Connect with us

Published

on

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

Continue Reading

Politics

House of Lords under fire for dropping rule that once caught out cricket legend and historian

Published

on

By

House of Lords under fire for dropping rule that once caught out cricket legend and historian

Campaigners have criticised a change to the rules around declarations of interest in the House of Lords as a “retrograde step” which will lead to a “significant loss of transparency”. 

Since 2000, peers have had to register a list of “non-financial interests” – which includes declaring unpaid but often important roles like being a director, trustee, or chair of a company, think tank or charity.

But that requirement was dropped in April despite staff concerns.

Tom Brake, director of Unlock Democracy, and a former Liberal Democrat MP, wants to see the decision reversed.

“It’s a retrograde step,” he said. “I think we’ve got a significant loss of transparency and accountability and that is bad news for the public.

“More than 25 years ago, the Committee on Standards in Public Life identified that there was a need for peers to register non-financial interests because that could influence their decisions. I’m confused as to what’s happened in the last 25 years that now means this requirement can be scrapped.

“This process seems to be all about making matters simpler for peers, rather than what the code of conduct is supposed to do, which is to boost the public’s confidence.”

Westminster Accounts: Search for your MP

MPs and peers alike have long faced scrutiny over their interests outside Westminster. File pic
Image:
MPs and peers alike have long faced scrutiny over their interests outside Westminster. File pic

Rules were too ‘burdensome’, say peers

The change was part of an overhaul of the code of conduct which aimed to “shorten and clarify” the rules for peers.

The House of Lords Conduct Committee argued that updating non-financial interests was “disproportionately burdensome” with “minor and inadvertent errors” causing “large numbers of complaints”.

As a result, the register of Lords interests shrunk in size from 432 pages to 275.

MPs have a different code of conduct, which requires them to declare any formal unpaid positions or other non-financial interests which may be an influence.

A source told Sky News there is real concern among some Lords’ staff about the implications of the change.

Non-financial interest declarations have previously highlighted cases where a peer’s involvement in a think tank or lobbying group overlapped with a paid role.

Please use Chrome browser for a more accessible video player

Protesters disrupt House of Lords

Cricket legend among peers to breach code

There are also examples where a peer’s non-financial interest declaration has prompted an investigation – revealing a financial interest which should have been declared instead.

In 2023, Lord Skidelsky was found to have breached the code after registering his role as chair of a charity’s trustees as a non-financial interest.

Lord Skidelsky. Pic: UK Parliament
Image:
Lord Skidelsky. Pic: UK Parliament

The Commissioner for Standards investigated after questions were raised about the charity, the Centre for Global Studies.

He concluded that the charity – which was funded by two Russian businessmen – only existed to support Lord Skidelsky’s work, and had paid his staff’s salaries for over 12 years.

In 2021, Lord Botham – the England cricket legend – was found to have breached the code after registering a non-financial interest as an unpaid company director.

The company’s accounts subsequently revealed he and his wife had benefitted from a director’s loan of nearly £200,000. It was considered a minor breach and he apologised.

Former cricketer Lord Botham. File pic: PA
Image:
Former cricketer Lord Botham. File pic: PA

‘Follow the money’

Lord Eric Pickles, the former chair of the anti-corruption watchdog, the Advisory Committee on Business Appointments, believes focusing on financial interests makes the register more transparent.

“My view is always to follow the money. Everything else on a register is camouflage,” he said.

“Restricting the register to financial reward will give peers little wriggle room. I know this is counterintuitive, but the less there is on the register, the more scrutiny there will be on the crucial things.”

Lord Eric Pickles
Image:
Lord Eric Pickles

‘I was shocked’

The SNP want the House of Lords to be scrapped, and has no peers of its own. Deputy Westminster leader Pete Wishart MP is deeply concerned by the changes.

“I was actually quite horrified and quite shocked,” he said.

“This is an institution that’s got no democratic accountability, it’s a job for life. If anything, members of the House of Lords should be regulated and judged by a higher standard than us in the House of Commons – and what’s happened is exactly the opposite.”

Public confidence in the Lords is already at a low ebb after the PPE controversy surrounding Baroness Michelle Mone, who took a leave of absence in 2022.

Michelle Mone attends the state opening of parliament in 2019. Pic: Reuters
Image:
Michelle Mone attends the state opening of parliament in 2019. Pic: Reuters

The government has pledged to reform the House of Lords and is currently trying to push through a bill abolishing the 92 remaining hereditary peers, which will return to the House of Commons in September.

But just before recess the bill was amended in the Lords so that they can remain as members until retirement or death. It’s a change which is unlikely to be supported by MPs.

Read more from Sky News:
Warning over water shortfall

Trump gaffe speaks volumes

MPs and peers alike have long faced scrutiny over their interests outside Westminster. File pic
Image:
MPs and peers alike have long faced scrutiny over their interests outside Westminster. File pic

A spokesperson for the House of Lords said: “Maintaining public confidence in the House of Lords is a key objective of the code of conduct. To ensure that, the code includes rigorous rules requiring the registration and declaration of all relevant financial interests held by members of the House of Lords.

“Public confidence relies, above all, on transparency over the financial interests that may influence members’ conduct. This change helps ensure the rules regarding registration of interests are understandable, enforceable and focused on the key areas of public concern.

“Members may still declare non-financial interests in debate, where they consider them directly relevant, to inform the House and wider public.

“The Conduct Committee is appointed to review the code of conduct, and it will continue to keep all issues under review. During its review of the code of conduct, the committee considered written evidence from both Unlock Democracy and Transparency International UK, among others.”

Continue Reading

UK

UK weather: Water shortfall declared ‘nationally significant’ – as amber heat health alert set to strike

Published

on

By

UK weather: Water shortfall declared 'nationally significant' - as amber heat health alert set to strike

The water shortfall situation in England has been described as a “nationally significant incident”, with five areas officially in drought ahead of an amber heat health alert coming into force for large parts of the country.

Six further areas are experiencing prolonged dry weather following the driest six months to July since 1976.

Many river flows and water reservoir levels in England continue to recede compared to June despite some storms and showers in July, which helped mask that it was still the fifth-warmest July on record.

A drone view from June shows vehicles using a bridge to pass over a dry section of the Woodhead Reservoir. Pic: Reuters
Image:
A drone view from June shows vehicles using a bridge to pass over a dry section of the Woodhead Reservoir. Pic: Reuters

A general view of Lindley reservoir near Otley in West Yorkshire with low water levels in June. Pic: PA
Image:
A general view of Lindley reservoir near Otley in West Yorkshire with low water levels in June. Pic: PA

Drier conditions have returned in August and now parts of the country are bracing for the fourth heatwave 2025, with today’s amber alert covering the East Midlands, West Midlands, East of England, London, and the South East.

Temperatures are forecast to rise above 30C (86F) in some areas, possibly even soaring past 35C (95F) in the south, threatening this year’s heat record of 35.8C (95.4F) in Faversham, Kent, on 1 July.

A milder yellow heat health alert is in place for the South West, North East, North West, Yorkshire and The Humber.

The alerts by the UK Health Security Agency (UKHSA) are due to be in place from 9am today until 6pm tomorrow, and put more pressure on struggling public water supplies and navigational waterways.

Check the weather forecast where you are

People enjoy the weather in Barnes on Monday. Pic: PA
Image:
People enjoy the weather in Barnes on Monday. Pic: PA

A man stands on a paddleboard with his dog near the beach at Rhos-on-Sea, Wales. Pic: Reuters
Image:
A man stands on a paddleboard with his dog near the beach at Rhos-on-Sea, Wales. Pic: Reuters

‘We are calling on everyone to play their part’

The National Drought Group (NDG), which includes the Met Office, government, regulators, water companies, the National Farmers’ Union, Canal & River Trust, anglers, and conservation experts, met at the start of the week to highlight the water-saving measures each sector is taking.

The group praised the public for reducing their daily usage, after Yorkshire Water reported a 10% reduction in domestic demand following the introduction of their hosepipe ban, which saved up to 80 million litres per day.

“The situation is nationally significant, and we are calling on everyone to play their part and help reduce the pressure on our water environment,” said Helen Wakeham, NDG chair and director of water at the Environment Agency.

“Water companies must continue to quickly fix leaks and lead the way in saving water.

“We know the challenges farmers are facing and will continue to work with them, other land users, and businesses to ensure everyone acts sustainably.”

Current drought situation in England

– Drought has been declared in: Yorkshire, Cumbria and Lancashire, Greater Manchester Merseyside and Cheshire, East Midlands, West Midlands.

– Areas in prolonged dry weather (the phase before drought) are: Northeast, Lincolnshire and Northamptonshire, East Anglia, Thames, Wessex, Solent and South Downs.

– Yorkshire Water has a hosepipe ban in place for all its customers, while Thames, South East, and Southern Water have postcode-specific bans.

– Reservoirs fell by 2% last week and are now 67.7% full on average across England. The average for the first week of August is 80.5%.

– The lowest reservoirs are Blithfield (49.1%), Derwent Valley (47.2%), Chew Valley Lake (48.3%), Blagdon (46.3%).

– Rainfall in July was 89% of the long-term average for the month across England. This is the sixth consecutive month of below-average rainfall.

– Across the country, 51% of river flows were normal, with the rest below normal, notably low or exceptionally low.

– Two rivers – Wye and Ely Ouse – were the lowest on record for July.

– There are currently navigation closures or restrictions across sections of the Leeds and Liverpool, Macclesfield, Trent and Mersey, Peak Forest, Rochdale, Oxford and Grand Union Canal.

The rainfall at the end of July was welcomed by growers, even though the dry weather is set to have an impact on the harvest, with the National Farmers Union (NFU) noting how water shortages have impacted the growing season.

“Some farms are reporting a significant drop in yields, which is financially devastating for the farm business and could have impacts for the UK’s overall harvest,” NFU vice-president Rachel Hallos said.

Ms Hallos urged that investment in water infrastructure and a more effective planning system was urgently needed “to avoid the swing between extreme drought and flooding and to secure water supplies for food production”.

Read more:
What is a heat-health alert?
How heatwaves affect your health

Where are hosepipe bans in place?

Please use Chrome browser for a more accessible video player

Drought in England explained

The dry weather also impacts the health of the waterways, as low water levels reduce oxygen levels in the water, which can lead to fish deaths, more algae growth, and could prevent wildlife from moving up or downstream.

Water minister Emma Hardy said the government is “urgently stepping up its response” to respond to dry weather, including investment in new reservoirs, but called on firms to do their bit.

“Water companies must now take action to follow their drought plans,” she said.

“I will hold them to account if they delay.”

Tips for staying cool from the UKHSA

  • Close windows and curtains in rooms that face the sun
  • Seek shade and cover up outside
  • Use sunscreen, wear a hat and sunglasses
  • Keep out of the sun at the hottest times, between 11am and 3pm
  • Restrict physical activity to the cooler mornings or evenings
  • Know how to respond to heat exhaustion and heatstroke

“We face a growing water shortage in the next decade,” the minister warned, which she said is why building new reservoirs – something the government has criticised the previous administration for not doing – is so important.

The hot and dry conditions have also led to warnings of wildfires, following blazes near Wimborne in Dorset and at Edinburgh’s Arthur’s Seat over the weekend.

Continue Reading

UK

House of Lords under fire for dropping rule that once caught out cricket legend and historian

Published

on

By

House of Lords under fire for dropping rule that once caught out cricket legend and historian

Campaigners have criticised a change to the rules around declarations of interest in the House of Lords as a “retrograde step” which will lead to a “significant loss of transparency”. 

Since 2000, peers have had to register a list of “non-financial interests” – which includes declaring unpaid but often important roles like being a director, trustee, or chair of a company, think tank or charity.

But that requirement was dropped in April despite staff concerns.

Tom Brake, director of Unlock Democracy, and a former Liberal Democrat MP, wants to see the decision reversed.

“It’s a retrograde step,” he said. “I think we’ve got a significant loss of transparency and accountability and that is bad news for the public.

“More than 25 years ago, the Committee on Standards in Public Life identified that there was a need for peers to register non-financial interests because that could influence their decisions. I’m confused as to what’s happened in the last 25 years that now means this requirement can be scrapped.

“This process seems to be all about making matters simpler for peers, rather than what the code of conduct is supposed to do, which is to boost the public’s confidence.”

Westminster Accounts: Search for your MP

MPs and peers alike have long faced scrutiny over their interests outside Westminster. File pic
Image:
MPs and peers alike have long faced scrutiny over their interests outside Westminster. File pic

Rules were too ‘burdensome’, say peers

The change was part of an overhaul of the code of conduct which aimed to “shorten and clarify” the rules for peers.

The House of Lords Conduct Committee argued that updating non-financial interests was “disproportionately burdensome” with “minor and inadvertent errors” causing “large numbers of complaints”.

As a result, the register of Lords interests shrunk in size from 432 pages to 275.

MPs have a different code of conduct, which requires them to declare any formal unpaid positions or other non-financial interests which may be an influence.

A source told Sky News there is real concern among some Lords’ staff about the implications of the change.

Non-financial interest declarations have previously highlighted cases where a peer’s involvement in a think tank or lobbying group overlapped with a paid role.

Please use Chrome browser for a more accessible video player

Protesters disrupt House of Lords

Cricket legend among peers to breach code

There are also examples where a peer’s non-financial interest declaration has prompted an investigation – revealing a financial interest which should have been declared instead.

In 2023, Lord Skidelsky was found to have breached the code after registering his role as chair of a charity’s trustees as a non-financial interest.

Lord Skidelsky. Pic: UK Parliament
Image:
Lord Skidelsky. Pic: UK Parliament

The Commissioner for Standards investigated after questions were raised about the charity, the Centre for Global Studies.

He concluded that the charity – which was funded by two Russian businessmen – only existed to support Lord Skidelsky’s work, and had paid his staff’s salaries for over 12 years.

In 2021, Lord Botham – the England cricket legend – was found to have breached the code after registering a non-financial interest as an unpaid company director.

The company’s accounts subsequently revealed he and his wife had benefitted from a director’s loan of nearly £200,000. It was considered a minor breach and he apologised.

Former cricketer Lord Botham. File pic: PA
Image:
Former cricketer Lord Botham. File pic: PA

‘Follow the money’

Lord Eric Pickles, the former chair of the anti-corruption watchdog, the Advisory Committee on Business Appointments, believes focusing on financial interests makes the register more transparent.

“My view is always to follow the money. Everything else on a register is camouflage,” he said.

“Restricting the register to financial reward will give peers little wriggle room. I know this is counterintuitive, but the less there is on the register, the more scrutiny there will be on the crucial things.”

Lord Eric Pickles
Image:
Lord Eric Pickles

‘I was shocked’

The SNP want the House of Lords to be scrapped, and has no peers of its own. Deputy Westminster leader Pete Wishart MP is deeply concerned by the changes.

“I was actually quite horrified and quite shocked,” he said.

“This is an institution that’s got no democratic accountability, it’s a job for life. If anything, members of the House of Lords should be regulated and judged by a higher standard than us in the House of Commons – and what’s happened is exactly the opposite.”

Public confidence in the Lords is already at a low ebb after the PPE controversy surrounding Baroness Michelle Mone, who took a leave of absence in 2022.

Michelle Mone attends the state opening of parliament in 2019. Pic: Reuters
Image:
Michelle Mone attends the state opening of parliament in 2019. Pic: Reuters

The government has pledged to reform the House of Lords and is currently trying to push through a bill abolishing the 92 remaining hereditary peers, which will return to the House of Commons in September.

But just before recess the bill was amended in the Lords so that they can remain as members until retirement or death. It’s a change which is unlikely to be supported by MPs.

Read more from Sky News:
Warning over water shortfall

Trump gaffe speaks volumes

MPs and peers alike have long faced scrutiny over their interests outside Westminster. File pic
Image:
MPs and peers alike have long faced scrutiny over their interests outside Westminster. File pic

A spokesperson for the House of Lords said: “Maintaining public confidence in the House of Lords is a key objective of the code of conduct. To ensure that, the code includes rigorous rules requiring the registration and declaration of all relevant financial interests held by members of the House of Lords.

“Public confidence relies, above all, on transparency over the financial interests that may influence members’ conduct. This change helps ensure the rules regarding registration of interests are understandable, enforceable and focused on the key areas of public concern.

“Members may still declare non-financial interests in debate, where they consider them directly relevant, to inform the House and wider public.

“The Conduct Committee is appointed to review the code of conduct, and it will continue to keep all issues under review. During its review of the code of conduct, the committee considered written evidence from both Unlock Democracy and Transparency International UK, among others.”

Continue Reading

Trending