Meet REDSIX, the Indonesian band crafting addictive, pop-punk classics – Happy

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Theres no denying that REDSIX are utterly addictive. From their 2018 debut until now, the Indonesian band have been releasing songs with the potential to fill stadiums, blending rich lyricism with riffs that will melt the heart of any pop-punk fan.

I have a steady craving for decorating dreams until they burst at the seams,frontman Denny sings at the start of the groups latest singleVessel.This line captures the essence of REDSIX; deeply poignant reflections on our everyday coiled around the sharp edges of alt-rock.

Sweeping onto the scene with their debut LPUproar,the group defined themselves from the get-go. Songs that revolve around pop-punk, emo, and alt-rock, all connected through their nuances, REDSIX were always going to be a force to be reckoned with.

REDSIX is a rock band from Jakarta, Indonesia, conceptualised in early 2017 by Denny (vocalist) andKevin (lead guitar) Wicak (rhythm guitar), Rizma (drums) and Ipang (bass), the band write in their bio. [We]hope to contribute to theIndonesian music scene by adding their varying influences to the forefront of theirsongwriting and composition to their unique sound.That they certainly do.

Crushing bass, acidic guitar riffs, and the undeniable energy of 00s-esque vocals combine into one all-consuming track that you cant help but jam to. However, the group still know how to tastefully pull away in the most needed moments. Championing the talents of particular members in these areas or using the space to frame defining lyrics, REDSIX are able to combine enthralling rock with sonic eloquence. What are audiences left with? Songs that you cant turn away from.

In their new releases, the group are reaching even further into their sound. Its new, fresh, but distinctly REDSIX.WhereBreak Inpunches through with pure emo-rock,Vesselsparkles with pop-adjacent inflictions andEndeavour emits earthycadence within the walls of pop-punk.

With their 2020 EP on the horizon, we are all waiting in anticipation to see what the band delivers next. REDSIX are truly ones who you should be watching out for.

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Meet REDSIX, the Indonesian band crafting addictive, pop-punk classics - Happy

Were the Rolling Stones Better in the ’60s or ’70s? Roundtable – Ultimate Classic Rock

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The Rolling Stones have gone through many phases on their way to becoming one of the biggest bands inrock history. From the start they were seen as an edgier alternative to the Beatles, focusing on American blues music. But they soon expanded their musical horizons, embracing everything from R&B to pop to psychedelia.

The band released much of its landmark work ina milestone runthat spanned the late 60s through the mid-70s,and which included such vaunted releases as Beggars Banquet (1968), Let It Bleed (1969), Sticky Fingers (1971) and Exile on Main St. (1972).

With so muchincrediblematerial, itsnot easy to pin downwhich decadeoffered fansthe best version of the Rolling Stones. Still, we challenged five writers tosort through it all and determine ifthe Rolling Stones were better in the '60s or '70s.

In theirformative years, the Stoneswere mainly influenced by American blues and R&B artists.By the '70s, they'd started infusing other genres and styles. Which version of the band do you prefer?Michael Gallucci: Their greatest run started in 1968 with Beggars Banquet and ran through 1972's Exile on Main St., with four classic albums split evenly between the two decades. But seeing that Exile is their all-time greatest LP, I'm going to give a slight edge to the '70s.

Nick DeRiso: They played way more blues before they really accepted its dangerous truths and made them their own into the '70s. Thats when the Rolling Stones finally became truly great. Conversely, as they moved away from that musics heart of darkness, there were times (some would say many times) when the Stones became decidedly average. The blues was, in no small way, their salvation.

Ryan Reed: This doesn't fall neatly along the decade lines, but my favorite Stones stretch is Beggars Banquet through Exile on Main St. For my fix, I often reach for their harder-hitting material from the late, late '60s and early '70s. It's not a purely sonic preference: I'm a big psych-rock fan, and I think Brian Jones' '60s experiments elevated the band's music that decade. (Imagine "Paint It, Black" without the sitar!) Mick Jagger and Keith Richards just didn't fully cement their own style until later on.

Dave Lifton: It's pretty much a consensus that the Rolling Stones' classic period straddled the decades, 1968-72. Even their own set lists suggest they know this is the case. Still, the blues has been at the heart of virtually everything the Rolling Stones have ever done, and even their most successful forays into other genres haven't strayed too far from it. And yet some of their worst records are their early blues and soul covers, which only shows how much life experience you need to be true bluesmen. Then again, some of that very same "life experience" also resulted in a lack of inspiration for a good chunk of the '70s.

Corey Irwin: Give me the later, more adventurous Stones. As the band matured, the subject matter became more engaging, the musical style more varied and the overall material improved. It was their willingness to bring new musical styles into their sound that took them from being a good band to one of rocks all-time greats.

Brian Jones died in 1969, less than a month after being fired from the band. Would the Stones have followed the same trajectory into the 70s had Jones been able to get sober and continue with the group?

Gallucci: I think, for the most part, yes, because his overriding influence was waning as the other members got more footing over the years. The Stones' evolution is pretty easy to trace from, say, "Satisfaction" to "Jumpin' Jack Flash" and then to "Brown Sugar." And from there "Tumbling Dice" and "Miss You" aren't that big of a stretch. Jones played on only two of those songs, and, frankly, is barely a presence on "Jumpin' Jack Flash."

DeRiso: Sure. But, in truth, his passing was just part of what toughened up the band as the new decade loomed. The promise of the '60s died, and the Rolling Stones found themselves in the nexus of that at Altamont. At the same time, this broader sense of dreams deferred played out in microcosm as they struggled to overcome the death of Jones, and extricated themselves from an absolutely terrible management deal. They were changed forever.

Reed: Jones was the sleeper genius on so many early Rolling Stones tunes, but it's clear from the band's evolution that their chemistry wasn't built for the long haul. It's hard to imagine Jones' role after the psychedelic era fully faded sure, he could have returned completely to the guitar, retiring his mellotrons and marimbas, but would he have felt creatively satisfied? The Stones could have continued to lean into a harder sound with Jones. But I think that relationship would have dissolved, tragedy or no.

Lifton: Yes and no. Those records with Mick Taylorwould have sounded very different because Taylor was such a great lead player, and it's hard to speculate how receptiveJones would have been to Keith Richards' burgeoning interest in country. But Jones was also the one who brought in more exotic instruments and textures, so they would have continued to find ways to experiment within their blues framework, and the Ron Wood part of their '70s output still would have had excursions into funk, reggae and disco.

Irwin: I cant imagine a scenario where the 70s Stones and Jones could have coexisted. His tenure with the band had run its course, regardless of his sobriety. Even though he unquestionably influenced the groups early work, his authority within the bands hierarchy had steadily declined. Jagger and Richards were the clear leaders, shouldering the majority of the songwriting. Had tragedy not struck, I like to imagine Jones would have gone on to a long and successful career outside of the Stones. He certainly had the talent to do so. But the band knew where they were going as the decade turned, and I imagine they would have gotten there with or without him.

You can only listen to one album from each decade. Which are you choosing? Explain your decision.

Gallucci: Beggars Banquet from the '60s; Exile on Main St. in the '70s. The band had shed most of its early blues roots by 1967's Between the Buttons and Their Satanic Majesties Request - both of which got caught up in psychedelic flavors of the era. Beggars Banquet took what they learned from those experiments and applied it to the music that made them want to start a band in the first place. That kicked off one of the all-time greatest runs in rock history, culminating in Exile on Main St., a double LP of late-night, sleep-deprived rock 'n' roll that still ranks as one of the best albums ever made.

DeRiso: It feels like cheating, since they came out sequentially, but Let It Bleed and Sticky Fingers. The first solidified their gutsy move away from found-object experiments and trend chasing. As for the second, I love the flinty confidence theyd gained to that point. That opens up Sticky Fingers to everything from cocksure rockers to paint-peeling soul shouts, from somnambulant ruminations to simmering blues. And on the album-closing Moonlight Mile, almost all of the above.

Reed: For the '60s, it's Let It Bleed. It isn't a perfect album does anybody count the fiddle-adorned "Country Honk" among their favorite tracks? but it's awfully close. "Gimme Shelter" could be the definitive Rolling Stones song the sound of amplified apocalypse. "You Can't Always Get What You Want" is a glorious miniature symphony. And underrated cuts abound. For the '70s, it has to be Sticky Fingers. The greasiness of those riffs in "Brown Sugar" and "Can't You Hear Me Knocking," the lonesome ache of "Wild Horses" it doesn't get much more memorable, this decade or otherwise.

Lifton: Can I go with hits compilations from both? No? In that case I have to go with the usual choices of Beggars Banquet and Exile on Main St. Their songwriting was never as consistent as during that period, in terms of how they were seeing the world, their confidence as musicians and willingness to go outside their comfort zone. On other days, my '60s choice could be Let It Bleed, but I'm writing this on a rainy morning and "No Expectations" fits the mood, so Beggars gets the nod.

Irwin:For the '60s, I'm takingLet It Bleed.My reasons: Gimme Shelter and You Cant Always Get What You Want, my two favorite songs from the Stones entire catalog. What can I say? Im a sucker for the hits. From the '70s, I chooseExile on Main St. From first note to last, its the most complete release in the Stones' history;the rare double LP that's actually worth its length.

The Stones released many of their biggest hits during these time periods. Pick their most underrated song from each decade and explain your choices.

Gallucci: "Out of Time" was released on the U.K. version of 1966's Aftermath, their first real successful album-length statement. It's a great pop song and should have been a single, but it doesn't even show up on the Stones' many multi-disc compilations. It's easy to name almost any song from Exile as underrated, since all you really hear about are the two singles, "Tumbling Dice" and "Happy," and occasionally LP opener "Rocks Off." But I'm going to pick a song from the album before that one, Sticky Fingers: "Sway." The entire album is great, but this Side One cut helps set the mood for everything to come: bluesy, soulful and mournful. Plus, Mick Taylor has a couple of great solos, maybe his all-time best.

DeRiso:From the 60s, Live With Me off Let It Bleed. The Stones first released song with Mick Taylor was Honky Tonk Women, but this was their first session together. His playing is already a wonder of nasty wit and sharp economy. A new era begins right here. From the 70s, Im sticking with the earlier-mentioned Moonlight Mile. I will never, ever tire of this songs many musical nooks and crannies. Then there's the utterly unselfconscious vocal: Its a crowning achievement for Jagger, who now more than ever is content simply to bark and bray.

Reed: My '60s pick is "Midnight Rambler," arguably the centerpiece of their best album that decade. Maybe you can trace it back to my love of prog-rock: This is the Stones at their least linear, utilizing unexpected grooves and tempo changes across the song's seven minutes. My '70s pick is "Bitch," a Sticky Fingers rocker with a snarling Richards riff and some tasty tenor saxophone.

Lifton: For the '60s, I'm going with "She Smiled Sweetly." It's clear that they were influenced by Bob Dylan's "Just Like a Woman" and they come really close to it. It lacks the misogyny of a lot of their early lyrics, and there's a vulnerability in Mick Jagger's vocals that he doesn't often show. I lovedhowWes Andersonused it in The Royal Tenenbaums- similar to theway he unearthed "I Am Waiting" in Rushmore. And it's hard to say that even a deep cut on Sticky Fingers or Exile is underrated, so I'm choosing "Memory Motel." A lot of my favorite Stones songs are the ones where there's a bit of longing, and the intertwining of Jagger's and Richards' vocals here is rather moving.

Irwin: From the 60s, Im going with Salt of the Earth, the ode to the common man found on Beggars Banquet. Theres a soulful earnestness to the track, and Richards vocals at the opening offer a raw vulnerability to its tone. From the 70s, I choose Shine a Light from Exile on Main St. Billy Prestons organ playing heightens this energetic gospel track, and the interplay between his work and Mick Taylors guitar offers one of my favorite Stones instrumental moments.

Which era made the Rolling Stones The Greatest Rock'n' Roll Band in the World?

Gallucci: The '70s. They planted that seed in the '60s, but once they made the turn with 1971's Sticky Fingers - following the equally excellent Beggars Banquet and Let It Bleed - was there any doubt? They were unstoppable.

DeRiso: The 70s, for good and for ill. They were part of a rabble of great bands in the 60s, but as a once-hope filled era drew to an awful close both in general and, after Altamont, specifically the Stones began to separate themselves. They would define the turbulent decade to come, again and again and again, from its early outbursts of violent emotions and drug-induced melancholy, to its descent into destructive appetite and malaise, and then into inevitable narcissism.

Reed: As my answer to the first question shows, I have a tough time picking between these two decades. But I have to go with the '60s here: I'd argue their legend was already cemented by 1965, the year of "(I Can't Get No) Satisfaction." They had more time to bolster an already impressive resume in the '70s. But if they'd stopped after 1969's Let It Bleed, just a few months before the Beatles' final LP, they'd still be considered a top-tier rock band.

Lifton: They never were. The Who could always blow them off the stage. But funny how the Stones didn't start calling themselves that until they started to go downhill.

Irwin: Apologies for using a lame analogy, but here we go: Imagine the Stones career like a cake. All the ingredients were there in the 60s and got mixed together well. Sure, the occasional person might argue that the batter tastes better than the cake, and Beggars Banquet and Let It Bleed offer two excellent reasons to do so. Still, the finished product - when the Stones were at their absolute best on Sticky Fingers and Exile on Main Street - came out of the oven in the 70s.

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Were the Rolling Stones Better in the '60s or '70s? Roundtable - Ultimate Classic Rock

5 extraordinary submerged sites that will make you believe Atlantis is real – BreakingNews.ie

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Theres something uniquely appealing about the prospect of a lost city at the bottom of the sea. We know nearly as much about the moon as we do the depths of our own oceans, and tall tales about the kraken and sea monsters survive because we dont know enough to fully disprove them.

But you neednt rely on your imagination for the mysteries of the deep, because theres enough reality beneath the waves to boggle most minds. These submerged settlements arent Atlantis, but they might as well be

1. Port Royal, Jamaica

Once nicknamed the Sodom of the New World, Port Royal was as close as there has ever been to a genuine pirate city. Awash with privateers and prostitutes and an infamously potent strain of rum the citys reputation for wickedness was known throughout the Caribbean, and notorious buccaneer Henry Morgan called Port Royal his home.

The debauchery continued unchecked until 1692, when, as if by divine justice, an earthquake levelled the city, plunging its brothels and taverns beneath the waves. Today Port Royal is one of the worlds most important underwater sites, and the ruins have sat undisturbed for centuries beneath the sea.

2. The Sunken City of Baia, Italy

Another famous den of hedonism, Baia was a playground for urban aristocrats in ancient Roman times. The city was described by philosopher Seneca as a harbour of vice, and entertained its clientele with free-flowing alcohol, limitless beach parties, and venues devoted to earthly pleasures, all with the understanding that the scandal would never reach Rome.

Eventually the city went the same way as Port Royal, though its fate was much less abrupt. Volcanic activity deep below ground saw the lower part of the city sink into the sea, and today divers can peruse crumbling columns, intricate statues, and elegant mosaics.

3. Reschensee, Italy(iStock/PA)

Reschensee differs from our other entrants in two important ways first, it was submerged deliberately, and second, its around 200 kilometres inland.

Known as the Atlantis of the Mountains, the Italian village of Reschen was flooded in 1950 to unify three lakes behind a large dam, despite the protestations of residents. Today only the church steeple remains, proudly protruding from the water like it was the most normal thing in the world. In winter the lake freezes over, and visitors can walk right up to the spire.

4. Ancient Alexandria, Egypt

Once one of the gems of civilisation, complete with a library said to contain hundreds of thousands of scrolls, the city founded by Alexander the Great remains one of the largest in North Africa, but its archaeological treasures were long thought lost to time.

In the last few decades wet-suited researchers have proved otherwise by meticulously probing the harbour floor. Since mapping began in the early-Nineties scientists have tagged dozens of columns, more than 30 sphinxes and five obelisks, as well as the submerged sister city of Heracleion. Best of all, French marine archaeologist Franck Goddio discovered a submerged palace with marble floors, which may well once have housed Cleopatra.

5. Pavlopetri, Greece

Pavlopetri is not among the worlds most visually impressive sites, but thats because its very, veryvery old. Dating back 5,000 years, this Mycenaean, Minoan, and late Bronze Age settlement is thought to have decayed beneath the waves since around 1000BC, and was only rediscovered in 1967.

Though heavily eroded, the town layout is exactly as it was, and researchers are using it as a guinea pig for 3D modelling techniques that should yield a digital reconstruction.

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5 extraordinary submerged sites that will make you believe Atlantis is real - BreakingNews.ie

Kylie Minogue Fans Have Broken The Internet At "Say Something" – Wonderland Magazine

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Stans have broken the internet at the singers disco-infused new drop.

The morale has been low. Spirits have been dampened. And dancefloors have remained bitterly empty and untrampled. But beloved queen Kylie Minogue is here to remind us that a time is coming a time of hedonism and body-shaking debauchery a less socially distanced future, when we will dance blissfully until the AM again.

Yes, Kylie has returned, and we have been blessed with a new album DISCO, which she put finishing touches to during lockdown slated for a 6 November release as well as pulsating lead track Say Something, out today. In honour of the new release, Kylie also joined TikTok. Our hearts!

There is no music video in sight yet, but we wait, entranced and not so patiently. The internet has obviously lost its mind at the euphoric news here are the best reactions.

Double Disco = Kylie Minogue + Jessie Ware albums

We live for an Eastenders crossover

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Kylie Minogue Fans Have Broken The Internet At "Say Something" - Wonderland Magazine

Purdue prof attracts cult following as expert on ‘The Black Death’ – Purdue Exponent

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Before COVID-19, Purdue University English professor Dorsey Armstrong was well known in a way that only other enthusiasts of medieval literature and culture might appreciate.

That is to say, she once got a discount on a replica of an Anglo-Saxon drinking horn - made from an actual cattle horn - because a guy at a conference recognized her.

"That's the only time I felt famous," said Armstrong, an expert in medieval studies who heads the English department at Purdue. "I got a really cool drinking horn. And whenever I teach 'Beowulf,' I bring it out and I pass it around."

But since the start of the pandemic, Armstrong, 49, has gained a whole new level of acclaim for her Old World expertise. She's the narrator of "The Black Death: The World's Most Devastating Plague," a video series that became must-see TV this spring when it aired on Amazon Prime, just as stuck-at-home 21st-century humans were reeling from the coronavirus crisis.

In 24 episodes, Armstrong introduced the devastation of the mid-14th century to doom-obsessed modern viewers. The flea-driven plague, also known as the "Great Mortality," overran Eurasia and North Africa from 1347 to 1353, killing tens of millions of people and wiping out half of Europe's population.

The series was filmed before the coronavirus pandemic, in 2016, as part of The Great Courses, a compendium of college-level audio and video lectures. But "The Black Death" has spurred a broad cult following for Armstrong, even as it underscores the dismaying parallels between the great plague and the deadly disease now circling the globe.

"I just wish that the course were not quite so relevant at the moment," said Armstrong, whose parents and siblings are among those who have contracted COVID-19 and recovered.

Since March, she has received a stream of daily emails from people who binge-watched "The Black Death," all wanting to know whether things are as bad now as they were back then.

The answer, thankfully, is no, Armstrong said. Though COVID-19 has infected more than 14.5 million people and killed at least 600,000 worldwide, the proportion of deaths doesn't compare with the devastation caused by the "Great Mortality."

The ferocious pandemic was dubbed the bubonic plague, reflecting the painful and (at the time) mysterious swellings, known as buboes, that developed in the lymph nodes of the neck, armpits and groin of those infected. The swellings oozed blood and pus, even as the unfortunate patients suffered other terrible symptoms: fever, chills, body aches, vomiting and diarrhea - often followed quickly by death.

As Armstrong notes, the disease could take other grisly forms: pneumonic plague, which infects the lungs, and septicemic plague, where the infection spreads to the blood, often causing skin on the fingers, toes and nose to blacken and die.

The Black Death originated in China and spread along trade routes, turning the Silk Road into a superhighway of infection. It arrived in many places via trading ships, long believed to be carried by the fleas on rats that coexisted closely with humans. A more recent theory contends that fleas and lice on humans themselves helped spread the disease widely. As deaths mounted, populations in region after region struggled vainly to understand its cause or cure.

"The good news is that, all things considered, we are in a much better position than those poor people who had to survive the Black Death," Armstrong said. "The mortality rate for the Black Death, for those who contracted it, was something like 80%. And we're still in single digits."

The modern world also has the advantage of seven centuries of scientific discovery that can root the current pandemic in a rogue coronavirus and target a treatment - and ultimately a cure - based on that understanding.

By contrast, humans suffering through the Black Death blamed an unfavorable conjunction of planets, "bad air," earthquakes and God's wrath. It wasn't until 1894 that Swiss scientist Alexandre Yersin discovered the bacillus that caused the plague: Yersinia pestis, named in his honor.

"During the Black Death, what was really terrifying about that is they really had no idea at all what it was," Armstrong said. "They had no good science to help them figure out how to cope with it."

Still, there are some unsettling similarities between societal responses to the plague and COVID-19. In both cases, some officials tried to downplay the severity of the outbreak and the far-reaching economic and social effects. In Florence, Italy, for instance, members of the elite ruling class, decimated by the plague, faced a rebellion from the newly powerful working class, Armstrong said.

Many people, from ordinary peasants to local religious leaders, took the plague seriously and tried to carry out their normal duties. Clergy members were called to the homes of the ill to provide last rites, often contracting the disease in the process. Others, however, ignored the calamity, turning to hedonism and debauchery, "figuring that if they were going to die, they might as well enjoy themselves," Armstrong said.

"What I would say is that people are the same then as now," Armstrong observed. "Humans, when they're together in a large group, often do dumb things. And it's frustrating that so many people don't seem to be learning lessons from the past."

It has been "particularly horrifying," Armstrong said, to see Asian Americans targeted as the presumed cause of the COVID-19 crisis. During the plague, Jews were scapegoated - and killed - as the possible source of the scourge.

"To think that anyone thinks you can call it 'kung flu,' which is so racist," Armstrong said, referring to President Donald Trump's characterization of COVID-19. "It's really distressing. We have international travel. Wherever it originated, it would have spread around the globe."

Amazon Prime officials wouldn't say how many people have viewed the series since March. But Cale Pritchett, vice president of marketing for The Great Courses, said tens of thousands of viewers have watched the show each month for the past four years. "It has been a constant in our top 10," he said. "For a while, it was No. 1."

Armstrong's clear mastery of the subject - her doctorate is in medieval literature - and her easygoing teaching style make for engaging TV. The show is set in an office decorated with replicas of skulls, bones and a distinctive beaked doctor's mask that was filled with sweet-smelling flowers to ward off the plague. Prop rats migrate around the set.

Armstrong, who favors brightly colored blazers, stands to talk through the 12 hours of lectures, striding back and forth across an ornate woven rug. She leavens the often-grisly subject matter with dark humor, reminding viewers, for instance, that the bodies of plague victims have been described as being layered in mass graves the way cheese is layered in lasagna.

In the more than 300 reviews of the series online, viewers note her approachable style. "She doesn't come across as an intellectual snob," one wrote. "I wish I would have had her as a professor when I was in college."

Because of her familiarity with the plague, Armstrong was alarmed this year when early reports emerged of an unknown virus spreading through China.

"I'm always worried about pandemic," said Armstrong, the mother of 13-year-old twin daughters. "I have peanut butter and toilet paper and water in a cabinet in the basement, even when there is no threat of a pandemic, because that is the situation that is the scariest. Especially if it's a novel virus, which is what this is."

In recent months, Armstrong's fears were realized as four family members - her parents in Seattle, her brother in New York and her sister in the San Francisco Bay area - contracted COVID-19. They've all since recovered, though her mother was hospitalized and received the antiviral drug remdesivir to aid her healing.

If there's one strong parallel between the Black Death and the current pandemic, it's the social upheaval spurred by both, Armstrong said. The 14th-century plague upended the rigid social structure of the era, which had confined people to narrow roles of clergy, nobility and peasant.

"Humanity came back after that," Armstrong said. "And some people would argue that it was that external pressure that changed society so radically that gave us eventually things like the Renaissance and the Protestant Reformation that all have their roots in that major event."

Perhaps current protests and calls for political, economic and social change may also have lasting impact.

"My hope is that we get something good out of this," Armstrong said.

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Purdue prof attracts cult following as expert on 'The Black Death' - Purdue Exponent

Is The West Repeating India’s Mistakes? Interview (Part II) – Eurasia Review

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Claudio Grass (CG): In such a vast and incredibly diverse country like India, can top-down measures and centralized policies like affirmative action or caste-based economic incentives effectively force social change and economic equality? Or can they be seen as merely symbolic moves, or perhaps just political maneuvers?(Note: Click here to read Part I of this interview)

Jayant Bhandari (JB):The government should get completely out of the business of social engineering. Even under a purely meritocratic system, India would be desperately short of leaders and competent bureaucrats. For example, India needs good doctors. Today, the best doctors emigrate, the second-best work for the Indian private sector, and those who knownothing, who usually get a certificate because of affirmative action policies, work for the public health system. This predicament is the reason why even the most impoverished Indians prefer to go to private hospitals, where their treatment costs might end up being more than what they earn in a year. Those who make, say, $50 a month, still find a way to send their children to private schools. The same dynamics are reflected in all institutions and all sectors. These policies have caused unimaginable destruction in the economy and in society at large.

You cannot elevate people, make them proud, self-respecting, and honorable by giving them free stuff and handouts. That way, you merely normalize begging and make it socially acceptable, thereby disincentivizing work and creating a permanent underclass. Decades of these policies have proven that you cannot force change from above, nor can you dismantle the caste system with a top-down approach. You can, however, make it lot worse and create new problems on top of the old ones, which is what the Indian government actually achieved. Today, free food, free electricity, and affirmative action in jobs, promotions, and university admissions have become such an integral part of the Indian state that they cannot be scaled back under Indias democratic setup.

So, what can be done about the caste system? As I said, tribalism afflicts the whole of Indian society, of which the caste system is only a part. Tribalism can go away only through an awakening in society. And that brings us to an extremely uncomfortable question: how to awaken people?

I returned to India in the early 90s, after my studies in the UK, hoping to participate in the Indian growth story. While I had a very successful career financially, and I contributed to bringing some well-paying jobs, I must admit that I failed to change the thinking of any Indian. How do you change people who are entrenched in materialism and hedonism? They have no interest in philosophy, in improving themselves, and in becoming a better member of society.

In this regard, the British were a blessing for India. Christian missionaries worked very hard, in challenging circumstances, to help awaken Indians, who were wallowing in magical thinking, superstition, the deeply entrenched caste system, and extremely backward social norms and customs, including the savage treatment of women in following the sati practice, i.e. the ceremonial burning of widows in the funeral pyre of their husbands.

A massive social shift happened in India under British guidance, mainly what is known as the Bengal Renaissance, which managed to make the sati system illegal, challenged the caste system, and encouraged great cultural, intellectual and artistic growth. Today, unfortunately, Bengal has erased all this progress and reverted to being a backwater, stagnant and regressive, mostly run by Marxist ideology.

Without the values the British brought and the work of the missionaries, the budding enlightenment fizzled out. I guess, even if the British had stayed put in India, there was a limit to which India could have been enlightened. Enlightening a society is not an easy job. It cannot be done in a couple of generations or even a couple of centuries. Europeans achieved very little cultural change in more than 300 years of their stay in India.

There is no moral and rational force present in the country today that can get rid of its tribalism. The vilest elements of India now run its institutions. It is this no wonder that tribalism is getting worse and all signs point to continuing degradation, eventually dragging India all the way back to its pre-British tribal dark ages.

If I had to make a recommendation, it would be to end all state-driven affirmative action policies in India altogether. The job of the government is to ensure law and order, not to force top-down social changes. The moral and legal boundaries set by a rational state, as was the case during the British times, would provide the right incentives and the structure for social awakening. But this is a utopian expectation, for how can you find good people and how can they reach positions of power, in a country where everyone votes on a tribal basis?

CG: In the Western world, were currently facing an extremely charged political climate, with protests, riots, and demands for change. Many of these demands are focused on the idea of different social and economic policies for different groups and on dividing society based on race, gender and other characteristics that one is simply born with and had no power over. Do you see a parallel there with the caste system?

JB:There certainly are a lot of parallels, except that the caste system in India is real, whereas racism in the US is very different from what it is made out to be. We simply cannot realistically compare the problems and the lack of opportunity one faces in US, no matter what the color of their skin might be, with the challenges that the majority of Indians face on a daily basis. BLM, Antifa and their ilk are idiotic movements run by losers who have no purpose in life, no skills, and who seek the thrill of destruction and abuse of others. Unable to build anything themselves, they derive a feeling of accomplishment by destroying civilization and its accumulated capital.

The radical Left has massively restricted freedom of speech in the US and the rest of the West. Anyone who does not go along with them is branded as racist and can expect to lose his job and social relationships. As a result, reasonable, sane people are afraid to speak up, to express their views or even to explore the validity of the claims made by BLM and similar groups, for they immediately risk getting doxxed, ostracized, and becoming a victim of cancel culture.

BLM, Antifa, and the radical Left are simply anti-civilization. Their members, by themselves, would have been ignored as nobodies and their ideas dismissed as lunatic. However, collectively, they managed to instill a climate of fear and intimidation and get tacit support from a sizeable part of the US population.

America is the greatest country on the planet. Life there is not perfect, but people who move to the US still see it as the land of opportunity. And it is. It is the most generous and open-minded country. Regardless of your skin color or your gender, you are very likely to get equal, or even preferential, treatment everywhere in the West, and particularly in the US. I have benefited hugely from this, and so have many people of color I have known.

The West is as good as it gets. I wasted many years of my life planning, and sometimes scheming, to get to the West. I can hardly think of anyone from Africa, Latin America, or the Indian sub-continent who would not sell himself into slavery or sell a kidney or two (if the latter were possible) just to get to the US.

Having lived in many different parts of the West over the last thirty years, I still cannot shake off the feeling of how much I could have achieved in life had I been born in the West. I feel sad for those who were extraordinarily lucky to have been born in the West, but still wasted their youth whining and fixating on its real or imagined failures to achieve total, utopian perfection, as if that is a realistic expectation.

As I mentioned earlier, affirmative action policies in India have achieved precisely the opposite of their stated goals. We see a parallel here with the situation in the US. The destruction of black families and incarceration among blacks has increased very significantly since the same sort of policies took hold there too, and since the adoption of a culture of low expectations from blacks and other people of color. Indeed, the worst kind of racism I have ever experienced is precisely this deeply offensive prejudice and this condescending assumption that I could not and should not be held to the same standards as everyone else. I find this attitude truly repulsive and I consider those who adopt it as hypocrites of the lowest order, as they like to cloak their racism in good intentions.

Finally, it is worth pointing out that there is likely no European today whose parents or grandparents did not suffer during the two great wars. Japanese-Americans and Japanese-Canadians were interned during WW-II. Japan, as a country, was flattened by the US at the same time. So was Germany. How many Japanese do you know who live in the US or Japan who still blame the US for what they did? Proud people move on, develop competencies, outgrow limitations set by other people, and emerge victorious. They certainly dont blame their failures on the real or imagined sufferings of their ancestors.

CG: You are in the rare position to have experienced the reality on the ground and the problems in both the Indian and western society. In your estimation, what are the biggest problems facing the US, Canadian or European society? Is it inequity and discrimination, or do we have other challenges that are perhaps being ignored?

JB:41% of Canadians and 49% of Australians are first- or second-generation immigrants. 46% of Americans are of non-European origin. A vast majority of ethnically non-Europeans tend to vote for the Left and for the nanny state, with its promises of free stuff and control over the lives of other people. Many simply fail to understand in their hearts what western civilization is. They are merely interested in the fruits of western prosperity. But without the roots, the tree that bears the fruit will eventually disappear.

I cannot see how the US can avoid a civil war once Trump is gone. In fact, it might have already started. What is, even today, the best country in the world will take a sharp turn to the Left once Trump is gone. The best-case scenario would be that the red states will revolt and secede. This will, of course, not be the end of the problem, as the blue states, being non-productive and dependent, will still ask for reparations and tributes. As for Canada, I cannot see how it can stand on its own feet without the support of the US.

Over in Europe, there is still some hope that people may wake up and realize the political and economic implications of the disastrous immigration policy of the last few years. Of course, I am not suggesting that they should not help out refugees, but this must be done rationally, with a realistic plan and with an understanding that even refugees who have suffered hugely from oppressive regimes can still bring that same virus of totalitarianism and intolerance with them. Once these toxic ideas enter the body politic and achieve a critical mass in a democratic system, they can present a very serious threat to the western culture, its values and its moral foundations.

CG: In our last conversation, you mentioned that youre more optimistic about East Asia, rather than the West, in your long-term view. What is the reasoning behind this position and what are the advantages you see there, especially from an investment point of view?

JB:East Asians have the highest average IQ in the world, they have an impressively strong work ethic, while civility and respect for others are ingrained in the culture and this is very clearly seen in everyday life. In Korea, Japan, Singapore, Hong Kong, and Taiwan, if your child is out by himself late at night, it is not a problem. Crime is virtually non-existent. In Singapore, for example, women leave their purses on the food court table to reserve their places while they go to pick up their order. These societies are incredibly polite, safe and respectful.

Social conformism is a part of their culture, but then, they are not busy-bodies. Their governments, quite in contrast to a wrongly held belief in the West, are largely non-intrusive. Overall, people there want to avoid confrontation and picking a fight as much as they can. No wonder I often feel freer in East Asia than I do in the West.

Also, technologically, East Asia is more advanced than the West. Hard work is a virtue, which is strongly encouraged, while begging is looked down upon. Complaining and expecting free stuff is seen as a sign of a lower culture, as it should be.

Although East Asians havent necessarily imported the philosophical values of the West, a lot of whats good in the region, especially in a more practical sense, is a direct import from the classical western civilization. Ideas around work, productivity, personal responsibility, law and order, respect for other people and their property, all formed a solid foundation that has produced healthy societies and economies, and this is why I consider East Asia to be the safest and most productive home for my money.

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Is The West Repeating India's Mistakes? Interview (Part II) - Eurasia Review

Cosmo the Bull Calf Has Been Genetically Engineered to Produce 75% Male Offspring – Interesting Engineering

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Genetic engineering can produce some pretty scary results. Just recently, scientists at the University of California, Davis, developed a bull calf, named Cosmo, who is capable of producing 75%male (or at least male-looking) offspring.

RELATED:11 FACTS ABOUT GENETIC ENGINEERING AND WHY IT'S IMPORTANT

The bull was genome-edited as an embryo using CRISPR technology. This method allows researchers to make targeted cuts to the genome or insert useful genes.

In Cosmo's case, scientists successfully inserted the cattle with the SRY gene. This gene controls the development of male features. The experiment marks the first demonstration of a targeted gene insertion for large sequences of DNA via embryo-mediated genome editing in cattle and it is made to produce cattle that look like males.

We anticipate Cosmos offspring that inherit this SRY gene will grow and look like males, regardless of whether they inherit a Y chromosome, said Alison Van Eenennaam, animal geneticist with the UC Davis Department of Animal Science.

As scary as the procedure may be, it could prove beneficial for the environment. Male cattle are about 15% more efficient at converting feed into weight gain, making them more fuel-efficient than females.

Ranchers could produce some females as replacements and direct a higher proportion of male cattle for market, said Joey Owen, a postdoctoral researcher in animal science who is leading the project with Van Eenennaam.

The project took two and a half years to develop the method to insert a gene into the developing embryo. It then saw another two years dedicated to successfully establishing a pregnancy.

And it is just the beginning of the researchers' work. When Cosmo reaches sexual maturity in a year, he will be bred to study if the experiment was indeed successful in producing offspring that will grow to look like males.

Cosmo and his offspring, however, will never enter the food supply. This is becausethe Food and Drug Administration regulates gene-editing of animals as if they were drugs.

What do you think of this initiative? Can it lead to a more environmentally friendly production of beef or is it a scary development in genetic engineering?

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Cosmo the Bull Calf Has Been Genetically Engineered to Produce 75% Male Offspring - Interesting Engineering

Impact of Covid-19 on Genome Editing Market 2020: set to witness adamant growth with Top Key Players Thermo Fisher Scientific Inc., Merck KGaA,…

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Global Genome Editing Market has been brewing up and influencing the international economy with respect to revenue, growth rate, sale, market share, and size. The Global Genome Editing Marketresearch report provides a rational explanation to the reader to understand fundamental attributes of the Genome Editing industry, which includes lucrative business strategies, market demands, leading players of the market, and growth prospects.

This is the only report that is inclusive of the current effect of the coronavirus on the market and its future trends. The coronavirus has widely impacted the world economy, and its aftereffects are elucidated in detail in the report for the Genome Editing market.

The sample of the report can be availed by [emailprotected] https://www.reportsanddata.com/sample-enquiry-form/1052

In the beginning, the report adds market properties, business stratagem, industry structure, issues, and industry effectiveness.The study ensures that the user is made aware of the prevailing market situations and the strategies that are employed for beneficial results. The report conducts a meticulous study of the past trends of the market; therefore, it provides very accurate and realistic speculations of the industry in the forecast period, i.e., from 2020-2026.

In market segmentation by manufacturers, the report covers the following companies-

Thermo Fisher Scientific Inc., Merck KGaA, GenScript, Horizon Discovery Group Plc, Integrated DNA Technologies, Inc, Lonza, New England Biolabs and Sangamo Therapeutics, Inc.

The Genome Editing report consists of streamlined financial data obtained from various research sources to draw specific and accurate projections, along with an in-depth analysis of the market trends of the Genome Editing industry and the factors that affect its functioning. Also, the factors are segmented into drivers and restraints for increased comprehensibility and understanding.

This research report has all the information you need to device optimum market strategies.

Type Outlook (Revenue, USD Million, 2016 2026)Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)Transcription Activator-Like Effector Nucleases (TALEN)Zinc Finger Nucleases (ZFN)Others

Application Outlook (Revenue, USD Million, 2016 2026)Animal Genetic EngineeringTherapeutic ApplicationGenetically Modified OrganismsPlant Genetic EngineeringCell Line Engineering

End User Outlook (Revenue, USD Million, 2016 2026)Pharma-Biotech CompaniesAcademic Institutes & Research CenterAgrigenomic CompaniesContract Research Organizations

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Major highlights of the global Genome Editing market report:

The report depicts all the analytical details in a well-structured manner, for example, in the statistics, graphs, tables, through which users can more easily grasp detailing. Moreover, it discusses accurate forecasts and gives a detailed research methodology.

Key Questions Answered in This Report Are:

Customization on the report is available according to the requirements of the user to ensure maximum utility to the reader and an increased level of comprehensibility.

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To summarize, the global Genome Editing market report studies the contemporary market to forecast the growth prospects, challenges, opportunities, risks, threats, and the trends observed in the market that can either propel or curtail the growth rate of the industry. The market factors impacting the global sector also include provincial trade policies, international trade disputes, entry barriers, and other regulatory restrictions.

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Impact of Covid-19 on Genome Editing Market 2020: set to witness adamant growth with Top Key Players Thermo Fisher Scientific Inc., Merck KGaA,...

Polymerase Chain Reaction (PCR) Market: Diagnostic Labs and Hospitals to Witness Promising Growth – BioSpace

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Increasing Investments to Help Market Development

There are several factors that are responsible for the overall development of the global polymerase chain reaction market. One of the key development factors for the global market has been increasing investments in the activities of research and development. In addition to this, emergence of digital polymerase chain reaction (PCR) technologies have become highly useful for cancer diagnosis as well treatment. This too has emerged as a key driving factor for the development of the global market. Furthermore, innovations and developments in pharmacogenomics and growing trend of self-diagnosis of disorders as a preventive measure are also expected to work in favor of the growth of the global polymerase chain reaction (PCR) market in the coming years of the forecast period. In recent years, the activities of research and development have considerably increased in the field of forensic science, genetic engineering, and advanced molecular biology. This too has worked in favor of the development of the global polymerase chain reaction (PCR) market.

However, there are a few factors that are projected to impede the development of the global polymerase chain reaction (PCR) market in the coming years. These challenges are projected to stop the market from reaching its full potential. One of the key restraining factors for the market development has been the emergence of alternative technologies. Some of the new and upcoming alternative technologies are next gen sequencing. In addition to this, high costs associated with some of the prominent polymerase chain reaction (PCR) technologies are also projected to impede the growth of the market in the near future.

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The global polymerase chain reaction (PCR) market is primarily segmented based on type of product and end user. Based on the type of product, the global market is mainly segmented into consumables, reagents, and instruments. Among these, the segment of reagents has been the most dominant one in terms of value. This dominance of the segment is due to its high levels of consumption. This segment is expected to witness a promising CAGR in the coming years of the forecast period because of the rising innovation in the field of specificity of reagents. In addition to this, large scale availability of different types of tests which need different types of reagents, increasing geriatric population, growing prevalence of infectious diseases, and increasing demand for innovation in specificity of reagents are some of the other factors helping to drive overall growth of the reagent segment.

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The segment of instruments is further segmented into digital polymerase chain reaction (PCR) systems, RT PCR systems, and standard polymerase chain reaction (PCR) systems. Of these, the sub segment of the RT polymerase chain reaction (PCR) is projected to account for a greater chunk in the global market in the coming years of the forecast period. However, the sub segment of digital polymerase chain reaction (PCR) is expected to witness a highly promising CAGR in the near future.

Diagnostic Labs and Hospitals to Witness Promising Growth

Based on end user, the global market for polymerase chain reaction (PCR) is segmented into research and academic organizations, clinical diagnostics hospitals and labs, and pharmaceutical and biotechnology industries. Among these, the segment of pharmaceutical and biotechnology industries account for a larger share in the global market. Constant advancements and developments in the fields of biotechnology as well pharmaceutical is projected to be the key driving factor for the development of the segment. These developments are also helping researchers and scientists to determine new gene expressions, genetic variations, and novel genes in the tissue of an organism.

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On the other hand, the segment of clinical diagnostics labs and hospitals is expected to witness a highly promising CAGR in the coming years of the forecast period. This growth of the segment is primarily driven due to the increasing prevalence of cancer and other prominent infectious disorders across the globe.

North America to Continue Leading Global Market

Based on geography, the global polymerase chain reaction (PCR) market is segmented into five key regions viz. North America, Latin America, Middle East and Africa, Europe, and Asia Pacific. Among these, the regional segment of North America has been the most dominant one in recent years. The growth of the regional market can be attributed to the matured and developed healthcare infrastructure present in North America. In addition to this, in recent years, there has been a growing advent of automated polymerase chain reaction (PCR) instruments. This, coupled with improved spending power and affordability of these instruments have also helped in improving the development of the regional market.

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Major players operating in the global PCR market include Bio-Rad Laboratories, Inc., QIAGEN N.V., F. Hoffmann-La Roche AG, Thermo Fisher Scientific, Inc. Becton, Dickinson and Company, Abbott, Siemens Healthcare GmbH (Siemens AG), bioMrieux SA, Danaher Corporation, and Agilent Technologies. Key players are expanding their product portfolio through mergers & acquisitions and partnerships & collaborations with leading pharmaceutical and biotechnology companies and by offering technologically advanced products.

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Polymerase Chain Reaction (PCR) Market: Diagnostic Labs and Hospitals to Witness Promising Growth - BioSpace

British people won’t thank the NFU for its stance on gene editing – The Grocer

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Farmers popularity has shot up. The general public used to ignore them or view them as subsidy junkies. Now, as the people who put food on our plates during the Covid-19 crisis, they are enjoying levels of gratitude unprecedented since the Second World War.

The NFU has increased this goodwill by campaigning to prevent the import of US foods produced in ways that would be illegal here. Its high-profile petition to that effect gathered over one million signatures, and certainly heaped pressure on the multiples to say no to chlorinated chicken.

But this tentative trust the public feels towards farmers would surely evaporate if consumers realised the NFU was lobbying hard for gene editing thats turbocharged genetic engineering, only with a less threatening name. The NFU backed an amendment to the Agriculture Bill that would allow ministers to change the law in England.

It would mean that gene editing would not be classified as genetic modification and could just be slipped quietly into our food system, even though it poses the same potential safety risks.

So if the NFU is truly defending higher UK food standards, why is it simultaneously enabling the introduction of this controversial technology that the British public, if asked, would surely reject? A 2017 survey for The Grocer by Harris Interactive showed 45% of Brits were concerned about genetically modified grain.

The NFU says gene editing will put the UK in a world-leading position to showcase sustainable climate-friendly farming. But as a national organisation, why is it lobbying for a change that would put England at odds with Scotland, Wales and Northern Ireland, which have banned GM crops and feel no need to change the definition?

Lawyers acting on behalf of the NFU have also been in court battling to overturn an EU ban on neonicotinoid pesticides, linked to harming bees. As lawsuits for alleged human health damage caused by glyphosate pile up in the US, NFU spokespeople take to the airwaves to say that they cant farm without it.

That petition? I signed it, but the NFU is no peoples food champion.

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British people won't thank the NFU for its stance on gene editing - The Grocer

Facing taboos: Conversation with GLP’s Jon Entine on sustainable agriculture, race and sports, ‘Jewish genetics’ and social investing – Genetic…

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Jon Entine is an American science writer. He is the founder and executive director of the Genetic Literacy Project, a nonprofit that educates the public about the revolution in biomedicine and agricultural biotechnology. He was formerly a fellow at the Institute for Food and Agricultural Literacy at the University of California, Davis, the Center for Health & Risk Communication at George Mason University, and at the American Enterprise Institute. After working as a network news writer, producer and head of documentaries for NBC News and ABC News from 1974-1994, Entine moved into scholarly research and print journalism.

Entine has written seven books, four on genetics and chemical risk, and hasaddressed a range of controversial issues,includingthe genetics of sports; the shared ancestry of Jews, Christians and Muslims; socially responsible investing; and why organic farming will not scale to produce sustainable food. He is a contributing columnist and writer for dozens of newspapers and magazines. He has was won 19 major journalism awards including two Emmys, three CINE, Ohio State Award, Chris Award, Best Feature Film Interntional Sport Film Festival, and a National Press Club Award for Consumer Journalism.Much of this interview appeared originally in European Scientist and was conducted by science journalist Grgoire Canlorbe.

Grgoire Canlorbe:You carefully investigated the genetic underpinnings of the over-representation of blacks in many high profile sports. Could you remind us of the fruits of your inquiry? Why do whites dominate strength related positions and events in so many sportsand why are blacks so poorly represented in some major sports, such as swimming?

Jon Entine:I think its phenomenal, really startling that if you look at the major sports around the world: track and field, football in Europe, American football, baseball, and basketball, which is an international sport, you see a very odd distribution of which athletes do the best in various sports. In many of the sports, the ones that require speed, quick reaction time, things like global and American football or basketball or sprinting, its utter dominance by athletes of West African ancestry.

In long distance running, which requires endurance, you see the dominance of East Africans and a few North Africans, whose ancestors evolved in higher altitudes, shaping their physique and physiology. You look at strength events, and you see dominance of East Europeans and Euro-Asians with very minimal representation of those of African ancestry. These arent just recent aberrations.

These patterns have persisted for decades and have actually become more pronounced as the playing field got more level, so natural talent could emerge and environmental factors were at a minimum. Once the influence of performance enhancement drugs during the 1960s, 70s, and 89s driven by Russia and the Eastern bloc dissipated, which distorted who were the best athletes, we saw these patterns become even more pronounced around the world. And I think the more you research this, the more you understand that at the elite level of athletic competition, we are very much a product of our genetics and the patterns reflect evolution in different geographical areas.

This is not a black/white issue or an issue of race as we have traditionally used the term. Its about regions of evolutionary origin. Phenotypes and genotypes are shaped by thousands of years of evolution. Although some characteristics seem to loosely correlated with traditional, folkloric notions of race, many do not. Just look at the difference in body types and athletic skill sets of distance running East Africans and elite athletes who trace their primary descent to West Africa. The differences in physiology and physique may be small in the case of some characteristics, and there is a great deal of overlap, but those differences are magnified at the elite level of sports competition where a fraction of a second can make the difference between winning a gold medal or being an also ran.

Social factors alone or even significantly cannot explain why the top two thousand all time 100 meter times are held by a person of West African ancestry yet West Africans are almost nonexistent at the elite level of medium and long distance running. I addressed many of these issues, along with the toxic history of race science, in my book Taboo: Why Black Athletes Dominate Sports and Why Were Afraid to Talk About It. Although the book is now 20 years old, and some data are dated, the arguments in the book are now mainstream science and genetics. It was actually based on a documentary that I wrote and produced back in 1989 with Tom Brokaw, Black Athletes: Fact and Fiction.

The idea that anybody can grow up and become an elite athlete with the proper training and opportunities is just not supported by what we know of genetics. Genetics is not destiny, but I would say that genetics is like designing a house. You can tidy up the rooms a little bit, you can move things around, but generally speaking, who you are is like the house itself. Once its built, its set, and these predeterminations are the result tens of thousands of years of evolution.

That said, there are always cultural and genetic factors in play. There is a biocultural feedback loop in sports in which culture helps magnify small but meaningful biologically-based differences. People say, Oh, there are few blacks in ice hockey, for instance. Well, ice hockey is played in northern climates, and there have been relatively few blacks in Canada or in Europe, historically. So, the number of blacks is almost representative of the number of blacks in those regions. And some sports, like gymnastics, for instance, or swimming, require a lot of training. They require facilities, pools that are very expensive. Social opportunity has largely excluded minorities. The more factors that cost a lot of money, like the availability of expensive facilities, then, cultural and social factors come into play.

The sports that I cited: running, football, soccer, and basketball which are usually state sponsored or sponsored by schoolssports like those represent a level playing field. It doesnt require special financial advantages to be a great long-distance runner or sprinter. Its really natural talent that comes to the fore. So, its best to think of sports as a biocultural phenomenonsports success. And the lower the cultural barriers to entry, the more genetic factors come into play. And those genetic based differences are not distributed equally among populations. In running, blacks of West African ancestry dominate the sprints, totally. In long distance running, blacks of East African ancestry dominate. And thats purely a result of our genetic history.

Grgoire Canlorbe:It has been hypothesized that race differences in intelligence and in psychopathy should be connected to the severity of encountered winters over tens of thousands of years of evolutionary timewith Caucasians and a fortiori Northeast Asians having faced the coldest winters and consequently evolved the higher IQs and the lower psychopathy levels necessary to navigate difficult environmental circumstances. Do you endorse this alleged connection?

Jon Entine:Well, there has been some speculation on that controversial issue by evolutionary psychologists and others, as well as some geneticists, that some people embrace and some people do not. One of the suppositions is that evolution does shape who we are physically, and there are group differencesoverlapping but real. We know that. And so, some people have asked, if genetics shape us physically, and we see the examples in sports, it must shape us psychologically and intellectually, as well. And theres belief among many scientists that there are patterns of differences based at IQ testsalthough many people like to dismiss them as unimportant or pseudo-science or racist. I think theres profound evidence and belief within the psychometric community that IQ tests are very real measurements of a kind of intelligence. But how much of the differences are the result of evolutionary factors versus environmental and cultural factors, including those that impact biology, such as natal and childhood development. Obviously environmental factors predominant in explaining patterns of differences.

But there is speculation that evolutionary factors are in play as well. As one theory goes, people who evolved in climates that were more rigorous, cold climates, northern climates, lets say East Asians, Northern Asians, whites from northern Europe, forged unique survival techniques. So that may be a form of intelligence, its claimed by some: how do you survive in that harsh environment? You have to build infrastructures that allow you to survive in harsh winters. But if youre living and evolving in an area where the temperature is more temperate, then you dont have the same survival challenges, and its not as challenging on your brain. Thats a theory anyway. So one of the theories in evolutionary psychology is that that there is some link between certain expressions of intelligence and whether you evolved in a cold climate versus a milder or warmer climate.

As for the alleged population-based differences in psychopathyI think its a very speculative claim. I think its something that science could explore over time, as all issues of what drives behavior is worth understanding. But were not in an area hard evidence. Were in the area of theories that make sense based on what we observe anecdotally

Stephen Gould, a famous, now deceased evolutionary biologist, believed that these kinds of stories were what he called just so stories, meaning that they sound good, but you really cant prove them. And I think this issue falls into that category. But on the other hand, it offers some reasonable explanation, like Occams razor, of why certain factors are reasonably like to be true. And so, I think its definitely something that, if you were someone trying to make sense of the unfolding of history, you would explore this is as a possibility. I dont think we have enough understanding of genetics, though, to say for sure that this his is a really persuasive argument in this case.

Grgoire Canlorbe:It is not uncommon to invoke the Beckerian claim that free competition between economic firms tends to evacuate racial discrimination in that those of firms which are basing recruitment on race rather than on work efficiency are allegedly disadvantaged with respect to their competitors. Does this line of thought capture the actual functioning of market economiesin America and elsewhere?

Jon Entine:I think it depends on what you want in a competition. If its purely an IQ competition, then, youd want the highest IQ people to win. If its a competition, like an athletic one, for the fastest runner, the person could be a total jerk and not be particularly smart, but if theyre the fastest runner, theyre going to win. Now, in a complex society, the leaders who emerge are going to have a whole suite of qualities, not just intelligence, not just drive, personality characteristics like sociability. And so, youre going be wanting many factors, many qualities, in leaders, besides just naked intelligence, if that even exists. And anytime you introduce a qualifying factor like race, it distorts the analysis. Now, you could argue that you want race as a factor because lack of racial diversity leads to a misperception of how the world really is.

So you can make an argument for it. But theres no question that if you only have the tallest people, or you only have the smartest people, or you only have some special factor that you tease out, youre going to leave out other, potentially hugely important, leadership and achievement qualities. I think its potentially dangerous that race be used as a factor if its over-exaggerated and it ends up discriminating against people who otherwise would be better able to perform in that situation.

Grgoire Canlorbe:Please tell us about the DNA of Abrahams Children. Do the genetic and historic data support Arthur Koestlers thesis that Ashkenazi Jews, instead of descending from the Ancient Hebrews, are the descendants of the pagan Khazars who purportedly converted en masse between the 8thand 10thcenturies? Or the claim that Jesus was ethnically Galilean and, also, partially Greekand virtually indistinguishable from Romans in appearance?

Jon Entine:I addressed this issue and the genetic history of Jews in my book,Abrahams Children: Race, Identity and the DNA of the Chosen People.

In the wake of World War II, during which many Jews were killed in the Holocaust, a well-known journalist of the 1950s, Arthur Koestler, tried to take the sting out of the idea that Jews were a race, which he believed was a driving factor behind the Nazi mass killings of Jews. The belief that Jews were a race was widely embraced by most everyone until the Holocaust, including who had long considered themselves a race, for many, many centuries. The word was viewed very loosely, however. It combined cultural factors with alleged physical and sociological factors, some clearly anti-Jewish. But obviously, this racialization of Jews led to a historic conflagration in World War ll.

The concept of Jews-as-race hinged somewhat on the ideas that most Jews of that time shared a common ancestry in ancient Israel. In his book The Thirteenth Tribe, Koestler propagated the idea that overwhelmingly majority of Jews, which were by then scattered around the world, actually were not descendants of the Israelites bur rather were mostly converts from Christianity, or more likely from paganism. He maintained that the large Jewish population in Eastern Europe and in parts of what is now Russia in an area once known as Khazaria were descendants of pagans who had converted to Judaism between the eighth and tenth centuries. That was the thirteenth Jewish tribe, he claimed, riffing on the Bible. Those converted Jews, primarily of Turkic origin, became the core of modern Judaism, gradually growing in number in Eastern Europe and, expanding westward, he maintained.

In essence, in an attempt to destroy the belief that Jews were a coherent race with links to the Middle East, which he believed propped up anti-Jewish racism and gave an excuse for Jews to live in a homeland in the Middle East (like many liberal Jews of the 1950s, he was opposed to the establishment of Jewish State in mostly Arab territories), he pushed the notion that Jews were converts with no ancient links. Koestlers view was widely embraced by many progressive Jews, traumatized by World War II, who wanted Judaism to shed the long-held belief that they were a race separate and apart. And, of course, many Middle Eastern Arabs embraced that theory as well, and went on to claim that it shut the door on the Jewish Biblical claim of a right of return that was a fundamental tenet for the creation of Israel. So you can guess the controversy it stirred then, and it reverberates even today. Far left Israeli radical, Shlomo Sand, wrote a best selling book, The Invention of the Jewish People, in an attempt to resurrect Koestlers speculative thesis. It became a best seller in some circles and has been cited in recent by anti-Israel groups inside and outside of Israel.

Koestler had understandable motivations, but they werent based on science. Genetic research has largely eviscerated his thesis. Weve been able to do a lot of genetic research on both Jewish men and Jewish women over the past two decades. And it appears that, although there is a sliver of truth to what Koestler claimed, its largely wrong. What we now know is that about 80 percent of males who claim Jewish ancestry, by looking at the Y chromosome and other parts of the genome of many thousand of people who claim Jewish ancestry, you can actually trace their ancestry back to the Near East, Middle East. So it indicates that they do have a history along the male lineage that traces back to what was biblical Israel.

On the female side, it looks like many females, about 50 percent, appear by their DNA to be converts, although the genetic evidence here is not as definitive. Its believed that a lot of the men left what was biblical Israel, Palestine, moved through Asia and what is now Italy to Europe and took local wives, either pagan wives or Christian converts. And so, there is a mix in Judaism. It appears there is, on the male line, mostly ancient Jewish ancestry, and on the female line, a mix.

We also do know that there were some converts from Khazaria, but it probably happened only among the elite few and numbered in the hundreds or thousands. Pagans were the majority, 98 percent of the population. By and large, its believed they were not affected by the religious practices of the elite. But the elite did convert to Judaism, based on sketchy historical evidence. It is speculated that many of them aspired to be part of the Jewish priesthood, the Aaronite line, those known as Cohanim. But because they were not born into that line, which is Biblically required, they were not accepted as priests. And so, they became essentially junior priests, a Jewish lineage known as Levites. And there is evidence of that in the genes of maybe, 12 to 15 percent of Jewish males, almost all with anecdotal evidence of Levite lineage, can trace their ancestry to Khazaria. So, there is some small truth in Koestlers claims, which is one reason it got such currency. Yes, there were some conversions in Eastern Europe, but it isnt the major seed of the Jewish population today.

As for the question about Jesus, I dont think we really have any understanding of what Jesus looked like. There are depictions of Jesus that go from light black to very Aryan looking. So, I think this is pure speculation about what Jesus actual genetic background was. But I think most of the biblical accounts, and theyre very sketchy, as I discuss inAbrahams Children, suggest that he was of Israelite ancestry, and that population was a mixture of locals and invading populations. The belief that Jews are a race is clearly flawed by there is powerful evidence that a significant portion of the modern day Jewish population traces their primary ancestry to what is now Israel. Thats science, however, not an endorsement of the Biblical claim.

Grgoire Canlorbe:While it has encountered periods of obscurantist and literalist remnants such as the burning of Maimonidess work in the 13thcentury, Judaismsince, at least, the times of the Jewish community of Alexandriahas been carrying within it Hellenizing principles such as the rationalization of the Torah, and such as the pursuit of knowledge through personal doubt and the confrontation of opinions. Has Islam gone down a similar route (towards interpretation and free inquiry) since the intellectual bubbling of Andalusia under Muslim rule?

Jon Entine:I think that many historical populations go through times of sophistication and then, fall into a retrograde period. It happened to the Egyptians, the Greeks and the Romans. You can actually see that trend in Judaism during the Middle Ages, into the 1600s, when Jews actually became very obscurantist and adopted many mystical beliefs, and were thought of as very irrational by the Christian majority. Now, they were very literate, but they were literate in a mystical kind of way; they read Jewish religious works but little else. The Christian enlightenment actually preceded the Jewish enlightenment by about a hundred years because Jews were caught into this mystical trap. But historically, Jews have always been a very literate culture.

Islam has had a much different history. It arose in the first millennial period. Muslims were by and large quite well educated in the early years of Islam. But over the centuries, theyve been back and forth between kind of a nomadic anti-intellectual history and one of intellectual inspiration. There was a period between the 8thand 14thcenturies when Islam was very dominant in Northern Africa and the Iberian peninsula, and at one point reaching to the area around Barcelona. The Muslims called their Caliphate and homeland Al Andalus. Their cities were great centers of Islamic learning, and the great libraries of the world were Islamic. Medicine advanced dramatically during this period. The period is sometimes referred to as the Ornament of History or The Ornament of the World, as for the most part, Jews and Christians lived mostly safely and in harmony with their Islamic neighbors.

The dominant intellectual group, the doctors, the legal positions of that time and the great intellectual thinkers were mostly Muslim. It is one of the only periods of tolerance among the three Abrahamic religions in history. But the rise of the Christian kingdoms ultimately crushed Muslim strongholds, and Islam never really recovered from there. Theres never been a Muslimsociety that performed at the level of Asian societies or European societies since the collapse of that era. So, there are definitely different traditions among different groups based on their cultural experiences.

Grgoire Canlorbe:As a fine connoisseur of chemophobia you cannot ignore the climate of mistrust surrounding the glyphosate. Why do you judge glyphosate and GMO farming to be far more sustainable, actually, than organic farming? What do you reply to the claim that the proponents of glyphosate should be ready to drink a glass wine of the latter if, truly, they think and intend to show that this product is half toxic as salt?

Jon Entine:Glyphosate was a product discovered literally by mistake in the 1970s, and its been used mostly as an herbicide. Scientists found that it has an ability to kill weeds inexpensively at modest toxic levels. Its toxicity is about equivalent to salt; its quite mild, not carcinogenic based on thousands of studies, and has little to no environmental footprint. Its quite a remarkable chemical concoction. Scientists in the 1980s figured out how to make commodity crops such as corn, soybeans and cotton, that tolerant to glyphosate. In other words, if you sprayed those crops, herbicide-tolerant glyphosate, originally developed and marketed as Roundup by Monsanto, which is now owned by Bayer, was one of two products that were the first out of the gate when GMO crops were introduced in the 1990s.

The other major commodity products were engineered to include a natural bacterium, Bacillus thuringiensis, actually used since the 1920s by organic farmers, that made certain crops resistant to many harmful insects. Those were the first two GMO products out of the gateherbicide tolerant and insect resistantand they were enormously successful, sparking the GMO boom that began in 1996. They also became the target of anti-GMO activists for many years, even though both of those products have been found extremely effective and safe.

A problem began to develop with glyphosate, though: many weeds developed a tolerance to it. Just because thats what happens. Evolution is evolution. And if you keep spraying weeds with a certain kind of weed killer, mutations eventually happen that allow the weed to survive. And after a few years, you have a whole bunch of weeds that arent being killed by glyphosate. So, we had this explosion of weed problems by 2010 or so. It was a real issue, although weed resistance is a fact of nature with non GMO-based weed killers as well. But the issue put conventional agriculture and genetic engineering on the defensive, absolutely.

And then, a controversial study came out in 2015 by a sub-agency of the United Nations called IARCInternational Agency for Research on Cancerand it concluded that glyphosate might cause problems for applicators, people who apply glyphosate, and that they could be subject to one specific kind of cancers, non-Hodgkins lymphoma. IARC was a relatively obscure agency before that finding. And its conclusions were contradicted by every other major regulatory and research organization in the world, 18 other international agencies from WHO itself, two other WHO sub-agencies, the European Food Safety Authority, the German Food Safety Authority, the Royal Academy in London, the United States EPA, and academies in Canada, Japan, Australia and New Zealand.All concluded that there was no convincing evidence that glyphosate causes cancer, and none recommended a ban.

Click here to review 19 reviews of glyphosate by independent global regulatory bodies

So, you had one agency, IARC, that said glyphosate might cause cancer under limited circumstances. They didnt say it affected humans when traces were in our food. And all the other major agencies, 18 of them, concluded that IARC was promoting flawed science, and that they selectively misrepresented the data. And many scientists also accused IARC of being politically motivated. Reuters documented that IARC appears to manipulated data. In fact, the main IARC scientist who guided and drafted the report secretly joined the litigation team that sued Monsanto, creating an undisclosed conflict of interest. But because IARC was linked to WHO, even though it was a sub-agency not WHO itself, its conclusions were widely publicized, especially by anti-GMO activists. Its monograph scared a lot of people and has led to a lot of lawsuits. And there have now been legal cases in the US in which juries rejected the overwhelming science evidence that shows that glyphosate is not harmful. But juries can do whatever they want. Plenty of people are convicted or found innocent, independent of what the evidence really shows. And, basically, Monsanto, now Bayer, was found guilty in multiple court cases of claims that it caused non-Hodgkins lymphoma in a number of workers who handled it.

So, basically, many people are now scared of glyphosate and politicians respond to public opinion, not science. There are moves afoot to ban it, even though its the most successful and one of the least toxic herbicides one can use. Its still in wide use. But over time, I think it will be phased out. And farmers are very upset about that. I think most farmers believe its extremely safe. Scientists overwhelmingly believe its safe. The myth that glyphosate is dangerous has been kept alive, and its really part of the ongoing war that exist about what kind of food system we want to have. Yet, there is no herbicide alternative in the organic world that is as safe as glyphosate. Theyre all as or more harmful. Many natural applications suffocate beneficial insects. So, we have a choice.

At some point, we can force farmers to abandon it, have less yield and kill more beneficial insects, or we can keep it, use it appropriately, follow guidelines that are endorsed by every major science organization in the world, from Health Canada to the European Food Safety Authority, and maintain a robust agricultural system. I think that all those people who are so ideological on agriculture and who despise biotechnology, in essence, are modern Luddites. They so reject modern technology that they will sacrifice our food supply for their ideological purism.

Grgoire Canlorbe:The fight against greenhouse gas emissions is most often put on an equal footing with that against nuclear poweras well as with the fight against GMOs and advanced agricultural technologies. Do you think that biotechnology and nuclear industry, on the contrary, should be jointly put at the service of depollution? That GMOs and nuclear technology are actually good for the climate?

Jon Entine:Well, I think organic farming is based on principles that are 100-150 years old, and I cant think of any technology that we embrace today thats 100 or 150 years old and believe that somehow its the cutting-edge way to do things. If you want agriculture to be sustainable; you need to have the most advanced and wisest science-based practices. Organic farming promotes soil health, and thats an emulatable goal. But there are so many other aspects to organic farming that are just outdated. Theres the belief that anything thats natural is betterthat if we put natural chemicals on plants, theyre going to be healthier. But organic farming uses copper sulfate, for instance, which is carcinogenic to humans and very dangerous: it kills beneficial insects. Thats clearly something that we wouldnt want to use if we had alternatives. Biotechnology, GMO farming, can be used inappropriately too, but it also offers many potential advantages because you can use weed killers that are focused specifically on weeds and preserve the crops and dont require tilling, which releases greenhouse gases into the atmosphere.

So, from a purely carbon preserving point of view, conventional farming using biotech seeds is much better than organic farming. Organic farming is also about 40 percent less yield efficient. Youre going to have to clear cut forests to get the same kind of yield globally that you can get in farming, using conventional means with GMOs. We cant afford, environmentally, to give up any more of our land to farming or urbanization. The newer gene-edited crops are designed to use fewer and fewer chemicals, and in some cases no chemicals. We are on the verge of developing crops that naturally create nitrogen to fertilize the soil without chemicals. We need fertilized soil to get the kind of yields that are appropriate for an industrial society. But nitrogen can cause all kinds of environmental problems. But gene editing is in the position to address those kinds of things. So, I guess the real question should be: Why dont we have a farming system that is based on sustainable principles rather than choosing organic and pitting it against conventional or GMO farming? We should pick the best elements from each system, and that should be the goal rather than ideologically proposing that either GMOs or organic is the best way to go.

I think nuclear energy and biotechnology are two of the most important tools to fight climate change. Nuclear energy is the only advanced technology we have right now in the energy sector that directly reduces carbon emissions to zero, with limited consequences. If you look at a lot of the renewable energies, they all have other consequences.

Wind towers chop up birds, and hydropower plants dam up rivers and cause mass death among fish. Nuclear energy, if its handled properly, especially the latest generation, is an essential tool in fighting climate change. And were naive to think that we could, through alternative energy alone, meet the challenge of reducing our carbon footprint. And GMOs and gene-edited crops do the same thing. If we have no till agriculture where carbon is not released, we can dramatically reduce the carbon footprint.

If we dont have to clear cut forests to grow organic food, we dramatically cut carbon. If we dont have cows, which burp methane gas, which is 20 times more carbon toxic than carbon itself, thenand we need those cows to generate fertilizer for agriculture or organics, and if we dont have those, we are very much in a better position to fight climate change. So, if we move in the direction of regenerative agriculture, organic agriculture, it will be a disaster in the long run. We need a mix of technologies in agriculture that are respectful of the cultural traditions of various communities, but also are sustainable. Otherwise, were courting long-term climate change disaster.

Grgoire Canlorbe:You openly distrust idiosyncratic ideological screens in investment decisionsand believe that such way of proceeding is more likely to harm people and the environment. Which sociopolitical system in the broad sense is the most immune to the siren voices of socially responsible investing?

Jon Entine:Im all for a system that encourages investments in socially or environmentally progressive activities, but if you become totally focused on systems that dont have an economic return, they ultimately cant survive over time. The socially responsible investing movement, the problem with that is twofold: one, the values that it promoted were very ideological. So, for instance, it determined among most social investment professionals that defense spending was something that should be avoided. I imagine that if people hadnt invested in defensive weapons, we could have lost World War II. So, I dont see avoiding defensive weaponry or protective weaponry necessarily as something thats not socially responsible; its too blunt an idea.

So, its really concerning that were going to develop an investing system based on peoples whims and their ideology. You could have a Muslim investing system, which is competing with a Jewish investing system, which is competing with a Christian one, each one thinking that their particular values are superior. And there are in fact Muslim and Christiana and Jewish social investing funds. Its pandering as it does not influence behavior; its evolved into a money-making gimmick by those who sell the funds. So, I think its best in investing to try to get the best return that we can and empower individuals and organizations to use their money in socially responsible ways. But I think rigging the system so that certain kinds of activities are rewarded, and theres not the economic incentives to provide checks and balances within the system, is a prescription for economic inefficiency.

Grgoire Canlorbe:Many expect neuro-augmentation and genetic manipulation to allow Homo sapiensthrough the taking over of their own biological evolution and the abandonment of the random processes of natural selectionto achieve the Cartesian project to render humans the masters and possessors of nature. Is transhumanism a reasonable dream?

Jon Entine:I think its reasonable to think that were going to change our human genome. Were already able to do that in small ways. Were already able to make micro-changes in the genome, and I think, over time, theres no doubt that well be able to rid ourselves of certain genetic disorders. Huntingtons disease would be one good example, as we know its linked to one gene. But the human genome is very complex. Human behavior and our chemical and genetic makeup are extremely complex. So, the fact that we can manipulate genes doesnt mean we can manipulate them precisely.

There always are consequences. Removing one gene or a suite of genes could have unintended consequences. I think, ultimately, over time, we are going to harness the genome and use it to develop many therapies that dont exist now. Whether we can develop the superhuman Sapiens Sapiens, I think that thats probably not necessarily in the cards. But I do think that we will make a lot of progress in coming decades in fighting many diseases that now seem out of the reach of the medical community.

Grgoire Canlorbe:Thank you for your time. Would you like to add anything else?

Jon Entine:I have nothing else, no. Thank you for the interview.

Grgoire Canlorbe is a journalist specializing in the scientific field. Find Grgoire on his website and on Facebook.

A version of this interview was originally published at the European Scientist@EuropeScientist

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Facing taboos: Conversation with GLP's Jon Entine on sustainable agriculture, race and sports, 'Jewish genetics' and social investing - Genetic...

The clonal repopulation of HSPC gene modified with antiHIV-1 RNAi is not affected by preexisting HIV-1 infection – Science Advances

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Abstract

Despite advances in hematopoietic stem/progenitor cell (HSPC) transplant for HIV-1infected patients, the impact of a preexisting HIV-1 infection on the engraftment and clonal repopulation of HSPCs remains poorly understood. We have developed a long terminal repeat indexing-mediated integration site sequencing (LTRi-Seq) method that provides a multiplexed clonal quantitation of both antiHIV-1 RNAi (RNA interference) gene-modified and control vector-modified cell populations, together with HIV-1infected cellsall within the same animal. In our HIV-1preinfected humanized mice, both therapeutic and control HSPCs repopulated efficiently without abnormalities. Although the HIV-1mediated selection of antiHIV-1 RNAi-modified clones was evident in HIV-1infected mice, the organ-to-organ and intra-organ clonal distributions in infected mice were indistinguishable from those in uninfected mice. HIV-1infected cells showed clonal patterns distinct from those of HSPCs. Our data demonstrate that, despite the substantial impact of HIV-1 infection on CD4+ T cells, HSPC repopulation remains polyclonal, thus supporting the use of HSPC transplant for anti-HIV treatment.

The widespread availability of combination antiretroviral therapy (cART) has significantly reduced AIDS-related mortality and morbidity. Thanks to improved cART and patient care, high-dose therapy and hematopoietic stem/progenitor cell (HSPC) transplantation, once considered too risky for HIV-infected patients, are now increasingly used in clinic to treat malignancies in patients with HIV (16). A number of recent studies have demonstrated a level of clinical efficacy for HSPC transplant in patients with HIV that is similar to the efficacy in noninfected patients (4, 7). Furthermore, two remarkable case studiesthe so-called Berlin patient, the first case cured of HIV after the allogeneic transplantation of HIV-resistant (CCR532/32) bone marrow (BM) (8), and the recent London patient, potentially the second cure with the same transplant strategy (9)have generated tremendous hope that HIV can be treated by the genetic engineering of a patients own HSPC (1013). Despite these recent clinical successes, however, our understanding of the functions of transplanted HSPC in HIV-infected patients remains unclear and controversial. In particular, a short-term cART interruption, recently recommended to minimize transplant-associated problems, often results in a marked increase in viral load in patients (2, 3, 14), but the impact of ongoing viral replication on HSPC engraftment and tissue repopulation remains poorly understood. It is noteworthy that numerous previous reports have shown both direct and indirect effects of HIV infection on BM niche cells, including HSPC (15), stromal cells (16), and possibly, CD4+ T cells (17). Furthermore, recent nonhuman primate studies have identified perturbations in the immune system following HSPC transplant in simian HIV (SHIV)infected, ART-suppressed animals (14, 18, 19). Nevertheless, most in vivo preclinical studies so far have tested HSPC transplant in the absence of HIV-1 infection (2026). All current and planned trials that include a cART interruption are moving forward without a full understanding of the effects of HIV infection on HSPC behaviors in vivo.

Retroviral tagging (cellular barcoding) has proven useful in evaluating HSPC transplant and the effects of genetic modification on HSPC behaviors in vivo (27). Previous human and nonhuman primate studies using the traditional reporter gene and polymerase chain reaction (PCR) assays have provided only limited information on HSPC behaviors in the presence of HIV-1 (2, 3, 5) or simian virus infection (14, 18, 19, 25), as these assays measure gene-marked cells as a whole population and thus overlook the clonal complexity that exists within the cells. In contrast, recent retroviral tagging studies, including our own, have shown highly coordinated repopulation by hundreds or thousands of individual repopulating HSPC clones at the systems level (2832). Quantitative sequencing of vector integration sites (ISs), in particular, has been an excellent means of studying the safety and functional diversities of gene-modified CD34+ HSPCs in gene therapy settings (3036). Although the importance of the tremendous regenerative potential and functional heterogeneity of individual hematopoietic stem cells has been well recognized (32, 37), all previous studies have tested hematopoietic reconstitution either in the absence of HIV infection or in the presence of suppressive ART. Thus, polyclonal HSPC repopulation, an important indicator of normal HSPC homing and in vivo function, in the presence of HIV-1 infection remains uncharacterized.

A humanized BM/liver/thymus (BLT) mouse model is arguably the most practical and functional small-animal model with which to test HSPC transplant (38). These mice enable human HSPCs to proliferate and populate the BM, generate various mature and functioning immune lineages, including mature, functional T cells through the transplanted thymic tissue, and repopulate all the lymphoid and nonlymphoid organs (20). We have previously demonstrated effective tissue repopulation and the anti-HIV efficacy of gene-modified HSPCs in BLT mice (2123) using several different types of anti-HIV lentiviral vectors, including dual-combination anti-HIV lentiviral vectors (dual-sh1005/sh516) expressing two anti-HIV short hairpin RNAs (shRNA), one directed at the HIV coreceptor CCR5 (sh1005) and the other at the viral long terminal repeat (LTR) (sh516). These dual-combination vectors showed antiviral efficacy against both R5- and X4-tropic HIV-1 in vivo (21). More recently, we developed a new preinfection BLT mouse model with which the HSPC transplant can be tested in the presence of HIV-1 infection (39). Gene-marking analysis in these mice has revealed the selective advantage of dual-sh1005/sh516engineered T cells over control (nonprotective) HSPCs cotransplanted in the same animal. However, it remains unclear whether and how HIV-1 infection affects polyclonal engraftment of nonprotective HSPC and whether selective repopulation by antiHIV genemodified HSPC occurs via normal polyclonal hematopoiesis.

Here, we have developed a novel LTR indexingmediated IS sequencing (LTRi-Seq) to directly compare and evaluate the homing/engraftment and tissue repopulation of anti-HIV (dual-sh1005/sh516) and control (nonprotective) HSPC in HIV-1preinfected humanized mice. The LTRi-Seq enables unbiased, simultaneous analysis of both anti-HIV and control HSPC clones and HIV-1infected cell clones in the same sample. With the new assay, our study provides novel insights into the competitive repopulation of HSPC clones in HIV-1-infected (HIV+) mice and the cellular proliferation and circulation of HIV-1infected cell clones in the same animals.

Two sets of independent preinfection humanized mouse experiments were performed at different times to evaluate and validate the impact of HIV-1 infection on HSPC transplant (Fig. 1A). The first set included five HIV+ mice and six uninfected (HIV) control mice; the second set included five infected and five HIV control mice. The details of the procedures and experimental results are described elsewhere (39). Briefly, human fetal liver CD34+ HSPCs were injected into irradiated neonatal (1 to 3 days old) nonobese diabetic.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. At 11 weeks after the first HSPC transplantby which point human CD45+ cells, including CD19 B cells and CD3+CD4+ and CD8+ T cells, had repopulated the bloodhalf of the mice were infected with CCR5-tropic HIV-1NFNSX, while the rest remained uninfected as a control (Fig. 1B). After 3 weeks of infection, the viral load reached 5.10 105 copies/ml (1.48 105 SD) for set 1 and 2.39 107 copies/ml (1.45 107 SD) for set 2 (table S1). Both groups of mice were then subjected to BLT surgery, in which an equal mixture of two pools of human HSPCone engineered with anti-HIV vectors (H1-EGFP-dual-shRNA) and the other with control vectors (H5-mCherry)and a piece of human thymus tissue, all from the same donor, were transplanted into each mouse following the administration of busulfan the previous day. The animals were followed for an additional 12 weeks and euthanized for tissue repopulation analysis. We and others have shown stable multilineage human cell engraftment and gene marking at 10 to 12 weeks following HSPC transplant in NSG and BLT mice (21, 4042).

(A) Cartoon representation of HIV preinfected BLT mouse model. (B) Bar plots showing comparative levels of reconstitution of different human cell populations before HIV-1 infection (10 weeks after the first CD34+ cell transplant) in the two groups of humanized mice. Group 1 animals were used as an HIV control and group 2 animals were infected with HIV-1 on week 11. Set 1 data are shown on the left and set 2 are on the right. Human cell reconstitution levels in groups 1 and 2 mice were similar; P values from Students t tests with Welch correction comparing groups 1 and 2 for human leukocytes (CD45), T cells (CD3), T helper cells (CD4), cytotoxic T cells (CD8), and B cells (CD19) are denoted in purple digits. (C) Gradual enrichment of anti-HIV (H1-EGFP-dual-shRNA)modified T cells in the peripheral blood of HIV+ mice. The line charts show the percentages of EGFP+ (circles connected with a solid green line) and mCherry+ cells (circles connected with a dotted red line) in CD45, CD3, CD4, CD8, and CD19 cells at 6, 8, and 10 weeks (w6, w8, and w10) after BLT surgery. HIV-1infected (HIV+, top) and mock-infected (HIV, bottom) mice for sets 1 and 2 are shown separately. The colors of the circles match the mouse IDs. The average of paired EGFP-to-mCherry marking ratios (H1/H5) for all samples is shown at the bottom of each line chart. The average value is not available (NA) when there is any missing sample data. (D) Boxplots show H1-EGFP/H5-mCherry marking ratios (H1/H5) in each tissue sample, including BM (squares), thymic organelle (TY; triangles), peripheral blood (PB; circles in set 1), and spleen (SP; circles in set 2), at the end point (12 weeks after BLT surgery). The P values in purple were calculated by comparing the H1/H5 ratios between HIV+ and HIV mice using mixed-effects gamma regressions (fig. S8A).

While showing marked mouse-to-mouse variations in human cell repopulation and experiment-to-experiment variations in the baseline gene markings, both sets 1 and 2 experiments demonstrated a gradual enrichment of enhanced green fluorescent proteinpositive (EGFP+) cells over time within the CD4+ T cell population in HIV+ mice (Fig. 1C). These experimental variations are common in BLT mouse studies likely due to donor variations and inherent technical inconsistency in performing the BLT surgery (38, 43). To effectively evaluate the impact of HIV-1 infection in our BLT mouse experiments, we compared paired EGFP versus mCherry marking levels in each sample, taking advantage of our cotransplantation strategy that provides an internal control (mCherry+ cells) in each mouse, using mixed-effects gamma regression models (see Materials and Methods for statistical analysis and fig. S8 for more details). At 12 weeks after the BLT surgery, despite the differences in the baseline data, HIV+ mice in both sets 1 and 2 showed a significant selective advantage for antiHIV geneengineered (EGFP+) cells over control (mCherry+) cells within the CD4+ T cell population (Fig. 1D and fig. S1E), demonstrating 7.6-fold higher EGFP/mCherry ratios than those of HIV mice for set 1 and 3.9-fold higher EGFP/mCherry ratios for set 2 (P = 0.005 for the first set; P = <0.001 for the second set). Other cell types, including human CD45+, CD19+ B cells, and CD3+CD8+ T cells, did not show such significant differences, indicating that the HIV-1NFNSXmediated selection was largely limited to mature CD4+ T cells (Fig. 1, C and D, and fig. S1). We have previously shown that CCR5+CD4+ T cells were primarily depleted in HIV-1NFNSXinfected mice (39). Lentiviral expression of dual-shRNA (sh516 and sh1005) had no obvious cytotoxicity in our previous tests (21). Most of the infected mice showed a similar or slightly increased viral load at the end point compared with their viral load before BLT surgery (table S1), indicating that viral replication continued until the end point.

The clonal-level evaluation of HSPC transplant, unachievable with conventional gene-marking or PCR assays, has been enabled by vector IS sequence analysis. The analysis of IS of multiple types of vectors in the same animal, however, remains challenging. To analyze the competitive clonal repopulation of anti-HIV and control HSPCs, we developed LTRi-Seq using two index sequences (H1 and H5) uniquely labeled at the U5 end of the LTR of the two lentivectors (see Fig. 2A). Distinguishing the unique LTR index sequences for these two vectors and the wild-type LTR of HIV-1 proviruses during IS sequence analysis enabled multiplexed and unbiased analysis of these two vectors and HIV-1 proviruses, all in parallel. The index sequences (H1 and H5) in the LTR did not induce a significant reduction in the efficiency of vector production or vector infectivity, nor did these indexes alter the genomic IS patterns of the lentiviral vectors (fig. S2).

(A) A diagram showing LTR index sequences for the 3-end U5 region (red box) of HIV, H1-EGFP-dual-shRNA, and H5-mCherry LTR. H1 and H5 indexes are two and three bases different, respectively, than the wild-type (WT) HIV-1. These unique index sequences will appear at the junction of the vector and host DNA after vector integration into the host genome and will thus serve as a marker with which to distinguish vector types during vector IS sequence analysis. The IS sequencing method is shown in the box. The LTR genome junction DNAs were PCR amplified by a linker-mediated PCR method using LTR- and linker-specific primers (blue-lined arrows) and then subjected to next-generation sequencing. (B) Table showing representative IS data with examples of collision events, a unique IS detected with multiple indexes (H1, H5, or WT). (C) Fold differences in IS sequence counts between the highest sequence count and the second highest sequence count for IS collision events detected in set 1 Roche 454-pyrosequencing and set 2 Illumina MiSeq sequencing data. For the sake of comparison, IS data from conventional IS sequencing (nonLTR indexed) using flow-sorted samples (flow sort) are shown. (D) A summary table for IS sequence analysis. Of the 729 unique ISs recovered in set 1 experiments and the 5727 unique ISs in set 2 experiments, approximately 7 and 8%, respectively, were collision events. Only five of the set 1 and none of the set 2 collision events showed a less than 10-fold difference; these remained unresolved even after we had conducted a selection process to determine the true index of the collision events.

In an effort to minimize the technical biases that can arise when comparing sample-to-sample IS profiles, we used the same amount of genomic DNA for all of the tissue samples (1 g for set 1 and 2 g for set 2 samples), with only a few exceptions (see table S2), and followed a well-established standard operating procedure for IS sequencing and quantitation (32, 44). Sets 1 and 2 samples were sequenced using different sequencing platforms, 454-pyrosequencing for set 1 and MiSeq sequencing for set 2, with the IS amplification and sequencing procedures kept identical for each set. A total of 729 and 5727 unique ISs were recovered at the end point (12 weeks after BLT surgery) from the tissues of sets 1 and 2 mice, respectively (Fig. 2D).

Given the semirandomness of lentiviral IS selection and the large human genome (3 billion bases), the likelihood of identical IS to occur with different vectors or different animals is negligible. We did, however, find that 51 ISs (7%) and 454 ISs (8%) in the 454-pyrosequencing and MiSeq datasets, respectively, were collision events (or sequence crossovers): In other words, the identical IS appeared in more than one vector (H1 or H5) or the HIV-1 IS sequence group (Fig. 2, B and D). These IS sequence collisions are a common problem for modern high-throughput sequencing (34, 45), likely occurring due to sample cross-contamination or demultiplexing errors (e.g., sequencing errors or mutations within the H1 and H5 index sequences). In contrast to our previous conventional (nonLTR indexing) IS analysis, where EGFP+ and mCherry+ flow-sorted cell pools were used for sequencing (fig. S3), the new LTR indexing approach does not require prior cell sorting and thus eliminates any potential sample cross-contamination that may occur during the cell-separation processes. The LTR indexing approach showed approximately 3.1- to 3.6-fold lower collision rates than those of conventional, nonLTR-indexing IS analysis (Fig. 2D).

LTR index read errors occurred at a rate of 1.92 and 0.73% for sets 1 and 2 individual sequences, respectively (fig. S4A). LTR index collisions can be effectively identified and corrected when identical IS sequences are available for the purpose of index sequence comparison (fig. S4B). A commonly used procedure for handling such collisions is to identify the correct IS by choosing the most frequent IS, that is, one showing a 10-fold higher detection frequency than any of the others (35, 36). The average IS frequency differences between correct and incorrect IS were 92-fold (160 SD) and 192-fold (157 SD) in the 454-pyrosequencing and MiSeq datasets, respectively (Fig. 2C). After applying these criteria, only five ISs (0.7%) in the first set (454-pyrosequencing data) and none in the second set (MiSeq dataset) remained unresolved, reflecting the higher sequencing depth of the MiSeq dataset (Fig. 2D). These unresolved ISs were excluded from the clonal profile analysis. The low-copy IS clones that did not show any LTR index collisions remained in our final data. There remains, however, a low level of uncertainty in LTR index identities for these low-copy IS clones: e.g., about 1.92 and 0.73% (or less) uncertainty due to potential read errors.

We compared IS sequence data with EGFP and mCherry gene-marking data shown above. To better present the frequencies of individual IS clones relative to the total repopulating cell pool, composed of both vector-marked (H1 and H5) and unmarked cells, we used IS clonal contribution data that factor in the % unmarked cells (see Materials and Methods for more details). The total combined IS clonal contribution for anti-HIV and control vectors showed a positive correlation with the marking levels of EGFP and mCherry in CD45+ cells (the Pearson correlation coefficients r = 0.88 and r = 0.84 for the first and second sets, respectively) (Fig. 3A). Similar positive trends between IS data and flow cytometry data have been reported in nonhuman primate studies, showing normal polyclonal reconstitution of HSPCs (32). The baseline H1 anti-HIV and H5 control sequence ratios in HIV mice differed from one set of experiments to the other, but both sets showed increased H1 clonal contribution and H1/H5 ratios in infected mice when compared with baseline H1/H5 ratios in HIV mice, indicating a selective expansion of HIV-protected cells in infected mice (Fig. 3B). Mixed-effects gamma regressions analysis comparing the H1/H5 ratios in HIV and HIV+ mice showed P = 0.039 for set 2 but, despite notable differences, set 1 showed P = 0.27, possibly due to the relatively low number of organ samples available for the set 1 analysis.

(A) A direct comparison of the total IS clonal contribution (sum of all IS frequencies, y axis) for H1-EGFP-dual-shRNA (H1-EGFP; green dots) and H5-mCherry clones (red dots) versus the corresponding EGFP or mCherry gene markings in CD45+ cells (flow cytometry results shown as % vector marking, x axis) from the same organ samples at the 12-week end point. Data indicate a strong correlation between the two parameters [Pearsons r = 0.88 for set 1 (left) and r = 0.84 for set 2 (right)]. The diagonal line is a reference line (r = 1). (B) The two-dimensional (2D) plots showing the total combined contribution of H5 IS clones (y axis) and H1 IS clones (x axis) in HIV+ organs (cyan squares, triangles, or circles) and HIV organs (black squares, triangles, or circles), including BM (squares), TY (triangles), PB (circles in set 1), and SP (circles in set 2). The outline colors of the squares, triangles, and circles correspond to the various mouse IDs. H1/H5 ratios are shown in the box. The P values were calculated using mixed-effects gamma regressions (ME) (fig. S8B). (C) The number of unique IS recovered from each sample was also positively correlated to the vector marking level of the sample. Scatter plots show a correlation between the number of total unique IS recovered from each organ sample (x axis) and respective EGFP or mCherry markings in CD45+ cells from the same organ samples (y axis). (D) Polyclonal repopulation by both H1-EGFP and H5-mCherryengineered cells. The 2D plots show the total number of unique IS of H1- (x axis) and H5-marked (y axis) cells in different organ samples. The shape and line color strategies are identical to those in (B). The average number of H1 IS clones increased approximately twofold in HIV+ samples relative to that in HIV samples, while H5 IS clone numbers remained relatively constant.

The number of unique IS recovered for these two vectors also reflected EGFP and mCherry markings (Fig. 3C). The HIV+ mice showed greater IS recovery rates for H1 anti-HIVprotected cells than did HIV mice, again indicating the potential selective advantage of HIV-protected cells (Fig. 3D). Mixed-effects gamma regressions comparing H1/H5 ratios in HIV+ and HIV mice showed P = <0.001 for set 2, but set 1 showed P = 0.63, probably for the same reasons addressed above. Notably, there was no significant reduction in the average number of unique IS for the control clones in the HIV+ animals; the unique IS showed a 1.3-fold increase in the first set and a 0.8-fold decrease in the second set when compared with those in HIV animals. This observation indicates that polyclonal repopulation by control H5-mCherry HSPCs occurs even in the presence of HIV-1 infection, in turn suggesting that, while HIV-1mediated selection of the anti-HIVmodified cells appears to be evident in infected mice, the impact of HIV-1NFNSX infection on HSPC BM homing and polyclonal hematopoiesis was insignificant in our humanized mouse study.

We next analyzed organ-to-organ clonal distributions in both HIV+ and HIV mice of set 2 (Fig. 4). Set 1 was excluded because of the lack of essential organ data (fig. S5). When we compared the H5 (control mCherry+ cell) IS profiles of BM, spleen, and human thymic implant of each HIV mouse, we found a unique organ-to-organ repopulation pattern (Fig. 4, B and D). BM and spleen showed relatively similar clonal profiles (average Pearson correlation coefficient r = 0.587 0.369 SD), whereas the correlation was poorer when thymic organelle was compared with BM or spleen (average r = 0.038 0.063 and r = 0.094 0.103 SD, respectively). Similar organ-to-organ IS distribution patterns were observed for the H1 anti-HIV vectors in HIV mice (Fig. 4B) and for both H5 and H1 in HIV+ mice (Fig. 4C). The observed organ-to-organ IS patterns, in which some of clones were notably expanded only in the thymic organelle, may reflect the unique clonal behaviors of thymocytes resulting from transient and extensive clonal expansion during normal T cell development, which manifest in unique clonal profiles in the thymic organelle, as previously demonstrated by Brugman et al. (46).

(A) Stacked bar plots show the relative clonal contributions (y axis) of H5-mCherry clones (H5) and H1-EGFP-dual-shRNA clones (H1) in the BM, SP, and human TY of HIV and HIV+ mice from set 2. Each color band in a stack represents individual IS clones, and its thickness corresponds to the relative clone frequency. Clones are stacked in a descending order of frequency with the highest frequency clone at the bottom and lowest to the top. The total number of IS clones (gray) and mouse IDs (black or blue) are shown at the top the chart. (B and C) 3D scatter plots show H5-mCherry (H5; red dots) and H1-EGFP-dual-shRNA clonal frequencies (H1; green dots) in BM, SP, and TY of HIV mice (A) and HIV+ mice (B). Each red or green dot represents the individual IS clones of H1-EGFP and H5-mCherry cells, respectively. These dots are positioned in 3D space based on IS clonal frequencies in BM, SP, and TY as three coordinates. Pearsons r values comparing the clonal profiles of BM and TY, BM and SP, and TY and SP are shown on the yellow, blue, and gray panels, respectively. (D) Boxplots of Pearsons r values between BM and SP (BM/SP), BM and TY (BM/TY), and SP and TY (SP/TY) for H5-mCherry (left) and H1-EGPF-dual-shRNA clonal profiles (right) in HIV and HIV+ mice.

To further address the impact of HIV-1 infection on HSPC function, we analyzed intra-organ clone-size variations. In a previous mathematical modeling study, we demonstrated that variations in the shapes of the clone-size distribution of blood repopulating cells reflect differences in the number of engrafted HSPCs and their functional parameters (birth, death, and differentiation rates) (47). All H1 anti-HIV and H5 control repopulating cells in the second set of animals showed highly variable IS clonal sizes (Fig. 4, A to C). When the shapes of clone-size distributions were compared, as described previously (47), all anti-HIV and control cells, particularly in the spleens of both HIV+ and HIV mice, showed similar shapes (fig. S6A). The observed clone-size patterns also mirrored those of the blood repopulating cells of rhesus macaques (32, 47). To further study the impact of HIV infection on clonal distribution, we used Rnyi diversity profiles (48) (details in Materials and Methods). Rnyi diversity plots show sloped diversity profiles in all organs, and the overlapping of Rnyi diversity curves from both HIV+ and HIV mouse samples (fig. S6B) signifies no particular ordering of clonal diversities, likewise suggesting that HIV-1 infection has no significant impact on normal clonal repopulation. These data thus consistently point to the efficient homing, hematopoiesis, and tissue repopulation of both HIV-protected and control HSPCs, even in the presence of HIV-1 infection, as demonstrated in our humanized mouse model.

In parallel with the IS of the two lentivectors (anti-HIV and control), a total of 111 and 1144 HIV-1 proviral DNA ISs were recovered in our first and second sets of experiments, respectively (see Fig. 2D). These HIV-1 ISs account for approximately 15.3 and 19.9%, respectively, of all the unique ISs (lentiviral vectors and HIV-1) recovered in each set. However, the total combined HIV-1 IS sequences constituted only 3.2% of all the IS sequences in set 1 and 2.1% in set 2, respectively, reflecting considerable frequency differences between the HIV-1infected and lentiviral vectorengineered cell clones. Although much smaller, on average, than lentiviral vector clones, individual HIV-1 IS clones also showed varying levels of detection frequencies. Of the 1144 HIV-1 unique ISs recovered from three different organs (BM, spleen, and human thymic implant) in the second set animals, 77 (6.7%) were recovered in at least two different organs.

A two to fourfold higher number of HIV-1 ISs were recovered in the human thymic implant (average 148 ISs, 84 SD) than in BM (33.4 ISs, 19 SD) or spleen (61.4 ISs, 40.4 SD). Overall, we found a consistent and unique organ-to-organ IS pattern, distinct from lentiviral vector IS patterns, in which the HIV-1 IS clones that were detected at a high frequency in one organ were undetectable or detected at a much lower frequency in any of other organs, resulting in poor statistical correlations in organ-to-organ IS comparison for all combinations of organs tested (average r = 0.050 0.131 SD to 0.233 0.091 SD) (see Fig. 5, A and C). This pattern suggests the limited proliferation and organ-to-organ circulation of HIV-1infected cell clones. For example, reflecting the development and circulation of T cells in various lymphoid organs, some of the high-frequency IS clones of the thymic organelle were detectable in the spleen at a lower frequency, whereas other high-frequency clones of spleen or BM were only rarely detectable in the thymic organelle (Fig. 5, B and D). Our data suggest that HIV-1infected human thymocytes may clonally expand and migrate to other organs, whereas HIV-1infected BM or spleen cells (mature T cells mostly) are not imported back to the thymic organelle. It is statistically highly unlikely that these organ-to-organ HIV-1 IS distribution patterns are simply the result of random contamination or sequencing errors (fig. S7). Our data thus provide novel insights into the limited circulation/migration abilities of HIV-1infected cells in different body organs, suggesting that the pathologic impacts of HIV-1infected clones may likewise be locally limited and confined, at least for the short term.

(A) 3D scatter plot shows HIV-1 IS clones (black dots) and their relative contributions (IS frequencies) in the BM, SP, and human TY of five infected mice. Pearson r values comparing the IS profiles of BM and TY, BM and SP, and TY and SP are shown on the yellow, blue, and gray panels, respectively. (B) The frequency differences among the common HIV-1 IS detected in multiple organs are shown. The relative frequencies of all IS clones recovered in BM, SP, and TY were plotted on BM, SP, and TY axes, respectively. Those ISs common to two different organs, linked by colored lines, showed significantly different frequencies in two comparative organs. The two IS clones detected in all three organs are shown in dash lines. The total number of unique IS for each organ is shown in a bracket. (C) Boxplots comparing Pearsons r values for the HIV-1 IS profiles of two organs. (D) Collective HIV-1 IS data from all infected mice. The number of IS common to two comparative organs is shown in blue. The arrows represent the quantities of common IS in the two comparative organs, with the direction of the arrows indicating the direction of higher to lower sequence frequencies of the IS common to the two organs. The two IS found in all three organs are in red. The black numbers indicate the total quantities of recovered IS for each organ.

In this study, the potential effects of HIV-1 infection on HSPC transplant, survival, and organ repopulation were investigated using HIV-1preinfected, humanized mice. LTRi-Seq enabled effective and unbiased analysis of two lentiviral vectors (anti-HIV H1-EGFP-dual-sh1005/sh516 and control H5-mCherry vectors) and HIV-1 proviruses. Our data provide novel insights into the behaviors of HSPC and HIV-1infected cell clones in vivo, insights with important implications for the repopulation of HSPC in the context of HSPC transplant and genetic therapy for HIV-infected patients.

We cotransplanted both H1 anti-HIVmodified and H5 control HSPC pools into the same host to better evaluate anti-HIV modifications in BLT mice, where host-to-host and experiment-to-experiment variations are common (38, 43). The analysis of vector IS in this type of competitive repopulation assay is challenging, particularly when testing lentiviral vectors in the presence of HIV-1 infection, due to the common LTR sequences shared by the therapeutic vectors and HIV-1 proviruses. LTRi-Seq enabled simultaneous analysis of anti-HIV vector (H1 indexed) and control vector (H5 indexed)marked HSPC clones and HIV-1infected cell clones (wild-type LTR) in the same mice. With the new approach, we have evaluated whether anti-HIV and control-engineered HSPC would efficiently engraft and repopulate the blood system in the presence of HIV-1 infection. ISs were sequenced and analyzed on the basis of methods proven effective in quantifying repopulating clones (32, 44).

Here, we demonstrate polyclonal and normal tissue repopulation of both types of HSPCs using our unique HIV-1 preinfection mouse model. By our analysis, we showed that HIV-1mediated selection primarily occurred at the mature T helper cell level and not significantly at the HSPC level. In clonal profile analysis, a complete absence of differences between HIV+ and HIV samples is not expected even if the HIV-1 infection has had no effect on HSPC; a small portion of mature cells will still be killed in infected mice. However, if HIV-1 infection affected HSPC homing and repopulation, then there would be notable differences in the clonal profile analysis. While a selective advantage for the H1 anti-HIV clones was evident, the control H5 cells (those with no anti-HIV modification) showed similarly high levels of polyclonal engraftment in both HIV+ and HIV mice. Despite the known limitations of xeno-transplant models (38, 43, 49), this polyclonal engraftment in infected hosts is noteworthy given the numerous previous reports on the direct and indirect effects of HIV infection on BM niche cells, including HSPC (15), stromal cells (16), and possibly, CD4+ T cells that reside in the niche (17). It appears that, following infusion, both types of HSPCs must have competed normally for the available HSPC niches, which remained functionally normal even in infected hosts, and HIV-1 infection had minimal impact on the polyclonal hematopoiesis and tissue repopulation of transplanted HSPCs.

Our data have important implications for the repopulation of gene-modified HSPC in the context of gene therapeutic clinical studies for the treatment of HIV-1 diseases. Our study is the first to experimentally evaluate the selective engraftment and clonal repopulation of gene-modified HSPCs in the presence of HIV-1 infection using a new preinfected mouse model. The premise of anti-HIV gene therapy is that anti-HIV gene engineering of a patients own HSPC can result in the selection of HIV-protected cells during hematopoietic reconstitution, long-term control of viral replication, and a favorable clinical course leading to a functional cure. Notably, virtually all of the T cells in the Berlin and London patients were replaced with CCR532/32 donor cells and cleared virus (8, 9, 50). Past anti-HIV clinical gene therapy trials, by contrast, have failed to demonstrate any clinical benefit in patients, primarily due to the scarcity of gene-engineered cells repopulating in the blood (1013). Even patients who have received myeloablative preconditioning, a treatment that significantly improves HSPC engraftment, have shown less than 0.32% anti-HIV gene marking in their peripheral blood mononuclear cells (11). Most salient among the many possible explanations for the low gene marking in patients with HIV is the possible impairment of HSPC function by direct HIV-1 infection and/or by an HIV-damaged BM microenvironment (15, 16). However, most in vivo preclinical studies so far have tested HSPC transplant in the absence of HIV-1 infection and only subsequently challenged the repopulating mature cells with HIV-1 to evaluate anti-HIV gene modification (2026). Recent nonhuman primate studies, testing HSPC transplant in SHIV-infected, ART-suppressed animals, have identified perturbations in the immune system following irradiation therapy (14, 18, 19). Our data demonstrate that the HIV-mediated selection for anti-HIVmodified cells was limited to a portion of mature cells, and the effects of viral infection on HSPCs homing and organ repopulation were insubstantial in our humanized mouse study.

Our study showing the normal polyclonal HSPC repopulation in the presence of HIV-1 infection supports the use of planned cART interruption during HSPC transplant. Although cART interruption has been recommended in recent studies (i) to minimize therapy-related toxicity and (ii) to improve anti-HIV gene marking in the peripheral blood, the safety and efficacy of cART interruption remain unclear and controversial (2, 3, 14, 19). Recent human and nonhuman primate studies have shown that autologous HSPC transplant in HIV-1infected patients is well-tolerated and feasible (4, 7, 18), but these studies evaluated HSPC transplant in the presence of cART and lacked a clonality analysis of the repopulating cells.

Furthermore, using LTRi-Seq, we have characterized HIV-1infected cell clones in comparison to repopulating HSPC clones. Clonal expansion of HIV-1infected cells has recently been reported in humans and humanized mice and is suspected to be an important mechanism of HIV-1 persistence (51). The clonal dynamics and organ-to-organ distribution of expended clones, however, remain poorly investigated. Our data demonstrate a unique pattern of organ-to-organ clonal profiles characteristic of HIV-1infected cells, distinct from the clonal patterns of gene-engineered HSPC clones, potentially suggesting strong organ confinement and limited circulation for at least 15 weeks after infection. A recent human case study, by contrast, showed wide anatomic distribution of a few infected clones, likely the result of the effects of years of HIV-1 infection and cancer in the patient (52). A recent humanized mouse study analyzing HIV-1infected cell clones at 15 weeks after infection has shown clonal patterns consistent with our results, identifying only a small fraction of HIV IS common in multiple organs (53).

Preinfection BLT mice are a practical and functional small-animal model with which to directly test therapeutic vectors in the presence of HIV-1 infection. In vitro experiments ignore the impact of complex tissue architecture, while in vivo studies in humans do not permit adequate experimental manipulation. Nonhuman primate models require the use of modified simian-version viruses. Both sets of our BLT mouse experiments have reproducibly demonstrated consistent polyclonal HSPC engraftment in all test animals and for all vector types. The clone-size distribution patterns, in particular, closely resemble those observed in our previous nonhuman primate studies of autologous HSPC transplant (32, 47), indicating the potential relevance of our data for HSPC transplant in HIV-infected individuals.

IS sequencingbased, lentiviral tagging approaches have been widely used to study HSPC clonal repopulation, and the data analytic procedures are well established (3036). Unlike other lentiviral tagging approaches that use short synthetic barcodes to distinguish among different cellular clones, IS sequencing approaches directly compare tagged sequences to the reference human genome and thereby enable highly accurate IS clone identification even for single-copy IS events (27, 54). In our study, IS clones and LTR indexes were determined by independent procedures. We found LTR index collision events in about 7 and 8% of IS clones due to read errors for LTR index sequences (average 1.92% for set 1 and 0.73% for set 2), but most of these index read errors can be readily identified and are correctable. Only 0.7% in set 1 and none in set 2 IS clones remained uncorrected after applying our 10 correction criteria; these were removed from clonal profile analyses. The low-copy IS clones, those that are not showing collision events, remaining in the final clonal profile data have approximately 1.92 and 0.73% (or less) uncertainty in their LTR index identities due to the potential read errors. The application of a threshold for low reads (27) would remove these uncertainties, but needs to be carefully applied as this would remove a significant amount of bona fide low-copy data.

In summary, our LTRi-Seq data provide new information on the repopulation of transplanted HSPCs in the presence of HIV-1 infection and the clonal profiles of HIV-1infected cells in key lymphoid organs. These results are particularly relevant to the issue of ART interruption in the context of HSPC transplant and anti-HIV gene therapy for HIV-infected individuals. The concepts and technological tools arising from this study will be critical for the development of future gene therapy protocols.

The H1-EGFP-dual-shRNA (sh1005/sh516) and H5-mCherry lentiviral vectors were derived from the FG12-sh1005/sh516 (21) and FG12-mCherry lentiviral vector (20), respectively, by introducing point mutations at the 3-end of the left LTR. The primers used for H1 were H1f (5-CAGTGTGGAAAATCTCCAACAGTGGC) and H1r (5-TGTTCGGGCGCCACTGTTGGAGATTT), and the primers for H5 were H5f (5-TGGAAAATATCCAACAGTGGCGCCCGAACAG) and H5r (5-CTGTTCGGGCGCCACTGTTGGATATTTTCCA). All lentiviral vectors were produced by the calcium phosphatemediated transient transfection of 293T cells described previously (20, 21, 55). Briefly, 293T cells, cultured in Dulbeccos modified Eagles medium with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 g/ml), were transfected with vesicular stomatitis virus glycoprotein (pHCMV-G), packaging (pCMVR8.2DVPR), and lentiviral vector plasmids. Virus supernatant was collected on days 2 and 3 after transfection, filtered through a 0.22-m pore-size filter and concentrated 100-fold by ultracentrifugation. Lentiviral vector stocks were titrated by infecting 293T cells (105) with various dilutions of the concentrated virus stock; this was followed by flow cytometry analysis of EGFP or mCherry expression 3 days after infection.

All the mouse experiments and HIV-1 infection procedures are described in detail in our previous publication (39). Briefly, neonatal (1 to 3 days old) nonobese diabetic.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were irradiated (125 centigrays) and transplanted with human fetal liver CD34+ HSPCs by intrahepatic injection; after 11 weeks, half of the mice were infected with CCR5-tropic HIV-1NFNSX (200 ng of p24 Gag). After 3 weeks of infection, both HIV+ and HIV mice were myeoablated with busulfan (35 mg/kg) and, the next day, transplanted with a piece of thymus and an equal mixture of H1-EGFP-dual-shRNA and H5-mCherry vectortransduced CD34+ HSPCs via a two-step procedure (implantation of the Matrigel-solidified CD34+ cell mix and infusion of the gel-free cell mix through the retro-orbital vein plexus on the same day). Human CD34+ HSPCs isolated from fetal livers and thymus pieces from the same donor were cryopreserved, as previously described (39). Human fetal thymus and fetal livers were obtained from Advanced Bioscience Resources, FPA Womens Health, and the University of California, Los Angeles (UCLA) Center for AIDS Research (CFAR) Gene and Cellular Therapy Core. The UCLA Institutional Review Board has determined that fetal tissues from diseased fetuses obtained without patient-identification information are not human subjects. Written informed consent was obtained from patients for the use of the tissue for research purposes. All mice were maintained at the UCLA CFAR Humanized Mouse Core Laboratory in accordance with a protocol approved by the UCLA Animal Research Committee. Flow cytometry and the viral load assay have been described in detail in a previous publication (39).

For IS sequencing, we followed the procedures described in our previous publication (32, 44). In this study, we focused on analyzing only the right LTR junctions. Briefly, 1 mg of genomic DNA for set 1 samples and 2 mg of genomic DNA for set 2 samples, with a few exceptions (see tables S1 and S2), were subject to a linker-mediated PCR method using RsaI and CviQI restriction enzymes. Linker-ligated IS DNA fragments were amplified by a nested PCR strategy using two LTR primers, 1R-primer (5-CTGGCTAACTAGGGAACCCACT-3) and 2R-primer (5-ACTCTGGTAACTAGAGATCC-3) that align 140 and 57 bases upstream of the 3-end of U5 LTR, respectively, and two primers that align on linker DNA. This strategy ensured that both the LTR indexes and vector-host junctions originating from cell clones could be PCR amplified and sequenced without any primer-associated bias. Set 1 samples were sequenced with Roche 454-pyrosequencing (Roche FLX genome sequencer) and set 2 samples were sequenced with a Illumina MiSeq sequencer. To process the 454-pyrosequencing data, we used a previously described python script (32, 44) with the additional feature of enabling identification and separation of IS sequences based on the LTR index sequence. Sequences that included both the 3-end U5 LTR DNA and 25-base host DNA (with 95% homology to the human genome) with the 3-end LTR sequence at the virus-host junction were considered a true IS sequence. Briefly, sequences that showed the 3-end LTR sequence joined to genomic DNA were identified using the Smith-Waterman algorithm [for 454 data, Emboss water tool http://emboss.sourceforge.net/ and for Illumina data, a modified version of SSW library (56) in C++] and then further tested for the presence of LTR indexes (H1, H5, or wild type). Genomic sequences shorter than 25 bases were removed. On the basis of the LTR index, IS reads were assigned to either H1 and H5 vectors or WT HIV-1. We followed similar procedures for the analysis of Illumina MiSeq data. IS sequences were mapped onto the human genome (Version hg19 downloaded from https://genome.ucsc.edu/) using either BLAT (BLAST-like alignment tool) for 454 data or Burrows-Wheeler Aligner software (57) for Illumina data. Sequence-mapping and IS-counting procedures were identical to those in our previous publications (44). To better present the IS clonal frequencies relative to the total repopulating cell pool, we calculated IS clonal contributions that factor in the % unmarked cells. The relative sequence frequencies of individual IS, initially normalized by the total count of H1 and H5 IS sequences combined, were multiplied by the fraction of total vector marked cells (both EGFP+ and Cherry+ cells combined) in CD45+ cells. This approach enabled direct comparison of IS data to flow cytometry gene marking data and direct comparison of IS clones in different organs.

Because of the semirandom nature of vector/virus integration into the host genome, events in which the same IS appeared in multiple animals or in different LTR index datasets were considered collisions or signal crossovers. To identify the correct and incorrect sample identities among the same IS collision events for both 454 and Illumina data, we used a commonly used criterion that identifies sequencing errors (or sample contamination) based on sequence count differences among the same IS collision events (3436). Correct sample identities were established when a sequence count was at least 10 times higher than the counts of all of the others in the dataset sharing the same IS (see Fig. 2C and fig. S4). IS data with an incorrect sample identity were removed from the dataset. Any signal crossover IS events that failed to show >10-fold sequence count differences were considered unresolved and removed from the dataset.

Students t tests with Welch correction were used to compare continuous variables between pairs of experimental conditions, including the levels of cell populations and the H1/H5 ratios between HIV and HIV+ humanized mice. An exact Mann-Whitney test was used to compare organ-to-organ IS crossover rates within the same animal. Pearson correlation (r) was used to compare pairs of continuous variables, including total clonal contribution versus % vector marking and number of unique IS versus % vector marking; clonal frequencies within pairs of organs (BM, spleen, and thymus) in HIV and HIV+ mice; and IS profiles within pairs of organs in HIV+ mice. The randomness of organ-to-organ IS distribution patterns was evaluated by three pair-wise 2 tests (fig. S7). For example, we tested whether the overlap of IS expressions with BM differed between SP and TY tissue types. A similar analytic framework was used for the other tissue pairs (spleen and BM and thymic organelle and BM). To evaluate the impact of HIV-1 infection on anti-HIV vector (H1-EGFP-dual-shRNA) and control vector (H5-mCherry)transduced cells (Fig. 1D and Fig. 3, B and D), we used mixed-effects gamma regression models with a log link and unstructured covariance matrix to compare cell% and IS frequency between H1 and H5 in HIV+ and HIV mice, adjusting for tissue type. Gamma regression was chosen over linear regression because of distribution skew among the cell% and IS frequencies. The models included an interaction term between H1 (versus H5) and HIV status to test whether the effect of H1/H5 differed by HIV status. For cell% and IS%, when the outcome measure was 0, a small number (0.1) was added to meet the range requirement of gamma regression. P values were reported from the models. Statistical significance was assessed at the 0.05 level, and analyses were implemented in R v.3.4.4 (58). More details on statistical analysis can be found in data file S1.

We investigated the impact of HIV-1 infection on the diversity of total clonal repopulation in different organs of HIV and HIV+ mice using Rnyis diversity profiles. The y axis of the Rnyis diversity plot, H, indicates species diversity, such that consistently higher values of H indicate a more diverse clonal sample. The x axis is , which ranges from 0 to infinity (59). If the lines or profiles for two groups cross, then their relative diversities are unknown. Diversity plots also indicate the evenness of the clones, where a horizontal line indicates equal expansion of each clone (i.e., uniform clonal expansion), and steeper slopes indicate greater nonuniform clonal expansion [see Kindt et al. (60), chapter 5 page 56]. Details of Rnyis diversity calculation are in Supplementary Text.

A. Renyi, in Proceedings of the Fourth Berkeley Symposium on Mathematical Statistics and Probability, Volume 1: Contributions to the Theory of Statistics (University of California Press, 1961), pp. 547561.

L. V. Bystrykh, M. E. Belderbos, Clonal analysis of cells with cellular barcoding: When numbers and size matter, in Stem Cell Heterogeneity: Methods and Protocols, K. Turksen, Ed. (Springer New York, New York, NY, 2016), pp. 5789.

R-Core-Team (R Foundation for Statistical Computing, Vienna, Austria, 2018).

R. Kindt, R. Coe, Tree Diversity Analysis. A Manual and Software for Common Statistical Methods for Ecological and Biodiversity Studies (Nairobi, World Agroforestry Centre, 2005).

Acknowledgments: We thank the technical support from the UCLA CFAR research cores, including the CFAR Gene and Cellular Therapy Core and the CFAR Humanized Mouse Core. Funding: This work was supported by grants from the NIH (5U19AI117941,AI028697,AI110297, AI100652,AI145038,AI18058, HG010108,HG010318,HL116234,HL125030,HL126544); CIRM (DR1-01431,TRX-01431-1), the James B. Pendleton Charitable Trust, and the McCarthy Family Foundation (I.S.Y.C); and a Junior Faculty Scholar Award from American Society of Hematology (Sa.K.). CIRM (DR1-01431,TRX-01431-1), the James B. Pendleton Charitable Trust and the McCarthy Family Foundation (I.S.Y.C). Author contributions: Sa.K. and I.S.Y.C. designed and supervised the study; G.W.S. and Sa.K. designed and carried out IS sequencing and data analysis; W.K., S.S., and D.-S.A. carried out the animal and cell biology experiments; J.W., H.A., Y.X., H.Y., and Sh.K. performed flow cytometry and IS sequencing data analysis. G.W.S., Sa.K., E.W., and N.K. developed computational workflow; H.C. generated H1 and H5 vectors; A.P.P., C.Z., and G.W.S. performed statistical analysis; Sa.K., I.S.Y.C., and G.W.S. wrote the manuscript and revision; A.P.P., D.-S.A., and N.K. provided critical appraisal of the manuscript. Competing interests: I.S.Y.C. has a financial interest in CSL Behring and Calimmune Inc. No funding was provided by these companies to support this work; D.-S.A. has a financial interest in Calimmune Inc. and CSL Behring that the University of California Regents have licensed intellectual property invented by D.-S.A, which is being used in the research, to Calimmune Inc. No funding was provided by these companies to support this work. All other authors declare that they have no competing interests. Sa.K., I.S.Y.C., and G.W.S. are authors on a patent application related to this work (no. 62/743,247. filed 9 October 2019). Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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The clonal repopulation of HSPC gene modified with antiHIV-1 RNAi is not affected by preexisting HIV-1 infection - Science Advances

COMMUNITY VOICES: Beyond the masks – The Bakersfield Californian

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It calls for a celebration that we as a nation can be unified in our response to the virus. Real festivities will await eventual triumph but a significant step has been taken to consolidate the message. A message supported by abundance of science. Its time to coalesce as a nation and aim to win.

At the risk of being redundant, its necessary to reinforce strong safety precautions under three conditions that enable the virus spread: closed spaces, crowds and close contacts the three Cs. Its especially important when loud speech, singing and longer contacts are involved.

Virus has a strike zone, and virus is avoidable.

The path forward is clear. Lets keep it simple. Safe distancing and hygiene will remain foundational. At the very least, these common sense measures will reduce the size of infective dose and mitigate chances of an adverse outcome while lending immunity against future infections. As the number of people recovering from infection increases, building herd immunity becomes a natural barrier against the spread of virus. Preventative measures help slow the dissemination of virus, sparing health care systems from being unduly stressed. COVID-19 related hospitalizations can crowd out non-COVID-19 health care, adding to preventable loss of life.

Pharmaceuticals are evolving rapidly, adding significantly to the toolbox against the virus. Pharmaceuticals can help reduce hospitalizations by safely managing the sick on an outpatient basis.

COVID-19 seems to have two phases. In the first phase, as virus multiplies, the immune system endeavors to battle the virus to submission. Thankfully, most battles end there.

If the battle enters the second phase, the immune system can go into hyperdrive and raise a cytokine storm, inflicting damage at a cellular level that may eventuate in respiratory and multiple system failure. A number of antiviral agents and anti-inflammatory agents are being used with varying degrees of success in both outpatient and in-hospital settings. Convalescent plasma, monoclonal antibodies and cytokine removal devices, akin to dialysis machines for kidney failure patients, are being used to help the critically sick. We have learned to use ventilators more effectively and discriminatingly.

The mortality rate has been falling significantly as doctors have learned to deal with this novel disease. Current evidence suggests that the infection related fatality rate is less than 1 percent. Mortality is mostly concentrated in the elderly and infirm. Fortunately, elderly have received the memo and are scrupulous in practicing preventative measures. This will likely lower the mortality rate further.

We still have unresolved mysteries of lingering sequelae of COVID-19 in many patients.

The real optimistic expectations are centered around successful development of effective vaccines that will offer durable immunity. Embracing existing knowledge and adopting state-of-the-art genetic engineering technologies, a number of viable vaccine candidates have emerged. Challenges to successful stage three trials abound, but hope looks realistic now. We may have more than one successful vaccine in the next six to eight months and put the virus in the rear view.

Its conceivable that based on current labors we may have a platform virus that can be successfully and expeditiously tweaked to defeat the next pandemic.

Its not a bridge too far now. We will hold hands on the other side. For now, lets join our efforts to keep virus at a distance.

Dr. Brij Bhambi specializes in cardiac and vascular intervention, nuclear cardiology, consultative and general cardiology and holds board certification in Interventional Cardiology, Cardiovascular Disease, and Internal Medicine. He is a chief medical officer at Bakersfield Heart Hospital.

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COMMUNITY VOICES: Beyond the masks - The Bakersfield Californian

UAE economy is strong and resilient to curb crises: Official – Gulf Today

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Younis Haji Al Khoori, Under-Secretary of Ministry of Finance. File

The national economy of the UAE is strong and resilient and has the ability to overcome such crises, he added.

The general framework of the economic strategy of the UAE in facing the pandemic is based on two main phases, he noted, explaining the first - in the short term - is the gradual opening of the economy and business activities while taking into account the health precautions. We also supported the most affected sectors with economic stimulus plans, which, totalled to date, AED282.5 billion, thereby protecting entrepreneurs and SMEs.

This links the funds to the beneficiary sectors based on well-defined plans and effective mechanisms, he said in an interview published in the MoF e-Newsletter for July.

As for the second stage, Al Khoori explained, it is a long-term stimulus plan for the economy to accelerate recovery and advance growth. It works to transform challenges into opportunities and achieve sustainable economic development by enhancing the flexibility and sustainability of the economic model. It also encourages financing and investment in sectors with high future potential.

The focus in future, he added, will be on a digital economy; including artificial intelligence, AI, 5G, Internet of Things, IoT, smart cities, and green economy concepts and industries such as renewable energy, electric cars, and circular economy by enhancing productivity by integrating 3D printing technologies and robotics, promoting food security using advanced technologies such as AI, biotechnology, and genetic engineering.

Speaking about programmes and projects the ministry will launch to manage the next stage, Al Khoori said, The Ministry of Finance is working on proposing the required policies, legislations, and incentives to support opportunities for the industry across the country to ensure self-sufficiency and preparation for any future challenges. We will also launch programmes to support the health, education, technology, and food security sectors - placing the human factor as the basis of comprehensive development. We have also continued to work with the international community to ensure that we build an economic and geopolitical system that addresses and contains health and environmental disasters and mitigates their effects. This is in addition to supporting and encouraging government innovation programmes that grow the UAEs economy and achieves comprehensive development for all sectors, as well as launching a fee-pricing guide for federal government services thus providing a framework that is transparent, consistent, and easy to implement for relevant federal entities, he added.

With regards to the ministrys priorities in the post-COVID-19 phases, he said, Based on the UAEs strategy for the post-COVID-19 phase, the ministrys priorities are to submit proposals for draft laws and legislation that address the effects of the global pandemic on the nation. We are also drawing action plans and setting specific goals to meet urgent development needs. This is in addition to addressing the current challenges posed by the novel coronavirus, and taking into account the developments in the economic, developmental, community, service, and technological sectors.

Furthermore, the ministry is formulating policies to deal with similar challenges, examining deficiencies in sectors that faced more difficulties than others in dealing with the crisis, as well as supporting those that have been more resilient. This has contributed to supporting production and providing services with great efficiency in light of difficult situations we currently face. As for new laws and legislations that can be modified to respond to the current crisis, Al Khoori said, The Ministry of Finance, in coordination with competent authorities, shall study the proposals for issuing new legislation, or make amendments to some texts of the articles of the laws in force, in a manner that supports comprehensive development across sectors. Among the important laws issued in light of the current crisis, he explained, is the UAE insolvency law for natural persons, which eases the burden on anyone facing financial difficulties in paying their debts. This includes both citizens and residents.

WAM

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UAE economy is strong and resilient to curb crises: Official - Gulf Today

How wild rice has sustained the Ojibwe people – MinnPost

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Wild rice is a food of great historical, spiritual, and cultural importance for the Ojibwe people. After colonization disrupted their traditional food system, however, they could no longer depend on stores of wild rice for food all year round. In the late 1950s and early 1960s, this traditional staple was appropriated by white entrepreneurs and marketed as a gourmet commodity. Native and non-Native people alike began to harvest rice to sell it for cash, threatening the health of the natural stands of the crop. This lucrative market paved the way for domestication of the plant, and farmers began cultivating it in paddies in the late 1960s. In the twenty-first century, many Ojibwe and other Native people are fighting to sustain the hand-harvested wild rice tradition and to protect wild rice beds.

Ojibwe people arrived in present-day Minnesota in the 1600s after a long migration from the east coast of the United States that lasted many centuries. Together with their Anishinaabe kin, the Potawatomi and Odawa, they followed a vision that told them to search for their homeland in a place where the food floats on water. The Ojibwe recognized this as the wild rice they found growing around Lake Superior (Gichigami), and they settled on the sacred site of what is known today as Madeline Island (Moningwanakauning).

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In the Ojibwe language, wild rice (Zizania palustris) is called manoomin, meaning good berry, harvesting berry, or wondrous grain. It is a highly nutritious wild grain that is gathered from lakes and waterways by canoe in late August and early September, during the wild rice moon (manoominike giizis).

Before contact with Europeans and as late as the early twentieth century, Ojibwe people depended on wild rice as a crucial part of their diet, together with berries, fish, meat, vegetables, and maple sugar. They moved their camps throughout the year, depending on the activities of seasonal food gathering. In autumn, families moved to a location close to a lake with a promising stand of wild rice and stayed there for the duration of the season. Men hunted and fished while women harvested rice, preparing food for their families to eat throughout the following winter, spring, and summer.

Ojibwe people harvested wild rice, and continue to harvest it today, in pairs, with one person pushing or paddling a canoe and the other knocking rice into it with sticks (bawaiganaakoog). When the wild rice is ripe, the grains fall easily into a canoe, and the grains that fall into the water lodge themselves into the mud, then grow into the following years stands of rice.

Freshly harvested manoomin is called green rice. When processed in the traditional way, it is parched (roasted) over a fire, then threshed by being stepped or danced on. This motion, called jigging, loosens and removes the fibrous outer covering of the grain. Finally, to separate the hulls from the grain, wild rice is winnowed or fannedtossed up in the air with birch bark trays (nooshkaachinaaganan) so that the hulls are blown away and only the edible grain is left behind.

The work that goes into preparing wild rice for eating and storage was traditionally carried out collectively. Women marked the areas designated for particular families by binding a number of heads together. This ensured that everyone in the community got the rice they needed, and it also made harvesting easier.

Taking care of the natural world that sustains us forms a central part of the Ojibwe peoples way of seeing the world. Traditional wild rice harvesting practices reflect this, protecting wild rice beds for the long-term wellbeing of the ecosystem as well as the community. Designated elders would, and on reservations still do, carefully monitor lakes to prevent premature or excessive harvests, opening and closing lakes to ricing as necessary, and leaving some mature grains unharvested for re-seeding.

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Loss of a traditional food system

As far back as the early fur trade in the mid-1600s, some Ojibwe families traded wild rice for goods. For the most part, however, they collected it for household consumption or trade between tribes. Processing wild rice is a labor-intensive activity, and families harvested only as much as they could process.

Colonization, land loss, the establishment of reservations, and dependence on government food and payments separated Ojibwe people from their way of life andwith some exceptionsthreatened their traditional food system. As people stopped eating their traditional diets in the mid-twentieth century, major health problems like diabetes emerged. Harvesting practices also changed as they lost access to the lakes and rivers in which wild rice grew, and as they adapted to the changing world around them. Men began to harvest wild rice together with women, gathering it in aluminum canoes rather than birch bark ones and processing it with machines. Until the 1950s, Ojibwe people remained the primary ricers.

Ojibwe people both on and off reservations faced a period of difficulty after World War II. Traditional lifeways could no longer sustain family needs, and jobs were difficult to find. Many people moved to cities; others suffered from poverty on their reservations. At the same time, the North American wild rice market shifted as a new marketing model began to demand products in large quantities to be sold across the nation. The price of wild rice rose as it gained in popularity, and both Natives and non-Natives began to harvest the crop for cash rather than for home consumption. Inexperienced non-Native harvesters used methods that began to put wild rice beds in danger. Technology, including parching, thrashing, and fanning machines, was further developed to process the rice with more ease. Processing plants were set up across the stateprimarily, but not exclusively, by white people.

As the national market for wild rice grew, more people became interested in figuring out how to cultivate it as a crop, like paddy rice. This led to domestication efforts at the University of Minnesota and the expansion of many acres of paddy rice in both Minnesota and California. Wild rice was made the Minnesota state grain in 1977. Unfortunately, however, the cheaper production costs of cultivated wild rice drove down demand for hand-harvested wild rice, leaving Ojibwe people without this source of income.

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As far back as the 1930s, the health of wild rice beds has been a serious concern. In 1939 Minnesota passed a law outlawing mechanized harvest and limiting how and when wild rice could be harvested. Since then, it has enacted other protective policies, including limiting the number of hours in the day during which it is permissible to rice and limiting the length of the canoe used for ricing. In the 1990s, wild rice was identified as an endangered food. The plant is sensitive to water levels altered by dams as well as road construction, pollution, poor harvesting practices, invasive species, genetic engineering (genetic contamination of the wild rice from the paddies), and climate change.

In response to these threats, Ojibwe and other Native people organized. For example, in 1994, the Fond du Lac and Bois Forte bands developed a Wild Rice Restoration Plan for the St. Louis River Watershed designed to restore lost stands of the crop and manage its harvest. In the same decade, the White Earth Land Recovery Project began to sell hand-harvested wild rice, and multiple bands formed reservation wild-rice committees to manage harvests.

In the 2020s, Ojibwe people continue to defend and protect this vital plant and the cultural, health, and spiritual importance that it holds. Individuals as well as tribes organize ricing camps to teach traditional practices of ricing, parching, and finishing. Others are actively fighting against the Enbridge Line 3 oil pipeline replacement project that would cross wild rice habitat, or collaborating in a movement for Native food sovereignty.

For more information on this topic, check out the original entry on MNopedia.

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How wild rice has sustained the Ojibwe people - MinnPost

Answers About Universes Age Could Be Found in the Dark – ScienceBlog.com

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In May 2009, the Planck Satellite was launched with a mission to discover the age of the universe. The goal was to measure cosmic background radiation (CMB), a source of light that traces back to around 380,000 years after the universes start at the Big Bang. Those measurements revealed the universe to be around 13.8 billion years old.

But in the past few years, astronomers have improved on classic observations of how fast distant galaxies move away from Earth. These measurements result in an age of the universe hundreds of millions of years younger than the Planck measurements indicated.

Arthur Kosowsky (Emily ODonnell)

In July, the research team behind the National Science FoundationsAtacama Cosmology Telescope(ACT), which includesDepartment of Physics and AstronomyChairArthur Kosowsky, graduate studentYilun Guanand 40 other participating institutions, jumped into the fray with their own estimates. The team made new maps of the slight variations in the microwave backgrounds temperature and polarization which are more sensitive and sharper than Plancks.

The simplest model of the universe that fits the new data from ACT, which is located in Chile, has an age of 13.77 billion years with an uncertainty of plus or minus 40 million yearsessentially confirming the Planck results.Papersfeaturing the findings were posted to the arXiv distribution service and have been submitted to the Journal of Cosmology and Astroparticle Physics.

Now weve come up with an answer where Planck and ACT agree, says Simone Aiola (A&S 14G, 16G), a lead author and researcher at the Flatiron Institutes Center for Computational Astrophysics who holds a PhD in physics from PittsKenneth P. Dietrich School of Arts and Sciences. It speaks to the fact that these difficult measurements are reliable.

Kosowsky said the findings add to the confusion surrounding the actual age of the universe but also provide a few clues about where cosmologists should look for answers.

He noted that ACT also essentially confirmed Planks estimated Hubble constant, which is the rate at which the universe is expanding. ACT found the universe is expanding at a rate of 67.6 kilometers per second per megaparsec, which means an object located a megaparsec away from Earth (approximately 3.26 million light years) is on average moving away from us at the rate of 67.6 kilometers per second due to the cosmic expansion.

Probably the most interesting possibility for this discrepancy is that our simple model of the universe is wrong.

Arthur Kosowsky

The Planck teams Hubble constant was 67.4 kilometers per second per megaparsec and the rates estimated by measuring galaxies range between 70 and 74 kilometers per second per megaparsec, significantly faster than the other results.

Probably the most interesting possibility for this discrepancy is that our simple model of the universe is wrong. So the inference were making about the Hubble parameter from our measurement is based on a model thats not quite right, said Kosowsky. Our basic model of the universe is based on some really simple assumptions about some really simple physics. If you change something youre probably saying theres new, undiscovered, fundamental physics that were seeing in this discrepancy.

Yilun Guan

Guan, who is pursuing a PhD in cosmology at Pitts Dietrich School, is investigating whether the flaw in the model lies in assumptions made about the nature of dark energy, which is what researchers call the unknown force that is accelerating the universes expansion.

In our current model of the universe we treat dark energy as a really simple component that has a simple evolutiontheres this amount of dark energy and it evolves in a certain way. But nobody knows what dark energy is doing, it could be that dark energy is changing from when we measure it at the CMB until we measure it in these local measurements, he explained. What were doing is thinking about if theres any way dark energy can be changing that is consistent with measurements from both our local measurements as well as measurements from CMB and other observations that have been made.

While Guan continues the dark energy research, he is also investigating ways to automate the process of finding the most useful data from telescopes and satellites. On the ACT project, he was responsible for the first levels of data quality assurance, a process that meant analyzing tens of millions of detector time streams collected by ACT each year to determine which ones are to be used to make sky maps.

Telling good data from bad data requires a lot of expert knowledge. In the past we used to have one expert who had a lot of experience in this area and if he looks at diagnostics and statistics he can tell if something is going wrong. But thats not a scalable solution, Guan said.

In 2022, ACT will officially shut down and its research team will shift attention to the Simons Observatory, also located in Chile, which will feature new telescopes, cameras and state of the art detector arrays. Those upgrades will give experts an order of magnitude more data to assess. In anticipation of the challenge, last year Guan started work on a machine learning algorithm designed to take on the task.

It turns out, this is a very easy problem for the machines. We have lots of data that experts told us is bad or good so we can train our algorithms to learn from what experts decide and have these algorithms make these decisions for us, he said.

For ACTs upcoming data release next year, which covers information gathered from 2017 through 2019 and will feature four times more data, experts will still lead the way in data quality assurance. However, Guan hopes to have an automated process in place before its time to analyze data from Simons.

Kosowsky said the data that comes from the Simons Observatory could hold precisely whats needed to resolve mysteries surrounding the age of the universe once and for all.

The fact that were doing measurements right now where the pieces are not all completely fitting together makes it really exciting to have the possibility of doing even better measurements of the universe with the next generation experiments, he said.

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Answers About Universes Age Could Be Found in the Dark - ScienceBlog.com

Happy Birthday, Sandra Bullock; Here are her 10 best movies – AL.com

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Sandra Bullock turns 56 today. As an actor and producer, the Oscar-winners films have made billions worldwide.

To wish the film icon and Virginia native a happy birthday, well share our 10 favorite films of hers.

Well cover plenty of the hits you know and love, but also include some lesser celebrated titles that deserve the love. Read our picks (and honorable mentions) below:

Love Potion No. 9 (1992)

Two scientists who are hopeless with the opposite sex experiment with a substance that makes them irresistible to anyone who hears them speak. So yeah, the premise (and much of the follow-through) are plenty problematic through a 2020 (or any) lens, and its cheesy. But it retains a certain 80s comedy aura and gave Bullock the opportunity she needed to show Hollywood she could carry a movie in any genre.

Demolition Man (1993)

A police officer (Sylvester Stallone) is brought out of suspended animation in prison to pursue an old ultra-violent nemesis (Wesley Snipes) who is loose in a non-violent future society. This clever sci-fi satire has plenty of the action and brawn fans expect from Stallone, but its comic strengths shine brightest thanks to Bullocks breakthrough performance as the naive but well-studied Officer Lenina Huxley. Hes finally matched his meet.

Speed (1994)

A police officer must prevent a bomb exploding aboard a city bus by keeping its speed above 50 miles per hour. Sound dumb? Well, it isnt. Its awesome. While Keanu Reeves leads the way as the heroic cop, stealing much of the spotlight is the equally brave passenger-turned-bus driver played by Bullock, in a star-making turn that gave this big dumb flick more charm than anyone expected. It was an instant action classic, but the takeaway was Whats next for Bullock?

While You Were Sleeping (1995)

A hopeless romantic Chicago Transit Authority token collector (Sandra Bullock) is mistaken for the fiance of a coma patient (Peter Gallagher). Bill Pullman co-stars in this hit comedy that helped make Bullock an even bigger star. Perfect for those lazy Saturdays if you can find it streaming.

Miss Congeniality (2000)

Bullock plays a straight-laced F.B.I. Agent who goes undercover in the Miss United States beauty pageant to prevent a group from bombing the event. Opposite Michael Caine, she plays against type, scoring big laughs and another huge box office hit, further cementing her status as an A-list draw and queen of the rom-com.

The Lake House (2006)

A lonely doctor, who once occupied an unusual lakeside house, begins exchanging love letters with its former resident, a frustrated architect. They must try to unravel the mystery behind their extraordinary romance before its too late. Unusual romantic fantasy gives us the Speed reunion (by way of this remake of a South Korean film Il Mare) we needed. And yes, its weird, but it kind of...works?

The Blind Side (2009)

Bullocks Oscar-winning performance as a housewife with a heart of gold won over American audiences two years ago with this touching tale about a family who takes in a homeless high schooler. Knock this film all you want for its cheese; you cant deny the fuzzy feelings you get just a half an hour into it when Mrs. Tuohy gives Michael Oher the first bed he ever had. While she has her strict moments, the tough love approach usually melts away, as the film brings us a proud new addition to our list of great movie moms.

The Proposal (2009)

Bullock plays a pushy boss who forces her young assistant (Ryan Reynolds) to marry her in order to keep her visa status in the U.S. and avoid deportation to Canada. Tack on the type of well-liked romantic comedy few like Bullock can pull off to her amazing 2009, when she scored a pair of box offices hits and an Oscar. Yet another reminder of her star power. Nevermind All About Steve, mkay?

Gravity (2013)

Bullock scored another Oscar nomination as one of two astronauts work together to survive after an accident leaves them stranded in space. Director Alfonso Cuaron (Children of Men, Roma) defies the laws of astro-physics and big-screen visual effects with another harrowing adventure, letting Bullock guide us through an unprecedented and immersive movie-going experience with a physically and emotionally daunting performance.

Oceans 8 (2018)

Debbie Ocean (Bullock) gathers a crew to attempt an impossible heist at New York Citys yearly Met Gala. While were huge fans of Steven Soderberghs Oceans Eleven trilogy that grouped A-list superstars like George Clooney and Brad Pitt to pull of ultra-cool high-tech shakedowns, we echo actress Rosamund Pikes female Bond sentiments that it also feels a bit like sloppy seconds for talented women like Bullock, Cate Blanchett, Anne Hathaway, Mindy Kaling, Sarah Paulson, Rihanna and Helena Bonham Carter. But the women get it done in this breezy heist flick that fits nicely within the franchise.

Honorable Mentions: The Net (1995), A Time to Kill (1996), 28 Days (2000), Crash (2004), Practical Magic (1998), Bird Box (2018)

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Happy Birthday, Sandra Bullock; Here are her 10 best movies - AL.com

First look at Elon Musks personal Tesla Model S with prototype color – Electrek.co

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Weve got a first look at Elon Musks personal Tesla Model S with a new prototype color that will apparently be possible thanks to a new paint shop.

Over the last year, Elon Musk has been talking about Tesla building the most-advanced paint shop at Gigafactory Berlin.

The CEO talked about new multi-layered paints:

Giga Berlin will have worlds most advanced paint shop, with more layers of stunning colors that subtly change with curvature.

Musk said that Tesla is working on a new deep crimson red that is becoming his favorite feature.

Now a Tesla Model S with a special color was spotted in the executive parking lot of SpaceXs headquarters:

It led people to speculate that the Model S his Musks personal Tesla and that the color might be the new deep crimson.

The CEO confirmed that it is a prototype of the color that Tesla is developing for the new paint shop.

It has been a while since Tesla has introduced new color options.

On the contrary, in recent years, Tesla has reduced color options in order to streamline its production and simplify service repairs.

We might have to wait a while to see the new colors though since Musk has linked them to Gigafactory berlin going online.

Tesla is currently building Gigafactory Berlin in Germany, and it is expected to be complete by the summer of 2021 to first produce the Model Y.

Musk says that the new paint shop and colors will make it to other vehicles, but it could take a while:

To be clear, Fremont and Shanghai will also be upgraded over time, but its hard to retrofit these improvements to an operating paint shop.

As we recently reported, Tesla built a new paint shop for Model 3 in Fremont, and it plans onrecommissioning its former paint shopas it increases Model Y production.

Unfortunately, the pictures are in the shade and we are not getting a great look at the color.

From what I can see, its similar to the signature red that Tesla had for early Model S vehicles, but it looks a bit darker.

Again, thats hard to tell because of the shade.

Also, Elon said that the color changes with the light so Ill hold off on judging it until we get a better look.

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First look at Elon Musks personal Tesla Model S with prototype color - Electrek.co

How Tesla defined a new era for the global auto industry – Reuters

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FRANKFURT/BEIJING/DETROIT (Reuters) - Tesla Incs (TSLA.O) rapid rise to become the worlds most valuable carmaker could mark the start of a new era for the global auto industry, defined by a Silicon Valley approach to software that is overtaking old-school manufacturing know-how.

FILE PHOTO: Tesla Inc CEO Elon Musk dances onstage during a delivery event for Tesla China-made Model 3 cars in Shanghai, China January 7, 2020. REUTERS/Aly Song/File Photo

Teslas ascent took many investors by surprise. But executives at Daimler AG (DAIGn.DE), the parent company of Mercedes-Benz, had a close-up view starting in 2009 of how Tesla and its chief executive Elon Musk were taking a new approach to building vehicles that challenged the established system.

Daimler, which bears the name of the man who invented the modern car 134 years ago, bought a nearly 10% Tesla stake in May 2009 in a deal which provided a $50 million lifeline for the struggling start-up.

That investment gave Mercedes engineers an inside view of how Musk was willing to launch technology that wasnt perfect, and then repeatedly upgrade it, using smartphone style over-the-air updates, paying little regard to early profitability.

Mercedes engineers helped Tesla develop its Model S luxury sedan in exchange for access to Teslas partially hand-assembled battery packs, but in 2014 Daimler decided to sell their stake amid doubts Teslas approach could be industrialized at scale.

Tesla would go on to pioneer new approaches in manufacturing, designs in software and electronic architecture which enable it to introduce innovations faster than rivals, leaving analysts to draw comparisons with Apple (AAPL.O).

Three people directly involved with the Mercedes side of the collaboration said the brief partnership highlighted the collision of old and new engineering cultures: the German obsession with long-term safety and control, which rewarded evolution, and the Silicon Valley carmakers experimental approach which embraced radical thinking and fast innovation.

Elon Musk has been walking on the edge of a razorblade in terms of the aggression with which he pushes some technologies, said a former Mercedes engineer who worked on the partnership.

By contrast, Mercedes and other established automakers are still not comfortable about releasing a new technology, such as partially automated driving, without years of testing.

Tesla did not respond to requests for comment.

Investors favor the Tesla model, in an industry undergoing fundamental and dizzying change even though the U.S. carmaker will face an onslaught of competing electric vehicles from established automakers during the next few years.

They are putting their money on Musk and his company, even though Mercedes-Benz alone sold 935,089 cars in the first half of 2020, dwarfing the 179,050 delivered by Tesla in the same period.

Today, Tesla is worth nearly $304.6 billion, more than six times Daimlers 41.5-billion-euro ($47.7 billion) market capitalization.

Tesla electrifies the auto industrys new era

TWO CULTURES COLLIDE

Daimler and Tesla began collaborating after Mercedes engineers, who were developing a second-generation electric Smart car, bought a Tesla Roadster. They were impressed by the way Tesla packaged batteries, so arranged a visit to Silicon Valley to meet Musk in January 2009 and ordered 1,000 battery packs.

The collaboration expanded. At a joint press conference in the Mercedes-Benz museum in Stuttgart in May 2009, Tesla said the partnership would accelerate bringing our Tesla Model S to production and ensure that it is a superlative vehicle.

For its part, Mercedes wanted to use Teslas batteries to power an electric version of its compact Mercedes-Benz B-Class. The Tesla Model S would hit the road in 2012. An electric B-Class, arrived in showrooms two years later.

Despite having batteries supplied by Tesla, the Mercedes had a shorter operating range after Daimler engineers configured the B-class more conservatively to address their concerns about long-term battery degradation and the risk of overheating, a second Daimler staffer who worked on the joint projects told Reuters.

German engineers found that Tesla engineers had not done long-term stress tests on its battery. We had to devise our own programme of stress tests, the second Daimler engineer said.

Before starting production of a new car, Daimler engineers specify a Lastenheft - a blueprint laying out the properties of each component for suppliers. Significant changes cannot be made once the design is frozen.

This is also the way you can guarantee that we will be profitable during mass production. Tesla was not as concerned about this aspect, the second Daimler source said.

Daimlers engineers suggested the underbody of the Model S needed reinforcing to prevent debris from the road puncturing a battery pack, the first Daimler engineer said.

To quash doubts about safety and security, following a series of battery fires, Tesla raised the ride height of its vehicles, using an over-the-air update, and a few months later, in March 2014, said it would add a triple underbody shield to new Model S cars and offered to retrofit existing cars.

Musk was able to make adjustments quickly thanks to Teslas ability to burn through more cash during development.

At Mercedes you can make such adjustments every three years at best, the engineer said.

The Model S, a four-door electric sedan would go on to outsell the flagship Mercedes-Benz S-Class in the United States in May 2013, and outstrip S-Class deliveries globally by 2017.

Musk's relentless focus on innovation explains, in part, why he has disrupted the traditional auto world. In an interview here at the 2020 Air Warfare Symposium, published on YouTube, he was asked about the importance of innovation among his employees.

We certainly need those that do advanced engineering to be innovative, Musk said. The incentive structure is set up ... such that innovation is rewarded. Making mistakes along the way does not come with a big penalty. But failure to try to innovate at all ... comes with a big penalty. You will be fired.

Established automakers are playing catch-up to Tesla, designing their own software operating systems and dedicated electric cars.

Mercedes will release its EQS next year - a four-door limousine built on a dedicated electric vehicle platform, with an operating range of 700 km. A new version of the Mercedes S-Class, which will have combustion and hybrid powertrains and semi-autonomous driver assistance systems, is due this year.

From an investor perspective, traditional players face billions of dollars in restructuring costs as they transform product lines and factories to move away from internal combustion technology

No one is going to give an OEM (established automaker) a five-year window to say ... you can totally retool your business, and I am going to buy in and fund this journey, said Mark Wakefield, co-leader of automotive and industrials practice at consulting firm AlixPartners.

Start-ups, however, get time from investors to learn, make mistakes and grow, he added.

Investors are betting on Teslas ability to scale up manufacturing just as they once backed Toyota Motor Corp (7203.T), which defined the auto industrys last era with its mastery of highly efficient, high-quality lean production.

Toyota overtook the market capitalization of former industry leader General Motors(GM.N) in 1996, though it wasnt until 2008 that it sold more vehicles than its Detroit rival.

The Japanese giant also cultivated ties with Tesla, with the U.S. startup helping it design an electrified RAV4 compact sports utility vehicle under a 2010 deal.

Toyota was impressed by the speed with which Tesla came up with the new design, but ultimately decided Teslas methods were not suitable for mass production by a mainstream manufacturer when Toyotas standards for product quality and durability were applied, two company insiders familiar with the partnership said.

Toyota said the joint project involved cooperation on the development of electric cars, parts and production system.

Toyota accomplished what the project set out to achieve, and it ended in October 2014 after Tesla delivered roughly 2,500 electric powertrain systems over three years for an electrified RAV4 crossover SUVs, a spokeswoman said.

Both the Toyota and Daimler collaborations were agreed before the Volkswagen (VOWG_p.DE) emissions-cheating scandal in 2015, which prompted a global regulatory backlash and forced carmakers to step up investments in electric cars.

That was all before dieselgate, which changed the economics of electric and combustion-engined cars, a senior Daimler manager said. Tesla has a lead. Lets see if they can scale up.

Reporting By Edward Taylor, Nori Shirouzu and Joe White; Additional reporting by Paul Lienert; Editing by Joe White and Pravin Char

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How Tesla defined a new era for the global auto industry - Reuters

Last Tesla Roadster ever built listed for more than $2 million – CarAdvice

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Only 2500 Lotus-based Tesla Roadsters were built, and the final one is for sale in Switzerland for more than AU$2 million.

The last original Tesla Roadster to be built has come onto the market in Switzerland for more than AU$2 million.

The 2012 car's Lotus-designed chassis is fitted with a 185kW electric motor that propels it from 0 to 100kmh in a claimed 4.0 seconds.

When new, a battery range of 393km was estimated.

The listed car is finished in sparkling white and has a white and black interior trim, full carbon package, carbon diffusor, and VIN 2500 badging (recognising that it was the 2500th and last Roadster built).

Dozens of Tesla employees also signed the car's battery pack on completion, and a scribble in the bottom left-hand corner strongly resembles the signature of company CEO Elon Musk.

According to the listing the car has been stored on tyre pillows on marble floor and was never registered.

It has just 200km on the odometer.

The vehicle is listed for 1,390,000 (approximately AU$2,082,220) which, if sold, would make it the world's most expensive electric car.

In Australia the 2012 Roadster was initially sold from $191,888 plus on-road costs slightly less than a Porsche 911 at the time.

Only one version of the car is currently listed for sale in Australia.

Located in Blackheath, NSW, the red MY2011 car is listed at $144,000.

The seller, Simon Crawford-Ash, told CarAdvice the Roadster represented a "pivotal moment" in automotive history.

"The rawness of it is what makes it so special. It is raw, unpolished and incredible to drive," he said.

"It is inspiring to touch and see how much had to be redesigned from an ordinary car. It is exposed and raw, and it feels very much like the prototype of future cars."

In 2017 Tesla announced plans for a new Roadster (pictured below), this time with a fixed roof, and claimed it would be capable of accelerating from 0 to 100kmh in 1.9 seconds faster than any production car available today.

After initially stating the car would be available by 2020, Tesla now says the Roadster will be launched in 2022.

Last Tesla Roadster ever built listed for more than $2 million

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Last Tesla Roadster ever built listed for more than $2 million - CarAdvice