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By Pooja Toshniwal Paharia Sep 22 2023 Reviewed by Danielle Ellis, B.Sc.

In a recent study published in Nature Communications, researchers proposed a simplified approach for analyzing messenger ribonucleic acid (mRNA) vaccines using long-read sequencing. Study: mRNA vaccine quality analysis using RNA sequencing. Image Credit: Jo Panuwat D/Shutterstock.com Background

Messenger RNA vaccines demonstrated safety and efficacy during the COVID-19 pandemic, but extensive quality and purity testing is required to verify their efficacy and safety. Manufacturing advances have enabled billions of doses to be manufactured with acceptable quality and safety.

Various approaches are now utilized to assess mRNA vaccines; however, the efficacy of novel therapies depends on speedy and safe manufacture. Rigorous analytics are required at each stage of the production process to detect impurities and assure the safety of mRNA vaccines. About the study

In the present study, researchers investigated the VAX-seq method for quality analysis of messenger RNA vaccines.

The researchers developed VAX-seq, a simplified procedure for analyzing mRNA vaccines and therapeutics using long-read sequencing. This procedure compares VAX-seq to industry standards, including chromatography, capillary and agarose electrophoresis, and immunoblotting. The researchers employed a variety of methodologies, including Illumina plasmid DNA sequencing, ONT cDNA-PCR sequencing, and Oxford Nanopore direct ribonucleic acid sequencing.

Key messenger RNA quality features assessed by VAX-seq were sequence similarity, integrity, 3'-poly(A) nucleotide tail dimension, and RNA and DNA contamination. To assist VAX-seq, a software toolbox was created that provides thorough and automated reporting on mRNA quality. An enhanced green fluorescent protein (eGFP) messenger RNA was created and generated as a reference to demonstrate the application and validity of the methodology,

The plasmid template was amplified in Escherichia coli, isolated, purified, and linearized as the initial stage in the preparation process. The linearized pDNA template was then employed as a template for synthetic mRNA transcription in vitro. To examine the isolated mRNA, the program was combined with complementary deoxyribonucleic acid (cDNA) sequencing. VAX-seq attached a reverse transcriptase primer to the 3' terminus of the poly(A) nucleotide tail, allowing the length of the tail to be measured.

The researchers used the tailfindr program to normalize deletion mistakes and the read-specific nucleotide translocation rate. As part of the VAX-seq process, the complementary DNA library preparation introduced two flanking-type adaptors to the messenger RNA's 5' and 3' ends.

To identify complete-length molecules of mRNA from truncated messenger RNA, sequencing reads contained both flanking adaptors. Off-target RNA contaminants were identified using VAX-seq, which was used to assess fragmented and off-target RNA contaminants in cDNA libraries. Results

The analysis revealed that VAX-seq, a technique for sequencing mRNA vaccines, can detect sequence, length, integrity, and purity. It also enabled the examination of linearized plasmid DNA templates and the detection of impurities from plasmid amplification. VAX-seq easily established the length and similarity of mRNA vaccine sequences. The eGFP mRNA size profile revealed a major peak (77%) that was within 5.0% of the predicted length [1,153 nucleotide (nt)-long], as well as a varied spectrum of smaller, fragmented mRNAs. Short-read sequencing provided insufficient and inconsistent coverage, while heterogeneous alignment coverage was highly repeatable across replicates. Related StoriesDoes IQ influence COVID-19 vaccination decision-making?Inactivated poliovirus vaccine elicits persistent immunity for up to 10 yearsModernas adapted COVID-19 vaccine that targets Omicron XBB.1.5 approved by MHRA

Most sequences were aligned with the on-target messenger RNA product, and only a few reads revealed Escherichia coli contamination. The remaining seven percent of ribonucleic acid species were off-target RNA molecules, with 0.3% presumably originating from initiation sites of cryptic transcription. Direct ribonucleic acid sequencing libraries produce lower yields than comparable complementary DNA sequencing genetic libraries and cannot be multiplexed at the moment.

The researchers did, however, identify biases particular to direct ribonucleic acid sequencing, such as inferior-quality poly(A) nucleotide tail deletion. Direct ribonucleic acid sequencing found changed nucleosides in messenger RNA vaccines, demonstrating that including modified nucleosides in mRNA vaccines might lessen the innate immunological response while improving stability and translation.

Modified nucleosides had minimal effect on messenger RNA quality features and complementary DNA sequencing errors between messenger RNAs, including native N1-methylpseudouridine and uridine, but direct ribonucleic acid sequencing had a larger error rate.

Complementary DNA and direct ribonucleic acid sequencing revealed that modified messenger RNA vaccines had more truncated-type transcripts, with 41% complete-length and 54% truncated messenger RNA molecules, especially those less than 500 nt in length. Direct ribonucleic acid sequencing discovered nucleosides of N1-methylpseudouridine with a distinctive base-calling mistake that miscategorized N1-methylpseudouridine into cytosines, skewing the messenger RNA length profile. Conclusions

Overall, the study findings showed that VAX-seq was a procedure based on sequencing long reads that assessed essential mRNA quality characteristics such as integrity, contamination, and sequence identity. This technique can potentially become important to developing and producing mRNA medicines, offering a thorough and integrated evaluation at various manufacturing stages. VAX-seq employed full-length complementary DNA sequencing using Nanopore chemistry, which allowed for accurate assessment of the poly(A) molecular tail length as well as various off-target readings.

The approach offered a sensitive and quantitative assessment of mRNA characteristics, making it a more efficient alternative to conventional analytical techniques. VAX-seq enabled real-time identification of antisense RNA and messenger RNA integrity, allowing for swift testing lasting a few hours post-manufacture.

It might also identify complicated off-target ribonucleic acid contaminants created during transcription in vitro, as well as the degradation or sharing of messenger RNA vaccines during manufacturing, storage, and transportation. VAX-seq needed only a small quantity of messenger RNA as input and may be integrated to allow for large-scale and low-cost validation of vaccination batches. Journal reference: Gunter, H.M., Idrisoglu, S., Singh, S. et al. mRNA vaccine quality analysis using RNA sequencing. Nat Commun 14, 5663 (2023). doi: https://doi.org/10.1038/s41467-023-41354-y https://www.nature.com/articles/s41467-023-41354-y

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Harvey Weinstein to be tried for third time in New York after mistrial on rape charge

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Harvey Weinstein to be tried for third time in New York after mistrial on rape charge

Disgraced US film producer Harvey Weinstein is to be tried for a third time in a sexual assault case.

A jury in New York could not reach a verdict in June against the 73-year-old who was accused of raping actress Jessica Mann, and a mistrial was declared.

Judge Curtis Farber has said he wants the new trial to happen before the end of this year.

The same jury found Weinstein guilty in June of sexually assaulting former Project Runway production assistant Miriam Haley in 2006 and not guilty of assaulting Polish former runway model Kaja Sokola the same year.

Weinstein will be sentenced for the guilty verdict in Ms Haley’s case on 30 September.

He denied all of the charges. Throughout the retrial, his lawyers insisted the sexual encounters with his three accusers were “transactional” and “consensual,” and labelled the women as opportunists.

Weinstein was originally convicted of rape and criminal sexual act by the same court in 2020 and sentenced to 23 years in prison for the crimes.

Last year, however, New York’s highest court overturned the conviction, prompting Weinstein’s retrial this summer.

Weinstein was once one of the most powerful people in Hollywood – the co-founder of film and television production companies Miramax and The Weinstein Company, who produced films such as the Oscar-winning Shakespeare In Love, Pulp Fiction, and The Crying Game.

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In 2017, a series of sexual misconduct allegations against him propelled the #MeToo movement.

Some of those accusations later led to criminal charges and his convictions in New York and California.

Before the retrial, Weinstein was also serving a 16-year prison sentence after being found guilty of rape in California in December 2022. He has also denied this charge.

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11 second-year players who could break out this season

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11 second-year players who could break out this season

Not every five-star recruit from the 2024 ESPN 300 was inserted into a starting role as a true freshman last fall, despite what their ranking might suggest. Numerous variables can take precedent over pure talent, and it often takes a year of seasoning or depth-chart movement before elite prospects break out in their second season.

Ohio State’s Jeremiah Smith and Alabama’s Ryan Williams became instant superstars as true freshmen in 2024, and we have a strong list of super sophomores ready to emerge in 2025, following the path of Notre Dame running back Jeremiyah Love, who made this list last year.

Note: These rankings omitted any player who started more than two games last season.

A heralded quarterback recruit in 2024, Sayin has the physical tools, poise and supporting cast in Columbus to be one of the top passers in the country. Yes, we understand he still has to win the job, but we are doubling down on the former five-star recruit. Sayin, who was the No. 2 QB behind DJ Lagway in the 2024 rankings, possesses outstanding arm talent, both in strength and accuracy, and he can move the chains with his legs. He is seasoned and polished, with arguably the best receiving corps in the country. We project he will win the job, opening the season with steady production and developing down the stretch into one of the best quarterbacks in the Big Ten.


We got a taste of what Marshall brings to the table at the ReliaQuest Bowl. The No. 7 back in the 2024 ESPN 300, he went for 100 yards on 23 carries in the first and only start of his young career. The former Ohio Mr. Football will team up with Alabama transfer Justice Haynes in the Wolverines backfield. Early in preseason camp, it appears the reps will be shared, with Haynes as 1a and Marshall 1b. Marshall is elusive in tight quarters, fast in the open field and powerful on contact with a low center of gravity. He boasted the top verified shuttle (5-10-5) out of high school with 4.1 seconds, which would have been second among running backs at this year’s NFL combine.


While many expected greater impact from Robinson as the No. 1-ranked player in the ESPN 300, Georgia’s defense had three players selected in the first round of the NFL draft. Even this season, Robinson is not projected to start on the outside, but he’ll see plenty of meaningful snaps at nickel and has the skills to take over the perimeter as a lockdown corner at any point. Word from Athens is that the light has come on. Robinson is seeing the game more clearly, allowing him to play faster. Reminder: Robinson possesses a rare blend of length, speed and ball skills. He just happens to be part of a defense where that’s the norm.


Miami fans may have expected more from Trader a season ago as the No. 6 receiver in the ESPN 300, but he was part of a very experienced and productive receiving room. He had only six receptions on the year but did start the Pop-Tarts Bowl and made three catches for 61 yards and a touchdown. That flash will become more consistent this year for the Canes. Trader is 6-foot-1 with smooth, fluid movements and quick hands to pluck the ball on the run for big gainers. He’s a legitimate three-level threat. Trader will team up with tight end Elija Lofton to give Miami two breakout stars on offense for Carson Beck to work with.


Yes, we are hedging our bet with this pick. We expect Matthews or Staley to break out this year as a top SEC receiver. Who that will be depends on who stays healthy, as both have been injury prone. They both have flashed as well. Matthews is as expected. Sudden and elusive after the catch, the No. 5 wide receiver in the ESPN 300 headlined the Vols’ No. 15 class. Staley was inside the top 300 but as the No. 21 receiver. A former state champion in the 200 meters and triple jump, Staley, who redshirted last season, has excellent short-area quickness, explosive movements and elite ball skills. This WR room needs to produce for the Vols to return to the College Football Playoff.


Lopa has one of the best blends of size and range of any back-end defender in the country, and the Ducks have production voids to fill at safety. The No. 13 safety in the 2024 ESPN 300 had limited reps last season, but in the Big Ten championship game against Penn State he was in third-down packages matching up with All-America tight end Tyler Warren. Lopa is 6-foot-5 and 210 pounds but covers ground fast with his long stride. He will come up inside the box and tackles soundly as well. In high school he played both sides of the ball, tallying four interceptions and 16 receiving touchdowns. Lopa will need to develop as the season progresses with his reads and recognition, but we project he will begin to reach his potential this fall.


There may not be a more highly scrutinized job in college sports than the starting quarterback at Notre Dame. Carr, the grandson of former Michigan coach Lloyd Carr, has been well prepared on and off the field and will be ready for the challenge. Carr is still locked in a battle with Kenny Minchey for the starting job coming off a strong spring practice, but we think he will be handed the keys before Week 1. Carr was ranked as the No. 2 pocket passer in the class of 2024 in part because of his great accuracy and acumen. With running backs Jeremiyah Love and Jadarian Price returning and an experienced offensive line, the Irish might not need Carr to break out with huge production. But he will need to anticipate, get the ball out and push it downfield to his targets. All of this is within his skill set, which is why he’s on this list.


Wisconsin didn’t dip into the portal to help replace Tawee Walker and Chez Mellusi’s production; it felt good about the underclassmen still in the running back room. So do we. Jones arrived in Madison with SEC offers and blue-chip skills. The No. 8 running back in the ESPN 300, Jones ran through his high school competition, and we project he’s ready to do the same in the Big Ten. He’s got an impressive size-to-speed ratio and good change-of-direction skills. Jones isn’t just a classic bruising back that Badgers fans are accustomed to. He can also hit the home run and make tackles miss in the open field. It’s a good RB unit and Jones won’t have to carry the load, which should keep him fresh and healthy as the Badgers look to bounce back from a disappointing 2024 season.


Expectations are sky high in Austin as Texas ranks No. 1 in the preseason AP Top 25 for the first time in program history. While the roster is loaded, there is turnover at key spots — and offensive tackle is one of them. Baker was a highly touted 2024 prospect (No. 2 OT in the ESPN 300) and was in a battle to start this season. Unfortunately for Texas, Andre Cojoe recently went down with a season-ending injury, which means Baker has more than likely won the starting spot. He has improved his strength this offseason and has worked hard at the technical points of the position. He will be tasked with protecting the most anticipated player in all of college football in Arch Manning.


The 11th-rated pocket passer in the ESPN 300, Brown saw limited action last year and was able to preserve a redshirt season. He is a winner above all his great physical traits. He’s the only quarterback from national power Mater Dei High School (Santa Ana, California) to win two state championships. He threw for more than 8,000 yards and 100 touchdowns, and now has the challenge of leading Stanford back to its storied levels. While young, he will have one of the better quarterback tutors in Frank Reich and a GM, Andrew Luck, who knows a thing or two about winning in Palo Alto. Brown has pro-style skills that fit well in Reich’s scheme. While experienced sixth-year transfer Ben Gulbranson was just named the starter in a close battle, we still expect Brown behind center early this season.


Boise State lost Heisman Trophy runner-up Ashton Jeanty to the NFL, but the Broncos have another under-the-radar recruit ready to emerge. Gaines was ranked the 45th running back out of high school and originally projected as a linebacker. He combines great downhill power and physicality between the tackles with 10.9 100-meter speed and polished receiving skills. He has gone from 6 feet, 195 pounds to close to 220, and early reports indicate he looks ultra-fast and explosive as he regains his form from an injury that kept him out most of last season and this spring. We got a glimpse of what he can do when he ran for 110 yards and added 44 yards on three receptions in his collegiate debut against Georgia Southern. Boise barely made the cut in the preseason AP Top 25 but Gaines could help them climb the poll.

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‘Game changer’: Kansas gets hefty $300M gift

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'Game changer': Kansas gets hefty 0M gift

The University of Kansas has received an unprecedented $300 million gift from donor David Booth, believed to be among the largest single gifts in the history of college athletics and the largest in school history.

Kansas plans to allocate $75 million of Booth’s gift toward launching the second phase of its ongoing transformation of David Booth Kansas Memorial Stadium and construction of the surrounding Gateway District, Kansas athletic director Travis Goff told ESPN.

Though school officials have not revealed a timetable for construction and completion of Phase 2, the funds will allow Kansas to move forward with renovating the east side of the stadium after the 2025 football season.

The remainder of Booth’s gift will establish an annual additional revenue stream for Kansas athletics, Goff said.

“I’d say it’s transformative and a game changer,” Goff told ESPN. “This gift makes an immediate impact on our top priority in a profound way, and it also provides us with an incredible revenue stream that gives us a chance to really invest in unique ways in the future of Kansas athletics.”

Kansas has already invested $450 million in the first phase of the Gateway District project, which included an overhaul of the southwest, west and north sides of the stadium and a major renovation of the Anderson Family Football Complex. Stadium construction got underway at the end of the 2023 football season and will be completed in time for the Jayhawks’ season opener later this month.

The second phase of the Gateway District project would also bring the development of a new hotel, outdoor event plaza, student housing, retail and restaurant spaces and parking located east of Kansas Memorial Stadium.

The total cost of Phase 2 — finishing the stadium and the mixed-use development — is estimated to be $360 million. Lawrence city commissioners voted Tuesday night to approve a package of financial and tax incentives worth around $94 million to support the project.

Kansas Memorial Stadium was named after Booth, a KU graduate and founder of global investment firm Dimensional Fund Advisors, in 2018. The Lawrence, Kansas, native previously provided a foundational gift of $50 million in 2017 to kick off renovations of Memorial Stadium, but the university didn’t move forward with renovating its more than 100-year-old stadium until Goff and chancellor Douglas Girod announced plans for the Gateway District in 2022.

“One of life’s greatest privileges is being able to give back to the people and places that gave so much to you,” Booth said in a statement. “KU and Lawrence are a big part of my story, and it means a lot to support the community that invested in me. Philanthropy, like investing, pays dividends over time. Each gift compounds, creating opportunities not just for today, but for years to come. This is really about the future we’re building.”

After playing their six home games in the Kansas City area during the 2024 season, the Jayhawks will open the season with their first home game inside the renovated Kansas Memorial Stadium on Aug. 23 against Fresno State.

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