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How ‘Gemini Man’ Gets Human Cloning Wrong – Hollywood Reporter

Films depicting clones, in showing us their versions of what two humans with identical genomes would be like, and how they would relate to each other, take their own stances on the nature vs. nurture debate. While a wide variety of clones have made it to screens over the years, the differences between them have not really followed trends or evolved in ways reflective of our ever-growing scientific understanding.

The scientific knowledge demonstrated by most clone films is elementary at best, and Gemini Man is no exception. In a pivotal scene, Henry (Smith) tries to appeal to his clone, Clay (also Smith), by emphasizing their connection through listing traits he presumes the other man must share. There are some that make sense. He, for instance, mentions a hatred of cilantro, which several investigations have linked to a particular allele (that is, a version) of the OR6A2 gene that causes individuals to taste a strong soapy flavor in cilantro that others do not. However, the shared trait which the film gives the utmost importance a severe bee sting allergy is actually one of the worst choices it could have made in terms of accuracy. While some studies have found evidence that certain alleles of particular genes might potentially predispose individuals to a bee sting allergy, it is a tentative link, and far more evidence has connected the development of allergies to environmental factors. Long story short, there is no bee sting allergy gene. In Gemini Man, Clay feels the need to test Henrys allergy in order to prove the man is truly his genetic identical, and while its a set-up that raises the stakes and makes for a tense scene, in actual fact it makes no sense at all. Allergies dont work that way.

But all of this still deals with what we actually do know, and writers not doing their researchor, more forgivingly, taking significant creative license. Putting inaccuracies aside, perhaps one of the most interesting things about cloning stories is how so many lean heavily on the side of genetic determinism the nature side of the nature vs. nurture debate. Most clone films, Gemini Man included, hold genes fully responsible for things, like allergies, in ways that are contrary to our current scientific understanding, so its perhaps not surprising that they also tend to credit genes for things for which scientists have no conclusive answers, like personality, taste, likes and dislikes.

Many of the most concerning narratives and prescient fears revolving around cloning and genetic engineering also depend on a high degree of genetic determinism. Neo-eugenics and the prospect of designer babies depend on traits like intelligence being genetically hard-wired. If whatever made Mozart Mozart and Einstein Einstein is environmental or some quality that defies quantification that is, anything other than genes these controversial schools of thought lose much of their steam.

Another intriguing trend regarding on-screen cloning is the frequency with which the very term cloning is misapplied. Clones are genetically identical. You take the DNA-housing nucleus from an existing individual and, for lack of a more apt analogy, cut and paste it into a fertilized egg that has had its nucleus removed. You dont mess with the DNA at all. Changing the DNA is genetic engineering. So, if you take someones DNA, modify it, and then use that DNA to create a new individual, that new individual is not a clone. Almost every major film and TV show to tackle cloning has in actual fact been about genetic engineering. In Moon, for instance, the short-lived Sam clones made to mine helium-3 on the moon are not really clones at all because they have been genetically modified to have a limited lifespan.

Gemini Man is somewhat unusual in that it does, to some extent, implicitly make a distinction between a clone and a genetically engineered individual in the difference between Clay, a genuine clone, and the unnamed adolescent super-soldier clearly modified from Henrys DNA. However, the film never really makes this distinction explicit and fails to clarify that clone isnt a catch-all umbrella term for individuals conceived deep in some secret laboratory.

Fictional depictions of cloning and genetic engineering matter because they shape the narrative on these subjects. They are the points of reference the general public uses to discuss these issues; the frames news media use to present new developments in these fields. From touch screen technologies to space travel, movies have long been a place to explore the possibilities of tomorrow, and in doing so play a role in the future that comes to pass. There are many fascinating possibilities and important debates to be had regarding the potential and concerns surrounding cloning and genetic engineering, but currently the way these issues are treated in film reflect more the presumptions of their writers than anything else. Movies present a great opportunity to ruminate and reflect on scientific frontiers, but unfortunately, particularly where genetics is concerned, they seem to have no interest in the reality of their subject matter.

Ciara Wardlow is a pop culture journalist and recent graduate of Wellesley College, where she studied Cinema and Media Studies as well as Biological Sciences.

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How 'Gemini Man' Gets Human Cloning Wrong - Hollywood Reporter

What it’s like to have Apple rip off your successful Mac app – Boing Boing

Companies that make successful Mac apps live in constant fear of being sherlocked -- having Apple release a feature-for-feature clone to compete with your product, bundling it in with Macos.

In June 2019, Astropad got sherlocked when Apple cloned its successful Astropad Studio and Luna Display apps in a new Macos feature that Apple called "Sidecar."

Astropad marketing director Savannah Reising describes how Apple had lionized their company and its products prior to cloning them, during a "false romance" that in which Apple "routinely invited us to demo our products at their headquarters, and offered to help us out with whatever business and engineering challenges we faced. They also ordered thousands of dollars worth of our hardware, and we naively thought it was because they were interested in our product. It turns out that they were just not in the way we were thinking."

What's more, Apple had bound over Astropad with nondisclosure agreements that limited how they could speak about their sherlocking, though Reising doesn't describe what happened to those NDAs now, she's pretty frank about the experience.

As Reising puts it, in platform capitalism, your main competitors aren't "other companies creating similar products to yours" -- it's the platform itself: "if your platform provider decides to step into your domain, its a tough battle to position your product against a free, native feature."

Reising has some advice for surviving a sherlocking (manage the PR carefully, have other products to fall back on, go cross-platform and don't get locked in with proprietary toolchains like Apple's APIs and Objective C).

What she doesn't touch on, though, is the real remedy for this kind of anticompetitive conduct, which is not within the scope of an individual entrepreneur or firm -- it's to demand the restoration of structural separation, the once-common antitrust measure that prohibited platforms from competing with their suppliers (for example, rail companies were banned from owning freight shipping companies that competed with their customers, and banks were not allowed to own businesses that competed with the businesses they loaned money to).

Elizabeth Warren's campaign platform includes structural separation for Big Tech.

I am a donor to both the Warren and Bernie Sanders campaigns.

When you take a birds-eye view, its the small startups and indie devs that are pushing innovation in the tech world. Its an imperfect synergy, though: with too much power, big tech like Apple eventually swallows up the innovators. The more sherlocking that happens, the more careful well be about dabbling in Apples playground. And the people that pay the ultimate price are the consumers.

What To Do When You Get Sherlocked By Apple [Savannah Reising/Astropad]

(via Four Short Links)

(Image: Astropad)

In early 2018, Apple SVP of internet software and services Eddy Cue and SVP of internet software and services Morgan Wandell instructed TV creators it had commissioned to produce content for Apple TV Plus to avoid plots and scenarios that held China and the Chinese state up in a critical light.

For decades, it was a commonplace in western business that no one could afford to ignore China: whatever problems a CEO might have with China's human rights record could never outweigh the profits to be had by targeting the growing Chinese middle-class.

Apple cant seem to figure out how to kowtow to China without losing face in the US.

Remember when the default state of your online presence was anonymity? Thats not so clear-cut anymore, and the worst part is you may not even know who is using your data or what theyre using it for. Small wonder that so many people are choosing to surf through virtual private networks. VPNs filter web access []

Get ready for the stream of your dreams, binge-watchers. Theres a contest afoot, and at stake is a lifetime subscription to Netflix. All you have to do is sign up, and youre entered to win this ultimate Netflix plan. When does it expire? Only when you do. And hey, just in case you need something []

Theres overwhelming support for clean energy, and the planet is giving us more reasons to invest in renewable power sources with every passing year. Even in the most inhospitable areas, wind and solar can provide a good chunk of our power, if not all of it. So why arent we all taking advantage of it? []

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What it's like to have Apple rip off your successful Mac app - Boing Boing

World’s first cloned cow Kaga dies aged 21 outliving twin who died last year – Daily Star

The world's first cloned cow has died at the age of 21 after living a healthy life.

Twins Kaga and Noto were the first ever cows to be born using the same technology that created Dolly the Sheep.

The clones were born as part of a research experiment at the Ishikawa Prefectural Livestock Research Centre in Japan.

Contrary to beliefs at the time, both of the cows proved to be healthy and lived as long as regular cows.

Noto was the first of the twins to die last year, being found unconscious in a barn on May 4 last year.

And now Kaga has become the last of the twins to die aged 21.

The bovine clone, which attracted worldwide attention when it was born, began having problems standing up in September.

After being given nutritional supplements and health drips, health officials confirmed the cow could no longer stand.

Kaga was pronounced dead yesterday, with its cause of death believed to be old age.

After the birth of the twins, 14 cloned cows were produced in 2006 to improve meat and milk production in Japan.

It was hoped the animals would pave the way for more cows to be cloned in Japan.

But these were quashed when the country outlawed cloned cow meat over fears of its safety.

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Since Kaga and Noto, a host of animals have received the cloning treatment.

This year, China cloned its first ever pet cat for a bereaved owner.

Scientists in the country are also thought to be able to clone dogs through their own urine.

Prehistoric beasts such as wooly mammoths are also thought to be on the horizon, due to cloning.

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World's first cloned cow Kaga dies aged 21 outliving twin who died last year - Daily Star

New Apple Card Fraud Case Shows Cloning Might Not Be the Only Concern – Softpedia News

An Apple Card fraud case that made the rounds last week was living proof that Apples new product is vulnerable to cloning just as much as any other credit card out there, but a new story indicates that Apple might have to deal with more serious concerns as well.

A report from 9to5mac highlights the case of an Apple Card owner who reported a fraudulent transaction despite never using the titanium card.

This means that skimming and cloning are not to blame for this incident, which raises questions as to how secure the Apple Card actually is in the first place.

According to this report, the Apple customer lives on the West Coast, but someone tried to make a transaction on behalf of his account from Chicago. The fraudulent charge was reported to Apple, who reportedly has a special department in charge of handling these incidents, but not even the staff here were able to determine what exactly happened.

I understand this can be concerning, especially regarding your financial security, however it is the most secure system of credit cards Ive ever seen. Not only is it extremely hard to get a hold of credit card information, but if somehow there are fraudulent charges, you will never be held responsible for unauthorized transactions on Apple Card, an Apple support engineer told the Apple customer as per the cited source.

Without the Apple Card owner ever using the physical titanium card, some wonder whether the fraudulent transactions are possible based on credit card details provided by other sources, including even insiders.

But as its always the case, taking such user reports with a healthy dose of skepticism is the right approach, especially because we never know if this is the full story or even if its real in the first place. As a matter of fact, online shopping with the Apple Card could also expose the credit card details if they are entered on shady websites, so even if not using the titanium card physically in a store, the account could end up being hacked eventually.

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New Apple Card Fraud Case Shows Cloning Might Not Be the Only Concern - Softpedia News

Tech company will pay $130K to put your face on a line of robots – New York Post

Heres your chance to be the literal face of a robotics company.

A tech firm is looking for the right person to lend their likeness to a new line of robot assistants for the elderly. And while it might sound like the plot to a bad sci-fi flick, the company will pay the chosen candidate about $130,000 for the privilege.

The privately funded firm has opted to remain anonymous due to the projects secretive nature, but they have hired robotics recruiter Geomiq to find the right face for the job, reports the Mirror. Ideal applicants will possess a kind and friendly face for the prototype, per the head, er, face hunters recruitment ad. Its a once-in-a-lifetime opportunity for the right person; lets hope we can find them, said a Geomiq spokesperson.

The lucky winner of the face-off will have their likeness reproduced on thousands of virtual friends la Will Smiths disturbing 2004 movie I, Robot as well as rake in the aforementioned big bucks. The project has been five years in the making.

Designers havent disclosed much beyond that, only that the robotic doppelgngers will hit the assembly line next year and will be readily available to the public upon completion.

On the application page, Geomiq acknowledges that licensing ones visage to an unnamed robotics company for eternity is potentially an extremely big decision.

The face-cloning campaign has drawn flack from social media skeptics, with many of them analogizing it to bad dystopian movie tropes. Janelle Mone warned us about this, cautioned one.

Others wondered why a supposedly tech-savvy robotics company needed a human face at all and couldnt just save money by using an online random-face generator. Have these people ever heard of GANs? asked one Twitter techie. There are datasets with 100k realistic (but not real) faces available already.

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Tech company will pay $130K to put your face on a line of robots - New York Post

Something exciting is coming with Ubuntu 19.10 – TechRepublic

Ubuntu 19.10 includes one feature that should have every user and admin overcome with the feels.

Hello, Ubuntu. It's been quite some time since you've brought us something truly exciting with a new release. Oh sure, a while back you shifted away from Unity and defaulted to GNOME, and that was a bold move--one I believe the majority of users are thankful for. Outside of that, the various releases over the past few years have been somewhat underwhelming.

But that all ends with 19.10 (Eoan Ermine). No matter what other new features find their way into the release, they are all overshadowed by one addition that is long overdue. That feature is ZFS.

SEE:10 free alternatives to Microsoft Word and Excel(TechRepublic download)

ZFS is a combined file system and logical volume manager that is scalable, supplying support for high storage capacity and a more efficient data compression, and includes snapshots and rollbacks, copy-on-write clones, continuous integrity checking, automatic repair, and much more.

So yeah, ZFS is a big deal, which includes some really great features. But out of those supported features, it's the snapshots and rollbacks that should have every Ubuntu user/admin overcome with a case of the feels.

Why? Imagine something has gone wrong. You've lost data or an installation of a piece of software has messed up the system. What do you do? If you have ZFS and you've created a snapshot, you can roll that system back to the snapshot where everything was working fine.

Although the concept isn't new to the world of computing, it's certainly not something Ubuntu has had by default. So this is big news.

Note: I've been working with the final release candidate, so the ZFS support is still in the experimental phase. Even so, it's worked tremendously.

When installing Ubuntu 19.10, you are given the option of using the ZFS file system (Figure A). Select that option and then click Install Now.

Figure A

Installing Ubuntu 19.10 with ZFS.

When the installation completes, reboot and log in. At first blush, you won't notice anything different with the ZFS system. In fact, everything just works, as you've come to expect with Ubuntu Linux.

I'm going to show you how to create a snapshot, make a change, and the roll back that snapshot.

The first thing you must do is find the name of the ZFS dataset you want to use. I'm going to make a snapshot of my home directory. To find the name of the home dataset, issue the command:

You should see a complete list of your datasets (Figure B).

Figure B

The dataset I'll be using is named rpool/USERDATA/jack_bwcn4u. It is important that you know the name of the dataset, as you cannot simply take a snapshot using the directory name or path. To create a snapshot named WED101619, the command would be:

The snapshots generally complete very quickly, regardless of how much data is stored in the location.

Now, let's make a change. We'll delete the Documents folder in my home directory with the command:

The Documents folder is now gone (Figure C).

Figure C

My Documents folder is gone!

Imagine that folder contained all of your work, school, or research documents? If you didn't have a backup (which you should), you might find yourself throwing a fit or 12. What do you do? Since you took a snapshot, you can roll it back with the command:

Give the command time enough to rollback the changes and viola! The Documents folder has returned (Figure D).

Figure D

My Documents folder is back!

Of course there is so much more ZFS can do (such as cloning snapshots and replication), but this gives you an idea of what's coming for the next release of Ubuntu Linux. The full release will be available on October 17, 2019. For those that are curious, the addition of ZFS for Ubuntu 19.10 means even greater things are yet to come.

ZFS is just the beginning of a much greater system, developed by Canonical, called Zsys. When Zsys is finally released, admins will be able to run multiple ZFS systems in parallel on the same machine, get automated snapshots, manage complex ZFS dataset layouts separating user data from system and persistent data, and more.

So yes, the addition of ZFS on Ubuntu should be cause for every Ubuntu user and admin to get very, very excited.

It's about time.

You don't want to miss our tips, tutorials, and commentary on the Linux OS and open source applications. Delivered Tuesdays

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Something exciting is coming with Ubuntu 19.10 - TechRepublic

‘Gemini Man’ Review: Uncle Kill and the Fresh Prince Star In Moron Clone Wars – Pajiba

Stories about twins can be powerful metaphors. They can be profound meditations on whether its nature or nurture that makes us who we are. They can be explorations of our darker halves made flesh. They can be mysteries about Arnold Schwarzenegger and Danny DeVito sharing most of their DNA. Or dramatic short films during the Super Bowl pondering why anyone would intentionally drink Coors.

Gemini Man is none of those things. Its a shittily shot action film that uses the CGI gimmick of a de-aged Will Smith as its sole point of creativity. No thoughtfulness, no contemplation, not even leaning into the humor of the concept. It is such a catastrophic waste of an insanely talented cast: Will Smith, Mary Elizabeth Winstead, Clive Owen, and Benedict Wong have so many better movies they could be making other than this.

The entire movie is in the trailer, so, well, spoilers follow which amount to telling you that in the movie about clones, the clones fight. And then they become friends. Sorry for ruining that for you. Theres old Will Smith and young Will Smith. Lets just go with calling them Uncle Kill and the Fresh Prince. Uncle Kill is the worlds bestest assassin but he wants to retire. Naturally, instead of giving him a pension to enjoy on his obligatory fishing boat on the gulf coast, his old bosses start sending death squads of SAG extras to die.

The bad guy is Clive Owen, playing the evil mercenary who not only runs a giant Blackwater style private army, but has the resources to have his own cutting edge genetics lab that does human cloning. When Uncle Kill turns down a job offer twenty years before this masterpiece of cinema is set, Clive clones him and secretly raises the Fresh Prince as his own adopted son. As one does.

As an aside, its more like the Fresh Prince of the Uncanny Valley, because this CGI is baaaaaaaaad. Most of the movie is dark enough that you cant tell, but sweet Jesus the mandatory epilogue set six months later in happy sunny days is painful to watch. Its like someone downloaded an animated GIF of Will Smith in 1995 on a spotty modem: noisy, spastic, and just disturbing. Also, they turned the dial up a bit too high on the de-aging algorithm, because the Fresh Prince looks at most fourteen despite them telling us hes 23. Like, he looks creepy young throughout.

So its basically The Bourne Identity with cloning instead of amnesia, and bad action. You see Ang Lee in the opening credits as director, and you at least expect the action to be decent. But no. The action is shot to make Fresh Prince and Uncle Kill move with superhuman speed and take blows that would sit Captain America down, which just adds to the cognitive dissonance of the bad CGI since this isnt a superhero movie. At one point the Fresh Prince beats Uncle Kill up with a motorcycle. On screen it makes even less sense than that sentence does on screen.

Its humorless except occasionally by accident when the stupidity of the script pushes an involuntary snort out of your bored catatonia. The movie is so absurdly and idiotically conceived that its only hope was to wink at the camera for the whole thing. One clone? Fuck it, make it fifty. I completely fail to understand why people keep casting Will Smith in movies and tell him to not have any fun. How do you take a men with more raw charm and humor than should be legal, stick him face to face with a CGI young version of himself, and have him mutter shit like its like looking in a mirror instead of a half dozen ad-libbed versions of damn son, I knew I was a good looking man?

A fantastically fun and surprisingly deep movie could have been made out of this premise. Take the old killer and see if the parts of what makes him a killer are built-in or were developed through experience. Theres a point at which Uncle Kill rattles off a whole laundry list of things he knows about the Fresh Prince because theyre the same person. It involves insights like having nightmares, being scared of opening up to anyone, still being a virgin at 23, and only being happy when shooting a gun. This just in, none of those things are genetic.

In fact, they use the fact that theyre both allergic to bee stings as a major plot point. The only problem is that like all of those other things, allergies to bee stings arent genetic. Ouch. Oops. But that would take a cursory google search, which is clearly beyond whatever idiot wrote this. *listens to earpiece for a moment* Yes, this just in, said idiot was David Benioff, master of such literary heights as the final season of Game of Thrones. Everything is making sense now. More like Castor and Pollsux, am I right?

Just go rewatch the Coors Light twins commercial if you need a clone fix. It has more depth and pathos than this mess.

Dr. Steven Lloyd Wilson is a hopeless romantic and the last scion of Norse warriors and the forbidden elder gods. His novel, ramblings, and assorted fictions coalesce at http://www.burningviolin.com. You can email him here.

Steven Lloyd Wilson is the sci-fi and history editor. You can email him here or follow him on Twitter.

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Fosmid Cloning Market 2019, Trend, CAGR Status, Growth, Analysis and Forecast to 2024 – TheFinanceTime

A research report on Fosmid Cloning Market 2019 Industry Research Report is being published by researchunt.com. This is a key document as far as the clients and industries are concerned to not only understand the competitive market status that exists currently but also what future holds for it in the upcoming period, i.e., between 2018 and 2025. It has taken the previous market status of 2013 2018 to project the future status. The report has categorized in terms of region, type, key industries, and application.

A sample of report copy could be downloaded by visiting the site:99marketresearch.com/global-fosmid-cloning-market-size-status-and-forecast-2019-2025/169040/#Free-Sample-Report

Global Fosmid Cloning revenue was xx.xx Million USD in 2013, grew to xx.xx Million USD in 2017, and will reach xx.xx Million USD in 2023, with a CAGR of x.x% during 2018-2023.

Major Geographical Regions

The study report on Global Fosmid CloningMarket 2018 would cover every big geographical, as well as, sub-regions throughout the world. The report has focused on market size, value, product sales and opportunities for growth in these regions. The market study has analyzed the competitive trend apart from offering valuable insights to clients and industries. These data will undoubtedly help them to plan their strategy so that they could not only expand but also penetrate into a market.

The researchers have analyzed the competitive advantages of those involved in the industries or in the Fosmid Cloningindustry. While historical years were taken as 2013 2017, the base year for the study was 2017. Similarly, the report has given its projection for the year 2018 apart from the outlook for years 2018 2025.

Key Players and Type

Like any other research material, the report has covered key geographical regions such as Europe, Japan, United States, India, Southeast Asia and Europe. Researchers have given their opinion or insights of value, product sales, and industry share besides availability opportunities to expand in those regions. As far as the sub-regions, North America, Canada, Medico, Australia, Asia-Pacific, India, South Korea, China, Singapore, Indonesia, Japan, Rest of Asia-Pacific, Germany, United Kingdom, France, Spain, Italy, Rest of Europe, Russia, Central & South America, Middle East & Africa are included.

Major players in the report included are :

Types covered in theFosmid Cloningindustryare :

Applications covered in the report are :

Report Aims

The objective of the researchers is to find out sales, value, and status of the Fosmid Cloningindustry at the international levels. While the status covers the years of 2013 17, the forecast is for the period 2018 25 that will enable market players to not only plan but also execute strategies based on the market needs.

Read Detailed Index of full Research Study at @99marketresearch.com/global-fosmid-cloning-market-size-status-and-forecast-2019-2025/169040/

The study wanted to focus on key manufacturers, competitive landscape, and SWOT analysis for Fosmid Cloningindustry. Apart from looking into the geographical regions, the report concentrated on key trends and segments that are either driving or preventing the growth of the industry. Researchers have also focused on individual growth trend besides their contribution to the overall market.

There are 15 Chapters to display the GlobalFosmid Cloningmarket.

Sections 1. Industry Synopsis of Global Fosmid Cloning Market.

Sections 2. Fosmid Cloning Market Organization Producers analysis and Profiles.

Sections 3. Fosmid Cloning Market Size by Type and Application.

Sections 4. Global Fosmid Cloning Market 2018 Analysis by key traders.

Sections 5. Europe Fosmid Cloning Industry Report Development Status and Outlook.

Sections 6. Japan Fosmid Cloning Industry Report Development Status and Outlook.

Sections 7. Development Status and improvements of Fosmid Cloning Market in the United States.

Sections 8. Southeast Asia Fosmid Cloning Market Improvement Status and Outlook.

Sections 9. China Fosmid Cloning Market Report Development Status and Outlook.

Sections 10. India Fosmid Cloning Market Development Status and Outlook.

Sections 11. Fosmid Cloning Market Figure by Aoplications, areas, and Sorts (2018-2023)

Sections 12. Fosmid Cloning Market Factors Analysis.

Sections 13. Fosmid Cloning Market Dynamics.

Sections 14. Research Findings and Conclusions of Fosmid Cloning Market.

Sections 15. Appendix.

Browse Detailed TOC, Tables, Figures, Charts And Companies Mentioned In Fosmid Cloning Market Research Report At@99marketresearch.com/global-fosmid-cloning-market-size-status-and-forecast-2019-2025/169040/#Buying-Enquiry

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Fosmid Cloning Market 2019, Trend, CAGR Status, Growth, Analysis and Forecast to 2024 - TheFinanceTime

Bacterial production and direct functional screening of expanded molecular libraries for discovering inhibitors of protein aggregation – Science…

Abstract

Protein misfolding and aggregation are associated with a many human disorders, including Alzheimers and Parkinsons diseases. Toward increasing the effectiveness of early-stage drug discovery for these conditions, we report a bacterial platform that enables the biosynthesis of molecular libraries with expanded diversities and their direct functional screening for discovering protein aggregation inhibitors. We illustrate this approach by performing, what is to our knowledge, the largest functional screen of small-size molecular entities described to date. We generated a combinatorial library of ~200 million drug-like, cyclic peptides and rapidly screened it for aggregation inhibitors against the amyloid- peptide (A42), linked to Alzheimers disease. Through this procedure, we identified more than 400 macrocyclic compounds that efficiently reduce A42 aggregation and toxicity in vitro and in vivo. Finally, we applied a combination of deep sequencing and mutagenesis analyses to demonstrate how this system can rapidly determine structure-activity relationships and define consensus motifs required for bioactivity.

The phenomenon of protein misfolding and aggregation is a defining feature of a wide range of human diseases with very high socioeconomic impact, including neurodegenerative disorders, type 2 diabetes, and cancer (1). Since aggregated proteins can cause disease, either because they can no longer efficiently perform their physiological function (loss of function) or because they form harmful aggregated species with cytotoxic properties (toxic gain of function) (1), compounds that prevent, delay, or reverse protein aggregation constitute valuable leads for the development of potential therapeutics. any such molecules are currently in preclinical and clinical development (2). As a proof of concept for the therapeutic value of this approach, tafamidis, a small molecule that prevents the misfolding and aggregation of the carrier protein transthyretin by binding and stabilizing its tetrameric native form, has been approved for the treatment of familial amyloid polyneuropathy in Europe and Japan (Vyndaqel, Pfizer) (3). More recently, migalastat, a chemical rescuer of the misfolding of the lysosomal enzyme -galactosidase (4), has been approved for the treatment of the lysosomal storage disorder Fabry disease in Europe and the United States (Galafold, Amicus Therapeutics). Despite these encouraging results, the vast majority of protein-misfolding diseases remain incurable, as no disease-modifying drug has reached the clinic in most cases. Among the reasons for the failure of current clinical trials, we mention an incomplete understanding of the exact molecular mechanism of action of the antiamyloid- peptide (A) candidates and the late treatment of the patients (5). Thus, it is imperative to develop systematic and robust approaches to discover previously unidentified and effective disease-modifying agents, which are urgently required for this type of disorders.

Advances in key scientific and technological areas are needed to increase the success rate with which effective drugs against these complex diseases are discovered. One such area is chemical library construction. The availability of molecular libraries with expanded diversities is expected to markedly increase the chances for identifying compounds with the desired properties (6, 7). Because of current limitations in organic synthesis and the isolation of natural products, however, the diversity of currently tested small-molecule libraries is typically not higher than 105 to 106 (8). Considering that the size of the chemical space for small molecules, i.e., the number of all possible lowmolecular weight structures has been estimated to be ~1060 (9), it is clear that drug screening efforts will benefit from increased diversity. In addition, even when chemical libraries with larger sizes are available, the majority of screening methodologies for the identification of drug-like compounds are not sufficiently high throughput to efficiently handle very large libraries. Functional screening assays in multiwell plate format, for example, become impractical for libraries with more than 106 to 107 members.

Genetically encoded combinatorial libraries can enable a marked expansion in the number and chemical complexity of lowermolecular weight compounds, which can be generated and subsequently tested for bioactivity (7, 10, 11). By using approaches of this type, molecular libraries with diversities ranging from many millions to even tens of trillions of test compounds have already been generated (6, 7, 10, 12, 13), and molecules with valuable biological activities have been discovered. These bioactivities include modulation of the aggregation process of misfolding-prone and disease-associated proteins, such as the A and huntingtin (11, 1417).

One important shortcoming when investigating DNA-encoded libraries for protein misfolding and aggregation diseases, however, is that they can only be screened for binding against immobilized protein targets (10). Despite their efficiency in identifying strong binders, these affinity-based selections cannot readily provide functional information regarding the aggregation inhibition activity of the identified hits (10). As a result, the selected binders need to be resynthesized chemically and evaluated again for aggregation-inhibitory activity in secondary assays. This procedure adds substantial time, complexity, and cost to the overall screening process and is regarded as a major bottleneck by the pharmaceutical industry (12). Furthermore, in many cases, the outcome of the selection process results in the identification of a large fraction of hits that are either completely inactive (12) or have opposite effects on protein misfolding and aggregation than the ones intended originally (15).

In an effort to generate new and efficient systems for discovering previously unidentified inhibitors of pathogenic protein aggregation, we have recently reported the development of a synthetic biology platform that enables the discovery of chemical rescuers of disease-associated protein misfolding (18). In this system, combinatorial libraries of lowermolecular weight peptide macrocycles are biosynthesized in Escherichia coli cells and are simultaneously screened for their ability to correct the problematic folding of misfolding-prone, disease-associated proteins using a flow cytometric ultrahigh-throughput genetic screen.

In the present work, we demonstrate how this bacterial discovery platform can be expanded to enable the production and direct functional screening of molecular libraries with greatly increased diversities, thus considerably surpassing the capabilities of other systems reported to date. We used this system to generate a combinatorial library of ~200 million peptide macrocycles and to perform simultaneous functional screening for aggregation inhibition activity against the 42-residue form of (42), which is associated with Alzheimers disease. Within a matter of only a few days, our bacterial platform enabled the production and screening of the complete library and identified hundreds of hits. Analysis of the selected macrocycles revealed that they form different clusters with distinct sequence characteristics. Selected macrocycles derived from the most dominant clusters were subjected to in vitro biochemical and biophysical testing and were found to be highly potent inhibitors of A42 aggregation at substoichiometric ratios. In vivo testing in established models of Alzheimers disease in the nematode Caenorhabditis elegans demonstrated that the selected macrocycles were effective in decreasing the deposition of A42 aggregates and in markedly reversing A42-induced pathogenic effects. We then used a combination of high-throughput sequencing and site-directed mutagenesis analyses to determine structure-activity relationships for the selected macrocycles and to define consensus motifs required for high bioactivity in these molecules. Overall, our discovery platform enables the simultaneous production and functional screening of molecular libraries with markedly expanded diversities for the identification of compounds with therapeutic potential for inhibiting the aggregation of disease-associated polypeptides.

The molecular libraries that we have chosen to use for the discovery of protein aggregation inhibitors are combinatorial libraries of head-to-tail cyclic heptapeptides, with an average molecular mass of about 770 Da. These macrocycles fall within the class of small molecules (molecular mass, <900 Da) but occupy an area of chemical space beyond the classical Lipinskis rule of five (bRo5 space; molecular mass, 500 to 1000 Da), where different rules for drug-likeness compared to classical small-molecule therapeutics apply (19, 20). The very large number of possible amino acid combinations comprising a peptide sequence (of seven amino acids in our case) enables greatly expanded levels of molecular diversity compared to available synthetic and natural small-molecule libraries (8). Furthermore, the cyclic nature of these molecules affords higher binding affinities for other proteins, enhanced ability to penetrate biological barriers, and enhanced resistance to proteolysis compared to their linear analogs (21).

Libraries of head-to-tail cyclic peptides can be conveniently produced in E. coli cells by the split inteinmediated circular ligation of peptides and proteins (SICLOPPS) method, where a circularly permuted split intein catalyzes the formation of a peptide bond between the termini of the target protein or peptide (22). SICLOPPS is a well-established technique, which has been previously used to identify cyclic peptides with different bioactivities (23). The only external requirement for the intein splicing reaction and peptide cyclization to take place is the presence of a nucleophilic amino acid (Cys, Ser, or Thr) as the first amino acid of the to-be-cyclized peptide (18). Thus, to maximize the diversity of our macrocycle library, we constructed a combinatorial heptapeptide library with the general formula cyclo-NuX1X2X3X4X5X6, where Nu is any one of the nucleophilic amino acids Cys, Ser, or Thr and X is any one of the 20 natural amino acids. The maximum theoretical diversity of this library is 3 206 = 192 million different sequences. The libraries of genes encoding these cyclic heptapeptide libraries were constructed using degenerate polymerase chain reaction (PCR) primers, in which the randomized amino acids (X) were encoded using randomized NNS codons, where N is A, T, G, or C and S is G or C (see Materials and Methods). The generated peptide-encoding gene libraries were cloned into the vector pSICLOPPS (18) to form the combined pSICLOPPS-NuX1X2X3X4X5X6 vector library (Fig. 1A). These vectors express a combinatorial library of tetrapartite fusion proteins comprising the following: (i) the C-terminal domain of the Ssp DnaE intein (IC), (ii) a NuX1X2X3X4X5X6 heptapeptide sequence, (iii) the N-terminal domain of the Ssp DnaE intein (IN), and (iv) a chitin-binding domain (CBD) for immunodetection and/or purification, under the control of the PBAD promoter and its inducer l(+)-arabinose (Fig. 1A). Cloning of the resulting gene libraries into the pSICLOPPS plasmid yielded a total of 1.2 109 independent transformants, as judged by plating experiments after serial dilutions.

(A) Left: Representation of the pSICLOPPS-NuX1X2X3X4X5X6 vector library encoding the combinatorial heptapeptide library cyclo-NuX1X2X3X4X5X6. Nu: Cys, Ser, or Thr; X: any of the 20 natural amino acids; NNS: randomized codons, where N = A, T, C, or G and S = G or C; IC: C-terminal domain of the Ssp DnaE split intein; IN: N-terminal domain of the Ssp DnaE split intein. Right: Peptide cyclization using the SICLOPPS construct. Upon interaction between the two intein domains IC and IN, the encoded IC-NuX1X2X3X4X5X6-IN-CBD fusions undergo intein splicing and peptide cyclization, leading to the production of the cyclo-NuX1X2X3X4X5X6 library. (B) Western blot analysis of 12 randomly picked individual clones from the combinatorial heptapeptide library cyclo-NuX1X2X3X4X5X6, showing the expression and processing of the precursor fusion protein IC-peptide-IN-CBD. The 25-kDa band corresponds to the unprocessed precursor and the 20-kDa band to the processed IN-CBD construct, and indicates, wherever present, successful intein splicing and peptide cyclization. Clone 10, for which the precursor is not expressed, was to contain a stop codon in its peptide-encoding region. (C) Heatmap representation of the amino acid distribution at each position of the constructed cyclo-CysX1X2X3X4X5X6 (left), cyclo-SerX1X2X3X4X5X6 (middle), and cyclo-ThrX1X2X3X4X5X6 (right) sublibraries, as demonstrated by the deep sequencing analysis results.

To assess the quality of our constructed library, we initially chose 150 randomly selected clones and performed colony PCR and SDSpolyacrylamide gel electrophoresis (SDS-PAGE)/Western blot. This analysis revealed that approximately 45% of the analyzed clones contained a DNA insert of the correct size and produced full-length IC-peptide-IN-CBD precursor fusion protein (molecular mass, ~25 kDa), which could undergo processing (appearance of a band with a molecular mass of ~20 kDa) (Fig. 1B). his processing is a prerequisite for successful intein splicing and indicates possible formation of a cyclic product. According to these results, the generated library contains approximately 5.6 108 clones that apparently produce cyclic heptapeptides, a number that covers the theoretical diversity of our combined library by almost threefold.

To characterize the constructed library further, we performed deep sequencing analysis of the peptide-encoding region of the pSICLOPPS-NuX1X2X3X4X5X6 vector library. Of the ~3.4 million plasmid sequences that we analyzed, ~75% were unique at the DNA level and ~95% of those were found to encode unique peptide sequences (table S1). All amino acids were found to be encoded at every position of the generated library, albeit with an overrepresentation of residues corresponding to Gly and Arg (Fig. 1C). Together, these results indicate that we have constructed a very high-diversity library encoding the vast majority, if not all, of the theoretically possible ~192 million cyclo-NuX1X2X3X4X5X6 heptapeptide sequences.

To perform direct functional screening of our vast library of cyclic heptapeptides and readily identify bioactive macrocyclic inhibitors of pathogenic protein aggregation, we used an ultrahigh-throughput system that we previously developed (18). Because of the high aggregation propensity of A, E. coli cells overexpressing A42green fluorescent protein (GFP) produce a misfolded fusion that eventually accumulates into insoluble inclusion bodies lacking fluorescence (24). Conditions that inhibit A aggregation, however, result in the formation of soluble and fluorescent A42-GFP, and bacterial cells expressing this fusion acquire a fluorescent phenotype (18, 24). On the basis of this, production of the macrocyclic peptide libraries under investigation and their screening for misfolding-rescuing activity in this system are carried out simultaneously in E. coli cells in an integrated fashion, by selecting and isolating the bacterial clones biosynthesizing the molecules that enhance the fluorescence of chimeric fusions of misfolding-prone proteins with the GFP (Fig. 2A).

(A) Schematic of the used bacterial platform for discovering inhibitors of protein aggregation and for the high-throughput analysis of the selected hits. pMisP-GFP: plasmid encoding a misfolded protein-GFP fusion; pSICLOPPS-NuX1X2X3X4X5X6: vector library encoding the combinatorial heptapeptide library cyclo-NuX1X2X3X4X5X6; Nu: Cys, Ser, or Thr; X:, any of the 20 natural amino acids; FSC-H: forward scatter; SSC-H: side scatter; P: sorting gate. (B) FACS of E. coli Tuner (DE3) cells overexpressing A42-GFP and the combined cyclic heptapeptide library. M: mean GFP fluorescence in arbitrary units. FITC-A: filter for fluorescein isothiocyanate. (C) Relative fluorescence of E. coli Tuner (DE3) cells overexpressing A42-GFP and 10 randomly selected cyclic heptapeptide clones isolated after the seventh round of FACS shown in (B) and using either the wild-type split Ssp DnaE intein (green bars) or the splicing-deficient variant H24L/F26A (white bars) (25). Two randomly picked cyclic peptide sequences (random 1 and 2) previously shown to have no effect on 42-GFP fluorescence and aggregation (18) were used as a negative control. The fluorescence of the bacterial population producing cyclic peptide random 1 was arbitrarily set to 100. Mean values SEM are presented (n = 3 independent experiments, each performed in three replicates). (D) Top: Western blot analysis of total (left) and soluble (right) lysates of E. coli Tuner (DE3) cells overexpressing A42-GFP and the 10 individual cyclic peptide sequences tested in (C). The predicted molecular mass of the A42-GFP fusion is ~32 kDa. Bottom: Western blotting using the anti-A antibody 6E10 (left) and in-gel fluorescence (right) analyses of total lysates following native PAGE of E. coli Tuner (DE3) cells coexpressing A42-GFP and the 10 individual cyclic peptide sequences tested in (C). (E) Emission spectra of E. coli Tuner (DE3) cells overexpressing A42 along with four of the selected cyclic heptapeptide sequences tested in (C) and stained with ThS. The maximum fluorescence of the bacterial population producing cyclic peptide random 1 was arbitrarily set to 100. Mean values SEM are presented (n = 1 experiment performed in three replicates).

Electrocompetent E. coli Tuner (DE3) cells carrying the expression vector pETA42-GFP (24), which produces A42-GFP under the control of the strong bacteriophage T7 promoter, were cotransformed with the pSICLOPPS-NuX1X2X3X4X5X6 vector library. Approximately 3 109 transformants carrying both vectors were harvested, pooled together, and grown in Luria-Bertani (LB) liquid medium containing 0.005% l(+)-arabinosethe inducer of cyclic peptide productionat 37C with shaking. When the optical density at 600 nm (OD600) of the bacterial culture reached a level of about 0.5, 0.1 mM isopropyl--d-thiogalactoside (IPTG) was added to the medium so as to induce overexpression of the A42-GFP reporter. After about 2 hours at 37C, ~3 109 cells were screened, and the population exhibiting the top 1 to 3% fluorescence was isolated using fluorescence-activated cell sorting (FACS) (fig. S1A). The isolated cells were regrown and screened for a total of seven rounds, at which point the mean fluorescence of the population displayed an almost sixfold increase compared to the starting library (Fig. 2B). No further substantial increase in fluorescence was observed after additional rounds of sorting.

After the seventh round of FACS screening, 10 individual clones were randomly chosen from the sorted population, and their peptide-encoding vectors were isolated and then retransformed into fresh E. coli Tuner (DE3) cells carrying pET A42-GFP. Protein production was induced from both plasmids, and the levels of A42-GFP fluorescence of these cultures were measured. A42-GFP fluorescence of the isolated peptide-expressing clones was found to be markedly increased compared to cells expressing the same A42-GFP fusion in the presence of two random cyclic peptide sequences previously found to have no effect on A42-GFP fluorescence and aggregation (Fig. 2C) (18). All isolated clones expressed a full-length intein-peptide fusion (~25 kDa), which could undergo processing to yield a lowermolecular weight band corresponding to excised IN-CBD (~20 kDa), thus suggesting successful intein processing and possible formation of a cyclic peptide product (fig. S1B). Furthermore, the observed phenotypic effects were dependent on the ability of the Ssp DnaE intein to perform protein splicing, as the double amino acid substitution H24L/F26A in the C-terminal half of the Ssp DnaE intein, which is known to abolish asparagine cyclization at the IC/extein junction and prevent extein splicing and peptide cyclization (25), was found to reduce A42-GFP fluorescence back to wild-type levels (Fig. 2C and fig. S1B). Last, the observed increases in fluorescence were found to be A42 specific, as the isolated pSICLOPPS-NuX1X2X3X4X5X6 vectors did not enhance the levels of cellular green fluorescence when the sequence of A42 was replaced in the same vector with that of the DNA-binding (core) domain of human p53 containing a tyrosine to cysteine substitution at position 220 [p53C (Y220C)], a protein whose misfolding and aggregation is associated with certain forms of cancer (fig. S1C) (26).

Analysis of the expressed A42-GFP fusions by SDS-PAGE and Western blotting revealed that the bacterial clones expressing the selected cyclic heptapeptides produce markedly increased levels of soluble A42-GFP compared to random cyclic peptides, despite the fact that accumulation of total A42-GFP protein remained at similar levels (Fig. 2D, top, and fig. S1D). Furthermore, when the same cell lysates were analyzed by native PAGE and Western blotting, we observed that coexpression of the selected cyclic peptides reduced the accumulation of higher-order A42-GFP aggregates, which could not enter the gel, and increased the amounts of species with higher electrophoretic mobility (Fig. 2D, bottom left). These higher electrophoretic mobility species correspond to the fraction of the total A42-GFP that exhibits fluorescence (Fig. 2D, bottom right). Since the solubility and fluorescence of bacterially expressed A42-GFP has been found to be inversely proportional to the aggregation propensity of A42 (18, 24, 27), the results described above suggest that A42 aggregation is markedly decreased in the presence of the selected cyclic heptapeptides. Similar results were acquired when A42 was produced in an unfused, GFP-free form. When we tested the effects of the selected cyclic heptapeptides on A42 aggregation with an in vivo assay using whole-cell staining of intracellular formation of A42 aggregates with thioflavin S (ThS) (28), we observed that coproduction of the selected peptides resulted in decreased levels of ThS fluorescence, further indicating a reduced aggregate formation (Fig. 2E).

DNA sequencing of the 10 selected clones revealed five distinct cyclic heptapeptide sequences: cyclo-CKVWQLL (present six times among the sequenced clones), cyclo-CRVWTEL, cyclo-CKVWMPL, cyclo-CIVVPSI, and cyclo-CRIVPSL (fig. S1E).

We previously found that lowmolecular weight peptide macrocycles are a rich source of chemical rescuers of disease-associated protein misfolding and aggregation (18). On the basis of that initial observation, and in combination with the fact that multiple distinct cyclic heptapeptide sequences were identified among the 10 selected clones initially tested (fig. S1E), we hypothesized that numerous A42-targeting macrocyclic sequences may exist among the selected peptide pool. To determine the entire ensemble of potentially bioactive cyclic heptapeptides present in our library, we performed deep sequencing analysis of the heptapeptide-encoding regions in >0.4 million pSICLOPPS-NuX1X2X3X4X5X6 vectors contained in the selected bacterial population after the seventh round of sorting (Fig. 2B). This analysis revealed 416 distinct cyclic heptapeptide sequences appearing at least 20 times within the sorted population, thus indicating that their presence in the selected pool is not coincidental. Cloning of three randomly chosen cyclic heptapeptide sequences appearing in the sorted pool only with very low frequencies revealed that they are also efficient in increasing the fluorescence of bacterially expressed A42-GFP (fig. S1F).

We next performed sequence analysis of the selected cyclic heptapeptides. We found that Cys was the nucleophilic amino acid that was present at position 1 in the vast majority of the selected cyclic heptapeptides (99.6% of all selected sequences) (Fig. 3A, left). Furthermore, we observed that the frequency of appearance of only a very small number of specific amino acids was enriched at each position among the selected sequences: Arg and Lys at position 2; Val at position 3; Trp and Thr at position 4; Ile, Gln, Cys, Met, Ser, Thr, and Pro at position 5; Ala, Leu, Val, Glu, Lys, and Pro at position 6; and Ile, Leu, and Pro at position 7 (Fig. 3A, right, and table S2). On the contrary, the majority of amino acids, including the ones that were present in higher abundance in the initial library, were strongly de-enriched (Fig. 3A, right, and table S2), thus indicating a highly efficient selection process.

(A) Left: Frequency of appearance of the 20 natural amino acids at each position of the heptapeptide sequences selected after the seventh round of sorting (Fig. 2B). Right: Enrichment of the 20 natural amino acids at each position of the heptapeptide sequences selected after the seventh round of sorting (Fig. 2B). Values represent the log2-fold change of the amino acid frequency of appearance of the peptides from the sorted pool compared to the initial library. (B) Visualization of the main clusters formed by the selected cyclic heptapeptides according to their sequence similarities. Nodes represent different cyclic peptide sequences, and solid lines connect pairs of peptides that share at least 70% sequence identity. The sequences of the members of the two most dominant clusters (clusters I and II) are shown in the corresponding dendrograms.

To identify potential relationships among the selected cyclic heptapeptides, we carried out sequence similarity analysis and hierarchical clustering. As the similarity analysis is performed using linear sequences, all possible circular permutations of each selected cyclic heptapeptide were taken into consideration (fig. S2A). From the 416 cyclic heptapeptides selected, 323 of them formed 1467 unique pairs with more than 70% sequence identity and formed 20 distinct clusters with similar sequence characteristics (Fig. 3B and fig. S2B). Clusters I and II were the most dominant, comprising 75.0 and 4.9% of the selected bacterial clones, respectively, as well as 25.7 and 6% of the unique cyclic heptapeptide sequences selected (Fig. 3B and table S3). The majority of peptides from clusters I and II appeared to belong to a cyclo-CxVWxxx and a cyclo-CxxVPSx motif, respectively, in agreement with our previous observations (fig. S1E).

Two of the selected heptapeptides, cyclo-CKVWQLL and cyclo-CRIVPSL, termed AC7-1 and AC7-14 (A-targeting cyclic 7-peptide number 1 and 14), respectively (Fig. 4A and table S4), were chosen for subsequent analysis and were synthetized chemically in milligram quantities (fig. S3A). These cyclic peptides were selected because they were both encountered in the postselection pool investigated initially (fig. S1E) and, more importantly, they were the most frequently encountered members among the two most dominant clusters (clusters I and II) (table S4).

(A) Chemical structures of the selected cyclic heptapeptides AC7-1 and AC7-14. (B) Kinetic profiles of the aggregation of 2 M A42 in the absence and presence of AC7-1 at different molar ratios (left) and the normalized t1/2, tlag, and tgrowth values of the corresponding aggregation reactions (right). (C) As in (B) for AC7-14. In (B) and (C), mean values SEM are presented (n = 1 experiment performed in three replicates).

AC7-1 and AC7-14 were initially evaluated by monitoring their effects on the kinetics of A42 aggregation by thioflavin T (ThT) staining using a highly reproducible approach previously described (29, 30). Monomeric A42 was purified after recombinant production in E. coli, and aggregation kinetic experiments were initiated using 2 M A42 in the absence and presence of AC7-1 and AC7-14. Both AC7-1 and AC7-14 inhibited A42 aggregation very effectively at substoichiometric ratios as low as 0.5 molar equivalents for AC7-1 and 0.1 molar equivalents for AC7-14 (Fig. 4, B and C). Specifically, we found that both the tlag (time required for the ThT fluorescence to reach 10% of the total amplitude) and tgrowth (transition time from 10 to 90% of the total ThT fluorescence amplitude) of the A42 aggregation reaction were increased in the presence of the two selected macrocycles, albeit to a different extent (Fig. 4, B and C, right). Furthermore, we found that the A42 fibrils formed after the completion of the aggregation reaction in the absence and presence of both AC7-1 and AC7-14 were similar in both size and morphology (fig. S3B). Thus, it is likely that these selected macrocycles are not binding irreversibly to A42 species and redirecting the aggregation process toward off-pathway aggregates. The observed deceleration of A42 aggregation by the selected macrocycles could also be observed in the absence of ThT, when the progress of the aggregation was monitored by extracting aliquots at different time points and probing fibril formation by dot blotting using the fibril-specific OC antibody (fig. S3C).

To evaluate the effects of AC7-1 and AC7-14 in vivo, we tested their impact on A42 aggregation and A42-induced pathogenicity in an established C. elegans model of Alzheimers disease. We used GMC101, a transgenic strain expressing human A42 in body wall muscle cells under the control of a heat-inducible promoter (31). Upon temperature upshift, these nematodes (hereafter referred to as A worms) exhibit muscle-localized A42 aggregation and eventually the emergence of a paralysis phenotype (31). Since the in vitro results suggested that the two compounds affect the early stages of A42 aggregation, AC7-1 and AC7-14 were administered to the A worms before aggregation was initiated. The fitness of the A wormsdefined as the frequency and speed of body bendswas monitored in the absence and presence of AC7-1 and AC7-14 and compared to wild-type nematodes, which do not express A42. Both peptides increased the motility and speed of the A worms throughout their lifetime (Fig. 5, A and B). Both peptides were able to restore the total fitness of the A worms to approximately the levels of the wild-type animals (Fig. 5C). Furthermore, A worms treated with either one of the selected cyclic peptides produced 50 to 60% fewer A42 aggregates, as determined by imaging of the worms using the amyloid-specific dye 2-{[5-(4-hydroxyphenyl)(2,2-bithiophen)-5-yl]-methylene}-propanedinitrile (NIAD-4) (Fig. 5, D and E).

(A) Normalized motility (left) and normalized speed of movement (right) of A42 and wild-type worms in the absence and presence of 40 AC7-1 and 5 AC7-14 during days 5 to 10 of adulthood. (B) Motility (left) and speed (right) of individual A and wild-type worms in the absence and presence of AC7-1 and AC7-14 at day 7 of adulthood. (C) Total fitness (51) of the worms as in (B). (D) Relative fluorescence of A42 and wild-type worms at day 7 of adulthood showing a 50 to 60% decrease in A42 aggregate formation in the presence of AC7-1 and AC7-14. (E) Representative images from (D). In (A) to (C), ~200 worms were analyzed on average, while in (D), 25 worms were analyzed in total. In all panels, mean values SEM are presented (n = number of worms tested in one experiment). Statistical significance is denoted by *P 0.05 and ****P 0.0001, for differences to the No peptide A worms sample.

To exclude the possibility of promoter- or strain-specific effects, we also treated the transgenic C. elegans strain CL4176 with AC7-1 and AC7-14, which expresses human A42 in its body wall muscle cells under a different promoter (32). Consistent with our previous observations, the administration of both cyclic peptides resulted in a significant delay in the emergence of its characteristic paralysis phenotype (fig. S4). These results demonstrate the protective effect of the two cyclic peptides in the context of an animal, as shown by decrease of A42 deposits, increased locomotion, delay of paralysis, and recovery of total fitness.

To identify the functionally important residues within the selected peptides, we performed nucleophile substitutions at position 1 and Ala-scanning mutagenesis at positions 2 to 7 for both AC7-1 and AC7-14. Then, we compared the effects of these amino acid substitutions on the levels of bacterially expressed A42-GFP fluorescence and aggregation with those of the selected sequences (positive control) and of random cyclic peptide sequences (negative control). For both AC7-1 and AC7-14, the substitution of Cys at position 1 with Ser resulted in ~50% reduction in fluorescence, while the substitution with Thr resulted in levels of A42-GFP fluorescence and aggregation similar to those corresponding to the selected sequence (Fig. 6, A and B). The latter observation is somewhat unexpected, considering the dominant appearance of Cys1 sequences among the selected cyclic heptapeptide pool (Fig. 3A, left), but it may be related to our previous results, where Thr played a crucial role in the identified cyclic peptides against A42 aggregation (18). Since the isolation of the bioactive sequences in our system requires repeated rounds of bacterial culturing, protein overexpression and FACS, the scarcity of Thr1-containing sequences in the isolated cyclic heptapeptide pool may be occurring because of a toxicity effect of these sequences on bacterial growth, which can result in de-enrichment of the clones that produce them, despite their efficiency in preventing protein aggregation.

(A) Relative fluorescence of E. coli Tuner (DE3) cells overexpressing A42-GFP and AC7-1 (left) or AC7-14 (right) or the indicated variants thereof as measured by flow cytometry. The fluorescence of the bacterial population coproducing the random cyclic peptide was arbitrarily set to 100. Experiments were carried out in triplicate (n = 1 experiment), and the reported values correspond to the mean value SEM. (B) Western blotting using the anti-A antibody 6E10 (top) and in-gel fluorescence (bottom) analyses following native PAGE of total lysates of E. coli Tuner (DE3) cells coexpressing A42-GFP and AC7-1 (left) or AC7-14 (right) along with the indicated variants thereof. (C) Heatmap representation of the amino acid distribution at each position of the peptide sequences corresponding to cluster I (Fig. 3B), as demonstrated by the deep sequencing analysis results. The total (left) or the unique (right) heptapeptide sequences were included in the analysis. (D) As in (C) for cluster II.

Furthermore, for both peptides, Ala-scanning mutagenesis at the majority of the positions 2 to 7 resulted in markedly A42-GFP fluorescence decrease and concomitant increase in aggregation (Fig. 6, A and B). Specifically, for AC7-1, substitutions at positions 2, 3, 4, and 7 resulted in a ~30 to 70% decrease in A42-GFP fluorescence, while for AC7-14, substitutions at all positions except Ser6 resulted in a ~45 to 80% decrease (Fig. 6, A and B). These observations indicate that a number of residues in both selected cyclic heptapeptides are important for optimal aggregation inhibition activity. When we performed sequence analysis of all the selected sequences belonging to either cluster I or cluster II, we found that the peptides appearing most frequently in each cluster have strong preferences for specific amino acids at each position. More specifically, for cluster I, Arg and Lys at position 2 appeared in >90% of the selected peptides, while Val at position 3, Trp at position 4, Gln, Cys, Ser, Met, and Thr at position 5, and Ile, Val, and Leu at position 7 appeared in >99% of the selected clones (Fig. 6C and table S4). Similarly, for cluster II, the frequency of appearance of Arg, Ile, Val, and Gln at position 2 was ~93%, whereas for Ile and Val at position 3, Val at position 4, Pro at position 5, Ser and Ala at position 6, and Ile, Leu, and Val at position 7, the frequency of appearance was >97% (Fig. 6D and table S4). Together, our results indicate that the most bioactive motifs against misfolding and aggregation in the investigated macrocycle library are cyclo-(C,T) (R,K)VW (,A,M)X (,P) and cyclo-(C,T) (I,V)VP (S,A) for clusters I and II, respectively, where X is any one of the 20 natural amino acids; is any one of the polar amino acids Q, C, S, and T; is R, I, V, or Q; and is any one of the aliphatic amino acids L, V, and I.

We have reported how a previously developed bacterial platform can be expanded to enable the simultaneous production and functional screening of molecular libraries with greatly increased diversities for the discovery of inhibitors of disease-associated protein aggregation. We have generated a complete combinatorial library of nearly 200 million head-to-tail cyclic heptapeptides in the cytoplasm of E. coli cells and have rapidly screened them to discover inhibitors of the pathogenic misfolding and aggregation of 42. We thus found head-to-tail cyclic heptapeptides that efficiently reduce A42 aggregation and toxicity both in vitro and in vivo. Our highly effective screening methodology, coupled with high-throughput sequencing analysis of the isolated hits, enabled the identification of >400 cyclic heptapeptide putative inhibitors of A42 aggregation. In addition, these results provide further support to our previous observations that lowmolecular weight peptide macrocycles are a very rich source of chemical rescuers of protein misfolding (18) and that they may constitute a promising class of potential therapeutics (33).

Our unbiased selection process yielded distinct groups of bioactive macrocyclic peptides with different sequence characteristics. For the two most dominant clusters, we used a combination of site-directed mutagenesis and deep sequencing analyses to rapidly define the sequence motifs providing optimal bioactivity. These were found to be cyclo-(C,T) (R,K)VW (,A,M)X (,P) for cluster I and cyclo-(C,T) (I,V)VP (S,A) for cluster II, where X is any one of the 20 natural amino acids; is any one of the polar amino acids Q, C, S, or T; is R, I, V, or Q; and is any one of the aliphatic amino acids L, V, or I. Our in vitro validation indicated that these macrocyclic peptides likely exert protective effects by interfering with microscopic reaction steps underlying the aggregation of A, which affect the generation of oligomers over time. In the context of an in vivo system, as observed in C. elegans, where aggregation proceeds on a far longer time scale, this delay in aggregation is much more pronounced and can be considered as effective as an overall arrest of the entire process (34).

To our knowledge, the present work describes the largest screen of small moleculelike molecular entities with the ability to perform direct functional screening beyond simple detection of binding to the target protein described to date. Compared to other reported functional compound screens for misfolding rescuing or other bioactivities in vitro or in vivo (8, 18, 25), we have demonstrated that the system that we described has the ability to generate and evaluate molecular libraries with 20 to 1000 higher diversity than what can be currently achieved. Furthermore, as the diversity of the generated peptide macrocycle libraries are limited only by the theoretical diversity of the library design and the transformation efficiency of E. coli cells, our system can allow the evaluation of libraries with tens or even hundreds of billions of members. Notably, E. coli can support the biosynthesis of not only head-to-tail cyclic peptides, as investigated here, but also side chaintotail cyclic peptides (35), bicyclic peptides (36), lasso peptides (37), -defensins (38), cyclotides (39), and other macrocyclic structures (40) that include both natural and noncanonical amino acids (41). Contrary to other approaches that allow the investigation of even wider areas of molecular space, such as mRNA display (10) and DNA-encoded libraries (7, 42), our technology goes beyond simple detection of binding to the target protein and, instead, selects directly for compounds rescuing aggregation. This is an important advantage, since compound resynthesis and testing for the desired bioactivity following affinity-based selections of DNA- and genetically encoded libraries is time consuming, expensive, and results in a high discovery rate of binders that do not exhibit the desired biological activity (42).

It is noteworthy that the sequences of the 42-targeting cyclic heptapeptide discovered here diverge completely from those isolated from our previous screen that included combinatorial libraries of shorter cyclopeptides (18). This result suggests that, apart from the specific amino acid residues in the primary sequence of the macrocyclic peptide interacting directly with the target protein and are necessary for bioactivity (18), there is probably a conformational component that is also important for molecular recognition between these macrocycles and their targets and that larger cyclopeptide scaffolds are not mere extensions of shorter bioactive sequences. Furthermore, the selected macrocycles bear no resemblance with the sequence of 42, and thus, their discovery would have been very challenging using rational or computationally guided design as, for example, in the case of classical sheet breaker peptides (43) and other designed peptide-based inhibitors of A aggregation (44, 45). Last, also note that the selected cyclopeptides have drug-like molecular characteristics, when compared to those of existing macrocyclic drugs and, in some aspects, to those of conventional drugs as well (table S5).

Our biotechnological approach for producing and evaluating molecular libraries with expanded diversities is not restricted to 42 but is highly versatile and can be applied broadly for targeting a variety of misfolding-prone proteins of both globular and intrinsically disordered nature, as we have shown previously (18). We are currently using this system to screen molecular libraries with expanded diversities, such as the ones described here, and have identified candidate macrocyclic rescuers of the misfolding and aggregation of variants of human Cu/Zn superoxide dismutase and p53, as well as of huntingtin, whose misfolding and aggregation are associated with amyotrophic lateral sclerosis, cancer, and Huntingtons disease, respectively (1).

The biosynthetic production of the lower-weight molecular libraries under investigation and their simultaneous screening for bioactivity in a simple bacterium like E. coli offer great simplicity and speed and reduces the overall cost of the discovery process markedly (7, 12). Once the peptide macrocycle library has been constructed, one can identify the entire repertoire of aggregation inhibitors for a target protein and, at the same time, acquire an initial understanding of structure-activity relationships for the acquired hits in less than a month. The simplicity, speed, and wide applicability of this approach could permit academic and industrial laboratories to simultaneously perform parallel screenings against multiple targets and to prioritize further compound development according to the number and nature of the hits uncovered by the screen. Overall, our approach represents a highly adaptable strategy for investigating molecular libraries with expanded diversities, which enables the discovery of New molecular entities that effectively target peptides and proteins associated with protein misfolding diseases.

The vector sublibraries pSICLOPPS-CysX1X2X3X4X5X6, pSICLOPPS-SerX1X2X3X4X5X6, and pSICLOPPS-ThrX1X2X3X4X5X6 (table S6) were generated as described previously (18). Briefly, the degenerate forward primers GS078, GS079, and GS080 were used together with the reverse primer GS035 and pSICLOPPS as a template (table S6). Cys, Ser, and Thr were encoded in these primers by the codons TGC, AGC, and ACC, respectively, while the randomized amino acids (X) were encoded using random NNS codons, where N = A, T, G, or C and S = G or C. A second PCR reaction was performed in each case to eliminate mismatches using the aforementioned amplified DNA fragments as templates and the forward primers GS069, GS070, and GS071 for each of the peptide sublibraries starting with Cys, Ser, or Thr, respectively, together with the reverse primer GS035. The resulting PCR products were then digested with Bgl I and Hind III for 5 hours and inserted into a similarly digested and dephosphorylated pSICLOPPSKanR vector (18). The ligation reactions were optimized at a 12:1 insert:vector ratio and performed at 16C for 4 hours. Approximately 10 g of the pSICLOPPSKanR vector was used for each sublibrary. The ligated DNA was then purified using spin columns, transformed into electrocompetent MC1061 cells, plated onto LB agar plates containing chloramphenicol (25 g/ml), and incubated at 37C for 14 to 16 hours. This process resulted in approximately 1.2 billion independent transformants, as judged by plating experiments after serial dilutions.

Electrocompetent E. coli Tuner (DE3) cells (Novagen, USA) carrying the expression vector pETA42-GFP (24) were cotransformed with the combined pSICLOPPS-NuX1X2X3X4X5X6 vector library. Approximately 109 transformants carrying both vectors were harvested, pooled together, and diluted to an OD600 of 0.1 in LB liquid medium containing 0.005% l(+)-arabinose to induce cyclic peptide production. Cultures were incubated at 37C with shaking until an OD600 of 0.4 to 0.5, at which point 0.1 mM IPTG was added to the medium to induce overexpression of the A42-GFP reporter. Fluorescence of 50,000 cells was recorder after 2 hours of induction at 37C using a BD FACSAria II system (BD Biosciences, USA) with a 488-nm solid-state laser for the excitation of GFP and a 530/30 band-pass filter for detection. Then, ~3 109 cells were gated on a side-scatter (SSC-H) versus forward-scatter (FSC-H) plot to eliminate noncellular events and were subjected to FACS for the isolation of the bacterial population exhibiting the top ~2% fluorescence. The isolated cells were regrown and screened for six additional rounds in an identical manner, at which point DNA was isolated from the enriched pool using a Qiagen Plasmid Mini Kit.

High-throughput sequencing analysis was performed at the Genomics Core Facility of the Biomedical Sciences Research Center Alexander Fleming (Athens, Greece) using an Ion Torrent high-throughput sequencing platform. Briefly, the combined pSICLOPPS-NuX1X2X3X4X5X6 vector library and the enriched peptide library after the seventh round of sorting were digested with Nco I and BsrG I, and the resulting ~250 base pair (bp) products that contained the variable peptide-encoding region were isolated and analyzed. Ion proton reads were aligned to a reference sequence using Bowtie2 (v2.2.8). The alignment information stored in the CIGAR string of the resulting Sequence Alignment Map file was parsed and mapped to matching and mismatching sequences using the tool Biostar59647 of the JVarkit utilities. From the resulting XML file, a custom awk script extracted the mismatching insert sequences, which were then clustered using the CD-HIT tool (v4.6.1) (46), together with their read counts. From the obtained data, only the 21-bp-long peptide-encoding sequences with NNS codons were subjected to further analysis. For the enriched peptide library, all sequences including stop codons were also discarded from subsequent analysis.

Sequence similarity analysis was performed using the Immune Epitope Database clustering tool (http://tools.iedb.org/cluster2/) and the fully interconnected clusters (cliques) method (47). This approach allows all peptides in a clique to share a minimal level of identity, while at the same time, one peptide can be part of multiple cliques (47). As sequence similarity analysis was performed using linear sequences, the circular permutants of each cyclic heptapeptide appearing at least 20 times within the sorted population were identified and taken into consideration, tallying up to 2912 linear representations for the 416 cyclic heptapeptides. From this analysis, 5087 cliques sharing at least 70% sequence identity were identified, and after reintegration of the different circular permutants to their original cyclic peptide sequence, 617 unique cliques remained. From the 416 distinct cyclic heptapeptides, 323 were covered in the cliques forming a total of 1467 unique pairs with more than 70% sequence identity. The remaining 93 cyclic peptides did not share a minimal level of 70% identity with any other of the peptides. The results were then presented in an undirected network graph using the Gephi graph visualization software (48), and cluster identification was performed using the Girvan-Newman Algorithm (49).

Kinetic experiments were performed as described previously (30). Briefly, appropriate amounts of the synthetic cyclic peptides were added to 2 M of monomeric A42 to obtain the desired cyclic peptide:42 molar ratios, and samples were supplemented with 20 M ThT, 1% (v/v) acetonitrile, and 0.025% or 0.1% (v/v) Tween 20 for C7-1 and AC7-14, respectively. Under these conditions, both C7-1 and AC7-14 remained stable in a monomeric state for the duration of the in vitro experiments, as judged by dynamic light scattering analyses. All samples were prepared in low-binding Eppendorf tubes on ice using careful pipetting to avoid introduction of air bubbles, and each sample was pipetted into three wells of a 96-well half-area, low-binding, clear-bottom, polyethylene glycol-coated plate (Corning 3881), at 80 l per well. The 96-well plate was then placed at 37C under quiescent conditions on a plate reader (Fluostar Omega, Fluostar Optima, or Fluostar Galaxy; BMG Labtech), and after excitation at 440 nm, ThT fluorescence was measured at 480 nm, through the bottom of the plate.

Strains. The following strains were used for this experiment: (i) GMC101, herein referred to as A worms; genotype dvIs100 [unc-54p::A-beta-1-42::unc-54 3-UTR + mtl-2p::GFP]; mtl-2p::GFP constitutively expresses the GFP in intestinal cells; unc-54p::A-beta-1-42 expresses A42 in body wall muscle cells, resulting in A42 aggregation and worm paralysis after temperature upshift from 20 to 25C (31). (ii) N2, wild-type C. elegans var Bristol, herein referred to as wild-type worms (50).

Propagation procedures. C. elegans worms were propagated using standard conditions and as described previously (30, 50). Briefly, the worms were treated with hypochlorite bleach, and eggs were hatched overnight in M9 buffer [KH2PO4 (3 g/liter), Na2HPO4 (6 g/liter), NaCl (5 g/liter), and 1 mM MgSO4] and then distributed on nematode growth medium (NGM) [1 mM CaCl2, 1 mM MgSO4, cholesterol (5 mg/ml), 250 mM KH2PO4 (pH 6), agar (17 g/liter), NaCl (3 g/liter), and casein (7.5 g/liter)] plates seeded with the E. coli OP50 cells and incubated at 20C. Upon reaching the L4 stage, ~700 worms were placed on NGM plates containing the desired concentration of the cyclic peptides in 1% (v/v) acetonitrile. Synthetic cyclic peptides were provided to the nematodes as is, without any additional steps to enhance their permeability. At that point, 75 M 5-fluoro-2deoxyuridine was also added to the plates to inhibit growth of offspring. The plates were then transferred to 24C to promote A42 expression and aggregation.

Motility assay. On days 5 to 10 of adulthood, worms were collected using M9 buffer and distributed on unseeded 9-cm NGM plates. The worms movements were recorded at 30 frames/s for 1 min using a homemade microscopic setup, and the body bends were quantified using a custom-tracking algorithm as described previously (30, 51). In total, ~2300 worms were analyzed per peptide with an average of ~200 worms per experiment. Total fitness refers to the sum of the mobility and speed of the worms.

Aggregate quantification. Staining and microscopy were performed as described previously (30). Briefly, live animals were stained by incubating with 1 M NIAD-4 [0.1% (v/v) dimethyl sulfoxide in M9 buffer] for 6 hours at room temperature and then transferred on NGM plates to allow destaining for about 16 hours. Stained worms were then anesthetized by adding 40 mM NaN3 and mounted on 2% agarose pads on glass microscope slides. Images were captured using a Zeiss Axio Observer D1 fluorescence microscope (Carl Zeiss Microscopy GmbH) with a 20 objective and a 49004 ET-CY3/TRITC filter (Chroma Technology Corp.), and fluorescence intensity was calculated using the ImageJ software (National Institutes of Health). Only the head region of the worms was examined because of the high background signal in the intestine.

Statistical analyses were performed using Prism (GraphPad Software Inc., La Jolla, CA, USA), and mean values were compared using unpaired two-tailed t tests. For animal experiments, group sizes were chosen on the basis of prior experience and literature precedence so that sufficient numbers remained at the endpoints of the experiment. No samples, worms, or data points were excluded from the reported analyses.

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaax5108/DC1

Section S1. Supplementary Materials and Methods

Fig. S1. Identification of potential A42 aggregation inhibitors using a bacterial genetic screen.

Fig. S2. Identification of different cyclic peptide clusters appearing in the sorted population.

Fig. S3. C7-1 and AC7-14 inhibit the aggregation of A42 in vitro.

Fig. S4. C7-1 and AC7-14 inhibit the aggregation of A42 in vivo.

Table S1. Deep sequencing analysis of the peptide-encoding regions of ~3.4 million clones from the constructed pSICLOPPS-NuX1X2X3X4X5X6 library.

Table S2. Enrichment (blue) and depletion (red) of the 20 amino acids in each position of the heptapeptide sequences.

Table S3. Distribution of the heptapeptide sequences in the different clusters identified.

Table S4. Sequences and frequency of appearance of cluster I and cluster II heptapeptide sequences as determined by high-throughput sequencing of the enriched library after the seventh round of sorting.

Table S5. Molecular properties of the selected cyclic heptapeptides AC7-1 and AC7-14 compared to those of conventional drugs, oral macrocyclic (MC) drugs, and nonoral MC drugs.

Table S6. Plasmids and PCR primers used in this study.

References (52, 53)

This is an open-access article distributed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Acknowledgments: Nematode strains used in this study were provided by the Caenorhabditis Genetics Center, supported by the National Center for Research Resources of the U.S. National Institutes of Health. We thank S. Casford (University of Cambridge, UK) for technical assistance with the in vivo experiments, V. Harokopos (Biomedical Sciences Research Center Alexander Fleming, Athens, Greece) for performing the deep sequencing, and E. Pappou (National Hellenic Research Foundation, Athens, Greece) for performing the flow cytometric phenotypic measurements. G. Georgiou (University of Texas at Austin, USA) is acknowledged for facilitating the flow cytometric sorting experiments. Funding: This work has received funding from the following: (i) the European Research Council (ERC) under the European Unions Horizon 2020 research and innovation program (Project ProMiDis; grant agreement no. 819934); (ii) the project STHENOS-b (MIS 5002398), which is funded by the Operational Programme Competitiveness, Entrepreneurship and Innovation (NSRF 2014-2020) and cofinanced by Greece and the EU (European Regional Development Fund); (iii) the project NEUROTHERAPY in the framework of the research grant Aristeia; and (iv) the project STHENOS in the framework of action KRIPIS, the last two financed by the Hellenic General Secretariat of Research and Technology (GSRT) and the National Strategic Reference Framework (NSRF 2007-2013). We also acknowledge support by a short-term scientific mission grant from COST Action BM1405 to D.C.D. Author contributions: G.S. conceived and coordinated the project. G.S., D.C.D., J.H., N.C., and M.P. designed the research. D.C.D., S.C., I.M., and N.P. performed the research. D.C.D., M.P., S.C., N.P., and G.S. analyzed the data. G.S., M.V., J.H., C.M.D., and N.C. supervised the research. G.S. and D.C.D. wrote the paper. All authors read and approved the final manuscript. Competing interests: G.S. is inventor on the patent application PCT/IB2018/000622 describing aspects of the herein described technology. G.S. and D.C.D. are inventors on a patent application for C7-1, C7-14, and other -targeting peptide macrocycles described in this article, which is currently in submission to the Hellenic Industrial Property Organisation. G.S. and D.C.D. are founders and equity holders of ResQ Biotech P.C. The authors declare no other competing interests. Data and materials availability statement: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Bacterial production and direct functional screening of expanded molecular libraries for discovering inhibitors of protein aggregation - Science...

3 Best Ways to Remove a Smiley from Pictures Online – Guiding Tech

A picture is modifiable in several ways. You can add text to it, crop it into shapes, add effects and filters, give emotions to it by adding emojis on it. Sometimes, after adding emojis on a photo, we want to view the original picture without the smiley. If you are still editing the photo, you can undo the changes, but if you have saved the copy and you dont have the original file, how do you remove the smiley from your image?

In this post, we will tell you how to remove smiley from your images. The same concept can be used to remove text and other objects without Photoshop.

However, its important to know that removing the smiley will not replace it with the original background. It will be swapped with the surrounding background that will make it look like an unedited photo.

Note: Sometimes, we use emojis to hide faces. Removing emojis will not show you the face behind the emoji.

Lets check out the three ways to remove emojis online from your pictures without downloading any software.

With these editors, you need to brush on the object that you want to delete. The tool will automatically replace the object with a nearby background. You can remove emojis, text, and even humans from photos. Here are two editors to help you.

Inpaint is one of the easiest tools usable to remove emojis. All you need to do is add a mask on the object that you want to remove and hit the Erase button. Voila! You will be astonished by the results. Here are the steps:

Step 1: Open the website using the link given below.

Visit Inpaint Online

Step 2: Click on Upload Image and add your image from which you want to remove the smiley.

Step 3: By default, the eraser marker will be selected. However, if you want to confirm the same, click on the red icon on the left side.

Step 4: Start brushing on the smiley that you want to remove. A red mask will be added to it. Color the entire object with the mask. You dont have to take care of the borders strictly. You add color outside the object too and the site will still detect the object automatically. Then hit the Erase button at the top.

Step 5: The tool will replace the emoji with the existing background.

Step 1: Launch the website and click on Upload file to add the image.

Visit GifGit

Step 2: Scroll down to view the items present in the left sidebar. Click on Clone.

Step 3: Just like the clone tool on any app, you need to select the cloning area. For that, press Alt key on your keyboard and click the area to select it. Once selected, hold the mouse button and brush the emoji with the cloning area.

Sometimes, the emoji can be easily cropped from the picture without affecting the actual image. Then you dont need to use either the heal or clone tool. However, since you are cropping the image, you will lose a part of the image. Use this method only when the emoji is at the borders. You can use online tools like BeFunky, LunaPic, Fotor, and more to crop the image.

Photopea, one of the best online replacements of Photoshop, can be used to remove emojis from photos without having any prior knowledge about photo editing. The tool offers both heal and clone mode, among many other photo editing features.

Visit Photopea

Open the website and click on the Healing brush icon present in the left sidebar. Click on Healing Brush tool from the menu.

Press the Alt key on your keyboard and click the mouse button simultaneously to select the source. Then stroke the image to replace it with the background.

To use the clone mode, click on the Clone icon and select the cloning area by hitting Alt key and mouse button.

The above methods help in removing the emoji by covering it with the surrounding background. As mentioned before, the faces under the emojis cannot be unmasked. The same is true for Twitter stickers. While you can swap it with the background, the underlying data be it text or face cannot be seen.

Next up: Want to add color to your old black and white pictures? Do it with these online tools.

Last updated on 15 Oct, 2019

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3 Best Ways to Remove a Smiley from Pictures Online - Guiding Tech

How ‘Gemini Man’ Gets Human Cloning Wrong – Hollywood Reporter

Films depicting clones, in showing us their versions of what two humans with identical genomes would be like, and how they would relate to each other, take their own stances on the nature vs. nurture debate. While a wide variety of clones have made it to screens over the years, the differences between them have not really followed trends or evolved in ways reflective of our ever-growing scientific understanding.

The scientific knowledge demonstrated by most clone films is elementary at best, and Gemini Man is no exception. In a pivotal scene, Henry (Smith) tries to appeal to his clone, Clay (also Smith), by emphasizing their connection through listing traits he presumes the other man must share. There are some that make sense. He, for instance, mentions a hatred of cilantro, which several investigations have linked to a particular allele (that is, a version) of the OR6A2 gene that causes individuals to taste a strong soapy flavor in cilantro that others do not. However, the shared trait which the film gives the utmost importance a severe bee sting allergy is actually one of the worst choices it could have made in terms of accuracy. While some studies have found evidence that certain alleles of particular genes might potentially predispose individuals to a bee sting allergy, it is a tentative link, and far more evidence has connected the development of allergies to environmental factors. Long story short, there is no bee sting allergy gene. In Gemini Man, Clay feels the need to test Henrys allergy in order to prove the man is truly his genetic identical, and while its a set-up that raises the stakes and makes for a tense scene, in actual fact it makes no sense at all. Allergies dont work that way.

But all of this still deals with what we actually do know, and writers not doing their researchor, more forgivingly, taking significant creative license. Putting inaccuracies aside, perhaps one of the most interesting things about cloning stories is how so many lean heavily on the side of genetic determinism the nature side of the nature vs. nurture debate. Most clone films, Gemini Man included, hold genes fully responsible for things, like allergies, in ways that are contrary to our current scientific understanding, so its perhaps not surprising that they also tend to credit genes for things for which scientists have no conclusive answers, like personality, taste, likes and dislikes.

Many of the most concerning narratives and prescient fears revolving around cloning and genetic engineering also depend on a high degree of genetic determinism. Neo-eugenics and the prospect of designer babies depend on traits like intelligence being genetically hard-wired. If whatever made Mozart Mozart and Einstein Einstein is environmental or some quality that defies quantification that is, anything other than genes these controversial schools of thought lose much of their steam.

Another intriguing trend regarding on-screen cloning is the frequency with which the very term cloning is misapplied. Clones are genetically identical. You take the DNA-housing nucleus from an existing individual and, for lack of a more apt analogy, cut and paste it into a fertilized egg that has had its nucleus removed. You dont mess with the DNA at all. Changing the DNA is genetic engineering. So, if you take someones DNA, modify it, and then use that DNA to create a new individual, that new individual is not a clone. Almost every major film and TV show to tackle cloning has in actual fact been about genetic engineering. In Moon, for instance, the short-lived Sam clones made to mine helium-3 on the moon are not really clones at all because they have been genetically modified to have a limited lifespan.

Gemini Man is somewhat unusual in that it does, to some extent, implicitly make a distinction between a clone and a genetically engineered individual in the difference between Clay, a genuine clone, and the unnamed adolescent super-soldier clearly modified from Henrys DNA. However, the film never really makes this distinction explicit and fails to clarify that clone isnt a catch-all umbrella term for individuals conceived deep in some secret laboratory.

Fictional depictions of cloning and genetic engineering matter because they shape the narrative on these subjects. They are the points of reference the general public uses to discuss these issues; the frames news media use to present new developments in these fields. From touch screen technologies to space travel, movies have long been a place to explore the possibilities of tomorrow, and in doing so play a role in the future that comes to pass. There are many fascinating possibilities and important debates to be had regarding the potential and concerns surrounding cloning and genetic engineering, but currently the way these issues are treated in film reflect more the presumptions of their writers than anything else. Movies present a great opportunity to ruminate and reflect on scientific frontiers, but unfortunately, particularly where genetics is concerned, they seem to have no interest in the reality of their subject matter.

Ciara Wardlow is a pop culture journalist and recent graduate of Wellesley College, where she studied Cinema and Media Studies as well as Biological Sciences.

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How 'Gemini Man' Gets Human Cloning Wrong - Hollywood Reporter

World’s first cloned cow Kaga dies aged 21 outliving twin who died last year – Daily Star

The world's first cloned cow has died at the age of 21 after living a healthy life.

Twins Kaga and Noto were the first ever cows to be born using the same technology that created Dolly the Sheep.

The clones were born as part of a research experiment at the Ishikawa Prefectural Livestock Research Centre in Japan.

Contrary to beliefs at the time, both of the cows proved to be healthy and lived as long as regular cows.

Noto was the first of the twins to die last year, being found unconscious in a barn on May 4 last year.

And now Kaga has become the last of the twins to die aged 21.

The bovine clone, which attracted worldwide attention when it was born, began having problems standing up in September.

After being given nutritional supplements and health drips, health officials confirmed the cow could no longer stand.

Kaga was pronounced dead yesterday, with its cause of death believed to be old age.

After the birth of the twins, 14 cloned cows were produced in 2006 to improve meat and milk production in Japan.

It was hoped the animals would pave the way for more cows to be cloned in Japan.

But these were quashed when the country outlawed cloned cow meat over fears of its safety.

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Since Kaga and Noto, a host of animals have received the cloning treatment.

This year, China cloned its first ever pet cat for a bereaved owner.

Scientists in the country are also thought to be able to clone dogs through their own urine.

Prehistoric beasts such as wooly mammoths are also thought to be on the horizon, due to cloning.

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World's first cloned cow Kaga dies aged 21 outliving twin who died last year - Daily Star

‘Gemini Man’ Review: Uncle Kill and the Fresh Prince Star In Moron Clone Wars – Pajiba

Stories about twins can be powerful metaphors. They can be profound meditations on whether its nature or nurture that makes us who we are. They can be explorations of our darker halves made flesh. They can be mysteries about Arnold Schwarzenegger and Danny DeVito sharing most of their DNA. Or dramatic short films during the Super Bowl pondering why anyone would intentionally drink Coors.

Gemini Man is none of those things. Its a shittily shot action film that uses the CGI gimmick of a de-aged Will Smith as its sole point of creativity. No thoughtfulness, no contemplation, not even leaning into the humor of the concept. It is such a catastrophic waste of an insanely talented cast: Will Smith, Mary Elizabeth Winstead, Clive Owen, and Benedict Wong have so many better movies they could be making other than this.

The entire movie is in the trailer, so, well, spoilers follow which amount to telling you that in the movie about clones, the clones fight. And then they become friends. Sorry for ruining that for you. Theres old Will Smith and young Will Smith. Lets just go with calling them Uncle Kill and the Fresh Prince. Uncle Kill is the worlds bestest assassin but he wants to retire. Naturally, instead of giving him a pension to enjoy on his obligatory fishing boat on the gulf coast, his old bosses start sending death squads of SAG extras to die.

The bad guy is Clive Owen, playing the evil mercenary who not only runs a giant Blackwater style private army, but has the resources to have his own cutting edge genetics lab that does human cloning. When Uncle Kill turns down a job offer twenty years before this masterpiece of cinema is set, Clive clones him and secretly raises the Fresh Prince as his own adopted son. As one does.

As an aside, its more like the Fresh Prince of the Uncanny Valley, because this CGI is baaaaaaaaad. Most of the movie is dark enough that you cant tell, but sweet Jesus the mandatory epilogue set six months later in happy sunny days is painful to watch. Its like someone downloaded an animated GIF of Will Smith in 1995 on a spotty modem: noisy, spastic, and just disturbing. Also, they turned the dial up a bit too high on the de-aging algorithm, because the Fresh Prince looks at most fourteen despite them telling us hes 23. Like, he looks creepy young throughout.

So its basically The Bourne Identity with cloning instead of amnesia, and bad action. You see Ang Lee in the opening credits as director, and you at least expect the action to be decent. But no. The action is shot to make Fresh Prince and Uncle Kill move with superhuman speed and take blows that would sit Captain America down, which just adds to the cognitive dissonance of the bad CGI since this isnt a superhero movie. At one point the Fresh Prince beats Uncle Kill up with a motorcycle. On screen it makes even less sense than that sentence does on screen.

Its humorless except occasionally by accident when the stupidity of the script pushes an involuntary snort out of your bored catatonia. The movie is so absurdly and idiotically conceived that its only hope was to wink at the camera for the whole thing. One clone? Fuck it, make it fifty. I completely fail to understand why people keep casting Will Smith in movies and tell him to not have any fun. How do you take a men with more raw charm and humor than should be legal, stick him face to face with a CGI young version of himself, and have him mutter shit like its like looking in a mirror instead of a half dozen ad-libbed versions of damn son, I knew I was a good looking man?

A fantastically fun and surprisingly deep movie could have been made out of this premise. Take the old killer and see if the parts of what makes him a killer are built-in or were developed through experience. Theres a point at which Uncle Kill rattles off a whole laundry list of things he knows about the Fresh Prince because theyre the same person. It involves insights like having nightmares, being scared of opening up to anyone, still being a virgin at 23, and only being happy when shooting a gun. This just in, none of those things are genetic.

In fact, they use the fact that theyre both allergic to bee stings as a major plot point. The only problem is that like all of those other things, allergies to bee stings arent genetic. Ouch. Oops. But that would take a cursory google search, which is clearly beyond whatever idiot wrote this. *listens to earpiece for a moment* Yes, this just in, said idiot was David Benioff, master of such literary heights as the final season of Game of Thrones. Everything is making sense now. More like Castor and Pollsux, am I right?

Just go rewatch the Coors Light twins commercial if you need a clone fix. It has more depth and pathos than this mess.

Dr. Steven Lloyd Wilson is a hopeless romantic and the last scion of Norse warriors and the forbidden elder gods. His novel, ramblings, and assorted fictions coalesce at http://www.burningviolin.com. You can email him here.

Steven Lloyd Wilson is the sci-fi and history editor. You can email him here or follow him on Twitter.

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'Gemini Man' Review: Uncle Kill and the Fresh Prince Star In Moron Clone Wars - Pajiba

From Gemini Man to Living with Yourself, Hollywood has an obsession with clones – digitalspy.com

In Gemini Man, Will Smith is forced to battle a younger version of himself who can predict his every move. Paul Rudd merely has to cohabit with his own better self in Netflix's imminent Living With Yourself. In doing so, they join a small group of stars, including Keanu Reeves, Arnold Schwarzenegger and Michael Keaton, whom Hollywood has cloned or otherwise duplicated for our entertainment.

Cinema has a long-held fascination with its actors taking on multiple roles. While some of English-language filmmaking's earliest stars, like William Bergman and Buster Keaton, would often play more than one role within any one picture, the first doppelgnger film was a 1913 German art film, A Student of Prague, co-directed by Stellen Rye and Paul Wegener.

A young college student, played by Wegener, trades his own mirror reflection to a sorcerer for gold so he may court a rich Countess. Eventually driven mad by the constant pursuit of his exact double, he tries to murder his twin, killing himself in the process. A macabre piece of fantasy-horror, the picture spearheaded technology that allowed the same actor to appear on screen with himself, creating a template for others to follow suit.

In 1936, British-made comedy The Man in the Mirror took a similar approach, having Edward Everett Horton's reflection begin talking to him before stepping out of the mirror and living the hard-partying life he never had the confidence for.

Though it's played more light-heartedly here, the existential terror of replacement would become a prevalent theme within the subgenre.

Works during the fifties like The Invasion of the Body Snatchers and I Married An Astro-Monster channeled the same fear through a political lens. Aliens trying to invade Earth through cloning or taking human identities were used as a stand-in for American paranoia over the 'invisible invasion' of Communism. The doubles acted suspiciously out of character and it was up to those who knew something was different to put things right.

1970's The Man Who Haunted Himself took this a step further, as Roger Moore's protagonist wakes from a car accident to find his life upended by someone everyone else thought to be him. In what Moore considers his best film, this Basil Dearden-directed thriller explores the psychological underpinnings of the concept by earnestly following Moore's perspective to the last, only revealing the truth in the climax.

The second, more well-known and distinctly darker version of Invasion of the Body Snatchers followed towards the end of the decade, whose morbid twist on the ending contains this heavily-giffed final shot. Filmmakers were shedding the inherent belief that someone would figure out a clone from the original, using the mystery to captivate audiences and dig into themes of conformity, humanity and personhood.

John Carpenter brought much of this to a head in The Thing, a tightly wound fusion of high-concept sci-fi and horror that turned Kurt Russell into a one-man army against a shape-shifting alien force that usurps his Antarctic colleagues one by one.

Thanks to movies such as Robocop, Evil Dead and Nightmare on Elm Street, science fiction and horror moved towards a more satirical, self-aware tone during the '80s and '90s.

Actors playing multiple roles came back in popularity, including cloning rom-com Multiplicity starring multiple Michael Keatons. Jackie Chan shouldered double duty in the quasi-doppelgnger action film involving estranged twins, Twin Dragons, as did Jean-Claude van Damme for Double Impact.

Director Sheldon Lettich commented that Van Damme enjoyed playing twins because they let him showcase his range, and the picture was so successful, Van Damme did it twice more for Maximum Risk in 1997 and Replicant in 2001. Jeremy Irons played less boisterous (but much weirder) twins in David Cronenberg's body horror Dead Ringers.

Arnold Schwarzenegger has played more Terminators than we can count (and to pull off its special effects T2: Judgment Day employed real-life identical twins Linda and Leslie Hamilton and Don and Dan Stanton). He returned to replication in The 6th Day awash in technological anxiety, this 2000 movie imagines a future where cloning anything except a human is legal and widely accepted.

Explosive and markedly less neurological than the likes of previously mentioned The Man Who Haunted Himself, there's still a poignancy to Schwarzenegger's turn as a family man stuck chasing bad guys rather than having a quiet night in. Being a performer for a living doesn't often include much down time, and the threat of being replaced by the next best version of you is forever looming.

Doppelgnger movies made a gradual resurgence later in the 2000s, particularly among indie and arthouse directors and actors. Duncan Jones' minimalist drama Moon contains a career-best pair of performances from Sam Rockwell as two conveyor belt clones who decide to burn down the factory, while Jesse Eisenberg is driven to madness when a sleeker, smoother model of him takes over his life before his very eyes in Richard Ayoade's The Double.

Similarly, in Denis Villeneuve's Enemy, Jake Gyllenhaal portrays a shy, introverted, emotionally closed off college lecturer who develops an obsession with a lookalike who proves to be his exact opposite.

These all use symbolism of clones and exact doubles to visualise difficult, deep-rooted fears surrounding identity, labour, personal value and desirability. They ponder the surreal mix of terror and curiosity that would result from seeing your fully formed self, but different, existing in the world. They contrast the life you have with the life you could have had, if you were just a tad more confident and a mite less neurotic; if you were raised in an altogether better environment.

Claudette BariusUniversal

That quandary forms the spine of Jordan Peele's Us, released earlier this year, where Lupita Nyong'o's family are terrorised by their clones. In Us, clones are "tethered" to the originals, forced to copy their every move as a population of copies live out their lives in an abandoned underground facility. As in Moon, Peele uses man-made doppelgngers to discuss the expendability of the lower class and lower-skilled labour.

One of the creepier moments features Elisabeth Moss' Tethered putting on lipstick in the mirror for the first time, finally getting to see herself the way her counterpart did. In Us, Peele shows us the Tethered's point-of-view, letting us feel the resentment and animosity and demonstrating that we would be as uncanny to any clone as they would be to us.

Will Smith has his work cut out keeping up with his double in Gemini Man. The former Fresh Prince and Man In Black is squaring up against his own youth, a terrifying prospect for anyone. Paul Rudd, meanwhile, only has to face his own inadequacy in Living With Yourself.

For better or worse, it seems Hollywood is finally taking a long hard look at itself.

Gemini Man is out in cinemas on Friday, October 11. Living with Yourself is on Netflix on October 18

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From Gemini Man to Living with Yourself, Hollywood has an obsession with clones - digitalspy.com

Theatre review: A Life Twice Given – E&T Magazine

This family drama about a couple who challenge Gods authority by cloning their deceased son has some enjoyable and moving moments, but fails to humanise the conflict at its heart.

A Life Twice Given is based on a novel by David Daniel, who explored his own fantasy of cloning his firstborn, David, after his death at the age of seven. The story has been adapted for the stage by playwright Gail Louw, in a production directed by John Burrows.

The play follows a mostly-secular Jewish couple living in rural Virginia, David (Johnny Neal) and Lisa (Natalia Campbell), who lose their young son David (Damian Reyes-Fox) in a car accident. While Lisa grieves, David quietly plots to replace his lost child with a clone, having had the foresight to scrape and preserve skin cells from his dying sons ear.

When he finally shares his plan with his wife, Lisa is appropriately horrified, insisting that they accept their loss and move on to have more children the old-fashioned way. Unfortunately for Lisa, Davids plan is already set in motion; he has the approval of a sympathetic rabbi who advises that man was intended to be Gods partner in creation. After rejecting Davids plans twice, Lisa relents when he takes her to meet a morally ambiguous academic in Prague, who uses both rational and otherworldly arguments suggesting that Young Davids spirit is wandering, desiring a second chance at life to convince her to carry a clone. The pushy academic is also portrayed by Reyes-Fox, decked out in spectacles, overcoat, and enjoyable accent, hinting at the possibility that Young Davids spirit is not so much wandering lost as much as sitting at the table opposite Lisa, prodding her towards agreement.

Reyes-Fox delivers convincing performances as three different characters, handling David 2s discovery of his origins with a burst of pain and anger at his parents selfishness which never spills into overacting.

There are some amusing moments, particularly Lisas incredulity when she discovers that David took cells from his sons ear (Did he cut off his ear? Did he store it in his wallet?), and the playful dance breaks performed by Reyes-Fox between scenes. The play also has its moments of clumsiness: an extended discussion between David and his son about new worms being regenerated from damaged bodies is as subtle as a brick through a window; Young Davids twin brother Noah vanishes halfway through the play never to be mentioned again; and the slightly wonky projections should probably have been abandoned in favour of a true black box.

A Life Twice Given could be accused of being regressive; it appears to conclude that technology cannot be harnessed to cheat Gods plans and the malach hamavet. However, the complications faced by David 2 and his parents in the plays final moments could just as legitimately be blamed on the hurried adoption of technology before it is fit for purpose; given the growing international pushback against tech bros and their move fast and break things culture, this interpretation feels more relevant and satisfying.

Despite intentions to explore the clash of deep-set religious tradition with biotechnology empowering humans to play God, A Life Twice Given does not plunge into the emotional stakes of this debate with the depth we should expect from a drama. Numerous references to tradition unavoidably bring to mind Fiddler on the Roof: the gold standard for theatrical depictions of conflict between traditional Jewish life and the exciting possibilities offered by the modern world (a conflict that tears Tevye and his family apart). In A Life Twice Given, however, David and Lisa exchange familiar arguments in the cloning debate, and in spite of the emotive context of a lost child, the conflict never felt truly personal.

A Life Twice Given is performed at London's Gatehouse, Guildfords Yvonne Arnaud Theatre, Londons Jermyn Street Theatre, Kents Astor Community Theatre, and Brightons Rialto Theatre. Tickets from 16.

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Theatre review: A Life Twice Given - E&T Magazine

What is Genetic Privacy and Does it Matter? – TechNadu

Genetic privacy is a topic that should be on everyones lips. Life is information and information is life, after all. With new technologies that allow us to sequence the genes of anyone quickly and cheaply, there are major ethical concerns popping up all the time. Modern and future genetic science as a whole holds immense promise. A promise to make our lives better, but at the same time, it can be abused towards horrific ends. Just like atomic energy or any other branch of scientific knowledge, its how we decide to use these powers that determine if they are ethical or not.

So what the heck is genetic privacy? Its a pretty complex topic, but before we can talk about the privacy aspect, we need to talk about the genes themselves.

The entire blueprint for who you are physically is in your genes. Every cell in your body (with some exceptions) contains a copy of your genome. The instructions that describe how a particular person should be put together. All human beings have genes that are 99% identical. We are, after all, a single species. However, the remaining 1% is responsible for some pretty dramatic differences between individuals. Your hair and skin color, your build and other physical traits are all determined by your genes.

Your genes even determine how you will react to certain environmental factors. Many problems that result from lifestyle choices, such as obesity or addiction to certain substances, have at least some link to your genes.

With the exception of identical twins, every person has a unique combination of genes. The odds of two people who are not twins (or clones) having identical DNA are astronomical. So much so that it is for all intents and purposes impossible.

You also leave your DNA around everywhere. Since its in every cell, wherever you shed cells there youll find copies of your DNA. With the cost of genetic sequencing falling rapidly, there are plenty of privacy issues to worry about when it comes to your genes.

What does it mean to have genetic privacy? As a whole, it seems we are still figuring this out. While some countries have laws in place that describe what third-parties are allowed to do with your genetic information, others still have to catch up.

Its pretty common that technology outpaces the ability of laws and policies to keep up with it. We dont know that we need certain laws until people do things with these new technologies that are harmful but couldnt be predicted.

One of the big problems is that you have no choice but to hand over your genes to other people. If you get a blood test, you have to give blood. That blood has pristine copies of your DNA in it. This goes for just about all medical waste. If you blow your nose and throw it away, it probably isnt illegal for someone to fish the tissue out of the bin and go sequence your genes.

So it should be obvious that genetic privacy is not about limiting whether someone can harvest your genetic material. It would be impossible to prevent it. Rather, genetic privacy is about limiting what others candowith your genetic information without your explicit consent.

Depending on where in the world you live, the legal protection of your genes can be rather good or decidedly spotty. The US, in particular, has large gaps in its genetic privacy laws. In 2017 a bill was approved that would allow your employer in the US to demand access to your genetic information.

The fact is that theres no adequate legal protection for your genetic information anywhere in the world. Even if you are resident in a part of the world with comprehensive genetic data protection, nothing can provide a 100% guarantee that your genes wont end up where they shouldnt.

In almost every country there are one or more medical certification boards. Anyone working in the medical field needs to be registered with this board in order to legally practice their profession. This obviously includes physicians but also includes people like radiologists and lab technicians who handle human materials.

These medical boards already have a significant role in protecting patient privacy. If your doctor breaches the strict rules of patient confidentiality they can be fined or lose their license to practice. With the advent of cheap, fast gene sequencing its important for such boards to update their conception and practice of patient privacy.

These boards have significant powers over what those in the medical field are allowed to do, but their scope is limited when it comes to who they can fine or punish. Which means medical boards alone arent enough to enforce genetic privacy.

Genetic privacy doesnt mean someone cant look at the contents of your genes. At least not in every context. Its also about controlling what they are allowed to do in that context.

Lets say you have a genetic predisposition to heart disease or diabetes. Life insurance companies may be unwilling to cover you if your genes say you are likely to get these diseases. Even if your lifestyle or other non-genetic factors meant you were never actually going to get the disorders. In the wrong hands, your genetic information can be used against you in a number of ways. Even for forms of prejudice based on your ancestry.

Its very important that strict rules are in place to dictate what an organization is allowed to do with genetic information about you. To ensure a free and fair society.

When routine samples are taken from your body that contains genetic information, should gene sequencing be done without your consent? If rapid DNA testing machines become commonplace and truly cheap, its not far-fetched to think that a given lab might just throw sequencing in as a normal part of the test battery.

The thing is, genetic sequencing isnt the same as cholesterol or blood sugar test. While clever cross-referencing of multiple test results mightpoint someone to a specific individual, these tests are just not unique enough to reveal someones identity.

A gene sequence is 100% specific to the individual by itself. If you have someones genetic information, you have their identity and procuring another sample to test against for confirmation is relatively trivial.

This absolutely means that you need the full informed consent of someone before running their samples through a sequencer and storing it on your computer systems. They need to know your policies and security measures before handing over that information.

As new medical tests and methods become affordable, there is a certain branch of thought that says we should afford every newborn the potential benefits of that technology. We had more or less the same conversation when the power of stem cells became apparent. Today we can make stem cells from other types of cells, but at first, scientists thought only stem cells harvested from newborns (or fetuses) could be used for practical therapy. So they proposed storing stem cells harvested at birth by freezing them. Then, one day when you need a new organ or other body parts, your own frozen stem cells could be used to regrow the missing or sickly bits.

So parents had to choose whether their child got this procedure at birth and some people are still doing it today. We also routinely screen unborn children for signs of specific diseases such as downs syndrome. Leaving the decision about what a positive test means to the parents.

Now imagine that every baby gets gene sequencing at birth. The idea would be to give the parents a heads up on whether their child is at higher risk to develop certain disorders. As we understand our genes more over time, the stored genetic information could even reveal new things not known when it was first taken. There are clearly many health benefits to getting a genetic screening at birth. However, at the same time universally digitizing the genes of newborns comes with a long list of ethical and privacy concerns. Especially since we dont know what the long term consequences of storing this information will be. If your genes are put on file at birth, is any sort of privacy possible for you?

Cloning is basically the act of artificially making an identical twin of someone. Scientists made history when they clone Dolly the sheep, but since then have cloned all sorts of animals. As far as we know, there arent any artificial human clones, but in principle, theres nothing special about cloning humans.

At the moment, cloning humans is illegal in most countries. You also need to get samples of the right cells to clone a person. So its not easy to do without their knowledge.

That might not be true for long, however. For example, scientists Craig Venter has created a protein printer where you can simply describe a novel protein sequence and create it from purely digital data. A more advanced version of this machine might one day print out DNA based on a file. That file could be the sequence of a persons DNA exactly as it was when sampled or it could be edited as easily as you can edit words in a document. Long after you are dead, someone in the future could call up your sequence from a computer database and create an identical twin based on your genes. You might not personally have a problem with that, but it would cross many lines.

So what is the answer to the issue of genetic privacy? Obviously, laws and policies have to be in place. Laws that, if enforced, provide effective protection against the misuse of our genetic data. Thats just one part of the puzzle though. The other is physically preventing the unauthorized use of your genetic information.

One approach to this is by having a shared, state-controlled database. One that uses encryption to protect the information from being stolen or copied. When a legitimate user (such as your doctor) needs access to the information, they can read it from the cloud, but not download it. Keeping all that genetic data under lock and key might be one way to prevent third-party agents from exploiting the information without your consent.

Another approach that may help ensure better genetic privacy is data that self destructs or policies that ensure deletion. For example, you may go for a genetic disease screening, but the data isnt kept. You just get a report that says whether you are susceptible or not. Individual people can, of course, choose whether they want their data destroyed and have to accept the possible medical consequences in the future. It would be similar to the Right to be forgotten that is now enshrined in EU law. Basically, you should have the right to destroy copies of your digital genetic data. And then simply sequence your genes again if you need to.

So back to our original question: does genetic privacy matter? In principle and in theory it certainly does. However, I have a feeling that the general public isnt going to be all interested in this issue until it starts to affect them. The problems are mostly theoretical and philosophical at this point. The thing is, its at the point of conception where we have the opportunity to avert some of the foreseeable privacy issues and their consequences.

So yes, itdoesmatter. Pay attention to the laws in your country that are being proposed. Do they favor you or do they protect biotech companies. Who stands to get rich off whats effectively your intellectual property? Whenever you are asked to hand over a copy of your genes for sequencing, make sure you know who stands to benefit. Gaining from that exchange and whether you are an equal partner in it.

Are you worried about your genetic privacy at all? Let us know down below in the comments. Lastly, wed like to ask you to share this article online. And dont forget that you can follow TechNadu on FacebookandTwitter. Thanks!

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Gemini Man’s Villain Is Surprisingly Engaging – Most of the Time – CBR – Comic Book Resources

WARNING: The following article contains spoilers for Gemini Man, in theaters now.

Gemini Man has a handful of problems with its overall narrative, often alternating from serious to almost comical at points. This makes it a very mixed film, with some strong sequences punctuated by bizarre story beats. Nowhere is this more evident with the film's chief antagonist, Clay Varis (Clive Owen), a former mentor to Henry Brogan (Will Smith) and a surrogate father to his clone. Naming him Clay Varis Jr. or Junior for short, Clay is actually given a very compelling relationship with his adopted son.

The problem is that the rest of the film portrays Clay without any of the nuances he gets in the handful of scenes he shares with Junior. It makes for a tonally confusing character, and a waste of a potentially complex villain.

RELATED:How Will Smith Killed DC's Wild, Wild West Comic - Accidentally

Clay is introduced as the head of Gemini, a black-ops unit working with the United States government. He's been in the military a long time, training a number of top soldiers. His best was Henry Brogan, an efficient and skilled killer. But as Brogan grew older, Clay realized a day would come where Brogan would retire or even die. Deciding his skills were necessary to the future survival of peace, Clay clones Brogan in secret. The first attempt produced Junior, who Clay raised as his own son.

When Brogan is used toassassinate a scientist who worked on the project, he begins digging into the organization. Clay attempts to have him killed to prevent him from learning the full truth. He eventually uses "Gemini" himself (Junior) to target the man he's cloned from. Junior almost kills Brogan, but he is able to survive and even reach out to the younger version of himself. Clay eventually reveals that he's been trying to build an army of Brogan clones, who could become the perfect soldiers for future conflicts. This way, he can save lives by never making anyone have to join the military. Instead, it'll be more clones, modified to lack personality, fears or even pain, who are sent into future conflicts unquestioningly.

RELATED:What Drew Gemini Mans Filmmakers to the Action Thriller

Two scenes elevate Clay as a character, however, giving his absurd plan a touch of humanity that makes it more engaging. In the first, an injured Junior returns home after failing to kill Brogan in Colombia. Clay helps patch him up, and actually shows a bit of a tender side. Although his son is still very respectful and dutiful, there's a touch of emotion in Clay here. He even offers to make him a big bowl of cereal, almost as if he was just seeing his son after coming home from college for the holiday.

Their next sequence is more intense, with Junior confronting Clay about being a clone. Clay defends himself and the practice, arguing that the Gemini soldiers can protect families from losing their loved ones to war. But when Junior asks if that's all he is, Clay becomes strangely comforting, almost hurt by the accusation. He genuinely sees Junior as his son, not a weapon, and he wants him to see how the other clone army is all to protect peoplelike Junior.

It adds a layer of complexity to both characters, and it's probably the single strongest moment in the film (largely thanks to Clive Owen and Will Smith's acting). It gives Clay a human connection to his argument that cloning an army of soldiers to fight wars saves parents from losing their children, a fear he suddenly is shown to be very aware of.

He argues that the unfeeling clones can be the necessary casualties of war, having no families to lose or emotion to distract them. If he has the ability to make that a reality, then why wouldn't he? In his mind, this will save an entire generation of young people, just like Junior.It all makes for a surprisingly complicated set of emotions and motives.

RELATED:Should You Watch Gemini Man In 3D & High Frame Rate?

The problem is that Clay doesn't remain that interesting throughout the film. Instead, he spends every scene away from Junior acting like a low-grade James Bond villain.He barks orders at other agents and has soldiers running drills constantly, who end up being nothing more than cannon-fodder in the third act. He casually orders the assassination of Brogan, and then plainly plans how he'll fake a Russian attack and a military funeral. He's a base-linebad guy the rest of the film, with no hint of the emotional core of the character.

For a career military man set on a mission, it makes sense for him to try and push those feelings aside somewhat. But the final sequence doesn't reflect the potential of a father trying to explain why he did what he did to protect his son.

Rather, he speaks to Brogan, making the point that he should be stressing to his son. It's an unsatisfying moment, as is the casual way Brogan shoots Clay so that Junior doesn't have to. It's a lackluster conclusion to a character who showed real potential to be an interesting villain. His argument has a core of a good idea in it, keeping families from losing their sons and daughters to war.

Something like a clone army is a morally questionable tactic, but framing it through his love for his son makes it at least understandable, if not acceptable. But the film doesn't pick up on that element as it could have, and we're left with a bad guy who feels like any other one-dimensional "playing god to take over" villain from action movies past.

In theaters now, Gemini Man is directed by Ang Lee from a script written by David Benioff, Billy Ray and Darren Lemke. The film stars Will Smith, Mary Elizabeth Winstead, Clive Owen, and Benedict Wong.

KEEP READING:In Gemini Man, When Will Smith Fights Will Smith, No One Wins

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El Camino: Every Breaking Bad Character Who Returns For the Movie

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Gemini Man's Villain Is Surprisingly Engaging - Most of the Time - CBR - Comic Book Resources

18 Things About Rick And Morty That Make No Sense – TheThings

From multiverse theory to inter-dimensional travel to cloning, there are lots of scientific theories (and pseudo-science) that Rick and Morty exploit to try to seem like the smartest show out there. And some of the science is actually backed up by evidence. But at the end of the day, the show is a narrative endeavorit often fits science around its plot-lines, to make the episodes work, whether the science is true or not.

Theres a reason why it took so long for season 3 (and now season 4) to come out: these pseudo-scientific ideas are complicated, and once Pandoras Box has been opened, its hard to close it. There are millions of Reddit theories out there explaining everything, but were here to point out a few things that simply dont make sense.

In a show with so much hoopla and head-scratching ideas, it isnt hard to come up with a nonsensical list. And no, you dont have to be a genius to understand these 18things in Rick and Morty that make no sense (just like you dont have to be a genius to enjoy or understand the show).

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Many scientists believe in some sort of multiverse theory these days, which basically says that there is an infinite number of universes where the actions we didnt take are being played out. In a nutshell, these parallel universes comprise of everything that exists. In Rick Potion #9, Rick explains that there is an infinite number of realities, but even multiverse theory calculated a huge number of possible universes, but not infinite. The number is roughly 1 with 500 zeroes, according to cosmologist Richard Matzner. The estimate derives from string theory.

In one strange episode, Morty accidentally impregnates an extraterrestrial doll (Gwendolyn), who successfully bears a human-alien hybrid. We already know that its impossible to crossbreed chimpanzees and humans, and they have a 98% DNA sequence similarity, so what are the odds of impregnating an alien with 0% DNA sequence homology? Not very likely, but it still made for a hilarious episode.

The main deus ex machina in Rick and Morty is Ricks portal gun, which allows him to jump to different dimensions at will. The problem is that we dont actually know how Ricks portal gun works. He showed it being made, and we assume it shoots wormhole-like blasts. Inter-dimensional travel isnt always as carefree as Rick would like it to be. Ricks portal gun might explain how quantum theory can be used, or we might be overcomplicating something that doesnt need a unified theory to form its basis.

In Rick Potion #9, Morty receives a love potion to have his high school crush instantly infatuated with him. The potion is a mixture of oxytocin extracted from a volea rodent that mates for lifeand some of Mortys DNA. And it works like a charm! But oxytocin wouldnt really work like that in real life. While it does play a part in social bonding and sexual attraction, it can also augment memories and induce crazed thoughts (which sort of happens, in the episode).

Just like Inception, Ricks dream hopper doesnt make a lot of sense. A device that lets someone enter someone elses dreams is so far off the realm of possibility, that even pseudo-science cant really explain this one. If we were all connected by means of digital devices, with a common server, maybe it could work (which many futurists believe in). But as it stands, wed need a neural implant (see: Black Mirror) for this to ever have a hope of working.

Ricks freeze gun showed up on the pilot episode, so maybe all the scientific kinks werent worked out yet. The gun is a high-powered liquid nitrogen weapon that turns Rick into Mr. Freeze, basically. Ricks gun works too fast and localized for modern technology, though, and liquid nitrogen wouldnt instantly freeze someone like in the show. This might be a bit nitpicky, but its the truth, and were sure Justin Roiland and Dan Harmon knew that.

Rick is supposed to be the smartest person in the multiverse, and hes also a jerk. Most people who become more knowledgeable of the world become wiser, and thus kinder, but this isnt true for Rickhis ego trumps all. He is the epitome of individualism but does contradictory things all the time that are counterintuitive to his own growth. Basically, he has no growthyet he can be kind, which we see him do on occasion (usually to Morty). It leads us to believe that the Rick we know and love isnt as wise or knowledgeable as he would have us believe.

Here are some examples of Ricks counterintuitive contradictions of character. He has no problem letting Unity take over an entire planet but tries to save Earth when a Cromulon appears. He destroys an entire universe (via throwing a battery on the ground), but saves a homeless man and creates a navigable park inside of him. Rick goes into Mortys teachers mind to manipulate him into giving Morty an A, but Rick also doesnt like the Devil trying to manipulate people. In a way, Rick encapsulates Absurdism at its finest (according to Medium.com).

For a man who seems so sure of himself all the time (one episode even hinges on this fact), we dont really know what Rick believes in. Hes ambiguous, purposefully, and were led to believe that maybe HE doesnt even know what he believes in. For instance, in one episode when he thinks hes going to die, he prays to Godimplying he believes in god, deep down. Rick knows he doesnt matterthat no one matters, and it makes him supremely arrogant, and sometimes even suicidal.

Even though Rick is supposedly the smartest man in the multiversethe idea of the multiverse alone refutes that thought. Rick knows that hes not a unique person, and he believes no one is. There is an infinite combination of events that can happen, infinite times, so the only thing that he resorts to his personal gain. Personal gain explains his motivations a bit, and also shows that Rick isnt the smartest person in the multiverse because hes not unique and there are infinite other Ricks out there who should be just as smart.

In the episode Mortynight Run, Rick drops off Jerry at the Jerryboree daycare center, and we make an interesting discovery when he fills out the form to admit Jerry. We know Rick is from dimension C-137 (he writes it on the form), but he puts N/A for Dimension of your Jerry. But Rick manually entered (into his portal gun) the dimension where he got his Jerry from, so he knows which dimension Jerry originates from. Why would he keep it a secret from us, unless theres something significant hes trying to hide?

The Bootstrap Paradox is a phenomenon where an object travels back in time and is trapped within an infinite cause-effect loop, wherein the item no longer has a point of origin. The item is self-created or uncaused, meaning its just always been. In the season 3 opener, Rick goes back in time to retrieve the secret to his portal gun technology, so he can invent (or reinvent) the portal gunbut that secret wouldnt exist, given the Bootstrap Paradox, in that timeline if that makes any sense.

Rick has been away from his family for an indeterminate amount of time at the start of the show after things spiraled out of control and he had to relocate to another dimension. We learned that he escaped to another dimension to find a Beth that is still alivethough is this Beth the one we follow on the show, or is this a fabricated memory of Ricks? Are the Morty, Beth, Jerry, and Summer from C-137 actually adopted from another dimension of Ricks?

And that brings us to another question: Which Morty is Evil Morty (given that hes the main antagonist at this point)? Evil Morty first appeared in S1E10, and theres a long-running theory that Evil Morty is actually the original Mortya.k.a Ricks Morty from C-137. The idea is that Rick returned to his family after 20 years, but Morty is only 14it doesnt take a mathematician to know that doesnt add up. So perhaps our Morty isnt the Mortiest Morty, and the Morty from Ricks dreams that he cared for in another dimension could be the one who later grew up to be Evil Morty.

Theres an episode where Mortys brainwaves cancel Ricks because hes as stupid as Rick is smart. But is Morty really that stupid? (Also, thats not how waves work.) Morty can think fast on his feet, is good at deducing situations (such as figuring out the weakness of the Parasites when Rick was clueless to it), and he has sensible reactions to Ricks insanity. And perhaps the most telling clue: Evil Morty is found out to be remote-controlling Evil Rick with a transmitter under his eye-patch. Is this malevolent, genius Morty smarter than all the Ricks?!

The shows logline tells us that Rick has returned after two decades to his family, after being in hiding in another dimension. But Morty is only 14, which means that the Morty were following in the show isnt Ricks original Morty. The relationship between Rick and Morty that we know is not all it seems to be! This is a big reason why people believe the Morty from Ricks dreams is the Original Morty, who grew up to become Evil Morty to get back at Rick for betraying his family, and is on a vengeance mission.

There have been hundreds of Ricks and Mortys weve run into throughout the showmost recently shown visiting the Citadel of Ricks. The show has had several subplots involving Doofus Rick, Pickle Rick, Cyclops Morty, The One True Morty, etc. The bottom line is that theres an infinite variety of Ricks and Mortys out there, enough to populate a governing council of alternate universes, so we might never really know who is who.

There are some scientific theories that Rick and Morty get right, but cloning humans is a bit of a stretch. In several episodes, Rick clones people: himself to send a younger Rick to hunt vampires in high school; Beth so she can abandon her family to replace Beths childhood friend Timmy, and to save Timmys father. A clone is an organism created from identical copies of genetic information, and while it would be biologically possible to clone a human, developmental deformities would be a high risk, and the brainwashing that Rick puts his clones through probably wouldnt happen.

References: medium.com, vulture.com, reddit.com, livescience.com

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18 Things About Rick And Morty That Make No Sense - TheThings

Embryo cloning, adult DNA cloning and therapeutic cloning

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It is unfortunate that the term "cloning" refers to three very different procedures with three very different goals. It is also unfortunate that the first thought many people have when they hear the term is of horror movies which have showed the creation of human monsters or of armies of superhuman soldiers with subhuman brains. The reality of cloning is very different.

The three different types of "cloning" are:

There are major ethical concerns about all three types of cloning, when applied to humans.

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Embryo cloning, adult DNA cloning and therapeutic cloning

Human Cloning Foundation | Your DNA does not have to die.

1.Cloning Xeroxes a person.Cloning merely re-creates the genes of the ancestor, not what he has learned or experienced. Technically, it re-creates the genotype, not the phenotype. (Even at that, only 99% of those genes get re-created because 1% of such a childs genes would come from those in the egg mitochondrial DNA). Conventional wisdom Read moreThe Top Ten Myths about Human Cloning

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Human Cloning Foundation | Your DNA does not have to die.


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