Category Archives: Evolution

SAN FRANCISCO, Aug. 22, 2022 /PRNewswire/ -- Global color cosmetics powerhouse KVD Beauty announces a new collection of liquid lipsticks sure to be their next cult classic. Introducing Everlasting Hyperlight Liquid Lipstick, a next generation, long-lasting, transfer-proof formula set to replace the original collection, Everlasting Liquid Lipstick. To celebrate the launch, brand partner Paris Jackson takes her partnership with KVD Beauty to the next level, serving not only as the face of the campaign, but also a co-creative director.

Paris Jackson, Global Campaign Face of KVD Beautys New Everlasting Hyperlight Liquid Lipstick

Everlasting Hyperlight Liquid Lipstick lasts like a liquid lipstick but feels like vapor. The evolution of their liquid lipstick revolution, KVD Beauty took the iconic, all-day, transfer-proof wear of brand's original formula and evolved it for an even better experience: extreme comfort and a hyper-lightweight feel. 100% of wearers said Everlasting Hyperlight Liquid Lipstick was comfortable even after 8 hours of wear (in a consumer study on 32 volunteers).

Powered by nourishing raspberry stem cells, this ultra-thin, flexible formula moves seamlessly with lips to help prevent caking, flaking and feeling dry. This pigment-packed formula is ultra-comfortable without compromising the impactful color payoff KVD Beauty is known for.

Dangerously bold and undetectably light, this evolved formula comes in 21 feather-resistant matte shades and features a slim, precision applicator for complete control without needing a lip linerjust line, fill and let it set. Pluck your favorite shade from a collection of petal-soft neutrals and intoxicating pops named after poisonous plants and bizarre blooms.

With 21 shades available at launch and more rolling out over the next year, Everlasting Hyperlight Liquid Lipsticks' shade names are inspired by a strange garden filled with beautiful, yet dangerous botanicals. Available shades include: Moonflower (terracotta rose), Moth Orchid (soft pink), Cobra Lily (rust red), Scorpiris (indigo blue) and more. Most notably, the brand's bestselling shade Lolita is undergoing an evolution of its own, relaunching with this new collection as Queen of Poisons.

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Back for her second campaign with KVD Beauty, multi-hyphenate Paris Jackson is the face of the new Everlasting Hyperlight Liquid Lipstick, an appropriate fit for this collection of both serene and statement shades. In addition to being featured in the campaign, Jackson was also involved in the creative direction of the shoot, where she contributed to location selection, guided the fairy-goth-meets-grunge aesthetic thanks to strategic styling realized by Jackson and her stylist, Danyul Brown, and collaborated with campaign makeup artist and KVD Beauty Global Veritas Artistry Ambassador Anthony Nguyen on the beauty looks showcased within the images.

"KVD Beauty has grown significantly over the past few years, and we're excited to continue to evolve both the brand and our cosmetics to fit the needs of our loyal consumers," says Tara Loftis, Global Vice President of Marketing and Public Relations at Kendo Brands. "With the new Everlasting Hyperlight Liquid Lipstick, and the incredible Paris Jackson as the face of the campaign, this new launch underscores our continued support of the artist community while also taking the bold collection of products you all know and love to the next level. We're confident this new collection will be well received by brand loyalists and new shoppers alike."

100% vegan and cruelty-free, KVD Beauty's Everlasting Hyperlight Liquid Lipstick ($22) is now available online at KVDBeauty.com, in store and online at Ulta.com, and in store and online at Sephora.comon August 21, 2022.

ABOUT KVD BEAUTY:

KVD Beauty stands for Kara, Veritas, DecoraValue, Truth, Beautybecause your truth is valuable and beautiful. We create high-pigment, high-performance makeup inspired by tattoo artistry, like our #1 award-winning Tattoo Liner. With long-wear, ink-like pigment and needle-sharp precision, it's the uncontested holy grail of liquid eyeliner. But we stand for more than just performance: Our makeup is 100% vegan and cruelty-free, we care about the planet, and we champion artistry of all kindsnot just makeup. We celebrate the individual through artistic self-expression and limitless creativity. And above all, we give you the tools to live your beauty truth.

KVD Beauty Introduces New Everlasting Hyperlight Liquid Lipstick launching in 21 shades

(PRNewsfoto/KVD Beauty)

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KVD Beauty Launches New Everlasting Hyperlight Liquid Lipstick: The Evolution of Their Iconic Liquid Lipstick Featuring Global Brand Face, Paris...

As of August 2022, DataDomes CAPTCHA has been used in production for several weeks with ~30 customers, including mostly e-commerce websites and mobile applications. Lets explore what we observed in the weeks following the deployment of our CAPTCHA on highly-targeted websites and mobile applications.

Each time we switched a customer to our CAPTCHA, we observed a significant drop in CAPTCHA-passing attempts coming from bots.It makes sense, given that bot developers need to update their bots (update the CSS selectors, etc.) to properly interact with our new CAPTCHA. However, we notice the decrease remains stable even after a month.

Malicious CAPTCHA forging attempts over time: We observe a significant drop following the activation of the DataDome CAPTCHA.

Our hypothesis, based on the data we collect, is that a majority of bot developers rely on popular open source projects/off-the-shelf tools (CAPTCHA farms) to forge CAPTCHAs. Thus, as long as the easily available tools dont offer any options to solve the DataDome CAPTCHA, we expect the number of malicious CAPTCHA passing attempts will remain lower than before.

It took between 6 hours to ~2 weeks, depending on the website and mobile application.

The fastest attempt to forge the CAPTCHA (6h after implementation) happened on a popular e-commerce platform heavily targeted by distributed scrapers. Six hours after switching to the new CAPTCHA, we detected bots trying to submit CAPTCHA challenges, though they were blocked for several reasons (such as inconsistent browser fingerprints linked to instrumentation frameworks and other bad behaviors). Thats how fast attackers will adapt to try to obtain data.

The good news is, since DataDomes primary purpose is to protect websites and mobile apps against fraudulent traffic, were used to continuously finding new bot signals and improving our ML models to stay ahead of bots. Weve been doing it for years to improve our real-time detection engine, and now it will also continue to strengthen our CAPTCHA.

Audio API: First, we observed evidence of a known issueaccessibility vs. security. We know that audio CAPTCHAs are often more exploited than their image-based counterparts, which was also evident with our CAPTCHA. However, with behavioral and fingerprinting signals, we can still invalidate a forged CAPTCHA, even when the response to the challenge is correct.

Non-Modified Puppeteer: Puppeteer is a popular automation framework to instrument (headless) Chrome. Its no surprise that we encounter it frequently among bots that try to forge our CAPTCHA. Bots use standard APIs provided by Puppeteer to mimic fake mouse movements and clicks. However, the behavior deviates from legitimate users, whichcombined with fingerprinting signalsallows us to invalidate CAPTCHAs passed by Puppeteer.

Puppeteer Extra Stealth: Puppeteer extra stealth is a popular bot automation framework that adds a layer of features on top of Puppeteer. Its API is compatible with Puppeteer, but includes features to spoof your fingerprint and simple integrations with CAPTCHA farm APIs, such as 2Captcha. The stealth plugin is popular among bot developers and bots as a service (BaaS).

Similarly to Puppeteer, our CAPTCHA collects behavioral and fingerprinting signals that enable us to invalidate CAPTCHAs passed by Puppeteer extra stealth bots, even if they submit a CAPTCHA with a valid response.

Users With 2Captcha Extension: Our CAPTCHA client-side JavaScript code has also detected the presence of instrumented browsers that use the 2Captcha auto solver browser extension. However, it doesnt help bots because 2Captcha doesnt support any integration for our CAPTCHA. It only makes it easier for us to invalidate forged CAPTCHAs.

So far, we dont see a significant volume of Selenium-based bots attempting to forge the DataDome CAPTCHA.

The graph below shows the evolution of bot forging attempts on DataDomes CAPTCHA. We see that bots try to adapt more and more over time as we protect more websites and mobile applications with DataDomes CAPTCHA.

In total, the graph shows more than 1.37M malicious CAPTCHA passing attempts stopped before the bots could go further.

Link:

The Evolution of Bots Forging CAPTCHAs, a Firsthand Report - Security Boulevard

MotoGP will introduce sprint races to its grand prix weekends from 2023, as first reported by Motorsport.com almost 24 hours ahead of the official announcement, in a move that has caused quite a stir but which was entirely predictable.

MotoGP, for all of its fire-fighting exercises in recent months, is facing a decline it is struggling to overturn. While Formula 1 has burst out of the niche bubble it once occupied with the rest of its motorsport counterparts, the once clear second-most-popular racing spectacle, MotoGP, has been left behind.

Largely, this has been blamed on the retirement of Valentino Rossi and the lengthy absence of an injured Marc Marquez. The guard has well and truly changed, following the likes of Jorge Lorenzo and Dani Pedrosa bowing out in recent years.

This was most highlighted at the Italian Grand Prix, where just over 43,000 people turned up to Mugello for an event that used to be one of the most popular stops on the MotoGP calendar. Not even an impromptu appearance from Rossi to celebrate the retirement of his famous #46 did much to boost the enthusiasm of fans to put their hands in their pockets and buy tickets that had gone up in price from previous years.

That has been a familiar tale all year at a number of events. But F1s soaring popularity has shown that people are willing to part with large swathes of hard-earned cash in the midst of a global financial squeeze if it means getting a front-row seat at one of the worlds hottest events.

Dyed-in-the-wool, long-time F1 fans have derided the celebrification of the series as Martin Brundle spends most of his grid walks tripping over seemingly disinterested famous people. But even if Brundle doesn't know who they are, their millions of fans on social media do, and that engagement is a goldmine that Liberty Media has been gleefully tapping into.

In a press conference on Saturday morning at the Austrian Grand Prix that raised a number of questions, FIM president Jorge Viegas made an incredibly honest assessment of why MotoGP has been forced into making its first radical format change since 2013 and the introduction of the split qualifying system.

FIM President Jorge Viegas was flanked by Dorna Sports boss Carmelo Ezpeleta and IRTA President Herve Poncharal at the press conference on Saturday outlining MotoGP's plans

Photo by: Dorna

After two years in COVID, we think that all of us have made incredible sacrifices to keep having this so important championship, he said. Its time to give more exposure in the TV but also to the spectators, we need more spectators, we need a better show and we need to fulfil the Saturdays.

The details of the sprint races that have been revealed so far are as follows:

When discussions were first had about this during the summer break between the FIM, Dorna, the International Race Teams Association (IRTA) and the Motorcycle Sport Manufacturers Association (MSMA), the reaction was one of positive unanimity.

That didnt extend to the riders, however. Opinions on sprint races were mixed when the riders were first asked about them on Friday, with reigning world champion Fabio Quartararo calling the idea totally stupid. He was unhappy that the physical strain of the riders had seemingly not been considered.

Most said they werent even aware sprint races were definitely happening, with some only hearing rumours from their teams. This was addressed in Saturdays press conference. Carmelo Ezpeleta, Dornas CEO, said he'd confirmed MotoGPs plans to the riders in Friday evenings safety commission meeting, adding that he thought he was only rubber-stamping something they already knew.

Reigning MotoGP champion Quartararo wasn't impressed by the proposal when asked on Friday prior to the official announcement

Photo by: Gold and Goose / Motorsport Images

Ezpeleta did absolve himself by stating that responsibility was with the teams to inform their riders, though IRTA president and Tech 3 boss Herve Poncharal said only so much highly confidential information could be shared to riders or else nothing would be secret. That doesnt exactly tally when riders are notorious for looking the media in the eye and either concealing the facts or telling half-truths.

First, a lot of teams dont have direct access to their riders because they are going to change riders, Poncharal said. Second, you cannot ask to keep highly confidential something and tell all the details before the official announcement, because then there is no official announcement. This is something you have to take into consideration. We have informed as much as possible that there will be some changes to the format.

This has renewed calls from riders for a proper union to be formed so that they can be much more involved discussions of topics that have a significant impact on them. This was largely dismissed by those chairing the press conference, with Ezpeleta saying no riders had ever talked to him about this. All three then went to great lengths to point out that MotoGP is one of the few championships in motorsport where the competitors are massively involved in decision-making.

That, ultimately, is a debate that extends much wider than that of sprint races. But it also leads onto the matter of safety, with several riders concerned that an extra race in the ultra-competitive era of MotoGP will only add to the risk. Viegas was adamant that, should any genuine safety concerns be raised over the new proposal, then the organising body would act on it.

We always have in our minds the limits of the riders, Viegas said. This is why we have this safety commission, so if the riders say we cannot go for all this so much time, so much laps on the limit, we will consider. You can be sure for us the safety of the riders comes first, of everything.

We need to improve our show because we want to be on top of the audiences. When you have good races, when you have real excitement, the media sells much more. So, this is what we want. We are all in the same boat, we cannot stay behind. But always in mind we have the safety of the riders, this is for sure.

Another matter raised was that of how an extra race per weekend assuming a 20-round calendar, that would lead to 40 races would impact rider contracts, many of which being signed before this proposal was put in place. Already this year riders have called for more support to come their way in dealing with contracts as salaries have dipped. This wasnt something that could be answered and it will likely become the centre of debate in the ensuing months.

All of that aside, the introduction of sprint races has a simple aim: to improve the show and give fans, sponsors and race promoters better value for their money. That in turn should boost television with Ezpeleta claiming that side of the business has taken well to the proposal which in turn feeds back into serving fans and bringing in more sponsorship.

The FIM has promised to respond to any safety concerns

Photo by: Gold and Goose / Motorsport Images

This is an entirely fair approach and has been driven largely by what has proven successful in F1 which introduced sprint races at select events in 2021 and World Superbikes, which introduced a Superpole sprint race in 2019 that decided the top nine grid slots for the main second race and counted as a race in its own right.

Ostensibly, the feedback gained from the Global MotoGP Fan Survey that was run by Dorna in conjunction with the Motorsport Networkplayed a factor. According to Carmelo Ezpeleta, the reception by fans to the sprint race idea and the plans to have it stand as an individual race was positive for the majority of respondents: "We have made a research and we will have the first results [of the survey], and practically the majority of the people are in favour to have sprint races and the majority of people are in favour of the sprint races not counting towards the grid. This is a result of that."

However, Dorna's Sporting Manager Carlos Ezpeleta contradicts this by saying the format change idea came before the survey results were seen.

"This project of changing, upgrading the format of the weekend started before we had the Fan Survey, so its something that we started before and fortunately the results of the Fan Survey confirm what we thought: our fans do not want the sprint race to result to decide the grid results for Sunday," he said. "They want the riders to be able to free to race. Other things from the survey we will be disclosing soon. But you know, in the end, the survey has been a huge success. Were very happy about the response that weve had, and we thank our fans for taking such a lot of time to respond to our questions.

However, what works elsewhere doesnt necessarily mean it will transfer to MotoGP. Sprint racing works in Superbike racing because, from day one, WSBK events have had at least two races. In F1, it was a hugely controversial decision that has generated more interest but the clunky designations by F1 in not calling the sprint race an actual race, while the polesitter in the qualifying for the sprint isnt actually a historical pole winner is confusing.

Then there is the general idea of a sprint MotoGP race. MotoGP races arent that long to begin with. At around 45 minutes, a grand prix is pretty much a sprint. Yes, there are many strategical factors that constrict a rider during a race. However, a cut-down hell-for-leather sprint MotoGP seems like it will be robbing Peter to pay Paul. If the sprint race is just a race that has no bearing on what will happen in the main grand prix, you run the risk of the sprint race just being a lite version of what you have seen in the main contest.

And with calendars already swelling beyond 20 races, at what point will fans really care who the winner of race 18 was when you have so many more to come? Like anything, overconsumption leads to weariness and fans naturally disappear to do other things safe in the knowledge they can catch up later.

In the press conference, the IRTA/Dorna/FIM trio made the point that more racing on offer would create better excitement and translate into the media benefitting from more readership. In reality, interest in MotoGP has dropped off considerably since 2020, its loss of interest easily seen by the fact it makes no impact in the UK national newspapers anymore because none of them turn up to races.

Devaluing something that already has waning interest is counterintuitive. And ultimately, a second race will do this to MotoGP.

It's hoped that the introduction of sprint racing will give fans and TV companies more for their money

Photo by: Gold and Goose / Motorsport Images

Poncharal is absolutely correct in saying MotoGP would be stupid not to look at what is working elsewhere and try to adapt MotoGPs own format, even if the racing right now is arguably the best on offer in the world.

However, simply copying what other series are doing well isnt necessarily going to work. That is evident in MotoGPs own Drive To Survive clone MotoGP Unlimited, which flopped when it was launched on Amazon Prime in March and led to the second series being shelved not long after.

The problem with MotoGP Unlimited was that it was aimed at existing MotoGP fans. The Global Fan Survey was aimed at existing MotoGP fans. Ultimately, these arent the fans you want to consider in efforts to grow the championship: its the ones still standing outside of the door. This is where F1 has succeeded with all of its efforts and what MotoGP is, to its credit, trying to do. But it must figure out how to do that in its own way, to have its own identity.

MotoGP is a fantastic product. Dorna has done an incredible job over the last 10 years to make it the ultra-competitive championship that it is now. The grid is littered with a young, fresh roster of characters that could so easily be marketed to bring in a new, young, diverse audience.

Introducing sprint races to grand prix weekends is a necessary revolution MotoGP needed to make to not get stuck in the mud. But its current proposal is inherently flawed by its lack of imagination.

F1 holds occasional sprint races - could making them part of every MotoGP weekend mean there is too much of a good thing?

Photo by: Gold and Goose / Motorsport Images

Read the original here:

The flawed MotoGP sprint evolution that is a necessary evil - Motorsport.com

Not all games lend themselves to a succinct elevator pitch. Imagine trying to secure finance for What Remains of Edith Finch: You wander around an old house watching family members die. Or Loop Hero: Its kind of a roguelike farming sim about making a circuit board. Payday 2, though, has one of the most delicious and succinct premises in the genre: do bank heists with your mates.

When Overkill Software released the action-packed sequel in 2013, that enticing premise had to do a lot of the work for it. This was an incredibly barebones game, in an era before games regularly stepped out half-dressed as were used to now. Steam had only just launched its Early Access program in March of that year, and the novelty of that model is probably the best explanation as to why Payday 2 wasnt released on it that and the incredible headache it would cause for the console versions.

This dewy-eyed, wobbly-kneed newborn Payday 2 featured just 10 heists the current version has 70 and the persistent systems it has today, like weapon purchasing and character abilities, were nearly unrecognizable. Story mode as we know it didnt exist just a campaign map and a few interstitial cutscene bits that barely established the main characters, and offered the briefest insight into their world.

Mechanically, Payday 2 felt like a proof of concept. If this wasnt a game about putting on masks, drilling into bank vaults and telling hostages to get down over and over and over again, it would probably have sunk into obscurity pretty soon after launch. It was wildly unbalanced, the stealth didnt work at all, and above all it was really, really hard.

Even understanding the difficulty levels themselves was hard. You couldnt select missions manually in those early days they just popped up randomly on the Crimenet map. It felt quite immersive, and consistent with the idea of these ragtag Slipknot understudies taking opportunities when they arose on the dark web. But, in practice, that meant a lot of simply staring at the Crimenet screen and waiting for the right heist to appear with the right difficulty rating attached to it for you to take on at your current level. There was the inherent difficulty of the mission in stars out of five then extra modifiers that ramped up the rating and the rewards further.

It would have felt a bit Early Access, if wed known what Early Access felt like back then. In 2013 we just called it playing the same 10 heists over and over without much incentive

In the absence of seasonal events, crossover content from John Wick and the like, or any structured narrative to put the heists in a sequence, it all felt... well, a bit Early Access. Or at least it would have done, if wed known what Early Access felt like back then. In 2013 we just called it playing the same 10 heists over and over without much incentive.

But despite these conceptual cable ties around its wrists, Payday 2 did well enough commercially for the Swedish studio to keep supporting it. And to reposition it as a more forgiving experience, closer to the hangout space vibe that co-op gamers demanded than the hardcore stealth-infused class shooter its predecessor offered, and which informed Payday 2s original form.

Not only have the intervening nine years filled Payday 2s content vault with stuff to do, weapons, every mask design ever imagined by humankind and revised upgrade trees theyve seen the game successfully pull off that repositioning. Its not Clicker Heroes now by any stretch, but its hardest difficulty mode is probably pitched around the middle of the original games levels, and its much easier to pull off stealthy heists. Its a game you can give as much or as little focus as you like, and still walk off smiling.

When you look at Overkills financial situation at the time, this turnaround of fortunes is all the more impressive. The studio needed to secure funding from fellow Swedish company Starbreeze in order to begin production of Payday 2 in 2012, although insiders have since described the situation as more like Overkills investors taking on Starbreezes assets the net result being that both studios could put out their next release (Brothers: A Tale of Two Sons in the latters case) and start generating revenue again.

For a game about psychologically exhausting shootouts that might last hours, and in which you could well walk away with absolutely zero reward, its surprisingly friendly

So much revenue, in fact, that Overkill was able to buy back publisher 505 Games share of the Payday IP in 2016 and maintain total ownership of the franchise. Had that premise not proved so irresistible that we looked past the heist sims considerable annoyances, it could all have been so different.

Its nearly impossible to grasp the full extent of the games lore now, so busy has Overkill been since release fleshing out its original roster of characters, adding to it, and then drawing confusing timelines of their exploits. Hoxton became Houston, John Wick is just another one of the gang, no biggie, and he might find himself fighting alongside Jacket from Hotline Miami. The safehouse has changed, expanded and filled itself with incredibly esoteric easter eggs that you need a YouTube iceberg video to decipher.

More importantly, Overkill has totally reworked every system within its heists, from AI detection behavior now vision cone based, rather than triggered by whether youre running, jumping etc to the way drills and saws work, and how you level your characters. For a game about psychologically exhausting shootouts that might last hours, and in which you could well walk away with absolutely zero reward, its surprisingly friendly.

And this has all happened without Payday 2 getting much fanfare along the way. Its been hiding in a sort of plain digital sight, always up there in Steams most played games in a given 24-hour period. It was there when Dota and CSGO looked impossible to topple, it was there when PUBG and Apex Legends toppled them, and its still there now. 17th by recent player count (opens in new tab), as Im writing this: 49,000 players in the last day.

Its a great example of traditional games coverages blind spot. People care about Payday 2 in a way that theyll probably never care about Stray, or the aforementioned Loop Hero, or Tunic, with the greatest respect to those brilliant titles. It slips through the cracks because its progress towards greatness has happened an inch at a time or, less poetically, over the course of 225 separate updates and as many DLC packs.

If you havent played it for a few years, just know that its moved forwards quite a lot, and without paying a penny on DLC itll feel like stepping into a dauntingly fleshed-out criminal sim. One in which deeply specialized builds, super-serious heisting, and run-and-gun sessions you barely have to pay attention to while chatting on Discord are all stuffed into the duffle bag.

Continued here:

The creeping evolution of Payday 2, the biggest game no one talks about - TechRadar

As a trusted partner in the life-sciences industry, Sartorius has contributed significantly to the evolution of biopharmaceutical manufacturing. To reflect on the rich history of the industry, we arranged a virtual roundtable. Based on shared questions, we corresponded about what we considered to be the most important scientific, technological, and operational developments in the past 20 years of bioprocessing. We also reflected on the emergence of new modalities and how such products are likely to shape the future of the biopharmaceutical industry.

What Scientific and Technological Advances Have Been Most Formative to the Biopharmaceutical Industrys Development?Horry: Many innovations have been implemented to increase upstream yields, improve product recovery, enhance product purity, and streamline manufacturing processes. In the early days of monoclonal antibody (MAb) production, for instance, protein yields from perfusion-mode cell-culture campaigns were around 100 mg/L.Now, perfusion has become the cornerstone of upstream process intensification, with process volumetric productivity reaching on average 6 g/L/day.

Dixit: Even 10 years ago, there was significant impetus to set up large-scale stainless-steel equipment e.g., stainless-steel production bioreactors of >10,000 L to produce several tons of MAbs annually. That strategy was meant to address indications with large patient populations. But the need to produce at such high volumes has transformed, with development pipelines showing greater numbers of MAbs and MAb variants with less annual demand (e.g., 50200 kg). Approval of several biosimilars in the European Union and in US markets has reinforced the need for drug companies to manufacture several products in smaller quantities out of each facility.

Nestola: Over the past 20 years, we also have witnessed strong uptake of single-use technologies (SUTs). That has helped the biopharmaceutical industry to become more efficient and flexible than ever before, especially by enabling multiproduct manufacturing environments. The shift to disposable equipment has been particularly recognizable in facilities for new modalities such as cell and gene therapies. The question of whether to go with single use or stainless steel is not asked anymore in that forum; SUTs have become the standard.

Horry: The biopharmaceutical industry generally has been conservative when implementing new technologies. But the status quo is being challenged today by the development of computational technologies, artificial intelligence (AI), and machine learning (ML). This revolution is happening while the biopharmaceutical industry is still in its infancy.

We already are witnessing significant acceleration in process development (PD) activities. In-depth understanding of critical process parameters (CPPs) and critical quality attributes (CQAs) through design-of-experiments (DoE) approaches and advanced analytics has enabled the construction of representative models that are paving the way to autonomous and predictive processes.

Nestola: Also of note is the industrys increased focus on process understanding and quality by design (QbD), prompted by pushes from the US Food and Drug Administration (FDA). This development has led to increasing investment and adoption of process analytical technologies (PATs), mainly for upstream production, but also increasingly for downstream processes. Process understanding and reproducibility both have improved dramatically because of closed-loop control of bioreactors using data collected by Raman spectroscopy probes.

Improved analytics programs have changed how data are collected and analyzed to monitor or control processes, both at PD and good manufacturing practice (GMP) scales. The industry also has much more data available than ever before thanks to adoption of high-throughput technologies, some of which have become standard equipment in even PD laboratories.

What Role Has Sartorius Played in the Industrys Development?Dixit: Sartorius has expanded its bioprocessing solutions portfolio significantly over the past 20 years. That stems from a combination of internal development work based on our core competencies in SUTs and separation membranes and strategic acquisitions that have complemented those strengths. We have developed an extensive line of single-use products for mixing, aseptic connection/disconnection, and freezethaw applications. We also have offered filter membranes for dead-end and tangential-flow filtration as well as scalable membrane-chromatography devices for MAb polishing steps and viral vector capture.

Our Ambr 15 and 250 automated minibioreactors are considered to be industry standards for early stage process development (PD) for microbial and mammalian cell cultures, and they are known to reduce PD timelines and thus decrease time to market. Our Biostat STR line of scalable single-use bioreactors (22,000 L) have been implemented at many biomanufacturing facilities to produce not only MAbs and other recombinant proteins, but also viral vectors that are used in vaccines and gene therapies.

We have strengthened our chromatography offerings through select acquisitions, such as that of BIA Separations. Now, Sartorius can provide chromatographic monoliths that can address a number of separation needs with solutions that already are available for commercial manufacturing. Sartorius also has acquired BioSMB and Novasep BioSC multicolumn chromatography (MCC) systems with single-use and reusable flow paths to address MAb process intensification needs. We plan to launch protein A affinity membranes later in 2022. Those could improve MAb capture significantly by providing solutions with capability for rapid flow, high dynamic binding capacity (DBC), good NaOH stability, and consistent performance over several hundred cycles.

Today, Sartorius also provides predictive and even prescriptive process monitoring tools. SIMCA software for multivariate process analysis has helped save many batches through early warnings about process perturbations.

How Has the Pandemic Changed Things?Dixit: The COVID-19 pandemic has ushered in a new era in the biopharmaceutical industry, with mRNA-based vaccines that hold much promise for changing the drug development landscape. Several million doses of a vaccine can be manufactured from a single batch with starting volumes of tens to hundreds of liters. Several biopharmaceutical companies are exploring this modality not only for vaccines against infectious diseases, but also for other indications such as certain cancers. Many leading companies in the mRNA space will have diverse pipelines focused on indications with few or no therapies available. Many drug companies will need to build PD expertise across different modalities, from MAbs to viral vectors to cell therapies and now mRNA.

Nestola: Although the biopharmaceutical industry has worked mainly with MAbs and other antibody products for the past two decades, we now are experiencing remarkable changes in the molecules that we work with. That development is causing a shift in mindsets and innovation pipelines of biomanufacturing suppliers, as well. Workflows for new modalities are quite different from those for MAbs. Thus, customer requirements for biomanufacturing equipment are changing, too. New products must be designed specifically for new modalities. As a process technology manager, my team needs always to stay up to date, reading the latest trends and anticipating customer needs to prepare the company for the future.

Horry: Its important to consider how the COVID-19 pandemic has influenced our work. Its difficult to present a general comment because everyone is experiencing the pandemic differently. But I observed that modern communication and networking tools computers, internet capabilities, shared cloud-based working spaces, and video conferencing tools kept the whole biopharmaceutical industry connected. Fortunately, communication and to a certain extent networking never stopped. Information continued to flow, and that kept the industry in motion. That was not necessarily the case for other sectors.

The pandemic forced us to pause and step aside from the work-related storm in which we found ourselves. Traveling stopped for many of us. That was significant for employees who spend 5060% of their work time taking flights, waiting in lobbies, and experiencing jet lag. Simply by being less tired, many people realized how exhausting our working style can be. That recognition has forced some of us to reconsider why we travel and network, how often, and where we might like to invest our time and resources instead. Simply put, we have become more selective.

We also have rediscovered the power of global capabilities, with colleagues from all around the world attending local events (when possible) and then sharing internally what they learned. We have remembered the importance of internal networking, work delegation, and empowerment of collaborators.

As we return to a relatively normal and stable work situation, companies need to find the right balance between local and external activities. Ive seen significant changes already. Now that we are visiting customers and suppliers again, we know how much we missed in-person interaction. Were also recognizing how valuable such interactions are for our companies. Business is not just numbers, facts, strategies, and tactics.

Habib Horry is manager of process technology at the Sartorius site in Strasbourg, France; habib.horry@sartorius.com. Piergiuseppe Nestola is manager of process technology at Sartorius in Zug, Switzerland; piergiuseppe.nestola@sartorius.com. Mandar Dixit is head of value-chain services at Sartorius in New York City, NY; mandar.dixit@sartorius.com.

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Reflections on the Evolution of Biopharmaceutical Manufacturing - BioProcess Insider

Introducing the d-xylose isomerase pathway into muconate-producing P. putida

Three xylose metabolic pathways were considered to enable the production of muconate from this substrate36, including the isomerase pathway in which xylose is metabolized to xylulose-5-P (X5P) in the pentose phosphate pathway (PPP)38, the Weimberg pathway that feeds xylose to the TCA cycle via -ketoglutarate38,39, and the Dahms pathway40, which shares the initial three steps with the Weimberg pathway, after which -ketoglutaric semialdehyde is converted by an aldolase into pyruvate and glycolaldehyde. Among these, the d-xylose isomerase pathway, in which xylose is metabolized via the d-xylose isomerase (xylA) and xylulokinase (xylB) to xylulose-5-phosphate (X5P), is ideal for achieving a high theoretical muconate yield since X5P can be further converted to E4P and subsequently enter the shikimate pathway (Fig.1a)35. We integrated the isomerase pathway into a strain previously engineered to produce muconate from glucose, CJ5223, by overexpressing codon-optimized versions of the E. coli d-xylose isomerase (xylA), xylulokinase (xylB), and d-xylose:H+ symporter (xylE), together with a transaldolase (tal) and a transketolase (tkt) to improve carbon flux within the PPP (Fig.1a)35. We also deleted hexR, which encodes a transcriptional regulator that controls expression of genes important for sugar metabolism, since we had previously found this to improve the conversion of glucose to muconate32.

Thompson et al. previously reported that employing both the asbF and aroE pathways can help to maximize net precursor assimilation and metabolite flux toward muconate25. Thus, an engineered chorismate pyruvate-lyase (ubiC-C22)41 with relieved product inhibition was integrated to enhance muconate production through the shikimate pathway via aroE (Fig.1a). We had previously deleted pgi-1 and pgi-2, which encode redundant glucose-6-P isomerases, to disrupt the EDEMP cycle, a combination of the Entner-Doudoroff, gluconeogenic Embden-Meyerhoff-Pernass, and the pentose phosphate pathways42. The purpose of disrupting the EDEMP cycle is to prevent it from cycling to generate pyruvate independent of PEP during growth on glucose, which could enable the cell to redirect carbon toward growth at the expense of muconate production, despite deletion of the genes encoding the pyruvate kinases (pykA, pykF) and PEP carboxylase (ppc)3. This strategy is beneficial for muconate production from glucose as the sole carbon source, but in this case, deletion of pgi-1 and pgi-2 would decrease the maximum theoretical muconate yield of both asbF- and aroE-catalyzed muconate biosynthesis pathways when xylose is converted via the PPP (Fig.1b).

Considering that the xylose fraction in the mixture of glucose and xylose (xylose/glucose+xylose%, moles) in corn stover hydrolysate ranges from 34 to 38% (Supplementary Fig.1), the modeling predicted maximum theoretical yield of muconate with pgi-1 and pgi-2 deleted to be lower than if one or both are present (Fig.1b). To test the hypothesis that glucose-6-phosphate isomerase (encoded by pgi-1 and pgi-2) activity is necessary for xylose flux to enter the EDEMP cycle, we built strains based on JE322635, a P. putida KT2440-dervied strain that was previously engineered to utilize xylose using the d-xylose isomerase pathway but is otherwise wild-type, generating strains LC041 (pgi-1), LC345 (pgi-2), LC347 (pgi-1 pgi-2). In plate reader cultivation on M9 medium containing xylose, LC347 failed to grow, whereas both LC041 and LC345 demonstrated reduced growth rates and increased growth lags (Supplementary Fig.2). LC345, with pgi-1 intact, exhibited a lower growth rate and longer growth lag compared to LC041, which contains only pgi-2, suggesting that Pgi-1 contributes less to the overall glucose-6-phosphate isomerase activity than Pgi-2. Since the EDEMP cycle would be expected to compete with muconate biosynthesis and reduce the muconate yield, we thus restored pgi-1 to enable xylose flux into the EDEMP cycle and improve the maximum theoretical yield, generating strain QP328 (Fig.1a and Table1).

Strain QP328 was cultivated in shake flasks on a mixture of glucose and xylose to examine their conversion to muconate. Although the xylose isomerase pathway has been shown to be efficient in wild-type P. putida35, the xylose utilization rate of QP328, however, was very low compared to that of glucose (Fig.1c). Since glucose and xylose can be utilized simultaneously in the P. putida KT2440 wild-type background upon introduction of the same xylose isomerase pathway35, we hypothesized that a bottleneck in xylose transport or metabolism was present in our muconate-producing strain.

To improve xylose utilization by QP328, we conducted ALE by serial passaging of the strain on M9 medium supplemented with 10mM xylose as a sole carbon and energy source. As the populations were passaged, higher OD600 values were achieved more rapidly. After 7 passages (~50 generations), all four lineages achieved turbidity in 24 days compared to 14 days at the beginning of the ALE, and the evolution was terminated. The evolved populations of the four lineages were plated onto an LB agar plate, and three isolates on each plate were chosen for shake flask pre-screening (12 isolates in total). In most cases, all triplicates from the same lineage exhibited similar growth and muconate production, so it was assumed that they likely represented the same genotype and only one from each lineage was saved. In lineage 1, however, one replicate performed differently, thus two isolates were saved (Supplementary Fig.3). To identify mutations that may contribute to improved xylose utilization, the genomes of all five isolates were sequenced. All five isolates had mutations in xylose transporter xylE (A62V, A62V and A455V, T34I, L214P, S205F, for isolates 1, 2, 3, 4, 5, respectively). Four of the isolates (1, 35) had mutations that likely inactivated aroG-D146N (frameshift +7bp, frameshift +2bp, M1N and L2H, frameshift 16 bp, for isolate 1, 3, 4, 5, respectively) (Fig.2a). The five isolates were then evaluated in shake flasks on glucose, xylose, and a mixture of glucose and xylose. As expected, strains with mutations in aroG-D146N grew better but produced less muconate. Isolate 2 achieved the highest muconate yield and the lowest biomass yield, and was designated QP478 (Supplementary Fig.4). QP478 demonstrated substantially improved growth on xylose compared to QP328 in a plate reader, in which the growth of QP328 was negligible while QP478 reached a OD600 of 0.5 in 72h (Fig.2b).

a Mutations identified in ALE by whole-genome sequencing of the five isolates (the Sankey diagram was built using SankeyMATIC online tool). b Growth curve of reverse engineered strains LC091 and LC100, with the unevolved strain QP328 and the evolved strain QP478 for comparison. A represents absolute growth rate, and all A presented here are the average values of three independent growth curves. c Shake-flask experiments of reverse engineered strains LC091 and LC100, comparing to QP478, on M9 medium supplemented with 30mM xylose. Yield of LC100 was compared to LC091 using two-tailed Student t test (P<0.0001). d Shake flask experiments comparing thereverse engineered strains LC091and LC100, and theevolved strain QP478 on M9 medium supplemented with 30mM glucose and 15mM xylose. % Molar yield was calculated as [mM muconate/mM (glucose+xylose)100], and% carbon yield was calculated as [mM muconate6/mM (glucose6+xylose5)100]. Error bars here represent the standard deviation of three biological replicates. Source data are provided as a Source Data file.

The mutations identified in QP478 are listed in Supplementary Data1. Of those, mutations we hypothesized might be related to the improved growth on xylose included: (1) two missense mutations in the xylose transporter gene, xylE, where alanine residues were replaced with valines, A62V and A455V; (2) a GA point mutation 10bp upstream of the 35 element of a putative promoter (Supplementary Fig.5) predicted by the BPROM 70 promoter prediction program43 upstream of PP_2569, which is annotated as a metabolite major facilitator superfamily (MFS) transporter in the Uniprot database; and (3) a 227.8 kB region of the genome from PP_5050 to PP_5242 that appeared to be duplicated (Fig.2a).

To understand the contribution of the mutations that led to improved growth on xylose during ALE, we created strains that individually restored the wild-type sequences into the evolved strain QP478. The A62V and A455V mutations were restored to wild type separately in xylE, generating LC093 and LC078, respectively. The GA mutation in the promoter region of PP_2569 was restored, generating LC061. In plate reader experiments, restoring either xylE-A455V or xylE-A62V led to decreased growth rate and increased growth lag of LC078 and LC093, respectively (Supplementary Fig.6ac). The restoration of the GA mutation in PPP_2569 led to slightly decreased growth rate (Supplementary Fig.6a, b). In shake flasks, only LC093 demonstrated significantly lower muconate yield compared to QP478 (Supplementary Fig.6f, g, j), but all three strains LC061, LC078, and LC093 exhibited slower growth and muconate production (Supplementary Fig.6gj), which is consistent with the results from the plate reader experiments (Supplementary Fig.6ac). The reduced muconate productivity, caused by decreased growth rates and/or increased growth lag, indicated that all these mutations contributed to improved cell growth on xylose of QP478 (Supplementary Fig.6).

We also performed the reverse experiment, engineering the ALE mutations into the parent strain QP328 to obtain a rationally engineered strain containing only mutations that contribute to improved production of muconate. We first reverse engineered the unevolved strain QP328 with the three point mutations. The A62V and A455V XylE mutations were introduced into the unevolved strain QP328, generating LC091. The GA mutation in PPP_2569 was engineered in QP328 and LC091, generating LC092 and LC100, respectively. Strains LC091, LC092, and LC100, together with QP328 and QP478, were evaluated in a plate reader containing M9 medium with 30mM xylose. Interestingly, introducing the two XylE mutations enabled cell growth on xylose in LC091 (Fig.2b), which exhibited a comparable growth rate but higher final biomass compared to QP478 on xylose alone and xylose and glucose mixture (Fig.2b). Introducing the GA mutation in PPP_2569 to QP328 also enabled cell growth of LC092 on xylose, while at a much lower rate compared to LC091 (Fig.2b). Unexpectedly, introducing the GA mutation in PPP_2569 to LC091 led to decreased growth and lower biomass of LC100 on both xylose and mixed substrates (Fig.2b).

We next evaluated LC091, LC100, and QP478 in shake flasks containing M9 medium with 30mM xylose. LC091 reached almost twice the biomass yield (OD600) but achieved lower muconate yield compared to QP478 (Fig.2c). Moreover, LC100 reached a comparable muconate yield to QP478, albeit at a lower rate (Fig.2c). RT-qPCR indicated that the GA mutation in PPP_2569 increased the expression of PP_2569 (Supplementary Fig.7). We also evaluated LC091, LC100, and QP478 on M9 medium with a mixture of glucose and xylose. Consistent with the results on xylose only, the muconate yield of LC091 is lower than LC100 and QP478 on the mixture; LC100 still exhibited much slower growth than QP478 on the mixture, though it utilized glucose and xylose simultaneously and reached comparable muconate yield (Fig.2d). The difference in these strains suggested that a gene or genes in the PP_5050PP_5242 duplicated region might be important for the improved performance of QP478.

The only genetic difference between strains LC091 and LC100 is the GA mutation in the promoter region that led to higher expression of a putative MFS transporter PP_2569 (Supplementary Fig.5). To rationalize how the mutation could occur in ALE, and to examine how metabolism could be affected by PP_2569, intracellular and extracellular metabolomics experiments were conducted with LC091 and LC100 grown on xylose. Selected metabolites from early log phase, mid-log phase, and late log phase were analyzed (Supplementary Fig.8), and Z-scores were plotted as Fig.3a. Generally, we observed that LC091 accumulated more metabolites in both the EDEMP pathway and the TCA cycle, both intracellularly and extracellularly, while LC100 demonstrated a greater accumulation of shikimate pathway-related metabolites (Fig.3a). There were three prominent exceptions of shikimate pathway-related compounds that were more abundant in LC091, namely l-tyrosine, l-phenylalanine, and l-tryptophan, which are all chorismate-derived aromatic amino acids (Fig.3bd). In Pseudomonas aeruginosa, it was previously reported that l-tyrosine and l-tryptophan could strongly inhibit the native DAHP synthases AroF-1 and AroF-244. Thus, we posit that AroF-1 and AroF-2 could be less inhibited in LC100 compared to LC091. Although a feedback-resistant DAHP synthase aroGD146N has been overexpressed in our strains, previous studies showed that the native DAHP synthases AroF-1 and AroF-2 alone led to ~30% PCA and phenol production compared to additional overexpression of aroGD146N, in P. putida45 and Pseudomonas taiwanensis46, respectively. We reasoned that the improved muconate production of LC100 compared to LC091 might be because of lower inhibition of AroF-1 and AroF-2 (Fig.3be), and the reduced PEP concentration and increased accumulation of DAHP in LC100 support this interpretation (Fig.3a).

a Heatmap of the selected intracellular and extracellular metabolites. Z-scores were calculated using the average intensities of intracellular and extracellular metabolites separately, and a time zero control from uninoculated medium was also included for extracellular metabolite analysis. be Intensities of selected metabolites from late log phase, In represents intracellular, Ex represents extracellular, and N.D. represents not detected. The intracellular intensity signal was collected using the cell pellet from a 1mL cell culture, and the extracellular intensity signal was collected using 20L of the corresponding supernatant. f Structure of XylE (PDB ID: 4GBY)48 with residues of ALE-derived mutation locations labeled in green. d-xylose is shown in black ball-and-sticks. g Growth curve of strain LC111 compared to JE3692 on M9 medium supplemented with 30mM xylose. Source data are provided as a Source Data file.

Moreover, a further explanation could be that PP_2569 is able to transport the aromatic amino acids extracellularlyhowever, we did not observe the extracellular accumulation of these amino acids (Fig.3bd). Instead, we found substantial extracellular accumulation of anthranilic acid in LC100 (Fig.3e). Anthranilic acid is a precursor of l-tryptophan and direct product of chorismate, which is an important node in the shikimate pathway from which all of the aromatic amino acids are derived. Chorismate was reported to be unstable intracellularly47 and was not detected in our intracellular metabolomics samples. Based on the current results, we posited that PP_2569 might be able to export anthranilic acid, which could lead to decreased l-tryptophan intracellular accumulation (Fig.3d, e). The increased accumulation of anthranilic acid may also reduce the flux from chorismate toward other aromatic amino acids, leading to the reduced accumulation of l-tyrosine and l-phenylalanine in strain LC100. Further work is warranted to investigate the mechanistic basis of this beneficial ALE-derived mutation.

Separately, to investigate the mechanism of mutations in XylE, five mutations from isolates 15 (A62V, A62V, and A455V, T34I, L214P, S205F, for isolates 1, 2, 3, 4, 5, respectively) were labeled in the structure (PDB ID: 4GBY)48, highlighting that the mutations were all located in the transmembrane domains (Fig.3f). Previously, Jiang et al. demonstrated that introducing two mutations (G58W and L315W) in XylE could prevent the binding of two inhibitors through conformational changes49. Notably, the G58W site is in the same transmembrane domain to A62V and close to T34I in the structure. Since the non-muconate-producing strain LC345 with wild-type XylE and similar genetic background to QP328 grew well on xylose (Supplementary Fig.2a), we hypothesized that the mutations in XylE that occurred in ALE might be induced by inhibitor(s) from the muconate-producing background strain. We also introduced the mutations xylE-A62V and A455V to strain JE3692, previously reported to grow on lignocellulosic hydrolysates, generating LC111. The two strains were evaluated in a plate reader on xylose. LC111 demonstrated improved growth with a reduced lag time and higher growth rate compared to JE3692 (Fig.3g). The slight improvement may be caused by the trace amounts of inhibitor(s) from the native pathways, and this may also suggest that introducing xylE-A62V, A455V can improve xylose utilization for the production of other non-shikimate pathway-related products.

The 227.8 kB duplication was identified based on approximately twofold higher sequencing coverage from PP_5050 to PP_5242 compared to the rest of the genome (Fig.4a). However, it was challenging to identify the exact location of the duplicated region based on the sequencing data due to the short read length of Illumina sequencing50,51. We thus deleted the original region of PP_5050PP_5242 in QP478, using the known sequences outside the region as homologous arms, to generate LC171, and deleted a portion of the duplication from PP_5084PP_5242 to generate strain LC173. On 30mM xylose in M9 medium, LC171 with the whole region deletion exhibited a lower growth rate, while LC173 with partial deletion showed comparable growth with a slightly longer growth lag and increased growth rate relative to QP478 (Fig.4b). Based on these results, we concluded that the duplication is important to the performance of QP478, and the potential beneficial gene(s) should be in the region PP_5050PP_5083, which remains intact in LC173. Thus, we next sought to identify the gene(s) in this region that contributed to the improved growth on xylose.

a Identification of the duplicated region from next-generation sequencing data, which presented ~2 the number of sequencing reads in QP478, and complete and partial deletions of these duplicated regions in LC171 and LC173, respectively. The graphs of coverage were generated in Geneious Prime 2020.0.4. b Growth curves of QP478, LC171, and LC173 on M9 medium with 30mM xylose. represents growth lag, A represents absolute growth rate, andboth are the average values of three independent growth curves. c Overexpressing candidate genes in reverse engineered strain LC100 at the pykF site. d Growth curves of QP478, LC100, LC199 and LC224 on M9 medium with 30mM xylose. e Maximum specific growth rates extracted from panel d. f Growth lag values extracted from panel d. gi Profiles in shake-flask experiments of strain LC224 on M9 medium with 30mM xylose, 30mM glucose+15mM xylose, and30mM glucose, respectively. For shake flask experiments, % molar yield was calculated as [mM muconate/mM (glucose+xylose)100], and% carbon yield was calculated as [mM muconate6/mM (glucose6+xylose5)100]. Error bars here represent the standard deviation of three biological replicates. Source data are provided as a Source Data file.

Since glucose and xylose were both utilized at similarly low rates in LC100 (Fig.2d), we reasoned that the slow growth might manifest in part(s) of the pathway shared by both sugars. Three candidate genes within PP_5050PP_5083 were selected for overexpression in LC100, including one related with sugar metabolism, PP_5056 (gpmI, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase), and two in the shikimate pathway, PP_5078 (aroB, 3-dehydroquinate synthase) and PP_5079 (aroK, shikimate kinase) (Fig.4c). Two other genes outside this region but related with sugar metabolism were also tested, including PP_5085 (maeB, malic enzyme B) and PP_5150 (rpiA, ribose-5-phosphate isomerase A). Overexpression cassettes of the five genes were then inserted individually at the pykF site, generating strains LC147 (gpmI), LC150 (maeB), LC151 (rpiA), LC199 (aroK), and LC224 (aroB). All of these genes were driven by the Ptac promoter except for gpmI, which was driven by Plac promoter after two unsuccessful attempts to insert the gene into the genome using Ptac. The resulting strains were then evaluated with LC100 and QP478 in a plate reader containing M9 medium and 30mM xylose (Fig.4df). Overexpression of the three genes related to sugar metabolism in LC100 did not reduce the growth lag, while strains LC150 and LC151 demonstrated higher growth rates and greater final biomass accumulation (Supplementary Fig.9). Considering that higher biomass yield may reduce muconate yield, as we observed with strain LC091 (Fig.2bd), we decided to not pursue these targets further. Strains LC199 and LC224, which overexpress aroK and aroB, respectively, both demonstrated improved growth rates and reduced growth lag compared to LC100 (Fig.4df). LC224 grew even faster than QP478 with a similar lag time and higher growth rate (Fig.4df).

To investigate the potential additive effect of overexpressing aroK and aroB, we also expressed aroK and aroB in an operon-like pattern as aroKB in LC100, generating strain LC168.Strains LC199, LC224, LC168, and QP478 were evaluated in shake-flask experiments with M9 medium containing glucose and xylose to examine muconate production. The aroB overexpression strain LC224 outperformed its evolved counterpart QP478 with a higher muconate yield and improved growth rate (Supplementary Fig.10a, c), suggesting that the reaction of DAHP to 3-dehydroquinate (3DHQ) was rate limiting in LC100. Overexpressing aroK in LC100 (generating LC199) increased the growth rate slightly (Supplementary Fig.10b, d). Strain LC168did not exhibit improvement compared to LC224 (Supplementary Fig.10c, e).

To investigate if aroB overexpression alone can lead to better strain performance, we overexpressed aroB in QP328, generating strain LC349. In the plate reader evaluation of strains LC349, QP328 and LC224, LC349 exhibited highest growth rate on glucose, and slightly lower growth rate on mixture of glucose and xylose compared to LC224, while not surprisingly much slower growth on xylose relative to LC224 (Supplementary Fig.11ac), probably due to the lack of mutations in xylE. Interestingly, in shake flasks experiment on mixture of glucose and xylose, LC349 outperformed QP328 with a much higher muconate yield, which was still significantly lower than LC224 (Supplementary Fig.11df). The muconate production of LC349 is slower than LC224, as it took up to 41h for LC349 and 26.5h for LC224 to reach maximum muconate titer(Supplementary Fig. 11e, f).

We next examined the performance of LC224 on M9 medium containing glucose, xylose, or a mixture of the two. Muconate yields were highest on xylose, lowest on glucose, and intermediate on the mixture (Fig.4gi), reflecting the benefit of introducing xylose into the pentose phosphate pathway to supply E4P. Interestingly, both glucose and xylose utilization rates were higher on the mixture of glucose and xylose compared to on glucose or xylose alone (Fig.4gi).

Bioreactor cultivations of LC224 and QP478 were conducted in fed-batch mode to maintain sugar (glucose and xylose) concentrations lower than 10gL1 (Supplementary Fig.12ac). Glucose and xylose were simultaneously utilized in both strains from the start of the cultivation (Fig.5a, b); however, sugar utilization rates were higher in LC224 than QP478. LC224 utilized 46gL1 glucose and 20gL1 of xylose by the end of the cultivation while QP478 utilized 34gL1 and 10gL1, respectively. The muconate titer was almost threefold higher in LC224 compared to QP478, a 26.8gL1 and 9.3gL1, respectively (Fig.5a, b). Muconate yields reached 50% (molar yield) in LC224 while the yields were 25.9% in QP478 (Fig.5a, b). These improvements in LC224 were also reflected in the overall muconate production rate (0.28gL1h1), which was substantially higher than that achieved in QP478 (0.10gL1h1) (Fig.5a, b).

a, b Bacterial growth, glucose and xylose utilization, and muconate titers, yields, and rates from QP478 and LC224 in 96.6-h cultivations. c Bacterial growth, glucose and xylose utilization, and muconate titer, yields, and rate from LC224 in 191-h cultivation. For a and b, data points represent the average values of biological duplicates, error bars represent the absolute difference between the data generated from duplicates at each time point; for (c), data points represent the values of singlets. Metabolic yields (mol%) at each time point were calculated as the amount of muconate (in moles) produced divided by the glucose and xylose (in moles) utilized. Metabolic yields (mol%) are corrected based on the dilution factor generated by the volumes of base and substratefeeding. Final carbon yields (carbon%) listed were calculated as [mM muconate6/mM (glucose6+xylose5)100]. Rates (gL1h1) at each time point were calculated as the muconate concentration divided by the cultivation time. All the titer (T), rate (R), and yield (Y) valueslisted were at the last time point. Final yields (mol%, carbon%) listed in (c) have alsobeen corrected based on the quantified evaporated volume. Source data are provided as a Source Data file.

The muconate titer, rate, and yield achieved in bioreactor cultivations were 26.8gL1, 0.28gL1h1, and 49.9% (Fig.5b), respectively, at 96.6h. This yield represents almost 100% of the maximum theoretical based on our strain design and metabolic modeling (vida supra). To explore whether the titer could be further improved, we conducted another bioreactor experiment where LC224 was cultivated for 191h (Fig.5c). The resulting muconate titer increased to 33.7gL1 and at a yield of 46%, 92% of the theoretical maximum when corrected for evaporation. It is also noteworthy that while LC224 reached stationary phase at ~54h, the cells continued utilizing sugars and producing muconate, which demonstrated that the muconate production here was not growth coupled, suggesting thatthe muconate titer and yieldcould be further improved if the experiment had continued (Fig.5b, c).

To better understand the differences between the unevolved parent QP328, the evolved strain QP478, and the rationally engineered strain LC224, intracellular and extracellular metabolomics experiments were conducted. Selected metabolites related to sugar metabolism and muconate production are presented in Fig.6. Compared to QP328, the strains QP478 and LC224 exhibited reduced accumulation of metabolites in the EDEMP cycle, and greater accumulation of metabolites in the shikimate and the muconate pathways (Fig.6a, b), which is consistent with the mutations that evolved in QP478 and were engineered in LC224 (Figs.2 and 4). The intensities of DAHP, the joint node of sugar metabolism and shikimate pathway, however, demonstrated theopposite pattern compared to other metabolites in shikimate pathway (Fig.6b). LC224 and QP478 accumulated less DAHP compared to QP328, which is consistent to our objective above regarding the duplication and aroB overexpression.

a Simplified metabolic pathway. Metabolites in the EDEMP cycle arelabeled blue, in the shikimate and muconate pathway are labeled green, the joint node DAHP is labeled purple, the extracellular anthranilic acid (ANA) is labeled brown. QA quinic acid, ANA anthranilic acid. b Intensity of selected metabolites in the three strains QP328 (blue), QP478 (orange), and LC224 (red). Intracellular intensities have been normalized by lyophilized biomass. Error bars represent the standard deviation of three biological replicates. Source data are provided as a Source Data file.

Specifically, the DAHP level in LC224 was much lower compared to QP478, which may suggest the aroB activity in LC224 driven by tac promoter was higher than that of QP478. Except for DAHP, LC224 accumulated a higher amount of metabolites in the shikimate pathway and fewer metabolites in the EDEMP cycle relative to QP478 (Fig.6a, b), suggesting greater flux entering the shikimate pathway in LC224 and enabling greater muconate biosynthesis.

Although its precursor 3DHQ was not detected in any samples, quinic acid (quinate, QA) was substantially accumulated in LC224 (Fig.6b), which may suggest an overflow of carbon resulting from overexpression of aroB. Shikimic acid (shikimate, SA) accumulation in LC224 is evidence of muconate biosynthesis through the aroE pathway, while SA accumulation was much lower in QP478 relative to LC224, which may be related to the aroK duplication in QP478. The accumulation of anthranilic acid in the culture media likely represents another case of overflow metabolism. More catechol (CAT) accumulated in LC224 (Fig.6b), which could represent new bottlenecks associated with increased flux to muconate. Together, these results illustrate that engineering to generate LC224 broadly recapitulated the evolved strain QP478 and suggest additional targets for further improvement.

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Muconic acid production from glucose and xylose in Pseudomonas putida via evolution and metabolic engineering - Nature.com

For both of you, what creative itch does doing voice work for animation scratch for you that live-action doesn't?

Parnell: Well, it's incredibly freeing to just have to worry about what's happening with your voice, I find. I don't have to worry about blocking, or this, or that. I usually gesture quite a lot, like I am now, in the booth. It's a challenge, because you're not looking at anybody. You're looking at the lines, and you might have a back and forth with the director of the episode, so it feels more like you're doing a scene and you can connect that way. But I don't know, it's just fun. It takes a lot less time. You can do it a lot of different ways without taking up much time. I just find it, I guess, freeing in a way that on-camera stuff isn't, but obviously I love doing the on-camera, too, when it comes along.

Chalke: Yeah. I love the experience of how different they both are, too. In terms of a creative itch, I completely agree with Chris. It's so creatively freeing, because you have your script, and you're going through it one line at a time, so there's the added challenge, that's kind of unique and interesting, of not getting to interact with someone else. But you can take a look at it and say, "Okay, I want to try this. I want to try that." The director throws you some ideas. You can take one line, and you can try it literally 20 different ways in two minutes. So there's really no constraints. Normally, you're recording in a studio with a big glass wall, and you can see whoever's directing you, whether in the early days when it was Dan [Harmon] and Justin [Roiland], or now it's Scott [Marder], and you can have that back and forth. Now you're in a vocal booth, and so I miss that piece of it. At the same time, there's something that's even more freeing about the fact that you're just because I'm like Chris, I gesticulate a lot. You're just completely in your own little world.

Parnell: It also doesn't matter what you look like. You can play a character, even though Jerry doesn't look that crazily different from me, but that's another freeing aspect of animation.

Jerry and Beth have experienced some wild evolution over the series, especially with the multiverse aspect being brought into the equation. How exciting and refreshing is it to get to play these different versions of the characters? Sarah, you've got Space Beth, and Chris, Jerry has a very interesting version of himself in the season 6 premiere.

Parnell: Yeah, whenever Jerry gets to go to a darker place, or more emotional place, it's always gratifying and fun to be able to go there and try to do justice to the material. Then when they went to the therapy planet and got to see Jerry as a worm as Beth envisioned him, it's just fun. It's great. Sometimes it's a little more challenging, but it's very satisfying to be able to play so many different aspects of the character.

Chalke: For me, with Space Beth, I was so pumped that it wasn't just a one-off for one episode that we saw Space Beth, that it was a situation that got to recur, and it's a very different experience recording Space Beth. Obviously, they're so different, and so in an episode, we'll record all of Home Beth, and then we'll do all of Space Beth right afterwards, so that they're completely separate. I think there's such a different swagger and confidence to Space Beth, and a different texture to her voice. So it's been a fun challenge to try and play two different characters that are also linked and yet one and the same.

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Rick And Morty's Chris Parnell And Sarah Chalke On The Evolution Of Jerry And Beth, Voice Acting Challenges & More [Interview] - /Film

Country

United States of AmericaUS Virgin IslandsUnited States Minor Outlying IslandsCanadaMexico, United Mexican StatesBahamas, Commonwealth of theCuba, Republic ofDominican RepublicHaiti, Republic ofJamaicaAfghanistanAlbania, People's Socialist Republic ofAlgeria, People's Democratic Republic ofAmerican SamoaAndorra, Principality ofAngola, Republic ofAnguillaAntarctica (the territory South of 60 deg S)Antigua and BarbudaArgentina, Argentine RepublicArmeniaArubaAustralia, Commonwealth ofAustria, Republic ofAzerbaijan, Republic ofBahrain, Kingdom ofBangladesh, People's Republic ofBarbadosBelarusBelgium, Kingdom ofBelizeBenin, People's Republic ofBermudaBhutan, Kingdom ofBolivia, Republic ofBosnia and HerzegovinaBotswana, Republic ofBouvet Island (Bouvetoya)Brazil, Federative Republic ofBritish Indian Ocean Territory (Chagos Archipelago)British Virgin IslandsBrunei DarussalamBulgaria, People's Republic ofBurkina FasoBurundi, Republic ofCambodia, Kingdom ofCameroon, United Republic ofCape Verde, Republic ofCayman IslandsCentral African RepublicChad, Republic ofChile, Republic ofChina, People's Republic ofChristmas IslandCocos (Keeling) IslandsColombia, Republic ofComoros, Union of theCongo, Democratic Republic ofCongo, People's Republic ofCook IslandsCosta Rica, Republic ofCote D'Ivoire, Ivory Coast, Republic of theCyprus, Republic ofCzech RepublicDenmark, Kingdom ofDjibouti, Republic ofDominica, Commonwealth ofEcuador, Republic ofEgypt, Arab Republic ofEl Salvador, Republic ofEquatorial Guinea, Republic ofEritreaEstoniaEthiopiaFaeroe IslandsFalkland Islands (Malvinas)Fiji, Republic of the Fiji IslandsFinland, Republic ofFrance, French RepublicFrench GuianaFrench PolynesiaFrench Southern TerritoriesGabon, Gabonese RepublicGambia, Republic of theGeorgiaGermanyGhana, Republic ofGibraltarGreece, Hellenic RepublicGreenlandGrenadaGuadaloupeGuamGuatemala, Republic ofGuinea, RevolutionaryPeople's Rep'c ofGuinea-Bissau, Republic ofGuyana, Republic ofHeard and McDonald IslandsHoly See (Vatican City State)Honduras, Republic ofHong Kong, Special Administrative Region of ChinaHrvatska (Croatia)Hungary, Hungarian People's RepublicIceland, Republic ofIndia, Republic ofIndonesia, Republic ofIran, Islamic Republic ofIraq, Republic ofIrelandIsrael, State ofItaly, Italian RepublicJapanJordan, Hashemite Kingdom ofKazakhstan, Republic ofKenya, Republic ofKiribati, Republic ofKorea, Democratic People's Republic ofKorea, Republic ofKuwait, State ofKyrgyz RepublicLao People's Democratic RepublicLatviaLebanon, Lebanese RepublicLesotho, Kingdom ofLiberia, Republic ofLibyan Arab JamahiriyaLiechtenstein, Principality ofLithuaniaLuxembourg, Grand Duchy ofMacao, Special Administrative Region of ChinaMacedonia, the former Yugoslav Republic ofMadagascar, Republic ofMalawi, Republic ofMalaysiaMaldives, Republic ofMali, Republic ofMalta, Republic ofMarshall IslandsMartiniqueMauritania, Islamic Republic ofMauritiusMayotteMicronesia, Federated States ofMoldova, Republic ofMonaco, Principality ofMongolia, Mongolian People's RepublicMontserratMorocco, Kingdom ofMozambique, People's Republic ofMyanmarNamibiaNauru, Republic ofNepal, Kingdom ofNetherlands AntillesNetherlands, Kingdom of theNew CaledoniaNew ZealandNicaragua, Republic ofNiger, Republic of theNigeria, Federal Republic ofNiue, Republic ofNorfolk IslandNorthern Mariana IslandsNorway, Kingdom ofOman, Sultanate ofPakistan, Islamic Republic ofPalauPalestinian Territory, OccupiedPanama, Republic ofPapua New GuineaParaguay, Republic ofPeru, Republic ofPhilippines, Republic of thePitcairn IslandPoland, Polish People's RepublicPortugal, Portuguese RepublicPuerto RicoQatar, State ofReunionRomania, Socialist Republic ofRussian FederationRwanda, Rwandese RepublicSamoa, Independent State ofSan Marino, Republic ofSao Tome and Principe, Democratic Republic ofSaudi Arabia, Kingdom ofSenegal, Republic ofSerbia and MontenegroSeychelles, Republic ofSierra Leone, Republic ofSingapore, Republic ofSlovakia (Slovak Republic)SloveniaSolomon IslandsSomalia, Somali RepublicSouth Africa, Republic ofSouth Georgia and the South Sandwich IslandsSpain, Spanish StateSri Lanka, Democratic Socialist Republic ofSt. HelenaSt. Kitts and NevisSt. LuciaSt. Pierre and MiquelonSt. Vincent and the GrenadinesSudan, Democratic Republic of theSuriname, Republic ofSvalbard & Jan Mayen IslandsSwaziland, Kingdom ofSweden, Kingdom ofSwitzerland, Swiss ConfederationSyrian Arab RepublicTaiwan, Province of ChinaTajikistanTanzania, United Republic ofThailand, Kingdom ofTimor-Leste, Democratic Republic ofTogo, Togolese RepublicTokelau (Tokelau Islands)Tonga, Kingdom ofTrinidad and Tobago, Republic ofTunisia, Republic ofTurkey, Republic ofTurkmenistanTurks and Caicos IslandsTuvaluUganda, Republic ofUkraineUnited Arab EmiratesUnited Kingdom of Great Britain & N. IrelandUruguay, Eastern Republic ofUzbekistanVanuatuVenezuela, Bolivarian Republic ofViet Nam, Socialist Republic ofWallis and Futuna IslandsWestern SaharaYemenZambia, Republic ofZimbabwe

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Memories reflect on the evolution of 2nd & Walnut St. since 1930 - Richland Source

Heigh-Ho, My Merry Rainbow Lovelies! Bobby Patrick, your RAINBOW Reviewer here. Putting the silent T in cabareT to bring you ALL the Tea!

Porter Carroll, Jr. returned to his NYC home base - 54 Below to bring us all THE EVOLUTION OF CABARET Wednesday night. Carrol, the founder of Atlantic Starr, a noted R&B/Pop hit producing company for three solid decades, now tours with Hall & Oats as their percussionist and backup vocalist. In his spare time, when he's not on stage with the number one selling duo in music history, he likes to get his solo on in clubs around the world. Porter's innovative shows, with his original takes on R&B, Rock & Roll, Jazz, funk, blues, and a few dashes of the American Song Book, have sold out everywhere he goes because that voice of his is as smashing as his movie star looks. Whether he is improv'ing jazz vocalese or bringing new blood to PURPLE RAIN - a particular highlight of the night - Porter Carroll, Jr. is a singer's singer and he proved it on Wednesday. Enjoy this PhotoPhlash from Little Bobby and keep a weather eye out for this man's next gig because he definitely gets...

5 Out Of 5 Rainbows.

Featuring a band that included Andy Abel (guitar), David Livolsi (bass), Danny Obadia (keyboard), and Joel Rosenblatt (drums).

Keep up with what's next at 54 Below HERE.

Keep Up With All Things Porter Carroll, Jr. On His WebbySite: HERE

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PHOTOS: Porter Carroll, Jr. Offers up Musical Missing Links In THE EVOLUTION OF CABARET At 54 Below - Broadway World

NFTs have suffered a severe non-fungible blow, yet as an asset class, they are still operating in record numbers, according to billionaire investor Mark Cuban, one of the many investors behind OpenSea, the leading NFT marketplace.

As cryptocurrency-rich speculators spent billions of dollars on these assets, driving up prices and profits, the market increased magnificently last year. Currently, six months into 2022, its looking a bit ugly.

OpenSea, the largest NFT marketplace, had a sharp decline in monthly sales value to $700 million in June from $2.6 billion in May, a far cry from the record of around $5 billion in January.

According to NonFungible.com, which records sales on the Ethereum and Ronin blockchains, the average NFT sale dropped to $412 by late June from $1754 at the end of April.

With central banks raising rates to fight inflation, the NFT market, along with cryptocurrencies, has fallen as consumers risk appetite waned.

However, analysts are hotly debating a new NFT project that they think might survive the market meltdown due to some properties that make it truly exciting.

Re-Evolution

Re-Evolution, an NFT project, consists of 5,000 planets that each have a unique set of qualities, resources, and rarities. Re-Evolution NFT includes access to the Unity Game Engine-based transformative play-to-earn.

Players can engage with their planets in the Re-Evolution game to create their civilizations and cultures. Players make decisions as the Supreme Rulers of their societies, impacting the level of happiness of their civilizations. Players will get extra $REV tokens if the civilization has a high satisfactory level.

Re-Evolution is not only an NFT project but also an initiative with a cause. The project will donate a portion of its sales to organizations that the community chooses. Re-Evolution establishes a legitimate DAO community of which holders can be a part, together with the governance token.

Learn more about Re-Evolution by visiting their website at re-evolution.io.

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Re-Evolution: NFTs to Stockpile During the Crypto Market Crash - The Ritz Herald