A Neglected Modern Masterpiece and Its Perverse Hero – The New Yorker

Imagine a novel about an ambitious, slightly coarse, provincial young man, determined to make his name in the capital city. He is tall and strong, with uncanny blue eyessea-cold, merman eyes. He talks too loudly. One of the capitals most polished journalists dismisses him as a swaggering farmboy. Even the rich heiress who almost marries him agrees with him that he is like a mountain troll from a fairy tale; her sister, on first meeting him, noticed his slightly provincial shoes. But he has brilliance and will, and others welcome this young engineer with a head full of projects as the prototype of the active man of the twentieth century, a figure from a different, luckier tale, an Aladdin (as one of his friends crowns him) who will surely prosper and triumph. The novel describes this journey.

Now imagine that the novel systematically subverts the swelling arc of the bildungsromanthat, on the cusp of each achievement, some ghostly hand pulls our hero back from victory. He is about to leave his mark in the capital city, but eventually withdraws. He is about to marry the rich heiress, but calls off the engagement. He returns to the country and starts a family with a modest country girl, but he isnt happy there, either: He was like a clock whose insides had been carefully removed, piece by piece. In fact, our Aladdin seems destined to follow the serial emaciations of Hans in Luck, one of the Grimms fairy tales, in which Hans, having been paid in gold by his master, is persuaded to exchange his gold for a horse, then his horse for a cow, then his cow for a pig, and so on, until finally he loses everything, and returns home happy and unencumbered. His luck is his reduction.

The hero of this novel comes to the conclusion that all worldly treasures lost their worth as he got closer to them. He spends his final years living in virtual isolation in a remote rural area in the north of the country. After his untimely death, a notebook of his is found, which contains these beautiful words of fatalism and rebellion:

When we are young, we make immoderate demands on those powers that steer existence. We want them to reveal themselves to us. The mysterious veil under which we have to live offends us; we demand to be able to control and correct the great world-machinery. When we get a little older, in our impatience we cast our eye over mankind and its history to try to find, at last, a coherence in laws, in progressive development; in short, we seek a meaning to life, an aim for our struggles and suffering. But one day, we are stopped by a voice from the depths of our beings, a ghostly voice that asks Who are you? From then on we hear no other question. From that moment, our own true self becomes the great Sphinx, whose riddle we try to solve.

This shattering, sometimes unbearably powerful novel, completed in 1904, was written by Henrik Pontoppidan, who won the Nobel Prize in 1917. It is considered one of the greatest Danish novels; the filmmaker Bille August turned the story into a nearly three-hour movie called, in English, A Fortunate Man (2019). The novel was praised by Thomas Mann and Ernst Bloch, and is effectively at the center of Georg Lukcss classic study The Theory of the Novel (1920). In Danish, it is called Lykke-Per; in German, it was given the title of the Grimm brothers fairy tale Hans im Glck. And in English? In English, it didnt exist, having gone untranslated for more than a century, until the scholar Naomi Lebowitz administered the translators equivalent of a magic kiss and roused it from shameful oblivion. Published nine years ago in academic format, Lucky Per has finally appeared in Everymans Library, in Lebowitzs fluent and lucid version, with an excellent introduction by the novelist and critic Garth Risk Hallberg. Our luck has caught up with everyone elses.

Have I spoiled the plot by revealing the ending? The critic only gives away in silver what the great novel eventually releases as gold. Besides, its almost impossible to discuss Lucky Per without discussing the shape of its plot, because the radical oddity of the book is so bound up with the heros final renunciations. At first sight, Lucky Per looks like a stolid work of realism. It is almost six hundred pages long. Through its ample halls moves a large cast of characters, from several layers of Danish societymiddle-class clergymen, rich merchants, lawyers and politicians, writers and intellectuals. There is much conversation about the coming century: the fate of the nation, the future of technology.

But one reason its generally unwise to talk about a single style called realism is that prose narrative is so often lured away from conventional verisimilitude by rival genres, notably allegory and fairy tale. The books opening chapter is at once familiarly realistic and heavy with the ironic fatalism of the folktale. In a small market town in East Jutland, Per Sidenius is one of eleven children growing up in an austerely religious family. His father is a pastor with an ascetic hatred of the body. His mother is bedridden. While his brothers and sisters mutter their prayers in a sort of underworld blindness to the light and full of a dread of life and its glory, Per is a singular, rebellious life force. He sneaks out of the house to go sledding, he flirts with a local girl. When a parishioner complains to the pastor that Per has been stealing apples from his garden, the wayward son is severely admonished at family dinner, warned that he could end up like Cain, the first murderer, whom God cursed thus: You will be a wandering fugitive in all the earth. His siblings weep in dismay, but Per silently scoffs. At the age of sixteen, he escapes this prison, and goes to Copenhagen to study engineering at the Polytechnic Institute. The coming-of-age novel, Pers sentimental education, will now begin in earnest, as the dark, religious family grotto recedes into the distance of legend.

Alas, the past cannot be escaped so easily. Fable and allegory curl themselves like creepers around our heros feet. Per has, in effect, been exiled from Eden, for the Adamic sin of stealing apples. But his home wasnt Edenic, and besides, he doesnt share his fathers Christian faith. If he hasnt committed a sin, how can he be cursed? All the secular energy of this noveland it has a magnificent, liberating secular powerpushes against the reality of the pastors Old Testament damnation. Yet Per is cursed: hes destined to wander, destined to quest, and destined to fail. With a steady, returning beat, closer to allegorical verse than to realist fiction, the novel reminds us of its guiding theme: the homelessness of its hero, condemned to spend his life in the lonely quest for a metaphysical safe harbor. So is Pers curse a religious curse or a fairy-tale curse? And what is the difference between the two?

Pers odd life path might simply be the result of being born into the Sidenius family. The Sideniuses, we learn at the novels opening, trace their lineage, through generations of ministers, all the way back to the Reformation. Its a family tree of unimpeachable piety and dreary episcopal conformity, with one exception. An ancestor, also a pastor, known as Mad Sidenius, somehow went off the rails. He drank brandy with the peasants, and assaulted the parish clerk. In a novel haunted by insanity and suicide, the memory of this family outcast is important. The potentially blasphemous question rears its head again: if its a curse to be a Sidenius, is Per cursed by generations of unerring piety, or by that ancestral aberrant flash of madness?

Henrik Pontoppidans life began much like his fictional heros. He was born in 1857, the son of a Jutland pastor, into a family that had produced countless clergymen. Unlike Per, Pontoppidan seems to have remained on friendly terms with his family, despite drifting away from his inherited Christianity. In his memoir, published in 1940, three years before his death, he declared himself to be an out-and-out rationalist, dismayed by the tenacity of religious superstition. Like Per, he left the provinces to study engineering at the Polytechnic Institute in Copenhagen.

Copenhagen of the eighteen-seventies and eighties has been described (by the critic Morten Hi Jensen) as the first real battleground of European Modernism. A parochially Protestant culture was beginning to do intellectual trade with the rest of Europe: French realism and naturalism, Darwinism and radical atheism were the imported goods. The two most talented conduits of these new freedoms were the novelist Jens Peter Jacobsen and the critic Georg Brandes, both of whom make appearances in fictionalized form in Lucky Per. Jacobsen translated Darwins major work into Danish, and wrote what is surely one of the most fanatically and superbly atheistic novels in existence, Niels Lyhne (1880). A lyrical aesthete and a Flaubertian prose polisher, he is pictured, in Lucky Per, as the sickly poet Enevoldsen, fussing with his lorgnette at a Copenhagen caf while worrying about where to put a comma. Jacobsen was championed by Brandes, whose lectures at the University of Copenhagen in 1871 were an inspiration for a generation of Scandinavian writers. (Brandes and Pontoppidan corresponded for decades.) Brandes had read Mill, Hegel, Feuerbach, Strauss. A fervent atheist, he introduced Danish readers to Nietzsche and, late in life, wrote a book entitled Jesus: A Myth (1925). He was an advocate of European naturalism, and of fiction that attended to the social and political moment. It was time, he argued, to open Denmark up to the outsidea movement that became known as the Modern Breakthrough. In Lucky Per, Brandes appears throughout the novel, more invoked than encountered, as the dominating Dr. Nathan, sometimes nicknamed Dr. Satan. Brandes was Jewish, and Pontoppidan, remarkably alert to European anti-Semitism throughout the novel, writes that Per had kept his distance from Dr. Nathan because of this: He simply didnt like that foreign race, nor did he have any leaning toward literary men.

But Pers life will soon be changed by another Jewish character, and one who shares the bulk of the novel with him: the fierce, brilliant, troubled Jakobe Salomon. Per meets Jakobe through her brother, Ivan, who decides, early in the novel, that Per has the potential of a Caesar on whose brow God has written I come, I see, I conquer! Pers imperial impulses are manifest in his vast utopian engineering project, which envisages a system of canals on the Dutch model that will connect Denmarks rivers, lakes, and fjords with one another, and put the cultivated heaths and the flourishing new towns into contact with the sea on both sides. His dream is a physical enactment of Brandess Modern Breakthrough. He also shares Brandess atheism. There was no hell, Per reflects, other than what mankind, afraid of loves joy and the bodys force, created in its monstrous imagination. The Anglophone reader is sometimes reminded of Thomas Hardy or D.H. Lawrence. Per exults in the healthy secularism of the body: The embrace of man and woman was the heaven in which there is oblivion for all sorrows, forgiveness for all sins, where souls meet in guiltless nakedness like Adam and Eve in the garden of paradise.

With the ruthlessness of the provincial hero, Per decides that marriage to an heiress of the vast Salomon merchant fortune will speed him on his way. At first, though, he stirs in Jakobe a deep-seated hatred of Christian culture, and she treats him with an insulting haughtiness. Bookish, sensitive, twenty-three, and already considered a bit of an old maid by her family, Jakobe had been a sickly child, and the target of anti-Semitic bullying. Per triggers in her a memory, at once sharp and hallucinatory, narrated with dreamlike indulgence by Pontoppidan, and one of the novels most potent scenes. Four years earlier, Jakobe had been in a Berlin railway station. Her eye was caught by a group of pitiable, ragged people surrounded by a circle of curious, gaping onlookers. When she asked a station official how to get to the waiting room, he replied that with her nose she should find it easy to smell her way there. On the floor of the waiting room were hundreds more desperate, emaciated paupers. Suddenly, she realized that they were Russian Jews, on their way to America via Germany. She had heard of the pogroms, and was astounded that this infamy crying out to heaven could happen right before Europes eyes with no authoritative voice raised against it! Pers Nordic frame and blue eyes make her think of two police officers she glimpsed in Berlin, who seemed the embodiments of the brutal self-righteousness of the Christian society she lives in.

With great ironic power, Pontoppidan convinces us that Jakobe and Per must inevitably hate each other, and then, soon enough, that these two damaged creatures could have found comfort only in each other. Their relationship is passionately erotic and ardently intellectual; Jakobe, again like some heroine out of D.H. Lawrence, is helplessly attracted to Per, despite the blaring correctives from her conscience. The couple have in common their committed atheism, their hatred of the established church, and a sense of being chosenby theology, by race, by similarly heroic notions of destiny.

Garth Risk Hallberg, in his introduction, says that Jakobe Salomon is as intelligent as anyone out of James, as bold as anyone out of Austen, as perverse as anyone out of Dostoyevsky, and adds that, with all due respect, the frankness and amplitude of Pontoppidans depiction of the Salomon household leaves George Eliots Daniel Deronda in the dust. I like it when writers are made to run races with one another, precisely because were supposed to be above such competitions, and I also think that Hallberg is right. Jakobe is utterly alive and complex, and burns at the living center of the book. Pontoppidan endows her with an extraordinary intellectual restlessness, and allows her some of the most movingly lucid secular proclamations I have ever encountered in fiction.

One of these statements, a long letter that she writes to Per, becomes an eloquent, scalding testament to her atheism and her faith in the known limits of our worldly existence. She excoriates Christianitys exaggerated anxiety about death and, following Nietzsche, complains about the link between the fear of death and slave morality:

Never will I forget the impression that some plaster casts of bodies excavated in Pompeii made on me. There were, among others, a master and his slave, both evidently caught by surprise in the rain of ash.... But what a difference in the facial expressions! On the slaves face, you could read the most confusing puzzlement. He was overturned on his back, his eyebrows were raised up to his hairline, the thick mouth open, and you could virtually hear him screaming like a stuck pig. The other, by contrast, had preserved his mastered dignity unto death. His almost-closed eyes, the fine mouth pressed shut, were marked by the proudest and most beautiful resignation in relation to the inevitable.

My primary complaint against Christianitys hope of eternal life is that it robs this life of its deep seriousness and, with that, its beauty. When we imagine our existence here on earth as only a dress rehearsal for the real performance, what remains of lifes festiveness?

The powerful secular argument of the novel resides in the freedom and intensity of Per and Jakobes brief relationship. Theres a marvellous scene in the Austrian Alps, where Per has travelled after the couples engagement, and where Jakobe has arrived without notice. The time they spend together in the Alps constitutes their true marriage, a new birth and baptism. One day, out walking, they come across a crude wooden cross, a simple hillside shrine with a rough painting of Jesus. Per tells Jakobe a fable that he heard as a child, about a farm boy who wants to become a great shot, a magic marksman. But in order to achieve this the boy must go out at night, find an image of Christ, and shoot a bullet through it. Every time the lad tries to do it, his confidence wavers, his hand shakes, and he fails the test. He remains a common Sunday hunter for the rest of his life.

Per turns back to the hillside shrine. Look at that pale man hanging there! he says. Why dont we have the courage to spit from disgust right in his face. Per takes out his revolver and fires at the image of Jesus, while yelling, Now I shoot in the new century! As the cross splinters, a second, hollow boom sounds through the valley, like infernal thunder. Per blanches, and then laughs, remembering the signposts he had seen earlier: Take notice of the echo!

Heavy, God-infested, magnificently metaphysical, unafraid to court ridicule, and playing for the highest possible stakesthey dont write like that anymore. They didnt write much like that in 1904, though Knut Hamsun, in 1890, and Jens Peter Jacobsen, in 1880, and above all Dostoyevsky, the great progenitor, had all sounded something like this, not so long before. Given the novels astonishingly raw atheism, how are we to read the religious renunciation of its ending? At the novels close, Jakobe and Per appear to be living alone, and each is now committed to a life of religious seriousness, though neither is a religious believer: Per in the remote north, living in monkish retreat, and Jakobe in Copenhagen, where she has founded a charity school for poor children.

Throughout, Per is hard to comprehend in his cloudy questing. At one momentaround the time of his mothers deathhe is pulled back toward his inherited faith, repenting his lust for worldly success and begging forgiveness from God. But fifty or so pages later his recoil from Christian self-sacrifice is palpable once again; he is repelled, for instance, by Thomas Kempiss lament, in Imitation of Christ, that truly, it is an affliction to live in the world. Per reflects that he is at home neither among ascetic Christiansthe piety of the Sideniusesnor among the children of the world: the luxury of the Salomons. And yet, troubled by this very homelessness, he feels that one must choose: on one side, renunciation; on the other, the world. Which is it to be? For it is necessary to take a stand, to swear fidelity... to the cross or champagne.

In the end, Per surrenders to the religious impulses of a faith he seems to stand outside of. We have been here before, in this world of a deformed and contradictory atheism. Raging heroes in Dostoyevsky, Jacobsen, and Hamsun enjoy denouncing a God they dont believe in. But Per Sidenius is stranger still, because he seems to want to imitate a Christ he doesnt believe in. Thomas Mann praised Pontoppidan as a kind of gentle prophet, for having judged the times and, like the true poet which he is, pointed toward a purer humanity. In a suggestive afterword, the novels translator, Naomi Lebowitz, notes how Per restlessly evicts himself from all those places which could offer him refuge. Subtler than Mann, she also sees Pers journey as the discovery of, finally, an authentic and transparent sense of self... the need to be himself, by himself.

The novel encourages such readings. Pers notebook, written in his final years, contains the following entry: Honor to my youths expansive dreams! And I am still a world conqueror. Every mans soul is an independent universe, his death the extinction of the universe in miniature. In this reading, Lucky Per, though rather Scandinavian in its religious intensity, is a still familiar version of the bildungsroman, in which our hero ventures out into the world, tastes success, tastes the ashes of success, and retreats to ponder, on his own authentic terms, the riddle of the self that has always preoccupied him. Fredric Jameson has suggested that we should see this as a happy ending, albeit an ironic one, in which Per has managed to get beyond success or failure.

Yet how can we accept the ironic wisdom of this ending without smothering the vital force of the novels earlier secularism? Where have the magic marksmen, willing not only to spit at Christ but to shoot at Christ, gone? Where has Jakobes proud Roman master scuttled away to? You dont have to be a fully paid-up Nietzschean to feel that if you no longer believe in the Christian God you should no longer believe in that Christian Gods slave morality. If you have rejected the content of the faith, why mimic its more self-punishing practices? Pers imagined choice between cross or champagne is not only a false choice but a mutilated one, posed by a reduced version of Christianity. In fact, Lucky Per emerges as a savage critique of the persistence, in Danish culture, of a certain Kierkegaardian masochism, in which all choices are made religious rather than secular, purifyingly negative rather than complicatedly affirmative. Kierkegaard said that one had to be a kind of lunatic in order to be a true Christian. Is there a difference between this form of religious madness and actual madness? Lucky Per inserts its secular, novelistic lever into just this question.

What if Pers final renunciation is a narrative false flag? Instead of looking at Per, we should perhaps look toward Jakobe, whose own renunciation takes her into the world, not away from it, and who seems to manage this turn without compromising her defiant secularism. She is the novels true hero. How do you get back to Eden? Back to the place you inhabited before the original religious curse? Back to a home before religion made it a home you could be exiled from? If you are a wandering, homeless Christian, scarred by original sin, the answer might be: in the arms of a wandering Jewbut one whose own itinerancy is unseduced by the lure of religion, whose own secularism is not tempted by the simplicity of religious masochism. In the strange switchback of their lives, Per and Jakobe each redefined the meaning of luck. The shame was that they could not share it. Lucky Jakobe, unlucky Per.

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A Neglected Modern Masterpiece and Its Perverse Hero - The New Yorker

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.

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‘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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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

Boeing forecasts 820 new aircraft deliveries in Oceania over next 20 years – Australian Aviation

Boeing 787s going through final assembly at the companys Everett facility. (Boeing)

Boeing says the airlines of Oceania will require 820 new aircraft in the two decades ahead to meet the demand for air travel led by passenger growth into and out of the region.

The manufacturers outlook covering Australia, New Zealand and the nations of the South Pacific is a decline of 50 aircraft from the 870 new aircraft deliveries for the region in the 2018-2037 Current Market Outlook (CMO).

Boeing Commercial Airplanes senior managing director for marketing Darren Hulst noted aviation played a key role in the the travel and tourism sectors, which have become an increasingly significant contributor to economic growth or gross domestic product (GDP).

About 11 per cent of Australias economy is travel and tourism 12 per cent of jobs and we see that growing faster than GDP growth over time, Hulst told reporters in Sydney on Wednesday.

Aviation will continue to have an increasing role in enabling economic growth but also supporting more and more tourism, more and more travel, whether that be for tourism or for leisure in this marketplace.

The Boeing forecast said the 820 new aircraft comprised 10 in the regional jets segment, 560 in the single-aisle segment, 250 in the widebody segment and nil in the freighters segment.

The only change from the prior years CMO was in the widebody segment, which was reduced by 50 aircraft from 300 previously.

While a 16.7 per cent reduction in widebody demand would seem, on the surface, statistically significant, Hulst said the bulk of the change was the decision to reallocate expected aircraft deliveries to other regions due to the way the CMO now treated airline affiliates.

He cited the example of the Jetstar group of carriers, which has operations in Australia and New Zealand, Japan, Singapore and Vietnam.

We used to forecast our aircraft based on where the parent company was domiciled, Hulst explained.

For example Jetstar in Japan and Vietnam would be considered Oceania. Now, weve adjusted that to really focus on where the airplanes will be flying.

Thats moved some of the demand out of the region but it hasnt changed the nature of the demand, it just moved it to its rightful home.

The improved operating performance of new single-aisle aircraft was also a factor as aircraft such as the Airbus A321LR and 737 MAX were being deployed on longer sectors.

Hulst noted more than half of the capacity today in Australia was on single-body aircraft such as 737 and A320 family of aircraft.

Both of the fleets have seen an increase in the last decade of over 50 per cent, Hulst said.

Widebodies are being de-emphasised regionally because of the flexibility of these single-aisle aircraft.

We will continue to see that because of the versatility of these fleets and going forward, both from an industry perspective and also here in the region, we continue to see a slow creep upward in terms of the average capacity.

A supplied image of Boeing 737 MAX 7, 8 and 9 artwork. (Boeing)

Hulst said the average number of seats on a single-aisle aircraft a decade ago was about 140 seats. Today the figure was closer to 155-160 seats.

This was achieved via upgauging and adding more seats onto existing aircraft.

In terms of the fleet composition, the number of aircraft in Oceania was expected to grow by about 60 per cent from 550 today to 920 by 2038.

Some 450 aircraft would be replaced, with 370 new aircraft joining the fleet for growth. Finally 100 aircraft flying today will still be in the skies in two decades time, according to Boeing estimates.

The split between growth and replacement for Oceania was at odds with the global CMO forecast, which showed 56 per cent of all aircraft deliveries would be for growth and 44 per cent to replace older airframes.

Market fragmentation is the main driver behind Oceanias modest passenger growth as airlines utilize new smaller and highly efficient widebody airplanes to increase city-pair growth to international destinations, Boeings CMO said.

The bulk of the growth in passenger traffic for Oceania, which Hulst described as a very stable, mature market, was likely to be for travel into and out of the region, which was expected to grow by between three and four per cent a year.

More inbound traffic, more outbound traffic, more medium and long-haul traffic, which is driving that growth rate higher, Hulst said.

The inter-regional markets to and from Oceania are the ones that have seen the biggest growth and likely will see the fastest growth going forward.

We see a lot of opportunity for continued expansion in these markets.

This compared with average annual growth of one to two per cent a year over the past five years in the domestic and trans-Tasman markets of Oceania.

The Boeing CMO, first published in June, estimated that over the next 20 years airlines would need 44,040 new aircraft worth some US$6.8 trillion. This was up from 42,730 aircraft in the prior years outlook.

A supplied image of the Boeing 787-10 on its first flight in April 2017. (Boeing)

Meanwhile, Hulst said Boeing was continuing to develop the specific timing of when the 777-8X would enter service, having pushed back the timetable for the program from 2022 to an unspecified future date in August.

Our timetable on the 777-8 obviously is still under consideration for when it actually enters service, Hulst said.

I would say we are absolutely committed to the 777-8 as a product and as a model but it is really more of when the combination of the demand from our customers and how it aligns with the design and production system for the 777-X as a program.

I dont have a specific date but we absolutely are committee to the 777-8 and think that it provides unique product capability that nothing else can in the market.

The 777-8X was one of the aircraft under consideration for Qantass Project Sunrise, which sought an aircraft able to opeerate nonstop from Australias east coast to London and New York, among other cities.

Airbus has put forward its A350 platform for Qantass consideration.

With the 777-8X not available by Qantass projected 2023 start date, Boeing has said previously it had offered a compelling offer to account for the new timetable for the aircraft.

The airline has said previously it would decide whether to proceed with Project Sunrise by the end of calendar 2019.

An artists impression of the Boeing777-9X and 777-8X. (Boeing)

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Boeing forecasts 820 new aircraft deliveries in Oceania over next 20 years - Australian Aviation

Oceania Cruises’ new itineraries in the Middle East and Asia – Traveller

Veranda Stateroom Oceania Sirena.Photo: Michel Verdure EXOTIC ENCOUNTERS

Oceania Cruises has produced a Sirena Exotic Collectionof cruises in the Middle East and Asia. The nine 10- to 24-day itineraries launch in November 2020 and continue into April 2021, kicking off with a 12-day voyage from Jerusalem to Istanbul. Sirena then sails through the Red Sea to explore the Emirates, Qatar, Bahrain and Oman. After that, it's onwards from Dubai to Mumbai, Yangon and Bangkok before Sirena traces its route back to Athens. All itineraries include overnight port calls, with some featuring as many as four overnight port stays.

Phone 1300 355 200. See oceaniacruises.com

Abercrombie & Kent has a new expedition cruise to Iceland and Greenland in August 2020 aboard the exclusively chartered Le Boreal. The cruise sails vast and spectacular landscapes such as the Westman archipelago, ice-covered Lindenow Fiord and Prince Christian Sound. Guests will explore the high Arctic tundra on foot to admire wildflowers and migrating seabirds, and will enjoy regular Zodiac excursions to glaciers, fiords and fishing villages. Cultural experiences are another highlight on visits to remote Greenlandic communities where guests learn first-hand about the local people, their cultures and ancient archaeological sites.

Phone 1300 590 317. See abercrombiekent.com.au

Latitude 33 has released a 26-night Golden Triangle & Rafflesjourney in Asia departing on November 13, 2020 that opens with an eight-night land tour in India and finishes with a stay in Singapore's Raffles Hotel. In between is a 16-night cruise from Mumbai to Singapore on Crystal Symphony, with stopovers in southern Indian spice-trading port Kochi, Sri Lankan capital Colombo, and two port nights in Yangon in Myanmar for an exploration of its golden temples, ancient pagodas and colonial heritage. Phuket, Penang and Port Kelang (Kuala Lumpur) round off the voyage.

Phone 1300 636 848. See latitude33.travel

Ponant is offering 62 cruises across Africa, Asia, the Indian Ocean and Polynesia in its 2020-21 season. Among them, a 10-night China & Vietnam cruise departs Shanghai on October 28, 2020 and visits Wenzhou, Xiamen, Hong Kong, Hainan Island and Vietnam's Ha Long Bay, while a 14-night Jewels of the Indian Oceancruise departing March 29, 2021 between Port Louis in Mauritius and Zanzibar in Tanzania visits Reunion Island and Madagascar. An eight-night Treasures of the Persian Gulfcruise has 10 departures between November 2020 and January 2021 and visits Abu Dhabi, Doha and Oman.

Get the latest news and updates emailed straight to your inbox.

Phone 1300 737 178. See au.ponant.com

A reminder that we're approaching the end of Cruise Month, organised each October by the member cruise lines and travel agencies of Cruise Lines International Association (CLIA) to showcase their cruise offerings. The good news is that the month finishes in style with a special emphasis on luxury cruising. If big-ship, budget-end cruise holidays aren't for you, head down to a cruise-focused travel agency and ask about the ever more impressive array of luxury ships that have floated out of the dockyards in recent years. To find a specialist CLIA travel agent in your area, see cruising.org.au

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Oceania Cruises' new itineraries in the Middle East and Asia - Traveller

City To Host 180 Riders For Oceania Track Champs – What’s On Invers

There will be plenty of world champions bling on display when the track cycling international season gets underway at the Oceania Championships in Invercargill.

Around 180 riders from Australia and New Zealand will compete in the four-day UCI continental championship from 16-19 October at the SIT Zero Fees Velodrome in Invercargill. As well as Oceania titles, the championship offers all-important qualifying spots for next years UCI World Championships.

It will also be a shakedown for riders ahead of the UCI Track World Cup season, which is highlighted with back-to-back events at Cambridge on 6-8 December and the following week in Brisbane.

The winners of 25 world championship rainbow jerseys will be competing at Invercargill to add some real sparkle to the event as the leading riders amp-up for a massive nine months ahead to the Tokyo Olympics.

The charge from across the Tasman comes from their sprinters led by four-time team sprint world champion Kaarle McCulloch and 2018 individual sprint world champion Matt Glaetzer, along with Sam Welsford, a three-time team pursuit world champion and the current scratch race rainbow jersey holder.

The Vantage New Zealand rainbow wearers include three-time team sprint victors Ethan Mitchell, Sam Webster and Eddie Dawkins, the 2017 individual pursuit winner Jordan Kerby and current omnium world champion, Campbell Stewart.

Current Australian team pursuit world champions Welsford and Kelland OBrien will pair-up to focus on the two-rider Madison, which is a new addition to the Olympic schedule.

The Vantage elite New Zealand team will be competing in force with their entire squad, with some riding for their centres as well.

Our endurance and sprint teams have worked hard over the winter in building a base for the upcoming track season. So this is a good opportunity for as many of our best riders, both elite and junior, to stretch their legs in very strong competition, said Cycling New Zealand high performance director, Martin Barras.

It is firstly a chance for us to find out where all the riders are at in terms of their development and what they need to focus on. It will give us information we need to select final combinations for the world cup season coming up, especially the home round in Cambridge, said Barras.

It also offers some important ranking points towards the world championships.

We will also be watching with real interest in the development of our junior riders, particularly the ones who have been prominent internationally this year.

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City To Host 180 Riders For Oceania Track Champs - What's On Invers

India optimistic Davis Cup tie will be shifted from Islamabad – Midwest Communication

Wednesday, October 16, 2019 3:55 a.m. EDT

By Amlan Chakraborty

NEW DELHI (Reuters) - India's tennis association will apply for visas for their Davis Cup tie against Pakistan next month but expects the fixture to be moved away from Islamabad, AITA Secretary General Hironmoy Chatterjee told Reuters on Wednesday.

The Asia/Oceania Group I tie, originally scheduled for Sept. 14-15 in Islamabad, was postponed by the International Tennis Federation (ITF) to late November following a security review.

"We've begun the visa procedure while waiting for a confirmation on the venue by the ITF," Chatterjee said by telephone.

"We've requested a change in venue and ITF has promised to get back to us by Nov. 3."

Pakistan was forced to host Davis Cup ties at neutral venues for more than a decade as teams refused to travel to the country citing security concerns.

They played their first home tie after a gap of 12 years against Iran in 2017, while Hong Kong was relegated and fined by the ITF after they refused to travel to Pakistan the same year.

Pakistan expelled India's ambassador and suspended bilateral trade and all public transport links with its neighbor in August after New Delhi removed "special status" from its portion of the contested region of Kashmir.

Chatterjee said it was premature to talk about a possible pullout by top players should the ITF stick to Islamabad as the venue and was optimistic the governing body would shift the tie.

"Based on the current situation, ITF should," he said.

(Reporting by Amlan Chakraborty in New Delhi; editing by Peter Rutherford)

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India optimistic Davis Cup tie will be shifted from Islamabad - Midwest Communication

Premier League legend invited to Fiji – Football in Oceania

Former Liverpool, Real Madrid, Manchester United and England striker Michael Owen has been invited to come to Fiji.

Owen will attend the 2019 Fiji Football Awards Night in February 2020.

The main guest is not yet fully finalised, so it could still change, but Fiji FA President Rajesh Patel says they are in discussions with FIFA over Owen coming to the country.

Owens appearance is made possible by the FIFA Legends Program, which saw former Manchester United and Arsenal full-back, Mikal Silvestre, come to Fiji earlier this year for the 2018 Awards Night.

The Fiji FA also says Owen will conduct clinics and visit schools and clubs during his stay.

This year we had Mikal Silvestre as the main guest during our annual awards function at the Sofitel Resort and next year we are getting ready to have Michael Owen, FFA President Rajesh Patel told the FAs website.

We want to reward our hardworking players and officials in a bigger and better way every year and having legends of the sport present at the event surely adds more colour and encourages everyone to contribute more to football, he said.

Michael Owen is most famous for playing over 200 matches for Liverpool and scoring 118 goals. He also played for Real Madrid, Newcastle and Manchester United.

For the English national team, he turned out 89 times and scored 40 goals. He is number 11 on the list of players with the most games for England and number five on the list of players with the most goals for Three Lions.

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Disaster Risk Reduction in the Republic of Fiji, Status Report (July 2019) – Fiji – ReliefWeb

Introduction

Fiji is one of the Pacific island groups, located within Melanesia which is one of the three major cultural areas within the geographic region of Oceania. The country is an archipelago state, consisting of 332 islands of mostly volcanic origin, of which 110 are inhabited permanently. The land area of Fijis islands covers 18,274 km2 , but the two largest islands of Viti Levu (where the capital Suva is located) and Vanua Levu encompass 85% of the total area (CBD, 2018). The overall geographic area covers almost 50,000 km2 , yet despite the size of the country, the majority of the population is concentrated into the two largest islands and urban centers. 75% of all people are inhabiting Viti Levu and 20% reside on the smaller Vanua Levu as of 2016 (Government of Fiji, 2016).

Administratively, the country is divided into Northern, Eastern, Central and Western divisions which are governed by a commissioner for the coordination of governmental activities at their respective regions (Rahman & Singh, 2011). These divisions consist of 14 provinces, headed by Provincial Officers (Roko Tui) at the local government levels, which are operating under the direction of the Ministry of Local Government, Housing and Environment. However, the governance responsibilities of the i-Taukei or indigenous Fijian villages are linked to the Ministry of i-Taukei Affairs at the national level (Winterford & Gero, 2018; Ministry of Finance, 2015). Issues related to community management are firstly discussed by the village councils, followed by district council before reaching the provincial councils and Roko Tuis.

Fijis islands have a rich ecological diversity; approximately 52% of the landmass is covered by forests, which contain unique biodiversity not found anywhere else on the planet. Half of the flora and 90% of all insect groups present are endemic to Fiji (CBD, 2018). The islands and surrounding reefs also support distinctive marine ecosystems, which are still not thoroughly researched.

In terms of economy, Fiji is one of the most developed and fastest growing countries among the Pacific Island Nations, with largest growth sectors in tourism and sugar cane exports (Jayaraman, et al., 2018). Tourism alone contributes to over 10% of the total GDP, and it has been estimated that the direct and indirect effects of tourism to economic growth could reach over 30% (Fiji Bureau of Statistics, 2016). It is also the regional hub for services, such as flights and shipping, among the Pacific islands. However, the economy is still facing challenges due to disasters and external shocks, impacts of which are enhanced by remoteness and by the reliance on vulnerable agricultural exports and visitors. Furthermore, and despite the positive development trends, 35% of Fijians live below the poverty line, with 44% of the rural populations living in poverty in 2016 (Government of Fiji, 2016).

Countries of the South Pacific are among the most disaster-prone in the world, and Fiji is no exception. The country is located in the vicinity of the Pacific Ring of Fire, meaning that earthquakes, earthquake-induced tsunamis and landslides are a possibility. There are also two active volcanoes located in the region, and the volcanic soil is inherently unstable, which adds to the landslide risks especially in areas where farming and settlement is spreading to fertile slopes. Cyclones, heavy rain and flooding are yearly occurrences, and the most common disasters in the past have been the result of high-impact hydrometeorological events. Majority of the population and infrastructure is located in the proximity of the ocean, which results in heightened exposure to sea-level rise and weather-related hazards.

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Disaster Risk Reduction in the Republic of Fiji, Status Report (July 2019) - Fiji - ReliefWeb

Elon Musk SpaceX Mars Missions, Space Travel Doomed To Fail Because Of ‘Space Junk’ – International Business Times

SpaceX CEO Elon Musks plans to bring humans to space and perhaps start a colony on the moon or planet Mars could be doomed to fail because of the real danger posed by space junk.

According to a report, space junk could clog up the Earths low orbit and trap future space ships from ascending and leaving our planets atmosphere. Based on recent data, the space on Earths upper atmosphere is now littered with 170 million pieces of floating junk. Whats alarming is that out of these, only 22,000 are actually being tracked.

Per the report, around 7,000 tons of space junk has been accumulated and currently going around the Earths atmosphere. This debris is a combination of old satellites, metals from rockets and even rock build-ups that are close to the proximity of Earth.

Previously, experts have already warned that it would be difficult for rockets to pass through the Earths orbit if the space junk around it increases. Colliding with any of this debris is very real and poses a possible danger. The theory is called Klesser syndrome.

Because of these, aggressive space plans such as SpaceX Mars and the moon colony could eventually fail if nothing will be done. Musk initially planned to take people out of Earth to start a livable community either on the moon or the Red Planet.

Even NASAs future moon missions could be in danger of failing with the danger of space junk wont be addressed.

Aside from space missions, even Earth-bound technologies such as mobile phones, GPS, weather instruments and television could be affected by this space junk. The instruments rely heavily on satellites and space junk could hinder the signals from getting through.

Now, an expert has actually issued a warning saying that theres so much space junk in low Earth orbit that in time, humanity could be trapped to do anything about it. According to Ralph Dinsley, founder and executive director of Northern Space and Security LTD, we are already approaching a time when it would be too late to leave Earth.

At the far end of the spectrum, worst-case scenario, it will wipe it out. We could have a major space junk event that will mean that we cant launch beyond low-Earth orbit and we trap ourselves on Earth. The simplest event is that it will slow down how we do space exploration. It has been ten years since the last satellite on satellite collision. The likelihood of it happening is very low but there is still that potential. But the catastrophic results could be huge, Dinsley said.

At any rate, its not like nothing is actually being done to clear our space junk and that companies have already been tapped to do the cleanup. One of these missions is the UK Space Agency's RemoveDEBRIS mission which is actually one of the pioneers attempts to address the build-up of dangerous space junk.

(Jan. 9, 2019) The SpaceX Dragon cargo craft is pictured attached to the International Space Station's Harmony module as the orbital complex flew 258 miles above the Indian Ocean off the eastern coast of South Africa. Photo: NASA/ JSC

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Elon Musk SpaceX Mars Missions, Space Travel Doomed To Fail Because Of 'Space Junk' - International Business Times

NASA Chief: First SpaceX (or Boeing) Crewed Space Mission Is Less Than 5 Months Away – Observer

NASA Administrator Jim Bridenstine (L) met with SpaceX CEO Elon Musk near the Crew Dragon clean room at SpaceX headquarters on October 10, 2019 in Hawthorne, California. David McNew/Getty Images

Both SpaceX and Boeing (through itspartnership with Richard Bransons Virgin Galactic) have bold plans to develop the first ever commercial space travel program targeting non-professional astronauts. But before either company sends tourists into space, their first actual manned flight will likely take place with NASA, as the two companies contract work with the federal agency approaches its final stages.

If everything goes according to the plan, SpaceX and Boeing could see astronauts shooting for the International Space Station (ISS) aboard their spacecraft as soon as early 2020, per NASA chief Jim Bridenstines latest estimate.

SEE ALSO: A Lunar Space Elevator Is Actually Feasible & Inexpensive

I think both systems could be ready in the first quarter of next year, NASA administratorBridenstine told CNBC in an interview last week, following a tour with SpaceX CEO Elon Musk at his companys headquarters in Hawthorne, Calif.

In 2014, NASA awarded two multibillion-dollar contracts each to SpaceX and Boeing to develop a commercial spacecraft under the agencys Commercial Crew program, an initiative created in 2010 to find alternatives to theRussian Soyuz spacecraft, currently the only supply of space capsules used on ISS-related missions.

The spacecraft developed by SpaceX and Boeing, Crew Dragon and Starliner, respectively, are required to be able to transport at least four astronauts to the ISS, stay docked for six months and bring the astronauts back to Earth after.

NASA initially expected the first manned flight to happen as soon as 2017. But the timeline was pushed back several times due to engineering hiccups and test failures. Now, both systems have only a few final tests to pass before astronauts can board the ship: Boeings Starliner has to complete a launchpad emergency test and an unmanned flight test scheduled for December, and SpaceX needs to re-certify Crew Dragons emergency rocket engines and complete a high-altitude emergency flight test.

Whether its Starliner or Crew Dragon, both these programs are still under development, Bridenstine cautioned. While we go through these test processes, we are going to learn things that need to be reviewed, where we need to take deeper dives, maybe make some modifications.

So when I say the first quarter of next year for human spaceflight, what I mean is if things all go right. Things all going right is not the history when you talk about development programs, he added.

In addition to flight tests, NASA is also in the process of conducting a safety and cultural assessment for both companies particularly SpaceX, after Musk was caught smoking marijuana on camera during an interview last September. The assessment involves interviewing hundreds of employees at both companies.

Bridenstine said hed be happy to make the final results of these reviews public if both SpaceX and Boeing agree to do so, noting that he doesnt think theres much [of] a story there. Quite frankly, I think both companies are operating really responsibly and making sure that their cultures are safe.

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NASAs New Spacesuits Unveiled, for Trips to the Moon and Beyond – The New York Times

WASHINGTON At NASA headquarters on Tuesday, officials introduced two prototype spacesuits to be used during upcoming moon missions.

The new spacesuits will offer improvements over existing models for the men and women expected to wear them, including greater comfort and movement. And as NASA races to meet the Trump administrations 2024 target for returning to the moon, the agency needs to make that astronauts have the technological capability to safely set foot on the surface, which no human has done since 1972.

Weve been working for a long time to build spacesuits that will do the job on the moon and going on to Mars, said Amy Ross, a spacesuit engineer at NASAs Johnson Space Center in Houston.

One spacesuit, with a colorful bright red, white and blue pattern, is called the exploration extravehicular mobility unit. That is what astronauts will wear as they explore the moons surface.

The moon suit, demonstrated by Kristine Davis, who works in the agencys spacesuit engineering efforts, provides additional mobility for an astronaut, making it easier to walk, bend and twist.

Astronauts will be able to reach across the suit and lift their arms above their heads. Which they cant do today and couldnt do during the Apollo program, Ms. Ross said.

You remember Neil Armstrong and Buzz Aldrin, they bunny hopped on the surface of the moon, Jim Bridenstine, NASAs administrator, said. Well now were actually going to be able to walk on the surface of the moon, which is very different than our suits in the past.

The suits are designed to work in temperatures ranging from 250 degrees Fahrenheit down to -250 degrees Fahrenheit and potentially even colder places around the lunar South Pole, where NASA is aiming to send astronauts.

The other suit, in a bright orange fabric, is to be worn by astronauts during launch and re-entry back to Earth while they are sitting inside the Orion crew capsule, the spacecraft for deep-space travel that NASA has been working on for 15 years. The suit provides protection and oxygen to the astronauts in case of an accident that causes the capsule to become depressurized. It is designed to keep astronauts alive for six days.

The primary use in the case of an accidental depressurization, said Dustin Gohmert, a spacesuit engineer who modeled the Orion suit. We can take safe haven in this suit. Well seek refuge in here.

Each outfit shared similarities with its utilitarian predecessors worn by earlier astronauts. Spacesuits being designed elsewhere have more futuristic and fashionable appearances.

SpaceX, which has a contract to fly astronauts to the International Space Station in the near future, sent a mannequin wearing its suit to orbit in March. It featured a 3D-printed helmet and a black and white design that seemed to take inspiration from motorcycle racing suits.

Boeing, whose Starliner will also travel to the station, debuted its blue, zippered suit in 2017. It is about 10 pounds lighter than what astronauts wore on the space shuttle.

NASAs new suits will be made in a wider range of sizes than had been previously available. In March, a spacewalk that was to be the first conducted by two women, Christina Koch and Anne McClain, had to substitute a male astronaut, Nick Hague, because there were not two spacesuits on the station that fit both women.

(Ms. Koch and another astronaut, Jessica Meir, are scheduled for the first all-female spacewalk this week.)

When the Trump administration announced its intent to send astronauts back to the moon by 2024, NASA was missing a few crucial pieces for the mission. In addition to updated spacesuits, the agency does not currently have a spacecraft capable of landing on the moon.

NASA had been planning to return to the moon in 2028. By moving up the date four years, the leisurely pace had to be greatly accelerated.

The space agency last month issued a call for commercial companies to propose a lunar lander that could be built that quickly. The deadline for proposals is Nov. 1.

NASA officials have insisted the spacesuits would be ready by 2024. That would avert the comical possibility of American astronauts landing on the moon and just looking out the windows without any garments they could wear to step outside.

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NASAs New Spacesuits Unveiled, for Trips to the Moon and Beyond - The New York Times