak to the dead people who once owned the artifacts you discover. It twists the haunted house investigation premise by giving its heroine ghost transformational powers. This adequately serves both the narrative and the gameplay, the latter comprising some of the most simplistic and easy exploration puzzles in recent memory. This is a minimal detriment to the game overall, which is well-supported with touching themes of regret, lost love, and one's spiritual connection to nature.

The Ecliptic by Benjamin Wood, review: 'playfully experimental'

Whispering Willows is an adventure in archaeology with a supernatural perk: it lets you speak to the dead people who once owned the artifacts you discover. It twists the haunted house investigation premise by giving its heroine ghost transformational powers. This adequately serves both the narrative and the gameplay, the latter comprising some of the most simplistic and easy exploration puzzles in recent memory. This is a minimal detriment to the game overall, which is well-supported with touching themes of regret, lost love, and one's spiritual connection to nature.

The Ecliptic by Benjamin Wood, review: 'playfully experimental'

In his assured debut, The Bellwether Revivals, Benjamin Wood explored how a charismatic man deceived both himself and others through sheer force of personality. Here, in his second novel, deception is also key. The narrator – known as “Knell” – is a young artist, thought to be among the greatest of her time. A subtle narrative system wrong-foots the reader, just as Knell wrong-foots herself when it comes to analysing her own motivations.

The narrator reveals herself gradually. She is hurled from impoverished Scotland to bohemian Sixties London, and then to the fruits of the success her art brings – a First Class cabin on a Transatlantic liner. In the ship’s sauna, Knell endures a cataclysmic experience which she must confront and unravel. It leads her to a Turkish island, on an artists’ retreat, where she finds herself unable to paint.

• 10:04 by Ben Lerner, review: 'I doubt I'll read a finer novel all year'

Wood is fascinated with creation and what drives it. Is it something apart from the self – a “transmission”? Or does it come firmly from within? And what happens to an artist when she becomes locked within her own process? It’s a terrifying prospect, and one that Wood examines with panache, alluding to a sinister Emily Dickinson poem, in which she describes the nightmarish corridors of the mind.

It is Knell’s final task to find the “ecliptic” of the title – an astrological term denoting an imaginary line – to lead her out of the labyrinthine corridors in which she has become trapped.

Knell's artistic success allows her to take a first class cabin aboard a Transatlantic liner (PHOTO: ALAMY)

Although the novel is statelier than Wood’s debut, he has lost none of his ability to combine suspense with striking images. At the centre of things are some blue, phosphorescent mushrooms, whose glow becomes an obsession for Knell. Unleashing their peculiar pigment becomes a test of her ability to produce a piece of art that has within it what she has been seeking all her life: clarity.

This is a concept which becomes increasingly difficult to pin down. Knell stumbles across a series of comic books belonging to a teenage boy she encounters on the Turkish island. These tell the tale of a man who wakes up chained to a pillar on an abandoned ship. But the pages have been torn up by the boy in an incomplete act of brutality. The cut-up narrative is a reflection of the novel’s own system, feeding us hints and suggestions that there is more, if only one can make the connections.

Wood’s fiction is playfully experimental, but never loses sight of its primary purpose: to entertain.

Philip Womack’s latest novel is The King’s Shadow (Troika)

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114.6.49.226 8080 139.0.16.202 8080 36.83.4.139 8080 202.77.123.38 5555 From the foundation of the colonies beginning with the founding of Jamestown until the beginning of the Revolutionary War, different regions of the eastern coast had different characteristics. Once established, the thirteen British colonies could be divided into three geographic areas: New England, Middle, and Southern. Each of these had specific economic, social, and political developments that were unique to the regions. Ads "New Crisis is Coming" www.ronpaulupdate.com 22-year Congressman speaks out and reveals #1 step to prepare. Metacomet's War : A Novel www.amazon.com A novel about King Philip and the war named for him. Re-edited. Why Men Pull Away catchhimandkeephim.com 10 Ugly Mistakes Women Make That Ruins Any Chances Of A Relationship

Colonial America
American History
American Colonies
King Philip 'S War
US History Timeline

New England

Colonies: New Hampshire, Massachusetts, Rhode Island, and Connecticut. These were known for being rich in forests and fur trapping. Harbors were located throughout the region. The area was not known for good farmland. Therefore, the farms were small, mainly to provide food for individual families. New England flourished instead with fishing, shipbuilding, lumbering, and fur trading along with trading goods with Europe. The famous Triangle Trade occurred in the New England colonies where slaves were sold in the West Indies for molasses. This was sent to New England to make Rum which was then sent to Africa to trade for slaves.


In New England, small towns were the centers of local government. In 1643, Massachusetts Bay, Plymouth, Connecticut, and New Haven formed the New England Confederation to provide defense against Indians, Dutch, and the French. This was the first attempt to form a union between colonies.

A group of Massasoit Indians organized themselves under King Philip to fight the colonists. King Philip's War lasted from 1675-78. The Indians were finally defeated at a great loss.

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Middle Colonies

Colonies: New York, New Jersey, Pennsylvania, and Delaware. This area was excellent for farming and included natural harbors. Farmers grew grain and raised livestock. The Middle Colonies also practiced trade like New England, but typically they were trading raw materials for manufactured items.

One important event that happened in the Middle Colonies during the colonial period was the Zenger Trial in 1735. John Peter Zenger was arrested for writing against the royal governor of New York. Zenger was defended by Andrew Hamilton and found not guilty helping to establish the idea of freedom of the press.

 
Southern Colonies

Colonies: Maryland, Virginia, North Carolina, South Carolina, and Georgia. Southern colonies grew their own food along with growing three major cash crops: tobacco, rice, and indigo. These were grown on plantations typically worked by slaves and indentured servants. The main commerce of the South was with England. Plantations kept people widely separate which prevented the growth of many towns.

An important event that occurred in the Southern Colonies was Bacon's Rebellion. Nathaniel Bacon led a group of Virginia colonists against Indians who were attacking frontier farms. The royal governor, Sir William Berkeley, had not moved against the Indians. Bacon was labeled a traitor by the governor and ordered arrested. Bacon attacked Jamestown and seized the government. He then became ill and died. Berkeley returned, hanged many of the rebels, and was eventually removed from office by King Charles II.

Part 2: The Thirteen Colonies

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