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The Democratic National Committee said the move to hand over the voter roll could violate federal election law.
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This story was originally published originally published by Votebeat, a nonprofit news organization covering local election administration and voting access.
Texas officials have turned over the state's voter roll to the U.S. Justice Department, according to a spokesperson for the Texas Secretary of State's Office, complying with the Trump administration's demands for access to data on millions of voters across the country.
The Justice Department last fall began asking all 50 states for their voter rolls — massive lists containing significant identifying information on every registered voter in each state — and other election-related data. The Justice Department has said the effort is central to its mission of enforcing election law requiring states to regularly maintain voter lists by searching for and removing ineligible voters.
Alicia Pierce, a spokesperson for the Texas Secretary of State's Office, told Votebeat and The Texas Tribune that the state had sent its voter roll, which includes information on the approximately 18.4 million voters registered in Texas, to the Justice Department on Dec. 23.
The state included identifiable information about voters, including dates of birth, driver's license numbers and the last four digits of their Social Security numbers, Pierce said.
Experts and state officials around the country have raised concerns over the legality of the Justice Department's effort to obtain states' voter rolls and whether it could compromise voter privacy protections. The Justice Department has said it is entitled to the data under federal law, and withholding it interferes with its ability to exercise oversight and enforce federal election laws.
The department has now sued 23 states and Washington, D.C., for declining to voluntarily turn over their voter rolls. Those states, which include some led by officials of both political parties, have generally argued that states are responsible for voter registration and are barred by state and federal law from sharing certain private information about voters. In an interview with “The Charlie Kirk Show” last month, Assistant Attorney General Harmeet K. Dhillon said 13 states, including Texas, had voluntarily agreed to turn over their voter rolls.
In a letter to Nelson dated Friday and obtained by Votebeat and The Texas Tribune, the Democratic National Committee said the move to hand over the voter roll could violate federal election law.
DNC Chair Ken Martin said the turnover of such data is tantamount to a “big government power grab” and would invite privacy violations and could result in eligible voters being kicked off the rolls. The DNC, he said in a statement, “won't stand idly by as the Trump DOJ tries to get access to Texas voters' sensitive information.”
In its letter, Daniel Freeman, the DNC's litigation director, requested records related to the Justice Department's request, and warned the party could take further action.
Some election officials and voting rights watchdog groups have raised concerns about what the Justice Department intends to do with the information provided by the states, with some suggesting it may be used to create a national database of voters.
Votebeat and The Texas Tribune have asked the Texas Secretary of State's Office for a signed copy of the agreement between the state and the Justice Department, known as a memorandum of understanding, governing how the sharing of the voter data would work and steps the state has agreed to take in response to any questions about voter eligibility raised by the Justice Department. The state has not yet released it.
In a proposed memorandum of understanding sent to Wisconsin officials last month and publicly released by state officials, the Justice Department said that upon receiving the state's voter data, it would check the state's voter roll for “list maintenance issues, insufficiencies, anomalies or concerns.” The department would then notify the state and give it 45 days to correct any problems. The state would then agree to resubmit the voter roll to the department. Wisconsin declined the agreement, and the Justice Department has since sued the state.
In his letter to Nelson, Freeman identified two potential legal violations associated with some of those clauses, though acknowledged he didn't yet know whether Texas had signed such an agreement and asked for records.
Freeman wrote that the 45-day removal period as laid out in the public versions of the memorandum would run afoul of a provision in the National Voter Registration Act that lays out specific conditions, such as having missed two elections after receiving a notice from the state, for states to remove registered voters from the rolls.
Freeman also wrote that federal law also bars states from doing systemic voter removals from the rolls within 90 days of a primary or general election. Because Texas has an upcoming March 3 primary, May 26 runoff and Nov. 3 general election, the state cannot conduct such list maintenance until after the runoff, Freeman wrote. The 90-day moratorium would then kick in again on Aug. 6, ahead of the November election.
Texas agreed to the memorandum of understanding and released the data, but told the department that it did so with the understanding it wouldn't “limit or affect the duties, responsibilities, and rights” of the state under either the NVRA or other federal laws, according to two letters the Texas Secretary of State's Office sent the Justice Department in December and released to Votebeat and The Texas Tribune.
Votebeat is a nonprofit news organization covering local election integrity and voting access. Sign up for their newsletters here.
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Natalia Contreras has covered a range of topics as a community journalist including local government, public safety, immigration, and social issues. Natalia comes to Votebeat from the Austin American-Statesman, where her reporting focused on impacts of government policies on communities of color. Natalia previously reported for the Indianapolis Star, where she helped launch the first Spanish-language newsletter, and at the Corpus Christi Caller-Times. Natalia was born in Tampico, Tamaulipas, Mexico, and grew up in Corpus Christi, Texas.
Gabby Birenbaum is the Washington correspondent for the Texas Tribune. She covers the Texas congressional delegation and the impact of federal policy on Texas. Gabby previously covered Washington for The Nevada Independent, where she wrote the weekly D.C. Download newsletter and covered the 2024 presidential election and competitive Senate race. Prior to The Nevada Independent, Gabby worked at The Washington Monthly and Vox. Gabby graduated from Northwestern University, where she studied journalism and political science. She is a native of Arlington, Virginia, and is based in Washington, D.C. In her free time, she enjoys playing tennis, watching movies and being a long-suffering fan of D.C. sports teams. Her work is supported by a partnership with Report for America.
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Iranian protesters shouted and marched through the streets into yesterday morning after a call by the country's exiled crown prince for demonstrations, despite Iran's theocracy cutting off the nation from the Internet and international telephone calls.
Short online videos shared by activists purported to show protesters chanting against Iran's government around bonfires as debris littered the streets in Tehran and other areas.
Iranian state media broke its silence over the protests, alleging “terrorist agents” of the US and Israel set fires and sparked violence.
Photo: AP
It also said there were “casualties,” without elaborating.
The full scope of the demonstrations could not be immediately determined due to the communications blackout, although it represented yet another escalation in protests that began over Iran's ailing economy and that has morphed into the most significant challenge to the government in several years.
The protests have intensified steadily since Dec. 28 and represented the first test of whether the Iranian public could be swayed by Crown Prince Reza Pahlavi, whose fatally ill father fled Iran just before the country's 1979 Islamic Revolution. Demonstrations have included cries in support of the shah, something that could bring a death sentence in the past, but now underlines the anger fueling the protests that began over Iran's ailing economy.
So far, violence around the demonstrations has killed at least 42 people while more than 2,270 have been detained, Human Rights Activists News Agency said.
Pahlavi, who called for protests Thursday night, similarly has called for demonstrations yesterday.
“What turned the tide of the protests was former Crown Prince Reza Pahlavi's calls for Iranians to take to the streets at 8pm on Thursday and Friday,” Washington Institute for Near East Policy senior fellow Holly Dagres said. “Per social media posts, it became clear that Iranians had delivered and were taking the call seriously to protest in order to oust the Islamic Republic.”
“This is exactly why the Internet was shut down: to prevent the world from seeing the protests. Unfortunately, it also likely provided cover for security forces to kill protesters,” Dagres added.
When the clock struck 8pm on Thursday, neighborhoods across Tehran erupted in chanting, witnesses said.
The chants included “Death to the dictator!” and “Death to the Islamic Republic!”
Others praised the shah, shouting: “This is the last battle! Pahlavi will return!”
Thousands could be seen on the streets before all communication to Iran cut out.
“Iranians demanded their freedom tonight. In response, the regime in Iran has cut all lines of communication,” Pahlavi said. “It has shut down the Internet. It has cut landlines. It may even attempt to jam satellite signals.”
He went on to call for European leaders to join US President Donald Trump in promising to “hold the regime to account.”
“I call on them to use all technical, financial, and diplomatic resources available to restore communication to the Iranian people so that their voice and their will can be heard and seen,” he added. “Do not let the voices of my courageous compatriots be silenced.”
Pahlavi had said he would offer further plans depending on the response to his call. His support of and from Israel has drawn criticism in the past. Demonstrators have shouted in support of the shah in some demonstrations, but it is not clear whether that is support for Pahlavi himself or a desire to return to a time before the 1979 Islamic Revolution.
The Internet cut also appears to have taken Iran's state-run and semiofficial news agencies offline as well.
US President Donald Trump on Friday said Washington was “locked and loaded” to respond if Iran killed protesters, prompting Tehran to warn that intervention would destabilize the region.
Protesters and security forces on Thursday clashed in several Iranian cities, with six people reported killed, the first deaths since the unrest escalated.
Shopkeepers in Tehran on Sunday last week went on strike over high prices and economic stagnation, actions that have since spread into a protest movement that has swept into other parts of the country.
If Iran “violently kills peaceful protesters, which is their custom, the United States of America will come to
Auschwitz survivor Eva Schloss, the stepsister of teenage diarist Anne Frank and a tireless educator about the horrors of the Holocaust, has died. She was 96.
The Anne Frank Trust UK, of which Schloss was honorary president, said she died on Saturday in London, where she lived.
Britain's King Charles III said he was “privileged and proud” to have known Schloss, who cofounded the charitable trust to help young people challenge prejudice.
“The horrors that she endured as a young woman are impossible to comprehend and yet she devoted the rest of her life to overcoming hatred and prejudice, promoting kindness, courage, understanding
‘DISRESPECTFUL':
Katie Miller, the wife of Trump's most influential adviser, drew ire by posting an image of Greenland in the colors of the US flag, captioning it ‘SOON' US President Donald Trump on Sunday doubled down on his claim that Greenland should become part of the US, despite calls by the Danish prime minister to stop “threatening” the territory.
Washington's military intervention in Venezuela has reignited fears for Greenland, which Trump has repeatedly said he wants to annex, given its strategic location in the arctic.
While aboard Air Force One en route to Washington, Trump reiterated the goal.
“We need Greenland from the standpoint of national security, and Denmark is not going to be able to do it,” he said in response to a reporter's question. “We'll worry about Greenland in
PERILOUS JOURNEY:
Over just a matter of days last month, about 1,600 Afghans who were at risk of perishing due to the cold weather were rescued in the mountains Habibullah set off from his home in western Afghanistan determined to find work in Iran, only for the 15-year-old to freeze to death while walking across the mountainous frontier.
“He was forced to go, to bring food for the family,” his mother, Mah Jan, said at her mud home in Ghunjan village.
“We have no food to eat, we have no clothes to wear. The house in which I live has no electricity, no water. I have no proper window, nothing to burn for heating,” she added, clutching a photograph of her son.
Habibullah was one of at least 18 migrants who died
Anti-government protests in Iran erupted for a thirteenth consecutive day on Friday, in a wave of nationwide unrest that marks the biggest challenge to the regime in years.
Authorities cut off internet access and telephone lines on Thursday – the biggest night of nationwide demonstrations so far – leaving Iran largely cut off from the outside world. Rights groups said dozens of people have been killed since the protests began.
US President Donald Trump has threatened to attack Iran if security forces respond with force. Supreme Leader Ayatollah Ali Khamenei has urged Trump to “focus on his own country” and blamed the US for inciting the protests.
As public anger continues to swell and demonstrations continue, here's what you need to know.
The protests began as demonstrations in Tehran's bazaars over rampant inflation but have spread across the country and morphed into more general protests against the regime.
Concerns over inflation came to a head last week, when the prices of basic goods like cooking oil and chicken dramatically spiked overnight, with some products vanishing from shelves all together.
Exacerbating the situation was a decision by the central bank to end a program allowing some importers to access cheaper US dollars compared to the rest of the market – which led shopkeepers to increase prices and some to shutter their doors, initiating the demonstrations.
The move by the bazaaris, as they are known, is a drastic measure for a group traditionally supportive of the Islamic Republic.
The reformist-ruled government attempted to alleviate the pressure by offering direct cash handouts of almost $7 per month, but the move has failed to quell the unrest.
The latest protests are the biggest in scale since 2022, when the death of 22-year-old Mahsa Amini while in custody of the religious police prompted the widespread “Woman, Life, Freedom” protests.
People across more than 100 cities have participated in the demonstrations, which began nearly two weeks ago.
The protests have spread to Iranian provinces as far west as Ilam, a Kurdish-majority region bordering Iraq, and Lorestan, both which have emerged as restive hotspots. Fueled by ethnic division and poverty, crowds set fire to the streets and chanted “Death to Khamenei,” directly challenging Khamenei, who holds ultimate authority over the nation's religious and state affairs.
Iranian state affiliated Fars news agency said 950 police forces and 60 personnel from the paramilitary Basij force have been injured in the protests mostly in confrontations with “rioters” in western provinces “equipped with firearms, grenades, and weapons.”
At least 45 protesters, including eight children, have been killed since demonstrations began, the Norway-based Iran Human Rights NGO (IHRNGO) reported Thursday. It said hundreds more have been injured and over 2,000 people detained. CNN could not independently verify the numbers of those killed and arrested, and Iranian state news organizations have sometimes reported individual deaths without reporting a comprehensive tally.
The fact that the recent protests began with the bazaaris – a powerful force for change in Iran's history and one seen as loyal to the regime – is notable.
The enduring alliance between the bazaaris and the clergy in Iran had the shopkeepers play a crucial role as kingmaker across Iran's history. It was their support to those very clergymen that eventually helped the Islamic Revolution of 1979 succeed, giving the rebels a financial backbone that led to the fall of the shah, or monarch.
“For more than 100 years of Iranian history, bazaaris have been key actors in all of Iran's major political movements. … Many observers do believe that the bazaaris are some of the most loyal to the Islamic Republic,” said Arang Keshavarzian, associate professor of Middle Eastern and Islamic studies at New York University and author of “Bazaar and State in Iran,” told CNN.
Their role as a major political force has since become more symbolic, but the impact of fluctuations in currency on their business is what led them to spark the protests that have since turned deadly.
Authorities have also sought to differentiate between economic protesters and those calling for regime change, branding the latter as “rioters” and foreign backed “mercenaries” while pledging a tougher crackdown against them.
Two experts who spoke with CNN said the protests could lead to significant change.
“These protests, whatever the outcome, will no doubt further damage an already fractured legitimacy for a state that I think is at the end of its life,” Sanam Vakil, the director of the Middle East and North Africa Program at Chatham House, told CNN's Eleni Giokos.
This round of protests feels different from prior ones because of a sense of frustration and exhaustion among people in Iran, said Dina Esfandiary, Middle East lead for Bloomberg Economics.
“It's reached a boiling point,” Esfandiary said. “I anticipate that the Islamic Republic that we're seeing today is one unlikely to see 2027. I really think there is going to be some change.”
Iran has been a theocracy since 1979, when clerics toppled a secular monarch allied with the West, leading to the formation of the Islamic Republic led by Khomenei.
Masoud Pezeshkian was elected president in 2024, promoting a more pragmatic foreign policy, but his powers are limited, and Khamenei calls the shots on all major matters of state.
“We should not expect the government to handle all of this alone,” Pezeshkian said in a televised speech Monday.
Pezeshkian previously positioned himself as a champion of the working class, promising economic relief through reduced government intervention in the currency market while also blaming US sanctions, corruption, and excessive money printing.
But corruption across all parts of government, mismanagement of funds and the convergence of environmental problems and stagnant leadership has the government on the brink.
More than a year after he was voted in, the very working class he vowed to protect and the middle class that form the backbone of the Iranian society, are struggling.
External factors like crippling sanctions and a potential new war with the United States and Israel, has left the state paranoid, and the population anxious.
On Friday, state media agency Tasnim reported that the Islamic Revolutionary Guard Corps had issued a statement warning that the preservation of the country's government is their “red line” and reserving the right to “retaliation.”
Reza Pahlavi, the exiled son of the late shah, has positioned himself as a viable alternative to the ruling regime, declaring support for the protests and issuing direct calls for coordinated nationwide action.
On Tuesday, Pahlavi called on Iranians to chant en masse.
At least some of the protesters appeared to be heeding his call. One of the slogans shouted by demonstrators Thursday was: “This is the last battle, Pahlavi will return,” according to video reviewed by CNN.
While pro-monarchy chants have been heard in videos from the demonstrations, the extent of monarchist support across the country remains unclear.
“None of Iran's political leaders have a blueprint to get Iran out the crises,” Keshavarzian told CNN.
“The only tool that the Islamic Republic truly has left is coercion and force. People have tried different methods to air their views,” he added. “But over the past 15 years large segments of the population have lost trust in the regime.”
Trump has warned Tehran of severe consequences if protesters are killed.
“I have let them know that if they start killing people, which they tend to do during their riots … we're going to hit them very hard,” Trump told conservative radio host Hugh Hewitt on Thursday.
Just six months ago, Israel and the US launched attacks on Iran for the first time, with Trump raising the prospects of new attacks just last week, days after meeting Israeli Prime Minister Benjamin Netanyahu.
In a televised address marking his first public comments since the demonstrations started, Khamenei called on Trump to “focus on the problems of his own country.”
“There are some agitators who want to please the American president by destroying public property. A united Iranian people will defeat all enemies,” he said.
“The Islamic Republic will not back down in the face of those who are looking to destroy us,” Khamenei added.
CNN's Kara Fox, Max Saltman, Adam Pourahmadi, Charlotte Reck and Aditi Sangal contributed reporting.
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NEW YORK, January 9. /TASS/. The US Southern Command has officially confirmed the seizure of the oil tanker Olina in its post on X, which included a video of the operation.
During the seizure, marines and sailors of the Southern Spear joint task force, acting in support of the US Department of Homeland Security, boarded the tanker Olina from the aircraft carrier USS Gerald R. Ford. The operation in the Caribbean Sea went smoothly.
The vessel was seized near the island of Trinidad (Republic of Trinidad and Tobago). According to Reuters, the tanker had previously made voyages to Venezuela under the flag of Timor-Leste.
Pre-dawn assault on the Olina oil tanker was carried out by US marines and navy sailors in the Caribbean near Trinidad
US politics live – latest updates
The US early on Friday boarded another oil tanker, the US military said, as part of efforts to target sanctions-busting vessels traveling to and from Venezuela.
US forces were seen in video footage that officials posted online landing on the ship's deck as the vessel named Olina was seized in the Caribbean near Trinidad. It is the fifth interdiction of such ships in recent weeks, separately from the series of previous US operations since the start of the fall to strike suspected drug boats off the coast of Venezuela.
The pre-dawn action against the oil tanker on Friday was carried out by US marines and navy sailors from the forces the US has built up in the Caribbean in recent months, according to US Southern Command, which declared “there is no safe haven for criminals” as it announced the seizure of the vessel. The assault was launched from the aircraft carrier the USS Gerald R Ford.
The Trump administration has moved to control the distribution of Venezuela's oil products globally after its ousting and capture of the Venezuelan president, Nicolás Maduro, less than a week ago from the capital city, Caracas, in a night-time raid which included airstrikes and US special forces deployed on the ground.
Southern Command also posted unclassified footage on social media of a US helicopter landing on the vessel and armed US personnel conducting a search of the deck, with the military forces acting in support of the US Coast Guard, which was in charge of the operation under the auspices of its parent agency the Department of Homeland Security (DHS).
The DHS secretary, Kristi Noem, on X said the ship was seized in international waters and added: “As another ‘ghost fleet' tanker ship suspected of carrying embargoed oil, this vessel had departed Venezuela attempting to evade US forces … The ghost fleets will not outrun justice.”
The Olina, according to public shipping database Equasis, was falsely flying the flag of the tiny south-east Asian nation of Timor Leste.
“The vessel's AIS [location] tracker was last active 52 days ago in the Venezuelan EEZ, north-east of Curacao,” Vanguard, a British maritime risk management company, said separately.
“The seizure follows a prolonged pursuit of tankers linked to sanctioned Venezuelan oil shipments in the region.”
The Olina left Venezuela last week fully loaded with oil as part of a flotilla shortly after Maduro's capture on 3 January, and the vessel was returning fully loaded to Venezuela after the US blockade of Venezuelan oil exports, the industry source said.
US government records show that the Olina was sanctioned for moving Russian oil under its prior name, Minerva M, and flagged in Panama.
While records show that the Olina is now flying the flag of Timor-Leste, it is also listed in the international shipping registry as having a false flag, meaning the registration it is claiming is not valid. In July, the owner and manager of the ship on its registration was changed to a company in Hong Kong.
The US government said it was part of the so-called shadow fleet of ships that sail with little regulation or known insurance.
According to ship-tracking databases, the Olina last transmitted its location in November in the Caribbean, north of the Venezuelan coast. Since then, however, the ship has been running dark with its location beacon turned off.
The ship has a listed cargo capacity of up to 890,000 barrels of oil, which at the current market price of about $60 a barrel would be about $53m.
Reuters and the Associated Press contributed reporting
Ayatollah Ali Khamenei calls protesters ‘vandals' and ‘saboteurs' and blames the US for instigating the unrest
Iran's supreme leader has vowed that authorities will “not back down” in the face of a rapidly growing protest movement, setting the stage for an intensified violent crackdown on the second day of a nationwide internet shutdown.
Protests have raged in cities and towns across the country in recent days, posing a threat to the authority of the regime, which has been significantly weakened since the last large protest movement in the country in 2022. Another round of demonstrations was called for Friday night.
In his first speech since demonstrations started on 28 December, Ayatollah Ali Khamenei described protesters as “vandals” and “saboteurs”, and accused them of working on behalf of foreign agendas.
Protesters are “ruining their own streets to make the president of another country happy … because he said he would come to their aid”, Khamenei said – a reference to Donald Trump, who has threatened American intervention in Iran if authorities kill protesters.
The protests started after a sudden depreciation in the value of the country's currency, but demands for political reform and an end to the regime's rule quickly emerged.
The US president, in an interview with Fox News on Thursday, suggested the supreme leader was preparing to flee Iran. “He's looking to go somewhere. It's getting very bad,” Trump said.
The demonstrations on Thursday were the biggest since the 2022-2023 rallies sparked by the death in custody of Mahsa Amini. Authorities look more vulnerable this time around because of the dire economic situation and the aftermath of last year's war with Israel and the US.
The head of Iran's judiciary, Gholamhossein Mohseni Ejei, said consequences for demonstrators would be “decisive, maximum and without any legal leniency”.
An internet blackout introduced on Thursday has sharply reduced the amount of information flowing out of the country. The Iranian rights group Hengaw reported that a protest march after Friday prayers in Zahedan, where the Baluch minority predominates, had been met with gunfire that wounded several people.
Videos showed crowds of thousands of people marching through the streets of Tehran on Thursday, setting fire to a building belonging to the Iranian state broadcasters and hoisting a flag bearing the lion and sun emblem –the flag of Iran before the 1979 revolution that brought the current regime to power.
Reza Pahlavi, the exiled son of the late shah, who had called for the protests on Thursday night, made another call for demonstrations to take place at 8pm (1600 GMT) on Friday. He also called on Trump to help the protesters, saying Khamenei “wants to use this blackout to murder these young heroes” .
Footage from Thursday showed protesters chanting in support of Pahlavi, including in Mashhad, Khamenei's home town. Protesters who went out on Thursday night said they were met with violence – part of what rights groups are calling an already brutal crackdown.
“They're aiming for the eyes,” Maryam, a 25-year-old artist who was at protests in Tehran in the early hours of Friday, told the Guardian via text message. “The Faraja [uniformed police], the Basij [paramilitary militia] and even plainclothes kill-squads are driving into the crowds with motorbikes. I don't know how long the internet will be working but we are thousands on the streets and I fear I will wake up to hundreds of casualties.”
At least 50 people have been killed in the violence surrounding protests, while more than 2,270 others have been detained, according to the US-based Human Rights Activists news agency.
Iranian state media acknowledged the protests for the first time on Friday, casting the unrest as violent riots instigated by “terrorist agents” of the US and Israel. State TV channels projected an air of normality, airing footage of pro-government demonstrations and insisting life went on as usual for most Iranians.
State media claimed Iran had caught agents from Israel's Mossad who had infiltrated protest movements. The Iranian-owned Press TV reported that an Israeli spy cell was planning a “false-flag killing operation aimed at blaming the state for civilian deaths”.
Speaking in Beirut on Friday, the Iranian foreign minister, Abbas Araghchi, echoed claims of overseas interference. “The protests that are happening in Iran of course are different from protests in other countries because of US and Israeli interventions in the protests,” he said. “You need to look at all the statements from the US and Israel to see how they are interfering.”
Authorities cut off internet to Iran at about 8pm local time (4.30pm UK time) on Thursday, around the same time as Pahlavi's call for protests. Understanding exactly what was happening in Iran and the true size of protests was difficult, with data and phone lines down. Human rights groups said documenting human rights violations was also hampered by the shutdown.
Students at a university in Tehran said they were trying to find a way to avoid the internet shutdown, relying on methods they developed during the war with Israel in the summer when authorities also shut down the internet.
Hossein, a 22-year-old university student, said: “Since June, we have been trying several ways to find these ‘secret tunnels' that can route our messages outside the country. A group of us are able to still chat but I can see mobile lines are also getting disrupted.”
Demonstrators appeared to respond to Pahlavi's call on Thursday, with anti-government chants ringing out at 8pm, as well as calls for the exiled crown prince to return.
A protester who responded to his call on Thursday night said that she believed that Pahlavi could assist with a transition to democracy. “We failed to unify under a strong opposition last time [2022] but we have learned our lessons,” said 46-year-old Mehnaz. “We have to rally for him because we are desperate to survive.”
Elements of the protest movement, largely leaderless until now, have rallied around the figure, though it was unclear if chants were in support of the crown prince or the pre-1979 rule.
“I am proud of each and every one of you who conquered the streets across Iran on Thursday night,” Pahlavi said in a post on X. “I know that despite the internet shutdown and communication, you won't leave the streets. Make sure that victory is yours!”
Pahlavi issued another call for demonstrations on Friday night at 8pm, which would be a further test for the exiled figure's popularity and the staying power of protests in the face of the authorities' crackdown.
Pahlavi's organisation also alleged that “tens of thousands” of security officers had signalled their intentions to defect via a platform it had set up, and that the organisation had been “inundated” by requests from officers.
Human Rights Watch and Amnesty International condemned on Friday what they said was authorities' use of force and mass arbitrary arrests. HRW has documented the deaths of 28 protesters who were shot by security forces between 31 December and 3 January, with instances of authorities using rifles and shotguns loaded with metal pellets.
“We have so far documented many of the harrowing patterns of human rights violations that authorities have repeatedly committed during the previous rounds of protest crackdowns, including in November 2019, and the Woman, Life, Freedom pretests of 2022,” said Bahar Saba, a senior HRW researcher on Iran and Kuwait.
The crackdown seemed only to harden protesters' resolve, many of whom described scenes of defiance, with rocks thrown at officers forcing them to retreat.
Ali, a 21-year-old student in Tehran, said via text message: “Fuck them! The cowards abandoned their vehicles and fled! We took over the streets tonight. We will burn their vans, the same that they use to drag our compatriots and kidnap our sisters from the streets. The country belongs to us!”
Anger at the regime and the clerics who form a backbone of the theocracy seemed to boil over throughout the week. On Wednesday, crowds of men streamed into a Shia seminary in the city of Gonabad, beating staff with sticks and damaging the facility, according to the director of the seminary, Ismail Tavakoli.
Another protester said unarmed protesters were confronting riot police, throwing rocks in response to bullets fired by officers.
“They are vulgar and are saying we are in bed with the Israelis and Americans,” said Farzad, a 37-year-old mobile shop owner in the city of Rasht in northern Iran. “They call us traitors. It's them that have betrayed the very sense of being an Iranian.”
The ad campaign ended in late 2025, the Swedish streaming giant confirmed, having previously said, despite protests, that it did not violate advertising policies
Spotify is no long running advertisements for the US Immigration and Customs Enforcement (ICE), the streaming service has confirmed, after the Trump administration campaign ended in late 2025.
“There are currently no ICE ads running on Spotify,” the Swedish company said in a statement. “The advertisements mentioned were part of a US government recruitment campaign that ran across all major media and platforms.”
Since April, the government ads have also run on Amazon, YouTube, Hulu and Max among other streaming companies, with the aim of recruiting more than 10,000 deportation officers by the end of 2025.
Previously, Spotify said that the ads, which encouraged US listeners to “fulfil your mission to protect America” and offered $50,000 in signing bonuses, did not “violate our advertising policies”.
“This advertisement is part of a broad campaign the US government is running across television, streaming, and online channels,” the company said in October. “However, users can mark any ad with a thumbs-up or thumbs-down to help manage their ads preferences.”
The ad campaign ended before an ICE agent fatally shot 37-year-old Renee Good in her car in Minneapolis on Wednesday. Last night, US border patrol agents shot two more people in Portland, whose condition is currently unknown.
It also ended before Indivisible, the grassroots movements behind the No Kings demonstrations, sent an open letter to Spotify's new CEOs, Alex Norström and Gustav Söderström, on their first day at work on 2 January petitioning them to drop the ads and make a public commitment to “reviewing and updating Spotify's advertising policy to prohibit government propaganda and hate-based recruitment ad campaigns”.
In September, it was announced that Spotify founder and former CEO Daniel Ek would step down to become executive chairman. Ek's €600m investment in the military AI company Helsing, of which he is also chairman, has also been a source of discontent among musicians and listeners: in September, UK trip-hop group Massive Attack withdrew their music from Spotify in protest.
Spotify has faced a number of high-profile musicians withdrawing their music from the platform in protest at Ek's investment in Helsing, with some motivated by the ICE ads, among them Australian psych-rock band King Gizzard and the Lizard Wizard, Canadian post-rockers Godspeed You! Black Emperor, US indie band Deerhoof and US singer-songwriter Kadhja Bonet. Numerous listeners have also reported quitting the service for political reasons.
The end of this specific advertising campaign certainly does not mean an end to ads of this nature, whatever platform might broadcast them: ICE has reportedly planned a $100m year-long media barrage for what it calls “wartime recruitment”, targeting conservative radio show listeners, gun rights aficionados, military affairs followers and men's interests enthusiasts.
“Want to deport illegals with your absolute boys?” one of the agency's ads says. Officials will also invest in deals with lifestyle influencers paid to push the administration's anti-immigration agenda.
Kyiv Mayor Vitali Klitschko called on residents of the capital, if possible, to leave the city after a Russian attack on Jan. 9 left half of the city's apartment buildings without heating.
As temperatures dropped below -10°C (14°F), Russia launched a mass attack on Ukraine overnight with 242 drones and 36 missiles, with Kyiv and its surrounding areas being the primary targets, the Air Force reported. The attack hit energy infrastructure facilities and multiple residential buildings in the capital, killing at least four people and injuring 19 others.
"We are doing everything we can to resolve this as quickly as possible. However, the combined attack on Kyiv last night was the most devastating for the capital's critical infrastructure," Klitschko said.
As of the time of publication, heating was knocked out in about 6,000 apartment buildings across Kyiv — nearly half of the city's total — following the Russian attack overnight, according to the mayor.
The city is also facing water supply interruptions, with emergency power outages being introduced.
"I appeal to the residents of the capital, who have the opportunity to temporarily leave the city for places where there are alternative sources of power and heat, to do so," Klitschko said.
The mayor said that hospitals and maternity wards have been reconnected to power and heating. Efforts to restore power and heating for residential homes in Kyiv is ongoing.
Mykola Kolisnyk, deputy energy minister, said that the fewer consumers there are, "the easier it is to handle the situation." If there is no heating supply, residents start using electricity to heat their homes, which, in turn, increases electricity consumption, the official added.
"This is why, when the population is more, let's say spread across villages and other regions, there will be less pressure specifically in one city," Kolisnyk told the Kyiv Independent.
Kyiv, home to around 4 million people, has endured multiple of Russian attacks on its energy infrastructure since the start of the full-scale war in 2022. Over past months, Moscow has continued pounding energy facilities, with Ukrainian officials warning that Russia is attempting to shut down the energy grid region by region.
In recent days, Russia has targeted regional capitals in the east and south, leaving Dnipropetrovsk and Zaporizhzhia oblasts without power on the evening of Jan. 7.
Russian forces also targeted critical infrastructure in the western Lviv Oblast overnight on Jan. 9, launching its new Oreshnik intermediate-range ballistic missile.
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President fears Russia is ‘playing for time' rather than seeking Ukraine peace say White House sources
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Donald Trump is growing increasingly frustrated with Vladimir Putin and sees him as a bigger obstacle to peace in Ukraine than Volodymyr Zelensky, according to sources.
They said the US president's decisions to seize a Russian-flagged oil tanker and support a new sanctions bill were a signal to Putin that he was running out of time to bring an end to the war.
The source, who is close to the US president's inner circle, said: “He works around a carrot and stick approach. And I think he's all out of carrots.”
Another source said Mr Trump's found the Russians' “two steps forward, one step back” strategy in talks increasingly “tiresome”.
Mr Trump has spoken warmly in the past about Putin and said Ukraine was to blame for the war. However, several current and former Washington officials said the Trump administration's thinking was becoming more in line with the European view that Putin was playing for time.
On Wednesday, American special forces risked a confrontation with Moscow when they boarded a Russian registered tanker flagged for sanctions violations.
Hours later, the White House confirmed that Mr Trump had “greenlit” a sanctions bill during a meeting with Lindsey Graham, one of the most high-profile Russia hawks in the Senate.
The legislation would give the president extraordinary powers to isolate Russia, including the ability to impose 500 per cent tariffs on goods imported from countries that purchase Russian oil, petroleum products or uranium.
One former official said: “What is tiresome to the administration is ... that the Russians use a two steps forward, one step back strategy ... they seem like they're ready, then when things come up that we thought they would be amenable to they either ramp things up, or they stop talking for a while.”
Ukraine had, he said, been more amenable to compromise.
Mr Trump has blown hot and cold with both sides in the Russia-Ukraine war as he tries to make good on a campaign promise to secure peace.
Frequent claims that he believes Putin is serious about finding peace in Ukraine have alarmed Mr Zelensky's allies in Europe. They point out that warm conversations between the two leaders were often followed by missile and drone attacks.
Russia launched ballistic missiles and drones on Kyiv, killing at least one person, a day before Mr Zelensky met Mr Trump at Mar-a-Lago at the end of last year.
A British official said the result was that the Trump administration's mindset was closer to the European view of the conflict than ever before. He said: “Putin's sustained brutality, nefarious behaviour and game playing do not go unnoticed by the administration.”
David Lammy, the British deputy prime minister, met vice president JD Vance at the White House on Thursday. Talks were expected to centre on a US offer to support security guarantees to protect Ukraine if Russia were to attack again.
Right on cue, Putin's foreign ministry denounced the plans and said any Western troops deployments in Ukraine would be viewed as “legitimate targets”.
A spokeswoman said: “The Russian Foreign Ministry warns that the deployment of military units, military facilities, warehouses, and other infrastructure of Western countries on the territory of Ukraine will be classified as foreign intervention.”
Russia then fired an Oreshnik hypersonic ballistic missile at Lviv. The city is hundreds of miles from the front line but, crucially, just 40 miles from the border with Nato and EU member Poland. Ukraine said it was a test for the transatlantic community.
Mr Trump signalled his personal irritation with Putin during a recent press conference at Mar-a-Lago, Florida. He said: “I'm not thrilled with Putin, he's killing too many people.”
A day later he contradicted Russian claims that Ukraine had targeted one of Putin's residences with drone. He told reporters on Air Force One: “I don't believe that strike happened. We don't believe that happened, now that we've been able to check.”
A former White House official said Mr Trump was also “emboldened” by the special forces raid to capture Nicolás Maduro, the president of Venezuela. He said: “It was the sort of operation that only the United States – or maybe Israel – could have carried out.”
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However, previous moves to get tough on Moscow have often been followed by a relaxing of pressure. And a member of Mr Trump's national security council during his first term said the latest twist was part of the usual ebb and flow of a policy still focused on talking with the Kremlin.
He said: “I think this is an operational versus strategic question. He may push back harder on certain operational things but his strategic desire to end the Ukraine war quickly seems unchanged.”
Anna Kelly, a White House spokeswoman, said Mr Trump's aim remained the same. She said: “President Trump is focused on getting this war settled and ending the senseless killing that was brought on by Joe Biden's incompetence.”
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MOSCOW, January 9. /TASS/. In response to Kiev's attack on President Vladimir Putin's residence, Russian servicemen launched a massive strike, including with the Oreshnik missile, on critical Ukrainian targets, the Defense Ministry reported.
"In the early morning hours, in response to the terrorist attack by the Kiev regime on the residence of the Russian president in the Novgorod Region, carried out in the early morning hours of December 29, 2025, the Russian Armed Forces launched a massive strike with high-precision long-range land-and sea-based weapons, including the Oreshnik intermediate-range ballistic missile, as well as strike drones against critical targets on the territory of Ukraine," the statement said.
According to the ministry, "the strike goals were achieved." "The targets were facilities for producing drones used in the terrorist attack, as well as energy infrastructure supporting the operation of Ukraine's military-industrial complex," the statement noted.
"Any terrorist acts by the criminal Ukrainian regime will continue to be met with a response," the ministry concluded.
PYONGYANG, January 9. /TASS/. North Korean leader Kim Jong Un has sent a reply letter to Russian President Vladimir Putin, in which he pledged to continue cooperation between Russia and the North Korea across various areas, the Korean Central News Agency (KCNA) reported.
"Our close cooperation will continue in various fields in line with the spirit of the comprehensive strategic partnership between the DPRK and the Russian Federation, the strategic interests of the two countries, and the aspirations and will of the two peoples," KCNA quoted the letter.
Kim Jong Un also emphasized the invaluable nature of his relations with the Russian president and pledged eternal support for Putin's policies. "I will absolutely respect and unconditionally support all the policies you pursue and the decisions you make, and I am ready to always stand together for you and for your Russia. This choice is unchanging and will remain eternal," KCNA reported.
MOSCOW, January 9. /TASS/. All issues related to the construction of the Rasht-Astara railway section, including land acquisition for the project, will be resolved by the end of March, Iranian Ambassador to Moscow Kazem Jalali said in an interview with TASS.
"Significant progress has been made in acquiring land plots for the project, and more than 100 km have already been allocated for construction," the ambassador said. "The acquisition process is moving forward daily, and according to the Iranian side, land purchases will be completed by the end of the current Iranian year, which corresponds to late March," he added.
According to him, the Rasht-Astara railway construction project, as the missing link of the western branch of the International North-South Transport Corridor, is "one of the most important projects in bilateral relations and is being actively advanced by the authorities of the two countries at the highest level."
"Fortunately, last year we saw good progress in advancing this project. An addendum to the engineering surveys was signed," the diplomat noted.
In 2023, Russia and Iran reached an agreement under the North-South project to jointly build the 160-km Rasht-Astara railway section, designed to ensure uninterrupted transit to ports in the Persian Gulf. The total cost of the project is estimated at 1.6 bln euro. Moscow and Tehran will jointly finance the design, construction, and the supply of goods and services.
The Syrian Defense Ministry has declared a ceasefire in three neighborhoods of Aleppo, the country's second-largest city, following clashes between government forces and Kurdish-led militias.
The ceasefire, which became effective at 3am local time on Friday, covers Sheikh Maqsoud, Ashrafieh, and Bani Zeid in the north of the city. The Aleppo security command has simultaneously imposed a curfew in several Kurdish-majority areas.
Fighting between the army and the Syrian Democratic Forces (SDF), which controls large parts of northern and eastern Syria, began last week, forcing an estimated 100,000 people to flee their homes.
On Thursday, government troops attempted to advance into Kurdish-held neighborhoods, while Damascus demanded the withdrawal of “militia groups.” The government blamed the SDF for “tensions and instability,” accusing the group of violating a previous accord.
The SDF, in turn, blamed Damascus, asserting that the militias had withdrawn “openly and in a documented manner” months ago and handed control of the neighborhoods to government forces.
The SDF, a longtime key US ally in the war-torn country, said the area was being “besieged” by the army.
Videos from Aleppo show heavy shelling and Syrian troops using drones against the SDF.
Kurdish forces were seen ambushing government troops and firing sniper rifles and machine guns from high-rise buildings.
The Syrian government reached a deal with Kurdish leaders in Aleppo in April granting them autonomy under Damascus' rule.
A broader agreement, meant to reintegrate Kurdish-led civil and military structures into the central government by the end of 2025, has never been implemented; the two sides blame each other.
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RFK Jr.'s new dietary guidelines ignore his own government's findings on the harms of alcohol.
How much alcohol should you drink? The US government now vaguely, in effect, says just don't drink too much. And what qualifies as too much? Well, that's up to you.
As part of the new federal dietary guidelines released this week, the Trump administration eliminated the previous specific recommended limits on alcohol consumption — two drinks or less per day for men and one drink for women. Now, the new guidelines say “consume less alcohol for better health. (It maintained the prior guidance discouraging a few certain groups — pregnant women and people who have a history of alcohol abuse — from drinking at all.) It's a major change that defies a growing public health consensus that people should drink as little alcohol as possible, because no amount of drinking is actually safe.
A newsletter for anyone trying to make sense of their health.
To justify the change, Dr. Mehmet Oz, who oversees the Medicare and Medicaid programs, argued that there was no scientific evidence to justify specific limits on drinking alcohol. “So there is alcohol in these dietary guidelines, but the implication is, don't have it for breakfast,” he said during the announcement of the new guidelines.
“The general move away from two glasses for men, one glass for women — there was never really good data to support that quantity of alcohol consumption,” he added.
That's not true.
There is such data — evidence commissioned by the federal government that the Trump administration itself tried to bury ahead of the dietary guidelines' release, as Vox reported a few months ago. But instead, Oz and Health Secretary Robert F. Kennedy Jr. have handed the alcohol industry a long-sought win in its battle against public health critics.
For the whole sordid saga, you can check out our feature story from September. But here is a brief recap: In early 2022, the Biden administration launched the Alcohol Intake & Health Study, a new report on alcohol and its health effects to inform the next dietary guidelines due in 2025, a response to the increasing evidence that no amount of alcohol is safe. The World Health Organization had made such a declaration in 2023; in the US, more than 170,000 people die every year from alcohol-related causes.
Almost as soon as that project began, the alcohol industry started pushing back and soliciting Congress in its efforts.
In response to this pressure, Congress approved in fall 2023 an alternate study to be overseen by the National Academies of Science and Medicine. Congressional hearings held by the lawmakers, who represented states where alcohol is a major industry, and letters they sent to the Department of Health and Human Services under President Joe Biden on behalf of their constituents framed the original report as a witch hunt against alcohol.
Nonetheless, both studies were undertaken, and their respective authors got to work. In December 2024, the National Academies report came out and stated that, with some very important limitations, the health effects of alcohol were marginal. But a draft version of the Alcohol Intake & Health Study was posted in January 2025, shortly before the end of the Biden administration, and it came to very different conclusions, as I wrote recently:
They broke out their findings by different drinking levels — from one drink per day to three — and focused on health outcomes that have been proven to be associated with alcohol use.
Their big-picture conclusion: Among the US population, the negative health effects of drinking alcohol start at low levels of consumption and begin to increase sharply the more a person drinks.
A man drinking one drink per day has roughly a one in 1,000 chance of dying from any alcohol-related cause, whether an alcohol-associated cancer or liver disease or a drunk driving accident. Increase that to two drinks per day, and the odds increase to one in 25.
That is precisely the kind of evidence that would suggest a specific limit on alcohol consumption would be appropriate — the kind of evidence that Oz claimed does not exist.
The final version of the Alcohol Intake & Health Study was shelved — and still has not been published by the Trump administration. They decided to squash its public release, as I reported last fall, even as they claimed it would be taken into consideration for the forthcoming dietary guidelines.
There was such a furor over that decision that even the authors of the National Academies report later published a commentary in the journal JAMA to make clear that their study should not be over-interpreted to justify more drinking or eliminating limits on drinking alcohol.
Nevertheless, that is exactly what happened in the new dietary guidelines — a policy victory cheered by beer, wine, and liquor manufacturers. The limits are…whatever you want them to be.
“Dr. Oz must have thrown back a few cocktails for breakfast before making that comment,” Mike Marshall, president and CEO of the US Alcohol Policy Alliance, told me. “The federal government's own report, the Alcohol Intake & Health study, made it clear that there is overwhelming evidence that reducing consumption to less than 2 drinks per day dramatically reduces the chance of dying due to alcohol. Just because the industry, via Congress, said ‘don't read it' doesn't mean the report never existed.”
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Live Updates
• Another tanker seized: The US has seized another oil tanker in the Caribbean Sea, according to a source, a move that comes after the US seized two Venezuela-linked tankers earlier this week. US President Donald Trump is set to meet with oil executives today as part of a efforts to persuade America's largest energy companies to return to Venezuela.
• Further attacks halted: Trump said he canceled a “second wave” of attacks on Venezuela, citing the country's cooperation with the US in the week since leader Nicolás Maduro was ousted. The Venezuelan government announced yesterday it will release a “significant number” of political prisoners, a key US demand. Sigue nuestra cobertura en español.
• US oversight in Venezuela: The US Senate yesterday advanced a measure to limit Trump's war powers in Venezuela as the country faces an uncertain future.
Venezuela announced on Friday the start of an “exploratory diplomatic process” with the United States.
The foreign ministry said the process is aimed at “re-establishing diplomatic missions in both countries,” as well as addressing the consequences of what they described as the “aggression and kidnapping” of President Nicolás Maduro and his wife, Cilia Flores.
The ministry confirmed that a delegation of diplomatic officials from the US State Department had arrived in the country, saying they would “conduct technical and logistical assessments.”
They also announced that a delegation of Venezuelan diplomats would travel to the United States “to carry out the corresponding duties.”
“Venezuela will confront this aggression through diplomatic channels,” the ministry concluded, referring to the US pressure campaign against Venezuela.
Colombian President Gustavo Petro says he has asked Venezuela's acting president, Delcy Rodriguez, to work together to fight drug trafficking.
He said on X that Latin America must defend itself from any actor that destabilizes it.
“I have invited the current president of Venezuela so that we can act together in this objective,” he said.
Some context: US President Donald Trump has previously said the US military campaign against Venezuela was aimed at fighting drug trafficking, and has repeatedly threatened to carry out operations against traffickers in Colombia, too, which Petro has opposed.
On Wednesday, US tensions with Colombia seemingly eased after Trump spoke with Petro and invited him to meet at the White House in the “near future”
The human rights organization Foro Penal says it has confirmed the release of eight political prisoners since yesterday's announcement by Venezuelan National Assembly President Jorge Rodríguez that “a significant number” of the prisoners would be released.
Alfredo Romero — who is president of the group, also known as the Venezuelan Penal Forum — said that as of 11 a.m. Caracas time (10 a.m. ET), eight people had been released, including five Spanish citizens, whom CNN has reported on previously.
Romero shared the list of eight individuals on his X account.
Venezuelan opposition leader Juan Pablo Guanipa is not yet on the list, but Romero said he is expected to be among those released.
Opposition leader weighs in: Venezuelan opposition leader Edmundo González wrote on X today that he held a call with Spanish Prime Minister Pedro Sánchez, in which he emphasized that the release of prisoners must be verified and “cannot be selective.”
González and María Corina Machado, the two most prominent figures in the Venezuelan opposition, have repeatedly called for the release of political prisoners following the US attack on Venezuela last weekend.
Members of the Trump administration told US lawmakers that they also made it clear to Venezuela it must release political prisoners, among other demands following the US capture of ousted leader Nicolás Maduro, a source previously told CNN.
This post has been updated with González's comments on X and additional background.
The largest oil reserves of any country on the planet, more than 300 billion barrels, are estimated to lie beneath the ground in Venezuela.
See how the country's oil infrastructure is mapped out below:
President Donald Trump's announcement this morning that Big Oil will invest “at least” $100 billion in Venezuela has caught at least some in the industry by surprise.
“We have no idea where this $100 billion figure is coming from,” one industry source told CNN. “That's the first we've seen of it. It could be that Trump is throwing this number out there to pressure the industry.”
Trump is scheduled to meet with executives from ExxonMobil, Chevron, ConocoPhillips and other companies later today as part of his mission to revive Venezuela's battered oil industry.
However, oil executives are reluctant to invest aggressively in Venezuela for a range of reasons, including security, rule of law and sanctions concerns.
Oil prices are so low that it's not a slam dunk to drill aggressively anywhere, let alone in a risky place with a difficult history like Venezuela.
“There are a lot of investable regions and there's a reason Venezuela is not at the top of the list,” the industry source told CNN.
While Trump may try to persuade oil CEOs to make specific investment promises, the source said companies will be reluctant to detail specifics out of fear of running afoul of antitrust laws.
Mexican President Claudia Sheinbaum described the recent statements made by her US counterpart, Donald Trump, about possible ground attacks on cartels as the US president's “way of communicating” and said that, if necessary, they will speak with him “to strengthen coordination.”
“Because of the statements President Trump has made in recent days, which we consider part of his communication style, I asked Foreign Minister Juan Ramón de la Fuente yesterday to make direct contact with the Secretary of State and, if necessary, speak with President Trump to strengthen coordination,” Sheinbaum said at her morning press conference.
Trump said on Thursday that his administration will soon begin actions to target cartels on the ground and made reference to Mexico.
“We've eliminated 97% of the drugs coming in by water, and now we're going to start attacking them on land, in relation to the cartels,” Trump said on Fox News.
“The cartels are controlling Mexico,” he added. “It's very, very sad to see what's happened to that country.”
A US State Department team traveled to Venezuela on Friday for the first time since the ouster of Nicolás Maduro, according to a US official.
The visit comes as the US looks to reopen its embassy in Caracas and underscores the administration's desire to re-establish a diplomatic presence within the country that President Donald Trump says the US is going to “run.”
The official said that US diplomatic and security personnel from the Venezuela Affairs Unit, which is based in Colombia, and the acting US ambassador to Colombia John McNamara, traveled to the Venezuelan capital “to conduct an initial assessment for a potential phased resumption of operations.”
The US withdrew its diplomats and suspended operations at the embassy in 2019. The Venezuela Affairs Unit has been operating with a team of US diplomats at the embassy in Bogota.
A senior State Department official said Monday the department was “making preparations to allow for a reopening” of its embassy in Venezuela “should the president make that decision.”
Former diplomats said that a lack of US presence on the ground would pose a challenge to rebuilding and ensuring accountability in Venezuela. President Donald Trump has said he wants to see US oil companies reopen their operations in the country and help with its rebuilding. He said earlier this week the US was “asked” to reopen the embassy but did not provide further details.
The US has seized another oil tanker in the Caribbean Sea, US Southern Command said on Friday.
US troops in coordination with the Department of Homeland Security “launched from the USS Gerald R. Ford and apprehended Motor/Tanker Olina in the Caribbean Sea without incident,” the command said in a social media post.
The Olina tanker was sailing near Venezuela under a Timor-Leste flag, according to Marine Traffic.
It comes after the US seized two Venezuela-linked tankers this week, including a Russian-flagged vessel in the Atlantic Ocean and another tanker in the Caribbean.
The White House said earlier this week that President Donald Trump was “not afraid” to continue seizing sanctioned oil tankers despite concerns that it could ratchet up tensions with Russia and China.
“He's going to enforce our policy that's best for the United States of America,” press secretary Karoline Leavitt told reporters at a press briefing on Wednesday. “That means enforcing the embargo against all dark fleet vessels that are illegally transporting oil.”
President Donald Trump has no public events today, but he will be meeting with some top administration members and oil executives.
The following are closed to the press, but we'll update you if we get any more information:
Trump will then head to Palm Beach, Florida, for the weekend.
President Donald Trump is meeting with more than a dozen oil executives at the White House today as part of his effort to convince the energy companies to invest in rebuilding Venezuela's oil infrastructure.
Secretary of State Marco Rubio, who has been leading the Trump administration's strategy toward Caracas; Energy Secretary Chris Wright, who has already met with some oil executives this week; and Interior Secretary Doug Burgum will join the president for the meeting, a White House official said.
“Following the announcement of President Trump's historic energy deal with Venezuela, American oil companies will come to the White House to discuss investment opportunities that will restore Venezuelan oil infrastructure,” White House spokesperson Taylor Rogers told CNN.
Among the companies sending representatives to the White House include Chevron, ExxonMobil, Conoco Phillips, Halliburton, Valero, Marathon and Shell, among others.
President Donald Trump said earlier today that he had canceled a “previously expected” second wave of attacks on Venezuela due to the country's cooperation with the United States.
It comes nearly a week after he ordered a military operation to oust Venezuelan President Nicolás Maduro, who is currently in US custody along with his wife Cilia Flores.
Shortly after that military operation, Trump said in a news conference, “We are ready to stage a second and much larger attack if we need to do so. … We actually assumed that a second wave would be necessary, but now it's probably not.”
In Friday's Truth Social post, Trump said the US and Venezuela are “working well together, especially as it pertains to rebuilding, in a much bigger, better, and more modern form, their oil and gas infrastructure.”
“Because of this cooperation, I have cancelled the previously expected second Wave of Attacks, which looks like it will not be needed, however, all ships will stay in place for safety and security purposes,” he added.
Trump went on to say that Venezuela was “releasing large numbers of political prisoners as a sign of ‘Seeking Peace,'” adding, “This is a very important and smart gesture.”
Venezuela began releasing the high-profile prisoners on Thursday, including opposition politicians in an effort to “seek peace,” the acting government said.
President Donald Trump said Thursday that his administration will soon begin actions to target cartels on land, following the operation to capture Venezuela's leader to face drug trafficking charges and months of strikes on alleged drug boats in the Caribbean and Eastern Pacific.
“We've knocked out 97% of the drugs coming in by water, and we are gonna start now hitting land, with regard to the cartels,” Trump said on Fox News.
“The cartels are running Mexico,” he added. “It's very, very sad to watch and see what's happened to that country.”
The president offered no further details. CNN has reached out to the White House for additional information.
Trump earlier this week said he had asked Mexican President Claudia Sheinbaum if she wanted the US military's help in rooting out drug cartels, warning that “Mexico has to get their act together.”
Sheinbaum has remained opposed to the effort, telling reporters Monday after the US operation in Venezuela: “We categorically reject intervention in the internal affairs of other countries.”
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FILE -A Meta logo is shown on a video screen at LlamaCon 2025, an AI developer conference, in Menlo Park, Calif., April 29, 2025. (AP Photo/Jeff Chiu, File)
Meta has cut a trio of deals to power its artificial intelligence data centers, securing enough energy to light up the equivalent of about 5 million homes.
The parent company of Facebook on Friday announced agreements with TerraPower, Oklo and Vistra for nuclear power for its Prometheus AI data center that is being built in New Albany, Ohio. Meta announced Prometheus, which will be a 1-gigawatt cluster spanning across multiple data center buildings, in July. It's anticipated to come online this year.
Financial terms of the deals with TerraPower, Oklo and Vistra were not disclosed.
The Mark Zuckerberg-led Meta said in a statement on Friday that the three deals will support up to 6.6 gigawatts of new and existing clean energy by 2035. A single gigawatt, according to a general industry standard for utilities, can power about 750,000 homes.
“These projects add reliable and firm power to the grid, reinforce America's nuclear supply chain, and support new and existing jobs to build and operate American power plants,” the company said.
Meta said its agreement with TerraPower will provide funding that supports the development of two new Natrium units capable of generating up to 690 megawatts of firm power with delivery as early as 2032. The deal also provides Meta with rights for energy from up to six other Natrium units capable of producing 2.1 gigawatts and targeted for delivery by 2035.
Meta will also buy more than 2.1 gigawatts of energy from two operating Vistra nuclear power plants in Ohio, in addition to the energy from expansions at the two Ohio plants and a third Vistra plant in Pennsylvania.
The deal with Oklo, which counts OpenAI's Sam Altman as one of its largest investors, will help to develop a 1.2 gigawatt power campus in Pike County, Ohio to support Meta's data centers in the region.
The nuclear power agreements come after Meta announced in June that it reached a 20-year deal with Constellation Energy.
Copyright 2026 The Associated Press. All Rights Reserved.
Copyright 2026 The Associated Press. All Rights Reserved.
Copyright 2026 The Associated Press. All Rights Reserved.
Russia said Friday it has used the new Oreshnik ballistic missile along with other weapons in a massive strike on Ukraine. (AP video by Vasilisa Stepanenko)
A residential building burns after a Russian strike in Kyiv, Ukraine, Friday, Jan. 9, 2026. (AP Photo/Efrem Lukatsky)
The dead body of a paramedic lies on the ground in front of a residential building damaged by a Russian strike on Kyiv, Ukraine, Friday, Jan. 9, 2026. (AP Photo/Evgeniy Maloletka)
This photo provided by the Ukrainian Security Service on Friday, Jan. 9, 2026, shows a fragment believed to be a part of a Russian Oreshnik intermediate range hypersonic ballistic missile that hit the Lviv region. (Ukrainian Security Service via AP)
A residential building is seen damaged after a Russian strike in Kyiv, Ukraine, Friday, Jan. 9, 2026. (AP Photo/Efrem Lukatsky)
President of Ukraine Volodymyr Zelenskyy, left, and British Defense Secretary John Healey talk in Kyiv, Ukraine, Friday, Jan. 9, 2026. (AP Photo/Danylo Antoniuk)
KYIV, Ukraine (AP) — Russia bombarded Ukraine with hundreds of drones and dozens of missiles in a large-scale overnight attack, officials said Friday, killing at least four people in the capital. For only the second time in the nearly 4-year-old war, it used a powerful, new hypersonic missile that struck western Ukraine in a clear warning to Kyiv's NATO allies.
The intense barrage and the launch of the nuclear-capable Oreshnik missile came days after Ukraine and its allies reported major progress toward agreeing on how to defend the country from further Moscow aggression if a U.S.-led peace deal is struck.
Europe's leaders condemned the attack as “escalatory and unacceptable,” and the European Union's top foreign policy envoy said Russian President Vladimir Putin's reply to diplomacy was “more missiles and destruction.”
The attack also coincides with a new chill in relations between Moscow and Washington after Russia condemned the U.S. seizure of an oil tanker in the North Atlantic. It comes as U.S. President Donald Trump signaled he is on board with a hard-hitting sanctions package meant to economically cripple Moscow, which has given no public signal it is willing to budge from its maximalist demands on Ukraine.
Ukrainian officials said four people were killed and at least 25 wounded in Kyiv as apartment buildings were struck overnight.
Those killed included an emergency medical aid worker, according to Kyiv City Military Administration head Tymur Tkachenko. Four doctors and one police officer were injured while responding to the attacks, authorities said.
About half of snowy Kyiv's apartment buildings — nearly 6,000 — were left without heat amid daytime temperatures of about minus 8 degrees Celsius (17.6 Fahrenheit), Mayor Vitali Klitschko said. Water supplies also were disrupted.
AP correspondent Charles de Ledesma reports Ukraine says it has proof Russia's latest barrage included an Oreshnik ballistic missile.
Municipal services restored power and heat to public facilities, including hospitals and maternity wards, using portable boiler units, he said
The attack damaged the Qatari Embassy in Kyiv, according to President Volodymyr Zelenskyy, who noted that Qatar has played a key role in mediating the exchange of prisoners of war.
He called for a “clear response” from the international community, particularly from the U.S., which he said Russia takes seriously.
“I am devastated to see the scale of the attacks that you've suffered overnight. Putin's attacks are brutal, they're cynical,” said U.K. Defense Secretary John Healey, who was visiting Kyiv and noted the Russian drones were aimed at residential blocks.
Ukraine's Security Service said it identified debris from the Oreshnik missile in the Lviv region in the country's west. It was fired from Russia's Kapustin Yar test range near the Caspian Sea in southwestern Russia and targeted civilian infrastructure, investigators said.
Russia's Defense Ministry said the attack was a retaliation to what Moscow claimed was a Ukrainian drone strike on one of Putin's residences last month. Both Trump and Ukraine rejected the Russian claim.
Moscow didn't say where the Oreshnik hit, but Russian media and military bloggers said it targeted an underground natural gas storage facility in the Lviv region. Western military aid flows to Ukraine from a supply hub in Poland just across the border.
Putin has previously said the Oreshnik streaks to its target at Mach 10, “like a meteorite,” and is immune to any missile defense system. Several of them used in a conventional strike could be as devastating as a nuclear attack, according to Putin, who has warned the West that Russia could use it against allies of Kyiv that allow it to strike inside Russia with longer-range missiles.
Ukrainian intelligence says the missile has six warheads, each carrying six submunitions.
Russia first used the Oreshnik missile on the Ukrainian city of Dnipro in November 2024. Analysts say it gives Russia a new element of psychological warfare, unnerving Ukrainians and intimidating Western countries that aid Ukraine.
Ukrainian Foreign Minister Andrii Sybiha said Ukraine would be initiating international action in response to the use of the missile, including an urgent meeting of the U.N. Security Council and a meeting of the Ukraine-NATO Council.
“Such a strike close to EU and NATO border is a grave threat to the security on the European continent and a test for the transatlantic community. We demand strong responses to Russia's reckless actions,” he said in a post on X.
Pope Leo XIV, speaking at the Vatican, urged the international community to keep pushing for peace and end the suffering in Ukraine.
“Faced with this tragic situation, the Holy See strongly reiterates the pressing need for an immediate ceasefire, and for dialogue motivated by a sincere search for ways leading to peace,” the pontiff told ambassadors to the Vatican from around the world.
The leaders of Britain, France and Germany said they spoke about the attack and deemed it “escalatory and unacceptable.”
EU foreign policy chief, Kaja Kallas, said the Oreshnik launch was “meant as a warning to Europe and to the U.S.”
“Putin doesn't want peace, Russia's reply to diplomacy is more missiles and destruction,” Kallas wrote on social media.
Several districts in Kyiv were hit in the overnight attack, according to Tkachenko, the city's military administration chief. In the Desnyanskyi district, a drone crashed onto the roof of a multistory building and the first two floors of another residential building were damaged.
In the Dnipro district, parts of a drone damaged a multistory building and a fire broke out.
Dmytro Karpenko's windows were shattered in the attack on Kyiv. When he saw that his neighbor's house was burning, he rushed to help him.
“What Russia is doing, of course, shows that they do not want peace. But people really want peace, people are suffering, people are dying,” the 45-year old said.
___
A previous version of this story corrected the style on Andriy Sadoviy to Andrii Sadovyi.
___
Vasilisa Stepanenko in Kyiv, Ukraine, and Kirsten Grieshaber in Berlin contributed.
___
Follow AP's coverage of the war in Ukraine at https://apnews.com/hub/russia-ukraine
Copyright 2026 The Associated Press. All Rights Reserved.
Copyright 2026 The Associated Press. All Rights Reserved.
Copyright 2026 The Associated Press. All Rights Reserved.
Hundreds of protesters marched down one of Minneapolis' major thoroughfares Thursday night to voice outrage over Wednesday's shooting of a woman by a U.S. Immigration and Customs Enforcement officer. (AP video by: Mike Householder)
Hundreds gathered outside a U.S. Immigration and Customs Enforcement building in Portland, Oregon, on Thursday in protest after authorities said federal immigration officers shot and wounded two people in a vehicle outside a hospital.
Federal officials claim an immigration agent, who has not been identified, acted in self-defense when shooting and killing a Minneapolis driver. A Chicago law professor offers context on when lethal force is legally justified. (AP video: Laura Bargfeld)
Minneapolis Mayor Jacob Frey said he's dismayed that the FBI and Justice Department have blocked the state out of the investigation into an Immigration and Customs Enforcement officer's fatal shooting of a Minneapolis woman.(AP video: Mike Householder and Mark Vancleave)
People gather at makeshift memorial for woman shot by ICE in Minneapolis; Bovino arrives on scene.
Protesters gather during a rally for Renee Good, Thursday, Jan. 8, 2026, in Minneapolis, after she was fatally shot by an ICE officer the day before. (AP Photo/Adam Bettcher)
Protesters chant and march during a rally for Renee Good, who was fatally shot by an ICE officer the day before, Thursday, Jan. 8, 2026, in Minneapolis. (AP Photo/John Locher)
Protesters sit on a barrier that is being assembled outside the Bishop Henry Whipple Federal Building as protesters gather in Minneapolis, Friday, Jan. 9, 2026. (AP Photo/Adam Bettcher)
Protesters are arrested by federal agents outside the Bishop Henry Whipple Federal Building in Minneapolis, Friday, Jan. 9, 2026. (AP Photo/Adam Bettcher)
Two protesters are lit by a police light as they walk outside the U.S. Immigration and Customs Enforcement facility on Thursday, Jan. 8, 2026, in Portland, Ore. (AP Photo/Jenny Kane)
An American flag burns outside the U.S. Immigration and Customs Enforcement facility on Thursday, Jan. 8, 2026, in Portland, Ore. (AP Photo/Jenny Kane)
MINNEAPOLIS (AP) — Hennepin County Attorney Mary Moriarty on Friday called on members of the public to send any video or other evidence in the fatal shooting of Renee Good directly to her office, despite the Trump administration's decision to hand the investigation solely to the FBI.
Moriarty said that although her office has collaborated effectively with the FBI in past cases, she is concerned by the Trump administration's decision to bar state and local agencies from playing any role in the investigation into Wednesday's killing of Good by an Immigration and Customs Enforcement officer in Minneapolis. Specifically, she said she's worried the FBI won't share evidence with state investigators.
Moriarty said she isn't sure what legal outcome the evidence her office receives from the public might produce. But she said her office is responsible for the investigation, despite the Trump administration's decision to assign it solely to the FBI.
“We do have jurisdiction to make this decision with what happened in this case,” she said. “It does not matter that it was a federal law enforcement agent.”
She also said that despite the Trump administration's insistence that the officer who shot Good has complete legal immunity, that isn't the case.
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Moriarty also said that her office would offer a link for the public to submit videos that captured the fatal shooting.
The prosecutor's news conference came as another round of protests were planned for Friday in Minneapolis over the Good's killing and a day after federal immigration officers shot and wounded two people in Portland, Oregon.
Hundreds of people protesting the Wednesday shooting of Renee Good marched in freezing rain Thursday night down one of Minneapolis' major thoroughfares, chanting “ICE out now!” and holding signs saying, “Killer ice off our streets.” The day began with a charged demonstration outside of a federal facility that is serving as a hub for the immigration crackdown in the Twin Cities of Minneapolis and St. Paul. Authorities erected barricades outside the facility Friday.
City workers, meanwhile, removed barricades made of old Christmas trees and other debris that had been blocking the streets near where the ICE officer shot Good as she tried to drive away. Officials said they would allow a makeshift shrine to the 37-year-old mother of three to remain.
The shooting in Portland, Oregon, took place outside a hospital Thursday afternoon. A man and woman, identified by the Department of Homeland Security as Venezuela nationals Luis David Nico Moncada and Yorlenys Betzabeth Zambrano-Contreras, were shot inside a vehicle, and their conditions were not immediately known. The FBI and the Oregon Department of Justice were investigating.
Portland Mayor Keith Wilson and the city council called on ICE to end all operations in the city until a full investigation is completed. Hundreds protested Thursday night at a local ICE building. Early Friday, Portland police reported that officers had arrested several protesters after asking the to move from the street to the sidewalk, to allow traffic to flow.
Just as it did following Good's shooting, DHS defended the actions of the officers in Portland, saying it occurred after a Venezuelan man with alleged gang ties and who was involved in a recent shooting tried to “weaponize” his vehicle to hit the officers. It wasn't immediately clear if the shootings were captured on video, as Good's was.
AP correspondent Ben Thomas reports the Trump administration's immigration crackdown is generating more protests and another shooting.
Homeland Security Secretary Kristi Noem, President Donald Trump and others in his administration have repeatedly characterized the Minneapolis shooting as an act of self-defense and cast Good as a villain, suggesting she used her vehicle as a weapon to attack the officer who shot her.
But state and local officials and protesters rejected that characterization, with Minneapolis Mayor Jacob Frey saying video recordings show the self-defense argument is “garbage.”
The Minneapolis shooting happened on the second day of the immigration crackdown in the Twin Cities, which Homeland Security said is the biggest immigration enforcement operation ever. More than 2,000 officers are taking part and Noem said they have made more than 1,500 arrests.
The government is also shifting immigration officers to Minneapolis from sweeps in Louisiana, according to documents obtained by The Associated Press. This represents a pivot, as the Louisiana crackdown that began in December had been expected to last into February.
Good's killing provoked an immediate response in the city where police killed George Floyd in 2020, with hundreds of people turning up to the scene to vent their outrage at the ICE officers and the school district canceling classes for the rest of the week as a precaution.
Her death — at least the fifth tied to immigration sweeps since Trump took office — has resonated far beyond Minneapolis, as protests happening in other places, including Texas, California, Detroit and Missouri.
In Washington, D.C., on Thursday, a woman held a sign that said, “Stop Trump's Gestapo,” as hundreds of people marched to the White House. Protesters in Pflugerville, Texas, north of Austin, banged on the walls of an ICE facility. And a man in Los Angeles burned an American flag in front of federal detention center.
A day before Moriarty called on the public to help her office investigate Good's killing, the Minnesota agency that investigates officer-involved shootings said it was told that the FBI and U.S. Justice Department would not work with it, effectively ending any role for the state to determine if crimes were committed. Noem said the state has no jurisdiction.
“Without complete access to the evidence, witnesses and information collected, we cannot meet the investigative standards that Minnesota law and the public demands,” Drew Evans, head of the Minnesota Bureau of Criminal Apprehension, said Thursday.
Minnesota Gov. Tim Walz demanded that the state be allowed to take part, repeatedly emphasizing that it would be “very difficult for Minnesotans” to accept that an investigation excluding the state could be fair.
Several bystanders captured video of Good's killing, which happened in a neighborhood south of downtown.
The recordings show an officer approaching an SUV stopped across the middle of the road, demanding the driver open the door and grabbing the handle. The Honda Pilot begins to pull forward and a different ICE officer standing in front of it pulls his weapon and immediately fires at least two shots at close range, jumping back as the vehicle moves toward him.
It is not clear from the videos if the vehicle makes contact with the officer, and there is no indication of whether the woman had interactions with agents earlier. After the shooting, the SUV speeds into two cars parked on a curb before crashing to a stop.
The federal agent who fatally shot Good is an Iraq War veteran who has served for nearly two decades in the Border Patrol and ICE, according to records obtained by AP.
Noem has not publicly named him, but a Homeland Security spokesperson said her description of his injuries last summer refers to an incident in Bloomington, Minnesota, in which court documents identify him as Jonathan Ross.
Ross got his arm stuck in the window of a vehicle whose driver was fleeing arrest on an immigration violation. Ross was dragged and fired his Taser. A jury found the driver guilty of assaulting a federal officer with a dangerous weapon.
Attempts to reach Ross, 43, at phone numbers and email addresses associated with him were not successful.
___
Associated Press reporters Steve Karnowski and Mark Vancleave in Minneapolis; Ed White in Detroit; Valerie Gonzalez in Brownsville, Texas; Graham Lee Brewer in Norman, Oklahoma; Michael Biesecker in Washington; Jim Mustian in New York; Ryan Foley in Iowa City, Iowa; and Hallie Golden in Seattle contributed to this report.
Copyright 2026 The Associated Press. All Rights Reserved.
Copyright 2026 The Associated Press. All Rights Reserved.
Copyright 2026 The Associated Press. All Rights Reserved.
A victim with burned hands and relatives attend the official commemorative ceremony for the victims of the deadly fire at the “Le Constellation” bar in Crans-Montana, in Martigny, Switzerland, Friday, Jan. 9, 2026. (Laurent Gillieron/Keystone/Pool via AP)
People gather to watch the official commemorative ceremony on a giant screen in front of the St. Christopher Chapel in Crans-Montana during the national day of mourning following the deadly fire at the “Le Constellation” bar in Crans-Montana, Switzerland, Friday, Jan. 9, 2026. (Alessandro della Valle/Keystone via AP)
Swiss Federal President Guy Parmelin, left, delivers his statement as Former Grand Duke of Luxembourg Henri, left, and Emmanuel Macron, right, President of France appear on the screen, during the official commemorative ceremony for the victims of the deadly fire at the “Le Constellation” bar in Crans-Montana, in Martigny, Switzerland, Friday, Jan. 9, 2026. (Laurent Gillieron/Keystone/Pool via AP)
People observe a minute of silence on the national day of mourning, at the main station in Zurich, Switzerland, Friday Jan. 9, 2026, following the deadly fire at the “Le Constellation” bar in Crans-Montana. (Andreas Becker/Keystone via AP)
The owners of the “Le Constellation” bar in Crans-Montana, where the deadly fire happened on New Year's Day, Jacques und Jessica Moretti from France, center, arrive with their lawyers Patrick Michod, Yael Hayat and Nicola Meier, to be auditioned by the Valais public prosecutor's office in Sion, Switzerland, Friday, January 9, 2026. (Jean-Christophe Bott/Keystone via AP)
MARTIGNY, Switzerland (AP) — Switzerland held a national day of mourning on Friday for the 40 people who died in an Alpine bar fire during a New Year's Eve celebration, as prosecutors requested one of the managers to be placed in pre-trial detention.
Valais region's chief prosecutor Beatrice Pilloud said in a statement the detention of the man was needed to avoid a “risk of flight.” The man's wife and co-manager will remain free under judicial supervision, the statement said.
A Swiss business register lists French couple Jacques and Jessica Moretti as the owners of Le Constellation bar, in the Alpine resort of Crans-Montana, where a fire broke out less than two hours after midnight on Jan. 1. As well as the fatalities, 116 people were injured, many of them seriously.
Local media reported that Moretti was being held in custody pending the court's decision after the couple were questioned by prosecutors in Sion on Friday morning.
Swiss authorities have opened a criminal investigation into the owners, who are suspected of involuntary homicide, involuntary bodily harm and involuntarily causing a fire.
A memorial service and a minute's silence marked Friday's national homage, while church bells across Switzerland rang out for five minutes, beginning at 2 p.m.. Across the country, people gathered to light candles, put down flowers for the victims and followed the national ceremony that was livestreamed on public television.
Speaking at the memorial ceremony in Martigny, Swiss President Guy Parmelin said that “the memory of that terrible night illuminates the faces of the 156 victims, their happy days, their carefree spirit.”
He added: “Our country is appalled by this tragedy. It bows before the memory of those who are no longer with us. It stands by the bedside of those who are about to embark on a long road to recovery.”
Investigators have said they believe sparkling candles atop Champagne bottles ignited the fire when they came too close to the ceiling. Authorities are looking into whether soundproofing material on the ceiling conformed with regulations and whether the candles were permitted for use in the bar. Fire safety inspections hadn't been carried out since 2019.
The severity of burns made it difficult to identify some victims, requiring families to supply authorities with DNA samples. Police have said many of the victims were in their teens to mid-20s.
An autopsy has been ordered for five of the six Italian victims and has been delegated to the prosecutors' offices in Milan, Bologna, and Genoa, where the bodies of the victims have been returned.
“What happened is not a disaster: It's the result of too many people who didn't do their job or who thought they were making easy money,” Italian Premier Giorgia Meloni said during a press conference on Friday. “Those responsible must be identified and prosecuted.”
Meloni said the State Attorney General's Office has contacted the Swiss Attorney General to follow the investigation. She also confirmed that the Rome Prosecutor's Office has started a separate probe.
“The families have my word that they will not be left alone while they seek justice,” she added.
The Paris prosecutor's office Monday announced that it was opening a probe to assist the Swiss investigation and make it easier for families of French victims to communicate with Swiss investigators. Nine French citizens were killed, the youngest of them aged 14, and 23 others were injured.
Copyright 2026 The Associated Press. All Rights Reserved.
U.S. officials are building a case that ongoing measles outbreaks in South Carolina and other states are unrelated to last year's major outbreak in Texas, as it tries to retain its status of having eliminated the disease after recording the highest number of confirmed infections in three decades.
To be considered measles-free by the World Health Organization, a country must have no locally transmitted cases of the same strain for 12 months or longer.
Scientists studying the issue say the elimination assessment may not be ironclad as it is difficult to be certain that all cases potentially linked to the Texas outbreak have been reported.
A large outbreak of measles in Texas kicked off 2025 in January and spread to several states, followed by large outbreaks along the Arizona-Utah border and in South Carolina that continue to cause infections.
As a measles outbreak burns through South Carolina, not enough people are getting vaccinated
In November, the Pan American Health Organization - part of the WHO - determined that Canada had lost its measles elimination status after nearly three decades due to its failure to curb a year-long outbreak of the vaccine-preventable disease.
PAHO this year will evaluate U.S. measles data, which involves 2,065 confirmed cases, to see if it can keep the measles elimination status it has had since 2000.
Former U.S. public health officials have described measles elimination status as a key indicator of a nation's health.
The United States will have to prove the ongoing measles cases are not related to the Texas outbreak, and were instead brought into the country from infected travelers, said William Moss, an epidemiology professor at the Johns Hopkins Bloomberg School of Public Health.
State public health officials have used traditional methods of interviewing infected individuals to identify chains of transmission, as well as comparing the viral genotype - a section of the virus' DNA - to see if cases are related. That still may not be sufficient to link cases in a large outbreak, experts said.
Kelly Oakeson, who leads next-generation sequencing at the Utah Public Health Laboratory, said none of the patients interviewed in Utah mentioned travel to Texas or contact with people from Texas, but many gave incomplete information.
Tracking measles cases in the United States
Based on the state's detailed genetic analysis, however, he said the Utah strain is “different enough” from the Texas outbreak to suggest they are unrelated.
“We don't think there is a direct link,” he said in an email, adding that the state has been working with CDC, Texas and Arizona to characterize transmission patterns.
In addition, the Centers for Disease Control and Prevention has been analyzing the entire genetic code of virus samples to determine if the outbreaks are related, a newer technique that has not traditionally been used to assess measles transmission.
Early evidence from the CDC analysis, which has not been made public, suggests the outbreaks are not related, according to two sources who have seen the data.
In a December 5 post on X, CDC Acting Director Jim O'Neill said there was no epidemiological evidence linking the Texas outbreak and the ongoing cases in South Carolina.
A U.S. Health and Human Services spokesman confirmed that the CDC has found no epidemiological evidence linking ongoing outbreaks to Texas, but said many recent U.S. cases share the same genotype and have no known source of infection, “which could indicate ongoing domestic transmission.”
Dr. Noel Brewer, an infectious disease doctor at the University of North Carolina who has seen the CDC data, said the preliminary evidence suggests that new cases in Texas stopped in July and there was a three-week gap before the Utah-Arizona outbreak began.
“No cases linked the two outbreaks in any clear way,” said Brewer, who chairs an independent committee that will analyze the U.S. data and make a report to PAHO on whether it agrees with the U.S. assessment.
The CDC's inability to link the outbreaks, Brewer said, may simply reflect that the virus is circulating broadly in the United States and that it is “not possible at this time to track all of the cases.”
There are many gaps in epidemiological knowledge in the U.S. outbreaks and not all cases are identified or reported, said Johns Hopkins' Moss, so you can miss cases linking two outbreaks.
In addition, many of the larger U.S. outbreaks are occurring in communities that don't trust the public health system and may be less likely to report cases or participate in an investigation.
That could lead scientists to incorrectly conclude that two viruses are different when it was just a sampling problem, Moss said.
Dr. Demetre Daskalakis, former CDC director of the National Center for Immunization and Respiratory Diseases who quit in August over concerns about U.S. vaccine policy under Health Secretary Robert F. Kennedy Jr., said PAHO will make the call based on the evidence it receives.
“If they don't believe in the strength of one part of the data, and there are other compelling factors that say that the U.S. has lost elimination, it means that the U.S. has lost elimination.”
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How ambitious is a MAGA woman allowed to be?
As President Donald Trump settles into his last term, his approval ratings sinking ever lower, an urgent new question has begun to coalesce at the center of MAGA: Would the movement survive without Trump's force of personality? What kind of person has the juice to lead it besides Trump?
While the question remains unanswered, a new archetype has abruptly acquired stature among the MAGA faithful, an archetype that is in some ways a photographic negative of crass, mud-slinging, macho Trump. As Erika Kirk strives to take over her husband's legacy at Turning Point USA, and Marjorie Taylor Greene remakes her image amid her retirement from the House of Representatives, both are building their image around the idea of a Christian woman who is notable for her godliness, grace, and mercy.
Erika Kirk rocketed to political stardom at the September memorial service for her late husband, Charlie Kirk, when she publicly forgave his assassin.
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“I forgive him because it was what Christ did and is what Charlie would do,” she said. “The answer to hate is not hate. The answer we know from the gospel is love and always love. Love for our enemies and love for those who persecute us.”
Trump, speaking immediately after Kirk, made a point of saying in response that he hated his enemies and would never forgive them.
The reaction from the mainstream media was polite and respectful. While Trump has profited from his alliance with the Christian right, he's never embodied any of the movement's traditional virtues. Kirk, outlets declared, showed what Christian forgiveness really could look like.
“The most admirable aspects of religion — mercy, charity, grace and contemplation — were found in Mrs. Kirk's words, not Mr. Trump's,” wrote Zaid Jilani for the New York Times. “I can only pray that today's Christian right finds more inspiration in her than him.”
On the right, the response went beyond polite respect. Kirk was suddenly in the conversation as a major leader on the MAGA right, like a funhouse mirror image of Barack Obama after his keynote speech at the 2004 DNC.
“With her powers of communication, moving story, and personal connection, Erika Kirk could end up the next Billy Graham. She could lead a generation to Christianity. She could be the first woman president,” wrote Matthew Continetti at the Free Press. A post on the r/Christianity subreddit described Kirk as “a new archetype of the conservative Christian woman: graceful, media-savvy, intellectually aligned, and unapologetically committed to a vision of biblical womanhood that is both traditional and powerfully public.” The question, the poster added, wasn't “if she will become a major leader, but how quickly.”
Among Kirk's new fans was Marjorie Taylor Greene. Speaking to the New York Times in December, Greene cited the contrast between Kirk and Trump at Charlie Kirk's memorial as the reason for her recent turn on Trump. “It just shows where his heart is,” Greene said. “And that's the difference, with her having a sincere Christian faith, and proves that he does not have any faith.”
Inspired, Greene reportedly texted a friend, “I wanted to be more like Christ.” She has since publicly apologized for “taking part in the toxic politics,” while remaining vague about what parts of her behavior she actually feels the need to apologize for.
As religious studies scholar Katherine Kelaidis has written for Vox, the forgiving Christian woman is a familiar archetype within faith narratives, going back to the medieval concept of the forgiving queen who intercedes with a vengeful king. “It is a model that allows the language of Christian mercy to coexist with the harsh realities of authoritarian rule, which is exactly what MAGA is aiming for,” Kelaidis explains. “It was also a model that allowed for women to have a significant public role while not transgressing normative ideas of femininity. Say, for example, Erika Kirk becoming the CEO of Turning Point USA.”
What gives the archetype its juice in this particular moment is that it is an inverse to everything Trump embodies: principled where Trump is vindictive, peaceful where he is violent, religiously motivated where he is plainly secular. In theory, the archetype of the forgiving Christian woman allows MAGA to make a show of installing women in highly visible roles that can appeal to young women, without necessarily threatening the power of the men at the center. Yet the fact that both Kirk and Greene have made a point of adopting this image during moments of transition in their image suggests — without making any claims as to the sincerity of their faith — that they have both made the calculation that MAGA wants a change from Trump as the movement develops beyond his presidency.
That their threat to Trump's power is real can perhaps be seen through the backlash both Greene and Kirk have faced as they make their moves. As Erika Kirk spends ever more time in public, a narrative has begun to brew that she is using her husband's death opportunistically to climb the political ladder. She's become the center of a derisive meme. “Everyone grieves differently,” someone will post, or “Normal widows: ‘I miss my husband.'” And then, “Erika Kirk:” above an image of a woman dancing and setting off fireworks.
Trump, meanwhile, has dubbed Greene “Marjorie ‘Traitor' Greene.“ She has never cultivated many congressional allies outside the MAGA faithful, and now she's isolated even from them. “I'm, like, radioactive” on both sides of the political aisle, Greene told the New York Times, adding that she plans to retire from politics for good.
The central question about MAGA since its emergence as a force in American politics has been: Is the movement simply a cult of personality built around Donald Trump and his whims? Or is it a genuine political coalition with real principles and a coherent ideology? If the movement embraces and elevates a figure whose image is built in opposition to Trump's, we may finally have an answer to that question.
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President Donald Trump announced he has canceled further military strikes planned on Venezuela on Friday morning.
Trump announced the decision in a social media post on his Truth Social account. The “second wave of attacks” was not publicly known, and it is currently unknown when they were supposed to have occurred. Trump said he gave the reprieve to Venezuela due to the synergy between collaborative efforts between his administration and the acting Venezuelan President, Delcy Rodriguez, and her administration. The impetus for Trump's decision appears to be Venezuela's release of political prisoners.
“Venezuela is releasing large numbers of political prisoners as a sign of ‘Seeking Peace,'” Trump said in his post. “This is a very important and smart gesture.”
Venezuela's interim government announced it would free an “important number” of political prisoners held under the former Maduro regime, the Washington Examiner previously reported. The freeing of these prisoners was announced by Jorge Rodriguez, the brother of Venezuela's new leader. He stressed the importance of freeing the prisoners as a matter conducive to “unity and coexistence.”
“In order to contribute and collaborate in the effort that all of us must make for national unity and peaceful coexistence, the Bolivarian government, together with the institutions of the state, has decided to free an important number of Venezuelan and foreign individuals,” Rodriguez said on Thursday.
Trump expressed optimism about the joint efforts in rebuilding Venezuela so far, stating that the interim administration and the U.S. are “working well together.” He then stated that it was due to these acceptable cooperative efforts that he decided to call off the previously unknown strikes.
“The U.S.A. and Venezuela are working well together, especially as it pertains to rebuilding, in a much bigger, better, and more modern form, their oil and gas infrastructure,” Trump stated. “Because of this cooperation, I have cancelled [sic] the previously expected second Wave of Attacks, which looks like it will not be needed, however, all ships will stay in place for safety and security purposes.”
ECONOMIC IMPLICATIONS OF MADURO OUSTER IN VENEZUELA ARE BROAD AND DEEP
He also added that oil corporations would be investing over $100 billion into new initiatives in Venezuela.
“At least 100 Billion Dollars will be invested by BIG OIL, all of whom I will be meeting with today at The White House,” Trump added.
The decision to call off further military strikes and the meeting of oil companies to invest in Venezuela's future come less than a week after Trump gave the approval for Operation Absolute Resolve, the military operation that led to the capture of former Venezuelan dictator Nicolas Maduro.
Somewhere between what had been a previously unimaginable football commute from Pasadena, California, to Bloomington, Indiana, the first day of January bled into the second.
The Indiana Hoosiers, fresh off eviscerating none other than Alabama at the high holy grounds of the Rose Bowl, pulled up to the John Mellencamp Pavilion around 3:30 in the morning, the business of the 2025 season still very much in front of them.
One door had not, per the cliché, closed. Yet, as the Hoosiers flew home, another door had, in fact, opened. The transfer portal, the door to either Alice's Wonderland or the gates of Hades, depending on your viewpoint, opened at midnight on January 2. And so while his players slept, his fanbase celebrated and a semifinal game loomed, Curt Cignetti went into the office and met with recruits to talk about the next season.
Meanwhile, in Oxford, Mississippi, an equally giddy and slightly more vindictive fanbase had all of about 30 minutes to celebrate their team's win. Then the clock struck midnight on New Year's Day, leaving Ole Miss fans to wonder if their offensive coachmen might turn back into mice. Or, more accurately, in this case, rats.
Jilted by Lane Kiffin, Ole Miss nonetheless beat Georgia in the Sugar Bowl but Pete Golding, the new man in charge, wasn't quite sure which of his coaches would be coming with him to prep for a semifinal date with Miami and who was making the short ride across the bayou to Baton Rouge.
As it turned out, Joe Cox and George McDonald abandoned ship. The tight ends coach and wide receivers coach joined Kiffin, allowing the Rebels to go on without them.
“Do they want to be here?” Golding said of his former co-workers. “Damn right they do.''
While all of this was going on across the country, retro recruits (also known as high school seniors) were saying goodbye to their families, their pals and their proms. Early college enrollment beckoned, even if high school graduation hadn't yet happened. They needed to get a jump on things to stake their claim on a roster, even if the same coaches who months earlier promised them the moon and the stars were currently recruiting over them by entertaining guys in the portal.
And in ordinary academic buildings and offices tucked up in the corners of the athletic department, university registrars and academic advisors were setting their hair on fire, trying to figure out how to approve transcripts and shoehorn new students into classes that were already full.
The college football calendar is a mess. That sentiment is now universal. But rather than merely try to undo what's been done, perhaps it's time to figure out what the point of it all is. Not only how college football got into this quagmire, but also if anyone is actually benefiting from it all. Rather than just fix the problem, the sport might want to identify what exactly the problem is.
“What I'll say is … it has made football teams and it's made coaches and players better at handling chaos,'' Oregon head coach Dan Lanning said.
And then he laughed, somewhat ruefully.
The transfer portal did not start the fire; it only added the accelerant.
If you untangle the mess that is now college football's January and try to figure out where it all got sideways, it might be wise to go back to 1991. That's when Georgia quarterback Eric Zeier decided to enroll in college a semester before graduating from high school. An exemplary student, Zeier took advantage of a dual enrollment program and made the jump from Marietta to Athens.
It was, however, easy to write Zeier off as an exception, not a norm, until a decade later, Maurice Clarett did the same at Ohio State. Clarett intentionally doubled down on classes in his junior season and took classes in the summer, all with the idea of jumpstarting his college progression.
When he led the Buckeyes to a national championship in 2003, the idea took root. By 2007, Florida brought nine kids in early and Clemson five more.
The argument for was simple: Enrolling early offered more time in the weight room and more time to acclimate to the rigors of college ball. Coaches could evaluate their whole team during spring ball, rather than wait to sort out starting rotations in the fall.
By 2017, the practice became so commonplace that the NCAA switched up the calendar, adding an early December signing day to the traditional February window.
This year, 32 players will get an early jump on college at USC.
Does it matter? Sometimes, yes. Trevor Lawrence enrolled at Clemson in January 2018 and started that September. And sometimes no. Arch Manning enrolled at Texas in January 2023. He started his first game this season, two years after he got to Austin.
Carter Smith arrived at Indiana in January 2022. An athletic offensive lineman who not only starred in the trenches but led his volleyball team to a state semifinal and 20 wins as a junior, he left Olentangy Liberty after his football season and went to Bloomington in January.
A few months later, his high school volleyball team won the state title.
“That wasn't the greatest,'' he says now. But Smith insists he would do it all over again.
“It really opened my eyes to how much more of the game I needed to learn,'' he said. “If you decide to come early, that's a big step in your career. There's really no downside to it.''
Smith wound up redshirting his freshman season.
Early commitments have never been guaranteed anything – not roster spots, not playing time – and getting recruited over is not new. A great player is great only until the next even greater player comes along at his position.
But the advent of the transfer portal has made the hope for a return investment even flimsier. Given the chance to grow a linebacker over time or plug in an experienced older one right now, coaches are going old every time.
There are only 105 spots on a college football roster, and little time to waste in a win-now culture. Consequently, while an early enrollment may show a player's commitment to the program, there is no promise that said commitment will be returned.
In January 2024, Parker Livingstone, a Texas native who long dreamed of wearing the burnt orange, happily left his high school for Austin. He wound up redshirting as a freshman but became a starter this year for the Longhorns, catching 29 passes from his roommate Arch Manning. Livingstone just transferred to Oklahoma.
“Never in a million years did I think I would be going into the portal looking for a new home,'' he wrote on his Instagram post. “Some things are out of my control. Such is the reality of the ever-changing landscape of college football.''
Two days later, Texas brought Auburn wide receiver Cam Coleman to campus for a visit.
Oregon started its winter semester on Monday. Indiana and Miami begin the semester on January 12, with Ole Miss commencing January 20. The portal closes on January 16.
So let's talk about the other calendar that nobody thinks about: the academic calendar.
College football players are still, by definition at least, students. To be eligible to compete, they have to be enrolled in classes and making progress toward a degree. Except at most schools, spring classes not only start before the portal begins, but the deadlines to drop or add classes are usually well before January 16.
A simple question then: how?
“It is,'' says Kyle Ross, the executive director of NACADA, a not-for-profit that offers networking for academic advisors, “a lift.''
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In a normal transfer world, one not involving a football player weighing multiple offers as or after the semester has started, an academic advisor will spend a few days reviewing a transcript.
Sometimes, it's easy. A state school move to another state school is fairly uncomplicated, with most courses already scrubbed and articulated into the system. A transcript for a student moving from one state to another, or from a select private school to a big state school, might need a little extra time to review.
It gets even trickier for upperclassmen who are trying to get upper-level coursework to count as credits. It's not unusual for a faculty member to get involved and review syllabi to make sure the classes stack up to the new school's standard.
“In the best scenario, we can get it done in 48 hours,'' Ross says. “But with what's being asked with football transfers, it can be as tight as 24 hours or less. People get frustrated because they need information and they need it fast.''
Approving transfer credits, though, is only the beginning. Transferring students – and even football players – have to have a class into which to transfer. By the time most football players are looking to transfer, regular students have long since crafted their new schedule.
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Most courses, Ross said, leave room for emergency additions – say five open places for a 30-person class – but a hefty transfer class can become a simple numbers crunch. An administrator at one Power 5 school who asked not to be named told CNN Sports that faculty at their institution had been asked to hold as many as 70 spots for possible new football players.
“The closer to the start of the semester you get, the tougher it is,'' the administrator said. “Miracles can happen and mountains, I know, have been moved, but it's not easy.''
And at what cost? No doubt few are concerned about such things as academic success, but Ross is. He worries about students enrolling in an 11-week class three weeks after it started.
“That, I know, creates even more frustration for the faculty,'' he said. “They allow someone into their class, but they're thinking to themselves, ‘I really shouldn't be letting them in now. I'm only setting them up for failure.'”
This is, of course, what started the current round of handwringing and – as usual – Lane Kiffin got the sport boiling.
He not only went through a messy divorce with Ole Miss, exiting just as the Rebels started their playoff march; he doubled down by then making it clear he expected the assistants who were joining him at LSU to choose portal recruiting over semifinal coaching.
The truth is, LSU and Kiffin remain the exception, not the norm. James Madison's Bob Chesney is the new UCLA head coach; he didn't leave for Westwood until after the Dukes lost. Oregon's offensive coordinator and defensive coordinator both accepted head coaching jobs elsewhere – Will Stein at Kentucky and Tosh Lupoi at Cal.
Both worked the portal for their new employers this weekend and both will be on the sidelines on Friday night when the Ducks face Indiana.
“The timing isn't perfect,'' Lanning said of his staff's fluidity, “but it's been done before.”
It is certainly not ideal that coaches have to split time between recruiting and game prep at such a critical part of the season, but that's hardly new. College basketball coaches routinely fly out on the heels of an afternoon practice to scout a recruit, and zip back the next day.
Does it look squishy? Sure. Is it impossible? No.
Besides all of this, the January crunch stems from the Wayback machine, to the coaches seeking a way to corral their entire roster on campus long before the first game kicks off.
“For me, it's been a philosophy of take the calendar, what fits best for us, how can we work the calendar to be an advantage to us in how we run our program,'' Minnesota's PJ Fleck told the New York Times six years ago. “For us, it's all about getting a jump-start on next year.''
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Fox News contributor Kellyanne Conway joins ‘America's Newsroom' to break down divisions within the Democratic Party as leaders search for a new direction.
FIRST ON FOX: The Democratic National Committee filed a Supreme Court amicus brief Thursday seeking to counter the Republican Party in a major election law case this year — setting the stage for a high-stakes court clash that could determine the fate of millions of mail-in ballots nationwide.
The Supreme Court agreed in November to take up the case, Watson v. Republican National Committee, which centers on states' ability to count mail-in ballots that are received within five days of an election.
At issue is a lawsuit filed by the Republican National Committee and Mississippi GOP in 2024, which seeks to overturn a state voting law that allows for the counting of late-arriving mail-in ballots that are postmarked on or before Election Day, so long as they arrive within five business days of the election. The RNC and state GOP have argued that they break with federal voting laws — a point vehemently disputed by other states and the DNC.
In the amicus brief, provided exclusively to Fox News Digital, lawyers for the party urged the Supreme Court to reverse a lower court ruling handed down by the U.S. Court of Appeals for the Fifth Circuit, noting the dozens of states that currently allow mail-in ballots to be counted, so long as they are postmarked before, or on Election Day. They also noted the long history of mail-in ballots in the U.S., which stretches back elections since the Civil War.
"Throughout this Nation's history, the term 'election' has been universally understood to refer to the voters' act of choosing an officeholder—not to the later administrative acts of receiving or counting ballots," the DNC said in its amicus brief.
SWING STATE'S SUPREME COURT ISSUES PIVOTAL RULING ON MAIL-IN BALLOTS SENT WITHOUT POSTMARK
The United Center is packed on the first night of the Democratic National Convention, as President Biden addresses the crowd, on August 19, 2024, in Chicago, Illinois. (Fox News - Paul Steinhauser)
Similar laws are in place in 30 other states, including the District of Columbia, prompting additional concerns about the outsize impact a ruling from the high court could have on millions of voters, including in the run-up to the midterm elections.
In their amicus brief, lawyers for the DNC emphasized the widespread use of mail-in ballots by many voters, including by seniors, voters with disabilities or members of the military.
"If the Supreme Court rules for the RNC, voters around the country will be disenfranchised by mail delays, and key protections for military and overseas voters could be eliminated," they noted in a press release sent alongside the brief.
"Republicans' continued assault on mail-in voting is an attack on our democracy and is wholly un-American," DNC Chairman Ken Martin told Fox News Digital in a statement.
COMEY SEEKS TO TOSS CRIMINAL CASE CALLING TRUMP PROSECUTOR 'UNLAWFUL' APPOINTEE
Democratic National Committee Chairman Ken Martin. (Bill Clark/CQ-Roll Call, Inc via Getty Images)
"Donald Trump and the RNC want to limit the rights of voters because they know that when more eligible voters make their voices heard, Republicans lose," he added. "Voting by mail is safe, secure, and empowers voters who would otherwise struggle reaching a ballot box, including seniors and people with disabilities, members of the military and their families, and working families who are unable to take the day off to vote."
Mississippi's attorney general appealed the case to the Supreme Court in June after the U.S. Court of Appeals for the Fifth Circuit ruled in 2024 that the state's mail-in ballot laws violated federal law, ruling that all mail-in ballots must be cast and received by Election Day in order to be legally counted.
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The Supreme Court building in Washington, D.C. (AP Photo/J. Scott Applewhite, File)
The court's decision to take up the case prompted outrage by some, who noted that a ruling could impact the fate of millions of ballots across the country. Critics also noted that the timing of a possible ruling could wreak havoc on the 2026 midterm elections, primarily for voters that live overseas, including members of the military.
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The RNC has urged the high court to leave the 5th Circuit ruling in place. They argued that upending the ruling before an election could yield "chaos and suspicions of impropriety," especially if "thousands of absentee ballots flow in after election day and potentially flip the results of an election."
Breanne Deppisch is a national politics reporter for Fox News Digital covering the Trump administration, with a focus on the Justice Department, FBI and other national news. She previously covered national politics at the Washington Examiner and The Washington Post, with additional bylines in Politico Magazine, the Colorado Gazette and others. You can send tips to Breanne at Breanne.Deppisch@fox.com, or follow her on X at @breanne_dep.
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HARRISBURG, Pennsylvania — Linda McMahon is having fun.
The education secretary, the 13th since the Cabinet department was founded during President Jimmy Carter's administration, has just left a classroom in Dauphin County doing the 2026 version of Schoolhouse Rock! with a class full of children clearly enjoying the civics game.
It is early December 2025, and McMahon has just kicked off a 50-state “History Rocks!” barnstorm in Pennsylvania as part of an effort to engage with schoolchildren across the country about civics as well as instilling the aspirations of patriotism.
The initiative is part of a nine-month effort ahead of this year's 250th anniversary of the signing of the Declaration of Independence. McMahon said in an interview with the Washington Examiner after her first of three events that the entire effort is a way to engage with young people to explain the importance and joy of patriotism, “as well as to draw us all together to enjoy our shared understanding of America's founding principles.”
Spending time with the schoolchildren has her buoyant and excited about the months ahead in the lead-up to the 250th anniversary and being able to engage with them on a personal level.
McMahon, who was also making stops in Delaware and New Jersey as part of this trip, said the experience of working with the pre-K through fifth grade children on this day has already inspired her.
“We played a couple of games that included questions about who were the signers of the Declaration of Independence and how our country was founded, and they were so enthusiastic to be part of it,” she said of the game that had two teams competing to get the answers right first.
McMahon, who was brought up in the South and started her school day with the Pledge of Allegiance and the Lord's Prayer, said she was stunned to find out in her new home state of Connecticut that there was no Pledge of Allegiance or national anthem to open her granddaughter's high school graduation ceremony.
“I was so struck by that,” she said. “And so then I went back to find out a little bit about just schools in Greenwich, Connecticut. Did children say the Pledge of Allegiance? And not all of them did.”
So when Flag Day approached last June, she had the Education Department get approval ahead of time to distribute little flags for the children to have.
“They all came outside on the school grounds, and all the kids were waving their flags, and they were saying the Pledge of Allegiance and singing the national anthem,” she said. “And the school itself was so impressed by what they saw, they said they would very much like to repeat that this year.”
McMahon's conclusion was not only that the school districts were not teaching patriotism, but they were also not teaching civics and the values of love of country.
“I recently saw a survey that was done, and it said that only 41% of 18- to 29-year-olds said that they love their country,” she said. “And I thought, ‘Wow, I really believe that they don't love their country because they don't know their country, or at least they haven't really been immersed in the history of the country, what our country has gone through, how it's evolved, and what a special place this is.'”
Her second stop of the day was in Delaware at St. Georges Technical High School, the first vocational technical high school she has visited in her role as the education secretary.
“I've been to community colleges, but this was the first 9-12 technology school that I've attended,” she said, adding that as part of her tour, she had her finger pricked by the students studying to be surgical technicians.
“They were also being trained in phlebotomy, so they offered to do a blood draw. I said, ‘I'd be very happy with just a finger prick today, thank you very much,'” she said, laughing.
McMahon said the trade school was made up of 10th, 11th, and 12th graders, with many of them set to walk out of school in May to join the workforce.
“I was talking an 11th grader that he said that when he graduated, he would have a certificate to go on into a professional position in a hospital as a tech person, and he was so thrilled to be able to have the opportunity to start a job, to start earning a living, which would help pay if he wanted to then expand his career,” she said.
Part of what she discussed at St. Georges was that its students are considered highly skilled and really sought after in the community once they graduate.
“In fact, a local Ford dealership donated a F-150 truck to the automotive repair class, and they will hire every student that passes through that course that works on the F-150,” she said. “They said the skills gap is so profound that they can't train students fast enough to meet the demand.”
That skills gap was one of the comments she made to President Donald Trump during a Cabinet meeting in December 2025. “I think that we're at a national emergency point with education in general in our country, but if we look at the skilled workforce, if we want to bring all of this technology and this manufacturing and all back to our country, we have to focus on training our skilled labor force,” McMahon said of her conversation with Trump.
McMahon said post-World War II families pushed their children to go to college because they didn't want them to have to rely on blue-collar jobs, and they wanted them to have better opportunities than they had.
Our culture is changing on that thought process, she said, “and that is really key now to understanding the fastest growing group of millionaires in the country are our skilled workforce.”
McMahon said the 50-state initiative will include an overlapping discussion with state legislatures about returning education to the states.
“That's a parallel tour that we're doing, but our History Rocks! and the Road to Independence tour is to really just revitalize civics education and our country and to show that it's fun,” she said.
While in the Pennsylvania State Capitol, McMahon met with Republican legislators to talk about giving states control of educational programming by dismantling the U.S. Department of Education.
In terms of Cabinet-level agencies, the Education Department is a relative newcomer. At its inception, it was, in fact, called the Department of Education during President Andrew Johnson's administration when he signed legislation creating it. However, its mission was to collect information and gather statistics. It was very different from the massive department it is today.
Within a year, it was demoted to the Office of Education in 1868 and remained small, lightly staffed, and often under the umbrella of other agencies, such as the Interior Department and the former Department of Health, Education, and Welfare, which is now known as the Department of Health and Human Services.
Social strife in the 1960s, along with massive government expansion under President Lyndon Johnson, led to a rallying cry for the federal government to have a larger hand in the education of American children. In October 1979, Congress passed the Department of Education Organization Act, and the department, as we now know it, began operations in May 1980.
The COVID-19 years cast a long shadow over the nation's students. American public education was turned on its head by school boards and teachers unions with prolonged shutdowns, remote learning, masking, and many school districts not reopening for an entire year. All of which led to not just a distinct lag in students' academic performances, but also emotional damage, as students have struggled to reconnect after the dramatic extended shutdowns.
During his campaign for a second term, Trump pledged to shut down the Education Department. Several weeks after he was sworn in last year, he signed an executive order calling for the dismantling of the department.
“People have wanted to do this for many, many years, for many, many decades, and … no president ever got around to doing it, but I'm getting around to doing it,” Trump said at the signing.
The inevitable question for McMahon is how this drawdown of the department is going.
“Well, it is very interesting,” she said, smiling, adding that she'll explain it through a story that Gov. Kevin Stitt (R-OK) told her not long ago.
“He said he was speaking to a conference of about 400-500 people, and he asked the question, ‘How many of you in here graduated before 1979?' and most of everyone's hands in the room went up,” she recounted. “He said, ‘Well, imagine that, we all graduated from high school and there was no Department of Education. Isn't that amazing?'”
“So, really, I think as the president has rightfully said, education that's closest to the child, he believes, is the best education, and I agree,” McMahon added. “What is best for each individual community or district or state can't be dictated from Washington. We've tried a national curriculum. We've tried and, I think, with the best of intentions.”
She rattled off efforts such as No Child Left Behind, Race to the Top, and the Common Core curriculum as those with the best of intentions that fell short.
“You just can't dictate it through a bureaucracy,” she said. “It has to be felt locally, and it has to be controlled locally. And so that is the goal.”
McMahon said they have started moving things out of the Education Department on what she calls a proof-of-concept basis. “Through the Authority of the Economy Act, we are able to utilize interagency agreements,” she said. “So our first one that we signed was with the Department of Labor, where we moved about 12 employees from the Department of Education over to the Department of Labor.”
The budget is still within the Education Department. The overall supervision of employees is with an Education Department supervisor, but they will now work in conjunction with a designated group of employees from the Labor Department to integrate education and labor.
“After a period of time when we believe we have done that successfully, then we will want to ask Congress to codify and make that move permanent,” she said, adding that is the larger goal.
McMahon has told every member of Congress whom she has had an opportunity to talk to that this is not a back-of-the-napkin, willy-nilly program.
“This is carefully thought through,” she said. “It is a careful look at the budgets, a very thorough proof of concept that lets us bring it to you and show you how it is more efficient.”
McMahon noted that the Labor Department has a more developed system for the distribution of grants that are awarded. It also distributes its own grants.
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“Our system at the Department of Education could best be described as being held together by bubble gum and rubber bands, so over at the Department of Labor, we've been able to utilize their system and already distribute grants,” she said.
“So why would we try to reinvent the wheel at the Department of Education when we can blend in and move things that were always in other agencies and not in the Department of Education and have them operate with greater expertise and more efficiency?” McMahon concluded. “It just is common sense.”
Salena Zito is a senior writer for the Washington Examiner and author of the New York Times bestseller Butler: The Untold Story of the Near Assassination of Donald Trump and the Fight for America's Heartland.
• Outrage in Minneapolis: Protesters are rallying a few blocks from the street where an ICE agent fatally shot a Minneapolis woman. Minnesota Gov. Tim Walz has authorized the state's National Guard to support local law enforcement.
• Portland shooting: In a separate incident, a husband and wife are hospitalized after they were shot by a Border Patrol agent. Agents had attempted to stop a vehicle with a suspected gang member, according to a senior law enforcement official.
• Scrutiny over investigation: Amid calls for a transparent investigation, the FBI has taken over the probe, with a Minnesota agency saying it has been blocked from accessing key materials. View CNN's video analysis of the incident here.
Two people are hospitalized after they were shot by federal agents, the Portland Police Bureau said in a statement.
It is not immediately clear what their conditions are. The FBI Portland office said in a post on X that the shooting involved Customs and Border Protection agents and they are leading the investigation.
Today at 2:18 p.m. PT, Portland police officers responded to the 10200 block of Southeast Main Street for a report of a shooting, the release said. Officers confirmed federal agents had been involved in the shooting, they said.
At 2:24 p.m., officers responded to a man and a woman who had been shot near Northeast 146th Avenue and East Burnside, where they were transported to the hospital, the release said. It is not immediately clear why the different locations were reported.
“We are still in the early stages of this incident,” said Chief Bob Day. “We understand the heightened emotion and tension many are feeling in the wake of the shooting in Minneapolis, but I am asking the community to remain calm as we work to learn more.”
CNN has reached out to the Department of Homeland Security for further information.
This post has been updated with additional information.
Across the street from a growing memorial where Renee Nicole Good's SUV came to rest, Francesca Taylor is still processing what she saw Wednesday.
“The doors were open, and it was covered in blood on the inside,” Taylor said. “That was here almost all day yesterday. In full view.”
At first, Taylor didn't know who was shot or whether the person would survive.“Once I could see inside the vehicle, I knew there was no chance she was alive. And that was just a really devastating feeling to see the blood of a dead woman from my front porch,” she said.
Taylor describes her neighborhood as artsy and eclectic, with friendly, welcoming neighbors.
As people lay flowers and chant in honor of the 37-year-old mother of three who was killed, one woman is handing out warm tea to strangers. She wasn't comfortable sharing her name, but told CNN she wanted to help her community heal.
“This is what we do. We show up,” she said. “That's what Renee did yesterday, that's what we did today with this small offering to people that are hurting so much, and that's just what we hope enough people are going to keep doing.”
“It's a really sad day,” the woman shared. “I'm just trying to be sad with other people that I love and people that I don't love yet, but might figure out a way to love.”
A few blocks away from where a woman was shot and killed by an ICE agent yesterday, a energized crowd of people has gathered protesting the presence of the agency in the city.
They are packed tightly on a street corner holding signs, banners and flags. Cars honk as they pass by. One large banner in the front appears to have a picture of President Donald Trump on it.
Many protesters are yelling or chanting.
The Department of Homeland Security is not naming the ICE agent who fatally shot Renee Good in Minneapolis yesterday, but did provide a little more background on the agent's experience.
The agent has over 10 years of experience as an ICE Deportation Officer and is on the agency's Special Response Team, which requires 30 hours of tryouts to be considered, Assistant Secretary Tricia McLaughlin said in a statement. She did not say how long the agent had been on the Special Response Team.
To qualify for the team, agents are required to participate in a basic operator course, maintain expert marksman qualification on all issued firearms, and have ongoing training in specialized skills like “breaching techniques, perimeter control, advanced firearms training, hostage rescue, and more,” she said.
CNN obtained multiple angles of the fatal shooting of a woman by ICE agents in Minneapolis yesterday. The video below walks through them, showing nuance in what happened.
What took place prior to the shooting remains unclear.
CNN obtained multiple angles of the fatal shooting by ICE agents in Minneapolis showing nuance in what happened. What took place prior to the shooting remains unclear.
Demonstrators have started to gather at the scene of the fatal shooting of a woman by an Immigration and Customs Enforcement agent ahead of a planned protest, aerial video from CNN affiliate KARE showed.
Dozens of people are standing near a growing memorial for the victim, Renee Nicole Good, the video showed.
The protest is slated to begin at 5:30 p.m. CT.
People in Minnesota have the right to protest in wake of the fatal ICE shooting of Renee Nicole Good but should do so safely and lawfully to avoid creating hazards, threatening safety or disrupting emergency services, said the state's department of public safety.
The agency outlined a list of lawful ways to “express your views,” including gathering in public areas where pedestrians are allowed, marching or walking — though not on roadways — as well as engaging in chants and speeches and displaying signs.
Under the list of illegal and unsafe demonstration activities, the agency said protesters should not walk on a freeway, throw objects, start or set a fire, damage property or create graffiti, use illegal fireworks or display or use illegal weapons.
“We encourage Minnesotans to rely on official information from local sources as they make plans,” the department said.
The Minnesota National Guard is on standby “out of an abundance of caution” as people protest in the city a day after an ICE agent there shot and killed a US citizen, Minnesota Lt. Gov. Peggy Flanagan said.
A short time ago, Gov. Tim Walz authorized the state's National Guard to help support local law enforcement.
“Making sure that folks are demonstrating peacefully is incredibly important. We keep each other safe and that is what is needed and necessary right now,” Flanagan told CNN.
She said so far the demonstrations have been peaceful, but emphasized that she understands people are demonstrating because they are angry.
“We all saw with our own eyes video footage from, you know, multiple vantage points, and now Donald Trump and Kristi Noem are trying to spin this tale that somehow that's not exactly what happened,” she said.
Flanagan said the administration's argument that ICE is in cities to make people safer is “complete and utter baloney.” She echoed other local officials in calling for ICE to leave the state.
“They should leave us alone because all we have seen is that they have caused chaos and terror in our communities,” she said. “It's time to go. Get out.”
Asked if she believes that ICE should be abolished, Flanagan didn't answer directly, but said that “how they are acting right now is unconstitutional.” The lieutenant governor said the “current model” of the agency should be “thrown out.”
This post has been updated with additional comments from Flanagan.
Federal agents are pushing protesters back near the Bishop Henry Whipple Federal building in the Minneapolis area.
As law enforcement deployed some kind of crowd repellant, protesters began running away. Smoke or gas can be seen in the air.
The officers are now walking through the crowd and at least one person is in handcuffs.
CNN's Omar Jimenez, who is on the scene, said it looked like someone threw a water bottle toward the agents just before they started moving into the crowd. Jimenez said agents were also tackling people.
Before the flare-up, agents had been lined up at the entrance of the building.
This post has been updated with additional details from our team covering the protest.
Minnesota Gov. Tim Walz has authorized the state's National Guard to help support local law enforcement as tensions have risen in Minneapolis following the fatal shooting of Renee Good.
The National Guard will help protect critical infrastructure and maintain public safety, a news release from Walzs' office said.
“Yesterday, I directed the National Guard to be ready should they be needed,” Walz said in a statement. “They remain ready in the event they are needed to help keep the peace, ensure public safety, and allow for peaceful demonstrations.”
He also thanked Minnesotans as they have “met this moment,” adding “we have every reason to believe that peace will hold.”
This post has been updated with additional information.
Protesters have started asking for milk as “something is in the air right now,” CNN's Laura Coates described from the scene of protests in the Minneapolis area. She was also seen coughing.
“It feels a little like you're choking in your chest, and it feels like you're trying to catch your breath. It inflames your nostrils. It's a burning sensation in your chest. It's a burning sensation in your nostrils as well,” she described.
The situation flared after a caravan of cars carrying Border Patrol agents arrived and agitated protesters, many of whom are already angry.
Protesters are carrying milk as they anticipate tear gas from the federal agents, as happened this morning.
People are distrustful of the FBI investigation into the shooting and killing of Renee Good, which the Minnesota Bureau of Criminal Apprehension says it was forced to withdraw from.
Homeland Security's assistant secretary called yesterday — when an Immigration and Customs Enforcement agent shot and killed a woman — “a heavy day.”
“This is so unfortunate that this happened and was ultimately incredibly preventable,” Tricia McLaughlin told Fox News' Martha MacCallum this afternoon.
She said the same officer involved in yesterday's fatal shooting was also the subject of another recent violent incident.
While the officer was making an arrest last summer, the agent reached his hand into the subject's car and the person then closed the window and drove away, McLaughlin recalled. She said the officer was dragged for 50 yards, had to get 33 stitches and “clung onto his life.”
“You can see why he would be in fear of his life and of the law enforcement officers around him's lives,” McLaughlin said of the officer.
She went on to maintain that shooting victim Renee Nicole Good, 37, “was stalking agents all day long” and “impeding our law enforcement” before the officer “had to fire a defensive shot.”
McLaughlin said officers then “immediately got medics” and an ambulance to the scene. Good, she added, “was immediately given aid.”
Protesters gathered at the Whipple Federal Building near Minneapolis today scuffled with a lone demonstrator showing support for ICE this afternoon.
Standing with federal agents — and US Border Patrol behind him — the man was holding an American flag and a bullhorn.
“You guys keep playing stupid games, you're guaranteed to get stupid prizes. You guys will continue to be killed if you continue to harm these men,” he yelled. “Don't you understand that?”
At one point, someone tried to grab his flag and another protester snatched his hat. ICE agents intervened at that point.
Protesters were also yelling back, telling him he, too, was an immigrant on stolen land, and that ICE wasn't helping him, either. The tension comes after an ICE agent shot and killed a woman in her car yesterday in Minneapolis.
One person was trying to get the press and other protesters to ignore him. “You're just talking to white supremacists. Do not give them your attention,” the person said.
“Do not feed the trolls. Don't feed the white supremacists. Go film something else,” they said.
Later, the man with the American flag was seen having what appeared to be a civil discussion with an anti-ICE protester who was trying to convince him to carefully watch the video of the deadly shooting.
Another pro-ICE counterprotester also joined him at one point.
The Hennepin County Attorney's Office, which prosecutes crime in the Minneapolis area, said it is “exploring all options to ensure a state level investigation can continue” after the FBI rescinded its cooperation agreement.
“If the FBI is the sole investigative agency, the State will not receive the investigative findings, and our community may never learn about its contents,” Hennepin County Attorney Mary Moriarty said in a statement. “We are speaking to our local partners on paths forward that will allow us to review the investigation and be transparent in our decision making.”
Aidan Perzana woke up yesterday to the sound of honking and whistles in his Minneapolis neighborhood — a sign that ICE agents were in the area. When he went over to his window, he saw “some sort of commotion” and a car partially blocking the road, he said.
What happened next has caused outrage and protests to sweep across the city today. The woman in the car, Renee Nicole Good, was shot and killed by an ICE agent. Now in the aftermath, the Trump administration has maintained the agent shot out of self defense, but state and local officials dispute that characterization.
Perzana said in the moments before the shooting, he saw two agents on either side of her car. He said his neighbor heard that the driver was getting conflicting orders from the agents: Some telling her to move her car, some telling her to get out of the vehicle.
When a third man approached the car and started pulling the door handle, Good backed up and changed directions, Perzana said.
“So it looked to me like she was attempting to escape, not attempting to hit anyone,” he said.
Good's car crashed off the road after she was shot. Perzana said he watched the agent who fired his gun “calmy” approach the vehicle. In the minutes before emergency responders arrived, Perzana said agents “didn't pull her out of the car or anything. She was left in there.”
“This is a terrible, terrible injustice,” he said.
The day after the fatal shooting of Renee Good in Minnesota by an Immigration and Customs Enforcement agent, the situation on the ground has been tense.
Here's a roundup of our reporting from the Twin Cities, where CNN teams have been watching protests since this morning:
CNN's Alisha Ebrahimji, Holly Yan, Brian Abel, Victor Ramirez, Karina Tsui, Rebekah Riess, Priscilla Alvarez and Boney Kapp contributed to this report.
General policy within Immigration and Customs Enforcement is to not chase vehicles or fire at them unless there is imminent danger, said John Amaya, a former deputy chief of staff for the agency during the Obama administration.
Agents are also trained to be aware of other people around them in these situations to avoid harming them, Amaya added.
The death of Renee Nicole Good has confirmed what Amaya said were some of worst the worst fears he and some agency employees had with the increase of immigration enforcement operations during the Trump administration.
“I have heard from a number of career professionals within the agency that one of their concerns before any of this ever happened was that these people were not going to be trained properly, they were going to be thrown into the field and collectively thrown into operations that they have no business conducting because they're not trained for crowd control,” he said.
“That someone was going to end up shot, either a citizen, either a migrant, someone from the press, some other innocent bystander, or another fellow agent,” Amaya said.
While the ICE agent who shot Good was experienced, Amaya emphasized agents are not trained in crowd control or to interact with residents, which makes certain interactions “a recipe for disaster.”
“This was something that we saw coming. And until some policies change, it's going to happen again,” he said.
The ICE officer who shot and killed a Minnesota woman as she was driving forward in her SUV Wednesday was dragged more than 50 yards in a separate incident over the summer, when he became trapped in a vehicle during an arrest, a senior Department of Homeland Security official told CNN.
According to court records on the summer incident, the officer used a window punch to open the car after the driver — who authorities say was a convicted child sex offender and was in the country illegally — refused to lower his window or open his car door.
As the officer reached over the broken glass to unlock the car, the driver, Roberto Munoz-Guatemala, started accelerating, dragging along the officer, who shot the driver with his Taser.
The officer eventually pulled his arm from the car and fell into the road. Munoz-Guatemala was later arrested after driving a mile further. Images from court filings show the officer on a hospital bed with dozens of stitches on different cuts on of his body.
Vice President JD Vance addressed the incident at a news conference today, saying it “led to over 30 stitches and very serious injuries to his legs.”
In December, a jury convicted Munoz-Guatemala of using a deadly weapon to inflict injury to an officer following a trial early last month.
The same ICE officer was seen in video yesterday walking around the back side of 37-year-old Renee Nicole Good's vehicle toward the front as another officer pulled on Good's car door.
The officer walked in front of Good's car before she began driving forward. He shot and killed her, authorities say.
One video of the incident obtained seems to show the car making contact with the officer before he fired his gun the first time.
We've been receiving updates on yesterday's fatal ICE shooting in Minneapolis from both state and federal authorities, whose descriptions of the incident have differed significantly at times.
If you're just joining us, here's a recap of the comments from officials so far:
CNN's Alisha Ebrahimji, Holly Yan, Brian Abel, Victor Ramirez, Karina Tsui, Rebekah Riess, Priscilla Alvarez and Boney Kapp contributed to this report.
The Department of Homeland Security said Thursday it was ramping up its investigations in Minneapolis.
In an X post, DHS shared a video showing several law enforcement officers entering a building.
“DHS investigations in Minneapolis are accelerating as more of our brave law enforcement enter the state,” the agency said in the post. “We will not be impeded from ending the FRAUD that has ABUSED our nation's generosity. See you on the streets!”
Context: Earlier this week, DHS said its agents were in Minnesota conducting a large scale investigation on fraud allegations at day care centers and other places.
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Philadelphia District Attorney Larry Krasner held a press conference on Thursday and issued a stark warning to Immigration and Customs Enforcement officers.
Krasner told ICE officers who are in his city, or planning to come to his city, to “get the F out of here” if they were coming to “commit crimes.” Krasner's press conference was in response to the death of Renee Good, who was shot after seemingly driving her car into an ICE officer in Minneapolis earlier this week.
“Let me be clear,” Krasner said. “If any law enforcement agent, any ICE agent, is going to come to Philly to commit crimes, then you can get the F out of here. Because if you do that here, I will charge you with those crimes, you will be arrested, you will stand trial, you will be convicted — whether it's in state or federal court, it's my office prosecuting it, that's how the law works — and you will do your time. Because Donald Trump cannot pardon you for a state court conviction.”
Krasner added that if any federal law enforcement officer committed a crime in his city, they would indeed face jail time.
“Do you hear me, ICE agents?” Krasner said. “Do you hear me, National Guard? Do you hear me, military? You're going to jail if you commit crimes in the city of Philadelphia.”
Krasner's words were the latest in his rebuke of President Donald Trump's immigration enforcement efforts throughout the country. In the past, Krasner has regularly objected to the deployment of National Guard troops in cities throughout the United States and has been an ardent critic of Trump, frequently calling him a “fascist” and a “Nazi” and often comparing him to Nazi leader Adolf Hitler.
“You will be accountable,” Krasner said. “The law applies to all of you.”
TRUMP SAYS MEETING WITH IRANIAN CROWN PRINCE CALLING FOR PROTESTS WOULD NOT BE ‘APPROPRIATE'
Krasner did specify that his comments were not for all law enforcement officers, but rather for those who allegedly break the law.
“There are honest, decent, moral law enforcement officers by the bushel. This is not for you,” Krasner said. “This is for any one of your colleagues who thinks they are above the law.”
Copyright 2026 The Associated Press. All Rights Reserved.
Copyright 2026 The Associated Press. All Rights Reserved.
“As far as we know, both of these individuals are still alive,” Elana Pirtle-Guiney, the President of Portland's City Council said during a meeting.
Hundreds gathered outside a U.S. Immigration and Customs Enforcement building in Portland, Oregon, on Thursday in protest after authorities said federal immigration officers shot and wounded two people in a vehicle outside a hospital.
Portland Mayor Keith Wilson and the city council called on U.S. Immigration and Customs Enforcement to end all operations in Oregon's largest city until a full investigation is completed following the shooting of two individuals. (AP video by: Claire Rush)
A protester yells at a Portland police officer outside the U.S. Immigration and Customs Enforcement facility on Thursday, Jan. 8, 2026, in Portland, Ore. (AP Photo/Jenny Kane)
Protesters and law enforcement stand outside the U.S. Immigration and Customs Enforcement facility on Thursday, Jan. 8, 2026, in Portland, Ore. (AP Photo/Jenny Kane)
Law enforcement officials work the scene following reports that federal immigration officers shot and wounded people in Portland, Ore., Thursday, Jan. 8, 2026. (AP Photo/Jenny Kane)
Law enforcement officials work the scene following reports that federal immigration officers shot and wounded people in Portland, Ore., Thursday, Jan. 8, 2026. (AP Photo/Jenny Kane)
A security guard stands at the scene following reports that federal immigration officers shot and wounded people in Portland, Ore., Thursday, Jan. 8, 2026. (AP Photo/Jenny Kane)
PORTLAND, Ore. (AP) — Federal immigration agents shot and wounded two people in a vehicle outside a hospital in Portland on Thursday, a day after an officer fatally shot a woman in Minnesota, authorities said.
The shooting drew hundreds of protesters to the U.S. Immigration and Customs Enforcement building at night, and Oregon Attorney General Dan Rayfield vowed to investigate “whether any federal officer acted outside the scope of their lawful authority” and refer criminal charges to the prosecutor's office if warranted.
The Department of Homeland Security said the vehicle's passenger was “a Venezuelan illegal alien affiliated with the transnational Tren de Aragua prostitution ring” who was involved in a recent shooting in the city. When agents identified themselves to the occupants during a “targeted vehicle stop” in the afternoon, the driver tried to run them over, the department said in a statement.
“Fearing for his life and safety, an agent fired a defensive shot,” it said. “The driver drove off with the passenger, fleeing the scene.”
There was no immediate independent corroboration of that account or of any gang affiliation of the vehicle's occupants. During prior shootings involving agents from President Donald Trump's immigration crackdowns in U.S. cities, including the fatal one Wednesday in Minneapolis, video evidence has cast doubt on the administration's characterizations of what prompted the shootings.
Law enforcement officials work the scene following reports that federal immigration officers shot and wounded people in Portland, Ore., Thursday, Jan. 8, 2026. (AP Photo/Jenny Kane)
Trump and his allies have consistently blamed the Tren de Aragua gang for being at the root of violence and drug dealing in some U.S. cities.
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The Portland shooting escalates tensions in a city that has long had a contentious relationship with Trump, including due to his recent failed effort to deploy National Guard troops there. The city saw long-running nightly protests outside the ICE building.
According to the Portland Police bureau, officers initially responded to a report of a shooting outside Adventist Health hospital at 2:18 p.m. Thursday.
A few minutes later, police received information that a man who had been shot was asking for help in a residential area a couple of miles away. Officers went there and found a man and a woman with gunshot wounds. Officers determined that they were injured in the shooting with federal agents, police said.
Law enforcement officials work the scene following reports that federal immigration officers shot and wounded people in Portland, Ore., Thursday, Jan. 8, 2026. (AP Photo/Jenny Kane)
Their conditions were not immediately known. Portland police said officers applied a tourniquet to one of them.
City Council President Elana Pirtle-Guiney said during a meeting that “as far as we know, both of these individuals are still alive, and we are hoping for more positive updates throughout the afternoon.”
At a nighttime news conference, Police Chief Bob Day said the FBI was leading the investigation and he had no details about the events that led to the shooting.
Mayor Keith Wilson and the City Council called on ICE to end all operations in Oregon's largest city until a full investigation is completed.
Multnomah County District Attorney Nathan Vasquez, center, speaks to the media following reports that federal immigration officers shot and wounded people in Portland, Ore., Thursday, Jan. 8, 2026. (AP Photo/Jenny Kane)
“We stand united as elected officials in saying that we cannot sit by while constitutional protections erode and bloodshed mounts,” they said in a statement. “Portland is not a ‘training ground' for militarized agents, and the ‘full force' threatened by the administration has deadly consequences.”
Wilson also suggested at a news conference that he does not necessarily believe the federal government's account of the shooting: “There was a time we could take them at their word. That time is long past.”
Democratic State Sen. Kayse Jama, who lives near where it took place, said Oregon is a welcoming state — but he told federal agents to leave.
“You are not welcome,” Jama said. “You need to get the hell out of Oregon.”
The city officials said “federal militarization undermines effective, community‑based public safety, and it runs counter to the values that define our region. We'll use every legal and legislative tool available to protect our residents' civil and human rights.”
They urged residents to show up with “calm and purpose during this difficult time.”
Several dozen people gathered in the evening near the scene where police found the wounded people.
“It's just been chaos,” said one, Anjalyssa Jones. “The community is trying to get answers.”
U.S. Sen. Jeff Merkley, an Oregon Democrat, urged protesters to remain peaceful.
“Trump wants to generate riots,” he said on the social platform X. “Don't take the bait.”
___
Johnson reported from Seattle. Associated Press writer Audrey McAvoy in Honolulu contributed.
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The Miami Hurricanes are heading to the College Football Playoff National Championship Game, coming away with a narrow victory over Ole Miss, 31-27, in an all-time postseason contest.
The Hurricanes will now await the winner of the other semifinal between the Indiana Hoosiers and Oregon Ducks to see who they will play on Jan. 19. But Miami will do so on their home turf, with the National Championship Game being played at Hard Rock Stadium – the site of their home games.
The game began slowly for both teams, with only Miami getting on the scoreboard in the first quarter with a field goal on their 13-play opening drive. But the fireworks came out from there for the Rebels thanks to the speed of running back Kewan Lacy.
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Charmar Brown of the Miami (FL) Hurricanes celebrates a run in the first quarter of the 2025 College Football Playoff Semifinal at State Farm Stadium on Jan. 8, 2026 in Glendale, Arizona. (Steve Limentani/ISI Photos)
On just the second play of the second quarter, Lacy was off to the race, finding a seam and busting out a 73-yard touchdown run to go up 7-3 after the extra point.
But this game was back and forth for quite some time, including the ensuing Hurricanes drive as quarterback Carson Beck led the way on a 15-play touchdown series with a CharMar Brown rushing score from four yards out.
The game was deadlocked at 10 apiece when Beck decided to air it out to Keelan Marion, and it was worth the risk. Marion made the grab for a 52-yard touchdown to help Miami go up 17-13 at halftime.
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The third quarter was an odd one for both squads, as their opening drives resulted in a missed field goal apiece. Then, after Beck threw an interception, the Rebels were able to cut the lead to 17-16 in favor of the Hurricanes heading into the fourth quarter for the ages.
There was no absence of electric plays when it mattered most in the final 15 minutes, as Rebels quarterback Trinidad Chambliss got his team downfield enough to take a 19-17 lead with a field goal.
But the speed of Malachi Toney changed the scoreboard for Miami in the best way possible, as he took a screen 36 yards to the house, capping a four-play, 75-yard answer drive for the Hurricanes right after Ole Miss took the lead.
Trinidad Chambliss of the Ole Miss Rebels celebrates a touchdown against the Miami Hurricanes in the second quarter during the 2025 College Football Playoff Semifinal at the VRBO Fiesta Bowl at State Farm Stadium on Jan. 8, 2026 in Glendale, Arizona. (Ronald Martinez/Getty Images)
With a 24-19 lead and five minutes left to play in the game, Chambliss and the Rebels' offense had quite enough time to retake the lead. He did just that, finding trusty tight end Dae'Quan Wright for 24 yards to send the Rebels faithful ballistic.
Ole Miss wanted to go for two in hopes of making it a three-point lead, and Chambliss came through again, finding a wide open Caleb Odom for the key score.
It was up to Beck and the Miami offense to keep the game alive with at least tying the game at 27 apiece. On a crucial third-and-10 just inside field goal range, Beck was confident with his pass to Marion to get well within range. Another pass to Marion made it first-and-goal, and it was clear Miami wasn't trying to force overtime. They wanted to win it all.
How fitting was it that Beck, scanning the field, found a seam to his left and just sprinted for the colored paint to score the game-winner with 18 seconds left.
But things got fascinating at the end, with Ole Miss going 40 yards in just a few seconds to set up a Hail Mary for the win. Chambliss had the space to loft a pass to the end zone, and though it hit off the hand of a teammate, it landed incomplete for the Miami victory.
Carson Beck of the Miami Hurricanes passes the ball against the Ole Miss Rebels in the first quarter during the 2025 College Football Playoff Semifinal at the VRBO Fiesta Bowl at State Farm Stadium on Jan. 8, 2026 in Glendale, Arizona. (Chris Coduto/Getty Images)
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In the box score, Beck was 23-of-37 for 268 yards with his two passing touchdowns and an interception. Marion was a key player in the victory with seven catches for 114 yards, while Mark Fletcher Jr. set the tone in the ground game with 133 yards rushing on 22 carries. Toney also tallied 81 receiving yards for Miami.
For Ole Miss, Chambliss also went 23-of-37 for 277 yards with his touchdown to Wright, who finished with 64 yards on three grabs. De'Zhaun Stribling was five for 77 through the air, while Lacy rushed for 103 yards on 11 carries.
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The Trump administration has said it will withdraw from dozens of international organizations, including the U.N.'s population agency and the U.N. treaty that establishes international climate negotiations, as the U.S. further retreats from global cooperation.
United National Secretary-General Antonio Guterres speaks during an event to mark the end of the U.N. political mission, in Baghdad, Iraq, Saturday, Dec. 13, 2025. (AP Photo/Hadi Mizban)
NEW YORK (AP) — The top United Nations official on Thursday said the United States has a “legal obligation” to keep paying its dues that fund U.N. agencies after the White House announced that it is withdrawing support from more than 30 initiatives operated by the world body.
Secretary-General Antonio Guterres said he regretted President Donald Trump's decision to withdraw from 31 U.N.-related agencies, including the U.N.'s population agency and the U.N. treaty that establishes international climate negotiations. The U.S. will also depart from dozens of other global organizations or initiatives not affiliated with the U.N.
“As we have consistently underscored, assessed contributions to the United Nations regular budget and peacekeeping budget, as approved by the General Assembly, are a legal obligation under the UN Charter for all Member States, including the United States,” Stephane Dujarric, a spokesperson for Guterres, said in a statement.
He added that despite the announcement, the U.N. entities targeted will keep doing their work: “The United Nations has a responsibility to deliver for those who depend on us.”
The strong retort from the U.N. comes after the world body has spent the better part of the past year in a somewhat hostile and fragile back-and-forth with U.S. officials, who, after Trump's return to office, zeroed in on eliminating billions of dollars in aid and funding to international organizations like the U.N. and humanitarian assistance at large.
Through many conciliatory public and closed-door appeals, U.N. officials, including Guterres, had been able to convince Trump and his allies not to completely abandon the institution the U.S. helped found on the ashes of World War II, including through a $2 billion agreement for humanitarian assistance announced last month. But America's retreat had already influenced other Western countries, including France and the U.K., to reevaluate humanitarian funding, with many shifting that money toward military spending.
But Wednesday's announcement surprised diplomats at the highest levels of the U.N., who said they learned about the withdrawal through news reports and the White House social media. There has been no formal communication from the Trump administration outlining the decision, Dujarric told reporters.
Many U.N. officials refused to comment on the impact it would have on their agencies because they had not been given details or official word from anyone in the U.S. government.
Following a yearlong review of participation in and funding for all international organizations, Trump signed an executive order suspending American support for 66 groups, agencies and commissions.
Many of the targets are U.N.-related agencies, commissions and advisory panels that focus on climate, labor, migration and other issues the Trump administration has categorized as catering to diversity and “woke” initiatives.
The administration previously suspended support for the World Health Organization, the U.N. agency for Palestinian refugees known as UNRWA, the U.N. Human Rights Council and the U.N. cultural agency UNESCO. It has taken a larger, à la carte approach to paying dues to the world body, picking which operations and agencies it believes align with Trump's agenda and those that no longer serve U.S. interests.
Some of the agencies impacted, including the U.N. Population Fund, an organization that provides sexual and reproductive health services worldwide, have long been a lightning rod for Republican opposition, and Trump cut funding for it during his first term.
The withdrawal from the U.N. Framework Convention on Climate Change, or UNFCCC, came as less of a surprise as Trump and his allies had previously withdrawn U.S. support from other climate initiatives.
The 1992 agreement between 198 countries to financially support climate change activities in developing countries is the underlying treaty for the landmark Paris climate agreement. Trump withdrew from that agreement soon after returning to the White House.
Simon Stiell, UNFCCC executive secretary, warned the U.S. that the decision to pull back will harm “the US economy, jobs and living standards, as wildfires, floods, mega-storms and droughts get rapidly worse.”
“The doors remain open for the U.S. to reenter in the future, as it has in the past with the Paris Agreement,” he said in a statement. “Meanwhile, the size of the commercial opportunity in clean energy, climate resilience, and advanced electrotech remains too big for American investors and businesses to ignore.”
The U.N.'s regular budget, which finances its day-to-day operations and primary activities, is funded by its 193 member nations, each paying a percentage based on the size of their economy. The U.S., the world's largest economy, is supposed to pay 22%, followed by China, with 20%. There is a separate budget to fund the U.N.'s peacekeeping operations, where the U.S. is required to pay 25%.
U.N. officials said the U.S. did not pay its annual contributions to the regular budget last year, an obligation outlined in the U.N. Charter. A member that is in arrears for two full years loses its vote in the General Assembly.
“The charter is not à la carte,” Dujarric said. “We're not going to renegotiate the charter.”
All four other veto-wielding permanent members of the U.N. Security Council — China, France, Russia and the U.K. — have paid in full. China paid over $685 million.
___ Associated Press writer Edith M. Lederer contributed to this report from the United Nations.
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The Missouri Attorney General's Office announced Wednesday that a Chinese national was arrested last month after allegedly photographing two U.S. Air Force bases and its military equipment, including B-2 Spirit stealth bombers.
Authorities identified the 35-year-old as Qilin Wu, who entered the U.S. illegally in 2023 near the Arizona-Mexico border. While Qu was initially arrested by immigration authorities, he was later released due to a lack of detention space and was given an immigration removal proceeding scheduled for 2027, officials said.
The attorney's office said investigators first caught Wu on Dec. 2 near Whiteman Air Force Base, home to the military's B‑2 Spirit stealth bomber fleet, an exceptionally sensitive asset due to its nuclear and stealth capabilities.
After initially receiving a warning, Wu reportedly returned the following day. Authorities then arrested Wu, who was allegedly found in possession of 18 photos and videos of the base. He also admitted to taking photos of another U.S. Air Force base, officials said, without naming the second location.
CHINESE ILLEGAL IMMIGRANT CROSSINGS THAT SURGED DURING BIDEN ADMIN A 'NATIONAL SECURITY CONCERN,' EXPERT WARNS
A B-2 Spirit stealth bomber takes off from a field. (Whiteman Air Force Base)
Wu has been charged in federal court in the Western District of Missouri. He faces one count of taking photographs of a vital military installation and military equipment without authorization.
Officials said that on Dec. 2, a suspicious minivan with a Massachusetts license plate was reported near the perimeter of Whiteman Air Force Base. Patrol officers then found Wu, who said he was there to observe the B‑2 Spirit aircraft, and informed him that he was not permitted to take photographs or record video of the military installation.
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A staff member directs a B-2 Spirit stealth bomber at Whiteman Air Force Base. (Whiteman Air Force Base)
The next day, patrol officers were notified of the same minivan nearby, the attorney's office said. Agents reportedly found Wu again, who admitted to taking videos of the B‑2 Spirit aircraft and numerous photographs of the base, including its perimeter fencing, a gate and military equipment. Wu handed over his phone, which contained 18 images and videos, and admitted to authorities that he photographed another U.S. Air Force base and its aircraft, officials said.
According to court documents, Wu is a Chinese national who illegally entered the United States on June 22, 2023, near Nogales, Arizona, but was released after promising to attend immigration removal proceedings scheduled for Feb. 9, 2027, officials said.
A fleet of B-2 Spirit stealth bombers are seen on a runway at Whiteman Air Force Base. (Whiteman Air Force Base)
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If convicted, Wu could face up to one year in federal prison.
The case was investigated by the U.S. Air Force Office of Special Investigations, the Federal Bureau of Investigation, the U.S. Air Force Security Forces and Immigration and Customs Enforcement's Enforcement and Removal Operations, officials said.
Bonny Chu is a Digital Production Assistant at Fox News Digital.
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JD Vance described Minnesota Gov. Tim Walz as ‘a joke' in response to Walz's comment after a fatal Minneapolis shooting that killed Renee Nicole Good.
Minnesota Gov. Tim Walz on Thursday authorized the Minnesota National Guard to be staged and ready to support local and state law enforcement in protecting critical infrastructure and maintaining public safety after the fatal shooting of Renee Nicole Good, 37, by an ICE agent in south Minneapolis.
Soldiers will serve in a support role, focused on protecting property, safeguarding critical infrastructure and allowing local law enforcement to remain focused on community safety and investigative responsibilities, according to a news release from the governor's office.
"Minnesotans have met this moment. Thousands of people have peacefully made their voices heard. Minnesota: Thank you. We saw powerful peace," Walz wrote in a statement. "We have every reason to believe that peace will hold.
"[Wednesday], I directed the National Guard to be ready should they be needed. They remain ready in the event they are needed to help keep the peace, ensure public safety, and allow for peaceful demonstrations."
Gov. Tim Walz speaks during a news conference after the deadly ICE-involved shooting Wednesday in Minneapolis. (Fox News/Pool)
NOEM ALLEGES WOMAN KILLED IN ICE SHOOTING 'STALKING AND IMPEDING' AGENTS ALL DAY
The staging, authorized by an executive order, allows National Guard personnel on state active duty to coordinate and support public safety and security services in Minnesota.
The Minnesota State Patrol has also mobilized 85 members of its Mobile Response Team to support law enforcement efforts in the Twin Cities.
During a news conference Wednesday, Walz said, "We've never been at war with our federal government," raising concerns he would use the National Guard to push back on federal immigration enforcement action in the state.
A demonstrator faces a Border Patrol agent during a protest outside the Whipple Building in Minneapolis Thursday. (Tim Evans/Reuters)
JONATHAN TURLEY SLAMS MINNEAPOLIS MAYOR'S 'RECKLESS' RESPONSE TO ICE SHOOTING
"We do not need any further help from the federal government. To [President] Donald Trump and Kristi Noem, you've done enough. I've issued a warning order to prepare the Minnesota National Guard," Walz said.
"We have soldiers in training and prepared to be deployed if necessary. I remind you, a warning order is a heads-up for folks. Minnesota will not allow our community to be used as a prop in a national political fight."
Republican lawmakers, including Rep. Mary Miller, R-Ill., subsequently urged Trump to invoke the Insurrection Act against Walz, calling for his arrest.
Border Patrol federal agents detain a demonstrator at a protest over the fatal shooting of Renee Nicole Good by a U.S. Immigration and Customs Enforcement agent during a rally outside the Whipple Building in Minneapolis Jan. 8, 2026. (Tim Evans/Reuters)
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"Someone remind him: Donald Trump is the Commander in Chief. And federal authority supersedes state authority," Rep. Nancy Mace, R-S.C., wrote in a social media post.
"That's not an opinion, that's the Constitution. What Walz is threatening has a name: insurrection. Mr. President, the law is on your side. Use it."
Walz's office did not immediately respond to Fox News Digital's request for clarification on his comments.
Alexandra Koch is a Fox News Digital journalist who covers breaking news, with a focus on high-impact events that shape national conversation.
She has covered major national crises, including the L.A. wildfires, Potomac and Hudson River aviation disasters, Boulder terror attack, and Texas Hill Country floods.
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Federal agents wounded two people in a shooting in Portland, Oregon, on Thursday.
The Portland shooting comes a day after an Immigration and Customs Enforcement officer in Minneapolis shot and killed a woman accused of trying to run him over.
The Department of Homeland Security said in a statement that Border Patrol agents were conducting a targeted stop to apprehend a Tren de Aragua-linked illegal immigrant when the driver of the vehicle tried to flee. Then the driver “weaponized” his vehicle and “attempted to run over” the officers.
“Fearing for his life and safety, an agent fired a defensive shot. The driver drove off with the passenger, fleeing the scene. This situation is evolving, and more information is forthcoming,” the department said.
The two were found by Portland police in a different location from where the shooting occurred after responding to a call from a man requesting help. The two, a male and a female, were hospitalized with gunshot wounds after Portland police officers applied a tourniquet on scene.
The shooting took place in the city's Hazelwood neighborhood.
Portland Police Chief Bob Day acknowledged the heightened tension in the community since the Minneapolis shooting.
“We are still in the early stages of this incident,” Day said. “We understand the heightened emotion and tension many are feeling in the wake of the shooting in Minneapolis, but I am asking the community to remain calm as we work to learn more.”
Rep. Janelle Bynum (D-OR), who represents southeastern Portland and parts of other areas, condemned the shooting and said to “stop f***ing with us.”
LIGHTFOOT ROLLS OUT ‘ICE ACCOUNTABILITY PROJECT' IN RESPONSE TO CHICAGO IMMIGRATION OPERATION
“This isn't law enforcement, it's state-sponsored terrorism. Stop f***ing with us. This is the second shooting this week by agents following the orders of a wannabe dictator who is trying to take over cities and rule by instilling terror in the hearts of the American people,” Bynum wrote in a statement.
“When the president deployed his guardsmen to our city earlier this year, we told him their presence was unwelcome and unhelpful. Today, we saw just how dangerous this is — we need these goons gone. Get to steppin',” she added.
Copyright 2026 The Associated Press. All Rights Reserved.
Copyright 2026 The Associated Press. All Rights Reserved.
A banner on the front end of a bus features images of Venezuelan President Nicolas Maduro and China's Xi Jinping with a message that reads in Spanish: “An example for the world,” during a government-organized rally opposing U.S. intervention, in Caracas, Venezuela, Saturday, Dec. 13, 2025. (AP Photo/Cristian Hernandez)
President Donald Trump waves as he walks off stage after speaking to House Republican lawmakers during their annual policy retreat, Tuesday, Jan. 6, 2026, in Washington. (AP Photo/Evan Vucci)
Venezuela's long time Foreign Minster Nicolas Maduro attends a ceremony declaring President Hugo Chavez official winner of the presidential elections at the Electoral Council in Caracas, Venezuela, Oct. 10, 2012, where Chavez announced he was naming Maduro as his new vice president. (AP Photo/Ariana Cubillos, File)
In this photo released by Xinhua News Agency, Chinese President Xi Jinping, also general secretary of the Communist Party of China Central Committee and chairman of the Central Military Commission, speaks at the New Year gathering held by the National Committee of the Chinese People's Political Consultative Conference (CPPCC) in Beijing on Wednesday, Dec. 31, 2025. (Yan Yan/Xinhua via AP)
WASHINGTON (AP) — When it comes to claiming that Venezuelan oil is now under his control, President Donald Trump is mincing no words. But no small part of that oil belongs to China under contracts it struck with Caracas years ago, setting the stage for a delicate diplomatic dance in the next few weeks.
Some experts expect Trump to work with China in an effort to stabilize trade relations. After all, Trump is expected to visit Beijing in April as part of an effort to protect the fragile trade truce he reached with Chinese President Xi Jinping late last year.
“The administration appears focused on avoiding unnecessary escalation or new irritants with Beijing while keeping leverage firmly on Washington's terms,” said Craig Singleton, senior director of the China program at the think tank Foundation for Defense of Democracies.
He added that he doubted Trump would risk turning Venezuela into a “flashpoint that complicates trade dynamics or Trump's personal engagement with Xi.”
China is owed at least $10 billion from Venezuela, according to various estimates, a debt that former Venezuelan President Nicolás Maduro had paid down by shipping oil to China. It is possible that the interim Venezuelan government complying with Washington's demands could question the legality of those loans-for-oil deals and cease payments.
Two major Chinese state-owned enterprises — China National Petroleum Corp. and Sinopec — are entitled to 4.4 billion barrels of oil reserves in Venezuela, the highest for any foreign country, according to a research note by the investment bank Morgan Stanley.
Stay up to date with the news and the best of AP by following our WhatsApp channel.
U.S. companies also have claims for tens of billions of dollars from when Caracas nationalized the oil industry, and it's not clear how these IOUs will be honored and in what order.
The U.S. seized two sanctioned oil tankers this week as part of a plan to assert control over Venezuelan oil shipments. Energy Secretary Chris Wright said the U.S. will handle the sales of Venezuela's oil “indefinitely,” depositing proceeds into U.S.-controlled accounts that will ultimately “flow back into Venezuela to benefit the Venezuelan people.”
The administration said this week that the U.S. would kickstart those sales with 30 million to 50 million barrels taken from the South American country's crude storage facilities. Asked for more detail, a Trump administration official not authorized to comment publicly and speaking on condition of anonymity said U.S. policy was to wind down “adversarial outside influence” in the Western Hemisphere.
The U.S. using such leverage over a crucial natural resource comes after Beijing flexed its muscles last year by choking off critical supplies of rare-earth magnets and weaponizing its purchase of American soybeans in the trade war with Washington. When Trump met Xi in South Korea in October, the two men agreed to a one-year truce, backing off from sky-high tariffs and export controls on each other.
Between 2000 and 2023, Venezuela was the fourth-largest recipient of Beijing's official credit, having received $106 billion worth of loans from China's official-sector creditors, according to AidData, a research lab at Virginia's College of William & Mary that tracks Beijing's overseas lending activities. But how much of the total that Caracas has paid off and what is still owed is unclear, said AidData executive director Brad Parks, because Caracas stopped reporting debt details several years ago.
While some estimates put the outstanding debt at $10 billion, Parks said the figure could be much higher because U.S. sanctions on Venezuelan oil might have delayed loan repayments. The loans from China, under an unusual arrangement, were set up to be paid down with proceeds from oil exports.
In China, the capture of Maduro evoked memories of another leader who had struck deals with Chinese companies and then suddenly lost power: Libya's Moammar Gadhafi.
After the 2011 fall of Gadhafi, Chinese businesses had to leave behind billions in investments. Cui Shoujun, professor of international studies at Renmin University in Beijing, told the Chinese news and commentary site guancha.cn that the transition government in Caracas could deem agreements under Maduro unlawful and the debt to China illegal.
As in Libya, Beijing's stakes in Venezuela have gone beyond oil. Chinese firms have invested in telecommunications, railways and ports in Venezuela, all now at risk, according to a report by the global financial firm Jefferies.
Still, the firm noted that Beijing will likely manage any disruption because Venezuelan oil counted for only a small percentage of China's oil imports and because Beijing has diversified its energy supplies and pivoted to electrification.
Hours before he was captured by U.S. forces, Maduro hosted a high-level Chinese diplomat at the presidential palace and praised the countries' ties that had prospered from the days of his predecessor, Hugo Chávez, and given Beijing a strong foothold in America's backyard.
Venezuela is the only Latin American country that has a high-level strategic partnership with China, on par with close friends like Pakistan, and the ouster of Maduro is expected to curtail China's influence in the Western Hemisphere — in line with one of the goals spelled out in the Trump administration's National Security Strategy.
Soon after Maduro was captured, Beijing said it was “deeply shocked” by the blatant use of U.S. force against a sovereign state and action against its president and said it “strongly” condemned the U.S. actions. It called for the immediate release of Maduro and his wife.
Chinese Ministry of Commerce spokesperson He Yadong said Thursday that no nation has the right to interfere with economic and trade cooperation between China and Venezuela, which he said is between two sovereign states and protected by international and domestic laws.
“No matter how the political situation in Venezuela evolves, China's willingness to deepen bilateral economic and trade cooperation will not change,” He said.
Singleton said Beijing does not wield the clout in the Western Hemisphere as touted.
“Beijing can protest diplomatically,” he said, “but it cannot protect partners or assets once Washington decides to apply direct pressure.”
___
Condon reported from New York.
Copyright 2026 The Associated Press. All Rights Reserved.
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Nationwide Iranian demonstrations intensified a 12th day as crowds target state symbols and security forces reportedly kill protesters. (Credit: NCRI.)
Iran has been plunged into a nationwide internet blackout as anti-regime protests intensify, severely restricting communication across the country as demonstrations enter their second week and the death toll reaches 44, according to reports.
Live network data from NetBlocks showed internet traffic collapsing in the troubled nation on Thursday evening, shortly after calls circulated for mass protests at 8 p.m. local time.
Before news of the latest killings came in, President Donald Trump, in an interview with Hugh Hewitt on Thursday, once again warned the regime if it starts killing people, "they will be hit very hard."
Asked by Hewitt if he had a message for the Iranian people, Trump said, "You should feel strongly about freedom. There's nothing like freedom. You're brave people."
PROTESTS SPREAD ACROSS IRAN AS REGIME THREATENS US FORCES AS 'LEGITIMATE TARGETS' AFTER TRUMP WARNING
The State Department's Persian feed on X also restated Trump's warning from his interview with Hugh Hewitt about the regime in Farsi.
Ali Safavi, a member of the National Council of Resistance of Iran (NCRI), told Fox News Digital the internet shutdown began earlier in the day.
"Around 1 p.m. local time, the internet traffic dropped," he said, adding there were widespread reports "that the regime had cut off the internet."
NetBlocks said the outage followed "a series of escalating digital censorship measures targeting protests across the country," while The Associated Press reported that telephone lines were also cut in parts of Iran.
Safavi said the blackout coincided with violent confrontations in several regions.
Protesters hold signs during a demonstration in Iran amid ongoing unrest, according to images released by the Iranian opposition group National Council of Resistance of Iran. (NCRI )
"The internet was cut off in Lordegan, Chaharmahal and Bakhtiari provinces as battles erupted," he said.
He also pointed to unrest in Isfahan, Iran's third-largest city with around 2.3 million residents, saying, "State radio and the TV station were set on fire by rebellious youth."
As the communications blackout deepened, the confirmed death toll from the unrest rose sharply.
The NCRI reported Thursday that at least 44 protesters had been killed by Iranian security forces since the uprising began.
IRAN'S KHAMENEI LASHES OUT AT PROTESTERS AS NATIONWIDE ANTI-REGIME UNREST GROWS
Iranian demonstrations intensified for a 12th day as demonstrators damaged government buildings across provinces. (The National Council of Resistance of Iran)
The group announced the names of 13 additional victims that day, describing them as "martyrs" of the nationwide uprising.
Seven of those newly identified were from Lordegan, including a woman and two teenagers.
According to the NCRI, the victims were killed by the Revolutionary Guard and other security forces using live ammunition.
The protests were sparked in December by the collapse of Iran's currency and soaring inflation, but they have since evolved into a broader movement demanding the overthrow of the Islamic Republic.
IRAN ON THE BRINK AS PROTESTERS MOVE TO TAKE TWO CITIES, APPEAL TO TRUMP
Iranian protesters try to take control of two cities in western Iran as nationwide unrest continues, with demonstrators chanting "Death to Khamenei" in the streets. (Getty)
The unrest entered its 12th day with general strikes spreading across major commercial centers and street clashes intensifying, particularly in western Iran.
Safavi described the scale of the demonstrations as unprecedented.
"Millions of Iranians from north to south and east to west have been out in the streets until nighttime," he said.
"Over the past 12 days, more young people have laid down their lives to free Iran."
In a statement shared on X, Reza Pahlavi wrote, "Millions of Iranians demanded their freedom tonight. In response, the regime in Iran has cut all lines of communication. It has shut down the Internet. It has cut landlines. It may even attempt to jam satellite signals."
IRAN CRACKDOWN RATTLES MIDDLE EAST AS ANALYSTS WEIGH US OPTIONS SHORT OF MILITARY INTERVENTION
Iranian protests intensified for a 12th day with cars turned upside down. (The National Council of Resistance of Iran)
In Lordegan, security forces had reportedly killed eight protesters in a single day, while clashes also left a regime colonel and two Basij members dead.
Elsewhere, protesters torched government buildings in cities, including Lumar in Ilam province. Safavi said symbols of the state have been targeted nationwide.
"Statues have been destroyed and set on fire," Safavi said, noting "the day before, buses were set alight in Mashhad and another torched."
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Footage circulating online and cited by Reuters showed protesters in Mashhad ripping an Iranian flag in half amid chants against the leadership.
In Tehran, Safavi said, demonstrators overturned a police car in Kaj Square, an affluent area near the Alborz Mountains, as crowds shouted, "Death to the oppressor!"
Emma Bussey is a breaking news writer for Fox News Digital. Before joining Fox, she worked at The Telegraph with the U.S. overnight team, across desks including foreign, politics, news, sport and culture.
Fox News' Antisemitism Exposed" newsletter brings you stories on the rising anti-Jewish prejudice across the U.S. and the world."
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Fox News senior national correspondent Kevin Corke has the latest on the shooting in Portland, Oregon involving Border Patrol agents and an alleged Tren de Aragua member on 'Hannity.'
The Department of Homeland Security (DHS) confirmed a U.S. Border Patrol agent shot two people in Portland, Oregon, after the driver of a car allegedly attempted to run over federal officers.
The incident occurred at approximately 2:19 p.m. local time, when Border Patrol agents stopped a vehicle and identified themselves as law enforcement, DHS said.
According to DHS, the driver – who is believed to be a member of the Venezuelan gang Tren de Aragua (TdA) – allegedly, "weaponized the vehicle and attempted to run over the law enforcement agents."
Fearing for his life and safety, an agent fired a defensive shot, according to DHS. The driver drove off with the passenger, fleeing the scene, officials said.
DHS said the driver was also allegedly involved in a recent shooting in the city.
NOEM CONDEMNS ALLEGED ATTACK ON ICE AGENTS STUCK IN SNOW IN MINNEAPOLIS AS 'ACT OF DOMESTIC TERRORISM'
FBI agents at the scene of an alleged shooting involving federal agents. (KPTV)
Following the incident, Portland Mayor Keith Wilson called on Immigration and Customs Enforcement (ICE) to "halt all operations" in the city until a full and independent investigation can take place.
"We know what the federal government says happened here," Wilson said during a news conference Thursday. "There was a time when we could take them at their word. That time has long passed."
Wilson added that ICE agents and DHS leadership "must fully be investigated and held responsible for the violence inflicted on the American people in Minnesota, in Portland, and in all the communities across America."
Oregon Gov. Tina Kotek reacted to the shooting, claiming it was "instigated by the reckless agenda of the Trump administration."
"While the details of the incident remain limited, one thing is very clear: when a president endorses tearing families apart and attempts to govern through fear and hate rather than shared values, you foster an environment of lawlessness and recklessness," she said.
Kotek said Oregon's attorney general and other leaders have raised concerns with the excessive use of force by federal agents in Portland, adding that "today's incident only heightens the need for transparency and accountability."
Oregon Attorney General Dan Rayfield announced the state DOJ has launched a formal investigation into the shooting.
Portland District Attorney Nathan Vazquez hosts a press conference outside a medical building in Portland after a shooting involving federal agents occurred. (KPTV)
Portland District Attorney Nathan Vazquez said Thursday he was "very concerned" by the incident and pledged a thorough investigation.
Vazquez said his office is working closely with Portland police and the FBI.
Portland Police Bureau (PPB) officers responded to reports of a shooting on the 10200 block of Southeast Main Street at about 2:18 p.m. local time and confirmed federal agents were involved, according to the city.
Fewer than 10 minutes later, at 2:24 p.m., officers were told a man who had been shot was calling and requesting help in the area of Northeast 146th Avenue and East Burnside.
Police responded and found a man and woman with apparent gunshot wounds, according to the city. They were taken to the hospital and their conditions are unknown.
The City of Portland released a map of where a shooting took place Thursday afternoon in Portland, Oregon. (City of Portland)
Both scenes were secured by the PPB pending an investigation, officials said.
No arrests have been confirmed.
"We are still in the early stages of this incident," PPB Chief Bob Day wrote in a statement. "We understand the heightened emotion and tension many are feeling in the wake of the shooting in Minneapolis, but I am asking the community to remain calm as we work to learn more."
Two people were allegedly shot by federal agents in an Oregon neighborhood. (KPTV)
MINNEAPOLIS ICE SHOOTING PROTESTERS SET UP CAMP, BARRICADE ROADS AS SCHOOLS, BUSINESSES CLOSE IN CITY ON EDGE
Audio released Thursday evening from a 911 call captures a request for emergency assistance after a man was shot twice in the arm and a woman, identified as his wife, was shot in the chest.
PPB officers were not involved in the incident, and "do not engage in immigration enforcement," according to city officials.
Sen. Ron Wyden, D-Ore., reacted to the shooting on X, blaming the Trump administration for "inflaming violence."
"I'm monitoring the first awful reports of two people shot in Portland by federal law enforcement," Wyden wrote in the post. "I'll keep you updated, but Trump's deployment of federal agents in my hometown is clearly inflaming violence--and must end."
Sen. Jeff Merkley, D-Ore., said he had "huge concern" about a reported shooting of two individuals by federal agents outside Portland Adventist Hospital.
"Please keep protests of Trump's ICE/CBP peaceful, as Trump wants to generate riots," Merkley said, adding, "Don't take the bait."
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Oregon state Sen. Kayse Jama forcefully rejected the presence of ICE and other federal agencies in the state, saying, "We do not need you."
"You are not welcome and you need to get the hell out of our community," Jama said at a news conference.
Alexandra Koch is a Fox News Digital journalist who covers breaking news, with a focus on high-impact events that shape national conversation.
She has covered major national crises, including the L.A. wildfires, Potomac and Hudson River aviation disasters, Boulder terror attack, and Texas Hill Country floods.
The hottest stories ripped from the headlines, from crime to courts, legal and scandal.
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In this article
New video that appears to show Renee Nicole Good in her car right before she was fatally shot by an ICE agent in Minneapolis was posted Friday on social media by the site Alpha News.
The 47-second-long video appears to have been taken by a phone held by the U.S. Immigration and Customs Enforcement agent who fired at the 37-year-old Good on Wednesday as she drove her SUV toward him.
"Ohhhh!" the agent apparently moans, as the Honda Pilot lurches by him.
"F---ing bitch," the agent apparently mutters after firing at Good.
Vice President JD Vance reposted the video on his X account.
"Watch this, as hard as it is," Vance wrote. "Many of you have been told this law enforcement officer wasn't hit by a car, wasn't being harassed, and murdered an innocent woman. The reality is that his life was endangered and he fired in self defense."
Minnesota Gov. Tim Walz, Minneapolis Mayor Jacob Frey and other Democrats have disputed the claim that Good was trying to intentionally hit the ICE agent.
Some observers have said Good, a mother of three, was trying to swerve around him after being approached by other ICE agents, who were directing her to get out of the SUV.
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DETROIT – Stellantis is scrapping its plug-in hybrid electric Jeep SUVs and Chrysler minivan amid slowing EV sales, quality issues and weakened federal fuel economy requirements.
The automaker on Friday said the decision to end production of the plug-in hybrid Jeep Wrangler, Jeep Grand Cherokee and Chrysler Pacifica was a result of waning customer demand and the need to focus on "more competitive electrified solutions, including hybrid and range‑extended vehicles."
"Stellantis continually evaluates its product strategy to meet evolving customer needs and regulatory requirements. With customer demand shifting, Stellantis will phase out plug‑in hybrid (PHEV) programs in North America beginning with the 2026 model year," the company said in an emailed statement.
The decision is an about-face for the automaker, which has touted its U.S. sales leadership of the models, known as PHEVs, for years. In 2024, then-Jeep CEO Antonio Filosa — who is now CEO of Stellantis — said the SUV brand planned to sell 160,000 to 170,000 PHEVs that year, and the company said it represented 41% of U.S. PHEV sales.
Aside from sales, Stellantis has been using PHEVs as a way to offset its production of gas-guzzling trucks and SUVs to attempt to meet federal fuel economy standards and avoid penalties. The goal has become less urgent been as the Trump administration eliminates or weakens aspects of those rules.
Chrysler first introduced its PHEV minivan in 2016. Jeep debuted the Wrangler PHEV, which it called a "4xe," in 2020, followed by a Grand Cherokee version in 2021.
PHEVs feature traditional internal combustion engines, but also have an all-electric range when charged like an EV. They have largely been viewed as a transitional technology from traditional vehicles to EVs; however, they are quite costly because of their two different propulsion systems.
The cancellation also comes amid a recall of the Jeep SUVs due to fire risk – the latest in a string of issues for the vehicles. The company is also reevaluating its product portfolio as part of its U.S. turnaround strategy.
The company said the recall, which included a stop-sale of the vehicles, "is in no way related" to the cancellation of the vehicles.
Jeep CEO Bob Broderdorf late last month told CNBC the brand was evaluating its electrification strategy since the end of up to $7,500 in federal incentives for EVs and PHEVs in September.
He said Jeep still had vehicles on the ground that it would continue to sell, but "all of us are waiting to see what the demand is, how it's going to continue to shake out, and what becomes steady state for 4xe and [battery] EVs in general."
A Jeep spokeswoman said the brand will continue to offer all-electric SUVs such as the Wagoneer S and Recon, which was officially revealed late last year.
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Industry watchers on both sides of the aisle say President Donald Trump's recently announced goal of banning "large institutional investors" from buying single-family homes won't address the root cause of high home prices: a shortage of homes.
Major investors, including hedge funds and private equity firms, own hundreds of thousands of single-family homes around the US, which has raised concerns that Wall Street-backed groups are outcompeting individual homebuyers, especially first-time buyers, and driving up home prices.
These mega landlords own only a tiny fraction of the overall US housing market, although they control a significantly larger share of single-family rental homes in certain markets, particularly in the Sun Belt. Studies have found that they may have contributed to increased home prices and rents in certain areas.
In the aftermath of the 2008 financial crisis, large investors bought thousands of distressed properties, particularly in Southeastern cities. They correctly predicted that home values and rents in many of the targeted areas would rise over time with population growth.
When the pandemic hit in 2020, and interest rates dropped, investors again snapped up thousands of homes, particularly in cities across the South and Sun Belt. This sparked alarm in communities where individual homebuyers struggled to compete with all-cash offers from major investors.
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Overall, large investors own only about 2% of the single-family rental housing stock, according to a 2024 analysis by the Government Accountability Office. And since 2022, large investors have slowed their purchasing as interest rates have spiked and home prices have remained high. Some major investors have shifted their business model to buying homes in bulk directly from homebuilders, rather than purchasing them one by one.
"You're seeing this huge increase in debt costs to buy a home, and rents have softened mostly or been flat, and then with prospects of potentially declining home prices, or at least flat home prices, it's just tough," said Glenn Hull, CEO of SFR Analytics, which provides software and data tools to real estate investors and others.
Institutional investors have tended to cluster their purchases, resulting in more concentrated investments in certain neighborhoods in a handful of markets, including Atlanta, Dallas, Phoenix, Houston, Charlotte, and Tampa. The 2024 GAO report found that they own 25% of single-family rental homes in Atlanta and 21% of those in Jacksonville. Researchers have found that mega landlords may have contributed to the rise in housing costs, particularly in areas where they're heavily concentrated.
"The studies reviewed by GAO indicate that institutional investment may increase rents and home prices, particularly in places with high rates of institutional ownership," the GAO report said. It added that there are some indications that big investors are more likely to file evictions.
Still, the evidence isn't particularly strong that big investors charge more than smaller landlords, or that they outcompete other buyers.
"It is unclear whether institutional investors crowd out individual home buyers, and evidence about whether they behave differently than other landlords is thin," a 2023 report by the Brookings Institution found.
To the extent that mega landlords are hurting affordability, it's a local problem in the most affected neighborhoods, industry watchers say.
"It's not a national story," said Jason Lewris, cofounder of Parcl Labs, which tracks institutional investors in housing. "If you look within those markets, it's a handful of zip codes."
Much remains unknown about what kind of ban Trump intends to pursue. The administration hasn't defined "large" institutional investor. Trump said he would ban these investors "from buying more single-family homes," but it's not clear whether they would also be required to sell their current portfolios.
A White House spokesman, Davis Ingle, said in an emailed statement, "President Trump is committed to making it easier and more affordable to achieve the American Dream of homeownership by eliminating unnecessary red tape, increasing supply, and lowering costs."
Trump's proposed ban is part of a recent White House effort to address housing affordability concerns. The president announced on Thursday that he's directing Fannie Mae and Freddie Mac to buy up to $200 billion in mortgage-backed bonds in an attempt to bring down mortgage interest rates.
Housing economists say that banning major investors from buying additional homes would do little to improve affordability.
"Targeting a small subset of landlords without addressing underlying market conditions and policy gaps will not meaningfully improve the well-being of renters and prospective homebuyers," Jenny Schuetz, a former senior fellow focused on housing at the Brookings Institution, told the House Financial Services Committee during a 2022 hearing on corporate investors in residential real estate.
That's in part because investors don't change the demand for or supply of housing.
"The housing demand is the same regardless of whether a household rents or buys a home, and housing supply is the same regardless of whether a landlord supplies the unit or a household owns it," researchers at the Urban Institute, a nonprofit think tank, wrote in a 2024 report in which they called institutional investors a "scapegoat" that distracts from the supply shortage driving higher prices.
"Periods of rapid rent increases correspond to periods of greater household formation coupled with supply shortages, not institutional investor buyers," they added. "It is hard to argue that institutional rental operators drive up home prices over any reasonable period."
Instead, economists argue that an undersupply of homes is the major underlying reason for rising prices.
"If we actually wanted to solve that problem, bringing down the cost of home ownership, what we should be doing is increasing the supply of housing," said Daryl Fairweather, the chief economist at Redfin.
Mom-and-pop landlords and other smaller investors own the vast majority of single-family rentals in the US. Stopping a large investor from buying a home doesn't necessarily mean a first-time homebuyer or other individual would end up purchasing the house, Fairweather said. Instead, many of these homes would be snapped up by the main competitors of big investors: smaller landlords.
From the tenant's perspective, having a mom-and-pop landlord isn't necessarily better than renting a home from a large investor. And when it comes to enforcing laws that protect tenants, including fair housing laws, it can be easier to prove a pattern of behavior by larger landlords, Fairweather said.
If the federal government succeeds in passing a ban, enforcing it could be tricky. Large investors could break themselves up into smaller shell companies to avoid the prohibition, Lewris said.
There are other ways to stop investors from buying up more homes that could be more effective. One alternative is to discourage the behavior by raising property taxes on homes owned by institutional investors, thereby making the practice less lucrative, Hull and Fairweather said.
"In general, bans don't really work, because people find a way around them," Fairweather said, "but if you tax something, at least you get tax revenue out of it that could be used to support things like first-time homebuyer programs or building more affordable housing."
Investors often argue that they actually boost quality housing options for lower-income people by offering rentals in neighborhoods dominated by single-family homes. Tenants tend to have far lower incomes and wealth than homebuyers. And the neighborhoods dominated by single-family homes often have attractive amenities, including good schools and jobs.
"The narrative today is that what my business does is wrong, that buying homes and renting them is a bad thing because I'm keeping people from buying homes," Sean Dobson, CEO of Amherst, a single-family rental company heavily invested in the Sun Belt, said at the real estate conference ResiDay in November 2025. "It couldn't be any further from the truth, but it's a powerful message."
Instead, he argued, his firm helps its tenants live in homes that they couldn't afford a down payment for or wouldn't qualify for a mortgage to buy.
Fairweather agreed that banning large landlords from owning single-family homes would mean fewer tenants could live in these wealthier neighborhoods. "In a way, it boxes out renters and exacerbates some of the inequalities that exist in the country," she said.
Madison Hoff contributed to this report.
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Someone made hundreds of thousands of dollars off a well-timed bet on Venezuelan President Nicolás Maduro's political future.
Now, a congressman wants to block government officials from being able to do the same.
Democratic Rep. Ritchie Torres of New York on Friday introduced a bill called the Public Integrity in Financial Prediction Markets Act of 2026, which would bar federal elected officials, political appointees, Executive Branch employees, and congressional staff from making trades on prediction markets when they have nonpublic information related to the transaction, or might be able to obtain it via their official duties.
30 House Democrats are cosponsoring the bill, including former Speaker Nancy Pelosi of California.
Last Friday, a newly created account on Polymarket, a prediction market platform, bet $30,000 that Maduro would be out of office by January 31, 2026.
The next day, after Maduro's capture, the user netted a $436,759.61 prize.
Polymarket does not currently have significant restrictions or rules against insider trading. In fact, CEO Shayne Coplan has argued that insider trading via prediction markets can be a public good.
"What's cool about Polymarket is that it creates this financial incentive for people to go and divulge the information to the market," Coplan said at an Axios Business event.
Polymarket did not respond to Business Insider's request for comment.
By contrast, Kalshi — another major prediction market platform — has rules barring insider trading.
According to the company, a government official would have been forbidden from making the Maduro trade. That's because the platform's rulebook bars "decision-makers" who "have any influence" on the outcome of an event from making trades related to it. The company also has safeguards in place to prevent insider trading.
Earlier this week, Kalshi CEO Tarek Mansour endorsed Torres's bill.
"Why? Because we already implement it," Mansour wrote on LinkedIn.
This isn't the first time that fears of insider trading have cropped up around a major Trump administration action.
In April, as President Donald Trump's "Liberation Day" tariff announcement caused a significant swing in the stock market, some administration officials and members of Congress made well-timed stock trades.
Below is the text of Torres's forthcoming bill, courtesy of his office:
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Intel stock climbed 7% Friday after CEO Lip-Bu Tan met with President Donald Trump, continuing a rally that has seen the stock more than double since the U.S. took a stake in the chipmaker in August.
"The United States Government is proud to be a Shareholder of Intel," Trump said in a Truth Social post Thursday following the meeting.
Trump lauded Tan as "very successful" and touted the launch of Intel's recent chip that was "designed, built, and packaged right here in the U.S.A."
Tan responded in an X post that he was honored and "delighted to have the full support and encouragement" of Trump and U.S. Secretary of Commerce Howard Lutnick.
The CEO also noted that Intel's latest Core Ultra Series 3 CPU processors, its first major product built on Intel 18A, is now shipping out.
In August, the White House negotiated an $8.9 billion investment in Intel, buying 433.3 million shares at $20.47 per share.
That stake is now worth about $19 billion. The stock is up nearly 20% since the start of the year.
The amicable alliance between Trump and Tan has taken a full turn since the two's rocky relationship prior to the administration's investment.
A few weeks before the stake announcement, Trump said in a Truth Social post that Tan "is highly CONFLICTED and must resign, immediately."
The demand came after Sen. Tom Cotton, R-Ark., expressed concern over the CEO's connection to multiple Chinese companies and the potential national security risk.
"Intel is required to be a responsible steward of American taxpayer dollars and to comply with applicable security regulations," Cotton wrote in a press release. "Mr. Tan's associations raise questions about Intel's ability to fulfill these obligations."
Intel responded in a statement that the company, its board of directors and Tan are "deeply committed to advancing U.S. national and economic security interests."
Tan also wrote in a memo to employees that the company was working with the administration to address the concern.
"I want to be absolutely clear: Over 40+ years in the industry, I've built relationships around the world and across our diverse ecosystem – and I have always operated within the highest legal and ethical standards," he said.
Tan was appointed CEO in March 2025 amidst declining sales and company instability under Pat Gelsinger's leadership.
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"Home is where you roam."
"Home is where you park it."
Those charming slogans look great on a bumper sticker. And at first, when my partner and I began our two years living full-time in a self-converted camper van, chasing mountains, cool climbing lines, and a cheaper, freer lifestyle, they felt true.
But over time, the charm wore thin. After the van came the tiny house, where we've now lived for five years.
Both have given us lessons in minimalism, self-reliance, and adaptation, but if I had to pick a favorite? The tiny house wins, even if it's not our ideal living situation.
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At first, van life was everything social media promised it to be: sunsets from mountain overlooks, coffee brewed with the back doors flung open, and a different backyard every few days.
It was pretty cheap, too. My partner and I bought a behemoth of a high-roof van new for about $40,000 in 2018. We spent three months and nearly $10,000 — nearly half of which went toward our fully off-grid solar setup — converting it to something we could live in.
We recouped our investment quickly, especially since we spent the following summer juggling several jobs while saving up to hit the road for an indefinite period.
We only had three relatively large recurring monthly expenses: the $400 loan payment on the van itself, $150 in insurance, and an average of about $200 a month in fuel, depending on how much we drove.
We utilized public land and the occasional Cracker Barrel parking lot for camping and boondocking, so we never spent a dime on campgrounds or RV parks.
Factoring in food and a slim recreational budget (which we often never used), our monthly cost of living hovered under $1,000.
The trade-off of free-wheeling, inexpensive living was everything else. First, there was the omnipresent stress of having everything you own in one compact wheeled thing that begs, "Steal me!"
It also wasn't easy living in a home that could break if you hit a pothole just wrong … or, in my case, misjudged the height of a stump questing for a backroad campsite.
Seeing friends and family meant long drives, taking showers meant going to public gyms, and having privacy meant … well, there wasn't any.
Nothing in van life was guaranteed, not even something as simple as having a safe place to sleep at night.
I certainly don't miss the unique stress of trying to find a place to park and not knowing whether we would wake up surrounded by cops or cows or an angry farmer because following a county road after dark unwittingly led us onto private land — all things that happened to us during our two-year tenure on the road.
Freedom, I learned, can feel a lot like instability.
And that was before the COVID-19 pandemic hit, and we suddenly felt like the community equivalent of an unwanted creepy uncle at the family reunion.
Though we practiced social distancing to the extreme, often spending weeks at a time parked in the middle of nowhere without encountering another soul, the second we pulled into a grocery-store parking lot with our out-of-state license plates for a biweekly supply restock, we were repeatedly given scornful looks and told to "stay home."
Never mind that the van was our home — the only one we had. All in all, it's hard to feel settled when your existence feels temporary in everyone else's eyes.
When those pressures finally got to us, we hung up our van-lifer jackets and parked the van for good on a rented lot in front of a brand-new 400-square-foot tiny house.
Suddenly, we had running water, electricity that didn't rely on a sunny day, and enough space to cook a full meal without bumping elbows.
The immediate financial stress, however, was undeniable.
Our mortgage and lot rent combined cost double our van payment (which we were also still paying off). Utility costs went from a $30 propane tank refill a month to electricity, water, gas, and internet bills that combined to cost a whopping $350 on average.
When adding the cost to furnish the house and buy a used vehicle to serve as our everyday all-season driver (a financial headache in its own right), our hard-earned reserves were quickly depleted.
At the same time, the tiny home gave us a tenuous sense of belonging and stability we didn't realize we were missing.
We now have an address to receive mail at, we know our neighbors, we can plant herbs in the yard, and we can sit on a porch that doesn't roll away with the wind.
If I could only keep one version of tiny living, it'd be the house — not because it's easier or perfect, but because it feels like ours.
I won't say I don't miss the untethered freedom to simply drive to warmer climates when I'm shoveling snow from the driveway or the near-viscous silence of a desolate campsite in the mountains when I'm listening to our neighbors' dogs bark incessantly at 3 a.m.
However, I can't say I'd go back on the road right now. I love the quiet, simple mornings of waking up to water tanks that aren't frozen and being able to sit in my rocking chair with a cup of coffee, thinking about the art I want to make that day.
If van life taught me anything, it's that part of the joy in traveling is the periods of stillness in between that make you restless for the road in the first place. Without a waking reality in between, the dream of perpetual motion becomes unspectacular.
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President Donald Trump suggested in a new interview that the U.S. military could launch land strikes on drug cartels in Mexico.
"We've knocked out 97% of the drugs coming in by water. And we are going to start now hitting land, with regard to the cartels," Trump told Fox News host Sean Hannity in an interview aired Thursday night.
"The cartels are running Mexico, it's very sad to watch and see what's happened to that country," Trump said.
Trump's comment comes less than a week after U.S. forces struck Venezuela and captured its authoritarian leader, Nicolás Maduro, and his wife, Cilia Flores.
The Venezuelan government has said more than 100 people were killed in the military operation, which came after a monthslong pressure campaign against Maduro.
The Trump administration has carried out 35 known strikes on alleged drug boats in the Caribbean, killing 115 individuals, according to The New York Times.
A White House spokeswoman, when asked by CNBC about Trump's comment on Fox News, said, in an email, "The administration is reasserting and enforcing the Monroe Doctrine to restore American preeminence in the Western Hemisphere, control migration, and stop drug trafficking."
"The President has many options at his disposal to continue to protect our homeland from illicit narcotics that kill tens of thousands of Americans every year," the spokeswoman, Anna Kelly, said.
Trump previously floated the idea of executing attacks on Mexico.
"Would I launch strikes in Mexico to stop drugs? OK with me, whatever we have to do to stop drugs," Trump told reporters in the Oval Office in November when asked if he would consider military action across the southern border.
Mexican President Claudia Sheinbaum pushed back on Trump after that, saying there would be no U.S. military action in Mexico without her permission.
This week, Sheinbaum condemned the U.S. capture of Maduro and reaffirmed her country's sovereignty.
"It is necessary to reaffirm that in Mexico the people rule, and that we are a free and sovereign country— cooperation, yes; subordination and intervention, no," Sheinbaum said, according to Reuters.
The Trump administration over the past week has also threatened military action in Cuba, Colombia, and Greenland, prompting backlash even from within the ranks of congressional Republicans.
On Thursday, the Senate passed the first phase of a measure to block Trump from further military action in Venezuela.
Five Senate Republicans voted in favor of the measure, known as a War Powers Resolution, indicating it has enough bipartisan support to clear a final vote.
One of those Republicans, Sen. Susan Collins of Maine, said that she supported Trump's removal of Maduro. But she also said that further action would require congressional approval.
"When the president raises the issue, as he has with not only Venezuela, but Greenland, of military force being used, then it does implicate the War Powers Act and Congress's constitutional role," Collins said.
Correction: Cilia Flores is Nicolás Maduro's wife. An earlier version misspelled her name.
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It's the week Sin City turned sci-fi.
Humanoid robots shadowboxed, danced and pretended to run small shops. Singapore-based Sharpa displayed a robotic hand playing table tennis and dealing blackjack hands.
Across Las Vegas, technology companies used the annual CES trade show to reveal their visions of the future and to loudly proclaim that physical artificial intelligence is poised for a breakout year.
"The humanoid industry is riding on the work of the AI factories we're building for other AI stuff," Nvidia CEO Jensen Huang said at a news conference on Tuesday.
Nvidia, which last year became the world's most valuable company, announced a new version of its vision language models called Gr00t for humanoid robots that can turn sensor inputs into robot body control, as well as a version of its Cosmos model for robot reasoning and planning.
Huang said he expects to see robots with some human-level capabilities this year.
"I know how fast the technology is moving," he said. His company highlighted partnerships with the likes of Boston Dynamics, Caterpillar and LG.
Science fiction writers have dreamed of this moment for decades. "The Jetsons" had Rosey, a robot maid. In "Star Wars," C-3PO helped Luke Skywalker save the galaxy. However, in real life, humanoids have so far been unable to demonstrate the intelligence or flexibility that would make them truly useful, a problem that's long eluded engineers.
Then came generative AI with the launch of OpenAI's ChatGPT in late 2022. The same deep learning technology that underpins ChatGPT can be used to teach the robots how to walk, use a hand or fold laundry. Many in the industry see self-driving cars as the first major commercial manifestation of physical AI.
Industry heavyweights are going big.
In addition to Nvidia, fellow chipmakers Advanced Micro Devices and Qualcomm made splashy robot-related announcements at CES. On Monday, Google's DeepMind said it would work with Hyundai's Boston Dynamics, formerly a division of Google, to develop new AI models for its Atlas robot.
McKinsey estimates that the market for what it calls general-purpose robotics could reach $370 billion by 2040, with top use cases including "warehouse logistics, light manufacturing, retail operations, agriculture, and healthcare."
But some analysts were quick to point out that it's a long way from the show floor to the factory floor, or to the home.
"Although the humanoids were the ones that grabbed everyone's attention, and it was the best kind of eye candy for the show, we're still a very, very long way from the commercial implementation of these," Ben Wood, chief analyst at CCS Insight, said in an interview.
According to CES' official exhibitor list, 40 companies at the event mentioned humanoid robots on the show's website. The Consumer Technology Association, which produces CES, hasn't said how many humanoid robot companies presented at this year's event, but CTA President Kinsey Fabrizio said the number of industrial and consumer robots at the show has been growing.
AMD CEO Lisa Su on Monday revealed a new humanoid robot from Italy's Generative Bionics, a company that it's backing financially. The robot, Gene.01, is scheduled be deployed later this year in industrial environments like shipyards.
Generative Robotics is using AMD's cloud-based graphics processing units to train and fine-tune its models.
"This allows us to customize the next generation of the models on their GPUs," said Generative Robotics CEO Daniele Pucci. "That is the brain."
For now, robot chip sales are a small fraction of Nvidia's business, and AMD reports them as "embedded" sales, a term for industrial chips. Qualcomm's "internet of things" revenue was about 18% of the company's sales in the latest fiscal year.
But they see an opportunity to win business from a new crop of robot makers by offering them not just chips, but an entire software ecosystem to make development easier.
"This is all about any of the major players establishing themselves as a one-stop shop for the robotics development community," Wood said.
While the tech industry has become enamored with large language models in the generative AI boom, many robots are being enabled by vision language models. They can pair sensor data from a robot with traditional AI models to allow for reasoning or planning, such as a route through a messy floor of obstacles.
Along with Nvidia's VLM announcements at CES, Qualcomm showed off a new line of robot chips called Dragonwing that can use the company's VLMs. Qualcomm is using tele-operations to teach its VLM specific skills like how to use actuators to grasp an object.
One particular area of excitement for Nvidia is medicine.
The company showcased a robot from a company called LEM Surgical using its Thor chip. The robot was described as a humanoid, but it didn't have legs. Rather, it had three arms, two for using tools and one that controlled a face-like module of cameras and sensors. Its sole function is to help doctors with spine surgery.
Down the exhibit hall, Nvidia demonstrated a Chinese humanoid robot called Agibot that used a large language model to chat with attendees, though it had trouble standing on the conference center's plush carpets.
Also at the show, Korea's LG showed off its wheeled humanoid CLOiD robot for the first time.
In the demonstration, CLOiD, which is designed for the home, promised to make breakfast and took a wet towel from the presenter and stuck it in a washing machine.
But it was slow. Folding a rectangular towel that was laid out by a presenter took CLOiD about 30 seconds.
Speed isn't the only concern. Experts are also worried about safety and the damage that could be caused by consumer robots.
"Home is very unstructured," said Jeff Burnstein, president of the Association for Advancing Automation. "You can't plan for a child running into the robot or the robot running over a pet."
Some of the first humanoid robots on the market could be more about fun and flash than productivity. China's Unitree Robotics displayed its $70,000 G1 robot at CES. Large crowds of onlookers were treated to a performance of boxing and dancing on the show floor.
The largest tech companies in the world are betting that the market is rapidly evolving. Nvidia's Huang said this week that robots are having their "ChatGPT moment."
Modar Alaoui, general partner at ALM Ventures, sees robots rapidly moving from novelty to reality.
"The next generation is just going to grow up with these machines whether we accept it or not," he said.
— CNBC's Katie Tarasov contributed to this story.
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It could be the year Main Street's appetite for cryptocurrency exposure meaningfully grows.
Although it's been two years since the first spot bitcoin ETFs began trading on U.S. exchanges, BlackRock's Jay Jacobs thinks they're a fairly new concept.
"It's still so early," the firm's U.S. head of equity ETFs told CNBC's "ETF Edge" this week. "Many investors have still just been starting their educational journey around what is bitcoin, [and] how might it fit in a portfolio .… We see this still being very early days for bitcoin and ethereum."
BlackRock manages iShares Bitcoin Trust ETF (IBIT) and iShares Ethereum Trust ETF (ETHA). As of Thursday's market close, IBIT is down more than 3% in the past year. The weakness comes after bitcoin prices hit a record high of around $126,000 last October. It's now trading in the low $90,000 range.
Meanwhile, ETHA is lower by almost 6%.
Cryptocurrencies have gained a lot of traction recently, and part of what's driving that is the fact that large-scale asset managers including BlackRock have expanded options for traders to invest in them, using equity-like formats, including ETFs.
"For many financial advisors, maybe they didn't have access to crypto before, or weren't able to buy IBIT before it was approved on their platforms," Jacobs said.
But for those who have taken the plunge, VettaFi's Todd Rosenbluth says the asset class is instilling a sense of loyalty — despite bouts of volatility.
"They're sticking with it, not necessarily selling out and looking for another alternative so quickly," Rosenbluth told CNBC in the same interview.
Rosenbluth finds crypto investors are basically staying put despite the uncertainty.
"It shows that investors that are moving into getting exposure to cryptocurrency through the ETF wrapper have some loyalty to the product, have confidence in the long-term trends," Rosenbluth said. "They're not necessarily moving in or moving out based on that volatility."
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President Donald Trump will meet Friday afternoon with more than a dozen oil companies at the White House to discuss plans for investment in Venezuela, less than a week after the U.S. ousted President Nicolas Maduro.
The CEOs of Exxon, ConocoPhillips, Shell and a representative from Chevron will attend, sources told CNBC's Brian Sullivan. Representatives from Halliburton, Valero and Marathon will also be there.
Secertary of State Marco Rubio, Energy Secretary Chris Wright, and Interior Secretary Doug Burgum are attending as well.
The White House called the 2 p.m. ET meeting, an industry source told CNBC. It was not scheduled at the request of the oil companies, the source said.
Trump said in the immediate aftermath of Maduro's overthrow that U.S. oil companies will invest billions of dollars to rebuild Venezuela's energy sector. But the industry has been mostly silent as the security situation and stability of the government in Caracas remains murky.
Venezuela has the largest proven crude oil reserves in the world at 303 billion barrels or about 17% of the global total, according to the U.S. Energy Information Administration.
But its oil sector is in dire disrepair. Production has declined from a peak of about 3.5 million barrels per day(bpd) in the 1990s to only around 800,000 bpd today, according to data from energy consulting firm Kpler.
It will cost tens of billions of dollars to return Venezuelan production to its historic levels, Energy Secretary Wright said at a Goldman Sachs' conference in Miami Wednesday. Rystad Energy estimates it will cost more than $180 billion through 2040 for Venezuelan production to reach 3 million bpd.
The Trump administration has provided few details on how it will encourage oil companies to make large investments in a country with history of nationalizing industry assets.
Chevron is the only U.S. oil company currently operating in Venezuela through a joint venture with state oil company Petróleos de Venezuela (PDVSA). Wright told CNBC Wednesday that the U.S. is working closely with Chevron.
"Chevron is on the ground so we're getting daily updates," Wright told CNBC. "They're actually working [under] this regime. So with them, how can we provide incremental tweaks or changes to allow their model to grow even more," the energy secretary said.
Venezuelan production could grow by several hundred thousand barrels per day in the short- to medium term with small capital deployments, Wright said.
But Exxon and Conoco will need reassurances to return to Venezuela, Wright said. The companies exited the country after former President Hugo Chavez seized their assets in 2007. They have billions of dollars in outstanding claims against the government they won in arbitration cases.
Wright said the debts Venezuela owes Exxon and Conoco need to be repaid at some point but are not an immediate priority for the Trump administration. The White House is focused on stabilizing Venezuela's economy through oil sales, the energy secretary said.
"We're trying to engineer a transition of Venezuela to a place that Americans want to do business in, want to invest new capital in, want to grow new partnerships with," Wright said.
But it is unclear whether the White House can convince companies like Exxon and Conoco to return to Venezuela without a dramatic change in government in Caracas.
"The big oil companies who move slowly, who have corporate boards, are not interested," Treasury Secretary Scott Bessent said Thursday at the Economic Club of Minnesota.
"I can tell you that the independent oil companies and individuals, wildcatters – our phones are ringing off the hook," Bessent said. "They want to get to Venezuela yesterday."
The U.S. has taken control of Venezuela's oil exports to pressure the government in Caracas, Wright said. Venezuela will ship tens of millions of barrels to the U.S., which the Trump administration will then sell, holding the proceeds in U.S.-controlled accounts, the energy secretary said.
"We need to have that leverage and that control of those oil sales to drive the changes that simply must happen in Venezuela," Wright said.
The energy secretary said the U.S. is not stealing Venezuela's oil. The proceeds from the sales will be used to benefit the nation of 30 million, he said. Trump said Wednesday that the revenue from the oil will be used to purchase U.S.-made products.
"I have just been informed that Venezuela is going to be purchasing ONLY American Made Products, with the money they receive from our new Oil Deal," the president wrote on social media Wednesday.
Purchases will include agricultural products, medicine, medical devices and equipment to modernize Venezuela's energy sector.
"In other words, Venezuela is committing to doing business with the United States of America as their principal partner," Trump said.
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Meta on Friday announced agreements with three nuclear power providers, including one backed by OpenAI CEO Sam Altman, as part of its efforts to secure necessary resources for its AI ambitions.
The arrangements with Vistra, TerraPower and Oklo, which are all working on nuclear power technologies, are for Meta's Prometheus supercluster computing system that's being built at a data center in New Albany, Ohio. No financial terms were disclosed.
Shares of Vistra and Oklo rocketed higher following the news, climbing 15% and 17%, respectively. Meta shares were flat.
Meta CEO Mark Zuckerberg announced Prometheus in July, describing the system as one of the keys to the development of the company's advanced artificial intelligence efforts. Meta has said it expects Prometheus to come online sometime in 2026.
In working with the three companies on energy production, Meta said the projects should add 6.6 gigawatts of power by 2035, exceeding the total demand of New Hampshire.
"State-of-the-art data centers and AI infrastructure are essential to securing America's position as a global leader in AI," Meta policy chief Joel Kaplan said in a statement.
The company said it will help fund Vistra's nuclear power plants in Ohio and Pennsylvania, extending the lifespan of those facilities and increasing their energy production. The other two companies' nuclear projects are still being developed.
Meta expects the agreements to create "thousands of construction jobs and hundreds of long-term operational jobs."
The deals mark the latest efforts by Meta to secure the energy needed to power its AI infrastructure as the company marches toward Zuckerberg's goal of developing superintelligence, a term used to describe AI that can greatly exceed the capabilities of humans on numerous tasks.
Meta's megacap rivals are also looking to nuclear power to help fuel their AI work. Meta, Amazon and Google signed a pledge in March supporting the tripling of global nuclear energy production by 2050.
In June, Meta announced a 20-year agreement with Constellation Energy so it could purchase purchase nuclear power from the company's Clinton Clean Energy Center in Illinois beginning in 2027.
Meta's deal with TerraPower will provide funding for two of the energy company's nuclear projects that are under development and could begin generating power by 2032, according to Friday's announcement. Meta said it could obtain rights for more energy from up to six of TerraPower's other nuclear energy projects that are targeted for delivery by 2035.
Meanwhile, Oklo's advanced nuclear technology campus is expected to come online as soon as 2030 in Pike County, Ohio, Meta said.
OpenAI's Altman is one of Oklo's biggest investors, owning a 4.3% stake worth about $650 million as of Thursday's close, according to FactSet. Oklo went public in 2024 through a special purpose acquisition company that Altman co-founded.
Altman stepped down as chairman of Oklo's board in April as a way to help the company secure more customers with companies that compete with OpenAI.
WATCH: AI hardware trade dominates in 2025.
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Semiconductor stocks rose on Friday after U.S. President Donald Trump praised Intel and its CEO.
Intel shares rose 2% in extended trading on Thursday after Trump said he had "a great meeting with the very successful Intel CEO, Lip-Bu Tan" in a post on Truth Social. It was last seen 2.63% higher in early pre-market trading Friday.
Other chip and AI-related names saw an uplift as investors digested Trump's post. Broadcom and Micron rose 1.66% and 1.36% in pre-market, respectively. AMD gained 0.36%.
"The United States Government is proud to be a Shareholder of Intel, and has already made, through its U.S.A. ownership position, Tens of Billions of Dollars for the American People — IN JUST FOUR MONTHS," Trump wrote.
"We made a GREAT Deal, and so did Intel. Our Country is determined to bring leading edge Chip Manufacturing back to America, and that is exactly what is happening!!!"
The U.S. government took a 10% stake in Intel through an $8.9 billion investment from the CHIPS and Science Act in August, which had otherwise been struggling.
Intel's share price has risen 75% since the Trump administration announced its stake in the company, which also made the U.S. government its majority shareholder.
Outside the U.S., European names ASML, which manufactures machines needed to make advanced computer chips, and ASMI, which makes semiconductor manufacturing equipment, advanced 5.37% and 4.53% respectively on Friday.
Elsewhere, memory chip firms also rallied at the start of the year as a core component to the training and running of artificial intelligence.
— CNBC's Jordan Novet contributed to this report.
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Taken from CNBC's Daily Open, our international markets newsletter — Subscribe today
U.S. President Donald Trump's ambitions around Greenland are rapidly gaining concrete form — that is, evolving from rhetoric into action.
Mining company Amaroq told CNBC the White House has been having discussions with the firm over investing in its mining projects in Greenland. If it's a purely business transaction, that might not be unwelcome: Aaja Chemnitz, the member of parliament representing Greenland in the Danish Parliament, and chair of the Greenland Committee, told CNBC that the island is "open for business."
Being open for business, however, does not mean welcoming a takeover bid. "It's been quite clear from the beginning that Greenland is not for sale and never will be," Chemnitz said.
But that could be on the agenda for U.S. Secretary of State Marco Rubio's planned meeting with Danish authorities next week. While the meeting was initiated by Denmark's foreign minister, Lokke Rasmussen, and his Greenlandic counterpart, Vivian Motzfeldt, Rubio will be looking to discuss how the U.S. can acquire the Arctic island.
In the very hypothetical scenario — let's call it a thought experiment — that the U.S. buys Greenland, the island would be valued at nearly $2.8 trillion, according to a center-right U.S. think tank, though other parties put that figure lower.
Rubio will also have to navigate thorny issues, such as dealing with Greenlander's desire for independence and Europe's response. As for other global powers, while Russia is conspicuously quiet — probably because it is more interested in seeing any division in NATO over this affair — China is keenly watching developments, having described itself as a "near-Arctic state" in 2018. Any transaction — or operation — on this scale will have global repercussions.
U.S. considering investing in mining projects in Greenland. Amaroq, which operates on the island, said the White House is talking to it about investment in its projects that extract or explore gold, gallium and other critical mineral deposits.
Trump administration races to come up with Greenland takeover plan. U.S. Secretary of State Marco Rubio is set to meet Denmark officials next week to discuss the situation. Here are four key issues surrounding the meeting.
Chinese inflation accelerates in December. The country's consumer price index rose 0.8% from a year earlier, as expected by a Reuters poll of economists. That's the highest level since Feb. 2023. However, inflation was flat for 2025 as a whole.
Divergence between U.S. indexes. The Dow Jones Industrial Average rose Thursday but the Nasdaq Composite slid as investors rotated out of tech. Asia-Pacific markets mostly advanced Friday. Australia's Rio Tinto had its worst day since 2022 on news it had entered buyout talks with Glencore. Meanwhile, Asian defense stocks broadly climbed.
[PRO] Venezuela bond bets pay off. Lee Robinson, founder and chief investment officer of distressed debt investor Altana Wealth, captured returns of 30% in Venezuelan bonds after the U.S. attack on the country. Robinson said there's "more upside" to come.
Trump is desperate to take Greenland. Can NATO defend itself — and is it willing to?
Europe spent much of 2025 scrambling to bolster its defenses against Russia — but just a week into the new year, it's being forced to rethink security once again amid U.S. President Donald Trump's threats to annex Greenland.
On Monday, Denmark's Prime Minister Mette Frederiksen warned an American takeover of Greenland would spell the end of NATO. Of NATO's 32 members, 23 – including Denmark – are also members of the European Union, which has been working extensively to ensure Trump's administration continues its support for Ukraine.
— Chloe Taylor
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Glencore's London-listed shares popped 10% on Friday, after it was confirmed a possible $260 billion takeover bid from Rio Tinto was back on the table.
Shares were last seen 9.9% higher. Meanwhile, London-listed shares of Rio Tinto fell 2.3%, after its Australian shares ended Friday's session 6.3% lower.
"Rio Tinto and Glencore have been engaging in preliminary discussions about a possible combination of some or all of their businesses, which could include an all-share merger between Rio Tinto and Glencore," Rio Tinto, the larger of the two companies, said in a statement early Friday morning.
"The parties' current expectation is that any merger transaction would be effected through the acquisition of Glencore by Rio Tinto by way of a Court-sanctioned scheme of arrangement."
If completed, the deal would create the world's largest mining company. Rio Tinto's market cap is around 209 billion Australian dollars ($139.7 billion), while Glencore's is around £48.5 billion ($65.1 billion) — a combined $204.8 billion.
Rio Tinto and Glencore discussed a merger in late 2024, but talks collapsed over issues such as valuation and the future of Glencore's coal mines.
European mining shares rose on Friday, with the Stoxx Europe Basic Resources index adding around 2%. Copper mining firm Antofagasta jumped 3.5%, while Anglo American was up 2.8%.
CNBC has approached both companies for further comment. Rio Tinto said it had until 5 p.m. London time (12 p.m. ET) on Feb. 5 to either announce a firm intention to make an offer for Glencore or announce that it does not intend to make an offer.
Back in August, Rio Tinto CEO Simon Trott announced a reorganization of the business. Trott promised to cut costs and unlock up to $10 billion from its asset base by making the company focus on three core product groups — iron ore, aluminium and lithium and copper.
A deal between Rio Tinto and Glencore would add to recent M&A activity in the mining sector, after Anglo American and Canada's Teck Resources agreed to merge in a $66 billion deal last September. The merger is expected to create one of the world's top five copper producers.
Renewed talks between Glencore and Rio Tinto have also been by rising demand for copper, with prices of the red metal hitting an all-time high of $13,000 a ton this week. Three-month copper prices on the London Metal Exchange were last seen trading 1.5% lower at $12,702 per metric ton.
Cole Smead, CEO of Smead Capital Management, told CNBC's "Squawk Box Europe" on Friday that he was not surprised the talks had resumed, and said that while Glencore's metals arms would likely be included in any merger, it was less certain what could happen to other divisions of the business.
Smead Capital Management holds Glencore, whose stock makes up around 5% of its international portfolio.
"The dirty, dirty business nobody wants to own is coal. So I wouldn't be surprised to see Glencore do a tax-free spin on the coal business," he said. "This is something that's been talked about, they asked shareholders about doing a U.S. coal business spin, that would fit with the Trump framework as well, he's talked a lot about coal businesses coming back, but I think the coal business is likely to end up on its own. They're in these talks, but there's nothing settled."
He said that this could lead to further consolidation, pointing to players in the coal sector like South Africa's Tendele and Australia's Whitehaven.
A merger with Rio Tinto would set markets up with one of the largest, most liquid public mining companies in the world, Smead added, creating an attractive opportunity for investors.
"So if an investor goes out and says, hey I want to find an attractive commodity-oriented business and I've got to put $10 billion to work, well there's very few securities they'd be able to go out and own, and this would be one of them," he told CNBC. "You'd see multiples go up on these businesses because that liquidity's out there for the large institutional investors of the world."
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LONDON — Europe-listed shares finished higher on Friday, as investors assessed key economic data out of the U.S. that could influence the Federal Reserve's monetary policy decisions this year.
The pan-European Stoxx 600 was almost 1% higher at the closing bell, with major regional bourses and most sectors in positive territory.
Data released by the U.S. Bureau of Labor Statistics on Friday showed nonfarm payrolls rose by 50,000 in December, lower than the downwardly revised 56,000 in November and short of the Dow Jones estimate of 73,000.
European stocks tracked morning gains on Wall Street, as the figures spurred hopes that the softening labor market will prompt an interest rate cut from the Fed at its meeting toward the end of this month.
Defense stocks extended gains for a fifth consecutive day on Thursday, adding 0.8% by the end of Friday's session. It follows President Donald Trump's call for U.S. military spending to rise and continued rhetoric on annexing Greenland.
Trump called for a 50% increase in U.S. military spending, eyeing a $1.5 trillion budget in 2027, in a Truth Social post late Wednesday.
He has also been ramping up calls for Greenland to be brought under Washington's control and is considering various options to make it happen — including military action. It could mean the end of NATO, given the U.S. and Denmark, which is responsible for the defense of Greenland, are both members.
Meanwhile, the CEO of mining company Amaroq told CNBC the U.S was mulling investing in critical minerals mining projects on Greenland. It comes ahead of high-stakes talks between Washington and Danish officials over the island's future as Trump maintains its importance to U.S. national security.
Looking at individual stocks, British aerospace group Rolls-Royce hit a fresh high on Friday, finishing the session 1.7% higher, buoyed by the defense sector and positive sentiment toward the U.K.'s FTSE 100.
It was confirmed Friday morning that British mining firm Rio Tinto is in preliminary talks to acquire Swiss firm Glencore, in what could create the world's largest mining company. Glencore shares closed 9.6% higher.
Shares of European oil companies fell this week as investors continue to react to Trump's action in Venezuela, but pared some gains during Friday's session. BP finished 2.4% higher, Shell added 3% and TotalEnergies gained 3.3%.
It's been a busy week for U.K. retailers, with reports from Tesco, Marks and Spencer and Sainsbury's. Each reported strong Christmas food sales.
Tesco lifted its end-of-year profit guidance for fiscal 2026, from £2.9bn ($3.9 bn) to £3.1bn. Its stock was 1.6% lower at the close of dealmaking.
Sainsbury's reported a 3.4% rise in underlying sales for the third quarter and reiterated its full-year guidance for profit of more than £1bn, roughly in line with its last fiscal year result. Shares in the UK's second largest supermarket chain finished 5.3% lower, sitting near the bottom of the European benchmark for its worst day since Dec. 8.
Elsewhere, European lawmakers provisionally approved a controversial trade agreement with South America's Mercosur bloc on Friday. The deal, which has been decades in the making, has seen strong opposition from farmers and some EU member states over concerns it could create unfair competition for European agriculture.
— CNBC's Olivia Levieux contributed to this report.
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Shares of China-based AI startup MiniMax Group doubled on its first day of trading in Hong Kong on Friday, becoming the second major Chinese developer of large language models to go public.
The company raised 4.8 billion Hong Kong dollars ($620 million) in its IPO, outperforming its local rival Zhipu AI, which had listed in Hong Kong just one day earlier and rose a modest 13% on its debut.
MiniMax shares closed at HK$345, up 109% from its offer price of HK$165.
Both MiniMax and Zhipu are part of China's so-called "AI tigers"— startups building large language models to rival American AI giants like OpenAI, which they've now beaten to going public.
Founded in 2022 and backed by investors such as Alibaba Group and Tencent Holdings, MiniMax specializes in AI applications, including chatbots, image generation and video synthesis.
Its founders, Yan Junjie, the company's chief executive officer, and Yun Yeyi, its chief operating officer, previously worked at SenseTime, a long-time player in China's AI space.
MiniMax's market debut comes as Chinese AI-related firms ramp up fundraising efforts to compete with U.S. rivals and navigate Washington's export curbs on advanced chips used for AI training to China.
According to the company's prospectus, it served over 200 million cumulative users across more than 200 countries and regions as of September last year.
It also reported revenue of $53.4 million in the nine months ended Sept. 30, 2025, up about 174% from a year earlier, though the company still posted a net loss of $512 million over the same period.
MiniMax said most of its revenue comes from subscriptions and in-app purchases for its AI products. It added that it remains in a "nascent stage in terms of monetization and commercialization," after years focused on developing its foundational models.
Minimax said it plans to use IPO proceeds for continued research and development.
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TikTok is preparing to carve off parts of its US business, but not all US workers are joining the new entity.
Some US staff were told this week that they will not work for the new joint venture, called TikTok USDS Joint Venture LLC, led by managing investors Oracle, Silver Lake, and MGX. Instead, they will work for a separate TikTok global entity that will remain under ByteDance's ownership, called TT Commerce & Global Services LLC.
The change, outlined in a memo sent to impacted employees, includes workers who focus on US products that will remain tied to TikTok's global operations after the deal closes. In December, TikTok's CEO Shou Chew told staff those business lines would include "certain commercial activities, including e-commerce, advertising, and marketing."
Other US workers, such as those focused on data protection or algorithm security, would work under the USDS Joint Venture entity, according to Chew's memo. He said the deal would close on January 22.
TikTok and ByteDance did not respond to Business Insider's request for comment about the staffing change.
If the proposed TikTok deal goes through, Oracle, MGX, and Silver Lake would each own 15% of the new US joint venture. Affiliates of existing ByteDance investors would get around 30%. Five percent would go to an unnamed group of new investors, and ByteDance would keep just under 20% of the business to comply with the ownership requirements of the US divestment law that sparked the deal.
The law, known as the Protecting Americans from Foreign Adversary Controlled Applications Act, requires companies based in countries the US has deemed a foreign adversary to divest from their US assets or face hefty fines.
Congress called out TikTok and China-based ByteDance in the law's text. TikTok challenged the law in court but lost its case in a Supreme Court ruling last January.
President Donald Trump allowed the app to continue to operate last year via a series of executive orders as his administration worked with TikTok and prospective buyers on a potential deal. In September, the White House announced a deal had been reached, which Vice President JD Vance said would value TikTok US at around $14 billion.
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TikTok is preparing to carve off parts of its US business, but not all US workers are joining the new entity.
Some US staff were told this week that they will not work for the new joint venture, called TikTok USDS Joint Venture LLC, led by managing investors Oracle, Silver Lake, and MGX. Instead, they will work for a separate TikTok global entity that will remain under ByteDance's ownership, called TT Commerce & Global Services LLC.
The change, outlined in a memo sent to impacted employees, includes workers who focus on US products that will remain tied to TikTok's global operations after the deal closes. In December, TikTok's CEO Shou Chew told staff those business lines would include "certain commercial activities, including e-commerce, advertising, and marketing."
Other US workers, such as those focused on data protection or algorithm security, would work under the USDS Joint Venture entity, according to Chew's memo. He said the deal would close on January 22.
TikTok and ByteDance did not respond to Business Insider's request for comment about the staffing change.
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If the proposed TikTok deal goes through, Oracle, MGX, and Silver Lake would each own 15% of the new US joint venture. Affiliates of existing ByteDance investors would get around 30%. Five percent would go to an unnamed group of new investors, and ByteDance would keep just under 20% of the business to comply with the ownership requirements of the US divestment law that sparked the deal.
The law, known as the Protecting Americans from Foreign Adversary Controlled Applications Act, requires companies based in countries the US has deemed a foreign adversary to divest from their US assets or face hefty fines.
Congress called out TikTok and China-based ByteDance in the law's text. TikTok challenged the law in court but lost its case in a Supreme Court ruling last January.
President Donald Trump allowed the app to continue to operate last year via a series of executive orders as his administration worked with TikTok and prospective buyers on a potential deal. In September, the White House announced a deal had been reached, which Vice President JD Vance said would value TikTok US at around $14 billion.
Jump to
Following the United States' capture of Nicolás Maduro over the weekend, a report came out claiming that Venezuela had $60 billion stored in Bitcoin—leading to speculation that the U.S. could lay claim to cryptocurrency as well as oil. Despite numerous reports of the huge Venezuelan Bitcoin stash, however, a crypto forensic firm is skeptical of the claims.
The news of Venezuela's Bitcoin holding began to bubble up last Saturday, the same day that Maduro was ousted. The digital publication Project Brazen reported that his regime could control $60 billion in the original cryptocurrency—but offered little in the way of proof.
“The article does not mention any addresses as a starting point, making it difficult to verify any of these speculated claims,” said Aurelie Barthere, principal research analyst at Nansen, about Project Brazen's report.
Barthere is not the first person to express skepticism about the country's purported crypto treasure trove. Mauricio di Bartolomeo, the Venezuelan co-founder of the financial services company Ledn, told Fortune on Wednesday that the level of the country's corruption makes the figure hard to believe. He expanded his argument in an opinion piece he wrote for Coindesk.
Estimates of Venezuela's crypto holdings vary wildly. Bitcointreasuries.net estimates that the country has $22 million worth of Bitcoin. That figure would make Venezuela the government entity with the ninth-most money tied up in the original cryptocurrency, just behind North Korea.
While the exact size of Venezuela's Bitcoin wealth is unclear, the country has long been a player in crypto. Maduro introduced a token called the Petro in 2018, which was shuttered six years later. Its citizens have also turned to stablecoins as a way to fight their currency's hyperinflation.
Trump has said that he will “run” Venezuela, and some have speculated that includes seizing the country's Bitcoin holdings. Andrew Fierman, head of national security intelligence at Chainalysis, said he could not speak to the likelihood of such a seizure. He did, however, explain what gaining control of assets might look like.
A freezing of assets could occur through centralized services, he says. These services would get a court order for an exchange or an issuer like Tether or Circle who could blacklist an address. The second method is through physical seizure. The U.S. could get control of wallets, devices, and keys through compelled cooperation.
For now, there is unlikely to be a full and accurate account of Venezuela's Bitcoin holdings until the political situation in the country becomes more stable.
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Crypto VC Giant Andreessen Horowitz Raises $15 Billion to Help America 'Win' Tech Race
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Leading American venture capital firm Andreessen Horowitz—a major crypto industry investor that also goes by a16z—announced Friday that it has raised over $15 billion across five separate venture funds to propel American technology.
The $15 billion represents more than 18% of all venture funds raised in the U.S. in 2025, according to firm co-founder Ben Horowitz.
“As the American leader in venture capital, the fate of new technology in the United States rests partly on our shoulders. Our mission is ensuring that America wins the next 100 years of technology,” Horowitz wrote in a post about the raise.
At Andreessen Horowitz, we just raised over $15B.
With these new funds including American Dynamism ($1.176B), Apps ($1.7B), Bio + Health ($700M), Infrastructure ($1.7B), Growth ($6.75B), and other venture strategies ($3B), we raised over 18% of all venture capital dollars… pic.twitter.com/KbtYvaH6Ed
— a16z (@a16z) January 9, 2026
“That starts with winning the key architectures of the future—AI and crypto. It continues with applying those technologies to the key areas that generate human flourishing: biology, health, defense, public safety, education, and entertainment,” he continued.
The raise netted funds for investments in American dynamism, apps, biology and health, infrastructure and growth, and “other venture strategies.”
Although crypto is not specifically allocated for in the fundraise announcement, the firm's crypto arm—a16z crypto—has led some of the largest fundraises in the crypto space over the last few years. It maintains investments in a long list of noteworthy projects and brands in the space, including Coinbase, Solana, Uniswap, OpenSea, Phantom, among others.
In the last year, those investments have included participation in a $300 million fundraise from prediction market Kalshi, a $70 million investment in Ethereum restaking protocol EigenLayer, and a token investment in Solana DeFi protocol, Jito.
The firm's investment in the crypto space dates back to at least 2018, when it built its first crypto fund with $350 million for investment into industry companies and protocols. In the years since then, the VC giant has raised at least three specific crypto funds, gathering more than $7 billion in total.
Andreessen Horowitz's newest raise more than doubles that amount, stressing the “fundamental importance for humanity that America wins.”
“There is no other country that comes close to giving everyone a chance to grab that opportunity and build. If America fails to win technologically, it will lose economically, militarily, geopolitically, and culturally. And the entire world will lose, as well,” Horowitz wrote.
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By Isabella Flores
Key Takeaways:
Andreessen Horowitz officially hit the mega capital milestone with billions to fund its next wave of innovation. The recent effort of the firm to raise money highlights a changing venture environment in which artificial intelligence and blockchain infrastructure are becoming more and more inseparable.
A16z is the name of the venture capital giant recently having completed a huge capital raise to the tune of 15.5 billion dollars. This flow of liquidity will be headed to a number of major “venture strategies,” in a refined manner of capturing the current tech cycle. The company has invested 6.75 billion in its Growth fund and has invested more in its Infrastructure and Apps funds with a total of 1.75 billion. The other tranches are of its strategy, American Dynamism amounting to one point two billion and Bio + Health at 700 million dollars.
The importance of this scale of fundraising is that it represents almost 20% of the total amount of venture capital funds distributed in the United States during the last year. The firm is establishing itself as a major architect of the next internet by acquiring this amount of dry powder. The plan shifts towards the specialized model of the present as compared with the generalist model of the past. All funds are headed by professionals who can offer extensive technical and regulatory assistance to startups in their areas of specialization.
Read More: a16z Makes Bold Asia Push: Opens Seoul Office as It Targets the World's No.2 Crypto Market
The most striking change in the investment direction of a16z is the combination of AI and blockchain. The company has just published a teaser and internal news about the fact that AI and crypto are not silos anymore. They are instead becoming dependent on each other. Blockchains must be transparent and verified to make AI work safely whereas crypto must use AI to enhance user experience and automatically carry out onchain tasks.
This vision is already being manifested through investments. Recently, the company made a $15 million token acquisition to collaborate with the Babylon protocol to convert Bitcoin into trustless collateral. This action indicates an urge to access the productive segment of the legacy assets. Moreover, a16z is peering at the emergence of the so-called AI agents that can also transact on their own. The use of cryptographically signed credentials is necessary to enable the functioning of these agents in financial systems without the involvement of humans.
Another important aspect of the future roadmap of the firm is not tokenization, but origination. In a recent update, a16z partners stated that it is not sufficient to move offchain assets onto a blockchain. The objective is to make both debt and credit as well as assets originated onchain. This makes the financial system more efficient, since the loan servicing acts are reduced and is more accessible worldwide.
The company also expects privacy to emerge as the most significant moat of the industry. The more the institutional capital markets join the market, the higher the quantum-resistant and decentralized messages become necessary. This is partly the reason why they have made privacy-preserving technology one of their 2026 pillars. They think that in the absence of strong privacy, global finance cannot entirely switch to open blockchains.
It's time to build. pic.twitter.com/wQoPalwFrZ
— a16z (@a16z) January 9, 2026
The huge increase of 15 billion is in an era where the regulatory climate in the United States is becoming more pronounced. The company has been an outspoken campaigner of legislative acts such as the CLARITY Act. They are convinced that transparent regulations will enable network tokens to make full economic circles. Such maturation is necessary to get the industry out of its adolescence phase and into a wider adoption phase.
The optimism is backed by market information. The overall crypto market cap has recently hit the 4 trillion mark and the amount of active mobile wallets is the highest ever. a16z is positioning itself to be at the forefront by having billions of new money to attract this mainstream era. They have a clear direction of movement: to support the most daring founders that are creating the infrastructure of a decentralized, AI-based future.
Read More: Trump Pledges to Sign CLARITY Act, Fast-Tracking U.S. Crypto Rules as China Accelerates
Isabella Flores
Blockchain Adoption Reporter
Isabella specializes in tracking how blockchain technology is transforming industries worldwide. She previously worked as a business analyst for a fintech startup before pivoting to journalism. Her pieces explore the real-world applications of blockchain, from supply chain to healthcare. Isabella is passionate about highlighting underrepresented use cases in the crypto space.
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Quick take:
Andreessen Horowitz (A16z), the Silicon Valley, CA-based venture capital firm, on Friday said it raised $15 billion in its latest funding secured across five funds.
According to a blog post published by Ben Horowitz on the A16z website, about $6.75 billion will go towards scaling startups, $1.7 billion has been allocated to infrastructure, $1.7 billion for various apps, $1.176 billion for American Dynamism (investing in national interests like defense, housing, and supply chain), and another $700 million for biotech and healthcare.
There is also an extra $3 billion set to go to “other venture strategies.”
According to Horowitz, this latest funding accounts for over 18% of all venture capital dollars allocated in the United States in 2025. The firm wants to power the next growth phase of the technology industry in the United States.
Highlighting the country's role in leading the world across various industries over the last 250 years, Horowitz said that given the current technological opportunity, presented by AI and crypto, “technological opportunity, it is fundamentally important for humanity that America wins.”
“Our mission is ensuring that America wins the next 100 years of technology. That starts with winning the key architectures of the future – AI and crypto,” he wrote.
The country will then continue applying those technologies to the key areas that generate human flourishing, including biology, health, defense, public safety, education, and entertainment.
Horowitz also highlighted the key steps the United States has taken towards ensuring America wins the tech race for the next 100 years, adding that if the country fails to push its policies of the country in the right direction, it will likely lose its position as the global leader in technology. “We have already seen the beginnings of this in both AI and crypto.”
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Ethereum co-founder Vitalik Buterin has thrown his public support behind Tornado Cash developer Roman Storm—who was convicted last August of a money transmitting charge—arguing that privacy-preserving software is a fundamental human right, and that Storm's work should not be criminalized simply because it can be misused.
“I have supported Roman Storm's work from the beginning both as a strong believer in the importance of privacy, and as an active user of privacy tools, including those developed by Roman,” Buterin wrote on X on Friday, sharing a letter of support he had written for Storm.
“In the 21st century, we are all faced with risks from all corners of the world, both online and offline. If someone has information about you, they have the [ability to] exploit you—socially, commercially, or even physically,” Buterin continued. “Being able to choose with whom we share information about our personal lives, our communications with friends and colleagues, our whereabouts and our finances, is an essential protection against this.”
Tornado Cash, a crypto mixer that helps obscure the path of transactions, was placed on the U.S. Treasury's sanctions list in 2022 after officials said it had been used by North Korea's Lazarus Group and other criminals to launder billions of dollars in stolen digital assets.
Tornado Cash was used to wash proceeds from major hacks, including the $622 million Ronin Bridge exploit and a $100 million theft from Harmony Bridge, with blockchain analytics firm Elliptic estimating that more than $1.5 billion in illicit crypto flowed through the mixer before it was sanctioned. Around $7 billion in total assets went through the platform.
The Office of Foreign Assets Control barred Americans from using the service, though the sanctions were lifted in March 2025.
Storm was charged in 2023 with conspiracy to commit money laundering, conspiracy to violate sanctions, and conspiracy to operate an unlicensed money transmitting business. Last August, a Manhattan jury convicted him on the unlicensed money transmitting count, but deadlocked on the others.
Several weeks later, Matthew Galeotti, head of the DOJ's criminal division, said software developers would no longer be convicted under the charge for which Storm had been convicted.
Storm has not yet been sentenced and has filed a motion for an acquittal, which prosecutors have opposed. He faces up to five years in prison.
Ethereum's Blockchain Trilemma 'Solved': Vitalik Buterin
Another Tornado Cash developer, Alexey Pertsev, also faced criminal proceedings. Dutch authorities arrested the Russian national in 2022, and a court later found him guilty of laundering $1.2 billion in illicit assets through the mixer, sentencing him to more than five years in prison. Pertsev has since been released to house arrest while he appeals.
The Ethereum Foundation pledged $1.25 million toward Pertsev's legal defense.
In Storm's case, Buterin added that privacy is necessary for many parts of our society—including culture and politics—to function without devolving into social games or outright coercion.
“Many have the implicit viewpoint that privacy from the public is fine, but surely governments and police and intelligence agencies should be able to see everyone's information to ensure safety,” he said.
The Year in Ethereum 2025: Institutions Embrace ETH as the 'Ivory Tower' Crumbles
Buterin said he strongly disagreed with this approach, as government databases can be hacked and information lands in the hands of foreign adversarial actors.
“Agencies routinely outsource their work to private corporations, who sell the data behind everyone's back. Cell phone companies, who have everyone's location data, often casually sell it to anyone who asks, which often leads it to end up in the hands of hostile foreign governments,” he explained.
“Roman, and I, want to see a world where basic protections of our rights, that were an unquestioned default in the previous millennium, stay with us in the next,” he added. “And for that, I support him.”
Altcoins Defy Bitcoin Slump as XRP, Solana Notch Double-Digit Gains
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Bitcoin and Ethereum consolidate after a strong start to 2026, while a select group of altcoins is posting significant weekly gains. Experts highlight this performance as a sign of capital rotation into higher-risk assets, as the broader market awaits key macroeconomic catalysts.
Majors like XRP and Solana are up nearly 10% and 7% over the past week, respectively, according to CoinGecko data. Other tokens, including Sui, Bittensor, and Shiba Inu, have notched gains between 14% and 17% in the same period.
This performance contrasts with Bitcoin's lackluster price action, whose year-to-date gain has halved to 4%, and Ethereum, which has similarly retraced from over 11% to nearly 4%.
“The altcoin rally reflects a classic rotation pattern—capital flowing toward perceived upside optionality when macro uncertainty peaks,” Marcin Kazmierczak, Co-founder of RedStone, told Decrypt.
Solana and XRP benefit from specific narrative tailwinds, but the move is “largely sentiment-driven rather than fundamental,” Kazmierczak added.
The narrative strength for leading altcoins includes continued inflows into the spot Solana ETF and market speculation about a potential XRP ETF approval in 2026.
“This may be why these tokens have seen more interest,” Nicolai Søndergaard, research analyst at on-chain analytics platform Nansen, told Decrypt. He added that smart money positioning remains in a “wait and see mode,” requiring further positive news to fuel a broader rally.
The altcoin moves occur against a backdrop of Bitcoin consolidation, with major directional catalysts on the horizon.
“Key near-term catalysts include the U.S. employment report on the 9th and the U.S. Consumer Price Index (CPI) on the 13th,” Yuya Hasegawa, crypto market analyst at Bitbank, told Decrypt.
Hasegawa noted that positive data could act as a catalyst for a Bitcoin breakout toward $98,000, a key technical level. However, he also pointed to a potential downside gap in CME futures near $88,000, which could be tested if data disappoints, though he expects “meaningful support” around that level.
Looking ahead, the sustainability of the altcoin surge is uncertain without support from the broader market.
“Heading into the weekend, we can expect continued volatility,” Kazmierczak said. “Alt jumps are quick to reverse without follow-through volume.”
The experts agree that early next week will be more telling, with the upcoming U.S. economic data sets to provide the next major signal for institutional risk appetite and the potential resumption of a market-wide uptrend.
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South Korea is moving to approve spot bitcoin ETFs this year as part of a broader crypto policy shift that pairs regulated market access with stricter stablecoin rules and expanded use of blockchain in public finance.
South Korea is reportedly preparing to open its financial markets to spot bitcoin exchange-traded funds (ETFs) this year, marking a shift in the country's long-standing approach to digital assets as regulators accelerate work on a comprehensive new crypto law.
The plan was outlined in the government's newly released 2026 Economic Growth Strategy, with the Financial Services Commission (FSC) taking the lead on implementation.
If approved, spot bitcoin ETFs would become available to domestic investors for the first time, placing South Korea alongside markets such as the United States and Hong Kong, where similar products have already attracted billions of dollars in inflows.
Until now, Korea's capital markets rules have not recognized cryptocurrencies like bitcoin or bitcoin ETFs as eligible underlying assets for ETFs, effectively blocking their launch. That stance is now changing as policymakers look to bring more crypto activity into regulated channels and reduce the flow of capital to offshore platforms.
The bitcoin ETF push is moving in parallel with a broader overhaul of digital asset regulation. The FSC is fast-tracking what it calls “Phase Two” digital asset legislation, a bill expected to focus heavily on stablecoins.
According to government plans, the law will introduce a licensing system for stablecoin issuers, minimum capital requirements, and strict reserve rules requiring at least 100% backing of issued tokens. Issuers would also be required to guarantee user redemption rights.
Regulators say the framework is designed to prevent failures like the 2022 Terra-Luna collapse, which wiped out roughly $40 billion and had deep ties to South Korea.
Alongside domestic rules, authorities are drafting standards for cross-border stablecoin transfers and transactions, reflecting growing use of digital tokens in trade and remittances. The effort is being coordinated between the FSC and the Ministry of Economy and Finance.
Officials point to global precedents as a key influence. Spot bitcoin ETFs in the U.S. and Hong Kong have seen strong demand, with major asset managers now treating the products as mainstream investment tools.
Korea's Financial Intelligence Unit estimates that more than 10 million people are eligible to trade digital assets domestically, underscoring the scale of potential demand.
Beyond private markets, blockchain is also moving into public finance. The government plans to digitize parts of the national treasury using so-called “deposit tokens,” a form of government-linked digital currency distinct from stablecoins, according to reports.
By 2030, up to 25% of treasury operations could be conducted via blockchain-based payments.
Pilot programs are already underway, and lawmakers are reviewing amendments to the Bank of Korea Act and the National Treasury Act to establish a legal foundation for these systems.
Officials say the goal is faster settlement, lower administrative costs, and improved transparency.
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Investors are piling into Polygon's POL token amid excitement surrounding a planned tech upgrade to the $1.5 billion blockchain.
On Thursday, Polygon Labs, the firm behind the blockchain, announced the Open Money Stack, a new stablecoin payment framework set to launch later this year.
It aims to let users instantly and reliably move money anywhere across the $162 billion DeFi ecosystem, providing an alternative to restrictive and often costly financial intermediaries like banks.
“Open and interoperable money ensures that it is usable everywhere, by everyone, on their own terms,” Marc Boiron, CEO of Polygon Labs, and Sandeep Nailwal, CEO of the Polygon Foundation, said in a joint post announcing the new technology.
“People don't need to understand settlement mechanics or lose sleep because they are worried about when money will arrive.”
The POL token, which is used to pay transaction fees on the network, among other things, has risen 13% over the past 24 hours. That adds to a 31% gain for the token over the past week, helped along by a broader crypto market recovery.
Polygon's announcement comes as both traditional finance firms and crypto upstarts rush to leverage the regulatory clarity surrounding stablecoins following the passing of the Genius Act in July.
But Polygon is far from the only firm hoping to capitalise on the situation.
Fintech giant Stripe is developing its own blockchain-based payments platform called Tempo, planned to launch later this year, which buy-now-pay-later firm Klarna will use to launch its own stablecoin.
Tempo has lured several well-respected crypto researchers, executives, and software engineers, including former Ethereum Foundation researcher Dankrad Feist, former Optimism Labs CEO Liam Horne, and Rice University Professor Mallesh Pai.
Elsewhere, stablecoin issuers Tether and Circle, as well as Visa, BVNK and Ripple are all actively building or expanding stablecoin-based payment networks and infrastructure.
A perennial problem in the crypto industry is that blockchains have had a hard time replicating the same functionality that traditional financial rails provide. Users eventually need to take their money offchain and rely on traditional financial infrastructure.
Polygon Labs' intent is to create a system where users can do everything while keeping their money in crypto.
The Open Money Stack is designed to work across different blockchains and allows financial institutions and fintech firms to integrate it into their own products.
Individual components include blockchain rails, wallet infrastructure, fiat on-ramps and off-ramps, stablecoin interoperability, compliance, and onchain identity verification, among other things.
The framework will also give users the ability to earn yield on their funds while they're not in use through DeFi, with different options depending on the user's risk appetite.
Tim Craig is DL News' Edinburgh-based DeFi Correspondent. Reach out with tips at tim@dlnews.com.
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Portugal's development bank Banco Português de Fomento (BPF) has signed an international financing contract with the Spain's Institute of Official Credit (ICO) for a line of financing with an initial injection of up to €50 million.
BPF has made “its first international financing agreement with the Official Credit Institute (ICO), the Development Bank of Spain, marking a relevant institutional milestone in BPF's internationalisation strategy and in strengthening financial cooperation between the two countries,” it said in a statement.
This agreement provides for “the provision of a financing line with an initial allocation of up to €50 million, on an ‘on-lending' basis” which aims to “directly finance Portuguese companies and projects in Portugal with an economic connection to Spain” and which will promote “investment initiatives with a cross-border impact”.
With this line, BPF believes that it will “expand its capacity to support the economy, ensuring favourable conditions for financing companies and business investment projects with an international dimension”, ensuring “high added value for the competitiveness” of the country.
The BFP has a goal to finance 20,000 companies overall with €8Bn in 2026.
The development's bank CEO, Gonçalo Regalado said “2026 brings a number of relevant challenges but with countless opportunities for companies and the Portuguese economy.
There is room for us to gain scale in global markets with the signing of the new EU-Mercosur agreement, with the strengthening of the European internal market and with the search for new markets in Africa, the Persian Gulf and the East.
Portugal is in a situation of economic and social stability, balanced public accounts and guarantees of a favorable investment environment”, said the CEO laying out the bank's goals for 2026.
He said that Portugal had a good foreign direct investment pipeline for industry and technology, artificial intelligence, software, tourism, in services, energy and infrastructure.
Companies, he added, relied on the development bank to be a driver of the economy, an accelerator for growth and a multiplier using financial instruments to support investment.
In 2025, BPF injected €6.5Bn into the economy, representing 2.2% of GDP, supporting more than €16,000 companies with more than 18,000 investment financing operations.
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Samson Mow, a vocal Bitcoin proponent and the JAN3 CEO, has taken to his account on the X social media platform to once again point out a key advantage of Bitcoin over altcoins to the global crypto community.
This statement occurred after the recent exodus of the Zcash core developer team.
Mow slams "s-coin developers quitting" and praises Bitcoin
The JAN3 boss addressed the community with a message, in which he praised the world's pioneer crypto, Bitcoin, and took a major jab at altcoins and their developer teams once again.
Mow emphasized that unlike with altcoins, whose developers can give up working on them and quit “all at once” all of a sudden, Bitcoin has 100% protection from that. Its initial developer, the mysterious Satoshi Nakamoto, voluntarily disappeared in December 2010.
Bitcoin is now believed to be completely decentralized, unlike altcoins, such as Ethereum, XRP, etc. Mow, along with other Bitcoin maxis, often slams these large-cap altcoins, calling them scams.
Bitcoin is insurance against shitcoin dev teams quitting all at once.
Aside from complete decentralization, Bitcoin maxis often name absolute scarcity — with 21 million being the finite supply of coins of BTC — from which more than 19 million have been mined already.
Zcash core devs' mass exodus
As reported by U.Today earlier, this week, the development team of Zcash's Electric Coin Company announced it was quitting after they massively clashed regarding governance issues with its nonprofit board, Bootstrap, over distractions from its mission of focusing on privacy.
Reacting to this unfortunate event, the price of the ZEC token immediately fell by roughly 20%, hitting $381 and erasing $1.6 billion in the coin's market capitalization value. This highlighted vulnerabilities in the governance of this decentralized project and boosted investor interest in the early privacy coin Monero (whose initial developer is no longer on the project either, like Satoshi).
The Zcash core team now has plans to set up a new company to sustain Zcash's core technological advancements.
When similar things happen in the Bitcoin developer community, such as in 2017 and 2018, they usually result in hard forks (Bitcoin Cash, Bitcoin SV), with a portion of the developers quitting, while the initial BTC protocol continues to function and dominate the cryptocurrency market.
Select market data provided by ICE Data Services. Select reference data provided by FactSet. Copyright © 2026 FactSet Research Systems Inc.Copyright © 2026, American Bankers Association. CUSIP Database provided by FactSet Research Systems Inc. All rights reserved. SEC fillings and other documents provided by Quartr.© 2026 TradingView, Inc.
Should investors trust the cryptocurrency trading for just $2, or the one trading for thousands of dollars?
XRP (XRP 1.99%) and Ethereum (ETH 0.90%) are two of the best high-upside cryptocurrency investment options. They both have demonstrated the ability to skyrocket in price, and both have strong potential catalysts. In the past, both have minted their fair share of millionaires.
So which one is more likely to be a millionaire maker? To answer that question, it's important to consider the following two factors.
A good starting point is historical performance. While past returns are no guarantee of future performance, they can help to provide a quick snapshot look at how a cryptocurrency does over time. Is the cryptocurrency a one-hit wonder, or is it capable of replicating its performance over an extended period of time?
From 2017 through 2025, XRP grew at a compound annual growth rate (CAGR) of 94%. Since its launch back in 2012, XRP is up an impressive 40,000%.
Based on those numbers alone, you might conclude that a single XRP token is trading for hundreds, if not thousands, of dollars. But you would be wrong. XRP still trades for a price of about $2.
There's a good reason for this low price. XRP's performance has been extremely uneven over the years. In fact, the standard deviation of XRP's returns from January 2017 to November 2025 is a head-spinning 334%. By way of comparison, the standard deviation of an S&P 500 stock is typically about 15%, while the standard deviation of a fast-growth tech stock is typically anywhere from 20% to 30%.
That's why it's hard to trust XRP. It's the type of cryptocurrency that's either soaring or collapsing in value. If you're trying to build a million-dollar nest egg, you might lose everything after one bad year. Or maybe one bad month.
That's not to say that Ethereum can be trusted either. It, too, is prone to intense periods of boom and bust. Case in point: Ethereum soared by 472% in 2020 and 395% in 2021, only to collapse in price by 68% in 2022.
But Ethereum has been much better at bouncing back from adversity and rewarding longtime investors. Since its launch back in 2015, Ethereum is up an incredible 115,000%. As a result of being able to piece together good years over an extended period of time, Ethereum currently trades for a price of about $3,150.
XRP and Ethereum are both vying to become important pieces of the modern global financial system. From stablecoins to real-world asset (RWA) tokenization, both XRP and Ethereum are at the forefront of exciting changes taking place on Wall Street.
Image source: Getty Images.
But Ethereum has a more diversified blockchain ecosystem than XRP, and that makes it more capable of growing at a smooth, steady rate. While Ethereum is a behemoth when it comes to decentralized finance (DeFi), it is also a market leader when it comes to other applications of blockchain technology as well. This includes everything from non-fungible tokens (NFTs) to blockchain gaming.
The XRP blockchain, for its part, is limited to use cases related to finance. After all, the XRP token is primarily just a bridge currency used to swap between different currencies. As such, it is a useful way to transfer value across borders. Once you get beyond cross-border payments, though, the number of use cases for XRP is relatively small.
I'm attaching a much higher growth multiple to Ethereum. It's simply capable of growing faster than XRP. And due to the diversification of its blockchain ecosystem, it's much more insulated from market peaks and valleys.
Based on the above, the clear winner is Ethereum. If you're trying to build a million-dollar crypto portfolio, this is where I'd start. It has a nearly unparalleled historical track record. And it is at the center of several important trends taking place within the global financial system. So much so, in fact, that the Trump administration thought about classifying Ethereum as a strategic asset.
Just keep in mind, though, that the future price trajectory of Ethereum is unlikely to be straight up. It's prone to intense cycles of boom and bust, so be prepared to hold through market volatility. But if you do so, you will likely be handsomely rewarded.
Dominic Basulto has positions in Ethereum and XRP. The Motley Fool has positions in and recommends Ethereum and XRP. The Motley Fool has a disclosure policy.
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Coinbase has updated its official asset listing roadmap to include four digital assets that the exchange has decided may be listed in the future.
According to an update on its blog, the assets newly added to the Coinbase roadmap include the Solana network Raydium (RAY) and Energy Dollar (ENERGY).
Two additional assets from the Base network are also featured on the roadmap: Elsa (ELSA) and Sport.fun (FUN).
Coinbase says asset transfers and trading for the listed assets are not supported until an official trading announcement is made.
The exchange also warns that depositing these assets into a Coinbase account before support is live may result in permanent loss of funds.
The company's roadmap update is part of its effort to increase transparency by publicly communicating when it has made a decision to list an asset.
Coinbase says that listing assets on the roadmap does not immediately enable trading; live support requires fulfillment of additional conditions, including sufficient technical infrastructure.
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Regulator to launch consultation on draft guidance for automated decision-making in 2026
The Information Commissioner's Office (ICO) has highlighted the potential for agentic AI to reshape how people live, work, and interact with public services over the next five years. The data protection body cautioned that developers and adopters must address significant data security challenges to ensure public trust.
In its latest Tech Futures report, the ICO described how agentic AI could bring both innovation and risk. The regulator warned that poorly designed systems could lead to unnecessary processing of personal data, unclear accountability, and heightened cybersecurity threats.
The UK Government is exploring the use of agentic AI for public services, with plans to automate administrative tasks such as updating addresses, electoral registration, or signing up for a new GP. In social services, agents could help users complete administrative tasks, freeing up professionals for more face-to-face interaction. While in healthcare, the sector could see teams of specialised AI agents supporting diagnosis, treatment planning, and care tasks. The report is positive about these developments, but warns that they must be professionally developed.
In cybersecurity, agentic AI presents a double-edged sword. Agentic AI could be used for large-scale automated attacks, but the technology also offers opportunities for advanced defences. These could proactively identify vulnerabilities or reactively safeguard networks, either by alerting humans or taking autonomous action.
“Agentic AI will have the capacity to make decisions and take actions independently,” said William Malcolm, executive director of regulatory risk and innovation at the ICO. “These systems can handle vast amounts of personal information, so both developers and adopting organisations must ensure transparency, security, and compliance is built in from the start.
“While the potential benefits could be transformational, the public needs assurance that their personal information is secure and well managed before placing their trust in agentic systems. Strong data protection foundations can help build that public trust and can help scale the fast and safe adoption of AI.”
The ICO plans to monitor advancements in agentic AI throughout 2026 and will launch a consultation on draft guidance for automated decision-making later this year. It continues to collaborate with other regulators and government bodies to address data privacy and AI development issues.
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Will crypto assets defend their key short-term support levels?
The cryptocurrency market has shed $120 billion this week after January's recovery curve stalled. In particular, Bitcoin's rebound, which was lifted by over $1 billion in ETF inflows, reversed by mid-week.
According to SoSo Value's data, U.S Spot ETFs saw $729 million in total outflows on Tuesday and Wednesday.
Source: SoSo Value
Over the same period, Bitcoin's price shed over $4,500 and dropped from $94,500 to $90,000 on the price charts.
The market sentiment was further soured by the expectation of a Fed rate pause at the meeting scheduled for 29 January. Over the past two days, the odds of a rate pause rose by 4% to 86.7%.
The Jobs report and inflation data scheduled for 9 and 14 January could further affect the rate cut outlook and drive the market sentiment for risk assets.
Source: CME FedWatch
However, the current rate pause outlook at 3.50%-3.75% further dragged crypto lower.
However, it must be noted that although BTC fell by 5%, major altcoins dumped even harder during the mid-week retreat.
XRP, for example, depreciated by 14% from $2.4 to $2, reversing nearly half of its significant January gains.
Source: XRP/USDT, TradingView
Near-term bulls could track the $2-support zone as a possible reversal point. The area also coincided with the 50-day Moving Average (MA) that could reinforce short-term bullish momentum if defended.
However, a break below it could send XRP's price to the recent lows near $1.80. Here, it's worth pointing out that the altcoin also saw massive whale interest during the early 2026 recovery – A trend that could trigger a swift reversal if market sentiment improves.
Like BTC, Ethereum's [ETH] price also dropped by about 6% from $3,300 to $3,000. December's price action chalked a symmetric triangle pattern that could go either way. However, in the event of a bullish breakout, the immediate target would be $3,600.
Source: ETH/USDT, TradingView
On the contrary, a dip below $2.9k would indicate a bearish breakout and likely lead to further price compression.
Even so, the altcoin season index reading jumped from 25 to a neutral reading of 57 at press time – A sign that alluded to a considerable rebound for the sector in January, despite the recent cool-off.
Disclaimer:
AMBCrypto's content is meant to be informational in nature and should not be interpreted as investment advice. Trading, buying or selling cryptocurrencies should be considered a high-risk investment and every reader is advised to do their own research before making any decisions.
© 2026 AMBCrypto
The monastery complex discovery comprised the remains of a church, residential halls, and communal dining spaces, in addition to numerous artifacts.
Here's what you'll learn when you read this story:
When archaeologists stripped away the desert sand in Upper Egypt's Sohag province, they uncovered a Byzantine-era monastery, complete with a church, residential halls, communal dining spaces, and artifacts up to 1,400 years old.
The discovery wasn't a one-off structure, but an entire integrated residential complex for monastic living, complete with a variety of mudbrick structures, according to a translated statement from Egypt's Ministry of Tourism and Antiquities, part of the government's Supreme Council of Antiquities.
Mohamed Ismail Khaled, secretary general of the Supreme Council of Antiquities, said the find adds new information to the understating of monastic life in Upper Egypt during the Byzantine era, while showing a pattern of settlement and everyday living. The Byzantine era was the height of Christian living in Egypt, stretching from the late Roman era of 330 C.E. to the Arab conquest in 641 C.E.
The popular Nile Valley construction method of using mudbrick for structures was clear, according to Mohamed Abdel Badii, head of the Egyptian Archaeological Sector at the Supreme Council of Antiquities. He said the excavation revealed mudbrick buildings stretching west to east, ranging in dimensions from 23 feet by 26 feet to 46 feet by 26 feet.
Buildings with rectangular halls also featured a semicircular vaulted apse on the eastern end and small, brick-vaulted alcoves throughout, likely used as individual worship retreats for monks.
Badii said the structures house trusses and wall beams, while including layers of tiling on the walls and floors. Some of the structures featured entrances on the southern side that were near the remains of small circular buildings. These lost structures may have been the monk's communal dining areas.
The team found a 46-by-32-foot mudbrick building they believe was the main church. It featured three key sections of a typical Coptic-style church design with a sanctuary nave, a home for the choir, and apse designs on both sides. Mudbrick pillars found in the nave suggest a central dome once covered the main sanctuary.
The excavation revealed ruins of buildings featuring red brick and limestone plaster basins covered with a layer of red tiles for waterproofing, which were likely used to store water or for an industrial activity that took place on the site.
Along with the buildings, the team located plenty of artifacts from when the monastery was inhabited. Amphorae found were once used for storage, some inscribed with Coptic letters, numbers, or names that could have delineated the type of writings stored inside. Limestone ostraca pieces featured the Coptic language and may have even featured architectural information.
Sharif Fathi, minister of tourism and antiquities, said such a find can deepen our understanding of Egyptian history across eras while helping develop a tourist attraction for those interested in the history of civilization and religions.
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People who consume higher amounts of food preservatives may face a greater risk of developing type 2 diabetes, according to a large new study. Preservatives are commonly added to processed foods and beverages to extend shelf life. The research was conducted by scientists from Inserm, INRAE, Sorbonne Paris Nord University, Paris Cité University and Cnam as part of the Nutritional Epidemiology Research Team (CRESS-EREN). The findings are based on health and diet data from more than 100,000 adults enrolled in the NutriNet-Santé cohort and were published in the journal Nature Communications.
Preservatives are part of the broader category of food additives and are widely used throughout the global food supply. Their presence is extensive. In 2024, the Open Food Facts World database listed around three and a half million food and beverage products. More than 700,000 of those products contained at least one preservative.
Two Major Types of Preservative Additives
In their analysis, Inserm researchers divided preservative additives into two main groups. The first group includes non-antioxidant preservatives, which slow spoilage by limiting microbial growth or slowing chemical reactions in food. The second group consists of antioxidant additives, which help preserve foods by reducing or controlling exposure to oxygen in packaging.
On ingredient labels, these additives typically appear under European codes between E200 and E299 (for preservatives in the strict sense) and between E300 and E399 (for antioxidant additives).
Why Researchers Are Investigating Preservatives
Earlier experimental research has raised concerns that some preservatives may harm cells or DNA and interfere with normal metabolic processes. However, direct evidence linking preservative intake to type 2 diabetes in large human populations has been limited until now.
To better understand this potential connection, a research team led by Mathilde Touvier, Inserm Research Director, examined long-term exposure to food preservatives and the incidence of type 2 diabetes using detailed data from the NutriNet-Santé study.
Tracking Diet and Health Over More Than a Decade
The study followed more than 100,000 French adults between 2009 and 2023. Participants regularly provided information about their medical history, socio-demographic background, physical activity, lifestyle habits, and overall health.
They also submitted detailed food records covering multiple 24-hour periods. These records included the names and brands of industrial food products they consumed. Researchers cross-referenced this information with several databases (Open Food Facts, Oqali, EFSA) and combined it with measurements of additives in foods and beverages. This allowed the team to estimate each participant's long-term exposure to preservatives.
Measuring Preservative Consumption
Across all food records, researchers identified a total of 58 preservative-related additives. This included 33 preservatives in the strict sense and 27 antioxidant additives. From this group, 17 preservatives were analyzed individually because they were consumed by at least 10% of the study participants.
The analysis accounted for many factors that could influence diabetes risk, including age, sex, education, smoking, alcohol use, and overall diet quality (calories, sugar, salt, saturated fats, fibre, etc.).
Diabetes Cases and Risk Increases
Over the study period, 1,131 cases of type 2 diabetes were identified among the 108,723 participants.
Compared with people who consumed the lowest levels of preservatives, those with higher intake showed a markedly increased risk of developing type 2 diabetes. Overall preservative consumption was linked to a 47% higher risk. Non-antioxidant preservatives were associated with a 49% increase, while antioxidant additives were linked to a 40% higher risk.
Specific Preservatives Associated With Risk
Among the 17 preservatives examined individually, higher intake of 12 was associated with an increased risk of type 2 diabetes. These included widely used non-antioxidant preservatives (potassium sorbate (E202), potassium metabisulphite (E224), sodium nitrite (E250), acetic acid (E260), sodium acetates (E262) and calcium propionate (E282)) as well as antioxidant additives (sodium ascorbate (E301), alpha-tocopherol (E307), sodium erythorbate (E316), citric acid (E330), phosphoric acid (E338) and rosemary extracts (E392)).
What the Researchers Say
"This is the first study in the world on the links between preservative additives and the incidence of type 2 diabetes. Although the results need to be confirmed, they are consistent with experimental data suggesting the harmful effects of several of these compounds," explains Mathilde Touvier, Inserm research director and coordinator of this work.
"More broadly, these new data add to others in favor of a reassessment of the regulations governing the general use of food additives by the food industry in order to improve consumer protection," adds Anaïs Hasenböhler, a doctoral student at EREN who conducted these studies.
"This work once again justifies the recommendations made by the National Nutrition and Health Programme to consumers to favor fresh, minimally processed foods and to limit unnecessary additives as much as possible," concludes Mathilde Touvier.
This work was funded by the European Research Council (ERC ADDITIVES), the National Cancer Institute, and the French Ministry of Health.
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Scientists at the University of Southampton have developed a new strategy designed to strengthen how the immune system responds to cancer. The approach aims to help immune cells recognize and attack tumors more effectively.
The findings were reported in the journal Nature Communications. In the study, researchers tested specially engineered antibodies designed to more strongly activate T cells, the immune cells responsible for killing cancer cells.
How Antibodies Can Boost Immune Signals
These antibodies work by grabbing and clustering several immune cell receptors at once, which increases the strength of the signal that tells a T cell to attack cancer. When these signals are stronger, T cells are more likely to launch a full immune response.
The research team from the University of Southampton's Centre for Cancer Immunology focused on a receptor called CD27. This receptor needs a matching key (ligand) to activate T cells. During infections, the body naturally produces this ligand, but cancer cells do not. Without it, T cells receive only a weak activation signal and struggle to attack tumors effectively.
Why Traditional Antibodies Fall Short
Antibodies can sometimes act like a master key, helping trigger immune responses. However, most antibodies used in medicine today have a Y shaped structure with two arms, which limits them to binding just two receptors at a time.
Although antibody based treatments have transformed cancer care, they do not work for every patient. In some cancers, T cells still fail to become fully active because they are missing the combination of signals needed to mount a strong attack.
A Four Pronged Antibody Design
The antibodies developed in this study were built with four binding arms instead of two. This allows them to attach to more receptors simultaneously. They also recruit a second immune cell, which forces all the CD27 receptors being held to gather together. This clustering greatly amplifies the activation signal and closely mimics how CD27 is triggered naturally in the body.
Professor Aymen Al Shamkhani at the University of Southampton, who led the research, said: "We already understood how the body's natural CD27 signal switches on T cells, but turning that knowledge into a medicine was the real challenge. Antibodies are reliable molecules that make excellent drugs. However, the natural antibody format was not powerful enough, so we had to create a more effective version."
Stronger Activation of Cancer Fighting T Cells
Laboratory tests using mice and human immune cells showed that the new antibodies were much better at activating CD8+ T cells than standard Y shaped antibodies. CD8+ T cells are often described as the special forces of the immune system because of their ability to directly destroy cancer cells. The enhanced activation led to a stronger anti tumor response.
By making CD27 easier to target with therapy, the research offers a roadmap for developing new immunotherapy treatments that better harness the immune system's natural power.
Professor Al Shamkhani added: "This approach could help improve future cancer treatments by allowing the immune system to work closer to its full potential."
The study was funded by Cancer Research UK and highlights the Centre for Cancer Immunology's role in advancing innovative approaches to cancer immunotherapy.
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Small nuclear RNAs (snRNAs) combine with specific proteins to generate small nuclear ribonucleoproteins (snRNPs), the building blocks of the spliceosome. U4 snRNA forms a duplex with U6 and, together with U5, contributes to the tri-snRNP spliceosomal complex. Variants in RNU4-2, which encodes U4, have recently been implicated in neurodevelopmental disorders. Here we show that heterozygous inherited and de novo variants in RNU4-2 and in four RNU6 paralogs (RNU6-1, RNU6-2, RNU6-8 and RNU6-9), which encode U6, recur in individuals with nonsyndromic retinitis pigmentosa (RP), a genetic disorder causing progressive blindness. These variants cluster within the three-way junction of the U4/U6 duplex, a site that interacts with tri-snRNP splicing factors also known to cause RP (PRPF3, PRPF8, PRPF31), and seem to affect snRNP biogenesis. Based on our cohort, deleterious variants in RNU4-2 and RNU6 paralogs may explain up to ~1.4% of otherwise undiagnosed RP cases. This study highlights the contribution of noncoding RNA genes to Mendelian disease and reveals pleiotropy in RNU4-2, where distinct variants underlie neurodevelopmental disorder and retinal degeneration.
While approximately2 million individuals worldwide are affected by retinitis pigmentosa (RP), it is estimated that 30% to 50% remain without a conclusive genetic diagnosis, even after exome or genome sequencing is performed1,2,3,4. This reflects high genetic heterogeneity, limited testing access and as-yet-unidentified disease genes, which in general carry pathogenic variants that are exceedingly rare in the control population5,6,7.
Noncoding RNAs are essential to many cellular processes, including pre-messenger RNA (pre-mRNA) splicing, which is ensured by the spliceosome, a macromolecular complex that in its major form is composed of five small nuclear RNAs (snRNAs), U1, U2, U4, U5 and U6, and ~300 proteins8. Each snRNA associates with a specific set of proteins to form a small nuclear ribonucleoprotein (snRNP), the functional unit of the spliceosome. Variants in RNU4-2, one of the two paralogs encoding U4, have been linked to a common neurodevelopmental disorder (NDD) known as ReNU syndrome (OMIM: 620851). These variants account for up to 0.4% of all NDD cases and lead to systematic misrecognition of donor splice sites by the spliceosome9,10,11. Likewise, RNU2-2 and RNU5B-1 have been recently associated with NDDs11,12,13.
Several spliceosomal proteins are also known to be involved in a wide range of hereditary diseases, including RP, as first noted by McKie and colleagues14. Specifically, of the ~100 genes that are currently associated with nonsyndromic RP5, the tri-snRNP splicing factor genes PRPF3, PRPF4, PRPF8, PRPF31 and SNRNP200 underlie the autosomal dominant form of the condition (adRP), with variants in PRPF31 accounting for 10–20% of all adRP cases3,15.
Here, we identify both inherited and de novo variants in RNU4-2 and four paralogs of RNU6, encoding the U6 snRNA, as the molecular cause of adRP in 153 individuals across 67 families. We demonstrate that all identified variants cluster within the U4/U6 duplex, in a region that binds directly to PRPF31 and PRPF3 and indirectly to PRPF6 and PRPF816,17. Furthermore, we show that such variants increase the association of U4 and U6 snRNAs with the splicing factors SART3 and PRPF31, suggesting impaired snRNP biogenesis.
We initially examined a nonconsanguineous family with adRP (Family M1-A; Supplementary Fig. 1), in which seven of eight siblings (II:1–II:7) and their father (I:1) displayed classical RP features (Supplementary Fig. 2 and Supplementary Data 1). Genome sequencing was negative for pathogenic variants in known retinal disease-associated genes, but selective DNA variant filtering and shared haplotype analysis revealed a total of 55 variants that were absent from gnomAD v.4.17,18 and co-segregated with RP. Of these, none was predicted to impact splicing (SpliceAI > 0.2)19 and only one was evolutionarily conserved (GERP = 4.03 and phyloP-vertebrate = 3.18)20,21, a single-nucleotide insertion in the gene RNU4-2 (NR_003137.2:n.18_19insA; Fig. 1a, Supplementary Fig. 1 and Supplementary Tables 1 and 2). This DNA change was present in one individual from the All of Us database22.
a, Two-dimensional structure of the U4/U6 duplex, with recurrent variants identified in RP cases (in red for U4 and in green for U6), all clustering within the three-way junction. Nucleotides affected by variants previously observed in NDD cases are underlined. b, Rare variants affecting RNU4-1, defined as AF < 0.1% in gnomAD v.4.1, identified in RP cases and in controls. c, Same as in b for RNU4-2, with recurrent pathogenic variants displayed in red. d, Same as b for all five RNU6 paralogs combined, with recurrent causative variants displayed in green. Significant P values for variants enriched in RP cases versus controls from gnomAD are indicated (two-sided Fisher's test with Bonferroni correction).
To find additional families, we first screened by Sanger sequencing a cohort of 1,891 individuals from the European Retinal Disease Consortium (www.erdc.info) with RP or Leber congenital amaurosis who remained undiagnosed after a large high-throughput screening using single molecule Molecular Inversion Probes23. This analysis led to the identification of three additional families comprising 15 affected individuals segregating the same pathogenic variant (Supplementary Fig. 1 and Supplementary Tables 1 and 2). The n.18_19insA allele was significantly enriched in the RP cohort compared with both the gnomAD and the All of Us databases (analyzed control genomes: 76,215 and 414,000, respectively; Bonferroni-corrected P values = 2.6 × 10−3 and 6.9 × 10−5, respectively, by two-sided Fisher's test; Supplementary Table 3). Additional screening of the RNU4-2 sequence in the same cohort led to the identification of 28 other variants, one of which (n.56T>C) recurred in eight individuals from four families (Fig. 1a, Supplementary Fig. 1 and Supplementary Tables 1 and 2), was absent in controls and was significantly enriched in patients versus controls (Bonferroni-corrected P values = 6.4 × 10−5 (gnomAD) and 7.9 × 10−8 (All of Us); Supplementary Table 3).
Additional screening of 2,830 RP cases without previous genetic diagnosis from our respective institutions' cohorts, the UK National Genomic Research Library (hosting data from the Genomics England 100,000 Genomes Project24 and from the NHS Genomic Medicine Service) uncovered an additional patient harboring n.18_19insA (for whom the variant was de novo) and six families (nine affected individuals) carrying the n.56T>C variant (Supplementary Fig. 1 and Supplementary Tables 1 and 2). Altogether, recurrent variants in RNU4-2 were identified in 41 affected individuals from 15 families (Supplementary Fig. 3 and Supplementary Tables 1 and 2). Of note, incomplete penetrance was observed for nine obligate carriers, without visual symptoms (Supplementary Fig. 1). One carrier of n.56T>C was asymptomatic, with subnormal electroretinogram, diffuse atrophic changes in the periphery and attenuated vessels. Another individual with the same variant showed no clinical signs of disease upon examination, and seven (among whom four were deceased) were not clinically evaluated to determine their disease status. Our combined screening of RNU4-2 also revealed 24 other unique rare DNA changes in 27 families, which were classified as variants of uncertain significance (VUS), as well as three benign changes (Supplementary Table 3).
Because U4 snRNA can also be transcribed from its paralog RNU4-1, which differs from RNU4-2 at only four positions (n.37, n.88, n.99 and n.113; Supplementary Table 4), we next examined its sequence in our initial cohort and identified 63 variants, none of which were significantly enriched in cases compared with controls; also, these changes did not include variants at sites corresponding to n.18_19 and n.56 of RNU4-2 (Fig. 1b and Supplementary Table 3). Notably, RNU4-1 appears to be more tolerant to variation compared with RNU4-2, as evidenced by the numerous and frequent variants that are present in genomes from the general population (cumulative allele frequency of 20.4% in RNU4-1 versus 1.2% in RNU4-2; gnomAD v.4.1) (Fig. 1b,c and Supplementary Fig. 4), as already noted previously9.
In the di-snRNP and the tri-snRNP complexes of the major spliceosome, U4 binds to U6 to form the U4/U6 RNA duplex. We therefore hypothesized that variants in U6 could also underlie adRP and extended our analysis to all five identical paralogous genes producing the U6 snRNA, scattered across the genome (RNU6-1, RNU6-2, RNU6-7, RNU6-8 and RNU6-9; Supplementary Table 4). A screening of these genes by Sanger sequencing in our initial cohort of 1,891 RP families revealed 94 DNA changes in total. The n.55_56insG insertion recurred at the exact relative position in RNU6-2, RNU6-8 and RNU6-9 (four families per gene, 34 cases in total; Supplementary Fig. 1 and Supplementary Tables 1 and 2) and was significantly enriched in cases versus controls, who were all negative for this change (Bonferroni-corrected P value = 2.6 × 10−18 (gnomAD) and 5.1 × 10−27 (All of Us); Supplementary Table 3). Since this variant was identical in three U6 genes, we reasoned that the specific DNA change, rather than any particular paralog, was relevant to the etiology of the disease. We therefore repeated our analysis by collapsing the five RNU6 genes and detected 66 unique variants. Another insertion, n.56_57insG, was identified in two unrelated families (once in RNU6-2 and once in RNU6-9, four cases in total; Supplementary Fig. 1 and Supplementary Table 2) and found to be significantly enriched in cases versus controls (Bonferroni-corrected P value = 1.8 × 10−3 (gnomAD, a single RNU6-2 positive individual of unknown status) and 2.1 × 10−5 (All of Us, no positive individuals); Supplementary Table 3). We then extended our analysis to the same international cohorts of patients that were previously analyzed (n = 2,830) and identified 74 additional cases from 38 families who were positive for either n.55_56insG or n.56_57insG (Supplementary Table 2).
In total, these two variants were detected in 112 affected individuals from 52 families, involving all RNU6 paralogs except RNU6-7. The n.55_56insG insertion was present in most cases (102 individuals from 47 families), occurring in four of the five RNU6 paralogs: RNU6-1, RNU6-2, RNU6-8 and RNU6-9, while n.56_57insG was present in ten individuals from five families, in RNU6-1, RNU6-2 and RNU6-9 (Supplementary Tables 1 and 2 and Supplementary Figs. 1 and 3). Notably, n.55_56insG was confirmed to be a de novo event in eight individuals, clinically identified as sporadic cases. In 14 additional pedigrees, it was also observed in individuals born to unaffected parents, for which de novo inheritance was suspected but could not be confirmed, due to the lack of parental DNA. In contrast, no de novo events could be detected for n.56_57insG, which was identified exclusively in families with adRP (Supplementary Fig. 1). Similar to the screening of the RNU4 paralogs, our analysis of RNU6 paralogs revealed 66 VUSs and 23 benign variants, validated by Sanger sequencing (Supplementary Table 3).
In summary, we identified variants in RNU4-2 or RNU6 paralogs that underlie de novo or inherited dominant RP in 67 families. The overall phenotype across all cases was consistent with classical RP, based on clinical examination and electrophysiological testing, with symptomatic onset predominantly in adolescence (Supplementary Table 5). In addition, other concurrent ocular disease features were noted across individuals in the cohort: cystoid macular edema (55.9%), non-age-related lens opacities (23.6%) and various vitreomacular complications (30.6%) (Supplementary Table 5). Based on our data from these 4,722 RP cases, mostly of European descent and lacking a genetic diagnosis, we estimate that RNU4- and RNU6-associated RP could be responsible for ~1.4% of all molecularly undiagnosed individuals with this disease. Furthermore, considering that approximately 30% of RP diagnoses correspond to adRP25,26 and that our positive families include 24 isolated individuals, we can further infer that these variants may account for approximately 3.0% of undiagnosed adRP families.
All RP variants are predicted to map in spatial proximity with each other, within the three-way junction delimited by stem-I and stem-II of the U4/U6 duplex and the 5′ stem-loop of U4 (Figs. 1a and 2a). In particular, they are located in a different region compared with those underlying NDD (Fig. 1a). In silico two-dimensional modeling of RNA secondary structure predicted as well that the RNU4-2 variant n.18_19insA inserts a nucleotide between stem-II and the U4 5′ stem-loop (Supplementary Fig. 5a,b), while n.56T>C disrupts the first base-pairing of the U4/U6 duplex within stem-I (Supplementary Fig. 5a,c). Both changes lead to the extension of the internal loop, an event that is predicted to impact the overall stability of the duplex. In addition, n.18_19insA slightly modifies the orientation of the 5′ stem-loop relative to stem-I and stem-II (Supplementary Fig. 5a,b).
In contrast, both n.55_56insG and n.56_57insG in RNU6 paralogs are predicted to extend the length of stem-I by three additional base pairs, reduce the size of the internal loop and drastically change the orientation of the 5′ stem-loop (Supplementary Fig. 5a,d,e). Interestingly, we observed that a benign insertion at the same position, n.55_56insT, was present in gnomAD v.4.1 in all five RNU6 paralogs with a cumulative frequency of 0.12% (n = 181) (Supplementary Fig. 5f). While these models provide a coherent structural rationale for the observed clustering, the precise effects of the variants on U4/U6 architecture remain to be experimentally verified.
Analysis of cryo-electron microscopy data (PDB 6QW6)27 confirmed that all RP variants identified reside in a region critical for binding of the U4/U6 duplex to the splicing factors PRPF31, PRPF3 and PRPF8, all previously associated with adRP16,17 (Fig. 2b). Specifically, this region first engages PRPF31 or the PRPF3/PRPF4 complex, initiating the assembly interface, and is subsequently stabilized in its native orientation upon the coordinated binding of additional tri-snRNP components, including PRPF6 and PRPF828. The mutated and neighboring U4 and U6 nucleotides detected in RP cases directly participate in the binding of PRPF31 and PRPF3 (Fig. 2c,d), via hydrogen bonds with eight and three residues of these proteins, respectively. Notably, by querying the ClinVar database29, we detected a missense variant affecting one of these residues, p.(Arg449Gly) of PRPF3, identified in a three-generation family with seven affected individuals having clinical features similar to those observed in most cases from our study30.
a, Naked U4/U6 pairing, showing the proximity of the causative variants identified (red and green). b, Same as in a with interacting PRPF proteins. c, Direct interactions of nucleotides of the U4/U6 duplex with PRPF31, via hydrogen bonds. d, Same as c but for PRPF3.
Since the human genome contains several RNU4 and RNU6 pseudogenes31, we investigated whether any of these might be incorrectly annotated and could instead produce functional RNA, potentially contributing to the disease. In addition, we sought to understand why the various U4 and U6 paralogs appear to be differentially mutated, with RNU4-1 and RNU6-7 displaying none of the recurrent pathogenic variants. We used RNA sequencing (RNA-seq) data from human neurosensory retina (NSR), retinal pigment epithelium (RPE) and choroid that were enriched for small RNAs, applying stringent and paralog-aware bioinformatics analyses designed to mitigate the complexities associated with reads aligning against multiple paralogs and/or pseudogenes (Methods). RNU4-2 was more highly expressed than RNU4-1 in all tissues (average ratio: 1.63; Fig. 3a). Conversely, individual expression of RNU6 genes and pseudogenes in the retina could not be reliably quantified by RNA-seq, since their sequences are identical, except for the last nucleotide. Therefore, we compared the total expression of RNU4 and RNU6, regardless of their respective paralogs and pseudogenes. RNU6 expression was on average 3.39× higher across the three tissues, compared with RNU4 (Fig. 3b). Of note, NSR and RPE had higher expression of RNU4 (2.51×) and RNU6 (6.09×) with respect to the choroid, an ocular tissue not directly involved in vision, used as a control (Fig. 3b). This observation is in agreement with previous data showing that snRNA expression in the retina is approximately sixfold higher compared with muscle, testis, heart and brain32, indicating a high demand for snRNAs in these two retinal layers.
a, Expression of RNU4-1 and RNU4-2 from RNA-seq of human donor choroid (n = 13), NSR (n = 4) and RPE (n = 16). For these boxplots, the tick line within boxes indicates the median (also expressed numerically), boxes represent the first and the third quartiles and whiskers indicate the largest observation smaller than or equal to the first quartile − 1.5 × IQR and the smallest observation greater than or equal to the third quartile + 1.5 × IQR. b, Same as in a for RNU4 genes (RNU4-1, RNU4-2 and pseudogenes) and for all RNU6 genes (RNU6-1, RNU6-2, RNU6-7, RNU6-8, RNU6-9 and pseudogenes). c, ATAC-seq and H3K27ac signals for RNU4-1, RNU4-2, RNU4ATAC (red) and 105 RNU4 pseudogenes (black). d, Same as in c for five RNU6 genes and RNU6ATAC (red), as well as for 1,312 RNU6 pseudogenes (black). IQR, interquartile range.
In addition, we analyzed the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and H3K27ac chromatin immunoprecipitation followed by sequencing (ChIP–seq) data from retinal tissues33 in genomic regions spanning all RNU4 and RNU6 sequences. ATAC-seq assesses chromatin accessibility across the genome, while H3K27ac ChIP–seq reveals the presence of active enhancers. These data, combined, indicate potential active transcription at promoter regions. Our analysis showed clear transcription marks in all paralogous RNU4 and RNU6 genes in the retina (Fig. 3c,d). Conversely, these signatures were absent from the 105 U4 pseudogenes and the 1,312 U6 pseudogenes, except for RNU4-8P, which displayed strong signals, but probably by virtue of its close proximity to the ACTR1B promoter. Of note, RNU6-92P and RNU6-656P had high ATAC-seq signals but very low H3K27ac signals at their respective promoters (Fig. 3d).
We performed the same analysis for other snRNA genes present in the human genome, which revealed a similar trend: all RNU genes, with the exception of RNU5F-1, had marks of active transcription and only a few among the thousands of RNU pseudogenes displayed signals compatible with potential expression, therefore representing plausible candidate genes for retinal disease (Supplementary Fig. 6). In addition, RNU2-2 was recently implicated in NDD, yet without evidence of ocular involvement13. Interestingly, the same type of analysis, based on conservation and expression data from GTEx, was recently performed by others, showing similar results34.
For RNU6-7, both ATAC-seq and H3K27ac signals were within the same range as those observed for other RNU6 genes (Fig. 3d), and, therefore, the absence of pathogenic variants could not be explained by a potential differential expression. We thus analyzed the genetic landscape of variations in healthy individuals in all five U6 paralogs and observed that RNU6-7 displayed a lower number of variants, compared with the others (Supplementary Fig. 7). We also identified the recurrent variant n.55_56insG in RNU6-7 in six control individuals of African or African American ancestry in gnomAD v.4.1 (allele frequency (AF) = 0.014%) and in 14 individuals of African origin in the All of Us database (AF = 0.013%). These seemingly contradictory observations merit further investigations in future studies.
We performed transcriptome analysis following the collection of RNA from circulating leukocytes in nine affected individuals carrying variants in RNU4-2, RNU6-1 and RNU6-9 (three individuals per gene), as well as from 14 healthy controls (Supplementary Table 6). To avoid systematic errors linked to the use of different collection kits35 across our cohort (Methods), we performed independent case–control tests for samples collected with PAXgene kits (three RNU4-2 cases, six controls) or Tempus kits (six RNU6 cases, eight controls). We identified 27 and eight differentially expressed genes in the two datasets, respectively, with no gene overlap (fold-change > 2 and false discovery rate (FDR) P value < 0.05; Supplementary Table 7), indicating no major differences in global gene expression in leukocytes in cases versus controls. We then further explored the data by investigating potential bias in pre-mRNA splicing, as performed in ref. 11. This analysis led to the identification of 107 upregulated and 67 downregulated 5′ splice sites in the PAXgene set, and 37 upregulated and 13 downregulated 5′ splice sites in the Tempus set, with only two sites in common between the datasets (in the genes CLEC2D and SNHG29; Supplementary Table 8). At these two sites the expression differences, although statistically significant, were below 10%.
We then examined the DNA sequences of the 224 differentially expressed splice sites, focusing on the occurrence of the ‘AG' dinucleotide at positions −2/−1, which was previously reported to be enriched in sites with increased splicing in RNU4-2 in patients with NDD11. No differences in ‘AG' frequency were observed between sites with increased versus decreased usage in either the PAXgene or Tempus samples (two-tailed Fisher's test, P = 0.63 and P = 0.52, respectively). We also compared nucleotide frequencies at each position between upregulated and downregulated splice sites, separately for the two groups, and found no significant differences (two-tailed Fisher's test with FDR correction, P < 0.05). A similar analysis of overlapping dinucleotides across the region flanking the 5′ splice site (for example, positions −4/−3, −3/−2, up to +7/+8) revealed no significant differences.
Since PRPF variants associated with RP affect primarily spliceosomal assembly32, we investigated whether the same phenomenon could be driven by the variants detected in this work. Specifically, we immunopurified ectopically expressed U4 and U6 snRNAs containing the RP variants and analyzed their association with specific markers for the U6 snRNP (SART3), the U4/U6 di-snRNP (SART3 and PRPF31), the U4/U6.U5 tri-snRNP (PRPF31 and SNRNP200) and the U5 snRNP (SNRNP200). The combined results showed an increased association of snRNA constructs with RP variants with SART3 and partially with PRPF31, while the interaction with SNRNP200 was unchanged or reduced (Fig. 4). For comparison, we included in our assays the U4 n.64_65insT variant, which causes NDD, and observed no significant alteration in the association with any of the proteins tested, compared with wild type (Fig. 4a). Additionally, no significant differences were detected between NDD and RP variants, pointing to the need for targeted functional studies to delineate their respective impacts on spliceosome dynamics. Similarly, U6 RNA bearing the n.55_56insT and n.57T>G variants, observed in healthy control individuals, presumably did not affect spliceosome formation, since the low amount of protein associated with them implies that they entered the spliceosome assembly process only minimally (Fig. 4b). Taken together, the results indicate that RP pathogenic variants have potentially a specific dominant effect on snRNP biogenesis and delay the assembly process at the di-snRNP stage.
a,b, Immunoprecipitation of U4-MS2 (WT and variants) (a) and U6-MS2 (WT and variants) (b). snRNPs were immunoprecipitated via MS2-YFP by anti-GFP antibodies and co-precipitated proteins were detected by western blotting. The position of the MS2 loop (green) in snRNAs is indicated. Four independent experiments were quantified. Immunoprecipitated proteins are normalized to input and U4 or U6 WT controls. Middle bars indicate average values and error bars the s.e.m. Statistical significance was analyzed by the two-tailed unpaired t-test and the P values were adjusted using the Benjamini–Hochberg FDR method to control for false discoveries. P values ≤ 0.05 are indicated. Full-length blots and antibody validation are provided as Source Data. Ctrl, control; IP, immunoprecipitation; WT, wild type.
Source data
The numerous genes associated with RP and allied diseases belong to a wide range of functional classes, from retina-specific biochemical pathways to ubiquitous cellular processes5. Yet, how these defects ultimately lead to retinal degeneration often remains unclear. The link between pathogenic variants in splicing factors of the tri-snRNP complex (RP-PRPFs), essential for survival in all eukaryotes, and RP, a phenotype limited to the eye, represents perhaps the most intriguing of these biological enigmas.
In this study, we identified recurrent heterozygous variants in RNU4-2, encoding U4 RNA, and in multiple paralogs of the U6 RNA as a cause of RP. Interestingly, these snRNAs are also an integral part of the di- and tri-snRNP and directly interact with some RP-PRPF proteins. In addition, similar to RP-PRPFs, they are also associated with the same specific phenotype: de novo or inherited adRP, with reduced penetrance for RNU4-2 variants. Importantly, the clinical presentation of patients with RNU4-2 and RNU6 variants overlaps with that of other spliceosome-related forms of adRP, particularly showing an earlier onset—contrasting with the generally milder prognosis observed in most other adRP types36,37—and a relatively high co-occurrence of features such as cataracts and cystoid macular edema, found in cases with PRPF3138,39, PRPF840 and SNRNP20041 variants. Prevalence estimations indicate that these snRNA pathogenic changes may account for an elevated number of undiagnosed cases, and it is therefore surprising that the RNU4 and RNU6 genes have escaped disease association until now. A partial explanation for this phenomenon is that mainstream sequencing approaches are biased towards DNA-capturing procedures that do not include snRNA genes. Furthermore, although genome sequencing is increasingly being adopted in routine diagnostics, variants in snRNA genes may have remained undetected because they affect noncoding transcripts, which are more challenging to interpret and are often overlooked or deprioritized by standard analytical pipelines.
An intriguing feature of pathogenic changes in RNU4-2 is their pleiotropy with respect to NDD (ReNU syndrome) and RP. Chen et al.9 described that more than half of the patients with ReNU also display some visual abnormalities, although only three were documented as having retinal phenotypes (one had an abnormal electroretinogram response, one had Leber congenital amaurosis and one presented with macular dysfunction). However, most cases were too young to display the typical symptoms of RP, which usually manifest during adolescence or early adulthood42. Although the exact mechanism for this phenotypic selectivity is unknown, RNU4-2 variants represent a clear and new allelic series involving noncoding RNA genes. A recent preprint highlighted a strong effect of ReNU variants in an RNU4-2 saturation genome editing experiment, while the RP variant n.56T>C in the same gene did not show any statistically significant effect43. In addition, the region of RNU4-2 containing RP variants showed function scores within the neutral range of the saturation genome editing assay43, suggesting a potentially milder pathogenic effect compared with ReNU changes. It is therefore plausible that the RP variants identified in RNU4-2 and RNU6 paralogs could lead to photoreceptor death and subsequent visual loss, while having no influence on the development of the brain. Additionally, ReNU variants are located in the stem-III and the T-loop of the U4/U6 duplex and interfere with the proper recognition of intronic 5′ splice signals, likely because these regions are involved in pairing pre-mRNA with U6 (ref. 9). In contrast, RP variants cluster in spatial proximity to the three-way junction, in regions not directly engaged in interactions with pre-mRNA but that are involved in the binding of various proteins, including RP-associated splicing factors.
Consistent with this evidence, we did not observe any major splicing anomalies in transcripts from patients with RP, with the only two significant events showing differences below 10% in expression. Conversely, our biochemical assays support a role for RP-associated variants in altering spliceosomal assembly. As the magnitude of the observed changes was in all instances rather moderate (less than 1.5-fold with respect to controls), we interpret these data as indicating that snRNA variants associated with RP are unlikely to prevent the assembly of spliceosomal complexes. Rather, they may cause a subtle alteration in snRNP dynamics, possibly affecting the efficiency of their biogenesis or recycling steps. In particular, the increased association with SART3 and, to a lesser extent, with PRPF31, together with unchanged or slightly reduced interaction with SNRNP200, may indicate a modest delay in the transition from the di-snRNP to the tri-snRNP form. Moreover, the pathogenic variants identified in this study lie within regions of the U4/U6 duplex that directly contact the PRPF3 and PRPF31 proteins, two splicing factors linked to adRP whose mutations also delay spliceosomal complex assembly32.
In terms of specific molecular effect, our functional data show that snRNAs bearing RP variants display enhanced interaction with di-snRNP protein markers, suggesting that pathogenesis could result from a gain-of-function or dominant-negative mechanism, rather than from haploinsufficiency. This hypothesis is strengthened by the evidence that molecularly similar but benign variants, commonly observed in the general population, seem not to bind efficiently to di-snRNP markers and potentially not to be incorporated into the spliceosome, supporting the idea that spliceosomal functions could be haplosufficient with respect to heterozygous and snRNA-depleting variants.
Although DNA changes associating RNU4-2 to ReNU syndrome have been primarily reported as de novo events9,10, in our study most families with RP (61%) bore RNU4-2 and RNU6 changes as inherited variants. In part, this difference can be explained by the reduced reproductive fitness associated with NDD versus RP. Unlike ReNU syndrome, symptomatic onset (night-blindness and peripheral vision loss) in nonsyndromic adRP begins later in life, with severe central vision loss usually occurring after the onset of reproductive age. Another difference involves the inheritance of dominant variants, which in ReNU seems to be almost exclusively of maternal origin9. We did not observe the same trend for RP, with variants being inherited from either of the parents, possibly indicating the absence of any sex-specific negative selection during gametogenesis or at the embryonic stage.
The human genome contains two RNU4 paralogs and five RNU6 paralogs. This indicates that, assuming equal expression within paralogs, the presence of only ~25% of mutant U4 (heterozygous genotype, over two copies) or ~10% of mutant U6 (heterozygous genotype, over five copies) is sufficient to lead to a disease phenotype, again in support of a gain-of-function or dominant-negative molecular mechanism. This could be a crucial consideration for the development of potential gene-based therapies, as gene-augmentation strategies may be suboptimal compared with gene correction or antisense oligonucleotide approaches. Our data also highlight the existence of mutational hotspots outside the coding regions of the human genome, emphasizing the need for further research into these parts of our genetic material, and show that the clustering of de novo pathogenic variants is not restricted to severe diseases with childhood onset44, but may extend to milder pathologies, such as RP.
In conclusion, we identified four recurrent pathogenic variants in RNU4-2 and in four of the five paralogs of the U6 snRNA as a frequent cause of de novo or inherited adRP. The immediate impact of these findings involves improved diagnosis and genetic counseling for patients with hereditary visual loss, especially for isolated cases who could potentially bear heterozygous de novo events. More fundamentally, this work substantially broadens our understanding of the genetic landscape of human disease, paving the way for the development of new molecular therapeutic approaches.
This study adhered to the tenets of the Declaration of Helsinki, and signed, informed consent was obtained from all participants. All procedures were conducted in accordance with Institutional Review Board-approved human research protocols and were approved by the ethics committees of the Radboud University Medical Center (Nijmegen, the Netherlands) and the Rotterdam Eye Hospital (Rotterdam, the Netherlands) (MEC-2010-359; OZR protocol no. 2009-32), and the local ethics committees of all other participating institutions.
Complete ophthalmic examinations were performed by a retinal specialist, which included measurement of best-corrected visual acuity and intraocular pressures, and examination of anterior segment and fundus (dilated). Color fundus photographs and montages were captured using the FF450plus Fundus Camera (Carl Zeiss Meditec) and Optos 200 Tx (Optos). Fundus autofluorescence images (488-nm excitation) and high-resolution spectral-domain optical coherence tomography (SD-OCT) scans were acquired using the Spectralis HRA+OCT module (Heidelberg Engineering). Hyper-autofluorescent ring contours were analyzed using a custom program in FIJI software (National Institute of Mental Health)45. Progression rates were calculated using linear mixed-effects regression in R (v.4.0.4) with time (years) since baseline as the primary independent variable, baseline ring size as a covariate and inter-ocular differences as a random effect. Photoreceptor+ thickness was assessed on horizontal SD-OCT scans through the fovea using a semi-automated procedure46. Photoreceptor+ was defined as the distance between the Bruch's membrane/choroid interface and the inner nuclear layer/outer plexiform layer boundary. Layer segmentation was performed in a semi-automated manner using a custom software in MatLab (MathWorks). Full-field electroretinogram recordings were conducted using the Espion Visual Electrophysiology System (Diagnosys) according to International Society for Clinical Electrophysiology of Vision (ISCEV) standards47.
Genomic DNA from probands was isolated from peripheral blood lymphocytes according to standard procedures. Sequencing was performed by BGI Tech Solutions using the DNBseq Sequencing Technology, with a minimal median coverage per genome of 30×. The processing of the sequencing data was performed by using BWA mem (v.0.7.17)48, Picard (v.2.14.0-SNAPSHOT) (http://broadinstitute.github.io/picard) and GATK (v.4.1.4.1)49 for mapping to the human genome reference sequence (build hg19/GRCh37) and variant calling2. For variant annotation, we used ANNOVAR50 with the addition of splicing predictions by MaxEntScan51 and SpliceAI19.
Human Genome Variation Society (HGVS) notations of the variants were retrieved using VariantValidator52 and American College of Medical Genetics and Genomics (ACMG) classification53 was applied according to the ACGS Best Practice Guidelines for Variant Classification in Rare Disease 202354. In particular, we used the PS4_strong criterion for variants significantly enriched in cases versus controls (gnomAD v.4.1 and All of Us), as assessed for each variant by two-tailed Fisher's exact test in R (fisher.test function), in agreement with the ACMG recommendations53 (odds ratio > 5.0, lower bound of the confidence interval > 1.0, corrected P value < 0.05), but only for variants present in at least three probands to avoid any bias from imbalanced case–control sets. This assessment was made using probands only (n = 1,891) for all variants, except for those in Supplementary Table 1, which were assessed in 4,722 individuals. PM6 and PP1 were applied according to ClinGen Sequence Variant Interpretation (SVI) recommendations. Specifically, PM6_sup was applied when two unrelated families had de novo variants without parental confirmation, given that RP is a ‘phenotype consistent with gene but not highly specific and high genetic heterogeneity'. PP1_sup, PP1_mod and PP1_strong were assigned when the variant segregated with disease in ≥1, ≥2 and ≥5 informative meioses, respectively55. We defined thresholds for PM2_sup and BS2 based on the frequency of RHO p.(Pro23His), the most prevalent variant causing adRP, which was detected once in gnomAD v.4.1 and 13 times in All of Us. Specifically, PM2_sup was assigned to variants that were present fewer than two times in gnomAD v.4.1 and fewer than 14 times in All of Us. BS2 was applied to variants that were observed more than four times in gnomAD v.4.1 or more than 28 times in All of Us, that is, twice the values of p.(Pro23His). PM2_sup was not applied to variants for which the PS4 criterion had already been used, to avoid double-counting evidence related to their low frequency in gnomAD. BA1 was considered for variants with allele frequency >5% in gnomAD v.4.1 or the All of Us databases, whereas BS1 was assigned to variants with allele frequencies greater than expected for disease (1/2,000 = 0.05%).
Genomic DNA was collected, and RNU4-1, RNU4-2, RNU6-1, RNU6-2, RNU6-7, RNU6-8 and RNU6-9 genes were amplified using standard PCR procedures. RNU4-1, RNU4-2, RNU6-1, RNU6-2, RNU6-7, RNU6-8 and RNU6-9 PCR fragments were sequenced using Sanger sequencing and screened for novel variants in these genes.
We utilized RNAfold WebServer to model the effect of variants with default parameters56 and RNAcanvas was used for drawing the structure57. We used ChimeraX with PDB file, using PDB file 6QW6 to draw three-dimensional representation of the U4/U6 duplex with and without surrounding PRPF proteins.
RNA was isolated from human donor eye tissue, which was collected and dissected according a reported procedure58 from an ethically approved Research Tissue Bank (UK NHS Health Research Authority reference no. 15/NW/0932). Total RNA was isolated from four NSR samples, 16 pelleted RPE samples and 13 choroid samples that had been stored in RNAlater (Thermo Fisher Scientific), using an Animal Tissue RNA Purification kit (Norgen Biotek), as per manufacturer's instructions. Sequencing libraries were prepared using the NEBnext multiplex small RNA library preparation kit, as per manufacturer's protocols, with size selection performed using Ampure beads. Paired-end sequencing (2 × 75 base pairs (bp)) was performed on an Illumina HiSeq 4000.
NEBnext adapters were removed from sequencing reads using trimmomatic (v.0.39) before alignment against the GRCh38 reference genome with bowtie59 (v.1.3). No mismatches between sequencing reads and the reference genome were allowed, and no restriction was set on multi-mapping reads. Sequence read counts were restricted to primary alignments using samtools (v.1.21)60, and therefore only counted once if they aligned to multiple RNU4 (n = 90) or RNU6 (n = 1,277) genes or pseudogenes. Calculations were drawn from read 1 datasets and normalized for the total read count achieved for the sample. Total RNU4 and RNU6 expression was based on all annotated genes and pseudogenes in GENCODE v.38.
ATAC-seq data from ref. 61 (eight different experiments) and H3K27ac ChIP–seq data from ref. 62 (five different experiments) were downloaded as bigwig files from the RegRet database (http://genome.ucsc.edu/s/stvdsomp/RegRet)63. For both data types, the signal (the genes and 500 bp on each side) was extracted using bedtools (v.2.27.1) after conversion using bigWigToWig (v.469). We quantified the signal for all RNU genes and pseudogenes first by normalizing the signal of each experiment to the maximum and then summing them. For RNU4, we quantified two genes and 105 pseudogenes, while for RNU6 we assessed five genes and 1,312 pseudogenes, in addition to RNU4ATAC and RNU6ATAC.
Peripheral blood samples were collected from affected individuals and controls using either Tempus Blood RNA tubes (Applied Biosystems) or PAXgene Blood RNA tubes (Qiagen). Total leukocyte RNA was extracted with the Tempus Spin RNA Isolation Kit (Applied Biosystems) or the Preserved Blood RNA Purification Kit II (Norgen Biotek), respectively, following the manufacturers' protocols. Following the quality assessment of RNA integrity and concentration, 100 ng of input RNA per sample was subsequently processed for library preparation using the KAPA RNA HyperPrep Kit with RiboErase (HMR) and KAPA Globin Depletion Hybridization Oligos (Roche). Sequencing was performed on an Illumina NovaSeq 6000 platform with 2 × 101-bp paired-end reads. To improve quality score calculations for the final base, one additional base was sequenced in both read 1 and read 2. The Q30 value for all RNA-seq data was ≥91.1%. Adapters were trimmed with Skewer (v.0.2.2)64.
Reads were aligned to reference transcripts from Ensembl (v.110, GRCh38) using STAR (v.2.7.11a) with the option --twopassMode Basic. DESeq2 (v.1.46.0) with default options was used for differential expression analysis between different groups according to sample origin (Tempus or PAXgene tubes) and presence/absence of the pathogenic RNU genotypes, with fold-change > 2 and FDR P value < 0.05. We used rMATS65 to assess differential alternative splicing, separately for the Tempus and PAXgene sets and with specific options (--allow-clipping --variable-read-length --anchorLength 1 --novelSS --task both --libType fr-unstranded -t paired --readLength 101). We further used the Python scripts from ref. 11 to process the rMATS output and filter the data according to a mean coverage > 7, an FDR P value < 0.1 and a deltaPSI value > 0.05. The R function fisher.test with default parameters was used to assess differences in base compositions at splicing sites, at each position, as well as differences for 2-mers (for example, positions −4/−3 to +7/+8).
U4 n.18_19insA, n.56T>C and n.64_65insT variants were introduced by site-directed mutagenesis into the plasmid expressing U4-MS266. The full-length U6 sequence, including 256 bp upstream and 93 bp downstream of the RNU6-1 gene, was inserted into the pcDNA3 plasmid lacking the CMV promoter. The MS2 loop was inserted between nucleotides 10 and 11. U6 n.55_56insG, n.55_56insT, n.56_57insG and n.57G>T variants were introduced by site-directed mutagenesis. U4- and U6-expressing plasmids were transfected into HeLa cells stably expressing MS2-YFP protein. At 24 h after transfection, snRNAs were immunoprecipitated using anti-GFP antibodies and co-precipitated proteins were analyzed by western blotting66.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Research on the de-identified patient data used in this publication from the Genomics England 100,000 Genomes Project and the NHS GMS dataset can be carried out in the Genomics England Research Environment subject to a collaborative agreement that adheres to patient-led governance. All interested readers will be able to access the data in the same manner that the authors accessed the data. For more information about accessing the data, interested readers may contact research-network@genomicsengland.co.uk or access the relevant information on the Genomics England website: https://www.genomicsengland.co.uk/research. Sharing of additional sequencing or blood RNA-seq data is subject to the European General Data Protection Regulation (GDPR) applicable in the countries of residence of the tested individuals and may become available upon a data transfer agreement approved by local ethical committees. Patient sample identifiers from this study can be released upon reasonable request from ‘M1-A to M9-B' to the corresponding local ‘DNA-number'. Specific variant requests or other data are available from the corresponding author (S.R.) upon reasonable request. The data generated during this study (causative variants from Supplementary Table 1) are submitted to the Leiden Open (source) Variation Database (LOVD) (http://www.lovd.nl) and ClinVar (accession codes SCV006562526 to SCV006562534). Sequences of primers used in this study are listed in Supplementary Table 9. Additional details regarding PCR conditions or primer design are available upon request. Detailed information on antibodies used are provided in Supplementary Table 10. Small RNA-seq datasets analyzed in this study are available at the NCBI Sequence Read Archive through accession PRJNA1256119 (https://www.ncbi.nlm.nih.gov/sra/PRJNA1256119). The genes and pseudogenes analyzed are present in Supplementary Table 11 and the read counts are available in Supplementary Table 12. Source data are provided with this paper.
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We thank all patients and their family members for their help and participation in this study. We thank S. Föhr for technical and administrative support; S. van der Velde-Visser, E. Blokland and M. Jacobs-Camps for sample registration and administration; and T. Rosseel (computational) and S. Van de Sompele (clinical reporting) for their support. The title page was formatted using AuthorArranger, a tool developed at the National Cancer Institute (National Institutes of Health, NIH). C.R. was supported by the Swiss National Science Foundation (SNSF) Grant No. 310030_204285 entitled ‘Genomics of inherited retinal diseases'. S.R. was supported by the Foundation Fighting Blindness Career Development Award (grant no. CD-GE-0621-0809-RAD), Radboudumc Starter grant (no. OZI-23.009) and NWO Aspasia (grant no. 015.021.028). S.R., C.R., E.D.B., M.B., S.B. and D. Stanek were supported by HORIZON-MSCA-2022-DN (grant no. 101120562, ProgRET). E.D.B., S.R. and C.R. were supported by the EJPRD19-234 Solve-RET. This work has been funded by a Foundation Fighting Blindness Program Project Award (grant no. PPA-0622–0841-UCL) (to A.J.H., S.R. and S.E.d.B.). S.R. and F.P.M.C. were supported by the Gelderse Blindenstichting, the Algemene Nederlandse Vereniging ter voorkoming van Blindheid, Oogfonds, Landelijke Stichting voor Blinden en Slechtzienden, Rotterdamse Stichting Blindenbelangen, Stichting Blindenhulp, Stichting tot Verbetering van het Lot der Blinden and Stichting Blinden-Penning. M.Q. was supported by the RetinAward 2021. S.H.T. – Jonas Children's Vision Care (JCVC) is supported by the National Institute of Health grant nos. U01EY030580, U01EY034590 R24EY028758, R24EY027285, 5P30EY019007, R01EY033770 and R01EY018213, R01EY024698; the Foundation Fighting Blindness grant no. TA-GT-0321-0802-COLU-TRAP; Richard Jaffe; the NYEE Foundation; the Rosenbaum Family Foundation; the Gebroe Family Foundation; the Piyada Phanaphat Fund; the Research to Prevent Blindness (RPB) Physician-Scientist Award; and unrestricted funds from RPB, New York, NY, USA. C.A. was supported by Instituto de Salud Carlos III (ISCIII) of the Ministerio de Ciencia e Innovación and Unión Europea – European Regional Development Fund (FEDER) (grant nos. PI22/00321 and IMP/00009), Centro de Investigación Biomédica en Red Enfermedades Raras (CIBERER, grant no. 06/07/0036), IIS-FJD BioBank (grant no. PT23/00114), the Organización Nacional de Ciegos Españoles (ONCE), the European Regional Development Fund (FEDER) and the University Chair UAM-IIS-FJD of Genomic Medicine. This work was performed by using the data contained in the ‘Programa Infraestructura de Medicina de Precisión asociada a la Ciencia y la Tecnología en Medicina Genómica (IMPaCT-GENóMICA)', coordinated by the CIBERER and founded by ISCIII. L.F.-C. was supported by Centro de Investigación Biomédica en Red (CIBER). R.A. was supported by the National Eye Institute (NEI) (grant nos. RO1 EY030591, RO1 EY031663, T32 EY026590 and P30 EY22589). C.C.W.K. was supported by the Combined Ophthalmic Research Rotterdam grant no. 8.2.0. S.B. was supported by the Italian Telethon Foundation and by the European Union HORIZON-MSCA-2021-DN-01 (grant no. 101073316, RETORNA). T.S.B. was supported by ZonMw Vidi (grant no. 09150172110002), and acknowledges support from Stichting 12q. G.J.F. and N.C. were supported by Fighting Blindness Ireland (grant nos. FB22FAR, FB16FAR), Fighting Blindness Ireland – Health Research Charities Ireland (grant no. MRCG-2016-14) and the Science Foundation Ireland (grant nos. 16/IA/4452 and 22/FFP-A/10544). J.M.E. was supported by the Macular Society (United Kingdom), the National Institute for Health and Care Research (NIHR) Manchester Biomedical Research Centre (BRC) (grant no. NIHR203308) and the University of Manchester Core Genomics Technology Facility. T.B.-Y. was supported by the Israel Science Foundation (grant no. 331/24). A.C.B.-J. is supported by the University of Melbourne Research Fellowship. K.M.B. was supported by the National Eye Institute (NEI) (grant nos. RO1 EY035717 and P30 EY014104 (MEE core support)), the Iraty Award 2023, the Lions Foundation and RPB (Unrestricted Grant). L.S.S., E.L.C. and S.P.D. were supported by grants from the Foundation Fighting Blindness (grant no. EGI-GE1218-0753-UCSD) and the Brett & Jane Eberle Foundation. E.D.B. and B.P.L. were supported by Ghent University Special Research Fund (grant no. BOF20/GOA/023) and E.D.B. (grant no. 1802220N) and B.P.L. (grant no. 1803816N) are Senior Clinical Investigators of the Research Foundation-Flanders (FWO). N.M. and S.S. are Ph.D. fellows of HORIZON-MSCA-2022-DN ProgRET (grant no. 101120562). R.A. was supported by the Foundation Fighting Blindness. J.L.D. was supported by the UCSF Vision Core shared resource of the NIH/NEI grant no. P30 EY002162, the Foundation Fighting Blindness, an unrestricted grant from RPB and the All May See Foundation. T.I. was supported by research grants from the Japan Agency for Medical Research and Development (AMED) (grant nos. 20ek0109493h0001, 21ek0109493h0002, 22ek0109493h0003, 23ek0109617h0002, 24ek0109617h0003). R.K.K. was supported by The Montreal Children's Hospital Foundation, The Vision Sciences Research Network (VSRN), The NIH (grant no. R01 EY030499-01, Dr. Lentz), The Canadian Institutes for Health Research (CIHR), Fighting Blindness Canada (FBC) and Fonds de Recherche du Québec - Santé (FRQS). R.K.K. participates in the NAC Attack clinical trial, which is funded by the NIH via grant nos. UG1EY033286, UG1EY033293, UG1EY033286 and UG1EY033292. T.M.L., T.L.M. and J.N.D.R. were supported by Retina Australia (awarded to the Australian Inherited Retinal Disease Registry and DNA Bank). O.A.M. was supported by the Wellcome Trust (grant no. 206619/Z/17/Z). M.P. was supported by the BrightFocus Foundation (grant no. M2024009N). E.A.P. was supported by the National Eye Institute (NEI) (grant no. R01 EY012910). R.R. was supported by Retina South Africa and the South African Medical Research Council (MRC). S.G.S. and E.M.V. were supported by the Italian Ministry of Health (grant no. PNRR-MR1-2023-12377314). D. Stanek was supported by the Project P JAC grant no. CZ.02.01.01/00/22_008/0004575 RNA for therapy, Co-Funded by the European Union. T.B.H. was supported by the European Commission (Recon4IMD – grant no. GAP-101080997) and the Deutsche Forschungsgemeinschaft (German Research Foundation, DFG, grant nos. 418081722 and 433158657 to T.B.H.). P.L. and L.D. were supported by a research grant (no. NW24-06-00083) from the Ministry of Health of the Czech Republic and grant no. UNCE/24/MED/022. V.R.d.J.L.-R. and J.C.Z. were supported by the Velux Stiftung Grant no. 1860. M.A., M.M., C.F.I., J.C.G., A.J.H. and C.T. are supported by Retina UK and Fight for Sight UK (RP Genome Project Grant no. GR586). A.J.H., J.C.G., M. Michaelides, O.A.M., A.R.W., G. Arno and S.L. were supported by the National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital and UCL Institute of Ophthalmology. S.L. was funded by a Medical Research Council (MRC) Clinician Scientist Fellowship (grant no. UKRI440). J.M.M. was supported by Instituto de Salud Carlos III (ISCIII) of the Spanish Ministry of Health (grant no. PI22/00213), CIBERER (grant no. 06/07/1030), grant no. CIPROM/2023/26 from the Generalitat Valenciana and IMPaCT-GENOMICA (grant no. IMP/00009) co-funded by ISCIII and FEDER. S.R., C.C.W.K., C.J.F.B. and A.G. were supported by the Dutch Ministry of Education, Culture and Sciences, Gravitation grant no. 024.006.034 Lifelong VISION. W.L. was supported by the National Institute of Health/National Eye Institute (grant no. 1K99EY036930-01). This work is supported by partners of the European Reference Network for Rare Eye Diseases ERN-EYE (Grant Agreement No. 101085439, C.J.F.B., E.D.B., C.B.H., S.K., B.P.L., P.L., L.H.-W., K. Stingl, L.I.v.d.B.). Novartis contributed funding for the preceding RP-LCA smMIPs panel design and subsequent sequencing (to F.P.M.C., S.R. and D.M.P.). Novartis was not involved in the study design; collection, analysis or interpretation of data; the writing of this article; or the decision to submit it for publication. This research was made possible through access to data generated by the 2025 French Genomic Medicine initiative and present in the National Genomic Research Library, which is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The National Genomic Research Library holds data provided by patients and collected by the NHS as part of their care, and data collected as part of their participation in research. The National Genomic Research Library is funded by the National Institute for Health Research and NHS England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure.
These authors contributed equally: Mathieu Quinodoz, Kim Rodenburg.
These authors jointly supervised this work: Susanne Roosing, Carlo Rivolta.
Ophthalmic Genetics Group, Institute of Molecular and Clinical Ophthalmology Basel (IOB), Basel, Switzerland
Mathieu Quinodoz, Karolina Kaminska, Ana Belén Iglesias-Romero, Mukhtar Ullah, Marc Folcher, Francesca Cancellieri & Carlo Rivolta
Department of Ophthalmology, University of Basel, Basel, Switzerland
Mathieu Quinodoz, Karolina Kaminska, Ana Belén Iglesias-Romero, Marc Folcher, Georg Ansari, Francesca Cancellieri, Nicolas Feltgen, Maximilian Pfau & Carlo Rivolta
Department of Genetics, Genomics and Cancer Sciences, University of Leicester, Leicester, UK
Mathieu Quinodoz & Carlo Rivolta
Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands
Kim Rodenburg, Suzanne E. de Bruijn, Erica G. M. Boonen, Nick Zomer, Lara K. Holtes, Zelia Corradi, Stefanida Shliaga, Daan M. Panneman, Rebekkah J. Hitti-Malin, Galuh D. N. Astuti, Ronny Derks, Alejandro Garanto, Christian Gilissen, Lonneke Haer-Wigman, Alexander Hoischen, Kornelia Neveling, Michiel Oorsprong, Frans P. M. Cremers & Susanne Roosing
Laboratory of RNA Biology, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic
Zuzana Cvackova & David Stanek
Department of Otorhinolaryngology, Hearing & Genes, Radboud University Medical Center, Nijmegen, the Netherlands
Suzanne E. de Bruijn
The Rotterdam Eye Hospital, Rotterdam Ophthalmic Institute, Rotterdam, the Netherlands
Erica G. M. Boonen & L. Ingeborgh van den Born
Vitreous Retina Macula Consultants of New York, New York, NY, USA
Jacques Bijon & K. Bailey Freund
Department of Ophthalmology, Rothschild Foundation Hospital, Paris, France
Jacques Bijon
Departments of Ophthalmology, Pathology & Cell Biology, Columbia Stem Cell Initiative, Vagelos College of Physicians and Surgeons Columbia University Irving Medical Center, New York, NY, USA
Stephen H. Tsang
Edward S. Harkness Eye Institute, Jonas Children's Vision Care (JCVC), Columbia University Irving Medical Center, New York-Presbyterian Hospital, New York, NY, USA
Stephen H. Tsang
Department of Ophthalmology, NYU Grossman School of Medicine, New York, NY, USA
K. Bailey Freund
Division of Molecular Medicine, Leeds Institute of Medical Research, School of Medicine, University of Leeds, Leeds, UK
Manir Ali, Chris F. Inglehearn, Martin McKibbin & Carmel Toomes
Molecular Biology Research Unit, St John Eye Hospital Group, Jerusalem, Palestine
Ala'a AlTalbishi
Department of Ophthalmology, University Hospital of Lund, Lund, Sweden
Sten Andréasson
JC Self Research Institute, Greenwood Genetic Center, Greenwood, SC, USA
Gavin Arno
UCL Institute of Ophthalmology, University College London, London, UK
Gavin Arno, Jessica C. Gardner, Alison J. Hardcastle, Omar A. Mahroo, Michel Michaelides & Andrew R. Webster
National Institute of Health Research Biomedical Research Centre, Moorfields Eye Hospital, London, UK
Gavin Arno, Omar A. Mahroo, Michel Michaelides, Andrew R. Webster & Siying Lin
Department of Genetics & Genomics, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain
Carmen Ayuso, Fiona Blanco-Kelly, Lidia Fernández-Caballero & Ana Isabel Sánchez-Barbero
Center for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Madrid, Spain
Carmen Ayuso, Fiona Blanco-Kelly, Lidia Fernández-Caballero & Ana Isabel Sánchez-Barbero
Department of Ophthalmology, Shiley Eye Institute, University of California San Diego, La Jolla, CA, USA
Radha Ayyagari & Pooja Biswas
Medical Genetics, Department of Precision Medicine, University of Campania “Luigi Vanvitelli”, Naples, Italy
Sandro Banfi, Marianthi Karali, Margherita Scarpato & Roberta Zeuli
Genomic Medicine, Telethon Institute of Genetics and Medicine, Pozzuoli, Italy
Sandro Banfi
Hadassah Medical Center, Division of Ophthalmology, The Hebrew University of Jerusalem, Jerusalem, Israel
Eyal Banin, Asodu Sandeep Sarma & Dror Sharon
Department of Clinical Genetics, Erasmus MC University Medical Center, Rotterdam, the Netherlands
Tahsin Stefan Barakat, Lies H. Hoefsloot & Virginie J. M. Verhoeven
Department of Ophthalmology, Semmelweis University, Budapest, Hungary
Mirella T. S. Barboni, Krisztina Knézy, Zoltán Zsolt Nagy & Viktória Szabó
Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium
Miriam Bauwens, Elfride De Baere, Marieke De Bruyne, Bart P. Leroy, Quinten Mahieu, Nelson Martins & Mattias Van Heetvelde
Department of Biomolecular Medicine, Ghent University, Ghent, Belgium
Miriam Bauwens, Elfride De Baere, Marieke De Bruyne, Quinten Mahieu, Nelson Martins & Mattias Van Heetvelde
Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel
Tamar Ben-Yosef
GCS Auragen, Plan France Medecine Genomique, Lyon, France
Virginie Bernard, Luke Mansard & Francis Ramond
Retina Foundation of the Southwest, Dallas, TX, USA
David G. Birch & Kaylie Jones
Institute for Neurosciences of Montpellier (INM), Montpellier University, INSERM, Montpellier, France
Beatrice Bocquet, Vasiliki Kalatzis, Gaël Manes, Luke Mansard & Isabelle Meunier
National Reference Centre for Inherited Sensory Diseases, University of Montpellier, Montpellier University Hospital, Montpellier, France
Beatrice Bocquet, Vasiliki Kalatzis & Isabelle Meunier
Department of Ophthalmology, Leiden University Medical Center (LUMC), Leiden, the Netherlands
Camiel J. F. Boon
Department of Ophthalmology, Amsterdam University Medical Center, Amsterdam, the Netherlands
Camiel J. F. Boon
Department of Ophthalmology and Human Genetics, University of Michigan, Ann Arbor, MI, USA
Kari Branham
Department of Ophthalmology, University Hospital Necker Enfants Malades, APHP, Paris Cité University, Paris, France
Dominique Bremond-Gignac
UMRS1138, Centre de Recherche des Cordeliers, Université Paris Cité, INSERM, Paris, France
Dominique Bremond-Gignac, Cyril Burin des Roziers & Sophie Valleix
Department of Optometry and Vision Sciences, The University of Melbourne, Melbourne, Victoria, Australia
Alexis Ceecee Britten-Jones
Ocular Genomics Institute, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA, USA
Kinga M. Bujakowska, Eric A. Pierce, Emily Place & Riccardo Sangermano
Service de Médecine Génomique des Maladies de Système et d'Organe, Hôpital Cochin, AP-HP Centre Université Paris Cité, Paris, France
Cyril Burin des Roziers & Sophie Valleix
Laboratoire de Biologie Médicale SeqOIA, Site Broussais, Paris, France
Cyril Burin des Roziers & Sophie Valleix
Department of Epidemiology and Human Genetics Center, School of Public Health, University of Texas Health Science Center at Houston, Houston, TX, USA
Elizabeth L. Cadena, Stephen P. Daiger & Lori S. Sullivan
Vista Vision Eye Clinic, Brescia, Italy
Giacomo Calzetti
Clinical Translation Group, Institute of Molecular and Clinical Ophthalmology Basel (IOB), Basel, Switzerland
Giacomo Calzetti
Neurogenetics Research Center, IRCCS Mondino Foundation, Pavia, Italy
Luca Cattaneo, Simone Gana & Enza Maria Valente
Institute of Genetics, School of Genetics and Microbiology, Trinity College Dublin, The University of Dublin, Dublin, Ireland
Naomi Chadderton, G. Jane Farrar & Laura Whelan
Department of Ophthalmology, TUM University Hospital, School of Medicine and Health, Technical University of Munich, Munich, Germany
Peter Charbel Issa
Oxford Eye Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, UK
Peter Charbel Issa
Serviço de Oftalmologia, Instituto de Oftalmologia Dr. Gama Pinto, Lisboa, Portugal
Luísa Coutinho-Santos & Cristina M. Santos
Department of Cell Therapy and Regenerative Medicine, Andalusian Molecular Biology and Regenerative Medicine Centre, CABIMER, Seville, Spain
Berta de la Cerda
Australian Inherited Retinal Disease Registry and DNA Bank, Department of Medical Technology and Physics, Sir Charles Gairdner Hospital, Perth, Western Australia, Australia
John N. De Roach, Tina M. Lamey & Terri L. McLaren
Department of Head and Skin, Ghent University, Ghent, Belgium
Julie De Zaeytijd & Bart P. Leroy
Department of Ophthalmology, Ghent University Hospital, Ghent, Belgium
Julie De Zaeytijd & Bart P. Leroy
Université de Lille, INSERM U1172 - LilNCog - Lille Neuroscience & Cognition, Lille, France
Claire-Marie Dhaenens
Department of Paediatrics and Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic
Lubica Dudakova, Petra Liskova & Marie Vajter
Department of Ophthalmology, University of California, San Francisco, Wayne and Gladys Valley Center for Vision, San Francisco, CA, USA
Jacque L. Duncan
Department of Medical Retina, Singapore National Eye Centre, Singapore, Singapore
Beau J. Fenner
Ophthalmology and Visual Sciences Academic Clinical Program, Duke-NUS Graduate Medical School, Singapore, Singapore
Beau J. Fenner
Singapore Eye Research Institute, Singapore, Singapore
Beau J. Fenner
Department of Ophthalmology, Federal University of São Paulo, UNIFESP, São Paulo, Brazil
Juliana M. Ferraz Sallum
Department of Pediatrics, Amalia Children's Hospital, Radboud University Medical Center, Nijmegen, the Netherlands
Alejandro Garanto
Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, Barcelona, Spain
Roser Gonzàlez-Duarte & Rebeca Valero
Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan
Kensuke Goto, Taro Kominami & Ai Fujita Sajiki
School of Biological Sciences, Division of Evolution, Infection, and Genomics, The University of Manchester, Manchester, UK
Sam Griffiths-Jones, Gillian I. Rice & Jamie M. Ellingford
Institute for Medical Genetics and Applied Genomics, University of Tübingen, Institute for Ophthalmic Research, University Hospital Tübingen, Tübingen, Germany
Tobias B. Haack & Theresia Zuleger
Center for Rare Disease, University of Tübingen, Tübingen, Germany
Tobias B. Haack
Genomics for Health in Africa (GHA), Africa-Europe Cluster of Research Excellence (CoRE), Ghana, South Africa
Tobias B. Haack
Department of Ophthalmology, The Jikei University School of Medicine, Minato-ku, Japan
Takaaki Hayashi & Kei Mizobuchi
Department of Ophthalmology and Vision Sciences, Ocular Genetics Program, The Hospital for Sick Children, Toronto, Ontario, Canada
Elise Héon
Department of Internal Medicine, Radboud University Medical Center, Nijmegen, the Netherlands
Alexander Hoischen
Department of Ophthalmology, Oslo University Hospital, Oslo, Norway
Josephine P. Holtan
Department of Ophthalmology, Radboud University Medical Center, Nijmegen, the Netherlands
Carel B. Hoyng & Caroline C. W. Klaver
Department of Ophthalmology, Section of Pediatric Ophthalmology, Strabismus, and Ocular Genetics, DOH Eye Center, East Avenue Medical Center, Quezon City, Philippines
Manuel Benjamin B. Ibanez IV
Section of Pediatric Ophthalmology, Strabismus, and Ocular Genetics, Makati Medical Center, Makati City, Philippines
Manuel Benjamin B. Ibanez IV
Division of Molecular and Cellular Biology, National Institute of Sensory Organs, NHO Tokyo Medical Center, Tokyo, Japan
Takeshi Iwata, Yang Pan, Akiko Suga & Kazutoshi Yoshitake
deCODE genetics, Amgen Inc., Reykjavik, Iceland
Brynjar O. Jensson, Kari Stefansson & Patrick Sulem
Department of Inherited Retinal Dystrophies, Ophthalmic Genetics Group, OMMA, Ophthalmological Institute of Athens, Athens, Greece
Smaragda Kamakari
Eye Clinic, Multidisciplinary Department of Medical, Surgical and Dental Sciences, University of Campania “Luigi Vanvitelli”, Naples, Italy
Marianthi Karali & Francesca Simonelli
Center for Rare Retinal Diseases, AugenZentrum Siegburg, MVZ Augenärztliches Diagnostik- und Therapiecentrum Siegburg GmbH, Siegburg, Germany
Ulrich Kellner
Department of Ophthalmology, Erasmus MC University Medical Center, Rotterdam, the Netherlands
Caroline C. W. Klaver, Alberta A. H. J. Thiadens & Virginie J. M. Verhoeven
Department of Pediatric Surgery, Division of Pediatric Ophthalmology, Montreal Children's Hospital, McGill University Health Center (MUHC), Montreal, Quebec, Canada
Robert K. Koenekoop & Irma Lopez
Institute for Ophthalmic Research, Centre for Ophthalmology, University Hospital Tübingen, Tübingen, Germany
Susanne Kohl & Bernd Wissinger
University Eye Hospital, Centre for Ophthalmology, University Hospital Tübingen, Tübingen, Germany
Laura Kühlewein & Katarina Stingl
Department of Ophthalmology, Rambam Health Care Campus, Haifa, Israel
Rina Leibu
Department of Ophthalmology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic
Petra Liskova & Marie Vajter
Department of Genetics, Institute of Ophthalmology “Conde de Valenciana”, Mexico City, Mexico
Victor R. de J. López-Rodríguez & Juan C. Zenteno
Section of Ophthalmology, King's College London, St. Thomas' Hospital Campus, London, UK
Omar A. Mahroo
Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK
Omar A. Mahroo
Molecular Genetics Laboratory, Montpellier University, CHU Montpellier, Montpellier, France
Luke Mansard
Department of Ophthalmology, Fundación Jiménez Díaz University Hospital, Madrid, Spain
M. Pilar Martín-Gutiérrez
Department of Clinical Genetics, Institute of Clinical Medicine, University of Tartu, Tartu, Estonia
Laura Mauring
Eye Clinic, Tartu University Hospital, Tartu, Estonia
Laura Mauring
Genetics and Personalized Medicine Clinic, Tartu University Hospital, Tartu, Estonia
Laura Mauring
Department of Ophthalmology, Leeds Teaching Hospitals NHS Trust, St James's University Hospital, Leeds, UK
Martin McKibbin & Rajarshi Mukherjee
Instituto de Investigación Sanitaria La Fe (IIS La Fe) and CIBERER, Valencia, Spain
José M. Millán
Department of Histology and Embryology, Medical University of Warsaw, Warsaw, Poland
Monika Ołdak
Department of Ophthalmology, School of Medicine, University of Crete, Heraklion, Crete, Greece
Anastasia Papachristou, Miltiadis K. Tsilimbaris & Pavlina Tsoka
Department of Medicine and Surgery, Medical Genetics, University of Parma, Parma, Italy
Antonio Percesepe
UCT/MRC Precision and Genomic Medicine Research Unit, Division of Human Genetics, Department of Pathology, Institute of Infectious Disease and Molecular Medicine (IDM), Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
Raj Ramesar, Lisa Roberts & Casey Valentine
Department of Medical Genetics, Saint-Etienne University Hospital, Saint-Etienne, France
Francis Ramond
Department of Ophthalmology, HUB-Erasme Hospital, Brussels, Belgium
Florence Andrée Rasquin
Department of Neuroscience, Biodonostia Health Research Institute, Donostia-San Sebastián, Spain
María Rodríguez-Hidalgo & Javier Ruiz-Ederra
Department of Genetic, Physical Anthropology and Animal Physiology, University of the Basque Country (UPV/EHU), Leioa, Spain
María Rodríguez-Hidalgo
Department of Ophthalmology, University of the Basque Country (UPV/EHU), San Sebastián, Spain
Javier Ruiz-Ederra
West Midlands Clinical Genetics Unit, Birmingham Women's and Children's NHS Foundation Trust, Birmingham, UK
Ataf H. Sabir
College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
Ataf H. Sabir
iNOVA4Health, NOVA Medical School, Faculdade de Ciências Médicas, NMS, FCM, Universidade NOVA de Lisboa, Lisboa, Portugal
Cristina M. Santos
Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria
Hendrik P. N. Scholl
Pallas Kliniken AG, Pallas Klinik Zürich, Zürich, Switzerland
Hendrik P. N. Scholl
European Vision Institute, Basel, Switzerland
Hendrik P. N. Scholl
Department of Child Neurology and Psychiatry, IRCCS Mondino Foundation, Pavia, Italy
Sabrina G. Signorini
Department of Medical Genetics, ULS Santa Maria, Lisboa, Portugal
Ana Berta Sousa
Laboratory of Basic Immunology, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal
Ana Berta Sousa
Department of Ophthalmology, School of Medicine, University of Ioannina, Ioannina, Greece
Maria Stefaniotou & Georgia G. Yioti
Faculty of Medicine, School of Health Sciences, University of Iceland, Reykjavik, Iceland
Kari Stefansson
Department of Ophthalmology, Medical University of Warsaw, Warsaw, Poland
Jacek P. Szaflik
SPKSO Ophthalmic University Hospital in Warsaw, Warsaw, Poland
Jacek P. Szaflik
Medical Genetics and Prenatal Diagnostics Clinic, Children's Clinical University Hospital, Riga, Latvia
Gita Taurina
Department of Ophthalmology; Oculogenetics Unit, Jules Gonin University Hospital; University of Lausanne, Lausanne, Switzerland
Viet H. Tran & Veronika Vaclavik
Centre for Gene Therapy & Regenerative Medicine, King's College London, London, UK
Viet H. Tran
Eye Disease Clinic, Children's University Hospital Riga, Riga, Latvia
Sandra Valeina
Department of Molecular Medicine, University of Pavia, Pavia, Italy
Enza Maria Valente
Department of Ophthalmology, Pediatric Ophthalmology and Ophthalmogenetics, University Hospitals Leuven, Leuven, Belgium
Joseph van Aerschot
Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
Andrea L. Vincent
School of Pharmacy and Biomolecular Sciences (PBS), RCSI University of Medicine and Health Sciences, Dublin, Ireland
Laura Whelan
FutureNeuro Research Ireland Centre, RCSI University of Medicine and Health Sciences, Dublin, Ireland
Laura Whelan
Department of Biochemistry, Faculty of Medicine, UNAM, Mexico City, Mexico
Juan C. Zenteno
Bonei Olam - Center for Rare Jewish Genetic Diseases, Brooklyn, NY, USA
Chaim Landau
Physicians Dialysis, Miami, FL, USA
Allan I. Jacob
Manchester Centre for Genomic Medicine, Manchester University NHS Foundation Trust, Saint Mary's Hospital, Manchester, UK
Siying Lin
Faculty of Biology, Medicine and Health, School of Biological Sciences, Division of Evolution, Infection and Genomics, University of Manchester, Manchester, UK
Siying Lin
Department of Ophthalmology, Columbia University Irving Medical Center, New York, NY, USA
Winston Lee
Genomics England Ltd, London, UK
Jamie M. Ellingford
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M.Q., F.P.M.C., S.R. and C.R. conceived and designed the study. M.Q., K.R., K.K. and M.U. analyzed pedigrees and genotypes. J.B., S.H.T., C.L., A.I.J., K.B.F. and W.L. contributed to the collection of material and clinical data for family M1-A. F.P.M.C., S.E.d.B., D.M.P., R.J.H.-M. and S.R. coordinated the collection of DNA samples. K.R., S.E.d.B., E.G.M.B., N.Z., L.K.H., Z. Corradi, S.S., D.M.P. and R.J.H.-M. performed the majority of the Sanger sequencing screening. K.K., A.B.I.-R. and M.F. carried out the molecular biology experiments. M.Q. and C.R. performed the statistical analyses. M.Q. and J.M.E. retrieved and analyzed large-scale data. K.R. and S.R. coordinated the genotyping of the cohorts. W.L. was responsible for clinical data analysis. Z. Cvackova and D. Stanek conducted all in vitro experiments. F.P.M.C., C.R. and S.R. supervised the project. M.Q., K.R., F.P.M.C., J.M.E., D. Stanek, S.R. and C.R. prepared the original draft of the manuscript. M.Q., K.R., S.R. and C.R. reviewed and edited the manuscript. M.A., A.A., S.A., G. Ansari, G. Arno, G.D.N.A., C.A., R.A., S.B., E.B., T.S.B., M.T.S.B., M.B., T.B.-Y., V.B., D.G.B., P.B., F.B.-K., B.B., C.J.F.B., K.B., D.B.-G., A.C.B.-J., K.M.B., C.B.d.R., E.L.C., G.C., F.C., L.C., N.C., P.C.I., L.C.-S., S.P.D., E.D.B., M.D.B., B.d.l.C., J.N.D.R., J.D.Z., R.D., C.-M.D., L.D., J.L.D., G.J.F., N.F., B.J.F., L.F.-C., J.M.F.S., S.G., A.G., J.C.G., C.G., R.G.-D., K.G., S.G.-J., T.B.H., L.H.-W., A.J.H., T.H., E.H., L.H.H., A.H., J.P.H., C.B.H., M.B.B.I., C.F.I., T.I., B.O.J., K.J., V.K., S. Kamakari, M.K., U.K., C.C.W.K., K.K., R.K.K., S. Kohl, T.K., L.K., T.M.L., R.L., B.P.L., S.L., P.L., I.L., V.R.d.J.L.-R., Q.M., O.A.M., G.M., L. Mansard, M.P.M.-G., N.M., L. Mauring, M. McKibbin, T.L.M., I.M., M. Michaelides, J.M.M., K.M., R.M., Z.Z.N., K.N., M. Ołdak, M. Oorsprong, Y.P., A. Papachristou, A. Percesepe, M.P., E.A.P., E.P., R.R., F.R., F.A.R., G.I.R., L.R., M.R.-H., J.R.-E., A.H.S., A.F.S., A.I.S.-B., A.S.S., R.S., C.M.S., M. Scarpato, H.P.N.S., D. Sharon, S.G.S., F.S., A.B.S., M. Stefaniotou, K. Stefansson, K. Stingl, A.S., P.S., L.S.S., V.S., J.P.S., G.T., A.A.H.J.T., C.T., V.H.T., M.K.T., P.T., V.V., M.V., S. Valeina, E.M.V., C.V., R.V., S. Valleix, J.v.A., L.I.v.d.B., M.V.H., V.J.M.V., A.L.V., A.R.W., L.W., B.W., G.G.Y., K.Y., J.C.Z., R.Z. and T.Z. were instrumental in acquiring funding, clinical data, validation of genetic or molecular data, and reviewing and editing the final manuscript. All authors approved the final content of this work.
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Susanne Roosing.
The authors affiliated with deCODE genetics/Amgen Inc. (B.O.J., K. Stefansson, P.S.) are employed by the company. The other authors declare no competing interests.
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Supplementary Figs. 1–7 and Data 1.
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Quinodoz, M., Rodenburg, K., Cvackova, Z. et al. De novo and inherited dominant variants in U4 and U6 snRNA genes cause retinitis pigmentosa.
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Nature Microbiology
(2026)Cite this article
In vitro antibiotic testing is important for guiding therapy and drug development. Current methods are focused on growth inhibition in bulk bacterial populations but often fail to accurately predict treatment responses. Here we introduce Antimicrobial Single-Cell Testing (ASCT), a large-scale live-cell imaging approach that quantifies bacterial killing in real time at single-cell resolution. By tracking over 140 million mycobacteria and analysing ~20,000 time–kill curves, we identify key determinants of antibiotic killing and its clinical relevance. For Mycobacterium tuberculosis, we found that drug-specific killing dynamics in starved bacteria, rather than growth inhibition or killing of growing cells, predict regimen efficacy in mice and humans. Extending this approach to Mycobacterium abscessus and comparing 405 bacterial strains, we show that antibiotic killing is also a genetically encoded bacterial trait (drug tolerance). We demonstrate that tolerance patterns cluster by antibiotic targets, identify a phage protein that modulates antibiotic killing, and show that strain-specific killing dynamics are associated with individual patient outcomes independent of drug resistance. Together, these findings establish a framework that reveals how drug properties and bacterial diversity shape treatment responses, offering a path to more effective and personalized therapies.
Bacteria have evolved diverse strategies to overcome toxic exposures. The best-known is antibiotic resistance, where genetic modifications alter the target site or change the effective drug concentration at the target, enabling bacterial growth during drug treatment1. Another bacterial strategy involves transient bacterial survival during antibiotic treatment, allowing bacterial regrowth once the drug is cleared. This phenomenon is known as drug tolerance, referring to delayed killing of the overall population, and persistence, when a highly tolerant subpopulation is present2. Despite decades of recognition and the identification of multiple tolerance mechanisms3,4,5,6, key aspects of tolerance biology, especially in human infections, and its impact on antibiotic treatment remain poorly understood7.
Even in the absence of antibiotic resistance, treatment outcomes for bacterial infections, including urinary tract, respiratory and bloodstream infections, are often poor8,9,10,11. This challenge is highlighted in mycobacterial infections, where limited treatment efficacy necessitates prolonged multidrug therapy lasting months to years. For drug-susceptible Mycobacterium tuberculosis, which caused more than 1 million deaths in 202312, it took decades of research to shorten treatment from 6 to 4 months13. These extended treatment durations are costly, associated with treatment-related toxicity and increase the risk of non-adherence, potentially driving relapses and transmission. The therapeutic challenge is even more acute in Mycobacterium abscessus infections, now one of the most prevalent mycobacterial infections in developed countries14. For M. abscessus, no consistently effective drug regimen exists, and treatment outcomes are often poor, with failure rates frequently exceeding 50% despite months or years of treatment with multiple in vitro active drugs15.
This gap between in vitro growth inhibition and in vivo efficacy motivated us to develop strategies beyond standard susceptibility testing to better predict treatment outcomes. Bacterial killing could be such a factor, which is not captured by minimum inhibitory concentrations (MICs) and has been challenging to study due to the labour-intensive, low-throughput nature of colony-forming unit (c.f.u.) assays16,17,18. To overcome this gap, we established Antimicrobial Single-Cell Testing (ASCT), a highly scalable method for quantifying bacterial viability at single-cell resolution. We first applied ASCT to Mycobacterium tuberculosis to examine whether the killing properties of different drug regimens are linked to in vivo treatment responses in mouse and human populations. Extending this approach to Mycobacterium abscessus, we then compared hundreds of clinical isolates to determine how bacterial strain variation shapes antibiotic killing dynamics and individual patient outcomes.
To quantify bacterial growth and antibiotic killing in bacterial populations and subpopulations at a scale comparable to that of MIC assessments, we developed ASCT (Fig. 1 and Supplementary Video 1), a workflow that simultaneously tracks the behaviour and viability of millions of single cells across diverse conditions in 1,536-well plates. In ASCT, bacteria are dispensed into multiwell plates, immobilized in agar pads containing the viability dye propidium iodide (PI) and exposed to drugs (Fig. 1a and Methods). High-content live-cell imaging captures brightfield and fluorescence images of over 10,000 fields every 2–4 h for up to 7 days, generating up to one million images per experiment. Time-lapse images of every field are sequentially analysed using sparse and low-rank decomposition to correct for background fluorescence19, supervised random forest classifiers for bacterial segmentation and viability classification20, drift correction and single-cell tracking based on object position and homology (Fig. 1b and Extended Data Fig. 1a–e). To analyse time–kill kinetics at the population level, single-cell data are pooled, and overall killing is quantified using the area under the time–kill curve (AUC; Fig. 1c). The AUC captures drug tolerance, representing the killing dynamics of the whole population, and persistence, reflecting the enhanced survival of smaller bacterial populations. ASCT showed no detectable effect on bacterial viability in antibiotic-free conditions (Extended Data Fig. 1f). M. abscessus time–kill kinetics were reproducible, drug- and concentration-specific, and largely consistent across gel volumes and concentrations (Fig. 1d,e and Extended Data Fig. 1g,h). Although ASCT can also quantify homogeneous and heterogenous bacterial growth (for example, resistance or hetero-resistance), single-cell tracking of growing cells is constrained by the small well size and overlapping bacteria. This study therefore focuses on the determinants and consequences of antibiotic killing.
a, Illustration of the ASCT setup. Bacteria (dead cells in red) are immobilized in agar pads containing the viability dye PI at the bottom of wells of a 1,536-well plate and exposed to drugs. Live-cell imaging (brightfield and fluorescence) of 9 fields per well and up to 1,536 wells was performed at 2–4-h intervals from the plate bottom. b, Schematic of the automated image analysis. Raw brightfield images (to assess cell morphology) and fluorescence images (Cy3 settings, to quantify PI accumulation) were processed to segment cells (lines indicate cell borders), classify bacterial viability (white, viable; red, dead) and track individual cells (track IDs indicated by numbers). c–e, ASCT-derived time–kill curves of M. abscessus (isolate number 328). c, Example of 4,212 single-cell trajectories during moxifloxacin exposure from a single imaging well, with mean PI intensity over time highlighted. Bacterial trajectories are ordered by the time of PI positivity. The white line represents the proportion of live bacteria across 29 timepoints and reveals population time–kill kinetics. The AUC quantifies overall killing, integrating drug tolerance and persistence phenotypes. Time–kill curves (mean ± s.e.m. of three replicates; s.e.m. barely exceeds the line thickness) across multiple antibiotics (d) and tigecycline concentrations (e). AMK, amikacin; AZM, azithromycin; FOX, cefoxitin; IPM, imipenem; LZD, linezolid; MIN, minocycline; MXF, moxifloxacin; TGC, tigecycline. The number of tracked bacteria per imaging well was 4,246 ± 1,227 (mean ± s.d.). f,g, Green fluorescent protein (GFP) induction after 12-h cefoxitin treatment and washout with PI fluorescence. f, Representative brightfield, GFP and PI fluorescence images with merged overlays acquired ~1 h after antibiotic removal and GFP induction. The experiment was repeated twice with similar results. g, Corresponding single-cell fluorescence intensities, with gating of GFP- and PI-positive and negative populations.
Source Data
Bacterial viability was assessed using PI, a cell-impermeable fluorescent dye that accumulates in cells upon membrane disruption and serves as a proxy for bacterial death, as previously demonstrated in multiple bacterial species21,22,23. To validate this method, we treated M. abscessus with a number of antibiotics for 24 h and tracked more than 30,000 single bacteria for a further 24 h post antibiotic washout. Only one PI-positive bacterium resumed growth (a misclassified doublet of a live and dead bacterium), while 1–11% of the PI-negative bacteria regrew in antibiotic-free media (Extended Data Fig. 2a and Supplementary Video 2), confirming that PI accumulation is a reliable marker of bacterial death.
To further assess the viability of PI-negative bacteria, we used an inducible M. abscessus reporter strain to test their capacity to synthesize new proteins. After 12 h of antibiotic exposure, drugs were removed and GFP expression was induced with anhydrotetracycline. Following cefoxitin or moxifloxacin treatment, most PI-negative bacteria expressed GFP shortly after induction (Fig. 1f,g and Extended Data Fig. 2b,c), indicating that they remained viable. Bacteria treated with amikacin, a direct inhibitor of protein synthesis, showed delayed GFP expression after washout. Importantly, the fraction of bacteria capable of GFP synthesis far exceeded the fraction of regrowing bacteria (4.2% for cefoxitin, 2.7% for moxifloxacin and 3.4% for amikacin), demonstrating that regrowth assays underestimate survival and that most metabolically competent cells fail to regrow in standard media conditions. Moreover, repeated assessments after antibiotic removal revealed a progressively increasing PI-positive population (Extended Data Fig. 2c and Supplementary Video 2), consistent with substantial post-exposure killing and explaining some of the mismatch between real-time ASCT and delayed c.f.u. measures24.
Using ASCT, we first investigated whether the drug's bactericidal properties correspond to in vivo outcomes in mice and humans. Specifically, we analysed the potential of ASCT to identify more effective treatments for tuberculosis. We tested antibiotic killing of 65 drug regimens in the two avirulent M. tuberculosis strains H37Ra and mc27000, in nutrient-rich and starvation conditions. Each drug was dosed at the maximum blood concentration (Cmax) achievable during therapeutic dosing in humans. Drug regimens containing isoniazid, rifampicin or ethambutol (all part of the standard M. tuberculosis regimen) were most effective in killing growing M. tuberculosis (Extended Data Fig. 3a–c). In contrast, combinations including bedaquiline, clofazimine or pretomanid, were more effective under starvation conditions, consistent with previous reports that these drugs retain activity in non-replicating M. tuberculosis (Fig. 2a,b and Extended Data Fig. 3d), and with clinical data demonstrating treatment-shortening potential of bedaquiline- and pretomanid-containing regimens25,26,27,28,29. Their efficacy during starvation is probably due to a disruption of energy metabolism, respiration or redox balance, which are essential processes for survival in nutrient-limited environments.
a, ASCT-based time–kill kinetics of PBS-starved M. tuberculosis (mc27000) exposed to 65 drug regimens, with the following regimens highlighted: isoniazid-rifampicin (HR, dark blue), isoniazid-rifampicin-ethambutol-pyrazinamide (HREZ, light blue), bedaquiline-pretomanid-linezolid (BPaL, red) and bedaquiline-pretomanid-pyrazinamide (BPaZ, purple). b, Areas under the kill curve for M. tuberculosis drug regimens, averaged across three M. tuberculosis starvation conditions (mean ± s.e.m.), with individual drugs indicated. c, Drug regimens were previously classified as similar to standard-of-care (SOC) or better than SOC on the basis of their performance in relapsing mouse models (RMM), bactericidal mouse models (BMM) of common mouse strains, the granulomatous C3HeB/FeJ mouse strain and in clinical studies30,31. Time–kill curves (AUC averaged across three M. tuberculosis starvation models) of similar-to-SOC drug regimens were compared with better-than-SOC regimens using a two-sided Mann–Whitney U-test. Each dot represents a drug regimen (colours indicate drug regimens in a). Boxplots show the median, interquartile range and total range (central line, box and whiskers, respectively) of AUC values (RMM: n = 46, P = 2.7 × 10−5; BMM common strains: n = 48, P = 0.0015; BMM C3HeB/FeJ: n = 15, P = 0.0047; clinical bactericidal activity: n = 14, P = 0.020). *P < 0.05, **P < 0.01, ****P < 0.0001; NS, not significant. d, Median MICs of drug regimens, calculated from the MICs of individual drugs (averaged from mc27000 and H37Ra strains), were compared between similar-to-SOC and better-than-SOC regimens. Sample sizes, statistical tests and box plots are identical to those described in c. BgMM, BMM C3HeB/FeJ.
Source Data
To evaluate ASCT's ability to predict infection outcomes, we used established classifications of M. tuberculosis drug regimen efficacy, categorizing them as either similar to standard-of-care or better than SOC, on the basis of mouse and clinical studies30,31. While time–kill kinetics of growing M. tuberculosis did not discriminate between similar-to-SOC and better-than-SOC drug combinations, we found strong associations between in vivo outcomes and in vitro killing under starvation conditions, independent of whether quantified by AUC or live-cell fractions at single timepoints (Fig. 2c, Extended Data Fig. 4a and Supplementary Table 1). These findings suggest that non-growing, metabolically less active bacteria, commonly found in intra- and extracellular infection niches, are more challenging to clear and probably responsible for long-term infection outcomes32,33,34,35.
For example, killing of pantothenate-starved mc27000 (the auxotrophic M. tuberculosis strain H37Rv ΔpanCD ΔRD1) was highly associated with outcomes in multiple mouse models, including the relapsing mouse model, the bactericidal model of common mouse strains and the C3HeB/FeJ strain, mirroring different outcomes (bacteriologic burden and relapses) and host pathobiology36,37. Moreover, in vitro mc27000 killing upon pantothenate starvation was associated with clinical outcomes in phase 2 clinical studies, as measured by bactericidal activity during treatment31. Similar results were obtained with 14-day phosphate-buffered saline (PBS) starvation of the mc27000 and H37Ra M. tuberculosis strains. Overall, 11 out of 12 associations between M. tuberculosis killing under starvation and clinical outcomes reached statistical significance.
In contrast, c.f.u.-based killing assessments were not associated with in vivo outcome measures (Extended Data Fig. 5a–c), and correlations of the lowest MIC, median MIC or mean MIC (mean MIC of the two most potent drugs) within each combination with in vivo outcome measures were poor (Fig. 2d and Extended Data Fig. 6). Because MICs within combinations may shift due to drug–drug interactions, we systematically measured all possible pairwise combinations among the 13 drugs tested (9 × 9 concentration matrices in quadruplicate for each of the 78 drug pairs, ~25,000 assays in total; Extended Data Fig. 7a). From these data we predicted high-order drug effects and compared them with Bliss expectations derived from single-drug activities38. Interactions were generally weak and not associated with in vivo outcomes, except for clinical trial data where antagonism correlated with regimens classified as better-than-SOC (Extended Data Fig. 7b,c). Logistic regression models combining MIC with interactions metrics showed only moderate predictive performance in the relapsing mouse model and no predictive value in the bactericidal mouse model (Extended Data Fig. 7d). Notably, all combinations contained at least one drug administered at concentrations above the MIC, highlighting the value of MICs for identifying active agents, even though regimens with very low MICs did not translate into improved outcomes. To complement ASCT, we also performed population-level resazurin assays as an alternative measure of antibiotic killing. As expected for a metabolic readout, starved M. tuberculosis showed lower fluorescence levels than growing cultures. Across all regimens, relative fluorescence changes correlated with ASCT-derived killing only under starvation, where modest associations with in vivo outcomes were also observed (Supplementary Fig. 1a,b).
The performance of ASCT in predicting clinical study outcomes using M. tuberculosis starvation models yielded areas under the receiver operating curve (AUC-ROC) between 0.76 and 0.94 (Extended Data Fig. 4b), suggesting that the killing activity of M. tuberculosis regimens upon starvation is a critical in vitro marker that corresponds to in vivo efficacy. These analyses, based on non-clinical M. tuberculosis strains, show that time–kill dynamics in starved conditions can quantify regimen performance at the population level. While these findings highlight the potential of ASCT to prioritize antibiotic treatments in development, they do not extend to predicting individual patient outcomes.
To investigate bacterial factors that influence antibiotic killing and individual infection outcomes, we next focused on different bacterial isolates. Due to the biosafety constraints of M. tuberculosis live-cell imaging, we studied M. abscessus, an emerging concern in developed countries. To evaluate how phenotypic and genetic diversity within a naturally evolved bacterial species correlate with antibiotic killing (that is, drug tolerance), we obtained a single bacterial isolate from each of 405 patients with M. abscessus lung infection across Europe and Australia39,40 (Fig. 3a). Using ASCT, we generated time–kill profiles for each clinical isolate and the M. abscessus laboratory strain against 8 antibiotics that are commonly used and recommended in clinical treatment41. Each drug was tested at 2 concentrations, and each condition was assessed in triplicate. In total, we assessed 18,244 time–kill curves and tracked ~130 million bacterial cells over 29 timepoints and 72 h. Because all clinical M. abscessus isolates of a given antibiotic condition were assessed simultaneously, they were frozen in logarithmic phase and exposed to drugs shortly after thawing, when they were probably in a lag phase. Isolate–drug pairs that exhibited bacterial growth (that is, drug resistance) or did not meet quality criteria were excluded from analyses (Methods). Our findings revealed highly diverse but reproducible time–kill kinetics under identical drug exposure, indicating that specific strain characteristics modulate antibiotic killing (Fig. 3b).
a, Study centres in the United Kingdom, Ireland, mainland Europe and Australia. Maximum-likelihood phylogenetic tree of 376 M. abscessus isolates obtained from different patients, with each centre indicated by colour. b, Time–kill curves of 406 M. abscessus isolates across 8 antibiotics (the lower drug concentrations are shown), with viability distributions at 72 h, excluding growing isolates. c, Gompertz function fitting to optical density (OD) measurements (black dots), illustrating the determination of bacterial growth rates and lag times. The insert highlights lag time estimation from the Gompertz function and inoculum size on a logarithmic scale. d, Distributions of lag times (top) and growth rates (bottom) from 226 fitted M. abscessus growth curves. e, Pearson correlation analyses between drug tolerance (high drug concentration, continuous line; low concentration, dashed line), bacterial growth rate, lag time and the corresponding MIC. Line colour indicates the direction of the correlation; line width represents the corresponding R2 value. AZM*, inducible azithromycin resistance.
Source Data
Drug tolerance and persistence have been linked to slow bacterial growth or prolonged lag times, associated with metabolic dormancy42,43. To evaluate how bacterial replication affects antibiotic killing, we measured optical densities of 226 M. abscessus strains during planktonic growth in nutrient-rich conditions and fitted Gompertz functions to provide model-based estimates of each isolate's lag time and growth rate (Fig. 3c,d). Apart from revealing a strong direct correlation between growth rate and lag time, reflecting a trade-off between bacterial growth and adaptation44, we identified correlations between growth rates or lag times and drug tolerance phenotypes (Fig. 3e and Supplementary Fig. 2). For instance, drug tolerance to imipenem and cefoxitin, which target cell wall synthesis specifically during bacterial replication, correlated with slow growth but not lag time. In contrast, moxifloxacin killing (targeting the DNA) was associated with longer lag times only. These associations were weak, explaining only up to 13% of the variation in the drug tolerance phenotype, suggesting that replication patterns were not the main driver of drug tolerance. However, potential technical factors, such as variability in growth assessments, curve fitting or isolate clumping, may have reduced the strength of these associations. We also found weak associations (R2 up to 0.11) between drug tolerance and resistance phenotypes (especially for cefoxitin and minocycline), mainly driven by outliers with MICs close to the tested concentration. Using a non-parametric test (Spearman correlation), most of these associations disappeared (Supplementary Fig. 2), supporting the idea that drug tolerance is a marker of antibiotic efficacy independent of drug resistance45. Drug tolerance phenotypes, especially for antibiotics targeting protein synthesis, were strongly associated, with some correlating weakly with MICs (Extended Data Fig. 8a), suggesting shared mechanisms or adaptive evolution reminiscent of collateral sensitivity and cross-resistance.
While drug resistance is well established as a genetic trait, drug tolerance has traditionally been viewed as a largely phenotypic characteristic. Recent studies, however, have identified multiple genetic mechanisms underlying drug tolerance in mycobacteria and other bacterial species42,46,47,48. We employed whole-genome sequencing and estimated the proportion of phenotypic variance attributable to genetic variability, known as heritability49. Specifically, we mapped 1.3 million M. abscessus unitigs (sequences of variable length), which capture most genomic variation arising from vertical inheritance, horizontal gene transfer and mutations including single nucleotide polymorphisms, insertions, deletions and gene presence–absence, to respective phenotypes using linear mixed models50,51. Resistance phenotypes for several antibiotics (particularly macrolide MICs) were strongly determined by bacterial genetics, whereas others, such as imipenem and cefoxitin, showed low heritability, probably reflecting chemical instability and limited biological variation in MIC measurements (Fig. 4a). Across all drugs, tolerance phenotypes demonstrated substantial heritability (32–97%), far exceeding random chance (1.1%, Student's t-test P < 2.2 × 10−16), indicating that killing phenotypes are strain-specific and largely genetically determined.
a, Heritability estimates for drug resistance (MIC; n = 221) and drug tolerance phenotypes (AUC; n = 371, excluding growing isolates) based on 1.3 million genetic M. abscessus variants (unitigs) derived from whole-genome sequencing. CLR, clarithromycin; CLR*, inducible clarithromycin resistance; L, low drug concentration; H, high drug concentration. b, Maximum-likelihood phylogenetic tree of 353 M. abscessus isolates, aligned with the tigecycline tolerance heat map (high concentration) and mutational macrolide and aminoglycoside (Aminogl.) resistance. Purple lines mark clades with reduced tigecycline tolerance.
Source Data
Mapping these phenotypes to the M. abscessus phylogeny (Fig. 4b and Extended Data Fig. 8b), we observed that some of the high- or low-tolerance phenotypes evolved in parallel, resulting in homoplastic traits, while others were conserved in phylogenetically related isolates, indicating inherited clades. One such example is a tigecycline low-tolerance clade within the dominant circulating clone of M. abscessus massiliense52. Many of these isolates carry high-level mutational aminoglycoside and macrolide resistance and have been linked to increased virulence40. Our finding of low tigecycline tolerance highlights a potential vulnerability in otherwise highly drug-resistant M. abscessus, where treatment success rates can fall below 20%.
We then investigated whether strain-specific kill kinetics influence individual patient outcomes, given the limited direct evidence linking drug tolerance to treatment failures7. Our analysis showed that, among eight tested drugs, only macrolide MICs were associated with clinical outcomes (Fig. 5a and Extended Data Fig. 9a,b). However, tolerance to amikacin, cefoxitin and imipenem, measured at low and high drug concentrations, correlated with M. abscessus clearance in patients (Extended Data Fig. 10). Specifically, rapid bacterial killing was associated with favourable clinical outcomes, whereas high tolerance was linked to poor outcomes. Notably, the effect sizes of drug tolerance in predicting infection outcomes were comparable to those of macrolide MICs, currently the main in vitro measure guiding M. abscessus treatment decisions. Incorporating a single drug tolerance measure alongside macrolide resistance improved clinical outcome prediction, increasing the AUC-ROC from 0.69 to 0.78 (Fig. 5b, and Supplementary Tables 2 and 3). These findings underscore drug tolerance as an independent and clinically relevant marker of antibiotic efficacy.
a, Comparison of M. abscessus drug tolerance (AUC, low concentration) and drug resistance (MICs) between isolates from patients with poor and favourable clinical outcomes, using a two-sided Mann–Whitney U-test (n = 135, excluding growing isolates). Boxplots show the median, interquartile range and total range (central line, box and whiskers, respectively) of AUC values. **P < 0.01, ***P < 0.001. b, Logistic regression predicting clinical outcomes from clarithromycin resistance (MIC) and imipenem tolerance (low concentration), individually and combined. Fifty ROC curves were generated by randomly selecting 80% of the samples for each condition. The blue line represents the mean ROC curve and the shaded area denotes 1 s.d. The mean AUC-ROC and 95% confidence intervals are shown.
By applying principal component analysis to 5,056 M. abscessus time–kill curves and mapping antibiotics in drug tolerance space, we identified drug clusters related to the drug's mode of action (Fig. 6a). These clusters indicate that bacterial survival mechanisms are linked to the drug's target, probably driven by shared downstream effects, such as conserved stress responses that modulate antibiotic killing53. To explore the genetic basis of drug tolerance, we mapped ~280,000 M. abscessus genotypes (single nucleotide polymorphisms, insertions and deletions) extracted from whole-genome sequences of each isolate to drug tolerance phenotypes, using mixed-effects models corrected for population structure. This analysis identified multiple genotypes and genes strongly associated with drug tolerance phenotypes (Fig. 6b). Tolerance-associated genes grouped by the antibiotic target, with specific gene sets linked to protein synthesis, DNA and cell wall-active drugs.
a, Principal component analysis of 8 drugs at 2 concentrations in drug tolerance space, based on Spearman correlations from 350 clinical M. abscessus isolates. b, Genome-wide association results for 16 drug tolerance phenotypes, highlighting the top 5 associated genes (moderate- or high-effect genotypes) per phenotype. The heat map shows association of 43 genes (top genotype per gene) with tolerance phenotypes using a mixed-effects model corrected for population structure (two-sided Wald test). Associations with P > 0.0001 are in white. c, Manhattan plot of 272,351 M. abscessus genotypes and their association with amikacin (low concentration) kill kinetics (two-sided Wald test), with the Bonferroni correction threshold (black line, 1.7 × 10−7). Multiple variants in MAB_0233 were strongly linked to amikacin killing (insert). d, Time–kill kinetics of control, ΔMAB_0233 and complemented strains (mean ± s.e.m., nine replicates per condition). AUC values of ΔMAB_0233 were compared with control or complemented strains using a two-sided Mann–Whitney U-test. P values for amikacin, linezolid and tigecycline were 4 × 10−5, except for tigecycline time–kill kinetics between ΔMAB_0233 and the control strain (P = 8 × 10−5). ****P < 0.0001.
Source Data
We further examined MAB_0233, a putative phage tail tape measure protein, in which several deletions were associated with drug tolerance to drugs targeting protein synthesis (Fig. 6c). As such, MAB_0233 could facilitate intracellular drug accumulation, enhance metabolic activity to potentiate drug effects, interfere with stress responses, or directly synergize with the drug. Using oligonucleotide-mediated recombineering followed by Bxb1 integrase targeting (ORBIT54), we generated a MAB_0233 knockout strain in M. abscessus, which showed increased tolerance for antibiotics targeting translation (amikacin, tigecycline and linezolid), but not the cell wall, while MICs remained stable (Fig. 6d and Supplementary Fig. 3a,b). MAB_0233 complementation restored the low-tolerance phenotype for amikacin and tigecycline. However, for linezolid, the complemented strain retained an altered time–kill profile, suggesting partial complementation or the involvement of additional mechanisms influencing linezolid tolerance.
Antibiotics that demonstrate activity in vitro may fail to achieve clinical success, highlighting a critical disconnect between microbiological testing and patient outcomes. This gap represents a major challenge in antibiotic development and patient care. To address it, we developed antimicrobial single-cell testing, a strategy that quantifies bacterial viability in real time and at large scale, providing new insights into an underexplored dimension of antibiotic activity. Applying ASCT, we demonstrate that drug-specific killing (in M. tuberculosis) and strain-specific killing (in M. abscessus) are key predictors of drug trial failures and poor clinical outcomes, and thus determinants of in vivo efficacy. This dual perspective of conserved drug effects and variable strain-specific responses shows how both components shape antibiotic killing and ultimately affect treatment outcomes.
Several large-scale approaches to quantify bacterial viability have been recently developed, typically coupling a viability marker with an appropriate readout technology. Markers include conventional regrowth, metabolic dyes, biosensors and fluorescent reporters, while readouts range from bulk optical or fluorescent measurements to live-cell imaging, flow cytometry and label-free biophysical methods16,17. Although these strategies move beyond classical culture assays, most remain constrained by limited throughput, sensitivity or single-cell resolution, leaving critical aspects of bacterial survival unresolved. To overcome these limitations, we established a platform for repetitive, large-scale, real-time, single-cell assessments of bacterial viability across diverse clinical isolates. We used PI as a viability marker and confirmed that PI-positive cells do not regrow and can therefore be considered dead23. To further characterize PI-negative cells, we employed a reporter strain with inducible GFP expression and found that most PI-negative bacteria retained the ability to synthesize new proteins, in contrast to the very small fraction of bacteria that regrew. Notably, many PI-negative cells that initially produced GFP later died after drug washout. This post-exposure killing probably reflects incomplete drug removal, classical post-antibiotic effects or nutrient shock24,55,56. These findings indicate that conventional methods underestimate bacterial survival and highlight the importance of real-time, single-cell measures of bacterial viability. Still, defining true bacterial viability remains challenging, and PI may only partially capture it. We therefore focused on identifying phenotypes predictive of in vivo outcomes, a goal achieved with greater performance and throughput by ASCT through real-time single-cell PI quantifications.
We applied ASCT to investigate the time–kill kinetics of multiple M. tuberculosis drug regimens across two strains and various experimental conditions. While combinations including the standard drugs isoniazid, rifampicin and ethambutol were most lethal under growth conditions, only killing under starvation predicted in vivo population outcomes. Antibiotic killing in these starvation conditions, reflective of metabolic inactivity in intra- and extracellular infection niches57, correlated strongly with outcomes in multiple in vivo models, including the relapsing and bactericidal mouse models as well as humans during phase 2 clinical trials. Thus, even using non-clinical, avirulent M. tuberculosis strains, we were able to predict in vivo outcomes of virulent tuberculosis at the population level, something not achievable with c.f.u. counts, MICs, drug interaction measures or resazurin-based assessments. Importantly, our data also show that not only the extent of killing matters, but also the physiological bacterial state in which killing is assessed, highlighting the importance of infection-relevant contexts when evaluating killing dynamics. Extending this approach to more host-like conditions may further improve its predictive performance. Given the high attrition rates in antibiotic development and clinical trials58, there is an urgent need for more efficient strategies to prioritize therapeutic regimens, which is a challenging task, considering the growing number of active lead compounds and possible combinations59. By enabling the evaluation of thousands of drug combinations in vitro—with demonstrated relevance in vivo—ASCT has the potential to identify more effective and much shorter treatment regimens against M. tuberculosis infection and disease. This advancement could accelerate global health initiatives, such as the World Health Organization's goal to eliminate most tuberculosis burden by 203512,60.
Highly effective antibiotics are the cornerstone of successful antibiotic treatments, yet clinical outcomes vary widely, with some patients responding to short courses and others failing despite prolonged multidrug treatment61. We show that antibiotic killing is highly variable across clinical M. abscessus isolates, further challenging the prevailing clinical view that bacterial killing is solely determined by the drug's bactericidal properties62. This variability in killing, different from drug resistance, highlights drug tolerance as a fundamental bacterial trait that may greatly affect treatment efficacy. Notably, there exists scarce evidence that drug tolerance, rather than limited drug penetration or reinfections, underlies antibiotic treatment failures63,64,65. We found that M. abscessus strains poorly killed by amikacin, cefoxitin or imipenem (in high and low drug concentrations) were linked to worse clinical outcomes in individual patients, providing some of the most compelling evidence so far that drug tolerance is a critical determinant of treatment success. Because drug treatments are complex and often change, actual drug exposure was unclear, probably underestimating the impact of drug tolerance. These findings indicate that tolerance phenotypes could complement conventional drug susceptibility testing to improve outcome prediction and clinical decision-making.
We also show that drug tolerance (such as resistance) is largely determined by the bacterial genetic background and, thus, a heritable and evolvable bacterial trait. For example, within the subspecies M. abscessus massiliense, we identified a low-tolerance clade among highly resistant strains, revealing vulnerabilities that could be exploited to enhance bacterial clearance. Tolerance phenotypes clustered with the drug's mode of action, suggesting conserved survival pathways among drugs targeting similar cellular functions. Using phenogenomic analysis, we identified numerous genes and genotypes associated with high- or low-tolerance phenotypes. Through gene knockout and complementation experiments, we further validated a phage protein that modulates bacterial killing. These tolerance mechanisms represent potential targets for sterilizing antibiotic treatments, while tolerance genotypes could be integrated into diagnostics, analogous to molecular susceptibility testing. Presumably, molecular testing, or phenotyping via ASCT, could facilitate individualized, tolerance-tailored antibiotic regimens and help improve patient outcomes beyond the development of new drugs and regimens.
We note several limitations. Because PI measures membrane compromise, it directly reflects killing by cell wall damage but only indirectly captures other killing mechanisms. Since all dead cells eventually lose membrane integrity, PI therefore detects killing by non-lytic antibiotics only after a delay. In M. abscessus (but not in M. tuberculosis) we observed a delay of ~3 h for such drugs. Given the extended duration of our experiments and in vivo validation, this effect is probably minimal in mycobacteria but may become more relevant when applying ASCT to other bacterial species. While our findings highlight bacterial killing as a key determinant of antibiotic activity, clinical treatment outcomes are influenced by many additional factors. These include host immune responses, fluctuating drug exposures and penetration into tissues, drug interactions, toxicity and adherence, as well as the complexity of infection environments, such as intracellular niches and granulomas in mycobacterial infections, or acidic compartments with altered drug activity (for example, pyrazinamide)34,66,67. ASCT does not capture these non-bacterial determinants and therefore cannot fully predict treatment outcomes, but it provides a scalable and biologically meaningful readout of bacterial susceptibility that complements existing tools. We validated ASCT in mycobacteria, but this approach is probably applicable to other bacterial species and multidrug-resistant pathogens. However, adjustments in experimental design, imaging setup and analysis will be required to account for the species-specific morphology and physiology, particularly in regard to viability assessments. The in vivo relevance of drug tolerance and nutrient limitation is also likely to vary across pathogens and infection sites.
In our study we interrogated antibiotic killing at two levels: first, whether drug-specific killing in M. tuberculosis correlates with in vivo outcomes at the population level; and second, whether strain-specific variation in killing across clinical M. abscessus isolates associates with individual patient responses. Although these approaches differ (drug regimen versus strain), both converge on the same principle that killing dynamics are critical determinants of treatment success. While we examined these factors independently, clinical treatment responses are shaped by the interplay of drug properties and bacterial behaviours. By studying millions of single-cell fates across hundreds of conditions, our approach provides a scalable framework to translate in vitro killing into in vivo efficacy, opening new avenues for drug development and personalized therapy.
We used the Mycobacterium abscessus laboratory strain ATCC-19977, the avirulent Mycobacterium tuberculosis strain H37Ra and the auxotrophic M. tuberculosis strain mc27000 (H37Rv ΔpanCD ΔRD1)68. Clinical M. abscessus isolates were obtained from respiratory samples (sputum or bronchoalveolar lavage fluid) collected from patients with pulmonary M. abscessus infection as described previously40. All work with clinical M. abscessus isolates and avirulent M. tuberculosis strains was conducted in a biosafety level 2 (BSL-2) facility, in accordance with institutional biosafety regulations. Included patients came from all major cystic fibrosis centres in the United Kingdom, the Republic of Ireland (Dublin), Sweden (Gothenburg), Denmark (Copenhagen and Skejby), the Netherlands (Nijmegen) and Australia (Queensland). M. abscessus isolates were retrieved from the original mycobacterial growth indicator tubes (MGIT) or, if otherwise unavailable, from subcultured isolates. The study was approved in England and Wales by the National Research Ethics Service (12/EE/0158) and the National Information Governance Board (ECC 3-03 (f)/2012) and in other centres by respective local review boards.
All consumables are provided in Supplementary Table 4. Planktonic growth rates and the lag times of M. abscessus isolates were assessed following suspension in nutrient-rich media. Approximately 105 c.f.u.s of M. abscessus were inoculated in 2.5 ml of Middlebrook 7H9 medium (supplemented with 0.4% glycerol, 10% OADC [oleic acid, albumin, dextrose, catalase] and 0.05% Tween 80) in 15 ml glass tubes, which were then incubated at 37 °C with 150 r.p.m. orbital shaking. Optical densities (OD565) were measured with a densitometer (DEN-1B; Biosan) and quantified in McFarland units. OD565 readings were performed every 12 h for a minimum of 5 days until the readings stabilized for at least 24 h (change in McFarland less than 0.5). Background-corrected OD565 measurements were used to fit Gompertz functions to estimate growth rates, where N is the number of bacteria at a given time (t), with A representing carrying capacity (maximum population size), B the initial growth factor affecting displacement along the y axis and C, the growth rate constant. Lag times were calculated from the fitted Gompertz function and the known inoculum size.
Given that conventional formulas are imprecise when considering that the growth rate is a proportion of the carrying capacity per time69, we calculated the growth rate (µ) with the following equation:
The lag time (λ) was calculated as:
Antibiotic resistance was quantified using MICs, following the Clinical Laboratory Standards Institute (CLSI; M24 3rd edition) guidelines70. B.W. received training at the National Center for Mycobacteria, Zürich (Switzerland), to align with standard clinical microbiology practices. M. abscessus isolates were cultured in Middlebrook 7H9, supplemented with 0.4% glycerol, 10% OADC and 0.05% Tween 80, for 3 days and then diluted in PBS to a McFarland standard of 0.5. A 140 µl aliquot of this suspension was mixed into 14 ml cation-adjusted Mueller–Hinton broth (CAMHB) to achieve an approximate density of 106 c.f.u.s ml−1. Antibiotics were tested in log2-fold concentration steps at the following ranges: amikacin (0.25–512 µg ml−1), azithromycin (3.9 ng l−1–512 µg ml−1), cefoxitin (0.5–256 µg ml−1), clarithromycin (3.9 ng l−1–512 µg ml−1), clofazimine (7.8 ng ml−1–16 µg ml−1), imipenem (0.13–56 µg ml−1), linezolid (0.25–128 µg ml−1), minocycline (15.6 ng ml−1–256 µg ml−1), moxifloxacin (15.6 ng ml−1–32 µg ml−1) and tigecycline (15.6 ng ml−1–8 µg ml−1). For each experiment CAMHB was freshly prepared. Antibiotics and CAMHB were added to 96-well plates to reach a volume of 50 µl per well. After mixing, the 50 µl bacterial suspension was added to each well to obtain a final bacterial concentration of ~5 × 105 c.f.u.s ml−1. Each plate included growth and media control wells. Mycobacterium peregrinum (ATCC-700686) MICs were assessed for quality control in each experimental batch. Plates were sealed and incubated at 30 °C and visually evaluated for growth after 3–5 days once growth was visible in the growth control wells. Azithromycin and clarithromycin conditions were reassessed after 14 days of incubation to evaluate inducible macrolide resistance. MICs were recorded as the lowest drug concentration preventing visible mycobacterial growth. For further analyses, we used either log2-transformed MIC values or applied previously reported M. abscessus resistance breakpoints (clarithromycin MIC ≥ 8 µg ml−1; amikacin MIC ≥ 64 µg ml−1 (ref. 71)).
M. tuberculosis drug susceptibility testing was done analogous to M. abscessus, except that M. tuberculosis isolates were cultured in 5 ml of Middlebrook 7H9 broth, supplemented with 0.4% glycerol, 10% OADC and 0.05% Tween 80 in 50 ml tubes (M. tuberculosis mc27000 was additionally supplemented with 100 µg ml−1 pantothenate), and that MIC plates were assessed after 14 days. Bedaquiline, clofazimine, delamanid, ethambutol, linezolid, moxifloxacin, pretomanid, rifampicin, rifapentin, SQ109 and sutezolid were assessed in log2-fold concentration steps between 2 ng ml−1 and 64 µg ml−1, whereas isoniazid and pyrazinamide were assessed between 4 ng ml−1 and 128 µg ml−1.
M. tuberculosis mc27000 was cultured in 10 ml of Middlebrook 7H9 broth supplemented with 0.4% glycerol, 10% OADC, 0.05% Tween 80 and 100 µg ml−1 pantothenic acid. Cultures were incubated at 37 °C with shaking (150 r.p.m.) until mid-log phase, then washed and resuspended in PBS before incubation at 37 °C for 14 days. PBS-starved bacteria where standardized to ~107 c.f.u.s ml−1, and 180 µl was dispensed into 96-well U-bottom-plates. Individual drugs or drug combinations were added (30 µl) to achieve maximum therapeutic blood concentrations (Cmax): bedaquiline 1.1 µg ml−1, clofazimine 1.25 µg ml−1, delamanid 0.5 µg ml−1, ethambutol 4 µg ml−1, isoniazid 4.5 µg ml−1, linezolid 19 µg ml−1, moxifloxacin 4 µg ml−1, pretomanid 7.8 µg ml−1, pyrazinamide 40 µg ml−1, rifampicin 16 µg ml−1, rifapentin 19 µg ml−1, SQ109 0.026 µg ml−1 and sutezolid 1.48 µg ml−1. Each condition was tested in triplicate. Plates were sealed with parafilm, covered with lids and incubated at 37 °C. At days 0, 3 and 7, 20 µl from each condition were transferred into 180 µl of PBS. Serial dilutions were performed, and 5 µl of each dilution were spotted onto 7H11 agar plates supplemented with 100 µg ml−1 pantothenic acid (in duplicate). In addition, 50 µl of undiluted samples were washed and directly plated on pantothenate-supplemented 7H11 agar. Plates were incubated at 37 °C until visible colonies emerged. Colonies were manually counted at dilutions where 2–200 colonies were present, and c.f.u.s ml−1 were calculated. The detection limit was 20 c.f.u.s ml−1.
The in vivo classifications of M. tuberculosis drug regimens ‘as good or worse than SOC (where isoniazid-rifampicin-pyrazinamide-ethambutol or isoniazid-rifampicin-pyrazinamide were considered SOC)' or ‘better than SOC' were obtained from two previous studies. In vivo classifications based on mouse models were obtained from ref. 30, and classifications based on phase 2a and 2b clinical trials (assessing bactericidal activity) were obtained from ref. 31. All similar-to-SOC or better-than-SOC drug combinations from the relapsing mouse model (n = 46) and clinical studies (n = 14) were assessed with ASCT. These drug combinations and classifications of single drugs were also used to evaluate the performance of ASCT in bactericidal mouse models (common mouse strains: n = 48; C3HeB/FeJ mouse strain: n = 15). Clinical metadata of patients with respiratory M. abscessus infection were available for a subset of patients. Treatment outcomes were assessed in patients meeting the ATS/IDSA criteria of NTM pulmonary disease72. Patients with positive cultures after 6 months of M. abscessus treatment were considered as having persisting infections, and patients with sustained culture conversion (microbiological clearance) as cleared infections. Growing isolates were excluded from the analysis of bacterial killing. The discriminative performance for predicting the success of drug regimens in mouse or patient populations (M. tuberculosis) and the clinical outcome in individual patients (M. abscessus) was assessed with logistic regression and quantified with AUC-ROC. For each condition, 50 bootstrap iterations were performed by randomly selecting 80% of the samples. Sensitivity, specificity, positive and negative predictive values, and F1 scores were calculated at the optimal cut-off determined by the Youden index.
M. abscessus and M. tuberculosis isolates were cultured in 5 ml of Middlebrook 7H9 broth, supplemented with 0.4% glycerol, 10% OADC and 0.05% Tween 80 in 50 ml tubes (M. tuberculosis mc27000 was additionally supplemented with 100 µg ml−1 pantothenate). The cultures were incubated at 37 °C with shaking at 150 r.p.m. until mid-log phase (McFarland: 5–8). In M. tuberculosis, we also assessed two starvation conditions: PBS starvation and pantothenate starvation. For PBS starvation, mid-log phase bacteria (H37Ra or M. tuberculosis mc27000) were washed and resuspended in PBS, the 50 ml tube filled with PBS and incubated for 14 days at 37 °C without shaking. For pantothenate starvation, M. tuberculosis mc27000 (H37Rv ΔpanCD ΔRD1) was incubated for 14 days with Middlebrook 7H9, supplemented with 0.4% glycerol, but not pantothenate.
After growth or starvation, mycobacteria were centrifuged at 3,000 g for 10 min, and the resulting pellet was resuspended in Middlebrook 7H9 (for growth conditions and pantothenate starvation), in Middlebrook 7H9 with pantothenate supplementation (for growth conditions in M. tuberculosis mc27000) or in PBS (for PBS starvation). To achieve single-cell suspensions, large clumps were removed by low-speed centrifugation (200 g, 3 min), followed by serial filtration of the supernatant through 5 µm and 1.2 µm filters (Sartorius Minisart). In every sample, bacterial densities were assessed using OD565 measurements, and the live-cell fraction was quantified using propidium iodide staining and imaging. Bacterial samples with a live-cell fraction below 95% were discarded and reprocessed. Single-cell suspensions were immediately used (M. tuberculosis) or frozen at −80 °C (M. abscessus clinical isolates).
ASCT uses a dual-layer approach: the first layer consists of an agar pad containing and immobilizing bacteria, and the second layer comprises drug-containing solutions (Fig. 1a). To prepare the agar pad, ultra-low gelling temperature agarose (ULGA; Lonza SeaPrep) and Middlebrook 7H9 were dissolved in hot dH2O. Additional dissolution was facilitated through heating, where any evaporated water was replaced to maintain constant 7H9 and agarose concentrations. Once the solution cooled to 50 °C or lower, OADC and glycerol were added. The mixture was then filtered through a 0.22 µm filter and propidium iodide was added. This agarose solution was aliquoted as needed. The final gel pad solution contained 0.4 agarose, 1× Middlebrook 7H9, 10% OADC, 0.4% glycerol, 8 µg ml−1 propidium iodide and ~5 × 106 bacteria per ml. For non-starving M. tuberculosis mc27000, the agar pad also contained 100 µg ml−1 pantothenate. In pantothenate-starvation conditions, the gel pad did not contain pantothenate, and in PBS-starvation media, all supplements were replaced with 1× PBS.
All preparations were made in 96-well plates using a 96-channel pipette (Mini96, Integra Biosciences). Of the isolate–agar–PI solution, 7 µl was dispensed, using liquid handling (I.DOT, Dispendix), into each well of a 1,536-well plate (Greiner, Screenstar). At least one column and row were left as buffer wells at the edges. All materials used in the agarose preparation, including chemicals, pipette tips, filters, the plate and so on, were preheated to 37 °C. The 1,536-well plate was centrifuged at 37 °C and 3,000 g for 30 min to position the bacteria at the bottom of the wells for imaging. This step was followed by cold centrifugation at 4 °C and 1,500 g for 20 min to solidify the ULGA. After leaving the plate for 30 min at room temperature, a defined drug solution (4 µl) dissolved in Middlebrook 7H9 or PBS (for PBS starvation) was added to each well. The plate was then sealed with parafilm, covered with a lid and remained for another 60 min at room temperature before being placed into the microscope's preheated live-cell chamber (37 °C; Life Imaging Services). To evaluate the impact of gel pad volumes and concentrations on antibiotic killing, we tested various volumes (4, 5, 6 and 7 µl) and concentrations (0.3, 0.4 and 0.5%; Extended Data Fig. 1g,h).
Images were captured with a Nikon Ti2-E inverted microscope using a CFI Plan Apo Lambda ×40 NA 0.95 or a CFI Plan Apo Lambda ×100 NA 1.45 objective, and the Perfect Focus System for maintenance of the focus over time. PI fluorescence was excited with a Spectra III Light Engine (Lumencor) at 555 nm and collected with a penta-edge 408/504/581/667/762 dichroic beam splitter and a 440/40, 520/21, 606/34, 694/34, 809/81 penta-bandpass filter. Images (×40 12-bit and ×100 (optionally with ×1.5 zoom lens) 16-bit) were acquired with a Photometrics Kinetics camera, controlled with the Nikon NIS acquisition software. For ×40, the fluorescence excitation light intensity was set to 10% and the camera exposure time to 20 ms, and the brightfield illumination light intensity was set to 50% and the camera exposure time to 8 ms. These settings were kept the same for all experiments and conditions for comparability. For ×100 acquisition, the excitation light intensity and camera exposure were adjusted accordingly.
The NIS JOBS module was used for image acquisition automation. Briefly, 9 equally spaced fields of view for each well and imaging timepoint were acquired. For M. abscessus, images were captured every 2.5 h for a total duration of 72 h and 29 timepoints; for M. tuberculosis, images were acquired every 4 h for a duration of 168 h (42 timepoints in total). Imaging one timepoint took ~130 min (1,260 wells with 9 fields of view per well). At the end of image acquisition, imaging data (M. abscessus: up to 11,340 movies and 7.8 TB per experiment; M. tuberculosis: up to 11 TB per experiment) were transferred to the high-performance computing cluster at the University of Basel (sciCORE) for subsequent image and data analysis.
To evaluate ASCT-based time–kill kinetics of M. tuberculosis, we exposed M. tuberculosis to previously published drug combination regimens30,31 at the maximum blood concentration (Cmax) achievable during therapeutic dosing in humans (9 replicates for each condition): bedaquiline 1.1 µg ml−1, clofazimine 1.25 µg ml−1, delamanid 0.5 µg ml−1, ethambutol 4 µg ml−1, isoniazid 4.5 µg ml−1, linezolid 19 µg ml−1, moxifloxacin 4 µg ml−1, pretomanid 7.8 µg ml−1, pyrazinamide 40 µg ml−1, rifampicin 16 µg ml−1, rifapentin 19 µg ml−1, SQ109 0.026 µg ml−1 and sutezolid 1.48 µg ml−1.
For drug tolerance profiling, ASCT was performed on all clinical M. abscessus isolates using a standardized panel of eight antibiotics, regardless of the treatment each patient received. M. abscessus isolates were assessed within a single ASCT plate for a single antibiotic condition in triplicate. The drug concentrations used were based on the MICs for ATCC-19977, determined similar to CLSI criteria71. In contrast to CLSI, they were assessed in 7H9 supplemented with 0.4% glycerol, 10% OADC and 0.05% Tween 80 and quantified using OD600. Growth inhibition at increasing drug concentrations was fitted with a four-parameter log-logistic model using the R drc package73. The MIC was defined as the drug concentration that prevented 90% growth (IC90). Antibiotic time–kill kinetics for all 8 drugs were evaluated at 2 different drug concentrations: a lower concentration (10-fold the MIC of ATCC-19977) and a higher concentration (20-fold the MIC of ATCC-19977). The specific drug concentrations (low and high) tested were as follows: amikacin 26 and 52.1 µg ml−1, azithromycin 63.5 and 127 µg ml−1, cefoxitin 63.1 and 126.2 µg ml−1, imipenem 37.7 and 75.4 µg ml−1, minocycline 67.2 and 134.4 µg ml−1, moxifloxacin 8.5 and 17 µg ml−1, linezolid 38.5 and 77.1 µg ml−1, and tigecycline 27.2 and 54.3 µg ml−1.
Image processing of every image time-lapse (all imaging timepoints of a single field) consists of five individual image analysis steps: background control, cell segmentation, cell classification, drift correction and cell tracking.
To increase the accuracy of fluorescence intensity quantifications, we applied BaSiC, a method that automates background correction through low-rank and sparse decomposition via Fiji19,74. This method adjusts for spatial and temporal variations in background fluorescence, which commonly arise from uneven and repetitive illumination. Employing the default BaSiC settings, we achieved consistent fluorescence signals across different conditions, well positions and time (Extended Data Fig. 1b).
Bacteria were segmented using a combined pixel and object classification approach implemented in ilastik20. In the pixel classification task, each pixel was subjected to supervised random forest classifiers (100 trees) to differentiate between the background and cellular structures. Within the object classification task, the pixel classification map, estimating the probability of each pixel belonging to the background or cellular class, was smoothed and thresholds were applied to generate object features such as intensity statistics and shape descriptors. These features were used to train the following object classifiers: PI+ single cells, PI− single cells and clumps. Pixel and object classifiers were trained on ~30 time-lapses, including different M. abscessus strains and antibiotic conditions. These classifiers were then applied to all data (over 200,000 time-lapses) in batch mode. To assess the fate of PI+ and PI− bacteria upon antibiotic washout, we trained bacteria with ambiguous PI signals as an additional object class. This class was excluded from single-cell growth assessments. To validate segmentation accuracy, 3 randomly selected time-lapse datasets were manually annotated (with automated support) to generate ground-truth pixel prediction maps. The same time-lapses were then processed by ASCT pixel classification. Ground-truth and ASCT pixel classifications were compared using the ImageJ CLIJ2 plugin74. Segmentation accuracy (that is, object overlap) was quantified with the Jaccard Index (Extended Data Fig. 1a). To quantify PI classification accuracy, 1,600 PI-positive and 1,600 PI-negative single cells were manually annotated within 5 timelapse datasets, including different M. abscessus isolates and treatment conditions. The ASCT PI classification algorithm was than compared with manually annotated objects using the caret R package75 (Extended Data Fig. 1c).
To optimize downstream analysis and especially bacterial tracking, we corrected for any drift in imaging fields, which mainly occurred at the beginning of each experiment due to thermal changes (thermal drift). The drift of fields was determined by identifying the maximum cross-correlation between two consecutive images by shifting the subsequent image along the x and y coordinates. Drift correction for large-scale time-lapse data is computationally expensive. Therefore, we used smaller field sections (600 × 600 pixels rather than the 2,400 × 2,400 pixels of the whole field of view) and adapted the process to distinct imaging timepoints (frames), given that the drifts were minor after the initial imaging frames. We extracted a window of 600 × 600 pixels from the binary map of segmented objects in frame 0 (centred at c1 = (800,800)), the very first image of a time-lapse. We also extracted a window of 600 × 600 pixels from frame 2 and shifted this section, one pixel at a time, up to 200 pixels away from c1 in each direction, creating 401 × 401 windows. The windows from frame 1 were overlaid on the frame 0 window to find maximum alignment. Given that cells are not always uniformly distributed or can lose focus, the same strategy was performed with two other windows (c2 = (1,300,1,300) and c2 = (1,700,1,700)). The maximum of the cross-correlation across the 3 locations was used. From frame 3 onward, windows were shifted by fewer pixels to achieve a 75% alignment likelihood between the frames. The shifts were initially set to 25 pixels and consecutively adjusted to 25, 50, 100 and 200 pixels if the alignment likelihood was not achieved.
To automatically follow the behaviours of individual cells during the experiment, we established a custom script to track segmented objects across imaging frames. For each frame, we extracted object features from the segmentation output obtained via brightfield imaging using the MATLAB function ‘regionprops'. Our cell-tracking algorithm aims to link corresponding objects between the initial frame 0 and subsequent frames, with nt denoting an object n at timeframe t. Object homology H between frames was determined by comparing features of individual objects. A graph of linking objects (nt, n0) at frame t was represented by a 3-dimensonal tensor H(n0, n0 + N, t), where N0 is the population of objects at frame 0. The H tensors at each frame were composed of dissociation weights based on the following features: (1) the distances between centroids of objects nt and n0; (2) the changes in the area of objects; (3) changes in object orientation; and (4) changes in the convex hull area of the object. We assigned higher weight to distances (raised to the power of 4) and a weight of one to the other features.
All possible homology values were integrated into an object matrix. Rather than comparing each frame's object to find the closest match, we employed a comprehensive local minimum strategy. The pair of two objects with the highest homology (lowest values in the matrix) was identified as a link in an array and subsequently removed from the matrix. This process was iteratively repeated to identify and remove further links until a homology threshold of 1 × 108 was reached. This local minimum strategy allowed individual objects to be linked throughout the time-lapse to generate single-cell trajectories. Objects of abnormal size, merging objects, moving objects, objects that increased or decreased in object area, objects with swapping labels, or objects that were not tracked for more than two frames within the time-lapse were discarded. Wells with less than 1,000 tracked bacteria for M. abscessus and less than 500 tracked bacteria for M. tuberculosis were discarded. The time of PI positivity of a single cell was defined as the first PI+ frame of two consecutive PI+ frames.
After quantifying the morphology and viability trajectories of each bacterium, the imaging data were further analysed. Bacterial growth in ASCT was defined as an increase in total object area greater than 3.1-fold during antibiotic exposure. This threshold was first defined by comparing growing and non-growing cells and then validated in M. abscessus isolates with and without inducible macrolide resistance during azithromycin exposure (Extended Data Fig. 1d). If growth was detected in two-thirds or more of the replicates for a given isolate–drug condition, the condition was considered growing and removed from the killing analyses.
The reproducibility of M. abscessus time–kill kinetics was assessed by comparing live-cell fractions at distinct timepoints (3, 6, 9, 12, 24, 36, 48, 60 and 72 h). Exact live-cell fractions at given timepoints were interpolated from overall time–kill kinetics. The coefficient of variation (CoV) was calculated for each triplicate at every timepoint. All triplicates were included in downstream analyses if the mean CoV of all 3 replicates fell within 3 s.d. of the overall mean triplicate CoV distribution per drug condition. Triplicates exceeding this threshold were reduced to the best-performing duplicate. These duplicates were retained if their mean duplicate CoV was within 3 s.d. of the overall mean duplicate CoV per drug condition; otherwise, the isolate–drug pair was excluded from further analyses. A minimum of 2 reproducible time–kill curves per isolate–drug condition was required for downstream analyses of M. abscessus kill analyses. To assess for outliers in our M. abscessus drug tolerance assessment, we performed principal component analyses for every antibiotic condition on the basis of live-cell fractions at 3, 6, 9, 12, 24, 36, 48, 60 and 72 h. Isolate–drug conditions with a distance of more than 3 s.d. from the mean, assessed with Mahalanobis distance and calculated using the first 2 principal components, were excluded from further analyses. In total, 957 (14.7%) M. abscessus isolate–drug pairs were excluded from time–kill analysis due to bacterial growth (mostly azithromycin and minocycline) and 156 (2.4%) due to poor reproducibility or being outliers. Due to the larger number of replicates, the strategies to account for reproducibility and outliers were not applied to M. tuberculosis.
Imaging data were acquired consecutively for each well at different timepoints. The live-cell fraction at 0 h (timepoint 0) was extrapolated by fitting a logistic function to the live-cell fractions from the first 5 imaging frames, corresponding to ~13 h (all conditions except imipenem and cefoxitin). Due to the rapid killing observed in imipenem and cefoxitin conditions, resulting in inaccurate fitting, the maximum live-cell fraction observed for each isolate in any of the other drug conditions was used as the isolate-specific live-cell fraction at 0 h. For M. abscessus, isolates with a live-cell fraction below 80% and isolates that had less than 1,000 cells per well across multiple wells were excluded from further analyses. Using the 0-h live-cell fraction, all frames were normalized to an initial live-cell fraction of 100%. Overall, antibiotic killing was quantified as the arithmetic mean of the area under the 72-h (M. abscessus) or 168-h (M. tuberculosis) time–kill curve, ranging from 1 to 0 (maximum survival to most rapid killing, respectively).
We assessed the relationship between M. abscessus drug tolerance phenotypes, growth rates, lag times and MICs using Pearson correlation and R2 values. Spearman correlation was employed to examine the contribution of outliers. For drug clustering in drug tolerance space, a Spearman correlation matrix was generated on the basis of pairwise comparisons of the area under the time–kill curves from 350 clinical M. abscessus isolates. Only isolates with 4 or less missing drug tolerance values (out of 16) were used. Principal component analysis was applied to the correlation matrix to visualize drug clustering.
We also applied the tracking algorithm to identify whether single cells are able to grow (form microcolonies) after antibiotic washout. To achieve tracking accuracy and computational efficacy, we employed a backward approach. We identified and categorized objects with a size 5–15 times the median size of single cells as microcolonies and recorded the area, appearance time and centroids. Analogous to the homology matrix to track single non-growing cells, we used the homology index to track microcolonies backwards to the final single-cell object, that is, the initial originating cell. We focused on two features: the object area, which is important particularly to track large objects, which grow and potentially move; and centroids, which are critical to track single non-moving cells. With this approach, we could determine whether specific objects generated microcolonies.
M. abscessus isolates were cultured on solid media and colony sweeps were collected40,76. DNA extraction was performed using the Qiagen QIAamp DNA mini kit. DNA libraries were constructed with unique identifiers for each isolate and sequenced using multiplexed paired-end sequencing. De novo genome assemblies were assessed for quality. Assemblies with a length longer than 6 Mb, more than 300 contigs, an average depth below 30×, a coverage of the reference genome below 50%, or presumed mixed infection were discarded. Sequence reads were mapped to the M. abscessus ATCC-19977 genome using BWA, followed by INDEL realignment77,78. Single nucleotide polymorphisms (SNPs) and small insertions/deletions (INDELs) were identified using bcftools and annotated with SNPeff79,80. SNPs were filtered to require a minimum base call quality of 50, a minimum mapping quality of 20, and at least 8 matching reads covering an SNP (3 per strand). To assess larger deletions, ATCC-19977 was partitioned into regions of 20 bp with 10 bp overlaps39. The coverage of these regions in clinical isolates was assessed with sambamba81. Large deletions were defined as 2 consecutive windows with a mean coverage of 5× or below, occurring in at least 5% of all genomes. Variants with identical distributions were collapsed into a single variant. Maximum-likelihood trees were generated using FastTree, inferred from core SNPs and visualized with iTOL82,83. Mutational aminoglycoside resistance was evaluated with mutations in the rrs genotype and macrolide resistance with mutations in rrl84,85.
Unitigs for all isolates were extracted using the unitig-caller tool, which utilizes an FM-index built around the Bifrost API51,86. A similarity matrix was generated from phylogenetic distances, which was then used to correct for population structure. Using pyseer, FaST linear mixed models were applied to estimate narrow-sense heritability (h2), representing the proportion of variance in the phenotype attributable to genetic variation50. Random chance was assessed by shuffling each drug tolerance phenotype across M. abscessus isolates and calculating heritability (10 times for each drug tolerance phenotype; 160 times in total).
We performed genome-wide association studies (GWAS) to analyse ~300,000 M. abscessus genetic variants, including SNPs, INDELs and large deletions, in relation to drug tolerance phenotypes. Variants were classified by presumed genetic effects: low effect (intergenic variants, synonymous SNPs), moderate effect (non-synonymous SNPs, inframe INDELs) and high effect (frameshift variants, start/stop alterations, large deletions). We applied linear mixed models to account for population structure, integrating a relatedness matrix, and quantified associations using the Wald test39,87. We used a Bonferroni threshold of 1.7 × 10−7 to control for multiple hypothesis testing. To summarize GWAS hits, we extracted the top 5 genes showing the strongest association per phenotype (for moderate- or high-effect variants). The top associations of these genes with all tolerance phenotypes were then shown in a heat map (moderate- or high-effect variants). Associations were plotted using LocusZoom88.
M. abscessus ATCC-19977 was transformed with pUV15tetORm (Addgene, plasmid 17975; gift from Sabine Ehrt) to generate a reporter strain expressing GFP under the control of the tetracycline-inducible promoter Pmyc1tetO89. Broth cultures were grown in Middlebrook 7H9 supplemented with hygromycin (1 mg ml−1) until mid-log phase, after which hygromycin was removed. Single-cell suspensions were generated as described for ASCT and treated for 12 h in non-shaking culture conditions with amikacin (26 µg ml−1), cefoxitin (63.1 µg ml−1) or moxifloxacin (8.5 µg ml−1). Following treatment, antibiotics were washed off and bacteria were transferred to the ASCT platform containing 2 µg ml−1 anhydrotetracycline to induce GFP expression. Brightfield and fluorescence images (PI and GFP) were acquired for 12 h. An untreated, induced control sample was used for GFP and PI gating. For each channel, log10-transformed fluorescence intensities were fitted with a 2-component Gaussian mixture model using the Mclust function. Cell counts and proportions were determined per treatment and timepoint. Data were visualized as hexbin density plots (100 bins) of PI versus GFP intensity with overlaid thresholds.
To assess M. tuberculosis drug combinations, 13 drugs were combined in every pairwise combination using 9 × 9 checkerboards with log√2-fold dilution steps. Each condition was assessed using 4 technical replicates. Drugs were diluted in 7H9 medium, transferred to 384-well plates and inoculated with ~1.2 × 105 c.f.u.s of the H37Ra strain per well. Plates were sealed and incubated at 37 °C with shaking at 250 r.p.m. After 21 days, OD600 was assessed and the bacterial fitness was determined as the background-corrected OD600 divided by the median OD600 of all growth control wells per plate. Single drugs and pairwise dose–response curves were fitted with a 4-parametric log-logistic model (drc R package). Pairwise interaction scores were determined using only the equipotent concentrations90 and further used to predict high-order interactions38. Bliss interaction scores of high-order combinations were determined by computing the deviation between the predicted high-order curve (derived from measured pairwise interactions) and the bliss-independent expectation of the single-drug effects. Deviations were expressed as log2 interaction strengths. To evaluate the combined predictive performance of interaction and MIC parameters (median MIC), a logistic regression model was implemented using the glm function in R. Models predicted binary outcomes (similar or better than SOC) in relapsing and bactericidal mouse models. Predictive performance was evaluated across 50 bootstrap iterations (randomly sampling 80% of the conditions), with mean ROC-AUC values assigned to each predictor. Statistical significance was evaluated using a z-test against the null hypothesis of random performance (AUC = 0.5).
As an alternative proxy of antibiotic killing, we measured the reduction of resazurin to resorufin in M. tuberculosis mc27000 across all drug regimens. M. tuberculosis growth and starvation conditions were prepared as described for ASCT. Approximately 5 × 106 bacteria suspended in 49.5 µl 7H9 were dispensed per well into clear, flat-bottom 384-well plates and exposed to 0.5 µl drug solution. Drug regimens and concentrations were identical to those tested in ASCT. Each condition was tested in quadruplicate and included medium only and untreated bacterial controls. Plates were sealed with parafilm and incubated at 37 °C in a non-shaking incubator. At days 0, 7 and 14, selected plates were removed and 5 µl of a 0.13% resazurin stock solution was added to each well. After further incubation at 37 °C for 12 h, plates were briefly opened to reduce condensation before fluorescence was measured (excitation 560 nm, emission 590 nm) on a Synergy H1 plate reader (BioTek). Mean relative fluorescence units (RFUs) were background corrected. Antibiotic killing was quantified as the RFU change between days 0 and 14, which was then compared with ASCT-derived killing and in vivo outcomes.
MAB_0233 knockout mutants (ΔMAB_0233) were generated on the M. abscessus ATCC-19977 background using ORBIT54. pKM444 (RecT-Int-expressing plasmid, Kanr) and pKM496 (for gene deletion by integration into target gene, Zeor) were gifted by Kenan Murphy (Addgene, plasmid 108319 and 109301)54. pMV261 (replicative vector for gene complementation) was obtained from NovoPro Biosciences91. To amplify plasmids, Escherichia coli DH5α cells were grown on LB agar plates supplemented with 50 μg ml−1 kanamycin (pKM444 or pMV261) or 50 μg ml−1 zeocin (pKM496).
Liquid cultures of M. abscessus ATCC-19977 were incubated at 37 °C and 150 r.p.m. for 2 days. Cells were diluted to an OD600 of 0.03 in 20 ml of Middlebrook 7H9, supplemented with 0.4% glycerol, 10% OADC and 0.05% Tween 80 in a 250 ml baffled flask. The culture was placed at 30 °C and 100 r.p.m. for 18–20 h. The culture was then washed 3× with 20 ml ice-cold sterile 10% glycerol supplemented with 0.05% Tween 80 (washing solution). Following the third wash, the cells were collected by centrifugation and resuspended in 200 μl of washing solution. The ORBIT plasmid pKM444 (200 ng) was mixed with competent M. abscessus and rested on ice for 5 min. The cells were electroporated (2.5 kV, 1,000 Ω and 25 μF) using a 2-mm-gap-width electroporation cuvette. Following electroporation, the cells were resuspended in 1.5 ml Middlebrook 7H9 medium and placed at 30 °C and 100 r.p.m. for 18–20 h. The recovered cells were pelleted at 3,000 ɡ (25 °C, 5 min), resuspended in 100 µl Middlebrook 7H9 medium and plated on 7H11 plates supplemented with 0.5% glycerol, 10% OADC and 250 μg ml−1 kanamycin. The plates were incubated at 30 °C for 3–4 days.
Colonies resulting from pKM444 transformation were picked and grown on Middlebrook 7H11 agar plates supplemented with 0.5% glycerol, 10% OADC and 250 μg ml−1 kanamycin. Cells were collected by centrifugation and resuspended in TRIzol (Thermo Fisher). Cell disruption was performed by bead beating using Lysing Matrix B beads (200 µl) in a FastPrep-24 Classic instrument at 6.5 m s−1 for 60 s, followed by 60 s incubation on ice. This process was repeated for 3 cycles. Total DNA was extracted using the DNA miniprep kit (Zymo Research). The presence of the pKM444 plasmid was verified by PCR, using DreamTaq DNA polymerase (Thermo Fisher). Primers specific to the plasmid (forward: CACGTTGTGTCTCAAA ATCTC; reverse: CGATAACGTTCTCGGCTC) were used to amplify a target segment of the plasmid. PCR products were resolved by agarose gel electrophoresis, and bands of the expected size were excised and submitted for Sanger sequencing to verify plasmid presence.
The oligonucleotide sequence consists of the 48 bp Bxb1 attP site (or the reverse complement) flanked by 60 bp upstream of the initiation codon and 60 bp downstream of the stop codon54. The targeting oligonucleotide (sequence for MAB_0233: CGGCCACATGTTTTGTGCCGCT AGGGGAAATCAGCTCGGCATCGTCCGTGTGCCGTTGTTGGTTTGTACCGTACACCACTGAGACCGCGGTGGTTGACCAGACAAACCCACATACCCTGATGCGAGTTCAACTGCCATCTGTTGCCCCCTTCGGGCAATCGGTGCAGC) was acquired from IDT with 4 nmole Ultramer DNA Oligo property and diluted in nuclease-free water to 1 μg μl−1.
M. abscessus ATCC-19977, harbouring the pKM444 plasmid, was cultured in Middlebrook 7H9 medium with 5 µg ml−1 anhydrotetracycline (ATc). A 20 ml culture was prepared in a 250 ml baffled flask wrapped in aluminum, washed as described above and resuspended in 200 μl washing solution. Oligonucleotide (2 μl, 1 μg μl−1) guiding the payload plasmid pKM496 to the target gene was aliquoted into 1.5 ml microcentrifuge tubes and denatured at 95 °C for 5 min to prevent secondary structure formation. The denatured oligonucleotides were cooled on ice for at least 5 min before adding 200 ng pKM496 (ref. 54). Subsequently, 200 μl of cells were added to the tube, gently mixed by pipetting and incubated for 5 min. Electroporation, recovery and plating were performed as described above, except that recovered cells were plated on 7H11 plates supplemented with 100 μg ml−1 zeocin. DNA extraction and PCR were performed as described using primers flanking MAB_0233 and pKM496 (forward: CGCTCACAACTGAATACCC; reverse: CCTGGTATCTTTATAGTCCTGTC).
To complement ΔMAB_0233, the gene was amplified by PCR (KAPA HiFi, high-fidelity enzyme) and cloned into pMV261 downstream of the mycobacterial strong constitutive promoter, pHSP60 (ref. 91). In addition, the T2 terminating region from E. coli rrnB was added to the end of the open reading frame (ORF) as an efficient terminator of transcription. To eliminate the pKM444 plasmid, which confers kanamycin resistance, the M. abscessus knockout strain ΔMAB_0233 was subjected to serial dilutions and growth cycles until cured. Cured strains were transformed with pMV261 following the same transformation protocol as described for pKM444.
As control, MAB-ATCC-pKM444 was used. The RNA of log phase cultures of the control strain, ΔMAB_0233 and ΔMAB_0233::MAB_0233 was isolated using the RNA miniprep kit (Zymo Research). Complementary (c)DNA was prepared using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). cDNA levels were quantified by quantitative real-time PCR (qPCR) on an Applied Biosystems qPCR machine using a PowerUp SYBR Green Master Mix (Thermo Fisher) and analysed by the ΔΔCt method. Gene expression was controlled using the housekeeping genes MAB_3009 (sigA) and MAB_ 3869c. Two sets of primers covering ~150 bp sequences of the beginning and the end of the MAB_0233 ORF were designed (primer_1_FWD: GCGAAGCCTTCGCCAAAGCTC, primer_1_REV: CCTCGTTGAGCTTTTCCAGCGC, primer_2_FWD: CGCAACGTCTCCGATGGGAAACC, primer_2_REV: GAGTTGAGCGGCGTCCATGC).
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Whole-genome sequence data are available at the European Nucleotide Archive (ENA). Representative images to test ASCT are available on Zenodo at https://doi.org/10.5281/zenodo.17232777 (ref. 92). The full raw imaging dataset is not publicly deposited due to size constraints but is available from the lead contact upon request. All downstream imaging data and other data are available in source data.
All original code has been deposited on GitHub at https://github.com/BoeckLab/ASCT and archived at Zenodo under https://doi.org/10.5281/zenodo.17775709 (ref. 93).
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We thank B. Schulthess and P. Sander for training in MIC assessments; A. Trauner, D. Pinschewer, M. Zampieri, A. Harms, D. Portevin, B. Aldridge, D. Bumann and U. Jenal for expert input; the scientific computing center at the University of Basel (sciCORE) and specifically R. M. Cabezón, I. M. de Ilarduya, G. Losilla and M. Jacquot for computational support; J. Sollier for critical reading and editing of the paper; the DBM microscopy core facility; and M. Roth and M. Tamm for lab space. Icons were obtained from PhyloPic (http://phylopic.org). L.B. received funding from the Swiss National Science Foundation (grant nos 177799, 185792, 215557), the Bangerter–Rhyner Foundation, Goldschmidt Jacobson Foundation, Helmut Horten Foundation, Swiss Society for Pneumology, Cloëtta Foundation, and NCCR AntiResist, a National Center of Competence in Research, funded by the Swiss National Science Foundation (grant no. 180541). R.A.F and D.M.G. received funding from the Wellcome Discovery award 226602/Z/22/Z and LifeArc/CF Trust Innovation Hub THUB01.
These authors contributed equally: Alexander Jovanovic, Frederick K. Bright, Ahmad Sadeghi.
Department of Biomedicine, University of Basel, Basel, Switzerland
Alexander Jovanovic, Frederick K. Bright, Ahmad Sadeghi, Basil Wicki, Santiago E. Caño Muñiz, Greta C. Giannini, Sara Toprak, Loïc Sauteur, Anna Rodoni, Andreas Wüst, Philippe Dehio, Michael Abanto & Lucas Boeck
Department of Medicine, McGill University, McGill International TB Centre, Montreal, Quebec, Canada
Andréanne Lupien
Swiss Tropical and Public Health Institute, Allschwil, Switzerland
Sonia Borrell & Sebastien Gagneux
University of Basel, Basel, Switzerland
Sonia Borrell & Sebastien Gagneux
Cambridge Centre for Lung Infection, Royal Papworth Hospital, Cambridge, UK
Dorothy M. Grogono & R. Andres Floto
Institute of Microbiology and Infection, University of Birmingham, Birmingham, UK
Nicole E. Wheeler
Department of Infectious Diseases and Hospital Epidemiology, University Hospital Zürich, Zurich, Switzerland
Johannes Nemeth
Pulmonary Medicine, University Hospital Basel, Basel, Switzerland
Hans Pargger & Lucas Boeck
Gallipoli Medical Research, Greenslopes Private Hospital, Brisbane, Queensland, Australia
Rachel Thomson
Greenslopes Clinical School, Faculty of Health, Medicine and Behavioural Sciences, The University of Queensland, Brisbane, Queensland, Australia
Rachel Thomson
School of Medicine and Dentistry, Griffith University, Southport, Queensland, Australia
Scott C. Bell
Gold Coast University Hospital, Southport, Queensland, Australia
Scott C. Bell
Wellcome Sanger Institute, Hinxton, UK
Josephine M. Bryant
Helmholtz Munich – German Research Center for Environment and Health, Munich, Germany
Tingying Peng
TASK, Cape Town, South Africa
Andreas H. Diacon
Molecular Immunity Unit, University of Cambridge Department of Medicine, MRC Laboratory of Molecular Biology, Cambridge, UK
R. Andres Floto
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L.B. conceptualized the project. A.J. and L.B. curated data. A.S., A.J. and L.B. conducted formal analysis. L.B. acquired funding. A.S., A.J., F.K.B., B.W., S.E.C.M., G.C.G., S.T., L.S., A.R., A.W., A.L., S.B. and L.B. conducted investigations. A.S., A.J., F.K.B. and L.B. designed the methodology. L.B. administered and supervised the project. A.S., A.J., T.P. and L.B. designed software. D.M.G., N.E.W., P.D., J.N., H.P., R.T., S.C.B., S.G., J.M.B., A.H.D., R.A.F., M.A. and L.B. procured resources. A.S., A.J., F.K.B. and L.B. performed validation. A.S., A.J., S.E.C.M. and L.B. performed visualization. L.B. wrote the original paper draft. A.S., A.J., F.K.B., B.W., S.E.C.M., G.C.G., S.T., L.S., A.R., A.W., A.L., S.B., D.M.G., N.E.W., P.D., J.N., H.P., R.T., S.C.B., S.G., J.M.B., T.P., A.H.D., R.A.F., M.A. and L.B. reviewed and edited the paper.
Correspondence to
Lucas Boeck.
The authors declare no competing interests.
Nature Microbiology thanks Minsu Kim and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
(a) Overlay of manually annotated “ground truth” bacterial segmentation with automated object segmentation (pixel classification) of M. abscessus brightfield images. The Jaccard index quantifies overlay accuracy across imaging time points during time-lapse acquisition of 5,887 bacteria. Boxplots show the median, interquartile range, and data range within 1.5 x IQR (central line, box, and whiskers, respectively) of Jaccard indices. The dashed line indicates high segmentation accuracy (Jaccard index of 0.8). (b) Changes in dynamic range following BaSiC background correction. Mean PI values per single M. abscessus bacterium before and after correction over 72-hour time-lapses (first imaging time point: blue, last time point: red) shown in density plots. (c) Accuracy of automated PI classification in predicting “ground-truth” classes of PI-positive and PI-negative single cells. (d) Changes in single-cell count (which declines during growth) and object area (assessed with ASCT) over 72 hours under azithromycin treatment across 406 M. abscessus isolates. Isolates with (red) and without (black) inducible macrolide resistance are highlighted. The dotted line indicates the ASCT growth threshold. (e) Number of single M. abscessus bacteria tracked over 72 hours per well within 1536-plates. Data from 406 M. abscessus isolates, and about 130 million tracked bacteria across eight drugs and two concentrations (data from Fig. 3). (f) Fraction of viable M. abscessus immobilised in ASCT under nutrient-rich and starvation conditions without antibiotic exposure (dividing and PI-negative cells were considered viable). Yellow dots indicate the fraction of PI-negative bacteria in starved broth culture at 24 and 48 hours (no single-cell tracking). Effect of different (g) gel pad volumes and (h) gel pad agarose concentrations on M. abscessus time-kill kinetics.
Source Data
(a) Fraction of bacteria (including numbers) showing regrowth after 24-hour antibiotic treatment and antibiotic washout across PI-negative and PI-positive bacteria. The single regrowing PI-positive bacterium is highlighted in the insert. (b-c) Induced GFP expression after antibiotic washout. (b) Image gating strategy for GFP and PI in untreated M. abscessus controls. (c) GFP and PI fluorescence intensities of M. abscessus populations treated with amikacin (AMK), cefoxitin (FOX), or moxifloxacin (MXF) following antibiotic removal. Time (hours) indicates imaging time points, with 0 set to the start of imaging ( ~ 1 hour after GFP induction).
Source Data
(a) ASCT-based time-kill kinetics of growing M. tuberculosis (H37Ra) exposed to 65 drug regimens, with the following regimens highlighted: isoniazid-rifampicin (HR; dark blue), isoniazid-rifampicin-ethambutol-pyrazinamide (HREZ; light blue), bedaquiline-pretomanid-linezolid (BPaL; red), and bedaquiline-pretomanid-pyrazinamide (BPaZ; purple). (b) Mean area under the kill curve for M. tuberculosis drug regimens under growth conditions, averaged across the two M. tuberculosis strains (H37Ra and mc27000). Each drug regimen was tested in nine technical replicates (poor-quality curves were excluded), and data are shown as mean ± SEM. (c-d) Killing dynamics (AUCs) of M. tuberculosis drug combinations were assessed in regard to the presence and absence of individual drugs. Drugs that increased killing under (c) growth conditions (averaged from mc27000 and H37Ra strains) or (d) starvation conditions (averaged from three starvation conditions) are shown. Each condition was tested in nine technical replicates each (poor-quality curves were excluded). Groups were compared using a two-sided Mann-Whitney U test. Each dot indicates a drug regimen. Boxplots show the median, interquartile range, and total range (central line, box, and whiskers, respectively) of AUC values.
(a) Time-kill curves (AUCs) upon growth and starvation conditions of two M. tuberculosis isolates were used to compare similar-to-SOC versus better-than-SOC classifications, based on outcomes in relapsing mouse models (RMM), bactericidal mouse models (BMM) of common mouse strains, the granulomatous C3HeB/FeJ strain and clinical studies30,31. Comparisons were performed using a two-sided Mann-Whitney U test (P values indicated). Each dot represents a drug regimen. Boxplots show the median, interquartile range and total range (central line, box, and whiskers, respectively) of AUC values. (RMM: n = 46, BMM common strains: n = 48, BMM C3HeB/FeJ: n = 15, clinical bactericidal activity: n = 14). PA indicates pantothenate, PBS phosphate-buffered saline. (b) Performance of time-kill kinetics (AUC averaged across three M. tuberculosis starvation models) for predicting in vivo M. tuberculosis outcomes (similar-to-SOC versus better-than-SOC drug regimens). Using logistic regression, fifty ROC curves were generated for every condition by randomly selecting 80% of the samples. The brown line represents the mean receiver operating characteristic (ROC) curve, the shaded area one standard deviation. Mean area under the ROC curves and 95% confidence intervals are presented.
(a) Colony forming unit (CFU) change of PBS-starved M. tuberculosis mc27000 during drug treatment. 65 drug regimens were tested. (b) Correlation of CFU-based killing of 65 drug regimens with ASCT-based time-kill kinetics, both assessed in the PBS-starved M. tuberculosis mc27000 strain and quantified with two-sided Pearson correlation. (c) CFU-based killing of PBS-starved M. tuberculosis mc27000 at day 3 and 7 was used to compare similar-to-SOC versus better-than-SOC classifications, based on outcomes in relapsing mouse models (RMM), bactericidal mouse models (BMM) of common mouse strains, the granulomatous C3HeB/FeJ strain and clinical studies30,31. Comparisons were performed using a two-sided Mann-Whitney U test (P values indicated). Each dot represents a drug regimen. Boxplots show the median, interquartile range and total range (central line, box, and whiskers, respectively; RMM: n = 46, BMM common strains: n = 48, BMM C3HeB/FeJ: n = 15, clinical bactericidal activity: n = 14).
Source Data
The minimum MIC or median MIC of all drugs within each combination, or the mean MIC of the two most potent drugs (mean*), was assessed and compared between similar-to-SOC and better-than-SOC, based on outcomes in relapsing mouse models (RMM), bactericidal mouse models (BMM) of common mouse strains, the granulomatous C3HeB/FeJ strain and clinical studies30,31. Comparisons were performed using a two-sided Mann-Whitney U test (P values indicated). Each dot represents a drug regimen. Boxplots show the median, interquartile range and total range (central line, box, and whiskers, respectively) of MIC values (RMM: n = 46, BMM common strains: n = 48, BMM C3HeB/FeJ: n = 15, clinical bactericidal activity: n = 14).
(a) Pairwise M. tuberculosis drug-drug interactions of 13 x 12 drugs, each performed in 9x9 checkerboards, highlighting expected and observed growth inhibition. Pairwise interactions are quantified by the Loewe interaction score. (b) Measured pairwise and predicted high-order drug interactions of 52 M. tuberculosis drug regimens. B indicates bedaquiline, C clofazimine, D delamanid, E ethambutol, H isoniazid, L linezolid, M moxifloxacin, Pa pretomanid, Z pyrazinamide, R rifampicin, P rifapentine, Sq SQ109 and Su sutezolid. (c) Drug interactions were compared across similar-to-SOC versus better-than-SOC classifications, based on outcomes in relapsing mouse models (RMM), bactericidal mouse models (BMM) of common mouse strains, the granulomatous C3HeB/FeJ strain and clinical studies30,31. Comparisons were performed using a two-sided Mann-Whitney U test (P values indicated). Boxplots show the median, interquartile range and total range (central line, box, and whiskers, respectively; RMM: n = 46, BMM common strains: n = 48, BMM C3HeB/FeJ: n = 15, clinical bactericidal activity: n = 14). (d) The performance of combined MIC (median regimen MIC) and drug interaction measures for predicting in vivo outcomes was assessed using logistic regression models. Fifty ROC curves were generated for every condition by randomly selecting 80% of the samples. The black line represents the mean receiver operating characteristic (ROC) curve, the shaded area one standard deviation. Mean area under the ROC curves and 95% confidence intervals are presented.
Source Data
(a) Pearson correlation of M. abscessus drug resistance and tolerance phenotypes. Correlation strength is indicated be circle size, correlation direction by colour. White central dots highlight significant correlations (two-sided Pearson test, p < 0.05). AUC indicates the area under the kill curve, MIC minimum inhibitory concentration, AMK amikacin, AZM azithromycin, FOX cefoxitin, IPM imipenem, LZD linezolid, MIN minocycline, MXF moxifloxacin, TGC tigecycline, L refers to low drug concentration, and H to high drug concentration. (b) M. abscessus phylogenetic trees (maximum-likelihood trees) aligned with drug tolerance heatmaps.
(a) MIC distributions of 229 clinical isolates and the frequency of isolates associated with cleared (n = 46) and persisting infection (n = 89). (b) Odds ratios of M. abscessus minimum inhibitory concentrations for predicting treatment failures (lack of culture conversion) in 135 patients. Odds ratios of individual MIC measures were assessed with logistic regression (clarithromycin: P = 0.0019; clarithromycin inducible: P = 0.0045). Each dot represents the odds ratio, and whiskers highlight the 95% confidence interval. ** indicates the two-sided P < 0.01.
Comparison of drug tolerance between M. abscessus isolates from patients with poor versus favourable clinical outcomes (n = 135, excluding growing isolates), using a two-sided Mann-Whitney U test (P indicated). Boxplots show the median, interquartile range and total range (central line, box, and whiskers, respectively) of AUC values. L refers to low drug concentration, and H to high drug concentration.
Supplementary Figs. 1–3 and Tables 1–4.
Automated image analysis in Antimicrobial Single-Cell Testing. Raw brightfield images (cell morphology; 100x objective) and fluorescence images (quantifying propidium iodide [PI] accumulation) are processed to segment cells (lines indicate cell borders), classify bacterial viability (white: viable; red: dead) and track individual cells (track indicated by colours). The video shows brightfield / fluorescence images of M. abscessus exposed to imipenem (high concentration), as well as cell segmentation, classification and tracking over 72 hours.
Growth of propidium iodide negative bacteria after antibiotic washout. M. abscessus ATCC-19977 was treated for 24 hours with imipenem (low concentration), washed and then imaged (100x objective) without antibiotic treatment for 24 hours.
Supplementary data.
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Jovanovic, A., Bright, F.K., Sadeghi, A. et al. Large-scale testing of antimicrobial lethality at single-cell resolution predicts mycobacterial infection outcomes.
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SLFN11 is epigenetically silenced and confers chemoresistance in half of all cancers. In response to replication stress, SLFN11 triggers translation shutdown and p53-independent apoptosis, but how DNA damage activates SLFN11 remains unclear. Here through CRISPR-based screens we implicate SLFN11 as the critical determinant of cisplatin sensitivity in cells lacking primase–polymerase (PrimPol)-mediated repriming. SLFN11 and the downstream integrated stress response uniquely promote cisplatin-driven apoptosis in PrimPol-deficient cells. We demonstrate that replication protein A (RPA) exhaustion and single-stranded DNA exposure trigger SLFN11 activation and cell death when PrimPol is inactivated. We further identify the USP1–WDR48 deubiquitinase complex as a positive modulator of SLFN11 activation in PrimPol-deficient cells, revealing an addiction to the Fanconi anaemia pathway to resolve cisplatin lesions. Finally, we demonstrate that rapid RPA exhaustion on chemical inhibition of DNA polymerase α activates SLFN11-dependent cell death. Together, our results implicate RPA exhaustion as a general mechanism to activate SLFN11 in response to heightened replication stress.
DNA replication is constantly challenged by numerous endogenous and exogenous sources of DNA damage yet is an inherently accurate and efficient process1,2,3,4. Replication stress invoked by DNA lesions is sensed by the ataxia telangiectasia and Rad3-related (ATR)-dependent intra-S-phase checkpoint, which is activated by the accumulation of RPA-bound single-stranded (ss)DNA at stalled forks and triggers a cascade of events that regulate origin firing, replication fork stabilization and lesion repair or bypass5,6,7,8. Indeed, multiple interdependent DNA damage, repair and tolerance pathways recognize, respond to and repair impediments at stalled replication forks. Recent work also uncovered the phenomenon of RPA exhaustion at stalled forks during heightened replication stress, which leads to exposure of ssDNA, fork breakage and irreversible replication catastrophe9,10. How cells respond to RPA exhaustion independently of ATR signalling is currently unknown.
Owing to their inherent plasticity, stalled replication forks can be remodelled or components of the replisome modified to differentially recruit factors required to cope with specific types of fork blockages2,11. Stalled replication forks can also be directly restarted by DNA primer synthesis downstream of fork blockages via the primase–polymerase PrimPol12,13,14,15,16,17. In vitro, PrimPol-mediated repriming can occur in as little as 14 nt downstream of a replication-blocking lesion15, which could limit ssDNA accumulation at stalled forks caused by helicase–polymerase uncoupling18,19. PrimPol reprimes downstream of diverse sources of replication fork blockage ranging from endogenous G-quadruplexes and R-loops to chain-terminating nucleotides, bulky base adducts induced by ultraviolet light, benzo[a]pyrene-diol-epoxide (BPDE) and interstrand and intrastrand crosslinks induced by mitomycin C (MMC) and cisplatin14,15,18,20,21,22,23,24,25,26,27.
Cisplatin is a commonly used chemotherapeutic that targets rapidly dividing cancer cells by creating intrastrand and interstrand DNA crosslinks that stall active replication forks28. The major pathway responsible for repairing cisplatin-induced interstrand crosslinks is the Fanconi anaemia (FA) pathway, which comprises over 20 gene products, many of which are shared with homologous recombination (HR) and translesion DNA synthesis (TLS)29. Initiation of the FA pathway depends on recruitment of the FA core complex comprising 14 proteins, followed by ATR-dependent phosphorylation and FANCL-dependent ubiquitination of the FANCD2–FANCI complex30,31. FANCD2–FANCI ubiquitination (and FA pathway activation) is tightly regulated by the deubiquitinase (DUB) complex USP1–WDR48 (also known as USP1–UAF1) (ref. 32). Intriguingly, a recent study proposed a potential role for PrimPol repriming in an interstrand crosslink tolerance pathway25. Paradoxically, despite evidence that PrimPol can reprime synthesis downstream of cisplatin DNA lesions in cells, loss of PrimPol alone does not broadly confer sensitivity to cisplatin14,20,22,33. A satisfactory explanation for this discrepancy is currently lacking and highlights the current challenge in understanding how PrimPol activity cooperates with other pathways at stalled forks.
Innate and acquired resistance to cisplatin occurs frequently in patients through various mechanisms, including increased drug efflux, increased DNA repair capacity and loss of proapoptotic pathways34,35. Indeed, previous work demonstrated a role for increased expression of PrimPol in the adaptive response to cisplatin treatment in BRCA-deficient tumours20. One of the strongest prognostic markers of cisplatin efficacy is the expression of Schlafen 11 (SLFN11), a transfer (t)RNA nuclease that induces p53-independent apoptosis in response to DNA damage36,37,38,39,40,41. Intriguingly, SLFN11 is epigenetically silenced in half of all treatment-naive cancer cell lines, leading to chemoresistance42,43,44. SLFN11 induces irreversible replication fork arrest through a range of proposed mechanisms, including prevention of origin firing via CDT1 degradation, increasing local chromatin accessibility and inhibition of HR or checkpoint responses37,45,46,47. After fork arrest, SLFN11 cleaves type II tRNAs, leading to ribosome stalling at rare UUA leucine codons38,48,49. Ribosome stalling results in activation of the general control non-derepressible 2 (GCN2)-dependent integrated stress response (ISR) and JNK-mediated ribotoxic stress response, triggering p53-independent apoptosis38,39. Importantly, the ssDNA-binding and tRNA nuclease activities of SLFN11 are absolutely required for activation of p53-independent apoptosis38. However, despite understanding how SLFN11 activation triggers apoptosis, the DNA lesion that activates SLFN11 remains unknown.
Here we sought to identify drivers of cisplatin cytotoxicity using the chronic myelogenous leukaemia eHAP cell line as a model system. Surprisingly, our targeted CRISPR–Cas9 screens identified loss of PrimPol as conferring cisplatin sensitivity. In contrast to FA, HR and TLS deficiencies, cisplatin sensitivity of PrimPol knockout (KO) cells uniquely depends on SLFN11. We show that cells deficient in PrimPol-mediated repriming or drugs that induce ssDNA accumulation at replication forks potently activate an SLFN11-dependent and GCN2-dependent cell death. Using PrimPol deficiency as a model system to study SLFN11 activation, we identified RPA exhaustion as the primary mechanism of SLFN11 activation in response to DNA damage. Furthermore, we implicated USP1-dependent downregulation of the FA pathway as the mechanism that induces RPA exhaustion and SLFN11-dependent cell death, specifically when PrimPol is inactivated. Finally, we identified DNA polymerase α inhibition as a potent inducer of RPA exhaustion and SLFN11-dependent cell death. We proposed that, through its activation by RPA exhaustion, SLFN11 maintains genome stability by triggering the elimination of cells experiencing heightened replication stress, a hallmark of early cancer cells.
To investigate the pathways required for cisplatin lesion metabolism at stalled replication forks, we performed a targeted CRISPR–Cas9 dropout screen in the diploid inducible Cas9 (iCas9) eHAP cell line (Fig. 1a). Two single guide (sg)RNA libraries (Supplementary Table 1) were utilized to interrogate genes associated with chromatin and telomere maintenance (pool 1) and the DNA damage response (DDR; pool 2). To determine the efficacy of our screens, we first compared sgRNA counts in wild-type (WT) cells infected with pool 2 treated with cisplatin to an untreated condition using the MaGeCK algorithm50 (Fig. 1b). As observed previously33, sgRNAs targeting genes belonging to the FA, nucleotide excision repair, TLS, DNA end-resection and HR pathways resulted in marked sensitivity to cisplatin (Fig. 1b). Surprisingly, we also identified loss of PrimPol, the primase–polymerase that catalyses repriming downstream of leading strand fork blockages, as also conferring sensitivity to cisplatin. This contrasts with previously published reports suggesting that loss of PrimPol alone does not confer sensitivity to cisplatin, despite its well-established role in DNA damage tolerance14,20,22,33.
a, An experimental scheme depicting how targeted CRISPR–Cas9 screens performed. Two separate screens were performed in biological triplicates utilizing pool 1 sgRNA library (1,117 genes targeted) or pool 2 sgRNA library (288 genes targeted). b, A volcano plot depicting a measure of statistical significance (−log10(MaGeCK score)) plotted against log2(fold-change) in abundance of sgRNAs targeting indicated genes in cisplatin-treated versus untreated conditions in eHAP iCas9 WT cells infected with the pool 2 sgRNA library. Labelled genes are coloured based on the DDR pathway in which they operate. c, A Venn diagram comparing genes scoring as significantly depleted in cisplatin-treated arms of the screen depicted in b performed in eHAP cells (yellow) and those in a genome-wide CRISPR–Cas9 dropout screen performed in RPE-1 p53 KO cells (blue) in ref. 33. d, A western blot depicting loss of PrimPol protein in an isogenic eHAP iCas9 PrimPol KO clone. e, Population doublings of eHAP iCas9 WT (grey) and PrimPol KO (red) cells plotted against time to demonstrate cell growth in the absence (squares) or presence of 450 nM (circles) or 550 nM (triangles) cisplatin. Growth curve experiments were performed in n = 2 biological replicates. Data are presented as means. f, Experimental scheme for a Cell Titer Glo viability assay to determine the cisplatin sensitivity of eHAP iCas9 WT or PrimPol KO cells complemented with WT PrimPol, PrimPol AxA (catalytic mutant Asp114Ala, Glu116Ala) or PrimPol CH (primase mutant Cys419Gly, His426Tyr). g, A western blot depicting PrimPol protein levels in eHAP iCas9 WT cells or PrimPol KO cells complemented with WT PrimPol, PrimPol AxA or PrimPol CH mutants. Exogenous protein levels were downregulated using the misFIT expression tuner system. h, Cell viability measured using the Cell Titer Glo assay on treatment of the indicated doses of cisplatin over 5 d. These experiments were performed in n = 4 biological replicates. Data are presented as mean ± s.d. i, Experimental scheme for a Cell Titer Glo assay to determine cell viability of eHAP iCas9 WT or PrimPol KO cells challenged with the indicated drugs. j, Cell viability measured after challenging eHAP iCas9 WT or PrimPol KO cells with the indicated doses of BPDE. Experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. k, Cell viability measured after challenging eHAP iCas9 WT or PrimPol KO cells with the indicated doses of MMC. Experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. l, A heatmap depicting sensitivity of eHAP iCas9 PrimPol KO versus WT cells to various DNA-damaging agents and inhibitors to DDR proteins. Sensitivities were determined by computing the log2(fold-change) between half-maximal inhibitory concentration (IC50) values of PrimPol KO and WT cells. Source numerical data and unprocessed blots are available in the source data. ATMi, ataxia-telangiectasia mutated (ATM) inhibitor; ATRi, ATR inhibitor; FC, fold-change; HU, hydroxyurea; MMS, methyl methane sulfonate; Rad51i, Rad51 inhibitor; TS, template switching.
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Comparing the dropout hits in our screen with those from previous screens in the RPE-1 p53 KO cell line33, we obtained three unique hits: (1) PrimPol, (2) the E2 ubiquitin ligase UBE2A and (3) the E2 ubiquitin ligase UBE2N (Fig. 1c). As UBE2A and UBE2N have well-established roles in protecting against cisplatin cytotoxicity51,52, we focused on understanding why loss of PrimPol confers cisplatin sensitivity specifically in eHAP cells. To this end, we generated an isogenic PrimPol KO clone in the eHAP iCas9 cell line, which retained doxycycline-induced Cas9 cutting as measured by a flow cytometry-based assay (Fig. 1d and Extended Data Fig. 1a–d). We validated our screen results by challenging eHAP WT and PrimPol KO cells with cisplatin in a growth curve experiment. PrimPol KO cells exhibited a marked loss in cell fitness when challenged with cisplatin compared with WT cells (Fig. 1e).
To ensure that this phenotype was specific to loss of PrimPol and not due to off-target effects from gene editing or clonal isolation, we complemented PrimPol KO cells with complementary DNAs expressing eGFP, WT PrimPol, catalytically dead PrimPol (AxA: Asp114Ala, Glu116Ala) or primase-dead PrimPol (CH: Cys419Gly, His426Tyr) (Extended Data Fig. 1e,f). To avoid artifacts associated with protein overexpression, we utilized the microRNA silencing-mediated fine-tuners (misFIT) system53 to decrease cellular expression of all cDNA constructs (Extended Data Fig. 1g,h). We selected a misFIT sequence that lowered WT PrimPol expression levels to near-endogenous levels (Extended Data Fig. 1h; 12C mutant). These constructs were stably expressed in the eHAP iCas9 PrimPol KO cell line and subsequently challenged with cisplatin (Fig. 1f,g). Only the WT PrimPol construct was able to complement cisplatin sensitivity observed in the PrimPol KO cell line, indicating that this phenotype is specific to loss of PrimPol (Fig. 1h). These data suggest that the repriming activity of PrimPol is required to tolerate cisplatin-induced DNA damage.
Next, we sought to understand whether PrimPol activity protects against other types of DNA damage in human cells. Previous reports have suggested that loss of PrimPol confers sensitivity to the bulky base adduct BPDE and interstrand crosslinking agent MMC21,25,33. We challenged both eHAP iCas9 WT and PrimPol KO cells with BPDE, MMC and ten other genotoxins or inhibitors of DDR proteins (Fig. 1i and Extended Data Fig. 1i–r). As previously reported, loss of PrimPol conferred cellular toxicity to both BPDE and MMC (Fig. 1j,k). However, no robust phenotype was observed when PrimPol KO cells were challenged with other types of DNA damage or inhibitors of DDR proteins (Fig. 1l and Extended Data Fig. 1i–r). Thus, loss of PrimPol-mediated repriming specifically confers cellular sensitivity to bulky base adducts induced by cisplatin, BPDE and MMC.
To identify drivers of cisplatin sensitivity in cells deficient in PrimPol-mediated repriming, we performed two targeted CRISPR–Cas9 dropout screens as previously described (Fig. 1a). We compared sgRNA counts between PrimPol KO and WT eHAP iCas9 cells in cisplatin-treated conditions, which identified loss of the tRNase SLFN11 as the top hit conferring cisplatin resistance in PrimPol KO cells in the pool 1 screen (Fig. 2a). In addition, we identified loss of the deubiquitinase USP1 as the top hit conferring cisplatin resistance in PrimPol KO cells in the pool 2 screen (Fig. 2b). Our screens also identified loss of FANCD2, RAD18 and the RPA trimeric complex as synthetic lethal with loss of PrimPol in cisplatin-treated cells (Fig. 2b).
a, A volcano plot measuring statistical significance against log2(fold-change) in sgRNA counts in eHAP iCas9 PrimPol KO cells versus WT cells in the cisplatin-treated arm of the pool 1 sgRNA screen on day 6. Labelled genes exhibited increased sgRNA counts in PrimPol KO cells versus WT cells. b, A volcano plot measuring statistical significance against log2(fold-change) in sgRNA counts in eHAP iCas9 PrimPol KO cells versus WT cells in the cisplatin-treated arm of the pool 2 sgRNA screen on day 6. Genes labelled in blue exhibited increased sgRNA counts whereas genes labelled in red exhibited decreased sgRNA counts in PrimPol KO cells versus WT cells. c, A histogram of curated messenger RNA sequencing data depicting the expression of SLFN11 in human cancer cell lines curated from the DepMap repository. d, A western blot (WB) showing SLFN11 and PrimPol expression levels in HeLa, U2OS, HCT116, A549, HEK293A, DU145, eHAP and A673 cells infected with lentiviruses harbouring either Cas9–AAVS1 or Cas9–PrimPol. e, Experimental scheme showing how HeLa, U2OS, HCT116, A549, HEK293A, DU145, eHAP and A673 cells infected with Cas9–sgAAVS1 or Cas9–sgPrimPol were challenged with cisplatin and cell viability measured using the Cell Titer Glo assay. f–n, Cisplatin dose–response curves for HeLa Kyoto (f), U2OS (g), HCT116 (h), RPE-1 p53 KO (i), A549 (j), DU145 (k), HEK293A (l), A673 (m) and eHAP (n) cell lines harbouring Cas9–AAVS1 or Cas9–PrimPol. All experiments were performed in n = 2 biological replicates. Data are presented as means. Source numerical data and unprocessed blots are available in the source data.
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We first sought to characterize the relationship between SLFN11 and PrimPol-mediated repriming in response to cisplatin treatment. Previous work identified SLFN11 activity as a potent sensitizer to a wide range of chemotherapies, including cisplatin36,42. Importantly, over half of cell lines catalogued in the DepMap repository do not express SLFN1144, including widely used non-cancerous RPE-1 and cancerous HeLa, U2OS and HCT116 (Fig. 2c). To explore whether loss of SLFN11 expression masks cisplatin sensitivity in PrimPol KO cells, we introduced a Cas9–sgRNA cassette targeting the AAVS1 safe harbour site or PRIMPOL in nine cell lines with varied tissue origin and differing PrimPol and SLFN11 expression levels (Fig. 2d).
Loss of PrimPol in cell lines expressing no or low levels of SLFN11 did not confer cisplatin sensitivity (Fig. 2e–k). Conversely, loss of PrimPol in the three cell lines expressing high levels of SLFN11—HEK293A, eHAP and A673—conferred mild-to-severe cisplatin sensitivity (Fig. 2l–n). Importantly, we also generated PrimPol KO clones in cell lines expressing low (HT-1080), medium (NCIH-460) or high (A673) levels of SLFN11. Loss of PrimPol did not confer cisplatin sensitivity in the HT-1080 cell line (Extended Data Fig. 2a–c) but did confer moderate-to-severe cisplatin sensitivity in NCIH-460 and A673 cell lines (Extended Data Fig. 2d,e). Together, these data suggest a correlation between SLFN11 expression levels and cisplatin sensitivity when PrimPol-mediated repriming is inactivated.
We sought to determine whether SLFN11 directly promotes cisplatin sensitivity on PrimPol inactivation (Fig. 3a). Inducible loss of SLFN11 conferred a growth benefit in both WT and PrimPol KO cells when challenged with cisplatin (Fig. 3b,c). Strikingly, loss of SLFN11 completely rescued cisplatin sensitivity observed in PrimPol KO cells to levels indistinguishable from WT cells (Fig. 3c). These results were recapitulated in an orthogonal colony formation assay (Extended Data Fig. 3a–c) and in the SLFN11-expressing A673 cell line (Extended Data Fig. 3f–h).
a, A schematic depicting how viability assays were performed. CTG, Cell Titer Glo. b, A western blot demonstrating transient knockout of SLFN11 in eHAP iCas9 WT or PrimPol KO cells. c, A dose–response curve of eHAP iCas9 WT or PrimPol KO cells in response to the indicated doses of cisplatin on loss of SLFN11. These experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. These experiments were performed using the same WT sgNTC and PrimPol KO sgNTC samples depicted in Fig. 5d. d, An experimental scheme for challenging eHAP iCas9 WT or PrimPol KO cells with cisplatin in the presence or absence of A-92, a chemical inhibitor of GCN2. e, A schematic depicting the signalling cascade connecting DNA damage to the ISR factor GCN2 and subsequent ribosome stalling and cell death. Inhibition of GCN2 prevents ribosome stalling and downstream cell death. f, Cisplatin dose–response curves for eHAP iCas9 WT or PrimPol KO cells in the presence or absence of 750 nM GCN2 inhibitor A-92. Experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. g, An experimental scheme for measuring apoptosis via cleaved caspase-3 using flow cytometry. h, A bar plot depicting the percentage of cleaved caspase-3 positive cells determined using flow cytometry as shown in g. Experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. A two-way ANOVA was performed to determine biological significance. P values: <0.0001 (WT versus PrimPol KO sgNTC + cisplatin); <0.0001 (PrimPol KO sgNTC versus PrimPol KO sgSLFN11 + cisplatin). i, An experimental scheme for measuring cell viability in response to cisplatin. j, A western blot showing SLFN11, PrimPol and GFP protein expression in SLFN11 WT, SLFN11 E209A (tRNA nuclease mutant), SLFN11 Lys652Asp (ssDNA-binding mutant) or SLFN11 Glu669Gln (helicase mutant) re-expression cell lines. k, Dose–response curves for SLFN11 re-expression cell lines challenged with cisplatin. These experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. l, An experimental setup depicting how chromatin-fractionation immunofluorescence experiments were performed. m, The eHAP iCas9 WT or PrimPol KO cells transiently depleted of SLFN11 mock treated or treated with 450 nM cisplatin for 24 h. Representative micrographs of chromatin-bound immunofluorescence of phospho-RPA32 Ser33, DAPI or merged are shown. n, Quantification of sum focal intensity of phospho-RPA32 Ser33 in the indicated cell lines. Data were normalized in each biological replicate to untreated WT samples. These experiments were performed in n = 5 biological replicates. Data are presented as mean ± s.d. A two-way analysis of variance (ANOVA) was performed to assess biological significance. P values: 0.0004 (WT versus PrimPol KO sgNTC + cisplatin); 0.2141 (WT sgNTC versus PrimPol KO sgSLFN11 + cisplatin); 0.1980 (PrimPol KO sgNTC versus PrimPol KO sgSLFN11 + cisplatin). The pRPA S33 staining was performed as a co-stain with γH2AX phospho-Ser139 depicted in Extended Data Fig. 4f–h. o, A representative western blot depicting levels of RPA phospho-Ser33, SLFN11 and GCN2 phospho-Thr899 in whole-cell extracts and chromatin fractions after treatment with 1 μM cisplatin for 24 h. This blot was performed in n = 2 biological replicates. Source numerical data and unprocessed blots are available in the source data. s.e., short exposure; l.e., long exposure.
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A recent landmark study implicated SLFN11 ssDNA-dependent tRNase activity as a trigger for downstream ribosome stalling and GCN2 (gene name EIF2AK4) activation38. To determine whether SLFN11-dependent activation of GCN2 confers cisplatin cytotoxicity in PrimPol KO cells, we challenged eHAP iCas9 WT or PrimPol KO cells with cisplatin in the presence of a fixed dose of A-92, a specific inhibitor of GCN2 (GCN2i)54 (Fig. 3d,e). Inhibition of GCN2 completely rescued cisplatin cytotoxicity in PrimPol KO cells to levels indistinguishable from WT cells (Fig. 3f). Importantly, this fixed dose of GCN2 inhibitor did not affect cell growth in untreated conditions, ruling out any effect on cell-cycle progression (Extended Data Fig. 3i). In addition, we confirmed that SLFN11 and GCN2 act epistatically to confer cisplatin sensitivity in PrimPol KO cells because concurrent loss of SLFN11 and GCN2 inhibition did not confer additive resistance to cisplatin in any genotype (Extended Data Fig. 3j,k).
Next, we examined whether SLFN11–GCN2-dependent cell death selectively confers cisplatin sensitivity in PrimPol KO cells or whether this is a generalized mechanism of cell death when other DDR pathways are compromised. Thus, we generated FANCD2 and RAD18 KO clones in eHAP iCas9 cells, then challenged these cell lines with cisplatin in the presence or absence of a fixed dose of GCN2i (Extended Data Fig. 3l,m). Inhibition of GCN2 completely rescued cisplatin sensitivity in PrimPol KO cells but did not provide any significant growth benefit in FANCD2 or RAD18 KO cells (Extended Data Fig. 3n). Similarly, loss of SLFN11 did not rescue cisplatin sensitivity when HR was inhibited using B02, a RAD51 inhibitor55 (Extended Data Fig. 3o–q). Together, these data suggest that the SLFN11–GCN2 axis selectively confers cisplatin sensitivity when repriming is inactivated.
Previous studies have determined that SLFN11 triggers p53-independent apoptosis in response to DNA damage, either through activation of the ribotoxic stress response or downregulation of the anti-apoptotic factor MCL138,39. We sought to determine whether the SLFN11–GCN2-dependent cell death that we observed was due to apoptosis (Fig. 3g). Cisplatin treatment modestly induced apoptosis in WT cells in an SLFN11-independent manner (Fig. 3h and Extended Data Fig. 4a). In the absence of PrimPol, cisplatin robustly induced apoptosis that was completely dependent on SLFN11 and GCN2 (Fig. 3h and Extended Data Fig. 4b–d). Together, these data demonstrate that cisplatin induces SLFN11-dependent and GCN2-dependent apoptotic cell death when PrimPol-mediated repriming is inactivated.
The ssDNA-binding, tRNA nuclease and helicase activities of SLFN11 have all been implicated as sensitizing cells to genotoxic stress37,38,39. To determine which functions of SLFN11 were required to confer cisplatin sensitivity in PrimPol KO cells, we generated an eHAP iCas9 PrimPol–SLFN11 double KO clone and re-expressed eGFP, WT SLFN11, SLFN11 Glu209Ala (tRNA nuclease mutant), SLFN11 Lys652Asp (ssDNA-binding mutant) or SLFN11 Glu669Gln (Walker B–helicase mutant) proteins to near-endogenous levels (Fig. 3i,j). All three activities of SLFN11 were required to confer cisplatin sensitivity in PrimPol KO cells (Fig. 3k).
As ssDNA binding was required to induce SLFN11-dependent cell death, we hypothesized that ssDNA accumulation at cisplatin-stalled replication forks could trigger SLFN11 activation. Levels of replication checkpoint activation and fork-associated ssDNA accumulation were determined by staining for ATR-dependent phosphorylation of RPA2 at Ser33 (RPA pSer33) in response to cisplatin (Fig. 3l). We observed a twofold increase in the mean focal intensity of RPA pSer33 in PrimPol KO cells treated with cisplatin compared with WT cells (Fig. 3m,n). This robust activation of the replication checkpoint was not completely suppressed on deletion of SLFN11 (Fig. 3m,n). We observed a similar induction of RPA pSer33 using western blotting techniques (Extended Data Fig. 4e). Neither γH2AX–pSer139 nor RAD51 levels increased after low doses of cisplatin treatment in PrimPol KO cells when compared with WT cells (Extended Data Fig. 4g–j). Finally, we observed a robust accumulation of SLFN11 on chromatin and activation of the ISR (as measured by phosphorylation of GCN2 at Thr899) after cisplatin treatment, particularly in PrimPol KO cells (Fig. 3o). Together, these data suggest that ssDNA accumulates at cisplatin-stalled replication forks in the absence of repriming, which could serve as a lesion for SLFN11 and GCN2 activation.
Having established a correlation between cisplatin-induced ssDNA accumulation at stalled forks and the induction of SLFN11-dependent cell death in PrimPol KO cells (Fig. 3k,l and Extended Data Fig. 4e), we sought to understand how SLFN11 is activated in response to these lesions. Our screens revealed that cells lacking repriming are particularly sensitive to loss of the RPA heterotrimeric complex when challenged with cisplatin (Fig. 2b). Under heightened replication stress, cells can undergo RPA exhaustion, where the available levels of RPA in the nucleus are not sufficient for the ssDNA generated in the cell9. We hypothesized that RPA exhaustion could result in uncoated ssDNA, which could serve as a template for SLFN11 binding to stalled forks, activating its tRNA nuclease activity and downstream apoptosis.
To determine whether cisplatin induces RPA exhaustion, we utilized quantitative image-based cytometry (QIBC) to simultaneously measure the levels of ssDNA (native 5-bromo-2ʹ-deoxyuridine (BrdU)) and chromatin-bound RPA at a cellular level, as previously described9 (Fig. 4a,b and Extended Data Fig. 5a). Cells undergoing RPA exhaustion were quantified by determining those cells where the ratio of ssDNA to chromatin-bound RPA signal deviated from linearity (Fig. 4c). In untreated conditions, <1.5% of cells were undergoing RPA exhaustion, with no significant differences in each genotype tested (Fig. 4c,d). On challenge with 2.5 μM cisplatin, the percentage of cells undergoing RPA exhaustion increased twofold in WT cells. Strikingly, the percentage of cells undergoing RPA exhaustion increased more than tenfold in cells deficient in PrimPol when compared with untreated conditions (Fig. 4c,d). In addition, knockout of SLFN11 resulted in a twofold increase in cells undergoing RPA exhaustion in both WT and PrimPol KO cells (Fig. 4c,d). Together, these data suggest that cells deficient in repriming are more prone to RPA exhaustion in response to cisplatin.
a, A diagram depicting how ssDNA was measured by detecting incorporated BrdU under non-denaturing conditions. b, An experimental scheme for measuring RPA exhaustion in eHAP iCas9 WT or PrimPol KO cells in which SLFN11 has been transiently knocked out using QIBC. c, Representative scatter plots depicting mean BrdU signal intensity per cell (y axis) plotted against mean chromatin-bound RPA32 (x axis). The linear relationship between BrdU and RPA32 signals is depicted as a light-blue line. Cells undergoing RPA exhaustion are depicted within the dashed box and coloured red. The percentage of cells undergoing RPA exhaustion is indicated in each panel. d, A bar plot depicting the percentage of cells undergoing RPA exhaustion in the experiments shown in a and b. These experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. A two-way ANOVA was performed to assess biological significance. P values: 0.0009 (WT sgNTC versus PrimPol KO sgNTC + cisplatin); <0.0001 (WT sgSLFN11 versus PrimPol KO sgSLFN11 + cisplatin); and 0.0009 (PrimPol KO sgNTC versus PrimPol KO sgSLFN11 + cisplatin). e, Representative scatter plots depicting mean γH2AX–pSer139 signal intensity per cell (y axis) plotted against mean chromatin-bound RPA32 (x axis). Cells with high RPA32 and γH2AX–pSer139 signals are coloured in red. The percentage of cells with high RPA32 and γH2AX–pSer139 signals is indicated in each panel. f, A bar plot depicting the percentage of cells exhibiting high RPA32–γH2AX–pSer139 signals in the experiments shown in a and e. These experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. A two-way ANOVA was performed to assess biological significance. P values: 0.0023 (WT sgNTC versus PrimPol KO sgNTC + cisplatin); 0.0003 (WT sgSLFN11 versus PrimPol KO sgSLFN11 + cisplatin); and 0.0001 (PrimPol KO sgNTC versus PrimPol KO sgSLFN11 + cisplatin). g, Experimental scheme for challenging eHAP iCas9 WT or PrimPol KO cells with cisplatin in the presence or absence of GCN2i after siRNA knockdown of RPA2. h, A western blot depicting RPA2, PrimPol, vinculin and total protein levels in eHAP iCas9 WT or PrimPol KO cells after transient knockdown of RPA2. i, A dose–response curve depicting cell survival in eHAP iCas9 WT or PrimPol KO cells (normalized to untransfected cells) after siRNA knockdown of RPA at indicated concentrations in the presence or absence of cisplatin and GCN2i. This experiment was performed in n = 3 biological replicates. Data are presented mean ± s.d. j, An experimental scheme for challenging eHAP iCas9 WT or PrimPol KO cells with two fixed doses of cisplatin in the presence or absence of DNA-PK inhibitor (DNA-PKi) after transient knockout of SLFN11. k, A bar plot depicting cell survival in the presence of 450 nM cisplatin with or without treatment with DNA-PKi. The experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. A two-way ANOVA was performed to assess statistical significance. P value: <0.0001 (PrimPol KO sgNTC versus PrimPol KO sgSLFN11 + cisplatin). l, A bar plot depicting cell survival in the presence of 1.5 μM cisplatin with or without treatment with DNA-PKi. The experiments shown were performed in n = 3 biological replicates. Data are presented as mean ± s.d. A two-way ANOVA was performed to assess statistical significance. P values: 0.0423 (WT sgNTC − DNA-PKi versus WT sgNTC + DNA-PKi); <0.0001 (WT sgSLFN11 − DNA-PKi versus WT sgSLFN11 + DNA-PKi); <0.0001 (PrimPol KO sgSLFN11 − DNA-PKi versus PrimPol sgSLFN11 + DNA-PKi).
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One consequence of RPA exhaustion is the generation of double-stranded breaks (DSBs) that manifest as high γH2AX–pSer139 signal in cells with a high burden of chromatin-bound RPA9. We repeated our QIBC experiments and stained for γH2AX–pSer139 and chromatin-bound RPA (Extended Data Fig. 5b). In untreated conditions, <1% of cells contained high γH2AX–pSer139 and chromatin-bound RPA signals (Fig. 4e,f). Although cisplatin treatment modestly increased the percentage of WT cells with high γH2AX–RPA signal, loss of SLFN11 resulted in a robust accumulation of cells with high γH2AX–RPA signal in both WT and PrimPol KO cells (Fig. 4e,f). Although SLFN11-proficient, PrimPol-deficient cells also exhibited a higher percentage of γH2AX–RPA double-positive cells, this is probably due to apoptosis56, because these cells are the most sensitive to cisplatin treatment (Fig. 4e). These data suggest that, although RPA exhaustion can occur in both SLFN11-proficient and SLFN11-deficient cells, the latter undergo replication catastrophe and DSB formation at higher rates when compared with the former.
Having established that cisplatin-induced RPA exhaustion occurs more readily when PrimPol is inactivated, we sought to understand whether RPA exhaustion could directly activate SLFN11–GCN2-dependent cell death. To test this possibility, we reduced RPA pools in cells by performing a titration of small interfering (si)RNA targeting the RPA2 gene in eHAP iCas9 WT or PrimPol KO cells (Fig. 4g,h). In the absence of cisplatin, siRNA-mediated depletion of RPA2 resulted in a dose-dependent loss in cell viability that was independent of SLFN11–GCN2 activity (Extended Data Fig. 5c). Strikingly, PrimPol KO cells exhibited an siRNA dose-dependent sensitization to cisplatin, which was completely rescued on inhibition of GCN2 (Fig. 4i). These results also explain why higher levels of RPA exhaustion are observed in SLFN11-negative cells, probably due to these cells no longer undergoing RPA exhaustion-induced apoptosis (Fig. 4c,d). Together, these data suggest that induced RPA exhaustion confers a SLFN11–GCN2-dependent cisplatin sensitivity when repriming is inactivated.
Having established that SLFN11-deficient cells undergo replication catastrophe and DSB formation at a higher rate after RPA exhaustion than SLFN11-proficient cells, we hypothesized that these DSBs could be suitable substrates for toxic repair via the non-homologous end-joining (NHEJ) repair pathway. To test this hypothesis, we transiently knocked out SLFN11 in eHAP iCas9 WT or PrimPol KO cells and subsequently challenged these cells with cisplatin in the presence or absence of a chemical inhibitor of the catalytic subunit of DNA-dependent protein kinase (DNA-PK), NU744157 (Fig. 4j). At a low dose of cisplatin, PrimPol KO cells exhibited an SLFN11-dependent loss in cell viability that was independent of DNA-PK activity (Fig. 4k). At a high dose of cisplatin, SLFN11-proficient WT or PrimPol KO cells exhibited a SLFN11-independent and DNA-PK-independent sensitization to cisplatin (Fig. 4l). However, in SLFN11-deficient cells, inhibition of DNA-PK resulted in a substantial rescue of cell viability in response to cisplatin, independent of PrimPol status (Fig. 4l). Together, these results suggest that DSBs that form after RPA exhaustion and replication catastrophe in SLFN11-deficient cells are suitable substrates for toxic DNA-PK activity.
Having established SLFN11 as a key determinant of cisplatin sensitivity in PrimPol KO cells, we sought to leverage this phenotype to identify modulators of SLFN11 activation in the absence of repriming. Our targeted CRISPR–Cas9 screens identified loss of the deubiquitinase USP1 as conferring resistance to cisplatin treatment in PrimPol KO cells (Fig. 2b).
Loss of USP1 or WDR48 rescued cisplatin sensitivity observed in PrimPol KO cells but had no effect in WT cells, as predicted by our screens (Fig. 5a–d). These results were recapitulated using a fixed dose of ML323, a specific inhibitor of the USP1–WDR48 complex58 (Extended Data Fig. 6a,b). These results were also observed in a second SLFN11-expressing cell line, A673 (Extended Data Fig. 6d,e). Importantly, WT or PrimPol KO cells treated with a fixed dose of USP1 inhibitor did not impact cell growth or viability in unchallenged conditions (Extended Data Fig. 6c). Together, these data suggest that loss of USP1–WDR48 complex activity confers resistance to cisplatin specifically in cells deficient in repriming.
a, A schematic depicting how cell viability assays were performed in eHAP iCas9 WT or PrimPol KO cells challenged with cisplatin after transient knockout of USP1 or WDR48. b, A representative western blot depicting PrimPol, USP1 and WDR48 protein levels on loss of USP1 or WDR48. This western blot was performed in n = 2 biological replicates. c, A cisplatin dose–response curve for eHAP iCas9 WT or PrimPol cells on loss of USP1. These experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. d, A cisplatin dose–response curve for eHAP iCas9 WT or PrimPol cells on loss of WDR48. These experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. These experiments were performed using the same WT sgNTC and PrimPol KO sgNTC samples depicted in Fig. 3c. e, An experimental scheme for measuring RPA exhaustion in cell pools after transient knockout of USP1 and SLFN11 using QIBC. f, A representative western blot showing USP1 and SLFN11 protein levels after transient knockout of each protein. This western blot was performed in n = 2 biological replicates. g, Representative scatter plots depicting mean ssDNA (BrdU) intensity (y axis) plotted against mean chromatin-bound RPA32 intensity (x axis) for each genotype and treatment depicted. The dashed boxes indicate RPA-exhausted cells where the ratio of mean ssDNA signal against mean chromatin-bound RPA32 signal has deviated from linearity (blue line). The percentage of cells undergoing RPA exhaustion in each genotype and treatment is shown. h, A bar plot depicting the percentage of cells undergoing RPA exhaustion in each genotype and treatment tested. These experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. A two-way ANOVA was performed to assess biological significance. P values: <0.0001 (WT sgSLFN11 versus PrimPol KO sgSLFN11 + cisplatin); and <0.0001 (PrimPol KO sgSLFN11 versus PrimPol KO sgSLFN11–USP1 + cisplatin). i, An experimental scheme depicting how apoptosis (cleaved caspase-3 signal) was measured using flow cytometry in eHAP iCas9 cells after transient knockout of USP1 or SLFN11. j, A bar plot depicting the percentage of cells undergoing apoptosis, as assessed by measuring the cleaved caspase-3 signal using flow cytometry. These experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. A two-way ANOVA was performed to assess biological significance. P values: <0.0001 (PrimPol KO sgNTC versus PrimPol KO sgUSP1 + cisplatin); <0.0001 (PrimPol KO sgNTC versus PrimPol KO sgSLFN11 + cisplatin); <0.0001 (PrimPol KO sgNTC versus PrimPol KO sgUSP1–SLFN11 + cisplatin); 0.0038 (PrimPol KO sgUSP1 versus PrimPol KO sgSLFN11 + cisplatin); and >0.9999 (PrimPol KO sgSLFN11 versus PrimPol KO sgSLFN11–USP1 + cisplatin). Source numerical data and unprocessed blots are available in the source data. DOX, doxycycline.
Source data
As loss of USP1–WDR48 complex activity rescued cisplatin sensitivity in PrimPol KO cells and this phenotype is entirely dependent on SLFN11-mediated and GCN2-mediated apoptosis, we hypothesized that USP1 promotes SLFN11 activation and induction of apoptosis by inducing RPA exhaustion. To test this hypothesis, we performed QIBC experiments to determine the levels of RPA exhaustion in eHAP iCas9 WT or PrimPol KO cells that have had USP1 or SLFN11 transiently knocked out (Fig. 5e,f). To avoid any effects of SLFN11-dependent apoptosis on masking RPA exhaustion levels, we performed all experiments in SLFN11-deficient cells. The level of cisplatin-induced RPA exhaustion in PrimPol KO cells was threefold to fourfold higher than in WT cells (Fig. 5g,h). Strikingly, loss of USP1 in PrimPol KO cells reduced the level of cisplatin-induced RPA exhaustion by twofold to threefold when compared with USP1-proficient PrimPol KO cells (Fig. 5g,h). Together, our data indicate that loss of USP1 significantly reduces the levels of cisplatin-induced RPA exhaustion in cells lacking repriming.
We hypothesized that the rescue of RPA exhaustion observed on loss of USP1 would lead to a reduction in SLFN11-dependent apoptosis and cell death. Loss of USP1 significantly rescued SLFN11-dependent apoptosis in PrimPol KO cells challenged with cisplatin and provided no benefit in SLFN11-deficient cells (Fig. 5i,j and Extended Data Fig. 7). As observed with RPA exhaustion and apoptosis, USP1 inhibition significantly rescued cell viability of PrimPol KO cells challenged with cisplatin in an SLFN11-dependent manner (Extended Data Fig. 6f,g). Together, these data suggest that loss of USP1 restricts activation of SLFN11 and subsequent apoptosis.
USP1 targets two major proteins in the DDR for deubiquitination: FANCD2–FANCI and PCNA59,60. Deubiquitination of FANCD2–FANCI and PCNA leads to downregulation of the FA and TLS pathways, respectively. As USP1 also drove cisplatin sensitivity in an SLFN11-dependent manner in PrimPol KO cells, we sought to determine whether downregulation of the FA or TLS pathways was responsible for driving RPA exhaustion and SLFN11-dependent cell death.
First, we wanted to determine whether loss of the FA or TLS pathway was synthetic lethal with loss of repriming in response to cisplatin as predicted in our targeted CRISPR–Cas9 screens (Fig. 2b). Indeed, loss of either RAD18 or FANCL led to an additive cisplatin sensitivity in cells lacking repriming (Extended Data Fig. 8a–e). Second, we wanted to determine whether upregulation of the FA or TLS pathway was responsible for rescuing cisplatin sensitivity in cells lacking repriming after USP1 depletion (Extended Data Fig. 8f–h). Loss of RAD18 did not reverse the cisplatin resistance observed on knockout of USP1 in PrimPol KO cells, whereas loss of FANCL completely reversed this phenotype (Extended Data Fig. 8i,j). Strikingly, loss of USP1 partially rescued the exquisite cisplatin sensitivity observed in double RAD18–PrimPol KO cells, underlining the importance of the FA pathway in repairing cisplatin lesions, particularly when TLS and repriming are inactivated (Extended Data Fig. 8i,j). Indeed, we observed an increase in the number of FANCD2 foci present in PrimPol KO cells challenged with cisplatin, suggesting a compensatory upregulation of the FA pathway when repriming is inactivated (Extended Data Fig. 8k–m). Together, our data implicate USP1-driven downregulation of the FA pathway as the mechanism behind the increased cisplatin-induced RPA exhaustion and SLFN11-dependent apoptosis in cells lacking repriming.
Having established that cells lacking repriming undergo RPA exhaustion and SLFN11-dependent cell death when challenged with cisplatin, we wanted to determine whether this mechanism is broadly applicable to other types of DNA damage, independent of PrimPol status. To address this, we transiently knocked out SLFN11 in WT eHAP iCas9 cells and challenged them with 12 different genotoxic agents or inhibitors of DDR pathways (Fig. 6a and Extended Data Fig. 9a–l). Strikingly, cells treated with ST1926, a selective chemical inhibitor of DNA polymerase α61, underwent a potent SLFN11-dependent death response (Fig. 6b and Extended Data Fig. 9a). Other drugs, such as the poly(ADP ribose polymerase) inhibitor, olaparib, or the topoisomerase I inhibitor, camptothecin, also induced a strong SLFN11-dependent death response (Fig. 6b and Extended Data Fig. 9b,c). Conversely, drugs targeting DSB repair pathways (RAD51 or ataxia-telangiectasia mutated (ATM) inhibitor) or inducing DSBs (bleomycin) did not elicit a strong SLFN11-dependent cell death (Fig. 6b). Surprisingly, hydroxyurea, a potent inducer of ssDNA at replication forks, failed to robustly activate SLFN11-dependent cell death (Fig. 6b). We hypothesized that this could be due to ST1926 and hydroxyurea inducing RPA exhaustion at different levels, as previously described9,10.
a, An experimental scheme depicting how cell survival was measured in cells with indicated genotypes after challenge with 12 different drugs targeting the DDR. b, A Forest plot depicting the log2(fold-change) of IC50 values derived from dose–response curves of the indicated drugs when SLFN11 was transiently knocked out versus a non-targeting control. Coloured dots represent calculated mean IC50 values, whereas black lines indicate 95% confidence intervals of each calculated mean IC50 value. c, An experimental scheme depicting how RPA exhaustion was assessed using QIBC after treatment with DNA polymerase α inhibitor (ST1926). d, A western blot depicting SLFN11 protein levels in eHAP iCas9 WT and an isogenic SLFN11 KO clone. e, A scheme of how SLFN11 activation was measured by staining cells for chromatin-bound ATF4 after ST1926-induced RPA exhaustion. f, Representative scatter plots measuring mean ssDNA or BrdU (y axis) versus mean chromatin-bound RPA32 signals (x axis). Linearity between the ssDNA and chromatin-bound RPA32 signals is depicted as a light-blue line. Cells undergoing RPA exhaustion are highlighted within the dashed box, with representative percentages of cells undergoing RPA exhaustion indicated. Cells with high levels of chromatin-bound ATF4 (ATF4high) are coloured red, whereas ATF4− cells are shown in grey. g, A bar plot depicting the percentage of cells with indicated genotype and treatment with ATF4high (high ATF4 activation). These experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. A one-way ANOVA was performed to assess biological significance. P values: 0.0024 (WT untreated versus WT 4 h ST1926); and 0.0010 (WT 4-h ST1926 versus SLFN11 KO 4-h ST1926). h, A bar plot depicting the percentage of eHAP iCas9 WT cells with high ATF4 activation that are undergoing RPA exhaustion. These experiments were performed in n = 3 biological replicates. A one-way ANOVA was performed to assess biological significance. P values: 0.0452 (untreated versus 1-h ST1926); and 0.0086 (untreated versus 4-h ST1926). i, A bar plot depicting the percentage of cells undergoing RPA exhaustion that exhibit ATF4 activation (ATF4high). These experiments were performed in n = 3 biological replicates. A one-way ANOVA was performed to assess biological significance. P values: 0.0008 (WT untreated versus WT 4-h ST1926); and 0.0006 (WT 4-h ST1926 versus SLFN11 KO 4-h ST1926). Source numerical data and unprocessed blots are available in the source data. Pol A, polymerase A.
Source data
As ST1926 potently induces RPA exhaustion and SLFN11-dependent cell death, we wanted to determine whether ST1926-induced RPA exhaustion could activate SLFN11. We utilized QIBC to simultaneously determine the levels of RPA exhaustion and SLFN11 activation in eHAP iCas9 WT cells or an isogenic SLFN11 KO clone (Fig. 6c,d). We were unable to detect SLFN11 chromatin binding under the rigorous pre-extraction conditions required for native BrdU or chromatin-bound RPA QIBC experiments. Thus, we stained for chromatin-bound ATF4, the master transcription factor activated by the GCN2-dependent ISR62, as a proxy for SLFN11 activation (Fig. 6e). Indeed, eHAP cells treated with ST1926 potently activated ATF4 in an SLFN11-dependent manner, providing confidence in our approach (Extended Data Fig. 10a,b). We utilized a conservative threshold for ATF4 activation by only scoring the top 25% of ATF4+ cells as ATF4high (Extended Data Fig. 10b). Given that only high or chronic levels of ISR activation are proapoptotic62,63, we wanted to ensure that both replicate reproducibility and accurate scoring of only the cells with the highest levels of ATF4. Low doses of ST1926 rapidly induced RPA exhaustion after 1 h of treatment in both WT and SLFN11 KO cells (Fig. 6f). After 4 h of ST1926 treatment, RPA exhaustion increased in both WT and SLFN11 KO cells; however, ATF4high cells were observed only in WT cells (Fig. 6e,f). Consistent with our model that RPA exhaustion activates SLFN11, >60% of WT cells with ATF4high were undergoing RPA exhaustion while 10% of WT cells undergoing RPA exhaustion exhibited high levels of ATF4 (Fig. 6f,h,i). Together, these data suggest that inhibition of DNA polymerase α results in robust RPA exhaustion that can activate the ISR in an SLFN11-dependent manner.
Having established ST1926 as a potent inducer of both RPA exhaustion and SLFN11-dependent cell death, we investigated whether this was broadly applicable to other SLFN11-expressing cell lines. We generated five cancer cell lines harbouring an inducible Cas9 that express different levels of SLFN11: HeLa Kyoto, HT-1080, TOV112D, A673 and eHAP. These cells were challenged with ST1926 treatment after transient knockout of SLFN11 (Fig. 7a,b). SLFN11-dependent cell death was observed only in cell lines that express high amounts of SLFN11 (Fig. 7c–g). Together, these data suggest that inhibition of DNA polymerase α induces SLFN11-dependent cell death through a mechanism that is conserved across different cancer cell types.
a, An experimental scheme depicting how cell viability was measured in indicated cell lines after transient knockout of SLFN11 and challenge with DNA polymerase α inhibitor (ST1926). b, A western blot depicting SLFN11 protein levels after transient knockout of SLFN11 in the indicated cell lines. c–g, Dose–response curves of HeLa Kyoto iCas9 (c), HT-1080 iCas9 (d), TOV112D iCas9 (e), A673 iCas9 (f) and eHAP iCas9 (g) cells challenged with the indicated doses of DNA polymerase α inhibitor (ST1926). Experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. h, In WT cells expressing SLFN11 (blue panel), PrimPol repriming activity restricts accumulation of ssDNA at cisplatin-stalled replication forks caused by uncoupling of the helicase and DNA polymerase, thereby preventing SLFN11 activation and cell death. Similarly, inhibition of DNA polymerase α uncouples DNA replication on each strand to drive ssDNA accumulation at forks in a PrimPol-independent manner. In PrimPol-deficient cells (green panel), cisplatin-induced DNA damage drives accumulation of ssDNA at replication forks in a USP1-dependent manner, leading to RPA exhaustion and SLFN11 activation. USP1-dependent downregulation of the FA pathway allows for full SLFN11 activation and subsequent ribosome stalling, ISR activation and cell death, as previously described38. In SLFN11-deficient cells (red panel), cisplatin-induced DNA damage or inhibition of DNA polymerase α causes ssDNA accumulation at forks and RPA exhaustion. Loss of SLFN11 prevents signal transduction through ribosome stalling and GCN2, preventing SLFN11-dependent cell death. Instead, prolonged RPA exhaustion leads to fork breakage and replication catastrophe as previously described9, leading to DSBs that are repaired by the NHEJ pathway, leading to cell death. Source numerical data and unprocessed blots are available in the source data. PolAi, Pol A inhibitor.
Source data
Our findings demonstrate that RPA exhaustion and exposure of ssDNA are the trigger for SLFN11 activation in response to replication-stalling lesions. Our study also reconciles a long-standing discrepancy between mechanistic observations that PrimPol reprimes downstream of DNA lesions while also being dispensable for cell viability in response to those same DNA lesions, which we attribute to the expression status of SLFN11 in cells.
Our serendipitous finding that chronic myelogenous leukaemia eHAP cancer cells lacking PrimPol-mediated repriming are sensitive to cisplatin (Fig. 1a–i) was initially surprising, because previous studies showed that other cell lines such as RPE-1, UWB1.289, HEK293T and U2OS do not exhibit this phenotype14,20,22,33. Our targeted CRISPR–Cas9 screens identified SLFN11 as a driver of cisplatin sensitivity, particularly in cells deficient in PrimPol-mediated repriming (Fig. 2a). Using a panel of 12 cancer and non-cancerous cell lines, we showed that SLFN11 expression level is broadly correlated with cisplatin sensitivity when PrimPol-mediated repriming is inactivated (Fig. 2d–n and Extended Data Fig. 2a–e). The lack of cisplatin sensitivity observed previously in other cell lines lacking repriming can be explained by the silencing of SLFN11 in these cell lines. Indeed, targeted knockout of SLFN11 or inhibition of the downstream ISR kinase GCN2 rescued both apoptosis and cisplatin sensitivity observed in eHAP PrimPol KO cells to levels indistinguishable from WT cells (Fig. 3a–h and Extended Data Fig. 4a–d). Importantly, our study implicates the GCN2-mediated ISR as being primarily responsible for SLFN11-dependent apoptosis, in contrast with previous work, which implicated the JNK-mediated ribotoxic stress response or impaired ribosome biogenesis38,39. This is probably due to a difference in dose and treatment time of genotoxic agents used in our work versus those used in other studies. Future work is needed to understand how different burdens of replication stress induce differential SLFN11-dependent responses.
Intriguingly, we found that the tRNA nuclease, ssDNA-binding and ATPase or helicase activities of SLFN11 were all required to confer cisplatin sensitivity in PrimPol KO cells (Fig. 3i–k), in agreement with previous work39. Although the requirements for the ssDNA-binding and tRNA nuclease activities are clear38,64, the purpose of the helicase activity of SLFN11 in promoting apoptosis has remained ambiguous37,39,45,47. We favour a model whereby the ATPase or helicase activity of SLFN11 is required to release SLFN11 from DNA to effectively cleave tRNAs. However, future studies focusing on further dissecting the distinct functions of SLFN11 and how each promotes p53-independent apoptosis are warranted.
SLFN11 preferentially binds ssDNA in vitro64 and this activity is required for translation shutdown, p53-independent apoptosis38,39, ssDNA-induced cell death65 and cisplatin sensitivity in PrimPol KO cells (Fig. 3i–k). However, the endogenous source of ssDNA that activates SLFN11 was previously unknown. To avoid spontaneous induction of apoptosis, the activation of SLFN11 must be tightly regulated. It is unlikely that every stalled replication fork would activate SLFN11-dependent cell death, because many drugs, such as hydroxyurea, induce fork stalling but do not robustly activate the SLFN11–GCN2 cell death pathway (Fig. 6b). We hypothesized that a potential source of ssDNA under heightened replication stress was RPA exhaustion, a rare yet catastrophic event that serves as a potent source of ssDNA in the cell9. Indeed, we observed more cells undergoing RPA exhaustion under conditions that induce SLFN11-dependent cell death (Figs. 4c,d, 6 and 7). Importantly, RPA exhaustion induced by siRNA targeting of RPA2 led to a SLFN11–GCN2-dependent sensitization to cisplatin when repriming was inactivated (Fig. 4h,i). RPA exhaustion triggering SLFN11-dependent apoptosis also explains why drugs like hydroxyurea do not potently activate an SLFN11-dependent response, whereas drugs like ST1926 do (Fig. 6b). Hydroxyurea alone does not induce RPA exhaustion even though it induces ssDNA accumulation at forks, probably through its potent induction of ATR and subsequent prevention of origin firing10. However, inhibition of DNA polymerase α induces rapid ssDNA accumulation, potent induction of RPA exhaustion10 and SLFN11 activation (Fig. 6e).
Another hallmark of RPA exhaustion is the generation of double-stranded DNA breaks after a replication fork catastrophe and fork breakage. Previous work connecting RPA exhaustion and replication fork catastrophe has been performed in U2OS, a SLFN11-negative cell line9. Strikingly, we observed RPA exhaustion-induced DSBs predominantly in SLFN11-negative cells that were preferentially acted on by DNA-PK, leading to cell death (Figs. 4e–f,k,l). Our interpretation of these data is that RPA exhaustion triggers SLFN11-dependent apoptosis before replication fork catastrophe (Fig. 7h). When SLFN11 is not expressed, cells undergoing RPA exhaustion do not undergo apoptosis but instead undergo canonical, replication fork catastrophe, DSB formation, potentially engaging the NHEJ pathway (Fig. 7h).
Our work also identified the USP1–WDR48 DUB complex as a modulator of SLFN11 activation at the replication fork. In cells lacking repriming, USP1 drives cisplatin-induced RPA exhaustion, resulting in induction of SLFN11-dependent apoptosis and cell death (Fig. 5). We identified upregulation of the FA pathway as responsible for preventing SLFN11-dependent apoptosis when USP1 is knocked out (Extended Data Fig. 7). Our interpretation of these data is that cells lacking PrimPol become addicted to the FA pathway to resolve cisplatin lesions. However, we cannot rule out that FA-mediated repair of cisplatin lesions is delayed in the absence of PrimPol25, potentially leading to a temporary increase in FANCD2 foci. However, as the FA pathway and PrimPol operate additively in response to cisplatin (Extended Data Fig. 7e) and upregulation of the FA pathway rescues cisplatin sensitivity in PrimPol KO cells (Extended Data Fig. 7j), we favour the model that PrimPol and the FA pathway act separately in response to cisplatin.
In conclusion, our findings describe a fundamental insight into the mechanism of SLFN11 activation at replication-stalling lesions, revealing a critical role in sensing and eradicating cells experiencing RPA exhaustion. We propose that the ATR-dependent, intra-S-phase checkpoint senses and signals replication stress by virtue of the targeting of ATR to RPA-coated ssDNA by ATR-interacting protein (ATRIP), which is required to stabilize the replisome, invoke repair and regulate origin firing. If this response is overwhelmed or RPA becomes limiting due to RPA exhaustion, this would create the dangerous scenario of unprotected ssDNA prone to nucleolytic attack and replication catastrophe. Our work reveals that SLFN11 has evolved to sense RPA exhaustion and, on activation, triggers a ‘pathway of last resort' to eliminate cells with heightened replication stress. Given that elevated replication stress is a hallmark of cancers, RPA exhaustion would create a strong selective pressure to epigenetically silence SLFN11 to allow cancer cell survival, which would explain its frequent inactivation in >50% of treatment-naive tumours.
The eHAP-inducible Cas9 (iCas9) cells were generated previously66 and maintained in Isocove's modified Dulbecco's medium (Gibco, catalogue number 12440053) supplemented with 10% tetracycline-free fetal bovine serum (FBS; Pan Biotech, catalogue number P30-3602) and penicillin–streptomycin (Gibco, catalogue number 15140122). HeLa Kyoto, U2OS, DU145, A549, HEK293A, HEK293 FT, HCT116, TOV112D and A673 cells were maintained in Dulbecco's modified Eagle's medium (Gibco, catalogue number 41966029) supplemented with 10% tetracycline-free FBS and penicillin–streptomycin. RPE-1 p53 KO cells were a kind gift of K. Vousden and maintained in Dulbecco's modified Eagle's medium/F12 medium (Gibco, catalogue number 11320033) supplemented with 10% tetracycline-free FBS and penicillin–streptomycin. HT-1080 iCas9 cells were generated as described in ‘Inducible Cas9 parental cell line generation' and maintained in Eagle's minimum essential medium (LGC, catalogue number 30-2003) supplemented with 10% FBS and penicillin–streptomycin. NCI-H460 iCas9 cells were generated as described in ‘Inducible Cas9 parental cell line generation' and maintained in Roswell Park Memorial Institute 1640 medium (Gibco, catalogue nnumber 11530586) supplemented with 10% FBS and penicillin–streptomycin. All cells utilized in this study were grown at 37 °C and 5% CO2 in humidified cell culture incubators. All cell lines in this study were obtained from the Francis Crick Institute Cell Science Scientific Technology Platform.
Cisplatin (Sigma-Aldrich, catalogue number P4394) was dissolved in phosphate-buffered saline (PBS) to a stock concentration of 5 mM and stored at room temperature, protected from light, for 2–3 weeks before making a fresh stock. Hydroxyurea (Sigma-Aldrich, catalogue number H627) was dissolved fresh in sterile water to a stock concentration of 100 mM before each experiment. Other chemicals used in this study were purchased as solid powder and reconstituted in dimethyl sulfoxide, aliquoted and stored at −20 °C or −80 °C according to the manufacturer's instructions before use: doxycycline (Sigma-Aldrich, catalogue number M0503), blasticidin (Thermo Fisher Scientific, catalogue number A1113903), puromycin (Thermo Fisher Scientific, catalogue number A1113803), BPDE (Santa Cruz, catalogue number sc-503767), MMC (Sigma-Aldrich, catalogue number M4287), olaparib (Selleckchem, catalogue number S1060), aphidicolin (Sigma-Aldrich, catalogue number A0781), ST1926 (Sigma-Aldric, catalogue number SML2061), bleomycin (Sigma-Aldrich, catalogue number B8416), methyl methane sulfonate (Thermo Fisher Scientific, catalogue number H55120.06), RAD51 inhibitor B02 (Selleckchem, catalogue number S8434), ATR inhibitor AZD6738 (Selleckchem, catalogue number S7693), camptothecin (Selleckchem, catalogue number S1288), ATM inhibitor KU-55933 (Selleckchem, catalogue number S1092), GCN2i A-92 (Axon Medchem, catalogue number 2720), USP1 inhibitor ML323 (Selleckchem, catalogue number S7529) and DNA-PKi NU7441 (Selleckchem, catalogue number S2638).
ON-TARGETplus SMARTPool non-targeting siRNA (catalogue number D-001810-10-05) or siRNA targeting human RPA2 (catalogue number L-017058-01-0005) was purchased from Horizon Discovery and resuspended in siRNA dilution buffer (Dharmacon, catalogue number B-002000-UB-100) to a concentration of 20 μM. The eHAP iCas9 WT or PrimPol KO cells were transfected with the indicated concentrations of siRNA targeting the RPA2 gene using lipofectamine RNAiMAX (Thermo Fisher Scientific, catalogue number 13778150) while keeping total siRNA concentration constant by buffering with non-targeting siRNA.
PrimPol cDNA was purchased from OriGene (catalogue number SC100629). PrimPol cDNA was amplified using Gateway primers PrimPol-gateway-F and PrimPol-gateway-R (Supplementary Table 2) and cloned into the pDONR221 plasmid (Thermo Fisher Scientific, catalogue number 12536017). PrimPol was subcloned into the pLEX_307 (pLenti EF1a) destination vector (Addgene, catalogue number 41392). PrimPol AxA (Asp114Ala, Glu116Ala) and CH (Cys419Gly, His426Tyr) mutants were introduced into this plasmid using the Q5 Site-Directed Mutagenesis kit (NEB), with primers shown in Supplementary Table 2.
SLFN11 cDNA was purchased from Dharmacon. SLFN11 cDNA was amplified and cloned into a pDONR221 plasmid as described above. SLFN11 Glu209Ala, Lys652Asp and Glu669Gln mutants were generated using the Q5 Site-Directed Mutagenesis kit with the primers shown in Supplementary Table 2. Finally, WT or mutant SLFN11 cDNAs were cloned into a Piggyback EF1a-driven Gateway plasmid. These plasmids were transfected into the indicated cells alongside the PiggyBac Transposase plasmid to generate stable cell lines expressing SLFN11.
PrimPol expression was downregulated by cloning misFIT sequences53 complementary to the mIR-17 microRNA with indicated mutations and spacer sequences into the 3ʹ-UTR downstream of WT PrimPol, PrimPol AxA or PrimPol CH mutants previously cloned into pLEX_307. A complete list of oligonucleotides used for misFIT cloning is shown in Supplementary Table 2.
The sgRNAs targeting the indicated genes were cloned into pLenti-sgRNA-puro, pLenti-sgRNA-hygro or pLentiCRISPRv2 using available protocols from the Zhang Lab (Addgene, catalogue nos. 52963, 139462 and 52961). The sgRNA sequences were sourced from the Brunello sgRNA library67 or designed in house (Supplementary Table 1).
Lentiviruses were generated by transfecting HEK293 FT cells with third-generation packaging plasmids VSVG, pLP1 (gag or pol) and pLP2 (Rev) and a lentiviral vector of interest. The medium was changed on transfected cells 18 h post-transfection; cells were left for a further 48 h, after which viral supernatants were harvested, filtered with a 0.45-μm filter, aliquoted and frozen at −80 °C.
Cells were prepared for transduction by changing the medium to a low volume of complete medium with added polybrene (Sigma-Aldrich, catalogue number H9268) to a final concentration of 8 μg ml−1. Cells were then transduced by adding viral supernatants and either left overnight or immediately spinfected by spinning cell or viral supernatant mixtures at 500g and 37 °C for 60 min.
Cell lines were selected using the following concentration of selected agents: eHAP (puromycin: 0.4 μg ml−1, hygromycin: 400 μg ml−1, blasticidin: 8 μg ml−1); HT-1080 (puromycin: 1 μg ml−1; blasticidin: 8 μg ml−1); NCIH-460 (puromycin: 1.0 μg ml−1, blasticidin: 8 μg ml−1); A673: (puromycin: 1.0 μg ml−1); HeLa Kyoto (puromycin: 1.0 μg ml−1); U2OS (puromycin: 1 μg ml−1); DU145 (puromycin: 1 μg ml−1); A549 (puromycin: 1 μg ml−1); HEK293A (puromycin: 1 μg ml−1); HCT116 (puromycin: 1 μg ml−1); and RPE-1 p53 KO (puromycin: 20 μg ml−1).
HT-1080 iCas9, NCIH-460 iCas9, TOV112D iCas9 and A673 iCas9 parental cell lines were generated by transducing lentiviral particles generated as described in ‘Lentivirus preparation and infection' harbouring the Edit-R Inducible Lentiviral Cas9 plasmid purchased from Dharmacon or Horizon (Horizon, catalogue number CAS11229). Transduced cells were enriched by selecting with blasticidin (8 μg ml−1) for 2–3 d until untransduced cells died. Single-cell clones were isolated from these pools of transduced cells and iCas9 activity was measured as described in ‘Inducible Cas9 activity assay'. Clones exhibiting <5% Cas9 activity in uninduced (no doxycycline) and >90% Cas9 activity in induced (with doxycycline) were used as parental iCas9 cell lines.
Inducible Cas9 clones were assessed for Cas9 activity by transducing lentiviral particles harbouring the pKLV2-U6gRNA5(gGFP)-PGKBFP2AGFP-W or pKLV2-U6gRNA5(gGFP)-PGKmCherry2AGFP-W plasmids (Addgene, catalogue nos. 67980 and 67982). Cells were split into two populations in the absence or presence of 1 μg ml−1 of doxycycline to induce Cas9 expression 24 h post-transduction. Cells were then maintained with or without doxycycline for 3 d (changing doxycycline as needed every 2 d). Cells were harvested and BFP–mCherry and GFP populations were assessed using flow cytometry. BFP+ cells were gated for GFP+ (no Cas9 activity) and GFP− cells (Cas9 activity).
To generate eHAP iCas9 PrimPol, FANCD2, SLFN11, RAD18 or USP1 KO clones, an sgRNA targeting PrimPol, FANCD2, SLFN11, RAD18 or USP1 was cloned into pLenti-sgRNA-puro vector. This vector was transfected into eHAP iCas9 WT or PrimPol KO parental cells while Cas9 was induced on addition of 1 μg ml−1 of doxycycline. Cells were enriched for those harbouring the vector by selecting with puromycin for 1–2 d while maintaining Cas9 expression with doxycycline. The resulting pool of transfected cells was seeded by limiting dilution and clones were picked and screened by purifying genomic DNA using the PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific, catalogue number K182001) and PCR amplifying and Sanger sequencing for the sgRNA cut site. Positive clones were further screened by western blotting to assess protein levels.
The indicated parental cell lines were transduced with lentiviral particles harbouring a vector containing either a constitutively expressed sgRNA (pLenti-sgRNA-puro or pLenti-sgRNA-hygro) or an all-in-one Cas9–sgRNA cassette (pLentiCRISPRv2-puro). Cells were selected with appropriate selection agents at the concentrations listed above. Selected pools of cells were then used directly in experiments or single-cell clones were isolated as indicated.
Whole-cell extracts were isolated by harvesting cells by centrifugation, washing once with PBS, spinning down again and freezing cell pellets at −80 °C. Cells were thawed and resuspended in radioimmunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecylsulfate (SDS), 0.5% deoxycholate, 1× phosphatase (Phos-Stop, Roche, catalogue number 4906845001) and 1× protease (cOmplete EDTA-free, Roche, catalogue number 11836170001) inhibitor cocktails). Cells were allowed to lyse for 15–20 min on ice, briefly sonicated to shear chromatin, then centrifuged at high speed (30,000g) to remove cellular debris. After centrifugation, lysates were quantified for protein concentration using the DC protein assay (BioRad). Cell lysates were normalized to equal protein concentration and mixed with appropriate volumes of NuPAGE LDS sample buffer (4×; Thermo Fisher Scientific, catalogue number NP0008) supplemented with 100 mM 2-mercaptoethanol. Lysates were boiled at 90 °C for 10 min and frozen at −20 °C until ready to run on SDS–polyacrylamide gel electrophoresis (PAGE).
Protein extracts were thawed at room temperature before loading 30–40 μg of total protein on to 4–12% Bis–Tris NuPAGE SDS–PAGE gradient gels or NuPAGE 3–8% Tris-acetate gradient gels (Thermo Fisher Scientific) with an appropriate number of wells. SDS–PAGE gels were run with an applied voltage of 120–180 V for 1–2.5 h.
For chromatin fractionations, whole-cell lysates were split in two and pelleted as above. One half of each sample was resuspended in radioimmunoprecipitation buffer as above. The other half of each sample was extracted with cytoskeleton (CSK) buffer (10 mM PIPES, pH 7.0, 100 mM NaCl, 300 mM sucrose, 1.5 mM MgCl2, 5 mM EDTA, 0.5% Triton, 1× phosphatase and 1× protease inhibitor cocktails) on ice for 5 min. Chromatin pellets were harvested by centrifuging at maximum speed for 15 s, after which pellets were washed in CSK buffer and centrifuged again. Chromatin pellets were resuspended in 1× NuPAGE LDS sample buffer, sonicated and boiled as with whole-cell lysates.
Proteins were transferred to nitrocellulose western blotting membranes (GE Healthcare or Amersham) by applying a constant amperage of 400 mA for 1 h. Membranes were stained with Ponceau S stain (Thermo Fisher Scientific) and imaged to assess protein loading. Membranes were then blocked using 5% non-fat milk powder dissolved in Tris-buffered saline with Tween 20 (TBS-T) for 1 h at room temperature. Primary antibodies diluted in 5% non-fat milk powder in TBS-T were applied overnight at 4 °C. Non-specific proteins were washed away with TBS-T 3× for at least 5 min per wash. Secondary horseradish peroxidase-conjugated antibodies were diluted 1:2,000 in 5% non-fat milk powder dissolved in TBS-T and applied to membranes for 1 h at room temperature. Membranes were then thoroughly washed at least 5× with TBS-T before the addition of chemiluminescence reagent (BioRad, Clarity or ClarityMAX). Membranes were then imaged using a BioRad ChemDoc imaging system.
Primary antibodies were utilized at the following concentrations: PrimPol17 (Proteintech, catalogue number 29824-1-AP, 1:1,000); PCNA (Santa Cruz, catalogue number sc-56, 1:1,000); vinculin (Sigma-Aldrich, catalogue number V9131, 1:5,000); RPA32 pS33 (Bethyl, catalogue number A300-246A, 1:1,000); RPA32 (abcam, catalogue number ab2175, 1:500); Chk1 pSer345 (Cell Signaling Technology, catalogue number 9664S, 1:1,000); Chk1 (Sigma-Aldrich, catalogue number C9358, 1:500); pGCN2 T899 (abcam, catalogue number 75836, 1:1,000); SLFN11 (Santa Cruz, catalogue number sc-374339, 1:500); glyceraldehyde 3-phosphate dehydrogenase (Abcam (6C5), catalogue number ab8245, 1:5,000); USP1 (Bethyl, catalogue number A301-699A, 1:1,000 or Proteintech, catalogue number 14346-1-AP, 1:1,000); WDR48 (Proteintech, catalogue number 16503-1-AP, 1:600); PCNA Ub K164 (Cell Signaling Technology, catalogue number 13439, 1:1,000); SMC1 (abcam, catalogue number ab21583, 1:1,000); RAD18 (Bethyl, catalogue number A301-340A, 1:1,000); FANCL (Santa Cruz, catalogue number sc-137067, 1:500); and FANCD2 (abcam, catalogue number ab108928, 1:1,000).
Indicated cell lines were grown for a total of 10 d in complete medium with the indicated concentrations of cisplatin, splitting every 2 d into fresh cisplatin as needed. Cells were counted every 2 d using a Countess 3 cell counter. Population doubling rates were determined using the following formula:
where n is population doubling, Xf the final cell count and Xi the initial cell count.
The indicated cell lines were plated in white, opaque, 96-well plates optimized for luminescence assays (Greiner, catalogue number 655098) at the following concentrations: eHAP 150 cells per well; HT-1080 400 cells per well; NCIH-460 400 cells per well; A673 1,000 cells per well; HeLa Kyoto 400 cells per well; U2OS 500 cells per well; RPE-1 p53 KO 400 cells per well; HCT116 1,000 cells per well; DU145 800 cells per well; A549 600 cells per well; HEK293A 300 cells per well; and TOV112D 800 cells per well. Cells were then exposed to selected drugs at indicated concentrations and grown for a further 5 d without changing the medium (7 d for A673). To read luminescence activity, growth medium was removed and replaced with a 1:1 ratio of 90 μl of complete medium: 90 μl of Cell Titer Glo One Solution Assay reagent (Promega, catalogue number G8462). The 96-well plates were agitated for 5 min at 120 rpm, after which the luminescence readings were read using a BMG LabTech ClarioStar plate reader. Dose–response curves were generated by normalizing the luminescence reading of all samples to an untreated internal control for each cell line.
Two customized sgRNA libraries containing four sgRNAs per gene were designed in house and ordered from Sigma-Aldrich (Supplementary Table 1). Pool 1 was designed to target genes found at human telomeres whereas pool 2 was designed as a supplemental sgRNA library to broaden the scope of pool 1 to cover additional targets involved in the DDR. Lentiviral particle preparations were obtained from Sigma-Aldrich and titered by infecting an equal number of eHAP iCas9 WT cells with increasing volumes of virus and determining the multiplicity of infection.
The eHAP iCas9 WT and PrimPol KO cells were infected with either pool 1 or pool 2 lentivirus in biological triplicate at a multiplicity of infection = 0.2 and an sgRNA representation of at least 500 cells per sgRNA. This representation was maintained throughout the rest of the screen. Cells harbouring the sgRNA libraries were enriched by selecting with puromycin for 2 d. Doxycycline was added to the medium to induce Cas9 expression over 6 d, splitting every 2 d with fresh doxycycline. Cells were then split into two arms per screen: untreated or treated with 450 nM cisplatin. Cells were grown for 6 d more, splitting every 2 d with fresh cisplatin in the treatment arm.
Cell pellets were collected throughout the screens with an appropriate number of cells to maintain 500 cells per sgRNA representation. Genomic DNA was isolated from these cells and subsequently used as templates for single-step PCR amplification of the sgRNA sequences present in each sample. PCR products were cleaned up using AMPure XP beads (Beckman Coulter, catalogue number A63800), according to the manufacturer's instructions and quantified using a QuBit system. PCR products were deep sequenced using Illumina Sequencing.
Sequencing reads were trimmed, sgRNA counts were calculated (Supplementary Tables 3 and 4) and comparisons between samples were performed using the MaGeCK algorithm50 with default settings (Supplementary Table 5).
The eHAP iCas9 cells were plated at a concentration of 200 cells per well in a 24-well plate in technical triplicates for each condition in each biological replicate. Cells were drugged with the indicated doses of cisplatin 18 h after plating. Colonies were allowed to form for 5 d after drugging, after which the medium was aspirated, colonies were washed with PBS, then stained with Crystal Violet stain (0.5% Crystal Violet in 20% methanol) for 15–30 min at room temperature. Excess Crystal Violet stain was removed and cells were washed with water and allowed to dry completely before imaging, using an Oxford Optronix GelCount colony counter. Colonies were quantified by automatically masking wells and adjusting CHARM settings to accurately detect colonies of varied sizes.
The eHAP iCas9 cells were seeded in 12-well plates and allowed to grow for 24 h before mock, cisplatin or GCN2 inhibitor treatment. After 40 h, cells were trypsinized and harvested in PBS, then transferred to 96-well round-bottomed plates (Falcon, catalogue number 353263). Cells were centrifuged at 500g for 5 min, after which the supernatant was aspirated. Cells were fixed in 100 μl of BD PhosFlow Fix Buffer I (BD Biosciences, catalogue number 557870) for 15 min at 37 °C. Cells were centrifuged again then permeabilized in 100 μl of BD PhosFlow Perm Buffer III (BD Biosciences, catalogue number 558050) at 4 °C for 30 min. Cells were centrifuged then blocked in 100 μl of blocking buffer (PBS + 10% goat serum) (Thermo Fisher Scientific, catalogue number G6767) for 30 min at room temperature. A 2× primary antibody dilution was made using rabbit anti-cleaved caspase-3 (BD Biosciences, catalogue number 570524) and added to blocked cells so that the final concentration of cleaved caspase-3 antibody was 1:1,000. After primary antibody incubation for 1 h, cells were washed once in PBS + 0.1% goat serum, then stained using goat anti-rabbit Alexa Fluor-647 (Thermo Fisher Scientific, catalogue number A-21244) at a final concentration of 1:1,000. After incubation with secondary antibody for 1 h at room temperature, cells were washed once in PBS + 0.1% goat serum, then resuspended in 200 μl of PBS + 0.1% goat serum. Cells were strained through a 35-μm mesh into round-bottomed flow cytometry tubes (Thermo Fisher Scientific, catalogue number 10585801). Doublets were removed from analysis by gating for forward scattering height versus area, after which cleaved caspase-3-positive cells were quantified.
The eHAP iCas9 cells were plated at a concentration of 3,000 cells per well in a black, 96-well PerkinElmer (now Revvity) PhenoPlate. Cells were allowed to attach overnight, after which they were mock treated or treated with the indicated doses of cisplatin for 24 h. Medium was aspirated and cells were washed once in PBS. Chromatin fractions were obtained by pre-extracting soluble proteins at the same time as fixation with 4% paraformaldehyde in PBS + 0.2% Triton X-100 for 15 min at room temperature. Fixed and extracted cells were washed twice with PBS and stored at 4 °C until ready to be stained.
When possible, all biological replicates were stained at the same time. PBS was aspirated from the 96-well plates, after which the cells were permeabilized by treating with PBS + 0.2% Triton X-100 for 15 min at room temperature. Cells were washed once in PBS and then blocked in blocking buffer (PBS + 3% bovine serum albumin + 0.1% Triton X-100) for 1 h at room temperature. Blocking buffer was removed and primary antibodies were applied at the following concentrations in blocking buffer for 1 h at room temperature: RPA32 phospho-Ser33 (Bethyl, catalogue number A300-246A, 1:2,500); RAD51 (Millipore, catalogue number ABE257, 1:500); γH2AX phospho-Ser139 (Millipore clone JBW301, catalogue number 05-636, 1:2,500); and FANCD2 (abcam, catalogue number ab108928, 1:2,000). After incubation with primary antibodies, cells were washed 3× for at least 5 min with PBS + 0.1% Triton X-100. Secondary antibodies and DAPI were diluted in blocking buffer to final concentrations of 1:1,000 (secondary antibodies) and 0.5 μg ml−1 of (DAPI), then incubated with cells for 1 h at room temperature. Goat anti-mouse Alexa Fluor-488 (Invitrogen, catalogue number A-11029) and goat anti-rabbit Alexa Fluor-647 (Invitrogen, catalogue number A-21245) were used in all experiments. After incubation with secondary antibodies, cells were washed 3× with PBS + 0.1% Triton X-100 for at least 5 min each. Cells were then washed with PBS twice and stored in PBS at 4 °C until ready to image.
Stained plates were scanned using the Operetta CLS confocal high-content system (×40). Maximum intensity projections were used for phenotypic analysis on 17 fields of view (FOVs); within each FOV, 5 planes were taken within each z-stack. Cell nuclei were identified using Harmony 5.1 software, followed by foci identification in the relevant imaging channels. Foci intensities were calculated as the sum intensity of foci per cell.
The eHAP iCas9 WT, PrimPol KO or SLFN11 KO cells with indicated genotypes were plated at a concentration of 10,000 (cisplatin treatment for 24 h) or 15,000 (ST1926 treatment time course) cells per well in a black, 96-well PhenoPlate in the presence of 10 μM BrdU. Treatments were added to cells as described in each figure. After completion of each treatment, cells were washed with PBS once. Chromatin-bound proteins were pre-extracted on ice in CSK buffer for 2 min. Cells were washed with PBS, then fixed in 4% paraformaldehyde in PBS for 15 min, washed again and stored in PBS until staining.
When possible, biological replicates for experiments were stained together. Cells were permeabilised in PBS + 0.2% Triton X-100 for 10 min and then blocked in blocking buffer (PBS + 3% BSA + 0.01% Triton X-100) for 45 min at room temperature. Primary antibodies targeting BrdU (mouse, BD Biosciences, catalogue number 555627, 1:250), RPA32 (rat, CST, catalogue number 2208 1:500) and either ATF4 (rabbit, CST, catalogue number 11815, 1:500) or γH2AX–pSer139 (rabbit, CST, catalogue number 2577, 1:500) were diluted at the indicated concentrations in blocking buffer and incubated with cells for 1 h at room temperature. Cells were washed 3× in PBS + 0.01% Triton X-100, after which secondary antibodies (goat anti-rat Alexa Fluor-647: Thermo Fisher Scientific, catalogue number A-21247, 1:1,000; HCA goat anti-mouse Alexa Fluor-488: Thermo Fisher Scientific, catalogue number A-11029, 1:1,000; HCA donkey anti-rabbit Alexa Fluor-568: Thermo Fisher Scientific, catalogue number A10042, 1:1,000) and DAPI (0.5 μg ml−1) were incubated with cells for 1 h at room temperature in the dark. Cells were washed twice with PBS + 0.01% Triton X-100 and once in PBS before imaging.
Stained plates were scanned using the Operetta CLS confocal high-content system (×20). Maximum intensity projections were used for phenotypic analysis on 49 FOVs. Within each FOV, five planes were taken within each z-stack. Cell nuclei were identified by DAPI staining using Harmony 5.1 software, followed by quantification of nuclear sum and mean intensities for each staining.
Raw mean BrdU–γH2AX–pSer139 and RPA32 intensities were plotted against one another in R Studio on log2(transformed) axes. Linear relationships between BrdU and RPA32 signals was derived from a linear transformation of untreated cells. RPA-exhausted cells were quantified by observing at which intensities the signal began to deviate from linearity. For experiments containing ATF4 staining, histograms of mean ATF4 nuclear intensities were generated for each condition. A threshold for all ATF4+ cells was drawn for each condition, after which the top quartile (25%) of ATF4+ cells was scored as ATF4high.
All statistical tests were performed using GraphPad Prism v.10.6.1 (799). The number of biological replicates and statistical test utilized for each figure panel are available in the corresponding figure legend. No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Raw Illumina sequencing FASTQ files related to the CRISPR–Cas9 screens performed in this study are publicly available in the National Center for Biotechnology Information Sequence Read Archive and BioProject Databases with accession number PRJNA1214387. Source data are provided with this paper.
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We thank members of S.J.B.'s lab for technical assistance, careful reading of the paper and helpful discussions. This work was supported by the Cell Science (grant number CC1079), Flow Cytometry (grant number CC1062), Genomics (grant number CC1064) and Screening and Automated Science (grant number CC1071) Scientific Technology Platforms at the Francis Crick Institute. We thank K. Vousden for providing the RPE-1 p53 KO cell line and J. Méndez for providing a PrimPol antibody. This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (grant number CC2057), the UK Medical Research Council (grant number CC2057) and the Wellcome Trust (grant number CC2057). Work in the S.J.B. laboratory is also funded by a Wellcome Trust Senior Investigator Grant (220808/Z/20/Z) and Advanced Investigator grants from the European Research Council (TelMetab 742437 and ChrEndProt 101053876).
DSB Repair Laboratory, The Francis Crick Institute, London, UK
Tyler H. Stanage, Shudong Li, Sandra Segura-Bayona, Aurora I. Idilli, Rhona Millar, Graeme Hewitt & Simon J. Boulton
Cancer Research UK Radnet City of London Centre, UCL Cancer Institute, London, UK
Rhona Millar
School of Cancer and Pharmaceutical Sciences, Comprehensive Cancer Centre, Kings's College London, London, UK
Graeme Hewitt
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T.H.S. and S.J.B. conceived the study and wrote the paper. T.H.S. performed all experiments and analysed all data. S.L. performed QIBC experimental design and conceptualization. S.L. and R.M. collected and analysed QIBC and high-content microscopy data. A.I.I., S.S.B. and G.H. helped with CRISPR–Cas9 screening design, execution and troubleshooting. A.I.I. designed CRISPR–Cas9 libraries. A.I.I., S.S.B. and G.H. generated some reagents and some iCas9 cell lines.
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Simon J. Boulton.
S.J.B. is co-founder and shareholder in Artios Pharma Limited, unrelated to this work. The other authors declare no competing interests.
Nature Cell Biology thanks Juan Mendez, Annabel Quinet and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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(a) DECODR analysis of Sanger sequencing of the PRIMPOL PR sgRNA cut site in eHAP iCas9 PRIMPOL KO C1 cells showing the cells habour a -2 bp deletion. (b) Experimental scheme for assessing inducible Cas9 activity using a lentiviral BFP/GFP flow cytometry reporter assay. (c) Representative gating strategy for all flow cytometry experiments conducted in this study. (d) Flow cytometry of eHAP iCas9 WT or PRIMPOL KO C1 cells infected with the BFP/GFP reporter shown in (B) in the presence or absence of doxycycline. (e) Sanger sequencing of the PRIMPOL AxA mutant (D114A/E116A) cDNA. (f) Sanger sequencing of the PRIMPOL CH mutant (C419H/H426Y) cDNA. (g) Scheme for introducing microRNA response elements (MREs) into the 3' UTRs of PRIMPOL expression constructs to fine-tune protein expression levels. (h) A Western blot showing PRIMPOL expression levels in eHAP iCas9 WT (lane 1) or PRIMPOL KO cells transiently transfected with PRIMPOL expression constructs containing indicated MREs. (i) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of the PARP inhibitor Olaparib. (j) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of the DNA polymerase inhibitor aphidicolin. (k) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of the DNA polymerase alpha inhibitor ST1926. (l) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of bleomycin. (m) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of the methyl methane sulfonate (MMS). (n) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of the RAD51 inhibitor B02. (o) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of the ribonucleotide reductase inhibitor hydroxyurea (HU). (p) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of the ATR inhibitor AZD6738. (q) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of the topoisomerase I inhibitor camptothecin (CPT). (r) Dose response curves for eHAP iCas9 WT or PRIMPOL KO cells challenged with indicated concentrations of ATM inhibitor KU-5593. All experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. Source numerical data and unprocessed blots are available in the source data.
Source data
(a) An experimental scheme demonstrating how viability assays were performed in indicated PRIMPOL KO cell lines. (b) A Western blot depicting PRIMPOL and SLFN11 protein levels in indicated cell lines and PRIMPOL KO clones. (c) A cisplatin dose response curve for HT-1080 ICas9 WT or PRIMPOL KO cells. Experiments were performed in N = 2 biological replicates. Data are represented as means ± SD. (d) Cisplatin dose response curves for NCIH-460 iCas9 WT or PRIMPOL KO clones. Experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. (e) A cisplatin dose response curve for A673 Cas9-AAVS1 or A673 Cas9-PRIMPOL KO clones. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. Source numerical data and unprocessed blots are available in the source data.
Source data
(a) A schematic depicting how viability assays were performed. (b) Representative images of colony formation assays used to assess viability of eHAP iCas9 WT or PRIMPOL KO cells in response to cisplatin upon loss of SLFN11. (c) Quantitation of colony formation assays as depicted in (b). These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was used to calculate biological significance. P-values: 0.0057 (WT sgNTC vs. PRIMPOL KO sgNTC + cisplatin). (d) Experimental scheme for a lentiviral-based mCherry/GFP Cas9 flow cytometry reporter. (e) Flow cytometry of A673 Cas9-AAVS1 C2 or A673 Cas9-PRIMPOL C2 cells showing Cas9 activity in these cells. (f) Experimental scheme for challenging A673 Cas9-AAVS1 C2 or A673 Cas9-PRIMPOL C2 cells transiently depleted of SLFN11 protein with cisplatin. (g) A Western blot showing PRIMPOL and SLFN11 protein levels in A673 Cas9-AAVS1 C2 and A673 Cas9-PRIMPOL cells transiently depleted of SLFN11 protein. (h) Bar blots showing cell survival of A673 Cas9-AAVS1-C2 or A673 Cas9-PRIMPOL C2 cells transiently depleted of SLFN11 protein in the presence of 58 nM cisplatin. Experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A one-way ANOVA was performed to assess biological significance. P-values: 0.0418 (A673 Cas9-AAVS1 sgNTC vs. A673 Cas9-PRIMPOL sgNTC); 0.0214 (A673 Cas9-PRIMPOL sgNTC vs. A673-Cas9 PRIMPOL sgSLFN11). (i) Bar plots showing cell survival of eHAP iCas9 WT or PRIMPOL KO cells in response to treatment with 750 nM GCN2 inhibitor in the absence of cisplatin. Experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess biological significance. P-values: >0.9999 (WT vs. WT + GCN2i); 0.7360 (PRIMPOL KO vs. PRIMPOL + GCN2i). (j) Experimental scheme for challenging eHAP iCas9 WT or PRIMPOL KO cells with cisplatin in the presence or absence of GCN2 inhibitor following transient knockout of SLFN11. (k) A dose response curve depicting cells with indicated genotypes challenged with cisplatin in the absence (solid lines, circles) or presence (dashed lines, triangled) of 750 nM GCN2 inhibitor. Experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. (l) An experimental scheme for challenging eHAP iCas9 WT, PRIMPOL KO, FANCD2 KO, or RAD18 KO cells with cisplatin in the presence or absence of 750 nM GCN2 inhibitor. (m) A Western blot showing PRIMPOL, FANCD2, and RAD18 protein levels in eHAP iCas9 WT, PRIMPOL KO, FANCD2 KO, and RAD18 KO cells. (n) A bar plot depicting cell survival of eHAP iCas9 WT, PRIMPOL KO, FANCD2 KO, and RAD18 KO cells at 450 nM cisplatin compared to an untreated control in each cell line in the absence (darker bars) or presence (lighter bars) of GCN2 inhibitor. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess statistical significance. P-values: 0.9939 (WT vs. WT + GCN2i); 0.0006 (PRIMPOL KO vs. PRIMPOL KO + GCN2i); 0.9901 (FANCD2 KO vs. FANCD2 KO + GCN2i); 0.2275 (RAD18 KO vs. RAD18 KO + GCN2i); 0.9998 (WT + GCN2i vs. PRIMPOL KO + GCN2i); <0.0001 (WT + GCN2i vs. FANCD2 KO + GCN2i); <0.0001 (WT + GCN2i vs. RAD18 KO + GCN2i). (o) An experimental scheme for challenging eHAP iCas9 WT cells with cisplatin in the presence or absence of RAD51 inhibitor (B02) following transient knockout of SLFN11. (p) A bar plot depicting cell survival in untreated conditions in the absence or presence of RAD51 inhibitor following transient knockout of SLFN11. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess statistical significance. P-values: >0.9999 (WT sgNTC vs. WT sgSLFN11); >0.9999 (WT sgNTC + 4.5 µM RAD51i vs. WT sgSLFN11 + 4.5 µM RAD51i); 0.9858 (WT sgNTC 9.0 µM RAD51i vs. WT sgSLFN11 + 9.0 µM RAD51i). (q) A bar plot depicting cell survival in cells treated with 450 nM cisplatin in the absence or presence of RAD51 inhibitor following transient knockout of SLFN11. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess statistical significance. P-values: >0.9999 (WT sgNTC vs. WT sgSLFN11); >0.9999 (WT sgNTC + 4.5 µM RAD51i vs. WT sgSLFN11 + 4.5 µM RAD51i); 0.9982 (WT sgNTC 9.0 µM RAD51i vs. WT sgSLFN11 + 9.0 µM RAD51i). Source numerical data and unprocessed blots are available in the source data.
Source data
(a) Representative flow cytometry experiments depicting cleaved caspase-3 signal plotted against forward scatter area. Cleaved caspase-3 positive cells were gated and quantitated as depicted. (b) A scheme depicting how apoptosis was measured in eHAP iCas9 WT or PRIMPOL KO cells using cleaved caspase-3 positive cells in flow cyomtetry experiments. (c) Representative dot plots depicting how the percentage of cleaved caspase-3 positive cells were determined using flow cytometry after challenge with cisplatin in the absence or presence of 650 nM GCN2 inhibitor. (d) A bar plot depicting the percentage of cleaved caspase-3 positive cells in each indicated genotype following challenge with cisplatin in the absence or presence of GCN2 inhibitor determined using flow cytometry in (b). These experiments were performed in N = 4 biological replicates. Data represented as means ± SD. A two-way ANOVA was performed to assess biological significance P-values: 0.0003 (WT sgNTC vs. PRIMPOL KO sgNTC + cisplatin); >0.9999 (WT sgSLFN11 vs. PRIMPOL KO sgSLFN11 + cisplatin); 0.0002 (PRIMPOL KO sgNTC vs. PRIMPOL KO sgSLFN11 + cisplatin). (e) Representative Western blots demonstrating induction of DNA damage response markers in eHAP iCas9 WT or PRIMPOL KO cells in response to indicated doses of cisplatin for 24 or 48 h. (f) Experimental setup of measuring chromatin-bound phospho-gamma H2AX serine 139 using immunofluorescence in eHAP iCas9 WT or PRIMPOL KO cells depleted of SLFN11 protein (top). (g) eHAP iCas9 WT or PRIMPOL KO Cells transiently depleted of SLFN11 were mock treated or treated with 450 nM cisplatin for 24 h. Representative micrographs of chromatin-bound immunofluorescence of γH2AX phospho serine 139, DAPI, or merged are shown. (h) Bar plots showing phospho-gamma H2AX serine 139 foci signal normalised to WT untreated cells in eHAP iCas9 WT or PRIMPOL KO cells depleted of SLFN11 protein. Experiments were performed in N = 5 biological replicates. Data are represented as means ± SD. A two-way ANOVA was used to assess biological significance. P-values: >0.9999 (WT sgNTC vs. PRIMPOL KO sgNTC + cisplatin); 0.4353 (PRIMPOL KO sgNTC vs. PRIMPOL KO sgSLFN11 + cisplatin); 0.2441 (WT sgNTC vs. PRIMPOL KO sgSLFN11). γH2AX phospho serine 139 staining was performed as a co-stain with pRPA S33 depicted in Fig. 4j-l. (i) eHAP iCas9 WT or PRIMPOL KO Cells transiently depleted of SLFN11 were mock treated or treated with 450 nM cisplatin for 24 h. Representative micrographs of chromatin-bound immunofluorescence of RAD51, DAPI, or merged are shown (j) Quantification of RAD51 foci in indicated cell lines. These experiments were performed in N = 5 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess biological significance. P-values: 0.0050 (WT sgNTC vs. PRIMPOL KO sgNTC + cisplatin); 0.0013 (PRIMPOL KO sgNTC vs. PRIMPOL KO sgSLFN11 + cisplatin); 0.9994 (WT sgNTC vs. PRIMPOL KO sgSLFN11 + cisplatin). Source numerical data and unprocessed blots are available in the source data.
Source data
(a) Representative micrographs depicting BrdU, RPA, and DAPI signals generated in QIBC experiments shown in Fig. 4c. (b) Representative micrographs depicting γH2AX pS139, RPA, and DAPI signals generated in QIBC experiments shown in Fig. 4e. (c) A dose response curve depicting cell viability in eHAP iCas9 WT or PRIMPOL KO cells treated with indicated doses of siRNA targeting the RPA2 gene in the presence or absence of 750 nM GCN2 inhibitor. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. Source numerical data are available in the source data.
Source data
(a) An experimental schematic describing how cell viability assays were performed in the presence of cisplatin and ML323 (USP1 inhibitor). (b) A cisplatin dose response curve for eHAP iCas9 WT or PRIMPOL KO cells with or without 4.5 μM ML323. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. (c) A bar plot showing how eHAP iCas9 WT (gray bars) or PRIMPOL KO cells (red bars) respond to 4.5 μM ML323. Survival is normalised to untreated conditions within each genotype. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess statistical significance. P-values: 0.4946 (WT vs. WT + USP1i); 0.7018 (PRIMPOL KO vs. PRIMPOL KO + USP1i). (d) A schematic showing how cell viability experiments were performed in A673 Cas9-AAVS1 or A673 Cas9-PRIMPOL cells in the presence of cisplatin and ML323 (16.5 μM). (e) A bar blot depicting cell survival for A673 Cas9-AAVS1 or A673 Cas9-PRIMPOL cells in the presence of 200 nM cisplatin -/+ 16.5 μM ML323. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess statistical significance. P-values: 0.9925 (WT vs. WT + USP1i); 0.0203 (PRIMPOL KO vs. PRIMPOL KO + USP1i). (f) An experimental scheme depicting how cell viability was measured after challenging cells with cisplatin and USP1 inhibitor (ML323) in eHAP iCas9 cells with indicated genotypes. (g) A bar plot depicting cell survival of eHAP iCas9 WT or PRIMPOL KO cells following challenge with 670 nM cisplatin in the absence (darker bars) or presence (light bars) of 4.5 μM USP1 inhibitor following transient knockout of SLFN11. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess statistical significance. P-values: >0.9999 (WT sgNTC vs. WT sgNTC + USP1i); >0.9999 (WT sgSLFN11 vs. WT sgSLFN11 + USP1i); 0.0032 (PRIMPOL KO sgNTC vs. PRIMPOL KO sgNTC + USP1i); 0.8134 (PRIMPOL KO sgSLFN11 vs. PRIMPOL KO sgSLFN11 + USP1i); 0.8522 (WT sgNTC vs. WT sgSLFN11); <0.0001 (PRIMPOL KO sgNTC vs. PRIMPOL KO sgSLFN11); 0.8160 (WT sgNTC + USP1i vs. PRIMPOL KO sgNTC + USP1i). Source numerical data are available in the source data.
Source data
(a) A series of representative dot plots depicting how apoptosis was measured in cells with indicated genotypes by quantifying the percentage of cells that stained positively for cleaved caspase-3. These dot plots are related to the quantification of apoptosis shown in Fig. 5j.
(a) A schematic showing how RAD18 or FANCL was knocked out in eHAP iCas9 WT or PRIMPOL KO cells and subsequently challenged with cisplatin. (b) A Western blot showing PRIMPOL and RAD18 protein levels in eHAP iCas9 WT or PRIMPOL KO cells upon loss of RAD18 (R18). (c) A cisplatin dose response curve for eHAP iCas9 WT or PRIMPOL cells upon loss of RAD18. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. These experiments were performed using the same WT sgNTC and PRIMPOL KO sgNTC samples depicted in (m). (d) A Western blot showing PRIMPOL and FANCL protein levels in eHAP iCas9 WT or PRIMPOL KO cells upon loss of FANCL (FL). (e) A cisplatin dose response curve for eHAP iCas9 WT or PRIMPOL KO cells upon loss of FANCL. eHAP iCas9 WT and PRIMPOL KO curves are the same as those shown in (d), as these experiments were performed at the same time. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. (f) A series of Western blots showing indicated protein levels in eHAP iCas9 WT or PRIMPOL cells upon loss of USP1 and/or RAD18 (R18). (g) A series of Western blots showing indicated protein levels in eHAP iCas9 WT or PRIMPOL KO cells upon loss of USP1 and/or FANCL (FL). (h) An experimental scheme for how USP1/RAD18/FANCL proteins were knocked out in eHAP iCas9 WT or PRIMPOL KO cells and subsequently challenged with cisplatin in a colony formation assay. (i) Representative images of colony formation assays in indicated cell backgrounds in untreated conditions or upon treatment with 450 nM cisplatin. (j) Quantification of colony survival in the colony formation assays represented in (d) normalised to untreated samples within each genotype. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to test statistical significance. P-values: 0.9964 (WT sgNTC vs. WT sgUSP1); 0.0098 (WT sgRAD18/NTC vs. WT sgRAD18/sgUSP1); 0.9998 (WT sgFANCL/sgNTC vs. WT sgFANCL/USP1); 0.0012 (PRIMPOL KO sgNTC vs. PRIMPOL KO sgUSP1); <0.0001 (PRIMPOL KO sgRAD18/NTC vs. PRIMPOL KO sgRAD18/USP1); >0.9999 (PRIMPOL KO sgFANCL/NTC vs. PRIMPOL KO sgFANCL/USP1). (k) An experimental schematic describing how chromatin-bound immunofluorescence was performed. (l) Representative micrographs depicting DNA staining (DAPI), FANCD2 staining, and a merged image of DAPI/FANCD2 staining. (m) A bar plot showing chromatin-bound FANCD2 foci numbers in eHAP iCas9 WT or PRIMPOL KO cells treated with 450 nM cisplatin. These experiments were performed in N = 5 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess statistical significance. P-values: 0.0071 (WT + cisplatin vs. PRIMPOL KO + cisplatin). Source numerical data and unprocessed blots are available in the source data.
Source data
(a) A dose response curve measuring cell viability in the presence of indicated concentrations of ST1926, a DNA polymerase alpha inhibitor. (b) A dose response curve measuring cell viability in the presence of indicated concentrations of olaparib, a PARP inhibitor. (c) A dose response curve measuring cell viability in the presence of indicated concentrations of camptothecin (CPT), a topoisomerase I inhibitor. (d) A dose response curve measuring cell viability in the presence of indicated concentrations of benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). (e) A dose response curve measuring cell viability in the presence of indicated concentrations of methyl methanesulfonate (MMS). (f) A dose response curve measuring cell viability in the presence of indicated concentrations cisplatin. (g) A dose response curve measuring cell viability in the presence of indicated concentrations of mitomycin C (MMC). (h) A dose response curve measuring cell viability in the presence of indicated concentrations of AZD6738, an ATR inhibitor. (i) A dose response curve measuring cell viability in the presence of indicated concentrations of hydroxyurea (HU), an inhibitor of ribonucleotide reductase. (j) A dose response curve measuring cell viability in the presence of indicated concentrations of bleomycin. (k) A dose response curve measuring cell viability in the presence of indicated concentrations of KU-5593, an ATM inhibitor. All experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. (l) A dose response curve measuring cell viability in the presence of indicated concentrations of B02, a RAD51 inhibitor. Source numerical data are available in the source data.
Source data
(a) Representative micrographs related to QIBC experiments shown in Fig. 4f. (b) Representative histograms depicting mean chromatin-bound ATF4 signal in indicated conditions.
Supplementary Table 1 SgRNA library sequences. Supplementary Table 2 Oligonucleotide sequences. Supplementary Table 3 CRISPR screen, pool 1, raw counts. Supplementary Table 4 CRISPR screen, pool 2, raw counts. Supplementary Table 5 MaGeCK gene summaries.
Unprocessed western blots.
Statistical source data.
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Stanage, T.H., Li, S., Segura-Bayona, S. et al. RPA exhaustion activates SLFN11 to eliminate cells with heightened replication stress.
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A restless night often leads to fatigue the next day, but it may also signal health problems that emerge much later. Scientists at Stanford Medicine and their collaborators have developed an artificial intelligence system that can examine body signals from a single night of sleep and estimate a person's risk of developing more than 100 different medical conditions.
The system, called SleepFM, was trained using almost 600,000 hours of sleep recordings from 65,000 individuals. These recordings came from polysomnography, an in-depth sleep test that uses multiple sensors to track brain activity, heart function, breathing patterns, eye movement, leg motion, and other physical signals during sleep.
Sleep Studies Hold Untapped Health Data
Polysomnography is considered the gold standard for evaluating sleep and is typically performed overnight in a laboratory setting. While it is widely used to diagnose sleep disorders, researchers realized it also captures a vast amount of physiological information that has rarely been fully analyzed.
"We record an amazing number of signals when we study sleep," said Emmanual Mignot, MD, PhD, the Craig Reynolds Professor in Sleep Medicine and co-senior author of the new study, which will publish Jan. 6 in Nature Medicine. "It's a kind of general physiology that we study for eight hours in a subject who's completely captive. It's very data rich."
In routine clinical practice, only a small portion of this information is examined. Recent advances in artificial intelligence now allow researchers to analyze these large and complex datasets more thoroughly. According to the team, this work is the first to apply AI to sleep data on such a massive scale.
"From an AI perspective, sleep is relatively understudied. There's a lot of other AI work that's looking at pathology or cardiology, but relatively little looking at sleep, despite sleep being such an important part of life," said James Zou, PhD, associate professor of biomedical data science and co-senior author of the study.
Teaching AI the Patterns of Sleep
To unlock insights from the data, the researchers built a foundation model, a type of AI designed to learn broad patterns from very large datasets and then apply that knowledge to many tasks. Large language models like ChatGPT use a similar approach, though they are trained on text rather than biological signals.
SleepFM was trained on 585,000 hours of polysomnography data collected from patients evaluated at sleep clinics. Each sleep recording was divided into five-second segments, which function much like words used to train language-based AI systems.
"SleepFM is essentially learning the language of sleep," Zou said.
The model integrates multiple streams of information, including brain signals, heart rhythms, muscle activity, pulse measurements, and airflow during breathing, and learns how these signals interact. To help the system understand these relationships, the researchers developed a training method called leave-one-out contrastive learning. This approach removes one type of signal at a time and asks the model to reconstruct it using the remaining data.
"One of the technical advances that we made in this work is to figure out how to harmonize all these different data modalities so they can come together to learn the same language," Zou said.
Predicting Future Disease From Sleep
After training, the researchers adapted the model for specific tasks. They first tested it on standard sleep assessments, such as identifying sleep stages and evaluating sleep apnea severity. In these tests, SleepFM matched or exceeded the performance of leading models currently in use.
The team then pursued a more ambitious objective: determining whether sleep data could predict future disease. To do this, they linked polysomnography records with long-term health outcomes from the same individuals. This was possible because the researchers had access to decades of medical records from a single sleep clinic.
The Stanford Sleep Medicine Center was founded in 1970 by the late William Dement, MD, PhD, who is widely regarded as the father of sleep medicine. The largest group used to train SleepFM included about 35,000 patients between the ages of 2 and 96. Their sleep studies were recorded at the clinic between 1999 and 2024 and paired with electronic health records that followed some patients for as long as 25 years.
(The clinic's polysomnography recordings go back even further, but only on paper, said Mignot, who directed the sleep center from 2010 to 2019.)
Using this combined dataset, SleepFM reviewed more than 1,000 disease categories and identified 130 conditions that could be predicted with reasonable accuracy using sleep data alone. The strongest results were seen for cancers, pregnancy complications, circulatory diseases, and mental health disorders, with prediction scores above a C-index of 0.8.
How Prediction Accuracy Is Measured
The C-index, or concordance index, measures how well a model can rank people by risk. It reflects how often the model correctly predicts which of two individuals will experience a health event first.
"For all possible pairs of individuals, the model gives a ranking of who's more likely to experience an event -- a heart attack, for instance -- earlier. A C-index of 0.8 means that 80% of the time, the model's prediction is concordant with what actually happened," Zou said.
SleepFM performed especially well when predicting Parkinson's disease (C-index 0.89), dementia (0.85), hypertensive heart disease (0.84), heart attack (0.81), prostate cancer (0.89), breast cancer (0.87), and death (0.84).
"We were pleasantly surprised that for a pretty diverse set of conditions, the model is able to make informative predictions," Zou said.
Zou also noted that models with lower accuracy, often around a C-index of 0.7, are already used in medical practice, such as tools that help predict how patients might respond to certain cancer treatments.
Understanding What the AI Sees
The researchers are now working to improve SleepFM's predictions and better understand how the system reaches its conclusions. Future versions may incorporate data from wearable devices to expand the range of physiological signals.
"It doesn't explain that to us in English," Zou said. "But we have developed different interpretation techniques to figure out what the model is looking at when it's making a specific disease prediction."
The team found that while heart-related signals were more influential in predicting cardiovascular disease and brain-related signals played a larger role in mental health predictions, the most accurate results came from combining all types of data.
"The most information we got for predicting disease was by contrasting the different channels," Mignot said. Body constituents that were out of sync -- a brain that looks asleep but a heart that looks awake, for example -- seemed to spell trouble.
Rahul Thapa, a PhD student in biomedical data science, and Magnus Ruud Kjaer, a PhD student at Technical University of Denmark, are co-lead authors of the study.
Researchers from the Technical University of Denmark, Copenhagen University Hospital -Rigshospitalet, BioSerenity, University of Copenhagen and Harvard Medical School contributed to the work.
The study received funding from the National Institutes of Health (grant R01HL161253), Knight-Hennessy Scholars and Chan-Zuckerberg Biohub.
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What happens here matters everywhere
by Todd Bishop on Jan 9, 2026 at 10:39 amJanuary 9, 2026 at 10:50 am
Bill Gates had a front-row seat for the rise of AI, from his longtime work at Microsoft to early demonstrations of key breakthroughs from OpenAI that illustrated the technology's potential. Now he's urging the rest of us to get ready.
Likening the situation to his pre-COVID warnings about pandemic preparedness, Gates writes in his annual “Year Ahead” letter Friday morning that the world needs to act before AI's disruptions become unmanageable. But he says that AI's potential to transform healthcare, climate adaptation, and education remains enormous, if we can navigate the risks.
“There is no upper limit on how intelligent AIs will get or on how good robots will get, and I believe the advances will not plateau before exceeding human levels,” Gates writes.
He acknowledges that missed deadlines for artificial general intelligence, or human-level AI, can “create the impression that these things will never happen.” But he warns against reaching that conclusion, arguing that bigger breakthroughs are coming, even if the timing remains uncertain.
He says he's still optimistic overall. “As hard as last year was, I don't believe we will slide back into the Dark Ages,” he writes. “I believe that, within the next decade, we will not only get the world back on track but enter a new era of unprecedented progress.”
But he adds that we'll need to be “deliberate about how this technology is developed, governed, and deployed” — and that governments, not just markets, will have to lead AI implementation.
More takeaways from the letter:
Job disruption is already here. He says AI makes software developers “at least twice as efficient,” and that disruption is spreading. Warehouse work and phone support are next. He suggests the world use 2026 to prepare, citing the potential for changes like a shorter work week.
Bioterrorism is his top AI concern. Gates warns that “an even greater risk than a naturally caused pandemic is that a non-government group will use open source AI tools to design a bioterrorism weapon.”
Climate will cause “enormous suffering” without action. Gates cautions that if we don't limit climate change, it will join poverty and infectious disease in hitting the world's poorest people hardest, and even in the best case, temperatures will keep rising.
Child mortality went backward in 2025. Stepping outside AI, Gates calls this the thing he's “most upset about.” Deaths for children under 5 years old rose from 4.6 million in 2024 to 4.8 million in 2025, the first increase this century, which he traced to cuts in aid from rich countries.
AI could leapfrog rich-world farming. Gates predicts AI will soon give poor farmers “better advice about weather, prices, crop diseases, and soil than even the richest farmers get today.” The Gates Foundation has committed $1.4 billion to help farmers facing extreme weather.
Gates is using AI for his own health. He says he uses AI “to better understand my own health,” and sees a future where high-quality medical advice is available to every patient and provider around the clock.
AI is now the Gates Foundation's biggest bet in education. Personalized learning powered by AI is “now the biggest focus of the Gates Foundation's spending on education.” Gates says he's seen it working firsthand in New Jersey and believes it will be “game changing” at scale.
Read the full letter here.
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by Lisa Stiffler on Jan 9, 2026 at 10:33 amJanuary 9, 2026 at 10:57 am
TerraPower, a Bill Gates-backed next-gen nuclear company, on Friday announced a deal with Meta to build up to eight small modular reactors in the U.S. with the first coming online as soon as 2032.
Tech companies have been aggressively pursuing new clean energy solutions as they race to build power-hungry data centers that support burgeoning AI services.
TerraPower's deal with Meta will provide the tech giant with up to 2.8 gigawatts of energy using its Natrium nuclear technology. The facilities include an energy storage system, which can provide shorter-term bursts of power that bring the total output to 4 gigawatts.
The contract with the Bellevue, Wash.-based company marks Meta's largest single nuclear deal to date. The companies did not indicate where the reactors would be built.
“To successfully address growing energy demand, we must deploy gigawatts of advanced nuclear energy in the 2030s. This agreement with Meta is designed to support the rapid deployment of our Natrium technology that provides the reliable, flexible, and carbon-free power our country needs,” Chris Levesque, TerraPower president and CEO, said in a statement.
Meta's announcement includes additional partnerships with Vistra and Oklo:
Meta in June signed a deal with Constellation that supports the relicensing and extends the operations of its nuclear plant in Illinois.
“Our agreements with Vistra, TerraPower, Oklo, and Constellation make Meta one of the most significant corporate purchasers of nuclear energy in American history,” said Joel Kaplan, Meta's chief global affairs officer, in a statement.
The tech sector has come under increased scrutiny for the utility ratepayer and environmental impacts of its scramble for power. Three Democratic senators last month sent letters to Amazon, Microsoft, Google, Meta and three data center firms informing them of an investigation into their effects on residential power bills.
Meta addressed these concerns in announcing the new partnerships.
“This work builds on our ongoing collaboration with electric utility companies and power providers to plan for and meet our energy needs years in advance of our data centers becoming operational. We pay the full costs for energy used by our data centers so consumers don't bear these expenses, and we support the broader grid through our energy agreements,” the company stated.
Amazon and Microsoft are likewise pursuing nuclear energy.
TerraPower is currently building its first commercial reactor in Kemmerer, Wyo., and plans to start splitting atoms by 2030. The reactor is located near a retiring coal plant.
In December, the company said it completed a key regulatory milestone, passing the Nuclear Regulatory Commission staff's final safety evaluation for its permit. Additional permitting hurdles remain, but the company hopes to be the first to deploy a utility-scale, next-gen reactor in the U.S.
The company launched in 2006 and is building on technology used in an experimental breeder reactor in Idaho that operated for nearly 30 years before shutting down. Its Natrium reactor includes technology from TerraPower and GE Vernova Hitachi Nuclear Energy.
In June TerraPower disclosed $650 million in new funding from Gates, who helped start TerraPower, as well as the venture arm of chip giant NVIDIA. It previously raised more than $1 billion, including investments from Gates as well as South Korea-based SK Inc. and SK Innovation, according to PitchBook. TerraPower has additionally been awarded roughly $2 billion from the U.S. Department of Energy.
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by Kurt Schlosser on Jan 9, 2026 at 9:57 amJanuary 9, 2026 at 9:57 am
Microsoft co-founder Bill Gates paid his ex-wife Melinda French Gates nearly $8 billion as part of the settlement from the former couple's 2021 divorce.
The $7.88 billion payout was revealed in new tax filings and first reported by The New York Times in its DealBook Newsletter on Friday.
The money shows up as a 2024 donation to French Gates' Pivotal Philanthropies Foundation and is called “one of the largest charitable contributions ever publicly recorded” by the Times.
French Gates announced in May 2024 that she was resigning as co-chair of the Seattle-based Bill & Melinda Gates Foundation (now just Gates Foundation). She left the philanthropic organization with $12.5 billion for her own initiatives as part of her divorce agreement with Gates, and said at the time that she would commit that money to her work “on behalf of women and families.”
Pivotal Philanthropies Foundation was established in 2022, a year after the divorce.
The Times noted that the balance of the agreement — about $4.6 billion — could still be coming and would show up in 2025 tax filings later this year. It's also possible that the missing amount “was not given to a charitable foundation, but to an entity, such as French Gates's limited-liability company, Pivotal Ventures, that does not file a tax return,” the Times reported.
Pivotal confirmed to the Times that the $12.5 billion agreement has been fulfilled.
GeekWire reached out to representatives for Gates and French Gates and will update when we hear back.
Gates and French Gates announced the decision to end their 27-year marriage in May 2021. The Microsoft co-founder began dating Melinda French after she started at Microsoft in 1987. The two married on Jan. 1, 1994, raised three children together and grew into one of the world's richest and most influential pairings.
Gates is 13th on the Forbes Billionaire List with a net worth of $108 billion. French Gates is No. 77 on the list with a net worth of $29.4 billion.
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Meta today announced three deals to provide its data centers with nuclear power: one from a startup, one from a smaller energy company, and one from a larger company that already operates several nuclear reactors in the U.S.
Oklo and TerraPower, two companies developing small modular reactors (SMR), each signed agreements with Meta to build multiple reactors, while Vistra is selling capacity from its existing power plants.
Nuclear power has become a favored power source for tech companies as their AI ambitions have grown, providing stable 24/7 electricity. Startups and existing reactors have benefited from the race for data center power, though in different ways.
Existing reactors tend to be the cheapest form of baseload capacity, but there are only so many to go around, which has pushed Meta and its peers toward SMR startups. Companies like Oklo and TerraPower are betting that by building a large number of smaller reactors, they'll be able to bring the cost down through mass manufacturing. It's a plausible hypothesis, though one that has yet to be tested. Meta's deal could give SMR startups a chance to prove it.
The deals are the result of a request for proposals that Meta issued in December 2024, in which Meta sought partners that could add between 1 to 4 gigawatts of generating capacity by the early 2030s. Much of the new power will flow through the PJM interconnection, a grid which covers 13 Mid-Atlantic and Midwestern states and has become saturated with data centers.
The 20-year agreement with Vistra will have the most immediate impact on Meta's energy needs. The tech company will buy a total of 2.1 gigawatts from two existing nuclear power plants, Perry and Davis-Besse in Ohio.
As part of the deal, Vistra will also add capacity to those power plants and to its Beaver Valley power plant in Pennsylvania. Together, the upgrades will generate an additional 433 MW and are scheduled to come online in the early 2030s.
Meta is also buying 1.2 gigawatts from young provider Oklo. Under its deal with Meta, Oklo is hoping to start supplying power to the grid as early as 2030. The SMR company went public via SPAC in 2023, and while Oklo has landed a large deal with data center operator Switch, it has struggled to get its reactor design approved by the Nuclear Regulatory Commission.
If Oklo can deliver on its timeline, the new reactors would be built in Pike County, Ohio. The startup's Aurora Powerhouse reactors each produce 75 megawatts of electricity, and it will need to build more than a dozen to fulfill Meta's order.
TerraPower is a startup co-founded by Bill Gates, and it is aiming to start sending electricity to Meta as early as 2032. It has designed a reactor that uses molten sodium to transfer energy from reactor to generator. When demand is low, the superheated salt can be stored in an insulated vat until more power is needed. The reactor can generate 345 megawatts of electricity, while the storage system can provide an additional 100 to 500 megawatts for more than five hours.
The company has navigated the NRC process more smoothly, and it is working with GE Hitachi to build its first power plant in Wyoming. Its first two reactors for Meta would provide 690 megawatts, and Meta said it has rights to buy another six units for a total of 2.8 gigawatts of nuclear capacity and 1.2 gigawatts of storage.
Meta did not disclose financial terms of the deals.
The power purchases from Vistra are certain to be the cheapest — electricity from already operating nuclear reactors is among the cheapest on the grid.
Costs for SMRs still have yet to be worked out. Several startups have aggressive cost targets: TerraPower has estimated that it can bring it down to $50 to $60 per megawatt-hour, while Oklo has said it is aiming for $80 to $130 per megawatt-hour. Those figures are for later power plants — the first examples are likely to cost more.
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De Chant is also a lecturer in MIT's Graduate Program in Science Writing, and he was awarded a Knight Science Journalism Fellowship at MIT in 2018, during which time he studied climate technologies and explored new business models for journalism. He received his PhD in environmental science, policy, and management from the University of California, Berkeley, and his BA degree in environmental studies, English, and biology from St. Olaf College.
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On Friday, Elon Musk's social media platform X added the lightest of restrictions on its AI chatbot Grok following backlash over the AI tool being used to alter users' photos on X to generate sexually degrading deepfakes of women and children. Not everyone is buying it.
The chatbot now appears to users from generating or editing images when they tag Grok in an X post, unless they're a premium subscriber.
“Image generation and editing are currently limited to paying subscribers. You can subscribe to unlock these features,” the Grok chatbot, which is not the same as a spokesperson, posted Thursday on X in response to a user.
However, as The Verge first pointed out, that statement isn't entirely true. Grok's image tools are still available for free when users access the chatbot through the Grok website and app or the Grok tabs on the X app and website. Users can also instruct Grok to alter images by using the “Edit image” button on X's desktop website or by long-pressing on any image on its mobile app.
The idea of limiting the chatbot's image tools to paid subscribers on X as a solution has drawn sharp criticism. A spokesperson for Downing Street called the move “insulting to victims of misogyny and sexual violence.” The U.K., along with many other governments, has been quick to call on X to address its deepfake problem.
“The move simply turns an AI feature that allows the creation of unlawful images into a premium service,” the spokesperson told The Guardian.
Since late last month, some X users have been using Grok to generate sexualized images from photos posted by other users on the platform without their consent, including images involving minors.
A social media and deepfake researcher found that Grok generated about 6,700 sexually suggestive or nudifying images per hour over a 24-hour period in early January, Bloomberg reported on Wednesday.
The U.K.'s online regulator, Ofcom, said earlier this week that it contacted the company over the issue and warned it could open an investigation into whether X is complying with the country's laws.
The European Commission is also looking into whether X is complying with its laws and has ordered X to retain all internal documents relating to Grok until the end of the year.
Meanwhile, Sen. Ron Wyden told Gizmodo that AI chatbots are not covered under Section 230, a law that shields online platforms from liability for illegal conduct by users.
“As I've said before, AI chatbots are not protected by Section 230 for content they generate, and companies should be held fully responsible for the criminal and harmful results of that content. States must step in to hold X and Musk accountable if Trump's DOJ won't,” Wyden said.
This isn't the first time Grok has caused problems for X. Last year, an update meant to address what Musk described as a “center-left bias” instead led Grok to generate antisemitic propaganda, even referring to itself as “MechaHitler.”
And these controversies don't appear to be helping the company's bottom line. Bloomberg reports that xAI, the parent company of X and Grok, reported a net loss of $1.46 billion for the quarter ending in September and burned through $7.8 billion in the first nine months of the year.
X has been facing its own financial fallout. The company's U.K. revenue fell nearly 60% in 2024 as advertisers fled the platform, according to The Guardian.
While it may seem bizarre that Musk hasn't taken stronger action to address the controversies, none of it appears to have slowed investor enthusiasm. xAI announced this week that it raised $20 billion in its most recent funding round.
Contacted for comment, xAI responded to Gizmodo with an email saying, “Legacy Media Lies.”
X, meanwhile, pointed to a statement it posted on January 3.
“We take action against illegal content on X, including Child Sexual Abuse Material (CSAM), by removing it, permanently suspending accounts, and working with local governments and law enforcement as necessary,” the company's Safety account posted on X. “Anyone using or prompting Grok to make illegal content will suffer the same consequences as if they upload illegal content.”
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by Kurt Schlosser on Jan 9, 2026 at 8:39 amJanuary 9, 2026 at 8:39 am
A bill introduced in the Washington state Legislature would ban employers from requiring or pressuring workers to be microchipped, a practice lawmakers want to prohibit before it ever becomes an issue.
House Bill 2303 was prefiled this week by Reps. Brianna Thomas (D-34) and Lisa Parshley (D-22).
The bill would prohibit employers from requiring, requesting or coercing employees to have microchips implanted in their bodies as a condition of employment, and would bar the use of subcutaneous tracking or identification technology for workplace management or surveillance.
It aims to protect worker privacy and bodily autonomy by establishing strict penalties for violations, including civil penalties starting at $10,000 and the right for aggrieved workers to sue for damages and injunctive relief.
While there's no known instance of an employer seeking such action, Thomas told GeekWire the bill is a preemptive move.
“We are getting out ahead of the problem because the practice of requiring these chips is too dangerous to wait for it to show up in Washington,” she said Thursday via email. “An employee with a microchip stops being an employee — they are essentially being dehumanized into corporate equipment.”
The Carnegie Council for Ethics in International Affairs reported that internationally, more than 50,000 people have elected to receive microchip implants to serve as their swipe keys, credit cards, and more. The organization noted that the technology is especially popular in Sweden, where chip implants are more widely accepted for gym access, e-tickets on transit systems, and to store emergency contact information.
HB 2303 would add a new section to Chapter 49.44 of the Revised Code of Washington (RCW), titled “Violations — Prohibited Practices.” The chapter serves as a catch-all for labor regulations that define and prohibit specific unfair or illegal activities by employers, employees, and labor representatives.
The legislation is similar to laws passed in Arkansas, California, Missouri, Montana, Nevada, New Hampshire, North Dakota, Oklahoma, Utah, Wisconsin, Indiana, Alabama, and Mississippi.
“Workers cannot legitimately consent to a program because of the power dynamic between them and the employer,” Thomas said. “Implanted chips have no place in a work environment.”
Nevada is “arguably the most restrictive” on microchip implants and permanent identification markers, according to the Carnegie Council. Its law prohibits people from voluntarily electing to receive such markers in Nevada.
Thomas said HB 2303 does not go as far as Nevada's restrictions, noting that workers would still be free to make their own choices outside the workplace.
Thomas said she believes companies will eventually pitch the technology to their employees by telling them it's more convenient and easier — you don't have to worry about forgetting your work access badge, etc.
“Many times convenience causes people to view things too narrowly and they don't see the big picture,” she said. “The power dynamic between an employer and an employee makes true, uncoerced consent impossible. This is about making sure workers not only have the option but also consider all the factors when these programs are presented to them.”
The Carnegie Council also reported on the privacy, data security, and health safety concerns that microchips present, including from technologists who worry about IoT vulnerabilities in sensors and network architecture that could be exploited by hackers.
While the Washington proposal targets simple Radio Frequency Identification (RFID) tags, a more sophisticated wave of “brain-computer interfaces” (BCIs) is rapidly moving toward the mainstream.
Elon Musk wants to ramp up production of his Neuralink brain‑computer interface chips in 2026. He envisions the technology helping people with neurological conditions while eventually enabling humans to interact directly with computers. The company plans to make the surgical implantation process nearly fully automated to scale the procedure.
Washington's HB 2303 is scheduled for a public hearing Jan. 14 in the House Committee on Labor & Workplace Standards.
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After creating thousands of “undressing” pictures of women and sexualized imagery of apparent minors, Elon Musk's X has apparently limited who can generate images with Grok. However, despite the changes, the chatbot is still being used to create “undressing” sexualized images on the platform.
On Friday morning, the Grok account on X started responding to some users' requests with a message saying that image generation and editing are “currently limited to paying subscribers.” The message also includes a link pushing people toward the social media platform's $395 annual subscription tier. In one test of the system requesting Grok create an image of a tree, the system returned the same message.
The apparent change comes after days of growing outrage against and scrutiny of Musk's X and xAI, the company behind the Grok chatbot. The companies face an increasing number of investigations from regulators around the world over the creation of nonconsensual explicit imagery and alleged sexual images of children. British prime minister Keir Starmer has not ruled out banning X in the country and said the actions have been “unlawful.”
Neither X nor xAI, the Musk-owned company behind Grok, has confirmed that it has made image generation and editing a paid-only feature. An X spokesperson acknowledged WIRED's inquiry but did not provide comment ahead of publication. X has previously said it takes “action against illegal content on X,” including instances of child sexual abuse material. While Apple and Google have previously banned apps with similar “nudify” features, X and Grok remain available in their respective app stores. xAI did not immediately respond to WIRED's request for comment.
For more than a week, users on X have been asking the chatbot to edit images of women to remove their clothes—often asking for the image to contain a “string” or “transparent” bikini. While a public feed of images created by Grok contained far fewer results of these “undressing” images on Friday, it still created sexualized images when prompted to by X users with paid for “verified” accounts.
“We observe the same kind of prompt, we observe the same kind of outcome, just fewer than before,” Paul Bouchaud, lead researcher at Paris-based nonprofit AI Forensics, tells WIRED. “The model can continue to generate bikini [images],” they say.
A WIRED review of some Grok posts on Friday morning identified Grok generating images in response to user requests for images that “put her in latex lingerie” and “put her in a plastic bikini and cover her in donut white glaze.” The images appear behind a “content warning” box saying that adult material is displayed.
On Wednesday, WIRED revealed that Grok's stand-alone website and app, which is separate from the version on X, has also been used in recent months to create highly graphic and sometimes violent sexual videos, including celebrities and other real people. Bouchaud says it is still possible to use Grok to make these videos. “I was able to generate a video with sexually explicit content without any restriction from an unverified account,” they say.
While WIRED's test of image generation using Grok on X using a free account did not allow any images to be created, using a free account on Grok's app and website still generated images.
The change on X could immediately limit the amount of sexually explicit and harmful material the platform is creating, experts say. But it has also been criticized as a minimal step that acts as a band-aid to the real harms caused by nonconsensual intimate imagery.
“The recent decision to restrict access to paying subscribers is not only inadequate—it represents the monetization of abuse,” Emma Pickering, head of technology-facilitated abuse at UK domestic abuse charity Refuge, said in a statement. “While limiting AI image generation to paid users may marginally reduce volume and improve traceability, the abuse has not been stopped. It has simply been placed behind a paywall, allowing X to profit from harm.”
The British government also said, according to reporting from the BBC, that the change to limit image generation to paid-only accounts is “insulting” to those who have been impacted. It said that it “simply turns an AI feature that allows the creation of unlawful images into a premium service.”
“While it may allow X to share information with law enforcement about perpetrators, it doesn't address the fundamental issue of the model's capabilities and alignment,” says Henry Ajder, a deepfake expert who has tracked harmful uses of the technology for years. “For the cost of a month's membership, it seems likely I could still create the offending content using a fake name and a disposable payment method.”
“They could have removed abusive material, but they did not,” AI Forensics' Bouchaud says. “They could have disabled Grok to generate images altogether, but they did not. They could have disabled the Grok application to generate pornographic videos.”
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Elon Musk's AI company has restricted Grok's controversial AI image-generation feature to only paying subscribers on X, after the tool invited heavy criticism from across the world for letting users generate sexualized and nude images of women and children.
In replies to users on Friday, Grok said only paying subscribers on X would be able to generate and edit images on the platform. Notably, these limits do not apply to the Grok app, which, at the time of publication was letting anyone generate pictures without having to pay for a subscription.
Initially available to anyone with daily limits, Grok's image-generation feature allowed users to upload anyone's picture and ask it to edit it or generate a sexualized or nude version. What ensued was a veritable flood of non-consensual sexualized images of children, actors, models and prominent figures, drawing the ire of multiple nations.
X and Musk have both publicly denounced the use of the tool to produce such images, writing that the company would stick to its policies against posting illegal content on the social media platform. “Anyone using grok to make illegal content will suffer the same consequences as if they upload illegal content,” Musk tweeted last week.
The U.K., the European Union, and India have all publicly denounced X and Grok for allowing such use of its capabilities. The EU on Thursday asked xAI to retain all documentation relating to the chatbot, and India's communications ministry last week ordered X to make immediate changes to stop the image-generation features from being misused or risk its safe harbor protections in the country. The U.K.'s communications watchdog said it's been in touch with xAI over the issue as well.
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Andreessen Horowitz just announced the firm has raised a little more than $15 billion in new funding. The haul represents over 18% of all venture capital dollars allocated in the United States in 2025, according to firm co-founder Ben Horowitz, but even more jaw-dropping is that it brings the organization to more than $90 billion in assets under management, putting it neck-and-neck with Sequoia Capital as among the largest venture firms in the world. Which is fitting, since a16z appears to be very friendly with actual sovereign wealth funds, including at least one from Saudi Arabia.
The firm, which employs approximately hundreds of people across five offices — three in California, plus New York and Washington, D.C. — has become a globe-spanning operation with employees on six continents. In December, it opened its first Asia office in Seoul for its crypto practice.
That newly committed capital breaks down across five funds: $6.75 billion for growth investments, $1.7 billion each for apps and infrastructure, $1.176 billion for “American Dynamism” (more on that shortly), $700 million for biotech and healthcare, and another $3 billion for other venture strategies. It's the kind of money that makes you wonder where it all comes from and, more importantly, where it all goes.
The “where it comes from” question is one the firm has historically declined to answer. When we asked a16z this week about its limited partners and its distributed-to-paid-in capital ratio — the DPI, or how much actual cash the firm has returned to investors over its 16-year history — the firm didn't respond. What we do know is that CalPERS invested $400 million in 2023, marking the first time in a16z's history it took money from a major California pension fund, probably because institutions with transparency requirements don't really align with the firm's preference for opacity. We also know that Sanabil Investments, the venture arm of Saudi Arabia's Public Investment Fund, lists Andreessen Horowitz among its portfolio holdings.
The Saudi connection isn't subtle. Back in 2023, Marc Andreessen and Ben Horowitz appeared onstage with WeWork co-founder Adam Neumann to discuss their $350 million investment in his then-new residential real estate venture, Flow. The venue was a conference backed by one of Saudi Arabia's largest sovereign funds. Horowitz praised Saudi Arabia as a “startup country,” adding that “Saudi has a founder; you don't call him a founder, you call him his royal highness.”
But Marc Andreessen has found another royal to admire. Since President Donald Trump's November 2024 election victory, Andreessen has logged a lot of hours at Mar-a-Lago, by his own account, helping shape policy on tech, business, and economics. Early last year, he became an “unpaid intern” at Elon Musk's Department of Government Efficiency, vetting candidates for the Trump administration — not just for tech roles but for positions in the Defense Department and intelligence agencies. Scott Kupor, a16z's first employee back in 2009, was sworn in as director of the U.S. Office of Personnel Management this past summer.
This matters because a16z's current strategy is heavily weighted toward what it calls “American Dynamism” — a practice that invests in defense, aerospace, public safety, housing, education, and manufacturing. The portfolio aligns remarkably well with Defense Department priorities: Anduril (autonomous defense systems), Shield AI (military drones), Saronic Technologies (autonomous naval vessels), and Castelion (hypersonic missiles). The bigger bet is that America needs to reindustrialize and reshore critical manufacturing, particularly since, as a16z itself notes, the U.S. would exhaust its entire missile inventory “in something like 8 days” in a conflict with China over Taiwan, then need three years to rebuild it.
Then there's the AI bet, which might be the firm's highest-risk, highest-reward play yet. a16z has positioned itself across every level of the AI stack: infrastructure (Databricks), foundation models (with stakes in Mistral AI, OpenAI and xAI), and applications (Character.AI, among many other portfolio companies).
The firm has wins to point to. Its $25 million investment in Coinbase turned into an $86 billion valuation at the 2021 IPO. There's Airbnb (public at over $100 billion), Slack (acquired for $27.7 billion), and GitHub ($7.5 billion to Microsoft). Its portfolio includes 115 unicorns, 35 IPOs, and 241 acquisitions, according to the market intelligence firm Tracxn. The firm has also made and lost money by snapping up cryptocurrency tokens, though there's less visibility into those numbers.
In a blog post published Friday morning, Ben Horowitz writes that “as the American leader in Venture Capital, the fate of new technology in the United States rests partly on our shoulders.” It's the kind of statement certain to cause agita at rival firms, some of which have been around closer to 50 years, compared with the much younger a16z. Horowitz frames a16z's mission as “ensuring that America wins the next 100 years of technology.”
Whether that happens remains to be seen. What's certain is that Andreessen Horowitz has mastered the art of raising money — $15 billion this time — to fund a vision of American technological dominance that runs through Riyadh, Mar-a-Lago, and the Pentagon. That's quite a pitch, and plainly, it works.
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Stranger Things‘ Upside Down was always a mysterious place—but fans were certain of a few key things. One is that the Upside Down is known to be crawling with vicious beasties, including the show's most famous pre-Vecna villain, the Demogorgon.
In season five we learned the Upside Down functions as a wormhole connecting Earth to the Abyss, and it's from that angry realm that the monsters actually originated. So fans wondered: where were all the demo-beasts when Mike, Nancy, Will, and company barged into their homeworld in the big finale?
It's not the most vexing question to linger after “The Rightside Up,” but it does feel like something that should be addressed. In a new interview with Variety, the director of Netflix's upcoming documentary about the making of Stranger Things season five explained the lack of Demogorgons wasn't an oversight—it was something the Duffer Brothers thought the fans would actually appreciate.
In fact, the documentary includes a scene in the writers' room where the issue is discussed outright.
“I like that moment, and I like the conversation, because it obviously changed, right? And so having them sit there and really explore that—to me, the writers' room is so fascinating, because you see them thinking,” director Martina Radwan said.
“I love that they were so sure about the Demogorgons in the final Abyss—except Ross was like, ‘Mmm, maybe there's some fatigue, Demogorgon fatigue.' And Kate [Trefry] agreed. And you're like, “Oh, OK—so this is not the last conversation. They disagree, and they will resolve it on some point.' We, in the doc, left it open because at the time the doc comes out, everybody knows there are no Demos in the Pain Tree.”
No Demos in the Pain Tree should have been the documentary title, but alas, it's called One Last Adventure: The Making of Stranger Things 5. It arrives on Netflix January 12.
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As age-verification laws continue to dismantle the adult industry—and determine the future of free speech on the internet—a Utah lawmaker proposed a bill this week that would enforce a tax on porn sites that operate within the state.
Introduced by state senator Calvin Musselman, a Republican, the bill would impose a 7 percent tax on total receipts “from sales, distributions, memberships, subscriptions, performances, and content amounting to material harmful to minors that is produced, sold, filmed, generated, or otherwise based” in Utah. If passed, the bill would go into effect in May and would also require adult sites to pay a $500 annual fee to the State Tax Commission. Per the legislation, the money made from the tax will be used by Utah's Department of Health and Human Services to provide more mental health support for teens.
Musselman did not respond to a request for comment.
A new age of American conservatism commands the political arena, and more US lawmakers are calling for additional restrictions on adult content. In September, Alabama became the first state to impose a porn tax on adult entertainment companies (10 percent) following the passage of age-verification mandates, which require users to upload an ID or other personal documentation to verify that they are not a minor before viewing sexually explicit content. Pennsylvania lawmakers are also eyeing a bill that would tax consumers an additional 10 percent on “subscriptions to and one-time purchases from online adult content platforms,” despite already requiring them to pay a 6 percent sales and use tax for the purchase of digital products, two state senators wrote in a memo in October. Other states have flirted with the idea of a porn tax in the past. In 2019, Arizona state senator Gail Griffin, a Republican, proposed taxing adult content distributors to help fund the border wall, a key priority during Donald Trump's first presidential term. So far, 25 US states have passed a form of age verification.
Although efforts to criminalize participants in the sex work industry have been ongoing for years—with new regulations unfolding at a moment of heightened online surveillance and censorship—targeted taxes have failed to gain widespread approval because the legality around such laws are up for debate.
“This kind of porn tax is blatantly unconstitutional,” says Evelyn Douek, an associate professor of law at Stanford Law School. “It singles out a particular type of protected speech for disfavored treatment, purely because the legislature doesn't like it—that's exactly what the First Amendment is designed to protect against. Utah may not like porn, but as the Supreme Court affirmed only last year, adults have a fully protected right to access it.”
Utah, Alabama, and Pennsylvania are among the 16 states that have adopted resolutions declaring porn a public health crisis. “We realize this is a bold assertion not everyone will agree on, but it's the full-fledged truth,” Utah governor Gary Herbert tweeted in 2016 after signing the resolution. One of Utah's earliest statewide responses to the proliferation of adult content happened in 2001, when it became the first state to create an office for sexually explicit issues by hiring an obscenity and pornography complaints ombudsman. The position—dubbed the “porn czar”—was terminated in 2017.
“Age restriction is a very complex subject that brings with it data privacy concerns and the potential for uneven and inconsistent application for different digital platforms,” Alex Kekesi, vice president of brand and community at Pornhub, told WIRED in a previous conversation. In November, the company urged Google, Microsoft, and Apple to enact device-based verification in their app stores and across their operating systems. “We have seen several states and countries try to impose platform-level age verification requirements, and they have all failed to adequately protect children.” To comply with the new age gate mandates, Pornhub has currently blocked access to users in 23 states.
Critics argue that age verification has never been about protecting children but rather scrubbing porn from the internet. A video leaked in 2024 by the Centre for Climate Reporting showed Russell Vought, a Trump ally and Project 2025 coauthor, calling age verification laws a “back door” tactic to a federal porn ban.
Sites like OnlyFans and Pornhub have brought platform-dependent sex work into the mainstream, but they have also made it easier to police adult entertainers and consumers. As more states begin to implement added tariffs on sex work, creators will bear the brunt of the new laws more than anyone.
The skewed ideology of cultural conservatism that is taking shape under Trump 2.0 wants to punish sexual expression, says Mike Stabile, director of public policy at the Free Speech Coalition, a trade association for the adult industry in the US. “When we talk about free speech, we generally mean the freedom to speak, the ability to speak freely without government interference. But in this case, free also means not having to pay for the right to do so. A government tax on speech limits that right to those who can afford it.”
According to company policy, OnlyFans complies with all tax requirements in the jurisdictions in which it operates. Creators are responsible for their own tax affairs. Pornhub, which is currently blocked in Utah and Alabama, did not respond to a request for comment.
Douek notes that following the Supreme Court's decision to uphold age-verification laws in Texas, states can legally regulate minors' access to sexually explicit material, “but a porn tax does nothing to limit minors' access to this speech—it simply makes it more expensive to provide this content to adults.” A 2022 report from Common Sense Media, a youth advocacy nonprofit, found that 73 percent of teens age 13 to 17 have watched adult content online. Today, young people regularly access NSFW content via social media, on platforms like X and Snap. Last year, a survey by the UK's Office of the Children's Commissioner reported that 59 percent of minors are being exposed to porn by accident, primarily via social media, up from 38 percent in 2023.
In Alabama, as would be the case with Utah, revenue raised by the tax is being used for behavioral health services, including prevention, treatment, and recovery support for young people.
Alabama state representative Ben Robbins, the bill's Republican sponsor, said in an interview last year that adult content was “a driver in causing mental health issues” in the state. It's a common argument among lawmakers pushing for a nationwide porn ban. Some scientific studies suggest that adolescent exposure to porn increases rates of depression, low self-esteem, and normalized violence, but health professionals have never reached a consensus on the matter.
With lawmakers working to reframe the issue around underage harm, Stabile says it's critical to remember that adult content isn't different from any other kind of protected speech, noting that content-specific taxes on speech have repeatedly been struck down by the courts as unconstitutional censorship.
“What if a state decided that Covid misinformation was straining state health resources and taxed newsletters who promoted it? What if the federal government decided to require a costly license to start a podcast? What if a state decided to tax a certain newspaper it didn't like?” he says. “Porn isn't some magical category of speech separate from movies, streaming services, or other forms of entertainment. Adult businesses already pay taxes on the income they earn, just as every other business does. Taxing them because of imagined harms is not only dangerous to our industry, it sets a dangerous precedent for government power.”
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Last February, a sick infant named KJ received a gene-editing treatment made just for him. Created in just six months, it was meant to correct a rare genetic mutation that was causing toxic ammonia to build up in his small body. The treatment likely saved his life, and baby KJ was discharged from the hospital in June.
Now, a new startup called Aurora Therapeutics, cofounded by gene-editing pioneer Jennifer Doudna, is aiming to scale such treatments to many more patients with rare diseases. Doudna is one of the inventors of the gene-editing system known as Crispr, and won a Nobel Prize in 2020 for her work on the technology.
Aurora plans to take advantage of a new regulatory pathway announced by Food and Drug Administration officials Marty Makary and Vinay Prasad in the fall. The new program, called the “plausible mechanism pathway,” allows the FDA to approve personalized treatments for rare and fatal diseases based on data from just a handful of patients, according to Makary and Prasad in a New England Journal of Medicine article.
Typically, new drugs must be tested in hundreds, if not thousands, of patients in order to get regulatory approval. For drug trials of rare diseases, it's difficult to recruit that many patients because so few people have the disease. The new FDA pathway provides a way for these types of drugs to be approved when a large, randomized trial isn't possible.
“Once a manufacturer has demonstrated success with several consecutive patients with different bespoke therapies, the FDA will move toward granting marketing authorization for the product,” Makary and Prasad say in their article. Drug companies will then be able to use data from those patients to get similar drugs approved that are based on the same underlying technology.
That is key for Aurora, which will initially focus on treating a metabolic disorder called phenylketonuria, or PKU, that's screened for at birth. The disease leads to toxic levels of phenylalanine, a building block of protein, in the blood. Patients with PKU must eat a highly restrictive low-protein diet. Without early treatment and monitoring, PKU can hinder brain development and impair cognitive functions. An estimated 13,500 people in the US are living with the disease.
“There are a lot of patients that could benefit from this therapy. But the problem is, you have many, many mutations—over a thousand—that cause this disease,” says Edward Kaye, CEO of Aurora Therapeutics and a pediatric neurologist.
Crispr works by using a guide RNA to deliver an editing molecule to a desired location in the genome. The guide RNA is like a car's GPS—it goes where it's programmed to go. In the case of baby KJ, scientists built a guide RNA to target his specific genetic mutation. It's why his treatment only works for him.
Aurora's strategy involves swapping out that guide RNA to make several versions of a PKU therapy that address different mutations. Previously, the FDA would have considered every version a totally new drug, each requiring its own clinical trial. But now, Aurora will be able to use the same technology platform to treat many mutations that cause PKU with less regulatory red tape.
Kaye says the company will use base editing, a more precise form of Crispr, and will have a standardized process to streamline the design and manufacturing of its therapies.
“We are very much about no mutation left behind,” says Fyodor Urnov, Aurora's cofounder and a genome editing scientist at UC Berkeley. Urnov and several of his colleagues at Berkeley's Innovative Genomics Institute, which Doudna established in 2015, were involved in designing baby KJ's treatment.
The Innovative Genomics Institute will continue to create bespoke gene-editing therapies for children with very rare diseases, while a trial at the Children's Hospital of Philadelphia and Penn Medicine, where baby KJ was treated, will test the same type of gene editor used in KJ's therapy in a group of similar disorders. He says establishing Aurora was necessary so that these treatments can eventually be available to many more patients.
So far, Crispr has yet to live up to its transformative potential. Several Crispr companies have downsized, and others have shut down in recent years. So far, there is only one approved drug on the market that uses Crispr technology. Called Casgevy, it debuted in December 2023 at $2.2 million to treat sickle cell disease and beta thalassemia, a related blood disorder.
Yet Urnov thinks the field is turning a corner as the technology matures.
“We are finally at a place where Crispr on demand has had all the technical problems worked out,” Urnov says. “I can say with reasonable certainty that, three to four years from now, there will be other children with their personalized editors.”
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Aurora Therapeutics' first target is the rare inherited disease phenylketonuria, also known as PKU.
Here at MIT Technology Review we've been writing about the gene-editing technology CRISPR since 2013, calling it the biggest biotech breakthrough of the century. Yet so far, there's been only one gene-editing drug approved. It's been used commercially on only about 40 patients, all with sickle-cell disease.
It's becoming clear that the impact of CRISPR isn't as big as we all hoped. In fact, there's a pall of discouragement over the entire field—with some journalists saying the gene-editing revolution has “lost its mojo.”
So what will it take for CRISPR to help more people? A new startup says the answer could be an “umbrella approach” to testing and commercializing treatments. Aurora Therapeutics, which has $16 million from Menlo Ventures and counts CRISPR co-inventor Jennifer Doudna as an advisor, essentially hopes to win approval for gene-editing drugs that can be slightly adjusted, or personalized, without requiring costly new trials or approvals for every new version.
The need to change regulations around gene-editing treatments was endorsed in November by the head of the US Food and Drug Administration, Martin Makary, who said the agency would open a “new” regulatory pathway for “bespoke, personalized therapies” that can't easily be tested in conventional ways.
Aurora's first target, the rare inherited disease phenylketonuria, also known as PKU, is a case in point. People with PKU lack a working version of an enzyme needed to use up the amino acid phenylalanine, a component of pretty much all meat and protein. If the amino acid builds up, it causes brain damage. So patients usually go on an onerous “diet for life” of special formula drinks and vegetables.
In theory, gene editing can fix PKU. In mice, scientists have already restored the gene for the enzyme by rewriting DNA in liver cells, which both make the enzyme and are some of the easiest to reach with a gene-editing drug. The problem is that in human patients, many different mutations can affect the critical gene. According to Cory Harding, a researcher at Oregon Health Sciences University, scientists know about 1,600 different DNA mutations that cause PKU.
There's no way anyone will develop 1,600 different gene-editing drugs. Instead, Aurora's goal is to eventually win approval for a single gene editor that, with minor adjustments, could be used to correct several of the most common mutations, including one that's responsible for about 10% of the estimated 20,000 PKU cases in the US.
“We can't have a separate clinical trial for each mutation,” says Edward Kaye, the CEO of Aurora. “The way the FDA approves gene editing has to change, and I think they've been very understanding that is the case.”
A gene editor is a special protein that can zero in on a specific location in the genome and change it. To prepare one, Aurora will put genetic code for the editor into a nanoparticle along with a targeting molecule. In total, it will involve about 5,000 gene letters. But only 20 of them need to change in order to redirect the treatment to repair a different mutation.
“Over 99% of the drug stays the same,” says Johnny Hu, a partner at Menlo Ventures, which put up the funding for the startup.
The new company came together after Hu met over pizza with Fyodor Urnov, an outspoken gene-editing scientist at the University of California, Berkeley, who is Aurora's cofounder and sits on its board.
In 2022, Urnov had written a New York Times editorial bemoaning the “chasm” between what editing technology can do and the “legal, financial, and organizational” realities preventing researchers from curing people.
“I went to Fyodor and said, ‘Hey, we're getting all these great results in the clinic with CRISPR, but why hasn't it scaled?” says Hu. Part of the reason is that most gene-editing companies are chasing the same few conditions, such as sickle-cell, where (as luck would have it) a single edit works for all patients. But that leaves around 400 million people who have 7,000 other inherited conditions without much hope to get their DNA fixed, Urnov estimated in his editorial.
Then, last May, came the dramatic demonstration of the first fully “personalized” gene-editing treatment. A team in Philadelphia, assisted by Urnov and others, succeeded in correcting the DNA of a baby, named KJ Muldoon, who had an entirely unique mutation that caused a metabolic disease. Though it didn't target PKU, the project showed that gene editing could theoretically fix some inherited diseases “on demand.”
It also underscored a big problem. Treating a single child required a large team and cost millions in time, effort, and materials—all to create a drug that would never be used again.
That's exactly the sort of situation the new “umbrella” trials are supposed to address. Kiran Musunuru, who co-led the team at the University of Pennsylvania, says he's been in discussions with the FDA to open a study of bespoke gene editors this year focusing on diseases of the type Baby KJ had, called urea cycle disorders. Each time a new patient appears, he says, they'll try to quickly put together a variant of their gene-editing drug that's tuned to fix that child's particular genetic problem.
Musunuru, who isn't involved with Aurora, does not think the company's plans for PKU count as fully personalized editors. “These corporate PKU efforts have nothing whatsoever to do with Baby KJ,” he says. He says his center continues to focus on mutations “so ultra-rare that we don't see any scenario where a for-profit gene-editing company would find that indication to be commercially viable.”
Instead, what's occurring in PKU, says Musunuru, is that researchers have realized they can assemble “a bunch” of the most frequent mutations “into a large enough group of patients to make a platform PKU therapy commercially viable.”
While that would still leave out many patients with extra-rare gene errors, Musunuru says any gene-editing treatment at all would still be “a big improvement over the status quo, which is zero genetic therapies for PKU.”
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Jonathan Ross, the Immigration and Customs Enforcement officer identified by multiple news outlets as the federal agent who shot 37-year-old Renee Good in Minneapolis on Wednesday, is a veteran deportation officer in ICE's Enforcement and Removal Operations division, according to sworn testimony from the federal district court in Minnesota obtained by WIRED. A member of a Special Response Team, ICE's version of a SWAT team, he's had duties as a firearms trainer and led teams drawn from multiple federal agencies including the FBI, Ross testified.
The testimony stems from a December 2025 trial related to a June incident with parallels to the interaction that led to Good's killing.
In June, according to Ross's testimony, he led a team seeking to apprehend a man named Roberto Carlos Muñoz-Guatemala, who was on an administrative warrant for being in the United States without authorization. Because the man's home was across from a school and immigration agents had no authority to enter his home, Ross testified, they instead trailed him in unmarked vehicles.
Muñoz-Guatemala's attorney did not immediately reply to a request for comment.
According to the December testimony and a New York Times account of an FBI agent's affidavit associated with the case, Ross approached Muñoz-Guatemala and asked him to roll down his window and open his door. Ross, who testified that he had been driving an unmarked vehicle, was dressed in ranger green and gray, and wore his badge on his belt, broke the driver's side back window and reached into the vehicle, at which point Muñoz-Guatemala pulled away.
While being dragged at a speed he claimed seemed like “40 miles an hour at least, if not more,” Ross pulled out his Taser and fired it at the driver. Muñoz-Guatemala continued to drive and succeeded in shaking Ross from the car. At trial, Ross testified that he suffered injuries that required 33 stitches.
According to the affidavit, Muñoz-Guatemala called 911 to report that he'd been assaulted by ICE, which led to his arrest. Last month, he was convicted of assault on a federal officer with a dangerous weapon.
Reports from the Minnesota Star-Tribune, The Intercept, and The Guardian identified Ross as the shooter who killed Good, a mother and recent transplant to Minneapolis, during an immigration enforcement action in the city. Video of the incident appears to show a federal agent firing shots into Good's vehicle as she attempted to leave the scene. The officer did not appear to have been struck by the vehicle, and Good appeared to be turning the wheel to avoid contact, video analysis by The New York Times and The Washington Post shows.
At Thursday's White House press briefing, vice president JD Vance answered questions about the incident, and his responses included numerous identifying details about Ross, mainly relating to his interaction with Muñoz-Guatemala. “That very ICE officer nearly had his life ended, dragged by a car, six months ago, 33 stitches in his leg,” said Vance, “so you think maybe he is a little bit sensitive about somebody ramming him with an automobile?”
Department of Homeland Security secretary Kirsti Noem has repeatedly described Good's actions as an intentional act of “domestic terrorism.” An FBI investigation into Good's killing is ongoing.
DHS spokesperson Tricia McLaughlin told WIRED in a statement that the department is “not going to expose the name of this officer. He acted according to his training.” McLaughlin added that federal immigration agents “are under constant threat from violent agitators” because of “doxxing” and that the Minnesota Star Tribune, which first published Ross' name, “should delete their story immediately.”
According to Ross' December testimony, he served in the Indiana National Guard and was deployed to Iraq from 2004 to 2005 as a machine gunner on a patrol truck, then joined Border Patrol in 2007 after finishing college, working near El Paso, Texas.
“I did normal Border Patrol duties,” Ross testified, “including line-watch operations, tracking, and I also was a field intelligence agent.” In that latter role, he testified, Ross “compiled and analyzed information from raw information, creating an intelligence product and focusing more so on the cartels and drug smuggling and also alien smuggling.”
In 2015, according to his testimony, Ross joined ICE, working in the ERO division, where he was tasked with targeting “higher-value targets” as a deportation officer in the Twin Cities area. A member of a joint anti-terrorism task force with the FBI, he testified that he's a “team leader” who, on a typical operation, oversees two FBI agents and an IRS or ATF agent.
“I develop the targets, create a target package, surveillance, and then develop a plan to execute the arrest warrant,” he testified. He also described what he termed “collateral” duties he's carried out in addition to deportation work.
“I am a firearms instructor, an active shooter instructor,” he testified. “I'm also a field intelligence officer, and I am a member of the SWAT team, the St. Paul Special Response Team.”
In his testimony, Ross said that he had made hundreds of vehicle stops in his career and generally described people who attempt to flee. “They do erratic behaviors,” he testified. “They take great risks, and they seem to not be aware of other people driving on the road. They usually—they make just extreme movements with their vehicles.”
In his testimony, Ross claimed that after he approached Muñoz-Guatemala, the man asked for his attorney.
In court, Eric Newmark, who was representing Muñoz-Guatemala, noted that even government lawyers hadn't previously heard this assertion. “I think he just made it up on the stand,” Newmark told the judge. “He never said it before. I think he said it for a particular reason”—allegedly to show that Muñoz-Guatemala was aware that he was being apprehended by law enforcement and not attacked by a masked carjacker. A prosecutor in the case conceded that the claim was “grounds for impeachment” against Ross because he “did not previously say that—as far I know.”
Under cross-examination by Newark, Ross testified that people he encounters often “act like they're confused,” implying that they do know he's a federal agent even if they appear not to.
“I believe it's—it seems to be something that some people just—just say to—to stall,” he testified. “I believe a lot of time people are on the phone and they're waiting for people to get—to show up, especially with our line of work.
“They've got phone trees where they call and then protesters show up.”
Updated 8:30 am ET, January 9, 2026: Added citation to The Intercept, which identified Ross following the Minnesota Star-Tribune and first reported on a Facebook photo captioned “Jon Ross in Iraq.”
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Zaida Álvarez Pinto, Principal Investigator at the Institute for Bioengineering of Catalonia (IBEC), has been awarded an ERC Consolidator Grant, one of the most prestigious and competitive sources of funding in the European Union. This research grant, awarded by the European Research Council (ERC) supports excellent scientists and scholars at the career stage where they are consolidating their independent research teams to pursue their most promising scientific ideas.
Álvarez is part of the 11,3% of selected candidates across Europe, chosen from among 3121 proposals received in this call.
She began her scientific trajectory at IBEC, completing her PhD under the supervision of Elisabeth Engel. She later moved to Northwestern University (USA), where she continued her work first as a Postdoctoral Researcher and subsequently as an Assistant Professor. In 2022, she returned to IBEC through a Ramón y Cajal fellowship, and just two years later, she was appointed Junior Group Leader. She currently leads the Biomaterials for Neural Regeneration group as a Ramón y Cajal Researcher.
The project to be developed with this new funding is named Engineered Humanized Spinal Cord Construct for Advancing Health and Disease Research (SPINECRAFT).
Current laboratory models of the central nervous system (CNS) struggle to replicate its extraordinary complexity, limiting progress in understanding neurological and neurodegenerative diseases. Traditional 2D cultures, 3D organoids and animal models fall short of capturing the intricate structure and dynamic function of the CNS, slowing advances in neuroscience. SPINECRAFT aims to change this. The project will create a cutting-edge, 4D human spinal cord construct that mirrors the architecture and functionality of the real spinal cord. By combining advanced imaging, computational modelling, bioprinting and innovative bioinks, researchers will fabricate a high-fidelity model populated with human neural and vascular cells. This platform will not only enable detailed studies of spinal cord biology but also integrate patient-derived cells to recreate disease-specific environments, such as those seen in amyotrophic lateral sclerosis (ALS).
This recognition marks a pivotal moment for our research and for the field of spinal cord regeneration. With SPINECRAFT, we expect to generate a high-fidelity human model that will allow us to study disease mechanisms with unprecedented resolution and pave the way for new therapeutic strategies. We finally have the tools to ask questions that were previously inaccessible - and to move closer to real treatments for patients."
Zaida Álvarez
This ambitious approach represents a paradigm shift in CNS research. SPINECRAFT will set new standards in tissue engineering and open doors to breakthroughs in neurodegenerative disease modelling and regenerative therapies-pushing the boundaries of what is possible in neuroscience.
The project's disruptive originality enabled it to secure an additional €800,000, beyond the standard €2 million from the call, dedicated to acquiring specialised equipment for multi-cellular bioprinting and advanced functional analysis. This strategic investment positions IBEC at the forefront of 4D human spinal cord modelling, accelerating the transformative potential of SPINECRAFT.
Institute for Bioengineering of Catalonia (IBEC)
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Breastfeeding may lower mothers' later life risks of depression and anxiety for up to 10 years after pregnancy, suggest the findings of an observational study, BMJ Open' page">published in the open access journal BMJ Open.
The observed associations were evident for any, exclusive, and cumulative (at least 12 months) breastfeeding, the study shows.
It is known that breastfeeding reduces the risk of postnatal depression and anxiety, but it's not clear if these lowered risks might persist in the longer term, say the researchers. To find out, they tracked the breastfeeding behaviour and health of 168 second time mothers who were originally part of the
Analysis of the data showed that women experiencing depression and anxiety at 10 years after pregnancy were less likely to have breastfed and had shorter durations of any or exclusive breastfeeding over their lifetime.
Each week of lifetime exclusive breastfeeding was associated with a 2% lower likelihood of reporting depression and anxiety, after accounting for potentially influential factors, including alcohol intake.
This is an observational study, and as such, no firm conclusions can be drawn about cause and effect, associations only can be relied upon.
Nevertheless, the team wrote: "We suggest there also may be a protective effect of successful breastfeeding on postpartum depression and anxiety, which in turn lowers the risk of maternal depression and anxiety in the longer term."
They said: "The likelihood is that the association is multifactorial, as many socioeconomic and cultural factors influence both breastfeeding and mental health. Additionally, women with a prior history of depression and anxiety are at risk of lower breastfeeding success, compounding the association."
They said: "We know that improving breastfeeding rates and duration can improve mothers' lifetime health outcomes in terms of less diabetes and heart disease and reduces at population level disease burden with resultant significant healthcare savings.
"The possibility that breastfeeding could further reduce the huge burden of depression on individuals, families, healthcare systems and economies only adds to the argument for policymakers to further promote breastfeeding."
UCD Research and Innovation
McNestry, C., et al. (2026). Breastfeeding and later depression and anxiety in mothers in Ireland: a 10-year prospective observational study. BMJ Open. doi: 10.1136/bmjopen-2024-097323. https://bmjopen.bmj.com/content/16/1/e097323
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Specialty laboratories provide advanced capabilities beyond routine diagnostics, positioning them at the forefront of infectious disease research and development.
The strength of these laboratories lies in their potential to provide a multifaceted approach, integrating molecular, virology, and immunology techniques to deliver comprehensive insights for antiviral drug discovery, vaccine development, and translational research.
Cell culture is a key foundation for drug screening, viral propagation, and physiologically relevant modelling, complementing immunological and genomic analyses. This ensures high specificity, sensitivity, and reproducibility, allowing their users to perform accurate immune profiling, pathogen detection, and functional characterization.
Single-modality testing is typically unable to capture the complexity of infectious diseases due to pathogens' capacity for rapid evolution. Specialty labs combine viral infectivity assays, genomic analysis, immune response profiling, and cell culture systems to:
This suite of tools is ideally suited to biomarker discovery, vaccine research, diagnostics development, and Phase I–III clinical trials. Techniques include:
This suite of tools is suitable for research on respiratory pathogens, antiviral drug development, and emerging infectious disease programs.
Techniques include:
The suite of tools is ideally suited to vaccine development, infectious disease research, immunogenicity studies, and cardiometabolic biomarker analysis. Techniques include:
These types of tools are suitable for a wide range of applications, including viral infectivity and neutralization assays, vaccine candidate evaluation aligned with relevant regulatory guidelines, and mechanistic studies of host-pathogen interactions; as well as antiviral drug screening and cytotoxicity testing, and neutralizing antibody assessments and biological potency measurements.
Techniques include:
There are several tangible benefits to working with a specialty laboratory that offers multifaceted capabilities.
For example, the laboratory can provide comprehensive insights across pathogen detection, infectivity, and immune response, as well as help to expedite drug and vaccine development.
Defensible, high-quality data is available for regulatory submissions, with global standardization made possible via powerful automation and LIMS integration.
Specialty laboratories like hVIVO will continue to be at the forefront of innovation, integrating these diverse and highly beneficial technologies into validated workflow designs to better ensure resilience against evolving pathogens.
Produced from materials originally authored by hVIVO.
hVIVO is a full-service early phase CRO offering end-to-end drug development services from preclinical consultancy through to Phase III clinical trials, including world-leading end-to-end human challenge trials services. With decades of experience in rapidly delivering data for our global client base, our team brings together strategic insight and operational expertise to deliver a variety of clinical study types across multiple locations.
To support rapid study start-up and reliable delivery, our dedicated recruitment teams in Germany and the UK provide direct access to both healthy volunteers and patient populations. This is complemented by our integrated drug development consultancy, as well as our infectious disease and immunology laboratories and biobanking services.
Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.net, which is to educate and inform site visitors interested in medical research, science, medical devices, and treatments.
Last updated: Jan 9, 2026 at 6:54 AM
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From the outside, the abandoned Family Dollar store in the Lower 9th Ward looks intimidating. It's covered in graffiti, with aluminum cans and trash dotting the parking lot. It sits on a street with other empty lots and decayed buildings — symbols of the lasting devastation this neighborhood, one of the city's poorest, has endured since Hurricane Katrina.
But inside, the store is a welcoming oasis. Twinkly string lights adorn racks of donated clothing. Shelves and bins overflow with children's books, allergy medications, and toiletries. Curtains cordon off one side of the room, where there's a stage for musicians and a neon sign depicting roller skates for weekly free skate nights.
The space is part free thrift store, part over-the-counter pharmacy, part punk show venue — and wholly "a radical community center," said Dan Bingler, who runs the place.
Bingler is a waiter and bartender in the city who founded a mutual-aid organization called the Greater New Orleans Caring Collective. He said the building owners allow him to use the space as long as he pays the water, electricity, and trash bills.
On Monday evenings, volunteers from other community organizations show up — some used to set up in the parking lot before Bingler opened the store. They offer free testing for sexually transmitted infections, basic medical care, hot meals, and sterile syringes and other supplies for people who use drugs.
The purpose of the space is simple, Bingler said: "We're going to make sure we provide for the community."
Although it's been open for a few years now, the space has become even more crucial to this community in recent months, with the Trump administration slashing funding for many social service organizations and taking an aggressive approach to homelessness and drug use. In Washington, D.C., the administration has bulldozed tents to push people living on the street to leave the city. Nationally, it has called for people who use drugs to be forced into treatment. It has decried harm reduction — practices that public health experts say keep people who use drugs safe and alive but that critics say promote illegal drug use.
The community space in New Orleans — named the Fred Hampton Free Store after the famous Black Panther activist known for bringing together diverse groups to fight for social reforms — aims to be a haven among this sea of changes.
It doesn't receive federal funding, state or local grants, or money from foundations, Bingler said. It's simply neighbors helping neighbors, he said, tearing up and adding, "It's a really beautiful thing to be able to share all this space."
All items inside are provided by people or organizations in the community. Bingler said one time a local hotel undergoing renovations donated 50 flat-screen TVs.
On nights the store is open, often more than 100 people visit, Bingler said.
One fall evening, dozens of people browsed for free clothing and over-the-counter medications. Others sat on the grass outside, chatting while keeping an eye on their bicycles or grocery carts full of possessions.
James Beshears stopped by the harm reduction group in the parking lot to get sterile supplies he uses to inject heroin and fentanyl. He said he'd been in treatment for years but relapsed after his doctor moved away and he was referred to a clinic that charged $250 a day. Street drugs were cheaper than treatment, he said.
He wants to stop. But until he can find affordable care, places like the free store keep him going. Without it, he said, he'd have "one foot in the grave."
Another man in the parking lot was waiting for the arrival of Aquil Bey, a paramedic and former Green Beret well known for helping people overcome obstacles to getting health care. As soon as the man spotted Bey's black Jeep, he ran up.
"I've got stage 4 kidney disease," the man said, adding that he was scheduled for treatments at a hospital but was struggling to get there.
"Do me a favor," Bey said as he unloaded folding tables and medical equipment from his car. "When our team gets here, come and see us. Maybe we can get you transportation."
Bey is the founder of Freestanding Communities, a volunteer-run organization that provides free basic medical care and referrals for people who are homeless, using drugs, or part of other vulnerable communities. The group has a steady presence at the free store.
That day, Bey and his team connected the man needing kidney disease treatment to reduced-cost transit programs. They also did blood pressure and blood sugar checks for anyone who wanted them, cleaned infected wounds, and called clinics to make appointments for patients without phones.
A man with a leg injury mentioned he was sleeping on the concrete floor of an abandoned naval base. Bey noticed the free store's furniture section had a mattress. He and another volunteer hauled it out, strapped it to the top of a car, and delivered it to where the man was sleeping.
"We're just trying to find all these barriers" that people face and "find ways to fix them," Bey said.
The clinic at the free store helped Stephen Wiltz connect with addiction care. He grew up in the Lower 9th Ward and had been using drugs since he was 10.
Fed up with discrimination from doctors who blamed him for his addiction, Wiltz said, he was reluctant to go to any treatment facility. But after years of knowing the volunteers at the free store, he trusted them to point him in the right direction.
At 56, Wiltz was in sustained recovery for the first time in his life, he said during a phone interview in the fall.
Those volunteers "cared for people who didn't have nobody to care for them," he said.
As the sun went down that fall evening at the store, a punk band started setting up for a show across the room from the medical clinic. Lights dimmed and music blared — a reminder that this was not your everyday clinic or community center.
Bey continued consulting with a patient who had gout.
"I get used to the sound," Bey said of the rapid drums and loud power chords. "I like it sometimes."
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Guillaume Bentzinger, Luis Carrillo, Philippe Robin, and Alejandro Bara-Estaún
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Many low-income Californians prescribed wildly popular weight loss drugs lost their coverage for the medications at the start of the new year.
Health officials are recommending diet and exercise as alternatives to heavily advertised weight loss drugs like Wegovy and Zepbound, advice experts say is unrealistic.
“Of course he tried eating well and everything, but now with the medications, it's better - a 100% change," said Wilmer Cardenas of Santa Clara, who said his husband lost about 100 pounds over about two years using GLP-1s covered by Medi-Cal, California's version of Medicaid.
California joined several other states in restricting an option they say is no longer affordable as they confront soaring pharmaceutical costs and steep Medicaid cuts under the Trump administration, among other financial pressures. Despite negotiated price reductions announced in November that the White House said would make the drugs available at a "dramatically lower cost to taxpayers" and enable Medicaid to cover them, states are going ahead with the cuts, which providers say may undermine patients' health.
"It will be quite negative for our patients" because data shows people typically regain weight after stopping the drugs, said Diana Thiara, medical director of the University of California-San Francisco Weight Management Program.
While California, New Hampshire, Pennsylvania, and South Carolina stopped covering adult GLP-1 prescriptions for obesity on Jan. 1, they continue to cover the drugs for other health issues, such as Type 2 diabetes, cardiovascular disease, and chronic kidney disease.
Michigan, Rhode Island, and Wisconsin are planning or considering restrictions, according to KFF's most recent survey.
That reverses a trend that saw 16 states covering the medications for obesity as of Oct. 1. Interest in providing the coverage "appears to be waning," the survey found, likely due to the drugs' cost and other state budget pressures. North Carolina pulled back GLP-1 coverage in October, but Gov. Josh Stein reinstated it in December, bowing to court orders despite a lingering budget shortfall.
Catherine Ferguson, vice president of federal advocacy for the American Diabetes Association and its affiliated Obesity Association, said it's not clear how states will adjust to the White House plan to lower the cost of several of the most popular GLP-1s through TrumpRx, an online portal for discounted prescription drugs. The price of Wegovy, for example, will be $350 per month for consumers, versus the current list price of nearly $1,350, and Medicare and Medicaid programs will pay $245, according to the plan.
"Many states are facing budgetary challenges, such as deficits, and are working to address the impacts of the changes to Medicaid and SNAP," Ferguson wrote, referring to the Supplemental Nutrition Assistance Program. "As more details become available for the Administration's agreements, we will see how state Medicaid responds."
The Department of Health and Human Services referred questions to the White House, which did not respond to requests for comment on states' termination of Medicaid coverage for the weight loss drugs.
California projected its costs to cover GLP-1s for weight loss would have more than quadrupled over four years to nearly $800 million annually if it didn't end Medi-Cal coverage for that use. Medi-Cal has covered weight loss drugs since 2006, but use of GLP-1s soared only in recent years. By 2024, more than 645,000 prescriptions were covered by Medi-Cal across all uses of the medications. The California Department of Health Care Services could not readily provide a breakdown of whether the drugs were for weight loss or other conditions.
When asked whether the state would reconsider its plans in light of the announced price cuts, Department of Finance spokesperson H.D. Palmer said it had no plans to do so. California's cut is written into the state's budget law.
California officials would not say how much it could save under the TrumpRx plan, citing federal and state restrictions on disclosing rebate information.
Health providers don't expect the Trump administration's negotiated price cuts to make much difference to consumers, because pharmaceutical companies already offer some discounts.
"The out-of-pocket costs will still be very cost-prohibitive for most, especially individuals with Medicaid insurance," Thiara said.
New Hampshire is among the other states that ended their coverage Jan. 1. Officials with the New Hampshire Department of Health and Human Services did not respond to requests for comment.
About 1 in 8 adults are now taking a GLP-1 drug for obesity, disease, or both, up 6 percentage points from May 2024, according to KFF poll results released in November. Over half of users said their GLP-1s were difficult to afford, and many who had stopped the treatment cited the cost.
Public and private payers have been trying to wean patients off to save costs. California health officials said Medi-Cal members and their health care providers should consider "other treatment options that can support weight loss, such as diet changes, increased activity or exercise, and counseling." That echoes advice from the New Hampshire Medicaid program.
California Department of Health Care Services spokesperson Tessa Outhyse said in an email that the official advice to try those other approaches now "is not meant to dismiss any past efforts, but to encourage Medi-Cal members to take a renewed, proactive, and medically supported approach with their healthcare provider that may appropriately include these additional options."
But that may be unrealistic, said Kurt Hong, founding director of the Center for Clinical Nutrition at Keck School of Medicine of the University of Southern California.
"We definitely want patients to do their part with the diet and exercise, but unfortunately, and from a practical standpoint, that itself frequently is not enough,” Hong said, adding that usually by the time patients see doctors they have already failed at achieving results through those means.
Hong understands why Medicaid programs, as well as private providers, want to cut back on covering the drugs, which can cost thousands of dollars per patient per year. However, they can produce twice the weight loss as the medications typically used previously, he said.
A school of medical thought supports patients' gradually ending their use, but Hong said obesity is generally considered a chronic condition that requires indefinite treatment.
“Once they reach their target weight, a lot of people will try to see whether or not they can wean off," Hong said. "We do see a lot of patients - when they try to get off, unfortunately, then the weight comes back."
Medi-Cal members under age 21 remain covered for purposes including weight loss, California officials said, citing a federal requirement.
Medi-Cal members are able to keep their GLP-1 coverage if they can demonstrate it is medically necessary for purposes other than weight loss, the department said. Members who are denied coverage can seek a hearing, the department said in a letter to members.
Members will still be able to pay for the prescriptions out-of-pocket and may be able to use various discounts to lower costs. Another option is new pills to treat obesity, which will be cheaper than their injectable counterparts. The FDA approved a pill version of Wegovy on Dec. 22, which will likely run $149 per month for the lowest dosage, and similar weight loss pills are expected to be available in the first half of the year.
While Cardenas said his husband, Jeffer Jimenez, 37, uses GLP-1s primarily for weight loss, Jimenez's prescription is for his diabetes, so the couple hoped to continue receiving coverage through Medi-Cal.
“He tried a thousand medications, pills, natural teas, exercise program, but it doesn't work like the injections,” Cardenas said. "You need both.”
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Dr Bryony Henderson
GLP-1 agonists are pivotal in obesity care, promoting weight loss and addressing related health issues, with a focus on personalized, holistic treatment.
Guillaume Bentzinger, Luis Carrillo, Philippe Robin, and Alejandro Bara-Estaún
Discover how AI, flow chemistry, and NMR come together in the PiPAC project to revolutionize scalable and autonomous API production.
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A new UCLA study reveals that a widely used federal hospital safety metric is fundamentally flawed when applied to emergency stroke care, potentially creating incentives that may discourage hospitals from performing lifesaving procedures for the sickest patients.
The research, published in the Journal of NeuroInterventional Surgery, examined Patient Safety Indicator 04 (PSI 04), a "failure-to-rescue" measure developed by the U.S. Agency for Healthcare Research and Quality (AHRQ) to track deaths following treatable complications in surgical patients. The study analyzed data from the Nationwide Inpatient Sample covering 73,580 stroke thrombectomy procedures between 2016-2019, along with detailed reviews of consecutive cases at UCLA.
While stating the metric is appropriate for elective procedures performed on relatively healthy patients, the study found the metric is inappropriate for endovascular thrombectomy, an emergency procedure to remove blood clots in stroke patients who are already gravely ill upon admission.
This metric was designed to identify preventable deaths, but when applied to emergency stroke care, it's flagging unavoidable complications of severe strokes rather than problems with the procedure itself. The unintended consequence is that hospitals providing excellent stroke care to the sickest patients may appear to have poor safety records."
Dr. Melissa Marie Reider-Demer, the study's first author and UCLA Health DNP
PSI 04 is triggered when patients develop any of five complications after a procedure (pneumonia, blood clots, sepsis, shock/cardiac arrest, or gastrointestinal bleeding) and subsequently die in the hospital. The metric is used nationally for public reporting, hospital quality ratings and pay-for-performance programs by Medicare and influential organizations like the Leapfrog Group.
The UCLA team analyzed both national data and detailed case reviews to assess the metric's appropriateness for stroke care. Their findings included:
At UCLA's Comprehensive Stroke Center, researchers examined every thrombectomy case flagged by PSI 04 between 2016-2018. An expert panel of neurointerventionalists and neurologists reviewed each case and found:
The study authors found the metric is flawed for two key reasons when applied to stroke thrombectomy:
"We're essentially penalizing hospitals for trying to save patients who are already dying from stroke," Dr. Reider-Demer said. "These procedures give severely affected patients their only chance at survival or functional recovery, but the current metric makes it look like the hospitals are providing poor care."
The researchers warn that inappropriate safety metrics can create harmful incentives. Previous research has shown that public reporting of surgical mortality rates led some heart surgeons to cherry-pick healthier patients to protect their performance ratings, limiting access for the sickest patients who need care most.
"There's a real concern that hospitals might be discouraged from performing thrombectomy on the most severe stroke patients, or that stroke centers with high volumes of critically ill patients could be unfairly penalized in quality ratings and reimbursement," said Dr. Jeffrey Saver, the study's senior author and vice chair for Clinical Research and the Carol and James Collins Chair of the Department of Neurology at UCLA Health.
This issue has become more pressing as recent clinical trials have expanded thrombectomy to patients with even larger strokes, who have high mortality rates even with intervention though still lower than without it.
The Centers for Medicare & Medicaid Services has proposed revising PSI 04 to exclude patients with acute conditions like stroke coded as the principal reason for admission, with implementation planned for fiscal year 2027.
In Dr. Saver's view, the revision addresses important shortcomings.
"This revision makes sense from a clinical perspective," Dr. Saver said. "The current metric doesn't identify preventable events in stroke care and has the potential to mislead the public about hospital quality while creating incentives that could harm the sickest patients."
University of California - Los Angeles Health Sciences
Reider-Demer, M. M., et al. (2025). Design limitations and unintended consequences of the AHRQ patient safety indicator for endovascular thrombectomy stroke care. Journal of NeuroInterventional Surgery. DOI: 10.1136/jnis-2025-023727. https://jnis.bmj.com/content/early/2025/10/12/jnis-2025-023727.full
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In our latest interview, News-Medical speaks with Rosanna Zhang from ACROBiosystems about utilizing organoids for disease modeling in the field of neuroscience research.
Dr Bryony Henderson
GLP-1 agonists are pivotal in obesity care, promoting weight loss and addressing related health issues, with a focus on personalized, holistic treatment.
Guillaume Bentzinger, Luis Carrillo, Philippe Robin, and Alejandro Bara-Estaún
Discover how AI, flow chemistry, and NMR come together in the PiPAC project to revolutionize scalable and autonomous API production.
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The worldwide prevalence of obesity has more than doubled in the last three decades, bringing with it an increase in weight-related diseases like diabetes, cardiovascular disease, and cancers. This public health crisis strains global healthcare systems and economies, but a new study co-led by investigators from Mass General Brigham could inform strategic programs to make GLP-1 medications part of the solution.
Mass General Brigham researchers and collaborators from Washington University School of Medicine in St. Louis and Emory University's Rollins School of Public Health pooled data from 99 countries and 810,635 adults to determine how many people worldwide may benefit from GLP-1 use. They found more than one-in-four adults would be eligible for GLP-1s for weight management, with women, older individuals, and low- and middle-income countries among the most eligible. These critical metrics could be formative in policy development to deploy GLP-1s around the world to tackle obesity and its comorbidities. Their results are published in a research letter in The Lancet Diabetes & Endocrinology.
There has never been such a potentially transformational and scalable tool for obesity, type 2 diabetes, and other health-related complications of obesity. For so many decades, we told everyone the problem was you-you need to move more and eat less, then you won't struggle with this problem. GLP-1 receptor agonists have allowed us to really understand that biology is much more powerful than that, and 'eat less, move more' is just an oversimplified way to think about things."
Jennifer Manne-Goehler, MD, ScD, co-senior author, physician in the Division of Infectious Diseases at Brigham and Women's Hospital and Department of Medicine at Mass General Brigham
The power and promise of GLP-1s has been recognized by the World Health Organization (WHO), as they actively are working to make them standard, accessible mediations. But scaling up production and rolling out application of GLP-1s globally starts with one big question: Exactly how many people need them?
"Given the steadily increasing prevalence of obesity, it's not surprising that our analysis found that more than one quarter of adults around the world may be eligible for this medication," said corresponding author Sang Gune K. Yoo, MD, who conducted this work as a research fellow in cardiology at WashU Medicine. "This medication has the potential to help many individuals, although further research is needed to better understand its long-term safety and sustainability. Access remains a major challenge as these medications are difficult to obtain in many settings. Most importantly, we must continue to invest in and develop effective non-pharmacological strategies for the prevention and treatment of obesity, an area where substantial gaps remain."
corresponding author Sang Gune K. Yoo, MD, who conducted this work as a research fellow in cardiology at WashU Medicine
The researchers started with household health survey data collected from 99 countries between 2008 and 2021. A total of 810,635 adults between 25 and 64 years old were pooled based on availability of diabetes biomarkers, blood pressure and BMI measurements, and hypertension and diabetes diagnostic history. Those that had a BMI of more than 30 or a BMI of more than 27 with additional hypertension, diabetes, or both, were deemed eligible for GLP-1 use.
Globally, 27% of the adults were eligible for GLP-1s for weight management-four-fifths of whom came from low- and middle-income countries. Eligibility rates were highest in Europe and North America (42.8%) and the Pacific Islands (41.0%). Women were also more likely (28.5%) than men to be eligible, as were older individuals more likely (38.3%) than their younger counterparts (17.9%).
"These socioeconomic and gender eligibility percentiles are especially staggering," added Manne-Goehler. "As of last year, type 2 diabetes was the top cause of death for women in South Africa. There are parts of the world where women can really benefit from these medicines, and it's our job to see through their implementation."
"Global access to GLP-1s is a question of health equity," said co-lead author Felix Teufel, MD, from Emory University's Rollins School of Public Health. "The goal is to ensure large-scale access for people who would benefit most – not just those easiest to reach."
Mass General Brigham
Gune, S., et al. (2026). GLP-1 receptor agonists for obesity: eligibility across 99 countries. The Lancet Diabetes & Endocrinology. DOI: 10.1016/S2213-8587(25)00356-0. https://www.thelancet.com/journals/landia/article/PIIS2213-8587(25)00356-0/fulltext
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Rosanna Zhang
In our latest interview, News-Medical speaks with Rosanna Zhang from ACROBiosystems about utilizing organoids for disease modeling in the field of neuroscience research.
Dr Bryony Henderson
GLP-1 agonists are pivotal in obesity care, promoting weight loss and addressing related health issues, with a focus on personalized, holistic treatment.
Guillaume Bentzinger, Luis Carrillo, Philippe Robin, and Alejandro Bara-Estaún
Discover how AI, flow chemistry, and NMR come together in the PiPAC project to revolutionize scalable and autonomous API production.
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Breastfeeding may lower mothers' later life risks of depression and anxiety for up to 10 years after pregnancy, suggest the findings of a small observational study, published in the open access journal BMJ Open.
The observed associations were apparent for any, exclusive, and cumulative (at least 12 months) breastfeeding, the study shows.
Breastfeeding is associated with lower risks of postnatal depression and anxiety, but it's not clear if these lowered risks might persist in the longer term, say the researchers.
To find out, they tracked the breastfeeding behavior and health of 168 second time mothers who were originally part of the ROLO Longitudinal Birth Cohort Study for 10 years.
All the women had given birth to a child weighing under 4 kg and they and their children had check-ups 3 and 6 months, and 2, 5, and 10 years after birth by which time the mothers' average age was 42.
At each check-up, the mothers completed a detailed health history questionnaire. This asked whether they had been diagnosed with, and treated for, depression/anxiety. They also provided information on potentially influential factors, including diet and physical activity levels.
At the check-ups, the mothers provided information on: whether they had ever breastfed or expressed milk for 1 day or more; total number of weeks of exclusive breastfeeding; total number of weeks of any breastfeeding; and cumulative periods of breastfeeding of less or more than 12 months.
Nearly three quarters of the women (73%; 122) reported having breastfed at some point. The average period of exclusive breastfeeding lasted 5.5 weeks and that of any breastfeeding for 30.5 weeks. More than a third (37.5%; 63) reported cumulative periods of breastfeeding adding up to at least 12 months.
Twenty two (13%) of the women reported depression/anxiety at the 10-year check-up, with a further 35 (21%) reporting depression or anxiety at any time point.
Those reporting depression/anxiety at the 10 year check-up were younger, less physically active, and had lower wellbeing scores at the start of the study than those who didn't report this. Those women reporting depression and anxiety at any time point differed only by age at the start of the study.
Analysis of the data showed that women experiencing depression and anxiety 10 years after pregnancy were less likely to have breastfed and had shorter periods of any or exclusive breastfeeding over their lifetime.
Each week of lifetime exclusive breastfeeding was associated with a 2% lower likelihood of reporting depression and anxiety, after accounting for potentially influential factors, including alcohol intake.
This is an observational study, and as such, no firm conclusions can be drawn about cause and effect. And the researchers acknowledge that participants were relatively few in number, not ethnically or socially diverse, and that the study relied on personal recall rather than objective measures of depression/anxiety.
Nevertheless, they write: "We suggest there also may be a protective effect of successful breastfeeding on postpartum depression and anxiety, which in turn lowers the risk of maternal depression and anxiety in the longer term."
They explain: "The likelihood is that the association is multifactorial, as many socioeconomic and cultural factors influence both breastfeeding and mental health in addition to the impact of health history. Additionally, women with a prior history of depression and anxiety are at risk of lower breastfeeding success, compounding the association but in the reverse direction."
They conclude: "We know that improving breastfeeding rates and duration can improve lifetime health outcomes, reducing population level disease burden and resulting in significant healthcare savings.
"The possibility that breastfeeding could further reduce the huge burden of depression on individuals, families, healthcare systems and economies only adds to the argument for policymakers to improve breastfeeding support."
BMJ Group
McNestry, C., et al. (2026). Breastfeeding and later depression and anxiety in mothers in Ireland: a 10-year prospective observational study. BMJ Open. DOI: 10.1136/bmjopen-2024-097323. https://bmjopen.bmj.com/content/16/1/e097323
Posted in: Medical Research News | Women's Health News
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Rosanna Zhang
In our latest interview, News-Medical speaks with Rosanna Zhang from ACROBiosystems about utilizing organoids for disease modeling in the field of neuroscience research.
Dr Bryony Henderson
GLP-1 agonists are pivotal in obesity care, promoting weight loss and addressing related health issues, with a focus on personalized, holistic treatment.
Guillaume Bentzinger, Luis Carrillo, Philippe Robin, and Alejandro Bara-Estaún
Discover how AI, flow chemistry, and NMR come together in the PiPAC project to revolutionize scalable and autonomous API production.
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UCLA has received four grants totaling $7.3 million from the California Department of Cannabis Control (DCC) to support research on a broad range of topics, from the therapeutic potential of cannabinoids to the cardiovascular risks of cannabis use and strategies for addressing California's unregulated cannabis market.
The funding will support research by faculty from the David Geffen School of Medicine at UCLA, the UCLA College of Letters and Science and the UCLA Fielding School of Public Health.
This achievement reflects UCLA's leadership in advancing cannabis science through innovative research. From the laboratory to real-world market dynamics, our faculty are generating findings that will shape evidence-based regulation that prioritizes public health."
Ziva Cooper, director of the UCLA Center for Cannabis and Cannabinoids and professor at the Jane and Terry Semel Institute for Neuroscience and Human Behavior at UCLA
The two-year grants awarded to UCLA researchers, who are members of the UCLA Center for Cannabis and Cannabinoids, represent a quarter of the $30 million that the DCC recently provided to nine academic institutions to further scientific understanding of cannabis and inform efforts to shape public policy related to the evolving legal cannabis market.
The UCLA studies funded by these awards include:
The studies are expected to begin in early 2026.
University of California - Los Angeles Health Sciences
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Rosanna Zhang
In our latest interview, News-Medical speaks with Rosanna Zhang from ACROBiosystems about utilizing organoids for disease modeling in the field of neuroscience research.
Dr Bryony Henderson
GLP-1 agonists are pivotal in obesity care, promoting weight loss and addressing related health issues, with a focus on personalized, holistic treatment.
Guillaume Bentzinger, Luis Carrillo, Philippe Robin, and Alejandro Bara-Estaún
Discover how AI, flow chemistry, and NMR come together in the PiPAC project to revolutionize scalable and autonomous API production.
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About one in nine adults suffer from chronic insomnia and its residual effects like drowsiness, cognitive issues, and irritability as well as increased health risks like diabetes and heart risks if left untreated. While many treatments are available, the challenge lies in determining how well a medication or other sleep aid works in individual patients.
Now a new study from the University of Maryland School of Medicine has found using real-time smartphone-based assessments can help to determine the effectiveness of sleep medications by detecting improvements in daytime insomnia symptoms including thinking, fatigue, and mood. Following a two-week course of treatment, this smartphone-based assessment approach detected treatment effects more powerfully than did traditional methods like recall questionnaires.
Results were published in JAMA Network Open.
The clinical trial involved 40 older adults ages 60 to 85 with chronic insomnia who were randomly assigned to take the sleep medication suvorexant or a placebo for 16 nights. Both groups used a smartphone app to record their daytime insomnia symptoms in real-time, four times per day throughout the study. Participants also completed traditional questionnaires assessing their sleep patterns and daytime symptoms both before and after treatment.
Daytime symptoms such as fatigue, cognitive impairment, and mood disturbances are core features of insomnia. Improving sleep is not enough. We need to determine how well treatments improve daytime functioning, which patients report matters most. In this study we found that retrospective questionnaires failed to detect subtle treatment-related changes that were detected via the smartphone assessment."
Emerson M. Wickwire, PhD, study corresponding author, faculty member at the University of Maryland School of Medicine and section chief of sleep medicine, University of Maryland Medical Center
This is the first randomized controlled trial to incorporate a smartphone EMA as an outcome measure in a sleep-focused clinical trial. It was able to detect clear treatment effects at various times of the day and was found to be easy to use and sustainable. Leveraging wearables and smartphones for real-time, multimethod assessment should be considered in future studies that evaluate treatment for insomnia as well as other sleep disorders including obstructive sleep apnea and excessive sleepiness that leads to prolonged sleep.
"These findings address a critical gap in sleep disorders clinical care and research," said Dr. Wickwire. "When viewed as a complement to traditional approaches, EMA offers a sensitive and patient-centered way to measure treatment effects throughout the day, in real-time. Such approaches could transform how we evaluate sleep treatments, personalize sleep medicine care, and ultimately improve outcomes for the millions of Americans with sleep disorders."
Shuo Chen, PhD, Professor of Epidemiology & Public Health at UMSOM, Avelino Verceles, MD, MS, Professor of Medicine at UMSOM, and University of Maryland graduate student Jingsong Zhou, MS were co-authors on this study. Funding for this study was supported in part by a research grant from the Investigator-Initiated Studies Program of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co, Inc.
"This research underscores the potential for innovative digital tools to be used in conducting comparative effectiveness studies," said UMSOM Dean Mark T. Gladwin, MD, who is the Vice President for Medical Affairs, University of Maryland, Baltimore (UMB), and the John Z. and Akiko K. Bowers Distinguished Professor and Dean at UMSOM. "Smartphone-based assessments can provide real-time insights that help improve patient outcomes across a range of common conditions."
University of Maryland School of Medicine
Wickwire, E. M., et al. (2026). Smartphone-Based Real-Time Assessment of Daytime Insomnia Symptoms With Suvorexant. JAMA Network Open. DOI: 10.1001/jamanetworkopen.2025.50186. https://jamanetwork.com/journals/jamanetworkopen/fullarticle/2843418
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Rosanna Zhang
In our latest interview, News-Medical speaks with Rosanna Zhang from ACROBiosystems about utilizing organoids for disease modeling in the field of neuroscience research.
Dr Bryony Henderson
GLP-1 agonists are pivotal in obesity care, promoting weight loss and addressing related health issues, with a focus on personalized, holistic treatment.
Guillaume Bentzinger, Luis Carrillo, Philippe Robin, and Alejandro Bara-Estaún
Discover how AI, flow chemistry, and NMR come together in the PiPAC project to revolutionize scalable and autonomous API production.
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Pioneering research led by Brazilians describes the immune system's reactions in detail in the first living patient to receive a genetically modified pig kidney transplant. This paves the way for the search for therapies that can prevent organ rejection.
The study demonstrates the feasibility of this type of graft but indicates that controlling initial rejection alone is insufficient. This is because even with immunosuppressants, continuous activation of innate immunity – the body's first line of defense, especially macrophages, which react to any threat – can compromise long-term survival.
Through transcriptomic, proteomic, metabolomic, and spatial analyses, the scientists have determined that new strategies are necessary to achieve long-term survival and favorable clinical outcomes. They recommend combining therapies that target innate immunity with advanced genetic engineering in donor pigs. They also suggest preventing early T lymphocyte-mediated rejection and implementing more sensitive monitoring approaches.
Xenotransplantation involves transplanting organs, tissues, or cells from one animal species – mainly genetically modified pigs – to humans. It is considered a promising solution to organ shortages, but rejection has been a major challenge.
In March 2024, the first living patient to receive a pig kidney was a 62-year-old man with end-stage kidney disease who underwent surgery at Massachusetts General Hospital, which is affiliated with Harvard Medical School in Boston. Brazilian nephrologist Leonardo Riella, one of the article's corresponding authors, led the team. The article was published on January 8 in the scientific journal Nature Medicine. The patient died two months later; the probable cause was previous chronic myocardial fibrosis.
According to data from the Brazilian Ministry of Health, kidney transplants are in the highest demand in Brazil. In 2025, about 6,670 surgeries of this type were performed in the country.
Additionally, it is estimated that between 10 and 12 million Brazilians have some form of kidney disease, a figure that could rise as the population ages and the number of people with diabetes, high blood pressure, and obesity increases. In more severe cases, dialysis may be a temporary treatment option. Dialysis is an artificial process that removes waste and excess fluids from the body when the kidneys are not functioning properly.
"The main finding of the study was the detailed, unprecedented, high-resolution characterization of the human immune response following the transplantation of a genetically modified pig kidney into a living patient. The results show that, for xenotransplantation to become a safe and lasting clinical option, controlling only adaptive immunity, as we traditionally do in transplants between humans, is insufficient. Specific strategies must also be developed to modulate the innate immune response and ensure the prolonged survival of xenogeneic grafts in humans," said Thiago Borges, a professor and researcher at Massachusetts General Hospital and Harvard Medical School, as well as the corresponding author of the article, in an interview with Agência FAPESP.
To comprehensively evaluate the response triggered by renal xenotransplantation, the researchers characterized the recipient's immune profile by cross-referencing information obtained from clinical analyses with information from proteomics and metabolomics, which includes sugars, lipids, amino acids, and other metabolites.
They observed that, in the first week after surgery, the patient's body recognized the transplanted organ as "foreign" and activated cellular rejection, a specific type of defense conducted mainly by T lymphocytes. This process can damage the transplanted organ and was identified and controlled with immunosuppressive drugs.
The study showed that although no more severe rejection (mediated by antibodies) occurred, the immune system remained partially active, especially in monocytes and macrophages. This reveals a central and hitherto underestimated role of innate immunity in xenotransplant rejection.
This rejection was not detected through blood tests. However, tests measuring DNA fragments from the transplanted organ in the bloodstream indicated kidney damage. Based on these results, the group suggests that levels of porcine donor-derived cell-free DNA (dd-cfDNA) could serve as a potential biomarker for this issue. In the case analyzed, the pig kidney had 69 genetic modifications to increase immune compatibility.
"We demonstrated that DNA fragments from the pig kidney circulating in the patient's blood can be used as a sensitive and noninvasive marker of rejection. This opens up the possibility of monitoring the graft in real time, which potentially reduces the need for biopsies," Borges explains.
Persistent activation of innate immunity was also observed, with signs of ongoing inflammation. Despite advances in treatment, the findings suggest that current treatments are still unable to fully control immune responses.
This study was important because it provided a broad view of all the molecular and cellular changes that occurred during the transplant. This can help guide and improve the efficiency of immunosuppression."
Helder Nakaya, senior researcher at the Albert Einstein Jewish-Brazilian Hospital and one of the authors of the article
Nakaya is a professor at the University of São Paulo's School of Pharmaceutical Sciences (FCF-USP) and receives support from FAPESP for the project "Integrative Biology Applied to Human Health." This project aims to develop innovative approaches to analyze epidemiological databases, map disease transmission hotspots, integrate transcriptome data with clinical and immunological information, and use machine learning to interpret and analyze microscopic images.
He is also the principal investigator at the Center for Research on Inflammatory Diseases (CRID), a FAPESP Research, Innovation, and Dissemination Center (RIDC).
"Due to our work developing various analytical tools, including single-cell analysis, we were invited by Harvard researchers to work on the integrated multiomic analysis of these thousands of molecules," adds Nakaya, who has advocated for creating an advanced school specializing in this type of analysis.
In November 2025, a different group of scientists affiliated with U.S. institutions published research evaluating the rejection of a pig kidney transplanted into a brain-dead person (read the article at www.nature.com/articles/s41586-025-09847-6).
São Paulo Research Foundation (FAPESP)
Ribas, G. T., et al. (2026). Immune profiling in a living human recipient of a gene-edited pig kidney. Nature Medicine. DOI: 10.1038/s41591-025-04053-3. https://www.nature.com/articles/s41591-025-04053-3
Posted in: Medical Procedure News | Medical Science News | Medical Research News
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Rosanna Zhang
In our latest interview, News-Medical speaks with Rosanna Zhang from ACROBiosystems about utilizing organoids for disease modeling in the field of neuroscience research.
Dr Bryony Henderson
GLP-1 agonists are pivotal in obesity care, promoting weight loss and addressing related health issues, with a focus on personalized, holistic treatment.
Guillaume Bentzinger, Luis Carrillo, Philippe Robin, and Alejandro Bara-Estaún
Discover how AI, flow chemistry, and NMR come together in the PiPAC project to revolutionize scalable and autonomous API production.
News-Medical.Net provides this medical information service in accordance
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Please note that medical information found
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Last Updated: Friday 9 Jan 2026
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Researchers at TU Graz have proven that espresso is a favourable alternative to the highly toxic and radioactive uranyl acetate in the analysis of biological samples.
To ensure that the tissue structures of biological samples are easily recognisable under the electron microscope, they are treated with a staining agent. The standard staining agent for this is uranyl acetate. However, some laboratories are not allowed to use this highly toxic and radioactive substance for safety reasons. A research team at the Institute of Electron Microscopy and Nanoanalysis (FELMI-ZFE) at Graz University of Technology (TU Graz) has now found an environmentally friendly alternative: ordinary espresso. Images of the samples treated with it were of equally good quality as images of comparative samples, which were prepared with uranyl acetate. The researchers have published their findings in the journal Methods.
"I got the idea of using espresso as a staining agent from the circular dried stains in used coffee cups," says Claudia Mayrhofer, who is responsible for ultramicrotomy at the institute. During preparation, she cuts tissue samples into wafer-thin slices and fixes them onto sample holders. Staining is the last step before examination under the electron microscope. "Initial tests have shown that coffee stains biological samples and enhances contrasts," says Mayrhofer.
Together with team leader Ilse Letofsky-Papst and graduate student Robert Zandonella, Claudia Mayrhofer investigated how well espresso performs in direct comparison with uranyl acetate. Under identical conditions, they treated ultra-thin sections of mitochondria with various staining agents and assessed the quality of the microscope images using special image analysis software. "Espresso provided comparatively very good contrast values, in some cases they were even better than with uranyl acetate," explains Claudia Mayrhofer.
Ilse Letofsky-Papst concludes: "Our results show that coffee is a serious alternative to uranyl acetate. However, further investigations on different types of tissues are still required to enable a broad application in life science electron microscopy."
Graz University of Technology
Mayrhofer, C., et al. (2025). Coffee – a ubiquitous substitute for uranyl acetate in staining of biological ultrathin sections for electron microscopy studies. Methods. DOI: 10.1016/j.ymeth.2025.08.009. https://www.sciencedirect.com/science/article/abs/pii/S1046202325001835?via%3Dihub
Posted in: Device / Technology News | Life Sciences News | Histology & Microscopy
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Rosanna Zhang
In our latest interview, News-Medical speaks with Rosanna Zhang from ACROBiosystems about utilizing organoids for disease modeling in the field of neuroscience research.
Dr Bryony Henderson
GLP-1 agonists are pivotal in obesity care, promoting weight loss and addressing related health issues, with a focus on personalized, holistic treatment.
Guillaume Bentzinger, Luis Carrillo, Philippe Robin, and Alejandro Bara-Estaún
Discover how AI, flow chemistry, and NMR come together in the PiPAC project to revolutionize scalable and autonomous API production.
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Julie Rovner KFF Health News @jrovner @julierovner.bsky.social
Julie Rovner is chief Washington correspondent and host of KFF Health News' weekly health policy news podcast, "What the Health?" A noted expert on health policy issues, Julie is the author of the critically praised reference book "Health Care Politics and Policy A to Z," now in its third edition.
Congress returned from its holiday break to the same question it faced in December: whether to extend covid-era premium subsidies for health plans sold under the Affordable Care Act. The expanded subsidies expired at the end of 2025, leaving more than 20 million Americans facing dramatically higher out-of-pocket costs for insurance.
Meanwhile, the Robert F. Kennedy Jr.-led Department of Health and Human Services announced an overhaul of the federal vaccine schedule for children, reducing the number of diseases for which vaccines are recommended from 17 to 11.
This week's panelists are Julie Rovner of KFF Health News, Sarah Karlin-Smith of Pink Sheet, Alice Miranda Ollstein of Politico, and Lauren Weber of The Washington Post.
Among the takeaways from this week's episode:
Plus, for "extra credit" the panelists suggest health policy stories they read (or wrote) this week that they think you should read, too:
Julie Rovner: KFF Health News' "Advertisements Promising Patients a 'Dream Body' With Minimal Risk Get Little Scrutiny," by Fred Schulte.
Alice Miranda Ollstein: SFGate's "A Calif. Teen Trusted ChatGPT for Drug Advice. He Died From an Overdose," by Lester Black and Stephen Council.
Sarah Karlin-Smith: ProPublica's "The End of Aid: Trump Destroyed USAID. What Happens Now?" by Anna Maria Barry-Jester and Brett Murphy.
Lauren Weber: The Washington Post's "How RFK Jr. Upended the Public Health System," by Rachel Roubein, Lena H. Sun, and Lauren Weber.
Also mentioned in this week's podcast:
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In our latest interview, News-Medical speaks with Rosanna Zhang from ACROBiosystems about utilizing organoids for disease modeling in the field of neuroscience research.
Dr Bryony Henderson
GLP-1 agonists are pivotal in obesity care, promoting weight loss and addressing related health issues, with a focus on personalized, holistic treatment.
Guillaume Bentzinger, Luis Carrillo, Philippe Robin, and Alejandro Bara-Estaún
Discover how AI, flow chemistry, and NMR come together in the PiPAC project to revolutionize scalable and autonomous API production.
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Last Updated: Friday 9 Jan 2026
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By Amin Touri, The Boston Globe
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Two of soccer's most successful men's national teams will meet at Gillette Stadium this spring, as Brazil will take on France in an international friendly on March 26.
The match will be part of “Road to 26,” a series of international exhibitions ahead of the 2026 FIFA World Cup, which kicks off in June.
“Gillette Stadium has a rich history as a host of global sporting events, and welcoming Brazil and France—two of the most recognized and influential national teams in the world—is an extraordinary way to kick off the Road to 26 series,” Jim Nolan, chief operating officer of Kraft Sports + Entertainment, said in a release. “As we prepare to host seven FIFA World Cup matches in 2026, including a quarterfinal, this event will provide fans a world-class preview.”
The four-game series features Brazil, France, Colombia, and Croatia, with two games to be played in Orlando, one in the Washington, D.C., area, and the marquee matchup to take place in Foxborough.
Tickets for the match will go on sale via Ticketmaster at 10 a.m. on Tuesday. Pre-sale access will be available at RoadTo26.com.
Brazil, winner of a record five FIFA World Cup titles, is currently fifth in the FIFA men's world ranking. France, World Cup winner in 2018 and runner-up in 2022, is third.
The two teams have met 18 times, with France winning seven, Brazil claiming five, and the other six played to a draw. The most notable meeting between the two came in the 1998 World Cup Final, when the French won its first World Cup in a 3-0 win on home soil.
Brazil and France last played in June 2024 in Paris, a 2-1 win for the hosts.
The March friendly will be a chance for the French to get acquainted with Gillette Stadium, where they'll take on Norway in group play on June 26.
Brazil's Group C will also play in Foxborough, but the group's marquee team will instead play one game each in New York, Philadelphia, and Miami.
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For the United States men's national team, much of 2025 was spent worrying about whether they would be up to the task at hand of performing in a World Cup on home soil. They were struggling to score, defend, and lacked any real identity under new manager Mauricio Pochettino. It wasn't until September that things began to change, when Tristan Blackmon, Tim Ream and Chris Richards all started together in a back three for the first time in a friendly against Japan. It would lead to a 2-0 victory which sparked a five match unbeaten run to close out 2025, a run where the USMNT faced five sides who will all be in action at the World Cup.
The shift is something that impressed Ream, who spoke about it on Call it What you Want, a CBS Sports Golazo network podcast.
"I think it was a stroke of genius to have three center backs. But in doing that, I think it unlocked the thought of guys being able to play in a fluid four for whatever reason, we went to three center backs, we seemed a lot more comfortable," Ream said.
He's right as the change in defending not only helped provide defensive cover, but it also allowed fullbacks to push further forward into the attack, giving support to Christian Pulisic and Folarin Balogun to put the ball in the net. It created a more repeatable system and added flexibility for the USMNT to go back to a back four if needed.
"Flexibility gives us incredible options to change on the fly in game... it gives guys a better understanding of what everybody needs, but it gives them a better understanding of positionally where they should be at all times," Ream said.
Without Tyler Adams, the USMNT tried out different midfielders to see if they could operate as adequate shields for the defense, and they came to the same conclusion that Bournemouth are coming to in the Premier League. Adams is irreplaceable in a one to one shift, so changing the system was needed to make it happen.
It shows Pochettino ensuring that he's creating the best system for the players at his disposal instead of trying to force something that isn't working on his team. Calling in 70 different players during his time so far as manager, Pochettino has taken a deep look at the squad, and at the moment, this is the best available setup. Adams is currently recovering from an MCL injury with Bournemouth, so even during the March international break, Pochettino could need to continue seeing how to operate without his best defensive midfielder.
On the bright side, the shift has given the USMNT an identity and made the system repeatable. Even with Pochettino changing the players in his XI from game to game, performance levels didn't drop off, and players knew what was expected from them tactically. It's a departure from when players sometimes didn't understand what their roles were during the end of Gregg Berhalter's tenure as manager. With time to spare ahead of the World Cup, Pochettino has been able to instill his tactics, which are about to be put to the test in upcoming March friendlies facing Belgium and Portugal. It's an important step to getting past the round of 16 and the USMNT are on the right track under the Argentine.
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Wesley Fofana has been slammed as a "habitual offender" after being fined for his tenth driving offence. The Blues defender has been injury-prone throughout his time in west London, but has managed to rack up an almost unbelievable haul of issues on the road, both during his time in west London and beforehand.
Per The Sun, Fofana has been fined for a tenth driving offence after he was caught travelling at 54mph in a 40mph zone in Twickenham on February 27 of last year. The centre-back was driving a £300,000 Rolls Royce and he pleaded guilty to one count of speeding in a 40mph zone. He was handed a two-year driving ban last year after a litany of offences. Having racked up eight speeding offences, he was given 38 points on his licence and fined £7,569. The defender was slammed by Magistrate Alan Jefferson, ahead of being handed down a £666 financial penalty, a £266 surcharge, and £130 court costs.
Jefferson said in court: “It's obvious that he's a habitual offender.
“We have taken into consideration his income and early guilty plea. His licence record will be endorsed with 3 penalty points.”
Kari Williamson, prosecuting, said: “He appeared before the court in May last year. He was disqualified as a totter.
“The offence on that occasion was dealt with by a two-year disqualification with 47 points.
“This offence occurred a couple of weeks after the most recent one. He will have to do another driving test before he gets another licence.
“He earns more than 200,000 a week.”
Fofana has struggled to stay fit since his move to Chelsea from Leicester. In the 2023/24 season, he sustained a cruciate ligament tear that kept him out for a year, and he then missed a further 36 games for a hamstring injury, and then surgery thereafter.
He has opened up, however, on how difficult his injury woes have been, telling the club's website: "In football it's hard to take your time because there is a game every three days, but he [Maresca] protects me and he pushes me in a good way,' says Fofana.
"I had hamstring surgery and you need to be careful when you come back. He doesn't take risks with me because he wants, step by step, for me to be able to play every three days.
"The target is to play every game; we need to carefully progress step by step towards that. Also, he protects me because of the type of player I am: aggressive, fast, not scared to have duels on the pitch. I understand it, and it's what I feel as well. I agree with it. The most important is to continue to progress."
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Chelsea face Charlton in the third round of the FA Cup, before meeting Arsenal in the first leg of their Carabao Cup semi-final.
Fofana has revealed how desperate he is to win a trophy after missing the Conference League triumph, adding: "I want more because I missed out. I am more concentrated on that. I want to feel those experiences and those moments. I am completely focused on that.
"Of course I want to make up for lost time, but you can't get the time you missed back. It's part of my life and my story, and it makes me hungrier to win.
"Now it's important we stay together because it's a long season as well. We have a lot of competitions to play for, and the objective is to go as far as we can in all of them and fight for everything. And my objective is just to stay with the team, and fight for everything with the team."
As the metro area makes preparations for the upcoming World Cup, Johnson County Park and Recreation District is celebrating the tournament with some new public art in the form of flags. They're taking proposals from local artists for the project until Sunday night.
The flags will be on display at Theatre in the Park and at Meadowbrook Park this summer.
“Flags are really something that globally people recognize and know that it's a place of gathering or meaning and celebration, so it just felt like the right thing at the right time to pursue that as a project,” said Angi Hejduk, public art specialist for Johnson County Park and Recreation District.
Theatre in the Park will host public watch parties of various World Cup matches, including the ones being played locally.
The call for proposals is open to both professional artists and hobbyists living in Cass, Clay, Douglas, Jackson, Johnson, Leavenworth, Miami, Platte and Wyandotte counties.
Proposals for the flags must include four different sketches inspired by nature, drawing from a color palette of yellow, dark blue, light blue, purple, light pink, hot pink, burgundy and light green.
Although the project administrators will provide most of the material in the form of new ripstop nylon kite fabric, some of it will be recycled from an old hot air balloon donated for this purpose.
Each flag must include a piece of the balloon's 2,400 square feet of pink, purple or burgundy fabric. All the flags will be three feet tall and six feet wide. The top and one side have to be straight to allow them to hang correctly on the poles, but there is room for curves on the other two sides.
Hejduk said that while artists' abstract designs should be inspired by nature, there's a lot of leeway . Artists aren't limited to what can be found locally in nature and might even use the flora and fauna of visiting nations as their starting point.
In addition to receiving the materials to make their flags, the 10 chosen artists will each get a $1,500 stipend.
So far, Hejduk said some submissions have come from groups of artists working together.
“Not all visual artists are fiber artists that can handle the sewing elements associated with it,” she said.
Meadowbrook Park will display one flag from each artist, and the other 30 flags will fly at Theatre in the Park. Some flags will be attached to lamp posts, but there is money in the project budget for temporary flag poles as well.
To encourage both visitors and locals to see not only the flag displays but all the other art on offer in Johnson County, the public art division will launch an art passport guide later this year that leads people to various local sites.
“We're going to have millions visiting the whole metro area. I think that we'll be very surprised by number of people who we see in our communities that are enjoying and seeking to learn what our culture is here,” Hejduk said.
“I think they said the average visitor will be in town for nine days. There's a lot of time between games when somebody's here that long.”
For more information on the project guidelines and how to submit a proposal, visit JCPRD's website.
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Manchester United interim coach Darren Fletcher has declared there is a huge amount of talent in the club's academy, in contrast to predecessor Ruben Amorim's derisory remarks about the Red Devils' youth players. Fletcher has been coaching United's under-18s this season but is temporarily in charge of the first team after Amorim was sacked on Monday.
Fletcher oversaw a disappointing 2-2 draw at Burnley in his first game but almost watched his side snatch victory thanks to academy graduate Shea Lacey. The Liverpool-born winger was given his United debut under Amorim at Aston Villa last month but his second appearance was more notable as he hammered the crossbar from outside of the area at Turn Moor and then let fly again in stoppage time, narrowly missing the target.
While Amorim also handed debuts to Fletcher's son Jack and Bendito Mantato as well as fielding Harry Amass, Tyler Fredricson and Chido Obi last season, he used academy players sparingly on the whole. He only turned to the youngsters during an injury crisis this term or as he rotated the squad heavily last term in preparation for Europa League knockout games. Amorim also angered the United fanbase with less than complimentary comments about players including Amass and Obi, and when those players responded by defending their own performances on social media, he said that young players in the club felt "entitled".
Fletcher, who came through the academy himself two decades ago and has another son in the first team squad in Tyler, said the club should be extremely proud of its tradition of blooding youngsters as well as proud of its current crop of players.
Fletcher told a press conference: "I think what I see is a lot of hard-working, humble young players who aren't perfect because they're young and they're learning and they've got a lot to do. I think we ask and expect too much of young people sometimes in society and in general, and that's not a criticism of anybody, that's just my beliefs.
"I think we have to let them learn, educate them, help them, understand they're going to make mistakes and they're going to do things that will frustrate you but through time with good guidance of coaches, family members, players in the first team squad, all of us play a part, that's our role, that's our job of developing them to be Manchester United players and Manchester United people.
"All I can say is my workings with those players as individuals and in general throughout the academy I deal with hard-working, honest, coachable, super talented kids and we've got a lot of talent in our academy and hopefully they can showcase themselves like Shea did when he came on against Burnley and so close to scoring a fantastic goal."
As well as rarely calling on the players in United's under-18 and under-21 squads, Amorim neglected some of their best known academy graduates in recent years. He forced Marcus Rashford and Alejandro Garnacho out of the club while he didn't start Kobbie Mainoo in any Premier League matches this season.
Mainoo returned to action against Burnley after missing the previous four games due to injury and Fletcher said the midfielder was in good spirits. He explained: "He seems in a good place. Kobbie doesn't give you much so you wouldn't know if he was in a good place or not. He's like that, it's just his natural way, I know him well and I have known him for a long time, I've seen him around the building and had conversations with him. He's in a good place, but I know Kobbie and he knows me and Travis Binnion and he's comfortable with this environment. He is in a good place, he's trained well and as I say he is hard to read, so that remains to be seen."
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United face Brighton in the FA Cup third round on Sunday at Old Trafford. The competition, which United have won 13 times and most recently in 2024, is their only chance of lifting a trophy this season and Fletcher stressed the importance of doing well in it. He said: "It's an important competition and Manchester United are about winning trophies. We've had good success in recent years, we won it a couple of years ago and lost in the final.
"It's an amazing competition, the first trophy I won as a player, it is a special tournament. Not in Europe, out of the League Cup, there is the Premier League obviously but the FA Cup, it's a trophy we should be vying to win and be giving ourselves every opportunity to win."
Fletcher expects to have the same squad available to him as against Burnley as he confirmed that neither Noussair Mazraoui nor Bryan Mbeumo will be in contention even though one of them will be heading out of the Africa Cup of Nations as Cameroon take on Morocco in the quarter-finals later on Friday.
The interim coach said that Harry Maguire could feature after he was unused substitute at Burnley but he stressed that he would have to carefully manage his minutes as he has not played since the 2-2 draw at Tottenham on November 8.
While New York has decided to charge for some of its fan activations, Philadelphia has stated that its fan festival at Lemon Hill will remain free.
There's been considerable discussion surrounding what many feel are exorbitant ticket prices to attend a match at this summer's FIFA World Cup.
But another recent announcement seemingly sent people over the edge. For the first time in the history of the tournament, FIFA would charge fans to attend its fan festivals across many of the 16 cities in North America selected to host games in the monthlong tournament.
And while that has been made public for at least one of the hosts, general admission to Philly's fan festival, scheduled for June and July on the grounds of Lemon Hill Mansion in the Brewerytown section of the city, will remain free, according to Philadelphia Soccer 2026, the committee responsible for the planning and execution of Philly's tournament footprint.
“Since our selection as a host city in 2022, Philadelphia Soccer 2026 has remained committed to making sure every fan can share in the excitement, culture, and community of this generational sporting event,” Meg Kane, host city executive of Philadelphia Soccer 2026, said in a statement to The Inquirer on Wednesday.
“Essential to that commitment, we made the decision to offer free general admission to FIFA Fan Festival at Lemon Hill, ensuring an inclusive and welcoming environment where fans from all backgrounds can come together to celebrate the world's game.”
» READ MORE: To kick-start a generation of city kids playing soccer, it will take more than just a place to play
While general admission will remain free for the scores of fans who are expected to descend upon Philly over the course of five group matches and a massive round of 16 game on July 4, there will be “optional VIP experiences,” including expedited entry into festival grounds, and are expected to be available for purchase at a later date.
Kane's announcement mirrors that of other cities, such as Kansas City and Vancouver, which also have stated their intention to keep admission free for their events.
But when the news of potential fees at fan festivals initially landed, it certainly didn't appear that would be the case.
Amid the news that FIFA plans to charge for its fan festivals, it was overlooked that only one delegation has formally announced its intent to charge an upfront entrance fee.
In fact, a spokesperson with knowledge of the proceedings told The Inquirer that any intention to add a fee to the festivals was not a blanket decision made by FIFA as soccer's world governing body; instead, it is left to host city committees to decide.
A FIFA spokesperson confirmed this and added on Thursday that while some host city delegations have begun relaying their fan festival plans, “FIFA will communicate the full suite of details [for all 16 host cities] in the first quarter of 2026,” where, in addition to what's to come at those sites, announcements of which ones might consider charging a fee will be made public.
“From the outset, FIFA has worked closely with host cities and local stakeholders to help shape meaningful fan experiences beyond the stadiums that are community-led, fan-oriented and aligned with the spirit of the FIFA World Cup,” a FIFA spokesperson said in a statement to The Inquirer. “It is important to recognize that there is no one-size-fits-all model for fan engagement across a tournament of this scale.”
FIFA's spokesperson also noted that “fan experiences can take many forms — from large-scale gatherings to more decentralized, community-driven activations,” which dovetails into the preliminary plan of attack of the New York-New Jersey delegation, which isn't viewing its overall fan engagement strategy as hosted one large site, but several.
» READ MORE: FIFA slashes price of some World Cup tickets to $60 after fan backlash
For soccer fans planning a trip for the World Cup final or New Yorkers who can't afford it but want in, tickets are available for New York's main fan festival at Liberty Park via Ticketmaster for $12.50.
But there's a methodology at play here.
According to a host city committee official, the move isn't as much a revenue driver as a crowd management strategy designed to regulate capacity and effectively coordinate staffing, security, and transportation.
Essentially, by putting a limit on the number of people expected to descend upon the area to watch a series of matches in June and July, the Liberty Park fan festival can be capped at a number, one anticipated to still be in the tens of thousands, daily.
To accommodate a global population, the delegation plans to bring in a scaled-down version of its festival, termed as “fan zones,” into all five New York boroughs. The first two have already been announced: Rockefeller Center in Manhattan will host a “fan village,” as will the Billie Jean King National Tennis Center, the home of the U.S. Open in Queens.
More are expected to be announced later, and the fan village at Rockefeller Center will be free to attend. As of now, New York-New Jersey is the only host city committee planning fan experience that's not situated in a single location.
“New York-New Jersey is building a regional fan experience unlike anything seen in World Cup history, Alex Lasry, CEO of the New York-New Jersey host committee, told The Inquirer. “We're proud to have announced three official NYNJ Host Committee fan experiences that will bring the World Cup far beyond the stadium.
“These spaces are essential to the World Cup experience, creating accessible and affordable places for people to come together and experience the biggest games in one of the world's most iconic venues. And this is just the beginning — we look forward to announcing additional fan engagement opportunities so the entire region can feel the impact of the World Cup.”
» READ MORE: Follow the Inquirer's complete coverage of World Cup soccer here!
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The Soweto giants are fully focusing on domestic duties after failing to reach the group stage of Africa's premier club competition. Having already won the MTN8 and Carling Knockout so far this season, the Buccaneers have managed to pacify their fans and at least have something to cheer about as they witness traditional rivals Mamelodi Sundowns and Kaizer Chiefs playing continental football.
GOAL runs through what the fans have been saying in reaction to Tshepang Moremi drawing positives from Orlando Pirates' early exit from the CAF Champions League.
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😂😂😂so in other words there's no ambitions of winning the Champions League at Orlando Pirates? Like there's no ambition to accumulate CAF rankings points to compete in serious tournaments like Club World Cup? The only ambitions they have is MTN 8 and Black Label 🤦🏽♂️- Bokang Ntsane Sibiya
Yeah like Sundowns. Busy wasting money for nothing. Every season dlala CAF but winning eludes them - Laqhasha Qhasho
Players from Europe play week in week out, and they don't complain. Stop drinking Savanna - Mduduzi Nkabinde
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Moremi took the words out of my mouth - Tsekiso Mpho Calvin Motaung
They tried to use AAFCON Bafana Bafana to travel now they are around South Africa once again 😂😂😂✌️✌️✌️- Linda Lee Linda
What about your team Cowza Chiefs is it doing well? What have your team achieved this season??? - Prince Bhuda Memela
You just arrived at Pirates and you already complaining - Ma Eiight Machidii
Pointless Khoza postponed games for them last season - Malesela Samuel
My favourite player talking nonsense 😭😭😭 we want CAF brother regardless - Mishack Ntiwane
We wanna win TotalEnergies CAF Champions League & Confederation Cup and compete at the FIFA Club Football, show ambition guys - Steghel Lebokoboko Pau Pau Nonsense
😂😂😂😂😂that's why European teams won't take our league serious - Lefa Strauss Lekitlane
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U.S. women's national team head coach Emma Hayes said she wasn't consulted before the NWSL announced its new High-Impact Player rule last month.
The mechanism allows teams to exceed the NWSL's salary cap by up to $1 million for certain "high-impact" players, with the aim of keeping some of the league's biggest stars from seeking out bigger paydays in Europe.
But the rule has proven controversial due to some of the criteria needed to be considered a high-impact player.
In addition to accomplishments like earning NWSL Best XI or MVP honors, a player could also meet the threshold by being named to the SportsPro Media Top 150 Most Marketable Athletes list, or being selected on the short list for awards like the Ballon d'Or that aren't considered representative of the top players in the world.
Another criteria is being in the top 11 for USWNT minutes over a two-year period for a field player, or the leader in minutes for a goalkeeper over a two-year period.
Though she now has some say over who qualifies for a bigger contract in the NWSL, Hayes revealed that she wasn't consulted prior to the rule's introduction.
"No, I didn't know about it," Hayes said on a call with media on Thursday.
The NWSL Players Association has come out strongly against the rule, advocating instead for an increase in the salary cap that will allow teams to spend money at their discretion.
"Under federal labor law, changes to compensation under the salary cap are a mandatory subject of bargaining — not a matter of unilateral discretion," the NWSLPA said in a statement after the rule was announced.
"Fair pay is realized through fair, collectively bargained compensation systems, not arbitrary classifications. A league that truly believes in the value of its Players would not be afraid to bargain over it."
The NWSLPA has said it is planning to take legal action.
Amid vociferous opposition from its players' union, Hayes said the NWSL may end up making some changes to the rule before all is said and done.
"I know that the NWSLPA and the league are trying to work through that criteria," she said. "I'd say it's between them to try and do that.
"But when it comes to whether that influences playing time, nothing will change with me and the way that I'm doing things, regardless of any ruling that's put in place. To be honest with you, it's probably going to be a little bit longer until they resolve what that criteria is."
University Announcements
Four-time World Cup Champions Arrive in Winston-Salem on June 8
January 9, 2026 | by University Marketing and Communications
The German Men's National Team has selected Winston-Salem as its home base for the 2026 FIFA World Cup, choosing Wake Forest University and The Graylyn Estate as its official training site and team headquarters – bringing one of the world's premier soccer programs to the Triad and placing Winston-Salem on an international stage.
As part of the partnership, the German team will reside at The Graylyn Estate and train at Wake Forest's W. Dennie Spry Soccer Stadium, one of the nation's premier collegiate soccer facilities. Together, Wake Forest and Graylyn will provide world-class training infrastructure and accommodations as the four-time World Cup champions prepare for and compete in one of the world's premier global sporting events.
“Wake Forest University is truly honored to host the German Men's National Team as its official training base for the FIFA World Cup 2026,” Wake Forest University President Susan R. Wente said.
“We are eager to welcome one of the world's premier soccer teams to Winston-Salem, our Triad community, and to Wake Forest's W. Dennie Spry Soccer Stadium – one of the top soccer facilities in the nation, and to The Graylyn Estate – one of the nation's most historic and highly sought-after boutique hotels. This global partnership reinforces our dedication to support a vibrant, connected region, and we are enthusiastic about the possibilities of making the World Cup experience available to our community.”
Germany is one of the most successful national teams in the history of the sport, having won FIFA World Cup titles in 1954, 1974, 1990, and 2014. The team is led by Coach Julian Nagelsmann, widely regarded as one of the most innovative coaches in international soccer.
Germany's selection of Winston-Salem reflects the region's growing reputation as a destination capable of hosting elite international events. The partnership is expected to generate international visibility, stimulate economic activity, and foster meaningful community engagement, while showcasing the strength of collaboration between Wake Forest, local leaders, and statewide partners.
Germany selected Wake Forest as its host training site due to its thriving collegiate environment, state-of-the-art athletics facilities, and reputation as one of the most beautiful campuses in the country. Germany also chose The Graylyn Estate as the team's base camp due to its national prominence as a premier boutique hotel and its secluded, historic 55-acre setting, which offers the privacy, focus, and comfort required at the highest level of international competition. Its proximity to Wake Forest's athletics facilities further positioned Winston-Salem as an ideal home base throughout the tournament.
“With our Home Ground in Herzogenaurach, we found a place that allows the team to come together regularly while still providing the necessary peace and privacy. We have discovered exactly the same conditions here at Graylyn. From a coaching and team perspective, the most important factor is the proximity to the training pitches,” German Men's National Team Head Coach Julian Nagelsmann said.
“The cooperation with Wake Forest University is unique. We already find many things available on site and don't have to bring everything from Germany. That makes logistics much easier. We have outstanding conditions here, both on the pitch and beyond.”
The 2026 FIFA World Cup will take place from June 11 to July 19 across the U.S., Canada, and Mexico. Germany is scheduled to open group play on June 14 at the Houston stadium, followed by matches at the Toronto stadium on June 20 and at the New York New Jersey stadium in East Rutherford, New Jersey, on June 25. The World Cup Final will be held July 19 in New Jersey.
This global partnership underscores Wake Forest's strategic role in delivering community impact for Winston-Salem, the Triad, and the state of North Carolina. The initiative is supported through collaboration with key partners, including the City of Winston-Salem, the Economic Development Partnership of North Carolina, Greater Winston-Salem, Inc., Visit North Carolina, and Visit Winston-Salem.
More information about a community engagement event with the team upon their arrival in Winston-Salem and the fan experience in general will be shared at a later date. For more information, please visit the University's website.
“Wake Forest University is truly honored to host the German Men's National Team as its official training base for the FIFA World Cup 2026. We are eager to welcome one of the world's premier soccer teams to Winston-Salem, our Triad community, and to Wake Forest's Spry Soccer Stadium – one of the top soccer facilities in the nation, and to The Graylyn Estate – one of the nation's most historic and highly sought-after boutique hotels. This global partnership reinforces our dedication to support a vibrant, connected region, and we are enthusiastic about the possibilities of making the World Cup experience available to our community.”
“Hosting Team Germany is a tremendous honor and highlight for Wake Forest and The Graylyn Estate. Our selection as their North American base validates both our university's global standing and the world-class hospitality delivered by the staff of The Graylyn Estate. We are excited about this partnership and the memorable experiences it will offer Wake Forest and our Winston-Salem community.”
“Wake Forest is proud to partner with Winston-Salem to serve as the German Men's National Team's home base during the 2026 FIFA World Cup. Hosting a four-time World Cup champion reflects the strength of our University soccer culture, which is truly world-class — most recently highlighted by our women's program finishing as the 2024 NCAA runner-up and our men's team capturing the 2024 ACC Championship. Under the leadership of Tony da Luz and Bobby Muuss, our programs have established a standard of excellence for Demon Deacon student-athletes and fostered deep engagement with the local soccer community, including a strong tradition of outstanding youth camps that will continue this summer. This is another winning moment for Wake Forest and Winston-Salem, and I am deeply grateful for the collaboration that made it happen!”
“Wake Forest and Winston-Salem being selected as the temporary home for a national powerhouse like Germany preparing for the World Cup is an incredible honor for our facilities, our university and our entire city. To welcome a World Cup-bound team and to play even a small role in their preparation is something our players, coaches and fans should be proud of. It puts a global spotlight on our campus and our city in a truly special way.”
“Hosting the German men's national team as they prepare for the World Cup is an honor and will bring immeasurable exposure to Wake Forest University and Winston-Salem. We've always promoted Spry Soccer Stadium as a world-class soccer facility, and this selection affirms that statement. This is a once in a lifetime opportunity to share Wake Forest with the world.”
“With our Home Ground at the adidas headquarters in Herzogenaurach, we have a place that allows the team to come together regularly while still providing the peace and privacy we need. We have found the same conditions here at The Graylyn Estate. From a coaching and team perspective, the most important factor is the proximity to the training pitches. We have three football fields in excellent condition that meet all of our sporting requirements, and they can be reached on foot or by bike in less than ten minutes. That is a decisive advantage. Given the vast distances across the United States, Canada and Mexico, we already have to cover long stretches when traveling, so we want to avoid additional journeys in our daily training routine. With this base camp, we have achieved exactly that.
“The cooperation with Wake Forest University is exceptional. Many of the things we need are already available on site, so we don't have to bring everything from Germany. That makes logistics much easier. We have outstanding conditions here, both on the pitch and beyond.”
“With this Team Base Camp, we are creating the best possible conditions for a successful tournament. The environment is ideal — both in sporting and infrastructural terms. The team behind the team at the DFB has, with great precision and passion, identified the perfect base camp for us through an extensive process across the three host nations. Our close and constructive cooperation with FIFA, as well as with the local authorities, Wake Forest University and the hotel management, has played a key role in making this possible. Thanks to a favorable draw, we were able to implement our preferred scenario.”
“Hosting the German Men's National Team is a big opportunity for the city of Winston-Salem to showcase our renowned hospitality, vibrant city, and first-class facilities. We are proud to welcome the team, their families, journalists, and fans who will call our beautiful city home during the World Cup. We encourage all visitors to enjoy our wonderful hotels, restaurants, and shops during their visit!”
“Welcoming the German national team to train at Wake Forest is an extraordinary honor for Winston-Salem and a proud moment for our community. Their presence showcases our city on the global stage and reflects the warm hospitality and passion for sports that define this destination.”
“We are excited for the German men's national team and their guests to get to explore Winston-Salem and feel that this is their ‘home away from home' during the World Cup. We are proud to be the host city for DFB and to further the strong business relationship between North Carolina and Germany. This opportunity brings Winston-Salem to the world's largest sporting event, and we are eager to showcase our city.”
“North Carolina is a destination for businesses and tourists from around the world, and the German national team's decision to establish its base in Winston-Salem this summer is another great endorsement of our state. This selection followed a thorough evaluation process, and it reflects the high standard of quality North Carolina offers — from our world-class facilities and hospitality to our global connectivity, including daily direct air service that positions Germany as one of our top international markets for visitation. We're proud to welcome a team known for its discipline and excellence as the world's attention turns to the 2026 World Cup.”
U.S. and international news media will be given access by the Deutscher Fusball-Bund's (DFB) press office, the sports organization managing the men's German National Soccer Team. For more information, visit https://www.dfb.de/.
Wake Forest University is known for its distinctive combination of world-class academics, unrivaled campus experience, intimate learning environment and Power 4 athletics in a top-growing metro market. A Charter member of the Atlantic Coast Conference, the Demon Deacons have won 59 conference titles and are one of nine ACC schools to win 11 or more national championships. Additionally, with 1.7 million people within 30 miles of campus, Wake Forest anchors the Winston-Salem and Triad market, which ranks as ESPN's seventh-best nationally from a viewership perspective.
Wake Forest's comprehensive excellence includes its highly regarded school of medicine, business school, law school, innovative department of engineering and its nationally renowned Program for Leadership and Character, which prepares students to live with purpose, integrity and courage. Additionally, Wake Forest has campuses across Winston-Salem, Charlotte and Washington, D.C. – providing many academic offerings to students from across the nation and around the world.
Learn more about Wake Forest University at wfu.edu and at GoDeacs.com.
The Graylyn Estate features 85 historic and modern guest rooms located throughout its beautiful 55-acre grounds. Originally built in 1932, the estate was once the private residence of Mr. Bowman and Mrs. Nathalie Bowman Gray. Bowman, the son of Wachovia founder James A. Gray Sr., served as President and CEO of RJ Reynolds Tobacco Company. Together, Bowman and Nathalie created their dream home, Graylyn, with each room inspired by their travels abroad.
In 1972, the property was donated to Wake Forest University by the family. After serving as student dorms, the property was transformed into a boutique hotel in 1984. Guests at Graylyn experience legendary hospitality through the staff's commitment to exceptional service, ensuring exquisite and unforgettable experiences. Graylyn has received national recognition as a top hotel in the United States, having been honored as a top 5 boutique hotel in the country for 2023, 2024 and 2025.
Learn more about The Graylyn Estate at graylyn.com.
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Roy Keane has sensationally accused Sir Alex Ferguson of "hanging on like a bad smell" at Manchester United. The legendary Scot retired from management in 2013 on the back of leading the Red Devils to a 13th Premier League title. He remains a regular presence at Old Trafford, with Darren Fletcher admitting that he spoke to his fellow countryman before agreeing to take charge of United on an interim basis following the dismissal of Ruben Amorim.
Another change in the dugout was made on January 5, with Fletcher becoming the 11th man to take charge of United - either permanently on in a caretaker post - since Ferguson brought his 27-year reign to a close.
It has been suggested that the Red Devils will look to make another appointment through to the end of the 2025-26 campaign - with former fan favourites Ole Gunnar Solskjaer, Michael Carrick and Ruud van Nistelrooy in the mix there - but Keane claims a lack of decisive leadership from the top is holding the club back.
The outspoken ex-United captain has told Sky Sports of the Red Devils' inability to find the right candidate to take them forward and recapture former glories: "What happens in these job interviews? I'm intrigued. Why do they keep giving certain people a job? What happens in the interview that they sit there and go, and 12, 14 months later, ‘he's not the guy for us'. Do you not suss that out when you speak to them? You look somebody in the eye and go…
"You see who's making the decisions at Manchester United... you still have Ferguson and David Gill [former chief executive] hanging on like a bad smell. Who's making the decisions? [Sir Jim] Ratcliffe, [Jason] Wilcox? Who's coming into this interview process, you're speaking to a manager, you get a feel for somebody and go ‘he's the guy for us?'
"Almost forget the CV. You need something on your CV, of course, that you've won a trophy or managed a long time. But you've got to look somebody in the eye and go ‘are you the man to get us places?'
"What happens when somebody walks in your dressing room, the top players sit and go, ‘what have you got for us?' That's what the top lads do. And if you haven't got the answers, the players are going to eat you alive."
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Keane went on to say, with Fletcher's first game at the helm delivering an uninspiring 2-2 draw away at relegation-threatened Burnley: "United-Burnley, the standard of the game was a joke. Everyone after the game was like, 'It wasn't bad, we did well' – nonsense! Absolute rubbish. We're going around in circles. Any ex-United player has got a chance."
Former Scotland international Fletcher is a product of United's fabled academy system and made his breakthrough at the age of 19 under Ferguson in 2003. He has said of continuing to seek the advice of an iconic mentor: "I don't like to make any major decisions or things without speaking to Sir Alex and that's something I've done since I've been at the club and since I've left the club in everything I do.
"I've got a really good relationship with Sir Alex, so he's probably the first person on the phone actually, so I wanted to speak to him first. And, ultimately, to get his blessing, to be perfectly honest with you."
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Fletcher may not be in charge of first-team affairs for long, with it being reported that 1999 Treble winner Solskjaer - who stepped in as cover for Jose Mourinho in 2018 before going on to spend almost three years at the helm - is being lined up for an emotional return.
Whoever gets the nod will be given the chance to stake their claim for a long-term stay, but Keane is among those to have suggested that the Red Devils need to make a play for Newcastle manager Eddie Howe, while Crystal Palace boss Oliver Glasner - who will be out of contract in the summer - is also said to be figuring prominently on the Old Trafford recruitment radar.
The 19-year-old centerback with German club Augsburg has all the physical and soccer tools to become big-time. If he keeps playing regularly, could there be room on the U.S. World Cup team for him?
You don't have to watch Noahkai Banks for long to see why so much hype surrounds him.
The 19-year-old centerback stands 6-foot-4 and around 200 pounds, and has been a regular starter for Augsburg in Germany's Bundesliga this season. Born in Hawaii and raised in Germany from a young age, he has been on the U.S. men's national team radar for a few years now, including the 2023 under-17 World Cup squad.
Last September, he earned his first call-up to the senior squad. Though he didn't play in that month's games and hasn't been called up again since, just that one invitation got a lot of attention.
“He is a player that is really young, but with amazing potential,” U.S. manager Mauricio Pochettino said at the time. “He's really young, but it's good to see him, because he can go fast to the next level.”
It might be too soon for Banks to make this year's World Cup team. Then again, the U.S. centerback depth chart isn't in ideal shape right now, and the 26-player roster might be big enough to have room for him. The starting trio looks likely to be Chris Richards, Tim Ream, and Mark McKenzie, but no one has yet locked down a backup spot.
That adds to the buzz around Banks right now, and he knows it's out there.
» READ MORE: Brenden Aaronson is on a hot streak with Leeds United at an ideal time for his World Cup hopes
“I don't read much, to be honest, but my mom always sends everything in the family chat because she uses a lot of Twitter and apps like this,” he said. “It's cool. It's a pleasure.”
He said he enjoyed his senior U.S. camp, though he hasn't had a one-on-one talk with Pochettino yet.
“I've already felt the excitement during my training sessions, and I can see the team playing a strong role,“ Banks said. ”The national team boasts some excellent players — [Christian] Pulisic, [Malik] Tillman, Richards — and, of course, a highly experienced coach who has managed some of Europe's top clubs. … It was immediately clear that Pochettino is a world-class coach, but I wouldn't presume to expect a personal one-on-one during my first training camp."
A touch of homespun warmth certainly won't hurt his standing with fans either. But asked if he's thinking about the World Cup, he quickly tapped the brakes.
“To be honest, I don't think about the World Cup at the moment, because we have a difficult situation here at my club,” Banks said. “So it's just about going from game to game here, and then let's see what happens.”
» READ MORE: Projecting the USMNT's World Cup roster
He was referring to Augsburg being in the thick of a relegation fight. The Fuggerstädter, whose ownership group includes 76ers part-owner David Blitzer, stand 15th in the 18-team table — the last spot where staying up is guaranteed.
Augsburg fired manager Sandro Wagner in early December after just 12 games. The current boss, Manuel Baum, is an interim who coached just three games before the Bundesliga's winter break arrived the weekend before Christmas.
That's enough for any player to handle, not just an 19-year-old. So perhaps you can take Banks' patience as a sign of maturity.
One of his best games this season so far was his next-to-last one before the winter break, on Dec. 13 at Eintracht Frankfurt.
Amid the hothouse atmosphere of Frankfurt's 59,500-seat stadium — perhaps you saw the NFL games it hosted in 2023, along with decades of big soccer tournaments — Banks was one of the best players on the field. Though Augsburg lost, 1-0, he had five tackles and six clearances, won eight of the 13 duels he contested, and completed 42 of 47 passes.
» READ MORE: Folarin Balogun is living up to the hype as a long-awaited top striker for the USMNT
It's worth noting, too, that Banks played mostly as the right centerback in a 3-4-2-1 formation — the same setup the U.S. national team is using heading toward the World Cup. He even would have scored a late equalizer had he not been just barely offside when a corner kick was flicked on to him near the goal line.
The Bundesliga season resumes this weekend, with Banks' Augsburg coincidentally visiting two other Americans: Borussia Mönchengladbach's Gio Reyna and Joe Scally (Sunday, 9:30 a.m., ESPN+). There are always lots of games around Europe for U.S. fans to watch, but that one might draw a little extra attention.
It might also remind those fans that Germany has long been the top port of call for U.S. players who move abroad.
Many aspire to play in England, and the doors there are much more open than perhaps they've ever been. Still, the Bundesliga's track record of being a place where Americans cannot just move but actually play and develop remains the best of any top European league.
» READ MORE: The USMNT, USWNT, and your kid's youth team are all different. U.S. Soccer is fine with that.
Eintracht knows this as well as any Bundesliga club, as Philadelphia fans have seen from its preseason tours here in recent years. Former World Cup outside back Timmy Chandler has long called Eintracht home, as did Medford's Paxten Aaronson for a while.
There's another young American playmaker in the club's pipeline in Marvin Dills, and Eintracht tried to sign much-touted Union striker prospect Malik Jakupovic before he decided to turn pro at home first.
“You have to give them the opportunity to grow and develop,” Eintracht sporting director Timmo Hardung said. “And I think this is what some of the clubs, us included, are trying to do: find players with top potential, with top talent, and ready to grow, ready to learn, ready to develop.”
Speaking of the U.S. specifically, he said: “The sportsmanship and the athleticism in the United States is top, and that should produce a lot of players out of the soccer landscape.”
» READ MORE: The USMNT will play Paraguay, Australia, and a European qualifier at the 2026 World Cup
Banks, who started in Augsburg's youth academy at the under-10 age level, said the club has brought him along well.
“FC Augsburg is a very well-run and family-oriented club,” Banks said. “The club gives me valuable playing time, which is crucial for my development.”
He praised the club's managing director, Michael Ströll, for having “a clear vision: Young players should make their way into the Bundesliga, and the club is also striving to further develop its playing style — with a focus on more active football.”
U.S. fans might remember that things didn't go as well there for Ricardo Pepi in 2022, or for Michael Parkhurst in 2013. But Ströll didn't take the job until after Pepi left, so we'll see if things are different now. One piece of evidence is that Augsburg reportedly looked at signing 21-year-old Tampa native Santiago Castañeda in the summer.
» READ MORE: The USMNT seems on course to do something it hasn't done in nearly a quarter century
Banks said he's also happy off the field at Augsburg, which is just over 70 miles from the town of Dietmannsried where he grew up. Both towns are in the German state of Bavaria, where the most famous city is Munich.
“I really appreciate being close to my family and the mountains,” Banks said. “Although I haven't lived abroad yet, it is certainly a goal I hope to pursue in the future.”
If he keeps playing this well, the odds of that will certainly go up. And so will the odds of another national team call-up.
The new year has so far been kind to American players, with many finding form across Europe's top leagues as the prospect of representing the U.S. men's national team at the 2026 World Cup looms.
While some players have locked themselves into roster spots, like in-form Juventus midfielder Weston McKennie and AC Milan attacker Christian Pulisic, others are still contending to secure their spots on manager Mauricio Pochettino's roster.
After a few quiet weeks for the holiday break, the Bundesliga and Eredivisie resume this weekend, while many of the Americans in England clash against others from the Stars and Stripes in FA Cup action.
Here's Sports Illustrated's look at who will look to turn heads in this weekend's action.
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Barring something exceptional, Cole Campbell won't be at the 2026 World Cup—but for those following the future of American soccer, he's one of the most important names to watch.
The 19-year-old recently moved to TSG Hoffenheim, currently sitting fifth in the Bundesliga, on loan, after struggling to get minutes at giants Borussia Dortmund.
Primarily a winger who can also play centrally, the former Atlanta United academy product moved to Europe at 15 and made his senior debut with Iceland's Hafnarfjarðar in 2021-22. Since then, he made five appearances with the Dortmund first team and scored 18 goals and 19 assists across 52 games at the U17 and U19 levels with Dortmund.
By making the move, he hopes to get more playing time in the top flight and that chapter begins on Saturday against Werder Bremen.
The Eredivisie returns for the first time in 2026 this weekend and plenty of eyes will be on Ricardo Pepi as the battle heats up for a forward spot on USMNT's 26-player roster.
Pepi, who turned 23 on Friday, enjoyed a stellar first portion of the season, scoring 10 goals across 21 appearances across all competitions for PSV Eindhoven—helping them to the top of the table by the time the calendar flipped. While his good form stands out, he will need to ensure it isn't just early-season success, but a maintainable level through to this summer's tournament.
He is likely to be in the starting lineup alongside USMNT and PSV teammate Sergiño Dest against Excelsior, who, until Wednesday, also had a young American on their roster in 21-year-old Zach Booth, before loaning him to MLS side Real Salt Lake for 2026.
With Brenden Aaronson, Patrick Agyemang, Christian Pulisic and other European-based American attacking players in form, it's vital for Pepi to keep scoring at an impressive clip.
The top three Americans to watch this weekend all find themselves in FA Cup competition, starting with Middlesbrough midfielder Aidan Morris, who, despite impressing in the fall friendlies, is still on the fringes of the USMNT's 2026 World Cup picture.
While he was an attacking presence in a transitional double pivot with the USMNT, his role at Boro this season has largely been in a defensive midfield role alongside Hayden Hackney —where he has helped them to second in the EFL Championship with 20 appearances.
This weekend he takes on Fulham in the third round of the FA Cup, with a chance to impress against a Premier Leageu side that contains USMNT fullback Antonee Robinson.
Morris is still looking for his first goal of the season and he's contributed just two assists during 2025–26. However, he's stood out in other areas, and completed an impressive 70 passes in last week's 4–0 beatdown of Southampton.
dark. Next. USMNT Best XI World Cup. Predicting USMNT's 2026 World Cup Best XI
Three straight games without a goal for Patrick Agyemang is worrying for a striker who bet on himself this season to impress in the Championship with Derby County, instead of remaining in MLS as a star striker with Charlotte FC.
Before his current dry streak, held goalless in 209 minutes against Wrexham, Middlesbrough and Leicester City combined, he had scored four goals in six games and looked a surefire lock for the USMNT.
Yet, his stat line has dipped to six goals in 22 appearances this season and the recent spell has seen him struggle more than he had in the early stages of his time with the English side, when he battled for a starting spot.
This weekend sees him take on one of the most in-form American attacking players, Brenden Aaronson, as Derby meet Leeds United in the FA Cup. At this point, Agyemang might not be on the final World Cup roster—but Aaronson is showing he could potentially create chances for whoever takes the starting forward roles.
It wasn't too long ago that Brenden Aaronson's World Cup dreams looked to be over. He was not making an impact with Leeds United in the Premier League and there was no guarantee he'd even remain at Elland Road. Now, he boasts three goals and an assist in his last four Premier League contests.
The 25-year-old has established himself as a key attacking midfield asset for Daniel Farke, and his reaping the rewards of a tactical change from the manager. Midweek action saw him bag a brace in Leeds' thrilling 4–3 defeat at Newcastle United, days after he netted against Manchester United. Prior to that, Aaronson had played a key role in a 12-pass buildup for one of Leeds' best goals of the season.
While attacking and central midfield are positions of strength for the USMNT, Aaronson's form puts him on track to start at the World Cup, a remarkable turnaround after he looked to be trending off the roster in the fall. Now, he just has to keep it up.
This weekend sees his Leeds side clash against Agyemang's Derby County in the third round of the FA Cup, where the West Yorkshire club will be heavy favorites as the Premier League side.
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Ben Steiner is an American-Canadian journalist who brings in-depth experience, having covered the North American national teams, MLS, CPL, NWSL, NSL and Liga MX for prominent outlets, including MLSsoccer.com, CBC Sports, and OneSoccer.
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Goalkeeper Matt Turner was part of the squad for the Gold Cup in June but did not play Omar Vega/Getty Images
Matt Turner's journey to the United States men's national team was as unexpected as it was unconventional.
He picked up the sport at the age of 14. He didn't play varsity soccer until his junior year and thought about quitting the game after a mistake for Fairfield University went viral. He wasn't drafted and made the New England Revolution as a trialist. Then, he proved himself to be the best goalkeeper in MLS and started for the U.S. at the 2022 World Cup in Qatar, posting two clean sheets in the tournament.
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Turner hasn't forgotten how he got here. The 31-year-old doesn't take anything for granted. Right now, he needs that strength.
It has been a difficult year for Turner, who lost his hold on the U.S. starting goalkeeper job after struggling for playing time in Europe. He has not played for the national team since a 4-0 loss against Switzerland in June. He came back to MLS in search of regular action, rejoining the Revolution as he sought stability and an international return.
Turner was solid for New England down the stretch — according to data from American Soccer Analysis, he was one of MLS' top-performing goalkeepers on a per-game basis — but was left out of Mauricio Pochettino's squad for international windows in September and November.
So while he remains a face of the program — he was at a U.S. Soccer event in Times Square alongside defender Tim Ream and fellow goalkeeper Matt Freese for the World Cup draw in December — Turner knows his spot for the 2026 World Cup this summer is far from guaranteed. Turner believes remaining patient and playing every week is the only real path back to the starting job with the U.S.
“The mentality has always been the same,” Turner told The Athletic Thursday. “Focus on what I can control. If I am looking too much into the future or harping too much on the past, I lose a lot of my ability to be here now, and that's something I learned in the last year.
“I wasn't playing consistently, and it was challenging to always have that rhythm and routine and readiness. I was putting a lot of pressure on myself, like, ‘Oh my God.' Getting really, really hyped up for the times that I had opportunities to play, rather than just being in that, ‘OK, every week is the same,' and ‘Every game is kind of the same.' When you're not called upon week-in, week-out to be the guy, it can be challenging to switch that on and off.
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“This season, my goals are clear. Everybody knows I want to be playing games, meaningful games, for this national team. But you have to be playing well if you want to play for the team. And they're not scared to give anybody and everybody an opportunity. They give players what they deserve when they're playing well. They'll get their chances with the national team. And that's been really, really nice. I appreciate and welcome and enjoy the competition.”
Being left off two of the last three rosters, Turner said, has only added motivation.
“I never feel secure,” Turner said. “I'm the type of person who thrives in this type of environment. I like feeling like I need to prove myself. Sometimes it can be exhausting, but I feel ready to fight tooth and nail to prove myself. I'm in a really good spot to do that.”
Turner insisted that his “relationship with the staff is strong,” and said they have communicated, but declined to go into specifics.
“I have a clear pathway in my head of what I need to do and what needs to be done to be on the field for the team,” he said.
Turner said he is clear-eyed going into the 2026 season.
He will get a full preseason for the first time since 2021. That allowed him to approach this offseason differently and get his body right. After heading to Crystal Palace, where he played on loan in the 2024-25 season, to train before the November window in a bid to stay fit and earn a call from Pochettino, Turner and his family took time to see London when he was ultimately left off the U.S. roster. Ever since, Turner has been focused on letting some small nagging injuries heal, and he said he feels mentally and physically sharp for preseason with New England.
His goal, he said, is to peak from March to May, before Pochettino selects his World Cup roster. The U.S.'s opening game against Paraguay in Los Angeles is on June 12.
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“I'm in a really good place. I'm in control of my own destiny, and I know that if I'm playing to my maximum capability, it'll be hard for them to keep me off the pitch,” Turner said. “The competition is good and healthy and I'm going to do whatever I can to help this team be best positioned in 2026.”
A battle to be No. 1 comes with awkwardness. The goalkeeper union is strong, but there is just one starting job and Freese looks to be firmly entrenched as Pochettino's first choice — though there has been a rotating door behind him. Turner said the competition is healthy and insisted he is ready for whatever role is asked of him.
“There are heated rivalries and intense rivalries, but when the team sheet comes out, I've always felt like I'm part of something so much bigger than myself,” he said. “That I have to do whatever the coaches are asking me to do to make the team the best.
“I trust in the coaches' decision-making processes. I've never been a me-first kind of guy. And I hope that people have seen that I've always put the national team first and foremost in my life and in my career.”
Paul Tenorio is a senior writer for The Athletic who covers soccer. He has previously written for the Washington Post, the Orlando Sentinel, FourFourTwo, ESPN and MLSsoccer.com. Follow Paul on Twitter @PaulTenorio
All you need to know about securing tickets to the World Cup 2026 opener
Players and fans are hoping to see their side lift the World Cup 2026 trophy aloft at the World Cup final at MetLife Stadium in New Jersey on July 19. However, it's Mexico City and the Estadio Azteca, which hosts the opening match of the tournament on June 11, that grabs the initial spotlight.
A trip to the Estadio Azteca in Mexico City is a lifetime's dream for many football fans. It's one of the most prestigious venues in international football, having staged two previous World Cup finals.
A huge crowd of 107,412 saw Brazil beat Italy to be crowned champions in 1970. The 4-1 victory was sealed by that unstoppable and unforgettable Carlos Alberto strike. Sixteen years later (1986), an even bigger crowd of 114,600 (the largest ever World Cup Final attendance) saw Diego Maradona lead Argentina to the title, after they came out on top against West Germany, with another five memorable goals scored during the match.
Let GOAL take you through all the latest information for the opening match of the World Cup 2026, including how to get your hands on a ticket, how much it will cost, and more.
Book World Cup 2026 opening match ticketsBuy tickets
Football fans have a number of opportunities to purchase World Cup 2026 opening match tickets via the ticket portal on the FIFA site between now and the big day next June.
The various sales phases, shown below, differ in terms of purchasing processes, payment methods, and ticket products.
This was considered the first opportunity to purchase World Cup 2026 tickets and ran from September 10-19. It was only open to those fans who had qualifying Visa cards.
The entry period for the second phase took place between October 27-31. Like the first phase, it involved an application process followed by a randomized selection.
Successful applicants received a designated time slot to purchase tickets, which commenced on November 17.
Shortly before then, there was a prioritized slot (November 12-15) for residents of the three host nations (the United States, Canada, and Mexico), which allowed them early access to buy tickets.
Following the FIFA World Cup 2026 draw on December 5, the next phase of ticket sales commences. During this phase, fans will be able to submit further applications.
Closer to the tournament (Spring 2026), fans will be able to purchase any remaining tickets on a first-come, first-served basis.
FIFA hasn't said how many tickets will be released, but you can expect limited availability and quick sellouts.
To purchase tickets, you must visit the official FIFA ticketing portal and register for an account. You can then log in to your FIFA account and check out ticket availability.
If you are looking for an official and secure way to resell/exchange your FIFA World Cup 26 tickets, the FIFA Resale/Exchange Marketplace is the official channel for doing so. The Marketplace opened on October 2 and be accessed via FIFA.com/tickets.
The FIFA Resale Marketplace is available to Canadian, American and international residents, while the FIFA Exchange Marketplace (Mercado de Intercambio de la FIFA) is intended for residents of Mexico.
One key point for resale buyers: availability can be very limited, and tickets may appear sporadically. Fans hoping to secure resale tickets should check the platform frequently, act quickly when tickets appear, and have payment details ready in advance.
Secondary marketplaces such as StubHub, will also have World Cup 2026 opening match ticket availability. Ticket prices on secondary platforms are subject to availability and demand.
Book World Cup 2026 opening match ticketsBuy tickets
FIFA previously announced that with dynamic pricing in place, tickets for some group matches may start from as low as $60. However, those matches involving tournament hosts, like the tournament opener, were always going to be substantially more, as a result of increased demand. Prices will fluctuate throughout the various ticket releases/sales phases, but seats for the opening encounter at the Estadio Azteca originally ranged from $370-$1825.
Ticket prices for the World Cup 2026 Final vary by seating category as follows:
On secondary sites such as StubHub, fans can secure FIFA World Cup opening match tickets from $2,200 upwards.
Estadio Azteca (officially known as Estadio Banorte for sponsorship reasons) is a football stadium located in Coyoacan, Mexico City. It's been the regular home ground of Liga MX team, Club America, as well as the Mexico national team, since opening in 1966. With a capacity of 87,523, it is the largest stadium in Latin America and the eighth-largest association football stadium in the world.
The Azteca famously staged both the 1970 and 1986 World Cup finals and although it won't be hosting the FIFA World Cup 2026 finale, it will make history in the summer, becoming the only stadium to stages matches at three different World Cups. Following recent renovations, the stadium is set to reopen in March, three months before the opening game of FIFA World Cup 2026.
Book World Cup 2026 opening match ticketsBuy tickets
Supporters have a number of opportunities to purchase World Cup 2026 opening match tickets, via the ticket portal on the FIFA site, between now and the big day in June. The various sales phases differ in terms of purchasing processes, payment methods and ticket products.
To buy tickets, you must visit the official FIFA ticketing portal and register for an account. You can then log in to your FIFA account and check out ticket availability.
If you are looking for an official and secure way to resell/exchange your FIFA World Cup 26 tickets, the FIFA Resale/Exchange Marketplace is the official channel for doing so. The Marketplace opened on October 2 and be accessed via FIFA.com/tickets.Third-party resellers, such as StubHub, will also have World Cup 2026 opening match ticket availability.
The World Cup 2026 finals draw will take place at 12pm ET on Friday, December 5 at the Kennedy Center in Washington, D.C.
The 48 teams will be divided into four pots of 12. Pot 1 will consist of the three hosts and the top nine teams in the FIFA World Rankings not including the hosts. Pots 2, 3, and 4 will consist of the remaining teams according to the world rankings. The 12 groups will randomly be formed by selecting one team from each of the four pots. Two teams from the same confederation cannot be placed into the same group, with the exception of UEFA teams, where up to two teams can be in the same group.
The three host nations have been pre-allocated to three groups for scheduling purposes. Mexico has been placed in Group A and will play the opening match of the tournament on June 11. Canada and the United States have been placed in Groups B and D, respectively. They will play the third and fourth matches of the tournament, which both take place on June 12.
The four winners of the UEFA playoffs and the two winners of the inter-confederation playoffs will not be known at the time of the draw, as these matches are scheduled to take place in March 2026.
Securing seats at FIFA World Cup matches can be difficult with millions of fans desperate to get their hands on tickets. This is where FIFA's Right-To-Buy (RTB) option can come in handy. An RTB is an official FIFA allocation for FIFA Collect users that secures them an opportunity to purchase a ticket to a FIFA World Cup 26 match.
Think of an RTB token as a paid reservation. You're not buying the ticket itself. You're buying the right to buy that ticket at full price during a dedicated window, meaning no lottery, no virtual queues, no guessing games. If you hold an RTB token and act within your purchase window, you're guaranteed to get tickets.
Users enter FIFA Collect (collect.fifa.com) and buy packs of collectibles with RTB opportunities included. RTB owners have been able to pay and receive their FIFA World Cup 26 tickets during the Conversion Window which opened on November 13.
FIFA will be using dynamic pricing during World Cup 2026 ticket sales. Dynamic pricing is a system where ticket prices are adjusted in real-time based on supply and demand, similar to airline or hotel pricing. It means seats will tend to vary in price match-to-match. The goal of dynamic pricing is to give fans fair and safe access to tickets, that are as close to market value as possible.As the system is based on demand, the price of tickets does not always increase. We saw this with the FIFA Club World Cup earlier this year, where tickets for some of the knockout games, including the semi-final between Chelsea and Fluminense, were available from as low as $13.
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Two giants in the world of men's soccer are set to face off in Foxboro as part of the final warm-ups ahead of the 2026 FIFA World Cup, taking place in the United States, Canada and Mexico.
The soccer governing bodies of Brazil and France announced on Tuesday, Jan. 8 that the two sides will take each other on in a friendly at Gillette Stadium on Thursday, March 26 with a 4 p.m. kickoff scheduled. It will be the first meeting between Seleção and Les Bleus, who faced each other in the 1998 World Cup final, since 2015.
The match is part of a four-game slate of friendlies called the "Road to 26" set to take place during the final international break ahead of this summer's tournament, with France also taking on Colombia at Northwest Stadium in Landover, MD on Sunday, March 29 and Brazil facing Croatia at Camping World Stadium in Orlando, FL on Tuesday, March 31. Croatia, who fell to France in the 2018 final, will also go up against Colombia in Orlando on March 26.
Tickets for the match are set to go for sale at 10 a.m. on Tuesday, Jan. 13 for the general public.
Brazil, led by stars such as Real Madrid forwards Vinícius Júnior and Rodrygo and captained by Paris Saint-Germain defender Marquinhos, are the most successful national team in the history of the men's World Cup with five titles (1958, 1962, 1970, 1994 and 2002).
France, meanwhile, has two World Cup titles (1998 and 2022) to its name, and the side led by Real Madrid star forward Kylian Mbappé finished runners-up to Argentina in the 2022 World Cup in Qatar. Both are currently ranked in the top five of FIFA Men's Rankings as of Dec. 22, with France at No. 3 and Brazil at No. 5.
This will be the first of at least two matches for Les Bleus at One Patriot Way in 2026, with France slated to take on Erling Haaland and Norway in a key World Cup group stage match on June 26. Brazil is not slated to visit Gillette, which will be referred to as Boston Stadium by FIFA during the tournament, this summer. In addition to the five group stage matches, a Round of 32 match, in which Seleção could feature, on June 2 and a Quarterfinal match on July 9 are also set to take place in Foxboro.
The friendly is the first international match to take place at Gillette since Lionel Messi and Argentina beat Venezuela 4-1 in the Copa América Centenario quarterfinals in 2016. It's also the first international friendly in Foxboro since Brazil defeated the United States 4-1 at Gillette a year prior.
Tickets go on sale for the general public on Tuesday, Jan. 11 at 10 a.m., but fans can sign up for exclusive presale access on RoadTo26.com and selecting the "Brazil vs. France" option. Once the sale opens to the general public, tickets will be available on Ticketmaster.com.
If you purchase through our links, the USA Today Network may earn a commission. Prices were accurate at the time of publication but may change.
Top-tier WTA talent leads the charge on Saturday at the United Cup, where Switzerland faces Belgium and the United States renews its high-stakes rivalry with Poland for a chance at the 2026 title.
It's the last scheduled match in a United Cup tie, but as we've seen repeatedly this past week Down Under, mixed doubles is of the very first importance.
On Friday night, the unheralded team of Katarzyna Kawa and Jan Zielinski defeated John-Patrick Smith and Storm Hunter 6-4, 6-0 to send Poland soaring past Australia 2-1 and into Saturday's semifinals against Team USA.
Switzerland, sporting its own brand of mixed magic, takes on Belgium in the first semifinal at Sydney's Ken Rosewall Arena.
Both Perth quarterfinals came down to the all-or-nothing co-ed challenge, a relatively rare phenomenon seen only in Grand Slams and the Olympics.
Coco Gauff and Christian Harrison rallied from a set down to Stefanos Tsitsipas and Maria Sakkari to win 10-8 in a match-tiebreak. The Greek team was down 6-1 but reeled off five straight points to make it 6-all. The Americans won four of the next six points to advance to Saturday's semifinals against Poland in Sydney.
“I think in those moments it's important because literally in tiebreaks every point matters more so than maybe in a three-set format,” Gauff noted. “It definitely came down to the wire. So, yeah, I appreciate the whole team talking to us, pumping us up and seeing things that maybe we couldn't see on the court.”
It was the third time in three matches that Team USA got the winning points from Gauff and Harrison.
Switzerland needed late wins from Belinda Bencic and Jakub Paul against Italy in group play and in the quarterfinals against Argentina's Maria Lourdes Carle and Guido Andreozzi.
Stan Wawrinka, Switzerland's captain and singles player, was hoping to play at least one match with Bencic, but in his last season at the age of 40, Wawrinka made the decision to go with Paul.
“I'm not even thinking too much about if I should play or not, because I'm happy to focus on my singles and I'm happy to see them playing so well,” Wawrinka said. “That's the beauty of this event, that we are a team and we can change. We can put different players on the court.
“So far they've been doing everything, so I'm happy on my bench.”
Bencic and Paul have also won all three of their mixed doubles matches.
Let's break down these sparkling matchups:
10 a.m. local; 6 p.m. ET
Sure, the United Cup is a team game but let it be said that the members of the WTA Tour Driven by Mercedes-Benz have done more than their share of the heavy lifting.
Switzerland's Bencic, who became a mother in 2024, has played three singles matches and three mixed -- and won all six.
“It's a great feeling to start a season well,” Bencic told reporters. “Of course, it's what you hope for to kind of keep going how last season ended. It gives you a lot of confidence that you come out of the blocks pretty good and you already have so much play before the Australian Open, which is always very important.”
Elise Mertens of Belgium has also won all three of her singles matches and is 1-1 playing with Zizou Bergs. She defeated two-time major champion Barbora Krejcikova in the quarterfinals, 5-7, 6-1, 7-5, firing 12 aces.
The two breadwinners -- both Top 20 players -- meet on Saturday in a match that could dramatically shape the final outcome. In over 1,400 career matches, they've only met once -- with Mertens winning a third-round match at the 2021 Australian Open, 6-2, 6-1. They were scheduled to meet last fall in Wuhan, but Mertens pulled out with a back injury.
Wawrinka holds a 1-0 head-to-head edge over Bergs.
If it comes down to mixed doubles, both teams are talented. Mertens partnered with Veronika Kudermetova to win the doubles title at the WTA Finals last November in Riyadh. She's played two matches with Bergs and is looking comfortable.
“We are growing as a team,” Mertens said. “I can really feel that.”
--Women's singles: Belinda Bencic vs. Elise Mertens--Men's singles: Stan Wawrinka vs. Zizou Bergs--Mixed doubles: Bencic and Jakub Paul vs. Mertens and Bergs
5:30 p.m. local; 1:30 a.m. ET
Rest versus momentum -- which team has the advantage?
The United States advanced to these semifinals on Wednesday and has enjoyed two off days but had to make the 4.5-hour cross-country flight from Perth to Sydney and navigate a three-hour time change.
Poland played its quarterfinal in Sydney on Friday night and launches right into Saturday's tie with the United States -- in a rematch of last year's final. No rest for the victors, but they are already intimately familiar with the conditions. Advantage?
It's Poland's fourth straight year as a semifinalist.
The crackling match between Iga Swiatek and Gauff features the No. 2 and No. 4 players on the WTA Tour Driven by Mercedes-Benz.
Swiatek, three years older, won 11 of their first 12 encounters, but Gauff has taken the past three -- all in straight sets. A year ago, Gauff beat Swiatek 6-4, 6-4 in a memorable United Cup match that saw Team USA win its second title in three years of the competition.
“Obviously we've played each other a lot of times,” Gauff said of Swiatek. “Last year I had a good match against her. Yeah, I'm looking forward to the battle. I feel like when I'm playing great tennis, it puts a lot of pressure on them. Yeah, I'm looking forward to that.”
Said Swiatek, “For sure it's a good match, like exciting for the fans I think. Obviously we know each other's game pretty well with Coco. The key for me will be just to focus on myself, try to implement the stuff that I worked on during the preseason and be brave with the decisions.”
Taylor Fritz leads the head-to-head with Hubert Hurkacz, 4-2, including a win in last year's final.
“Taylor, he's such a great player,” Hurkacz said. “It's going to be a fun challenge to play against him. I mean, we played in the finals last year. It was a close battle. Will try to be a bit better this year.”
And while Team USA has the undefeated Gauff and Harrison lined up for mixed doubles, Kawa and Zielinski are fire-tested -- and also a spiffy 3-0.
--Men's singles: Taylor Fritz vs. Hubert Hurkacz--Women's singles: Coco Gauff vs. Iga Swiatek--Mixed doubles: Gauff and Christian Harrison vs. Katarzyna Kawa and Jan Zielinski
Top-tier WTA talent leads the charge on Saturday at the United Cup, where Switzerland faces Belgium and the United States renews its high-stakes rivalry with Poland for a chance at the 2026 title.
Katarzyna Kawa and Jan Zielinski defeated Australia in a deciding mixed doubles match Friday to clinch the tie for Poland.ByTENNIS.comPublished Jan 09, 2026 copy_link
Published Jan 09, 2026
© 2026 Getty Images
At two of the first three United Cups, the U.S. dashed Poland's hopes of standing on top.Come Saturday, Poland will get another crack at the American squad after ending Australia's bid.In a rare winner-take-all mixed doubles match that did not feature Iga Swiatek and Hubert Hurkacz, Katarzyna Kawa and Jan Zielinski rose to the occasion in securing a 6-4, 6-0 victory over Storm Hunter and John-Patrick Smith. Kawa and Zielinski had previously won a pair of dead rubbers against Germany and the Netherlands during Group F action.
Come Saturday, Poland will get another crack at the American squad after ending Australia's bid.In a rare winner-take-all mixed doubles match that did not feature Iga Swiatek and Hubert Hurkacz, Katarzyna Kawa and Jan Zielinski rose to the occasion in securing a 6-4, 6-0 victory over Storm Hunter and John-Patrick Smith. Kawa and Zielinski had previously won a pair of dead rubbers against Germany and the Netherlands during Group F action.
In a rare winner-take-all mixed doubles match that did not feature Iga Swiatek and Hubert Hurkacz, Katarzyna Kawa and Jan Zielinski rose to the occasion in securing a 6-4, 6-0 victory over Storm Hunter and John-Patrick Smith. Kawa and Zielinski had previously won a pair of dead rubbers against Germany and the Netherlands during Group F action.
📲🖥️ Stream the 2026 United Cup on the Tennis Channel App!Swiatek raced to the finish line to kick off the tie by dispatching 19-year-old Maya Joint, 6-1, 6-1. The world No. 2 converted five of her eight break chances en route to closing out a 57-minute victory.“I had a clear plan of what I wanted to do. I just focused on that,” Swiatek told press. “Wanted to be intensive from the beginning, put pressure on Maya, and it worked.”Home favorite Alex de MInaur gave locals hope when he held off Hurkacz, 6-4, 4-6, 6-4, to level the scoreboard, before the No. 9-seeded nation regrouped to claim the quarterfinal battle.
Swiatek raced to the finish line to kick off the tie by dispatching 19-year-old Maya Joint, 6-1, 6-1. The world No. 2 converted five of her eight break chances en route to closing out a 57-minute victory.“I had a clear plan of what I wanted to do. I just focused on that,” Swiatek told press. “Wanted to be intensive from the beginning, put pressure on Maya, and it worked.”Home favorite Alex de MInaur gave locals hope when he held off Hurkacz, 6-4, 4-6, 6-4, to level the scoreboard, before the No. 9-seeded nation regrouped to claim the quarterfinal battle.
“I had a clear plan of what I wanted to do. I just focused on that,” Swiatek told press. “Wanted to be intensive from the beginning, put pressure on Maya, and it worked.”Home favorite Alex de MInaur gave locals hope when he held off Hurkacz, 6-4, 4-6, 6-4, to level the scoreboard, before the No. 9-seeded nation regrouped to claim the quarterfinal battle.
Home favorite Alex de MInaur gave locals hope when he held off Hurkacz, 6-4, 4-6, 6-4, to level the scoreboard, before the No. 9-seeded nation regrouped to claim the quarterfinal battle.
In each of Poland's prior two defeats to the Americans, Swiatek and Hurkacz were unable to put points on the board. Swiatek dropped her respective singles matches to Jessica Pegula in 2023 and Coco Gauff last year, while Hurkacz twice came up short against Taylor Fritz.Revenge is on the table, with Gauff and Fritz running it back this week for the defending champions.“For sure it's a good match, like exciting for the fans I think. But I'll focus on my singles. Obviously we know each other's game pretty well with Coco,” said Swiatek.“The key for me will be just to focus on myself, try to implement the stuff that I worked on during the pre-season and be brave with the decisions. Yeah, we'll see.”
Revenge is on the table, with Gauff and Fritz running it back this week for the defending champions.“For sure it's a good match, like exciting for the fans I think. But I'll focus on my singles. Obviously we know each other's game pretty well with Coco,” said Swiatek.“The key for me will be just to focus on myself, try to implement the stuff that I worked on during the pre-season and be brave with the decisions. Yeah, we'll see.”
“For sure it's a good match, like exciting for the fans I think. But I'll focus on my singles. Obviously we know each other's game pretty well with Coco,” said Swiatek.“The key for me will be just to focus on myself, try to implement the stuff that I worked on during the pre-season and be brave with the decisions. Yeah, we'll see.”
“The key for me will be just to focus on myself, try to implement the stuff that I worked on during the pre-season and be brave with the decisions. Yeah, we'll see.”
Honestly after last year's tournament, I didn't really analyze this match that much. I was already kind of focused on Australian Open. For sure I'm going to sit with Wim tomorrow and he will watch it and give me the tips. Iga Swiatek
Hurkacz, who missed seven months of competition prior to making a winning return at this event, was similar in his sentiment.“It's going to be a fun challenge to play against him,” the two-time ATP Masters 1000 champion said. “We played in the finals last year. It was a close battle. Will try to be a bit better this year.”Switzerland faces Belgium in the first semifinal on the back of Stan Wawrinka being announced as the final men's singles wild card recipient for this month's Australian Open.
“It's going to be a fun challenge to play against him,” the two-time ATP Masters 1000 champion said. “We played in the finals last year. It was a close battle. Will try to be a bit better this year.”Switzerland faces Belgium in the first semifinal on the back of Stan Wawrinka being announced as the final men's singles wild card recipient for this month's Australian Open.
Switzerland faces Belgium in the first semifinal on the back of Stan Wawrinka being announced as the final men's singles wild card recipient for this month's Australian Open.
Australian captain Lleyton Hewitt had not regrets about resting Alex de Minaur in the decisive mixed doubles against Poland in the United Cup quarter-finals Friday night in Sydney.
After his two-hour, 18 minute victory over Hubert Hurkacz to send the tie to a deciding mixed doubles, de Minaur did not front up for mixed with Storm Hunter, like he did when Australia needed the mixed point to beat Czechia Tuesday to advance through the group stages.
Hunter teamed with John-Patrick Smith in a 6-4, 6-0 defeat to Jan Zielinski and Katarzyna Kawa.
“Alex was feeling a few tweaks the last few days,” Hewitt said. “He's been doing a lot of training in the off-season to get ready for five-set matches. We decided the best thing for his body moving forward was not to put his hand up to play mixed tonight.
“Also, if you are that second match, to turn around straightaway and switch on. You just don't want to risk injury as well at certain times, especially the amount of moving he had to do tonight against a bloody quality player out there."
De Minaur produced a heroic performance in the singles, saving nine break points across his first four service games before going on a streak of winning 21 consecutive points on serve through the late stages of the second set.
He won 82 per cent of first serves for the match, a vast improvement from 55 per cent and eight double faults in a scrappy opening-tie loss to Casper Ruud earlier in the tournament.
“I think what was a little bit strange for me was the amount of double-faults,” de Minaur said. “That's something I don't normally hit. Obviously that was what felt like an anomaly. We got back to the practice court, just worked on a couple things, a couple different feels.
“I'm very happy with my serving performance the following two matches, kind of showing all the work that I put in. Of course, if I can land first serves and play with a lot of first serves, it makes my life a whole lot easier. Makes me definitely a more dangerous opponent."
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Poland has earned the chance to avenge last year's United Cup final loss to the United States after winning a decisive mixed doubles against Australia in Friday night's quarter-final in Sydney.
After Iga Swiatek and Alex de Minaur claimed contrasting singles wins for their countries earlier in the evening, Jan Zielinski produced a masterclass with Katarzyna Kawa to seal Poland's victory with a 6-4, 6-0 win over John-Patrick Smith and Storm Hunter.
Advancing to the semi-finals for the fourth consecutive year, Poland will get a shot at defending champion United States Saturday night when the singles match-ups – Swiatek against Coco Gauff and Hurkacz against Taylor Fritz - are a repeat of the 2025 final. Last year Gauff won 6-4, 6-4 while Hurkacz suffered a heartbreaking third-set tie-break loss to Fritz.
In a dramatic tie Friday in front of a highly partisan Australian crowd, De Minaur produced one of the grittiest opening-set performances of his career Friday night in Sydney to set up a 6-4, 4-6 6-4 win over Hurkacz to send the last United Cup quarter-final to a deciding mixed doubles.
The World No. 6 saved all nine break points he faced across his first four service games – including four in the second game of the match – to keep Australia alive after former World No. 1 Iga Swiatek brushed aside Aussie teen Maya Joint 6-1, 6-1.
“Nights like these… these are just the best,” De Minaur said. “I love playing here. The atmosphere from the first ball to the last was amazing.
“I just had to fight him off from the first point to the last. It was a huge mental effort and we're still alive. I'm glad I was able to bounce back after losing that second set. I had some dark thoughts in my head.”
The tie will now be decided by the mixed doubles, with the winner advancing to Saturday night's semi-final against defending champion United States. None of the four singles players will compete in the mixed doubles. Australians John-Patrick Smith and Storm Hunter will take on Jan Zielinski and Katarzyna Kawa.
Playing his first tournament since last year's grass season, Hurkcaz was near untouchable in his first three service games when he dropped just one point on serve. But growing increasingly frustrated by his inability to cash in on his chances in return games, his unforced error count ticked up in the closing stages of the set.
After De Minaur saved three break points to level at 4-4, the 26-year-old claimed the decisive break and then won 21 consecutive points on serve before unexpectedly dropping serve at the end of the second set.
But inspired by deafening roars from Aussie fans inside Ken Rosewall Arena, De Minaur went into lockdown mode with just two unforced errors in the final set en route to his eighth victory in 11 United Cup singles appearances.
Earlier, Swiatek overpowered Australia rising star Joint, capturing a convincing 6-1, 6-1 win in just 57 minutes at the United Cup quarterfinals.
From start to finish, the World No. 2's experience prevailed as the Swiatek earned her second career win over Joint: Swiatek won 6-0, 6-2 in the Seoul semifinals, a tournament she'd go on to win. Swiatek also earned her 15th singles win at the United Cup.
“I think the intensity -- the balls get quite heavy so I'm happy that I was always pushing forward,” Swiatek said on court. “I got pretty confident at the end so for sure it was a good match.”
Joint held serve to have an initial 1-0 lead, but after, the Swiatek show ensued. The six-time Grand Slam champion overwhelmingly controlled the rallies, often forcing Joint to scramble from side-to-side just to keep the rally alive.
Swiatek won six straight games to win the first set with a breadstick in 26 minutes and had garnered “Iga” chants from her Polish supporters early on into the match. Her set point, a cross-court forehand winner that sped past an outstretched Joint, summed up the match best.
In the second, Swiatek and Joint traded holds, the latter of which earned some encouragement from the home crowd. Similarly, Swiatek recaptured the lead with a break, and didn't look back, en route to the double breadstick win.
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Karolina Muchova was able to put a stop to Elena Rybakina's 13-match win streak with a three-set victory in the Brisbane quarterfinals. As a result, she earned the chance to extend her head-to-head advantage over Aryna Sabalenka in the semis after the World No. 1 defeated Madison Keys in straight sets in her quarterfinal match.
If Friday's first Brisbane quarterfinal had been a boxing match, Karolina Muchova would have taken the first round, Elena Rybakina the second and the third -- or the decider -- would have been considered too close to call.
Brisbane: Scores | Draws | Order of play
But despite the two Top 20 players trading blows for more than two hours, this was tennis, not boxing, and there were no split decisions to determine the winner. Instead, Muchova definitively snapped Rybakina's 13-match winning streak and earned her first win over the former Wimbledon champion since 2019 with a 6-2, 2-6, 6-4 victory at Pat Rafter Arena.
“I'm just happy to be playing again, to be out here,” Muchova said after the match. “I missed a few years in Australia that I didn't even come because of injury, so it's just great that I'm here and enjoying playing with my team and friends here.”
With the win, she booked a semifinal showdown with Aryna Sabalenka, after the World No. 1 defeated Madison Keys 6-3, 6-3 a few hours later to secure her own spot in the final four.
It marks Muchova's first WTA Tour Driven by Mercedes-Benz semifinal appearance since last February in Dubai.
Talent has never been the issue for Muchova. Availability has. The Czech has long been snakebit by injuries, but when she's healthy, or close to it, she's capable of giving anyone trouble. On Friday, that “anyone” was Rybakina.
Muchova began her offensive early, earning two break points -- the first of 11 she would generate throughout the match -- in the opening game. Rybakina escaped with timely serving, but her serve faltered at 2-all when a double fault on break point handed Muchova a 3-2 lead.
It was the first of four straight games won by the 29-year-old, who closed out the first set in 35 minutes by playing patient, disciplined tennis. She finished the first set with just five unforced errors to Rybakina's 18.
Rybakina flipped the script in the second set to force a decider, where neither player could separate in the first eight games. But Muchova seized control with a break to love for a 5-4 lead, then served out the match, sealing the win with a forehand winner.
It's hard to pinpoint why, but there's something undeniably pure about watching Sabalenka and Keys mash the heck out of the ball, point after point. Even on a day when neither was at her best, the sheer power on display -- forehand, backhand, first serve, it didn't matter -- was unmistakable.
But with great power comes great responsibility, and few players harness their strength more effectively than Sabalenka.
Despite Keys throwing everything at her, the Belarusian never blinked. After Keys held for a 3-2 lead, Sabalenka responded by reeling off four straight games to take the first set, closing it out with her trademark backhand down the line on her third set point.
In the second set, Keys appeared to dial back the aggression after seven costly double faults in the opener, just as Sabalenka decided to crank hers up. Sabalenka took more risks on serve, firing five aces after hitting none in the first set, and pounced on Keys' increasingly conservative second serves. On one such occasion, Sabalenka vultured Key's second serve and crushed a forehand winner down the line for a 3-1 lead.
“I was trying to put as much pressure as I can on her serve,” Sabalenka said in her on-court interview. “That's all I was thinking about, and I think I did it well. I put so much pressure back on her because yeah, she's aggressive, but then I tried to put all that speed back at her.”
Keys kept at it, though, breaking back for 3-2 and staying in the mix with several highlight-reel winners of her own. She actually finished with more winners than Sabalenka (25 to 20), but the damage caused by the vulnerability of her second serve proved too much to overcome, allowing Sabalenka to run away with the victory and advance to the semifinals to face Muchova.
There aren't many players who have found consistent success against Sabalenka, but Muchova is one of them.
Muchova leads the head-to-head 3-1, having won each of their last three meetings dating back to 2023. Sabalenka's lone victory in the series came in their first matchup in 2019.
Their most recent meeting came in the 2024 Beijing quarterfinals, where Muchova won in three sets en route to the final. Coincidentally, that remains her most recent WTA final, but that could change if she manages to pull off the unthinkable and defeat the World No. 1 for a fourth straight time.
Karolina Muchova was able to put a stop to Elena Rybakina's 13-match win streak with a three-set victory in the Brisbane quarterfinals. As a result, she earned the chance to extend her head-to-head advantage over Aryna Sabalenka in the semis after the World No. 1 defeated Madison Keys in straight sets in her quarterfinal match.
The world No. 1 earned a straight-sets win in their Australian Open final rematch.ByTENNIS.comPublished Jan 09, 2026 copy_link
Published Jan 09, 2026
© 2026 Getty Images
World No. 1 Aryna Sabalenka passed her first big test of the season—an Australian Open final rematch against No. 5 seed Madison Keys—to book her spot in the Brisbane International semifinals.Sabalenka, the top seed and defending champion in Brisbane, needed an hour and 30 minutes to claim revenge, winning 6-3, 6-3 in an entertaining battle between two of the WTA's biggest hitters.“She's a great player, always tough battles against her,” Sabalenka said, speaking to Jelena Dokic on court after improving to 6-2 in her head-to-head against Keys.“The whole match I was trying to stay focused, I was trying to stay in control on my serve and put as much pressure as I can on her serve.“That's all I was thinking about, and I think I did it well. She's aggressive, but I tried to put all that speed back on her.”Read More: Aryna Sabalenka's Australian Open outfit is a stylish nod to Serena Williams and Sharapova
Sabalenka, the top seed and defending champion in Brisbane, needed an hour and 30 minutes to claim revenge, winning 6-3, 6-3 in an entertaining battle between two of the WTA's biggest hitters.“She's a great player, always tough battles against her,” Sabalenka said, speaking to Jelena Dokic on court after improving to 6-2 in her head-to-head against Keys.“The whole match I was trying to stay focused, I was trying to stay in control on my serve and put as much pressure as I can on her serve.“That's all I was thinking about, and I think I did it well. She's aggressive, but I tried to put all that speed back on her.”Read More: Aryna Sabalenka's Australian Open outfit is a stylish nod to Serena Williams and Sharapova
“She's a great player, always tough battles against her,” Sabalenka said, speaking to Jelena Dokic on court after improving to 6-2 in her head-to-head against Keys.“The whole match I was trying to stay focused, I was trying to stay in control on my serve and put as much pressure as I can on her serve.“That's all I was thinking about, and I think I did it well. She's aggressive, but I tried to put all that speed back on her.”Read More: Aryna Sabalenka's Australian Open outfit is a stylish nod to Serena Williams and Sharapova
“The whole match I was trying to stay focused, I was trying to stay in control on my serve and put as much pressure as I can on her serve.“That's all I was thinking about, and I think I did it well. She's aggressive, but I tried to put all that speed back on her.”Read More: Aryna Sabalenka's Australian Open outfit is a stylish nod to Serena Williams and Sharapova
“That's all I was thinking about, and I think I did it well. She's aggressive, but I tried to put all that speed back on her.”Read More: Aryna Sabalenka's Australian Open outfit is a stylish nod to Serena Williams and Sharapova
Read More: Aryna Sabalenka's Australian Open outfit is a stylish nod to Serena Williams and Sharapova
Competing in her first tournament of the 2026 season, Sabalenka has shaken off any lingering rust after a series of off-season exhibition events. So far, she's passed with flying colors, yet to drop a set at the WTA 500 event.Against Keys, Sabalenka weathered 16 winners from her opponent in the first set (she hit only six herself) and stayed patient. It took nearly 30 minutes for the pair to reach 3-3, but from there Sabalenka took control and began swinging more freely.The second set was closer than the scoreline suggested, with Keys breaking Sabalenka's serve twice as her opponent's frustration mounted.Keys was attempting to bounce back after a grueling quarterfinal the day before, when the American edged No. 12 Diana Shnaider 7-6 (5), 6-7 (5), 7-6 (4). She was sporting medical strapping on her left thigh during the match on Pat Rafter Arena, and couldn't match Sabalenka's firepower as the match unfolded.
Against Keys, Sabalenka weathered 16 winners from her opponent in the first set (she hit only six herself) and stayed patient. It took nearly 30 minutes for the pair to reach 3-3, but from there Sabalenka took control and began swinging more freely.The second set was closer than the scoreline suggested, with Keys breaking Sabalenka's serve twice as her opponent's frustration mounted.Keys was attempting to bounce back after a grueling quarterfinal the day before, when the American edged No. 12 Diana Shnaider 7-6 (5), 6-7 (5), 7-6 (4). She was sporting medical strapping on her left thigh during the match on Pat Rafter Arena, and couldn't match Sabalenka's firepower as the match unfolded.
The second set was closer than the scoreline suggested, with Keys breaking Sabalenka's serve twice as her opponent's frustration mounted.Keys was attempting to bounce back after a grueling quarterfinal the day before, when the American edged No. 12 Diana Shnaider 7-6 (5), 6-7 (5), 7-6 (4). She was sporting medical strapping on her left thigh during the match on Pat Rafter Arena, and couldn't match Sabalenka's firepower as the match unfolded.
Keys was attempting to bounce back after a grueling quarterfinal the day before, when the American edged No. 12 Diana Shnaider 7-6 (5), 6-7 (5), 7-6 (4). She was sporting medical strapping on her left thigh during the match on Pat Rafter Arena, and couldn't match Sabalenka's firepower as the match unfolded.
Read More: Aryna Sabalenka calls season schedule "insane," plans to skip tournaments to avoid burnoutUp next for Sabalenka is a tricky semifinal clash against Karolina Muchova, who defeated No. 3 seed Elena Rybakina 6-2, 2-6, 6-4.Muchova owns a 3-1 edge in their head-to-head. Sabalenka won their first meeting in 2019, but Muchova has taken the next three encounters, most recently in the quarterfinals in Beijing in 2024.“It doesn't matter what's the score between us, the past stays in the past,” Sabalenka stated. “I'm just trying to bring everything I've been working throughout my whole career, and I just try to be better every time.”
Up next for Sabalenka is a tricky semifinal clash against Karolina Muchova, who defeated No. 3 seed Elena Rybakina 6-2, 2-6, 6-4.Muchova owns a 3-1 edge in their head-to-head. Sabalenka won their first meeting in 2019, but Muchova has taken the next three encounters, most recently in the quarterfinals in Beijing in 2024.“It doesn't matter what's the score between us, the past stays in the past,” Sabalenka stated. “I'm just trying to bring everything I've been working throughout my whole career, and I just try to be better every time.”
Muchova owns a 3-1 edge in their head-to-head. Sabalenka won their first meeting in 2019, but Muchova has taken the next three encounters, most recently in the quarterfinals in Beijing in 2024.“It doesn't matter what's the score between us, the past stays in the past,” Sabalenka stated. “I'm just trying to bring everything I've been working throughout my whole career, and I just try to be better every time.”
“It doesn't matter what's the score between us, the past stays in the past,” Sabalenka stated. “I'm just trying to bring everything I've been working throughout my whole career, and I just try to be better every time.”
"I'm fit and back on court, but five-setters are a different beast and I'm not quite ready to go the distance yet,” said the Aussie.ByTENNIS.comPublished Jan 09, 2026 copy_link
Published Jan 09, 2026
© 2026 Getty Images
Nick Kyrgios has taken himself out of the running for a main-draw wild card at this month's Australian Open, clearing the way for three-time major winner Stan Wawrinka to get his farewell Down Under.The former Wimbledon finalist shared the decision Friday morning (Thursday evening in the U.S.) on his Instagram story, noting that playing best-of-five set isn't where he's at yet in his comeback from knee and wrist injuries.“After some good conversations with TA (Tennis Australia), I've made the call to focus on doubles for this year's AO. I'm fit and back on court, but five-setters are a different beast and I'm not quite ready to go the distance yet,” he said.
The former Wimbledon finalist shared the decision Friday morning (Thursday evening in the U.S.) on his Instagram story, noting that playing best-of-five set isn't where he's at yet in his comeback from knee and wrist injuries.“After some good conversations with TA (Tennis Australia), I've made the call to focus on doubles for this year's AO. I'm fit and back on court, but five-setters are a different beast and I'm not quite ready to go the distance yet,” he said.
“After some good conversations with TA (Tennis Australia), I've made the call to focus on doubles for this year's AO. I'm fit and back on court, but five-setters are a different beast and I'm not quite ready to go the distance yet,” he said.
This tournament means everything to me but I'd rather give my spot to someone who's ready to make their moment count.
Kyrgios partnered long-time friend Thanasi Kokkinakis to an opening doubles win in Brisbane ahead of a 6-3, 6-4 defeat in singles to Aleksandar Kovacevic.In not pursuing the 128-player field at the season's first major, it further opened the door for 2014 champion Wawrinka to make one final appearance in his last season on tour. A few hours after Kyrgios' note went up, the Swiss was announced as the wild card recipient by the tournament.“This tournament means everything to me but I'd rather give my spot to someone who's ready to make their moment count,” added Kyrgios. “It's all building blocks and I'll be back next year and pumped to compete. See you out there.”
In not pursuing the 128-player field at the season's first major, it further opened the door for 2014 champion Wawrinka to make one final appearance in his last season on tour. A few hours after Kyrgios' note went up, the Swiss was announced as the wild card recipient by the tournament.“This tournament means everything to me but I'd rather give my spot to someone who's ready to make their moment count,” added Kyrgios. “It's all building blocks and I'll be back next year and pumped to compete. See you out there.”
“This tournament means everything to me but I'd rather give my spot to someone who's ready to make their moment count,” added Kyrgios. “It's all building blocks and I'll be back next year and pumped to compete. See you out there.”
The 40-year-old Wawrinka, who has been more than competitive in Switzerland's run to the United Cup semifinals, previously noted he had also been in discussions with tournament director Craig Tiley.Former Top 30 player Jordan Thompson had been a potential candidate to receive the wild card as well, having slipped outside of the Top 100 following an up-and-down year that brought its share of health setbacks.Kyrgios and Kokkinakis will reunite for another run at Melbourne Park, with the latter working his way back from a devastating pectoral injury. The two memorably captured the trophy at their home major four years ago in an all-Australian final over Matthew Ebden and Max Purcell.
Former Top 30 player Jordan Thompson had been a potential candidate to receive the wild card as well, having slipped outside of the Top 100 following an up-and-down year that brought its share of health setbacks.Kyrgios and Kokkinakis will reunite for another run at Melbourne Park, with the latter working his way back from a devastating pectoral injury. The two memorably captured the trophy at their home major four years ago in an all-Australian final over Matthew Ebden and Max Purcell.
Kyrgios and Kokkinakis will reunite for another run at Melbourne Park, with the latter working his way back from a devastating pectoral injury. The two memorably captured the trophy at their home major four years ago in an all-Australian final over Matthew Ebden and Max Purcell.
Average Joes will take on pros including Carlos Alcaraz, Coco Gauff and Iga Swiatek for a $1 million cash prize.ByTENNIS.comPublished Jan 09, 2026 copy_link
Published Jan 09, 2026
© 2025 Getty Images
The Australian Open player field is expanding in 2026 ... by amateurs who have a simple objective: Win a point, just one, against the best players in the world.At the new "1-Point Slam" event at Melbourne Park later this month, "Average Joes" representing tennis clubs from all the states and territories in Australia will take on ATP and WTA stars including Carlos Alcaraz, Coco Gauff and Iga Swiatek for a $1 million cash prize. Also competing are Jannik Sinner, Naomi Osaka, Jasmine Paolini, Daniil Medvedev, and Nick Kyrgios.📲🖥️ Stream the AO 1 Point Slam at 3:30 a.m. ET on Jan. 14 on the Tennis Channel App!Each match will last just one point, with rock-paper-scissors deciding who serves or receives. The winner of each point advances, and the final will be played on Rod Laver Arena—where the last player standing will walk away with the prize.The "1-Point Slam" is one of the signature new innovations of an expanded first week in Melbourne, and followed the smash-hit success of the revamped US Open mixed-doubles tournament last summer that initially courted controversy.
At the new "1-Point Slam" event at Melbourne Park later this month, "Average Joes" representing tennis clubs from all the states and territories in Australia will take on ATP and WTA stars including Carlos Alcaraz, Coco Gauff and Iga Swiatek for a $1 million cash prize. Also competing are Jannik Sinner, Naomi Osaka, Jasmine Paolini, Daniil Medvedev, and Nick Kyrgios.📲🖥️ Stream the AO 1 Point Slam at 3:30 a.m. ET on Jan. 14 on the Tennis Channel App!Each match will last just one point, with rock-paper-scissors deciding who serves or receives. The winner of each point advances, and the final will be played on Rod Laver Arena—where the last player standing will walk away with the prize.The "1-Point Slam" is one of the signature new innovations of an expanded first week in Melbourne, and followed the smash-hit success of the revamped US Open mixed-doubles tournament last summer that initially courted controversy.
📲🖥️ Stream the AO 1 Point Slam at 3:30 a.m. ET on Jan. 14 on the Tennis Channel App!Each match will last just one point, with rock-paper-scissors deciding who serves or receives. The winner of each point advances, and the final will be played on Rod Laver Arena—where the last player standing will walk away with the prize.The "1-Point Slam" is one of the signature new innovations of an expanded first week in Melbourne, and followed the smash-hit success of the revamped US Open mixed-doubles tournament last summer that initially courted controversy.
Each match will last just one point, with rock-paper-scissors deciding who serves or receives. The winner of each point advances, and the final will be played on Rod Laver Arena—where the last player standing will walk away with the prize.The "1-Point Slam" is one of the signature new innovations of an expanded first week in Melbourne, and followed the smash-hit success of the revamped US Open mixed-doubles tournament last summer that initially courted controversy.
The "1-Point Slam" is one of the signature new innovations of an expanded first week in Melbourne, and followed the smash-hit success of the revamped US Open mixed-doubles tournament last summer that initially courted controversy.
This week at the United Cup, Gauff sheepishly confirmed her participation, saying "I think it's great for the sport to have these different things going on."“We'll see how I do," she joked. "I'm not very confident in myself. But yeah, I think it's great. Just promotes the more fun side of tennis.“Obviously it's going to get pretty serious later on, but I think it's great for fanfare. Especially because our sport is really, be quiet and don't move around too much. It gives the fans a little different perspective on the sport.“Yeah, I try to make sure it's just the right amount of things. I like to just do one thing and then after that I try to focus on the two weeks.”
“We'll see how I do," she joked. "I'm not very confident in myself. But yeah, I think it's great. Just promotes the more fun side of tennis.“Obviously it's going to get pretty serious later on, but I think it's great for fanfare. Especially because our sport is really, be quiet and don't move around too much. It gives the fans a little different perspective on the sport.“Yeah, I try to make sure it's just the right amount of things. I like to just do one thing and then after that I try to focus on the two weeks.”
“Obviously it's going to get pretty serious later on, but I think it's great for fanfare. Especially because our sport is really, be quiet and don't move around too much. It gives the fans a little different perspective on the sport.“Yeah, I try to make sure it's just the right amount of things. I like to just do one thing and then after that I try to focus on the two weeks.”
“Yeah, I try to make sure it's just the right amount of things. I like to just do one thing and then after that I try to focus on the two weeks.”
Tennis Australia seeks to capitalize on a 2025 tournament that saw 1.2 million fans through the gates."That momentum brings a new challenge: how do we give even more people a spectacular AO experience?" said Tennis Australia CEO Craig Tiley in October.Three days of music festivals, and the first-ever Australian pop-up of New York City's famous Shake Shack, are also among the featured events.
"That momentum brings a new challenge: how do we give even more people a spectacular AO experience?" said Tennis Australia CEO Craig Tiley in October.Three days of music festivals, and the first-ever Australian pop-up of New York City's famous Shake Shack, are also among the featured events.
Three days of music festivals, and the first-ever Australian pop-up of New York City's famous Shake Shack, are also among the featured events.
It's not new to note that the fortunes of America's movie theaters rest, pretty precariously, on a thick, greasy cloud of popcorn, soda, and nacho cheese dip; as the U.S. box office has seemingly plateaued in its recovery from the COVID-19 shutdowns over the last few years, theaters have had to lean more and more heavily on concessions in order to make ends meet. Even with that understanding, though, we'll admit to a bit of shock at a planned promotion that theater chain Cinemark is rolling out for this year's “National Popcorn Day,” set by the food advertising minds that cook this kind of thing up for January 18 and 19. The company's marketing has its own language for it, but if it's all the same to you, we're going to just go ahead and keep thinking of the promotion like this: Welcome, one and, all, to the “Fuck it, bring your own goddamn bucket” plan!
As noted by THR, this is the second consecutive year that Cinemark has run the FIBYOGBP, in which consumers—and the word has rarely felt more apt—are invited, for that 48-hour period, to bring in any container they like, up to 400 fluid ounces, and get it filled with popcorn for just five bucks. The twist this year (and it's a powerful one, to our mind) is that the chain has now gone one step further and struck a promotional deal with hardware company Lowe's: If you show up with a 5 gallon bucket that you bought at Lowe's, that can also be filled, netting you an extra 250 ounces of indigestion, kernels-in-teeth, and the looming specter of heart disease. The THR piece employs the phrase “Food-grade bucket liners will be available upon request,” which is the kind of thing that's going to haunt us almost as much as 5 full gallons of popcorn are bound to haunt our large intestines in the months and years to come.
Meanwhile, it's also noted that non-bucket-owners—i.e., chumps—will still be able to buy a 200-ounce “XL” popcorn for the same price, if your FIBYOGBP FOMO isn't too intense at the thought of leaving all that hot, slick salted corn on the table. Oh, and the promotion doesn't require a ticket purchase, so no reason to stay put: Wander America's decaying malls and crumbling streets with your big ol' paint bucket of popcorn clutched in your greasy mitts, fending off birds and raccoons as you go! It's National Popcorn Day, baby! Food-grade bucket liners are available on request!
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Between Sisu and Ready Or Not 2, the world of John Wick is finding common cause with the influence of Quentin Tarantino. And there's no shortage of comedic kung fu, ironic bloodletting, and stylish hotel rooms in the first trailer for They Will Kill You. Produced by It sibilings Andy and Barbara Muschietti and directed by Kirill Sokolov, the Russian action-comedy filmmaker behind Why Don't You Just Die, the trailer makes no attempt to hide its influences before veering off in its own Satanic course. Still, while it may take people a second to realize that this isn't the trailer for Ready Or Not: Here I Come, it still gives Zazie Beetz, the Bullet Train star who's no stranger to this type of thing, an excuse to square off against Patricia Arquette, Tom Felton, and Heather Graham.
Here's the logline:
A young woman must survive the night at the Virgil, a demonic cult's mysterious and twisted death-trap of a lair, before becoming their next offering in a uniquely brazen, big screen battle of epic kills and wickedly dark humor.
They Will Kill You opens in theaters on March 25, 2026.
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The following article is an excerpt from the new edition of “In Review by David Ehrlich,” a biweekly newsletter in which our Chief Film Critic and Head Reviews Editor rounds up the site's latest reviews and muses about current events in the movie world. Subscribe here to receive the newsletter in your inbox every other Friday.
There comes a time in everyone's life when they have to plan and host a glitzy movie awards show, and for me, that time came earlier this week when I — in my role as chair of the New York Film Critics Circle —emceed this year's NYFCC awards dinner at Tao Downtown in Manhattan. In addition to corralling luminaries like Ben Stiller, Lupita Nyong'o, and Mikhail Baryshnikov to present the awards to our winners, my responsibilities also included kicking things off with some introductory remarks about the evening's purpose.
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Every chair approaches that part of the program in their own way; some have done schtick, others have been more straightforward. As anyone familiar with my work knows all too well, it's a lot more comfortable for me to ramble on for thousands of words than it is to be concise, and so the only way I could steady myself in a situation as nerve-wracking as this one was to hold half of Hollywood hostage for a long, long speech about the relationship between critics and filmmakers. So long, in fact, that Jafar Panahi and Paul Thomas Anderson both later roasted me about it from the stage, with the latter declaring that, for the next calendar year, I've forfeited the right to criticize movies for their length. Just to make sure that I can never repress the memory, I've conveniently been provided with a picture of the exact moment that happened:
The only silver lining is that the speech was so long that IndieWire Editorial Director Kate Erbland, knowing that I would be too exhausted to write anything else this week, suggested that I just publish the text as the lead piece of this week's “In Review” newsletter. And so that's what I've done.
My guess is that it's less punishing to get through in print, but if you want to recreate the vibe of sitting through it live at the NYFCC dinner, feel free to read it aloud to yourself in an extremely crowded restaurant full of your colleagues and heroes.
***
Hello! My name is David Ehrlich, and it's my great pleasure and profound anxiety to welcome you to the 91st New York Film Critics Circle Awards. You would never know it from my Victorian skin pallor, a physique that makes Marty Supreme look like The Smashing Machine, or from the fact that I own the entire Criterion Collection despite living paycheck-to-paycheck, but I am indeed a film critic; to my wonderful mother, who is in the room tonight, I'm so sorry that you had to find out this way. If it's any consolation, mom, had I become a doctor or a lawyer I would never have been able to lend you a heavily watermarked For Your Consideration DVD of Brendan Fraser's fish-out-of-water drama “Rental Family,” so at least there's something you can still brag about to your friends.
Of course, we are not here this evening to discuss my disappointments as a human being — there are truly so many different spaces on the internet where you can do that on your own time. But this is my time, for better or worse, and I'm afraid to say that I'm going to drag it out for another 10 to 12 minutes; like several of our winners tonight, I simply don't know how to do anything short, but my promise to you is that this won't be the worst thing that's ever happened in this country on January 6th. Top five, maybe.
Anyway, back to the topic at hand. Many of the year's best movies and performances were unexpected in one respect or another. After firmly establishing herself as one of the great comedic talents of her generation, Rose Byrne blew us all away with her nerve-shredding dramatic performance in “If I Had Legs I'd Kick You.” After 13 years of absence following her unforgettable 2011 narrative film “The Loneliest Planet,” Julia Loktev returned with a monumental documentary about the last independent journalists in Russia. And after several decades of shamelessly appealing to the masses with multiplex slop like “Phantom Thread,” “Inherent Vice,” and “There Will Be Blood,” Paul Thomas Anderson finally made something for the critics. Well done, Paul, thank you for throwing us a bone at last.
Of course, “One Battle After Another” would be a fitting descriptor of what the movies have faced since their first invention. Cinema as a medium has always been defined by its persistence: the persistent illusion of motion that allows a quick succession of still images to assume the quality of a dream, the persistent hold that it maintains over our imaginations in the face of invasive new technologies, and the persistent faith that it projects onto the power of communal experiences, even at a time when reality itself is being siloed into a series of discrete echo chambers (shoutout to “Eddington”).
I'm reminded of that persistence as I reflect on a year of tumult, loss, and potentially catastrophic self-destruction within the film community. This doesn't feel like the right time to elaborate on that last point, but — on a completely unrelated note — I would like to congratulate Netflix and Warner Bros. for combining to win almost half of our awards tonight.
In 2025, we lost David Lynch, Robert Redford, and Souleymane Cissé. We lost Diane Keaton, Diane Ladd, and Tatsuya Nakadai. We lost the war against the word “casted,” the fight against taking pictures of the screen, and whatever remaining foothold documentaries had in the theatrical marketplace. Indeed, for the second consecutive year, our winner for Best Non-Fiction Film is more likely to win the Academy Award for Best Documentary Feature than it is to receive proper distribution. Yes, “My Undesirable Friends: Part I — Last Air in Moscow” is five-and-a-half hours long and filled with some of the ugliest and most upsetting things you'll ever see on screen, but neither of those factors stopped “Wicked: For Good” from grossing more than $500 million, and so I can't fathom why they would be such dealbreakers for Julia Loktev.
Most of all, too many of my colleagues lost their jobs as a result of employers who failed to appreciate their value, and the depth of their readers' connection to their voices, and the entire community is poorer for that.
And yet, this was also a year that epitomized cinema's tradition of persistence. Specifically, the persistence of historical memory, as we saw in films like “It Was Just an Accident,” “The Secret Agent,” and, in its own way, the elegiacally quotidian baseball drama “Eephus,” whose writer/director, Carson Lund, has worked as a critic himself, and has actually reviewed the work of some of our other winners tonight. Quite well, I might add. And positively, which might be even more important.
Try as I might, it's hard to be all doom and gloom about a year that gave us the single most unstoppable movie soundtrack since “Saturday Night Fever,” two Bronstein family classics, and “a few small beers” for good measure. So while it may seem a bit “rearranging deck chairs on the Titanic” to get dressed up and celebrate film in the growing shadow of technocratic greed, unabated genocide, and the AI-generated perversion of image-making itself, I don't think it's wholly unimportant that the movies of 2025 were really, really fucking great. Or that we are gathering here tonight to acknowledge them as such in the face of these imperiled times.
That is what the New York Film Critics Circle has done through thick and thin for the last 91 years, which some might choose to see as a remarkable display of persistence in its own right. We bravely gave our awards during periods of strife as painful and varied as World War II, the Great Recession, and the Isiah Thomas era of the New York Knicks, and I don't think it's a coincidence that all of those things ended on our watch.
Perhaps I'm giving critics too much credit for our role in shaping modern history, and in shaping the art that it's produced. Then again, perhaps the rest of you aren't giving us enough. That film critics and filmmakers depend on each other is self-evident, but I think that critics are unfairly often seen as the barnacles rather than the whales.
The sacred task of the critic is to provoke and entertain the masses, to introduce them to new worlds, to stoke the imaginations of children, to inspire generations of dreamers, to excite people about the possibility of being alive, and to challenge them to engage with the world in a variety of radically empathic new ways. And the simple job of the filmmaker is just to give the critic something to write about. And I don't mean to diminish that! Somebody has to do it. You kindly provide us with crude eruptions of light and sound, and we do the hard — some would say holy — work of shaping that chaos into layers of meaning.
Allow me to illustrate my point. Much as I enjoyed “Eephus,” we all know that movie was really just 99 minutes of under-employed character actors standing around on a field until Nick Schager called it “a new sports-movie classic, as sneakily effective as the pitch which gives it its title.” Beautifully shot as it was by our Best Cinematography winner Autumn Durald Arkapaw, “Sinners” was really just an IMAX-sized version of that sex dream we've all had about Michael B. Jordan trying to bite himself, at least until Kelli Weston hailed it as “a dynamic testament to Ryan Coogler's populist impulses, his proclivity for grandiose portraiture, and his unerring insistence that no one who embraces the American project may escape its grotesque transformations.” “The Secret Agent” was just a thrilling and deeply felt display of the movies' ability to manufacture a meaningful history of their own — an unforgettable argument that cinema can be even more valuable as a vehicle for exhuming the truth than it is as a tool for burying it — until David Sims wrote on his Letterboxd that it “whips ass.” That was the entire review.
Of course, determining the exact nature of the relationship between critics and artists has become a recurring theme at this dinner, as any number of chairs have taken the opportunity to lament the fact that critics and artists are spiritually aligned but also somehow irreconcilable; that we're both fighting against the common enemy of mediocrity, but are fated to do so while facing directly towards each other from separate and unequal vantage points. It was just a few years ago that the great Alison Willmore stood at this very podium and quoted the art critic Peter Schjeldahl, who said that “Closeness is impossible between an artist and a critic. Each wants something from the other — the artist's mojo, the critic's sagacity — that belongs strictly to the audiences for their respective work; it's like two vacuum cleaners sucking at each other.”
But with all due respect to the late Mr. Schjeldahl, who belonged to a different and more clearly demarcated era, as the once-monolithic film industry continues to shrink into a glorified niche, it's become increasingly undeniable that artists and critics ARE the audience for each other's respective work. Which isn't to say that filmmakers make movies for critics, or that critics write their reviews for filmmakers, but rather to observe that we have each become the last remaining failsafes to ensure that anyone still gives a shit about the work that either of us do, and the medium that both of us still love.
Whether it's streaming executives who insist that going to the movies is an outmoded practice (despite the overwhelming data that young audiences are the most AMC-friendly demographic), or movie theaters that refuse to mask their screens, or AI evangelists who preach that grotesquely Frankensteining stolen work into a soulless chimera of some kind is a worthy replacement for the magic of motion pictures, the viewing public has never been so aggressively conditioned to expect less — to surrender more, to put convenience before fulfillment, to be passively sated rather than actively engaged.
And yet, in spite of those obstacles, it's because of the critics and the filmmakers in this room that the movies still remain among the most persistent bulwarks against the forces of enshittification despite all of the medium's diminishments and defeats. And in the shadow of such enshittification, it's never been more obvious that film without critics would be as doomed as critics without film. It's never been more obvious that no one would challenge us if we failed to challenge each other. Perhaps even more importantly, no one would reward us either.
The future of movies — which is a microcosm for the future of everything — is a fight between people who give a shit and people who don't. At the end of the day, I really think it's that simple. And everyone in this room, whatever their discipline, is someone who gives a shit. Love it or hate it, “Marty Supreme” is not the kind of movie that someone makes by accident while trying to churn out content. By the same token, Richard Brody got so mad when the latest Wes Anderson movie failed to win any prizes this year that he beat a fellow NYFCC member to death with a bagel, and is only here tonight because critic-on-critic violence is legally considered to be a victimless crime.
Which brings us to the purpose of tonight's dinner. Maybe this is as close as critics and artists are ever likely to ever get — sitting at different tables in the same oversized pan-Asian restaurant in front of a giant Quan Yin statue that Adrien Brody so confidently misidentified as Shiva last year. But, contrary to Peter Scheldahl's words, a night like tonight reminds me that it's not what critics and artists want from each other, but what we give to each other that matters. Yes, we give you awards in return for you giving us things to write about, but really, what we give to each other are reasons to continue giving a shit about the things we love, and the courage to insist that the things we love are still capable of giving us something meaningful in return.
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No intellectual property is ever really gone forever in Hollywood, and a new Peacock series is preparing to breathe some new life into a cult classic from 1989.
Joe Dante's “The ‘Burbs” was ahead of its time, lampooning the monotony of American suburbia and the darkness that sometimes hides beneath it with a story about a young couple (Tom Hanks and Carrie Fisher) who encounter a Satanic cult in their otherwise unremarkable neighborhood. The film eventually built a fanbase that surpassed its $49 million box-office haul, and audiences will soon have a chance to experience a new iteration of the property. The new Peacock series is a new story set in the present day that takes inspiration from the ideas and imagery of Dante's film.
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Per an official synopsis, Peacock's “The ‘Burbs” follows “a young couple who have reluctantly relocated to the husband's childhood home. Their world is upended when a new neighbor moves in across the street, bringing old secrets of the cul-de-sac to light, and new deadly threats shatter the illusion of their quiet little neighborhood.”
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The series sees Keke Palmer and Jack Whitehall stepping into the lead roles (though they're not playing the same characters as Hanks and Fisher), flanked by a supporting cast that includes Julia Duffy, Paula Pell, Mark Proksch, and Kapil Talwalkar.
The limited series hails from creator Celeste Hughey, who has writing and producing experience on shows such as “Palm Royale” and “Dead to Me.” Executive producers include Seth MacFarlane, Brian Grazer, and Dana Olsen, who wrote the original film.
“The ‘Burbs” was named one of IndieWire's most anticipated TV series of 2026, with our film critic Ben Travers explaining that fans have two reasons to be excited about the show.
“Two good reasons: You loved the original film (starring Tom Hanks) and believe its premise is well-suited for a modern interpretation,” Travers wrote. “That, or Keke Palmer. She's great! She deserves her own show! And with a supporting cast including Paula Pell and Mark Proksch, she may have found one funny enough to earn her skills.”
All eight episodes of “The ‘Burbs” will stream on Peacock on Sunday, February 8. Watch the trailer below.
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By
Brenna Ehrlich
Heated Rivalry creator Jacob Tierney is flustered when he joins our Zoom call, pacing around under a clear blue sky. “I just realized I'm wearing an REM T-shirt to a Rolling Stone interview. That's so on-the-nose!” he declares, gesturing at his gray concert T-shirt. It doesn't take long for him to get over that and start gushing about Michael Stipe and Co., though. “I would actually physically fight somebody over them,” he adds, laughing. Good thing he has a whole pack of hot hockey players in his corner — or, at least, that's what they play on TV.
Tierney's HBO drama exploded at the end of 2025, an adaptation of a popular book series by Rachel Reid that follows a pair of hockey players as they go from rivals to something steamier — a secret romance. The show premiered on Canadian subscription service Crave in late November, and after getting picked up by Max, it barreled into the public consciousness like a hockey player… doing hockey player things. Stars Hudson Williams (who plays shy Canadian Shane Hollander) and Connor Storrie (in the role of brooding Russian Ilya Rozanov) were suddenly everywhere — a dizzying turn from waiting tables and sparse IMDB pages.
The show's soundtrack — a mix of early-to-mid aughts nostalgia — has also found a foothold in the zeitgeist, giving artists like t.a.t.u and Wolf Parade sizable streaming bumps even decades after their songs originally dropped. And that's largely due to Tierney's vision. “I've never made TV the normal way,” he says. “Music is so important to me. [Wolf Parade's 2005 track] ‘I'll Believe in Anything' was so built into the show — I wanted to clear it before we even started filming. That's kind of like an indicator of how music is crucial to the way that I like to tell stories.”
Despite the fact that the show takes place from roughly 2008 to 2018, Tierney says the throwback soundtrack wasn't intentional. “People always say to me, ‘You really tried to do this accurate to the period.' No, the [songs] just happened to largely be from a playlist I made,” he says. “It's the music of my early 20s.” His stars, who are currently in that age range, immersed themselves in the soundtrack, despite not really knowing any of the artists of Tierney's youth.
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“They made me feel really old,” he says. “They were like, ‘What is a wolf and why is it on parade?' And I was like, ‘I hate both of you.' And I was like, ‘Feist? 1,2,3,4?' And they're like, ‘Oh yeah, we watched that on Sesame Street.' And I was like, ‘Again, I'd like to push you down a flight of stairs. Your youth enrages me.'”
Age matters aside, Tierney has been overwhelmed by musicians' reactions to the show; artists like Miley Cyrus have already been vying to soundtrack season two. “It's been crazy who has reached out, because there's a bunch of people that I shouldn't even say who have DMed to me,” Tierney says, sadly declining to elaborate. “I'm like, ‘Holy shit. I can't believe I can even think about music like this. It's like dreaming.'” But that doesn't mean he's abandoning those aughtie faves. His current writing soundtrack? TV on the Radio and the New Pornographers.
As the hype for Heated Rivalry continues to grow — composer Peter Peter dropped two tracks for the soundtrack, “Rivalry” and “It's You” Friday — Tierney broke down his favorite needle drops from the cultural phenomenon.
Tierney sees this track as the theme song for hockey player Scott Hunter (François Arnaud) and his boyfriend, Kip (Robbie G.K.).
That was a big, hugely important needle drop for me, because I also wanted to use it multiple times. I wanted to make it like Scott's subconscious. It's just this big, romantic, beautiful song. That's what I want for him and Kip. And I just love a love song. Needle drops, to me, are so important, because they do trigger a subconscious reaction. I mean, music in general, right? For me, if I can make three people feel the way I feel when I hear this song, then I'll have accomplished what I want to share.
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I'm also a big Wolf Parade fan. I was at an Arcade Fire show and they were the openers. Their first EP was out. I bought that EP that night, and was like, “Who the fuck is this? Oh, my God, I love this band so much.” In the early 2000s, I was talking to Spencer [Krug] on and off about scoring something else. So, this was kind of a big, “I'll circle back to you.” Yes, I circled back. It just took a long time.
“My Moon, My Man” opens episode four when there's a massive, long montage of the boys texting and fucking, being in love and being apart from each other. And “Sealion” is in episode one when Ilya is going to see Shane for the first time in the hotel, and he runs into his mother in the elevator.
“My Moon, My Man” is such a good song; it's so poppy and smart and beautiful and all that shit. I loved it so much. We literally looped it and played it twice because my editor was like, “You know, this song is nowhere near long enough.” I was like, “Great, loop it. Just play it twice.” That's how much I love this song.
And “Sealion” — I just felt like it had the right nervous energy song for that moment. Boys getting ready to meet for the first time. There's something kind of propulsive about it. “My Moon, My Man” was in the script, but “Sealion” wasn't, because I really wasn't sure if I could afford it. And then I was like, “I can't. The replacement song was not working.” So we went back to her people and we figured it out.
This French track scores a scene in which Hollander dates actress Rose Landry (Sophie Nélisse).
I love that there's this kind of female energy that is so exquisite to me. I love this for Rose, and I love this for that montage in episode four where Ilya is looking at all of this press coverage of Shane and his new girlfriend. It felt exactly like the right cheeky, cute, poppy earworm. I believe that my Apple Music told me it was the song I listened to the most last year.
Heated Rivalry's de facto “Running Up That Hill” moment, this mix of the original and remix serves to underscore the agony of hidden love.
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I wanted some Russian in there. Even though I know that there's controversy about the queerness of it, it was queerest Russian pop hit that I can think of. Other than “Rasputin” [by Boney M.]. I did not think I could afford it, but as soon as we put it in, I was like, “Oh, we need this.” I liked the idea of doing a song and then a cover of it, and then found that Harrison cover. And I was like, “Oh, God, we've got to make this work, because these two songs together are really doing the heavy lifting.” I don't mind telling you, it took a lot of figuring out how to make that work, not just financially, but content-wise. I'm so grateful it did, because I had a feeling that it could be really iconic. Or, at least, iconoclastic.
The song ends the show. My friend Michelle Mylet, who was on Letterkenny, she sent me that song. What I loved about it for that moment was, it's another love song, but it's also horny and naughty. And I was like, “Yes, please.” I didn't want to let people think we've forgotten about being horny.
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The creator of the hit dystopian series said he compares crafting each season to making an album: "What haven't we done yet, and what tone am I looking for?"
By
Lily Ford
The cogs in Charlie Brooker‘s brain are at work on the next installment of Black Mirror.
The hit dystopian series is returning for an eighth season. Netflix confirmed the news on Friday by releasing a transcript of a conversation with Brooker, in which he beamed about the show's three Golden Globe nominations.
The seventh edition of Brooker's dystopian anthology series immediately topped Netflix charts upon its April 2025 release, accruing 10.6 million views in its first full week on the platform. The six episodes included a sequel to what is regarded as one of the most popular episodes in the program's history: USS Callister.
The episodes landed Brooker a nomination for best television limited series, anthology series or motion picture made for television, as well as performance nods for stars Rashida Jones and Paul Giamatti in episodes Common People and Eulogy, respectively.
“It was a pleasant surprise and an honor,” said the British writer-producer about the Globes recognition. “Obviously, I don't experience human emotion, but as much as I can emulate it, I was pleased,” he joked.
Amid Brooker's analysis on how season seven was crafted and which spine-chilling sci-fi moments still linger in his mind, he also discussed the future of Black Mirror, telling the streamer: “Well, luckily it does have a future, so I can confirm that Black Mirror will return, just in time for reality to catch up with it. So, that's exciting. That chunk of my brain has already been activated and is whirring away.”
When asked about his describing each season as an individual album and what kind of tune the next one will be, Brooker responded: “It's a useful thought experiment when approaching a new story. I'll often think of, ‘Well, what haven't we done yet, and what tone am I looking for? … Where does this track come on the album, and what musical direction are we going to go into?' We'll find out. [It's] very unlikely you'll ever see a Black Mirror hoedown.”
In an August, 2025 conversation with The Hollywood Reporter, Brooker said he's got “plenty more Black Mirror stories ready to go.”
“The way that technology is improving, there are new Black Mirror-y ideas you can see all the time,” he said. “I was reading the other day about people's mental delusions being reinforced by large language models and AIs who chat with you, and you can almost immediately see several story ideas start leaping out at you. There's plenty more horrible oil in the tank, basically.”
Brooker also teased a little about his new, untitled Netflix series, which he said is a “profoundly serious crime thriller” and “very much not Black Mirror.”
“There's definitely something I'm not saying about it at the moment just yet. I can't even tell you what it's called yet, but it's very much not Black Mirror. I can say that. It's a very, very different thing. The most detective show of all time. It's a deeply profound and profoundly serious crime thriller,” he adds, listing The Bridge, The Killing and Knives Out among his favorites of the genre.
The 2026 Golden Globe Awards are set for Sunday.
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By
Josh Crutchmer
Zach Bryan introduces With Heaven on Top, his first album since mid-2024, with a spoken-word story of a Manhattan apartment in the winter, a fire, and the New York Fire Department dousing him with water that runs down his back, down the floor, and ultimately downstream and to the ocean.
The 25-track record finds Bryan, one of country music's most successful and most polarizing figures, seeking to embody the person in that story. He dwells on the people and places that got him to the pinnacle, and the cost of life at the top.
Now that With Heaven on Top is out, fans and social media lurkers alike are working overtime to assess how successful Bryan was in his quest. Here are our five takeaways from the record to help move that process along.
After three years in the center of country music's orbit, Bryan spent at least part of 2025 away from the spotlight. He played a limited number of stadium shows — plus a pair of blowout weekends in Dublin and London — but nothing like the schedule he kept during 2024's Quittin' Time Tour. He also sparred publicly with Gavin Adcock (not surprisingly) and John Moreland (quite surprisingly). But he spent a long stretch of time off the grid for the first time in his career, and just about all of With Heaven on Top came from it. He all but spells this out in “Anyways” as he laments the difference in his life between the summers of 2024 and 2025. The song speaks to burnout, frustration, and spending his time “underneath the covers, trying to hide from the world outside” before getting some advice: “If you quit now you let those greedy bastards win somehow.” By the end of the song, Bryan's outlook has come around: “I ain't feeling empty lately. I'm gonna go and make them scenes.” It's hard not to hear this album as a collection of life experiences that led Bryan to such a turnabout.
The record is full of self-awareness on Bryan's part, which is likely to intensify feelings toward him. People who like him are all but certain to like him more, and those who do not like Bryan are likely to come away liking him even less after a listen. There's no moral to the stories he's telling and no quest for personal growth (which aligns with his Adcock feud), but the person singing on With Heaven on Top matches up with the Zach Bryan the public did see in 2025. When he sings “I've been working on myself all fall. Six beers a week ain't bad, a little boring is all,” in “Slicked Back,” he's not singing about some abstract character. It's him. When the album takes Bryan to New York time and again, he's baring a soft spot for the Big Apple. And when he sings that he's never been to Spain, he's doing so from a plane bound for Spain. This has always been Bryan's approach to songwriting, and to his credit, he doubled down.
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Fans and foes of Bryan alike who latched on to his public relationship with Brianna LaPaglia and its equally public, and tumultuous, ending will be largely disappointed in With Heaven on Top. Certainly, there are moments when Bryan picks at those wounds. “Skin,” in particular, is a veritable diss track referencing matching tattoos he and LaPaglia got as a couple, and taking a razor to his to remove it. Along the same lines, though, Bryan does not go overboard with tunes about his new love and wife, Samantha Leonard. But the lyric, “When I get to hell or heaven, can I bring my girl? 'Cause she likes romance, good sex, music and ruling the world,” still goes a long way toward covering those particular bases in Bryan's life.
Bryan released a snippet of “Bad News” on social media in October. The lyric “ICE is gonna come bust down your door, try and build a house no one builds no more,” directly referenced the ongoing crackdown by the federal government and set off a fresh round of chatter over Bryan's intentions. Bryan played it coy at the time, imploring people to wait until the entire song drops to make up their minds. Well, now that the song has dropped, it's political. It would have been political in any week, but especially in a week in which the ICE he sang about shot and killed a citizen in Minneapolis. But it is also a reminder that singing about a political flashpoint does not make one political. Bryan is a Navy veteran, and that comes with its own set of frustrations, and he makes it clear, too, before getting back to his point that “right's turned red, and the left's all woke.”
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No single artist shoulders more credit for ushering in the era of stripped-down, lyrics-first country music than Bryan — or more blame for the endless debates over “authenticity” that followed. On first listen, With Heaven on Top is Bryan emphasizing musically and lyrically what got him to this point. If there's anything new to the sound, it's an obvious influence from Bruce Springsteen. This record takes Bryan's acoustic medleys — and occasional waltzes — and adds just enough harmonica, horn, and string accompaniments to evoke the Boss without cribbing from him. And, for all of Bryan's travels, his lyrics return him to his roots time and again. He sings about his late mother in “DeAnn's Denim.” He sings about his Oklahoma home in roughly a quarter of the songs, name-checking Rogers County and the Red River, and references Oklahoma's Turnpike Troubadours — one of Bryan's favorite bands — in a passing mention of “Kansas City Southern” for those listening close enough to hear it.
Josh Crutchmer is a journalist and author whose book (Almost) Almost Famous will be released April 1 via Back Lounge Publishing.
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With so much good music being released all the time, it can be hard to determine what to listen to first. Every week, Pitchfork offers a run-down of significant new releases available on streaming services. This week's batch includes new albums from Dry Cleaning, Max B and French Montana, Winged Wheel, Zach Bryan, the Cribs, and Jenny on Holiday. Subscribe to Pitchfork's New Music Friday newsletter to get our recommendations in your inbox every week. (All releases featured here are independently selected by our editors. When you buy something through our affiliate links, however, Pitchfork earns an affiliate commission.)
With each album serving as one big swing above home plate, Dry Cleaning use their third LP, Secret Love, to confirm they've got one of the most consistent batting averages in modern post-punk. The South London quartet allows each member to lean closer to the forefront and show off their sleek form, from Florence Shaw's crisp deadpan quips in “Cruise Ship Designer” to Lewis Maynard's polished, wooden-like bass tone across “Hit My Head All Day.” Produced by Cate Le Bon and composed by the band inside studios owned by Jeff Tweedy and Gilla Band, Secret Love would be a return to form, but Dry Cleaning never fell out of shape in the first place.
Listen on Apple MusicListen on SpotifyListen on TidalListen on Amazon MusicListen/Buy at BandcampBuy at Rough Trade
It's been a long time since French Montana and Max B launched their Coke Wave mixtape series—17 years, to be exact. Now that Max B has been released from prison following a 16-year-long sentence, the close friends and collaborators have picked up where they left off in the form of Coke Wave 3.5: Narcos. For those keeping track, no, you're not losing your mind about the chronology; their latest collaboration is the fourth mixtape in the series, following 2009's Coke Wave and Coke Wave 2, as well as 2019's Coke Wave 4.
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Everyone in Winged Wheel knows their grouping doesn't make sense, least of all geographically; hence why members of Sonic Youth, Tyvek, Matchess, and more turned to remote file-trading from their different abodes to pull it off. All six members of the band—Whitney Johnson, Cory Plump, Matthew J. Rolin, Steve Shelley, Lonnie Slack, and Fred Thomas—poke and prod one another to keep their sound evolving through experimental indie rock, post-punk, krautrock, and beyond, often refining arrangements after tracking the music itself. Their third album, Desert So Green, is more aggressive than last year's Big Hotel, yet it's arguably the best jumping-off point for their discography, welcoming new listeners into the fold to see where and how they get where they're going.
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We're barely a week into 2026 and Zach Bryan is already having one of his most memorable years. Following a New Year's Eve marriage ceremony in Spain, the country star has dropped his sixth full-length album, With Heaven on Top. The follow-up to last year's The Great American Bar Scene is a sprawling 25 songs long, alternating between rustic confessionals and trumpet-dotted uptempo numbers. On the whole, it sounds like it cuts straight to the heart and strips away frilly layers – more or less in line with its rural, pond-side cover art.
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Exactly 25 years into their long career as one of England's consistently charting rock bands, the Cribs are not only still going strong, but know the strongest currency is hooks with simple immediacy. Welcome their ninth album and its winking title, Selling a Vibe, which shoots the band's guitar riffs through a sunny filter, giving listeners their daily vitamins in the form of bubbly earworms like “Never the Same” and “Summer Seizures.” Produced by former Chairlift musician Patrick Wimberly, Selling a Vibe embraces its pop construction, relaxed attitudes, and desire to readjust the Cribs' rock status to leave room for a softer peace of mind.
Listen on Apple MusicListen on SpotifyListen on TidalListen on Amazon MusicBuy at Rough Trade
Let's Eat Grandma spent nearly a decade making surreal synth-pop. Now, under the moniker Jenny on Holiday, Jenny Hollingworth peels off as one half of that duo to release her own solo debut, Quicksand Heart. Drawing inspiration from Prefab Sprout, the Replacements, and Cyndi Lauper, Jenny on Holiday uses her expressive vocals to stamp an impression on life's flickering moments. Born from Let's Eat Grandma's brooding synth-pop but still light on its toes to match the alt-pop of the present, Quicksand Heart sounds like a refreshing commitment to greet the day despite its shadows.
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CN Entertainment
By Anthony D'Alessandro
Editorial Director/Box Office Editor
Paramount/18Hz's Primate rang up $1.4M from both Thursday night showtimes and previous previews. Meanwhile, Lionsgate/STX's Greenland 2: Migration did $900K. Triple note on that Gerard Butler movie: Canada isn't going this weekend. Primate and Greenland 2 were forecast to make $8M-$10M heading into this weekend.
Despite the entry of two older-male-skewing titles, Avatar: Fire and Ash is expected to hold on to No. 1 in its fourth weekend with $20M-$22M. The James Cameron-directed threequel grossed $2.5M yesterday, ending its third week at $55M for a running cume of $321.2M.
The Johannes Roberts-directed, Walter Hamada-produced $24M Primate opens today in 2,964 locations. Internationally, the film opens this week in 26 markets including Mexico. That 90% fresh Rotten Tomatoes critics score has simmered down to 78% fresh, which is still notable for an R-rated horror movie. Oz Perkins' The Monkey last year had a 77% certified fresh RT score in reviews. Speaking of which, Primate‘s previews are about $500K shy of Monkey‘s $1.9M (which turned into a $5.8M Friday/previews and $14M opening).
Watch on Deadline
RELATED: The Movies That Have Made More Than $1 Billion At The Global Box Office
Butler's Lionsgate movie from a year ago, Den of Thieves: Pantera, did $1.35M in previews before a $5.7M Friday and a $15M No. 1 debut. The first Greenland was 77% certified fresh with audiences during the pandemic (remember, it only got a PVOD play), and Part 2 is lower at 59%. Greenland 2 was made for $90M, but Lionsgate only took U.S. for $10M. Elevation will be releasing the sequel in Canada as PVOD via Amazon.
Per Comscore, 76% of colleges are on break today, a number that declines to 42% on Monday but spikes back up to 94% the following Monday on the Martin Luther King Jr. holiday in addition to K-12 being 94% on break.
RELATED: The 25 Highest-Grossing Animated Films Of All Time At The Global Box Office
Top pics of the week:
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Avatar 3 continues to lag way behind 2, and I think it's mostly due to it being an inferior product. Still, way ahead of last year's Marvel movies. From Disney's POV, there's too much money on the table to walk away. But when we get Avatar 4, it may be a while and it may not be directed by James Cameron.
The maker's of Greenland were probably hoping recent news would boost grosses. Doesn't seem to be the case.
Avatar is doing better overseas where the real money is.
Uh, the studio gets back only 25-45% of ticket sales from overseas and 50-55% domestically.
Not only that, the first two Avatars made $2.1 billion and $1.6 billion overseas, and this one won't even make it to $1.6 billion overseas and domestic combined.
There is no way Avatar 4 and 5 are getting made.
Avatar 2 – 1st week 197
2nd week 160
3rd week 113
Avatar 3 did 154, 112, 55
James Cameron isn't stupid, he can read the writing on the wall.
As someone who would really like to see 4 and 5, I have to say that I agree with you.
There is no way to justify two more $400m movies. The fourth one might be able to eek out a profit, but the 5th one would be one of the biggest money losers of all time, surpassed only by Mission Impossible: Dead Reckoning.
You don't understand the biz, bro: Avatar 4 also would pump up 3 other Avatar movies plus theme parks etc.
It was released earlier, though, in 2022. They missed out on a lot of money by releasing on Dec 19th which allowed only 2 weekends in 2025 and the 3rd in 2026 as people were going back to work and prepping for school to start back up.
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By Ted Johnson
Political Editor
On the day that Warner Bros. Discovery‘s board announced its rejection of Paramount‘s latest bid, the David Ellison-led conglomerate took its argument to Capitol Hill.
In a letter filed with a House Judiciary antitrust subcommittee on Wednesday, Paramount's chief legal officer, Makan Delrahim, wrote to lawmakers that the Netflix–WBD combination was “presumptively unlawful.”
Delrahim wrote that Netflix's proposed acquisition of Warner Bros. Discovery assets was “presumptively unlawful,” arguing that it would “further cement its dominance in streaming video on demand.”
The letter was filed with the committee on the same day that it held a hearing on the streaming market, with the sale of WBD a primary topic of discussion among lawmakers and the expert witnesses. Paramount representatives did not address the subcommittee in person, but the lawmakers have been taking written comments.
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A frequent point made during the hearing was that much will depend on how the government ultimately defines the relative market, i.e. whether Netflix competes in a narrow world of subscription streaming for premium content, or a much wider landscape that includes YouTube and social media.
Delrahim said that the broader market definition was “tortured and absurd” and something that “no serious regulator would ever accept.”
He wrote, “It asserts, for example, that free, user-generated videos on YouTube and TikTok should be considered an adequate substitute for premium produced content available on Netflix or HBO Max. This is what some call ‘psychedelic antitrust' — it has no ground in market or legal reality.”
Delrahim argued that Netflix had previously dismissed the idea that YouTube was a rival. pointing to securities filings in which “it compared itself to actual competitors in streaming video on demand.”
Delrahim led the Justice Department's antitrust division during Donald Trump's first term.
A Netflix spokesperson did not immediately respond to a request for comment. The Wrap first reported on the filing, which was made with little fanfare as attention focused on what may be Paramount's next step as it seeks to win a hostile bid for all of WBD.
Last month, Warner Bros. Discovery entered into a deal with Netflix, with the latter buying the studio and streaming assets, and the WBD cable channels spun off into a separate company.
Congressional lawmakers have oversight over the Justice Department, but no direct authority in whether the transaction is approved or rejected. The regulatory review also will include European regulators and state attorneys general.
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Dude, she's just not into you! Move on, for the sake of the kids!!
Two things:
1) The idea that merging Netflix and HBO Max would give them some huge market share is mostly nonsense, based on the idea that that would be combining two completely different groups of people when in reality there would be a huge amount of overlap and it probably wouldn't even increase their market share by very much.
2) Did they use AI for writing that letter? Given that the quote acts like it's a thing people say, “psychedelic antitrust” literally seems like an AI hallucination or something; it finds nothing on Google other than articles about this.
Then the lawmakers should be good to go.
and the law is, of course, whatever their daddy trump says it is.
Imagine being as rich as an Ellison and still being willing to debase yourself by acting like a little baby because you can't have even more
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She's all about the base, no trouble. Meghan Trainor is suggesting that she had no idea about the scathing drama surrounding her mom group after Ashley Tisdale blasted them as “toxic.” The hitmaker took to TikTok to share her surprised reaction while reading the drama online in a video paired with her 2025 track “Still Don't Care.” Trainor's husband, Daryl Sabara, said he has no bad feelings towards Ashley Tisdale despite all the drama.
By
Andy Greene
A little over a year after telling a live audience that “nobody will release my music anymore because I'm a chief exponent of free speech,” Morrissey has indeed found a label willing to release his new LP, Make-Up Is A Lie. The album will arrive March 6 via Sire/Warner Bros., and the title track is available right now.
The album was cut with producer Joe Chiccarelli, who Morrissey previously worked with on 2020's I Am Not a Dog on a Chain, 2019's covers collection California Son, 2017's Low in High School, and 2014's World Peace Is None of Your Business. They recorded it at the Studio La Fabrique in the Saint-Rémy-de-Provence region of Southern France with help from musicians Jesse Tobias, Camila Grey, Carmen Vandenberg, Juan Galeano, Alain Whyte, Gustavo Manzur, and Brendan Buckley.
Eleven new songs were written for Make-Up Is a Lie, and they also recorded a cover of the 1973 Roxy Music classic “Amazona.” He's never performed the song in concert, but Morrissey is a lifelong Roxy Music fan, and he sang their 1973 tune “Street Life” on his 2006 summer tour of Europe.
Morrissey originally called the new album You're Right, It's Time. Production was completed three years ago, but Morrissey said he was unable to find a distributor. Throughout that time, he toured throughout Europe and America, playing sets packed with Smiths classics and solo tunes. But in typical Morrissey fashion, many of the shows were canceled at the last minute.
He was supposed to kick off an American tour on Jan. 3 in Rancho Mirage, California. It was postponed a day before due to “an adverse reaction to a prescription medication.” Not long afterward, he yanked a planned Jan. 6 show in San Diego for reasons that weren't explained. As of now, the tour is supposed to begin on Jan. 10 in San Antonio. But nothing will be official until the lights dim and Morrissey walks onto the stage. (And even then, there's always a chance he'll call things off midway through the night.)
In 2024, Morrissey said that he agreed to a Smiths reunion tour, but guitarist Johnny Marr “ignored the offer.” Marr usually ignored Moz's provocations, but he responded to this one. “I didn't ignore the offer,” he said. “I said no.”
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The track listing for Make-Up Is a Lie:
1. “You're Right, It's Time”2. “Make-Up Is a Lie”3. “Notre-Dame”4. “Amazona” (Roxy Music cover)5. “Headache”6. “Boulevard”7. “Zoom Zoom the Little Boy”8. “The Night Pop Dropped”9. “Kerching Kerching”10. “Lester Bangs”11. “Many Icebergs Ago”12. “The Monsters of Pig Alley”
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The Hollywood Reporter caught up with the Texas native at back-to-back events on Wednesday and asked how he's dealing with the crush of newfound fame. (Hint: Deleting Instagram from his iPhone.)
By
Chris Gardner
Connor Storrie had one of those days that can only happen for a Hollywood star.
His Wednesday began early with a high-profile stop at SAG-AFTRA headquarters on Wilshire Boulevard where he teamed with Abbott Elementary's Janelle James to announce the nominees for The Actor Awards. Standing at a podium inside the Tom Hanks and Rita Wilson Theater, he delivered a line that nailed his star-is-born arc thanks to a role as smoldering Russian hockey star Ilya Rozanov in Jacob Tierney's Heated Rivalry: “Eight months ago, I was literally waiting tables, so this is pretty wild. Now, instead of reading specials, I am reading nominations,” he said in a moment that went viral for encapsulating the meteoric rise with only six episodes.
After facing a crush of press in a Dolce & Gabbana suit and Hublot watch, he exited SAG-AFTRA flanked by his team of publicists from Shelter PR en route to a meeting with another new roster of reps on his side — a team of power agents at CAA.
By night, Storrie, then in a black satin Giuliva Heritage ensemble with a watch and diamonds by Chanel, landed on the cobblestone driveway at Chateau Marmont for Chanel's Coco Crush campaign launch celebration, headlined by Gracie Abrams and performer Lily Allen. But it was clear early on in the intimate evening that amid a sea of singers, artists and actors, it was Storrie stealing all the stares. “Everyone wants to meet you,” Tessa Thompson confirmed, microphone in hand, while addressing Chanel-clad dinner guests as Storrie looked on and blushed. For proof of just how high the fever for the Heated Rivalry phenom is, look no further than Instagram. When Abrams and Vogue recapped the night with carousels of pics, both led with images of Storrie.
It would be an overwhelming day for the most veteran of stars, but Storrie, 25, was calm, cool and beaming throughout. Rather than stay glued to his phone, he clutched a pack of blue American Spirits while focusing on conversations or Allen's mesmerizing performance of three songs off the critically acclaimed West End Girl about the dissolution of her marriage to David Harbour. “I just keep moving and be mindful about where I'm putting my attention,” Storrie told me about his laser focus amid a demanding schedule that includes reading new scripts from CAA, shepherding an experimental film he shot on an iPhone and juggling the demands of newfound stardom. I ran into him at both events Wednesday and asked how he's holding up.
How are you staying sane right now?
It's been so much that I honestly don't have time to really process or think about it, which I think is kind of healthy. I try to stay off of comments and too much online stuff because our algorithms, especially now, are so geared toward whatever you intake that if you go online, it could just be all me. It's like, OK, that's not healthy and that's not human to only look at yourself. I just keep moving and be mindful about where I'm putting my attention.
I wrote about you signing CAA this week. Have you thought about want to do next or what this year looks like?
A big part of my career and what I want to do right now with the show is I write and direct myself. I'm in the middle of doing an iPhone feature, this super indie kind of Sean Baker-esque film because I'm making it on an iPhone. I just want to develop as a filmmaker, too, alongside Heated Rivalry. I consider myself somewhat of a character actor in the sense that I really like to transform, as seen on the show. I want to be able to do that again too, see what that's all about.
What's the strategy with your agents?
They're bringing me a bunch of cool opportunities that I can't even believe I'm having. I'm just trying to do things that are cool and bold and swing big. I'm just talking to people and feeling it out. I'm very big on vibes too, because I think that even though you want to work with the best of the best or have parts that are ideally really cool, at the end of the day, it comes down to vibe. It's really about meeting people and seeing if you're on the same creative wavelength. That's why I think something like Heated Rivalry did what it did. Me and Hudson before this weren't even nothing, but we just had such a good rapport with each other and Jacob.
And are you reading scripts already?
Yeah. A lot. I mean, I don't really have time, to be honest.
I was going to say, when have you had the time?
I have a lot of plane time coming up. I'm probably going to [then]. I can't really sleep on planes.
I'm interviewing you while you're standing next to Gracie Abrams. You've said that you just dreamed of being able to do the work but there's a lot around it now. How are you processing these experiences?
I've said this before, I don't really have the time to process it, and that's true. We were doing things every single day from Nov. 19 until Dec. 20. I had a little break for the holidays. To be honest with you, I just really try to center myself on the creative projects that I'm actively writing. I'm doing this iPhone feature that I've also been trying to tackle on top of doing all of this. I have a few events in the next few nights and the days leading up, from like 7 a.m. until 4 p.m., so I'm shooting my movie, going home, getting ready and then going to whatever meetings or events I have. Having something active to do is what makes it doable.
Seeing you this morning and then again tonight, I'm thinking about style since it was Dolce & Gabbana in the morning and now you're here at Chanel. Is there an overall Connor Storrie style strategy with your stylist James Yardley?
Fashion was never really on my mind before getting into this world. The thing is about nice things is you don't know why you like nice things until you start being around nice things. I was never a car guy and then the first time you get in a really nice car, you're like, “Oh, I get why people pay hundreds of thousands of dollars to get a nice car.” It's kind of the same thing with clothes. Getting to wear designer clothes, you're like, “Oh, I understand why people spend the money if they have it.” Nice things are nice and it feels good and sexy to be in nice things.
Speaking of nice things, you're going to the Golden Globes as a presenter and it's cool that you and Hudson Williams get to do it together.
I'm excited. I think that's a really good platform for Hudson and I to be ourselves. Just think of the iconic duos that have been there and to be one of those is kind of … it's making me think of Kristen Wiig, Will Ferrell, Amy Poehler, Tina Fey. I'm excited that Nikki Glaser is doing it. It's going to be great.
In between seeing you, I saw some clips online of the sheer pandemonium outside Jimmy Fallon today when Hudson arrived. Did you see that?
No, I didn't have Instagram on my phone today because I had a lot going on, so I usually will get rid of the app when I really need to lock in. I texted Hudson, “How did it go?” And then I forgot. He was like, “It was really great. How did your thing go?” I was like, “It was really great.”
The fandom is so proud and protective of you guys. One of the reports that came out stated that you had already signed on for three years, which led people online to speculate if you were getting paid enough or getting a bonus. Has there been a contract renegotiation or a nice bonus with the show's massive success?
To be honest, I have a lawyer and a business manger now, so I have someone who kind of gets to do the deals for me. I'm surviving. I'm happy. I'm happy I get to make money doing what I want to do and I'm just happy that I'm not waiting tables anymore. That's all I'll say, money-wise.
What has been the most meaningful text, email or DM you've received over the past month?
From my mom. [This is] going to be emotional. My mom said something really interesting, like, “With you starting to do well in your career that you always wanted to do,” she said, “I feel this massive weight lifted off of me or a sense of relief.” She feels that part of her purpose in life or her life's journey or calling was to support me in this. Now that it's gaining momentum, she said, “I feel this sense of relief for myself.” That made me cry.
Interview edited for length and clarity. See below for views inside Storrie's Wednesday in L.A.
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The Hollywood Reporter caught up with the Texas native at back-to-back events on Wednesday and asked how he's dealing with the crush of newfound fame. (Hint: Deleting Instagram from his iPhone.)
By
Chris Gardner
Connor Storrie had one of those days that can only happen for a Hollywood star.
His Wednesday began early with a high-profile stop at SAG-AFTRA headquarters on Wilshire Boulevard where he teamed with Abbott Elementary's Janelle James to announce the nominees for The Actor Awards. Standing at a podium inside the Tom Hanks and Rita Wilson Theater, he delivered a line that nailed his star-is-born arc thanks to a role as smoldering Russian hockey star Ilya Rozanov in Jacob Tierney's Heated Rivalry: “Eight months ago, I was literally waiting tables, so this is pretty wild. Now, instead of reading specials, I am reading nominations,” he said in a moment that went viral for encapsulating the meteoric rise with only six episodes.
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Hudson Williams Teaches Jimmy Fallon Hockey Stretches and Teases 'Heated Rivalry' Season 2 on 'Tonight Show' Debut
After facing a crush of press in a Dolce & Gabbana suit and Hublot watch, he exited SAG-AFTRA flanked by his team of publicists from Shelter PR en route to a meeting with another new roster of reps on his side — a team of power agents at CAA.
Related Video
By night, Storrie, then in a black satin Giuliva Heritage ensemble with a watch and diamonds by Chanel, landed on the cobblestone driveway at Chateau Marmont for Chanel's Coco Crush campaign launch celebration, headlined by Gracie Abrams and performer Lily Allen. But it was clear early on in the intimate evening that amid a sea of singers, artists and actors, it was Storrie stealing all the stares. “Everyone wants to meet you,” Tessa Thompson confirmed, microphone in hand, while addressing Chanel-clad dinner guests as Storrie looked on and blushed. For proof of just how high the fever for the Heated Rivalry phenom is, look no further than Instagram. When Abrams and Vogue recapped the night with carousels of pics, both led with images of Storrie.
It would be an overwhelming day for the most veteran of stars, but Storrie, 25, was calm, cool and beaming throughout. Rather than stay glued to his phone, he clutched a pack of blue American Spirits while focusing on conversations or Allen's mesmerizing performance of three songs off the critically acclaimed West End Girl about the dissolution of her marriage to David Harbour. “I just keep moving and be mindful about where I'm putting my attention,” Storrie told me about his laser focus amid a demanding schedule that includes reading new scripts from CAA, shepherding an experimental film he shot on an iPhone and juggling the demands of newfound stardom. I ran into him at both events Wednesday and asked how he's holding up.
How are you staying sane right now?
It's been so much that I honestly don't have time to really process or think about it, which I think is kind of healthy. I try to stay off of comments and too much online stuff because our algorithms, especially now, are so geared toward whatever you intake that if you go online, it could just be all me. It's like, OK, that's not healthy and that's not human to only look at yourself. I just keep moving and be mindful about where I'm putting my attention.
I wrote about you signing CAA this week. Have you thought about want to do next or what this year looks like?
A big part of my career and what I want to do right now with the show is I write and direct myself. I'm in the middle of doing an iPhone feature, this super indie kind of Sean Baker-esque film because I'm making it on an iPhone. I just want to develop as a filmmaker, too, alongside Heated Rivalry. I consider myself somewhat of a character actor in the sense that I really like to transform, as seen on the show. I want to be able to do that again too, see what that's all about.
What's the strategy with your agents?
They're bringing me a bunch of cool opportunities that I can't even believe I'm having. I'm just trying to do things that are cool and bold and swing big. I'm just talking to people and feeling it out. I'm very big on vibes too, because I think that even though you want to work with the best of the best or have parts that are ideally really cool, at the end of the day, it comes down to vibe. It's really about meeting people and seeing if you're on the same creative wavelength. That's why I think something like Heated Rivalry did what it did. Me and Hudson before this weren't even nothing, but we just had such a good rapport with each other and Jacob.
And are you reading scripts already?
Yeah. A lot. I mean, I don't really have time, to be honest.
I was going to say, when have you had the time?
I have a lot of plane time coming up. I'm probably going to [then]. I can't really sleep on planes.
I'm interviewing you while you're standing next to Gracie Abrams. You've said that you just dreamed of being able to do the work but there's a lot around it now. How are you processing these experiences?
I've said this before, I don't really have the time to process it, and that's true. We were doing things every single day from Nov. 19 until Dec. 20. I had a little break for the holidays. To be honest with you, I just really try to center myself on the creative projects that I'm actively writing. I'm doing this iPhone feature that I've also been trying to tackle on top of doing all of this. I have a few events in the next few nights and the days leading up, from like 7 a.m. until 4 p.m., so I'm shooting my movie, going home, getting ready and then going to whatever meetings or events I have. Having something active to do is what makes it doable.
Seeing you this morning and then again tonight, I'm thinking about style since it was Dolce & Gabbana in the morning and now you're here at Chanel. Is there an overall Connor Storrie style strategy with your stylist James Yardley?
Fashion was never really on my mind before getting into this world. The thing is about nice things is you don't know why you like nice things until you start being around nice things. I was never a car guy and then the first time you get in a really nice car, you're like, “Oh, I get why people pay hundreds of thousands of dollars to get a nice car.” It's kind of the same thing with clothes. Getting to wear designer clothes, you're like, “Oh, I understand why people spend the money if they have it.” Nice things are nice and it feels good and sexy to be in nice things.
Speaking of nice things, you're going to the Golden Globes as a presenter and it's cool that you and Hudson Williams get to do it together.
I'm excited. I think that's a really good platform for Hudson and I to be ourselves. Just think of the iconic duos that have been there and to be one of those is kind of … it's making me think of Kristen Wiig, Will Ferrell, Amy Poehler, Tina Fey. I'm excited that Nikki Glaser is doing it. It's going to be great.
In between seeing you, I saw some clips online of the sheer pandemonium outside Jimmy Fallon today when Hudson arrived. Did you see that?
No, I didn't have Instagram on my phone today because I had a lot going on, so I usually will get rid of the app when I really need to lock in. I texted Hudson, “How did it go?” And then I forgot. He was like, “It was really great. How did your thing go?” I was like, “It was really great.”
The fandom is so proud and protective of you guys. One of the reports that came out stated that you had already signed on for three years, which led people online to speculate if you were getting paid enough or getting a bonus. Has there been a contract renegotiation or a nice bonus with the show's massive success?
To be honest, I have a lawyer and a business manger now, so I have someone who kind of gets to do the deals for me. I'm surviving. I'm happy. I'm happy I get to make money doing what I want to do and I'm just happy that I'm not waiting tables anymore. That's all I'll say, money-wise.
What has been the most meaningful text, email or DM you've received over the past month?
From my mom. [This is] going to be emotional. My mom said something really interesting, like, “With you starting to do well in your career that you always wanted to do,” she said, “I feel this massive weight lifted off of me or a sense of relief.” She feels that part of her purpose in life or her life's journey or calling was to support me in this. Now that it's gaining momentum, she said, “I feel this sense of relief for myself.” That made me cry.
Interview edited for length and clarity. See below for views inside Storrie's Wednesday in L.A.
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Season 13 of Hallmark's “When Calls the Heart” got off to a fiery start with the January 4 premiere. Kevin McGarry, who plays Nathan Grant, shared some intriguing teasers about what lies ahead during a recent podcast appearance.
McGarry revealed that the wildfire will impact this entire season of “When Calls the Heart.” In addition, the actor shared, he is confident everybody will be satisfied with how it is wrapped up.
McGarry chatted with the podcasters behind “Hallmark Mysteries & More,” “Suspenders Unbuttoned,” and others during a group December 22 podcast.
It was mentioned that during The Hallmark Christmas Experience, fellow “When Calls the Heart” star Jack Wagner provided a teaser that drew attention.
Apparently, Wagner told fans that the character of Nathan would “really command more of the scenes throughout the season.” McGarry admitted he thought it was a very nice thing for Wagner to say.
According to McGarry, this emergence of a more commanding Nathan begins during the episode airing on January 11.
“We really get to see him specifically in episode two kind of step into this leader role where he is kind of leading the pack in the rescue,” McGarry explained.
He shared that “Nathan really kind of takes the lead” in organizing the community to get people to the woods to fight the fire. As viewers know, his daughter, Allie is out there, but so are others from Hope Valley.
Luckily, it sounds as if everybody escapes relatively unharmed. However, that doesn't mean there's not more to come regarding the wildfire.
Once the fire is put out, McGarry revealed that Nathan will also initiate the initial investigation into the fire. The big questions of who started the fire and why will become an arc that “When Calls the Heart” viewers will watch throughout most of the season.
McGarry loves the fact that the fire storyline becomes a longer arc this season compared to what the show typically incorporates.
He explained, “Sometimes stories get wrapped up pretty quickly, and they kind of feel unfinished and you wanna dive into it more.” In resolving the who and why questions behind the fire, everybody gets to do that deep dive.
The fire, McGarry shared,” is something that really, pardon the pun, but sparks and catches fire very early in the season. This sends an arc that affects every character right up until the final effort.”
Of course, McGarry couldn't reveal anything juicy regarding the identity of who started the fire, or why. However, he did share that once answers emerge, “It's sad. It's not a great thing, but it brings out the best in a lot of the characters.”
Ultimately, McGarry foreshadowed, “The town really comes together to solve a massive problem and doing so everyone grows a little bit in certain ways.”
He thinks that everybody, both the fans and the “When Calls the Heart” characters, will find the conclusion very satisfying.
The arc revolving around the fire investigation will affect nearly every character in Hope Valley, McGarry suggested. He believes that it's all true to the roots of the show and Hope Valley community. It seems that “When Calls the Heart” fans have a lot to look forward to in the episodes ahead
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When film scholar Elena Gorfinkel first discovered Barbara Loden‘s “Wanda” 20 years ago, the format was a far cry from the restoration by UCLA and The Film Foundation so lovingly released by Criterion.
“The film had an active following among cinephiles, but it was difficult to see,” Gorfinkel told IndieWire. She found a bootleg DVD-R online that had been sourced from a VHS copy taken from a well-worn print, and in spite of the grainy, murky transfer, Gorfinkel was transfixed. “The force of the film still came through, and I think it has maintained that force and power over the years.”
Released in 1970, “Wanda” was Loden's first and only fiction feature as a writer and director, but its vérité depiction of a woman (played by Loden herself) on the margins drifting through life after leaving her husband and children was riveting enough to win the film a prize at the Venice Film Festival the year of its release — and to place it on Sight and Sound's 2022 list of the 100 greatest films ever made as chosen by an international critics' poll. As Gorfinkel points out, ever since its release, “Wanda” has inspired new waves of interest among cinephiles thanks to its singular heroine and a style that stands apart from other New Hollywood movies of the era.
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In fact, Gorfinkel says that to place Loden in context alongside New Hollywood auteurs like Bob Rafelson and Robert Altman is, to a certain degree, inaccurate.
“‘Wanda' is really not a Hollywood film at all,” Gorfinkel said. “It's very antithetical to it in that Loden talked about Hollywood as an albatross and a ship made of lead that wouldn't float anymore. She was very influenced by experimental film and the films of Andy Warhol and Jonas Mekas, and her very artisanal, low-budget approach is much more aligned with the American cinema of that stripe than, say, Arthur Penn, or what we think of as the New Hollywood.”
Placing “Wanda” in its proper context has been a mission for Gorfinkel in the 20 years since she first discovered it, and her research on the film has yielded a series of revelations about the myths surrounding its production and reception — several of which she debunks in her superb recent book on the film. Next weekend, Gorfinkel will host “Wanda and Beyond,” a weekend of films and lectures at the Joshua Tree Cultural Center designed to deepen and expand our understanding of Loden, her work, and the complicated tributaries of film history.
“A myth that I've come to find interesting and troublesome is the idea of the film as a forgotten work,” Gorfinkel said. “It didn't have wide distribution, but it was in circulation. Many people did know about it.” Although prominent critics like Pauline Kael dismissed “Wanda,” Gorfinkel also disputes the notion that the film was poorly reviewed.
“75% of the criticism I read about the film was really positive,” she said. “There's a sort of seizing on ways of trying to ‘rescue' the film from history that we all have as part of the process of being cinephiles, but I think the romance of referring to it as overlooked clouds our understanding of how the film actually moved through film culture and who did see it and talk about it.”
In fact, Loden traveled extensively with “Wanda” at film festivals, and even in its early days, it was well known and highly regarded in serious film circles. “It's often repeated that it only showed once in a theater in New York, which was not the case,” Gorfinkel said. “It was never meant to be a big-budget Hollywood film that would blanket all cinemas, but as an independent film, it was screening quite a bit in various cities and met with quite a lot of success and critical celebration.”
A more truly forgotten film is a movie Gorfinkel has programmed to screen alongside “Wanda” at the Joshua Tree Cultural Center, “Fade In.” Shot in 1968 but not released until 1973 — and then only dumped to television, not released theatrically as originally intended — “Fade In” marked Loden's starring debut as a film actress following supporting roles in her husband Elia Kazan's “Wild River” and “Splendor in the Grass” and her triumphant stage work in Arthur Miller's “After the Fall.”
“Fade In” stars Loden as a film editor on location in Moab, Utah, where she has an affair with a local driver (Burt Reynolds, four years before “Deliverance” would make him a star) hired for the production. It's a charming, beautiful romance with cinematography by the legendary William Fraker, an odd lineage; the movie was shot to piggyback on the production of the Western “Blue,” which was already shooting in Moab. “Blue” is the movie within the movie, and its stars Terence Stamp and Ricardo Montalban weave in and out of “Fade In” as themselves.
For whatever reason, “Fade In” was a doomed production; director Jud Taylor took his name off of it after disagreeing with studio recutting, and Paramount executive Robert Evans hated the movie so much that he never bothered to release it. For Gorfinkel, the movie asks tantalizing questions about what might have been since it was shot before “Wanda” but released after it. Had “Fade In” come out when intended and raised Loden's profile as an actress, would it have impacted the way she made “Wanda?” Would she still have made “Wanda” at all?
“I think putting ‘Wanda' and ‘Fade In' together is an interesting way of thinking about different trajectories through film history,” Gorfinkel said. “It's unclear what would have happened in Loden's career if ‘Fade In' had been released.” What is clear is that Loden had an oppositional relationship to Hollywood that might or might not have come from her experience on “Fade In,” the only time she was ever the star of a major studio production.
“She took a very critical, anti-Hollywood [approach] in the way that she presented and framed ‘Wanda,'” Gorfinkel said. “She really reduced the number of people on set, down to having four people on the crew.” Whether or not Loden's stripped-down approach to shooting was a direct response to “Fade In” is a tantalizing question that will probably never be definitively answered (Loden died in 1980 at the age of 48), but the opportunity to see it on the big screen in conversation with “Wanda” is one that should not be missed by any cinephile able to make the trek.
“The Joshua Tree setting will bring its own set of questions,” Gorfinkel said, noting that one of the reasons she programmed “Fade In” was its status as a gorgeous desert-set movie that would resonate in the Joshua Tree surroundings. Mostly, however, she just wants people to explore Loden in her totality, and in terms of both the films she made and the ones she didn't. “I think this is a way of thinking more expansively about the ways this single great work of cinema, ‘Wanda,' really opens up the questions of someone's life. How does one tell the history of the striving and all the potential and the creative imagination?”
“Wanda” and “Fade In” will screen as part of the Joshua Tree Cultural Center‘s “Wanda and Beyond” weekend on January 16 and 17.
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By
Andy Greene
Fifty-five years after recording the Doors classic “Riders on the Storm,” surviving band members John Densmore and Robby Krieger have come back together to recreate the song with musicians from across the globe, including Lukas Nelson, Micah Nelson, Don Was, Rami Jaffee, Sierra Ferrell, and Aaron White, as part of Playing For Change's annual Songs Around The World project. Check out the video right here, which was helmed by Playing For Change founder Mark Johnson.
“Vocally, Lukas Nelson filled Jim Morrison's leather pants quite well!” Densmore says in a statement. “Ray would be proud of the way Rami Jaffee ‘channeled' the piano solo. Mark Johnson masterminded a beautiful video.”
Adds Krieger: “Seeing musicians from different cultures come together around this song means a lot. Playing For Change is doing important work to unite people across the globe.”
Other artists on the track and video include William Barton (Didgeridoo), Boboulaye Sissokho (Kora), Macarena Montesinos (Cello), Guarani Andeva Group (Percussion), Iron Cult Dancers (Dancers), Izzana Jaa (Vocals), Ezequiel Acosta (Bandoneón), and Erik Prevost David (Trumpet).
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“Much has been said about the poetic side of The Doors,” says Don Was, who plays bass on the recording. “However, I can also testify to the depth of their swing and the swagger of their pocket. John and Robby's groove is the secret sauce of their success.”
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Over the past two decades, Playing For Change has teamed up with icons like Keith Richards, Robbie Robertson, Bono, Buddy Guy, David Crosby, Steve Miller, Tom Morello, Ringo Starr, Bunny Wailer, and many others, racking up over two billion YouTube views. Songs to get the Playing For Change Treatment have included “Fly Like An Eagle,” “The Weight,” “No Woman No Cry,” “Peace Train,” and “When The Levee Breaks.”
The Doors, meanwhile, spent the past year celebrating the group's 60th anniversary. Densmore is largely retired from the road, but Krieger continues to keep the band's music alive on concert stages. On March 28, he's holding a special 80th birthday show at the Whisky a Go Go in Los Angeles.
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Rolling Stone is a part of Penske Media Corporation. © 2026 Rolling Stone, LLC. All rights reserved.
Kate Middleton gave Mother Nature a shoutout on her 44th birthday.
The Princess of Wales posted a serene video via Instagram Friday of herself strolling outdoors as she spoke in a voiceover about enduring the “coldest, darkest season.”
The royal told her followers, “Winter has a way of bringing us stillness, patience and quiet consideration. The stream slows just enough for us to see our own reflection, to discover the deepest parts of ourselves alongside the whispers and the pulse of every living thing.”
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Middleton noted that she has recently been “reflecting on how deeply grateful” she is.
As for what she's thankful for, Middleton explained, “The rivers within us flow with ease, fears washed away [as they] cleanse and purify. [We] come to peace with our tears and discover what it means to be alive, to be at one with nature.
“[Nature is] a quiet teacher and a soft voice that guides in memory, helping us to heal,” she concluded.
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In the caption, Middleton called her quarterly video series on Mother Nature this year a “deeply personal, creative reflection on how nature has helped [her] heal.”
Middleton, who is in remission after battling cancer, added, “But it is also a story about the power of nature and creativity in collective healing. There is so much we can learn from mother nature, as we look to build a happier, healthier world.”
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She, notably, made headlines for her cancer diagnosis in March 2024.
She stepped back from the public eye to undergo treatment, revealing six months later that she had completed chemotherapy.
Middleton, who has since returned to her royal duties, confirmed in January 2025 that she is in remission.
Her husband, Prince William, gave rare insight into Middleton's health struggles on Eugene Levy's “The Reluctant Traveler” after their “hardest year” as a family.
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The Prince of Wales, who shares Prince George, 12, Princess Charlotte, 10, and Prince Louis, 7, with his wife, said, “Things are good. Everything is progressing in the right way, which is all good news.”
William, 43, admitted in October 2025 that while he tried to maintain a “positive outlook” during the scare, his mind went to some “pretty not great places.”
He continued, “Life is sent to test us as well and being able to overcome that is what makes us who we are.”
By
Emily Zemler
Ejae, Audrey Nuna, and Rei Ami, the singing voices from KPop Demon Hunters, stopped by Jimmy Kimmel Live to perform “Golden (Glowin' Version).”
The trio, accompanied by a group of string musicians, appeared on the late night-show's stage in a circle of candles. They showcased a reimagined version of “Golden,” the hit song from the Netflix animated film, which dropped on streaming at midnight.
The trio performed the original version of “Golden” at Dick Clark's New Year's Rockin' Eve in New York's Times Square on New Year's. In October, they brought the song to The Tonight Show stage, marking their first live performance together.
In the film, Ejae, Audrey Nuna, and Rei Ami are the singing voices of fictional K-pop girl group Huntr/x, performing the roles of Rumi, Mira, and Zoey respectively. Arden Cho, May Hong, and Ji-young Yoo voice the characters' spoken lines. The movie follows the K-pop sensation as they moonlight as the mystical destroyers of an invisible demon realm and are soon faced with their biggest battle yet when rival band Saja Boys turn out to be demons intent on stealing Huntr/x's fans.
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“Golden,” was a breakout hit, topping Billboard's Hot 100 for 18 nonconsecutive weeks. It was also Number One on charts around the world. The RIAA certified it double platinum in the United States. KPop Demon Hunters also became the most-watched movie in Netflix's history.
“I feel like I experienced all facets of K-pop,” Ejae told Rolling Stone after “Golden” became a runaway hit. “I was a trainee, I became a songwriter, I also vocal directed for K-pop idols. So I really resonated with [the KPop Demon Hunters character] Rumi. She's such a perfectionist and trying to hide her shame, hide her flaws. And that's a huge part of when you're training. You're trying to put your best foot forward. ‘Golden' really means a lot to me, because I feel like at that time, I needed a song like that.”
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Rolling Stone is a part of Penske Media Corporation. © 2026 Rolling Stone, LLC. All rights reserved.
The pop supernova's fourth studio album arrives Feb. 27.
By
Michael Saponara
Bruno Mars is back. The Grammy-winning singer returned Friday (Jan. 9) to launch his The Romantic era with the new single “I Just Might.”
Dropping at the stroke of midnight, the slick new song channels Leo Sayer's disco classic “You Make Me Feel Like Dancing,” and is accompanied with a music video which sees Mars, dressed in a green suit, fronting a band of Brunos. Daniel Ramos and Mars direct the clip.
The single will land on Bruno's fourth studio album, with The Romantic slated to arrive on Feb. 27. It's the pop supernova's first solo LP in nearly a decade, which came with 2016's Grammy-winning 24K Magic album.
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Bruno shook up the music landscape with a simple four-word announcement on Jan. 5. “My album is done,” he wrote to X.
In the time between solo projects, Bruno has been selective with his releases. He teamed up with Anderson .Paak for An Evening With Silk Sonic in 2021, which dominated at the Grammy Awards.
Bruno Mars also made his presence felt on the charts with collaborations like the dynamic Billboard Hot 100-topper “Die With a Smile” featuring Lady Gaga and “APT.” alongside BLACKPINK's Rosé, which topped the year-end Billboard Global 200 chart for 2025.
The 40-year-old will also embark on his first global stadium outing. The Romantic Tour is set to invade North American stadiums this spring and summer, as the trek will kick off in Las Vegas on April 10.
Anderson .Paak as DJ Pee. Wee, RAYE, Victoria Monét and Leon Thomas will be taking turns as supporting acts at various stops on the run. In addition to major North American markets, The Romantic Tour is heading across the pond for European shows in Paris, Milan, London, Berlin, Amsterdam, Madrid and Paris.
Listen to “I Just Might” and watch the official music video below.
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Billboard is a part of Penske Media Corporation. © 2026 Billboard Media, LLC. All Rights Reserved.
By Glenn Garner
Associate Editor
SPOILERS: This post contains details about the 9-1-1, Season 9 episode ‘Secrets'
With almost nine seasons of playing fun-loving fireman Buck under his belt, Oliver Stark is growing with his 9-1-1 character.
Ahead of this week's episode ‘Secrets', the actor spoke to Deadline about the character's unexpected bisexual love triangle with a married couple he meets during a night out, who happened to be played by “a real husband and wife,” Dean Geyer and Jillian Murray.
“It made it a little awkward, I can't lie,” he said. “And it didn't feel awkward until we were shooting the intimate scenes, and I was like, ‘Oh, that's your real-life husband right there,' or, ‘oh, that's your real life wife right there.' But they were so great and coaching each other, like, ‘Do this a little bit more, arch a little bit more.' And I was like, ‘Wow, I wish I had that, somebody to like coach me through this.'”
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Stark's behind-the-scenes experience came as Buck begins to realize “he just wants his person” and that his wild life is “not always that fulfilling.”
“He sees that around him, right? He has Hen and Karen, his sister Maddie and Chimney. They have such great relationships, they have their person, they know they're safe with them,” explained Stark. “And so, while this wild life may seem appealing, I think at the end of the day, what you want is to go home and have your safety there, the person that sees you and understands you. So, I think that's what this teaches Buck, is that he needs to find his way to that, some way, somehow.”
*SPOILER ALERT! Do not listen or read if you haven't seen the '9-1-1', Season 9 episode 'Secrets''9-1-1's Oliver Stark discusses Buck's bi love triangle (featuring a real-life married couple), plus 'Buddie' shippershttps://t.co/0v8ToiTSq0 pic.twitter.com/dzYFCkXAej
Meanwhile, two seasons after Buck came out as bisexual, Stark is fully aware of social media's devoted ‘Buddie' shippers and understands why fans want to see the character end up with Ryan Guzman‘s Eddie, who remains Buck's best friend after returning to Los Angeles and the 118.
“Tim Minear, who writes the show, said in an interview a few days ago, he's actively a fan of the ship of Buddie, and he understands it and roots for it, and doesn't not like the idea of it, which, I get it as well,” admitted Stark. “I've spoken about it in interviews. I say happily that I see the moments that other people see, and you see fan edits and there's nice romantic music playing over the top of it or whatever it is, and you go, ‘Oh yeah, those do feel like charged moments.'”
Read on about Buck's growth on 9-1-1, how Bobby's death has affected him and the upcoming 9-1-1: Nashville crossover.
DEADLINE: Buck came out a couple seasons ago. What's the LGBTQ fan response been since that?
OLIVER STARK: You know, it was so overwhelmingly lovely when that storyline first came around, and I spoke about it a bit at the time, but it's always nice to look back on it because it really struck me how overwhelmingly positive it was, and I didn't know how it would go down. You never know what the audience of the show is, demographically, and how they're going to react to something, and it was just overwhelmingly positive. I think that is probably because that is the theme of the show. So much is about inclusivity, and there's so much positive representation across the show already, that I think the fan base in general was one that understood and was built around love. So, it was really an honor to get to be a part of it and to get to tell this story, and I've really loved being able to continue to do that.
DEADLINE: This week's episode is very fun for Buck because he has multiple partners, and he gets to explore his bisexuality. But by the end of it, he's turning down the whole situation, and it almost feels somewhat out of character for Buck to turn down something like that. But at the same time, I feel like he's grown a lot since the first season. What is it he's kind of looking for in life at this point?
STARK: I think it's a case of, this wild, free life is nice in theory, but it's not always that fulfilling when it comes to it. And I think this situation teaches him that more than anything, he just wants his person. He sees that around him, right? He has Hen and Karen, his sister Maddie and Chimney. They have such great relationships, they have their person, they know they're safe with them. And so, while this wild life may seem appealing, I think at the end of the day, what you want is to go home and have your safety there, the person that sees you and understands you. So, I think that's what this teaches Buck, is that he needs to find his way to that, some way, somehow.
DEADLINE: Dean Geyer and Ryan Guzman look so much alike, I'm wondering if that was a conscious choice to cast someone who kind of looks like Eddie, almost like that's Buck's type.
STARK: I don't believe so, no. I don't know, actually. So, did you know that that is a real husband and wife?
DEADLINE: Oh, really?
STARK: Yeah. I don't know about this, this is what I heard, that they were cast completely independently of each other, and they just turned out to be real husband and wife. That feels a little too coincidental, but that's the story I was told. So, I don't think it had anything to do with that. I just think that this casting lined up and they both were great for their roles and got the jobs.
DEADLINE: That's funny. I love that.
STARK: It made it a little awkward, I can't lie. And it didn't feel awkward until we were shooting the intimate scenes, and I was like, “Oh, that's your real-life husband right there,” or, “Oh, that's your real life wife right there.” But they were so great and coaching each other, like, “Do this a little bit more, arch a little bit more.” And I was like, “Wow, I wish I had that, somebody to like coach me through this.”
DEADLINE: That's so funny. That's goals right there. Of course, I asked about Eddie because all the fans have been shipping Buck and Eddie, even long before Buck came out. And I'm curious if that's something that you think the writers will ever entertain or do you think it's maybe a little too unrealistic?
STARK: Honestly, and this is may be a little of a cop out, but I don't know. I do know, however, that Tim Minear, who writes the show, said in an interview a few days ago, he's actively a fan of the ship of Buddie, and he understands it and roots for it, and doesn't not like the idea of it, which, I get it as well. I've spoken about it in interviews. I say happily that I see the moments that other people see, and you see fan edits and there's nice romantic music playing over the top of it or whatever it is, and you go, “Oh yeah, those do feel like charged moments.” So yeah, I'm just happy to keep telling the story of these characters, and if that's the way that, authentically, Tim decides the story goes, then I think that's beautiful.
DEADLINE: And beyond the Buck romance storyline of this episode, one part that was hilarious was the chastity belt scene. There's been a lot of crazy stuff like that on this show, what would you say has been the craziest call or scene for you to shoot?
STARK: There's crazy in different ways on this show. In some respects, the first thing that flashes into my head is way back in Season 1, where I pulled a tapeworm out of a guy's ass. That was pretty crazy, just in terms of the quirkiness of it, and I loved that when we did that, Buck was just into it. That's just a great one. It was so free and easygoing. Then obviously, there's crazy in terms of scale, when you look at space or a tsunami or whatever it is, and then crazy in terms of the peril that it puts us in. Some of my favorite moments aren't necessarily the biggest spectacle emergencies, but they're the ones I had a fire truck dumped on my leg, or Eddie got shot, the ones that really put our characters that we've come to know and love right at the edge. I think those are probably the most meaningful ones and feel the most epic to shoot, because it feels like one of our own, and over the years, we have genuinely become a family. So, it's very easy to bring the urgency and feel that in those moments.
DEADLINE: Yeah, I was gonna say, like the time Buck got electro—or almost electrocuted.
STARK: Yeah—oh no, I did in fact, I believe I was dead for a few minutes. I think I died for a few minutes, but they brought me back.
DEADLINE: Bobby's death is obviously such a big moment for the show in general, but how would you say that's affected Buck when it's someone who really saw the potential in him from the beginning?
STARK: Yeah, I think it's really interesting this season to explore the way grief comes in waves. Some episodes, you don't see it in the characters at all, and then there will suddenly be an episode where he is brought up, and this feeling rushes over them. Or we had this Halloween episode earlier in the season, where Buck thought that Bobby was trying to communicate to him. So, I think it's nice the way that character will continue to literally haunt the narrative. And he was such a grounding figure for Buck, that when you remove that piece, you're left with a choice of, do you revert back to your old ways because the thing that was keeping you steady is gone, or do you learn to be that grounding piece for yourself? And I think we've actually seen Buck really step up, through his mentorship of the Harry character as well. He's almost getting to be what Bobby was for him. He's getting to be that for Harry. And so, I think he's continuing that legacy in a really positive and lovely, fulfilling way.
DEADLINE: Do you and the cast still talk to Peter much?
STARK: Yeah. Listen, after eight years of this show—and I know every show is obviously an intense experience—but I do feel like there have been things that we've gone through as a cast that have particularly bonded us, just time spent. And it's not like we speak every day anymore, but over Christmas, we spoke, and New Year's. We still have a group chat with all of us, and seeing his name pop up in it is always lovely. He's spoken about on set a lot and missed. We just shot something earlier today from episode 10 with this little kid, and he came up to me. He's a kid in the emergency, and he said, “Can I ask you a question? Do you miss Bobby?” And I say, “Yeah, we all miss him.” He will always be missed. He was such a big part of the show, and it's about finding ways to move forward without him on screen, but also off screen.
DEADLINE: Being on the show since the beginning, for nine seasons, Buck has changed on so many levels. Can you tell me anything about what the future holds for Buck or what you hope to see for Buck?
STARK: It's funny you say that, because yes, it has been a long time, and so we've watched Buck grow up a little bit, which is something that as we move deeper into the season, he's going to start to erestle with a little bit, that he's not the young guy anymore and maybe he doesn't bounce back from things so fast as he moves into his mid-30s, into late 30s. He doesn't feel the same way that he was feeling in Season 1, and we do have other people around us that are young and spright, and that's going to be an adjustment of identity for Buck that he's going to have to deal with as we go forward. And I like that because I think that's part of the journey to maturing and understanding that it's OK to not still be the 20-year old version of yourself, and so I look forward to continuing that story and watching him grow into this current form.
DEADLINE: Also I've been watching 9-1-1: Nashville, and I'm curious what's to come with the upcoming crossover.
STARK: Buck's going. Buck and Eddie are going, we'll actually be there for episode 12. I can say that much. Oh yeah, we're going. I can't tell you why we're going. But it's a good time.
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The fashion house toasted fine jewelry line Coco Crush with its campaign star and an intimate performance from Allen off her ‘West End Girl' album while the ‘Heated Rivalry' breakout had stars swooning.
By
Chris Gardner
Directly across from Chateau Marmont, looming large above Sunset Boulevard, is a billboard with the face of Gracie Abrams for a new Chanel campaign featuring the house's fine jewelry line Coco Crush.
It was no coincidence that Chanel selected the famed Hollywood hotel for a starry campaign celebration Wednesday night. It was obviously planned months in advance, just like the expert Chanel touches in so many of the Chateau's corners. Like, in the garage with a neon Coco Crush sign or a vintage convertible with Chanel license plates. Or the many rooms that hosted a revelers during a cocktail hour or for photo shoots and gossip sessions between buzzy stars, singers, artists, actors, designers and stylists.
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A crush of moments from the larger-than-life to the intimate could be felt from the cobblestone driveway to the velvet curtain-draped dining room that hosted a dinner and piano-side performance by Lily Allen. Even Abrams had some experience to share from the awe to the small. “Oh my god, the billboard. Holy shit, I know,” she said, looking over toward the sky. “That is a big picture of my face right there.” She was fresh from a quiet and reflective New Year's Eve. “We did a burning of wishes for the new year. Secret resolutions and hopes for loved ones.”
Abrams, in Chanel threads and pieces from Coco Crush, said she was honored to be the cause for celebration. “I've enjoyed this campaign process so much and wearing these pieces because whether it's on stage or at home in my day-to-day life, it's very comfortable and just feels like me,” she explained. ““I appreciate so much what Chanel is doing in this very moment and they people they are inviting in.”
Chanel welcomed past the velvet ropes a long list of stars and a certain phenomenon from Heated Rivalry who happened to be thee crush of the night: Connor Storrie. “We met upstairs. Everyone wants to meet you,” Tessa Thompson said in addressing dinner guests before dessert (and following plates of branzino and rib-eye) as she eyed Storrie at the main table in the center of the room. “Here's your chance.”
Plenty of people took that chance as Storrie, fresh from announcing the Actor Award nominations earlier in the day, fielded a steady stream of selfie requests and compliments for his work as Ilya Rozanov opposite Hudson Williams' Shane Hollander in the Jacob Tierney-created series, which became a certified phenomenon over the holiday break. While Williams was experiencing Harry Styles-like pandemonium outside New York's 30 Rock for a taping of The Tonight Show With Jimmy Fallon, Storrie was tucked away inside the celebrity hideout catching up with Abrams (who placed their photo as the No. 1 slide in her Instagram carousel), Maggie Rogers, Conan Gray and others like “the most famous person in the room,” whispered one attendee.
Other than eyeing Storrie, Thompson had official duties she accepted only moments before by toasting Abrams and introducing Allen. And she nailed it.
“Tonight you have the chance to meet people that you probably have a little Coco Crush on or that you admire and I love that. I had the chance to do that many times over tonight. One of those people is the extraordinary Gracie Abrams. We should raise a glass and toast her for being the new face of Coco Crush. She is indeed most crush worthy,” she said before turning her attention to Allen and her critically acclaimed new album West End Girl. “I recently slid into someone's DMs, which I hadn't done in a while. It's cool to do it. Everyone's making their in-and-out lists in 2026 so slide into more DMs. I did so because I listened to this record and I listened to it in the way that I used to listen to records, which is front to back, dedicated. I didn't do anything else. I was late to the party and I listened to this record in bed alone and I was so transported into the world of her experiences into her amazing, beautiful, brilliant, brave mind.”
Those experiences have been splashed across the globe as fodder for countless articles as Allen reportedly wrote the album inspired by the dissolution of her marriage (in very NSFW ways) to actor David Harbour. Even with, again, the larger-than-life narrative, Allen delivered a most intimate and at times cheeky performance of such songs as “Pussy Palace,” “Madeline” and “Tennis.”
Before the latter, she said, “Luckily, they are not based on real life experiences and these things definitely did not happen at this hotel.”
When it was over, one of the night's boldfaced names said, “It felt like watching a therapy session. I was mesmerized.”
Many people were, whether it was the music, the moment, the story or the Storrie of it all. Speaking of a good story, Thompson had the best one for how she was going to end her evening by ducking into a cozy hotel suite to watch her new Netflix show, His & Hers, opposite Jon Bernthal. “I have friends coming here to watch it with me upstairs at midnight and I'm so excited. I've never watched something in real time that I'm on and I'm nervous,” admitted the Hedda star. “I have seen it because I produced the show, so of course I saw the cuts, but it'll be very special tonight to watch at midnight with some friends.”
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The year's best nonfiction filmmaking on the big screen and TV shows were awarded at the New York Academy of Medicine in East Harlem on Thursday, January 8 — at the 19th annual Cinema Eye Honors.
The 2026 Cinema Eye Honors, comprised of a voting body of more than 800 documentary film experts, gave top honors to Ryan White's Apple documentary “Come See Me in the Good Light,” which follows poets Andrea Gibson and Megan Falley as they face Gibson's cancer journey. It won Outstanding Nonfiction Feature, Original Music Score, and Unforgettables Honors (the equivalent of career or life achievement) for Gibson and Falley.
That film is on the Oscar shortlist for Best Documentary Feature. Two other high-profile candidates, “The Perfect Neighbor” and “Seeds,” took two honors each. Geeta Gandbhir's Netflix-acquired documentary “The Perfect Neighbor” — which uses the true-crime format as a bait-and-switch to lure audiences into a more probing examination of injustices in our police system — won Outstanding Direction for Gandbhir and Outstanding Editing for Viridiana Lieberman. “Seeds,” a documentary about Black farmers in the South that premiered at the 2025 Sundance Film Festival, took the prizes for Debut Feature and Cinematography for Brittany Shyne.
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There was a tie for Outstanding Production, with two Oscar contenders, “The Alabama Solution” and “Apocalypse in the Tropics,” both honored. Other Oscar contenders abound on the list, from documentary short film shortlister “All the Empty Rooms” to “Cutting through Rocks,” about an Iranian woman elected to council in her rural village. But not “The Tale of Silyan,” which won the audience award from Cinema Eye Honors but was left off the Oscar shortlist.
See the full list of winners across film and TV from Cinema Eye Honors below.
Feature“Come See Me in the Good Light”Ryan White, Jessica Hargrave, Tig Notaro, Stef Willen, Brandon Somerhalder, BereniceChávez, Blake Neely, Dave Richards, Brent Kiser, Andrea Gibson and Megan Falley
DirectionGeeta Gandbhir“The Perfect Neighbor”
EditingViridiana Lieberman“The Perfect Neighbor”
Production(tie)Andrew Jarecki and Charlotte Kaufman“The Alabama Solution”andPetra Costa and Alessandra Orofino“Apocalypse in the Tropics”
CinematographyBrittany Shyne“Seeds”
Original Music ScoreBlake Neely“Come See Me in the Good Light”
Sound DesignJames LeBrecht, Greg Francis and Nina Hartstone“Deaf President Now!”
Visual DesignSara Gunnarsdóttir, Josh Shaffner and Kevin Eskew“It's Never Over, Jeff Buckley”
Debut“Seeds”Directed By Brittany Shyne
Audience Choice Prize“The Tale of Silyan”Directed By Tamara Kotevska
Nonfiction Short“All the Empty Rooms”Directed by Joshua Seftel
Spotlight Award“To the West, in Zapata”Directed By David Bim
Heterodox Award“The Voice of Hind Rajab”Directed By Kaouther Ben Hania
Unforgettables HonoreesNoam Shuster-Eliassi“Coexistence, My Ass!”
Andrea Gibson and Megan Falley“Come See Me in the Good Light”
Seymour Hersh“Cover-Up”
Sara Shahverdi“Cutting Through Rocks”
Pavel Talankin“Mr. Nobody Against Putin”
Jacinda Ardern“Prime Minister”
Fatma Hassona“Put Your Soul On Your Hand and Walk”
Broadcast Film“Pee-wee as Himself”Directed by Matt WolfHBO | Max
Nonfiction Series“Social Studies”Directed by Lauren GreenfieldFX on Hulu
Anthology Series“Conan O'Brien Must Go”Executive Producers Conan O'Brien and Jeff RossHBO | Max
Broadcast Editing“Social Studies”Edited by Alyse Ardell Spiegel, Helen Kearns, Catherine Bull and Charles Little IIFX on Hulu
Broadcast Cinematography“Omnivore”Director of Photography Tom Elliott, Sy Turnbull and Jurgen LisseApple TV+
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Earlier today, we reported on news that Johnny Knoxville was preparing to roll out the loosely connected assemblage of bone fragments and bad decisions he calls a body for a fifth Jackass film, set to arrive in the summer of 2026. But while Jackass has become a sort of gleefully violent, and even oddly joyful, nostalgia exercise in recent years—harkening back to halcyon days when the world was young, and its skull as-yet un-pummeled by relentless Butterbeans—there has been at least one major cloud hanging over proceedings since the production of 2022's Jackass Forever: The split between the Knoxville camp and long-time associate and co-star Bam Margera.
The details are both well-documented and pretty depressing: Margera filmed for a few days on the fourth movie before being fired, after (by his own admission) breaking a sobriety pledge he says he was forced to sign as a condition of appearing in the film. He then went on a long series of rants against Knoxville and various other people involved in the franchise, eventually launching a lawsuit, dismissing it, and then settling with the producers of the movie on undisclosed terms. (In a 2024 interview, Knoxville said that while he loves Margera and wishes him the best, he hasn't spoken to him since the firing.)
With some executive somewhere presumably hoping to get out ahead of things this time, Variety reports that news of Jackass 5 was accompanied by sources stating that Margera has signed a deal to allow archival footage of himself to appear in the movie. (The Variety piece states that, while Paramount hasn't commented on things either way, sources state it's extremely unlikely that Margera will film any new footage for the movie.) For his part, Margera hasn't commented publicly on the news of the new film, but he gave a Newsweek interview in 2025 in which he stated that “The damage has been done with that… It's not the same anymore, and you couldn't offer me enough money to be a part of that again.”
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Brooklyn Beckham reportedly wants no contact with his parents, David and Victoria Beckham.
As their family feud rages on, the Daily Mail reported Thursday that the 26-year-old chef and his parents exchanged letters last summer, but only through their legal teams.
Brooklyn reportedly said he didn't want his parents to contact him, or make public statements about him on social media, and that they should only contact him through his lawyers.
He reportedly made the request after he was offended by briefings about his wife, Nicola Peltz, which suggested that he was being “controlled” by her and that he was a “hostage.”
“David was told to speak to them via [law firm] Schillings,” a source told the outlet. “That was the only way for them to communicate.”
Reps for Brooklyn and David didn't immediately respond to Page Six's request for comment.
Last month, David and Victoria's 20-year-old son, Cruz, claimed Brooklyn blocked both him and their parents on Instagram while refuting reports that David, 50, and Victoria, 51, were the ones to block Brooklyn.
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“NOT TRUE. My mum and dad would never unfollow their son,” Cruz wrote.
“Let's get the facts right. They woke up blocked … as did I,” he added.
Although Brooklyn was absent from David's Instagram roundup of his 2025 year which included all of his other children — Cruz as well as Romeo, 23, and Harper, 14 — the soccer star did post a picture of him and his eldest son on his Instagram Story on the same day.
David wrote that he was grateful for his family and shared his love for all of his four kids.
“You are my life,” he wrote in part. “I love you all, Love Daddy.”
Victoria then re-posted her husband's Instagram Story with Brooklyn.
Last October, a source told Us Weekly that Brooklyn had “no interest” in making amends with the Beckham family after their clear falling out last year.
He's “really focused on living a peaceful, drama-free life” with his wife for the time being, the insider said.
This story was featured on a recent episode of Page Six Radio, a daily morning show serving up the hottest celebrity headlines, exclusives, and behind-the-scenes buzz. Catch Danny, Evan and Ian chat with celebrity guests every weekday on SiriusXM from 8 a.m. to 10 a.m. ET on Stars Ch. 109.
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A woman says she never agreed to let the Puerto Rican megastar include her voice on the songs "Solo de Mi" and "EoO."
By
Rachel Scharf
Bad Bunny has been sued by a woman who claims her voice is sampled on two hit songs from the Puerto Rican megastar without permission.
The lawsuit, filed on Monday (Jan. 5) in Puerto Rico, alleges that Tainaly Serrano Rivera can be heard speaking on both “Solo de Mi,” a 2018 song off Bad Bunny's debut album X 100pre, as well as the track “EoO” from his latest No. 1 set, Debí Tirar Más Fotos.
The sample in question supposedly features Rivera saying in Spanish, “Mira, puñeta, no me quiten el perreo,” which translates roughly in Puerto Rican slang to, “Listen, damn it, don't take away my vibe.” Rivera says she recorded this phrase at the request of Bad Bunny's producing partner, La Paciencia, when they were in college together back in 2018.
“At the time of the request, the purpose of the audio was not explained, nor was the plaintiff informed that her identity would be used and commercially exploited,” reads the lawsuit, originally filed in Spanish and translated by Billboard using the service DeepL. “No compensation of any kind was discussed. No contract or agreement was signed, nor was any license or authorization granted.”
Rivera is now suing for violations of Puerto Rico's right of publicity statute and seeking $16 million in damages from Bad Bunny (Benito Martínez Ocasio), La Paciencia (Roberto Rosado) and the star's record label Rimas Entertainment.
According to the lawsuit, Bad Bunny and his collaborators improperly used Rivera's voice not just in the commercial releases of “Solo de Mi” and “EoO” — which hit No. 93 and No. 24 on the Billboard Hot 100, respectively — but also in performances during his record-breaking San Juan residency last year.
Reps for Bad Bunny, La Paciencia and Rimas did not immediately return requests for comment on the lawsuit.
Rivera is represented in the case by attorneys Jose Marxuach Fagot and Joanna Bocanegra Ocasio. This is the same legal team that sued Bad Bunny back in 2023 for allegedly featuring the voice of his ex-girlfriend on the tracks “Pa' Ti” and “Dos Mil 16” without her consent.
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As legalized sports betting from the casino in everyone's pocket continues to financially bankrupt our morally bankrupt society, MoviePass hopes that the few online bettors might have some money left over to bet on the Avatar movies. In the latest installment of LetterBoxd and Ankler's newsletter, Crowd Pleaser, per Vulture, MoviePass CEO Stacy Spikes announced that the company, which has long evaded the “wait, is that a scam?” allegations, is joining the online gambling boom. The company is testing out whether “entertainment speculation” is something movie fans are into through a new site called “Mogul,” which it advertises as “the first fantasy league for entertainment.” (Apologies to Vulture‘s Movie Fantasy League players).
Mogul wouldn't work exactly like Kalshi or Polymarket, the online speculation marketplaces that seem to make millionaires of people who happen to know when Venezuela will be invaded, or who know exactly when White House Press Secretary Karoline Leavitt will finish her press conferences. At least not yet. In the future, Spikes wants “to build the first true hub for entertainment speculation.” For now, players would buy proprietary “Mogul Coin,” create a studio of chosen industry names, and earn points on their box office success. “You can know so much detail and data, and we see the only other place that is as data-rich is sports,” Spikes said. “Well, the closest business that has that level of data that people are predicting and guessing about every Friday is movies.”
Of course, sites like Kalshi, which, according to its CEO, aims to “financialize everything and create a tradable asset out of any difference in opinion,” are already betting on the Academy Awards, Rotten Tomatoes scores, and even the opinions of specific critics. Gambling on box office returns, however, is still illegal in the U.S.—though we're quite confident that will change, or one of these sites will just start doing it because there are no consequences for any bad behavior so long as the extremely wealthy get a little bit wealthier. Still, Spikes hopes Mogul will help turn entertainment speculators into MoviePass cardholders. He even had “a lot of fun,” prompting an AI slop video together for it. See you at the movies, speculators!
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Kaley Cuoco's celeb mom pals are praising their close bond in light of Ashley Tisdale's essay about her own “toxic” mom group going viral.
Cuoco's friend, actress Ashley Jones, shared pictures of their close-knit group Wednesday, which includes Lacey Chabert, Ali Fedotowsky and Amy Davidson.
The pictures included moments of the group getting together with their kids, solo shots of just the gal-pals, as well as throwbacks of when various members of the group were pregnant.
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“Mom groups are having a real moment on the interweb this week,” Jones wrote. “Shoutout to my village, without whom I could be very lost and lonely.”
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“Tag your supportive ride or die mom group!” she continued. “I miss them all this little, but so grateful we had each other during this stage —-and every stage. #lifteachother #womensupportingwomen **not all are pictured, but all who are pictured are loved #momgroup.”
Tisdale made waves when she criticized her former mom group in an essay for The Cut. While the “High School Musical” star didn't name names, fans quickly zeroed in on past pics of her with Hilary Duff, Meghan Trainor and Mandy Moore.
In Tisdale's essay, she claimed she was excluded from hangouts from the group, which made her feel “not cool enough” and “lost” as to why she was being left out.
The actress, 40, said she eventually texted the group, “This is too high school for me and I don't want to take part in it anymore.”
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But a source familiar with the drama told Page Six that Tisdale is “insufferable,” and that the friend breakup “has been a long time coming.”
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Members of the mom group have since publicly supported one another on social media.
Duff raved about fellow mom group member Janice Gott in an Instagram Story Wednesday, after her husband, Matthew Koma, savagely hit back at Tisdale in a more direct way.
Koma posted a fake mocked-up cover of himself on the cover of The Cut, with a headline that read, “A mom group tell all through a father's eyes: When You're the Most Self-Obsessed Tone Deaf Person on Earth, Other Moms Tend to Shift Focus To Their Actual Toddlers.”
Moore then gushed of Koma in an Instagram Story post just hours later, “@matthewkoma happens to be one of the most talented and generous humans I'm lucky to know.”
Meanwhile, Trainor humorously shared her surprised reaction while reading the drama online in a TikTok video set to her 2025 track “Still Don't Care.”
“Me finding out about the apparent mom group drama,” she wrote over the clip she posted Thursday.
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In a time when many might be mourning the passing of the holidays or longing for refuge from cold climates, Sony, Temple Hill and Netflix‘s collaboration to adapt Emily Henry's People We Meet on Vacation provides all kinds of escape as it finds a spot for itself on the map of romantic comedies.
Star duo Emily Bader and Tom Blyth play the friends-to-lovers pair Poppy Wright and Alex Nilsen, opposites who manage to attract despite a tense first encounter in a carpool ride — not exactly a meet-cute moment — that shows just how different they are and leaves viewers wondering how they could have ever recovered from it.
But Poppy's controlled chaos, clumsiness and reactions make her endearing, and Alex anchors her while matching her wild imagination. Their chemistry creates a pull toward each other to give what some may find an impossible opposites-attract story a solid foundation for the movie to work. It's helped by Bader, who brings the comedy and genuineness to Poppy with her expressive eyes and face, and Blyth, who expertly fleshes out a rigid, routine-loving Alex, who can only really be brought out of his shell with the help of Poppy.
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With a dual timeline that weaves between the current summer of Alex's brother David's (Miles Heizer) wedding in Barcelona and various previous “summer trips” to Squamish, BC, and New Orleans, context clues help differentiate the before and after of a major fallout between Poppy and Alex that hangs over them as they arrive in Spain.
Poppy's wanderlust and Alex's willingness to go along for the ride chart a course through spur-of-the-moment decisions like saying yes to a water-taxi ride from Buck (Lukas Gage) to pretending they are engaged to get free beignets. Of course, a detailed life story for engaged Poppy and Alex's alter egos, Gladys and Keith Vivant, comes with that pretense, and a resulting vibrant dance scene, choreographed by Maya Taylor, captures the spirit of Henry's book and the spark between Poppy and Alex, who fluctuate between the best of friends and the “what if?” of something more.
The back-and-forth, will-they-won't-they dynamic combined with the timeline jumps might drag for some, especially those who read the book and weren't fans of the stretched-out miscommunication (and at times, no communication) between Poppy and Alex; the simplified past summer culmination scene cuts that down a bit. Without spoiling what exactly goes down, the script writing and dialogue between the pair on David's wedding day still hint at a rub when they basically swap their “wing it” and planned out itinerary moods from the start of the day to its end. But this also shows how they have influenced each other. The ending is well worth it and earned, but of course everything it takes to get there is the fun part.
The humor, which is so key to Henry's novel, translates, even if it means less or no space for iconic lines from the book such as “It speaks to me” and “Do I have slow loris hands?” The simplest of scenes — like an early-on conversation in a diner — let Bader and Blyth shine. The great casting doesn't stop there, with the ensemble also boosting the story as each guest star plays their part, from Gage's aloof water taxi driver to Molly Shannon and Alan Ruck as Poppy's parents.
Haley infuses the film with color as he did with the film adaptation of Jennifer Niven's All the Bright Places. People We Meet on Vacation has a much lighter tone than that YA film starring Elle Fanning and Justice Smith, which traces themes of mental health.
As the first adaptation of a Henry novel (there are four more in the works) to cross the finish line, People We Meet on Vacation sets the bar high for future projects, visually, sonically and via its star-studded cast. Needle-drop selections, the likes of which were teased with Robyn's “Hang With Me” in the film's trailer, elevate big scenes. If only it could have had a theatrical release for book readers and moviegoers to experience together.
Title: People We Meet on VacationDistributor: Netflix Release date: January 9, 2026 (streaming)Director: Brett HaleyScreenwriters: Yulin Kuang, Amos Vernon & Nunzio RandazzoCast: Emily Bader, Tom Blyth, Lucien Laviscount, Sarah Catherine Hook, Lukas Gage, Molly Shannon, Alan Ruck, Miles Heizer, Tommy Do, Alice LeeRating: PG-13Running time: 1 hr 57 mins
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After years of delays and debate, the United States Congress has passed a NASA budget that omits any plan to retrieve several rock and soil samples collected and stored by the Perseverance Rover, including Martian rocks that may contain signs of past or present extraterrestrial life on the Red Planet.
According to the new compromise spending bill for the current fiscal year, it appears Congress has given in to the White House's demands that resources needed to complete the planned Mars Sample Return (MSR) program be explicitly excluded from any approved budget. While the bill still awaits a final vote in Congress and the President's signature, NASA officials are sending signals that the MSR program is effectively dead.
“The agreement does not support the existing Mars Sample Return program,” the bill states.
Victoria Hamilton, a planetary scientist at the Southwest Research Institute (SwRI) and chair of NASA's Mars Exploration Program Analysis Group, described the death of MSR as “deeply disappointing” while also questioning the veracity of the current administration's stated goals.
“When we've got memos coming out saying we want to be the dominant power in space, I wonder how we leave something this ambitious behind,” Hamilton said.
Before NASA launched Perseverance in July 2020, mission planners had struggled with the concept of retrieving the samples the rover was tasked with collecting through its mission. Since then, the interplanetary explorer has collected dozens of samples and stored them for the planned future return.
During that time, the cost for MSR has continued to rise, with 2024 estimates reaching $11 billion. Regardless of the potential scientific value, the extraordinarily large share of NASA's science budget that the single mission was projected to consume left planetary scientists working on other stalled or underfunded projects wondering whether the MSR was worth the broader scientific cost.
Repeated threats from Congress to scrap the ambitious yet costly program altogether resulted in a new, stripped-down plan released in January 2025 that came closer to the original $7 billion estimate. Now, it appears even that plan was still too rich for the current administration's taste, resulting in its explicit exclusion from the new bill.
As advocates of the MSR program try to regroup and seek other alternatives to bring the Martian rocks back to Earth to study, NASA's proposed $7.25 billion science budget offers some hope that a future mission may not be completely off the table.
For example, the bill, which cuts roughly 1% from the 2025 budget, allocates $110 million for a “Mars Future Missions” program to continue developing the key technologies needed for any future sample-return mission. These include further development of landing systems that can survive the thin atmosphere during descent, which has proven challenging and costly for previous missions.
Assuming the bill is signed into law, other missions that have waited patiently for MSR to run its course may see new signs of life. For example, recent discoveries on the moons of Jupiter and Saturn and in the atmosphere of Venus have led to plans for missions to further explore those tantalizing findings. These include plans to explore potential subsurface oceans on Enceladus and Europa for extraterrestrial life.
Although the newly proposed NASA science budget adds hope to the search for life on other moons and planets, proponents of MSR note the tantalizing cache of samples already collected by the rover since its 2021 arrival.
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In 2024, mission operators spotted a surface feature containing mineral deposits called “leopard spots” in a dry riverbed that leads to Jezero crater. NASA scientists note that on Earth, these types of leopard spots are often left by microbial organisms interacting with the rock, making the Chevaya Falls sample arguably the best candidate for containing signs of past life on Mars.
Bethany Ehlmann, a planetary scientist at the University of Colorado Boulder, said scientists eagerly await the opportunity to examine these samples in Earth labs equipped to determine if they do hold signs of extraterrestrial life. Elhman also pointed to the potential for other scientific discoveries that the dozens of samples gathered by Perseverance may hold.
“A rock with a potential biosignature is awaiting return now, and other rocks hold breakthrough discoveries,” the UC Boulder scientist explained.
Unless Congress has an unexpected last-minute change of heart, the new budget has left mission planners wondering about the fate of the samples, as the rover has nearly finished filling its collection tubes.
“We'd really like to hear from NASA sooner than later that they will work with the community on a plan to get these samples,” Hamilton said.
Christopher Plain is a Science Fiction and Fantasy novelist and Head Science Writer at The Debrief. Follow and connect with him on X, learn about his books at plainfiction.com, or email him directly at christopher@thedebrief.org.
We might not be looking in the right places, scientists warn
ADVANCED alien civilisations might be talking to each other across the vast reaches of space using a “firefly” technique.
That's according to scientists who reckon spotting aliens might be far easier if we change how we look for them.
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Aliens might actually be communicating with flashing lights – similar to fireflies here on Earth.
Scientists at Cornell University say that the search for aliens is usually inspired by “human centric ideas” of what intelligence and technology look like.
But they point out that humans aren't the only example of intelligent species communicating on Earth.
That led scientists to look at fireflies, which communicate through light.
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Part of the problem for the current Search for Extraterrestrial Intelligence, or SETI, is what we're looking for.
The scientists say that we've been too focused on human tech.
“For instance, the earliest indicators of technological activity from our planet came from the invention of radio transmission by the human species,” the researchers explained.
“The significance of our own radio broadcasts, and the fact that radio can be transmitted readily over cosmic distances, led to those wavelengths becoming a prime target for early SETI efforts.”
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To help broaden our horizons, the researchers analyses the flashes of dozens of pulsars.
These are fast-spinning (and highly magnetised) neutron stars that release beams of electromagnetic radiation from their poles.
They used these as a way of examining what the alien signals might look like.
Researchers don't think that these pulsars are linked to alien signals, but noted that detecting them could be a similar process to spotting firefly-style E.T communications.
“The firefly's distinct flash sequence provides a means of identifying members of the same species for mating,” the researchers said.
What's interesting is that the scientists think this sort of communication would actually be linked to a much more advanced civilisation.
“Any extraterrestrial intelligence using this method to broadcast their presence is likely to be far more advanced than the current level of technology on Earth,” they said.
They linked this to changing habits here on Earth.
“Earth has become less radio loud, not more,” they write.
“Especially due to the transition from analogue TV to cable TV and the internet.
“This example highlights a challenge associated with looking for short-lived human technologies as a window into alien technologies.”
So the researchers say we need to look for other signals – like firefly-style flashes – to “cast as wide a net as possible” for alien life.
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That way we can “broaden the range” of types of alien intelligence we're looking for.
Sadly, until we find alien life, it will be impossible to know exactly what we're looking for.
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A peculiar metal shard that sparked UFO enthusiasts' imaginations as potential alien technology has been debunked by scientists.
The metal shard had fascinated UFO enthusiasts, some speculating that it could be a piece of alien technology capable of levitation. However, after thorough analysis by the All-Domain Anomaly Resolution Office (AARO) and Oak Ridge National Laboratory (ORNL), scientists concluded that the metal was not extraterrestrial but instead an unusual alloy made on Earth.
While its properties raised questions about possible alien technology, the shard was ultimately shown to have been created using materials and processes familiar to human manufacturing.
The shard gained attention due to its alleged connection to the 1947 Roswell incident, where many believed that a UFO had crashed near Roswell, New Mexico. While the U.S. Air Force later confirmed that the debris found was from a weather balloon, conspiracy theories surrounding the incident have persisted.
The shard in question had long been considered a potential remnant of alien technology, sparking interest from figures like Tom DeLonge, the frontman of Blink-182 and UFO enthusiast, whose To the Stars Academy began investigating the sample.
Initially, the metal seemed to exhibit unusual properties that could suggest an extraterrestrial origin. It was believed that the metal might be part of a device capable of reducing inertial mass, a technology that could allow for levitation. This theory captured the imagination of UFO enthusiasts, who hoped that the shard could be a clue to the advanced technology of an alien civilization. However, new findings have ruled out this hypothesis, showing the metal to be a product of Earthly manufacturing, reports Popular Mechanics.
To confirm the true nature of the object, the AARO turned to Oak Ridge National Laboratory (ORNL) for further analysis in 2022. The team at ORNL tested the shard for signs of alien biosignatures and technosignatures, potential markers of extraterrestrial life or technology. What they found was a sample that matched the isotopic signatures of magnesium and lead commonly found on Earth, ruling out the possibility that it was from beyond our planet.
The magnesium in the sample was found to have undergone typical separation of lighter and heavier isotopes, likely caused by heat and mechanical stress during manufacturing processes. The analysis also confirmed that the lead had isotopic signatures specific to Earth, further eliminating the possibility that the metal had extraterrestrial origins. These findings led scientists to conclude that the shard likely originated from Earth, possibly linked to research into magnesium alloys used in the construction of aircraft after World War II.
One of the more intriguing theories surrounding the metal was the idea that it could be part of a terahertz waveguide, a device capable of channeling electromagnetic waves to levitate objects. This hypothesis arose from the metal's unusual composition, which some believed could be used in advanced technology to manipulate energy. However, the analysis by ORNL scientists revealed that the sample was not suitable for such use.
To qualify as a proper waveguide capable of levitation, the shard would need a single layer of pure bismuth placed between layers of magnesium alloy. While the metal did contain bismuth, the material did not meet the necessary specifications for such technology. The bismuth was mixed with lead and was not in the required single-layer form, meaning it could not function as a waveguide to channel terahertz radiation. This discovery further dispelled the notion that the object was part of alien technology capable of levitation.
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Lavika Gupta, a resident of Gaur City Avenue-1, in central Noida, recently filed a police complaint against her husband and four in-laws, alleging that she was deceived when she agreed to marry her spouse in 2024. Among her accusations, the woman claims that she was promised a man with lush, healthy hair, but married a bald man wearing a wig.
According to the First Information Report (FIR) filed at Bisrakh police station, Lavika Gupta and Sanyam Jain were married on January 16, 2024. The wife claims that she was deceived at the time, with Jain and his family concealing various details about his physical appearance, education, and financial status.
Photo: Unsplash
“After the marriage, I also found that my husband had only passed Class 12, while his biodata showed that he had a BCom (Bachelor of Commerce) degree. He also lied that his income was Rs 18 lakh ($20,000). In this way, I was seriously defrauded.”
Gupta added that when she agreed to marry Sanyam, she was promised a man with “thick hair”, but she later learned that her spouse was bald and wearing a patch to conceal his lack of hair.
“The woman has also alleged that her husband assaulted her during a trip abroad and pressured her to bring marijuana from Thailand to India,” an officer from Bisrakh police station told journalists.
To test this hypothesis, the research team analyzed more than 150
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The victim was homeless and suffered from mental health issues, his family said. Hetty Chang reports for the NBC4 News at 4 p.m. on Thursday, Jan. 8, 2026.
A late-night SWAT operation at a Buena Park business Wednesday led to the arrest of a man suspected of a deadly assault, authorities said.
Police arrested the 57-year-old man, who they say may have been living inside a psychic business on La Palma Avenue just a few feet away from where the deadly assault took place.
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The business sits right across the street from Knott's Berry Farm and near a parking lot where there's a memorial for 29-year-old Elton Harrell. On Dec. 29, investigators say Harrell was assaulted and later died at the hospital from his injuries.
"He loved being here so much. This is always where we will come to see him now," said Cindy Salazar, a friend of Harrell.
Friends and family told NBC4 that the 29-year-old called the parking lot home. Harrell's mother, Barbara Campbell, says her son suffered from mental illness and was homeless.
"Everyone loved my son, he was a good guy," said Campbell. "Never hurt anybody, never bothered anybody."
Campbell says she still has the blankets and bags of clothes she would bring to him every day.
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"Every day as long as he didn't disappear," said Campbell.
For a week and a half, the family searched for answers and so did Buena Park investigators, who say a search warrant led them right across the street from where Harrell was attacked.
On Wednesday, authorities say they detained a woman and arrested 57-year-old Barney Lee on suspicion of murder. The woman was later released.
"We're unsure what the relation is to the business. We are unsure of the status of the business that's still part of the ongoing investigation as to the relationship to the location," said Buena Park Police Sgt. Martin Tomsick. "It did appear they may be living in the location we are unsure."
Harrell's loved ones say they still have a lot of unanswered questions, like why anyone would hurt him.
"We're never going to be the same. I want to know why someone would take my son's life like he was nothing. He was something and somebody, and we loved him. He was somebody to us," said Campbell.
Police say they are still investigating whether Harrell knew the man they arrested. The motive for the assault is unclear.
By Chris Snellgrove
| Updated 1 hour ago
The Stranger Things series finale left the fandom largely divided, with many wanting an episode that would deliver both a better final fight and more answers to our most burning questions. However, some fans were convinced that we haven't actually seen the final episode…that we would get a secret ninth episode to Season 5 that would drop on January 7. These fans are part of the Conformity Gate conspiracy, and now that the 7th came and went without any new episode, it's clear that these fans set themselves up for serious disappointment with a conspiracy theory that never made sense from the beginning.
So, what the heck is “Conformity Gate,” and how does it tie to Stranger Things suddenly giving us a bonus episode? The short answer is that some fans thought that the show's final episode was a little too happy and that what we saw in that extended epilogue was, in fact, a mental illusion created by Vecna, who is very much alive. Believers in this conspiracy theory claim that this will all be made clear with the release of a ninth episode that serves as the true Stranger Things finale, one that was supposed to drop on Netflix on January 7, 2026.
As you might imagine, the “evidence” for this theory was all circumstantial, though some of it is admittedly interesting. For example, many Conformity Gate believers point to the Stranger Things finale's graduation scene and how several of the characters are holding their hands in the same distinctive way that Henry Creel does.
It's neat to consider, but Creel's hand pose isn't all that unique…it's really just holding his hands together. That's all the audience of this graduation is doing, too. Isn't it far likelier that people sitting down at a long ceremony simply got comfy (like, what else do you do with your hands on a chair with no arms?) rather than Netflix spending half of its $80+ million episode on a fake ending?
That same graduation scene features other circumstantial evidence, like someone holding up a blank yellow poster that you might expect to have, say, a student's name on it. Some think this is a sign that our favorite characters are in a dream-like state where things don't entirely make sense. But isn't it likelier that this was a sloppy production error, possibly even something they intended to fix in editing but forgot to do?
For some Conformity Gate believers, the most convincing evidence comes from two innocuous background details: first, look closely in the final Stranger Things scene, and you will notice multiple boxes of the WHATZIT board game. When Henry Creel was recruiting children for his evil plan, he went by the name “Mr. Whatsit.” Therefore, many in the fandom think that these games are a clue that the entire happy ending is a Vecna illusion lulling our heroes into a false sense of security.
However, WATZIT was a real game that was popular in the ‘80s, and it makes perfect sense for dorky Mike to have copies of this kicking around the same basement space where he and his friends play D&D. More sense, at least, than all of this being a complex illusion meant to fool fans and characters alike. Plus, this whole conspiracy theory falls apart once you realize that the biggest piece of ‘evidence” is a complete and total lie.
The second major background detail that Conformity Gate believers point to is Mike's D&D books…they are all carefully arranged, and some are convinced they spell out the phrase “X a lie.” The theory goes that this is saying that what we saw in Dimension X was a lie, which would explain why the final fight against a cosmic god and his superpowered flunkie was over in just a few minutes. It would also hint that the real final battle is yet to come, possibly in a new Stranger Things episode dropping on January 7.
However, this is a blatant lie, as you can tell from the image above. Conformity Gate believers love to share an image showing that a message is hidden in the D&D books that are arranged in this order: Max, Lucas, Will, Dustin, Mike. There's a meme going around showing this order and highlighting how it spells out “X A Lie.”
But the meme is a fabrication, and in the actual Stranger Things episode, the books are arranged in the following order: Max, Lucas, Dustin, Will, Mike. Squint hard, and all this spells out is “X Aile,” which isn't exactly a secret message. Unless, you know, you have a mean case of dyslexia!
Why did the conspiracy believers focus on January 7? Previously, Netflix posted a weirdly cryptic teaser that read “Your future is on the way” and prominently featured this date. Fans convinced themselves this was the streamer hinting at a big reveal on that date, but now that no new Stranger Things episode dropped, it's now clear that Netflix was likely just doing advertising for its own shows (like the upcoming season of Bridgerton) rather than teasing us about more Stranger Things content.
While I love a good conspiracy theory, Conformity Gate is pretty bonkers…like, why would Netflix, after spending nearly half a billion dollars to make Season 5, deliberately disappoint fans with a fake series finale and then try to redeem themselves with a new episode that has absolutely zero marketing? This would be the quickest way for the streamer to ruin its biggest IP, so I wouldn't hold my breath on Episode 9 ever seeing the light of day.
It seems clear that this entire conspiracy theory is just an outsized reaction to disappointment, giving fans a way to cope with a final episode that didn't really meet their expectations. Netflix wants to keep Stranger Things alive for future spinoffs, and the powers that be know quite well how such a fake-out would destroy everything the Duffer Brothers have built. They might be willing to blow up the Upside Down, but they'd never blow up their biggest cash cow!
Sadly, the brothers are ultimately responsible for this conspiracy blossoming in the first place…when you go out of your way to leave so much (from Vecna's motivations to Eleven's death) up to interpretation, you can't act surprised when millions of fans glom onto conspiracy theories. When Conformity Gate turned out to be the dumbest hoax of the year (so far, at least) and a certain segment of fervent fans went absolutely insane, the whole controversy became further proof that Stranger Things had one of the most divisive finales in television history.
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Barsha Dutta has been covering the NFL since 2024, bringing a fresh and engaging perspective to the game. With Masters in Literature, she blends passion with insight, making football stories both relatable and exciting for readers. When she's not writing about the gridiron, Barsha enjoys tending to her garden and immersing herself in the world of K-pop. She also occasionally covers entertainment and pop culture news. With her unique mix of interests, she connects sports fans to the NFL in a way that feels both personal and vibrant.Read More