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With the pedigree of a person who has seen his share of bust-ups in the boardroom and on the ice, Canadian American businessman Graeme Roustan is blunt about the looming trade war and what it will mean for the two countries he calls home.
“It's totally ridiculous,” says Roustan, a prolific entrepreneur and owner of Roustan Hockey in Brantford, Ontario. “This business here has been in place for 178 years and it's been selling product and trading product with the United States since before Canada was a country. It's just ridiculous to insult your neighbor, and as a dual citizen, Canadian American, I don't understand it from the American point of view either, why would we insult Canadians?”
His wooden hockey stick company is one of the last manufacturers of its kind in North America, based in the proud hometown of the man widely seen as hockey's G.O.A.T. (greatest of all time), Wayne Gretzky, known here as simply, “The Great One.”
Roustan's business, the city, and even the hockey legend himself have all been caught in the crossfire of the trade war declared by US President Donald Trump that from April 2 will see the US impose widespread tariffs against Canada and other once-friendly trading nations.
For Roustan, what the president is calling “liberation day,” feels more like “disaster day.” Business has already been impacted and customers are jittery, he says.
“All these are going to the Miracle on Ice Team USA 45th Anniversary Fantasy Camp,” Roustan says, while holding a red, star-spangled hockey stick in his hand. The stick, he says, needs to be shipped to the US in a hurry to avoid tariffs. “The customer wants this to cross the border as soon as possible because they don't want to get a 25% tax on their invoice.”
While similar concerns are being voiced by many in Brantford's business community, the looming disaster for the town's arguably most famous export is about reputation rather than the bottom line.
Hockey legend Gretzky, who has nurtured a long-standing and very public relationship with Trump and has lived in the US for decades, has been taking flak from his fellow Canadians since Trump first announced he wanted America's northern neighbor to be reduced to the 51st state.
Now, with the looming tariffs deepening the sense of betrayal felt across Canada, many of Gretzky's countrymen are directing and distilling their anger toward their once-untouchable hockey hero.
Words like “Traitor,” “MAGA junkie,” and “sell-out” now proliferate in online rants and news columns. Even in Edmonton where Gretzky won four NHL Stanley Cups, a statue honoring him was smeared with fecal matter, according to CNN affiliate CTV News.
Roustan calls Gretzky a friend, and he is astounded that some would think he would ever be a traitor to his Canadian roots.
“To drop Wayne Gretzky's name into the middle of this,” says Roustan, clearly incredulous, “It's a drive-by assassination of a name, a good quality Canadian name, it's just been just completely ridiculous.”
It started with Gretzky attending an election night party with Trump, a social media post of him wearing a MAGA hat, capped with a happy snap of him and his wife, Janet, attending Trump's inauguration in January. Trump, for his part, boasted he counseled Gretzky to run for Prime Minister of Canada, and then quipped he'd rather see Gretzky as “governor” of Canada as America's “51st state.”
Since then, the Gretzky reckoning has been chronicled in Canada with social media riffs, memes and newspaper editorials.
The pages of Canada's Globe and Mail newspaper have weighed in on much of it, starting weeks ago with opinion columnist Cathal Kelly writing, “He'll show up for any gala dinner, but when his best buddy the president is threatening to annex the country? Oh, you wouldn't believe how busy he is then.”
Kelly wrote again last week, wondering why Canadians are so obsessed with Gretzky, concluding, “What most of these people feel is betrayal. Many countries have a great turncoat in their history. Gretzky has become ours.”
As parents and players headed into the Wayne Gretzky Sports Centre on a recent weekend here in Brantford, passing a triumphant statue of the hockey prodigy lifting the Stanley Cup, the anger and contempt does not square with the devotion and donations Gretzky and his family have made to this community over the years.
Rick Mannen took his seat aside the rink to watch the local Brantford Titans take to the ice. He says he wishes the hockey legend he still admires would say something to his friend, President Trump.
“He's kind of a voice of Canada, he has been that way in the past and he is now if he chose to do that. So I really would like to see Wayne do that, but I still don't feel any ill against Wayne just because he's a friend of Donald Trump.”
When asked what Mannen wishes Gretzky might tell the president: “I wish he would say to Donald ‘back off and treat Canada as a partner instead of trying to take over.'”
Junior hockey coach Terry Corbin has a different take, saying Gretzky hasn't really been a part of Brantford for a long time.
“He hasn't lived here for how many years. I mean, I almost see him as kind of somebody with dual citizenship, but who has chosen United States of America,” says Corbin.
The highway leading to this gritty, working-class city bears the name of its hockey icon, but the Wayne Gretzky Parkway might as well be a free-trade expressway.
Hundreds of warehouses and manufacturing facilities dot the landscape. The city is a little more than an hour's drive from both Toronto and Buffalo and has become a convenient crossroad for Canadian companies and US subsidiaries.
For Brantford, the recessions of the 1980s and 90s gave way to a thriving business and commuter corridor that led to substantial growth in both employment and income.
A recent Canadian Chamber of Commerce analysis found that Brantford is one of the top five cities vulnerable to American tariffs.
The city's mayor, Kevin Davis, says some businesses here sell up to 80% of what they make in the US, but they also buy many of their raw materials from American factories. He describes Brantford as tough and resilient but he says there is no doubt tariffs will affect livelihoods.
“Our local economy is very intertwined and integrated with that in the US and not just in the auto parts industry. We have a lot of food processing here, plastics, pharmaceuticals, that is the essence of the economy here in Brantford. It's a mutually beneficial relationship with the United States,” he says, adding that it would take the city four to 10 years to reimagine its industrial strategy if tariffs are punishing and long-lasting.
“There'll be industries in Brantford that may shut down, they may reduce production, they may have to retool, and – for a year or two – not be producing much and employing less,” warns Davis. “That'll happen and there will be people in our community that will suffer.”
But, he promises, the city will fight back and bounce back.
“You know, we're nice until we're not. And yeah, if you want a war, then it's a war. But it's a, it's a totally meaningless war from my perspective. I really, frankly don't understand it.”
Many bewildered workers and consumers in this city are already preparing by cutting back on spending and cutting out most American products.
Buying American now seems like an act of treason here. Restaurants are scratching Caesar salads from their menus – they won't buy American romaine lettuce – and alcohol from the US, no wines from California.
“Even in our store, we get asked all the time, you know, are these products local? Have they been made in Canada?” says Ines Kowal of family-owned and operated Uniqpol, a grocery, deli and food processer in Brantford.
Before learning of the tariff threat, Uniqpol invested in a significant expansion that is set to come online in a few months.
Kowal says they're already seeing cautious consumers cut back even on staples like groceries, afraid of the impact tariffs will have on the family budget.
“Unfortunately, it's very difficult to absorb all these kinds of costs, so that will have to eventually be reflected in some of our prices as well,” she says.
Back rinkside at Wayne Gretzky Sports Centre, Karen Robb is at her son's game. She wishes Gretzky would say something to the president but acknowledges, like many here, how much he and his family have already done for the Brantford community.
“I think it's just about, you know, we don't want anybody to get hurt,” says Robb adding that some good has come of this. “The upside is we're thinking more Canadian. We're starting to think a little bit more about Canada, you know, supporting our businesses.”
Correction: This story has been updated to correct the spelling of Ines Kowal's name
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Audrey Hale felt no hatred against anyone at the school where the former student gunned down six people. In fact, the 28-year-old relished fond memories of The Covenant School and wanted “to die somewhere that made her happy,” Nashville police said.
“Hale bore no grudge against the school or staff” and considered them to be “‘innocents' and victims on par with herself,” the Metropolitan Nashville Police Department said.
The revelation came in a trove of new details released in a report by Nashville police Wednesday – two years after Hale randomly slaughtered three teachers and three 9-year-old children at the private Christian school.
Shortly after the massacre on March 27, 2023, Hale was gunned down by police – leaving myriad questions about the inexplicable killings that remained unanswered until now.
After conducting interviews and reviewing Hale's digital devices, online accounts, artwork and writings, detectives managed to piece together the killer's preparation and motivation behind the gruesome attack.
Hale attended The Covenant School in the early 2000s, from kindergarten through fourth grade. The former student denied suffering any emotional or physical abuse during this period, investigators said in the report obtained by CNN.
“She felt safe and accepted at The Covenant and made friends with other students,” the report said. “She considered her family life during this time as happy, with a positive relationship with both of her parents and her brother.”
But more than 16 years after leaving the school, Hale targeted the beloved alma mater “due to the notoriety she would obtain” and “because she had a personal connection to the school from earlier in her life and felt she had to die somewhere that made her happy,” police said.
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While the killer “identified as a male and used he/him as preferred pronouns,” Nashville police said, “Under Tennessee law, a person's gender identity must correspond with their biological sex or with information present on their certificate of live birth.”
As a result, authorities described Hale as a female in their 40-plus-page report.
Investigators discovered Hale left “material behind intentionally to be found and analyzed,” including “her detailed plan to commit carnage in a school, with timelines, diagrams, etc.,” Nashville police said.
“The publication of the writings would serve three purposes: to document her struggles with her mental health; to show how … her mental health conditions helped her ‘to execute the perfect plan'; and to show others like her how to best plan and carry out an attack,” the investigative report states.
In the days leading up to the attack, Hale started to transfer writings onto a thumb drive so “they could be found and studied,” the report states.
“Notoriety was the motive,” the report summary says. “It is known that Hale, and other mass shooters, studied material from Columbine High School prior to committing their attacks.”
Hale rated other mass shooters based on the number of people they murdered and considered those who killed a low number of people to be “amateurs” who weren't worthy of respect, Nashville police said. But Hale “made exceptions for those offenders with documented mental health histories showing they had serious mental health diseases or disorders, as she believed they were ‘too sick' to formulate an effective plan to kill an adequate number of victims.”
Hale wanted books, documentaries and movies “to be made about her life and her attack,” police said. The killer also wanted “her firearms to be placed in a museum” and “wanted her bedroom to be left as it was when the attack occurred as a memorial to her.”
Investigators determined Hale was sane, but evidence suggested worsening anxiety, depression and rage.
Before the massacre, Hale's parents assisted their child “with obtaining mental healthcare despite them not being legally required to do so,” police wrote.
But “Hale chronicled that she withheld information from providers to prevent her from being stopped,” authorities said.
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The killer often wrote about the level of secrecy needed to carry out the attack, including withholding information from the therapist, taking steps to delete browsing history, and concealing guns and ammunition, police said.
Even though Hale's “disappointments in relationships, career aspirations, and independence fueled her depression, and even though this depression made her highly suicidal, this doesn't explain the attack,” police said.
“As Hale wrote on several occasions, if suicide was her goal then she would have simply killed herself,” the report states. “Throughout the writings and videos, Hale frequently commented that her death needed to matter and be remembered.”
Investigators learned Hale felt chronic loneliness and disappointment.
“She felt abandoned and ignored by those she longed to befriend and engage with romantically, which angered her more than anything else,” the report says.
“She believed that by simply committing suicide, she would be quickly forgotten and not even worthy of a footnote in history. She craved the notoriety Harris and Klebold attained following Columbine.”
Eric Harris and Dylan Klebold were the teen gunmen who killed 13 people at Columbine High School in Littleton, Colorado. The 1999 massacre has been cited as an inspiration for other school shooters in the decades since.
Hale focused on how the Columbine killers' “documented mental health history and societal views were similar to her own and how the notoriety they achieved following their deaths led them to becoming ‘gods,'” Nashville police said.
Hale “sought to become a ‘god' like Harris and Klebold by killing victims nobody would forget: children.”
Like Harris and Klebold, Hale “longed for her name and actions to be remembered long after she was dead,” police said.
“She openly acknowledged none of those she would kill were guilty of anything and denied any personal motivation for targeting them,” Nashville police wrote. Instead, the killer “felt their deaths were necessary to give her death meaning.”
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“She wanted her mental health to be a prominent topic of discussion and debate,” investigators said.
“Most disturbingly, she wanted the things she left behind to be shared with the world so she could inspire and teach others who were ‘mentally disordered' like her to plan and commit an attack of their own.”
Yet Hale “often complained how she didn't know enough about the mental health history or motives of most mass killers, as nothing was publicly documented regarding their struggles,” the reports says.
Nonetheless, “Hale felt she would be a failure if she killed less than 10 people during the attack,” the report said.
Hale took a guided tour of the school in 2021, claiming to be “an alum who wanted to reminisce about her time in elementary school,” the report said. In reality, Hale was casing the school and “discreetly photographed different locations,” police said.
CNN has reached out to the school for comment.
Less than two years after that visit, Hale burst into her beloved former school wielding an assault-style rifle and gunned down six people. The victims included three children, a substitute teacher, a custodian and the head of the school.
This is a developing story and will be updated.
CNN's Karina Tsui contributed to this report.
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Tyrannosaurus rex ancestors and their plant-eating dinosaur prey would have congregated to drink water from a lagoon in what is now Scotland, new research suggests.
Despite the fact that the carnivorous megalosaurs would have hunted the long-necked sauropods 167 million years ago, newly identified footprints show that both types of dinosaur would have milled around the edge of the lagoon, much like how modern-day animals congregate at watering holes, researchers from the University of Edinburgh say.
Lead study author Tone Blakesley, a Masters graduate at the Scottish university, told CNN that he was among a small group that recognized an initial three footprints at the remote site on the Isle of Skye's Trotternish Peninsula in 2019.
“It was very exciting,” said Blakesley, who went on to document a total 131 footprints for the study, using a drone to take thousands of overlapping images of the site before producing digital 3D models of the footprints using specialist software.
Because of their flatness, the footprints had previously been mistaken for fish resting burrows. Blakesley explained that this was due to the fact that there would have only been a thin layer of sand on top of a much harder layer of mud, leaving only a shallow indentation.
They are preserved in “exquisite detail,” he added.
The footprints were made 167 million years ago, during the Middle Jurassic period, an important time in dinosaur evolution, but little rock remains from the era, Blakesley said.
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As a result, the site in northern Scotland provides invaluable insights into the life of dinosaurs at the time.
In stark contrast to the generally cold and blustery weather on Skye today, the area would have had a warm and humid subtropical climate during the Middle Jurassic, with a series of lagoons on a huge river estuary, Blakesley said.
The sauropods were “big lumbering giants which would have plodded along,” said Blakesley, who used the spacing of the footprints to estimate that they would have moved at speeds of around 2.5 kilometers per hour (1.55 miles per hour), around half the average human walking speed.
They would have used their long necks to feed from the top of conifers and other trees, he added.
The “jeep-sized” megalosaurs, which are a kind of theropod, would have moved around the lagoon on their way from one area of vegetation to another — in search of prey or to seek shelter and rest — traveling much faster, at around 8 kilometers per hour (5 miles per hour), he said.
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“It would have been quite a surreal place to stand in,” Blakesley said.
But while the dinosaurs would have been in the area at around the same time, the footprints do not demonstrate any evidence that they interacted by the lagoon, and it is unlikely that they would have been side by side.
“That would be a disaster for the sauropods if that happened,” he said. “The temptation for lunch… would have been too much for the theropods.”
Blakesley continues to work at the site and discovered more dinosaur footprints on Tuesday, he told CNN.
“There's more footprints to find,” he said, adding that he is also investigating other dinosaur track sites on Skye as well as in the south of England.
The study was published Wednesday in the journal PLOS One.
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The speed with which US democracy is being dismantled is dizzying, but if we organise resistance now we can stop this
Everything is moving too fast. The Trump-Musk administration is tearing through US government, universities and health organisations, firing tens of thousands of employees, eliminating billons in funding. The scope and speed of the attack is dizzying. It is almost impossible to keep up with the ongoing destruction, let alone to organise the resistance. None of this is accidental.
We need to understand the origins of the Trump blitzkrieg to counter it in the US and prevent it from spreading abroad. The speed of the attack can be traced to Trump strategist and “accelerationist” Steve Bannon, and aligns with his information warfare tactic to “flood the zone” to confuse, disengage and disorient. Whether on climate or Covid, rumours, lies and conspiracy theories create a chaotic cacophony, leaving the public disoriented, fearful and prey to oversimple Trumpist messages: blame the woke, migrants, transgender people, Muslims, doctors, scientists. Musk's purchase of Twitter/X supports Bannon's agenda.
What we are seeing is the coming together of two major forces of the Trump world: the fossil fuel-funded Heritage Foundation and the world of tech. The former authored Project 2025, a plan for Trump's first year in office. Curtis Yarvin, PayPal billionaire Peter Thiel's favoured theorist, has expressed the desire to “reboot” a whole country, substituting the outmoded democracy software with something far less accountable and more business-friendly. Together, both industries poured hundreds of millions into Trump's campaign.
So far, regulatory positions have been eliminated, cryptocurrency will be able to bypass democratic oversight, budgets have been cut to allow Musk to cash in with SpaceX and Starlink contracts, and fired government employees are to be replaced with AI products.
Many industries sense the winds of change and no longer even bother with greenwashing or climate commitments, as they drop all pretence of responsibility for a livable world. The climate and ecological implications of this shift are as disastrous as they are deliberate. We need an appropriate name for this new era of fossil and tech bros accelerating attacks on democracy and the planet: cataclysm capitalism.
Cataclysm capitalism is the worthy heir to neoliberalism and its disaster capitalism. As Naomi Klein described in her book The Shock Doctrine, neoliberal economic ideology took advantage of crises to deregulate and privatise public services, hobble trade unions and civil society, and generally create conditions ideal for private wealth accumulation and disastrous for equality, work and welfare. Cataclysm capitalism does all this, and goes further. The pace of change is accelerated, the dismantling of public institutions more complete, the attack on democracy more overt. Perhaps the most frightening aspect is that the industries laughing in the face of planetary and social destruction have made a clear calculus: they don't need prosperous economies to profit. Neoliberalism at least claimed to be serving a form of greater good via winner-takes-all market competition. Cataclysm capitalism dispenses with this illusion altogether.
The fossil fuel companies, the rightwing tech magnates and the financial companies hurrying in their wake have somehow convinced themselves that they don't need prosperous economies to prosper themselves. They have learned to profit from disruption and destruction. They know from experience that immiserated populations will endure exploitative working conditions and go deep into debt to keep themselves and their families alive.
Paradoxically, the creation of vast economic insecurity favours far-right politics. Voters in a constant state of fear and stress, without a clear understanding of the system creating hardships, are an easy prey for far-right rhetoric blaming migrants, woke and trans people. Sadly, since neoliberal ideology has devoured previously centre-left parties (UK Labour and US Democrats), we are left with much less of an organised opposition, and much more of a pipeline to accelerating disaster.
The picture is grim. We are faced with an organised hostile takeover of democracy, coupled with a dismantling of the economy in favour of the sectors and industries most beneficial to the fossil-fuel and tech magnates, to our detriment and the detriment of all life on Earth. What can we do? I propose a three-pronged plan, short and schematic – enough to get started.
First, understanding is power. We need to learn more about the devourers of our world, from the fossil fuel thinktanks to the far-right tech accelerationists. We need to explain to our fellow citizens who we are facing, and what their ultimate plan is. Replace helpless fear with knowledgable anger.
Second, we need to organise, come together, in trade unions, in neighbourhood groups, in any collectives we can form. At this point, we were all raised in neoliberal cultures of individualism and isolation. Organising sounds foreign and difficult. Indeed, our learned organisational helplessness is integral to the endeavour of disaster capitalism.
However, as an exceptionally cooperative species, in reality, we all have impressive innate capacity. Quite literally, organising is what we, as social animals, were born to do. At its most basic forms, organising consists of gathering people, raising awareness of the causes of our common problems, discussing possible avenues of action, putting them into operation. Rinse, repeat; make it part of your life's hobbies and work.
Third, we need to respond to the Trump-Musk project at the strategic level, not blow by blow. We know we can expect nothing but destruction and corruption from them: we have to put forward a positive vision, worth fighting for. From my research, I would describe it as scientifically informed democratic decision-making for the common good. This also means creating our own organisations for mutual aid and protection of vulnerable people. We have everything to lose if we don't act, and everything to gain if we do.
Julia Steinberger is professor of societal challenges of climate change at the University of Lausanne
State police anti-terrorism unit investigating whether blaze in Torre Angela was started by anarchists
Italy's interior ministry has written to police forces across the country to increase security at Tesla dealerships after 17 of the electric cars made by Elon Musk's company were destroyed in a fire in Rome.
Italy's state police anti-terrorism unit, Digos, is investigating whether the fire at the Tesla dealership in Torre Angela, a suburb in the east of the capital, was started by anarchists.
Firefighters worked for hours to put out the blaze in the early hours of Monday. Drone images showed a row of the burnt-out remains of the vehicles in a parking area of the dealership. Using his social media platform, X, Musk referred to it as “terrorism”.
There are 13 Tesla dealerships in Italy, all managed by the parent company, the majority of them in Rome, but also in other cities including Florence and Milan.
An interior ministry source said the circular was aimed at “raising awareness” of possible anti-Tesla protesters amid a global wave of vandalism in response to Musk's political activities in the US. If needed, surveillance of dealerships would be increased, it said.
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Since the inauguration of Donald Trump as US president in January, Musk has been slashing the federal workforce as chief of the administration's so-called “department of government efficiency”, prompting the emergence of “Tesla Takedown”, a boycott movement that started in the US before spreading to Europe.
Although most protests have so far been peaceful, Tesla dealerships and cars have increasingly been targets of vandalism. Seven vehicles were set alight at a dealership in Ottersberg, Germany, on Saturday, and two Tesla stores in Sweden, one in the capital, Stockholm, and the other in the coastal city of Malmö, were vandalised with orange paint on Monday.
Musk has nurtured close relations with European far-right party leaders including the Italian prime minister, Giorgia Meloni, who in an interview in early January described him as “a brilliant man”.
Matteo Salvini, who leads the far-right League, a member of Meloni's ruling coalition, expressed solidarity with Musk after the Rome incident.
“Too much unjustified hatred against the Tesla car company,” Salvini wrote on X. “The season of hate and conflict must come to an end as soon as possible. My solidarity goes out to Elon Musk and to all the workers who have been threatened and attacked.”
The ruling military junta in Myanmar say the death toll after last week's devastating earthquake now stands at almost 2,900 people.
The actual death toll is feared to be much higher, with many people still stuck under the rubble in affected cities.
The ruling military junta declared that it will not let foreign journalists into the country after the earthquake making it hard to report from the country.
The BBC's Yogita Limaye has become the first foreign journalist to enter the country since the disaster struck and travelled to the city of Mandalay.
You can read her full report here.
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The BBC's Stephen McDonell breaks down what to look out for as China's leaders meet this week.
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Manila says it will file a formal protest over the latest incident in the South China Sea.
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It comes as Sheikh Hasina, who was ousted in protests last year, announced she would address the country via social media from India.
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Louise Haigh calls for end to two-tier system over complaints of sexual misconduct or harassment
Bosses should be banned from the “improper” use of non-disclosure agreements (NDAs) for low-paid workers in the service, retail or hospitality sectors, a former cabinet minister has said, as she calls for an end to a two-tier system for victims.
Louise Haigh, the former transport secretary, has urged MPs to look beyond high-profile cases linked to the #MeToo movement and advocate for workers in insecure employment who may not have “the means and the confidence to pursue their employers through the courts” to be able to challenge the NDAs.
Haigh is due to tell her colleagues in a Westminster Hall debate on Wednesday: “If mental health charities are exploiting this practice to discriminate against people with mental health issues then we have to accept that it is a serious problem in every type of workplace in this country.”
Last month, the Labour MP tabled an amendment to the employment rights bill that would bring an end to the widespread misuse of NDAs. She invoked parliamentary privilege during the debate on the legislation, to reveal the use of NDAs at ITN to cover up cases of workplace harassment.
Haigh is campaigning for the government's policy on NDAs to be changed so it aligns with existing protections those employed in higher education institutions receive.
She noted the Higher Education Act 2023 prevented universities from entering into NDAs with staff, students or visiting speakers in regards to complaints of sexual misconduct or harassment.
Haigh was due to share chilling testimonies from low-paid workers who have been affected by the practice.
She would discuss: “The woman who was raped by a colleague at work but had signed a confidentiality clause that prevented her from discussing the issue even with medical professionals made it impossible to recover from her trauma.
“The employee who signed an NDA on leaving her workplace and has since been effectively blacklisted as her former employer is undermining her to prospective employers whilst she cannot tell her side of the story.
“Or the woman who told about the mental health charity she works for, which has discriminated on disability grounds against at least four people in the last year that she is personally aware of. Three of them have signed an NDA, she is bravely pursuing them through the courts because she believes it's the only way to get justice.”
Statistics from the Can't Buy My Silence campaign found 100% of NDAs or confidentiality clauses were written broadly. It means workers who may not think twice about signing their contracts may unwittingly overlook the broadly drafted confidentiality clauses that Haigh says “go far further than is require to protect commercial confidentiality or trade secrets”.
Haigh is due to say: “Let's not let this opportunity pass us by. Let this Labour government lead the way on protecting victims and survivors in the workplace and finally bring an end to legalised abuse.”
The co-founder of Can't Buy My Silence Zelda Perkins said: “The government has an opportunity to put Labour values into practice by legislating to protect workers' rights and remove barriers to work by taking action on abusive NDAs. The current two-tiered situation, where only workers in higher education are protected from abusive NDAs is untenable – all workers deserve protection from being silenced in the workplace.”
Senior doctor who saw bodies says men appeared to have been ‘executed', adding to evidence of potential war crime
Some of the bodies of 15 Palestinian paramedics and rescue workers, killed by Israeli forces and buried in a mass grave nine days ago in Gaza, were found with their hands or legs tied and had gunshot wounds to the head and chest, according to two witnesses.
The witness accounts add to an accumulating body of evidence pointing to a potentially serious war crime on 23 March, when Palestinian Red Crescent ambulance crews and civil defence rescue workers were sent to the scene of an airstrike in the early hours of the morning in the al-Hashashin district of Rafah, Gaza's southernmost city.
International humanitarian teams were only allowed access to the site this weekend. One body was recovered on Saturday. Fourteen more were found in a sandy grave at the site on Sunday and were brought back for autopsies in the nearby city of Khan Younis.
Dr Ahmed al-Farra, a senior doctor at the Nasser medical complex in Khan Younis, witnessed the arrival of some of the remains.
“I was able to see three bodies when they were transferred to the Nasser hospital. They had bullets in their chest and head. They were executed. They had their hands tied,'' Farra said. “They tied them so they were unable to move and then they killed them.”
He provided photographs he said he had taken of one of the dead on arrival at the hospital. The pictures show a hand at the end of a long-sleeved black shirt with a black cord knotted around the wrist.
Another person, an eyewitness who took part in the recovery of remains from Rafah on Sunday, also said they saw evidence of one of the dead having been shot after being detained.
“I saw the bodies with my own eyes when we found them in the mass grave,” the witness, who did not want his name used for his own safety, told the Guardian in a telephone interview. “They had signs of multiple shots in the chest. One of them had legs tied. One was shot in the head. They were executed.”
The accounts add to allegations made by a senior Palestinian Red Crescent official, the Palestinian Civil Defence and the Gaza health ministry that some of the victims had been shot after they were detained and put in restraints by Israeli troops.
The incident came after the Israeli government ended a two-month-old ceasefire and resumed its military campaign against Hamas and other militant groups in Gaza on 17 March, with heavy aerial bombing and ground operations. The people of Rafah were ordered to leave the town on Monday, before Israeli ground operations there.
The international criminal court issued arrest warrants for war crimes in November against the prime minister, Benjamin Netanyahu, and his former defence minister Yoav Gallant, and the ICC prosecutor has said he is still investigating Israeli forces and Hamas for suspected atrocities.
The victims are believed to have been killed on 23 March, two of them in the early hours when their ambulance came under Israeli fire while on the way to collect injured people from an earlier airstrike. The remaining 13 of the dead were in a convoy of ambulances and civil defence vehicles dispatched to retrieve the bodies of their two colleagues. One of the dead was a UN employee. A Red Crescent paramedic, named as Assad al-Nassasra, is still missing.
The UN said the ambulances and other vehicles were buried in sand by bulldozers alongside the bodies of the dead, in what appears to have been an attempt to cover up the killings. UN video footage taken by the recovery team showed a crushed UN vehicle, ambulances and a fire truck that had been flattened and buried in the sand by the Israeli military.
“This is a huge blow to us … These people were shot,” Jens Laerke, a spokesperson for the UN aid coordination office, said on Tuesday. “Normally we are not at a loss for words and we are spokespeople, but sometimes we have difficulty finding them. This is one of those cases.”
Israel's military has said its “initial assessment” of the incident had found that its troops had opened fire on several vehicles “advancing suspiciously toward IDF troops without headlights or emergency signals”, and has claimed, so far without evidence, that Hamas fighters and other militants had been using the ambulances for cover.
The Israel Defense Forces have so far not responded to questions, first posed on Monday, about the reports that the bodies and their vehicles had been buried or to the allegations that some had been shot after being detained.
Dr Bashar Murad, the Red Crescent's director of health programmes in Gaza, said at least one of the recovered bodies of the paramedics had had his hands tied, and that one of the paramedics had been on a call to the ambulance dispatcher when the attack took place.
On that call, Murad said, gunshots fired at close range could be heard as well as the voices of Israeli soldiers on the scene speaking in Hebrew, ordering the detention of at least some of the paramedics.
“The gunshots were fired from a close distance. They could be heard on the call between signal officer and of the medical crews that survived and phoned the ambulance centre for help. The soldiers' voices were clearly audible in Hebrew and very close, as well as the sound of the gunfire.”
“Gather them at the wall and bring some restraints to tie them,” was one of the lines that Murad said could be heard by the dispatcher. He said the call had not been recorded.
Mahmoud Basal, a spokesperson for the Palestinian Civil Defence in Gaza, said the bodies had been found with at least about 20 gunshots in each of them and confirmed that “at least one of them had their legs bound”.
In a statement released on Monday, Gaza's health ministry said: “They were executed, some of them handcuffed and had sustained head and chest injuries. They were buried in a deep hole to prevent their identities from being identified.”
On Monday the IDF issued evacuation orders covering most of Rafah, indicating it could soon launch another major ground operation, eight days after the paramedics and rescue workers were killed.
According to the Red Crescent, an ambulance was dispatched to pick up the casualties from the airstrike in the early hours of 23 March and called for a support ambulance. The first ambulance arrived at hospital safely but contact was lost with the support ambulance at 3.30am. An initial report from the scene said it had been shot at and the two paramedics inside killed.
The Palestinian Red Crescent president, Dr Younis al-Khatib, said the IDF had impeded the collection of the bodies for several days. The IDF said it had facilitated the evacuation of bodies as soon as “operational circumstances” allowed.
“The bodies were recovered with difficulty as they were buried in the sand, with some showing signs of decomposition,” the Red Crescent said.
Their burial had been put off pending autopsies at the Nasser hospital in Khan Younis. The autopsies have now been carried out, according to hospital sources, and a full report is due to be delivered to the Gaza health ministry within 10 days.
The Real Madrid coach, Carlo Ancelotti, who is on trial for allegedly defrauding Spain's tax office of more than €1m in undeclared earnings from image rights, has told a court he believed his financial affairs were in order and “never thought a fraud could have been committed”.
Prosecutors allege the 65-year-old former Chelsea and Everton manager used shell companies outside Spain to conceal “the real beneficiary of the income from the exploitation of his image rights” in 2014 and 2015. They are seeking a jail term of four years and nine months and a fine of €3.2m.
Giving evidence on Wednesday, the first morning of the two-day trial in Madrid, Ancelotti said he had focused on his salary and had left other financial matters in the hands of his advisers. “I've never been bothered about image rights,” he told the court. “Coaches aren't that important – it's the players who sell shirts. I was only bothered about getting the €6m net for three years and I never realised that something wasn't right and I didn't receive any notification that I was under investigation by tax prosecutors.”
The manager said that when Real Madrid had proposed a payment scheme under which 15% of his salary would be paid from his image rights, he had said yes and had referred the matter to his British adviser. “I thought it was quite normal,” he said, adding that all the players and the previous coach had done the same thing.
“I got in touch with my British adviser and I didn't think any more about it because it all seemed right,” he said. “I never thought a fraud could have been committed. But, given that I'm here, I guess things weren't done that right.”
Prosecutors allege that, despite his status as a Spanish resident for tax purposes, Ancelotti declared only his personal earnings from the club and omitted the income he received from the sale of his image rights. The prosecutors allege that he failed to pay a total of €1,062,079 in tax on the sale of those rights, which amounted to €1.24m in 2014 and €2.96m the following year.
Ancelotti, the most successful manager in Champions League history, denies the charges. “I have total confidence in the law and in justice,” he said on Friday. “I'm not worried but I am obviously annoyed if they say that I've committed fraud, but, once again, I have total confidence in justice.”
The Italian is the latest high-profile football figure to find himself pursued by Spain's tax authorities in recent years. In July 2016, Lionel Messi and his father, Jorge, were sentenced to 21 months in prison for evading tax on Lionel's image rights during his time at Barcelona, with more than €4m owed in back payments. Both were spared jail time because sentences of under two years in Spain do not have to be served, and neither man had a criminal record.
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Six years ago, Cristiano Ronaldo admitted committing tax fraud while playing for Real Madrid and agreed to pay an €18.8m fine after striking a deal with prosecutors and tax authorities in return for a 23-month suspended prison sentence. In February 2019, José Mourinho was given a one-year suspended prison sentence and agreed to pay €2.2m in fines after admitting tax fraud while he was the manager of Real Madrid.
The following year, the Atlético Madrid forward Diego Costa paid a fine of €543,208 after pleading guilty to defrauding the tax authorities of more than €1m by not declaring payments of more than €5.15m from his 2014 move to Chelsea.
Plan to quit Aim after ‘review of benefits and drawbacks' is further blow to capital's junior market
The leisure group that owns the 126-year-old Brighton Palace Pier is planning to delist from the London stock market and return to life as a private company, in the latest blow to the capital's junior market.
Brighton Pier Group, which also owns several bars and mini-golf sites around the country, told investors it intended to cancel its listing on the capital's Alternative Investment Market (Aim) after more than 11 years, blaming bad weather, falling consumer spending, rising wage costs and higher interest rates.
The news led to shares in the company tumbling by around 60% on Wednesday.
The group, which is chaired by the business veteran and former Pizza Express and Patisserie Valerie boss Luke Johnson, said it had faced “persistent challenging trading conditions” since the coronavirus pandemic, forcing it to cut costs and sell off underperforming assets.
In a statement to the market, the group said it had carried out “a careful review of the benefits and drawbacks” to the company and its investors of remaining listed on Aim, adding that it believed it was in the “best interests” of the business to return to private hands.
Shareholders will be given the chance to vote on the plans at a meeting on 22 April, where approval of at least 75% of investors is required. If they vote in favour, Brighton Pier Group will delist from the London Stock Exchange on 2 May.
Johnson owned 27% of the company's shares, according to its most recent annual report, for the year to 24 December 2023, and was paid a salary of £20,000 for the role.
The delisting would be another setback for the London stock market, which has been hit by a string of departures in recent months.
According to the latest figures from the accountancy group UHY Hacker Young, 71 companies delisted from Aim in the year to 12 February, leaving fewer than 700 companies listed on the market, the lowest level since 2001.
The model train maker Hornby said in March it would cancel its listing and go private, saying it would help to cut costs and reduce its regulatory burden.
Brighton Pier Group, which also owns Lightwater Valley Family Adventure Park in Yorkshire, said it was looking to delist partly because of the “disproportionate” annual costs of between £250,000 and £300,000 connected with remaining listed on the stock market, and said it could use the money to invest directly into growing the business.
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The group also blamed lack of liquidity on Aim, which it said meant shareholders could not trade in “meaningful” volumes, and which also resulted in market volatility.
It came as Brighton Pier Group said its results for the year to 29 December 2024 were in line with market expectations.
However, it said the cost of living crisis was having a “material impact” on consumer spending, as people cut back on non-essential purchases. Sales fell slightly over the past 12 weeks compared with a year earlier, although Brighton Pier reported an increase in visitor numbers thanks to warm weather in March.
Comments come after it was revealed judge ignored DoJ recommendations against expelling survivors from panel
Clergy abuse survivors who were ousted from a committee trying to negotiate a settlement resolving the New Orleans Catholic archdiocese's bankruptcy say they have lost their confidence in the judge presiding over the case.
Their comments come after it was revealed that Judge Meredith Grabill ignored federal justice department recommendations against expelling the survivors from the group, delivering the latest twist in a child sexual abuse scandal roiling one of the oldest Roman Catholic dioceses in the US.
“I can honestly say that my faith in … Grabill is greatly diminished,” James Adams said about decisions that at first were largely made in secret against him as well as three of his fellow survivor committee members. “It's shattered.”
Grabill removed Adams, Jackie Berthelot, Theo Jackson and Eric Johnson from the group in question after tasking a justice department office which assists with administering bankruptcy cases to investigate an attorney for the survivors. The lawyer, Richard Trahant, drew Grabill's attention after taking steps to tip off a local high school that its chaplain, Paul Hart, was an admitted molester in early 2022.
Investigators concluded that they believed Trahant had violated the terms of an applicable confidentiality order in what he maintained was an attempt to protect children – but their report to Grabill said the clients should not be punished.
Grabill nonetheless ruled that she was “forced” to kick his four clients off the survivors committee; imposed a $400,000 fine on Trahant; and then ordered the findings of the investigation by the US trustee's office sealed from public view.
The Guardian joined news partner WWL Louisiana in recently obtaining the US trustee's investigative report through a successful public records request seeking files pertaining to the criminal prosecution of serial child molester Lawrence Hecker, who pleaded guilty to child rape and kidnapping charges in December and died in prison a few weeks later.
An article published on Monday by the outlets marked the first time that many learned of the findings of the US trustee report that preceded Grabill's punishments against Trahant and his clients, widely seen as one of the most consequential decisions issued during the New Orleans archdiocese's unusually tortuous chapter 11 reorganization.
Not even Adams, Berthelot, Jackson and Johnson had seen the document, having been barred from accessing it even as they unsuccessfully appealed their removals.
Asked to comment on Grabill's decision to remove them in light of the exposed US trustee investigation, Jackson said: “We were just blindsided … with something that we [had] nothing to do with.”
Johnson added: “It hurts because you're trying to do good for the other survivors. Yet [they're] blaming you for everything.”
Grabill also ignored a US trustee recommendation to hold a court hearing before taking any action after the completion of the Hart investigation. The judge ordered the committee removals one business day after receiving the US trustee report. She has not commented on the Guardian and WWL's recent reporting on Hart.
Trahant's four clients all had Catholic upbringings in the New Orleans area and reported enduring child sexual molestation at the hands of clergymen or lay religious personnel amid the worldwide church's decades-old clerical abuse scandal.
In May 2020, the New Orleans archdiocese filed for bankruptcy protection in an attempt to limit its liability from such abuse claims. It is one of about 40 US Catholic institutions to do so.
The cases reported by Adams and the others became ensnared in the bankruptcy, with Trahant representing them. And they all successfully applied to serve on a committee of abuse survivor claimants whose job it was to aid in settlement negotiations.
In his role as the committee members' attorney, Trahant learned that Hart had been allowed to serve as chaplain of New Orleans' Brother Martin high school despite previously admitting to having sexual contact in the early 1990s with a 17-year-old girl whom he met at a prior assignment.
In 2002, US bishops adopted a policy making 18 the age of consent under canon – or church – law. Before that, 16 was the age of consent. So New Orleans Archbishop Gregory Aymond declined to substantially punish Hart and assigned him to work at Brother Martin beginning in 2017.
Hart was forced to retire after Brother Martin learned of his past, the bulk of which was meant to be kept secret because of confidentiality rules in the archdiocesan bankruptcy.
An investigation into how Brother Martin learned the truth about Hart concluded that Trahant violated the bankruptcy's confidentiality rules simply by making reference to the name and position of the chaplain while communicating with the school's principal. Brother Martin's principal, Ryan Gallagher, was Trahant's cousin and knew Trahant represented clergy abuse survivors. Trahant later testified he knew his cousin would infer Hart was likely an abuser from the mere mention of his name.
Furthermore, the investigation asserted that Trahant violated the secrecy order by telling a journalist who later reported on the circumstances surrounding Hart's forced retirement to keep the clergyman on his “radar”.
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But the investigation found it was actually the church itself that informed Brother Martin about the particulars of Hart's abusive past. And it essentially ruled out Trahant as having been the source of the information that the journalist reported about Hart, who has since died.
Trahant asserted to investigators and Grabill that he was careful only to share information about Hart that was publicly available, such as his name and job. That's because the bankruptcy confidentiality order allowed him to do at least that, he argued.
Despite his assertions, Grabill decided Trahant had violated the confidentiality order – and that he and his clients deserved to be sanctioned.
Trahant and his clients appealed. The appeal pursued by Trahant, who had access to the US trustee report but could not legally discuss it with his clients, remained pending as of Tuesday in the federal fifth circuit court. The clients' appeal failed after Grabill's seal on the report impeded them from viewing it.
While the US trustee report opined that only Trahant merited punishment, Grabill said she was “forced” to remove his clients from the survivors committee to ensure the smooth functioning of that entity. Adams, who had been the group's chairperson, pointed out that the US trustee never publicly indicated that the committee removals were against its recommendation.
The expulsions were unnecessary, Adams added. He said virtually every one of the committee's votes during his two years of participation was unanimous.
He said he wondered whether he and his colleagues were ousted – on the morning that they were scheduled to address Aymond as part of settlement negotiations – because they had expressed a commitment to “genuinely investigating how the archdiocese ended up in bankruptcy”.
Even with their removals, Adams and his former colleagues noted how the bankruptcy was unresolved as of Tuesday, nearly three years after their June 2022 removals.
Berthelot, a survivor of Hecker as well as of another abusive clergyman, said he “absolutely” believes Grabill “needs to step down” from the case. He described experiencing health problems and losing friendships amid the bankruptcy's various twists. By Monday, it had all worn him down so much that he resigned his elected office from the city council in the New Orleans suburb of Gretna, which he first joined in 2013.
“Judge Grabill has been compromised,” said Berthelot, whose council term was supposed to last until June. “To me, as a victim, a survivor of clergy abuse, it was extremely shocking.”
Adams said part of him wanted to see Grabill step down from the case after the exposure of the sealed Hart report. But he worried about how much longer something like that might delay the bankruptcy.
Figures associated with Catholic institution bankruptcies settled elsewhere relatively recently suggest the New Orleans archdiocese's reorganization could cost the church and its insurers more than $300m to settle.
As it continued efforts to finance a settlement with survivors, the archdiocese earlier in March agreed to sell a suburban orphanage with a lengthy history of child abuse to local government officials for $3.8m.
Johnson survived abuse at a sister orphanage across the street from the one being sold. He said he hoped officials followed through on stated goals of redeveloping the property into a multi-use facility and building a memorial to all those who survived abuse on those grounds.
“Use it for something positive,” Johnson said. “Use it for the good of the community.”
Latest security flap again focuses scrutiny on Waltz after he earlier added journalist to Yemen war-planning chat
Michael Waltz, the embattled national security adviser to Donald Trump, and other members of the national security council have reportedly used personal Gmail accounts to conduct government business.
The apparent use of Gmail, a relatively insecure method of communication for high-level government officials, places further scrutiny upon Waltz, who is already under pressure after adding a journalist to a group chat on the commercial Signal app, where top US officials then planned and celebrated a US airstrike in Yemen last month.
The revelations of the Gmail use come from the Washington Post, which said it has reviewed documents and interviewed three unnamed officials about the apparent security lapse.
Waltz had “potentially exploitable information” sent to his Gmail, such as his schedule and other work documents, the Post reported.
One of Waltz's aides on the national security council, meanwhile, allegedly used Gmail for more sensitive material, such as discussing military positions and weapons systems with colleagues in other government agencies who used their government-issued accounts.
A spokesperson for the national security council denied to the Post that Waltz had used Gmail improperly. “Waltz didn't and wouldn't send classified information on an open account,” the spokesperson said.
This latest episode, however, risks further damage to the standing of Waltz following the revelation last week that he added Jeffrey Goldberg, editor-in-chief of the Atlantic magazine, to a Signal group chat that involved senior figures such as JD Vance, the US vice-president, and Pete Hegseth, the defense secretary, that discussed details of an airstrike upon the Houthi rebel group in Yemen.
Waltz has said that Goldberg's number was somehow “sucked in” to his phone, a comment that drew scorn from Goldberg and others. Trump has, however, declined to fire Waltz, instead calling the incident a “glitch”.
Prior to his role as the president's national security adviser, Waltz was highly critical of Hillary Clinton for her use of a private email server when she was secretary of state.
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Democrats have been pushing for Waltz and Hegseth to resign amid the scandal. “Mike Waltz is totally and completely unqualified to be in a sensitive national security position, as is the case with the Trump national security team,” Hakeem Jeffries, the Democratic leader in the House of Representatives, told Axios.
Jeffries said that Hegseth and Waltz should be fired if they did not resign immediately.
Nigeria's president has appointed Bayo Ojulari - a former Shell executive - to lead the state-owned oil company, as part of sweeping reforms aimed at cleaning up the sector dogged by allegations of corruption, pollution and decades-long inefficiency.
Mr Ojulari was picked in a "crucial" overhaul of the Nigerian National Petroleum Company (NNPC), the presidency said on Wednesday.
It added that the restructure - which also involved replacing the entire board - was necessary to drive economic growth in Africa's biggest oil exporter.
President Bola Tinubu's time in power has seen a series of economic shocks, with food and fuel prices rocketing over the past couple of years.
The NNPC has also been under financial strain - last September it admitted to having debts of around $6bn (£4.5bn).
In its statement announcing the NNPC restructure, the presidency said Tinubu wished to boost Nigeria's oil output and refining capacity.
Despite its massive oil sector, most of the crude oil Nigeria produces is exported, while it has to import the fuel it uses from other countries because it does not have enough oil refineries.
The cost of fuel used to be subsidised by the government - until this was removed by President Tinubu when he came to office nearly two years ago.
He said the country could no longer afford to pay for it, leading the price of fuel to skyrocket.
The subsidy was overseen by the NNPC and was shrouded in mystery leading to allegations of massive corruption.
However, oil marketers say the price at which fuel is imported is still higher than the pump price, leading many to believe that the government is still subsiding the cost of fuel through the NNPC, which it has denied.
Nigeria's oil production slowed to less than a million barrels per day in 2023, news agency AFP reported.
Tinubu's administration wants to hit two million barrels per day of oil by 2027 and three million barrels per day by 2030.
Tasked with executing this mission, Mr Ojulari replaces former NNPC boss Mele Kyari.
Some oil industry experts have expressed confidence in his appointment owing to his decades of experience in the oil sector, however they fear the challenges he inherits.
"The commission is indebted. Even the NNPC Ltd does not know how much it owes," energy expert Henry Adigun told the BBC.
"Its accounting has been marred by corruption and inefficiencies. I think it's bloated. It's a case of the more you read, the less you understand."
Mr Ojulari joined Shell Nigeria 1991 and rose to become managing director, a position he held for six years. He left the company in 2021 to join the investment advisory organisation BAT Advisory and Energy Company.
He then moved to Renaissance Africa Energy Company last year.
Along with trying to boost Nigeria's oil production, Mr Olujari will no doubt also seek to improve the NNPC's poor public image.
For many years, under previous governments, much of the company's profits never reached the treasury.
It is only in the last five years that the NNPC has been publishing accounts.
The new board appointed by President Tinubu comprises oil industry experts with decades of experience.
The hope is the team will be free of politics and restore transparency in the country's murky oil sector.
"If he doesn't succeed, it would be because of government interference," Mr Adigun added.
Go to BBCAfrica.com for more news from the African continent.
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President Mohamed Bazoum has been under house arrest since the 2023 military takeover.
The Americans are among 37 people sentenced to death last September by a military court.
Our correspondent enters Khartoum just days after Sudan's army recaptured it from the Rapid Support Forces after a six-month offensive.
A total of 95 people are charged with promoting "public violence" and for "breaches of peace".
Hakainde Hichilema says some are dozing off because of "indulgence" and a lack of self-control.
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This live blog is now closed
French President Emmanuel Macron has commented for the first time since Marine Le Pen was found guilty of embezzlement and barred from running in France's next presidential election.
Opening a weekly meeting of French ministers, Macron addressed the court's decision by recalling the three things government spokesperson Sophie Primas said.
They were “that the judiciary is independent”, “that the threats made against judges are absolutely unbearable and intolerable”, and that “the law is the same for everyone”, Politico reports.
Macron also stated that “all defendants have the right to legal recourse,” Primas said.
We are now closing the blog. Here is a round-up of today's events.
Europe is braced for US president Donald Trump to impose sweeping tariffs on global trading partners on Wednesday, threatening cost increases and likely drawing retaliation from all sides. Details of Trump's tariff plans are due to be announced in the White House Rose Garden at 4 pm Eastern Time (10pm CET, 9pm BST).
Donald Trump's planned tariffs will be negative across the world, with the damage depending on how far they go, how long they last and whether they lead to successful negotiations, European Central Bank head Christine Lagarde said on Wednesday.
The effect of US tariffs on Italian companies will be massive – with fashion, pharmaceuticals and the food industry the hardest hit, the head of national industry lobby Confindustria said on Wednesday.
Benedicte de Perthuis, the judge who barred far-right leader Marine Le Pen from running in France's 2027 presidential election is under police protection after facing death threats, a source with direct knowledge of the matter told Reuters.
French President Emmanuel Macron said “that the threats made against judges are absolutely unbearable and intolerable” and that “the law is the same for everyone”, Politico reports. Macron also stated that “all defendants have the right to legal recourse.”
European visitors to the UK will need a new online entry permit from Wednesday as the British government shakes up longstanding travel rules. Travellers from Europe will now need a digital Electronic Travel Authorisation (ETA) permit.
Russian attacks damaged energy facilities in Ukraine's Sumy and Dnipropetrovsk regions, President Zelenskyy said adding that Russia was intentionally attacking Ukrainian energy infrastructure and called on allies to mount pressure on Moscow to halt its invasion.
Russia's Defence Ministry said on Wednesday that Ukraine had attacked Russian energy facilities twice during the past 24 hours. The Guardian could not verify the reported strikes.
The Kremlin said on Wednesday that a visit to the US by President Vladimir Putin's envoy, Kirill Dmitriev, was “possible”, and that contacts with the US were continuing.
Israeli prime minister Benjamin Netanyahu was due to visit Hungary on Wednesday defying an arrest warrant from the International Criminal Court (ICC) issued over allegations of war crimes in Gaza.
Denmark's prime minister Mette Frederiksen began her three-day visit to Greenland on Wednesday for talks with the territory's incoming government, following US President Donald Trump's repeated expressions of interest in controlling the Arctic island, Reuters reports.
President Emmanuel Macron convened key ministers and experts on Wednesday to discuss Iran, including its nuclear programme, amid growing tensions between Tehran and US President Donald Trump, three diplomatic sources told Reuters.
Italy's interior ministry has written to police forces across the country to increase security at Tesla dealerships after 17 of the electric cars made by Elon Musk's company were destroyed in a fire in Rome.
Nato allies have pledged $21.65bn (more than 20bn euros) in military support for Ukraine in the first three months of the year, Nato Secretary General Mark Rutte said on Wednesday.
Norway will not withdraw from the global convention banning anti-personnel landmines as all the other European countries bordering Russia have done, the country's foreign minister said on Wednesday.
Norway will not withdraw from the global convention banning anti-personnel landmines as all the other European countries bordering Russia have done, the country's foreign minister said on Wednesday.
Finland on Tuesday said it planned to quit the 1997 Ottawa Convention as a way to mitigate the military threat posed by Russia, following Poland and the Baltic countries, which announced similar moves last month.
Norway's foreign minister Espen Barth Eide told Reuters: “This particular decision [by Finland] is something that we regret.
“If we start weakening our commitment, it makes it easier for warring factions around the world to use these weapons again, because it reduces the stigma.”
He added: “We have a very modern advanced defence system. We have purchased extremely advanced systems that can attack from land, the air and sea.”
Nato allies have pledged $21.65bn (more than 20bn euros) in military support for Ukraine in the first three months of the year, Nato Secretary General Mark Rutte said on Wednesday.
Foreign ministers from the alliance meet in Brussels on Thursday and Friday to discuss further support for Ukraine against Russia's three-year-old invasion.
French President Emmanuel Macron has commented for the first time since Marine Le Pen was found guilty of embezzlement and barred from running in France's next presidential election.
Opening a weekly meeting of French ministers, Macron addressed the court's decision by recalling the three things government spokesperson Sophie Primas said.
They were “that the judiciary is independent”, “that the threats made against judges are absolutely unbearable and intolerable”, and that “the law is the same for everyone”, Politico reports.
Macron also stated that “all defendants have the right to legal recourse,” Primas said.
Hungary has deployed soldiers and launched new disinfection measures to help contain an outbreak of foot-and-mouth disease in a northwestern area bordering Slovakia and Austria, the agriculture minister said on Wednesday.
Hungary reported its first case in more than 50 years of the disease, which often leads to trade restrictions, on a cattle farm early last month, Reuters reports.
Since then, neighbouring Slovakia has detected outbreaks at five locations after reporting its first cases in March. It has also stepped up measures to contain the disease's spread.
Hungarian agriculture minister Istvan Nagy said foot-and-mouth disease was found at two additional farms in Gyor-Moson-Sopron county by Wednesday morning, affecting 3,500 cattle.
Italy's interior ministry has written to police forces across the country to increase security at Tesla dealerships after 17 of the electric cars made by Elon Musk's company were destroyed in a fire in Rome.
Italy's state police anti-terrorism unit, Digos, is investigating whether the fire at the Tesla dealership in Torre Angela, a suburb in the east of the capital, was started by anarchists.
Firefighters worked for hours to put out the blaze in the early hours of Monday. Drone images showed a row of the burnt-out remains of the vehicles in a parking area of the dealership. Using his social media platform, X, Musk referred to it as “terrorism”.
There are 13 Tesla dealerships in Italy, all managed by the parent company, the majority of them in Rome, but also in other cities including Florence and Milan.
An interior ministry source said the circular was aimed at “raising awareness” of possible anti-Tesla protesters amid a global wave of vandalism in response to Musk's political activities in the US. If needed, surveillance of dealerships would be increased, it said.
French president Emmanuel Macron was convening key ministers and experts on Wednesday to discuss Iran, including its nuclear programme, amid growing tensions between Tehran and US President Donald Trump, three diplomatic sources told Reuters.
Such a cabinet meeting dedicated to a specific subject is rare and highlights mounting concern among Washington's European allies that the United States and Israel could launch air strikes on Iran's nuclear facilities unless there is a quick negotiated deal on its nuclear programme.
US defence secretary Pete Hegseth has reinforced US military capability in the Middle East with more warplanes, the Pentagon said on Tuesday, amid a US bombing campaign against the Houthis who control much of Yemen and are supported by Iran.
A senior European official said European strategists were asking themselves whether the campaign could be a precursor to a US strike on Iran in the coming months.
Three Spanish citizens and one Australian have been detained attempting to reach the Russian-operated Baikonur cosmodrome in Kazakhstan, Russian state news agency RIA reported on Wednesday, citing a source.
The outsized role of the United States in the global economy means that economic policy moves there can influence economic decisions in Europe, European Central Bank board member Isabel Schnabel said on Wednesday in Paris.
The judge who barred far-right leader Marine Le Pen from running in France's 2027 presidential election is under police protection after facing death threats, a source with direct knowledge of the matter told Reuters.
Benedicte de Perthuis - the head of a three-judge panel that found Le Pen guilty of embezzling EU funds and handed her a five-year ban on seeking public office - is now receiving police protection at work and at home.
De Perthuis also received threats on social media, with her photo plastered all over X and far-right sites.
Paris police confirmed an investigation was underway into the threats, referring further queries to the Paris prosecutor's office, which did not respond.
Denmark's prime minister is visiting Greenland on Wednesday for talks with the territory's incoming government, following US President Donald Trump's repeated expressions of interest in controlling the Arctic island, Reuters reports.
Mette Frederiksen begins her three-day trip less than a week after a visit to the territory by US vice-president JD Vance received a frosty reception from authorities in Denmark and Greenland.
Greenland's incoming prime minister Jens-Frederik Nielsen, who won last month's general election and will form a coalition government, has said he welcomes Frederiksen‘s trip, stating on Monday that Denmark remains “Greenland's closest partner”.
Nielsen told Reuters on Monday that Greenland would strengthen its ties with Denmark until it could fulfil its ultimate wish to become a sovereign nation.
Meanwhile, Greenland wishes to establish a respectful relationship with the United States, he said.
“Talking about annexation and talking about acquiring Greenland and not respecting the sovereignty is not respectful, he said. “So let's start by being respectful to each other and build up a great partnership on everything.”
The introduction of new US tariffs would hit Italian producers hard, Italian prime minister Giorgia Meloni said in Rome on Wednesday, adding that every effort should be made to avoid a trade war, Reuters reports.
Israeli prime minister Benjamin Netanyahu is due to visit Hungary today, defying an arrest warrant from the International Criminal Court (ICC) issued over allegations of war crimes in Gaza.
During the visit, which runs until Sunday, Netanyahu will meet his Hungarian counterpart Viktor Orbán, who invited him in November, soon after the ICC issued the arrest warrant.
Orbán said at the time that the warrant would “not be observed”.
All European Union member states, including Hungary, are members of the ICC, which means they are required to enforce its warrants.
Orbán, a right-wing nationalist, has often been at odds with the EU over democratic standards and human rights in Hungary.
The Times of Israel reported a source as saying Netanyahu and Orbán will discuss potential Hungarian support for US President Donald Trump's Gaza plan.
The Kremlin said on Wednesday that a visit to the US by President Vladimir Putin's envoy, Kirill Dmitriev, was “possible”, and that contacts with the US were continuing.
Reuters reported that Dmitriev is expected in Washington this week for talks with US President Donald Trump's administration.
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The Supreme Court sided with a former commercial truck driver Wednesday who was fired after failing a drug test he said was caused by a “CBD-rich medicine” in a decision that could make it easier to sue companies under organized crime laws.
Douglas Horn sued the manufacturers of a CBD product called “Dixie X” that the companies proclaimed included no THC, the psychoactive ingredient in marijuana. Horn took the product to manage debilitating pain after a serious accident, but he later failed a drug test and was fired. The Supreme Court's decision will allow Horn's case to move forward in lower courts.
Justice Amy Coney Barrett wrote the opinion for a 5-4 majority that included both liberal and conservative justices.
“When all is said and done, Medical Marijuana is left fighting the most natural interpretation of the text – that ‘injured' means ‘harmed' – with no plausible alternative in hand,” Barrett wrote. “That is a battle it cannot win.”
Horn sued under the Racketeer Influenced and Corrupt Organizations, or RICO, Act, a 1970 law targeted at organized crime that also authorizes civil lawsuits – and allows plaintiffs to collect triple damages – for harm to their business or property. The question for the Supreme Court was whether Horn's loss of employment qualified as a business harm.
Medical Marijuana, Inc., and other companies involved in distributing Dixie X, argued that Horn's injury was a personal one – and therefore not a business or property harm as required in the law. Congress, the companies argued, didn't intend for Americans to be able to collect triple damages under the RICO law for run-of-the-mill injury claims.
Horn sued in the Western District of New York in 2015, alleging in part that Medical Marijuana Inc. engaged in mail and wire fraud. The district court ruled against Horn, but the New York-based 2nd US Circuit Court of Appeals allowed his suit to move forward.
The companies appealed to the Supreme Court.
Allowing Horn to sue, the companies said, would vastly expand the number and type of “civil RICO” lawsuits permeating through federal courts. Concern over a flood of new lawsuits appeared to resonate with several of the court's conservatives during the oral arguments in October.
But other justices suggested during arguments that a loss of employment would fall under what most Americans view as an ordinary definition of “business.”
“If you're harmed when you lose a job, then you've been injured in your business, haven't you?” Justice Elena Kagan, a member of the court's liberal wing, asked the lawyer for the companies.
The law, Kagan said, “just says if you've been injured by a RICO violation in your business, which includes your employment, then you're entitled to threefold damages.”
President Richard Nixon signed the federal RICO Act in 1970 to give prosecutors more power to go after the heads of organized crime families. Several states have enacted their own versions of the law. In Georgia, prosecutors were attempting to use a state RICO law to prosecute Donald Trump for his efforts to overturn the 2020 election results there.
This story has been updated with additional details.
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Jon Hamm excels in Apple TV+'s "emotionally real but absurdist" drama about a desperate corporate man who resorts to stealing from his neighbours.
Jon Hamm has his best role since Mad Men and brings all his sharp comic timing and dramatic nuance to it as Andrew Cooper, known as Coop, a hedge-fund manager who loses his job and manoeuvres to maintain his elaborate lifestyle and sense of himself. If you took Don Draper and dropped him into 2025 he might be Coop, a corporate man with a broken marriage and two teenaged children, a decent guy and a charmer with all-around bad judgement. Your Friends and Neighbors is emotionally real and affecting but also absurdist and funny, a rare combination.
The show begins with Coop waking up in a pool of blood next to a man's dead body in the foyer of a neighbour's mansion. He cleans up the blood – a bad move – and in one of the many mordant voiceovers that run through the series, looks back four months to reflect on "the swirling hot mess of my life". His worst choice is replacing his lost income by stealing loot from friends so rich they'll never miss it. The robberies are the comic vein in the story, but also a Trojan horse for its drama. Coop is hurt and distraught over the loss of his marriage, but that theme exists next to the caper-like thefts. The series is edgier and smarter than its entertaining surface suggests, as it deftly takes on issues of family, class privilege, vapid materialism and toxic masculinity.
Like most series, this could start a bit faster, as it sets up the life Coop is accustomed to in a suburban New York community of huge houses and country clubs, where $200,000 cars and $60,000 donations to charity are ordinary. That was his life until he found his wife Mel (Amanda Peet) in bed with his so-called friend, Nick (Mark Tallman), a former basketball star still in great shape. The divorce has sent Coop out of his expansive home into a smaller rental house nearby.
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But soon enough the first episode gets to its boldest turn as we see why Coop lost his job. A woman who works at his firm, whom he has never met and who doesn't report to him, comes on to him in a bar. Months later, that consensual hookup leads to his being fired for violating the company's HR rules. Pop culture has at times gingerly dipped a toe into the theme of sexual harassment. On The Morning Show, Steve Carell's character, a toxic predator, was sent off a cliff to die. But there has been nothing as complicated as this. The show doesn't deny Coop's bad judgement but also makes it clear through the woman herself and Coop's ruthless boss that the HR rule was a convenient excuse to get rid of him while keeping his clients. The show doesn't go far beyond that in exploring the issue, but it sets up the series' complex realism about social standards.
A non-compete clause makes Coop unhireable, but he insists on keeping up appearances. His burglaries are treated like adventures, punctuated by mock ads as he describes the luxury items he's lifting, with voiceovers that slyly echo Hamm's in all those Mercedes-Benz commercials. "The Patek Phillippe Nautilus: sealed 18-carat white gold..." Coop says, talking about an item no one really needs.
The series' real strength, though, is in his relationships, most of them driven by his wounded pride, and the way his identity has been tied to his role as a successful man of the world. Hamm smoothly navigates the shifts in tone, never allowing the humour to flag while letting us see the emotional pain Coop won't reveal to anyone around him. There is a lot of residual affection between Coop and his cheating ex-wife, whom Peet manages to make sympathetic. When Mel worries and asks him what's wrong, he displays a classic alpha-male inability to share feelings. "I'm fine," he tells her when clearly he is not. Olivia Munn has less to do as Sam, a divorced neighbour and Coop's occasional fling. And Lena Hall is brilliant as Coop's sister, Ali. Briefly off her meds, she stalks her ex-fiancé, sitting on his lawn with her guitar and singing. Hall, who won a Tony for her role in the Broadway revival of Hedwig and the Angry Inch in 2014, sings occasionally through the series – an asset, not an intrusion. She makes Ali empathetic, funny and warm, and the bond between the brother and sister is touching. After she is back on her meds, her romantic subplot suggests that theirs really is a family full of bad choices.
A second season of the show has already been ordered, so the question of whether Coop will pay for his crimes will linger. That plot is an effective hook, but his resonant feelings and increasing self-knowledge set the show apart. At a party of male friends hosted by Nick – why is Coop even there? more bad choices – he looks around at the men who still have their jobs and money, and sees the person he might have become. "Their future was already written, and so the quest to stave off the emptiness began," he says in voiceover, observing their devotion to "Scotch and cigars", to "custom golf clubs and high-end escorts". He recognises that there are "entire industries built to cash in on the quiet desperation of rich middle-aged men". By even acknowledging that desperation, through a hero who is sympathetic – however wrong-headed his path to self-awareness – Your Friends and Neighbors is radically different from any other show around.
★★★★★
Your Friends and Neighbors is on Apple TV+ from 11 April.
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Even by Nikola Jokić's absurdly lofty standards, his performance on Tuesday night was something special.
The three-time NBA MVP put up 61 points, 10 rebounds and 10 assists, but it wasn't enough to prevent the Denver Nuggets from losing 140-139 to the Minnesota Timberwolves in a double overtime classic.
Jokic's 61 points are the most scored in a triple-double in NBA history and mark a new career-high, while it is also the most points any player has scored in a game this season.
The Serbian star also becomes only the second player in NBA history after Wilt Chamberlain in 1963 to lose a game when posting at least 50 points in a triple-double, per ESPN.
It's not even the first time Jokić has recorded a historic triple-double within the last month, after posting the first 30-20-20 triple-double in league history against the Phoenix Suns in early March.
After the game, Nuggets coach Michael Malone called Jokić “Superman.”
A wild finale to the contest concluded with Nickeil Alexander-Walker hitting two free throws to seal the win for Minnesota.
Moments earlier, Russell Westbrook had stolen the ball back for Denver but then missed an open layup. Westbrook then fouled Alexander-Walker on a three-point attempt at the other end as the game clock expired, giving the Timberwolves guard three free throws to win the game.
Alexander-Walker made the first two and then deliberately missed the third to ensure the clock expired again with just 0.2 seconds remaining.
“I feel awful for him,” Malone said of Westbrook. “When you put forth that kind of an effort, you put so much into and you don't get the result, I just feel bad for our guys.
“It never comes down to who deserves to win, but our guys did everything within their power to win that game and it didn't go our way.
“We've just got to regroup and find a way to get one tomorrow night.”
Jokić's record-breaking night further emphasized his MVP credentials and again highlighted the frequent lack of help he endures on a consistent basis. Though fellow Nuggets star Aaron Gordon did add 30 points, eight rebounds and five assists on the night in the losing effort.
For the Timberwolves, Anthony Edwards scored a team-high 34 points to go with 10 rebounds and eight assists, while Julius Randle and Alexander-Walker had 26 points each as six Minnesota players hit double figures.
The Timberwolves have now won three straight to climb to the No. 7 seed in the Western Conference as they bid to avoid the play-in, while the Nuggets remain the No. 3 seed.
Steph Curry cooked up a vintage performance on Tuesday, dropping 52 points as the Golden State Warriors beat the Memphis Grizzlies 134-125.
The 37-year-old hit 12 three-pointers in the contest. It is the 15th 50-point game of Curry's career, tied for sixth all time with Damian Lillard.
Curry's final line was 52 points, 10 rebounds, eight assists, five steals and a block, and he surpassed Jerry West to move up to 25th in the NBA's all-time scoring charts.
“Fifty-two points with people draped all over him, all game long,” Kerr said, per ESPN. “I've been watching this for 11 years, and actually longer before I became his coach.
“You get a real sense of just the magnitude of his talent. The guy is amazing to watch.”
Jimmy Butler added 27 points, six rebounds, four assists and three steals for the Warriors, who have won three straight and jump ahead of the Grizzlies into the No. 5 seed in the West.
Ja Morant had a team-high 36 points for Memphis, whose slide down the standings continues with a fourth consecutive loss.
Away @ home (winners in bold)
Phoenix Suns 123-133 Milwaukee Bucks
Philadelphia 76ers 91-105 New York Knicks
Portland Trail Blazers 127-113 Atlanta Hawks
Golden State Warriors 134-125 Memphis Grizzlies
Toronto Raptors 118-137 Chicago Bulls
Orlando Magic 116-105 San Antonio Spurs
Minnesota Timberwolves 140-139 (2OT) Denver Nuggets
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Jeffrey Epstein and Prince Andrew accuser Virginia Giuffre says she's in critical condition after a school bus hit her car, according to her Instagram page. Giuffre says she's gone into kidney failure, and a spokesperson for her tells CNN she is receiving treatment in a hospital.
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Prime minister says Israel will build a new security corridor to isolate parts of the strip in major escalation
Benjamin Netanyahu has said Israel is “seizing territory” and intends to “divide up” the Gaza Strip by building a new security corridor, amid a major expansion of aerial and ground operations in the besieged Palestinian territory.
“Tonight, we have shifted gears in the Gaza Strip. The [Israeli army] is seizing territory, hitting the terrorists and destroying the infrastructure,” the prime minister said in a video statement on Wednesday evening.
“We are also doing another thing – seizing the ‘Morag route'. This will be the second Philadelphi route, another Philadelphi route,” he said, referring to an Israeli-held corridor along the Egypt-Gaza border.
“Because we are currently dividing up the strip, we are adding pressure step by step, so that our hostages will be given to us,” Netanyahu added.
The Israel Defense Forces (IDF) have seized buffer zones around Gaza's edges totalling 62 sq km, or 17% of the strip, since the war began in October 2023, according to the Israeli human rights group Gisha.
The Netzarim corridor, named for a defunct Israeli settlement, now cuts off Gaza City from the south of the strip.
Morag was a Jewish settlement that once stood between Rafah and Khan Younis, so the use of the name suggests the new corridor is designed to separate the two southern cities.
The Israeli prime minister's announcement follows remarks on Wednesday from his defence minister, Israel Katz, who said the Israeli army would “seize large areas” of Gaza, necessitating large-scale civilian evacuations.
Neither Netanyahu nor Katz elaborated on how much Palestinian land Israel intended to capture in the renewed offensive, but the move is likely to complicate ceasefire talks and inflame fears that Israel intends to take permanent control of the strip when the war ends.
Israel's newly stated intentions to establish another military corridor followed a night of intense airstrikes on Khan Younis and Rafah in southern Gaza, which hospital officials said had killed at least 21 people.
An airstrike on Wednesday afternoon on a Jabaliya health clinic housing displaced people killed at least 19 people, including nine children, according to the civil defence agency.
The IDF said in a statement it had taken precautions to avoid civilian casualties in the bombing of what it said was a Hamas control centre in the Jabaliya refugee camp in the north of the strip. It later said it was aware the target was located in the same building as the clinic.
Unrwa, the UN agency for Palestinian refugees, said in a statement that the strike hit “two rooms on the first floor of an Unrwa destroyed health centre” that had been used as a shelter for 160 displaced families.
“Many displaced families have not left the site, simply because they have absolutely nowhere else to go,” the statement said. The agency had shared the building's coordinates with the IDF, it added.
In Khan Younis, the bodies of five women, one of them pregnant, two children and three men from the same family were brought to Nasser hospital on Wednesday morning, medics said. The IDF said it was examining the reports of civilian deaths.
Palestinian media reported bombing and shelling along the Egyptian border, at least two airstrikes on Gaza City, and Israeli troop movement in the Rafah area. The IDF said it had deployed an extra division to southern Gaza early on Wednesday.
The Israeli military issued sweeping evacuation orders last week telling people in Rafah and a swath of land stretching northwards towards Khan Younis to move to al-Mawasi, an area on the shore that Israel has designated as a humanitarian zone but has repeatedly bombed.
Israel renewed intensive bombing across Gaza on 18 March, followed by the redeployment of ground troops, abruptly ending the two-month ceasefire and exchanges of Israeli hostages held by Palestinian militant groups and Palestinians in Israeli jails.
According to the terms of the truce, the sides were supposed to negotiate implementing further phases of the deal during the first 42-day stage, but the Israeli government repeatedly postponed the talks.
The latest UN estimate, from 23 March, suggested approximately 140,000 people had been displaced since the end of the ceasefire. More than 90% of the strip's population of 2.3 million have been forced to flee their homes during the conflict, many of them multiple times.
Hundreds of people have been killed in Israeli airstrikes since it ended the ceasefire. Israel has also cut off humanitarian aid, food and fuel to the strip in an effort to pressure Hamas. The month-long blockade is now the longest in the war to date.
Efforts led by Qatari and Egyptian mediators to resume talks aimed at ending the war have not yet led to a breakthrough. The resumption of fighting in Gaza has also fuelled protests in Israel against the government from supporters of the remaining hostages and their loved ones.
The Hostages and Missing Families Forum, which represents most captives' relatives, said it was “horrified to wake up this morning to the defence minister's announcement about expanding military operations in Gaza”.
The group said: “Our highest priority must be an immediate deal to bring ALL hostages back home – the living for rehabilitation and those killed for proper burial – and end this war.”
The 7 October 2023 Hamas attack on southern Israel, in which Israel says 1,200 people, the majority of them civilians, were killed and a further 250 taken captive, was the trigger for the conflict in Gaza, the worst war between Israel and the Palestinians in more than 70 years of fighting.
Israel's retaliatory military campaign has killed at least 50,357 people, the majority of them civilians, according to the territory's health ministry.
Court hears arguments on whether state can block clinics that provide abortions from receiving Medicaid funds
In its first abortion-related case since Donald Trump retook control of the White House, the US supreme court on Wednesday heard oral arguments in a case challenging South Carolina's attempt to effectively “defund” Planned Parenthood because the reproductive health giant performs abortions.
The case, Medina v Planned Parenthood South Atlantic, deals with a 2018 executive order from the South Carolina governor, Henry McMaster that blocked clinics that provide abortions from receiving reimbursements via Medicaid, the US government's healthcare program for low-income people, despite the fact that those reimbursements don't actually cover abortions. “Payment of taxpayer funds to abortion clinics, for any purpose, results in the subsidy of abortion and the denial of the right to life,” McMaster said at the time.
However, federal law bans Medicaid from covering the vast majority of abortions, which only comprised about 4% of Planned Parenthood's activities in fiscal year 2022. Instead, people use Medicaid to cover Planned Parenthood's other services. In the same year, the organization performed nearly half a million Pap tests and breast exams as well as 4.6m STI tests and treatments. It also provided birth control services to more than 2.2 million people.
Planned Parenthood South Atlantic, a Planned Parenthood affiliate that operates two clinics in South Carolina, teamed up with a patient who sought birth control, Julie Edwards, to sue over McMaster's order. Lower courts have since kept the order from going into effect.
The supreme court is currently controlled 6-3 by conservatives. If the court sides with South Carolina, it could pave the way for other states to exclude Planned Parenthood from their Medicaid programs as well as devastate South Carolinians' ability to access reproductive healthcare and family planning services.
Of the 2.4 million people treated at Planned Parenthood nationwide each year, nearly half are on Medicaid. About four in 10 women who have sought care at family-planning clinics say those clinics are their only recent source of healthcare.
At the heart of the case is a federal provision that guarantees that people insured by Medicaid can freely choose their own providers as long as they accept the program and are qualified to provide care. The case hinges on a technical argument: South Carolina – which is being represented by the powerhouse Christian legal group Alliance Defending Freedom (ADF) – says Medicaid beneficiaries have no right to sue if they believe that guarantee has been violated.
Oral arguments zeroed in on this technicality, as ADF senior counsel John Bursch argued that the guarantee lacks “clear rights-creating language”.
“It does not use the word ‘right' or its functional equivalent,” Bursch said. “Nor does it use word with a deeply rooted, rights-creating pedigree, like the fifth amendment's ‘no person shall'.”
The three liberal justices on the supreme court – Elena Kagan, Sonia Sotomayor and Ketanji Brown Jackson – appeared ready to reject that argument. It's impossible to describe Medicaid's guarantee, Kagan said, without calling it a “right”.
“The obligation is to ensure that individuals can choose their doctor,” Kagan said. “There's a correlative right. There's an obligation, there's a right. And the right is the right to choose their doctor.”
The conservative wing of the court seemed more split. Justice Amy Coney Barrett appeared particularly worried about leaving patients unable to seek help if their preferred qualified provider was kicked out of Medicaid. “If I want to go see Dr Jones and that's the provider of my choice and the state has disqualified Dr Jones … there's no mechanism, am I right, for the beneficiary to say: ‘Well, you're depriving me of my ability' – we won't call it right, we won't use the loaded word – ‘but the ability to see the provider of my choice'?” she asked.
During arguments, Bursch suggested that, rather than suing, patients could ask the government to intervene administratively.
Ultimately, these seemingly narrow arguments, legal experts warn, conceal the broader consequences of the case. If people can't sue when they believe a state is violating Medicaid, then it will become far more difficult to keep states from discriminating against certain kinds of care, said Nicole Huberfeld, a health law professor at Boston University's School of Public Health.
“Even though the state is trying to claim that it has sole authority to decide who's a qualified provider, this isn't really about whether Planned Parenthood is a qualified provider. It's about a political calculation on abortion,” Huberfeld said. “Really, what's happening here is states making politically driven decisions about access to medical care.”
A decision in the case is expected in June.
More than 1 million people in South Carolina use Medicaid and the Children's Health Insurance Program (Chip) – which is closely linked to Medicaid – for their insurance. Almost 40% of South Carolina counties are believed to be “contraceptive deserts”, where there are not enough providers to meet the needs of the people who live there. South Carolina bans abortion past six weeks of pregnancy.
Because Texas terminated Planned Parenthood from its state Medicaid program in 2021, it offers a window into what happens when people lose access to medical provider. The termination affected the healthcare of some 8,000 Texans.
“Planned Parenthood, we heard again and again, was a medical home for people with very few options for providers,” said Anna Chatillon, a research scientist at Resound Research for Reproductive Health, who led a study that interviewed Texans about how the termination affected their lives. “In several different interviews, we heard people talking about showing up for an appointment, finding out that they couldn't use Medicaid any longer, not having any idea where else to go, and experiencing real disruptions to their care based on that very quickly, but also serious emotional ramifications.”
The case is part of a broader, longstanding anti-abortion campaign against Planned Parenthood, which includes an ongoing lawsuit that could bankrupt the organization over allegations that it defrauded Medicaid.
On Tuesday, Planned Parenthood announced that the Trump administration – which helped South Carolina make its case in oral arguments – had notified nine of its affiliates that they would lose funding from Title X, the nation's oldest and largest family-planning program. Politico reported the funds were being withheld as punishment for the organization's diversity, equity and inclusion efforts.
A ‘derivative' of Quentin Tarantino's original, rather than a sequel, will be produced by Netflix, and continues Pitt's relationship with the director of Seven and Fight Club
Brad Pitt is to star in a follow-up to Quentin Tarantino's Once Upon a Time in Hollywood. But, having written the script, Tarantino will hand directing duties to David Fincher, according to a report in the Playlist and subsequently confirmed by Deadline.
In Tarantino's 2019 alternative-history drama about the Manson “family” and Sharon Tate, Pitt starred as stunt performer Cliff Booth opposite Leonardo DiCaprio (as washed-up actor Rick Dalton) and Margot Robbie (as Tate). While no plot details of the new project have been officially announced, the Hollywood Reporter is saying it will be “derivative” of the original, rather than a sequel. Tarantino published a “novelisation” of the film in 2021, which included further details of Booth's backstory and life episodes.
Tarantino is also believed to have decided against directing the film, and allowed Pitt to take the script to Fincher, with whom he has had a long working relationship, including starring in Fincher's early films Seven, Fight Club and The Curious Case of Benjamin Button. While Tarantino is now renowned as a director, he sold scripts earlier in his career, notably True Romance (directed by Tony Scott) and Natural Born Killers (directed by Oliver Stone).
In another departure from the original Once Upon a Time in Hollywood, the project will not be produced by Sony Pictures but has been acquired by Netflix, with whom Fincher has made two feature films (Mank and The Killer) and several TV series (including House of Cards, Mindhunter and Voir).
It is not clear whether the project is related to Tarantino's scrapped “final” movie The Movie Critic, in which Booth's character was rumoured to have appeared. It is also unclear if the new script makes room for appearances by DiCaprio and Robbie.
Blanche Hoschedé-Monet has barely been acknowledged in art history. But not only did she help her stepfather Claude, she created her own fine works – often of the same scenes as him.
Haystack at Giverny, Poplars at the Water's Edge, Morning on the Seine. These painting titles bring only one name to mind – the great Claude Monet, whose flickering evocations of light and atmosphere are the cornerstone of Impressionism.
But while Monet painted these very subjects, the paintings belong to the oeuvre of his stepdaughter, and subsequently daughter-in-law, Blanche Hoschedé-Monet (1865–1947). She learned to paint at Monet's shoulder, and exhibited and sold her work through the leading Parisian dealers of the time. Her finest paintings suggest an artist of such flair that you wonder how she has slipped from history's grasp.
A new exhibition and accompanying monograph – the artist's first – seeks to restore her reputation. Blanche Hoschedé-Monet in the Light at The Sidney and Lois Eskenazi Museum of Art, Indiana, brings together 40 of her paintings, along with sketchbooks, photographs and letters, establishing not just her impressionist credentials, but her role as Monet's assistant and companion on plein air painting expeditions – the only one of his children, blood-related or otherwise, whose passion for painting mirrored his own.
The difficulty of extracting Hoschedé-Monet from history has been compounded by the fact that few of her 300-ish works are in public collections. In her native France, the former Musée Municipal de Vernon, near the Normandy village of Giverny, where the Monet family lived for decades, holds the largest number – eight paintings and one pastel – and was valiantly renamed the Musée Blanche Hoschedé-Monet last year to coincide with the 150th anniversary of Impressionism. Meanwhile Paris's Musée d'Orsay (widely regarded as the best collection of Impressionist art) has only a further two – neither of which, at the time of writing, is on display.
There are no Hoschedé-Monet paintings in British public collections at all, and in the US, only one, The Weeping Willows on the Lily Pond at Giverny (c 1893-7) at the Columbus Museum of Art in Ohio. The Indiana exhibition is the artist's first solo show on US soil, in fact, and an indication that her reputation is finally growing.
"While Hoschedé-Monet's work remains relatively unknown and somewhat underappreciated in the marketplace, it is clear that she is gaining more recognition with collectors and institutions," says Julia Leveille, head of modern-day auctions at Sotheby's New York." Strong prices have been set for her work at auction recently, and we are excited to see where her market goes from here."
Hoschedé-Monet was the daughter of Eugene Hoschedé, a wealthy businessman and connoisseur of avant-garde painting. Édouard Manet, Auguste Renoir, Gustave Caillebotte, Mary Cassatt and of course Monet were regular guests at his beautiful homes on the Boulevard Haussmann in Paris, and at his estate at Montgeron, on the city's edge.
Hoschedé-Monet was 11 when she first met Claude Monet in 1876. The then-35-year-old had been commissioned by her father to paint a quartet of panels for the dining room at Montgeron. Hoschedé was an important early patron of Monet's – in fact, it was he who bought Impression, Sunrise (1872), the painting that gave Impressionism its name.
"I remember his arrival. He was introduced to me as a great artist, and he had long hair," she recollected in autobiographical notes she wrote towards the end of her life, which her brother Jean-Pierre then used as a basis for a personal and intimate account of Monet's life and family at Giverny. " That struck me, and I immediately had sympathy for him because we could tell he was fond of children."
In her notes, Hoschedé-Monet also singled out Monet's Springtime or The Reader (1872) as the painting she loved most in her father's considerable collection. She was "deeply struck", she said, by the touches of sunlight on the skirt of the woman – Monet's first wife, Camille.
The six months Monet spent at Montgeron in 1876 led to a magnificent set of paintings that includes the much-loved Turkeys and Willows at the River's Edge, though they did not remain in Hoschedé's possession for long. Dire financial pressures forced him to sell most of his collection in 1878, along with both of his homes.
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Monet brought Hoschedé, his wife Alice and their six children (Marthe, Blanche, Suzanne, Jacques, Germaine, and Jean-Pierre) to live with him, Camille and their two sons Jean, 11, and newborn Michel, at his home in Vétheuil, northwest of Paris.
Camille's health deteriorated post-partum, however, and in 1879 she died, after which Alice and Monet entered into "what we believe was a domestic partnership," says Haley Pierce, co-curator of Blanche Hoschedé-Monet in the Light. But they didn't marry until 1892, after Hoschedé had passed away. [Hoschedé] had been estranged from the family for a while at this point."
Juvenalia aside, the earliest works in the show date from 1882, when the newly-merged family rented a house in the seaside resort of Pourville, near Dieppe. Monet's painting Cliff Walk at Pourville features the then-17-year-old Hoschedé-Monet with her elder sister, Marthe, and Hoschedé-Monet filled a sketchbook with her own sunlit views.
In spring 1883, Monet moved the family to Giverny, where Hoschedé-Monet began to paint in earnest. Delighted by her enthusiasm, he began taking her with him on his outdoor painting expeditions. According to the biography of Hoschedé-Monet's which her brother Jean-Pierre also wrote, "she helped him in all circumstances… transporting his canvases and easel as well as her own. She did it all with the help of a wheelbarrow, following elusive paths, across fields and meadows sometimes drenched in dew. That was the case, for example, with his morning views of the Seine. Here again, she would help her stepfather by taking up the oars of the canoe."
Monet also suggested exhibitions she might enjoy. In 1891, for instance, he asked his friend Gustave Geffroy, an influential critic, to obtain a pass for her to the Salon of the Société Nationale des Beaux-Arts, and later that year, took her with him to dine at the home of the British artist James McNeill Whistler in London (they were visiting her brother Jacques in Lymington) and to see the famous Peacock Room that Whistler created for shipping magnate Frederick Leyland's mansion near Hyde Park.
Monet's 1887 painting In the Woods at Giverny: Blanche Hoschedé at Her Easel with Suzanne Hoschedé Reading, confirms that he and Hoschedé set up their easels within spitting distance of each other, or even, if she is the figure in the white dress at an easel in John Singer Sargent's oil sketch, Monet in his Bateau Atelier (also 1887), squashed together in the floating studio which Monet designed to paint while drifting along the river.
Unsurprisingly, then, Hoschedé-Monet's paintings often share her mentor's visual vocabulary: Shadows on the Meadow (Giverny, the Ajoux plain) matches his Spring Landscape at Giverny (1894) for instance, and The Small Grainstacks (Les Moyettes) (c 1894) his Stacks of Wheat (End of Summer) (1890-91). Julie Manet, daughter of the impressionist Berthe Morisot and Manet's brother Eugène, would also describe two of Blanche's paintings of "trees reflected in the Epte [that] are very like Monsieur Monet's painting" while visiting Giverny in 1893.
As Hoschedé-Monet's great-nephew, the art historian Philippe Piguet, further explains, "her touch is more emphatic, out of concern for capturing what she saw on her canvas rather than what she felt" – meaning that Hoschedé-Monet's paintings can be described as more solid or direct in their rendering and composition, and less atmospheric, as compared to Monet. She also preferred to paint a subject from varying viewpoints, he adds, rather than, as Monet did, at different times of day.
Consider her painting Haystack at Giverny alongside the Monet Haystacks at Giverny (1893) that was sold at Sotheby's last year. "They're at the same or approximate location, but she has distinctly chosen a different view," says Pierce. "Her painting is also more solid. She has less interest in the quality of atmosphere, and more in really getting down her subject. Her compositions are very well thought out."
By now, Hoschedé-Monet was blooming. The previous year, the US artist Theodore Robinson noted in his diary: "I saw some things by Mlle Blanche, she has improved greatly since I saw her work last – a spring landscape, sold to [the Chicago millionaire] Potter Palmer quite charming." Her rapid progress excited Monet, too: away in Norway in 1895, he wrote to Alice, "I can't wait to see what she has done in my absence, especially from what you tell me".
In 1897, Hoschedé-Monet married Jean and they moved to Rouen, but returned to Giverny at weekends. For a time, her upward trajectory continued – in 1905, she began exhibiting with the Salon des Indépendants in Paris, and in 1907 the Salon de la Société des Artistes Rouennais – but when Jean had a serious fall in 1912, she gave up painting to care for him.
When Jean died two years later, Hoschedé-Monet returned to live at Giverny, where the recently widowed Monet was panic-stricken that he was losing his sight and finding it difficult to work. Her decision delighted his friends. Geffroy told her he was "happy for him and happy for you. Your mutual misfortune makes your reunion stronger."
Her reassuring presence gave Monet the strength he required to begin the monumental compositions of water lilies that occupied his final years and which he hoped would create "the illusion of an endless whole". The exhibition includes a photograph of him working on these "Grand Decorations", as he called them, with Hoschedé Monet at his side.
Monet's close friend, Prime Minister Georges Clemenceau, later wrote that she had prepared the canvas grounds, but Hoschedé-Monet refuted the claim. "Contrary to what has been said and what has been written, I didn't make a brushstroke on [Monet's] canvases. It would have been a sacrilege." She also described Monet's death in 1926: "It was the soul of the house who left… Everything here was illuminated by him."
With Michel's approval, Hoschedé-Monet remained at Giverny for the rest of her life, caring for the house and studio, and tending to Monet's beloved gardens. She resumed painting again almost immediately, and in 1927 had her first solo exhibition at Galerie Bernheim-Jeune in Paris. Critics took great interest in her renaissance, with one from newspaper Le Peuple proclaiming her "more than the heiress of a great name".
Impressionism lost its grip on the art world almost as soon as Monet died, but Hoschedé-Monet always remained loyal to her mentor's painting style. We might see her as the comet's tail of that hugely influential artistic movement, though perhaps her greatest contribution to it was her steadfast support. "Without her, Claude Monet would have lived in an isolation that would have killed him," wrote the art dealer René Gimpel. "It was she who kept him alive for us, posterity must not forget her."
Blanche Hoschedé-Monet in the Light is at the The Sidney and Lois Eskenazi Museum of Art, Indiana until 15 June.
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The German military has begun its first permanent deployment of troops on foreign soil since World War II. The 45th Armored Brigade is being positioned in Lithuania, near Russian ally Belarus, as Berlin prepares for a potential conflict in the coming years.
On Tuesday, a ceremony was held outside Vilnius, with Brigadier General Christoph Huber assuming command of the newly established unit, as reported by the German Bundeswehr Association (DBwV) lobby group and state media.
”We have a clear mission. We have to ensure the protection, freedom, and security of our Lithuanian allies here on NATO's eastern flank,” the general said during the ceremony.
Military personnel who arrived in Lithuania last year to prepare for the deployment have been formally integrated into the brigade. Once fully staffed and combat-ready by 2027, the unit will comprise approximately 5,000 military and civilian personnel, equipped with around 2,000 heavy weapons, according to German Army plans.
Moscow views NATO as a hostile entity, describing its expansion in Europe as a direct threat to Russian national security. The bloc's pledge to admit Ukraine as a member and its increasing involvement with the nation have been cited by Russian officials as key causes of Moscow's conflict with Kiev. German Defense Minister Boris Pistorius, a staunch advocate for troop deployment in Lithuania, has claimed that Russia could launch an attack on NATO by 2029 or 2030 – a claim that Moscow categorically rejects.
In 2023, Berlin and Vilnius signed a stationing agreement, initially designating the new German unit as the 42nd Brigade. Two of its battalions will consist exclusively of German soldiers, while the third will incorporate personnel from other NATO countries. Portions of the brigade will be stationed at the Rudninkai training ground in southeastern Lithuania, only 20 kilometers (12 miles) from the Belarusian border, while additional units will be stationed near the village of Rukla between Vilnius and Kaunas.
Post-Nazi Germany previously restricted military deployments abroad to temporary assignments, such as contributing to NATO forces following the occupation of Afghanistan in 2001.
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When Hillary Clinton and Joe Biden faced intense scrutiny for their handling of classified material, top officials now serving in Trump's Justice Department and FBI demanded criminal probes and severe penalties.
Yet today, those same figures – including Attorney General Pam Bondi, FBI Director Kash Patel, Deputy FBI Director Dan Bongino, and DC interim US Attorney Ed Martin – have all declined to publicly criticize senior Trump officials who used Signal to share military attack plans in a chat that inadvertently included a journalist.
The Trump administration has denied any classified information was discussed in the text messages released by The Atlantic about plans to bomb rebels in Yemen, but CNN reported that information shared in the chat was highly classified at the time it was sent.
Bondi, now the country's highest ranking law enforcement official, vigorously defended the officials who participated in the Signal chat, including Vice President JD Vance and Defense Secretary Pete Hegseth, and suggested it was unlikely their actions would be investigated criminally. But previously, Bondi argued that both Clinton and her aide Huma Abedin needed to face charges after emails that contained classified information were found on the computer of her ex-husband, former Democratic Rep. Anthony Weiner.
“This has everything to do with the security of our country,” Bondi said in January 2018 on Fox News. “When you have the top-secret security clearance that Huma Abedin had – you know when you send those emails that you are violating the law, and there is no objective law enforcement officer in this country that would not charge her based on that. Alright? No one.”
Bondi repeatedly framed the Clinton email investigation as a clear-cut legal issue in her appearances on Fox News reviewed by CNN's KFile, saying that “we live in a nation of laws” and that Clinton “jeopardized our national security.”
On Thursday, Bondi defended the officials in the Signal chat, saying the chat included “sensitive information, not classified.”
“What we should be talking about is it was a very successful mission,” she told reporters. “If you want to talk about classified information, talk about what was at Hillary Clinton's home, the classified documents in Joe Biden's garage.”
A spokesperson for the Department of Justice said Bondi's public comments last week “speak for themselves.”
Similarly, Patel has previously called for harsh prison sentences for leaking classified information.
“A leak of classified information — that's a federal offense punishable by, I think, over a decade in prison, off the top of my head,” Patel said in 2021 when discussing leaks to the media during the investigation into Russian interference in the 2016 election. “It's a pretty serious matter. But no one's been prosecuted for a leak of classified information.”
In another interview in September 2024, Patel criticized the decision by special counsel Robert Hur not to charge Biden for allegedly mishandling classified documents.
“They let the guy off the hook,” he said of Biden, who was investigated over classified documents found at his private residence in Delaware. “They just said it doesn't matter … you've made up a new legal standard that applies to exonerate him and to not even charge him.”
Bongino, a former Secret Service agent and now the FBI's second-in-command, frequently also accused Democrats and the Biden administration of mishandling classified information and using encrypted apps to avoid accountability, including the app Signal.
“This was a travesty of justice, there's no other way to look at it,” Bongino said regarding the recommendation by former FBI Director James Comey not to charge Hillary Clinton in 2016. “And having worked as a federal agent for 12 years of my life and as a local police officer before that, I'm intimately familiar with the elements of crimes, the elements of an investigation, and good, solid, packaged federal cases. This thing was in a bow.”
“What Jim Comey laid out yesterday that for 15 minutes what Mrs. Clinton did — in the federal system, you can put forth information in a complaint or an indictment. If that went in front of a judge and jury, he could have convicted Mrs. Clinton just on that information alone.”
In since-deleted tweets, Bongino also took aim at Abedin and questioned if former FBI Deputy Director Andrew McCabe may have used unsecure communications to handle classified information.
“Either Huma knew she was sending classified emails to Weiner, & was doing so illegally, therefore she committed a crime. Or, Huma didn't know sending classified emails was a crime & had absolutely no business having access to the Clinton political machine & insiders in our govt,” he said in a deleted tweet from late 2017.
In a February 2018 tweet Bongino wrote, “Here's the next shoe to drop -> was Andy McCabe using secure comms to send/receive classified information? Someone should check on that.” McCabe, a longtime target of Bongino's, was a part of the FBI investigation into Russian interference in the 2016 election.
Patel and Bongino have not publicly criticized the officials involved in the Signal chat. The FBI declined to comment to CNN.
Ed Martin, now the acting US Attorney for the District of Columbia and the official who could oversee any prosecution related to the Signal chat, has previously taken a hardline stance on the handling of classified material.
“When he took them he couldn't have done anything but broken the law, period,” Martin said on his radio show in 2023, referring to Biden's possession of classified documents.
In a 2018 tweet, Martin wrote that “almost anyone” who leaked classified information would “go to jail.” And in 2024, he asked, “Was it not a crime for a vice president to remove top secret and classified documents from the White House and store them unsecured in his garage…? If it's not, it should be.”
Martin has not commented publicly on whether the Signal chat warrants investigation.
A spokesperson for the US Attorney's Office for the District of Columbia declined to comment to CNN.
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Editor's Note: This story contains description of sexual assaults.
To reach Diane Swick's rural California homestead, you have to drive 70 miles north of Los Angeles and traverse a rocky road best suited for an ATV. Few come here without a good reason.
But a man dubbed the “Pillowcase Rapist” had no trouble getting to Pearblossom — in fact, he got a court-mandated escort to the community nearly two weeks ago. Christopher Hubbart, 73, is now Swick's next-door neighbor.
“We are women and that's his prey,” Swick said. The 61-year-old had heart surgery last year and says she was depressed when she first heard Hubbart was coming.
“Now I'm mad,” she told CNN from her back porch, within eyeshot of Hubbart's rented home. “I've never had to lock my doors for the 28 years I've lived here, and now that's been taken away from us.”
Hubbart's release has reignited a debate over how the state should handle the most extreme sexual offenders in its custody, including those like Hubbart who attacked again after serving multiple sentences.
Hubbart was charged with dozens of sexual assaults dating back to the 1970s and spent seven years in a state mental hospital after pleading guilty to one count of rape and three counts of sodomy.
Court records show he was released in 1979 to an outpatient program in the San Francisco Bay area, and “within a few months” sexually assaulted about 15 women after breaking into their homes. He would “bind the victim's hands, cover her head, and commit forcible sex acts,” court records allege.
In 1982, he was sentenced to 16 years in prison and released in 1990 after serving about half of his time. Once free, he raped again. “Within a two-day period, Hubbart attempted to sexually assault one woman and actually assaulted another,” court records state. His parole was revoked, and he was returned to prison.
Until last month, he was held in a state hospital awaiting placement into a home as part of California's “Sexually Violent Predator (SVP) Conditional Release Program.” It's designed for predators in state hospitals “to safely transition back to being a fully functional member of the community,” according to a fact sheet from the California Department of State Hospitals (DSH).
“I have no problem with someone who has gone to prison, done their time and paid their debt to society,” said Linda Adams, who lives across the street from Hubbart. “But how can you pay a debt for (that many) rapes? It's not something that just goes away.”
Rachel Purcell lives down the street from Hubbart's new home and says she now walks with bear spray.
“Any male Caucasian that I see coming up to me, I'm going to be wary,” she told CNN. “And if he gets within 30 feet of me, he's getting sprayed.”
It's not just residents who are irate. Politicians have voiced opposition since last September when the Antelope Valley was identified as Hubbart's next home by Liberty Healthcare, a state contractor that oversees SVP cases.
“A man who has admitted to raping over 40 women and suspected of raping dozens more is not fit for release or community reintegration at any level,” wrote Los Angeles County Supervisor Kathryn Barger in a letter to the Superior Court. The district attorney's office also opposed the move.
Hubbart did not respond to a letter sent from CNN prior to his release from the Coalinga State Hospital. Outside his new home, a representative from Liberty declined to submit CNN's request to speak with Hubbart. A Liberty spokesperson referred all questions to DSH.
Hubbart is now neighbor to a mix of about two dozen suburban-style homes and dusty ranches, flanked by the San Gabriel mountains, where many enjoy the peace and pitch black of a country night. “It's the whole reason I moved up here,” Purcell said.
But that isolation now seems like a liability, she admits, as the nearest law enforcement station is at least 15 miles away.
“Half of us don't have reliable cell (service) at all,” Adams agreed. “Our landline goes out frequently, and there's no urgency in repairing it. If (Hubbart) approached my property none of my neighbors would hear me if I needed assistance.”
Neighbors here don't dispute that Hubbart's release happened through proper legal channels. The 1996 Sexually Violent Predator Act allows for treatment and rehabilitation for SVPs who have completed their prison sentence. In Hubbart's case, a Santa Clara County court first deemed him suitable for release from a state hospital in 2014. Placement into his current home has been approved and overseen by a Los Angeles County judge — even if some residents scoff at the process.
“The judge said he would make the decision based on whether he would put (Hubbart) across from his own family,” Adams said. “So, he must hate his family.”
The case played out in a mental health court in hearings livestreamed to the public. However, the case files remain sealed.
In its fact sheet on the SVP program, DSH says a court can only approve conditional release when it has determined “the individual would not be a danger to the health and safety of others in that it is not likely that the person will engage in sexually violent criminal behavior due to the person's diagnosed mental disorder if under supervision and treatment in the community.”
It continued that treatment was given after someone seeking release had completed their criminal sentences. “In making its determination, the court generally considers treatment records, assessments, and information provided by mental health professionals and forensic evaluators.”
Others who live in this high desert section of the county say what stings even more is that this is the third sex criminal released to this region.
The Los Angeles County District Attorney, Nathan Hochman, accused the state of treating the Antelope Valley as a “dumping ground” for sexually violent predators.
DSH countered, telling CNN it must legally consider factors like proximity to schools and parks, which “significantly limit(s)” suitable options to “outside of dense, urban areas.”
In an email, the department said: “DSH does not place individuals designated as SVP into the community until a court has determined it is safe and appropriate to do so. It is also the court that ultimately approves the placement location.”
That's not good enough for Mary Jeters, a local resident who operates the Facebook page “No SVP's in the Antelope Valley.”
“My frustration has gone past the point where I'm going to scream about it,” Jeters said. She disputes the notion that rural areas equate to safer locations.
“These SVPs are not going to be restricted to being in their house all the time,” Jeters said. “They bring them out to go to doctors' appointments, they go shopping. People don't know who they're standing next to in line, you don't know who's talking to your daughter.”
I just don't trust that he can be trusted
Mary Jeters, resident
Citing patient privacy laws, DSH declined to speak specifically about Hubbart's case but confirmed there are several safeguards at the home of any released prisoner placed in the community. They include 24-hour GPS monitoring, covert surveillance, unannounced and scheduled home visits, and advanced approval — including routes of travel — for scheduled outings.
“The close supervision is there to guard against the individual committing any new crimes,” DSH said in a statement.
Security cameras are also visible at Hubbart's home and residents say a private security guard has been posted at the home around the clock.
“They're just watching that we don't bother him,” said Purcell, a neighbor, adding her view was that “the security is to protect him, not to keep him locked up.”
Jeters said some residents have been the target of restraining orders by other former inmates in the area who felt harassed, but that the goal is to protest peacefully. She held a public gathering Sunday on vacant land next to Hubbart's new home.
“I just don't trust that he can be trusted,” Jeters said, noting this is Hubbart's second attempt at a successful release in the area.
The first came in 2014, but two years later Hubbart was sent back to a state hospital for violating his release terms. DSH declined to confirm details of the violation, again citing privacy laws.
In 2016, Hubbart's former parole officer praised his return to custody.
“Christopher Hubbart is a waste of a human heart,” John Bays, then retired, told the Los Angeles Times. “He never should have gotten out.”
Swick, the next-door neighbor, wonders why this time would be any different. She feels her community is being piled on and that in addition to safety issues, the neighborhood has been affected by depressed property values in the wake of 2020's Bobcat fire.
“We lost our barn, we lost a tractor, we lost a shed. And I would much rather go through a fire than what's going on here,” Swick said. “This sense of not knowing what's going to happen. Nobody wishes a fire on anybody, and I feel this is worse.”
Los Angeles County is not the only area dealing with backlash from the SVP conditional release program. Just over 160 miles to the south of the Antelope Valley, in Borrego Springs, residents were informed in February by the sheriff that another rapist, Alvin Ray Quarles, was being placed in their community. Quarles served time for multiple rapes in the San Diego area dating back to 1988.
“Now that there's two or three sexually violent predators in that small community of 3,000 people, with one grocery store … how do you protect that community?” asked Republican state Senator Brian Jones of San Diego.
Jones believes sex criminals should only be released to structures such as prefab homes on state-owned and operated land.
“Outside the perimeter of a state institution… in a fire camp, any other area that the state is responsible for the safety and condition of that area,” Jones said.
Very few make it through, it's hard to do
Michael Aye, lawyer who has represented some sexual predators seeking release
But in its statement to CNN, DSH said placing released inmates on state-run property would not fulfill its legal obligation under the SVP Act to “safely reintegrate” individuals so they can achieve “unconditional release.”
Jones wants to change the law. He's proposing two state Senate bills that would ultimately force DSH to sign off on placement locations selected by its contractor, Liberty. “It basically puts it back on the desk of the director of state hospitals and holds (them) accountable,” Jones said.
The bills passed a Senate public safety committee Tuesday though a similar proposal from Jones failed last year. Among the opposition at a committee hearing last April was lawyer Michael Aye, who has represented sexual predators seeking release through the system. He worried a new law would stifle the program altogether.
“Our fear is that… giving the director (of state hospitals) the responsibility, that no one will be authorized release,” Aye told the state Senate standing committee on public safety. “Very few make it through, it's hard to do.”
Those that do make it through to community placement have first spent “an average of 12 years or more” working toward successful completion in the hospital, DSH told CNN.
State audit figures also show fewer crimes committed by people in the SVP release program. Of 56 participants in the last 21 years, two were convicted of criminal acts. The same audit found that of 125 sex criminals released unconditionally by the courts, 24 were convicted of subsequent crimes.
Jones, the state Senate minority leader, says the state's current procedure ignores common sense.
“It's not fair for a community, a mom and a dad, a grandma and grandpa, to live next door to these monsters, and be responsible for keeping an eye on them,” he said. “That's the state's job and the state should do its job.”
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On Tuesday morning, Rajesh Nayak stepped outside the firecracker warehouse in India's Gujarat state where he worked to drink water.
Some moments later, an explosion ripped through the building, killing 21 people, including Mr Nayak's brother.
"Some of my other relatives have also died. I had come to work here only from Sunday," a distraught Nayak, who is in hospital with minor injuries, said.
Most of the victims were from neighbouring Madhya Pradesh state and had recently come to work at the warehouse, located in an industrial estate in Banaskantha district in Gujarat.
Their families lived in huts close to the building and some of them were also killed from the force of the explosion. Banaskantha District Collector Mihir Patel told BBC Gujarati that the victims included four women and three children.
It's not clear yet what caused the explosion, but officials are investigating if firecrackers were being manufactured illegally at the warehouse.
"Primary information has been received that the explosion took place when firecrackers were being made here," said Mr Patel, the collector.
India has strict rules around firecracker production but these are often not enforced strongly on the ground. Accidents are regularly reported, especially at illegal factories.
The incident in Gujarat came a day after eight people were killed in an explosion at an illegal firecracker factory hundreds of miles away in West Bengal state.
Police in Gujarat have arrested two men, owners of the warehouse, in connection with the explosion and are searching for one more person. A special investigation team has been set up to look into the incident.
Banaskantha district police chief Akshay Raj Makwana said a preliminary investigation showed that aluminium powder was stored in the building.
"This powder is non-explosive but flammable and easily available in the market. We are investigating the supply chain and how the accused sourced such material," said Mr Makwana.
Mr Patel told reporters that the building had been registered as a warehouse for storing firecrackers, but its licence had expired in December. When a team went to inspect the area in March, he said, the building was empty.
When BBC Gujarati reached the area on Tuesday, the air smelt strongly of sulphur.
The explosion caused extensive damage, destroying the warehouse and a wall of the adjacent factory. Large concrete slabs were thrown up to 300ft away.
Mr Makwana, the police chief, said a slab in the building collapsed, trapping workers underneath.
The powerful blast also destroyed surrounding huts and killed some family members of the workers.
A sanitation worker told BBC Gujarati that he carried out four bodies on stretchers from the site. "My heart sank when I saw a child's body," he said.
Prime Minister Narendra Modi, who is from Gujarat, has expressed his condolences to the victims' families and announced financial assistance.
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The United States has approved the potential sale of 20 F-16 fighter jets to Manila, giving the key US ally in the Indo-Pacific a major upgrade to its air force just days after US Defense Secretary Pete Hegseth vowed to counter “China's aggression.”
The US Defense Security Cooperation Agency (DSCA) announced the proposed sale of the F-16s and related equipment, worth an estimated $5.58 billion, in a statement on Tuesday.
“This proposed sale will support the foreign policy and national security of the United States by helping to improve the security of a strategic partner that continues to be an important force for political stability, peace, and economic progress in Southeast Asia,” DSCA said.
The announcement comes less than a week after Hegseth visited the Philippines, his first trip to Asia as defense chief, and said Washington will enhance its military alliance with Manila as it aims to “reestablish deterrence” to counter “China's aggression” in the Indo-Pacific region.
On Wednesday, China cautioned Manila on the deal.
“Any defense and security cooperation that the Philippines engages in with other countries should not target or harm the interests of any third party, nor should it threaten regional peace and security or escalate tensions in the region,” Chinese Foreign Ministry spokesperson Guo Jiakun said.
“As for who is fueling the flames, who is provoking military confrontation, and who is turning Asia into a powder keg, we believe that regional countries can see the situation clearly.”
The Philippines has been on the front lines of China's increasingly aggressive posture in Asia. Beijing seeks to assert its claim over the bulk of the South China Sea, despite an international ruling denying its sovereignty over the waterway.
Hegseth said Friday the US would deploy additional advanced military capabilities to the US ally for joint training, enhance interoperability for “high end operations” and prioritize defense industrial cooperation.
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US defense chief Hegseth vows to counter ‘China's aggression' on first Asia visit
In its statement, DSCA said Manila had requested to buy 16 F-16Cs – single-seat, single-engine fighter jets – and four F-16Ds, dual-seat jets that are usually used for training purposes.
The F-16s are the block 70/72 newest variant of the workhorse military warplane, which entered service with the US Air Force in the late 1970s.
Manufacturer Lockheed Martin says the new F-16s are the world's most advanced fourth-generation fighter, touting a “structural service life” of more than 12,000 hours.
The F-16s, along with advanced avionics, radar and weaponry included in the deal, are a significant upgrade to the Philippine Air Force's fighter fleet. Currently, it has only 12 South Korean-made FA-50 jets, a lighter ground attack and fighter jet.
The F-16s have a top speed of more than 1,500 miles per hour, Lockheed Martin says, about 350 mph faster than the FA-50.
Speaking alongside Philippine Defense Secretary Gilbert Teodoro on Friday, Hegseth called the US-Philippine relationship an “ironclad alliance, particularly in the face of Communist China's aggression in the region.”
The Trump administration has vowed to “truly prioritize a shift” to the Indo-Pacific, Hegseth said, with the “recognition that for the 21st century to be a free century, America needs to stand alongside our allies and partners shoulder to shoulder.”
The American military presence in Asia is seen by allies as a critical counterbalance in a fractious region where China has been rapidly expanding its military might and a belligerent North Korea has been empowered by closer ties with Russia.
Trump has repeatedly questioned the structure of US military alliances and whether the US was getting enough out of such partnerships and basing arrangements, including those in Asia where tens of thousands of troops are stationed in sprawling bases in Japan and South Korea.
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Val Kilmer, a movie star who throughout his career proved he was up for any task, from playing a cocky naval pilot to wearing the Batman suit, died on Tuesday, according to his daughter Mercedes Kilmer, who released a statement to the New York Times and the Associated Press. He was 65.
The cause of death was pneumonia, Mercedes Kilmer told the media outlets.
CNN has reached out to representatives for Kilmer and his family for comment.
Kilmer had recovered from a 2014 throat cancer diagnosis that required tracheotomy surgery that altered his voice.
In recent years, Kilmer largely stepped away from acting, but made a brief return to the craft in “Top Gun: Maverick,” the sequel to the original film that launched him and that cast into superstardom.
The sequel, which released in 2022 after a two-year delay, deftly blended Kilmer's off-screen health issues into the film's story, with his Tom “Iceman” Kazansky, the smug but cool rival to Tom Cruise's Pete “Maverick” Mitchell, ultimately succumbing to an ailment in the film.
As one of Kilmer's last film roles, it concluded one of the most interesting — and bankable — careers in Hollywood history.
Born and raised in California's San Fernando Valley, Kilmer studied at the Hollywood Professional School before heading to New York where, at 21, he became the youngest student at the time to be accepted into Julliard School's drama department.
He began his film career in the 1984 comedy “Top Secret!” and acted in several movies throughout the ‘80s including his breakout role in 1986's “Top Gun.”
Then came an incredible streak of successful genre-spanning movies in the 1990s: A superhero film (with 1995's “Batman Forever,” in which he played the Dark Knight), a Western (1993's “Tombstone,” where he played Doc Holliday), a biopic (1991's “The Doors,” in which he portrayed Jim Morrison) and a crime film (Michael Mann's 1995 masterpiece “Heat”).
One of his less lucrative pursuits included his one-man stage show “Citizen Twain,” about Mark Twain, whose work he deeply admired and referenced frequently in interviews.
In all, Kilmer's films made nearly $2 billion at the global box office, according to Comscore.
Throughout his career, the roles that Kilmer played possessed an iconic quality while underscoring the way his career frequently zigged at the point where it could have zagged, to his detriment if not in terms of the quality of the work but how Hollywood and the public perceived him.
Kilmer enthusiastically agreed to star as Batman, for example, but quickly soured on that experience, passing on another sequel in favor of the forgettable reboot “The Saint.”
Similarly, he took a role in “The Island of Dr. Moreau” in order to play opposite Marlon Brando, only to clash with director John Frankenheimer (exchanges caught on camera) and be disappointed when a detached Brando periodically refused to come to the set, letting a stand-in take his place.
He reflected on his career and life in the 2021 documentary “Val” through interactions he had videotaped with his family and on film sets for years, including behind-the-scenes footage from “Tombstone” and the “Top Gun” cast partying after hours (“We were all at the start of our careers,” Kilmer recalled).
At the time, Kilmer was recovering from throat-cancer surgery, so his son, Jack, read the actor's written narration — sounding uncannily like his father.
Kilmer frequently expressed about his desire to work with certain directors, showing audition videos that he shot for parts he failed to land in “Goodfellas” and “Full Metal Jacket,” seeking to impress directors Martin Scorsese and Stanley Kubrick, respectively. He also documented his laborious auditioning and preparation process for portraying Jim Morrison in “The Doors.”
Ultimately, in the documentary, he expressed excitement about all that would come next.
“I have behaved bizarrely to some. I deny none of this and have no regrets because I have lost and found parts of myself that I never knew existed. I am blessed,” he said in the movie.
Kilmer identified as a Christian Scientist, opening up in interviews about his faith and beliefs.
In a 2020 interview with Men's Health, the actor spoke about illness through the lens of his faith.
“Well, something that was reaffirmed to me – on such a level, it was almost shocking – was a sense of universal love, a kind of power and a different sense of love. It was coming into my consciousness and my body while I was at the hospital,” he said, going on to add that he didn't “believe in death.”
Kilmer is survived by his two adult children, Jack and Mercedes, from his marriage to “Willow” co-star Joanne Whalley.
The younger Kilmers are both actors and have been involved in projects with their father.
Kilmer co-starred with Mercedes in the 2020 film “Paydirt” and, in addition to voicing the “Val” documentary, Jack lent his voice to his father's character on the Disney+ “Willow” series.
Despite his condition at the time of the 2021 documentary, and tragedies that included the death of his younger brother at age 15, Kilmer spoke of leading a “magical life.”
He said that he found the silver lining even as he endured fans asking for the same “Top Gun”-inspired “You can be my wingman” autograph over and over at Comic-Con.
“I feel grateful,” he said.
CNN's Tricia Escobedo and Frank Pallotta contributed to this reporting.
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The Pentagon has sent at least six B-2 bombers – 30% of the US Air Force's stealth bomber fleet – to the Indian Ocean island of Diego Garcia, in what analysts have called a message to Iran as tensions once again flare in the Middle East.
The deployment comes as US President Donald Trump and his defense chief Pete Hegseth warn of further action against Iran and its proxies, while US jets continue to attack the Tehran-backed Houthi rebels in Yemen.
Images taken by private satellite imaging company Planet Labs on Tuesday show the six US bombers on the tarmac on the island, as well as shelters that could possibly conceal others. Tankers and cargo aircraft are also at the island airbase, a joint US-British base which is 3,900 kilometers (2,400 miles) from Iran's southern coast.
Planet Labs images from Sunday show four B-2s and six support aircraft on the Diego Garcia tarmac.
Without mentioning the B-2s directly, Pentagon spokesperson Sean Parnell confirmed that the US military is sending additional aircraft and “other air assets” to the region to improve America's defensive posture in the region.
“The United States and its partners remain committed to regional security … and are prepared to respond to any state or non-state actor seeking to broaden or escalate conflict in the region,” Parnell said.
CNN military analyst Cedric Leighton said the placement of the highly sophisticated, $2 billion warplanes was a signal to US adversaries.
“The deployment of these B-2s is clearly designed to send a message – perhaps several messages – to Iran,” said the former US Air Force colonel.
“One of them could be a warning to cease supporting the Houthis in Yemen. Another message the Trump administration might be sending to Iran is that it wants a new nuclear deal (to replace the ‘bad' deal Trump withdrew the US from in his first term) and if Iran doesn't start to negotiate with the US the consequences could be the destruction of Iran's nuclear weapons program,” Leighton said.
Trump began ramping up military action against the Houthis in mid-March, with airstrikes that killed at least 53 people and wounded almost 100 others in Yemen, according to the Houthi-run Health Ministry.
Strikes have continued since, as Houthis threaten US warships in the region, in attacks that the militants say are in solidarity with Gaza as it faces bombardment by Israel, a key US ally.
In disclosures that caused a major controversy for the Trump administration, Hegseth shared sensitive information last month on pending military strikes against Houthi militants on an unsecured group chat with top national security officials — a chain to which The Atlantic's editor-in-chief Jeffrey Goldberg was accidentally added.
Trump, in a Tuesday post on his social media platform Truth Social, on Tuesday threatened more could be coming.
“Stop shooting at U.S. ships, and we will stop shooting at you. Otherwise, we have only just begun, and the real pain is yet to come, for both the Houthis and their sponsors in Iran,” Trump posted.
Trump has also been pushing Iran to make a deal over its nuclear capabilities, saying on March 19 that he would give Tehran two months to come to an agreement or face the consequences.
There “are two ways Iran can be handled: militarily, or you make a deal. I would prefer to make a deal, because I'm not looking to hurt Iran,” Trump told Fox News last month.
But Iran this week rejected any direct negotiations.
Defense Secretary Pete Hegseth “continues to make clear that, should Iran or its proxies threaten American personnel and interests in the region, the United States will take decisive action to defend our people,” Parnell said.
Military aviation analyst Peter Layton told CNN that the six-bomber deployment to Diego Garcia is likely focused beyond possible Houthi targets.
“Six is a serious number. For Houthi deeply buried targets, two or maybe three, but six B-2s is a major effort,” Layton said.
Leighton, the CNN military analyst, said the B-2 can carry the Massive Ordnance Penetrator, “a 30,000-pound bomb that is designed to destroy what we would call hardened and deeply buried targets.”
“Such targets would potentially include Iranian nuclear and weapons storage facilities,” the former US Air Force officer said.
Layton, the aviation analyst, noted that the six B-2s likely represent the entire deployable fleet of the aircraft.
“I assume there are one or two at home for training and another few standing nuclear alert. Rest in maintenance,” said Layton, a former Royal Australian Air Force pilot and now visiting fellow at the Griffith Asia Institute.
Parnell, the Pentagon spokesperson, said the aircraft carrier USS Harry S. Truman, which has been carrying out strikes on Houthi rebels in Yemen, will stay in the region through this month, though its deployment there was scheduled to have ended at the end of March.
CNN also previously reported that the USS Carl Vinson aircraft carrier would move to the Middle East after finishing an exercise in the Asia-Pacific region.
Hegseth “also ordered the deployment of additional squadrons and other air assets that will further reinforce our defensive air-support capabilities,” Parnell said. It's unclear what squadrons or assets will be moving to the region.
Parnell added that the Nimitz Strike Group is deploying to the Western Pacific “to preserve our warfighting advantage in the Indo-Pacific.”
Leighton said that the presence of the B-2s on Diego Garcia would be noted across Asia.
“It's unlikely the deployment of six B-2s to Diego Garcia is meant to deter actions by other powers, such as China or Russia, but they are surely taking note of this deployment as well. Of course, we can't forget that Iran is an ally of those two countries,” he said.
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The Environment Agency (EA) has launched a comprehensive review into shipments of waste tyres from the UK to India.
Last week, BBC File on 4 Investigates heard that millions of these tyres - sent for recycling - were actually being "cooked" in makeshift furnaces, causing serious health problems and environmental damage.
The pressure group Fighting Dirty has threatened legal proceedings against the EA over what it called a "lack of action" over the issue of tyre exports.
The EA has asked the group to wait until its own review is complete, and it has also asked File on 4 Investigates to share the evidence from its investigation.
The UK generates about 50 million waste tyres (nearly 700,000 tonnes) every year. According to official figures, about half of these are exported to India, supposedly to be recycled.
But BBC File on 4 Investigates revealed that some 70% of tyres exported to India from the UK and the rest of the world are being sent to makeshift industrial plants, where they are "cooked" in order to extract steel, small amounts of oil as well as carbon black - a powder or pellet that can be used in various industries.
Conditions at these plants - many of which are in rural backwaters - can be toxic and harmful to public health, as well as potentially dangerous.
In January, two women and two children were killed in an explosion at a plant in the western state of Maharashtra, where European-sourced tyres were being processed.
A BBC team visited the site and saw soot, dying vegetation and polluted waterways around. Villagers complained of persistent coughs and eye problems.
Following the broadcast, the Department for the Environment, Food & Rural Affairs (Defra) told BBC File on 4 Investigates that officials and lawyers within the EA were "very keen" to investigate the claims made in the programme, including any potential criminal activity.
In a letter seen by the BBC, lawyers for the EA said that our investigation would be carefully considered as part of a review it has launched into its approach to waste tyre shipments.
They added that the EA has been working to engage the relevant environmental authorities in India on this issue and is taking steps to arrange a delegation to meet with officials later this year.
Fighting Dirty founder Georgia Elliott-Smith, who has been in correspondence with the EA over this issue since 2023, said it was a "major victory" for the group and that "the government must stop turning a blind eye to the illegal and immoral activity".
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The bill seeks to change how properties donated by Indian Muslims over centuries are governed.
Bin collections are currently delayed due to strike action that has been ongoing for weeks.
Twenty-one people were killed in an explosion in a firecracker warehouse in Gujarat state.
Bajinder Singh, a self-styled pastor with millions of followers, was convicted for raping a woman in 2018.
India is one of the countries which is most vulnerable to heat, with hundreds dying each year.
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Violent protests took place in Nepal last week as pro-monarchy groups demanded the return of former King Gyanendra Shah to power, 16 years after the monarchy was abolished in the mountainous nation.
Kathmandu, the capital of Nepal, saw twin protests on March 28. The rally led by the United People's Movement Committee, a coalition supporting monarchy, devolved into clashes with security forces and resulted in the deaths of at least two people, including a journalist.
Another rally, organized by several republican political parties that formed the Socialist Alliance to “defeat regressive and reactionary forces,” processed peacefully.
Two different locations were allocated for rallies. However, the pro-monarchy rally got out of control. Protesters burned private homes, vehicles, and media houses, and attacked journalists as well as the offices of political parties. Pro-monarchy groups also looted supermarkets, homes, and department stores.
According to the Ministry of Home Affairs, at least 80 people were injured during the protest, including 15 police officers.
The Nepal Police arrested around 111 people, including Rabindra Mishra, the former editor-in-chief of the BBC Nepali Service, who entered Nepalese politics in 2017 and formed the Rastriya Prajatantra Party (RPP).
Mishra and RPP General Secretary Dhawal Shamsher Rana, who was also arrested during the protest, have been charged with treason by the Kathmandu District Court. Both Mishra and Rana were accused of inciting the protesters.
The chief coordinator of the pro-royalist movement, Nawaraj Subedi, an 87-year-old politician who was a leader in the country's Panchayat system, which was established by King Mahendra in 1960 and remained in force until the 1990s, was placed under house arrest.
Security agencies also initiated a search of Durga Prasai, labeled by local media as the chief commander of the ongoing royalist movement. Prasai, a controversial businessman who has been implicated in a bank loan fraud, allegedly provoked the crowd and triggering widespread stone-pelting, according to witnesses. Prasai later released a video apologizing for the incident, claiming he was not hiding.
The committee behind the royalist movement was formed earlier in March, after thousands of people joined a demonstration in the capital in support of restoring the monarchy with the former king, Gyanendra Shah, as its head.
On February 18, the eve of Nepal's Democracy Day, Shah issued a statement from the city of Pokhara where he was staying, expressing concern that the country's long-term peace, stability, and sovereignty have started to erode in recent years. “Even though the reins of the state power have been in the hands of many repeatedly, or even though it has changed, our existing state system does not seem to be able to make any profound contribution to the happiness, peace, and prosperity of the nation,” he was quoted by local media as saying.
Upon his arrival in Kathmandu, pro-monarchy supporters gathered outside of Tribhuvan International Airport in and welcomed the former royal, giving pro-monarchy forces a signal for a coordinated political action.
Following the violence, the government led by K.P. Sharma Oli, who was sworn in as Nepal's prime minister in May last year, has signaled its intent to take action against pro-monarchy groups.
The government has reduced the security detail assigned to Gyanendra Shah and reshuffled the security team. Before the Kathmandu incident, 26 police officers were assigned to his security, but after the violence, the government reduced the number to 16.
According to government sources, further action may be taken against Shah. “The government is taking former King Gyanendra's activities seriously and might take further action within the next few days,” a senior government official told RT.
The ‘Arrest Gyanendra' campaign has gained momentum in social media following the protests. Despite differences on various issues, major political parties of the country share the same stance regarding Gyanendra's activities.
Ram Prasad Shrestha, a former member of the Judicial Council called for investigating former king's involvement in the movement and held accountable for the violence that occurred, if found guilty. “The current crisis has emerged because of the immunity he was granted,” he said.
Journalist Harihar Birahi, who served as a member of the Commission of Inquiry into the suppression of the second people's movement in 2006, also believes that these circumstances provide sufficient grounds to investigate Gyanendra's role in the movement.
Major political parties have demanded legal action against Shah. The Nepali Congress party, the main coalition partner in the government, has accused him of being responsible for the March 28 violence in Kathmandu. “The kind of activities that took place in the name of royalism – former King Gyanendra Shah is fully responsible for them. He should take responsibility for the actions carried out under his direct influence,” Ram Saran Mahat, a leader of the Nepali Congress, said.
Pushpa Kamal Dahal ‘Prachanda' – the leader of the Communist Party of Nepal (Maoist Centre) and former prime minister – stated that the former king was directly involved in the events that unfolded over the past six months. He added that political parties and the government could not take a lenient stance on him. “Gyanendra Shah cannot be allowed to go unpunished. The Nepali people are not ready for that. The government must take this seriously. It is now very clear that Gyanendra Shah is behind everything that has been happening,” Prachanda said, according to the Kathmandu Post.
Political analyst Jhalak Subedi, in a conversation with RT, suggested that the main plan was orchestrated under the leadership of the former king, who “has been preparing for the restoration of the monarchy for a couple of years.” Subedi also claimed that Gyanendra has support by “India's Hindus” and that Indian media has been “consistently giving priority” to the movement for the restoration of the Hindu state and monarchy in Nepal.
Indeed, a political controversy arose in Nepal in March, after Gyanendra supporters flashed posters with the image of India's Uttar Pradesh state chief minister Yogi Adityanath during their rallies in Kathmandu. Adityanath is known for viewing the neighboring country as a “Hindu state.” According to Indian media, ties between Nepal's royal Shah dynasty and the Goraksh Peeth, a monastery of the Nath tradition, which is currently lead by monk-turned-chief minister Adityanath, date back centuries. On April 1, days after the deadly unrest Kathmandu, Nepal's ambassador to India, Shankar Sharma, met with Adityanath in India's Lucknow to discuss bilateral relations.
Gyanendra Shah assumed the crown in 2001 after his brother, then-King Birendra, was killed in a massacre at the Narayanhiti Royal Palace. The attack, allegedly carried out by Crown Prince Dipendra, resulted in the assassination of much of the royal family before Dipendra took his own life.
In February 2005, Gyanendra seized power from the civilian government – a move that made him deeply unpopular. He dissolved the government, banned news reporting, and authorized the army to arrest senior political leaders, journalists, trade unionists, human rights activists, and civil society members. All telephone and internet connections were cut.
In response, the parliamentary political parties and the Maoist rebels – then engaged in armed conflict – joined forces and launched a united movement against the monarchy. Following a 19-day people's uprising, Gyanendra relinquished power to political parties in 2006. Two years later, the Constituent Assembly formally abolished the monarchy, forcing Gyanendra to vacate the Narayanhiti Palace.
The government subsequently allocated Nagarjuna Palace, located seven kilometers west of the capital, as his residence. Since then, he has traveled to various districts, organizing public engagement programs. In recent months, however, he has become increasingly active in campaigning for the restoration of the monarchy.
Only around 5% of Nepal's parliamentarians advocate for monarchism. They belong to the right-wing Rastriya Prajatantra Party, which campaigns for reinstating Nepal as a Hindu kingdom.
Political analysts suggest that public frustration with the government's poor service delivery, corruption, and unemployment has created dissatisfaction. Yubaraj Ghimire, a senior Nepali journalist, suggested that in recent times, political parties had failed to deliver and have become extremely corrupt.
Tanka Karki, a political analyst and former Nepali ambassador to China, believes, however, that former king wrongly believed he had widespread public support. “The only alternative to democracy is democracy itself; the restoration of the monarchy is not possible,” Karki told RT. “Shah made a daring but unsuccessful attempt.” He suggested that the events of the past week provided lessons to all political parties in the country.
Subedi believes that former King Gyanendra and his movement weakened after the March 28 Kathmandu incident. “The restoration of the monarchy is not possible. I see it facing suppression once again,” he said.
By Mukesh Pokhrel, a journalist based in Kathmandu, Nepal
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US Senator Cory Booker has broken the record for the longest speech ever delivered in the Senate.
The New Jersey Democrat's marathon address, a symbolic protest against President Donald Trump, in which he warned of a "grave and urgent" moment in American history, ended after for 25 hours and four minutes.
Although it was not a filibuster - a speech designed to obstruct passage of a bill - it held up legislative business in the Republican-controlled Senate. The rules for such speeches require a speaker to remain standing and forgo bathroom breaks.
The previous record was held by Republican Senator Strom Thurmond who, when still a Democrat in 1957, spoke for 24 hours and 18 minutes against the Civil Rights Act.
Booker said he would speak for as long as he was physically able as he began his address at around 19:00 local time on Monday evening. He concluded at 20:06 on Tuesday.
The 55-year-old, who is the fourth-ranking Democrat in the chamber, filled some of the time reading letters from constituents, who said they had been harmed by President Trump's policies.
The former presidential candidate also ran out the clock by discussing sports, reciting poetry and taking questions from colleagues.
Booker, who is African-American, spoke of his roots as the descendant of both slaves and slave-owners.
"I'm here because as powerful as he was, the people are more powerful," he said, referring to segregationist Thurmond's record-setting address 68 years ago.
As he reached the milestone, Booker said he was going to "deal with some of the biological urgencies I'm feeling".
He was able to give his jaw much-needed respite during the speech by taking questions from colleagues, including Senate minority leader Chuck Schumer of New York, Senator Dick Durbin of Illinois and Senator Kirsten Gillibrand of New York.
The Democratic Party, currently out of power in the White House, Senate and House of Representatives, rallied behind Booker's symbolic act of protest.
Booker's speech is also the longest in the Senate since a 21-hour filibuster in 2013 by Texas Senator Ted Cruz, a Republican, against Obamacare.
Cruz told CBS, the BBC's US partner, that a filibuster is a challenging physical feat.
For his own protest, he wore comfortable shoes and tried to drink as little water as possible - an approach he described as "nothing in, nothing out".
Kirill Dmitriev is the most senior Russian official to visit Washington since the war began.
The ex-president of Costa Rica has compared Donald Trump's behaviour with that of a Roman emperor.
The result dashes Democrats' hopes of pulling off a major upset but they closed the gap.
More than $100m (£77m) was spent by the candidates and their allies, including $20m from Elon Musk.
Tariffs are a key part of Trump's political vision, but economists fear they could spark a trade war.
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A new lawsuit filed Tuesday accuses tech billionaire Elon Musk and his super PAC of failing to pay canvassers who helped his pro-Trump political operation in 2024.
During the final stretch of the 2024 campaign, Musk and his political group, America PAC, offered $100 to registered voters in battleground states who signed a petition supporting free speech and gun rights, and another $100 for every additional voter they convinced to sign the petition. The goal was to excite conservative voters and elect then-candidate Donald Trump – who narrowly won Pennsylvania in November.
A man from the Philadelphia suburbs – who filed the lawsuit anonymously because he says he fears retribution – claims he is owed $20,000 for gathering signatures in Pennsylvania. The lawsuit claims there could be at least another 100 victims of Musk's alleged breach-of-contract. This comes after press reports about allegedly late or missing payments from the super PAC around the presidential election.
“This lawsuit is about keeping promises,” the man's attorney, Shannon Liss-Riordan, told CNN. “He was expecting to be able to pay his bills because of this promise. He was pounding the pavement during the campaign because Elon Musk asked him too. He believed in Elon Musk.”
America PAC spokesman Andrew Romeo denied wrongdoing when asked about the lawsuit.
“America PAC is committed to paying for every legitimate petition signature, which is evidenced by the fact that we have paid tens of millions of dollars to canvassers for their hard work in support of our mission,” Romeo said in a statement, adding that the pro-Trump group is “committed to rooting out fraud” and has “the right to withhold payments to fraudsters.”
In the past week, Musk revived his 2024-style giveaways in Wisconsin, offering $1 million cash prizes as he campaigned in support of a conservative candidate for the state Supreme Court.
Musk and his super PAC denied claims during the 2024 campaign that their cash giveaways to voters violated federal laws against vote-buying or that it operated an illegal state lottery.
The Justice Department warned the super PAC in October about its $1 million sweepstakes for registered voters. And the Philadelphia district attorney, a Democrat, tried and failed in state court to get the daily giveaways shut down because, he claimed, it was an unlawful operation.
Liss-Riordan, who filed the latest lawsuit Tuesday in Pennsylvania federal court, has previously sued X, the Musk-owned social media platform formerly called Twitter, in a case about Musk's takeover of the company. She was also involved in an unsuccessful effort to remove Trump from the 2024 ballot in Massachusetts because of the US Constitution's “insurrectionist ban.”
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The Rev. W. J. Mark Knutson, right, walks with an El Salvadoran immigrant at the Augustana Lutheran Church on Thursday, Jan. 9, 2025, in Portland, Oregon. (AP Photo/Jenny Kane, File)
As many as four in five immigrants at risk of deportation from the United States are Christian, according to a new report that calls on their fellow believers to consider the impact of the Trump administration's aggressive deportation policies.
The report says about 10 million Christians are vulnerable to deportation and 7 million U.S. citizens who are Christian live in households where someone is at risk of deportation.
The report, under the auspices of major Catholic and evangelical organizations, draws on a range of data, including percentages of religious affiliation in various migrant and national populations and on an advocacy group's analysis of U.S. census data on migrants.
“Though we're deeply concerned about fellow Christians, we're not exclusively concerned with immigrants who happen to share our faith,” said Matthew Soerens, vice president of advocacy and policy at World Relief, an evangelical humanitarian organization that cosponsored the report.
“As Christians, we believe that all people, regardless of their religious tradition or nationality, are made in God's image with inherent dignity,” Soerens said in a video statement. But he added that many Christians in the U.S. may not realize that most of those who could be deported share their faith.
Other groups that helped produce the report include the National Association of Evangelicals, the U.S. Conference of Catholic Bishops' Committee on Migration and the Center for the Study of Global Christianity at Gordon-Conwell Theological Seminary in Massachusetts. While the report doesn't advocate any political positions, it mainly seeks to raise awareness of the issue among Christians, and some of its sponsoring groups have individually advocated for reforms that would give some categories of immigrants a path to legal status.
Immigrants at risk of deportation range from those who crossed the border illegally to those who may have some sort of legal status that could be revoked. For example, the Trump administration has taken steps to end temporary protected status, held by many from Venezuela and Haiti, as well as humanitarian parole that had been granted for others from those troubled countries as well as Cuba and Nicaragua.
President Donald Trump enjoyed wide support from certain Christian blocs in all three of his campaigns. In 2024, he was supported by about 8 in 10 white evangelical Christian voters, about 6 in 10 white Catholics and just over half of Latino evangelicals, according to AP VoteCast, a sweeping survey of more than 120,000 voters.
While the report doesn't directly refer to that support, it says it seeks to raise awareness of the potential impact of Trump's immigration crackdown.
Even the fear of deportation could cause people to avoid going to public places — such as worship services. In an era when a growing number of people in the U.S. don't have a religious affiliation, many immigrants who are Christian have helped reenergize churches and spur their growth, said Walter Kim, president of the National Association of Evangelicals.
“They're coming from parts of the world where the church is actually thriving,” Kim said. “Not only are they bringing that thriving faith and contributing to America, they're also contributing to the vibrancy of the church in America.”
Mass deportation would amount to a government-fostered “church decline strategy,” Kim said.
Kim said his organization has long advocated for reforms that would distinguish between those convicted of violent crimes and “the much larger share of immigrants who are contributing to our communities and to our churches, and who are serious and eager” to stay in the country.
Many studies have found immigrants are less drawn to violent crime than native-born citizens.
The recent report said Catholics in particular represent more than half of all those vulnerable to deportation in the United States, noted Bishop Mark Seitz, chair of the Committee on Migration of the bishops' conference.
The deportations would likely separate family members, Seitz said.
“We know the impact of tearing apart the family unity and also the tremendous threats that are faced by people who are summarily deported to their home countries, which they fled in the first place because of the tremendous threats they were living under there,” said Seitz, who heads the Diocese of El Paso, Texas.
They face danger from government oppression and organized crime in their home countries, Seitz said.
“People are going to die if this deportation effort continues at the level it is,” he said.
The report's methodology included calculating the percentages of Catholics, evangelicals and other Christian groups in the countries from which immigrants originated, based on self-reported affiliations. The report then applied those percentages to immigrant populations within various categories of immigrants.
While such methods include numerous assumptions, many regions of origin for major immigrants and refugee groups, including Latin America, sub-Saharan Africa and Ukraine, have large Christian populations.
Associated Press religion coverage receives support through the AP's collaboration with The Conversation US, with funding from Lilly Endowment Inc. The AP is solely responsible for this content.
Copyright 2025 The Associated Press. All Rights Reserved.
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A massive wave of job cuts got underway at US health agencies Tuesday, with some employees receiving early-morning emails saying their jobs were eliminated and some unable to access the building when they arrived at work.
It was not immediately clear how many employees had received notice Tuesday morning. The US Department of Health and Human Services has not responded to CNN's request for comment.
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HHS Secretary Robert F. Kennedy Jr. said last week that 10,000 full-time employees would be cut on top of thousands who had already left and probationary employees currently on leave. He said the changes would make fighting chronic disease the priority and reduce “bureaucratic sprawl.” Kennedy promised that the department would do more with less.
After weeks of worry from agency staffers, job cuts — known as a reduction in force, or RIF — were sweeping across offices at multiple agencies, hitting leadership, longtime staffers, scientists, administrators and communications staff.
“It's a bloodbath,” one US Food and Drug Administration employee said.
“These cuts to agency experts and programs leave our country less safe, less prepared and without the necessary talent and resources to respond to health threats,” Dr. Mandy Cohen, director of the US Centers for Disease Control and Prevention during the Biden administration, said Tuesday.
In a post on LinkedIn, former US Food and Drug Administration Commissioner Dr. Robert Califf said, “The FDA as we've known it is finished, with most of the leaders with institutional knowledge and a deep understanding of product development and safety no longer employed.”
Califf told CNN on Tuesday that “If we let down our guard and don't do a good job reviewing, we're going to unleash some things that are really dangerous into the population. Unless there's some super plan, there's going to be an effect on safety, because it takes whole teams of people to monitor safety of products, and the timetables for product review will probably be delayed.”
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Califf said he was dismayed to see how federal workers were being treated.
“This is a sad and inhumane way to treat people,” he said. “It's different when you're a company and you're out of money and you can't pay people, but the federal government can pay people and do things in an orderly, respectful fashion – and not have them end up in line trying to get to work and have their badges not work as a way to fire them.”
Sens. Bill Cassidy, R-Louisiana, and Bernie Sanders, I-Vermont, of the Senate Health, Education, Labor, and Pensions Committee announced Tuesday that they were inviting Kennedy to a hearing April 10 about the restructuring of HHS.
“This will be a good opportunity for him to set the record straight and speak to the goals, structure and benefits of the proposed reorganization,” Cassidy said in a statement.
Sanders was also among dozens of senators who wrote a letter to Kennedy on Tuesday to outline areas of the health care system that will be affected by the cuts and to request answers about the number of employees affected, the method of communication and any analysis on the impacts to programs and activities.
“You continue to deny visibility to the American public, despite your oft-repeated commitment to ‘radical transparency.' You have promised that HHS will do more for the American people, at a lower cost to the taxpayer, yet you have not provided anything to substantiate these claims, despite repeated requests from Congress to do so,” the letter says.
Kennedy said in a social media post that the agency is being “recalibrated to emphasize prevention, not just sick care.”
“This is a difficult moment for all of us at HHS. Our hearts go out to those who have lost their jobs,” he wrote. “But the reality is clear: what we've been doing isn't working.”
The totality of the cuts was not clear Tuesday, with many workers waiting for emails — or waiting to get into buildings, uncertain whether their badges would work. Even supervisors weren't sure who was being eliminated, one senior official at the CDC said.
Cuts at the CDC slashed divisions that work on workplace health and safety, HIV, injury prevention, reproductive health, smoking and violence prevention, among others. Despite Kennedy's focus on chronic diseases, CDC employees described broad cuts at the agency's National Center for Chronic Disease and Health Promotion.
The offices at the CDC and the National Institutes of Health that handle Freedom of Information Act requests were also cut. The FDA lost many of its FOIA officers, but a few remained, including some who were working on litigation, according to a source familiar with the circumstances who spoke on the condition that they not not be named for fear of reprisal.
Several senior leaders at the CDC were reassigned to other roles, and some were told they would have to relocate. At NIH, Dr. Jeanne Marrazzo, director of National Institute of Allergy and Infectious Diseases, was also offered reassignment to the Indian Health Service and would have to move to an area such as Alaska, New Mexico or Minnesota.
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At the Centers for Medicare and Medicaid Services, the entire Office of Equal Opportunity and Civil Rights – which handles issues of diversity, civil rights and a supportive work environment – was let go, according to a former HHS employee.
The notices sent to staffers in the office suggested that they contact the former director of the office if they had a complaint — but that director died late last year, Karen Shields, a former CMS executive, posted on LinkedIn on Tuesday morning. CNN has obtained a copy of the notice.
“The dissolution of the office is disturbing but was expected. The referral to a woman who died over four months ago is a result of a significant lack of collaboration and consultation with the career team at the agency who would have helped to prevent this disgrace,” Shields wrote.
Also, CMS cut all or nearly all of the Office of Minority Health, the former HHS employee said. The website of the office, which aims to improve the health of racial and ethnic minority groups through health policies and programs, was not working Tuesday, although it had been operational as recently as Saturday, according to web archives.
The office, which was created by Congress, also focused on rural health, former CMS Administrator Chiquita Brooks-LaSure said Friday.
“We have health disparities, and we need to be measuring what is happening so that we can address those,” she said, noting that the office looked at issues such as diet and diabetes.
At the FDA, staffers in the Office of New Drugs, the Office of Policy & International Engagement and the Office of Regulatory Programs were among those receiving notice. The agency's entire press office - the group that shares material with news media - received notice and was put on administrative leave, according to a person familiar with the cut who declined to be named because they weren't authorized to speak publicly about it.
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A RIF notice received by one FDA employee on Tuesday included performance ratings that were incorrectly low, meaning the severance pay offered was lower than it would be for the ratings they previously received, the employee told CNN.
Multiple other colleagues who received the RIF had the same issue, the FDA employee said, making them uncertain whether they should sign the inaccurate paperwork. The employee said they couldn't tell if the inaccuracy was deliberate or an honest mistake.
“Everybody's got families and mortgages and real-life concerns,” the source said, speaking on the condition of anonymity because they were concerned that being named would hurt their job prospects.
Also terminated was the entire staff of the Low Income Home Energy Assistance Program, or LIHEAP, according to Mark Wolfe, executive director of the National Energy Assistance Directors Association. The program provides about $4 billion to help millions of Americans with their heating and cooling bills.
“It will definitely hamper program operations,” Wolfe said, noting that he doesn't see how the agency can “allocate the remaining $387 million in funds for this year without federal staff. The program could well grind to a halt.”
Staff of the HHS Administration for Children and Families – which provides support for child care, child welfare, early childhood development, family violence prevention, refugee resettlement, homeless teens and Head Start programs, among others – also received notice Tuesday.
“We're devastated for our colleagues and friends who have lost their careers, and we're really feeling primarily devastated for our programs that we care so much about. In the regional offices, we're the ones who work directly with the people — the states, the territories, the tribes, the people that we serve — and we're just abruptly cut off from that work and our relationships,” said one employee in the administration who received notice Tuesday.
“Our concerns are that by gutting these offices, it can make child care less safe, less affordable.”
HHS announced last week that its reduction in force would cut 10,000 full-time employees in addition to 10,000 employees who've left voluntarily, shrinking the workforce from about 82,000 full-time employees to 62,000. About 5,200 probationary workers who have been in their positions less than a year or two were also terminated last month. Most are on temporary administrative leave as their fate winds its way through federal courts.
HHS said last week cuts would include:
Kennedy announced the cuts as part of a sweeping reorganization that would move or cut several parts of the federal health workforce, including the creation of a new Administration for a Healthy America, which it said would combine the Office of the Assistant Secretary for Health, the Health Resources and Services Administration, the Substance Abuse and Mental Health Services Administration, the Agency for Toxic Substances and Disease Registry and the National Institute for Occupational Safety and Health.
CNN's Ross Levitt and Rene Marsh contributed to this report.
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A Chinese national has been sentenced to federal prison for exploiting a little-known loophole in an effort to ferry a dozen illegal migrants to a U.S. territory.
Zhongli Pang, 36, pleaded guilty to conspiracy to transport illegal immigrants and conspiracy to defraud the United States after attempting to smuggle 12 Chinese nationals to the territory of Guam, the Department of Justice (DOJ) announced on Tuesday.
"The arrest of Mr. Pang is a testament to HSI's continued efforts to deter the extremely dangerous and unlawful movement of illegal aliens within the CNMI," Homeland Security Investigations (HSI) Special Agent in Charge Lucy Cabral-DeArmas said. "By holding criminals accountable, HSI works with state and local authorities to thwart future violations, ensuring the safety of our communities through continuing partnerships."
INFLUX OF ILLEGAL CHINESE MIGRANTS THREATEN US TERRITORY, ISLAND MUST SHOW ‘STRENGTH OF THE NATION'
The island of Saipan is approximately 100 miles from Guam by sea. (Fox News Graphics)
Pang attempted to exploit a little-known loophole allowing Chinese visitors to travel to the Northern Mariana Islands without a visa, according to prosecutors.
However, Chinese migrants will occasionally take advantage of the visa waiver to sail to Guam, where a U.S. visa is required.
In June 2024, Pang, along with several co-conspirators, purchased a boat for $33,000 and plotted to ferry 12 Chinese nationals from the island of Saipan to Guam. Due to Pang's limited boating experience and the risk of traveling approximately 100 miles by sea, the overcrowded vessel ran out of fuel and required rescue by the U.S. Coast Guard.
TOP REPUBLICAN SOUNDS ALARM ON US TERRITORY BEING 'HIGHLY VULNERABLE' TO CHINESE MISSILES
The U.S. territory of Guam is an attractive target for Chinese migrants given its proximity to the Northern Mariana Islands. (iStock)
The DOJ and an attorney for Pang did not immediately respond to Fox News Digital's request for comment.
A federal judge sentenced Pang to three months in federal prison with credit for time served and 50 hours of community service. The case comes as U.S. officials are cracking down on the flow of illegal immigrants through U.S. territories, such as Guam.
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In 2023, HSI announced the launch of a Mariana Islands Border Enforcement Security Task Force in an effort to combat threats looking to enter the U.S. through the islands. The Mariana Islands and Guam can be an attractive entry point for criminals given their proximity to Asia.
HSI did not immediately respond to Fox News Digital's request for comment.
"We will continue to target illegal aliens unlawfully traveling between the CNMI and Guam," U.S. Attorney Shawn Anderson said. "The risk to personal safety is substantial. Those interdicted also face imprisonment and immigration penalties. We urge PRC nationals to fully comply with the CNMI's parole program and return to China as agreed upon entry to Saipan. Enhanced enforcement efforts by DHS will result in the apprehension of those attempting to evade detection."
Julia Bonavita is a U.S. Writer for Fox News Digital and a Fox Flight Team drone pilot. You can follow her at @juliabonavita13 on all platforms and send story tips to julia.bonavita@fox.com.
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Denise Richards is opening up about the highs and lows of raising children with an ex.
During a recent appearance on the "Whine Down with Jana Kramer" podcast, the 54-year-old star discussed what it was like raising her two daughters with her ex-husband, Charlie Sheen, admitting on the show that their relationship could be better.
"Truthfully, it wasn't co-parenting. I parent my way. He parents his way, and there was no co-parenting," she said. "I have kids with him, and I'm not really — I'm friendly with Charlie, but I wish we were friends and that we could talk all the time and be able to be there really for each other with our girls. But that's just not the relationship we have. Maybe one day. But, I mean, right now, there's nothing. There's no discord between he and I, which is great."
The former couple tied the knot in 2002 and were married for just under three years before Richards filed for divorce in March 2005, when she was pregnant with their second daughter, Lola. They welcomed their first daughter, Sami, one year prior.
DENISE RICHARDS LETS HUSBAND CONTROL ONLYFANS CONTENT, SAYS SITE HELPS HER LAND MORE 'SEXY' ACTING ROLES
Denise Richards shared she and Charlie Sheen do not have a good co-parenting relationship. (Getty Images)
Following their divorce, Sheen went on to marry Brooke Mueller from 2008 to 2011, welcoming twin sons, Bob and Max, with her in March 2009. Despite no longer being married to Sheen, Richards made it a priority to make sure her daughters had a relationship with their dad and siblings.
"I was always an advocate of wanting everything to be great and be friends and blend the family because he had two boys with his wife after me," she continued. "I just didn't want the girls to feel like they had to choose sides."
She went on to say she always made the effort to invite Sheen to their daughters' birthday parties and family holiday celebrations, explaining she "thinks it's good for the kids to see that too."
Although their marriage did not work out, she said she "never talked bad about" Sheen when her children were around but says "now that they're older, they're discovering things" from the past on their own.
Denise Richards shares daughters Sami and Lola with Charlie Sheen. (Charles Sykes/Bravo via Getty Images)
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Richards and her family have recently put their lives on display in the new reality show, "Denise Richards & Her Wild Things." Sheen appeared on an April 1 episode of the show, when he sat down to dinner with Richards and their daughter Lola, during which she shared her fears about becoming a public figure.
"I'm scared of being famous. I'm scared of what people have to say," Lola told her parents.
"Don't read anything," Sheen said. "Don't read anything about it. Because if you don't read it, it doesn't exist."
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Denise Richards and her daughters are sharing their lives on a new reality show on Bravo. (Photo by Bruce Glikas/Getty Images)
"Denise Richards & Her Wild Things" airs Tuesday nights on Bravo.
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Lori Bashian is an entertainment production assistant for Fox News Digital.
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In a race that could have changed the makeup of the Wisconsin State Supreme Court, the liberal candidate prevailed with major judicial decisions coming.
MILWAUKEE — Democrats are celebrating a larger-than-expected victory in a high-profile and historically expensive election in battleground Wisconsin, in the first statewide ballot box contest since President Donald Trump's return to power in January.
Liberal-leaning Judge Susan Crawford topped conservative-leaning Judge Brad Schimel by roughly 10 percentage points – with some votes still being tabulated – to preserve the liberal majority on the Wisconsin Supreme Court, which is likely to rule going forward on crucial issues like congressional redistricting, voting rights, labor rights and abortion.
With a massive infusion of money from Democratic-aligned and Republican-aligned groups from outside Wisconsin, which turned the race into the most expensive judicial election in the nation's history, the contest partially transformed into a referendum on Trump's sweeping and controversial moves during the opening months of his second tour of duty in the White House.
Also front and center in the technically nonpartisan showdown was someone who, along with Trump, was not on the ballot: billionaire Elon Musk, the president's top donor and White House advisor, who inserted himself into the race.
LIBERAL-LEANING CANDIDATE WINS FIRST MAJOR STATEWIDE ELECTION OF THE YEAR
Elon Musk speaks during a town hall on Sunday, March 30, 2025, in Green Bay, Wisconsin. (AP Photo/Jeffrey Phelps)
"The people of Wisconsin squarely rejected the influence of Elon Musk, Donald Trump, and billionaire special interests," Democratic National Committee chair Ken Martin claimed.
And the DNC, looking ahead to next year's bigger contests in the 2026 midterm elections, called the showdown in Wisconsin a "bellwether race."
But Republicans came out on top in Tuesday's other marquee contests, holding control of two vacant congressional seats in twin special elections in red state Florida. The double-digit victories by the Republican candidates will give the GOP a little bit of breathing room in the House of Representatives, where the party is holding onto a very fragile majority as it aims to pass Trump's agenda.
REPUBLICANS HOLD CONTROL OF TWO VACANT CONGRESSIONAL SEATS IN THIS RED STATE
"The American people sent a clear message tonight: they want elected officials who will advance President Trump's America First agenda, and their votes can't be bought by national Democrats," Republican National Committee chair Mike Whatley argued.
The Democratic candidates in the two special congressional elections vastly outraised their Republican counterparts – a sign that the party's base is angry and energized – which forced GOP-aligned outside groups to pour money and resources into the races during the final stretch. And the Democratic candidates ended up losing by 15 and 14 points in districts that Trump carried by 37 and 30 points in last November's presidential election.
Democrats quickly spotlighted how the party "overperformed" in Florida. And the House Majority PAC, the top super PAC supporting House Democrats, touted that the results showed "that the political headwinds are firmly at our backs heading into 2026."
But Mike Marinella, spokesman for the National Republican Congressional Committee, argued that "Democrats just lit over $20,000,000 on fire in a doomed-to-fail effort to make two deep-red Florida districts competitive – and got blown out of the water in the most embarrassing way."
Republican Randy Fine, center, won the April 1, 2025, special election to fill the vacancy left by Mike Waltz's resignation to be Trump's national security advisor. (AP Photo/Phelan M. Ebenhack)
But the results in Florida, and especially Wisconsin, will likely give the Democrats a jolt, and validate their efforts to target Musk.
Musk, the chief executive of Tesla and SpaceX, who has taken a buzz saw to the federal government workforce as he steers Trump's recently created Department of Government Efficiency, dished out roughly $20 million in the Wisconsin race through aligned groups in support of Schimel.
And Musk, in a controversial move, handed out $1 million checks at a rally in Green Bay on Sunday evening to two Wisconsin voters who had already cast ballots in the contest and had signed a petition to stop "activist judges."
"I never could have imagined that I'd be taking on the richest man in the world, for justice in Wisconsin. And we won," Crawford said in her election night victory speech.
Senate Minority Leader Chuck Schumer, the top Democrat in the chamber, argued that Wisconsin voters "sent a decisive message to Elon Musk, Donald Trump, and DOGE by rejecting an extreme Republican for their Supreme Court: our democracy is not for sale."
"Anyone who counted Democrats out was dead wrong," he emphasized.
But Democrats have a serious brand issue right now.
The party's favorable rating sank to all-time lows in separate national polls conducted last month by CNN and NBC News. Those numbers followed a record low for Democrats in a Quinnipiac University survey in the field in February.
Additionally, the latest Fox News National poll indicated that congressional Democrats' approval rating is at 30%, near an all-time low. And Democratic activists are irate over their party's inability to blunt Trump's agenda.
And when it comes to normally low-turnout off-year elections and special elections, the party in power – which in the nation's capital is clearly the Republicans – often faces political headwinds.
"We'll get up to fight another day. But this wasn't our day," Schimel said in his concession speech.
Judge Brad Schimel concedes his election loss in Pewaukee, Wisconsin, on April 1, 2025. (Fox News - Paul Steinhauser)
And Wisconsin GOP chair Brian Schimming noted that "coming off a successful November, we knew the April elections would be challenging."
Republicans note that Democrats enjoyed a slew of special election victories in 2023 and 2024 before suffering serious setbacks in last November's elections.
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"Special elections are special for a reason, and not always useful canaries in the coal mines for what lies ahead," veteran Republican strategist Colin Reed told Fox News Digital. "While they can be used as a barometer for energy, they are also a reflection of the individual candidates whose names are on the ballots."
Reed argued that "the bigger challenge for the Democrats looking ahead is the lack of a vision or governing agenda beyond reflexive and blanket opposition to the White House and their continued positioning way outside the mainstream on a slew of commonsense issues."
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An American ICU nurse had a harrowing brush with death after her rental moped crashed during a vacation to Thailand, leaving her with a brain bleed, a broken collarbone and shattered teeth.
Sierra Fairhurst, 23, arrived at Boston Logan International Airport in Massachusetts on March 27 after two weeks in Thailand. According to a GoFundMe, Fairhurst was in a moped crash during a vacation in Thailand.
"Despite the long road ahead for her, this could have been completely different, and I'm so grateful to have her here, alive and her brain in working order," Zoe Rose, Fairhurst's mother, said in the most recent GoFundMe update.
"Her face can be fixed, her wounds will heal, bones can be repaired," Rose said. "She has the strength to overcome all of this."
MOTHER, 2 CHILDREN DEAD IN NYC AFTER SUSPENDED DRIVER ALLEGEDLY MOWS THEM DOWN
Sierra Fairhurst, 23, was severely injured in a moped crash during a trip with friends in Thailand. (GoFundMe)
Fairhurst rented a moped during her travels in the Southeast Asian country. She told Boston 25 that at one moment they were on the road, and the next they weren't.
She awoke in a foreign hospital with devastating injuries.
DELTA PLANE, AIR FORCE JET NEARLY CRASH IN ‘LOSS OF SEPARATION' DURING ARLINGTON NATIONAL CEMETERY FLYOVER
"I remember going to see the elephants, and we went zip-lining and all the excursions that we did, but that's it," she said. "My eye socket is broken, I almost lost my eye, my cheek bones and nose were broken, so that's all metal now."
After the accident, the 23-year-old nurse was taken to Krabi, on southern Thailand's west coast, for treatment. She was eventually transferred to a facility in Bangkok before returning home, according to GoFundMe.
Sierra Fairhurst was taken to Krabi, on southern Thailand's west coast, for treatment. (GoFundMe)
Sierra Fairhurst was taken via plane to Bangkok for additional surgery after her moped crash. (GoFundMe)
Rose and Fairhurst's brother, J, arrived in Thailand after the accident to help her. Her identification was lost in the aftermath of the accident, which complicated the process.
"In the confusion of the accident and her friends packing luggage to transfer with her to the next hospital, all of Sierra's identification is missing," Rose said. "She has no ID, social security card, or passport. It is imperative that once she is stable she gets to come home to Boston and receive the best care and recover at home with her family."
PLANE CRASH NEAR MINNEAPOLIS SENDS HOME UP IN FLAMES
With the help of the U.S. State Department, Fairhurst was given proper documentation to travel back to the U.S.
"I know she's very self-conscious in regard to the scarring on her face and missing teeth, but she still came out of this alive, and her brain is healing and doing well, and she's still beautiful," she told Boston 25.
Sierra Fairhurst suffered facial injuries, a brain bleed and multiple broken bones. (GoFundMe)
When she returned from her trip, Fairhurst went directly to a Boston hospital to be evaluated, and it was determined she did not need inpatient care.
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Fairhurst visited the wound clinic and oral maxillofacial surgeon and has appointments scheduled with her primary care provider, neurologist, ocular plastic surgeon, orthopedic surgeon and cosmetic dentist, according to the GoFundMe.
Sarah Rumpf-Whitten is a U.S. Writer at Fox News Digital.
Sarah joined FOX in 2021, where she has assisted on coverage of breaking and major news events across the US and around the world, including the fallout following the "Defund the police" movement, the assassination attempts on President Donald Trump's life and illegal immigration.
She has experience reporting on topics including crime, politics, business, lifestyle, world news and more. You can follow her on Twitter and LinkedIn.
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‘All-Star' panelists Mary Katharine Ham, Sabrina Singh and Josh Holmes discuss the potential impact of President Donald Trump's tariff agenda on ‘Special Report.'
President Donald Trump called out GOP Sens. Mitch McConnell and Rand Paul of Kentucky, Susan Collins of Maine and Lisa Murkowski of Alaska in an effort to apply public pressure before the Senate votes on a measure to scuttle his Canadian tariff policy.
The joint resolution would terminate the national emergency Trump declared regarding illicit drugs and Canada; in his executive order, Trump called for slapping tariffs on America's northern neighbor.
In a lengthy Truth Social post shortly before 1 a.m. on Wednesday, the president suggested that the four GOP senators have "Trump Derangement Syndrome."
"Mitch McConnell of Kentucky, Susan Collins of Maine, Lisa Murkowski of Alaska, and Rand Paul, also of Kentucky, will hopefully get on the Republican bandwagon, for a change, and fight the Democrats wild and flagrant push to not penalize Canada for the sale, into our Country, of large amounts of Fentanyl, by Tariffing the value of this horrible and deadly drug in order to make it more costly to distribute and buy," Trump declared.
TRUMP'S 11TH WEEK IN OFFICE SET TO FOCUS ON TARIFFS AS PRESIDENT TOUTS ‘LIBERATION DAY'
President Donald Trump walks toward Marine One on the South Lawn of the White House in Washington, D.C., on Friday. (Andrew Harnik/Getty Images)
"They are playing with the lives of the American people, and right into the hands of the Radical Left Democrats and Drug Cartels. The Senate Bill is just a ploy of the Dems to show and expose the weakness of certain Republicans, namely these four, in that it is not going anywhere because the House will never approve it and I, as your President, will never sign it. Why are they allowing Fentanyl to pour into our Country unchecked, and without penalty," he continued.
Trump blasted the four lawmakers as "disloyal" to the GOP.
"What is wrong with them, other than suffering from Trump Derangement Syndrome, commonly known as TDS? Who can want this to happen to our beautiful families, and why? To the people of the Great States of Kentucky, Alaska, and Maine, please contact these Senators and get them to FINALLY adhere to Republican Values and Ideals. They have been extremely difficult to deal with and, unbelievably disloyal to hardworking Majority Leader John Thune, and the Republican Party itself. MAKE AMERICA GREAT AGAIN!"
CANADIAN PRIME MINISTER MARK CARNEY SAYS ‘OLD RELATIONSHIP' WITH US ‘IS OVER' AMID TENSION OVER TRUMP TARIFFS
Paul is a cosponsor of the joint resolution.
Murkowski reportedly informed Politico's Lisa Kashinsky that she will vote for the resolution, while Collins has said she is "very likely" to back it, according to the outlet.
RAND PAUL ENDORSES TRUMP 3 MONTHS AFTER ELECTION DAY, ADMITS ‘I WAS WRONG'
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McConnell declared in an op-ed earlier this year that "tariffs are bad policy."
Alex Nitzberg is a writer for Fox News Digital.
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‘MAHA Mom' and former Sports Illustrated model Kristen Louelle Gaffney expresses her excitement about Robert F. Kennedy Jr. getting confirmed as Health and Human Services secretary on ‘Jesse Watters Primetime.'
MAHA mom and Sports Illustrated model Kristen Louelle Gaffney has been outspoken about her support for the confirmation of Robert F. Kennedy Jr. as HHS secretary.
Gaffney told Fox News Digital in an interview that she's excited for RFK Jr. to take a more active approach to healthcare, calling him the "white horse for moms."
She said the MAHA movement has created a community of Americans and parents who are "fed up with the system and seeing their kids sick, having diseases, behavioral issues and want to get to the common denominator of it."
EVERYTHING TO KNOW ABOUT MAHA
Gaffney is married to Ty Gaffney, a former NFL running back.
The couple has three children.
Kristen Louelle Gaffney revealed to Fox News Digital why she considers herself a MAHA mom. (Rodrigo Varela/Getty Images for Sports Illustrated Swimsuit)
"How can we live a proactive lifestyle to avoid things like dementia or cancer or any other diseases relating to behavioral or mental illnesses?" said Gaffney.
"What can we proactively do to be better so that it's not too late?"
The key to living longer, she said, lies in loving oneself by taking care of the mind, body, soul and the gut.
"Everything is connected. You have to follow your gut and heal your gut," Gaffney said.
FOOD SOLD AT GAS STATION EXPLODES INTO HEALTH-FOCUSED RESTAURANT CHAIN
"That's through food and supplementing as well," she said.
"I think a good supplement routine [is] staying active every day, getting sunlight and breathing fresh air. It really is a simple recipe, but the hard part is consistency."
Gaffney joked that after having three babies, she will never be in the same shape again, but said that healing begins on the inside and impacts other aspects of life.
Gaffney is married to Ty Gaffney, a former NFL running back. The couple has three children. (Kristen Louelle Gaffney)
"When I put in the work, I feel like a better person," she said. "I have more energy. I'm a better mom. I'm a better wife."
Gaffney said she stays as active as possible and always continues moving.
"If you don't use it, you lose it," she said.
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Gaffney is a big proponent of cooking at home for herself and her family.
"I think we overcomplicate things and think we need a chef and meal prep and expensive equipment. You just need to get sun and go walk outside for 20 minutes and cook your own dinner."
"When I put in the work, I feel like a better person."
Gaffney said that when she is grocery shopping, she avoids seed oils and dyes, making sure to look at each label of products she is purchasing.
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"I tell people, 'If you can't pronounce something on the ingredients, you should probably put it back," she advised.
Gaffney decided to lead the effort to combat ultra-processed foods by developing her own line of snacks for kids called Super True.
Gaffney developed her own line of healthy snacks for kids called Super True. (Super True; Kristen Louelle Gaffney )
The chocolate chip brownie and peanut butter banana chocolate chip bars are gluten-free, non-GMO, free of dairy and artificial ingredients or dyes and colored by nature.
Each bar has protein and fiber — and is sweetened with monk fruit and Stevia extract.
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Gaffney said it is critical to educate children about the different foods they are putting in their bodies.
"I always try to educate them [about] the 'why' instead of, 'No, you can't have soda.' [I say], 'No, you can't have soda because there's 60 grams of sugar in it and because that's going to lead to yucky cells growing in your body.'"
A look at the top-trending stories in food, relationships, great outdoors and more.
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Authorities are working to identify the people who were caught on video driving ATVs and dirt bikes inside the World War II memorial on the National Mall in Washington, D.C. (Credit: @MAalcofribas/X)
People on dirt bikes and ATVs were seen driving inside the World War II memorial on the National Mall in Washington, D.C., over the weekend.
Video posted on X showed at least three people driving around the outdoor memorial late Saturday night.
The United States Park Police is aware of the video, according to FOX 5 DC.
NYC VANDALS WHO DEFACED WWI STATUE, BURNED AMERICAN FLAG SHOULD 'LEARN THEIR HISTORY,' VETERANS GROUP SAYS
Authorities are working to identify the people who were caught on video driving ATVs and dirt bikes inside the World War II memorial on the National Mall in Washington, D.C., over the weekend. (@MAalcofribas/X)
"We are currently reviewing security footage to identify potential suspects. The National Park Service strongly condemns activities like the ones seen in the video that disrespect the solemnity of the National Mall's memorials," said National Park Service's Mike Litterst.
Veteran David Fields told FOX 5 that the acts seen in the video were "disgraceful."
PORTLAND MEMORIAL DEDICATED TO CITY'S FALLEN POLICE OFFICERS VANDALIZED: 'STARTLING AND UGLY'
"This is just as sacred as Arlington National Cemetery. And to have people riding motorcycles around there, was just a lack of respect," Fields said.
Laws in D.C. prohibit the use of ATVs on public streets and in the National Mall area, but the Metropolitan Police Department said officers won't pursue the vehicles because of the department's chase policy.
The World War II memorial in Washington, D.C., is located on the National Mall, which specifically prohibits ATVs. (Fox News)
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Earlier this month in the nation's capital, two Metro police officers were hit by an ATV rider in northeast D.C. That driver fled the scene.
Fox News Digital has reached out to the National Park Service for comment.
The hottest stories ripped from the headlines, from crime to courts, legal and scandal.
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A Maryland bill to establish a commission to study reparations – including financial restitution – is moving forward, as it is expected to clear its final hurdle in the House of Delegates, while the governor attempts to dodge questions about whether he supports the proposal.
The bill, a priority for the Legislative Black Caucus of Maryland, passed the Senate in the middle of last month before Crossover Day, which marks the unofficial deadline for legislative leaders in the General Assembly to move bills to the other legislative chamber that they plan to send to the governor for final approval.
The bill received a favorable vote in its assigned House committee on Friday, WBFF reported. It is expected to be voted on in the full House before the legislative session adjourns next week.
Senate Minority Whip Justin Ready, a Republican, told WBFF he does not understand why the reparations bill is being advanced as the state faces a $3.3 billion deficit, which is expected to increase even more to $6.7 billion by fiscal year 2028.
NTSB SAYS LACK OF 'VULNERABILITY ASSESSMENT' BY MARYLAND OFFICIALS PRECEDED DEADLY KEY BRIDGE COLLAPSE
Maryland Gov. Wes Moore has attempted to dodge questions about whether he supports the proposal to establish a commission to study reparations. (Andrew Harnik/Getty Images)
"We don't have the money right now to be exploring these options, period," Ready told the outlet. "[T]he issue of reparations, I'm sure elicits strong opinions, but the fact is, it's just something that's not financially feasible, whether you think it's a good idea or not."
"I question whether using taxpayer money would ever be appropriate in this context," he added. "Even going back to when reparations were paid to survivors of the Holocaust, they went after companies that were involved. Not after taxpayers."
The proposed commission is expected to initially cost Maryland taxpayers $54,500 annually, according to the nonpartisan Maryland Department of Legislative Services.
Similar reparations commissions have been created by state governments in California, Colorado, Massachusetts, New York and Illinois.
DEM GOV SAYS MD, WITH $3B DEFICIT, HAS BEEN DOING DOGE 'BEFORE ANYONE KNEW WHAT [IT] WAS'
Similar reparations commissions have been created by state governments in California, Colorado, Massachusetts, New York and Illinois. (Justin Sullivan/Getty Images)
Last year, the California Reparations Task Force released a report following a two-year study in which the state was called on to issue a formal apology for slavery and other racial injustices and to offer financial payouts. The report recommended a financial restitution formula that would provide eligible recipients with up to $1.2 million each, although state lawmakers have not yet held a vote to authorize the first payments.
Maryland Gov. Wes Moore, a Democrat, has dodged questions about his state's bill since it was introduced in January. He was asked at the time if he supported the measure, but turned his focus to economic priorities.
"Nah, we are going to work with the Maryland General Assembly on a whole collection of different issues," Moore told WBFF at the time. "Our focus is economic advancement; our focus is economic growth. It's making sure we can really meet people where they are, make life more affordable. That we are modernizing of state government."
The governor was asked again about the issue during his visit to celebrate the Baltimore Orioles' home opener on Monday afternoon at Pickles Pub. WBFF attempted to speak with him, but the requests were denied. The outlet reached out to the governor's office after his appearance at the pub, but his spokesperson's response did not address the questions about the reparations bill.
The proposed commission is expected to initially cost Maryland taxpayers $54,500 annually. (AP Photo/Steve Ruark)
Ready told WBFF that the bill could harm the national attention Moore has received over the past year.
"I don't think Gov. Moore would want this on his desk because I think it is a distraction to trying to get real problems solved," Ready said. "[T]here may be some people it motivates, in some way, but a lot of those are kind of on the fringes."
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If the law is enacted in its current form, the commission must provide its preliminary report by January 1, 2027, and a final report by November 1, 2027.
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Copyright 2025 The Associated Press. All Rights Reserved.
Toronto residents Douglas Bloomfield, left, and his son Phoenix, right, who are on vacation in Washington, hold a Canadian flag and an ice hockey stick to show their support for Canada regarding trade tariffs as they pose with with another visitor to the city wearing a mask of President Donald Trump in front of the White House in Washington, Thursday, March 13, 2025. (AP Photo/Ben Curtis)
Anger over the Trump administration's tariffs and rhetoric will likely cause international travel to the U.S. to fall even further than expected this year, an influential travel forecasting company said Tuesday.
Tourism Economics said it expects the number of people arriving in the U.S. from abroad to decline by 9.4% this year. That's almost twice the 5% drop the company forecast at the end of February.
At the beginning of the year, Tourism Economics predicted a booming year for international travel to the U.S., with visits up 9% from 2024.
But Tourism Economics President Adam Sacks said high-profile lockups of European tourists at the U.S. border in recent weeks have chilled international travelers. Potential visitors have also been angered by tariffs, Trump's stance toward Canada and Greenland, and his heated White House exchange with Ukraine President Volodymyr Zelenskyy.
“With each policy development, each rhetorical missive, we're just seeing unforced error after unforced error in the administration,” Sacks said. “It has a direct impact on international travel to the U.S.”
The decline will have consequences for airlines, hotels, national parks and other sites frequented by tourists.
Tourism Economics expects travel from Canada to plummet 20% this year, a decline that will be acutely felt in border states like New York and Michigan but also popular tourist destinations like California, Nevada and Florida.
The U.S. Travel Association, a trade group, has also warned about Canadians staying away. Even a 10% reduction in travel from Canada could mean 2.0 million fewer visits, $2.1 billion in lost spending and 14,000 job losses, the group said in February.
Other travel-related companies have noted worrying signs. At its annual shareholder meeting on Monday, Air Canada said bookings to the U.S. were down 10% for the April-September period compared to the same period a year ago.
Sacks said he now expects foreign visitors to spend $9 billion less in the U.S. compared to 2024, when international tourism to the country rose 9.1%.
“The irony is that the tariffs are being put in place to help right the trade deficit, but they're harming the trade balance by causing fewer international travelers to come and spend money here,” Sacks said.
Sacks said international arrivals had been getting close to returning to 2019 numbers, before the coronavirus pandemic halted most travel. Now he thinks they won't get back to that level until 2029.
Copyright 2025 The Associated Press. All Rights Reserved.
Copyright 2025 The Associated Press. All Rights Reserved.
Watch live as U.S. Senator Cory Booker addresses the Senate floor. Booker, a Democrat from New Jersey, has promised to speak “as long” as he's physically able on the Senate floor Monday night into Tuesday morning, criticizing the Trump administration.
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New Jersey Democratic Sen. Cory Booker held the Senate floor with a marathon speech that lasted 25 hours and 5 minutes, setting the record for the longest continuous Senate floor speech in the chamber's history. His feat of endurance was aimed at showing Democrats' resistance to President Donald Trump's sweeping actions.
Booker broke a record previously held by Strom Thurmond, a segregationist who filibustered for 24 hours and 18 minutes against the Civil Rights Act of 1957, according to the Senate's records.
Other news we're following today:
Florida Chief Financial Officer Jimmy Patronis speaks during a meeting between Gov. Ron DeSantis and the state cabinet at the Florida capitol in Tallahassee, Fla., on March 5, 2025. (AP Photo/Rebecca Blackwell, File)
The state's chief financial officer and Trump-backed candidate fended off a challenge from Democrat Gay Valimont.
Patronis was far outraised and outspent by Valimont, who benefited from the outrage of national donors alarmed by Trump's aggressive second term.
Patronis will fill the northwest Florida seat vacated by former Rep. Matt Gaetz. Gaetz had been tapped to be Trump's attorney general but dropped out amid allegations of sexual misconduct, which he has denied.
The senator wrapped his speech at 8:05 p.m. Tuesday, 25 hours and 5 minutes after he began.
The chamber had exploded in applause when Senate Democratic Leader Chuck Schumer announced that Booker had broken the record, which was previously held by segregationist Strom Thurmond at 24 hours and 18 minutes.
The arrest rate fell 14% in March from a month earlier, the White House said.
Arrests totaled 7,181 last month, down from 8,347 in February and down 95% from 137,473 in March 2024. The daily average of 232 arrests is the lowest since 1967.
Traffic slowed sharply last year after Mexican authorities increased enforcement within their own borders and the Biden administration introduced severe limits on asylum. Numbers plummeted more when Trump took office and restricted asylum even more as part of a broad immigration crackdown.
Rep. Randy Fine, R-Palm Bay, answers a question about his House Bill 3-C: Independent Special Districts in the House of Representatives April 20, 2022, at the Capitol in Tallahassee, Fla. (AP Photo/Phil Sears, File)
He beat Democrat Josh Weil, who had outraised and outspent his counterpart.
Fine ran in the reliably conservative district with the endorsement of Trump and former U.S. Rep. Mike Waltz. Waltz resigned from the seat to become Trump's national security adviser.
Fine had faced growing pressure during the race's final days as some Republicans publicly criticized his campaign and fundraising efforts. His victory ends Democratic hopes to score a huge upset in a district that was heavily supportive of Trump in November.
Booker took to the Senate floor Monday evening, saying he would remain there as long as he was “physically able.” More than 24 hours later, the 55-year-old senator is still going.
He has set the record for the longest continuous Senate floor speech in the chamber's history, though he was assisted by fellow Democrats who gave him a break from speaking by asking him questions.
It was a remarkable show of stamina as Democrats try to show their frustrated supporters that they are doing everything possible to contest Trump's agenda.
Yet Booker also provided a moment of historical solace for a party searching for its way forward: By standing on the Senate floor for more than a night and day and refusing to leave, he had broken a record set 68 years ago by then-Sen. Strom Thurmond of South Carolina, a segregationist, to filibuster the advance of the Civil Rights Act in 1957.
Sen. Cory Booker has held the Senate floor for 24 hours and is closing in on the record for the longest continuous floor speech held by Strom Thurmond, a segregationist.
Thurmond filibustered for 24 hours and 18 minutes against the Civil Rights Act of 1957, according to the Senate's records. As Booker's speech rolled past 24 hours, anticipation is growing in the Capitol.
“Thank you for all the interest in my dinner with the president last night,” TV host Bill Maher posted Tuesday on X. He vowed that “all will be revealed” during his show “Real Time” on April 11, adding that if he offered details on April Fool's Day, “no one would believe what I said.”
That followed Kid Rock, who was at the White House on Monday, offering to help arrange a dinner. He told Fox News Channel that “Bill's obviously a very big liberal” but Trump was nonetheless “so gracious.”
Sen. Tim Kaine, D-Va., right, listens as Senate Minority Leader Chuck Schumer, D-N.Y., speaks during a news conference regarding President Donald Trump's pending tariffs on Canada, at the Capitol, Tuesday, April 1, 2025, in Washington. (AP Photo/Rod Lamkey, Jr.)
The Democrats are under pressure to show their voters they are doing all they can, even as the minority party in Congress, to block Trump's agenda.
“We are standing together against the GOP tax scam and in defense of the American people,” House Democratic Leader Hakeem Jeffries said at the Capitol steps with Senate Democratic Leader Chuck Schumer.
Schumer praised fellow Democratic Sen. Cory Booker as a “tour de force” for seizing the Senate floor in a landmark speech as they work to stall action on the GOP agenda.
“Florida and Wisconsin, it's not too late. Go VOTE,” former President Joe Biden posted on X — offering a rare political pronouncement since leaving office.
Wisconsin has a state Supreme Court election, while two Florida districts are holding special elections to replace Republican congressmen who vacated House seats after Trump tapped them for administration posts.
Biden has kept a low profile since leaving office. His words echoed Trump's White House press secretary, Karoline Leavitt, who also urged Wisconsin residents and Floridians to vote during her briefing with reporters.
The Rev. W. J. Mark Knutson, right, walks with an El Salvadoran immigrant at the Augustana Lutheran Church on Thursday, Jan. 9, 2025, in Portland, Oregon. (AP Photo/Jenny Kane, File)
As many as four in five immigrants at risk of deportation from the United States are Christian, according to a new report.
The report, under the auspices of major Catholic and evangelical organizations, says about 10 million Christians are vulnerable to deportation.
“Though we're deeply concerned about fellow Christians, we're not exclusively concerned with immigrants who happen to share our faith,” said Matthew Soerens, vice president of advocacy and policy at World Relief, an evangelical humanitarian organization that cosponsored the report.
Trump enjoyed wide support from certain Christian blocs in all three of his campaigns. While the report doesn't directly refer to that support, it says it seeks to raise awareness of the potential impact of Trump's immigration crackdown.
▶ Read more about the report on Christians at risk of deportation
The billionaire government adviser told voters that the battle is so close that it could be decided by a single vote.
He made clear the political reasons behind supporting Brad Schimel, the candidate backed by Republicans.
“A judge race, election in Wisconsin will decide whether or not the Democrats can gerrymander Wisconsin in order to remove two House seats from Republican to Democrat,” Musk said during a Fox News interview Tuesday afternoon.
If Republicans lose control of the U.S. House, Musk asserted that Democrats would do “everything possible to stop the agenda that the American people voted for,” Musk said.
“If you know people in Wisconsin, call them right now,” he added.
Trump will hold a Wednesday meeting with aides about possible investors who could buy a stake in TikTok, a deal that could potentially stop the social media site from being banned in the U.S.
The details of the meeting were confirmed by a person familiar with the situation who insisted on anonymity to discuss internal deliberations.
The icon for the TikTok video sharing app is seen on a smartphone in Marple Township, Pa., on Tuesday, Feb. 28, 2023. (AP Photo/Matt Slocum, File)
There has been uncertainty about the popular video app after a law took effect on Jan. 19 requiring its China-based parent, ByteDance, to divest its ownership because of national security concerns. After taking office, Trump gave TikTok a 75-day reprieve by signing an executive order that delayed until April 5 the enforcement of the law requiring a sale or effectively imposing a ban.
Among the possible investors are the software company Oracle and the investment firm Blackstone.
CBS News first reported on the meeting.
Another major international law firm has reached a deal with the White House to dedicate at least $100 million in free legal services to causes including veterans support and combating antisemitism.
The agreement announced Tuesday makes Willkie Farr & Gallagher the third law firm in the last two weeks to cut a deal with the White House. As part of the arrangement, Willkie agreed to disavow the use of diversity, equity and inclusion considerations in their hiring decisions.
The firm is home to Doug Emhoff, the husband of 2024 Democratic presidential nominee Kamala Harris, and Tim Heaphy, who was chief investigative counsel to the House of Representatives committee that investigated the Jan. 6, 2021, riot at the U.S. Capitol.
The firm had been bracing for an executive order like the one leveled at nearly a half-dozen other major firms over the last month. Those orders have generally targeted firms over their association with attorneys Trump regards as adversaries.
The orders have threatened the security clearances of attorneys at the firms as well as the termination of the firms' federal contracts and access by employees to federal buildings.
The Trump administration has paused $27.5 million for organizations that provide family planning, contraception, cancer screenings and sexually transmitted infection services as it investigates whether they're complying with federal law.
The money isn't allowed to be used for abortion, but much of it goes to organizations that also provide abortions — and that's long made the funding a target for conservatives.
About one-fourth of the grant recipients were told this week that their funding is on hold. That includes all the groups that receive the money for seven states.
In this image provided by Senate Television, Sen, Cory Booker, D-N.J. speaks on the Senate floor, Tuesday morning, April 1, 2025. (Senate Television via AP)
As Sen. Cory Booker's speech rolled past 21 hours, it marked the fourth-longest in Senate history.
Booker, a New Jersey Democrat, has surpassed the longest speech time for a sitting senator — the 21 hours and 19 minutes that Sen. Ted Cruz, a Texas Republican, held the floor to contest the Affordable Care Act in 2013.
This image from Senate video show Sen. Ted Cruz, R-Texas, speaking on the Senate floor at the U.S. Capitol in Washington, Tuesday, Sept. 24, 2013. (AP Photo/Senate TV)
Throughout his determined performance Tuesday, Booker has repeatedly invoked the civil rights leader Rep. John Lewis of Georgia.
The longest speech time in Senate records is that of Strom Thurmond of South Carolina, who filibustered for 24 hours and 18 minutes against the Civil Rights Act of 1957. Booker would need to hold the floor for almost three more hours to beat that record.
Sen. Bill Cassidy, a doctor from Louisiana, summoned Kennedy to testify about his restructure of the U.S. Department of Health and Human Services before the Senate health committee next week.
Cassidy backed Kennedy for the health secretary job after obtaining “serious commitments” from the administration, including that the health secretary would regularly appear before Senate lawmakers.
Cassidy's request comes on the same day thousands of staffers were laid off from the health department.
Dr. Richard Besser, who also previously served as acting director of the Centers for Disease Control and Prevention, said those carrying out the mass firings at Health and Human Services are “systematically and cruelly dismantling our nation's public health system and workforce, which threatens the health and wellbeing of everyone in America.”
The firings “represent an abdication of the department's essential responsibility to promote and protect health,” Besser said in a statement on Tuesday. “And they present a fundamentally different vision of what government can and should do to improve people's lives. Americans deserve better.”
He concluded his statement by saying: “It is clear that political leaders in this administration are neither committed to nor serious about improving everyday people's lives and health.”
Five regional offices for Head Start were closed Tuesday as part of a workforce reduction at the Department of Health and Human Services, according to the National Head Start Association.
Head Start is a preschool program that serves some of the nation's neediest families and children. Parents who otherwise would not be able to afford child care often rely on the program when they go to work or school.
Children play during aftercare for the Head Start program at Easterseals South Florida on Wednesday, Jan. 29, 2025, in Miami. (AP Photo/Rebecca Blackwell)
The association's statement said the move was taken without a “clear plan for how the administration intends on supporting Head Start.”
The Office of Head Start had 12 regional offices to provide oversight and support administration of grants. It was not immediately clear which regional offices were closed.
Liberal leader Mark Carney speaks during a campaign stop, Tuesday, April 1, 2025 in Winnipeg. (Adrian Wyld/The Canadian Press via AP)
Canada Prime Minister Mark Carney says he had a call with Mexican President Claudia Sheinbaum to discuss the “importance of building upon the strong trading and investment relationship between the two countries.”
The discussion came in the lead up to Trump's anticipated tariffs on Wednesday. If enacted, the tariffs would deal a hefty economic blow to both nations, which are both in a free trade agreement with the U.S.
In the call, the two leaders spoke about the “challenging times ahead,” safeguarding economic competitiveness in the region and calls by both nations for the U.S. to respect their sovereignty, according to the Canadian government.
The leaders they would remain in “close contact,” and that top government officials would work together to boost trade between the two countries.
A federal judge in California held off Tuesday on granting the request from legal aid groups.
The Republican administration on March 21 terminated a program that provides legal services for unaccompanied migrants under 18, which plaintiffs say puts 26,000 children at risk of losing their attorneys.
Defendants say taxpayers have no duty to pay for direct legal services and the contract has expired.
But plaintiffs said Tuesday they're asking not for contract restoration but for the government to come up with a plan so children as young as five months old will have legal representation in immigration courts as required by Congress.
U.S. District Judge Araceli Martínez-Olguín of San Francisco requested further briefing.
Speaker of the House Mike Johnson, R-La., takes questions on tariffs while meeting with reporters at a news conference, at the Capitol, in Washington, Tuesday, April 1, 2025. (AP Photo/J. Scott Applewhite)
House Speaker Mike Johnson on Tuesday exercised his power of the gavel — and tried to bring it down with an unusually aggressive effort to squash a proposal for new parents in Congress to able to vote by proxy, rather than in person, as they care for newborns.
His plan failed, 206-222.
In an unprecedented move, the House Republican leadership had engineered a way to quietly kill the bipartisan plan from two new moms — Republican Rep. Anna Paulina Luna of Florida and Democratic Rep. Brittany Pettersen of Colorado. Their plan has widespread support from a majority of House colleagues. Some 218 lawmakers backed the new moms, signing on to a so-called “discharge petition” to force their proposal onto the House floor for consideration.
But Johnson, like GOP leaders before him, rails against proxy voting, as President Trump pushes people back to work in the aftermath of the COVID-19 pandemic work from home trend.
▶ Read more about proxy voting
Melania Trump arrives to speak during the International Women of Courage award ceremony, Tuesday April 1, 2025, held at the State Department in Washington. (AP Photo/Jacquelyn Martin)
Courage is rooted in love, the first lady said as she helped recognize eight women from around the world with the 2025 International Women of Courage Award.
At a ceremony at the State Department, which created the award, Trump said the “exceptional assembly of brave women” shows their love by refusing to be defined by fear or hardship.
Trump sought parallels with them, saying that, in her own life, she has “harnessed the power of love” as a source of strength during challenging times.
The eight women receiving awards hail from Burkina Faso, Papua New Guinea, the Philippines, Romania, South Sudan, Sri Lanka, Yemen and Israel.
The honorees include Amit Soussana, who was taken hostage by Hamas after the militant group's Oct. 7, 2023, attack on Israel. Soussana was released after 55 days.
They call the recent executive order unconstitutional and say millions of voters could be disenfranchised.
The “Defending America's Future Elections Act” was filed Tuesday and is likely to face opposition as Republicans maintain majorities in both the House and Senate.
Trump's March 25 order called for several election-related changes, including a documentary proof of citizenship requirement for voter registration and revised voluntary standards for voting machines to prohibit the use of barcodes on ballots. Lawsuits challenging the order have been filed.
Sen. Alex Padilla, a California Democrat who sponsored the bill, calls Trump's order an “illegal and unconstitutional power grab.” Ten other senators co-sponsored the legislation.
They note that proving citizenship would be difficult for millions of Americans who don't have easy access to their birth certificates or who haven't obtained U.S. passports.
It was just hours after thousands of his employees began receiving emailed notices or were told to turn in their badges at HHS offices around the country.
Health and Human Services Secretary Robert F. Kennedy Jr. posted the videos on social media celebrating the swearing in of his two latest hires: Jay Bhattacharya, the NIH director, and Martin Makary, the FDA commissioner.
“The revolution begins today!” Kennedy wrote.
HHS hasn't provided additional details or comments about Tuesday's mass firings.
Hey, Rose. Thanks for your question. New Jersey Sen. Cory Booker has been holding the Senate floor since Monday night with a marathon speech that's lasted into Tuesday afternoon. It was a remarkable show of stamina as Democrats try to show their frustrated supporters that they are doing everything possible to contest Trump's agenda.
As Booker's speech rolled past 18 hours, it became the sixth longest in Senate history.
AP's Mike Catlini and Stephen Groves wrote about Booker's speech and what it represents. Here's some of what they said:
Booker's speech was not a filibuster, which is a speech meant to halt the advance of a specific piece of legislation. Instead, Booker's performance was a broader critique of Trump's agenda, meant to hold up the Senate's business and draw attention to what Democrats are doing to contest the president. Without a majority in either congressional chamber, Democrats have been almost completely locked out of legislative power but are turning to procedural maneuvers to try to thwart Republicans.
As Democrats search for a next generation of leadership, frustrated with the old-timers at the top, Booker's speech could cement his status as a leading figure in the party's opposition to Trump.
While it's not required for members of Congress to attend, Booker got some help from his Democratic colleagues, who gave him a break from speaking to ask him a question and praised his performance. Booker yielded for questions but made sure to say he would not give up the floor. He stayed standing to comply with Senate rules.
Watch live as U.S. Senator Cory Booker addresses the Senate floor. Booker, a Democrat from New Jersey, has promised to speak “as long” as he's physically able on the Senate floor Monday night into Tuesday morning, criticizing the Trump administration.
The Princeton University campus is in Princeton, N.J., Oct. 8, 2024. (AP Photo/Ted Shaffrey, File)
The Ivy League school received notifications Monday and Tuesday that grants were being suspended by agencies including the Department of Energy, NASA and the Defense Department, according to a campus message from President Christopher Eisgruber.
The rationale wasn't fully clear, but Princeton will comply with the law, Eisgruber said.
“We are committed to fighting antisemitism and all forms of discrimination, and we will cooperate with the government in combating antisemitism,” he wrote.
Princeton is the latest Ivy League school to have its federal funding come under scrutiny from the Trump administration amid investigations into campus antisemitism. Columbia University last week agreed to several demands from the administration after the government cut $400 million and threatened to slash billions more.
A federal task force on antisemitism announced Monday that Harvard was facing a “comprehensive review” of almost $9 billion in federal grants and contracts.
▶ Read more about Princeton University
Arizona Sen. Ruben Gallego announced Tuesday he'll block the confirmation of top leaders at the Veterans Affairs Department, raising the stakes in Democrats' bid to get the Trump administration to back off plans to cut jobs from the sprawling agency that serves millions of military veterans.
Gallego, a Marine Corps veteran, made the announcement just hours before the Senate Committee on Veterans' Affairs was scheduled to hear testimony from three nominees for the VA who are military veterans themselves. His move was a significant escalation in the Democrats' efforts to counter Trump's plans to slash federal agencies and a sharply partisan move on a committee that's often seen cooperation between Republicans and Democrats.
“Talking to veterans, people that I served with as well as seeing some of what's happening in Arizona, I decided that whatever tool I have to fix the situation, I'm going to use it,” Gallego told The Associated Press. “And this is one of the few tools I have at this point.”
▶ Read more about the Veterans Affairs Department
The White House says Trump will head to Saudi Arabia in May. Press secretary Karoline Leavitt offered no further details on the trip.
A TikTok sign is displayed on top of their building in Culver City, Calif., on Tuesday, Dec. 3, 2024. (AP Photo/Richard Vogel)
Trump has signaled he's confident his administration can broker an agreement with ByteDance, the social media app's China-based parent company.
Speaking with reporters on Air Force One late Sunday, Trump said “there's tremendous interest in Tiktok.” He added that he would “like to see TikTok remain alive.” The president's comments came less than one week before an April deadline requiring ByteDance to divest or face a ban in the United States.
“We have a lot of potential buyers,” Trump said.
Trump also said the administration is “dealing with China” who “also want it because they may have something to do with it.” Last week, Trump said he would consider a reduction in tariffs on China if that country's government approves a sale of TikTok's operations in the U.S.
▶ Read more about the upcoming TikTok ban
The White House is asserting that the man with protected legal status who was mistakenly sent to an El Salvador prison was a member of the MS-13 gang and alleged he'd been involved in human trafficking.
Officials with U.S. Immigration and Customs Enforcement, as well as White House press secretary Karoline Leavitt, acknowledged an “administrative error” in efforts to remove Kilmar Armando Abrego Garcia from the U.S.
“The administration maintains the position that this individual, who was deported to El Salvador and will not be returning to our country, was a member of the brutal and vicious MS-13 gang,” Leavitt told reporters at a Tuesday briefing.
Leavitt claimed “credible intelligence” showed he was involved in human trafficking and that he was a leader of the notorious MS-13 gang.
“Foreign terrorists do not have legal protections in the United States of America anymore,” Leavitt said.
In a complaint, Abrego Garcia's lawyers disputed the government's claims.
“Although he has been accused of general ‘gang affiliation,' the U.S. government has never produced an iota of evidence to support this unfounded accusation,” the complaint stated.
▶ Read more about the deportation
A protester holds the flags of Canada and the United States outside on Parliament Hill in Ottawa, on Saturday, Feb. 1, 2025. (Justin Tang/The Canadian Press via AP)
With Trump's so-called “Liberation Day” of tariff implementation fast approaching, Senate Democrats are putting Republican support for some of those plans to the test by forcing a vote to nullify the emergency declaration that underpins the tariffs on Canada.
Republicans have watched with some unease as the president's attempts to remake global trade have sent the stock market downward, but they have so far stood by Trump's on-again-off-again threats to levy taxes on imported goods.
Even as the resolution from Democratic Sen. Tim Kaine of Virginia offered them a potential off-ramp to the tariffs levied on Canadian imports, Republican leaders were trying to keep senators in line by focusing on fentanyl that comes into the U.S. over its northern border. It was yet another example of how Trump is not only reorienting global economics, but upending his party's longtime support for ideas like free trade.
▶ Read more about the Senate vote on tariffs
Sen. Tammy Duckworth, a Democrat from Illinois, pressed retired Lt. Gen. Dan Caine on whether he would follow orders from the president to use the military in domestic matters like law enforcement.
Duckworth accused Trump of engaging in “threat inflation” of domestic protests to justify military involvement on issues like protests, immigration or free speech.
“I think there's strong systems in place, legal systems in place, that prevent any missteps there,” said Caine.
Sen. Elissa Slotkin, a Michigan Democrat, asked Caine whether he would push back on an order by Trump “to use the military in a way that was unconstitutional.” She argued that Trump had made such orders during his first term and promised to do so during his reelection campaign.
“I will senator. I don't expect that to happen, but I will,” Caine said.
Booker, who's 55, started speaking Monday evening and hasn't left the Senate floor since. As it rolled into Tuesday afternoon, Booker's performance is currently the sixth longest in Senate history.
The record for the longest individual speech belongs to Strom Thurmond of South Carolina, who filibustered for 24 hours and 18 minutes against the Civil Rights Act of 1957.
Only one other sitting senators has spoken for longer than Booker. In 2013, Sen. Ted Cruz, a Republican of Texas, held the floor for 21 hours and 19 minutes to contest the Affordable Care Act.
The agency is losing more than 1,000 employees.
NIOSH is based in Cincinnati but also has people in Pittsburgh; Spokane and Morgantown, West Virginia.
Micah Niemeier-Walsh, vice president of the union local representing NIOSH employees in Cincinnati, said the union had heard reports that around 850 of the center's employees are receiving notices, including the center's director.
The cuts are hitting mining safety research, work on developing personal protective technology, a firefighter cancer registry, and a lab that's key in the certification of respirators for industry. Niemeier-Walsh called the cuts “a very pointed attack on workers in this country.”
FILE - In this April 20, 2019 file photo Democratic presidential candidate Sen. Cory Booker, right, speaks with Astrid Silva, left, and others after a round table with Dreamers and immigrant activists, in Las Vegas. (AP Photo/John Locher,File)
Booker, 55, was born in Washington, D.C., and moved to northern New Jersey when he was a boy. He's spoken about growing up in a Black family in a predominantly white neighborhood and how his parents faced opposition when they tried to buy a house.
He played football in college at Stanford University before attending Yale Law School and then worked as an attorney in nonprofits, giving legal aid to poorer families. Elected to the Newark City Council and then as mayor of the state's biggest city, he served there until 2013.
His time in office coincided with Facebook founder Mark Zuckerberg's $100 million donation to the city's public schools, a boon that burnished his status as a Democratic rising star at the time.
Booker also ran an unsuccessful presidential campaign in 2020.
▶ Read more about Sen. Cory Booker
In a Tuesday post on his social media platform, Trump said the pair discussed U.S. military operations against the Iran-backed Houthis in Yemen, as well as the Israel-Hamas war in Gaza and “possible solutions” to the conflict, as well as “military preparedness.”
Egyptian President Abdel Fattah el-Sisi, right, watches Foreign Minister of Egypt Sameh Shoukry during a ceremony after talks with his Serbian counterpart Aleksandar Vucic at the Serbia Palace in Belgrade, Serbia, Wednesday, July 20, 2022. Abdel Fattah el-Sisi is on a three-day official visit to Serbia. (AP Photo/Darko Vojinovic)
The Tuesday sanctions were against a network of six firms and two people based in Iran, the United Arab Emirates, and China who are allegedly responsible for procuring drone components on behalf of Iranian drone manufacturers.
They mark the second round of sanctions targeting Iranian weapons proliferators since President Trump signed an executive order in February imposing a “restoring maximum pressure” campaign on Iran meant to deny Iran all paths to a nuclear weapon.
Treasury Secretary Scott Bessent said Treasury will continue to target Iranian drones “missiles, and conventional weapons that often end up in the hands of destabilizing actors, including terrorist proxies.”
Mexican President Claudia Sheinbaum gives her daily morning press conference at the National Palace in Mexico City, Monday, Feb. 3, 2025. (AP Photo/Marco Ugarte)
After previously saying Mexico would seek “preferential treatment” with Trump and his tariffs, Mexican President Claudia Sheinbaum shifted her tone Tuesday to note the tariffs set to go into effect on Wednesday weren't targeting Mexico.
“What they're going to announce on April 2 isn't against Mexico, it's not against Canada. It's a policy of the United States to the entire world,” Sheinbaum said in her morning news briefing.
That said, if tariffs go into effect, Mexico would be dealt a particularly hard blow, as much of its economy is intertwined with the U.S., especially the auto sector.
While other leaders have butted heads with Trump, Sheinbaum has assumed a less confrontational approach, following through on U.S. demands in the hope that doing so will offset the bulk of American populist's threats.
“You have to trust the president's instincts on the economy,” Johnson said.
He said he expects the tariffs to go forward as Trump promised.
“We'll see how it all develops,” he said. “It may be rocky in the beginning. But I think that this will make sense for Americans and help all Americans.”
House Speaker Mike Johnson of La., right, and Vice President JD Vance, left, listen as President Donald Trump addresses a joint session of Congress at the Capitol in Washington, March 4, 2025. (AP Photo/Ben Curtis)
“There's a constitutional path. You have to amend the constitution to do this, and that's a high bar,” said Johnson, a lawyer who specialized in constitutional issues.
“I think he recognizes the constitutional limitations,” he said.
The Republican speaker says Trump has joked with him about the idea. He said he takes the president “at his word.”
Tuesday's special elections for two Florida congressional seats in heavily pro-Trump districts have become an unexpected source of concern for national Republicans as Democrats have poured millions in fundraising into the races.
Both seats opened when Trump chose their representatives for jobs in his second administration. Matt Gaetz was briefly nominated to be Trump's attorney general before withdrawing, while Mike Waltz became national security adviser.
Florida state Sen. Randy Fine, running for Waltz's seat, and state Chief Financial Officer Jimmy Patronis, running to replace Gaetz, are widely expected to hold the seats in their reliably conservative districts, which would give Republicans a 220 to 213 advantage over Democrats in the U.S. House.
But both have been outraised by their Democratic counterparts, and Republicans in Florida and Washington have begun trying to distance themselves from any potential underperformance.
▶ Read more about Florida's special elections
As the trade wars launched by U.S. President Donald Trump continue to escalate, all eyes are on Wednesday. Trump has repeatedly called April 2 “Liberation Day,” with promises to roll out a set of tariffs, or taxes on imports from other countries, that he says will free the U.S. from a reliance on foreign goods. Associated Press reporter Seung Min Kim explains.
HHS employees wait outside the department's Washington office to find out if they were laid off. (AP Photo/Amanda Seitz)
As a biting spring wind whipped around them, staffers waited for as long as an hour outside the health department's Washington offices to get scanned into the building.
As many as 10,000 workers are expected to lose their jobs Tuesday and some are finding out as they try to enter the building that they no longer have jobs. Laid off staffers are being asked to immediately turn in their badges and cellphones at the door.
One staffer waiting in line loudly joked: “Is this an April Fool's joke?”
During retired Lt. Gen. John Caine's confirmation hearing for joint chiefs chairman, Sen. Jack Reed asked him whether top uniformed military leaders should have participated in a controversial Signal chat in which U.S. officials discussed battle plans.
“From what I understand of that chat, it was a partisan political chat and so the joint force should not have been represented in there,” Caine said.
Caine declined to comment on whether senior U.S. officials, including the vice president, defense secretary, secretary of state and national security advisor, should have discussed battle plans on an unclassified, commercial application.
“What I will say is we should always preserve the element of surprise,” Caine said. He noted that the Senate Armed Services Committee had requested an inquiry into the matter.
President Trump has told a story about retired Air Force Lt. Gen. Dan Caine saying he wore one of the hats when the two met some years ago.
When asked about the story during the Senate Armed Services hearing on his confirmation, Caine said, “For 34 years, I've upheld my oath of office and my commitment to my commission. And I have never worn any political merchandise.”
He added that he thinks Trump must have been “talking about somebody else.”
Copyright 2025 The Associated Press. All Rights Reserved.
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This is CNBC's live coverage of President Donald Trump's announcement Wednesday of new tariffs targeting U.S. trade partners.
What you need to know
CNBC's reporters are covering the tariffs and their impact, live all day on air and online from our bureaus in Washington, London, Singapore, San Francisco, and Englewood Cliffs, New Jersey.
Treasury Secretary Scott Bessent is attending Senate Republicans' weekly lunch today before Trump's expected tariff announcement.
The Treasury secretary's visit to Capitol Hill comes a day after he visited with House Republicans.
There, Rep. Kevin Hern, Okla., told CNBC that Bessent said the tariff rates being announced today would be the top rates, and will go down from here, not up..
— Erin Doherty
Democratic Rep. Josh Gottheimer (N.J.) is warning that Trump's tariffs are "regressive" and will hit "people on things they buy every day."
"I don't think people are seeing this, including a lot of Republicans, as a day of liberation, but ... a day of higher costs and a day of chaos," Gottheimer told CNBC's 'The Exchange.'
"Regardless where people are on the political spectrum, what they don't like is a lot of uncertainty," said Gottheimer, who is currently running for governor of New Jersey.
"I think people are hearing all this noise, they're seeing every day the news change, and they just want some certainty," he added.
"They want less chaos in their lives."
— Erin Doherty
Major averages are trading modestly higher in the volatile session as investors await details about tariffs on key U.S. trading partners.
The S&P 500 last traded up 0.2% after swinging significantly between gains and losses. At its session high, the benchmark was up 1.1%; at its session low, the S&P 500 declined 1.1%.
The 30-stock Dow Jones Industrial Average rose 1just 60 points, while the Nasdaq Composite added 0.3% as Tesla shares jumped more than 4%.
"Any 'relief' bounce is likely to be short-lived as the absolute effect of the trade agenda remains decidedly negative with downside pressure on growth/earnings and upside risks to inflation," Adan Crisafulli, founder of Vital Knowledge, said in a note.
— Yun Li
The Trump administration is adding imports of beer and empty aluminum cans to its 25% tariffs on derivative aluminum products, according to a notice posted Wednesday in the Federal Register.
The notice from the Commerce Department said the adjusted tariffs would take effect after midnight Friday.
One of the two product tariff codes referenced in the notice applies to "beer made from malt." The other applies to aluminum casks, drums, cans, boxes and similar containers with a capacity no greater than 20 liters.
The notice does not include a separate code that would encompass beer in glass containers.
The Commerce Department did not immediately respond to CNBC's request for comment.
— Kevin Breuninger
Uncertainty surrounding Trump's tariff policy has created panic for smaller businesses grappling with the likelihood of added operations costs and potential supply chain bottlenecks.
"I'm going to run out of appliances," said Robin Liss, founder of kitchen gadget company Suvie, ahead of her two-week trip to Taiwan and Vietnam to work out a new business plan. "I've got to figure this out."
Trump slapped additional tariffs on China and is threatening to strain America's relationships with other longtime trading partners.
Read the full story here.
— Samantha Subin
Mexican President Claudia Sheinbaum says she does not plan to implement tit-for-tat tariffs on American goods in response to Trump's import duties.
"It's not about tit-for-tat, but about what is best for Mexico and how to face this situation," she said during a morning press conference.
"We do not believe in an eye-for-an-eye, a tooth-for-a-tooth because that always leads to a bad situation," she said.
— Erin Doherty
The Senate is poised to vote later today on a Democratic-led resolution to undo the "national emergency" that Trump declared in order to create a legal rationale for Trump's tariffs on Canada.
Sen. Tim Kaine (D-Va.) told reporters earlier that Sen. Mitch McConnell (R-Ky.) indicated that he plans to back the resolution. At least three other Republican senators — Lisa Murkowski (Alaska), Susan Collins (Me.) and Rand Paul (Ky.) — have also signaled plans to support the measure.
The resolution is largely symbolic, because it still needs to move through the Republican controlled House and would require Trump's signature to become law.
Nonetheless, Senate passage with the support of some Republicans — on the same day Trump unveils his tariff plan — would mark a rebuke to Trump's tariff policy.
— Erin Doherty
Sen. Peter Welch, a member of the Senate Finance Committee, is slamming Trump's tariff policies, warning that his "half-baked trade war will only raise prices for consumers."
"Trump's so-called 'liberation day' will throw the global economy into turmoil and leave Americans holding the bag," the Vermont Democrat Welch says in a statement hours before the unveiling of so-called reciprocal tariffs at the White House.
"We should not impose sweeping tariffs on our allies and longtime partners in trade. America's close economic ties with our trading partners are based on trust," Welch says.
"These on-again, off-again tariffs are extremely destructive and totally unnecessary," he says. "President Trump is sticking it to our farmers, our businesses, and everyday working people."
— Kevin Breuninger
Trump has made a final decision on how he wants to proceed with his sweeping reciprocal tariff plans, a White House official told CNBC's Megan Cassella.
The update marks an apparent end to the internal deliberations that were reportedly ongoing in the hours before the Wednesday afternoon announcement event.
— Kevin Breuninger and Megan Cassella
Trump will be joined in the Rose Garden by workers from industries that are likely to be affected by his reciprocal tariffs.
The guest list includes "steel workers, autoworkers, oil and gas workers, steam fitters, truck drivers, and hardworking Americans from a variety of trades," a senior White House official told NBC News.
— Kevin Breuninger and Peter Alexander
Trump has said tariffs on pharmaceutical products imported into the U.S. were coming soon, but it is not clear if they will be announced at the White House event.
Those potential tariffs would likely drive up U.S. drug prices for patients because even if companies moved to produce those medications domestically, it would take years and cost more than producing medicines abroad, Leerink Partners analyst David Risinger said in a note last week.
Predicting the potential impact of tariffs on pharmaceutical companies is difficult since they have vast and complex manufacturing networks with multiple steps, sometimes in different geographies, TD Cowen analyst Steve Scala said in a note.
But Scala said Eli Lilly, Bristol Myers Squibb and AbbVie appear better positioned than others to weather tariffs because they have more major manufacturing plants in the U.S. than internationally.
The majority of their sites responsible for producing the active ingredients in drugs are also in the U.S., he added.
Meanwhile, Novartis and Roche "look more at risk" because they have few U.S. plants and a higher share of active ingredient sites that are international, Scala said.
- Annika Kim Constantino
Some tequila makers have been warning about how tariffs could hit their businesses, but Colorado-based Suerte Tequila said it won't raise prices to offset tariffs.
"Tequila margins are stronger than ever," said Laurence Spiewak, Suerte Tequila CEO.
Still, the industry could see a hit with tariffs on Mexico.
In 2024, the U.S. imported $5.2 billion worth of tequila and $93 million worth of mezcal from Mexico, according to the Distilled Spirits Council of the U.S.
— Brandon Gomez and Michele Luhn
Trump is targeting Mitch McConnell and three other Republican senators in a critical Truth Social post, urging them to oppose a bill that would undo U.S. tariffs on Canada.
McConnell, Rand Paul, Ky., Sen. Susan Collins, Me. and Lisa Murkowski of Alaska, "will hopefully get on the Republican bandwagon, for a change," Trump writes.
He presses the senators to "fight the Democrats wild and flagrant push to not penalize Canada for the sale, into our Country, of large amounts of Fentanyl, by Tariffing the value of this horrible and deadly drug in order to make it more costly to distribute and buy."
It is not immediately clear what Trump means by "tariffing the value" of fentanyl.
Trump calls the Senate bill introduced by Sen. Tim Kaine, D-Va., a "ploy" intended to "show and expose the weakness of certain Republicans, namely these four."
The president also calls on Kentucky, Alaska and Maine voters to call these senators' offices.
"They have been extremely difficult to deal with and, unbelievably disloyal to hardworking Majority Leader John Thune, and the Republican Party itself," Trump adds
— Kevin Breuninger
Ontario Premier Doug Ford said he stands ready to remove retaliatory tariffs on U.S. products if Trump cancels heavy American import duties on Canadian goods.
"We're willing to take these tariffs off, like in the next minute, if he said he's taking their tariffs off," Ford told CNBC's "Squawk Box."
Canada has imposed 25% tariffs on more than $20 billion worth of U.S. goods in retaliation for the Trump administration's steel and aluminum duties.
In addition, Ottawa has slapped 25% counter-tariffs on $30 billion worth of U.S. goods, in response to Trump's imposition of broad-based tariffs on imports from Canada.
— Yun Li
Trump is hyping his reciprocal tariffs in a Truth Social post declaring the day of their unveiling "LIBERATION DAY IN AMERICA!"
Trump commonly refers to his tariffs in historic terms, raising expectations and concerns about how severe the duties could be.
— Kevin Breuninger
Slovakia, the landlocked country east of Austria, could suffer the most from the new auto tariffs that Trump said will start to take effect Thursday.
"Germany's car industry is in the eye of the storm and by far most exposed in terms of value, with major players like Volkswagen, BMW, Mercedes, and Porsche likely getting hit by tariffs," economists Inga Fechner and Rico Luman of Dutch bank ING said in a recent research note.
"But Slovakia — home to several car plants — is most exposed in terms of total US export volume," they said.
The nation of 5.4 million people produces more cars per capita than any other country in the world. And the "Detroit of Europe" relies heavily on U.S. trade, with autos comprising a major chunk of its U.S. exports.
— Kevin Breuninger and Sam Meredith
The U.S. stock market was poised to open lower Wednesday as Wall Street continues to struggle in the face of the looming tariff policies from the Trump administration.
The S&P 500 is now down 4.2% over the past month and is 8.4% below its record high. The tech-heavy Nasdaq Composite has dropped 7.4% over the past month and is 13.6% below its record high.
Uncertainty about tariffs is not the only reason the stock market has struggled, as some tech stocks that are thought to be relatively insulated from trade war concerns have also fallen.
Other stock moves seem to have a more direct line to concerns about tariffs and their economic impact, such as the 12% decline in the past month for Stellantis.
On the other hand, gold and Treasury bonds have both been rallying in recent days, a sign that investors may be looking to reduce risk. Gold was trading near a record high Wednesday morning.
— Jesse Pound
The scope of new tariffs has yet to be finalized by the White House, the Bloomberg news service reported.
"The White House has not reached a firm decision on their tariff plan," Bloomberg reported, citing people familiar with the plan.
Several options are under consideration at the White House. One is a universal flat tariff rate applied to all trade partners, while another would tailor tariffs to each trade partner.
A third option prepared by the U.S. Trade Representative's office would apply a flat rate to a select group of countries.
— Dan Mangan
Democratic Sen. Peter Welch of Vermont said the tariff disagreements between Trump and Democrats go beyond typical party differences, calling it a "very, very serious situation."
"We believe what President Trump is doing is, in many cases, lawless and really beyond any norms," he said on "Squawk Box."
Welch said Congress has tariff authority that can be traced back to the Constitution.
Trump's actions are an "overreach" in service of a personal agenda, he said.
"There's a real abdication by Congress of its own authority," he said.
— Laya Neelakandan
The U.S. Trade Representative's office has prepared a third option for tariffs, The Wall Street Journal reports.
This tariff structure would set a rate below 20% and apply it only to a small group of trade partners.
There are two other plans under consideration at the White House.
Read more at The Wall Street Journal.
— Erin Doherty
— Christina Wilkie
Trump's new tariffs will take effect "immediately" after he announces them in the Rose Garden, White House press secretary Karoline Leavitt said Tuesday.
The timeline offered a morsel of clarity about the reciprocal tariff plan, which remains highly opaque just hours before it is set to be unveiled.
— Kevin Breuninger
Canadian Prime Minister Mark Carney and Mexican President Claudia Sheinbaum spoke on the phone in advance of looming U.S. tariffs to reaffirm their "strong trading and investment relationship," Carney's government in Ottawa said.
"With challenging times ahead," the two leaders "emphasized the importance of safeguarding North American competitiveness while respecting the sovereignty of each nation," Ottawa said in a statement after Tuesday's call.
Carney also laid out his "plan to fight unjustified trade actions against Canada" by the Trump administration.
In separate remarks to reporters Tuesday, Carney said Canada is prepared to retaliate against whatever actions the U.S. takes Wednesday.
"We have held back, but we will not disadvantage Canadian producers and Canadian workers relative to American workers," he said.
— Kevin Breuninger
Trump is about to drop a sweeping tariff policy on an economy that already seems to be showing cracks.
Several top analysts have lowered economic growth projections in recent weeks and warned of persistent inflation, dragging the term stagflation back into the conversation.
Strategists and fund managers have also recently raised the probability of a recession, a shift that stems in large part from concerns about the Trump administration's fiscal policies.
These fears coincide with a highly volatile stock market and souring sentiment from consumers and businesses alike.
— Kevin Breuninger
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In this article
The Trump administration will implement a 25% tariff of all imported canned beer and empty aluminum cans starting Friday, according to a notice from the Department of Commerce.
The expansion of U.S. aluminum tariffs comes shortly before President Donald Trump is expected to announce sweeping new levies on imported goods at a Rose Garden event at 4 p.m. ET.
Industry analysts expect the tariffs on canned beer imports to weigh most heavily on Constellation Brands. Constellation imports all of its beer from Mexico, including Modelo and Corona; beer accounted for 82% of the company's sales in its most recent quarter. While Corona is best known for coming in glass bottles, Modelo — the bestselling beer in the U.S. — most commonly comes in cans.
Constellation's shares were down less than 1% in afternoon trading on Wednesday, but concerns about tariffs have weighed on the stock for months. The company's shares have fallen 22% since Trump's election in November.
The updated notice for aluminum tariffs published on Wednesday does not mention levies for imported beer packaged in glass bottles. Aluminum cans accounted for 64.1% of beer distribution in 2023, compared with glass bottles' 26.9% share, according to the Beer Institute.
For years, canned beer has been gaining market share against its bottled counterpart. Brewers can produce and transport cans more easily than glass bottles, which are heavier, leading to cheaper prices on canned beer for consumers.
The U.S. imports most of its aluminum from Canada. China and Mexico, the two other main targets of Trump's trade ire, are also major exporters of aluminum to the U.S.
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When President Donald Trump made it clear that he was serious about implementing dramatic tariffs on top U.S. trading partners, Robin Liss knew her Suvie machines were in trouble.
Suvie's products — kitchen gadgets that can whip up dinner in a matter of minutes — are built in a facility in one of China's largest manufacturing hubs and are comprised of more than 500 components sourced throughout the country.
After running the financial models and tallying the costs associated with the new levies, Liss set off to Asia in March to search for an alternative business plan.
"I'm going to run out of appliances," Liss said, ahead of her two-week trip to Taiwan and Vietnam. "I've got to figure this out."
Suvie is among the scores of gadget makers scrambling to stay afloat while managing through President Trump's tariff plans and the uncertainty that they bring. The day-to-day changes in rhetoric from the White House has created volatility on Wall Street, where tech stocks just wrapped up their worst quarter since 2022, and a sense of panic for smaller businesses that have less of a cushion to deal with the added costs of operations and potential supply chain bottlenecks.
Trump slapped additional tariffs on China earlier this year and is now going much bigger, threatening to strain America's relationships with other longtime trading partners. On Wednesday afternoon, President Trump is expected to deliver remarks at a "Make America Wealthy Again Event" in the Rose Garden, and to announce reciprocal tariffs that follow a bevy of other import duties on goods from China, Canada and Mexico. Those three countries accounted for over $1.3 trillion, or about 40%, of total imports last year, according to the U.S. Census Bureau.
"Folks who thought they had safely moved their supply chain out of China suddenly find themselves wondering if that was a great decision," said Peter Hanbury, a partner at consulting firm Bain. "There's a lot of different options about where you could move things, but you don't want to make that decision if you don't know exactly where the tariff structure is going to land."
Liss said her company, based in Cambridge, Massachusetts, is eating the costs — for now — rather than passing them on to customers. For Suvie, those costs aren't just tied to its appliances, which are about the size of a microwave but offer more than 10 different cooking modes.
She also has to deal with higher food prices, as part of Suvie's business is selling meal kits starting at $11.49. Suvie delivers meals to customers weekly, but also has plans for users who prefer deliveries every two to four weeks.
The problem for Suvie and other consumer-focused businesses is that just as their costs are skyrocketing due to tariffs and other inflationary pressures, Americans are losing their buying power.
Federal Reserve Chair Jerome Powell acknowledged a "moderation in consumer spending" during the central bank's March policy meeting and said tariffs could put upward pressure on prices. Financial markets have also sold off in recent weeks in response to the uncertainty.
A recent analysis from the Yale Budget Lab estimates that tariffs could cost the average American household an additional $1,600 to $2,000 a year. Target CEO Brian Cornell told CNBC in March that consumers could see imminent price increases on produce items such as strawberries and avocados.
Liss launched Suvie in 2015 and began shipping products in 2019, a year after a Kickstarter campaign for a "kitchen robot with multi-zone cooking and refrigeration." The company has 20 U.S. employees and has avoided layoffs thus far, thanks to 80% growth last year that Liss said brought annual revenue to between $20 million and $30 million.
If not for recent China tariffs, which hiked the levies she pays on goods to 23% from 3%, Liss said Suvie would be operating at a profit.
Liss said that no matter what Trump announces on Wednesday, in what he's calling "Liberation Day," the existing tariffs on goods from China have made it imperative that she find a new country for production. But where the company lands depends on what gets thrown into the mix.
Wherever Suvie goes, the company estimates it can scale up within six months.
"That is incredibly fast and almost unheard of," Liss said. "But if we don't pull it off, we might not have products for the holidays, which is our main sales season."
For Austere founder Deena Ghazarian, tariffs on products from China as well as Mexico have put her business in peril.
Austere, based in Wilsonville, Oregon, south of Portland, makes cable, cleaning, and surge-protected power products. The 12-person company, founded in 2018, previously moved about half of its operations to Taiwan and Vietnam from China and was in talks to shift production to Mexico. Those discussions stalled in November.
Depending on the item, up to 50% of Ghazarian's components come from China. Half of her products are still made there, while the rest are manufactured in Taiwan and Vietnam. For her cleaning product solution, which is the most reliant on China, she estimates it would take over a year to move production to Thailand.
Ghazarian began stockpiling products last year for the company to get ahead of potential tariffs. She says others have done the same. The inventory investment has diverted many resources she'd otherwise use for hiring, marketing and scaling.
"I'm buying time to figure out my next move," she said. "If I keep having to spend this money to shift to get around it – at some point it's not going to have a return that's worth it financially."
The final step would be hiking prices for consumers, which Ghazarian said many partners she works with are already planning to implement later this week.
The whole electronic devices market is feeling the pain.
The Consumer Technology Association estimated in January that new tariffs could hike laptop and tablet prices by as much as 68%, and boost smartphone prices by as much as 37%. Video game consoles could rise as much as 58%. Tariffs would reduce consumer spending power by $90 billion to $143 billion annually, it estimates.
"This is just devastating to the U.S. economy," said CTA CEO Gary Shapiro. "It's tremendously inflationary."
Andrew Wilson, deputy secretary general and global head of policy at the International Chamber of Commerce, said that the cost implications from a 20% to 25% tariff could be severe enough to wipe out a company's entire operating margin, while also complicating it ability to operate abroad.
"There is a risk if retaliation takes hold in the global economy, that we see a severe worsening of the business environment for the tech sector, and for American firms," he said.
The logistics of moving are unrealistic for many companies. Over the years, Chinese cities such Shenzhen, Guangzhou and Dongguan have established themselves as key manufacturing meccas for technology and electronics production.
Those markets have steadily accrued the best component supply base, labor expertise and cost structures, which are difficult to replicate elsewhere, said Terry Arbaugh, chief commercial officer at SEACOMP, which designs and manufactures electronics such as thermostats and video game devices for customers. The company operates facilities in China and Mexico.
"Talk of tariffs is sending more business outside of China, but in a lot of cases, it's also not coming into the U.S.," Arbaugh said.
Many larger corporations have managed to pivot production to areas like Taiwan, Thailand and India over the years, but have also exhausted much of the existing supply in those smaller markets, Arbaugh said. And making lower cost consumer and medical electronics in volume in the U.S. is not a viable option for many businesses, he added.
Suvie isn't considering onshoring its production at the moment, and is currently focused on finding an alternative spot in Asia.
Liss has already booked a flight back to Taiwan in a couple weeks. From there, she'll hop around to other countries in the region for more answers.
"Maybe I'll book the next flight in the air," she said from the Detroit airport on the way home from her initial Asia trek.
WATCH: Economists estimate how much revenue tariffs could bring in
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A canvasser says Elon Musk and the billionaire's super PAC never paid up after promising Pennsylvania voters cash for their efforts around the 2024 presidential campaign.
The Bucks County, Pennsylvania, voter, who is using the pseudonym John Doe for what he said in court papers was for his "safety and security," filed a class-action lawsuit Tuesday in the US District Court for the Eastern District of Pennsylvania.
The man says in the lawsuit he's owed at least $20,000 for signatures he collected as part of Musk's massive push to help the then-Republican candidate — and now president — Donald Trump get elected to a second term in the White House.
In the lead-up to the November election, Musk and his America PAC offered to pay registered voters in the swing state of Pennsylvania $100 for signing a petition backing free speech and gun rights — and an additional $100 for each referral of a registered voter who signed the petition.
The offer, which initially started at $47, was also made to registered voters in the other six battleground states.
"Plaintiff and Class Members accepted Defendants' offers by signing or successfully referring Pennsylvania registered voters to the America PAC petition," the lawsuit against Musk and his political action committee says. It adds that Musk and the PAC "have since failed to pay Plaintiff and Class Members in full for their signatures and referrals."
The lawsuit says the plaintiff worked as a canvasser for Musk's America PAC ahead of the 2024 election and got many voters to sign the petition along the way.
The canvasser alleges that while he was paid his hourly rate for the work and for some referrals for the petition signatures he obtained, he says hasn't been paid at least $20,000 he's owed for his referrals.
"This case is about a broken promise: Elon Musk promised supporters that they would be paid for signing a petition and referring others to do the same," the man's attorney, Shannon Liss-Riordan, told Business Insider. "Our client relied on that promise because he believed in Elon, but unfortunately, that promise was not kept. It appears the promise was broken for many others as well."
The Pennsylvania man has repeatedly contacted Musk's super PAC, the lawsuit says, "making multiple attempts to receive full payment for his referrals, but to no avail."
The complaint says this lack of payment has caused the man "significant emotional and physical distress."
"He relied on these payments to pay his bills and suffered damage to his credit and health when the payments did not arrive as expected," the court papers say.
The suit, which alleges breach of contract and seeks a jury trial, says more than 100 others could be owed money and that the amount at issue may be over $5 million.
"Plaintiff is in communication with numerous others who referred voters to sign the America PAC petition, who are likewise frustrated that they did not receive full payments for their referrals," the lawsuit says.
Musk did not immediately respond to a request for comment.
An America PAC spokesperson, Andrew Romeo, told BI in a statement that the super PAC "is committed to paying for every legitimate petition signature, which is evidenced by the fact that we have paid tens of millions of dollars to canvassers for their hard work in support of our mission."
"While we don't yet know who this 'John Doe' plaintiff is and can't speak to their specific circumstances, we can say that we are also committed to rooting out fraud and have the right to withhold payments to fraudsters," Romeo said.
During the 2024 campaign, Musk also launched a $1 million-a-day giveaway to encourage voter registration and push swing-state residents to the polls. The move raised legal questions.
At the time, the Department of Justice sent Musk a letter saying that the contest could violate federal law, and Philadelphia's district attorney filed a lawsuit, calling the petition an "unlawful lottery."
A Philadelphia judge ultimately allowed the program to move forward, saying it did not meet the criteria for a lottery.
Meanwhile, on Sunday, ahead of a high-stakes state Supreme Court race in Wisconsin, Musk doled out $1 million checks to two voters who had signed his super PAC's petition against "activist judges."
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Newsmax (NMAX) stock dropped 45.7% midday Wednesday after a massive post-IPO surge saw shares soar from $10 to $233.
Newsmax is a conservative cable news outlet and Fox News alternative that was founded in 1998 by CEO Christopher Ruddy, a media mogul and friend of US President Trump. The company — which also owns subsidiaries that sell nutritional supplements and insurance advertised in Newsmax's newsletters — raised $75 million in its IPO Friday, with shares priced at $10. Newsmax had previously raised $225 million in a private offering in February.
Read more about Newsmax's stock moves and today's market action.
The stock surged a staggering 735% Monday and another 180% Tuesday, rocketing its market cap from $1.2 billion upon its market debut to more than $20.8 billion at Tuesday's close. It showed the Trump trade still has room to run in some cases, just as the president's tariff policies slam the market at large.
Newsmax's market cap at Tuesday's close was higher than those of Wall Street Journal parent News Corp (NWSA) and AI server maker Super Micro Computer (SMCI).
Newsmax is unprofitable. While the company's revenue jumped more than 26% to $171 million in 2024 from the prior year's $135 million, its loss increased nearly 73% to $72 million from the prior year's loss, according to Newsmax's 10-K SEC filing. The company also said in its filing that it has identified "material weaknesses" in its financial reporting controls such that there may be "a material misstatement" in its financial statements that it may not detect "on a timely basis."
Newsmax's massive upswing has prompted various media outlets to draw comparisons to the meme stock craze of 2020 as well as the notoriously volatile stock of Trump Media & Technology Group (DJT). On Monday, trading of Newsmax stock was halted at various points due to volatility.
Newsmax's drop Wednesday sent its market cap down to $11.3 billion, with shares trading around $127. Meanwhile, DJT stock fell more than 5%.
Newsmax is facing an ongoing lawsuit from Dominion Voting Systems seeking $1.6 billion in damages related to false claims it made in its coverage of the 2020 election, which Newsmax cited among risk factors to its business in its latest 10-K filing to the SEC. Newsmax settled another lawsuit with another election tech company, Smartmatic, in 2024 for similar claims and has paid $20 million of the $40 million settlement thus far, according to the filing.
Laura Bratton is a reporter for Yahoo Finance. Follow her on Bluesky @laurabratton.bsky.social. Email her at laura.bratton@yahooinc.com.
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The Trump 2.0 stock market is off to one of the worst starts of any presidency since 1950.
New data crunched by SunDial Capital Research strategist Jason Goepfert on Wednesday shows the market's response to President Trump since the inauguration is among the bottom of all presidents since 1950. In the first 50 trading sessions since Inauguration Day, the S&P 500 (^GSPC) has dropped about 6.4%.
The only two worse starts were Richard Nixon's first 50 days (-7.2%) and George W. Bush's (-13.6%).
The best 50-day starts are held by John F. Kennedy (+9.4%), Barack Obama (+5.7%), and Bill Clinton (+4.2%).
Rough starts are a "bad open" for stocks when zooming out, warns Goepfert.
The S&P 500's median return six months after negative returns in the first 50 days was -1.9%. Even a year later, it was flat. Only four of the 10 instances showed a larger maximum gain than loss over the following year.
"While it is generally foolhardy to assign political motives to stock movements, the correlation seems pretty clear in this case. The new administration's take on tariffs has not impressed the stock market, which overshadows what is perceived as a bevy of more business-friendly policies. Whether the tariffs will take effect in announced form is guesswork," said Goepfert.
Trump is expected to unveil his new tariffs on what he calls "Liberation Day" after the market closes today around 4:00 p.m. ET. How tariffs shake out is anyone's guess.
The administration has floated everything from a 20% universal tariff to ones that are more sector-based. Administration officials said Tuesday the tariffs would take effect immediately but hinted Trump would be open to negotiating them lower.
Watch: AMD CEO warns about impact of tariffs on semis
A 25% US tariff on imports of steel and aluminum from all countries already took effect on March 12.
Fresh tariffs would arrive as other countries have fired back.
China has implemented a 15% tariff on US chicken, wheat, corn, and cotton products and an additional 10% tariff on sorghum, soybeans, pork, beef, seafood, fruits, vegetables, and dairy products. Canada has announced a 25% tariff on 30 billion Canadian dollars of US imports.
Market pros think tariffs could squeeze the economy and send stock prices even lower.
"I don't think the effect of tariffs is fully priced into markets," BCA Research chief strategist Peter Berezin told me on Yahoo Finance's Opening Bid podcast (clip above). "If you look at what's happened to stocks this year, they have gone down, but they've gone down primarily because of the Magnificent Seven stocks. If you look at the other 493 companies, they're basically flat for the year. That's not what you would expect from a market that has priced in a recession."
Brian Sozzi is Yahoo Finance's Executive Editor. Follow Sozzi on X @BrianSozzi, Instagram, and LinkedIn. Tips on stories? Email brian.sozzi@yahoofinance.com.
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Tesla shares rose Wednesday after Politico reported that Elon Musk could leave his post at the so-called Department of Government Efficiency, paving the way for the CEO to return his focus on the struggling electric vehicle maker.
The White House later called the report "garbage."
The stock was last up about 5%. At its session lows, it had dropped as much as 6.4% on the back of weaker-than-expected vehicle deliveries for the first quarter.
The report — which cites Trump insiders — noted that, while President Donald Trump is pleased with Musk and the DOGE spending cuts that have been pushed through, the two decided in recent days that the billionaire would soon return to his businesses.
NBC News is reporting that Trump told the Cabinet that Musk could leave in the coming months. A senior government official told NBC News that Musk would leave at the end of a 130-day stint as a special government employee.
Wednesday's report comes during a tough stretch for Tesla. Despite Wednesday's gains, the stock has dropped more than 5% over the past month. Year to date, it has tumbled more than 31%. The shares also shed 36% in the first quarter, marking their biggest quarterly drop since 2022.
Musk's role in the White House is one factor weighing on Tesla's stock. It has sparked waves of protests, boycotts and violent attacks on Tesla stores and vehicles around the world. Trump's automotive tariffs are also a concern as they involve Tesla's key suppliers — notably in Mexico and China.
"My Tesla stock and the stock of everyone who holds Tesla has gone, went roughly in half," Musk said Sunday night at a rally he held in Green Bay, Wisconsin, to promote a Republican judge he backed in Tuesday's state supreme court election, Brad Schimel. "This is a very expensive job is what I'm saying."
In addition to holding the rally in Wisconsin, Musk spent millions and frequently posted about the race on his social network X. Judge Susan Crawford, who won the seat on the Wisconsin Supreme Court, was backed by Democrats and progressive groups who criticized Musk, his money and influence on the race as well as his DOGE work in their campaigns.
Separately, New York City Comptroller Brad Lander urged the city to sue Tesla on behalf of NYC pension funds citing Musk's work for the White House.
In a Tuesday statement, Lander's office said: "The basis of the potential litigation are the material misstatements from Tesla claiming that CEO Elon Musk spends significant time on the company and is highly active in its management, despite his helming the Trump Administration's DOGE initiative, spending little of his time actually managing Tesla, and promoting policies that are actively harmful to Tesla's business."
CNBC reached out to the White House for comment.
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A man accused of being a corporate spy for a rival HR software company admitted to secretly working for Deel while he was an employee at Rippling.
The now-former employee, Keith O'Brien, wrote in an affidavit made public in an Irish court Wednesday that he was recruited as a corporate spy by Alex Bouaziz, Deel's founder and CEO.
"Alex told me he 'had an idea.' He suggested that I remain at Rippling and become a 'spy' for Deel, and I recall him specifically mentioning James Bond," O'Brien wrote in the affidavit.
"About thirty minutes later, I called Alex back over WhatsApp and told him that I was onboard with the plan," O'Brien added.
The affidavit is part of legal proceedings in Ireland, where O'Brien worked in one of Rippling's satellite offices.
The San Francisco-based workforce management software company has a long and bitter rivalry with Deel. Each company has been valued at north of $10 billion.
In March, Rippling sued Deel in San Francisco federal court, alleging Deel turned O'Brien — who was not named in the lawsuit — into a corporate spy.
Rippling said O'Brien had collected company secrets on the company's Slack, Salesforce, and Google Drive networks and then passed them on to Deel, giving the rival a competitive edge.
O'Brien said in the newly filed affidavit that he collected the information at Bouaziz's direction. His first conversations with Bouaziz are recounted in a section titled "I Begin Spying for Deel."
"Alex was particularly interested in Rippling's strategies around global payroll and expansion efforts, as well as reviewing specific sales, marketing information, and customer details," O'Brien wrote. "Alex would give me direction for what terms to search on Rippling's Slack system in order to yield the information he wanted."
O'Brien, who was hired in Rippling's Dublin office to oversee payroll issues in Europe, wrote that he first met Bouaziz after he created a payroll consulting company as a side job.
O'Brien, while offering to consult for Deel, told Bouaziz he was considering quitting his day job and consulting full time, he wrote.
O'Brien says Bouaziz suggested he continue working for Rippling and "spy" for Deel.
For his work, O'Brien received a monthly payment of $6,000, mostly in cryptocurrency, he wrote. He also communicated with Philippe Bouaziz, Alex's father and Deel's CFO, he wrote in the affidavit.
O'Brien's attorneys at the Irish law firm Fenecas Law attached 47 pages of exhibits to the affidavit, showing records of crypto transactions, notifications of deleted messages, and examples of documents O'Brien says he stole from Rippling on Deel's behalf.
Representatives for Deel didn't immediately respond to a request for comment.
The company previously said it denies "all legal wrongdoing" and would file a counterclaim against Rippling.
Rippling said in its lawsuit last month that O'Brien was caught after the company devised an elaborate honeypot operation that led him to search for information in a phony Slack channel called "#d-defectors," where Rippling employees purportedly discussed information Deel would find embarrassing.
In the new affidavit, O'Brien wrote his handlers at Deel realized Rippling had set the trap — but not until it was too late.
"I ran the search immediately and began to look at the results," O'Brien wrote. "Within minutes, Alex messaged me again and told me not to run the search because he believed it was a 'trap.'"
"I told Alex that by the time I got his message, I had already done the search and he said 'oh shit,'" O'Brien continued.
Rippling said in its lawsuit that it brought O'Brien's spying to the attention of an Irish court, which appointed a lawyer to Rippling's office to seize his electronic devices for preservation in potential future litigation. O'Brien's affidavit, dated April 1 and made public Wednesday, came in a court case related to those proceedings.
When O'Brien was confronted by the court-appointed lawyer, he hid in the bathroom and appeared to try to flush his phone down the toilet, Rippling alleged.
In O'Brien's affidavit, he said the lawyer's appearance made him realize "how serious this was" and he "panicked."
He performed a factory reset on his phone while in the bathroom and "flushed the toilet a couple of times," he wrote.
O'Brien left the Rippling office and contacted Alex Bouaziz, he wrote. A Deel lawyer instructed him to get rid of his phone, he wrote.
"He told me to destroy my old phone by breaking it up and throwing it in the canal," he wrote.
The next day, O'Brien "smashed my old phone with an axe and put it down the drain at my mother-in-law's house, as Deel's lawyer Asif had advised," he wrote.
Deel also offered to move O'Brien's entire family to another country, he wrote.
"Deel's lawyer Asif said they would move me and my family to Dubai, and would figure out a mechanism to cover my legal costs," O'Brien wrote.
O'Brien said he became frustrated with the people at Deel, who he said wanted to promote a false story accusing Rippling of violating Russian sanctions to "shift the narrative."
Deel's attorneys also deleted their messages and made it hard to reach them, he wrote.
After a few days, O'Brien agreed to cooperate with Rippling, he said.
"I was getting sick concealing this lie," he wrote. "I realised that I was harming myself and my family to protect Deel."
"I was concerned, and I am still concerned, about how wealthy and powerful Alex and Philippe are, but I know that what I was doing was wrong," he continued.
April 2, 2025: This story has been updated with more details from the court documents.
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Trump Media's stock slid on Wednesday after the company disclosed in a securities filing the possibility of significant stock sales, including by insider shareholders such as the president's trust.
The parent company of Truth Social said in a filing dated April 1 that the company could soon sell roughly 8.4 million shares of common stock related to existing warrants issued during the initial public offering. The company said insiders and major stakeholders could also sell up to about 134 million shares "from time to time."
That includes the more than 114 million shares held by the Donald J. Trump Revocable Trust.
The stock was down about 5% in midday trading.
President Donald Trump has previously said he does not plan to sell his stake in the company. His son Donald Trump Jr. is in charge of the revocable trust.
"The sale of the Resale Securities being offered pursuant to this prospectus, or the perception that these sales could occur, could result in a significant decline in the public trading price of our Common Stock," the prospectus warned.
The company said the total shares available for resale amounted to 129.2% of the company's public float of shares, which is a measure of the amount of stock currently available for trade.
In a statement, the company said the filing does not indicate that insider sales are planned.
"Legacy media outlets are spreading a fake story suggesting that a TMTG filing today is paving the way for the Trump trust to sell its shares in TMTG. To be clear, these shares were already registered last June on an S-1 form, and today TMTG submitted a routine filing that re-registers them on an S-3 form in order to keep the Company's filings effective. In fact, there currently is no open window for any affiliate to sell shares," the statement said.
Trump Media stock has been highly volatile since it went public through a combination with a special purpose acquisition company last year. The stock is down about 70% from its postmerger highs.
Still, the shares are worth a lot of value on paper, especially relative to the company's meager revenue. Shares were trading at around $19 per share Wednesday morning, putting the notional value of the trust's position at more than $2 billion.
The company warned in the filing that insiders could profit from a sale of stock even if doing so drives down the price.
"Selling Securityholders may still experience a positive rate of return on the shares of Common Stock purchased by them due to the lower price per share at which such shares of Common Stock were purchased as referenced above, but public stockholders may not experience a similar rate of return on the Common Stock they purchased if there is such a decline in price and due to differences in the purchase prices and the current market price," the prospectus said.
The underlying business of Trump Media is much smaller than other public social media companies. The firm reported less than $4 million in total sales in 2024, with a net loss of about $401 million. The company said earlier this year it plans to expand into financial services.
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Listen and subscribe to Opening Bid on Apple Podcasts, Spotify, Amazon Music, YouTube or wherever you find your favorite podcasts.
Investors should brace for a leaner stock market and economy as Trump tariffs and retaliations from trading partners like Canada, Europe, and China take hold.
Those are the gloomy calls from BCA Research chief strategist Peter Berezin.
BCA Research is an independent research firm that has been in business since 1942 and is among the largest independent macroeconomic forecasters to institutions. And Berezin has been an economist for more than 30 years, with stints at the International Monetary Fund (IMF), Goldman Sachs, and now BCA Research.
Berezin gained attention this year for being the lone bear on Wall Street coming into 2025. Further, he correctly called that in 2022 there would be no US recession — despite most on the Street bracing for one.
In a new episode of Yahoo Finance's Opening Bid podcast (see video above), Berezin doubled down on his recent call that there is a 75% chance of a US recession this year. In a new wrinkle shared with me, Berezin believes the US may already be in a mild recession.
He forecasts slight negative economic growth for the year as consumers pull back amid a more inflationary environment, thanks to a global trade war.
Read more: What Trump's tariffs mean for the economy and your wallet
Assuming his economic call holds true, Berezin thinks the S&P 500 (^GSPC) is destined for 4,450 — down about 21% from current levels. The best places for investors to hide out this year may continue to be gold and consumer staples and soon, bonds.
"I don't think the effect of tariffs is fully priced into markets," Berezin said. "If you look at what's happened to stocks this year, they have gone down, but they've gone down primarily because of the Magnificent 7 stocks. If you look at the other 493 companies, they're basically flat for the year. That's not what you would expect from a market that has priced in a recession."
Others on the Street have become more cautious about stocks and the economy as Trump tariffs come into focus.
Goldman Sachs chief economist Jan Hatzius said Monday he now sees US gross domestic product (GDP) growth averaging 1.5% in 2025. That's down from previous expectations for growth to average 1.9%.
In addition, he sees a 35% chance of a US recession in the next 12 months, compared with 20% previously.
Meanwhile, Hatzius's colleague David Kostin slashed his 2025 S&P 500 target to 5,700 from 6,200. He cited a higher recession risk and tariff-related uncertainty.
Kostin joins the likes of SocGen and Yardeni Research in lowering S&P 500 targets within the last few weeks.
Berezin explained, "I don't expect a very deep recession because the imbalances in the economy are not as severe as they were in say, 2008. Nevertheless, I think we'll probably get a fairly nasty recession, especially as financial markets are concerned. You think about the 2001 recession, that was a very mild recession. We didn't even have two consecutive quarters of negative growth, and yet stocks still fell 49% peak to trough because they were so expensive going into that recession."
The full Opening Bid episode with Berezin will air at 8:30 a.m. ET on April 7.
Three times each week, I field insight-filled conversations and chats with the biggest names in business and markets on Opening Bid. You can find more episodes on our video hub or watch on your preferred streaming service.
Brian Sozzi is Yahoo Finance's Executive Editor. Follow Sozzi on X @BrianSozzi, Instagram, and LinkedIn. Tips on stories? Email brian.sozzi@yahoofinance.com.
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Coinbase CEO Brian Armstrong spoke out this week on the need for interest-bearing stablecoins ahead of the House Financial Services Committee's Wednesday markup of its version of stablecoin legislation.
Specifically, he said stablecoins should be able to pay interest to consumers, and that banks and crypto companies should be able to do the same.
"We're supportive of the draft bill that's going through the Senate as it's written but there is a part of it that does concern me a bit, which is this idea that consumers cannot get interest on stablecoins," he said on CNBC's "Money Movers" Tuesday. "Many Americans, they're only earning about 0.14% in their savings account, but in Treasurys, you can earn 4.5% right now. That seems unfair to me … we'd like to see legislation that allows that."
The House STABLE (Stablecoin Transparency and Accountability for a Better Ledger Economy) Act specifies that permitted issuers "may not pay interest or yield to holders" of the stablecoin. Similarly, the Senate's GENIUS (Guiding and Establishing National Innovation for U.S. Stablecoins) Act's definition of a "payment stablecoin" specifies that it "does not offer a payment of yield or interest." The latter bill was passed in the Senate in March.
Armstrong's comments follow a lengthy post on his X account that goes into more detail about his view.
"Unlike interest-bearing checking and savings accounts, stablecoins do not currently benefit from the same exemptions under the securities laws that allow issuers to pay interest to users," he wrote. "Stablecoins should be able to pay interest just like an ordinary savings account, without the onerous disclosure requirements and tax implications imposed by securities laws."
Stablecoins like Tether (USDT) or USD Coin (USDC) are backed 1-to-1 by the U.S. dollar and their issuers typically hold reserve assets in cash and short-term Treasury securities. Historically these types of assets have acted as a bridge for traders between traditional financial rails and crypto systems, and crypto investors watch them closely for evidence of demand, liquidity and activity in the market.
In recent years, stablecoins have also become popular for their ability to pay users interest for simply holding them – however, that interest payment is typically an incentive offered by exchanges like Coinbase or Kraken or wallet operators rather than the stablecoin issuer itself.
Coinbase has an agreement with the stablecoin issuer Circle – the company behind USDC, which filed Tuesday evening for an initial public offering with the SEC – to share 50% of the revenue of USDC. A section of the company's prospectus echoed Armstrong's concern:
"Absent federal regulations, there is a possibility that Circle stablecoins may be classified as 'securities,'" the filing said. "Any classification of Circle stablecoins as a 'security' would subject us to additional regulation and could materially impact the operation of our business."
The Securities and Exchange Commission typically views assets as securities if they constitute an investment of money with the expectation of profit from others' efforts. Security status often implies being subject to greater regulatory scrutiny and compliance obligations that are very expensive for companies.
Ben Kurland, CEO at crypto research platform DYOR, said allowing issuers to pay users interest would also mark a big shift in who holds profits in crypto.
"Right now, exchanges and platforms capture the yield from stablecoins by investing user deposits and keeping most of the return," he said. "If stablecoin issuers start paying interest directly to users, it flips that model: users benefit more, middlemen get squeezed, and stablecoins become a lot more attractive to hold."
Traditional, non-yield-bearing, dollar-backed stablecoins have grown their market cap by more than 46% in the past year, according to CryptoQuant. But yield-bearing stablecoins like Ethena's USDe or Ondo Finance's USDY are the fastest-growing cohort within the stablecoin universe, according to JPMorgan.
"This universe of yield-bearing stablecoins has been growing exponentially post the U.S. election, with the market cap of the five biggest surpassing $13 billion, or 6% of the total stablecoin universe," JPMorgan's Nikolaos Panigirtzoglou said in a note last Wednesday.
Over time, yield-bearing stablecoins could replace most of the "idle cash" sitting in traditional stablecoins, he added, saying the 6% share it currently has of the total stablecoin market cap will grow "to a much higher percentage but not more than 50%."
"Traditional stablecoin issuers such as USDT and USDC do not share reserve yields with users, a practice that could significantly reduce their revenues but more importantly would make stablecoins classified as securities," he added. "That would have subjected stablecoins to additional regulatory restrictions and require compliance with securities law, thus hindering their current seamless and permissionless use as source of collateral in the crypto ecosystem."
—CNBC's Michael Bloom contributed reporting
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Take it easy on your kitchen's 20-year-old tile floors. New ones likely won't come cheap if the Trump administration imposes new tariffs today.
New research from Bank of America analyst Rafe Jadrosich found that 40% of the ceramic tile market is imported. About 80% of the luxury vinyl tile market is imported. Roughly 35% of ceramic imports come from Italy and Spain, followed by 14% each from Mexico and India. China accounts for 50% of the imports of luxury vinyl tile.
Those tile floors, along with bathroom fixtures and dishwashers, were highlighted as three common household items that could see higher prices with new tariffs.
Read more: What Trump's tariffs mean for the economy and your wallet
Jadrosich added that plumbing fixtures, valves, and rough plumbing are manufactured in China, in addition to upstream components imported from Asia.
Plumbing supplier play Fortune Brands Innovations (FBIN) has disclosed that 50% to 60% of its input costs come from outside the US, per Jadrosich.
Home improvement products supplier Masco (MAS) has said a 10% tariff on China would have an annualized impact of $45 million on profits before any mitigating actions like price hikes.
Jadrosich estimated that about 40% of the US appliance industry's sales are imported. About 43% of the industry's products are sourced from China, 20% from Mexico, and 11% from South Korea. Whirlpool (WHR) leans on Mexico and China for 20% of its cost of goods sold.
The most exposed building materials stocks to new tariffs include Whirlpool, Fortune Brands Innovations, Masco, and flooring player Mohawk Industries (MHK), Jadrosich said. All four stocks are down for the year and trailing the S&P 500 (^GSPC), with Whirlpool leading the way with a 20% tumble.
"The impact to housing stocks will depend on the details," Jadrosich warned.
Markets are on edge as worries about Trump's tariffs ripple through corporate America, with the threat of retaliation by trading partners adding to the potential impact of hiked duties.
Trump is expected to unveil his new tariffs on what he calls "Liberation Day" after the market closes today around 4:00 p.m. ET. How tariffs shake out is anyone's guess.
The administration has floated everything from a 20% universal tariff to ones that are more sector-based. Administration officials said Tuesday the tariffs would take effect immediately but hinted Trump would be open to negotiating them lower.
Watch: AMD CEO warns about impact of tariffs on semis
A 25% US tariff on imports of steel and aluminum from all countries already took effect on March 12.
Fresh tariffs would arrive as other countries have fired back.
China has implemented a 15% tariff on US chicken, wheat, corn, and cotton products, and an additional 10% tariff on sorghum, soybeans, pork, beef, seafood, fruits, vegetables, and dairy products. Canada has announced a 25% tariff on 30 billion Canadian dollars of US imports.
"The bigger picture is U.S. economic growth [GDP] revisions are being revised lower for the first time in years. Our view is there are downside risks to earnings given the lowering in economic growth estimates alongside risks to profit margins posed by tariffs," Truist co-chief investment officer Keith Lerner warned.
Brian Sozzi is Yahoo Finance's Executive Editor. Interact with Sozzi on X @BrianSozzi, Instagram, and LinkedIn. Tips on stories? Want to share your views on stocks and leaders? Email brian.sozzi@yahoofinance.com.
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The fashion industry is gearing back up to navigate Trump tariffs that could hit profits for the crucial spring and holiday shopping seasons.
“It's impossible [to deal with], so we're not dealing with it this time around,” said noted handbag and accessories designer Rebecca Minkoff to Yahoo Finance Executive Editor Brian Sozzi during an episode of the Opening Bid podcast (see the video above; listen below). “We dealt with it the last time tariffs were instituted. I think that year we thought we were going to make $3 million. We lost $3 million.”
Minkoff has had a robust career designing everything from handbag color pallets to supply chains.
She is the founder of the Female Founder Collective, as well as her namesake fashion company, and recently published a book called "Fearless: The New Rules for Unlocking Creativity, Courage, and Success."
Her fashion business is still rebounding from losses suffered during COVID-19, a time she estimates hampered the company's sales by 70%. Although she sold the company three years ago, she remains a “very happily paid employee.”
Watch: How Build-a-Bear's CEO is navigating Trump tariffs
Minkoff said she went to testify on behalf of the fashion industry during the first Trump administration. Those efforts didn't help sway policymakers on tariffs, which can leave companies with a slew of matters to solve.
“It was impossible with the timeline set forth to change [the] supply chain,” said Minkoff, who noted that fashion runs on a nine-month life cycle from concept to hitting the floor.
To stay in business back then, Minkoff relocated operations out of China, a move that “saved us a lot of money in the long run, but it's pain we didn't need."
The company has been manufacturing out of Vietnam and Indonesia for the past three years, which has helped keep costs under control.
Tariffs have proven to be more than a buzzword for the consumer lately.
Read more: What Trump's tariffs mean for the economy and your wallet
Since Trump took office, he has announced a 25% tariff on Mexico and Canada, though goods under the United States-Mexico-Canada Agreement (USMCA) were exempt until April 2. In March, another 10% duty on Chinese goods was levied on top of the previous 10%.
To put it into context, China's 2024 foreign textiles and apparel trade reached $322.4 billion, with $301.1 billion in exports and $21.27 billion in imports.
This is before Trump's "Liberation Day" today when he is expected to unwrap fresh tariffs on individual countries.
“We're going to have to spend more on everything,” said Minkoff of the new tariffs. “Everything is going to be more expensive.”
In fashion, one move to offset costs can be to adjust the quality of materials, such as zippers or buttons. “I think a lot of brands are going to do that, and it's going to hurt them in the end,” said Minkoff. “It might be a quick fix for now, but the customer sees it.”
An outsider might think the easiest way to avoid tariff-based fees is to move operations back to the US full-time. But US-made goods will sport heftier price tags, Minkoff warned.
Just the cost to make one T-shirt varies greatly by region based on rules and regulations. According to the website successfulfashiondesigner.com, one shirt made in New Jersey costs an average sample fee of $250 plus $7 for shipping, while the same shirt made in China costs $50 to make and $30 to ship.
Minkoff, who started designing out of a shop on 38th Street in New York City, has fielded this advice before. “I would love manufacturing to come back, but it has to come back on a scale that is not just the make,” she said.
Three times each week, Yahoo Finance Executive Editor Brian Sozzi fields insight-filled conversations and chats with the biggest names in business and markets on Opening Bid. You can find more episodes on our video hub or watch on your preferred streaming service.
Grace Williams is a writer for Yahoo Finance.
Click here for the latest stock market news and in-depth analysis, including events that move stocks
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United Airlines plans to add daily flights to Vietnam and Thailand in October, further expanding the network for the U.S. carrier that already has the most Asia service.
In the expansion, United is using a tactic that's unusual in its network: Its airplanes from Los Angeles and San Francisco that are headed for Hong Kong will then go on to the two new destinations. The Bangkok and Ho Chi Minh City, Vietnam, service is set to begin on Oct. 26.
On Oct. 25, United plans to add a second daily nonstop flight from San Francisco to Manila, Philippines, and on Dec. 11, it will launch nonstops from San Francisco to Adelaide, Australia, which will operate three days a week.
The carrier has aggressively been adding far-flung destinations not served by rivals to its routes, like Nuuk, Greenland, and Bilbao, Spain, which start later this year. Getting the mix right is especially important as carriers seek to grow their lucrative loyalty programs and need attractive destinations to keep customers spending.
Bangkok, in particular, "is in even more demand now given the popularity of 'White Lotus,'" Patrick Quayle, United's senior vice president of network and global alliances, said of the HBO show.
He said the carrier isn't planning on cutting any international routes for its upcoming winter schedule.
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Germany's armed forces, the Bundeswehr, announced on Tuesday that they'd created a new brigade, the 45th Armored Brigade, to be stationed in Lithuania.
It will be the first long-term deployment of German soldiers to another country since World War II, the Associated Press reported.
Brig. Gen. Christoph Huber, the commander of the 45th Armored Brigade, said that with its creation, "we're not only moving toward operational readiness, we're taking responsibility."
"For the alliance, for Lithuania, for Europe's security," he said. "As a sign of our determination to defend peace and freedom with our partners."
Russia's invasion of Ukraine has sparked a surge of defense agreements and spending among European countries.
When plans were first made for the 45th Armored Brigade in 2023, Germany described it as part of growing efforts by NATO members to boost both their own security and the security of NATO's eastern borders.
Germany's defense minister, Boris Pistorius, previously said, "With this war-ready brigade, we're taking on leadership responsibility on NATO's eastern flank."
The new brigade is made up of several battalions and will have about 5,000 soldiers and civilian staff, the Bundeswehr said this week.
It added that the brigade's command facility was already fully operational and that the aim was to have it at full wartime readiness by 2027.
Lithuania — which borders the Russian enclave of Kaliningrad and the close Russian ally Belarus — is one of the countries that has sounded the alarm the loudest that Russia could move beyond Ukraine to attack elsewhere in Europe.
It's also one of NATO's biggest defense spenders by proportion of GDP and one of Ukraine's biggest allies, describing Ukrainian troops as the ones who are protecting all of Europe.
There are already NATO troops in Lithuania, on a rotating basis, with a multinational battle group led by Germany. NATO countries have also deployed assets such as fighter jets and air defenses there.
US troops are among those stationed in Lithuania, though their long-term future is less clear, with President Donald Trump critical of US allies, of NATO, and of assistance to Ukraine.
Dovilė Šakalienė, Lithuania's defense minister, told Business Insider in February that her country wanted US troops to stay and that she expected the US could see "eye to eye" with countries who pay their part when it comes to defense.
"We do our part," she said, adding that she expected the US to do its part, too.
Lithuania has also been strengthening its border with Russia.
Germany's new brigade is the latest in a series of measures introduced by the country since Russia's full-scale invasion of Ukraine in February 2022.
It spent 1.51% of its GDP on defense in 2022, which jumped to an estimated 2.12% in 2024, according to NATO.
The rise in defense purchases by Germany, and Europe more broadly, has been a boon for the continent's defense industries.
The German arms manufacturer Rheinmetall said in March that it expected sales this year to rise by 25% to 30%.
Germany's defense spending has increased less than some of its allies: It ranked 15th out of 31 NATO members for defense spending as a proportion of GDP in 2024, according to NATO estimates.
But it has vowed to do more.
After grappling with its World War I and World War II legacies, which led to an avoidance of heavy militarism, Germany has committed to major military moves.
Lawmakers this month voted to alter the German constitution in a way that would unlock billions of dollars that could be used for defense spending.
Šakalienė, Lithuania's defense minister, told BI in February that Europe "needs to up our defense spending very fast and very significantly."
She said Europe needed to be able to match the US and to match Russia, which was escalating its own defense production: "We need to catch up to the speed of Russia," she said.
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TKO Group's UFC has struck a multimillion-dollar, multiple-year partnership deal with Meta that will bring the mixed martial arts league closer to Mark Zuckerberg's technology company, UFC told CNBC.
UFC's integration with Meta will span the company's portfolio, including Meta AI, Meta Glasses, Meta Quest, Facebook, Instagram, WhatsApp and Threads. Specific financial terms weren't disclosed.
Meta will become the "official fan technology partner" of UFC and will have its branding featured in UFC's Octagon ring for pay-per-view and "Fight Night" events.
"Mark and his team at Meta are going to do things that will blow away UFC fans," UFC President and CEO Dana White said in a statement to CNBC.
The partnership with Meta is separate from the UFC's media rights discussions, which are set to kick off later in April. UFC's exclusive negotiating window with its current partner ESPN ends April 15. ESPN doesn't plan to renew its deal before the window's expiration, CNBC has previously reported.
The companies first started working on a sponsorship deal in the second half of 2024, according to Grant Norris-Jones, TKO's head of global partnerships. UFC held discussions with a number of potential partners in different business units and realized Meta could provide the league with much of what it wanted, Norris-Jones said in an interview.
"Meta will be our official marketing partner, our official AI glasses partner, our official wearable partner, an official social media partner," said Norris-Jones. "They're making a significant investment into our ecosystem."
Meta's Threads will feature exclusive UFC content and will be referenced in live UFC broadcasts, Norris-Jones said. Both companies are already working on a series of follow-on announcements to come in the next three to nine months, including more details around a new UFC fighter rankings system that will draw on Meta technology, he said.
While White and Zuckerberg, Meta's founder and CEO, didn't personally hammer out terms of the deal, it's "nice to have the air cover" of the executives' close relationship, Norris-Jones said. White joined the Meta board in January.
"I love this sport and I'm looking forward to working with UFC to let fans experience it in new ways," Zuckerberg said in the statement to CNBC.
The Meta CEO has attended a number of UFC events and personally participates in mixed martial arts.
Zuckerberg said on Joe Rogan's podcast in January that corporate culture would benefit from more "masculine energy." The Meta CEO said in July that President Donald Trump's reaction after getting shot in the ear was "badass." Trump is also friendly with White, who endorsed Trump and spoke at the 2024 Republican National Convention.
"Having a culture that celebrates the aggression a bit more has its own merits," Zuckerberg said on the Rogan podcast.
Meta noted Zuckerberg's love of combat sports in its annual report as a potential risk factor.
"We currently depend on the continued services and performance of our key personnel, including Mark Zuckerberg," the company said in a corporate filing. "Mr. Zuckerberg and certain other members of management participate in various high-risk activities, such as combat sports, extreme sports, and recreational aviation, which carry the risk of serious injury and death. If Mr. Zuckerberg were to become unavailable for any reason, there could be a material adverse impact on our operations."
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Uncertainty surrounding upcoming U.S. tariffs has weighed on global markets over the last month — but President Donald Trump's highly-anticipated Wednesday announcement may not end the turmoil, analysts say.
Trump is expected to flesh out many unknowns in an address on Wednesday at 4 p.m. ET (9 p.m. BST), including exactly which countries and sectors will be impacted, when the measures will come into effect, how high they will be or how they will be calculated, and whether there will be any or many notable exemptions.
Investors have been attempting to parse rhetoric from reality in the run-up to the presidential address. Global stocks suffered their worst month for a year and a half in March, according to the MSCI World Index. Europe's Stoxx 600 dropped 4.18% while the U.S. S&P 500 tumbled 5.75%. Asia-Pacific stocks were slightly more resilient, though China's CSI 300 dipped 0.07%.
Zoe Gillespie, chartered wealth manager at RBC Brewin Dolphin, said the Wednesday announcement is "unlikely" to contain full details — and would still leave a lack of clarity over the outlook.
"The danger is actually, what's going to happen after the announcement, whether we're going to see the European Union coming back with some retaliatory tariffs, and really the kind of the outlook from that," she told CNBC's "Squawk Box Europe" on Wednesday.
"There could still be more uncertainty as this plays out, and [questions on] whether it becomes a bit like the inflation story, more entrenched in the longer term. And I think the danger is that we don't have much clarity for a longer period of time, and the impact on growth, and if we start getting some negativity coming through then it could actually delay any real 'this is the peak bottom' signals."
The EU — which, as a bloc, is the U.S.'s biggest trading partner across goods, services and investment — has already announced a first tranche of retaliatory tariffs in response to Trump's initial wave of duties on steel and aluminum imports. European Commission President Ursula von der Leyen on Tuesday said the EU would "take firm counter-measures if necessary" and that "all instruments are on the table" in response to further U.S. action.
The U.K., which is hoping to leverage its more balanced trade relationship with the U.S. to strike an economic deal and avoid the worst impact of tariffs, is also keeping all options on the table, Prime Minister Keir Starmer said Tuesday.
While Trump has branded April 2 "Liberation Day," a better descriptor might be "Carnage Day," Ozan Ozkural, founding managing partner at Tanto Capital Partners, told CNBC on Tuesday.
"This is just classic Trump shock and awe in trying to get counterparts to the negotiation table to try to get a better deal for United States," he said.
"The way he is going about doing that, and the fact that there is a continuous news cycle which is sometimes contradictory, is what's making it very difficult to price any asset right now."
"If you take a look at the commodities markets, it's crazy, because on the one hand you're talking about secondary sanctions on Venezuela oil, potentially a Russia-Ukraine deal, which may bring Russian crude back into the market, which is a completely different game, and now potential secondary sanctions on Russian oils. It is very difficult to price anything at the moment, so we are taking it day by day and hour by hour, sometimes," Ozkural said.
He added that he was nonetheless betting on the U.S. outperforming Europe in the long term, despite its rougher start for 2025 in markets. While Europe will benefit from tailwinds including regional commitments to rearmament and Germany's huge new defense, infrastructure and climate fund, the continent will continue to suffer from its innovation gap, he argued.
RBC Brewin Dolphin's Gillespie also said the wealth manager saw the U.S. eventually outperforming, even if it had trimmed its U.S. allocation recently.
"Because of the U.S.'s broad nature and the quality of companies, it's quite difficult to look elsewhere, longer term, and avoid the U.S.," she said, citing the impact from artificial intelligence.
But further uncertainties clouding the near- to medium-term outlook for investors include what impact tariffs will have on U.S. growth, inflation and the path for interest rates, Gillespie continued — which in the short term could spur investors to look at more domestically-focused companies avoiding global supply chains.
However, Arnaud Girod, head of economics and cross asset strategy at Kepler Cheuvreux, said Wednesday may mark "peak uncertainty" for markets.
He also noted that the U.S. had seen a huge pullback in the first quarter, logging one of the biggest outperformances in Europe on record.
"I'm hoping that there is a sense of moderation tonight, we'll see. Maybe that's too naive, after everything we saw from Trump," he told CNBC's "Street Signs Europe."
"What's for sure is that a lot of numbers have been shared by analysts, they have been modeling every kind of scenario. So I'm hoping tonight is the worst, is the peak of uncertainty, and from there there will be negotiations, discussions, and hopefully the impact will be slightly less than the worst case... and so hopefully that can help the U.S. markets recover."
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Mortgage rates have barely budged in the last month. That, and a broader concern among consumers about the state of the economy, has also kept mortgage demand muted.
Total mortgage application volume dropped 1.6% last week compared with the previous week, according to the Mortgage Bankers Association's seasonally adjusted index.
The average contract interest rate for 30-year fixed-rate mortgages with conforming loan balances, $806,500 or less, decreased to 6.70% from 6.71%, with points increasing to 0.62 from 0.60, including the origination fee, for loans with a 20% down payment.
Applications to refinance a home loan dropped 6% for the week and were 57% higher than the same week one year ago. The annual comparison is so large because the total volume is so low. Mortgage rates were 21 basis points higher the same week one year ago. Given how low rates were during the first few years of the Covid pandemic, which sparked a massive refinance boom, there are now precious few borrowers who can benefit from a refinance at the current rates.
Applications for a mortgage to purchase a home rose 2% for the week and were 9% higher than the same week a year ago. Demand from buyers was at its highest level since the end of January, driven by a 3% increase in conventional purchases. Demand for government loans, favored by lower-income borrowers, was down 2%.
"Overall purchase activity has shown year-over-year growth for more than two months as the inventory of existing homes for sale continues to increase, a positive development for the housing market despite the uncertain near-term outlook," said Joel Kan, vice president and deputy chief economist at the MBA.
Mortgage rates started this week slightly lower but are still moving in a very narrow range as uncertainty over tariffs keeps the markets guessing.
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U.S. President Donald Trump's tariffs on auto imports is expected to hit hard for car brands and countries around the world, with economists warning a small landlocked nation in Europe could feel the worst of the impact.
Trump is poised to unveil the details of his latest round of sweeping tariffs at around 9 p.m. London time (4 p.m. ET) on Wednesday. The measures, which are separate from Washington's levies on the global automotive sector, appear designed to rebalance the economic order in America's favor.
Trump has already announced a 25% tariff on all imported vehicles, with charges on businesses importing vehicles coming into effect as of Thursday. The White House has said a tax for automobile parts is set to start in May.
The president's back-and-forth announcements on tariffs have alarmed global investors, corporate executives and U.S. trading partners in recent months. Germany slammed Trump's 25% auto tariffs as bad news for the U.S., the European Union and global trade.
Inga Fechner and Rico Luman, economists at Dutch bank ING, have said that, while Germany is most exposed to Trump's 25% auto tariffs in terms of value, it is Slovakia that may feel the biggest impact.
"Germany's car industry is in the eye of the storm and by far most exposed in terms of value, with major players like Volkswagen, BMW, Mercedes, and Porsche likely getting hit by tariffs," ING said in a research note published March 28.
"But Slovakia – home to several car plants – is most exposed in terms of total US export volume," they added.
Nicknamed the "Detroit of Europe" due to its thriving automotive industry, Slovakia produces more cars per capita than any other country in the world.
The nation of just 5.4 million people relies heavily on U.S. trade, with autos accounting for a sizable chunk of its U.S. exports and the sector indirectly employing more than 250,000 people.
ING's Luman said there has been a frontloading of car shipments to the U.S. ahead of Trump's auto tariffs and that "this will unwind" after the measures are imposed.
"Supply chains won't be immediately redirected and redesigned amid high uncertainty for investors, but if it holds a while production will start to shift to the US," Luman told CNBC by email on Wednesday.
On the other hand, Luman said Slovakia still has relatively low production costs, as local wages are significantly lower than in Germany.
"So, VW and Stellantis will likely seek to keep occupation rates of their factories in Slovakia up (for ICE models) and as such the export setback could also weigh on production in elsewhere in Europe, which will be replaced," Luman said.
Vladimir Vaňo, chief economist at Globsec, a think tank based in Slovakia's capital of Bratislava, has previously described the prospect of Trump tariffs as "worrisome" for Slovakia, while suggesting there appeared to be "very little" the country could do in the short term.
A spokesperson for Slovakia's government was not immediately available to comment.
Slovakia is Europe's joint-third-largest exporter of passenger cars to the U.S., alongside Sweden, with 4 billion euros ($4.3 billion) worth of exports to the U.S. in 2023, according to ING.
Notably, ING said more than 73% of Slovakia's total exports to the U.S. consist of car and car parts, leaving the country acutely exposed to Trump tariffs.
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Ten major firms are under the spotlight as the Trump administration continues its consulting crackdown — but one is taking the most heat.
At least 127 of Deloitte's government contracts have been cut or modified since January, about double the total for the second-hardest-hit consultancy on the Trump administration's list, a Business Insider analysis of data on the White House DOGE office's website found.
The Big Four firm is one of the federal government's 10 highest-paid consultancies, which have come under scrutiny amid the White House's push to cut waste and improve efficiency. The list includes Accenture, Booz Allen Hamilton, IBM, and General Dynamics.
DOGE estimates the Deloitte cuts will save taxpayers about $371.8 million.
This includes $51.4 million in savings from a contract providing IT services to the Centers for Disease Control and Prevention and $1.1 million in savings from a contract for training on diversity, equity, inclusion, and accessibility that has been running since 2020.
Deloitte US contracts with federal agencies were worth $3.3 billion a year, or almost 10% of its most recent annual revenues, the firm said in a recent earnings report.
Booz Allen Hamilton, which generates almost all of its $11 billion in annual revenue from the public sector, is the second most affected firm on the list, with 61 contracts cut, BI's analysis found.
At least 30 Accenture contracts have been cut, saving $240.2 million, per DOGE's data.
In an annual earnings call last month, Accenture CEO Julie Sweet said that DOGE's cost-cutting efforts had already hit the firm's revenues, and staffers have told BI they're worried about layoffs.
CGI Federal declined to comment and Leidos said it would continue discussions with the administration. The other firms on DOGE's list did not respond to requests for comment about their government contracts.
The General Services Administration, the government's largest procurement arm, is leading the effort to reevaluate federal consulting spend.
The agency, which operates separately from DOGE, said that consulting contracts with the 10 firms were set to generate more than $65 billion in fees in 2025 and beyond.
In March, the GSA asked the consultancies to submit a scorecard containing a detailed breakdown of their pricing and suggestions for where they could reduce costs.
It told the firms to identify which contracts were "mission critical" and to use simple terms to do it: "A 15-year-old should be able to understand what service you provide and why it is important."
Responses were due by Monday.
A person at the agency told BI that the GSA and federal bodies were now reviewing the scorecards and would decide on further cuts. The goal was to cut waste and move toward a more outcome-based approach instead of open-ended contracts, they said.
CEOs and senior executives at the consultancies appeared aligned with the administration's priorities, the person added.
Here's the list of contracts cut and savings made, according to the DOGE website:
Science Applications International Corp.: five contracts, $7.5 million.
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In this article
WASHINGTON — Boeing CEO Kelly Ortberg told senators on Wednesday that he's happy with the company's progress improving manufacturing and safety practices following several accidents, including a near catastrophe last year.
Ortberg faced questioning from the Senate Commerce Committee about how the company will ensure that it doesn't repeat past accidents or manufacturing defects, in his first hearing since he became CEO last August, tasked with turning the manufacturer around.
Sen. Ted Cruz, R.-Texas, the committee's chairman, said he wants Boeing to succeed and invited company managers and factory workers to report to him their opinions on its turnaround plan. "Consider my door open," he said.
Ortberg acknowledged the company still has more to do.
"Boeing has made serious missteps in recent years — and it is unacceptable. In response, we have made sweeping changes to the people, processes, and overall structure of our company," Ortberg said in his testimony. "While there is still work ahead of us, these profound changes are underpinned by the deep commitment from all of us to the safety of our products and services."
Boeing executives have worked for years to put the lasting impact of two fatal crashes of its best-selling Max plane behind it.
Ortberg said Boeing is in discussions with the Justice Department for a revised plea agreement stemming from a federal fraud charge in the development of Boeing's best-selling 737 Maxes. The previous plea deal, reached last July, was later rejected by a federal judge, who last month set a trial date for June 23 if a new deal isn't reached.
Boeing had agreed to plead guilty to conspiring to defraud the U.S. government, pay up to $487.2 million and install a corporate monitor at the company for three years.
"We're in the process right now of going back with the DOJ and coming up with an alternate agreement," Ortberg said during the hearing. "I want this resolved as fast as anybody. We're still in discussions and hopefully we'll have a new agreement here soon."
Asked by Sen. Maria Cantwell, the ranking Democrat on the committee, whether he had an issue with having a corporate monitor, Ortberg replied: "I don't personally have a problem, no."
Ortberg and other Boeing executives have recently outlined improvements across the manufacturer's production lines, such as reducing defects and risks from so-called traveled works, or doing tasks out of sequence, in recent months, as well as wins like a contract worth more than $20 billion to build the United States' next generation fighter jet.
But lawmakers and regulators have maintained heightened scrutiny on the company, a top U.S. exporter.
"Boeing has been a great American manufacturer and all of us should want to see it thrive," Sen. Ted Cruz, a Texas Republican and chairman of the committee, said in a statement in February announcing the hearing. "Given Boeing's past missteps and problems, the flying public deserves to hear what changes are being made to rehabilitate the company's tarnished reputation."
The Federal Aviation Administration last year capped Boeing's production of its 737 Max planes at 38 a month following the January 2024 door plug blowout. The agency plans to keep that limit in place, though Boeing is producing below that level.
Ortberg said at the hearing Wednesday that the company could work up to production rate of 38 Max planes a month or even higher sometime this year, but said Boeing wouldn't push it if the production line isn't stable.
Acting FAA Administrator Chris Rocheleau said at a Senate hearing last week that the agency's oversight of the company "extends to ongoing monitoring of Boeing's manufacturing practices, maintenance procedures, and software updates."
Correction: Chris Rocheleau is acting FAA administrator. An earlier version misstated his title.
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Americans' credit card debt is at an all-time high. Soaring interest rates on credit cards coupled with other economic factors could make the situation worse.
In March, Representatives Alexandria Ocasio-Cortez and Anna Paulina Luna introduced a bipartisan House bill that aims to cap annual credit card interest rates at 10% a year.
"Credit cards with high interest rates regularly trap working people in endless cycles of debt," Ocasio-Cortez said in a press statement. "At a time when families are struggling to make ends meet, we cannot allow big banks to shake down our communities for profit."
The average annual percentage rate on credit balances has nearly doubled in the past decade to 21% in 2024 from 12% in 2003, per the Consumer Financial Protection Bureau. Credit card interest rates can fluctuate with an individual's credit score, but they are ultimately determined by market conditions.
As credit card interest rates have risen, so has the amount of consumer debt, as well as delinquencies on payments.
The total amount of credit card debt has ballooned to $1.2 trillion in Q4 of 2024 up from $720 billion in the same quarter of 2004, per the Federal Reserve Bank of New York. More people are falling behind on their monthly payments and the number of active credit card users only making the minimum payment on their monthly credit card statement has reached a record high, "showing signs of consumer stress," per the Federal Reserve Bank of Philadelphia's 2024 Q3 report.
President Donald Trump ran on campaign promises last year that he would temporarily cap credit card interest rates at 10%, to allow Americans to "catch up" on their balances, but he hasn't taken executive action or spoken about interest rate caps since entering office.
The bill is now referred to the House Committee on Financial Services, but has yet to be put on the calendar for a vote in the House of Representatives.
Ocasio-Cortez and Luna did not respond to requests for comment.
Do you have a story to share about being delinquent on a student loan or credit card payment? Contact this reporter via email at jdeng@businessinsider.com.
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Companies have warned of rising prices. Markets have plunged. Bad vibes are back. President Donald Trump's tariff plans might not be in full force yet — but Americans are feeling the strain.
Trump's so-called "Liberation Day" is here. It's the name he gave April 2, when he's expected to unveil a range of broad tariffs on key trading partners. Leading up to the announcements, the University of Michigan's consumer survey in late March found that Americans' outlook on the economy took a dive — consumer sentiment was down 11.9% from February and 28.2% from one year ago.
Inflation fears largely drove the downturn, and consumers are worried about "the potential for pain amid ongoing economic policy developments," the survey said. One of those developments is Trump's tariff plans; Trump has maintained that his trade policies would bring in revenue and restore jobs but acknowledged in February that Americans would feel "some pain" as a result.
That pain has already started taking shape. Federal Reserve Chair Jerome Powell said during a March press conference that American consumers' gloomy outlook — particularly around high grocery prices — "probably has to do with turmoil at the beginning of an administration that's making big changes in policy."
Powell also said that "a good part" of the Federal Open Market Committee's latest inflation projections, which showed higher inflation than its December forecast, was a response to Trump's shifting tariff announcements.
"I do think with the arrival of the tariff inflation, further progress may be delayed," Powell said.
Trade policy experts have said companies might absorb some of the cost of Trump's planned tariffs, but Americans are likely to see higher prices on goods like groceries and cars. While Trump has also said that his plans would promote production in the US, the uncertainty is leaving markets, businesses, and Americans on edge.
Alex Jacquez, an advisor on the National Economic Council under former President Joe Biden, told reporters on a Tuesday press call that uncertainty around Trump's trade policy makes it "hard for the American people to see that they are going to have a strategy long term for how they should be spending their money and investing."
"When the president doesn't have a clear strategy or direction, it is extremely difficult for businesses in particular and consumers as well, to plan for the future, and that's why you're seeing so much uncertainty in the consumer market right now and so much uncertainty in the business community," Jacquez said.
Kush Desai, a White House spokesperson, told Business Insider that "fearmongering by the media and Democrats about President Trump's America First economic agenda isn't going to change the fact that industry leaders have already made trillions in investment commitments to make in America."
"President Trump used tariffs to deliver historic job, wage, and economic growth with no inflation in his first term, and he's set to restore American Greatness in his second term," Desai said.
Joseph Dennis, 73, voted for Trump but previously told BI he was concerned about the administration's decisions on tariffs and federal job cuts and wished they were less extreme. He's one of many retirees watching their investments fluctuate in value under Trump.
"I hope he knows what he's doing, but I'm not so sure," Dennis said.
Stock markets worldwide have swung wildly in anticipation of Trump's Liberation Day. Major US stock indexes dropped sharply to start the week before staging a rebound and ended Tuesday's session mostly higher as investors braced for the tariff announcement.
It's not just Liberation Day — markets have been fluctuating for weeks in response to Trump's shifting tariff announcements. Additionally, some companies have been preparing to raise prices in anticipation of Trump's tariff plans. Target CEO Brian Cornell told CNBC in early March that Trump's tariff policy could lead the company to raise prices on produce.
Trump has previously said a range of tariffs would go into effect on April 2, including levies on goods from Canada and Mexico, automobiles, and reciprocal tariffs on the countries that have imposed tariffs on US goods. However, the president told reporters on March 23 that he "may give a lot of countries a break."
"I don't change. But the word flexibility is an important word," Trump said.
That "flexibility" is making it difficult for businesses and consumers to prepare for the future, Heather Boushey, an economist who served on former President Joe Biden's Council of Economic Advisors, told reporters.
"Both businesses and consumers are getting shaken by this approach," Boushey said. "They see it as chaotic. They don't see what the plan is, but there's a concept of a plan, and that expectations are falling and falling rapidly."
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President Donald Trump's big tariff day is here — and it could influence various products Americans rely on.
For weeks, Trump has said that Wednesday will be "Liberation Day" for America, during which he intends to impose new tariffs on imports from various countries.
"April 2nd is Liberation Day in America!!! For DECADES we have been ripped off and abused by every nation in the World, both friend and foe," Trump wrote in a post on Truth Social. "Now it is finally time for the Good Ol' USA to get some of that MONEY, and RESPECT, BACK. GOD BLESS AMERICA!!!"
Trump is expected to hold a formal event to announce his tariff plans on Wednesday afternoon, so it's unclear what exactly the president will unveil. He has previously suggested that new tariffs might not be as expansive as originally planned. Trump told reporters on March 23 that tariffs on autos, semiconductors, and lumber would be announced "down the road," adding that while reciprocal tariffs were planned for Wednesday, he "may give a lot of countries a break."
"I don't change. But the word flexibility is an important word," Trump said.
Here's what we know about the tariffs Trump might have planned for Wednesday.
In early March, Trump paused 25% tariffs on goods from Canada and Mexico covered by the US-Mexico-Canada Agreement.
The USMCA covers a wide variety of imports from both Canada and Mexico, including agricultural products, clothing, fruits, vegetables, and dairy. Trump said the tariffs on Mexican goods are intended to crack down on drug and border policy.
Canada's finance minister, Dominic LeBlanc, said on March 6 in an X post that Canada would delay retaliatory tariffs until Wednesday "while we continue to work for the removal of all tariffs."
Treasury Secretary Scott Bessent told Fox Business on March 18 that Trump was planning to retaliate against countries that had imposed tariffs on US goods.
"On April 2, we are going to produce a list of other country's tariffs," Bessent said, adding that the Trump administration would tell those countries why it disagrees with their tariff levels. If countries roll back their tariffs, Bessent said, "we will not put up the tariff wall."
But if they maintain current policies, Bessent said, "then we will put up the tariff wall to protect our economy, protect our workers, and protect our industries."
He didn't provide details other than saying: "For some countries, it could be quite low. For some countries, it could be quite high."
And Trump has said that depending on how the countries respond, his administration may not enact the proposed tariffs.
Trump wrote on Truth Social on March 3 that tariffs on "external" agricultural products would begin.
"To the Great Farmers of the United States: Get ready to start making a lot of agricultural product to be sold INSIDE of the United States," he said.
It's unclear which products would be affected or whether these tariffs would be a part of Trump's broader reciprocal tariffs.
In a Truth Social post on March 24, Trump said he'd impose a "secondary tariff" on oil and gas from Venezuela, adding that Venezuela had sent "criminals" into the US.
"Any Country that purchases Oil and/or Gas from Venezuela will be forced to pay a Tariff of 25% to the United States on any Trade they do with our Country," he said. "All documentation will be signed and registered, and the Tariff will take place on April 2nd, 2025, LIBERATION DAY IN AMERICA."
Trump signed an executive order on March 26 to place a 25% tariff on all cars and car parts imported into the US. The order said the tariff would go into effect on or after Thursday and no later than May 3.
"This will continue to spur growth like you haven't seen before," Trump said in the Oval Office before signing the order. "We'll effectively be charging a 25% tariff. But if you build your car in the United States, there is no tariff."
The order said that cars and car parts were being imported into the US "in such quantities and under such circumstances as to threaten to impair the national security of the United States."
Have a tip or story to share? Contact this reporter via Signal at asheffey.97 or via email at asheffey@businessinsider.com. Use a personal email address and a nonwork device; here's our guide to sharing information securely.
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A new AI trend has taken the internet by storm, and at least one Big Tech giant stands to make a killing.
Last week, OpenAI rolled out GPT-4o, an upgraded image generator that users piled into to create images in the Japanese animation firm Studio Ghibli's style. The trend is helping fuel a record spike in users, so much so that OpenAI is struggling to keep up.
The surge in ChatGPT users should benefit Microsoft because it boosts OpenAI's growth, computing needs, and valuation, Jefferies analysts led by Brent Thill said in a note on Tuesday.
Microsoft is a major investor in the artificial intelligence firm and its primary cloud provider. The company also integrates OpenAI's large language models into its products. Last week, Microsoft CEO Satya Nadella said the company may build its own generative-AI capability to complement its partnership with OpenAI.
"We would glean that surging ChatGPT user growth likely indicates surging ChatGPT+ growth i.e. revenue growth for OpenAI," the analysts wrote.
The analysts also said OpenAI's most recent funding round was beneficial to Microsoft. On Monday, OpenAI announced it closed the largest private tech funding round on record. The company raised $40 billion from SoftBank and other investors, bringing the company's valuation to $300 billion. The ChatGPT maker was last valued at $157 billion in October.
Jefferies has a buy rating on Microsoft and a price target of $500. The stock closed at $382 on Tuesday. It's down 9.3% over the past year.
ChatGPT has become a household name since its launch in November 2022, but last week's animation trend sent demand soaring.
On Monday, OpenAI CEO Sam Altman wrote on X that ChatGPT had added 1 million users in the past hour. At its launch two years ago, it took five days to reach that number of users.
Last week, ChatGPT's weekly app downloads, weekly active users, and revenue from subscriptions and in-app purchases reached an all-time high — increasing 11%, 5%, and 6%, respectively, week over week — according to data from the market intelligence firm Sensor Tower.
Besides Microsoft, producers of graphics processing units stand to benefit from the Ghibli hype. Nvidia, AMD, and Intel are some of the biggest GPU companies.
"Working as fast we can to really get stuff humming; if anyone has GPU capacity in 100k chunks we can get asap please call!" Altman wrote Tuesday on X.
The Jefferies analysts said few vendors could deliver this much capacity and estimated that any deal with OpenAI could bring in $1 billion to $2 billion a year for the GPU provider.
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For Wisconsin Republicans, regaining a conservative majority on the state's highest court was a top priority.
Elon Musk, the world's richest man, funneled more than $12 million via his America PAC to sway the pivotal judicial race in one of the country's premier swing states.
It wasn't enough.
On Tuesday, Musk's big bet on the Wisconsin Supreme Court race fell apart, with the Associated Press reporting that Susan Crawford, a liberal judge in Dane County, had defeated Brad Schimel, a conservative judge in Waukesha County, in the most expensive judicial race in US history.
Musk, the face of President Donald Trump's White House DOGE office, has been met with increasingly vocal opposition by voters over the task force's cost-cutting efforts. And the fallout from DOGE is also affecting Tesla, the company that catapulted Musk to international prominence.
Crawford's victory is a significant blow for Musk as DOGE's work continues to face increased scrutiny from the public and could lead to electoral gains for Democrats in the 2026 midterm elections.
Here's how Crawford's win is set to upend Musk's political playbook:
For weeks, scenes of frustrated voters sharply questioning and booing GOP members of Congress have become a defining narrative of DOGE, as many lawmakers have had to defend waves of staffing cuts.
As a guiding force behind efforts to cut costs at critical federal departments — along with efforts to eliminate the US Agency for International Development — Musk has faced mounting pushback over the task force's aggressive tactics to rein in spending.
Musk went all in for Schimel, arguing that the Wisconsin race was "important for the future of civilization."
Referring to the potential for Democrats to make gains through a new congressional map, Musk argued that if the Wisconsin Supreme Court "is able to redraw the districts, they will gerrymander the district and deprive Wisconsin of two seats on the Republican side."
"Then they will try to stop all the government reforms we are getting done for you, the American people," he added.
In a state that narrowly backed Trump over Kamala Harris in November, voters this week made a new choice.
Crawford's win keeps the liberal bloc in the majority. The court could revisit the state's congressional maps, with a redraw likely to offer Democrats an opportunity to pick up additional seats.
With Republicans clinging to a razor-thin 218-213 majority in the US House ahead of what could be a tough midterm cycle, holding the lower chamber will be key for Musk and Trump — especially as it relates to future oversight over DOGE's work.
Musk campaigned heavily for Trump in Pennsylvania in last year's election, with America PAC pouring millions of dollars into the state, much of it for canvassing and other digital-related efforts.
The tech mogul's decision to hand out $1 million checks to select voters who signed petitions at town hall events — similar to what he employed in Wisconsin this time around — drew many people out as he criticized Harris and the media. Trump would go on to win Pennsylvania in the 2024 general election.
Schimel's loss, on the other hand, is a setback for Musk.
Wisconsin Supreme Court races in recent years have become increasingly polarized, with issues like abortion rights, union collective bargaining rights, and voting regulations being used to drive up turnout among base voters. This week, conservatives fell short in their efforts to take the court in a different direction.
Musk is poised to wade into other contests ahead of the midterms, especially with Trump's agenda on the line. But the latest results in Wisconsin show that there's a limit to such an influence.
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Kyrgyzstan continues to solidify its position as a regional crypto hub. The country is advancing its digital asset regulation, testing legal frameworks, and launching licensed platforms. One of the key steps in this direction is the launch of A7A5 — a stablecoin pegged to the Russian ruble within the cryptocurrency ecosystem. The token was issued by the Kyrgyz company Old Vector, in full compliance with local regulatory requirements and with the support of the Kyrgyz government.
As part of the strategic course set by the country's president, Kyrgyzstan has adopted a comprehensive package of laws regulating the cryptocurrency market. For the first time, the country has introduced full legislation on digital assets, covering all major aspects of the industry — from exchanges to token issuers. This has created a new institutional infrastructure that did not previously exist in the market.
Among the unique innovations is the mechanism for registering token issuances under official state supervision. Regulators ensure that token emissions comply with regulatory requirements, have fiat backing, undergo regular audits, and meet obligations to token holders. In essence, Kyrgyzstan provides one of the most transparent and secure tokenization models in the world.
The first issuance of A7A5 (mint) was carried out in complete accordance with the new national legislation — under the control of regulatory authorities and directed to an officially registered, regulated broker.
The A7A5 token is now available for trading on the regulated exchange Meer Exchange and is expected to be listed on decentralized platforms in the future. Its fiat backing is stored in bank accounts, and its volume is audited by an independent firm on a quarterly basis. The key advantage of A7A5 is the opportunity to earn up to 20% annually, driven by its link to the refinancing rate of the Central Bank of the Russian Federation and additional income strategies in DeFi.
The digital asset market is moving toward the integration of traditional finance with decentralized technologies. The emergence of stablecoins has enabled users to:
However, despite the overall growth of the segment, stablecoins denominated in other currencies are still in their early stages.
Although the segment has seen significant capitalization, stablecoins other than the dollar still have very limited trading volumes:
This limits the potential for building robust currency strategies, including FX and carry trades, which are at the core of the global financial market with a daily volume exceeding $7 trillion.
To execute a traditional carry trade strategy in the digital space, several key elements are still missing:
The launch of A7A5, followed by its listing on both CEX and DEX, marks the first step in expanding the range of tools available to crypto investors, including:
A7A5 is designed for investors who are ready to leverage next-generation technologies to achieve higher returns, given the limited alternatives in the world of traditional finance.
The listing on Meer Exchange ensures liquidity, transparency, and institutional access to a new class of digital assets tied to the Russian economy and emerging markets.
Disclaimer. This publication is sponsored. Cointelegraph does not endorse or assume responsibility for the content, accuracy, quality, advertising, products, or other materials on this web page. Readers are advised to conduct their own research before engaging with any company mentioned.
Please note that the featured information is not intended as, and shall not be understood or construed as legal, tax, investment, financial, or other advice. Nothing contained on this web page constitutes a solicitation, recommendation, endorsement, or offer by Cointelegraph or any third party service provider to buy or sell any cryptoassets or other financial instruments.
Crypto assets are a high-risk investment. You should consider whether you understand the possibility of losing money due to leverage. None of the material should be considered as investment advice. Cointelegraph shall not be held liable, directly or indirectly, for any damages or losses arising from the use or reliance on any content, goods, or services featured on this web page.
Representative Maxine Waters suggested that any stablecoin bills in Congress should receive no support unless Republicans address Donald Trump's conflicts of interest.
California Representative Maxine Waters, ranking member of the US House Financial Services Committee, used her opening statement at a markup hearing to criticize President Donald Trump's business and ethical entanglements with the crypto industry, including the launch of a stablecoin by a family-backed company.
Addressing lawmakers at an April 2 hearing, Waters said Trump had used his position as president to leverage “multiple crypto schemes” for profit, including a US dollar-pegged stablecoin launched by World Liberty Financial (WLFI) — the firm backed by his family.
The California lawmaker pointed to Trump's memecoin launched in January, his plans to establish a national cryptocurrency stockpile, and “his own stablecoin,” referring to WLFI's USD1 token launched in March.
Rep. Maxine Waters addressing the House Financial Services Committee on April 2. Source: GOP Financial Services
“With this stablecoin bill, this committee is setting an unacceptable and dangerous precedent, validating the president and his insiders' efforts to write rules of the road that will enrich themselves at the expense of everyone else,” said Waters, adding:
Waters does not stand alone in her criticism of Trump's crypto ventures, with many lawmakers and experts across the political spectrum suggesting potential conflicts of interest.
Committee Chair French Hill, who spoke on stablecoins before Waters, also reportedly said that the Trump family's involvement in the industry makes legislation “more complicated.”
“If there is no effort to block the President of the United States of America from owning his stablecoin business [...] I will never be able to agree on supporting this bill, and I would ask other members not to be enablers,” said Waters.
Related: Crypto has a regulatory capture problem in Washington — Or does it?
Representative Bryan Steil, who introduced the Stablecoin Transparency and Accountability for a Better Ledger Economy, or STABLE Act, did not immediately address Waters' concerns about Trump's stablecoin but referred to establishing safeguards for consumers.
Hill did not mention Trump in his opening statement but said there needed to be a “clear federal framework” for payment stablecoins.
The committee will consider amendments to the STABLE Act, as well as bills to combat illicit finance using emerging financial technologies and blocking the US government from issuing a central bank digital currency, or CBDC.
The markup hearing was a necessary step before the committee could vote on whether to advance the bills to the House of Representatives.
Magazine: Trump's crypto ventures raise conflict of interest, insider trading questions
Bitcoin bulls are trying to clear a critical overhead hurdle at $88,000. Will large-cap altcoins follow?
Bitcoin (BTC) bulls have pushed the price above the $87,000 level even as US trade tariffs are slated to kick in on April 2. Bitcoin may remain volatile in the near term, but analysts remain bullish for the long term.
According to Fidelity analyst Zack Wainwright, Bitcoin is currently in an acceleration phase, which “can conclude with a sharp, dramatic rally” if history repeats itself. If that happens, Wainwright expects $110,000 to be the starting base of the next leg of the upmove.
Crypto market data daily view. Source: Coin360
BitMEX co-founder and Maelstrom chief investment officer Arthur Hayes said in a post that if the Federal Reserve pivots to quantitative easing, then Bitcoin could rally to $250,000 by year-end.
Could Bitcoin break above the $89,000 overhead resistance, starting a rally in select altcoins? Let's analyze the charts of the top 10 cryptocurrencies to find out.
Bitcoin has risen close to the resistance line, where the sellers are expected to pose a solid challenge.
BTC/USDT daily chart. Source: Cointelegraph/TradingView
The flattening 20-day exponential moving average ($85,152) and the relative strength index (RSI) just above the midpoint signal the bears are losing their grip. That improves the prospects of a rally above the resistance line. If that happens, the BTC/USDT pair could climb to $95,000 and eventually to $100,000.
Alternatively, if the price turns down sharply from the resistance line and breaks below $81,000, it will suggest that the bears are back in the driver's seat. The pair may then tumble to $76,606.
Ether (ETH) rebounded off the $1,754 support on March 31, signaling that the bulls are attempting to form a double-bottom pattern.
ETH/USDT daily chart. Source: Cointelegraph/TradingView
The bears will try to stall the relief rally at the 20-day EMA ($1,965). If the price turns down from the 20-day EMA, the possibility of a break below $1,574 increases. The ETH/USDT pair may then collapse to $1,550.
Contrarily, a break and close above the 20-day EMA opens the doors for a rise to the breakdown level of $2,111. If buyers pierce this resistance, the pair will complete a double-bottom pattern, starting a rally to the target objective of $2,468.
XRP's (XRP) weak bounce off the crucial $2 support suggests a lack of aggressive buying by the bulls at the current levels.
XRP/USDT daily chart. Source: Cointelegraph/TradingView
That heightens the risk of a break below $2. If that happens, the XRP/USDT pair will complete a bearish head-and-shoulders pattern. This negative setup could start a downward move to $1.27. There is support at $1.77, but it is likely to be broken.
On the upside, a break and close above the 50-day SMA ($2.39) suggests solid buying at lower levels. The pair may then rally to the resistance line, where the bears are expected to mount a strong defense. A break and close above the resistance line signals a potential trend change.
BNB's (BNB) recovery attempt stalled at the moving averages on April 1, indicating that the bears are selling on rallies.
BNB/USDT daily chart. Source: Cointelegraph/TradingView
The bears will try to strengthen their position by pulling the price below $587. If they can pull it off, the BNB/USDT pair could descend to the 50% Fibonacci retracement level of $575 and later to the 61.8% retracement of $559. The deeper the pullback, the greater the time needed for the pair to recover.
A break above the moving averages is the first sign that the selling pressure has reduced. The pair may rally to $644 and then to $686, which is likely to attract sellers.
Solana (SOL) is getting squeezed between the 20-day EMA ($132) and the $120 support, signaling a possible range expansion in the short term.
SOL/USDT daily chart. Source: Cointelegraph/TradingView
If the price breaks and closes above the 20-day EMA, it suggests that the buyers have overpowered the sellers. The SOL/USDT pair may rise to the 50-day SMA ($145) and, after that, to $180.
This positive view will be invalidated in the near term if the price turns down from the moving averages and breaks below $120. That could pull the price to $110, where the buyers are expected to step in.
Dogecoin (DOGE) remains pinned below the 20-day EMA ($0.17), indicating that the bears continue to sell on minor rallies.
DOGE/USDT daily chart. Source: Cointelegraph/TradingView
The first sign of strength will be a break and close above the 20-day EMA. The DOGE/USDT pair may climb to $0.21, which could act as a strong barrier. If buyers pierce the $0.21 resistance, the pair may rally to $0.24 and later to $0.29.
Sellers are likely to have other plans. They will try to defend the moving averages and pull the price below $0.16. If they manage to do that, the pair could descend to the $0.14 support. A break and close below the $0.14 level may sink the pair to $0.10.
Buyers are trying to push Cardano (ADA) back above the uptrend line, but the bears are likely to sell near the moving averages.
ADA/USDT daily chart. Source: Cointelegraph/TradingView
The downsloping 20-day EMA ($0.71) and the RSI just below the midpoint signal that bears have the edge. If the price turns down and breaks below $0.63, the ADA/USDT pair could plunge to $0.58 and thereafter to $0.50.
Buyers will have to drive and maintain the price above the 50-day SMA ($0.75) to signal a potential trend change in the near term. The pair could rally to $0.84, which may act as a hurdle.
Related: Is Bitcoin price going to crash again?
Toncoin (TON) broke above the $4.14 resistance on March 1, but the bulls could not sustain the breakout.
TON/USD daily chart. Source: Cointelegraph/TradingView
A minor positive in favor of the bulls is that they have not allowed the price to slip much below $4.14. That increases the possibility of a break above the overhead resistance. The TON/USDT pair could rally to $5 and later to $5.50.
The 20-day EMA ($3.71) is the critical support to watch out for on the downside. If the support cracks, it will signal that the bulls are losing their grip. The pair may slide to the 50-day SMA ($3.48) and then to $2.81.
Chainlink (LINK) tried to rise above the 20-day EMA ($14.32) on April 1, but the bears held their ground.
LINK/USDT daily chart. Source: Cointelegraph/TradingView
Sellers will try to pull the price to the support line of the descending channel pattern, which remains the key short-term level to keep an eye on. If the price breaks below the support line, the LINK/USDT pair could descend to $10.
If buyers want to prevent the downside, they will have to push and maintain the price above the 50-day SMA ($15.47). If they manage to do that, the pair could rally to $17.50 and subsequently to the resistance line.
UNUS SED LEO (LEO) turned down from the overhead resistance of $9.90 and plunged below the uptrend line on March 30.
LEO/USD daily chart. Source: Cointelegraph/TradingView
However, the bears could not sustain the lower levels, and the bulls pushed the price back into the triangle on April 1. The recovery is expected to face selling at the 20-day EMA ($9.60). If the price turns down from the 20-day EMA and breaks below the uptrend line, it increases the risk of a fall to $8.
Instead, if the LEO/USD pair breaks above the 20-day EMA, it suggests that the markets have rejected the breakdown. A breakout and close above $9.90 will complete an ascending triangle pattern, which has a target objective of $12.04.
This article does not contain investment advice or recommendations. Every investment and trading move involves risk, and readers should conduct their own research when making a decision.
In an exclusive interview with Cointelegraph, the chief investment officer discusses Bitcoin's risks, institutional adoption and why now could be the best time to invest.
If you've ever wondered when is the right time to invest in Bitcoin (BTC), you won't want to miss our latest interview with Matt Hougan. As the chief investment officer at Bitwise, Hougan provides an in-depth analysis, explaining why, from a risk-adjusted perspective, there has never been a more opportune time to buy Bitcoin.
In our discussion, Hougan lays out a compelling argument: Bitcoin's early days were filled with uncertainty — technology risks, regulatory threats, trading inefficiencies, and reputational concerns. Fast forward to today, and those risks have significantly diminished. The launch of Bitcoin ETFs, adoption by major institutional investors, and even the US government's strategic Bitcoin reserve have all cemented its place in the global financial ecosystem.
“Bitcoin is only 10% of gold. So just to match gold, which I think is just a stopping point on its long-term journey, it has to ten-x from here,” he said.
But that's just the beginning. Hougan also touches on Bitcoin's long-term price potential, why institutional adoption is about to accelerate, and how market fundamentals could push Bitcoin to new heights.
“There's just too much structural long-term demand that has to come into this market against a severely limited new supply,” he said.
Watch the full interview now on our YouTube channel, and don't forget to subscribe!
FDUSD, the stablecoin issued by Hong Kong-based First Digital, has wobbled from its $1 price peg as investor concerns mounted over its reserves, though the company said Wednesday that it was "completely solvent."
FDUSD has dropped to 0.87 against Tether's USDT stablecoin and 0.76 against Circle's USDC on Binance, the main exchange where FDUSD is listed. Notably, bitcoin (BTC) also nearly hit 100,000 against FDUSD. The token has stabilized around $0.98-$0.96 later, still trading below its supposed price anchor.
The sudden price action happened as CoinDesk earlier Wednesday reported that some of the TrueUSD stablecoin's reserve assets were stuck in illiquid investments, according to filings. Tron founder Justin Sun bailed out the issuer company. First Digital Trust, a trust company affiliated to First Digital, was appointed to manage TUSD reserves.
"First Digital Trust (FDT) is effectively insolvent and unable to fulfill client fund redemptions. I strongly recommend that users take immediate action to secure their assets," Tron founder Justin Sun claimed in a Wednesday X post. First Digital refuted the allegations in an X post, saying that "First Digital is completely solvent" and "every dollar backing FDUSD is completely, secure, safe and accounted for with US backed T-Bills."
"This is a typical Justin Sun smear campaign to try to attack a competitor to his business. As we told the reporter at CoinDesk, we have not yet had the opportunity to defend ourselves and instead of letting the TUSD matter be dealt with in court, Justin has instead resorted to a coordinated social media effort to try to damage FDUSD as a business competitor," the company said. "FDT will pursue legal action to protect its rights and reputation."FDUSD's latest monthly reserve report showed that the $2 billion of reserve assets were held mostly in U.S. Treasury bills and a lesser part in repo facilities and fixed deposits.Stablecoin analytics firm Bluechip told CoinDesk it gave FDUSD a C grade (on a scale from F as least safe to to A+ being safest) and expressed concerns over reserve assets being bankruptcy remote from the issuer company."FDUSD appears to be backed by reasonably sound reserves. But we concluded that in case of bankruptcy, the reserves could be used to pay off the parent company's debt," Garett Jones, Bluechip's chief economist, said in a message. "It's hard to predict what would really happen in a crisis, but safer stablecoins do exist."
UPDATE (Apr. 2, 17:58): Added analyst comment from Bluechip. Updated FDUSD price action.
Krisztian Sandor is a U.S. markets reporter focusing on stablecoins, tokenization, real-world assets. He graduated from New York University's business and economic reporting program before joining CoinDesk. He holds BTC, SOL and ETH.
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The spot bitcoin ETFs saw sizable inflows in the first quarter despite the lame price action and at least one analyst sees the next three months as even bigger even if the prices don't recover.
“Even if current market conditions persist in the second quarter, we are seeing strong traction from financial advisors and institutional investors," said Juan Leon, senior investment strategist at Bitwise (whose BITB is among the bitcoin ETFs).
"While retail interest is weak due to the fixation on price action, professional investors are recognizing the global adoption momentum spurred by the Trump administration's embrace of bitcoin, and many are seeing these market conditions as an opportunity to start or increase an allocation," Leon added.
The ETFs saw over $1 billion in inflows in the first quarter of the year despite a challenging macro situation that sent the S&P 500 Index into its biggest quarterly loss since 2022 and bitcoin's 13% plunge.
Leon expects inflows to be even stronger in the second quarter — as much as $3 billion or even more as wirehouse platforms unlock and legislative policy progresses.
ETF inflows possibly less than meets the eye
The $1 billion in first quarter net flows — and whatever the second quarter brings — doesn't necessarily reflect investor interest in buying the bitcoin dip. That's because of the so-called basis trade (also known as cash-and-carry). In this, institutional players buy the spot bitcoin ETF while shorting CME bitcoin futures, picking up yield without exposure to price movement.
That yield was well into the double-digits in late 2024 and remained nicely above the risk-free rate throughout much of the first quarter. It's collapsed to the 5% area of late, suggesting arbitrage-related ETF inflows may dry up.
Back to bull case: It's still early
“While a favorable price environment would certainly be a boost, it's important to remember that adoption of spot bitcoin ETFs by these groups is still in its infancy," said Nate Geraci, president of the ETF Store, who is also bullish on the outlook for inflows throughout the rest of the year.. "As they grow more comfortable allocating to bitcoin, this should provide a meaningful tailwind for inflows,” he added..,
While many institutions have indeed already made their first allocations into bitcoin in the past year, it represents only a small fraction of ETF investment, with most of the money still coming from retail investors — something recently noted by BlackRock CEO Larry Fink, whose IBIT is the asset-gathering leader among the spot ETFs. The more favorable regulatory stance toward the industry, not to mention the government's own potential allocation into bitcoin, however, means that ratio could soon shift significantly.
During an ETF conference in Las Vegas earlier this month, a survey showed that 57% of advisors plan on increasing their allocations into crypto ETFs this year as crypto has lost its “reputational risk” attribute among advisors.
The view that bitcoin could serve as a “safe haven” in times of an economic decline, which investors remain anxious about, could also boost confidence in the asset, especially as fears of a potential recession grow.
“If we see continued rate cut expectations, signs of economic uncertainty, or deepening fears of a potential recession in the US, Bitcoin's role as 'digital gold' will likely support additional inflows,” said David Siemer, CEO of Wave Digital Assets. “While some short-term traders may rotate out if price weakness persists, long-term players will continue to keep inflows strong, especially as institutional adoption takes off and drives demand throughout the year."
Helene is a New York-based markets reporter at CoinDesk, covering the latest news from Wall Street, the rise of the spot bitcoin exchange-traded funds and updates on crypto markets. She is a graduate of New York University's business and economic reporting program and has appeared on CBS News, YahooFinance and Nasdaq TradeTalks. She holds BTC and ETH.
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According to RWA.XYZ, the total stablecoin market cap is currently over $227 billion, with over 155 million holders worldwide.
Sumitomo Mitsui Financial Group (SMBC), a Japanese banking and financial services conglomerate, along with business systems firm TIS Inc, Ava Labs — the developer of the Avalanche network — and digital asset infrastructure company Fireblocks, have signed an agreement to explore a framework for commercializing stablecoins in Japan.
Under a Memorandum of Understanding, the companies will focus on developing strategies around issuing and circulating stablecoins pegged to the US dollar and Japanese yen, according to a joint announcement.
Additionally, the collaboration will explore stablecoins as a settlement mechanism for tokenized real-world assets such as stocks, bonds, and real estate.
Stablecoins continue to be a major focus of crypto regulatory frameworks worldwide, and one of the sectors venture capitalists are eyeing in 2025 as nation-states push stablecoins to the forefront of their digital asset strategies.
Stablecoin total market overview. Source: RWA.XYZ
Related: Stablecoins, tokenized assets gain as Trump tariffs loom
Speaking at the White House Crypto Summit on March 7, US Treasury Secretary Scott Bessent said that comprehensive stablecoin regulation was central to President Donald Trump's stated goal to become the worldwide leader in crypto.
Bessent said stablecoins would help protect US dollar hegemony in global markets by expanding the use and scope of the dollar across the world.
Centralized overcollateralized stablecoins rely on short-term US Treasury instruments and fiat money held in banks to back the value of the tokenized real-world assets.
According to Paolo Ardoino, the CEO of stablecoin issuer Tether, the company is now the seventh-largest buyer of US Treasury bills, beating out sovereign countries such as France, Singapore, Belgium, and the United Kingdom.
Stablecoin issuer Tether is now the seventh-largest buyer of US Treasury bills. Source: Paolo Ardoino
Stablecoin issuers like Tether and Circle accumulate the yield from holding US debt instruments as part of their profit from issuing tokenized fiat assets to buyers.
Recently, calls to share stablecoin yield with customers have escalated, with industry leaders like Coinbase CEO Brian Armstrong proposing that stablecoin laws change in the US to allow firms to distribute yield to clients onchain.
US Senator Kirsten Gillibrand disagreed with those proposals and warned against stablecoin issuers sharing yield with clients, arguing that it would displace the banking industry and disrupt home mortgage loans, small business loans, and local bank lending.
Magazine: Unstablecoins: Depegging, bank runs and other risks loom
Fidelity Investments rolled out an IRA plan that invests directly in crypto on Wednesday, giving investors another method for tapping this asset class.
The brokerage firm offers bitcoin (BTC), ethereum (ETH) and Litecoin (LTC) to any U.S. citizen over the age of 18. The assets are custodied by Fidelity Digital Assets and held in a cold wallet. The crypto IRA product has no fees, and customers can invest in a Roth IRA, traditional IRA or rollover IRA, according to Fidelity's website.
The new product comes as financial advisors are increasingly offering crypto to their clients. A survey by TMX Vetta Fi recently showed that 57% of advisors plan on increasing their allocations into crypto ETFs, although their biggest focus is in crypto equity ETFs.
“Fidelity is committed to offering investment products and solutions to meet the changing needs and interests of our customers, accompanied by education and support,” a spokesperson told CoinDesk.
Clients of the brokerage firm have increasingly voiced interest in a tax-advantaged way to trade and hold crypto, a person familiar with the matter, said.
Fidelity already offers a number of crypto exchange-traded funds, which let investors track the prices of digital assets without directly investing in them. The company recently filed to list a Solana ETF on the Cboe Exchange.
UPDATE (April 2, 16:15 UTC): Corrects first paragraph to state that the plan was introduced on Wednesday, not Thursday, and clarifies that assets are custodied by Fidelity Digital Assets.
Helene is a New York-based markets reporter at CoinDesk, covering the latest news from Wall Street, the rise of the spot bitcoin exchange-traded funds and updates on crypto markets. She is a graduate of New York University's business and economic reporting program and has appeared on CBS News, YahooFinance and Nasdaq TradeTalks. She holds BTC and ETH.
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Ripple, an enterprise-focused blockchain service closely tied to the XRP Ledger (XRP), said on Wednesday it has integrated its stablecoin to the company's cross-border payments system to boost adoption for Ripple USD (RLUSD).
Select Ripple Payments customers including cross-border payment providers BKK Forex and iSend are already using the stablecoin to improve their treasury operations, the company said. Ripple plans to further expand the token's availability of its token to payments customers.
Additionally, crypto exchange Kraken added RLUSD to its platform, following recent listings on LMAX and Bitstamp.
Ripple entered the rapidly growing stablecoin market with its short-term U.S. government bond-backed cryptocurrency after receiving regulatory approval from the New York New York Department of Financial Services in December.
Since then, RLUSD reached a $244 million market capitalization, growing 87% over the past month and reaching a monthly transfer volume of $860 million, rwa.xyz data shows.
Jack McDonald, Ripple's senior vice president of stablecoins, said in a statement that RLUSD's growth is "outpacing our internal projections" with adoption spanning multiple financial sectors. Ripple is also working with NGOs exploring stablecoins for more efficient aid distribution, he added.
Krisztian Sandor is a U.S. markets reporter focusing on stablecoins, tokenization, real-world assets. He graduated from New York University's business and economic reporting program before joining CoinDesk. He holds BTC, SOL and ETH.
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Bitcoin could return to $76,000 lows, analysis warns as risk assets prepare for US trade tariff volatility, but a daily chart breakout is on the cusp of confirming.
Bitcoin (BTC) reached new April highs at the April 2 Wall Street open as markets braced for US “Liberation Day.”
BTC/USD 1-hour chart. Source: Cointelegraph/TradingView
Data from Cointelegraph Markets Pro and TradingView showed local highs of $86,444 on Bitstamp, the best performance for BTC/USD since March 28.
Volatility remained in the run-up to US President Donald Trump announcing a sweeping round of reciprocal trade tariffs.
The measures would be unveiled in an address from the White House Rose Garden at 4 pm Eastern Time, with Trump then holding a press conference.
While US stocks traded slightly down after the open, Bitcoin managed to claw back lost ground, acting in a key area of interest filled with long-term trend lines.
As Cointelegraph reported, these include various simple (SMA) and exponential (EMA) moving averages, among them the 200-day SMA — a classic bull market support line currently lost.
BTC/USD 1-day chart with 200 SMA. Source: Cointelegraph/TradingView
In his latest observations, popular trader and analyst Rekt Capital made additional reference to the 21-week and 50-week EMAs.
“The consolidation between the two Bull Market EMAs continues. However, the 21-week EMA (green) represents lower prices as it declines,” he wrote in a post on X alongside an illustrative chart.
BTC/USD 1-week chart with 21, 50 EMA. Source: Rekt Capital/X
Rekt Capital flagged more bullish news in the making, thanks to BTC/USD attempting to break out of an extended downtrend on daily timeframes.
He confirmed:
BTC/USD 1-day chart. Source: Rekt Capital/X
Last month, Bitcoin's daily relative strength index (RSI) metric broke free from its own downtrend that had been in place since November 2024.
Continuing on the macro picture, however, trading firm QCP Capital was uninspired.
Related: Bitcoin sales at $109K all-time high 'significantly below' cycle tops — Glassnode
Risk assets, it told Telegram channel subscribers on the day, were likely to “remain under pressure” following the tariffs announcement.
“In crypto, sentiment remains broadly subdued. BTC continues to trade without conviction, while ETH is holding the line at $1,800 support. Across the board, crypto markets are showing signs of exhaustion with numerous coins down 90% YTD, with some shedding over 30% in the past week,” it summarized.
Previous tariff moves in Q1 almost unanimously delivered downward BTC price reactions.
Other industry participants were more hopeful, including asset management firm Swissblock, which argued that “no sign of an imminent collapse” occurred on Bitcoin.
“Will $BTC hold as a hedge, or follow TradFi into a pullback?” it queried in an X thread on March 31, describing BTC price action as being “at a crossroads.”
Bitcoin price momentum chart. Source: Swissblock/X
Swissblock saw the potential for a return to $76,000 multimonth lows in the event of a negative reaction — a drop of 11% versus current levels.
This article does not contain investment advice or recommendations. Every investment and trading move involves risk, and readers should conduct their own research when making a decision.
Justin Sun bailed out Techteryx's TrueUSD stablecoin after nearly half a billion dollars of its reserves were rendered illiquid, people close to the matter confirmed, and the stablecoin issuer said in Hong Kong court documents.
After acquiring TrueUSD from TrueCoin in December 2020, Techteryx appointed First Digital Trust (FDT), a Hong Kong-based fiduciary, to manage its stablecoin reserves.
According to documents prepared by U.S. law firm Cahill Gordon & Reindel, FDT was instructed to invest the reserves in the Aria Commodity Finance Fund (Aria CFF), a Cayman Islands-registered vehicle. However, court filings allege that approximately $456 million was instead improperly diverted into Aria Commodities DMCC, a separate, unauthorized entity based in Dubai.
Court documents identify Matthew Brittain as controlling Aria Commodity Finance Fund (Aria CFF) through Aria Capital Management Ltd and Cecilia Brittain as the sole shareholder of the separately owned Dubai-based entity Aria Commodities DMCC.
However, emails from Aria's Matthew Brittan are signed with an address in Dubai.
Court documents say that Cecilia is Matthew's wife.
ARIA DMCC engages in trade finance, asset development, and commodity trading, while ARIA CFF finances commodity traders, including ARIA DMCC and third parties, according to Matthew Brittain, who described the relationship between the two companies in an email to CoinDesk.
Attestations produced by Moore CPA Limited show, first published by Protos, that FDT managed $501 million of TrueUSD's reserves by November 2024.
Hong Kong court filings also say Vincent Chok, First Digital's CEO, allegedly directed around $15.5 million in undisclosed commissions to an entity called "Glass Door" and separately structured approximately $456 million in unauthorized trade finance loans from FDT to Aria DMCC, later retroactively mischaracterizing them as legitimate fund investments in actions plaintiffs describe as fraudulent misrepresentation and misappropriation.
"The remittances to Aria DMCC were blatant misappropriation and money-laundering," a statement of claim reads. "They were made without the knowledge, authorization or approval of the Plaintiff."
These statements have not been tried in court as of press time.
Aria DMCC invested funds in global projects that they described as relatively illiquid, such as manufacturing plants, mining operations, maritime vessels, port infrastructure, and renewable energy ventures.
When Techteryx attempted to redeem its investments from Aria CFF between mid-2022 and early 2023 it received little or no funds back, with Aria entities allegedly defaulting on payments and failing to fulfill redemption requests, the court documents say.
Techteryx then took full operational control of TUSD in July 2023, after TrueCoin terminated its involvement. As part of a transitional period following the December 2020 sale, TrueCoin continued running the day-to-day operations of TUSD.
According to the complaint Cahill prepared for the U.S. DOJ, Sun stepped in around this time to provide emergency liquidity support, which was structured as a loan.
The Techteryx team then quarantined 400 million TUSD so that retail redemptions could continue and token holders wouldn't be affected, despite the stablecoin issuer's empty coffers, the court filings said.
In response to a request for comment from CoinDesk, First Digital's Chok, categorically denied any wrongdoing or participation in fraudulent schemes.
Chok told CoinDesk that First Digital Trust acted strictly as a fiduciary intermediary, executing transactions precisely according to instructions provided by Techteryx and its representatives. He asserted that his company was not responsible for independently evaluating or advising on these investment decisions.
"It is our understanding that one of the main blockers voiced by ARIA for early redemptions of funds (as requested by Techteryx) has been their AML/KYC concerns regarding the deal between TrueCoin and Techteryx and the true identity of the ultimate beneficial owner of Techteryx," Chok said in an email to CoinDesk, adding that he believed nobody named in the case considers Aria illiquid.
"We have not yet had the opportunity to fully defend ourselves," Chok said in an email to CoinDesk. "We are fully committed to clarifying these matters in due course as the legal and arbitration process continues."
Aria Group's Matthew Brittain said to CoinDesk that he "completely rejects Techteryx's claims against ARIA DMCC and any related entities," adding that "a number of false allegations were made in the court proceedings."
Techteryx was fully aware of term commitments, Brittain said, and these were outlined in contracts that subscribers have agreed to when investing in ARIA CFF, which are clearly set out in the Offering Memorandum.
Brittain also echoed Chok's concerns about Techteryx's beneficial ownership, pointing to Wall Street Journal coverage of the topic.
The Hong Kong writ identifies Li Jinmei as the ultimate beneficial owner of Techteryx. A spokesperson for Techteryx confirmed that this is not the same person as Jennifer Yiyang — the previous ultimate beneficial owner of the company — despite some media reporting to the contrary.
"The subscriber has not resolved these issues," Brittain continued, referring to the beneficial ownership concerns.
While this was happening, TUSD's challenges continued in the form of a collapsing banking partner and regulatory scrutiny in the U.S.
In mid-2023, Prime Trust, an independent crypto custodian based in Nevada that is not connected to this case, but which TrueUSD used for its fiat ramps, was put into receivership by state regulators.
State regulators alleged Prime Trust had improperly used customer funds to cover withdrawal requests, raising serious concerns about its financial stability.
Court filings from Nevada showed that Prime Trust owed around $85 million in fiat obligations with only about $3 million available.
This wasn't the last headache for the stablecoin issuer.
In September 2024, TrueCoin and TrustToken (the stablecoin's owners before Techteryx) settled with the SEC over allegations they falsely marketed TrueUSD as fully dollar-backed while secretly investing reserves in risky offshore funds.
Without admitting wrongdoing, or detailing the nature of their offshore investments with Aria's companies, both TrueCoin and TrustToken agreed to pay civil penalties and disgorge profits to the tune of just over $500,000 to resolve charges of fraud and unregistered securities offerings.
For his part, Aria's Brittain said that investing in Aria wasn't the right move to begin with for a stablecoin's reserves.
"ARIA CFF has never held [its] strategy out as highly liquid, or appropriate for the reserves of a stablecoin," he said in an email. CORRECTION (April 2, 2025, 16:09 UTC): Corrects reference to court filing versus Department of Justice filing.CORRECTION (April 2, 2025 16:09 UTC): Corrects amount of loans made between Glass Door and FDT.
Sam Reynolds is a senior reporter based in Asia. Sam was part of the CoinDesk team that won the 2023 Gerald Loeb award in the breaking news category for coverage of FTX's collapse. Prior to CoinDesk, he was a reporter with Blockworks and a semiconductor analyst with IDC.
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Coreum's blockchain framework is designed for institutional use. It features deterministic fees, compliance integration and interoperability for regulated asset tokenization. A new report by Cointelegraph Research examines the blockchain ecosystem.
A new report by Cointelegraph Research explores Coreum's role in institutional blockchain adoption. It analyzes the project's technical architecture, compliance framework and its potential impact on regulated asset tokenization. The report presents insights into transaction efficiency, security mechanisms and crosschain interoperability. It also evaluates how Coreum fits into the evolving financial landscape.
The adoption of blockchain technology by financial institutions has been increasing in lockstep, with the value locked in tokenized real-world assets (RWA). The latter grew by 85% in 2024.
Our report examines how third-generation blockchains, such as Coreum, are addressing the challenges of scalability, regulatory compliance and interoperability. Improvements in the infrastructure on the base layer will lead to more seamless institutional adoption in the future.
Read the full version of the report for free here.
Coreum is structured to support applications that require predictable transaction costs, regulatory oversight and seamless integration with financial infrastructure. Network data indicates that Coreum achieves a transaction throughput in excess of 7,000 TPS and a time to finality of about 1.2 seconds. This positions Coreum well in a crowded and highly competitive layer-1 blockchain landscape.
Coreum integrates most of its compliance features at the protocol level, a critical factor for institutional adoption. The network includes onchain KYC and AML monitoring in collaboration with AnChain.ai, an AI-driven compliance provider.
This is unlike conventional blockchains, where compliance tools are third-party application-layer software. Coreum puts compliance at its foundation together with real-time risk assessment and fraud detection.
Our report also analyses Coreum's decentralized exchange (DEX) infrastructure. While many layer-1 blockchains rely on liquidity pools, Coreum features a built-in onchain order book. There are important differences between the models.
Coreum's order book DEX allows for deterministic trade execution with minimal slippage, which makes it well-suited for institutional trading strategies. In contrast, AMM-based DEXs rely on liquidity pools that sometimes lead to price inefficiencies and higher exposure to impermanent loss.
Coreum's DEX architecture also supports high-frequency trading, with transaction processing speeds comparable to traditional financial exchanges.
A notable aspect of Coreum's DEX is its advanced API, which enables integration with institutional trading systems. The API is designed to provide low-latency access to order book data, market execution tools and automated trading strategies.
This infrastructure allows financial firms and market makers to integrate Coreum's DEX into their existing trading workflows. It ensures compliance with industry standards and benefits from blockchain-based settlement efficiencies.
Read the full version of the report for free here.
Coreum's interoperability strategy includes connections with the XRP Ledger (XRPL) and the Cosmos/IBC network. These integrations enable crosschain liquidity and asset transfers, which creates support for financial applications that require seamless movement between blockchain ecosystems.
This integration allows institutional users to leverage XRPL's efficiency in payments and Cosmos' modular interoperability framework with over 100 connected chains. The ability to interact with multiple networks without sacrificing security or compliance aligns with institutional requirements for blockchain adoption.
Networks designed for institutional adoption will need to address compliance, scalability and interoperability challenges. Coreum's technical structure and regulatory considerations provide a case study for how blockchain networks may evolve to meet these requirements.
With its deterministic fee structure, built-in compliance framework and high-speed trading infrastructure, Coreum represents an example of how third-generation blockchains are positioning themselves at the intersection of crypto and regulated financial markets.
Read the full version of the report for free here
Disclaimer. This article does not contain investment advice or recommendations. Every investment and trading move involves risk, and readers should conduct their own research when making a decision.
This article is for general information purposes and is not intended to be and should not be taken as legal or investment advice. The views, thoughts, and opinions expressed here are the author's alone and do not necessarily reflect or represent the views and opinions of Cointelegraph.
Cointelegraph does not endorse the content of this article nor any product mentioned herein. Readers should do their own research before taking any action related to any product or company mentioned and carry full responsibility for their decisions.
Crypto asset manager Grayscale has listed two new exchange-traded funds (ETFs) that offer investors a differentiated source of income through bitcoin's (BTC) characteristic volatility.
The two New York Stock Exchange-listed funds will start trading on Wednesday.
The Bitcoin Covered Call ETF (BTCC) and Bitcoin Premium Income ETF (BPI) offer covered call writing strategies, which involves selling call options to generate income on the premium received.
Call options are derivatives contracts betting on the price of an asset rising. They give the holder the right, but not the obligation, to buy the asset at a predetermined price within a define period of time.
BTCC will write calls very close to spot prices to deliver income for investors seeking regular cash flow, with the options premiums possibly also providing a cushion against market downturns.
BPI meanwhile will target options with strike prices that are well out-of-the-money, meaning the price is much higher than the spot price. This would allow investors to participate in much of BTC's upside potential while possibly benefiting from some dividend income, according to an emailed announcement from Grayscale on Wednesday.
The options contracts that both ETFs use will track other bitcoin ETFs, including Grayscale's own Bitcoin Trust (GBTC) and Bitcoin Mini Trust (BTC).
Despite the surge in institutional investment into BTC through spot ETFs since their introduction in January 2024, bitcoin's volatility does not appear to be going anywhere for the time being.
After gaining nearly 48% in the fourth quarter, the largest cryptocurrency kicked off 2025 by losing 12% in the historically bullish first quarter. It rose by 72% and 69% in the first quarters of 2023 and 2024, respectively, according to data tracked by Coinglass.
Therefore, as institutional investors increase their exposure to bitcoin, there may be more demand for products such as Grayscale's that can offer differentiated sources of income to cushion against this volatility.
Jamie has been part of CoinDesk's news team since February 2021, focusing on breaking news, Bitcoin tech and protocols and crypto VC. He holds BTC, ETH and DOGE.
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The Trump administration's formation of a U.S. Strategic Bitcoin Reserve might have China re-thinking its hardline stance against crypto and that could be key for accelerated global adoption of BTC, asset manager Grayscale said in a research report Monday.
"The most important country to watch in this regard is China," said Grayscale, adding that if the country eases its crypto restrictions "it could significantly boost global adoption."
President Trump last month directed his administration to form a Strategic Bitcoin Reserve to — at a minimum — hold the assets that have been seized by the government.
Grayscale noted that current Chinese government policy bans most crypto activities, such as trading and mining, but permits the holding of digital assets.
Still, "policymakers have allowed an expansion of crypto-related activity in Hong Kong under the 'one country, two systems' framework," Grayscale said.
Local regulators may be taking another look at the legal treatment of cryptocurrencies in the country. China's Supreme Court and other judicial bodies had a discussion in February about how to treat digital assets in future legal cases, the report noted.
Read more: U.S. Strategic Bitcoin Reserve, Crypto Stockpile a 'Pivotal Moment' for Industry: KBW
Will Canny is an experienced market reporter with a demonstrated history of working in the financial services industry. He's now covering the crypto beat as a finance reporter at CoinDesk. He owns more than $1,000 of SOL.
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AVAX lost over half its value during the past year despite the new liquidity, but the crypto market may find its bottom by June amid tariff negotiations.
Avalanche saw a significant surge in stablecoin supply over the past year, but the onchain deployment of this capital points to passive investor behavior, which may be limiting demand for the network's utility token.
The stablecoin supply on the Avalanche network rose by over 70% over the past year, from $1.5 billion in March 2024, to over $2.5 billion as of March 31, 2025, according to Avalanche's X pos
Market capitalization of stablecoins on Avalanche. Source: Avalanche
Stablecoins are the main bridge between the fiat and crypto world and increasing stablecoin supply is often seen as a signal for incoming buying pressure and growing investor appetite.
However, Avalanche's (AVAX) token has been in a downtrend, dropping nearly 60% over the past year to trade above $19 as of 12:31 pm UTC, despite the $1 billion increase in stablecoin supply, Cointelegraph Markets Pro data shows.
AVAX/USD,1-year chart. Source: Cointelegraph Markets Pro
“The apparent contradiction between surging stablecoin value on Avalanche and AVAX's significant price decline likely stems from how that stablecoin liquidity is being held,” according to Juan Pellicer, senior research analyst at IntoTheBlock crypto intelligence platform.
Related: Bitcoin can hit $250K in 2025 if Fed shifts to QE: Arthur Hayes
A “substantial portion” of these inflows consists of bridged Tether (USDT), the research analyst told Cointelegraph, adding:
The AVAX token's downtrend comes during a wider crypto market correction, as investor sentiment is pressured by global uncertainty ahead of US President Donald Trump's reciprocal import tariff announcement on April 2, a measure aimed at reducing the country's estimated trade deficit of $1.2 trillion.
Related: Michael Saylor's Strategy buys Bitcoin dip with $1.9B purchase
Nansen analysts predict a 70% chance that the crypto market will bottom in the next two months leading into June as the ongoing tariff-related negotiations progress and investor concerns are alleviated.
“Once the toughest part of the negotiation is behind us, we see a cleaner opportunity for crypto and risk assets to finally mark a bottom,” Aurelie Barthere, principal research analyst at the Nansen crypto intelligence platform, told Cointelegraph.
Both traditional and cryptocurrency markets continue to lack upside momentum ahead of the US tariff announcement.
BTC/USD, 1-day chart. Source: Nansen
“For the main US equity indexes and for BTC, the respective price charts failed to resurface above their 200-day moving averages significantly, while lower-lookback price moving averages are falling,” wrote Nansen in an April 1 research report.
Magazine: Bitcoin ATH sooner than expected? XRP may drop 40%, and more: Hodler's Digest, March 23 – 29
Need evidence of the drastic change in the crypto market sentiment lately? Look no further than Deribit's options market, where the $80,000 bitcoin (BTC) put option, offering downside protection below the said level, is now the most popular bet.
It's a 180-degree change from early this year when call options at six-figure levels garnered the most interest among traders.
As of writing, the number of open positions in the $80,000 put option tallied 10,278 contracts, equating to a notional open interest of $864.26 million, according to data source Amberdata. That makes it the most popular options play on Deribit, where one contract represents one BTC.
It also marks a significant positioning shift from early January when the call option at the $120,000 strike was the most popular bet with an open interest of nearly $1.5 billion. Last month, the $100,000 call took the crown.
The positioning shift indicates traders have reassessed upside expectations amid the market swoon and lingering economic uncertainty. BTC fell 11.66% in the first quarter, with prices slipping below $80,000 at one point as President Donald Trump's tariffs shook Wall Street. Additionally, disappointment over the lack of fresh purchases in the U.S. strategic reserve weighed over prices.
Later Wednesday, Trump is expected to announce sweeping reciprocal tariffs on its trading partners, which could lead to a full-blown trade war. That has BTC traders chasing downside protection.
"BTC volatility smiles have shifted sharply towards OTM puts, reaching levels not seen since the US Banking Crisis in March 2023. ETH's short-tenor volatility smile skews have partially recovered from their strong tilt toward OTM put," analytics firm Block Scholes said in its market update Wednesday.
Omkar Godbole is a Co-Managing Editor on CoinDesk's Markets team based in Mumbai, holds a masters degree in Finance and a Chartered Market Technician (CMT) member. Omkar previously worked at FXStreet, writing research on currency markets and as fundamental analyst at currency and commodities desk at Mumbai-based brokerage houses. Omkar holds small amounts of bitcoin, ether, BitTorrent, tron and dot.
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By James Van Straten (All times ET unless indicated otherwise)
President Trump's so-called Liberation Day has arrived, and markets are nervously awaiting developments on U.S. tariffs. Even within the administration, the mood appears far from optimistic.
Commerce Secretary Howard Lutnik is reportedly in the crosshairs, with suggestions he may become the scapegoat for favoring overly aggressive tariffs if the U.S. were to head into a recession, according to The Independent, a U.K. online newspaper.
A recession looks likely according to the Atlanta Fed's GDPNow model, which is projecting a first-quarter contraction of -3.7% for U.S. real GDP. That is a dramatic downward revision from earlier estimates: +3.9% two months ago, +2.3% one month ago and -1.8% just two weeks ago.
While Trump has yet to disclose which country the tariffs will target, an announcement is scheduled for after the stock market closes at 4 p.m.
Bitcoin (BTC), meanwhile, remains unfazed, trading little changed on the day and holding above $85,000. U.S. equities finished higher on Tuesday, although futures are pointing slightly negative heading into Wednesday.
Currently, bitcoin is 25% below its Jan. 20 all-time high of $109,000. This places it in the middle of the performance range of the "Magnificent 7" tech stocks. Here's how they compare to their respective all-time highs: Apple is down 17%, Microsoft 22%, Amazon 24%, Meta 25%, Google 26%, NVIDIA 32% and Tesla 50%.
The cryptocurrency's resilience stands out when compared to past cycles. In 2022, BTC fell 75% from its peak to a low of $15,500, more than twice as much as the Nasdaq-100 ETF (QQQ)'s 34%. This year, bitcoin has dropped 30% versus 16% for QQQ — a relative drawdown of 1.87 times. This relative performance suggests bitcoin has become more resilient over time, even as volatility remains a defining trait.
Still, a lot hinges on the tariff announcement and how markets react. Stay alert!
By Shaurya Malwa
Spot BTC ETFs:
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Source: Farside Investors
James Van Straten is a Senior Analyst at CoinDesk, specializing in Bitcoin and its interplay with the macroeconomic environment. Previously, James worked as a Research Analyst at Saidler & Co., a Swiss hedge fund, where he developed expertise in on-chain analytics. His work focuses on monitoring flows to analyze Bitcoin's role within the broader financial system.
In addition to his professional endeavors, James serves as an advisor to Coinsilium, a UK publicly traded company, where he provides guidance on their Bitcoin treasury strategy. He also holds investments in Bitcoin, MicroStrategy (MSTR), and Semler Scientific (SMLR).
Shaurya is the Co-Leader of the CoinDesk tokens and data team in Asia with a focus on crypto derivatives, DeFi, market microstructure, and protocol analysis.
Shaurya holds over $1,000 in BTC, ETH, SOL, AVAX, SUSHI, CRV, NEAR, YFI, YFII, SHIB, DOGE, USDT, USDC, BNB, MANA, MLN, LINK, XMR, ALGO, VET, CAKE, AAVE, COMP, ROOK, TRX, SNX, RUNE, FTM, ZIL, KSM, ENJ, CKB, JOE, GHST, PERP, BTRFLY, OHM, BANANA, ROME, BURGER, SPIRIT, and ORCA.
He provides over $1,000 to liquidity pools on Compound, Curve, SushiSwap, PancakeSwap, BurgerSwap, Orca, AnySwap, SpiritSwap, Rook Protocol, Yearn Finance, Synthetix, Harvest, Redacted Cartel, OlympusDAO, Rome, Trader Joe, and SUN.
Omkar Godbole is a Co-Managing Editor on CoinDesk's Markets team based in Mumbai, holds a masters degree in Finance and a Chartered Market Technician (CMT) member. Omkar previously worked at FXStreet, writing research on currency markets and as fundamental analyst at currency and commodities desk at Mumbai-based brokerage houses. Omkar holds small amounts of bitcoin, ether, BitTorrent, tron and dot.
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XRP price is holding above $2.05 after modest gains on April 1, while the RSI's bullish divergence signals a potential bottom.
XRP (XRP) price fell 22% between March 19 and March 31, potentially forming a local bottom at $2.02. The price then increased by 9% to $2.20 before retracing to current levels.
Has the popular altcoin finally bottomed out, or is there a deeper retracement in the cards?
The XRP relative strength index (RSI) displays bullish divergence conditions in lower timeframes, according to analyst CasiTrades.
A bullish divergence is when the asset's price prints lower lows and the RSI produces higher lows, indicating that downward momentum is waning.
“After reaching the 0.786 retrace at $2.05, XRP is printing bullish divergences from the 15-min all the way up to the 4-hour chart,” the analyst said in a March 31 post on X.
CasiTrades notes that these signals are a positive indicator both for short-term bounces and potential macro recovery.
XRP/USD hourly chart. Source: CasiTrades
She added that $2.25 remains a key resistance level to watch, as breaching it with strong momentum would signal a convincing bullish breakout.
“If we break above $2.25 with strong momentum, that would invalidate the need for another support retest, a very bullish sign,” CasiTrades said, adding that the demand zone between “$2.00 and $2.01 remains a support if the $2.05 doesn't hold.”
The analyst projects a bullish month for XRP in April, with targets of $2.70 and $3.80 in the short term.
Related: XRP funding rate flips negative — Will smart traders flip long or short?
Despite XRP's recent recovery from local lows, the risk of a deeper correction remains, according to veteran trader Peter Brandt.
Last week, Brandt said the presence of a “textbook” head-and-shoulders pattern (H&S) could see XRP price as low as $1.07.
This potential H&S pattern is still in play on the daily chart (see below) and will be completed on a break and close below the neckline at $1.90.
If the price stays below the neckline, the pair could plummet to $1.50 and then to the pattern's target of $1.07.
Brandt said:
XRP/USD daily chart with H&S pattern. Source: Cointelegraph/TradingView
Brandt said this bearish chart pattern would be invalidated if buyers push and maintain the price above $3.00.
Meanwhile, macroeconomic headwinds from US tariffs on April 2 may spook traders, pulling the XRP price toward $1.31.
Not everyone agrees. Analyst Dark Defender shared a positive outlook, saying that XRP price is likely to revisit the last Fibonacci level at $2.04 before bouncing back again.
According to the analyst, a key resistance level for XRP is $2.22, which “should be broken” to ensure a sustained recovery toward the Wave 5 target at $8.
XRP/USD daily chart. Source: Dark Defender
This article does not contain investment advice or recommendations. Every investment and trading move involves risk, and readers should conduct their own research when making a decision.
Wednesday could be a pivotal day for financial markets, including cryptocurrencies, as President Donald Trump is expected to announce sweeping reciprocal tariffs to "liberate" the U.S. from the supposed unfair practices of its trading partners.
Ahead of the pivotal announcement, signs of downtrend exhaustion have emerged in the ether (ETH) market. Yes, you read that right: after having lagged BTC by a large margin through the two-year bull crypto bull run, ETH may take the lead if the looming tariffs are more measured than expected.
Ether fell along with bitcoin last week, but the bears failed to penetrate the 16-month low of $1,755 hit on March 11. That's the first sign of seller fatigue or downtrend exhaustion.
Since then, prices have bounced to $1,880, teasing a double bottom formation with the neckline resistance at $2,104. A move through that would confirm the bullish breakout, opening doors for $2,400, the level identified as the next resistance per the measured move method.
While prices revisited the low from March 11 last week, the histogram that represents the spread between the price and its 50-day simple moving average (SMA) did not follow suit and carved put a higher low.
The divergence suggests that although prices fell, the momentum behind the downward price movement weakened.
After a prolonged downtrend that saw prices halve to $2,000, the three-line break chart has now flipped bullish, marking a significant shift in market sentiment. This change is illustrated by the appearance of a green bar on the daily time frame, indicating a potential reversal in price momentum.
The line break's previous bullish signal from early March was short-lived and turned out to be a bear trap. Still, the latest bullish flip appears more reliable as it is accompanied by signs of downtrend exhaustion on candlestick charts discussed above.
That said, macro factors can single handedly make or break charts, meaning broad-based risk aversion on the back of Trump tariffs could invalidate all the bullish signals discussed above, potentially leading to deeper losses in ether.
Omkar Godbole is a Co-Managing Editor on CoinDesk's Markets team based in Mumbai, holds a masters degree in Finance and a Chartered Market Technician (CMT) member. Omkar previously worked at FXStreet, writing research on currency markets and as fundamental analyst at currency and commodities desk at Mumbai-based brokerage houses. Omkar holds small amounts of bitcoin, ether, BitTorrent, tron and dot.
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North Korean “IT workers” are increasing illicit cyber activity across Europe with an eye on blockchain projects, Google Cloud warned in a Wednesday report.
Projects built on the popular Solana network, including applications and job boards, are getting hit by the rising attacks. Democratic People's Republic of Korea (DPRK) operatives pose as legit remote workers to infiltrate companies, take over critical systems and steal sensitive data which is likely sold to “generate revenue for the regime.”
The increased threat in Europe is a shift from a U.S.-heavy focus as DPRK-linked entities faced heat from DOJ indictments and tighter hiring scrutiny stateside.
The report reveals that one such worker juggled 12 fake personas across the U.S. and Europe and sought employment by fabricating references, building a rapport with job recruiters, and using additional personas they controlled to vouch for their credibility.
It's not like the workers lack coding chops either: Workers were found taking projects ranging from token hosting platform using Next.js, React and CosmosSDK, and Golang, and even created an entire Solana-based job marketplace.
More blockchain-related projects involved Anchor and Rust smart contract development. One worker even developed an artificial intelligence (AI) web application using Electron, Next.js, and blockchain applications.
A key culprit may be workplaces that let employees use their own devices.
“(Google Cloud) believes that IT workers have identified BYOD environments as potentially ripe for their schemes, and in January 2025, IT workers are now conducting operations against their employers in these scenarios,” the report said.
“Global expansion, extortion tactics, and the use of virtualized infrastructure all highlight the adaptable strategies employed by DPRK IT workers.”
DPRK entities and hacking groups are one of the biggest threat actors in the crypto ecosystem, stealing an estimated $1.3 billion from projects in 2024 and conducting a $1.5 billion hack on crypto exchange Bybit in February alone.
Shaurya is the Co-Leader of the CoinDesk tokens and data team in Asia with a focus on crypto derivatives, DeFi, market microstructure, and protocol analysis.
Shaurya holds over $1,000 in BTC, ETH, SOL, AVAX, SUSHI, CRV, NEAR, YFI, YFII, SHIB, DOGE, USDT, USDC, BNB, MANA, MLN, LINK, XMR, ALGO, VET, CAKE, AAVE, COMP, ROOK, TRX, SNX, RUNE, FTM, ZIL, KSM, ENJ, CKB, JOE, GHST, PERP, BTRFLY, OHM, BANANA, ROME, BURGER, SPIRIT, and ORCA.
He provides over $1,000 to liquidity pools on Compound, Curve, SushiSwap, PancakeSwap, BurgerSwap, Orca, AnySwap, SpiritSwap, Rook Protocol, Yearn Finance, Synthetix, Harvest, Redacted Cartel, OlympusDAO, Rome, Trader Joe, and SUN.
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Sony customers in Singapore can now use the USDC stablecoin in its online store.
Sony Electronics' Singapore (SES) has integrated cryptocurrency payments in partnership with crypto exchange Crypto.com, the two firms said Wednesday.
Stablecoins are cryptocurrencies whose value is pegged to a real-world asset, such as the U.S. dollar or gold. USDC, issued by Circle, is the second-largest dollar-backed token, trailing only Tether's USDT in size.
Headquartered in Singapore, Crypto.com won approval as a provider of Digital Payment Token (DPT) services in the city state in June 2023, allowing it to provide crypto payments to clients.
Jamie has been part of CoinDesk's news team since February 2021, focusing on breaking news, Bitcoin tech and protocols and crypto VC. He holds BTC, ETH and DOGE.
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7-Eleven is participating in the country's CBDC test phase, which started on April 1 and will end on June 30.
South Korea's 7-Eleven stores will accept payments in the country's central bank digital currency (CBDC) until June, as the retailer participates in the test phase of its CBDC project.
The convenience store chain will reportedly provide a 10% discount on all products paid for with CBDC during the test period. According to Moon Dae-woo, head of 7-Eleven's digital innovation division, the company is making an effort to incorporate digital technology advancements in its operations.
The executive added that the company's participation in the CBDC test will help accelerate the firm's digital transformation.
Many stores will participate in South Korea's CBDC testing phase, which runs from April 1 to June 30. The project also involves 100,000 participants who will be allowed to test payments using CBDC issued by the central bank.
Central bank digital currencies are digital assets issued by government agencies. Like other digital assets, CBDCs offer faster and more modernized payment features. However, unlike Bitcoin and other privacy-focused tokens that offer certain levels of anonymity, CBDCs are controlled and monitored by governments.
Related: Over 400 South Korean officials disclose $9.8M in crypto holdings
On March 24, government agencies including the Bank of Korea, the Financial Services Commission (FSC) and the Financial Supervisory Service (FSS) announced the CBDC test.
Participants can convert their bank deposits into tokens stored in a distributed ledger during the test period. The tokens hold the same value as the Korean won.
The government agencies said citizens aged 19 or older with a deposit account in a participating bank could apply to take part. Registrations were limited to 100,000 participants. KB, Koomin, Shinhan, Hana, Woori, NongHyup, IBK and Busan are among the banks participating in the CBDC tests.
Apart from 7-Eleven, participants can use their CBDCs in coffee shops, supermarkets, K-Pop merchandise stores and delivery platforms. However, users will be limited to a total conversion limit of 5 million won ($3,416) during testing.
The Bank of Korea first announced the retail CBDC testing for 100,000 users in November 2023 and was originally scheduled to begin in the fourth quarter of 2024. The FSS said the country's CBDC test represents a step toward creating a prototype for a “future monetary system.”
Magazine: Ridiculous ‘Chinese Mint' crypto scam, Japan dives into stablecoins: Asia Express
Decentralization involves spreading control and decision-making across a network instead of a single authority. Unlike centralized systems, where one entity controls everything, decentralized blockchains distribute data among participants (nodes). Each node holds a copy of the ledger, ensuring transparency and reducing the risk of manipulation or system failure.In blockchain, a decentralized network provides significant advantages:Security: Decentralization reduces vulnerabilities associated with central points of attack. Without a single controlling entity, malicious actors find it more challenging to compromise the network. Transparency: All transactions are recorded on a public ledger accessible to all participants, fostering trust through transparency. This openness ensures that no single entity can manipulate data without consensus. Fault tolerance: Decentralized networks are more resilient to failures. Data distribution across multiple nodes ensures that the system remains operational even if some nodes fail. So, decentralization is good, but it's not a fixed state. It's more of a spectrum, constantly shifting as network participation, governance structures and consensus mechanisms evolve.And yes, there's a ruler for that. It's called the Nakamoto coefficient.
Decentralization involves spreading control and decision-making across a network instead of a single authority.
Unlike centralized systems, where one entity controls everything, decentralized blockchains distribute data among participants (nodes). Each node holds a copy of the ledger, ensuring transparency and reducing the risk of manipulation or system failure.
In blockchain, a decentralized network provides significant advantages:
So, decentralization is good, but it's not a fixed state. It's more of a spectrum, constantly shifting as network participation, governance structures and consensus mechanisms evolve.
And yes, there's a ruler for that. It's called the Nakamoto coefficient.
The Nakamoto coefficient is a metric used to quantify the decentralization of a blockchain network. It represents the minimum number of independent entities — such as validators, miners or node operators — that would need to collude to disrupt or compromise the network's normal operation. This concept was introduced in 2017 by former Coinbase chief technology officer Balaji Srinivasan and was named after Bitcoin's creator, Satoshi Nakamoto. A higher Nakamoto coefficient indicates greater decentralization and security within the blockchain network. In such networks, control is more widely distributed among participants, making it more challenging for any small group to manipulate or attack the system. Conversely, a lower Nakamoto coefficient suggests fewer entities hold significant control, increasing the risk of centralization and potential vulnerabilities. For example, a blockchain with a Nakamoto coefficient of 1 would be highly centralized, as a single entity could control the network. In contrast, a network with a coefficient of 10 would require at least 10 independent entities to collude to exert control, reflecting a more decentralized and secure structure.Did you know? Polkadot's high score on the Nakamoto coefficient is largely due to Polkadot's nominated proof-of-stake (NPoS) consensus mechanism, which promotes an even distribution of stakes among a large number of validators.
The Nakamoto coefficient is a metric used to quantify the decentralization of a blockchain network. It represents the minimum number of independent entities — such as validators, miners or node operators — that would need to collude to disrupt or compromise the network's normal operation.
This concept was introduced in 2017 by former Coinbase chief technology officer Balaji Srinivasan and was named after Bitcoin's creator, Satoshi Nakamoto.
A higher Nakamoto coefficient indicates greater decentralization and security within the blockchain network. In such networks, control is more widely distributed among participants, making it more challenging for any small group to manipulate or attack the system. Conversely, a lower Nakamoto coefficient suggests fewer entities hold significant control, increasing the risk of centralization and potential vulnerabilities.
For example, a blockchain with a Nakamoto coefficient of 1 would be highly centralized, as a single entity could control the network. In contrast, a network with a coefficient of 10 would require at least 10 independent entities to collude to exert control, reflecting a more decentralized and secure structure.
Did you know? Polkadot's high score on the Nakamoto coefficient is largely due to Polkadot's nominated proof-of-stake (NPoS) consensus mechanism, which promotes an even distribution of stakes among a large number of validators.
Calculating this coefficient involves several key steps:Identification of key entities: First, determine the primary actors within the network, such as mining pools, validators, node operators or stakeholders. These entities play significant roles in maintaining the network's operations and security.Assessment of each entity's control: Next, evaluate the extent of control each identified entity has over the network's resources. For instance, in proof-of-work (PoW) blockchains like Bitcoin, this involves analyzing the hashrate distribution among mining pools. In proof-of-stake (PoS) systems it requires examining the stake distribution among validators.Summation to determine the 51% threshold: After assessing individual controls, rank the entities from highest to lowest based on their influence. Then, cumulatively add their control percentages until the combined total exceeds 51%. The number of entities required to reach this threshold represents the Nakamoto coefficient.Consider a PoW blockchain with the following mining pool distribution:Mining pool A: 25% (of the total hashrate)Mining pool B: 20%Mining pool C: 15%Mining pool D: 10%Others: 30%To determine the Nakamoto coefficient:Start with mining pool A (25%).Add mining pool B (25% 20% = 45%).Add mining pool C (45% 15% = 60%).In this scenario, the combined hashrate of mining pools A, B and C reaches 60%, surpassing the 51% threshold. Therefore, the Nakamoto coefficient is 3, indicating that collusion among these three entities could compromise the network's integrity. Did you know? Despite Bitcoin's reputation for decentralization, its mining subsystem is notably centralized. The Nakamoto coefficient is currently 2 for Bitcoin. This means that just two mining pools control most of Bitcoin's mining power.
Calculating this coefficient involves several key steps:
Consider a PoW blockchain with the following mining pool distribution:
To determine the Nakamoto coefficient:
In this scenario, the combined hashrate of mining pools A, B and C reaches 60%, surpassing the 51% threshold. Therefore, the Nakamoto coefficient is 3, indicating that collusion among these three entities could compromise the network's integrity.
Did you know? Despite Bitcoin's reputation for decentralization, its mining subsystem is notably centralized. The Nakamoto coefficient is currently 2 for Bitcoin. This means that just two mining pools control most of Bitcoin's mining power.
While the Nakamoto coefficient serves as a valuable metric for assessing blockchain decentralization, it possesses certain limitations that warrant careful consideration. For example: Static snapshotThe Nakamoto coefficient provides a static snapshot of decentralization, reflecting the minimum number of entities required to compromise a network at a specific point in time. However, blockchain networks are dynamic, with participant roles and influence evolving due to factors like staking, mining power shifts or node participation changes. Consequently, the coefficient may not accurately capture these temporal fluctuations, potentially leading to outdated or misleading assessments. Subsystem focusThis metric typically focuses on specific subsystems, such as validators or mining pools, potentially overlooking other critical aspects of decentralization. Factors like client software diversity, geographical distribution of nodes and token ownership concentration also significantly impact a network's decentralization and security. Relying solely on the Nakamoto coefficient might result in an incomplete evaluation.Consensus mechanism variationsDifferent blockchain networks employ various consensus mechanisms, each influencing decentralization differently. The Nakamoto coefficient may not uniformly apply across these diverse systems, necessitating tailored approaches for accurate measurement. External InfluencesExternal factors, including regulatory actions, technological advancements or market dynamics, can influence decentralization over time. For example, regulatory policies in specific regions might affect the operation of nodes or mining facilities, thereby altering the network's decentralization landscape. The Nakamoto coefficient may not account for such externalities, limiting its comprehensiveness.To sum up, the Nakamoto coefficient is useful for assessing certain aspects of blockchain decentralization. It should be used alongside other metrics and qualitative assessments to gain a comprehensive understanding of a network's decentralization and security.
While the Nakamoto coefficient serves as a valuable metric for assessing blockchain decentralization, it possesses certain limitations that warrant careful consideration.
For example:
The Nakamoto coefficient provides a static snapshot of decentralization, reflecting the minimum number of entities required to compromise a network at a specific point in time.
However, blockchain networks are dynamic, with participant roles and influence evolving due to factors like staking, mining power shifts or node participation changes. Consequently, the coefficient may not accurately capture these temporal fluctuations, potentially leading to outdated or misleading assessments.
This metric typically focuses on specific subsystems, such as validators or mining pools, potentially overlooking other critical aspects of decentralization. Factors like client software diversity, geographical distribution of nodes and token ownership concentration also significantly impact a network's decentralization and security.
Relying solely on the Nakamoto coefficient might result in an incomplete evaluation.
Different blockchain networks employ various consensus mechanisms, each influencing decentralization differently. The Nakamoto coefficient may not uniformly apply across these diverse systems, necessitating tailored approaches for accurate measurement.
External factors, including regulatory actions, technological advancements or market dynamics, can influence decentralization over time. For example, regulatory policies in specific regions might affect the operation of nodes or mining facilities, thereby altering the network's decentralization landscape.
The Nakamoto coefficient may not account for such externalities, limiting its comprehensiveness.
To sum up, the Nakamoto coefficient is useful for assessing certain aspects of blockchain decentralization. It should be used alongside other metrics and qualitative assessments to gain a comprehensive understanding of a network's decentralization and security.
Bitcoin midterm holders — even those with a cost basis at around $3,600 — are still refusing to sell despite major profits and BTC price volatility.
Bitcoin (BTC) investors who bought BTC in 2020 or later are still waiting for higher prices, new research says.
In findings published on X on April 1, onchain analytics firm Glassnode revealed that $110,000 was not high enough to make many hodlers sell.
Bitcoiners who entered the market between three and five years ago have retained their holdings despite significant BTC price upside.
According to Glassnode, this investor cohort, with a cost basis between the 2020 lows of $3,600 and the 2021 highs of $69,000, is still hodling.
“Although the share of wealth held by investors who bought $BTC 3–5 years ago has declined by 3 percentage points since its November 2024 peak, it remains at historically elevated levels,” it said.
Bitcoin Realized Cap HODL Waves data. Source: Glassnode
An accompanying chart shows data from the Realized Cap HODL Waves metric, which splits the BTC supply into sections based on when each coin last moved onchain.
Using this, Glassnode is able to draw a distinction between the 2020-22 buyers and those who came immediately before them.
“In contrast, over two-thirds of those who had bought $BTC 5–7 years ago exited their positions by the December 2024 peak,” it reveals, reflecting their lower cost basis.
As Cointelegraph reported, more recent buyers, who form the more speculative investor cohort known as short-term holders (STHs), have proven much more sensitive to recent BTC price volatility.
Related: Bitcoin sellers 'dry up' as weekly exchange inflows near 2-year low
Episodes of panic selling have occurred throughout the past six months as BTC/USD hit new record highs and then fell by up to 30%.
Continuing, Glassnode said that current STH participation does not suggest a speculative frenzy — something common to previous BTC price cycle tops.
“Short-Term Holders currently hold around 40% of Bitcoin's network wealth, after peaking near 50% earlier in 2025,” it said, alongside Realized Cap HODL Waves data on March 31.
Bitcoin Realized Cap HODL Waves. Source: Glassnode
This article does not contain investment advice or recommendations. Every investment and trading move involves risk, and readers should conduct their own research when making a decision.
Sony Electronics Singapore partnered with Crypto.com to enable USDC payments, highlighting a growing trend of stablecoin adoption in the region.
The online store of a Singapore-based subsidiary of Japanese tech behemoth Sony is now accepting USDC payments through Crypto.com.
According to an April 2 announcement, Sony Electronics Singapore now accepts USDC (USDC) stablecoin payments through an integration with the Crypto.com exchange. Crypto.com Singapore general manager Chin Tah Ang said:
The Sony subsidiary is not the only high-profile partnership Crypto.com is involved in. At the end of 2024, the mobile-first crypto exchange partnered with Deutsche Bank to provide corporate banking services across Asian-Pacific markets, covering regions such as Singapore, Australia and Hong Kong.
Related: CFTC mulling probe of Crypto.com over Super Bowl contracts: Report
Still, the Singaporean Sony subsidiary allowing stablecoin payments may be the start of a new trend in the region. Late February reports indicated that Metro, a publicly listed department store chain in Singapore, had enabled its customers to pay for products using stablecoins like Tether's USDt.
The initiatives also follow January reports that Singapore is becoming a key destination for Web3 companies after it issued twice as many crypto licenses in 2024 as the previous year. William Croisettier, chief growth officer of ZKcandy, told Cointelegraph at the time:
Related: Singapore Exchange to list Bitcoin futures in H2 2025: Report
In late November, the crypto-friendly digital bank Singapore Gulf Bank reportedly sought a fund injection of at least $50 million as it planned to acquire a stablecoin payments company in 2025. The firm was motivated to pursue the effort, with plans to sell up to 10% of its equity to fund it.
A study published at the end of 2024 revealed that its approach to regulation has made Singapore a global champion of blockchain technology. The country scored highest among all considered jurisdictions based on multiple factors.
The top blockchain jurisdictions ranked based on patents, jobs, and exchanges. Source: ApeX Protocol
Magazine: Singapore ‘not ready' for Bitcoin ETFs, sneaky crypto mining rig importer: Asia Express
XRP, the token closely related to Ripple Labs, is showing signs of a potential breakout as its price chart forms a symmetrical triangle pattern alongside other bullish technical indicators.
XRP has been consolidating within the pattern shown below, characterized by two converging trend lines that suggest a buildup of momentum. Technical analysis from well-followed X user @DefendDark indicates that a breakout from this formation could propel XRP toward a short-term target of $6.
The symmetrical triangle has been forming over recent weeks, with XRP's price action tightening as it approaches the apex of the triangle. This setup, commonly observed in crypto markets, often precedes a sharp move — either upward or downward — depending on the direction of the breakout.
#XRP is heavily compressing. This breakout will create many new millionaires! pic.twitter.com/IIT3XMaMxy
Fibonacci retracement analysis further supports this outlook, identifying $2.04 as a key support level and $2.2 as a critical resistance. A break above $2.2 could confirm the bullish trend, potentially driving XRP toward the $5 to $8 range in the mid term, per @DarkDefend.
Hi all! I hope you are well! #XRP has touched our lowest Fibonacci level. As stated, $2.222 is the key level and should be broken upwards.We set $2.04 as our last Fibonacci level, and we can visit there again! April-May will be hot, and our Targets of Wave 5 stand at… pic.twitter.com/j2JunirCkN
The Elliott Wave framework forecasts price movements in five distinct “waves” based on repeating price patterns, called "waves." The five-wave pattern reflects growing optimism, while the three-wave correction shows profit-taking or pessimism. These waves are driven by collective investor psychology and can occur across different timeframes and are considered by followers to be a way to map market behavior.
As such, a bullish technical outlook coincides with growing optimism in the crypto market, driven in part by regulatory developments. The U.S. House will hold a crypto hearing on April 9, termed “The Future of American Innovation and Digital Assets: Adjusting U.S. Securities Law for the Digital Age,” with plans to develop a regulatory framework for digital assets.
That could boost eyes on tokens perceived to be close to the U.S. government, such as XRP and Cardano's ADA, which were named as part of potential crypto stockpile by President Donald Trump earlier in January.
Shaurya is the Co-Leader of the CoinDesk tokens and data team in Asia with a focus on crypto derivatives, DeFi, market microstructure, and protocol analysis.
Shaurya holds over $1,000 in BTC, ETH, SOL, AVAX, SUSHI, CRV, NEAR, YFI, YFII, SHIB, DOGE, USDT, USDC, BNB, MANA, MLN, LINK, XMR, ALGO, VET, CAKE, AAVE, COMP, ROOK, TRX, SNX, RUNE, FTM, ZIL, KSM, ENJ, CKB, JOE, GHST, PERP, BTRFLY, OHM, BANANA, ROME, BURGER, SPIRIT, and ORCA.
He provides over $1,000 to liquidity pools on Compound, Curve, SushiSwap, PancakeSwap, BurgerSwap, Orca, AnySwap, SpiritSwap, Rook Protocol, Yearn Finance, Synthetix, Harvest, Redacted Cartel, OlympusDAO, Rome, Trader Joe, and SUN.
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The Bitcoin-Japanese yen (BTC/JPY) pair faced a setback at key trendline resistance Wednesday, as Goldman Sachs (GS) cited the anti-risk yen as the leading hedge against rising U.S. tariff and recession risks.
The BTC/JPY trading on the Japan-based bitFlyer fell 1% after failing to take out the trendline drawn off the record high reached on Jan. 20, data from charting platform TradingView show.
BTC's USD-denominated price faced similar losses. Meanwhile, Asian equity indices and the U.S. equity futures treaded water ahead of President Donald Trump's sweeping new “Liberation Day” reciprocal tariffs on Wednesday that could trigger a global trade war.
The tariff uncertainty has spurred several investment banks, including JPMorgan and Goldman Sachs, pencil in a higher chance of U.S. recession or consecutive quarterly contractions in the growth rate.
Some crypto observers expect investors to treat bitcoin (BTC) as a haven asset should a tariff-led economic swoon materialize. Goldman, however, sees the Japanese yen, a long-preferred safe haven, as the top hedge against U.S. risks.
"The yen offers investors the best currency hedge should the chances of a US recession increase," Kamakshya Trivedi, head of global foreign exchange, interest rates and emerging market strategy at Goldman Sachs, said late Tuesday, according to Bloomberg.
Trivedi added that the yen is also a "very good hedge" against U.S. labor market weakness and tends to do best when U.S. real rates [inflation-adjusted yields] and U.S. equities fall together.
While BTC is widely seen as a digital gold or haven asset by crypto market participants, the cryptocurrency has historically moved in tandem with technology stocks. In other words, tariffs-led risk-off on Wall Street could spill over into the crypto market.
Additionally, the yen's strength could prompt the unwinding of risk-on bullish trades financed by inexpensive yen-denominated loans, contributing to overall risk aversion in financial markets. The crypto market experienced this in early August last year when the yen carry trade unravelled, leading to declines in both stocks and BTC. During that period, bitcoin plummeted from approximately $65K to $50K within a week.
Goldman expects the Japanese yen to rise to the low 140s against the U.S. dollar this year. The USD/JPY pair traded at 149.77 at press time. The exchange rate is known to closely track the differential between yields on the 10-year U.S. and Japanese bonds.
The latter recently dropped to its lowest since August 2022, offering yen-bullish cues.
Omkar Godbole is a Co-Managing Editor on CoinDesk's Markets team based in Mumbai, holds a masters degree in Finance and a Chartered Market Technician (CMT) member. Omkar previously worked at FXStreet, writing research on currency markets and as fundamental analyst at currency and commodities desk at Mumbai-based brokerage houses. Omkar holds small amounts of bitcoin, ether, BitTorrent, tron and dot.
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GameStop's board had greenlit an investment in Bitcoin and US-dollar-pegged stablecoins using the convertible debt offering and cash reserves on March 25.
Video game retailer GameStop Corporation (GME) has finished a convertible debt offering that raised $1.5 billion, with some proceeds earmarked for buying Bitcoin.
The offering was initially set to raise at least $1.3 billion, but purchasers opted for an additional $200 million aggregate principal amount of notes, GameStop said in an April 1 filing with the Securities and Exchange Commission.
"The company expects to use the net proceeds from the offering for general corporate purposes, including the acquisition of Bitcoin in a manner consistent with the Company's Investment Policy," GameStop added.
The convertible notes are debt that can later be converted into equity and are scheduled to mature on April 1, 2030, unless earlier converted, redeemed or repurchased.
The conversion rate for the notes will initially be 33 shares of Common Stock per $1,000 principal amount of notes, according to the filing.
GameStop shares didn't see a significant move following the close of the convertible debt offering. GME closed the April 1 trading day up 1.34% at $22.61 and only saw an extra 0.5% bump after the bell, Google Finance data shows.
GameStop's share price barely moved after sharing it closed the convertible debt offering. Source: Google Finance
Positive shareholder sentiment saw the stock jump nearly 12% to $28.36 on March 26, the day after GameStop announced its Bitcoin (BTC) plan, but its fortunes reversed the next day, with GME shares dropping nearly 24% to $21.68.
Analysts at the time suggested the chilly reception reflected shareholders' fear of GameStop's deeper problems with its business model.
On March 25, GameStop confirmed that it had received board approval to invest in Bitcoin and US-dollar-pegged stablecoins using the notes and its cash reserves. Those reserves stood at $4.77 billion as of Feb. 1, compared with $921.7 million a year earlier, according to its 2024 fourth-quarter financial statements.
GameStop is a relative latecomer among public companies creating Bitcoin treasuries. A slew of others have already added Bitcoin to their balance sheets in a playbook popularized by Micheal Saylor's Strategy.
Related: Metaplanet adds $67M in Bitcoin following 10-to-1 stock split
The video game retailer previously made forays into the crypto space with a crypto wallet for its users, which it eventually shut down in November 2023 due to regulatory uncertainty.
GameStop is also considered the first example of meme stock success after a short squeeze in 2021 that sent the stock surging over 1,000% in a month as traders flipped the table on hedge funds that had been making money shorting on the company.
Magazine: SEC's U-turn on crypto leaves key questions unanswered
Crypto enthusiasts might have heard of the ERC-20 token standard, which provides guidelines to ensure that tokens created on the Ethereum smart contract blockchain are compatible and can interact with other tokens and applications within the network.
A similar standard for data-backed tokens, called VRC-20, has now emerged.
Vana, an EVM-compatible Layer 1 blockchain that helps users monetize personal data by bundling it into DataDAOs for AI model training, introduced the new standard early this week to boost trust and transparency in the market for data-backed digital assets.
"For data markets to work, tokens must be reliable, secure, and useful. As a universal standard for data-backed tokens, VRC-20 delivers this by ensuring fair and transparent data token trading," Vana announced on X.
The VRC-20 standard design includes specific criteria such as fixed supply, governance, and liquidity rules while ensuring real data access by tying tokens to actual data utility. Additionally, it promotes continuous liquidity through rewards that ensure market stability.
"This isn't speculation. This is real financialization of data," Vana noted.
Vana launched its mainnet in December, with VANA as its native cryptocurrency. Since then, the network has onboarded over 12 million data points through multiple DataDAOs, reflecting strong demand for user-owned data.
DataDAOs or data liquidity pools are decentralized marketplaces that bring data onchain as transferable digital tokens. DLPs are where data is contributed, tokenized and made ready for use in applications such as AI model training.
Monday's announcement replaced VANA emissions as DataDAO inventive with a new feature that calls for DAOs to issue VRC-20-compliant tokens to receive liquidity support.
Additionally, the protocol introduced data validator staking, where VANA holders can lock their coins in data validators instead of individual DataDAOs.
"Rewards are based on network security and usage. Stakers earn proportionally to their contribution to network uptime and data availability. No more idle staking. Earnings are tied to real network utility and reliability," Vana said.
The VANA token changed hands at $5.58 at press time, the lowest in over two weeks, extending the decline from the recent price high of $8.78 on Binance, according to data source TradingView.
Omkar Godbole is a Co-Managing Editor on CoinDesk's Markets team based in Mumbai, holds a masters degree in Finance and a Chartered Market Technician (CMT) member. Omkar previously worked at FXStreet, writing research on currency markets and as fundamental analyst at currency and commodities desk at Mumbai-based brokerage houses. Omkar holds small amounts of bitcoin, ether, BitTorrent, tron and dot.
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The Securities and Exchange Commission and Gemini have asked for a 60-day stay of the regulator's lawsuit to “explore a potential resolution.”
The US Securities and Exchange Commission and crypto exchange Gemini have asked to pause the regulator's suit over the exchange's Gemini Earn program, saying they want to discuss a potential resolution.
In an April 1 letter to New York federal court judge Edgardo Ramos, lawyers representing the SEC and Genesis requested a 60-day hold on the case and that all deadlines be pulled “to allow the parties to explore a potential resolution.”
“In this case, the parties submit that it is in each of their interests to stay this matter while they consider a potential resolution and agree that no party or non-party would be prejudiced by a stay,” the letter states.
The lawyers added that a stay was in the court's interest as “a resolution would conserve judicial resources” and proposed that a joint status report be submitted within 60 days after the entry of the stay.
The SEC sued Gemini and crypto lending firm Genesis Global Capital in January 2023, alleging they offered unregistered securities through the Gemini Earn program.
In March 2024, Genesis agreed to pay $21 million to settle charges related to the lending program, but the enforcement case against Gemini remains outstanding.
Letter from SEC and Genesis Global requesting extension of stay. Source: CourtListener
The letter did not specify what a possible resolution would entail, but the SEC has dropped several lawsuits it launched against crypto companies under the Biden administration, including against Coinbase, Ripple and Kraken.
Related: Will new US SEC rules bring crypto companies onshore?
In February, Gemini said the SEC closed a separate investigation into the firm as the regulator winds back its crypto enforcement under President Donald Trump.
“The SEC cost us tens of millions of dollars in legal bills alone and hundreds of millions in lost productivity, creativity, and innovation. Of course, Gemini is not alone,” Gemini co-founder Cameron Winklevoss said at the time.
OpenSea, Crypto.com and Uniswap, among others, have also recently reported that the SEC had closed similar probes into their companies that were investigating alleged breaches of securities laws.
Magazine: Bitcoin ATH sooner than expected? XRP may drop 40%, and more: Hodler's Digest, March 23 – 29
Crypto-friendly Republicans Jimmy Patronis and Randy Fine have won Florida's special elections for the 1st and 6th Congressional Districts of the US House.
Two Republicans who received a combined $1.5 million from the crypto-backed political action committee (PAC) Fairshake will enter the US House after winning special elections in Florida.
Republican Jimmy Patronis won the vacant seat in Florida's 1st Congressional District to replace Matt Gaetz, taking 57% of the vote to defeat Democrat Gay Valimont, according to AP News data.
Randy Fine also took Florida's 6th Congressional District with 56.7% of the vote to beat his Democratic rival, public school teacher Josh Weil, and fill a seat left vacant by Mike Waltz, who took a job as White House national security adviser.
Florida's 1st and 6th Congressional Districts — located in Florida's western panhandle and along the state's northeast coast — have been controlled by Republicans for roughly 30 years, but their lead has narrowed in recent years.
Fairshake, a PAC backed by crypto industry giants including Coinbase, Ripple and Andreessen Horowitz, gave Fine around $1.16 million in advertising spending and funneled $347,000 to Patronis to support his campaign.
Both Republicans have expressed support for the crypto industry, with Fine stating in a Jan. 14 X post that “Floridians want crypto innovation!”
Source: Randy Fine
Fairshake and its affiliates poured around $170 million into the 2024 US presidential and congressional elections to back candidates who committed to supporting the crypto industry.
The wins by Patronis and Fine increased Republican representation in the House to 220 seats, with the Democrats holding 213 seats.
There are two vacant seats to be filled after Texas and Arizona Democrats Sylvester Turner and Raúl Grijalva died on March 5 and March 13, respectively.
The victories for Patronis and Fine likely mean that crypto legislation will continue to see support in the US capital.
The Republican Party would have maintained its House majority even if it lost both seats in Florida, but it would have made it more difficult for some of the recently introduced Republican-backed crypto bills to pass through the House and Senate.
Related: Florida bill proposes strict rules against online gambling
At the Digital Assets Summit on March 18, Democratic Congressman Ro Khanna said he believes Congress “should be able to get” both a stablecoin and crypto market structure bill done this year.
Bills that could eventually make their way to the House include the Guiding and Establishing National Innovation for US Stablecoins (GENIUS) Act, which passed the Senate Banking Committee in an 18-6 vote on March 13.
Senator Cynthia Lummis also reintroduced a Bitcoin reserve bill about a week after the Trump administration announced the establishment of a Strategic Bitcoin Reserve on March 6, with the legislation referred to the Senate Banking Committee on March 11.
Magazine: Trump's crypto ventures raise conflict of interest, insider trading questions
Blockchain and tech trade associations want UK Prime Minister Keir Starmer's office to follow the US and appoint a special envoy dedicated to crypto.
Several British trade associations have asked Prime Minister Keir Starmer's office to appoint a special envoy dedicated to crypto and for a dedicated action plan for digital assets and blockchain technology.
In a March 31 letter, the coalition of six UK digital economy trade bodies urged Starmer's special adviser on business and investment, Varun Chandra, for a “greater strategic focus and alignment to deliver investment, growth and jobs” for the crypto industry.
The group, which consisted of the UK Cryptoasset Business Council, Global Digital Finance, The Payments Association, Digital Currencies Governance Group, the Crypto Council for Innovation and techUK, noted the US policy shift on crypto under President Donald Trump and his appointment of a crypto czar.
Britain's commitment to an economic trade deal focused on technological cooperation with the US “presents a significant opportunity to mirror the United States' ambition in fostering leadership in blockchain, digital assets, and other emerging financial technologies,” the letter stated.
The group recommended that the UK appoint a blockchain special envoy, similar to the US, to coordinate policy, foster innovation, and position the country competitively in global markets.
The trade bodies also called for the development of a dedicated government action plan for crypto and blockchain technology, including a concierge service to attract high-potential firms.
They added that the government should acknowledge and leverage the commonalities between blockchain, quantum computing and artificial intelligence technologies, including potential applications for government services.
Another recommendation was to create a high-level industry-government-regulator engagement forum to ensure informed decision-making and cross-sector collaboration.
The UK crypto and tech associations lobbying the government for a policy shift. Source: LinkedIn
“With deep pools of talent, access to capital, world-class academic institutions, and sophisticated regulators, the UK provides an environment where digital assets and blockchain innovation can thrive,” they stated.
Related: UK should tax crypto buyers to boost stock investing, economy, says banker
The coalition argues that crypto and blockchain technology could boost the UK economy by 57 billion British pounds ($73.6 billion) over the next decade, with the sector potentially increasing global gross domestic product by 1.39 trillion pounds ($1.8 trillion) by 2030.
Tom Griffiths, the co-founder and managing partner of crypto compliance advisory firm BitCompli, said in response to the letter on LinkedIn that the Financial Conduct Authority “has a lot of talent and a good sight of future plans, but the UK is definitely losing pace with Dubai, Singapore, and other EU jurisdictions.”
“Now is the time for the FCA to act, or the UK will lose out on this huge opportunity, which is digital assets and all the benefits this sector can bring, not only now but over the next 20 years,” he added.
Magazine: Bitcoin ATH sooner than expected? XRP may drop 40%, and more: Hodler's Digest, March 23 – 29
Google threat intelligence says North Korea's fake workers have also increased extortion attempts as they're under pressure to maintain money streams amid a US crackdown.
Fraudulent tech workers with ties to North Korea are expanding their infiltration operations to blockchain firms outside the US after increased scrutiny from authorities, with some having worked their way into UK crypto projects, Google says.
Google Threat Intelligence Group (GTIG) adviser Jamie Collier said in an April 2 report that while the US is still a key target, increased awareness and right-to-work verification challenges have forced North Korean IT workers to find roles at non-US companies.
“In response to heightened awareness of the threat within the United States, they've established a global ecosystem of fraudulent personas to enhance operational agility,” Collier said.
“Coupled with the discovery of facilitators in the UK, this suggests the rapid formation of a global infrastructure and support network that empowers their continued operations,” he added.
Google's Threat Intelligence Group says North Korea's tech workers expanded their reach amid a US crackdown. Source: Google
The North Korea-linked workers are infiltrating projects spanning traditional web development and advanced blockchain applications, such as projects involving Solana and Anchor smart contract development, according to Collier.
Another project building a blockchain job marketplace and an artificial intelligence web application leveraging blockchain technologies was also found to have North Korean workers.
“These individuals pose as legitimate remote workers to infiltrate companies and generate revenue for the regime,” Collier said.
Along with the UK, Collier says the GTIG identified a notable focus on Europe, with one worker using at least 12 personas across Europe and others using resumes listing degrees from Belgrade University in Serbia and residences in Slovakia.
Separate GTIG investigations found personas seeking employment in Germany and Portugal, login credentials for user accounts of European job websites, instructions for navigating European job sites, and a broker specializing in false passports.
At the same time, since late October, the North Korean workers have increased the volume of extortion attempts and gone after larger organizations, which the GTIG speculates is the workers feeling pressure to maintain revenue streams amid a crackdown in the US.
“In these incidents, recently fired IT workers threatened to release their former employers' sensitive data or to provide it to a competitor. This data included proprietary data and source code for internal projects,” Collier said.
Related: North Korean crypto attacks rising in sophistication, actors — Paradigm
In January, the US Justice Department indicted two North Korean nationals for their involvement in a fraudulent IT work scheme involving at least 64 US companies from April 2018 to August 2024.
The US Treasury Department's Office of Foreign Assets Control also sanctioned companies it accused of being fronts for North Korea that generated revenue via remote IT work schemes.
Crypto founders have also been reporting an increase in activity from North Korean hackers, with at least three founders reporting on March 13 that they foiled attempts to steal sensitive data through fake Zoom calls.
Having audio issues on your Zoom call? That's not a VC, it's North Korean hackers. Fortunately, this founder realized what was going on.The call starts with a few "VCs" on the call. They send messages in the chat saying they can't hear your audio, or suggesting there's an… pic.twitter.com/ZnW8Mtof4F
In August, blockchain investigator ZachXBT claimed to have uncovered a sophisticated network of North Korean developers earning $500,000 a month working for “established” crypto projects.
Magazine: Lazarus Group's favorite exploit revealed — Crypto hacks analysis
Kentucky's Department of Financial Institutions is the third state-level regulator in the past month to drop its action over Coinbase's staking rewards.
Kentucky's finance watchdog has dismissed its lawsuit against Coinbase over the exchange's staking rewards program, following its peers in Vermont and South Carolina.
Kentucky's Department of Financial Institutions filed the stipulation to dismiss jointly with Coinbase on April 1, ending the state's legal action against the exchange first filed along with 10 other state regulators in June 2023.
Coinbase chief legal officer Paul Grewal posted to X on April 1, calling for Congress “to end this litigation-driven, state-by-state approach with a federal market structure law.”
Source: Paul Grewal
Financial regulators from 10 states launched similar suits against Coinbase in June 2023, on the same day the Securities and Exchange Commission sued the exchange — a lawsuit the SEC dropped last month.
Alabama, California, Illinois, Maryland, New Jersey, Washington and Wisconsin are the seven states that are still continuing with their lawsuits, which all allege Coinbase breached securities laws with its staking rewards program.
Vermont was the first state to end its suit against Coinbase, with its Department of Financial Regulation filing an order to rescind the action on March 13, noting the SEC's Feb. 27 decision to drop its action against the exchange and the likelihood of changes in the federal regulator's guidance.
The South Carolina Attorney General's securities division followed Vermont days later, dismissing its lawsuit in a joint stipulation with Coinbase on March 27.
Related: South Carolina dismisses its staking lawsuit against Coinbase, joining Vermont
Kentucky's decision to drop its case against Coinbase follows just days after the state's governor, Andy Beshear, signed a “Bitcoin Rights” bill into law on March 24 that establishes protections for crypto self-custody and exempts crypto mining from money transmitting and securities laws.
The axed state-level lawsuits come amid a stark policy change at the SEC, which has dropped or delayed multiple lawsuits against crypto companies that it filed under the Biden administration.
The federal securities watchdog has also created a Crypto Task Force that is engaging with the industry on how it should approach cryptocurrencies.
Magazine: SEC's U-turn on crypto leaves key questions unanswered
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Anthropogenic biodiversity decline threatens the functioning of ecosystems and the many benefits they provide to humanity1. As well as causing species losses in directly affected locations, human influence might also reduce biodiversity in relatively unmodified vegetation if far-reaching anthropogenic effects trigger local extinctions and hinder recolonization. Here we show that local plant diversity is globally negatively related to the level of anthropogenic activity in the surrounding region. Impoverishment of natural vegetation was evident only when we considered community completeness: the proportion of all suitable species in the region that are present at a site. To estimate community completeness, we compared the number of recorded species with the dark diversity—ecologically suitable species that are absent from a site but present in the surrounding region2. In the sampled regions with a minimal human footprint index, an average of 35% of suitable plant species were present locally, compared with less than 20% in highly affected regions. Besides having the potential to uncover overlooked threats to biodiversity, dark diversity also provides guidance for nature conservation. Species in the dark diversity remain regionally present, and their local populations might be restored through measures that improve connectivity between natural vegetation fragments and reduce threats to population persistence.
Direct detrimental effects of anthropogenic activity on the biodiversity of natural ecosystems have been extensively documented3,4. For example, conversion of natural forest into urban landcover5 or transformation of grassland into cropland6 causes conspicuous declines in biodiversity. Biodiversity may also decline in ecosystems that are not directly modified but occur in regions in which human activities have caused habitat fragmentation7 or exert diffuse effects on natural areas—through pollution, for example8. Although compelling case studies show the influence of human activities on surrounding natural vegetation, beyond a direct area of impact8,9,10, there is no empirical evidence demonstrating the generality of regional-scale anthropogenic effects on local biodiversity in natural vegetation. Comparisons of relatively undisturbed vegetation inside and outside protected areas have revealed no discernible differences in local biodiversity11, but this overlooks the possibility that biodiversity has declined systematically in both settings12,13. The lack of empirical evidence might stem from the masking effect of high variation in biodiversity across regions and along ecological gradients14,15,16. We hypothesize that anthropogenic impoverishment of natural ecosystems can be revealed by the dark diversity—species that are ecologically suitable and present in a region but currently absent from a given site2. Dark diversity allows estimation of community completeness, a biodiversity metric that represents the proportion of all suitable species in a region that are actually present at a site17. This metric is globally comparable because it accounts for natural variation in potential biodiversity. Estimating the ecological suitability of species that are absent from a site is challenging, but methodological advances offer a solution based on species co-occurrences18.
The notion of dark diversity aligns with Whittaker's classic alpha–beta–gamma diversity framework19—a cornerstone of modern biodiversity research (Fig. 1). In Whittaker's work, alpha diversity represented the number of species at a particular site, gamma diversity comprised all species found in the surrounding region and beta diversity described changes in community composition along environmental gradients. The dark diversity concept is taxon-oriented, because it considers the suitability of each absent species for a study site. When aggregated, alpha and dark diversity together constitute the site-specific species pool, which includes only those species from the region that are suitable for a given site on the basis of its ecological conditions. In this context, beta diversity, as first defined by Whittaker, can be articulated as the change in site-specific species pools within gamma diversity. This is sometimes referred to as ‘structured' beta diversity, whereas ‘unstructured' beta diversity represents the variation in species composition among sampled sites within an ecologically similar area20,21. The dark diversity concept enhances the alpha–beta–gamma framework by providing a site-specific toolbox that complements alpha diversity at a site with the set of suitable yet absent species (dark diversity), the biodiversity potential of the site (species pool size) and the degree to which this potential is realized (community completeness).
a, Data included a local study site where certain species were present, but many species sampled elsewhere in the region were absent. To estimate the probability that a species that is absent from the site but present in the region belongs to the dark diversity of the site, we used information about species co-occurrences at other sites in the region. b, We calculated an indicator matrix in which each present species indicated the ecological suitability of each absent species for the study site. We compared the observed number of co-occurrences with the number of co-occurrences expected at random (according to the hypergeometric distribution) and standardized the difference using the standard deviation from the hypergeometric distribution. c, By averaging across all observed species, each absent species was assigned a probability of belonging to the dark diversity for the study site. Consequently, the dark diversity was a fuzzy set to which species belonged to varying degrees. d, Several biodiversity metrics were characterized for each site in the region. Alpha diversity was the number of species recorded at the site, and gamma diversity was the total number of species recorded in a region. The size of dark diversity was estimated as the sum of the probabilities of absent species belonging to the dark diversity of the study site. Alpha and dark diversity together formed the site-specific species pool, and gamma diversity not falling into this category was considered the unsuitable part of gamma diversity; that is, belonging to the species pools of other sites. We investigated the percentage of the species pool that was present among the alpha diversity (community completeness) and the turnover of species pools in the region, expressed as the percentage of gamma diversity that was unsuitable for the study site (beta diversity).
Alpha diversity is the most commonly used biodiversity metric, but it depends on variation in natural biodiversity potential between regions (for example, boreal versus temperate regions; North America versus East Asia) and ecological conditions within regions (for example, wetlands versus forests; south-facing versus north-facing slopes). Speciation, large-scale dispersal, species sorting and stochastic variation have produced site-specific species pools of considerably different sizes22. Community completeness accounts for such variation by quantifying the extent to which the biodiversity potential (that is, the site-specific species pool) is realized locally17. Even in natural ecosystems, some suitable taxa might be absent owing to natural processes that cause local extinction or limit recolonization. Such limiting processes vary along environmental gradients, reflected in the global patterns of plant persistence strategies23 and interactions with other organisms; for example, seed predators24. Consequently, there is likely to be natural variation in community completeness across broad environmental gradients25. In addition, regions with high geodiversity or a mosaic of vegetation types (that is, high structured beta diversity) might have lower community completeness because the isolation of natural habitat fragments and the likelihood of local extinction increase26. Furthermore, climatic conditions leave some regions prone to extreme events, such as natural fire, that cause local species loss27,28. Nevertheless, in addition to natural variation, human activities might strongly influence community completeness by reducing the persistence of local populations; for example, by promoting highly competitive taxa (through eutrophication, for instance8) or by restricting mutualistic interactions (reducing pollinators, for instance29). Similarly, human activities might hinder the recolonization of suitable sites through habitat fragmentation7 and loss of seed-dispersing animals30.
To determine whether anthropogenic impoverishment of natural vegetation is a worldwide phenomenon, we established DarkDivNet, a global collaborative research network31. Using a standardized methodology, we assessed both the alpha and the dark diversity of vascular plants across 5,415 sites with relatively intact natural or semi-natural vegetation, in 119 regions, spanning a wide range of vegetation types and representative of most global climatic conditions on all vegetated continents (Extended Data Figs. 1 and 2).
In our study, ‘site' refers to a 100-m2 area in which vegetation was sampled, and ‘region' represents the surrounding area of approximately 300 km2. Each region encompasses at least 30 sites, representing the natural and semi-natural vegetation typical of the region. We first confirmed that the sampling area of 100 m2 provided highly similar estimates of dark diversity to those obtained from a considerably larger area of 2,500 m2 (Extended Data Fig. 3). We assessed alpha diversity as the number of all vascular plant species found at each site. To estimate dark diversity, we used a fuzzy set approach in which all species occurring in the region but absent from the site were assigned a probability of inclusion in the dark diversity on the basis of an established co-occurrence methodology18. The use of probabilities maximizes the amount of information used for estimating dark diversity. Specifically, co-occurrences were based on the species composition of 30 randomly selected sites in the region (Fig. 1a). Using a subset of regions in which 60 sites were available yielded highly similar outcomes, indicating that 30 sites were sufficient for estimating co-occurrence patterns among species (Extended Data Fig. 3). We estimated the degree to which each species present in a region but absent from a site co-occurred with species found at the site, and compared it with random expectation, mathematically described by the hypergeometric distribution (Fig. 1b). If an absent species co-occurred with a present species more than would be randomly expected, they probably shared ecological requirements, and the present species provided a positive indication of the site's suitability for the absent species. The overall suitability of the site for the absent species was estimated by averaging the suitability indications from all species present at the site (Fig. 1c). The magnitude of dark diversity at a site was then estimated as the sum of these suitability estimates (probabilities of absent species belonging to the dark diversity of the site, ranging between 0 and 1) across all absent species. The unsuitable fraction of gamma diversity reflects the species belonging to different site-specific species pools in the same region. Using alpha diversity, dark diversity and the unsuitable diversity found in the region, we calculated other biodiversity metrics for each site to have a full description of biodiversity (Fig. 1d): site-specific species pool size as the sum of alpha and dark diversity; gamma diversity as the total set of species found in a region (this value was the same for each site within a region); community completeness as the proportion of the site-specific species pool size represented by alpha diversity; and beta diversity as a quantification of the extent to which gamma diversity exceeds the site-specific species pool size (that is, the proportion of gamma diversity that is unsuitable for the study site and is more likely to be associated with different site-specific species pools in the region). In this way, we specifically quantified the ‘structured' beta diversity, or turnover in site-specific species pool composition due to environmental gradients. In the statistical analyses, community completeness and beta diversity were included as log-ratios (logit transformation of percentages) to improve the distribution of the data. We used two independent datasets (expert assessments and examination of species found in the close vicinity of the site) to ensure that the co-occurrence method provided consistent estimates of species suitability for dark diversity (Extended Data Fig. 2). We also determined that, for this particular dataset, the hypergeometric method outperformed an alternative approach—joint species distribution modelling32 (see Supplementary Methods).
The median community completeness of sites across all regions was 25% (95% confidence interval 15–46%), highlighting a frequent absence of suitable species despite their presence in surrounding regions (Fig. 2a). The existence of relatively high dark diversity is clearly a general phenomenon, but the large variation meant that sometimes much fewer species were present locally than might be expected from the specific site conditions. To understand how much variation in alpha diversity was explained by community completeness besides beta and gamma diversity, we used variation partitioning. We found that 33% (26–43%) of the variation in alpha diversity was explained by community completeness. Consequently, if human activities reduce the colonization and persistence of suitable species, resulting in lower community completeness, this could substantially affect alpha diversity. The largest proportion of variation in alpha diversity, 52% (40–61%), was explained by gamma diversity, reflecting the well-known match of local and regional diversity33, whereas 14% (9–21%) was explained by beta diversity, reflecting how gamma diversity is distributed across different site-specific species pools. The strong dependence of alpha diversity on regional richness is clearly sufficient to mask the negative effect of human activities on alpha diversity.
a, Relationship between community completeness in natural vegetation and the human footprint index in the surrounding area, defined by a radius of 300 km. The prediction line from a multiple linear regression model is shown with the 95% confidence intervals. Note that community completeness values on the y axis are back-transformed from the logit scale. The symbol tones indicate forest cover (0–100%). R² value of the model and two-tailed P value of the relationship are shown; n = 116 regions. The distribution of community completeness is shown in the histogram on the right (median, 25%). b, Left, model summaries linking community completeness to the human footprint index and its components across spatial scales. Human influence was averaged over various spatial scales around the study regions (radii 10 km, 50 km, 100 km, 200 km, 300 km and 400 km), and the respective models were compared using the Akaike information criterion (AIC). Filled symbols indicate significant relationships (P < 0.05), and the large symbol indicates the set of best significant models (ΔAIC < 2). Right, from the best model (the smallest scale at which ΔAIC < 2), the effect of the human footprint index or one of its components is shown as a standardized coefficient (dot) with a 95% confidence interval (CI; line); n = 116 regions. Filled symbols and bold confidence interval lines indicate significant effects. c, Map of sampling regions, with community completeness indicated by symbol size and the underlying map showing the global variation in the human footprint index34 (the highest value within each grid cell of around 0.25° × 0.25°). The inset shows part of Europe containing a large number of study regions. Triangles indicate regions in which only woody species were sampled. Symbol tones indicate the percentage of forests in regions.
We tested the hypothesis that impoverishment of natural vegetation is related to anthropogenic influence in the surrounding region by building a series of models with various biodiversity metrics (community completeness, alpha diversity, beta diversity, gamma diversity, dark diversity and species pool size) as response variables. To estimate the intensity of human activities in the surrounding regions, we used the human footprint index from 2018 (the year our sampling began)—a well-established cumulative metric of human influence34—along with all of its eight components, including human population density and various human infrastructure layers. We averaged human influence at various spatial scales around the study region (radii from 10 km to 400 km), because human influence can reach far from mapped features. For example, poaching and logging can occur tens of kilometres from human settlements35 and are facilitated by many unmapped ‘ghost roads' that start from documented roads and lead into natural areas36. Similarly, anthropogenic ignition of fires can occur hundreds of kilometres from main roads37. Aerial pollution is often deposited several hundreds of kilometres from its source35, and land use can change local climate over similar scales38. To account for the effects of natural processes on biodiversity (for example, geodiversity, habitat patchiness and likelihood of natural fires), we included in our statistical models variables describing climatic, soil and topographic conditions, which we derived from global GIS layers and summarized using four principal component axes. Using fivefold spatial block cross-validation, we determined that linear models produced lower prediction errors with test data, compared with nonlinear alternatives (around 20% versus 40%). We therefore used linear models in further analyses.
The human footprint index and community completeness exhibited a robust negative linear relationship (Fig. 2a), which was already significant when the average human footprint index within a 50-km radius around the site was used, but became even more pronounced when radii of 300 km or larger were considered (Fig. 2b and Extended Data Table 1). In the sampled regions with minimal human footprint index values (close to zero), an average of 35% of suitable species were found in the 100-m2 sites, but this proportion declined to less than 20% in regions with high human impact. However, there was still variation in community completeness at both the low and the high ends of the human footprint index, showing that sites do not respond uniformly. In contrast to community completeness, alpha diversity was not strongly related to the human footprint index, and nor were the other tested metrics, except beta diversity (Extended Data Fig. 4 and Extended Data Table 1). These results are consistent with our hypothesis that local biodiversity is lower in natural vegetation surrounded by regions with more human activity, but this effect was evident only when we considered community completeness. Raw estimates of alpha diversity were strongly influenced by the wide natural variation in diversity potential determined by the specific biogeographical history of each region. Our results were consistent for six of the eight individual components of the human footprint index: human population density, the extent of electric infrastructure, railways, roads, built environments and croplands all exhibited negative relationships with community completeness (Fig. 2b and Extended Data Table 2). The extent of pasture was an exception to this pattern, because it was not negatively related to community completeness. This could be due to the influence of semi-natural grasslands, in which long-term moderate human influence, including grazing of domestic animals, cultural burning and haymaking, has resulted in highly diverse and well-functioning ecosystems, exemplifying how certain human activities can actually promote native biodiversity39. We found that the effect of the human footprint index was strongest when averaged over a range of several hundred kilometres. Besides incorporating far-reaching human influence, larger scales might also more accurately capture cumulative human influence in a particular region over long time periods40. However, including in the model a variable representing change in the index between 2000 and 2013 did not reduce the Akaike information criterion (AIC) by more than two units, which suggests that anthropogenic effects have operated over longer timescales. To account for the effects of natural processes on community completeness, our models included environmental variables. We found that community completeness decreased along the first principal component (Extended Data Table 1). Thus, suitable species are more likely to fall into the dark diversity in regions characterized by acidic organic soils and higher precipitation (see correlations of principal component axes in Extended Data Fig. 5). Dark diversity, gamma diversity and species pool size increased along the first axis (representing higher soil carbon content, acidity and precipitation; Extended Data Table 1). Alpha and beta diversities showed no significant relationships with the environmental axes.
The negative effect of human activities on community completeness might be associated with several phenomena. Human activities might have led to the fragmentation or reduction of suitable habitats, resulting in smaller populations that are more susceptible to random extinction9. In addition, habitat loss is likely to have decreased connectivity between remaining patches of natural vegetation, making it difficult for species to move between areas41, and defaunation might have disrupted plant seed dispersal networks30. Beyond habitat loss, some anthropogenic disturbances, such as tree cutting, illegal harvesting of plants and human-induced wildfires, can cause local extinctions in natural vegetation10,42. Moreover, regional human impact can affect natural ecosystems through pollution from roads and other human infrastructure; eutrophication is the most serious threat to plant diversity, because it disproportionally favours a few competitively superior species at the expense of a greater number of other species8.
Using average human influence as an explanatory variable can mask differences between regions. For example, regions that comprise both highly modified areas (for example, cities) and nature reserves, as well as those experiencing moderate human influence throughout (for example, agricultural landscapes with smaller settlements), might both exhibit an intermediate level of average human influence. We therefore tested how the distribution of the human footprint index within regions affected community completeness. Notably, we found that community completeness had an even stronger negative relationship with anthropogenic influence when we used the 30% quantile of the human footprint index values found within regions (Extended Data Fig. 6). This result suggests that completeness is determined mainly by the extent to which the most natural areas in a region already experience human influence. The idea that 30% coverage of natural vegetation in a landscape supports the persistence of many specialist taxa was proposed previously43, and aligns with the global target of the Convention on Biological Diversity to protect 30% of land by the year 2030. Our results therefore underscore the importance of devising regional-scale conservation strategies that include maintaining well-preserved natural areas44.
The turnover of site-specific species pools within regions (structured beta diversity) was significantly positively associated with the human footprint index (Extended Data Fig. 4 and Supplementary Table 1). This might reflect a human preference for naturally diverse regions with a range of different resources45. Alternatively, human activities could have promoted plant diversity over millennia by expanding semi-natural habitats and modifying natural ecosystems39. Most components of the human footprint index generally exhibited similar relationships, except for the extent of navigable waterways and pastures, which were negatively related to beta diversity (Supplementary Table 1). It is likely that coastal and riverine regions, and those suitable for livestock grazing, naturally exhibit relatively low variation in vegetation types.
The finding that high human footprint index values in a region are associated with low community completeness persisted in several other robustness tests (Supplementary Methods). Statistical interactions between the human footprint index and environmental gradients did not improve the model. Because naturally high beta diversity might decrease community completeness owing to the spatial separation of ecologically similar sites, and because beta diversity was correlated with human influence, we used structural equation modelling to examine the direct and indirect effects of human influence on community completeness. The negative direct effect of the human footprint index on community completeness persisted even if there was an additional negative direct effect of beta diversity. In addition, the effect of the human footprint index on community completeness was consistent across sampling scales (2,500 m2 or twice as many sites for species co-occurrences), when we excluded alien or very rare species, when regions with only woody species records were included and when we considered the proportion of forest cover in regions. Community completeness was slightly lower in more forested regions. The most parsimonious explanation for this might be a scaling effect—fewer large plant individuals can fit into a fixed area46. We also examined the possible effect of geographically uneven sampling by selecting a single study region from each ecoregion (the anthropogenic effect was always negative), adding the European continent as a factor to the model (the negative relationship remained significant) and investigating model residuals (no significant spatial autocorrelation was apparent). Community completeness was slightly lower in Europe than in other regions, which could reflect a cumulative effect of long-term human influence40.
This global-scale study reveals general patterns, and linkage to specific drivers is based on ecological interpretation rather than experimentation. It is also clear that the human footprint index does not provide a proxy for all potentially important processes, such as the disruption of biotic interaction networks, increasingly frequent climate extremes or the habitat destruction and fragmentation caused by war. The plethora of processes affecting biodiversity certainly contributes to variation around the general trends revealed by our models. The significant relationships we identified apply to the sampled range of the human footprint index, whereas index values outside this range might produce different relationships. In addition, even if the uneven distribution of study regions did not produce an effect in statistical models, the under-representation of several parts of Africa, the Americas and Asia might mean that some human impacts on biodiversity were not well represented. Future work should examine the exact patterns and processes of natural vegetation impoverishment in these undersampled regions.
Our finding of a globally consistent negative relationship between human influence and local plant diversity in relatively natural vegetation is alarming, because plants form the foundation of all terrestrial ecosystems. Reduced community completeness indicates that many species present in the region do not inhabit suitable sites, and this can affect local ecosystem functioning47. Although vegetation functioning depends mainly on the traits of co-existing taxa, the presence of a larger proportion of suitable taxa increases the chance that essential functions are represented48. We also found that negative human influence was most evident when considered at a scale spanning several hundred kilometres; in other words, biodiversity in natural ecosystems is reduced far beyond human infrastructure. Therefore, conservation actions and land-use planning should consider not only the observed alpha diversity of a site, but also a broader regional context. Ecology has a rich history of conceptual frameworks for biodiversity across scales, such as species–area relationships49, alpha–beta–gamma diversity19, community saturation and assembly33 and the meta-community concept50. Building on this collective knowledge, the dark diversity concept offers a species-oriented toolkit for evaluating community patterns and explaining the underlying processes. By allowing the estimation of a site's biodiversity potential (site-specific species pool) and its realization (community completeness), it fosters the comparative study of biodiversity across regions, ecosystem types and taxonomic groups2. This improved understanding could help conservation biologists, land managers and policymakers to prevent further losses of biodiversity51. Moreover, while site-specific species pools are not depleted, dark diversity offers a narrow window of opportunity for restoration because it indicates which missing species are still regionally present52,53,54.
In 2018, we launched a global collaborative research consortium to sample both locally observed alpha diversity and dark diversity of terrestrial plant communities using a standardized methodology. A detailed sampling protocol was produced before fieldwork began31. Each study region covered an area of approximately 300 km2, defined by a circle of 20-km diameter with the available area influenced by geographical and practical limitations (coastline, private ownership and other access restrictions). This spatial scale was selected on the basis of the authors' expertise, in the expectation that it would incorporate areas with a relatively uniform biogeographical history while still exhibiting variation in natural vegetation. In addition, mechanisms of long-distance seed dispersal can operate at this scale55. In each region, we defined at least 30 sites, in which we sampled a 100-m2 (10 m × 10 m) area by recording all vascular plant species. Where feasible, we sampled more sites in the region to examine how sampling intensity might affect the results. The sites were selected to proportionally represent the typical natural vegetation types of the region without major human influence. These included semi-natural grasslands, representing habitats that have developed over thousands of years through grazing by domestic animals and mowing, and forests that had been managed with low intensity and had species composition and tree-layer structure similar to old-growth forests. Here we report the results from 5,415 sites in 119 regions for which sampling was completed by 1 February 2024 (Supplementary Table 2).
To assess whether dark diversity methods could predict species that were absent from the 100-m2 area but present in its immediate vicinity, and to estimate the effect of spatial scale on dark diversity, we selected one to three sites per region in which we sampled vascular plants in a 2,500-m2 (50 m × 50 m) area within which the 100-m2 area was nested. In four regions, sampling of the larger area was not possible or the large area had no new taxa, so these regions were omitted from the respective test. In addition, in 76 regions, we had sufficient expertise to assess which of the species found in the region were ecologically well-suited for a selected site (that is, belonging to the site-specific species pool). This information allowed us to test the applicability of dark diversity methods within our sampling framework (see below).
Biodiversity metrics were determined for each site in each region (Fig. 1). Alpha diversity A was defined as the number of vascular plant species found in the 100-m2 area describing a site (Fig. 1a). Dark diversity D was quantified for each site k by examining species co-occurrences within the surrounding region using the hypergeometric method, implemented in the R package DarkDiv (ref. 18). This technique uses information about how each species i that is absent from the study site but present in the surrounding region co-occurs with species j that is present at the study site. If an absent species co-occurs more frequently with observed species than it would do under random expectation, it is likely to belong to the dark diversity. The expected number of co-occurrences is mathematically defined by the hypergeometric distribution. For each pair of absent and present species, we compared the observed number of co-occurrences Mij with the expected value, which is defined as the mean of the hypergeometric distribution:
where ni and nj are the total number of occurrences of species i and j, respectively, and N is the total number of sites sampled in that region. The standardized effect size (SES) was used as an indicator of the suitability of absent species i for site k on the basis of co-occurrences with present species j (Fig. 1b), and was calculated as the difference between the observed and the expected numbers of co-occurrences divided by the standard deviation of the expected number of co-occurrences, as derived from the hypergeometric distribution:
We estimated the suitability of site k for all species i absent from the site but present in the region, by averaging suitability indicator values from all present species j using the number of species found in site k (nk):
The SESki values were subsequently transformed to a 0–1 scale by applying inverse probit transformation, which places the SESki value within the cumulative normal distribution function with mean = 0 and standard deviation = 1 (Fig. 1c):
This estimate expressed the probability that species i belonged to the dark diversity of site k. Our estimated dark diversity probabilities were supported by two independent tests, one investigating which absent species were found in the immediate vicinity of a site and another using expert assessment (Extended Data Fig. 2). We also considered how the suitability of absent species might be estimated using an alternative technique—joint species distribution modelling (JSDM)56 (Supplementary Methods).
Dark diversity size for a study site was the sum of the probabilities Pki of all locally absent species found elsewhere in the region (Fig. 1d). For co-occurrences, we always considered 30 sites (each described by a 100-m2 area) within the same region (Fig. 1a), which is the minimum number sampled and generally sufficient for the method18. For regions with more than 30 sampled sites, we used an iterative procedure, each time randomly selecting 30 sites for species co-occurrences. Dark diversity size in those regions was estimated as the median from 100 iterations. Similarly, estimates of gamma diversity G were obtained using iteration, taking the median cumulative species number from 30 sites in a region. To test whether 30 sites was sufficient to estimate the variation in regional richness, we estimated species richness with complete sample coverage using incidence-based extrapolation based on the Bernoulli product model57, implemented within the R iNEXT package58. Gamma diversity from 30 sites correlated strongly with the extrapolated value (Spearman r = 0.95; Extended Data Fig. 3a)
Using alpha, dark and gamma diversities for each site, we calculated: species pool size as the sum of alpha and dark diversity: P = A + D; community completeness as the percentage of alpha diversity among all suitable species for that site: C = A/(A + D) × 100%; and beta diversity as the percentage of gamma diversity belonging to other species pools in the region and unsuitable for the specific site: B = (G – A – D) / G × 100% (Fig. 1d). This metric is identical to Whittaker's effective turnover at the species pool level, expressed as a percentage rather than a ratio (G/P) – 1. In analyses, all biodiversity metrics were transformed to improve distributions: those based on counts or sums (alpha, dark and gamma diversity, species pool size) were log-transformed, and those based on percentages (community completeness and beta diversity) were logit transformed. To aid intuitive understanding, we show untransformed values on graph axes. Because several of the diversity metrics are either subsets or calculated from each other, it is expected that these are closely related. However, bivariate relationships between our study variables (Extended Data Fig. 7) showed that all metrics bear some independent information, and the variability among and within regions is large.
All of our biodiversity metrics depend on the sampling scheme, including characteristics such as sample area or number of sites. To investigate how much our biodiversity metrics change if using a larger sample area, we used 1–3 sites in each region where both 100-m2 and 2,500-m2 areas were sampled. Similarly, we examined the effect of using a larger number of sites to characterize co-occurrences; using 60 sites from 27 regions where they were available. Overall, global variation in our metrics was highly correlated regardless of sample area and the number of sites considered (Spearman correlation > 0.8; Extended Data Fig. 3b–k).
According to the DarkDivNet protocol, in very diverse tropical regions we only sampled woody vascular plant species. Although alpha diversity, dark diversity, species pool size and gamma diversity are evidently smaller when herbaceous species are omitted, community completeness and beta diversity should still be relatively comparable with other regions because these metrics are unitless. To ensure full comparability between biodiversity metrics, we used only the 116 regions in which all vascular plants were sampled in the main analyses, but repeated the main tests for community completeness with all 119 regions within robustness analyses (Supplementary Methods).
Alpha diversity can be seen as a subset of gamma diversity in which the species pool has been filtered according to beta diversity, and the realization of the species pool is defined by community completeness (Fig. 1). We examined how much of the variation in alpha diversity is determined by variation in gamma diversity, beta diversity (these two define the site-specific species pool size) and community completeness. We randomly selected one site from each region in order to have independent local and regional variables (gamma diversity is the same for all sites in a region). The contribution of each source of variation was calculated using hierarchical variation partitioning (function varpart in the vegan package59 in R). This procedure was repeated 100 times to obtain a median and confidence interval.
In further statistical analyses, we used the medians of biodiversity variables across sites per region. We related community completeness and other calculated biodiversity variables (alpha diversity, beta diversity, gamma diversity, dark diversity and species pool size) to the human footprint index from the year 201834. The index ranges from 0 to 50 and is calculated from eight components (human population density, electric infrastructure, railways, roads, navigable waterways, the extent of built-up land, pastures and croplands). The resolution of the human influence data layers was 100 m, and we calculated average values over various spatial extents around the centre of each region (radii 10 km, 50 km, 100 km, 200 km, 300 km and 400 km). The averaging did not include areas representing water bodies. Because all regions included at least some areas less affected by humans, the total range of the averaged human footprint index values used in our analyses was somewhat lower than the maximum value. To test how well our sampled regions captured global variation in the human footprint index, we generated 500 random points worldwide using the discrete global grid system (which maintains uniform point density across the globe). From random points, we omitted glaciated regions of Antarctica and Greenland. We averaged the human footprint index in the surroundings of these random points in the same manner as we did with our empirical data. This revealed a high degree of correspondence between the average human footprint index ranges around sampled and randomly generated points at different scales: at radii of 50 km (sampled range 1.1–25.4, random 0.0–24.5), 200 km (sampled range 0.3–20.7, random 0.0–20.7) and 400 km (sampled 0.2–17.7, random 0.1–16.6).
To account for natural processes affecting community completeness, we included environmental variables in the multiple linear regression models. We used mean annual temperature and annual precipitation from the CHELSA database (resolution 1 km)60,61, soil pH, organic carbon content, sand fraction proportion from SoilGrids (resolution 250 m)62 and the topographic ruggedness of the terrain (resolution 250 m)63. Environmental factors were averaged within a 100-km radius to describe the broader region and consolidated through principal component analysis (PCA). For PCA, variables with only positive values were log-transformed if this resulted in a distribution closer to normal, and all variables were standardized. We kept the four first principal components, which described more than 90% of the variation. The first component was positively correlated with soil organic carbon content, acidity and precipitation; the second with temperature; the third with soil sand content; and the fourth with topographic ruggedness (Extended Data Fig. 5).
We fitted both linear and nonlinear (generalized additive models, function gam in the R package; ref. 64) models, incorporating the 116 regions in which all vascular plants were sampled. The estimates of the human footprint index at the different spatial scales were inherently strongly related to each other. Therefore, we constructed models for each scale at which the human footprint index (or its components) was averaged. We examined which scales produced the best models (ΔAIC < 2) and selected the smallest scale, which is most directly related to the study region. We compared linear and nonlinear models using spatial block validation, implemented in the R package blockCV (ref. 65). We used fivefold cross-validation across hexagons (Extended Data Fig. 2). To estimate the variation in model predictive power we further implemented a bootstrap approach66 by selecting bootstrap samples within each fold and then performing cross-validation. We used the normalized root mean square error (normalized by minimum and maximum values) to compare the predictive error of linear and nonlinear models, and found that linear models had much lower error in test sets (around 20% of the range compared with around 40% of the range; see Extended Data Fig. 8). Linear models were therefore used as a more general option.
We report the results of the best linear model (the smallest spatial scale at which ΔAIC < 2) for each biodiversity metric and note significant relationships (P < 0.05). We used the variance inflation factor (VIF) to confirm that correlations between environmental gradients and human impact (Extended Data Fig. 5) were not confounding in the models (VIF < 2). We applied type III model testing. Consequently, the effect of human impact was tested only after the environmental effects were accounted for. We visualized the results of the fitted models in terms of how the predictor variable human footprint index affects the outcome of community completeness using the visreg function and package67 in R. Model summary tables can be found in Extended Data Tables 1 and 2.
Besides the human footprint index from 2018, we also examined whether including change in the human footprint index during recent years improved the model68. Specifically, we tested whether a model including human footprint index change yielded a lower AIC value (by more than two units) compared with the model without change. We derived the measure of human footprint index change from a source that used a consistent methodology69 during a temporal range 2000–2013. Change in human footprint index was quantified as log(human footprint index value from 2013/human footprint index value from 2000).
We tested whether community completeness is better described by certain quantiles of the human footprint index at different scales around study regions. Compared with the mean, considering quantiles allowed us to determine the extent to which it is important to maintain a certain proportion of area with lower human influence. We compared models incorporating as predictor variables the 10–90% quantiles of the human footprint index using AIC and recorded cases in which the quantiles yielded a better model than the mean (models with AIC lower by more than two units were considered superior).
We also tested the robustness of the relationship between community completeness and the human footprint index by looking at statistical interactions between human influence and the environment, indirect effects, the role of sampling scale, alien or rare species; by including areas in which only woody species were recorded and considering forest cover in regions; and by examining the effect of geographically uneven sampling (see Supplementary Methods).
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
All data supporting the findings of this study, along with the R scripts to handle them, can be found in Figshare: https://doi.org/10.6084/m9.figshare.25158059 (ref. 70). We also used published data for the human footprint index34,69; from the CHELSA database60,61 for annual mean temperature and annual precipitation; from SoilGrids62 for soil pH, organic carbon content and sand fraction proportion; and from the Geomorpho90m database63 for the topographic ruggedness of the terrain.
Ceballos, G. et al. Accelerated modern human-induced species losses: entering the sixth mass extinction. Sci. Adv. 1, e1400253 (2015).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Pärtel, M., Szava-Kovats, R. & Zobel, M. Dark diversity: shedding light on absent species. Trends Ecol. Evol. 26, 124–128 (2011).
PubMed
Google Scholar
Jaureguiberry, P. et al. The direct drivers of recent global anthropogenic biodiversity loss. Sci. Adv. 8, eabm9982 (2022).
PubMed
PubMed Central
Google Scholar
Newbold, T. et al. Global effects of land use on local terrestrial biodiversity. Nature 520, 45–50 (2015).
ADS
PubMed
MATH
Google Scholar
Williams, N. S. G. et al. A conceptual framework for predicting the effects of urban environments on floras. J. Ecol. 97, 4–9 (2009).
MATH
Google Scholar
Le Provost, G. et al. Grassland-to-crop conversion in agricultural landscapes has lasting impact on the trait diversity of bees. Landsc. Ecol. 36, 281–295 (2021).
PubMed
MATH
Google Scholar
Haddad, N. M. et al. Habitat fragmentation and its lasting impact on Earth's ecosystems. Sci. Adv. 1, e1500052 (2015).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Stevens, C. J., Thompson, K., Grime, J. P., Long, C. J. & Gowing, D. J. G. Contribution of acidification and eutrophication to declines in species richness of calcifuge grasslands along a gradient of atmospheric nitrogen deposition. Funct. Ecol. 24, 478–484 (2010).
Google Scholar
Chase, J. M., Blowes, S. A., Knight, T. M., Gerstner, K. & May, F. Ecosystem decay exacerbates biodiversity loss with habitat loss. Nature 584, 238–243 (2020).
ADS
PubMed
Google Scholar
Barlow, J. et al. Anthropogenic disturbance in tropical forests can double biodiversity loss from deforestation. Nature 535, 144–147 (2016).
ADS
PubMed
MATH
Google Scholar
Gray, C. L. et al. Local biodiversity is higher inside than outside terrestrial protected areas worldwide. Nat. Commun. 7, 12306 (2016).
ADS
PubMed
PubMed Central
Google Scholar
Santangeli, A. et al. Mixed effects of a national protected area network on terrestrial and freshwater biodiversity. Nat. Commun. 14, 5426 (2023).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Jones, K. R. et al. One-third of global protected land is under intense human pressure. Science 360, 788–791 (2018).
PubMed
MATH
Google Scholar
Cai, L. et al. Global models and predictions of plant diversity based on advanced machine learning techniques. New Phytol. 237, 1432–1445 (2022).
PubMed
MATH
Google Scholar
Sabatini, F. M. et al. Global patterns of vascular plant alpha diversity. Nat. Commun. 13, 4683 (2022).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Fraser, L. H. et al. Coordinated distributed experiments: an emerging tool for testing global hypotheses in ecology and environmental science. Front. Ecol. Environ. 11, 147–155 (2013).
MATH
Google Scholar
Pärtel, M., Szava-Kovats, R. & Zobel, M. Community completeness: linking local and dark diversity within the species pool concept. Folia Geobot. 48, 307–317 (2013).
Google Scholar
Carmona, C. P. & Pärtel, M. Estimating probabilistic site-specific species pools and dark diversity from co-occurrence data. Glob. Ecol. Biogeogr. 30, 316–326 (2021).
MATH
Google Scholar
Whittaker, R. H. Vegetation of the Siskiyou Mountains, Oregon and California. Ecol. Monogr. 30, 279–338 (1960).
MATH
Google Scholar
Harrison, S., Vellend, M. & Damschen, E. I. ‘Structured' beta diversity increases with climatic productivity in a classic dataset. Ecosphere 2, 1–13 (2011).
Google Scholar
Anderson, M. J. et al. Navigating the multiple meanings of beta diversity: a roadmap for the practicing ecologist. Ecol. Lett. 14, 19–28 (2011).
ADS
PubMed
MATH
Google Scholar
Vellend, M. Conceptual synthesis in community ecology. Q. Rev. Biol. 85, 183–206 (2010).
PubMed
Google Scholar
Salguero-Gómez, R. et al. Fast–slow continuum and reproductive strategies structure plant life-history variation worldwide. Proc. Natl Acad. Sci. USA 113, 230–235 (2016).
ADS
PubMed
MATH
Google Scholar
Peco, B., Laffan, S. W. & Moles, A. T. Global patterns in post-dispersal seed removal by invertebrates and vertebrates. PLoS One 9, e91256 (2014).
ADS
PubMed
PubMed Central
Google Scholar
Riibak, K. et al. Drivers of plant community completeness differ at regional and landscape scales. Agric. Ecosyst. Environ. 301, 107004 (2020).
MATH
Google Scholar
Ben-Hur, E. & Kadmon, R. Heterogeneity–diversity relationships in sessile organisms: a unified framework. Ecol. Lett. 23, 193–207 (2019).
PubMed
MATH
Google Scholar
González-Trujillo, J. D., Román-Cuesta, R. M., Muñiz-Castillo, A. I., Amaral, C. H. & Araújo, M. B. Multiple dimensions of extreme weather events and their impacts on biodiversity. Clim. Change 176, 155 (2023).
ADS
Google Scholar
Gallagher, R. V. et al. High fire frequency and the impact of the 2019–2020 megafires on Australian plant diversity. Divers. Distrib. 27, 1166–1179 (2021).
MATH
Google Scholar
Outhwaite, C. L., McCann, P. & Newbold, T. Agriculture and climate change are reshaping insect biodiversity worldwide. Nature 605, 97–102 (2022).
ADS
PubMed
MATH
Google Scholar
Dirzo, R. et al. Defaunation in the Anthropocene. Science 345, 401–406 (2014).
ADS
PubMed
Google Scholar
Pärtel, M. et al. DarkDivNet—a global research collaboration to explore the dark diversity of plant communities. J. Veg. Sci. 30, 1039–1043 (2019).
MATH
Google Scholar
Tikhonov, G. et al. Joint species distribution modelling with the R-package HMSC. Methods Ecol. Evol. 11, 442–447 (2020).
PubMed
PubMed Central
MATH
Google Scholar
Ricklefs, R. E. Community diversity: relative roles of local and regional processes. Science 235, 167–171 (1987).
ADS
PubMed
Google Scholar
Gassert, F. et al. An operational approach to near real time global high resolution mapping of the terrestrial Human Footprint. Front. Remote Sens. 4, 1130896 (2023).
MATH
Google Scholar
McDonald, R. I. et al. Urban effects, distance, and protected areas in an urbanizing world. Landsc. Urban Plann. 93, 63–75 (2009).
MATH
Google Scholar
Engert, J. E. et al. Ghost roads and the destruction of Asia-Pacific tropical forests. Nature 629, 370–375 (2024).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Fusco, E. J., Abatzoglou, J. T., Balch, J. K., Finn, J. T. & Bradley, B. A. Quantifying the human influence on fire ignition across the western USA. Ecol. Appl. 26, 2390–2401 (2016).
Google Scholar
Lawton, R. O., Nair, U. S., Pielke, R. A. & Welch, R. M. Climatic impact of tropical lowland deforestation on nearby montane cloud forests. Science 294, 584–587 (2001).
ADS
PubMed
Google Scholar
Bengtsson, J. et al. Grasslands—more important for ecosystem services than you might think. Ecosphere 10, e02582 (2019).
MATH
Google Scholar
Ellis, E. C. Land use and ecological change: a 12,000-year history. Annu. Rev. Environ. Resour. 46, 1–33 (2021).
MATH
Google Scholar
Taylor, P. D., Fahrig, L., Henein, K. & Merriam, G. Connectivity is a vital element of landscape structure. Oikos 68, 571–573 (1993).
ADS
MATH
Google Scholar
van Wees, D. et al. The role of fire in global forest loss dynamics. Glob. Chang. Biol. 27, 2377–2391 (2021).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Hanski, I. Habitat loss, the dynamics of biodiversity, and a perspective on conservation. Ambio 40, 248–255 (2011).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Aavik, T. & Helm, A. Restoration of plant species and genetic diversity depends on landscape-scale dispersal. Restor. Ecol. 26, S92–S102 (2018).
MATH
Google Scholar
Araujo, M. B. The coincidence of people and biodiversity in Europe. Glob. Ecol. Biogeogr. 12, 5–12 (2003).
MATH
Google Scholar
Oksanen, J. Is the humped relationship between species richness and biomass an artefact due to plot size? J. Ecol. 84, 293–295 (1996).
MATH
Google Scholar
Hooper, D. U. et al. Effects of biodiversity on ecosystem functioning: a consensus of current knowledge. Ecol. Monogr. 75, 3–35 (2005).
MATH
Google Scholar
Reich, P. B. et al. Impacts of biodiversity loss escalate through time as redundancy fades. Science 336, 589–592 (2012).
ADS
PubMed
MATH
Google Scholar
Storch, D. The theory of the nested species–area relationship: geometric foundations of biodiversity scaling. J. Veg. Sci. 27, 880–891 (2016).
MATH
Google Scholar
Leibold, M. A. et al. The metacommunity concept: a framework for multi-scale community ecology. Ecol. Lett. 7, 601–613 (2004).
MATH
Google Scholar
Leclère, D. et al. Bending the curve of terrestrial biodiversity needs an integrated strategy. Nature 585, 551–556 (2020).
ADS
PubMed
MATH
Google Scholar
Lewis, R. J. et al. Applying the dark diversity concept to nature conservation. Conserv. Biol. 31, 40–47 (2017).
PubMed
MATH
Google Scholar
Deschênes, É., Santala, K. R., Lavigne, J. & Aubin, I. Using a trait-based dark diversity approach to evaluate natural recovery potential in forests. Restor. Ecol. 32, e14251 (2024).
Google Scholar
Moeslund, J. E. et al. Using dark diversity and plant characteristics to guide conservation and restoration. J. Appl. Ecol. 54, 1730–1741 (2017).
MATH
Google Scholar
Nathan, R. et al. Mechanisms of long-distance seed dispersal. Trends Ecol. Evol. 23, 638–647 (2008).
PubMed
MATH
Google Scholar
Pollock, L. J. et al. Understanding co-occurrence by modelling species simultaneously with a Joint Species Distribution Model (JSDM). Methods Ecol. Evol. 5, 397–406 (2014).
MATH
Google Scholar
Chao, A. et al. Rarefaction and extrapolation with Hill numbers: a framework for sampling and estimation in species diversity studies. Ecol. Monogr. 84, 45–67 (2014).
MATH
Google Scholar
Hsieh, T. C., Ma, K. H. & Chao, A. iNEXT: an R package for rarefaction and extrapolation of species diversity (Hill numbers). Methods Ecol. Evol. 7, 1451–1456 (2016).
MATH
Google Scholar
Oksanen, J. et al. vegan: Community Ecology Package. R version 4.2.2 https://CRAN.R-project.org/package=vegan (2022).
Karger, D. N. et al. Climatologies at high resolution for the earth's land surface areas. Sci. Data 4, 170122 (2017).
PubMed
PubMed Central
MATH
Google Scholar
Karger, D. N. et al. Data from: Climatologies at high resolution for the earth's land surface areas. Dryad Digital Repository https://doi.org/10.5061/dryad.kd1d4 (2017).
Hengl, T. et al. SoilGrids250m: global gridded soil information based on machine learning. PLoS One 12, e0169748 (2017).
PubMed
PubMed Central
Google Scholar
Amatulli, G., McInerney, D., Sethi, T., Strobl, P. & Domisch, S. Geomorpho90m, empirical evaluation and accuracy assessment of global high-resolution geomorphometric layers. Sci. Data 7, 162 (2020).
PubMed
PubMed Central
Google Scholar
Wood, S. N. Fast stable restricted maximum likelihood and marginal likelihood estimation of semiparametric generalized linear models. J. R. Stat. Soc. B 73, 3–36 (2011).
MathSciNet
MATH
Google Scholar
Valavi, R., Elith, J., Lahoz-Monfort, J. J. & Guillera-Arroita, G. blockCV: an R package for generating spatially or environmentally separated folds for k-fold cross-validation of species distribution models. Methods Ecol. Evol. 10, 225–232 (2019).
MATH
Google Scholar
Fu, W. J., Carroll, R. J. & Wang, S. Estimating misclassification error with small samples via bootstrap cross-validation. Bioinformatics 21, 1979–1986 (2005).
PubMed
MATH
Google Scholar
Breheny, P. & Burchett, W. Visualization of regression models using visreg. R J. 9, 56–71 (2017).
MATH
Google Scholar
Di Marco, M., Venter, O., Possingham, H. P. & Watson, J. E. M. Changes in human footprint drive changes in species extinction risk. Nat. Commun. 9, 4621 (2018).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Williams, B. A. et al. Change in terrestrial human footprint drives continued loss of intact ecosystems. One Earth 3, 371–382 (2020).
ADS
MATH
Google Scholar
Pärtel, M. et al. Supporting data for ‘Global impoverishment of natural vegetation revealed by dark diversity'. Figshare https://doi.org/10.6084/m9.figshare.25158059 (2025).
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We thank the Estonian Research Council (PRG609, PRG1065, PSG293 and PRG2142) and the Estonian Ministry of Education and Research (Centre of Excellence AgroCropFuture, TK200) for funding the DarkDivNet steering committee, and we thank the many students and volunteers who helped with the fieldwork. Acknowledgements related to specific authors can be found in the Supplementary Notes.
Institute of Ecology and Earth Sciences, University of Tartu, Tartu, Estonia
Meelis Pärtel, Riin Tamme, Carlos P. Carmona, Kersti Riibak, Mari Moora, Maarja Öpik, John Davison, Junichi Fujinuma, Nele Ingerpuu, Madli Jõks, Ene Kook, Tatjana Oja, Bruno Paganeli, Triin Reitalu, Enrico Tordoni, Diego P. F. Trindade, Oscar Zárate Martínez & Martin Zobel
Department of Plant Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Jonathan A. Bennett
Department of Biological, Geological and Environmental Sciences, Alma Mater Studiorum —University of Bologna, Bologna, Italy
Alessandro Chiarucci, Silvia Del Vecchio & Arianna Ferrara
Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, Czech Republic
Milan Chytrý
CIDE, CSIC-UV-GVA, Valencia, Spain
Francesco de Bello, Daniel A. Rodríguez Ginart & Diego P. F. Trindade
Department of Botany, Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic
Francesco de Bello, Jiri Dolezal, Eva Janíková, Marie Konečná, Aleš Lisner & Mercedes Valerio
Department of Ecology, Environment and Plant Sciences, Stockholm University, Stockholm, Sweden
Ove Eriksson
Department of Environmental Science and Policy, University of California Davis, Davis, CA, USA
Susan Harrison
Norwegian Institute for Nature Research, Bergen, Norway
Robert John Lewis
Evolution and Ecology Research Centre, School of Biological, Earth and Environmental Sciences, UNSW Sydney, Sydney, New South Wales, Australia
Angela T. Moles
Gulbali Institute, Charles Sturt University, Albury, New South Wales, Australia
Jodi N. Price
Plant Ecology Group, Institute of Evolution and Ecology, University of Tübingen, Tübingen, Germany
Vistorina Amputu
Independent researcher, Tehran, Iran
Diana Askarizadeh
Department of Reclamation of Arid and Mountainous Regions, University of Tehran, Tehran, Iran
Diana Askarizadeh
Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran
Zohreh Atashgahi
Great Lakes Forestry Centre, Canadian Forest Service, Natural Resources Canada, Sault Ste Marie, Ontario, Canada
Isabelle Aubin & Laura Boisvert-Marsh
Terrestrial Ecology Group, Department of Ecology, Universidad Autónoma de Madrid, Madrid, Spain
Francisco M. Azcárate, Irene Guerrero & Begoña Peco
Centro de Investigación en Biodiversidad y Cambio Global (CIBC-UAM), Universidad Autónoma de Madrid, Madrid, Spain
Francisco M. Azcárate
Australian Tropical Herbarium, James Cook University, Smithfield, Queensland, Australia
Matthew D. Barrett
Department of Range and Watershed Management, Faculty of Natural Resources and Environment, Ferdowsi University of Mashhad, Mashhad, Iran
Maral Bashirzadeh
Department of Ecology, University of Szeged, Szeged, Hungary
Zoltán Bátori, András Kelemen & Csaba Tölgyesi
Centre for Environmental Sciences, Hasselt University, Hasselt, Belgium
Natalie Beenaerts
Plant Ecology and Nature Conservation, University of Potsdam, Potsdam, Germany
Kolja Bergholz, Florian Jeltsch, Michael Ristow & Lina Weiss
Department of Biological Sciences, University of Bergen, Bergen, Norway
Kristine Birkeli, Ruben S. Thormodsæter & Vigdis Vandvik
Bjerknes Centre for Climate Research, University of Bergen, Bergen, Norway
Kristine Birkeli & Vigdis Vandvik
Department of Plant Biology and Ecology, University of the Basque Country UPV/EHU, Bilbao, Spain
Idoia Biurrun & Juan A. Campos
Department of Evolutionary Biology, Ecology and Environmental Sciences (Botany and Mycology), Universitat de Barcelona, Barcelona, Spain
José M. Blanco-Moreno & Aaron Pérez-Haase
Biodiversity Research Institute (IRBio), Universitat de Barcelona, Barcelona, Spain
José M. Blanco-Moreno & Aaron Pérez-Haase
Department of Biology, University of North Carolina at Greensboro, Greensboro, NC, USA
Kathryn J. Bloodworth & Kimberly J. Komatsu
Department of Biology, National University of Mongolia, Ulaanbaatar, Mongolia
Bazartseren Boldgiv & Khaliun Sanchir
Department of Forest Sciences, Luiz de Queiroz College of Agriculture, University of São Paulo, Piracicaba, Brazil
Pedro H. S. Brancalion & Joannès Guillemot
Re.green, Rio de Janeiro, Brazil
Pedro H. S. Brancalion
Department of Natural Sciences, Manchester Metropolitan University, Manchester, UK
Francis Q. Brearley
Département de biologie, Université de Sherbrooke, Sherbrooke, Quebec, Canada
Charlotte Brown
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada
Charlotte Brown, James F. Cahill, Raytha A. Murillo, Karina Salimbayeva & Viktoria Wagner
Instituto Pirenaico de Ecologia, CSIC, Jaca, Spain
C. Guillermo Bueno & Pablo Tejero
Department of Environmental Sciences, Informatics and Statistics, Ca' Foscari University of Venice, Venice, Italy
Gabriella Buffa, Edy Fantinato & Giulia Silan
Department of Life, Health and Environmental Science, University of L'Aquila, Coppito, L'Aquila, Italy
Giacomo Cangelmi
Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parma, Italy
Michele Carbognani, T'ai G. W. Forte, Alessandro Petraglia & Marcello Tomaselli
École Pratique des Hautes Études, Paris Sciences Lettres University (EPHE-PSL), Paris, France
Christopher Carcaillet
University Claude Bernard Lyon 1, LEHNA UMR5023, CNRS, ENTPE, Villeurbanne, France
Christopher Carcaillet
Department of Biotechnology and Life Science, University of Insubria, Varese, Italy
Bruno E. L. Cerabolini & Michele Dalle Fratte
Conservatoire d'espaces naturels Centre–Val de Loire, Orléans, France
Richard Chevalier
Research Group Plants and Ecosystems (PLECO), University of Antwerp, Wilrijk, Belgium
Jan S. Clavel, Jonas J. Lembrechts & Dajana Radujković
Centre for Functional Ecology, Associate Laboratory TERRA, Department of Life Sciences, University of Coimbra, Coimbra, Portugal
José M. Costa, Ruben H. Heleno & Daniel Montesinos
Department of Physical Geography, Stockholm University, Stockholm, Sweden
Sara A. O. Cousins
Department of Invasion Ecology, Institute of Botany, Czech Academy of Sciences, Průhonice, Czech Republic
Jan Čuda, Martin Hejda, Jiří Sádlo, Hana Skálová, Kateřina Štajerová, Michaela Vítková & Martin Vojík
Instituto de Biociências, Lab of Vegetation Ecology, Universidade Estadual Paulista (UNESP), Rio Claro, Brazil
Mariana Dairel & Alessandra Fidelis
Independent researcher, Kirovsk, Russia
Alena Danilova & Natalia Koroleva
Lendület Seed Ecology Research Group, Institute of Ecology and Botany, HUN-REN Centre for Ecological Research, Vácrátót, Hungary
Balázs Deák, András Kelemen, Katalin Lukács & Orsolya Valkó
Institute of Environmental Biology, Faculty of Biology, University of Warsaw, Warsaw, Poland
Iwona Dembicz, Łukasz Kozub & Nadiia Skobel
Vegetation Ecology Research Group, Institute of Natural Resource Sciences (IUNR), Zurich University of Applied Sciences (ZHAW), Wädenswil, Switzerland
Jürgen Dengler
Institute of Botany, Czech Academy of Sciences, Průhonice, Czech Republic
Jiri Dolezal & Miroslav Dvorsky
CREAF (Centre for Ecological Research and Forestry Applications), Bellaterra, Spain
Xavier Domene
Universitat Autònoma de Barcelona, Bellaterra, Spain
Xavier Domene
Quantitative Plant Ecology and Biodiversity Research Lab, Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashdad, Iran
Hamid Ejtehadi & Sahar Karami
Instituto Multidisciplinario de Biología Vegetal (CONICET-UNC), Córdoba, Argentina
Lucas Enrico & Melisa A. Giorgis
FCEFyN, Universidad Nacional de Córdoba, Córdoba, Argentina
Lucas Enrico & Melisa A. Giorgis
Independent researcher, Moscow, Russia
Dmitrii Epikhin
Department of Ecology and Genetics, University of Oulu, Oulu, Finland
Anu Eskelinen & Risto Virtanen
German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Leipzig, Germany
Anu Eskelinen, Lotte Korell & Soroor Rahmanian
Division of BioInvasions, Global Change and Macroecology, University of Vienna, Vienna, Austria
Franz Essl
State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing, China
Gaohua Fan & Houjia Liu
Chair of Plant Ecology, University of Bayreuth, Bayreuth, Germany
Fatih Fazlioglu
Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Ordu University, Ordu, Turkey
Fatih Fazlioglu
Biodiversity Research Institute (IMIB), University of Oviedo–CSIC–Principality of Asturias, Mieres, Spain
Eduardo Fernández-Pascual & Borja Jiménez-Alfaro
Department of Organismal and Systems Biology, University of Ovidedo, Oviedo, Spain
Eduardo Fernández-Pascual & Borja Jiménez-Alfaro
Institute of Plant Sciences, University of Bern, Bern, Switzerland
Markus Fischer & Lena Neuenkamp
Department of Agricultural and Food Chemistry, Universidad Autónoma de Madrid, Madrid, Spain
Maren Flagmeier & Eduardo Moreno-Jiménez
Department of Natural Resource Sciences, Thompson Rivers University, Kamloops, British Columbia, Canada
Lauchlan H. Fraser
Graduate Program in Botany, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
Fernando F. Furquim
Independent researcher, Mancelona, MI, USA
Berle Garris
Au Sable Institute of Environmental Studies, Mancelona, MI, USA
Heath W. Garris
Department of Geological, Biological and Environmental Sciences, University of Catania, Catania, Italy
Gianpietro Giusso del Galdo
Departamento de Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén, Jaén, Spain
Ana González-Robles & Rubén Tarifa
Instituto Interuniversitario del Sistema Tierra de Andalucía, Universidad de Jaén, Jaén, Spain
Ana González-Robles
School of Agriculture, Food and Ecosystem Sciences, University of Melbourne, Melbourne, Victoria, Australia
Megan K. Good
Unit of Botany, Department of Animal and Plant Biology and Ecology, Universitat Autònoma de Barcelona, Bellaterra, Spain
Moisès Guardiola
Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Palermo, Italy
Riccardo Guarino
CIRAD, UMR Eco&Sols, Montpellier, France
Joannès Guillemot & Agnès A. Robin
Eco&Sols, University Montpellier, CIRAD, INRAE, Institut Agro, IRD, Montpellier, France
Joannès Guillemot & Agnès A. Robin
Biology Education, Dokuz Eylül University, Buca, Turkey
Behlül Güler
Key Laboratory of Soybean Molecular Design Breeding, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun, China
Yinjie Guo
Department of Earth and Environmental Sciences, KU Leuven, Leuven, Belgium
Stef Haesen, Karlien Moeys & Koenraad Van Meerbeek
KU Leuven Plant Institute, KU Leuven, Leuven, Belgium
Stef Haesen & Koenraad Van Meerbeek
Department of Ecoscience, Aarhus University, Aarhus, Denmark
Toke T. Høye & Jesper Erenskjold Moeslund
Arctic Research Centre, Aarhus University, Aarhus, Denmark
Toke T. Høye
Institute of Botany, Plant Science and Biodiversity Center, Slovak Academy of Sciences, Bratislava, Slovakia
Richard Hrivnák & Ivana Svitková
State Key Laboratory of Black Soils Conservation and Utilization, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun, China
Yingxin Huang & Xuhe Liu
Jilin Songnen Grassland Ecosystem National Observation and Research Station, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun, China
Yingxin Huang & Xuhe Liu
Jilin Provincial Key Laboratory of Grassland Farming, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun, China
Yingxin Huang & Xuhe Liu
School of Environmental and Rural Science, University of New England, Armidale, New South Wales, Australia
John T. Hunter
Institute of Biological Sciences, University of Zielona Góra, Zielona Góra, Poland
Dmytro Iakushenko
F. Falz-Fein Biosphere Reserve Askania Nova, Kyiv, Ukraine
Dmytro Iakushenko
Departamento de Biología Ambiental, Facultad de Ciencias, Universidad de Navarra, Pamplona, Spain
Ricardo Ibáñez & Mercedes Valerio
Biogeography and Biodiversity Lab, Institute of Physical Geography, Goethe-University Frankfurt, Frankfurt am Main, Germany
Severin D. H. Irl
Faculty of Agricultural and Environmental Sciences, University of Rostock, Rostock, Germany
Florian Jansen
Department of Disturbance Ecology, Bayreuth Center of Ecology and Environmental Research (BayCEER), University of Bayreuth, Bayreuth, Germany
Anke Jentsch & Andreas von Hessberg
Department of Range and Watershed Management, Faculty of Natural Resources, Islamic Azad University Nour Branch, Nour, Iran
Mohammad H. Jouri
Chair of Sensor-based Geoinformatics, Faculty of Environment and Natural Resources, University of Freiburg, Freiburg, Germany
Negin Katal
Independent researcher, Kazan, Russia
Bulat I. Khairullin, Maria V. Kozhevnikova & Vadim E. Prokhorov
Centre for Biodiversity and Taxonomy, Department of Botany, University of Kashmir, Srinagar, India
Anzar A. Khuroo & Sajad A. Wani
Department of Species Interaction Ecology, Helmholtz Centre for Environmental Research—UFZ, Leipzig, Germany
Lotte Korell
Department of Functional Ecology, Institute of Botany, Czech Academy of Sciences, Třeboň, Czech Republic
Kirill A. Korznikov & Vojtech Lanta
Chair of Biodiversity and Nature Tourism, Estonian University of Life Sciences, Tartu, Estonia
Lauri Laanisto & Petr Macek
Kalmar County Administrative Board, Färjestaden, Sweden
Helena Lager & Michael Tholin
Instituto Nacional de Tecnología Agropecuaria (INTA), Río Gallegos, Argentina
Romina G. Lasagno & Pablo L. Peri
Ecology and Biodiversity (E&B), Utrecht University, Utrecht, The Netherlands
Jonas J. Lembrechts
Aerospace Information Research Institute, Chinese Academy of Sciences, Beijing, China
Liping Li & Yichen Tian
State Key Laboratory of Grassland and Agro-Ecosystems, School of Life Sciences, Lanzhou University, Lanzhou, China
Kun Liu & Sa Xiao
School of Grassland Science, Beijing Forestry University, Beijing, China
Xuhe Liu
Higher Technical School of Agricultural and Forestry Engineering, Castilla-La Mancha University, Albacete, Spain
Manuel Esteban Lucas-Borja & Pedro Antonio Plaza-Álvarez
Applied Plant Ecology, Institute of Plant Science and Microbiology, University of Hamburg, Hamburg, Germany
Kristin Ludewig
Netzwerk für Angewandte Ökologie, Hamburg, Germany
Jona Luther-Mosebach
Institute of Hydrobiology, Biology Centre of the Czech Academy of Sciences, Ceske Budejovice, Czech Republic
Petr Macek
Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy
Michela Marignani
Univ. Bordeaux, CNRS, Bordeaux INP, EPOC, UMR 5805, Pessac, France
Richard Michalet & Blaise Touzard
ÖMKi—Research Institute of Organic Agriculture, Budapest, Hungary
Tamás Miglécz
Australian Tropical Herbarium, James Cook University, Cairns, Queensland, Australia
Daniel Montesinos
College of Science and Engineering, James Cook University, Cairns, Queensland, Australia
Daniel Montesinos
Institute for Advanced Research in Chemical Sciences (IAdChem), Universidad Autónoma de Madrid, Madrid, Spain
Eduardo Moreno-Jiménez
Department of Botany, Kherson State University, Kherson, Ukraine
Ivan Moysiyenko & Nadiia Skobel
Harry Butler Institute, Murdoch University, Perth, Western Australia, Australia
Ladislav Mucina & James L. Tsakalos
Department of Geography and Environmental Studies, Stellenbosch University, Stellenbosch, South Africa
Ladislav Mucina
Laboratorio de Biodiversidad y Funcionamiento Ecosistémico Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS), CSIC, Sevilla, Spain
Miriam Muñoz-Rojas
Centre for Ecosystem Science, UNSW Sydney, Sydney, New South Wales, Australia
Miriam Muñoz-Rojas
Department of Agriculture and Natural Resource Sciences, Namibia University of Science and Technology, Windhoek, Namibia
Sylvia M. Nambahu
Institute of Landscape Ecology, University of Münster, Münster, Germany
Lena Neuenkamp
Center for Sustainable Landscapes Under Global Change, Department of Biology, Aarhus University, Aarhus, Denmark
Signe Normand
Botanical Garden, Center for Biological Diversity Conservation, Polish Academy of Sciences, Warszawa, Poland
Arkadiusz Nowak & Sebastian Świerszcz
Greenpeace España, Madrid, Spain
Paloma Nuche
Shenzhen MSU-BIT University, Shenzhen, China
Vladimir G. Onipchenko
Department of Ecology and Environmental Protection, Faculty of Biology, Sofia University St Kliment Ohridski, Sofia, Bulgaria
Kalina L. Pachedjieva
Departemento Biología y Geología, Física y Química Inorgánica, Universidad Rey Juan Carlos, Móstoles, Spain
Ana M. L. Peralta
Universidad Nacional de la Patagonia Austral (UNPA), CONICET, Río Gallegos, Argentina
Pablo L. Peri
Department of Civil and Environmental Engineering, University of the Andes, Bogotá, Colombia
Gwendolyn Peyre
Swedish Biodiversity Centre, Department of Urban and Rural Development, Swedish University of Agricultural Sciences, Uppsala, Sweden
Jan Plue
Department of Biology, Lund University, Lund, Sweden
Honor C. Prentice
Remote Sensing Centre for Earth System Research (RSC4Earth), Leipzig University, Leipzig, Germany
Soroor Rahmanian
Institute of Geology, Tallinn University of Technology, Tallinn, Estonia
Triin Reitalu
Department of Soil Sciences, Luiz de Queiroz College of Agriculture, University of São Paulo, Piracicaba, Brazil
Agnès A. Robin
Estación Experimental del Zaidín (CSIC), Granada, Spain
Ana Belén Robles
Department of Agronomy, University of Almería, Almería, Spain
Raúl Román
Norwegian Institute for Nature Research, Oslo, Norway
Ruben E. Roos
Faculty of Environmental Sciences and Natural Resource Management, Norwegian University of Life Sciences, Ås, Norway
Ruben E. Roos
Scuola di Scienze Agrarie, Forestali, Alimentari e Ambientali, Università della Basilicata, Potenza, Italy
Leonardo Rosati
Departamento de Biodiversidad, Ecología y Evolución, Facultad de Ciencias Biológicas, Universidad Complutense de Madrid, Madrid, Spain
Rut Sánchez de Dios & Enrique Valencia
School of Natural Sciences, Macquarie University, Sydney, New South Wales, Australia
Cornelia Sattler & Julian Schrader
Department of Ecosystem Science and Management, Laramie Research and Extension Center, University of Wyoming, Laramie, WY, USA
John D. Scasta
Institute of Plant Science and Microbiology, University of Hamburg, Hamburg, Germany
Ute Schmiedel
Future Regions Research Centre, Federation University Australia, Ballarat, Victoria, Australia
Nick L. Schultz
CIRAD-UMR EcoFoG, Kourou, French Guiana
Giacomo Sellan
Botanical Institute of Barcelona (CSIC-CMCNB), Barcelona, Spain
Josep M. Serra-Diaz
Department of Ecology, University of Debrecen, Debrecen, Hungary
Judit Sonkoly
Institute of Agroecology and Plant Production, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
Sebastian Świerszcz
Ecosystems and Global Change Group, School of the Environment, Trent University, Peterborough, Ontario, Canada
Andrew J. Tanentzap
Department of Plant Sciences, University of Cambridge, Cambridge, UK
Fallon M. Tanentzap
Estación Experimental de Zonas Áridas (EEZA-CSIC), Almería, Spain
Rubén Tarifa
Independent reseacher, Teberda, Russia
Dzhamal K. Tekeev
Yuriy Fedkovych Chernivtsi National University, Chernivtsi, Ukraine
Alla Tokaryuk
HUN-REN-UD Functional and Restoration Ecology Research Group, Department of Ecology, University of Debrecen, Debrecen, Hungary
Péter Török
HUN-REN-UD Biodiversity and Ecosystem Services Research Group, Department of Ecology, University of Debrecen, Debrecen, Hungary
Béla Tóthmérész
Centre de Recherche sur la Biodiversité et l'Environnement (CRBE), UMR 5300 UPS-CNRS-IRD-INP, Université Paul Sabatier–Toulouse 3, Toulouse, France
Aurèle Toussaint
School of Biosciences and Veterinary Medicine, University of Camerino, Camerino, Italy
James L. Tsakalos
Department of Mathematics and Science Education, Faculty of Education, Ordu University, Ordu, Turkey
Sevda Türkiş
Departamento de Ciencias de la Vida, Universidad de Alcalá, Alcalá de Henares, Spain
Jesus Villellas
Department of Applied Ecology, Faculty of Environmental Sciences, Czech University of Life Sciences, Prague, Czech Republic
Martin Vojík
Nature Conservation Agency of the Czech Republic, Prague, Czech Republic
Martin Vojík
Department of Biology, Aarhus University, Aarhus, Denmark
Jonathan von Oppen
Department of Environmental Sciences, University of Basel, Basel, Switzerland
Jonathan von Oppen
Key Laboratory of Mountain Surface Processes and Ecological Regulation, Institute of Mountain Hazards and Environment, Chinese Academy of Sciences, Chengdu, China
Ji-Zhong Wan
Sichuan Academy of Forestry, Chengdu, China
Chun-Jing Wang
National Monitoring Centre for Biodiversity Germany, Leipzig, Germany
Lina Weiss
Deakin University, Burwood, Victoria, Australia
Tricia Wevill
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The steering committee members (M.P., R. Tamme, C.P.C., K.R., M. Moora and M.Z.) initiated and coordinated DarkDivNet, analysed the data and wrote the manuscript. The advisory board members (J.A.B., A.C., M. Chytrý, F.B., O.E., S. Harrison, R.J.L., A.T.M., M.Ö. and J.N.P.) contributed to the study design and to the initial versions of the manuscript. V.A., D.A., Z.A., I.A., F.M.A., M.D.B., M.B., Z.B., N.B., K. Bergholz, K. Birkeli, I.B., J.M.B.-M., K.J.B., L.B.-M., B.B., P.H.S.B., F.Q.B., C.B., C.G.B., G.B., J.F.C., J.A.C., G.C., M. Carbognani, C.C., B.E.L.C., R.C., J.S.C., J.M.C., S.A.O.C., J.Č., M. Dairel, M.D.F., A.D., J. Davison, B.D., S.D.V., I.D., J. Dengler, J. Dolezal, X.D., M. Dvorsky, H.E., L.E., D.E., A.E., F.E., G.F., E.F., F.F., E.F.-P., A. Ferrara, A. Fidelis, M. Fischer, M. Flagmeier, T.G.W.F., L.H.F., J.F., F.F.F., B. Garris, H.W.G., M.A.G., G.G.G., A.G.-R., M.K.G., M.G., R.G., I.G., J.G., B. Güler, Y.G., S Haesen, M.H., R.H.H., T.T.H., R.H., Y.H., J.T.H., D.I., R.I., N.I., S.D.H.I., E.J., F. Jansen, F. Jeltsch, A.J., B.J.-A., M.J., M.H.J., S.K., N. Katal, A.K., B.I.K., A.A.K., K.J.K., M.K., E.K., L.K., N. Koroleva, K.A.K., M.V.K., Ł.K., L. Laanisto, H. Lager, V.L., R.G.L., J.J.L., L. Li, A.L., H. Liu, K. Liu, X.L., M.E.L.-B., K. Ludewig, K. Lukács, J.L.-M., P.M., M. Marignani, R.M., T.M., J.E.M., K.M., D.M., E.M.-J., I.M., L.M., M.M.-R., R.A.M., S.M.N., L.N., S.N., A.N., P.N., T.O., V.G.O., K.L.P., B. Paganeli, B. Peco, A.M.L.P., A.P.-H., P.L.P., A.P., G.P., P.A.P.-Á., J.P., H.C.P., V.E.P., D.R., S.R., T.R., M.R., A.A.R., A.B.R., D.A.R.G., R.R., R.E.R., L.R., J. Sádlo, K. Salimbayeva, R.S.D., K. Sanchir, C.S., J.D.S., U.S., J. Schrader, N.L.S., G. Sellan, J.M.S.-D., G. Silan, H.S., N.S., J. Sonkoly, K. Štajerova, I.S., S.Ś., A.J.T., F.M.T., R. Tarifa, P. Tejero, D.K.T., M. Tholin, R.S.T., Y.T., A. Tokaryuk, C.T., M. Tomaselli, E.T., P. Török, B. Tóthmérész, A. Toussaint, B. Touzard, D.P.F.T., J.L.T., S.T., E.V., M. Valerio, O.V., K.V.M., V.V., J.V., R.V., M. Vítkova, M. Vojík, A.H., J.O., V.W., J.-Z.W., C.-J.W., S.A.W., L.W., T.W., S.X. and O.Z.M. provided data and were involved in the interpretation of the results and manuscript preparation.
Correspondence to
Meelis Pärtel.
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Nature thanks Marta Jarzyna, Alejandro Ordonez and Moreno Di Marco for their contribution to the peer review of this work.
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Lines indicate ranges within a radius of 100 km. Approximate broad biomes are shown. Triangles indicate regions in which only woody species were sampled.
We used two tests. In the Vicinity test, we examined whether species absent from the site (100 m2) but present in the immediate vicinity (2500 m2) have higher estimated suitabilities than absent species found further away. The sample area and vicinity area are assumed to share relatively similar ecological conditions. In the Expert test, we compared whether species absent from the site but assessed by expert opinion to be ecologically suitable (i.e., belong to the site-specific species pool) have higher calculated suitabilities than those absent species that were evaluated as unsuitable. In both cases, we calculated the log response ratio of the mean suitability of species in the respective groups. Positive log response ratios indicate agreement between assessments of suitability calculated from co-occurrences and from the independent information considered in the tests. The length of the lines (vertical for the Vicinity test and horizontal for the Expert test) shown at study region locations indicates the magnitude of the log response ratio; negative values are in red and positive values are in blue. Both tests comprised data from a subset of study regions. The box plot on the left (centre line, median; box limits, upper and lower quartiles; whiskers, the range, excluding outlying points that exceed the quartiles by more than 1.5× the interquartile range) shows the results of single-sample two-sided t-tests (difference from zero), with log response ratios significantly larger than zero in both cases, n = 115 regions for the Vicinity test and n = 76 regions for the Expert test. Hexagons on the map (made with Natural Earth; free vector and raster map data; https://www.naturalearthdata.com/) delimit the spatial blocks used in cross-validation.
a, Gamma diversity from 30 sites compared with extrapolation up to complete sampling coverage. b–f, Sites described in a 2500 m2 area in addition to the DarkDivNet standard of 100 m2. g–k, Biodiversity metrics when 60 sites were used to estimate co-occurrences in addition to the DarkDivNet standard of 30. The scatter plots show mean values for regions where the respective sampling scheme was applied (n = 119 regions for a, n = 116 regions for b–f and n = 27 for g–k). The 1:1 lines are shown as diagonals. Estimates of Spearman correlation for each comparison are shown above the panels. Comparisons where the alternative sampling method did not influence the metric (i.e. gamma diversity when using a larger sample area or alpha diversity when using more sites) are not shown.
a, Alpha diversity. b, Beta diversity. c, Gamma diversity. d, Dark diversity. e, Species pool size. A similar graph for community completeness is shown in Fig. 2a. For each metric, the relationships from the spatial scale producing the best multiple linear regression model is shown (n = 116 regions, see Extended Data Table 1 for details of the models). The prediction lines are shown with 95% confidence intervals. The solid line indicates a significant relationship (two-tailed p < 0.05); the dashed lines indicate non-significant trends. Note that the range of the human footprint index varies when averaged at different spatial scales. Diversity values on y-axes are back-transformed from log or logit scales.
Correlations with raw environmental variables (used in the PCA; top) and the human footprint index or its components (not used in the PCA; bottom).
Filled symbols indicate significant relationships (n = 116 regions, two-tailed p < 0.05), and the large symbols indicate models where the Akaike information criterion (AIC) is lower than the minimal value among the models using the mean human footprint index value (Extended Data Table 1). The asterisk indicates a combination of quantile and spatial scale that yielded a considerably better model (AIC value lower by more than 2 units).
a-e, Alpha diversity. a, f-i, Dark diversity. b, f, j-l, Species pool size. c, g, j, m-o, Gamma diversity. d, h, k, m, o, Community completeness. e, i, l, n, o, Beta diversity. Lines indicate variation within regions (99% quantiles, i.e. omitting outliers), crossing at median values within regions. Several metrics are inherently related (see Fig. 1), and strong relationships are expected. However, variation within regions can be large, indicating the importance of site-specific metrics. The colours of lines reflect the different biodiversity metrics (see Fig. 1) to facilitate comparisons across panels.
a, Community completeness. b, Alpha diversity. c, Dark diversity. d, Species pool size. e, Gamma diversity. f, Beta diversity. We used fivefold spatial cross-validation (see Extended Data Fig. 2) with bootstrapping to estimate the variation. The box plots (centre line = median; box limits = upper and lower quartiles; whiskers = the range, excluding outlying points that exceed the quartiles by more than 1.5× the interquartile range) show that while the nonlinear models had lower errors for the training set, the test data were predicted with lower error by the linear models.
This file contains Supplementary Tables 1 and 2, Supplementary Methods and Supplementary Notes.
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Pärtel, M., Tamme, R., Carmona, C.P. et al. Global impoverishment of natural vegetation revealed by dark diversity.
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A new NASA space telescope has turned on its detectors for the first time, capturing its first light in images that contain tens of thousands of galaxies and stars.
The Spectro-Photometer for the History of the Universe, Epoch of Reionization, and Ices Explorer (SPHEREx) arrived in orbit atop a SpaceX Falcon 9 rocket on March 11.
The six released images, collected by the space telescope on March 27, were each snapped by three different detectors. The top three images span the telescope's complete field of view, and are captured again in the bottom three which are colored differently to represent varying ranges of infrared wavelengths.
Within each image's full field of view — an area roughly 20 times wider than the full moon — roughly 100,000 light sources from stars, galaxies, and nebulas can be glimpsed.
"Our spacecraft has opened its eyes on the universe," Olivier Doré, a SPHEREx project scientist at Caltech and NASA's Jet Propulsion Laboratory, said in a statement. "It's performing just as it was designed to."
Related: Euclid space telescope: ESA's groundbreaking mission to study dark matter and dark energy
Costing a total of $488 million to build and launch, the new telescope has been in development for roughly a decade, and is set to map the universe by observing both optical and infrared light. It will orbit Earth 14.5 times a day, completing 11,000 orbits during its lifetime to filter infrared light from distant gas and dust clouds using a technique called spectroscopy.
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Once it is fully online in April, SPHEREX will scan the entire night sky a total of four times using 102 separate infrared color sensors, enabling it to collect data from more than 450 million galaxies during its planned two-year operation. This amounts to roughly 600 exposures a day, according to NASA.
This dataset will give scientists key insights into some of the biggest questions in cosmology, enabling astronomers to study galaxies at various stages in their evolution; trace the ice floating in empty space to see how life may have begun; and even understand the period of rapid inflation the universe underwent immediately after the Big Bang.
—Our entire galaxy is warping, and a gigantic blob of dark matter could be to blame
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SPHEREx's wide panorama view makes it the perfect complement for the James Webb Space Telescope, flagging regions of interest for the latter to study with greater depth and resolution.
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Psilocybin is a serotonergic psychedelic with therapeutic potential for treating mental illnesses1,2,3,4. At the cellular level, psychedelics induce structural neural plasticity5,6, exemplified by the drug-evoked growth and remodelling of dendritic spines in cortical pyramidal cells7,8,9. A key question is how these cellular modifications map onto cell-type-specific circuits to produce the psychedelics' behavioural actions10. Here we use in vivo optical imaging, chemogenetic perturbation and cell-type-specific electrophysiology to investigate the impact of psilocybin on the two main types of pyramidal cells in the mouse medial frontal cortex. We find that a single dose of psilocybin increases the density of dendritic spines in both the subcortical-projecting, pyramidal tract (PT) and intratelencephalic (IT) cell types. Behaviourally, silencing the PT neurons eliminates psilocybin's ability to ameliorate stress-related phenotypes, whereas silencing IT neurons has no detectable effect. In PT neurons only, psilocybin boosts synaptic calcium transients and elevates firing rates acutely after administration. Targeted knockout of 5-HT2A receptors abolishes psilocybin's effects on stress-related behaviour and structural plasticity. Collectively, these results identify that a pyramidal cell type and the 5-HT2A receptor in the medial frontal cortex have essential roles in psilocybin's long-term drug action.
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Details for sample sizes and statistical tests for all experiments are provided in Supplementary Table 1. Data associated with the study are available at GitHub (https://github.com/Kwan-Lab/shaoliao2025). The RNA-seq dataset was obtained from publicly available sources at the Allen Institute (https://doi.org/10.1016/j.cell.2021.04.021). Source data are provided with this paper.
Code for data analysis associated with the study are available at GitHub (https://github.com/Kwan-Lab/shaoliao2025), and from the corresponding author on request.
Goodwin, G. M. et al. Single-dose psilocybin for a treatment-resistant episode of major depression. N. Engl. J. Med. 387, 1637–1648 (2022).
PubMed
MATH
Google Scholar
Davis, A. K. et al. Effects of psilocybin-assisted therapy on major depressive disorder: a randomized clinical trial. JAMA Psychiatry 78, 481–489 (2021).
PubMed
MATH
Google Scholar
Carhart-Harris, R. et al. Trial of psilocybin versus escitalopram for depression. N. Engl. J. Med. 384, 1402–1411 (2021).
PubMed
MATH
Google Scholar
Bogenschutz, M. P. et al. Percentage of heavy drinking days following psilocybin-assisted psychotherapy vs placebo in the treatment of adult patients with alcohol use disorder: a randomized clinical trial. JAMA Psychiatry 79, 953–962 (2022).
PubMed
PubMed Central
MATH
Google Scholar
Ly, C. et al. Psychedelics promote structural and functional neural plasticity. Cell Rep. 23, 3170–3182 (2018).
PubMed
PubMed Central
MATH
Google Scholar
Jones, K. A. et al. Rapid modulation of spine morphology by the 5-HT2A serotonin receptor through kalirin-7 signaling. Proc. Natl Acad. Sci. USA 106, 19575–19580 (2009).
PubMed
PubMed Central
Google Scholar
Shao, L. X. et al. Psilocybin induces rapid and persistent growth of dendritic spines in frontal cortex in vivo. Neuron 109, 2535–2544 (2021).
PubMed
PubMed Central
MATH
Google Scholar
de la Fuente Revenga, M. et al. Prolonged epigenomic and synaptic plasticity alterations following single exposure to a psychedelic in mice. Cell Rep. 37, 109836 (2021).
PubMed
MATH
Google Scholar
Jefferson, S. J. et al. 5-MeO-DMT modifies innate behaviors and promotes structural neural plasticity in mice. Neuropsychopharmacology 48, 1257–1266 (2023).
PubMed
PubMed Central
MATH
Google Scholar
Kwan, A. C., Olson, D. E., Preller, K. H. & Roth, B. L. The neural basis of psychedelic action. Nat. Neurosci. 25, 1407–1419 (2022).
PubMed
PubMed Central
Google Scholar
Duman, R. S. & Aghajanian, G. K. Synaptic dysfunction in depression: potential therapeutic targets. Science 338, 68–72 (2012).
PubMed
PubMed Central
Google Scholar
Cameron, L. P. et al. A non-hallucinogenic psychedelic analogue with therapeutic potential. Nature 589, 474–479 (2021).
PubMed
MATH
Google Scholar
Lu, J. et al. An analog of psychedelics restores functional neural circuits disrupted by unpredictable stress. Mol. Psychiatry 26, 6237–6252 (2021).
PubMed
PubMed Central
MATH
Google Scholar
Anastasiades, P. G. & Carter, A. G. Circuit organization of the rodent medial prefrontal cortex. Trends Neurosci. 44, 550–563 (2021).
PubMed
PubMed Central
MATH
Google Scholar
Baker, A. et al. Specialized subpopulations of deep-layer pyramidal neurons in the neocortex: bridging cellular properties to functional consequences. J. Neurosci. 38, 5441–5455 (2018).
PubMed
PubMed Central
MATH
Google Scholar
Shepherd, G. M. Corticostriatal connectivity and its role in disease. Nat. Rev. Neurosci. 14, 278–291 (2013).
PubMed
PubMed Central
MATH
Google Scholar
Li, N., Chen, T. W., Guo, Z. V., Gerfen, C. R. & Svoboda, K. A motor cortex circuit for motor planning and movement. Nature 519, 51–56 (2015).
PubMed
Google Scholar
Musall, S. et al. Pyramidal cell types drive functionally distinct cortical activity patterns during decision-making. Nat. Neurosci. 26, 495–505 (2023).
PubMed
PubMed Central
Google Scholar
Tang, L. & Higley, M. J. Layer 5 circuits in V1 differentially control visuomotor behavior. Neuron 105, 346–354 (2020).
PubMed
MATH
Google Scholar
Garcia, A. F., Crummy, E. A., Webb, I. G., Nooney, M. N. & Ferguson, S. M. Distinct populations of cortical pyramidal neurons mediate drug reward and aversion. Nat. Commun. 12, 182 (2021).
PubMed
PubMed Central
Google Scholar
Davies, M. F., Deisz, R. A., Prince, D. A. & Peroutka, S. J. Two distinct effects of 5-hydroxytryptamine on single cortical neurons. Brain Res. 423, 347–352 (1987).
PubMed
Google Scholar
Araneda, R. & Andrade, R. 5-Hydroxytryptamine2 and 5-hydroxytryptamine1A receptors mediate opposing responses on membrane excitability in rat association cortex. Neuroscience 40, 199–412 (1991).
Google Scholar
Avesar, D. & Gulledge, A. T. Selective serotonergic excitation of callosal projection neurons. Front. Neural Circuits 6, 12 (2012).
PubMed
PubMed Central
Google Scholar
Elliott, M. C., Tanaka, P. M., Schwark, R. W. & Andrade, R. Serotonin differentially regulates L5 pyramidal cell classes of the medial prefrontal cortex in rats and mice. eNeuro https://doi.org/10.1523/ENEURO.0305-17.2018 (2018).
Amargos-Bosch, M. et al. Co-expression and in vivo interaction of serotonin1A and serotonin2A receptors in pyramidal neurons of prefrontal cortex. Cereb. Cortex 14, 281–299 (2004).
PubMed
Google Scholar
Savalia, N. K., Shao, L. X. & Kwan, A. C. A dendrite-focused framework for understanding the actions of ketamine and psychedelics. Trends Neurosci. 44, 260–275 (2021).
PubMed
Google Scholar
Puig, M. V., Celada, P., Diaz-Mataix, L. & Artigas, F. In vivo modulation of the activity of pyramidal neurons in the rat medial prefrontal cortex by 5-HT2A receptors: relationship to thalamocortical afferents. Cereb. Cortex 13, 870–882 (2003).
PubMed
Google Scholar
Kim, Y. et al. Whole-brain mapping of neuronal activity in the learned helplessness model of depression. Front. Neural Circuits 10, 3 (2016).
PubMed
PubMed Central
MATH
Google Scholar
Davoudian, P. A., Shao, L. X. & Kwan, A. C. Shared and distinct brain regions targeted for immediate early gene expression by ketamine and psilocybin. ACS Chem. Neurosci. 14, 468–480 (2023).
PubMed
Google Scholar
Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S. & Roth, B. L. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc. Natl Acad. Sci. USA 104, 5163–5168 (2007).
PubMed
PubMed Central
Google Scholar
Matho, K. S. et al. Genetic dissection of the glutamatergic neuron system in cerebral cortex. Nature 598, 182–187 (2021).
PubMed
PubMed Central
Google Scholar
Nagai, Y. et al. Deschloroclozapine, a potent and selective chemogenetic actuator enables rapid neuronal and behavioral modulations in mice and monkeys. Nat. Neurosci. 23, 1157–1167 (2020).
PubMed
Google Scholar
Halberstadt, A. L., Chatha, M., Klein, A. K., Wallach, J. & Brandt, S. D. Correlation between the potency of hallucinogens in the mouse head-twitch response assay and their behavioral and subjective effects in other species. Neuropharmacology 167, 107933 (2020).
PubMed
PubMed Central
Google Scholar
Malenka, R. C., Lancaster, B. & Zucker, R. S. Temporal limits on the rise in postsynaptic calcium required for the induction of long-term potentiation. Neuron 9, 121–128 (1992).
PubMed
MATH
Google Scholar
Bittner, K. C., Milstein, A. D., Grienberger, C., Romani, S. & Magee, J. C. Behavioral time scale synaptic plasticity underlies CA1 place fields. Science 357, 1033–1036 (2017).
PubMed
PubMed Central
Google Scholar
Lee, S. J., Escobedo-Lozoya, Y., Szatmari, E. M. & Yasuda, R. Activation of CaMKII in single dendritic spines during long-term potentiation. Nature 458, 299–304 (2009).
PubMed
PubMed Central
Google Scholar
Friedrich, J., Zhou, P. & Paninski, L. Fast online deconvolution of calcium imaging data. PLoS Comput. Biol. 13, e1005423 (2017).
PubMed
PubMed Central
MATH
Google Scholar
Ali, F. et al. Ketamine disinhibits dendrites and enhances calcium signals in prefrontal dendritic spines. Nat. Commun. 11, 72 (2020).
PubMed
PubMed Central
Google Scholar
Chen, T. W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).
PubMed
PubMed Central
MATH
Google Scholar
DeFelipe, J., Arellano, J. I., Gomez, A., Azmitia, E. C. & Munoz, A. Pyramidal cell axons show a local specialization for GABA and 5-HT inputs in monkey and human cerebral cortex. J. Comp. Neurol. 433, 148–155 (2001).
PubMed
Google Scholar
Jun, J. J. et al. Fully integrated silicon probes for high-density recording of neural activity. Nature 551, 232–236 (2017).
PubMed
PubMed Central
MATH
Google Scholar
Cameron, L. P. et al. 5-HT2ARs mediate therapeutic behavioral effects of psychedelic tryptamines. ACS Chem. Neurosci. 14, 351–358 (2023).
PubMed
MATH
Google Scholar
Hesselgrave, N., Troppoli, T. A., Wulff, A. B., Cole, A. B. & Thompson, S. M. Harnessing psilocybin: antidepressant-like behavioral and synaptic actions of psilocybin are independent of 5-HT2R activation in mice. Proc. Natl Acad. Sci. USA 118, e2022489118 (2021).
PubMed
PubMed Central
Google Scholar
Sekssaoui, M., Bockaert, J., Marin, P. & Bécamel, C. Antidepressant-like effects of psychedelics in a chronic despair mouse model: is the 5-HT2A receptor the unique player? Neuropsychopharmacology 49, 747–756 (2024).
PubMed
PubMed Central
Google Scholar
Choi, W. et al. Serotonin signals through a gut-liver axis to regulate hepatic steatosis. Nat. Commun. 9, 4824 (2018).
PubMed
PubMed Central
MATH
Google Scholar
Yao, Z. et al. A taxonomy of transcriptomic cell types across the isocortex and hippocampal formation. Cell 184, 3222–3241 (2021).
PubMed
PubMed Central
MATH
Google Scholar
Aghajanian, G. K. & Marek, G. J. Serotonin induces excitatory postsynaptic potentials in apical dendrites of neocortical pyramidal cells. Neuropharmacology 36, 589–599 (1997).
PubMed
MATH
Google Scholar
Kaplan, A. L. et al. Bespoke library docking for 5-HT2A receptor agonists with antidepressant activity. Nature 610, 582–591 (2022).
PubMed
PubMed Central
Google Scholar
Cao, D. et al. Structure-based discovery of nonhallucinogenic psychedelic analogs. Science 375, 403–411 (2022).
PubMed
MATH
Google Scholar
Dembrow, N. C., Zemelman, B. V. & Johnston, D. Temporal dynamics of L5 dendrites in medial prefrontal cortex regulate integration versus coincidence detection of afferent inputs. J. Neurosci. 35, 4501–4514 (2015).
PubMed
PubMed Central
MATH
Google Scholar
Anastasiades, P. G., Collins, D. P. & Carter, A. G. Mediodorsal and ventromedial thalamus engage distinct L1 circuits in the prefrontal cortex. Neuron 109, 314–330 (2021).
PubMed
Google Scholar
Silberberg, G. & Markram, H. Disynaptic inhibition between neocortical pyramidal cells mediated by Martinotti cells. Neuron 53, 735–746 (2007).
PubMed
Google Scholar
Wu, S. J. et al. Cortical somatostatin interneuron subtypes form cell-type-specific circuits. Neuron 111, 2675–2692 (2023).
PubMed
PubMed Central
MATH
Google Scholar
Suzuki, M. & Larkum, M. E. General anesthesia decouples cortical pyramidal neurons. Cell 180, 666–676 (2020).
PubMed
MATH
Google Scholar
Bharioke, A. et al. General anesthesia globally synchronizes activity selectively in layer 5 cortical pyramidal neurons. Neuron 110, 2024–2040 (2022).
PubMed
PubMed Central
MATH
Google Scholar
Wilkinson, S. T., Holtzheimer, P. E., Gao, S., Kirwin, D. S. & Price, R. B. Leveraging neuroplasticity to enhance adaptive learning: the potential for synergistic somatic-behavioral treatment combinations to improve clinical outcomes in depression. Biol. Psychiatry 85, 454–465 (2019).
PubMed
Google Scholar
Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).
PubMed
MATH
Google Scholar
Tervo, D. G. et al. A designer AAV variant permits efficient retrograde access to projection. Neuron 92, 372–382 (2016).
PubMed
PubMed Central
MATH
Google Scholar
Shamash, P., Carandini, M., Harris, K. D. & Steinmetz, N. A. A tool for analyzing electrode tracks from slice histology. Preprint at bioRxiv https://doi.org/10.1101/447995 (2018).
Wang, Q. et al. The Allen Mouse Brain Common Coordinate Framework: a 3D reference atlas. Cell 181, 936–953 (2020).
PubMed
PubMed Central
MATH
Google Scholar
Claudi, F. et al. Visualizing anatomically registered data with brainrender. eLife 10, e65751 (2021).
PubMed
PubMed Central
MATH
Google Scholar
Pologruto, T. A., Sabatini, B. L. & Svoboda, K. ScanImage: flexible software for operating laser scanning microscopes. Biomed. Eng. Online 2, 13 (2003).
PubMed
PubMed Central
Google Scholar
Thevenaz, P., Ruttimann, U. E. & Unser, M. A pyramid approach to subpixel registration based on intensity. IEEE Trans. Image Process. 7, 27–41 (1998).
PubMed
MATH
Google Scholar
Holtmaat, A. et al. Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window. Nat. Protoc. 4, 1128–1144 (2009).
PubMed
PubMed Central
MATH
Google Scholar
Mitrić, M. et al. Layer- and subregion-specific electrophysiological and morphological changes of the medial prefrontal cortex in a mouse model of neuropathic pain. Sci. Rep. 9, 9479 (2019).
PubMed
PubMed Central
MATH
Google Scholar
Radnikow, G. & Feldmeyer, D. Layer- and cell type-specific modulation of excitatory neuronal activity in the neocortex. Front. Neuroanat. 12, 1 (2018).
PubMed
PubMed Central
Google Scholar
Pnevmatikakis, E. A. & Giovannucci, A. NoRMCorre: an online algorithm for piecewise rigid motion correction of calcium imaging data. J. Neurosci. Methods 291, 83–94 (2017).
PubMed
Google Scholar
Ali, F. et al. Inhibitory regulation of calcium transients in prefrontal dendritic spines is compromised by a nonsense Shank3 mutation. Mol. Psychiatry 26, 1945–1966 (2021).
PubMed
MATH
Google Scholar
Lutcke, H., Gerhard, F., Zenke, F., Gerstner, W. & Helmchen, F. Inference of neuronal network spike dynamics and topology from calcium imaging data. Front. Neural Circuits 7, 201 (2013).
PubMed
PubMed Central
Google Scholar
Friedman, A. et al. A corticostriatal path targeting striosomes controls decision-making under conflict. Cell 161, 1320–1333 (2015).
PubMed
PubMed Central
MATH
Google Scholar
Boudreau, E. et al. Intraperitoneal catheter placement for pharmacological imaging studies in conscious mice. Lab. Anim. 39, 23–25 (2010).
MATH
Google Scholar
Siegle, J. H. et al. Open Ephys: an open-source, plugin-based platform for multichannel electrophysiology. J. Neural Eng. 14, 045003 (2017).
PubMed
MATH
Google Scholar
Buccino, A. P. et al. SpikeInterface, a unified framework for spike sorting. eLife 9, e61834 (2020).
PubMed
PubMed Central
Google Scholar
Pachitariu, M. et al. Kilosort: realtime spike-sorting for extracellular electrophysiology with hundreds of channels. Preprint at bioRxiv https://doi.org/10.1101/061481 (2016).
Liu, R. J. & Aghajanian, G. K. Stress blunts serotonin- and hypocretin-evoked EPSCs in prefrontal cortex: role of corticosterone-mediated apical dendritic atrophy. Proc. Natl Acad. Sci. USA 105, 359–364 (2008).
PubMed
PubMed Central
Google Scholar
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We thank L. Sun for help with analysing the slice electrophysiology data. Psilocybin was provided by Usona Institute's Investigational Drug & Material Supply Program. The Usona Institute IDMSP is supported by A. Sherwood, R. Kargbo and K. Kaylo. This work was supported by NIH grants R01MH121848, R01MH128217, R01MH137047, U01NS128660, One Mind–COMPASS Rising Star Award (A.C.K.); NIH training grants T32GM007205 (P.A.D. and N.K.S.), T32NS041228 (C.L.); NIH fellowships F30DA059437 (P.A.D.) and F30MH129085 (N.K.S.); Source Research Foundation student grant (P.A.D.); NIH instrumentation grants S10RR025502 and S10OD032251 (Cornell Biotechnology Resource Center Imaging Facility); NIH grants R00NS114166, R01NS133434 and R01DA059378 (A.C.); State of Connecticut, Department of Mental Health and Addiction Services (A.C. and R.-J.L.).
These authors contributed equally: Ling-Xiao Shao, Clara Liao
Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
Ling-Xiao Shao, Clara Liao, Pasha A. Davoudian, Neil K. Savalia, Quan Jiang, Cassandra Wojtasiewicz, Diran Tan, Jack D. Nothnagel, Samuel C. Woodburn, Olesia M. Bilash & Alex C. Kwan
Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA
Ling-Xiao Shao, Rong-Jian Liu, Alicia Che & Alex C. Kwan
Interdepartmental Neuroscience Program, Yale University School of Medicine, New Haven, CT, USA
Clara Liao, Pasha A. Davoudian & Neil K. Savalia
Medical Scientist Training Program, Yale University School of Medicine, New Haven, CT, USA
Pasha A. Davoudian & Neil K. Savalia
Graduate School of Medical Science and Engineering, KAIST, Daejeon, Republic of Korea
Hail Kim
Department of Psychiatry, Weill Cornell Medicine, New York, NY, USA
Alex C. Kwan
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L.-X.S., C.L. and A.C.K. planned the study. L.-X.S. and C.L. conducted and analysed the imaging and behavioural experiments. P.A.D. and Q.J. conducted and analysed the electrophysiological experiments. N.K.S. and O.M.B. analysed the dendritic calcium imaging data. R.-J.L. and A.C. conducted slice electrophysiology experiments. Q.J. assisted in animal surgery. Q.J., D.T., C.W. and J.D.N. assisted in behavioural experiments and histology. S.C.W. and C.W. conducted pilot studies to validate the protocols for the behavioural assays. H.K. generated and provided the Htr2af/f mice. L.-X.S., C.L. and A.C.K. drafted the manuscript. All of the authors reviewed the manuscript before submission.
Correspondence to
Alex C. Kwan.
A.C.K. has been a scientific advisor or consultant for Boehringer Ingelheim, Empyrean Neuroscience, Freedom Biosciences and Xylo Bio. A.C.K. has received research support from Intra-Cellular Therapies. The other authors declare no competing interests.
Nature thanks Adema Ribic, Bryan Roth and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
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a, To express GFP in IT neurons, we injected AAV-CAG-FLEX-eGFP in the ACAd and medial MOs portion of medial frontal cortex and low titre of the retrogradely transported AAVretro-hSyn-Cre in the contralateral striatum of adult C57BL/6J mice. Post hoc histology and imaging of the GFP fluorescence in coronal sections shows ipsilateral and contralateral projections to various striatal and cortical regions. CP, caudoputamen. b, To express GFP in PT neurons, we injected AAV-CAG-FLEX-eGFP in the ACAd and medial MOs portion of medial frontal cortex and low titre of the retrogradely transported AAVretro-hSyn-Cre in the ipsilateral pons of adult C57BL/6J mice. Post hoc histology and imaging of the GFP fluorescence in coronal sections shows ipsilateral projections to striatum and subcortical regions including the pons (lower rightmost image). c, In vivo two-photon images of apical dendrites from PT and IT neurons targeted to express GFP using retrogradely transported viruses. d, Baseline spine density for all imaged dendrites on Day -3 prior to any psilocybin or saline administration. PT neurons have lower spine density than IT neurons (P < 0.001, two-sample t-test). Yellow, PT neurons. Purple, IT neurons. PT: n = 160 branches from 17 mice. IT: n = 142 branches from 16 mice. e, Similar to (d) for spine head width. PT: n = 1071 spines from 6 mice. IT: n = 615 spines from 5 mice. PT neurons have larger spine head width than IT neurons (P < 0.001, two-sample t-test). ***, p < 0.001. Detailed sample size n values are provided in Methods. Statistical analyses are provided in Supplementary Table 1.
Source Data
a, b, Potential differential effect of psilocybin on dendritic spine head width in frontal cortical PT and IT neurons. Spine head width in the apical tuft of PT neurons (a) after psilocybin (yellow; 1 mg/kg, i.p.) or saline (grey) across days, expressed as fold-change from baseline in first imaging session (day -3). b, Similar to (a) for IT neurons after psilocybin (purple) or saline (light purple). There was cell-type difference in psilocybin's effect on spine head width (interaction effect of treatment × time × cell type: P = 0.007, mixed effects model). These results show that PT neurons have a more pronounced increase in spine head width than IT neurons, indicative of a strengthening of excitatory connections in addition to gaining new inputs for PT neurons. This enlargement in spine head for PT neurons was less durable than the changes in spine density, returning to baseline after 35 days. We note the results should be interpreted with the consideration that the spine head width is near the spatial resolution limit for in vivo two-photon microscopy. c, Density of dendritic spines in the apical tuft of PT neurons after psilocybin (yellow; 1 mg/kg, i.p.) or saline (grey) across days. d-f, Similar to (c) for spine head width, formation rate, and elimination rate. g-j, Similar to (c-f) for IT neurons. These figure panels correspond to Fig. 1i–n and panel (a,b in this figure), except here across-dendrite values are shown, without taking advantage of the longitudinal data for within-dendrite baseline normalization. n = 8 mice (PT neurons, saline), n = 9 mice (PT neurons, psilocybin) in (a, c-f). n = 8 mice (IT neurons, saline), n = 8 mice (IT neurons, psilocybin) in (b, g-j). k-r, Psilocybin effects on structural plasticity in PT and IT neurons by sex of the animals. k, Density of dendritic spines in apical tuft of PT neurons in female (top row) and male mice (bottom row) after psilocybin (1 mg/kg, i.p.) or saline across days, expressed as fold-change from baseline in first imaging session (day -3). l, Similar to (k) for spine head width. m, Spine formation rate determined by number of new and existing spines in consecutive imaging sessions across two-day interval, expressed as difference from baseline in first interval (day -3 to day -1). n, Similar to (m) for elimination rate. n = 4 mice (PT neurons, saline, female), n = 4 (PT neurons, psilocybin, female), n = 4 (PT neurons, saline, male), n = 5 (PT neurons, psilocybin, male) in (k-n). o-r, Similar to (k-n) for IT neurons in female (top row) and male mice (bottom row). n = 5 mice (IT neurons, saline, female), n = 5 (IT neurons, psilocybin, female), n = 3 (IT neurons, saline, male), n = 3 (IT neurons, psilocybin, male). We did not detect effect of sex for any of the measures (interaction effect of treatment × sex × cell type, indicated in plots, mixed effects model). s-v, Psilocybin has no effect on spine protrusion length. s, Protrusion length of dendritic spines in apical tuft of PT neurons for all mice (left), or separately plotted for females (middle) and males (right), after psilocybin (1 mg/kg, i.p.) or saline across days, expressed as fold-change from baseline in first imaging session (day -3). t, Similar to (s) for IT neurons. Psilocybin had no detectable effect on spine protrusion length (main effect of treatment: P = 0.309, mixed effects model). u, Protrusion length of dendritic spines in apical tuft of PT neurons for all mice after psilocybin (1 mg/kg, i.p.) or saline across days, without taking advantage of the longitudinal data for within-dendrite baseline normalization. v, Similar to (u) for IT neurons. n = 8 mice (PT neurons, saline, 4 females, 4 males), n = 9 (PT neurons, psilocybin, 4 females, 5 males) in (s, u). n = 8 (IT neurons, saline, 5 females, 3 males), n = 8 (IT neurons, psilocybin, 5 females, 3 males) in (t, v). Data are mean and s.e.m. across dendrites. *, p < 0.05. ***, p < 0.001, post hoc with Bonferroni correction for multiple comparisons. Detailed sample size n values are provided in Methods. Statistical analyses are provided in Supplementary Table 1.
Source Data
a, Volumetric reconstruction from z-stack images of GFP-expressing IT neurons. IT neurons can be divided into two groups based on the laminar position of their cell body (200–400 μm or 450–600 μm). For the deep-lying IT neurons, sometimes the cell body could not be imaged due to the depth limitation of two-photon microscopy, but nonetheless the apical trunk was observed at >450 μm. b, Density of dendritic spines (left), spine head width (middle), and spine protrusion length (right) in apical tuft of IT neurons residing in layer 2/3 (top row) or layer 5 (bottom row) after psilocybin (1 mg/kg, i.p.) or saline across days, expressed as fold-change from baseline in first imaging session (day -3). The same mice were used for both depth ranges (200–400 μm and 450–600 μm). c, Left: spine formation rate determined by number of new and existing spines in consecutive imaging sessions across two-day interval, expressed as difference from baseline in first interval (day -3 to day -1). Right: similar to left for elimination rate. d-e, Similar to (b) for density of dendritic spines and spine head width but further divided the data based on the sex of the animal. n = 8 mice (IT neurons, saline, 5 females, 3 males), n = 8 (IT neurons, psilocybin, 5 females, 3 males) in (b-e). The analysis was motivated by the question: is psilocybin-evoked increase in spine size specific to cell type (IT versus PT), or specific to laminar position (layer 2/3 versus layer 5)? This is because IT neurons can be both superficial and deep, but PT is only found in deep layer. We detected no significant depth dependence for spine size for layer 2/3 and deep layer 5 IT neurons (interaction effect of treatment × soma depth, indicated in plots, mixed effects model). Data are mean and s.e.m. across dendrites. Detailed sample size n values are provided in Methods. Statistical analyses are provided in Supplementary Table 1.
Source Data
a, For head-twitch response assessed 10 min after drug administration, effect of chemogenetic inactivation in male (left) and female (right) Fezf2-creER mice during psilocybin (1 mg/kg, i.p.) or saline administration. Circle, individual animal. n = 14 (mCherry_psilocybin, 6 males and 8 females), n = 13 (mCherry_saline, 6 males and 7 females), n = 20 (hM4DGi_psilocybin, 12 males and 8 females), and n = 15 (hM4DGi_saline, 7 males and 8 females). b, Similar to (a) for Plxnd1-creER mice. n = 15 (mCherry_psilocybin, 8 males and 7 females), n = 12 (mCherry_saline, 6 males and 6 females), n = 11 (hM4DGi_psilocybin, 6 males and 5 females), and n = 13 (hM4DGi_saline, 7 males and 6 females). c-d, Similar to (a-b) for learned helplessness assessed 24 h after drug administration. For both male and female mice, there were significant effect of PT inactivation interfering with psilocybin's impact on learned helplessness (interaction effect of treatment and DREADD: P = 0.001 for males and P = 0.009 for females, two-factor ANOVA). Fezf2-creER mice: n = 13 (mCherry_psilocybin, 8 males and 5 females), n = 15 (mCherry_saline, 8 males and 7 females), n = 16 (hM4DGi_psilocybin, 9 males and 7 females), and n = 13 (hM4DGi_saline, 5 males and 8 females). Plxnd1-creER mice: n = 14 (mCherry_psilocybin, 7 males and 7 females), n = 11 (mCherry_saline, 5 males and 6 females), n = 11 (hM4DGi_psilocybin, 6 males and 5 females), and n = 11 (hM4DGi_saline, 5 males and 6 females). e-f, Similar to (a-b) for tail suspension test assessed 24 h after drug administration. For male mice, there was significant effect of PT inactivation interfering with psilocybin's impact on tail suspension (interaction effect of treatment and DREADD: P = 0.002 for males and P = 0.08 for females, two-factor ANOVA). Fezf2-creER mice: n = 14 (mCherry_psilocybin, 7 males and 7 females), n = 10 (mCherry_saline, 5 males and 5 females), n = 13 (hM4DGi_psilocybin, 6 males and 7 females), and n = 12 (hM4DGi_saline, 8 males and 4 females). Plxnd1-creER mice: n = 14 (mCherry_psilocybin, 7 males and 7 females), n = 9 (mCherry_saline, 5 males and 4 females), n = 9 (hM4DGi_psilocybin, 5 males and 4 females), and n = 9 (hM4DGi_saline, 5 males and 4 females). *, p < 0.05. ** p < 0.01. ***, p < 0.001, post hoc with Bonferroni correction for multiple comparisons. Data are mean and s.e.m. across mice. Detailed sample size n values are provided in Methods. Statistical analyses are provided in Supplementary Table 1.
Source Data
a, Chronic stress-induced resistance to fear extinction. b, Time spent freezing during conditioning. c, Time spent freezing during extinction session. Chemogenetic inactivation of frontal cortical PT neurons significantly diminished psilocybin's facilitating effect (interaction effect of treatment × DREADD: P = 0.003, two-factor ANOVA). d-e, Similar to (c) for extinction retention sessions. n = 13 (mCherry, saline), n = 17 (mCherry, psilocybin), n = 15 (hM4DGi, saline), n = 13 (hM4DGi, psilocybin) in (b-d). n = 13 (mCherry, saline), n = 16 (mCherry, psilocybin), n = 15 (hM4DGi, saline), n = 13 (hM4DGi, psilocybin) in (e). Data are mean and s.e.m. across mice. *, p < 0.05. ** p < 0.01. ***, p < 0.001, post hoc with Bonferroni correction for multiple comparisons. Detailed sample size n values are provided in Methods. Statistical analyses are provided in Supplementary Table 1.
Source Data
a, An example in vivo two-photon image of GCaMP6f-expressing apical dendrite and spines from a PT neuron. Dashed lines, the dendritic branch. Arrows, spines attached to the branch. b, Top, ΔF/F0 trace of a PT dendritic branch (ΔF/Fbranch). Middle, ΔF/F0 trace recorded from a dendritic spine attached to that branch (ΔF/Fspine). Bottom, the branch-subtracted spine calcium signal (ΔF/Fsynaptic), subtracting the scaled version of ΔF/Fbranch signal from the ΔF/Fspine signals. The synaptic calcium signals of dendritic spines shown and analysed in the paper are branch-subtracted ΔF/Fspine transients. Note that negative values can appear due to the subtraction procedure; however, negative transients are not detected by the peeling algorithm and therefore do not impact the subsequent analyses. Left, ΔF/F0 signals before psilocybin. Right, ΔF/F0 signals after psilocybin. c, Similar to (b) for ΔF/F0 traces in an IT neuron. d, Left, ΔF/F0 from two different dendritic branches of PT neurons before and after saline. Right, ΔF/F0 from two different dendritic branches of PT neurons, before and after psilocybin (1 mg/kg, i.p.). e, Similar to (d) for dendritic branches of IT neurons. f-g, Similar to (d-e) for dendritic spines.
a, Schematic illustrating how fluorescent transients are processed and analysed to derive event rate, amplitude, and frequency. b, Fractional change in frequency detected in dendritic branches of PT neurons (top row) and dendritic branches of IT neurons (bottom row) after psilocybin (PT, yellow; IT, purple) or saline (PT, grey; IT, light purple). c, Similar to (b) for amplitude. d, e, Similar to (b, c) for dendritic spines. PT: n = 140 branches from 4 mice (Saline), n = 149 branches from 4 mice (Psilocybin). IT: n = 90 branches from 3 mice (Saline), n = 95 branches from 3 mice (Psilocybin). ** p < 0.01. ***, p < 0.001, post hoc with Bonferroni correction for multiple comparisons. Detailed sample size n values are provided in Methods. Statistical analyses are provided in Supplementary Table 1.
Source Data
a, The three quality metrics, including the empirical distribution of recorded units and thresholds used for curation of single units to include for the analysis of the electrophysiology data. b, Mean spike waveform features for all opto-tagged neurons and other untagged cells in Fezf2-2A-creER and Plxnd1-2A-creER mice. There is no single feature of the spike waveform that can reliably classify the two types of opto-tagged neurons or from the untagged cells. c-g, More examples of opto-tagged neurons and untagged neurons. c, d, Spike raster of neurons classified as opto-tagged in Fezf2-2A-creER (left) and Plxnd1-2A-creER (right) mice. e-g, Spike raster of neurons classified as untagged in Fezf2-2A-creER (left) and Plxnd1-2A-creER (right) mice.
Source Data
a, Example sEPSC traces from 5 GFP+ layer 5 pyramidal neurons, with each set of 3 traces coming from recording of the same cell, including baseline (black), after bath application of 20 μM 5-HT (red), and after bath application of 20 μM 5-HT with 100 nM MDL100,907 (grey). Cells 1 and 2 are from control animals. Cells 3, 4, and 5 are animals with local 5-HT2A receptor knockout. b, Mean sEPSC amplitude from GFP+ layer 5 pyramidal neurons for baseline, 20 μM 5-HT, and 20 μM 5-HT + 100 nM MDL100,907 conditions, for control mice (grey) and local 5-HT2A receptor knockout mice (blue). Circle, individual cell. Mean and s.e.m. across cells. n = 22 cells (Baseline), n = 22 cells (+5-HT), n = 6 cells (+5-HT + MDL) from 4 mice in the Control condition, n = 23 cells (Baseline), n = 23 cells (+5-HT), n = 7 cells (+5-HT + MDL) from 4 mice in the 5-HT2AR local KO condition. c-e, Constitutive Camk2acre;Htr2af/f mice had reduced Htr2a transcripts and fewer psilocybin-induced head-twitch response. c, Breeding scheme to generate Camk2acre;Htr2af/f mice. d, Transcript expression via qPCR from whole-brain tissue. n = 2 (Control), n = 3 (Camk2acre;Htr2af/f). e, Head-twitch response induced by psilocybin (1 mg/kg, i.p.). Mean and s.e.m. across mice. n = 11 (Control), n = 12 (Camk2acre;Htr2af/f). ** p < 0.01. Detailed sample size n values are provided in Methods. Statistical analyses are provided in Supplementary Table 1.
Source Data
a, HTR2A antibody staining shows colocalization of 5-HT2A receptors and GFP-expressing cell bodies and neurites in the medial frontal cortex of a Thy1GFP line M mouse. b, To image dendrites from neurons without 5-HT2A receptors, we injected a low titre of AAV-hSyn-Cre-P2A-dTomato into the medial frontal cortex of Thy1GFP; Htr2af/f mouse. The subset of neurons with viral-mediated transgene expression would have Cre recombinase for knockout of 5-HT2A receptors and have dTomato for identification. Only dendrites that expressed both dTomato and GFP were scored. Control animals were Thy1GFP; Htr2a+/+. c, Density of dendritic spines in the apical tuft of dTomato- and GFP-expressing neurons after psilocybin (green) or saline (grey) in Thy1GFP; Htr2af/f mice or after psilocybin in Thy1GFP; Htr2a+/+ mice (orange), expressed as fold-change from baseline in first imaging session (day -3). n = 38 dendrites from 2 mice (Thy1GFP; Htr2a+/+, psilocybin), n = 49 dendrites from 5 mice (Thy1GFP; Htr2af/f, psilocybin), n = 98 dendrites from 3 mice (Thy1GFP; Htr2af/f, saline). d, HTR2A antibody staining shows colocalization of 5-HT2A receptors and PT neurons targeted to express GFP using retrogradely transported virus in a C57BL/6J mouse (left). For PT neuron-targeted 5-HT2A receptor knockout, 5-HT2A receptors were absent in PT neurons targeted using retrogradely transported virus in a Htr2af/f mouse (right). e, From two-photon microscopy, spine head width across days, expressed as fold-change from baseline in first imaging session (day -3), in wild type mice after saline (light grey) or psilocybin (yellow) and in mice with PT neuron-targeted 5-HT2A receptor knockout after saline (grey) or psilocybin (blue). Post hoc test compared WT:saline and WT:psilocybin groups. f, Similar to (e) for spine protrusion length. g, Density of dendritic spines, spine head width, and spine protrusion length in the apical tuft of PT neurons across days, in wild type mice after saline (light grey) or psilocybin (yellow) and in mice with PT neuron-targeted 5-HT2A receptor knockout after saline (grey) or psilocybin (blue), without taking advantage of the longitudinal data for within-dendrite baseline normalization. h, Similar to (g) for formation rate and elimination rate. n = 7 mice (WT, saline), n = 5 (WT, psilocybin), n = 5 (PT_5-HT2AR KO, saline), n = 6 (PT_5-HT2AR KO, psilocybin) in (e-h). i, Spine head width on day 1 plotted separately for pre-existing versus newly formed spines in different conditions. n = 7 mice (WT, saline), n = 5 (WT, psilocybin), n = 4 (PT_5-HT2AR KO, saline), n = 5 (PT_5-HT2AR KO, psilocybin). j, From confocal microscopy, spine head width in the apical tuft of PT neurons in wild type mice after saline (light grey) or psilocybin (yellow) and in mice with PT neuron-targeted 5-HT2A receptor knockout after saline (grey) or psilocybin (blue, interaction effect of treatment × genotype: P = 0.025, two-factor ANOVA). Circle, individual dendritic segment. n = 7 mice (WT, saline), n = 5 (WT, psilocybin), n = 3 (PT_5-HT2AR KO, saline), n = 5 (PT_5-HT2AR KO, psilocybin). k, Similar to (j) protrusion length of dendritic spines. n = same as in (j). Data are mean and s.e.m. across dendrites. * p < 0.05. **, p < 0.01. ***, p < 0.001, post hoc with Bonferroni correction for multiple comparisons. Detailed sample size n values are provided in Methods. Statistical analyses are provided in Supplementary Table 1.
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Detailed statistics for all data. This table provides comprehensive statistical details for all datasets reported in the Article, including statistical analyses, P values, error estimates and sample sizes.
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Shao, LX., Liao, C., Davoudian, P.A. et al. Psilocybin's lasting action requires pyramidal cell types and 5-HT2A receptors.
Nature (2025). https://doi.org/10.1038/s41586-025-08813-6
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A close look at air flow around high-speed shapes reveals surprising turbulence, according to a new study. The findings, published March 7 in the journal Physical Review Fluids, could inform the design of future high-speed vehicles.
In the study, researchers used three-dimensional simulations to reveal unexpected disturbances around fast-moving cones.
At hypersonic speeds — above Mach 5, or more than 5 times the speed of sound (3,836 mph or 6,174 kilometers per hour) — the flow of air around a vehicle's surface becomes complex and bumpy. Most simulations assume that the flow is symmetrical around the whole cone, but until recently, studies of the transition from streamlined to turbulent were only possible in two dimensions so we couldn't be sure that there weren't any asymmetries in flow around a three-dimensional structure.
The findings could help engineers design stronger, faster vehicles able to withstand the extreme temperatures, pressures and vibrations felt during hypersonic flight.
"Transitioning flows are 3D and unsteady in nature, regardless of the flow geometry," study co-author Irmak Taylan Karpuzcu, an aerospace engineer at the University of Illinois Urbana-Champaign, said in a statement. "Experiments were conducted in 3D in the early 2000s [but they] didn't provide enough data to determine any 3D effects or unsteadiness because there weren't enough sensors all around the cone-shaped model. It wasn't wrong. It was just all that was possible then."
Using the Frontera supercomputer at the Texas Advanced Computing Center, Karpuzcu and aerospace engineer Deborah Levin simulated how air flow around a cone-shaped object — often used as a simplified model for hypersonic vehicles — changes in three dimensions at high speed. They studied both a single cone and a double cone, which helps scientists study how multiple shock waves interact with each other.
"Normally, you would expect the flow around the cone to be concentric ribbons, but we noticed breaks in the flow within shock layers both in the single and double cone shapes," Karpuzcu said.
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These breaks were particularly prevalent around the tip of the cone. At high speeds, the shock wave lies closer to the cone, squeezing air molecules into unstable layers and amplifying instabilities in the airflow. The team confirmed their findings by running a program that tracks each simulated air molecule and captures how collisions between the molecules affect air flow.
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The disturbances also seem to develop at high speeds. "As you increase the Mach number, the shock gets closer to the surface and promotes these instabilities. It would be too expensive to run the simulation at every speed, but we did run it at Mach 6 and did not see a break in the flow," Karpuzcu said.
The breaks could affect design considerations for hypersonic vehicles, which could be used for shipping, weapons and transportation, Karpuzcu said, as engineers will need to account for the newly observed discontinuities.
Skyler Ware is a freelance science journalist covering chemistry, biology, paleontology and Earth science. She was a 2023 AAAS Mass Media Science and Engineering Fellow at Science News. Her work has also appeared in Science News Explores, ZME Science and Chembites, among others. Skyler has a Ph.D. in chemistry from Caltech.
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Neurotropic herpesviruses may be implicated in the development of dementia1,2,3,4,5. Moreover, vaccines may have important off-target immunological effects6,7,8,9. Here we aim to determine the effect of live-attenuated herpes zoster vaccination on the occurrence of dementia diagnoses. To provide causal as opposed to correlational evidence, we take advantage of the fact that, in Wales, eligibility for the zoster vaccine was determined on the basis of an individual's exact date of birth. Those born before 2 September 1933 were ineligible and remained ineligible for life, whereas those born on or after 2 September 1933 were eligible for at least 1 year to receive the vaccine. Using large-scale electronic health record data, we first show that the percentage of adults who received the vaccine increased from 0.01% among patients who were merely 1 week too old to be eligible, to 47.2% among those who were just 1 week younger. Apart from this large difference in the probability of ever receiving the zoster vaccine, individuals born just 1 week before 2 September 1933 are unlikely to differ systematically from those born 1 week later. Using these comparison groups in a regression discontinuity design, we show that receiving the zoster vaccine reduced the probability of a new dementia diagnosis over a follow-up period of 7 years by 3.5 percentage points (95% confidence interval (CI) = 0.6–7.1, P = 0.019), corresponding to a 20.0% (95% CI = 6.5–33.4) relative reduction. This protective effect was stronger among women than men. We successfully confirm our findings in a different population (England and Wales's combined population), with a different type of data (death certificates) and using an outcome (deaths with dementia as primary cause) that is closely related to dementia, but less reliant on a timely diagnosis of dementia by the healthcare system10. Through the use of a unique natural experiment, this study provides evidence of a dementia-preventing or dementia-delaying effect from zoster vaccination that is less vulnerable to confounding and bias than the existing associational evidence.
Recently, evidence has grown that neurotropic herpesviruses may have a role in the pathogenesis of dementia1,2,3,4,5. One approach to targeting herpesviruses is vaccination. However, vaccines are also increasingly being recognized as eliciting a broader immune response that can have important off-target effects, particularly in the case of live-attenuated vaccines6,7,8,9. Such effects have frequently been observed to differ strongly by sex7.
To date, studies in cohort and electronic health record data on the effect of vaccination receipt on dementia have simply compared the occurrence of dementia among those who received a given vaccination and those who did not11. These studies have to assume that all characteristics that are different between those who are vaccinated and those who are not (and that are also related to dementia) have been sufficiently well measured and modelled in the analysis, such that no factors confound the relationship between vaccination receipt and dementia12. This assumption of no confounding bias is often implausible because it has to be assumed that the study has detailed data on factors that are difficult to measure, such as personal motivation or health literacy13. It is also an assumption that cannot be empirically verified.
We used a fundamentally different approach that takes advantage of the fact that, in Wales, starting on 1 September 2013, those born on or after 2 September 1933 were eligible for herpes zoster vaccination for at least 1 year, while those born earlier never became eligible14. Using detailed large-scale electronic health record data, we were able to compare adults who were ineligible for the vaccine because they were born immediately before the eligibility cut-off date with those born immediately after who were eligible. Importantly, individuals who are only a few weeks apart in age are not expected to differ systematically from each other. That is, all potential confounding variables are in expectation balanced between our comparison groups. By taking advantage of this unique natural experiment, we were able to avoid confounding more credibly than all existing studies on the topic15,16,17,18,19,20,21,22,23,24, which have simply compared vaccine recipients to non-recipients while trying to control for the myriad of differences between these groups.
Adults born immediately after the 2 September 1933 date-of-birth eligibility cut-off had a 47.2 percentage point higher probability (from 0.01% to 47.2%) of ever receiving the herpes zoster vaccine than those born immediately before this cut-off date. As expected, other than this abrupt change in herpes zoster vaccination uptake, patients were balanced across the 2 September 1933 date-of-birth eligibility threshold in their uptake of other preventive health services, past common disease diagnoses and educational attainment. We then used this ‘quasi-randomization' in a regression discontinuity analysis to first replicate the known finding from clinical trials that the herpes zoster vaccine reduces new diagnoses of shingles. Second, we extended this approach to an outcome—dementia—that was never assessed in clinical trials of the herpes zoster vaccine, and find that the vaccine reduces the probability of a new dementia diagnosis over a seven-year follow-up period by approximately one-fifth. Third, we show that the herpes zoster vaccine did not affect the occurrence of any other common causes of mortality or morbidity other than shingles and dementia. Similarly, we show that receipt of the herpes zoster vaccine did not lead to increased uptake of other vaccinations or preventive health measures. Fourth, we provide evidence that no other intervention (such as health insurance eligibility) in Wales used the identical date of birth (2 September 1933) as eligibility cut-off as was used to define eligibility for the herpes zoster vaccine. Fifth, we show that all findings remain similar when using a different analysis approach. Sixth, we show that changes in healthcare pathways as a result of a shingles episode are unlikely to explain our findings. Seventh, we provide exploratory evidence from our electronic health record data on the mechanism through which herpes zoster vaccination could affect dementia. Our study focuses on the live-attenuated herpes zoster vaccine (Zostavax; hereafter, zoster vaccine), because the newer recombinant subunit zoster vaccine (Shingrix) became available in the UK only after our follow-up period ended25.
We used the Secure Anonymised Information Linkage (SAIL) Databank26, which contains detailed electronic health record data on primary care visits from approximately 80% of primary care providers in Wales, linked to secondary care records and the country's death register data. The study population for our primary analyses consisted of all adults born between 1 September 1925 and 1 September 1942 who were registered with a primary care provider (which is the case for over 98% of adults residing in Wales27), resided in Wales and did not have a diagnosis of dementia at the time of the start of the zoster vaccine program in Wales (on 1 September 2013). Basic sociodemographic and clinical characteristics of the sample of 282,541 adults in our primary analysis cohort are shown in Supplementary Table 1.
In Wales, individuals born between 2 September 1933 and 1 September 1934 (16,595 adults in our data) became eligible for the zoster vaccine for at least 1 year on 1 September 2013. Eligibility was then progressively extended to younger, but not older, age cohorts annually on the basis of their exact date of birth (Methods).
We find that being born just 1 week after 2 September 1933, and therefore being eligible for the zoster vaccine for at least 1 year, caused an abrupt increase in the probability of ever receiving the zoster vaccine from 0.01% to 47.2% (P < 0.001; Fig. 1). This provides a unique opportunity to avoid confounding concerns because it is unlikely that individuals born immediately around the date-of-birth eligibility threshold systematically differ from each other by anything but a one-week difference in age and a large difference in the probability of receiving the zoster vaccine. We substantiate this empirically by showing that, at the time of the start date of the zoster vaccination program, neither the prevalence of common disease diagnoses (including having been diagnosed with dementia before the vaccination program rollout), dementia risk as predicted from a series of clinical and sociodemographic variables, nor the prevalence of preventive behaviours (other than zoster vaccine uptake) display a discontinuity at the date-of-birth eligibility threshold for the zoster vaccine (Fig. 1 and Supplementary Figs. 1–4). Thus, after flexibly controlling for age, our two comparison groups (one with a low and one with a high probability of receiving the zoster vaccine) born immediately on either side of the 2 September 1933 date-of-birth eligibility threshold are probably exchangeable with each other on all observed and unobserved potential confounding variables.
a–f, The date-of-birth eligibility cut-off led to a large discontinuity in zoster vaccine receipt (a), but there is baseline exchangeability across the cut-off for uptake of other preventive interventions (flu vaccine (d), pneumococcal polysaccharide vaccine (PPV) (e) and statin medications (f)) as well as past shingles (b) and dementia (c) diagnoses. The data source for this analysis was the SAIL database for Wales. All analyses were run on the same sample as those for the effect of the zoster vaccine on dementia occurrence. The exception is c, for which we did not exclude individuals with a diagnosis of dementia before the start of the zoster vaccine program. The grey dots show the mean value for each 10-week increment in week of birth. The grey shading of the dots is proportionate to the weight that observations from this 10-week increment received in the analysis.
Our analysis approach primarily compares those who were ineligible for zoster vaccination because they had their 80th birthday immediately before the program's start date with those who were eligible because they had their 80th birthday immediately after the start date. As is standard practice in regression discontinuity analyses28,29, the effect of actually receiving the vaccine (as opposed to merely being eligible) was determined using a two-stage least-squares regression, which divides the magnitude of the abrupt change in the outcome at the date-of-birth eligibility threshold by the magnitude of the abrupt change in vaccine uptake at this threshold (Methods). Thus, the fact that not all those who were eligible received zoster vaccination does not bias our analysis.
We first demonstrate that our approach successfully reproduces the known causal effect from clinical trials that the vaccine reduces the occurrence of shingles30. Specifically, using a regression discontinuity design28,29, we compared the occurrence of shingles between adults born close to either side of the date-of-birth eligibility threshold for the zoster vaccine. Consistent with the approach used by clinical trials of the zoster vaccine30, our outcome was whether or not an individual had at least one shingles diagnosis during the follow-up period. During our follow-up period of 7 years, a total of 14,465 among 296,324 adults in our sample had at least one diagnosis of shingles. Over the same follow-up time, we find that being eligible for the vaccine reduced the probability of having at least one shingles diagnosis by 1.0 (95% CI = 0.2–1.7; P = 0.010) percentage point (Fig. 2a), corresponding to a relative reduction of 18.8% (95% CI = 8.8–28.9). When calculating the effect of actually receiving the zoster vaccine, we find a reduction in the probability of having at least one shingles diagnosis of 2.3 (95% CI = 0.5–3.9; P = 0.011) percentage points over the seven-year follow-up period (Fig. 2b); an effect (37.2% (95% CI = 19.7–54.7) in relative terms) that is similar in size to that observed in clinical trials of the live-attenuated zoster vaccine (Zostavax)30.
a–c, Effect estimates of being eligible for (a), and having received (across different follow-up periods (b) and across different grace periods (c)), the zoster vaccine on the probability of having at least one shingles diagnosis during the follow-up period. For a, the MSE-optimal bandwidth is 145.7 weeks (95,227 adults). The grey dots show the mean value for each 10-week increment in week of birth. The grey shading of the dots is proportionate to the weight that observations from this 10-week increment received in the analysis. For b and c, the MSE-optimal bandwidth for our primary specification is 116.9 weeks (76,316 adults). The triangles (rather than points) depict our primary specification. The red (as opposed to white) fillings denote statistical significance (P < 0.05). Grace periods refer to time periods since the index date after which the follow-up time is considered to begin. The grey vertical bars show the 95% CIs around the point estimate of the regression coefficient (two-sided t tests).
We show that our estimated effect is not sensitive to the chosen functional form of the regression used to model the relationship of shingles occurrence with week of birth (Supplementary Fig. 5), the width of the week-of-birth window (bandwidth) around the date-of-birth eligibility cut-off that defines our analysis sample (Supplementary Fig. 6a) or to different grace periods (Fig. 2c). With ‘grace periods', we refer to time periods since the index date after which the follow-up time is considered to begin (Methods). There was also a strong indication that the zoster vaccine reduced the probability of having at least one diagnosis of postherpetic neuralgia (a common complication of shingles), although this effect did not reach statistical significance in all specifications (Supplementary Fig. 7).
Given the neuropathological overlap between dementia types and the difficulty in distinguishing dementia types clinically31, as well as our reduced statistical power when studying less-common outcomes, we defined dementia as dementia of any type or cause as our outcome. We considered an individual to have developed dementia if there was a new diagnosis of dementia in our electronic health record data (which includes all diagnoses made in primary or secondary care) or dementia was listed as a primary or contributory cause of death on the death certificate. The Read and ICD-10-codes used to define dementia are listed in the Supplementary Codes. During our seven-year follow-up period, 35,307 among 282,541 adults in our sample were newly diagnosed with dementia.
Using our regression discontinuity approach, we estimate that the effect of being eligible for the zoster vaccine is a 1.3 (95% CI = 0.2–2.7; P = 0.022) percentage points absolute and 8.5% (95% CI = 1.9–15.1) relative reduction in the probability of a new dementia diagnosis over 7 years (Fig. 3a). Scaled to account for the fact that not all those who were eligible received the vaccine, we find that actually receiving the zoster vaccine reduced the probability of a new dementia diagnosis by 3.5 (95% CI = 0.6–7.1; P = 0.019) percentage points, corresponding to a relative reduction of 20.0% (95% CI = 6.5–33.4) (Fig. 3b). The effect estimates were generally not sensitive to different grace periods (Fig. 3c), the functional form of our regressions (Supplementary Fig. 8) nor the width of the week-of-birth window (bandwidth) drawn around the date-of-birth eligibility cut-off (Supplementary Fig. 6b). We also find significant effects of the zoster vaccine on reducing dementia diagnoses if a diagnosis is defined solely as a new prescription of a medication (donepezil hydrochloride, galantamine, rivastigmine or memantine hydrochloride) that is frequently prescribed to slow the progression of Alzheimer's disease (Supplementary Table 2 (column 2)). Similarly, the effects remain similar when adjusting for all input variables to the Dementia Risk Score32 (as recorded before 1 September 2013) (Supplementary Table 2 (column 7)).
a–c, Effect estimates of being eligible for (a), and having received (across different follow-up periods (b) and across different grace periods (c)), the zoster vaccine on new diagnoses of dementia. For a, the MSE-optimal bandwidth is 134.4 weeks (83,167 adults). The grey dots show the mean value for each 10-week increment in week of birth. The grey shading of the dots is proportionate to the weight that observations from this 10-week increment received in the analysis. For b and c, the MSE-optimal bandwidth for our primary specification is 90.6 weeks (56,098 adults). The triangles (rather than points) depict our primary specification. The red (as opposed to white) fillings denote statistical significance (P < 0.05). Grace periods refer to time periods since the index date after which the follow-up time is considered to begin. The grey vertical bars show the 95% CIs around the point estimate of the regression coefficient (two-sided t tests).
The key strength of our study is that a confounding variable can bias our analysis only if the variable changes abruptly at the 2 September 1933 date-of-birth threshold28,29. Thus, confounding bias could occur if another intervention also used the date of birth cut-off of 2 September 1933 as an eligibility criterion. Such an intervention is unlikely to affect only the risk of developing dementia without also influencing other health outcomes. We therefore implemented the same regression discontinuity approach as we did for shingles and dementia for the ten leading causes of disability-adjusted life years and mortality for the age group 70+ years in Wales in 201933, and all conditions that are part of the Charlson Comorbidity Index34. As shown in Supplementary Figs. 9 and 10, we generally do not detect effects of zoster vaccination on new diagnoses of these other common health outcomes.
We undertook four additional types of analysis, all of which provide evidence against another intervention having used the identical day-month-year combination (2 September 1933) as was used as the date-of-birth eligibility threshold for the zoster vaccine rollout. First, we show that the 2 September 1933 date-of-birth threshold does not affect the probability of taking up other preventive health interventions (Supplementary Fig. 11). Second, we examined whether the day–month (that is, 2 September) date-of-birth cut-off used for zoster vaccine eligibility was also used by other interventions that affect dementia risk. We did so by implementing the identical analysis as for 1 September 2013 (the actual date on which the zoster vaccine program started) for 1 September of each of the three years before and after 2013. Thus, for example, when shifting the start date of the program to 1 September 2012, we compared those around the 2 September 1932 date-of-birth threshold with the follow-up period starting on 1 September 2012. As an additional check that enabled us to maintain the length of the seven-year follow-up period used in our primary analyses, we shifted the program start date to 1 September of each of the 6 years preceding (but not after) 2013. As expected, for both of these checks, we find a significant effect on dementia occurrence only for the date-of-birth cutoff (2 September 1933) that was actually used by the zoster vaccination program (Supplementary Figs. 12 and 13). Third, we find that there is no difference in the seven-year incidence of dementia between age cohorts around the 2 September 1933 date-of-birth threshold for the seven-year period before the zoster vaccine rollout (Supplementary Fig. 14). Fourth, using data from the 2011 Census, we show in Supplementary Figs. 15–17 that there are no discontinuities across the 2 September 1933 threshold in the proportion of individuals in Wales who reached a particular level of education.
As an additional test of the robustness of our findings, we implemented all primary analyses using a difference-in-differences instrumental variable analysis (DID-IV) that takes advantage of the fact that the only 2 September date-of-birth threshold at which we would expect an abrupt change in the outcome is the 2 September threshold in 1933 (that is, the day–month–year combination that was used as eligibility cut-off by the zoster vaccination program). In doing so, our analysis relaxes the continuity assumption of regression discontinuity (that is, the assumption that potential confounding variables do not display a sudden change at the 2 September 1933 date-of-birth eligibility threshold), and instead assumes that (in the absence of the zoster vaccination program) a possible discontinuity in the outcome at the 2 September 1933 threshold is not different in size from a discontinuity at the 2 September threshold in previous birth years. Details of our approach are provided in the Methods. Encouragingly, the effect of zoster vaccine receipt on the probability of a new dementia diagnosis during our seven-year follow-up period is remarkably similar between the DID-IV and regression discontinuity approach (−3.1 (95% CI = −5.8 to −0.4, P = 0.024) versus −3.5 (95% CI = −7.1 to −0.6, P = 0.019) percentage points) (Fig. 4). This is also the case for the outcomes of shingles and postherpetic neuralgia (Fig. 4). We conducted the same checks for balance in health characteristics between our comparison groups for the DID-IV as we implemented for our regression discontinuity analyses (Supplementary Fig. 18). We also verified that our DID-IV approach yields significant effects only for the outcomes of dementia, shingles and postherpetic neuralgia, but not for any other common health outcomes (Supplementary Fig. 18).
Comparison of absolute effect estimates of having received the zoster vaccine on new diagnoses of dementia, shingles and postherpetic neuralgia between the DID-IV and the regression discontinuity analyses. The data source for this analysis was the SAIL database for Wales. The sample size for the dementia outcome is 96,767 adults and the sample for the shingles and postherpetic neuralgia outcomes is 105,258 adults. P values were calculated using two-sided t-tests. The P value for the DID-IV effect on shingles is 0.001. The error bars depict the 95% CIs around the point estimate of the regression coefficient (two-sided t-tests).
A protective effect of zoster vaccination on dementia diagnoses could arise from three (non-mutually exclusive) mechanisms: (1) changes in healthcare pathways as a result of a shingles episode; (2) a reduction in reactivations of the varicella zoster virus (VZV); and (3) a VZV-independent immunomodulatory effect (for example, one mediated through heterologous adaptive immunity or trained innate immunity). In this section, we present evidence to examine each of these mechanisms.
Reduced healthcare use resulting averted shingles episodes from zoster vaccination receipt could have translated to fewer opportunities for the health system to (1) diagnose dementia (ascertainment bias); or (2) implement care changes (for example, initiation of a new medication) that increase the risk of being diagnosed with dementia in the future. It is important to point out that this mechanism is unlikely to fully explain our findings, because the size of our effect estimates for reductions in shingles episodes from zoster vaccination were considerably too small to plausibly account for the observed reduction in dementia diagnoses.
We nonetheless conducted five types of analysis to examine this potential mechanism further. First, if shingles episodes presented an opportunity for the health system to diagnose dementia, then they would probably also present an opportunity to diagnose other chronic conditions. We therefore applied the same regression discontinuity approach as for shingles and dementia to all chronic conditions that are either among the ten leading causes of disability-adjusted life years and mortality for the age group 70+ years in Wales in 201933 or part of the Charlson Comorbidity Index34. We plotted our estimates across one-year increments in the follow-up period. With the exception of rheumatological diseases, we show that being eligible for the zoster vaccine did not have an effect on new chronic disease diagnoses (Supplementary Fig. 19). Second, we adjusted our regressions for the frequency of health service use (the number of primary care visits, outpatient visits, hospital admissions and influenza vaccinations received) during the follow-up period, which did not substantially change our effect estimates (Supplementary Table 2 (column 4)). Third, we implemented our analyses when restricting the analysis cohort to the 247,784 (87.6% of the analysis cohort for our primary analyses) patients who visited their primary care provider at least once a year during each of the 5 years before the start of the zoster vaccine rollout. The rationale for this analysis is that, among patients who already interact frequently with the health system, a reduction of one further contact with the health system due to an averted shingles episode is less likely to affect the probability of detecting undiagnosed dementia. The effect sizes among this cohort of frequent healthcare users remain similar to those in our primary analytical cohort (Supplementary Table 2 (column 3)). Fourth, we added whether individuals experienced a shingles episode during the follow-up period as a covariate in our primary regression discontinuity analysis. We found that adjusting our analysis for shingles episodes did not substantially change our point estimate (Supplementary Fig. 20). Fifth, we implemented an event study among those participants in the mean-squared-error (MSE)-optimal bandwidth of our primary regression discontinuity analysis for dementia who received a shingles diagnosis during the follow-up period. To investigate whether episodes of shingles led to changes in healthcare received by patients, we examined the effect of the shingles diagnosis on the following outcomes in each of the 36 months after the diagnosis: (1) the probability of receiving a new dementia diagnosis; (2) a set of indicators of health service use; (3) the probability of receiving a new medication prescription for antiviral drugs, opioid medications, gabapentin or pregabalin, and any of 216 medications that were associated with an increased risk of dementia in another analysis in the SAIL database23; and (4) the probability of being diagnosed with any of the chronic conditions that are part of the Charlson Comorbidity Index34. We found that shingles diagnoses did not increase the probability of receiving a new dementia diagnosis in the months after the shingles diagnosis, and led to only short-term increases in healthcare service use and new medication prescriptions (Supplementary Fig. 21). The increase in the probability of receiving a gabapentin or pregabalin prescription in the months after the shingles episode, while more sustained, was small in magnitude. Similarly, the increase in the probability of being diagnosed with any chronic condition in the month of a shingles episode compared with the month before the episode was less than one percentage point (Supplementary Fig. 21).
As the effect of zoster vaccination on shingles episodes is moderate (Fig. 2), and the five types of analysis in this section document only small and short-lived effects of shingles episodes on healthcare pathways, even the most conservative assumptions about the effect of these care paths on dementia imply that changes in healthcare as a result of a shingles episode cannot explain our findings.
As described in the previous section, adjusting our regression discontinuity analysis for whether a patient had a record of at least one shingles episode during the follow-up period did not change our point estimate substantially (Supplementary Fig. 20). However, conclusions from this analysis regarding reductions in VZV reactivations as the effect mechanism are limited by the fact that (1) zoster vaccination probably reduces both clinical as well as subclinical reactivations of VZV30,35; and (2) having a shingles episode may be an unreliable indicator of the degree of subclinical VZV reactivations experienced during the entire follow-up period, given that shingles episodes may boost immunity for VZV30,35. We therefore conducted the following analyses to further examine reductions in VZV reactivations as the effect mechanism.
First, we examined the time during the follow-up period at which the effect of zoster vaccination on dementia appears to begin. Specifically, among patients who were born in close proximity to the 2 September 1933 date-of-birth threshold, we plotted the Kaplan–Meier and cumulative incidence curves for dementia for those who were eligible versus ineligible for zoster vaccination (Methods). If the effect mechanism is through a reduction in VZV reactivations, then one would expect that the effects of the vaccine on reductions in clinical and subclinical reactivations of the virus would begin before observing an effect on dementia. The live-attenuated zoster vaccine is thought to begin being efficacious within weeks after vaccine administration30,36. Consistent with the principle that the effect on VZV reactivations should precede the dementia effect, we observe that the reduction in the incidence of dementia begins to emerge only after more than one year, both among the full population as well as among women only (Supplementary Fig. 22).
Second, while a shingles episode may boost VZV immunity and, therefore, reduce subsequent subclinical VZV reactivations30,35, individuals who experience multiple episodes as opposed to a single shingles episode during the follow-up period probably experience a greater degree of both clinical and subclinical VZV reactivations during the follow-up period30. Using propensity score matching (Methods), we therefore compared the association with dementia from experiencing multiple versus a single shingles episode. We find a higher incidence of dementia among those who experienced multiple shingles episodes (Supplementary Fig. 23).
Third, if VZV reactivations increase the risk of dementia, then limiting the degree of replication of the virus during a shingles episode through antiviral medication could be expected to decrease dementia incidence. Using a multivariable Cox proportional hazards model (Methods), we therefore compared the association with dementia between individuals whose shingles episode was treated with antiviral medication and those for whom the episode was untreated. We find that antiviral treatment of a shingles episode is associated with a reduced incidence of dementia (Supplementary Fig. 23).
To probe this mechanism, we take advantage of two observations on pathogen-independent immunomodulatory effects from vaccination in the literature: they tend to (1) vary strongly by sex, with beneficial effects from live-attenuated vaccination often seen only in female but not male individuals6,7,8; and (2) depend on the receipt of other vaccines before, or at the same time as, receipt of the vaccine in question6,7,8. Consistent with these observations, we find that the effect of zoster vaccination on new diagnoses of dementia was markedly greater among women than men (Fig. 5 and Supplementary Table 3 (column 1)). There was no significant difference between women and men in the effect of the zoster vaccine on diagnoses of shingles and postherpetic neuralgia (Supplementary Table 3 (columns 2 and 3)). Similarly, the magnitude of the abrupt increase in vaccine uptake at the 2 September 1933 date-of-birth eligibility threshold was comparable between women and men (Supplementary Fig. 24), with a slightly larger magnitude among men.
a–f, Effect estimates of being eligible for (a (women) and d (men)) and having received (b and c (women) and e and f (men); across different follow-up periods (b and e) and across different grace periods (c and f)) the zoster vaccine on new diagnoses of dementia, separately for women and men. The data source for this analysis was the SAIL database for Wales. The triangles (rather than points) depict our primary specification. Red (as opposed to white) fillings denote statistical significance (P < 0.05). Grace periods refer to time periods since the index date after which the follow-up time is considered to begin. The grey vertical bars depict the 95% CIs around the point estimate of the regression coefficient (two-sided t-test). The grey dots show the mean value for each 10-week increment in week of birth. For a, among women, the MSE-optimal bandwidth is 95.5 weeks (32,601 women). For b and c, among women, the MSE-optimal bandwidth for our primary specification is 149.1 weeks (50,816 women). For d, among men, the MSE-optimal bandwidth for our primary specification is 121.3 weeks (33,725 men). For e and f, among men, the MSE-optimal bandwidth for our primary specification is 91.8 weeks (25,563 men). The grey shading of the dots is proportionate to the weight that observations from this 10-week increment received in the analysis.
We also find strong effect heterogeneity by receipt of previous influenza vaccination. Specifically, the protective effect of zoster vaccination for dementia was larger among those who did not recently receive the influenza vaccine (Supplementary Fig. 25). Influenza vaccination is the only vaccine that was provided within the 5 years preceding zoster vaccination eligibility to a substantial proportion of individuals in our study population (pneumococcal vaccination is already provided at age 65 years in the United Kingdom37).
Finally, we examined the differences in the effect of the zoster vaccine on dementia incidence between those with versus without an autoimmune or allergic condition. Our reasoning for this analysis was based on the observation that the incidence of shingles is increased among individuals with an autoimmune or allergic condition38,39,40,41, while there do not appear to be major differences in vaccine immunogenicity and its relative effectiveness for shingles prevention between those with versus without such conditions30. Thus, if the protective effect of zoster vaccination for dementia is mainly driven through a reduction of clinical and subclinical virus reactivations, then those with an autoimmune condition will likely benefit equally or more than those without such a condition. However, because autoimmune and allergic conditions are generally characterized by a heightened activation of the (adaptive) immune system42,43, individuals with such a condition might benefit less from further activation of more generalized, VZV-independent, immune system pathways than those without such a condition. Consistent with this second hypothesis, we observe suggestive evidence for stronger effectiveness of the zoster vaccine for dementia among those without an autoimmune or allergic condition than those with such a condition (Supplementary Fig. 25). The patterns that we observe remain largely unaffected by whether or not patients were taking any immunosuppressive medications in the year preceding the start of the zoster vaccination program.
Thus, overall and with the caveat that these exploratory analyses are suggestive only, our analyses indicate that both a mechanism of action through a reduction in clinical and subclinical reactivations of VZV as well as through a VZV-independent immunomodulatory effect are plausible. Importantly, these two mechanisms are not mutually exclusive.
Here we found that the zoster vaccine reduced the probability of a new dementia diagnosis by approximately one-fifth over a seven-year follow-up period. By taking advantage of the fact that the unique way in which the zoster vaccine was rolled out in Wales constitutes a natural experiment, and examining each possible remaining source of bias, our study provides evidence that is more likely to be causal in nature than the existing, exclusively associational15,16,17,18,19,20,21,22,23,24, evidence on this topic. Our substantial effect sizes, combined with the relatively low cost of the zoster vaccine, imply that, if these findings are truly causal, the zoster vaccine will be both far more effective as well as cost-effective in preventing or delaying dementia than existing pharmaceutical interventions.
Our quasi-experimental approach reduces the probability of confounding compared with more standard associational analyses. Moreover, we have provided evidence from a series of analyses against any of the possible remaining sources of bias being a likely explanation of our findings. Nonetheless, it is possible (even if statistically unlikely) that our findings are due to chance. Confirmation of our findings in other populations, settings and data sources is therefore critical. Importantly, we have successfully confirmed our findings using country-wide death certificate data from England and Wales10. Specifically, because England rolled out the zoster vaccine in an almost identical way to Wales44, we were able to use the same quasi-experimental approach as in our electronic health record data from Wales to determine the effect of eligibility for zoster vaccination based on one's date of birth on deaths for which the underlying cause was recorded as being dementia. We found that, over a nine-year follow-up period, approximately 1 in 20 such deaths were averted from being eligible for zoster vaccination. This study constitutes an important confirmation of our results because it analysed a different population (England's population accounts for approximately 95% of England's and Wales's combined population45), type of data (death certificates as opposed to electronic health records) and outcome (deaths due to dementia). In addition to this confirmation of our results in mortality data, the probability of a chance finding is further reduced by the fact that we successfully replicate our main findings using a second analysis approach (DID-IV) and that our effect sizes remain stable across a multitude of analysis choices, including choice of grace periods, follow-up periods, study population definitions (for example, restriction to frequent healthcare users), functional form of our regressions, width of the week-of-birth window drawn around the date-of-birth eligibility cut-off and index date definitions.
We observed large differences in the effect of zoster vaccination on dementia between women and men, with women benefitting more than men. In our view, these large differences between women and men are plausible for several reasons. First, we cannot exclude the possibility of substantial reductions in new dementia diagnoses from zoster vaccination among men, especially given the lower incidence of dementia in older age among men than women in our data and, therefore, our wider confidence intervals for analyses among men. Second, off-target effects of vaccines have often been observed to be far stronger among female than male individuals, with female individuals benefiting more from live-attenuated vaccines in particular6,7. Third, there appear to be important sex differences in the immunological response to vaccines more generally46. Lastly, there is a growing body of evidence that there may be differences in the pathogenesis of dementia between women and men47.
Other than investing into randomized trials, investments into basic science research on the potential role of VZV and the immune response to the zoster vaccine in the pathogenesis of dementia could provide critical mechanistic insights. There are already several lines of evidence on plausible mechanistic pathways that link VZV reactivations to dementia. Specifically, VZV reactivations have been found to lead to long-lasting cognitive impairment through vasculopathy48,49, amyloid deposition and aggregation of tau proteins50, neuroinflammation51,52,53,54, as well as a similar spectrum of cerebrovascular disease as seen in Alzheimer's disease, including small to large vessel disease, ischaemia, infarction and haemorrhage51,52,53,54,55,56. As suggested by a recent study57, it may also be the case that reducing subclinical and clinical reactivations of VZV reduces reactivations of the herpes simplex virus-1 in the brain through neuroinflammatory pathways. This mechanism would link VZV to the body of literature on the role of herpes simplex virus-1 in the pathogenesis of dementia1,2,3,4,5. Nonetheless, our exploratory analyses on the effect mechanism that links zoster vaccination to dementia suggest that both a mechanism through reducing clinical and subclinical reactivations of VZV as well as a pathogen-independent immune mechanism are plausible. Some of these possible pathogen-independent immune mechanisms have recently been detailed elsewhere58.
Our study has several limitations. First, our outcome ascertainment probably suffers from some degree of under-detection, both in whether and in how timely a fashion dementia is diagnosed. Importantly, because the probability of under-detecting dementia, as well as the delay in doing so, is unlikely to change abruptly at the 2 September 1933 date-of-birth eligibility threshold for zoster vaccination, this outcome misclassification is most likely non-differential. Our effect estimates are therefore likely to be conservative (that is, our absolute effect sizes would be an underestimate of the true absolute effect magnitude). Similarly, changes in the accuracy and timeliness of dementia ascertainment over the years of our follow-period, such as due to changing clinical practice or health system incentives to detect and record dementia, affected those born immediately before versus immediately after 2 September 1933 equally. We would therefore not expect these changes to be a source of bias in our analyses. Second, we are unable to provide estimates for the effectiveness of the zoster vaccine for reducing dementia occurrence in age groups other than those who were weighted most heavily in our regression discontinuity analyses (primarily those aged 79 to 80 years). Third, the COVID-19 pandemic probably affected the timeliness with which dementia was diagnosed. However, the follow-up period used in our primary analyses ended before the start of the COVID-19 pandemic. Moreover, because the pandemic affected those born just before versus just after 2 September 1933 equally, pandemic-related under-detection of dementia is unlikely to bias our relative effect estimates. Fourth, we were limited to a maximum follow-up period of 8 years. Our study can therefore not inform on the effectiveness of the zoster vaccine for reducing dementia occurrence beyond this time period. Lastly, because the newer recombinant subunit zoster vaccine (Shingrix) replaced the live-attenuated zoster vaccine (Zostavax) in the United Kingdom only in September 202325, which is after our follow-up period ended, our effect estimates apply to the live-attenuated zoster vaccine only.
The live-attenuated zoster vaccine (Zostavax) was made available to eligible individuals in Wales through a staggered rollout system starting on 1 September 2013. Under this system, individuals aged 71 years or older were categorized into three groups on 1 September of each year: (1) an ineligible cohort of those aged 71 to 78 years (or 77 years, depending on the year of the program), who became eligible in the future; (2) a catch-up cohort, consisting of individuals aged 79 years (or 78 years, again depending on the year of the program); and (3) those who were ineligible as they were aged 80 years or older and who never became eligible.
Our analysis focused on adults born between 1 September 1925 (88 years old at program start) and 1 September 1942 (71 years old at program start). Those born between 1 September 1925 and 1 September 1933 never became eligible, whereas those born between 2 September 1933 and 1 September 1942 became progressively eligible in a catch-up cohort. Specifically, the vaccine was offered to those born between 2 September 1933 and 1 September 1934 in the first year of the program (1 September 2013 to 31 August 2014); those born between 2 September 1934 and 1 September 1936 in the second year (1 September 2014 to 31 August 2015); those born between 2 September 1936 and 1 September 1937 in the third year (1 September 2015 to 31 August 2016); and those born between 2 September 1937 and 1 September 1938 in the fourth year (1 September 2016 to 31 August 2017). As of 1 April 2017, individuals become eligible for the vaccine on their 78th birthday and remain eligible until their 80th birthday. Our analysis principally compared individuals born on or shortly after 2 September 1933, to individuals who never became eligible as they were born shortly before 2 September 1933. We show in Supplementary Figs. 26–28 that most eligible individuals, especially in the first two eligibility cohorts, which are the focus of our analysis, took up the vaccination during their first year of eligibility (as opposed to during later years) and that vaccination uptake in these first two eligibility cohorts was of a similar magnitude.
Healthcare in Wales is provided through the Welsh National Health Service (NHS), which is part of the United Kingdom's single-payer single-provider healthcare system59. NHS Wales and Swansea University created the SAIL Databank26,60,61,62,63,64, which includes full electronic health record data for primary care visits linked to information on hospital-based care as well as the country's death register data.
SAIL generates a list of all individuals who have ever been registered with a primary care provider in Wales (which is the case for over 98% of adults residing in Wales27) from the Welsh Demographic Service Dataset65. SAIL then links this universe of individuals to each of the following datasets. Electronic health record data from primary care providers is made available in SAIL through the Welsh Longitudinal General Practice dataset66, which contains data from approximately 80% of primary care practices in Wales and 83% of the Welsh population. These electronic health record data use Read codes, which provide detailed information on patients and their care encounters, including diagnoses, clinical signs and observations, symptoms, laboratory tests and results, procedures performed and administrative items67. Zoster vaccination was defined using both codes for the administration of the vaccine as well as product codes (Supplementary Table 1). Diagnoses made and procedures performed in the hospital setting (as part of inpatient admissions or day-case procedures) are provided in SAIL through linkage to the Patient Episode Database for Wales68, which begins in 1991 and contains data for all hospital-based care in Wales as well as hospital-based care provided in England to Welsh residents. Procedures are encoded using OPCS-4 codes69 and diagnoses using ICD-10 codes70. Attendance information at any NHS Wales hospital outpatient department is provided through linkage to the Outpatient Database for Wales71, which starts in 2004. ICD-10-encoded diagnoses of cancers are identified through linkage to the Welsh Cancer Intelligence and Surveillance Unit72, which is the national cancer registry for Wales that records all cancer diagnoses provided to Welsh residents wherever they were diagnosed or treated. This dataset begins in 1994. Finally, cause-of-death data are provided for all Welsh residents (regardless of where they died in the United Kingdom) through linkage to the Annual District Death Extract73, which begins in 1996 and includes primary and contributory causes of death from death certificates. Dates for deaths were those on which the death was registered, as opposed to when it occurred. Cause-of-death data use ICD-9 coding until 2001 and ICD-10 coding thereafter.
When testing for any discontinuities in educational attainment across the date-of-birth eligibility threshold, we used a dataset provided by the Office for National Statistics (ONS)74. This dataset was generated by the ONS from the 2011 UK Census for all usual residents aged 16 or over, born in Wales between January 1925 and December 1950, regardless of their employment status. The data were categorized by the ONS by sex, month and year of birth (January 1925 to December 1950), highest level of qualification and occupation.
Ethics approval was granted by the Information Governance Review Panel (IGRP, application number, 1306). Composed of government, regulatory and professional agencies, the IGRP oversees and approves applications to use the SAIL databank. All analyses were approved and considered minimal risk by the Stanford University Institutional Review Board on 9 June 2023 (protocol number, 70277).
Our study population consisted of 296,603 individuals born between 1 September 1925 and 1 September 1942 who were registered with a primary care provider in Wales on the start date of the zoster vaccine program rollout (1 September 2013). As we only had access to the date of the Monday of the week in which an individual was born, we were unable to determine whether the individuals born in the cut-off week starting on 28 August 1933 were eligible for the zoster vaccine in the first year of its rollout. We therefore excluded 279 individuals born in this particular week. Among the remaining individuals, 13,783 had a diagnosis of dementia before 1 September 2013 and were therefore excluded from the analyses with new diagnoses of dementia as outcome. The size of our final analysis cohort for all primary analyses for new dementia diagnoses was therefore 282,541. This analysis cohort was used for all analyses except those with incidence of dementia before zoster vaccination program start, shingles and postherpetic neuralgia as outcomes; analyses for which we did not exclude individuals with a dementia diagnosis before 1 September 2013.
We followed individuals from 1 September 2013 to 31 August 2021, which allowed for a maximum follow-up period of 8 years. In our primary specification, we selected a follow-up period of 7 years (that is, until 31 August 2020) because this enabled us to include grace periods of up to 12 months while still keeping the follow-up period constant for individuals on either side of the date-of-birth eligibility cut-off. However, we also show all results for follow-up periods from one to eight years in one-year increments. Owing to the unique anonymized NHS number assigned to each patient, we were able to follow individuals across time even if they changed primary care provider. Patients were therefore only lost to follow-up in our cohort if they emigrated out of Wales or changed to one of the approximately 20% of primary care practices in Wales that did not contribute data to SAIL. Over our seven-year follow-up period, this was the case for 23,049 (8.2%) of adults in our primary analysis cohort, with no significant difference in this proportion between those born just before versus just after the 2 September 1933 eligibility threshold. In total, 92,629 (37.8%) of adults in our primary analysis cohort died during the seven-year follow-up period. Our primary analysis approach does not adjust for any competing risk of death for three reasons. First and foremost, in the absence of a zoster vaccination program, there is no reason that the competing risk of death should differ across the 2 September 1933 date-of-birth eligibility threshold. Second, not adjusting for competing risk of death in our setting is a conservative choice because eligibility for zoster vaccination may reduce (but is very unlikely to increase) all-cause mortality10,75. Thus, those eligible for zoster vaccination will, on average, be exposed to a longer time period during which they could become newly diagnosed with dementia. Third, to date, no well-established approach exists for survival analysis in a regression discontinuity framework, including the ability to determine the CACE and optimal bandwidth76.
Owing to the neuropathological overlap between dementia types and difficulty in distinguishing dementia types clinically77,78,79, we chose to define dementia as dementia of any type or cause. Dementia was defined as a diagnosis of dementia made either in primary care (as recorded in the primary care electronic health record data), specialist care or hospital-based care, or dementia being named as a primary or contributory cause of death on the death certificate. The date of the first recording of dementia across any of these data sources was used to define the date on which the patient was diagnosed with dementia. Similarly, all other outcomes were defined using a diagnosis made in any care setting or mentioning as a primary or contributory cause of death. For chronic conditions, the date of the first recording across any of these data sources was used to define the date on which the chronic condition occurred. For non-chronic conditions or events (that is, shingles, postherpetic neuralgia, stroke, lower respiratory tract infections, falls, lower back pain, medication prescriptions, influenza vaccination and breast cancer screening), the date of first recording after the program date across any of these data sources was used for defining the occurrence of the outcome during the follow-up. The Read and ICD-10 codes used to define all outcomes are detailed in the Supplementary Codes.
The two authors who analysed the data (M.E. and M.X.) have coded all parts of the analysis independently. Occasional minor differences, resulting from different data coding choices, were resolved through discussion.
We used a regression discontinuity design to analyse our data, which is a well-established method for causal inference in the social sciences80. Regression discontinuity analysis estimates expected outcome probabilities just left and just right of the cut-off, to obtain an estimate of the treatment effect. We used local linear triangular kernel regressions (assigning a higher weight to observations lying closer to the date-of-birth eligibility threshold) in our primary analyses and quadratic polynomials in robustness checks. This is the recommended and most robust approach for regression discontinuity analyses even in situations in which the relationship between the assignment variable (here, date of birth) and the outcome is exponential81. An important choice in regression discontinuity analyses is the width of the data window (the bandwidth) that is drawn around the threshold. Following standard practice, we used an MSE-optimal bandwidth82, which minimizes the MSEs of the regression fit, in our primary analyses. We determined this optimal bandwidth separately for each combination of sample and outcome definition. In robustness checks, we examined the degree to which our point estimates vary across different bandwidth choices ranging from 0.25 times to two times the MSE-optimal bandwidth. We used robust bias-corrected standard errors for inference83.
In the first step, we determined the effect of being eligible for the zoster vaccine (regardless of whether the individual actually received the vaccine) on our outcomes. To do so, we estimated the following regression equation:
where Yi is a binary variable equal to one if an individual experienced the outcome (for example, shingles or dementia). The binary variable Di indicates eligibility for the zoster vaccine and is equal to one if an individual was born on or after the cut-off date of 2 September 1933. The term (WOBi − C0) indicates an individual's week of birth centred around the cut-off date. The interaction term Di × (WOBi − C0) allows for the slope of the regression line to differ on either side of the threshold. The parameter β1 identifies the absolute effect of being eligible for the vaccine on the outcome. Wherever we report relative effects, we calculated these by dividing the absolute effect estimate β1 by the mean outcome just left of the date-of-birth eligibility threshold, that is, the estimate of α.
In the second step, we estimated the effect of actually receiving the zoster vaccine on our outcomes. This effect is commonly referred to as the complier average causal effect (CACE) in the econometrics literature84. As is standard practice84, we used a fuzzy regression discontinuity design to estimate the CACE. Fuzzy regression discontinuity analysis takes into account the fact that the vaccine is not deterministically assigned at the week-of-birth cut-off. Instead, a proportion of ineligible individuals still received the vaccine and a proportion of eligible individuals did not receive the vaccine. To account for this fuzziness in the assignment, the fuzzy regression discontinuity design uses an instrumental variable approach, with the instrumental variable being the binary variable that indicates whether or not an individual was eligible to receive the vaccine, that is, is born on or after 2 September 1933. As we verify in our plot of vaccine receipt by week of birth (Fig. 1a), individuals who were born immediately after the date-of-birth eligibility threshold had a far higher probability of receiving the zoster vaccine compared with those born immediately before the threshold. Other than the abrupt change in the probability of receiving the zoster vaccine, there probably is no other difference in characteristics that affect the probability of our outcomes occurring between those born immediately after versus immediately before the date-of-birth eligibility threshold. Thus, the indicator variable for the date-of-birth eligibility threshold is a valid instrumental variable to identify the causal effect of receipt of the zoster vaccine on our outcomes. To compare the probability of experiencing the outcome between those who actually received the zoster vaccine versus those who did not, the instrumental variable estimation scales the effect size for being eligible for the zoster vaccine by the size of the abrupt change in the probability of receiving the vaccine at the date-of-birth eligibility threshold. The size of the jump is estimated through the following first-stage regression equation:
where Vi is a binary variable indicating whether the individual received the zoster vaccine and θ1 identifies the discontinuous increase in vaccine receipt at the date-of-birth eligibility threshold. All other parameters are the same as in regression (1).
The CACE estimated by rescaling the effect of eligibility with the first-stage effect from equation (2) can be represented as an IV estimate for μ1 from the following second-stage regression:
where \({\hat{V}}_{i}\) is the predicted probability of zoster vaccine receipt obtained from the first-stage estimation from equation (2). This CACE, μ1, represents the (absolute) average causal effect of receiving the vaccine among compliers, that is, patients who take up the vaccine if and only if they are eligible.
To compute relative effect sizes, we first introduce some notation. Let R0,c be the mean outcome among unvaccinated compliers and R1,c the mean outcome among vaccinated compliers just at the threshold. By definition, the absolute CACE is μ1 = R1,c − R0,c and the relative effect is \(\frac{{\mu }_{1}}{{R}_{0,c}}\). To estimate the relative effect, we need an estimate for R0,c. While it is impossible to identify compliers individually, we can estimate means of their observable characteristics, including R0,c (ref. 85). Let R1 denote the mean outcome among all vaccinated individuals (including compliers) at the cut-off. Assuming no defiers exist (patients who get vaccinated if and only if they are not eligible), all vaccinated people are either compliers or always-takers (patients who get vaccinated irrespective of their eligibility). Thus, R1 is equal to the population-weighted average of the mean outcomes among vaccinated compliers and always-takers: R1 = Pc × R1,c + Pa × R1,a, where Pc and Pa are the population share of the compliers and always-takers and R1,a is the mean outcome among always-takers at the cut-off. Solving for R1,c yields \({R}_{1,c}=\frac{{R}_{1}-{R}_{1,a}\times {P}_{a}}{{P}_{c}\,}\). All right-hand-side quantities in this equation can be estimated from data. First, R1 and R1,a can be estimated, respectively, as α + β1 and α from re-estimating regression (1) only among vaccinated individuals. Second, Pa and Pc can be estimated, respectively, as \(\frac{\gamma }{{\theta }_{1}+\gamma }\) and \(\frac{{\theta }_{1}}{{\theta }_{1}+\gamma }\) from regression (2). Finally, we estimate R1,c by the above formula and R0,c = R1,c − μ1. The relative effect is estimated as \(\frac{{\mu }_{1}}{{R}_{0,c}}\). All regressions involved in these steps can be stacked and jointly estimated, so that the relative effect is expressed as a differentiable function of known estimators a 95% confidence interval of the relative CACE can be estimated using the delta method86.
Our analysis can only be confounded if the confounding variable changes abruptly at the 2 September 1933 date-of-birth eligibility threshold such that individuals very close to either side of this threshold would no longer be exchangeable with each other. The most plausible scenario of such a confounding variable would be the existence of an intervention that used the exact same date-of-birth eligibility threshold as the zoster vaccine rollout and that also affected the probability of a dementia diagnosis during our follow-up period. We conducted five analyses to demonstrate that the existence of such an intervention is unlikely, by establishing that measures of outcomes and behaviours that would be affected by such an intervention are smooth across the date-of-birth eligibility cut-off.
First, across a range of birthdates around the 2 September 1933 eligibility threshold, we plotted the probability of having received the following diagnoses or interventions before the start of the zoster vaccine program (on 1 September 2013): diagnosis of shingles, influenza vaccine receipt in the preceding 12 months, receipt of the pneumococcal vaccine as an adult, current statin use (defined as a new or repeat prescription of a statin in the 3 months preceding program start), current use of an antihypertensive medication (defined as a new or repeat prescription of an antihypertensive drug in the 3 months preceding the program start), participation in breast cancer screening (defined as the proportion of women with a record of referral to, attendance at or a report from breast cancer screening or mammography), each of the ten leading causes of disability-adjusted life years and mortality for Wales in 2019 as estimated by the Global Burden of Disease Project33, and all comorbidities (except for AIDS, which falls under privacy-protected diagnoses not made available by the SAIL database) that are included in the widely-used Charlson Comorbidity Index34. Moreover, we used each of these conditions, gender, decile of Welsh Index of Multiple Deprivation56, as well as all input variables to the Dementia Risk Score (as recorded before 1 September 2013)32, to predict the probability (while imputing a fixed age) of a new dementia diagnosis for each patient in the MSE-optimal bandwidth in our primary regression discontinuity analysis for dementia. In addition to plotting these predicted probabilities across a range of birthdates around the 2 September 1933 eligibility threshold, we also plotted the distribution of these predicted probabilities for patients who were eligible versus patients who were ineligible for zoster vaccination. The Read codes for each of these variables are provided in Supplementary Tables 1 and 2. As is the case for balance tables in clinical trials, these plots provide reassurance that individuals close to either side of the 2 September 1933 eligibility threshold are likely to be exchangeable with each other.
Second, we conducted the same analysis as we did for individuals with birthdays on either side of the 2 September 1933 threshold also for people with birthdays around 2 September of each of the three years of birth preceding and succeeding 1933. For example, when moving the start date of the program to 1 September 2011, we started the follow-up period on 1 September 2011 and compared individuals around the 2 September 1931 eligibility threshold. To ensure the same length of follow-up in each of these comparisons, we had to reduce the follow-up period to 5 years for this set of analyses. Thus, as an additional check, we shifted the start date of the program to 1 September of each of the six years preceding (but not succeeding) 2013, which enabled us to maintain the same seven-year follow-up period as in our primary analysis. If another intervention that affects dementia risk also used the 2 September threshold to define eligibility, we may then expect to observe effects on dementia incidence for these comparisons of individuals just around the 2 September thresholds of other birth years.
Third, we conducted the identical comparison of individuals around the 2 September 1933 date-of-birth threshold as in our primary analysis, except for starting the follow-up period 7 years before the start of the zoster vaccine program rollout. If there was an intervention that used the 2 September 1933 date-of-birth eligibility threshold but was implemented before the rollout of the zoster vaccine program, then we may expect to see an effect of the September 1933 threshold on dementia incidence in this analysis.
Fourth, we verified that the effects that we observed in our analyses for dementia incidence appear to be specific to dementia. If an intervention that used the exact same date-of-birth eligibility threshold as the zoster vaccine program indeed existed, it would be unlikely to only affect dementia risk without also having an influence on other health outcomes. We therefore conducted the same analysis as for when using dementia incidence as the outcome but for each of the ten leading causes of disability-adjusted life years and mortality in Wales in 2019 for the age group 70+ years33, as well as all conditions that are part of the Charlson Comorbidity Index34.
Fifth, we tested for discontinuities in educational attainment at the 2 September 1933 date-of-birth threshold using data from the 2011 census in Wales74. If an educational policy had used a 2 September (or specifically 2 September 1933) date-of-birth threshold and the policy was effective in increasing educational attainment, we would then expect discontinuities at the 2 September 1933 threshold in the attained education level between eligible and ineligible individuals. We used the identical analysis approach for this balance test as for our primary analyses in the SAIL database, except that we computed ‘honest' confidence intervals based on the approach by Armstrong and Kolesár because the assignment variable (month of birth) in these data was monthly, and therefore coarser than the assignment variable (week of birth) in our analyses in the SAIL database87,88. This approach guards against potential vulnerability to model misspecification and resulting under-coverage of confidence intervals computed with more standard methods. These honest confidence intervals are conservative in the sense that they have good coverage properties irrespective of whether the functional form in the regression discontinuity analysis is misspecified, provided that the true functional form falls within a certain class of functions. For this class, we considered a function class defined by bounds on the second derivative of the conditional expectation function mapping date of birth to the probability attaining a certain educational level. We used conservative bounds of the respective curvatures by relying on global estimation of higher-order polynomials as proposed by Armstrong and Kolesár88.
We additionally used a difference-in-differences instrumental variable approach (DID-IV) to confirm the findings from our regression discontinuity design because, in contrast to the regression discontinuity analysis, this approach does not rely on the continuity assumption (that is, the assumption that potential confounding variables do not abruptly change at precisely the date-of-birth eligibility threshold for the zoster vaccine program). To do so, we restricted our sample to patients born between 1 March 1926 and 28 February 1934. This sample consists of 96,767 adults, of whom 7,752 (8.0%) were eligible for, and 3,949 (4.1%) actually received, zoster vaccination. We then divided our sample into yearly cohorts centred around 1 September (that is, a cohort is all patients born between 1 March of one year and 28 February of the following year). Finally, we divided each yearly cohort into a pre-September birth season and a post-September birth season. Using a difference-in-differences approach, we then compared the outcome (new diagnoses of dementia) between patients born in pre- and post-September birth seasons and across yearly cohorts. More precisely, we tested whether the difference in outcomes across birth seasons is different for the 1933/1934 cohort than for the other cohorts. In doing so, we exploit the fact that zoster vaccination eligibility only differs between the two birth seasons in the 1933/1934-cohort but not in other cohorts, while accounting for the possibility that pre-September and post-September birth seasons may be systematically different for other reasons.
Our difference-in-differences setup implies that the interaction between the post-September birth season indicator and the 1933/1934-cohort indicator constitutes an instrumental variable for receipt of the zoster vaccine, enabling us to estimate the CACE (that is, the effect of actually receiving the vaccine among the compliers). This DID-IV approach relies on two important assumptions. As per the standard exclusion restriction assumption of IV analyses, the IV component of our DID-IV approach assumes that vaccine eligibility affects the outcome solely through a change in actual vaccine receipt. The DID component of our DID-IV approach assumes that, in the absence of the vaccine eligibility rule, the between-birth-season difference in vaccine uptake and in dementia incidence would have been the same in the 1933/1934 cohort as in the other cohorts. To investigate the validity of this assumption, we plotted the mean vaccine uptake and dementia incidence with 95% CIs by birth cohorts and birth seasons (Supplementary Fig. 29). As expected, we find that the between-birth-season differences in vaccine uptake diverge only in the 1933/1934 birth cohort. The absence of a between-birth-season difference in other birth cohorts supports the validity of our DID assumption.
To estimate the CACE in this DID-IV framework, we used two-stage least-squares regression. In the first stage, we identify the vaccine uptake due to the exogeneous change in vaccination eligibility by the following regression equation:
where Vi is a binary variable indicating patient i actually received the zoster vaccine. Si and Ci are binary variables indicating that patient i is born in the post-September birth season and in the 1933/1934 birth cohort, respectively. γ identifies the vaccine uptake due to the change in eligibility. θ, ηm and ηc are the constant term, birth month (January, February, …, December) and birth cohort (1926/1927, 1927/1928, …, 1933/1934) fixed effect, respectively. ϵi is the error term.
In the second stage, we estimate the effect of vaccine receipt by the following regression:
where Yi is the outcome of patient i. \({\widehat{V}}_{i}\) is the probability of vaccine receipt predicted from the first-stage regression (4). The coefficient β identifies the CACE. α, ηm and ηc are the constant term, birth month and birth cohort (1926/1927, 1927/1928, …, 1933/1934) fixed effect, respectively. ϵi is the error term.
We conducted a series of additional robustness checks. First, instead of starting the follow-up period for all individuals on 1 September 2013, we adjusted the follow-up period to account for the staggered rollout of the program by beginning the follow-up period for each individual on the date on which they first became eligible for the zoster vaccine (as described in the ‘Description of the zoster vaccine rollout in Wales' section) (Supplementary Fig. 30). We controlled for cohort fixed effects in these analyses to account for the one- to two-year (depending on the year of the program) differences between cohorts in the calendar year in which this moving follow-up window started. That is, we defined one cohort fixed effect for ineligible individuals and the first catch-up cohort and then included additional cohort fixed effects for each group of patients who became eligible at the same time. Second, we varied our definition of a new diagnosis of dementia by implementing our analysis when defining dementia as a new prescription of donepezil hydrochloride, galantamine, rivastigmine or memantine hydrochloride. Third, we tested whether our results for the effect of being eligible for zoster vaccination on new diagnoses of dementia, shingles and postherpetic neuralgia hold across grace periods (that is, time periods since the index date after which follow-up time is considered to begin to allow for the time needed for a full immune response to develop after vaccine administration) of 0, 2, 4, 6, 8, 10 and 12 months (Supplementary Fig. 31). Fourth, we show our results with bandwidth choices of 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75 and 2.00 times the MSE-optimal bandwidth (Supplementary Fig. 32). Fifth, we verified that our results are similar when using a local second-order polynomial specification instead of local linear regression.
We conducted four analyses to examine this potential effect mechanism, the first three of which are described in detail in the main text. The fourth analysis was an event study that focused on the date of a shingles diagnosis during the follow-up period. Our event study compared the mean outcome in each month relative to the month before the date of the shingles diagnosis. Our regression model controls for changes over time (such as due to ageing of the study population or seasonal patterns in healthcare provider visits) using month-level fixed effects.
To implement our event study, we restricted our study population to those 56,098 individuals born within the MSE-optimal bandwidth of our primary regression discontinuity analysis for dementia. We then aggregated our event-level data into monthly longitudinal data, spanning September 2013 to March 2022. For each outcome of interest (as described in the main text), we then estimated the following event-study regression:
where Yit is the outcome of interest for individual i in period t; shingles is a binary variable equal to one if the individual was diagnosed with shingles during the follow-up period; Dk are indicator variables for the k months before and after the shingles diagnosis (with k = −36, −35,…, 35, 36, and set to zero for individuals who were never diagnosed with shingles during the follow-up period); γk are the coefficients of interest, which capture the difference in the outcome in month k relative to the month before the shingles diagnosis; ηi is an individual-level fixed effect capturing time-invariant differences across individuals; and λt is a month-level fixed effect, capturing differences across periods. We used standard errors that allowed for clustering at the individual level, and therefore for autocorrelation.
We conducted four analyses to examine this potential effect mechanism. First, we implemented the identical regression discontinuity as in our primary analysis, except that we included a binary variable for being diagnosed with shingles during the follow-up period as a covariate. For the resulting estimate to be an unbiased measure of the degree to which the effect of zoster vaccination on dementia incidence is mediated by shingles diagnoses, there must be no variables that are related to both new dementia diagnoses and the probability of being diagnosed with shingles (that is, no confounding of the mediator-to-outcome relationship)89.
Second, to examine when during the follow-up period the dementia-delaying or dementia-preventing effect of zoster vaccination begins to emerge, we plotted Kaplan–Meier plots and (to account for competing risks) cumulative incidence curves among individuals born close to 2 September 1933. This analysis was based on the concept of local randomization28,29, which relies on exchangeability of individuals born immediately before versus immediately after 2 September 1933. To define the bandwidth for our analysis in which we could reasonably assume exchangeability across the threshold while maximizing statistical power, we used the widest bandwidth for which we achieved balance in baseline demographic and clinical characteristics of individuals eligible versus ineligible for zoster vaccination. We evaluated bandwidths ranging from 100% to 10% of the MSE-optimal bandwidth (90.6 weeks) in our primary regression discontinuity analysis in 10% decrements. The variables we used for our balance tests were the 14 variables listed in Supplementary Figs. 1 and 2 (except for the more sex-specific variables of past breast cancer screening, breast cancer and prostate cancer diagnoses) using a significance threshold of P < 0.05, while controlling for the false-discovery rate using the Benjamini–Hochberg procedure90. The largest bandwidth that achieved balance across all variables was 54.4 weeks.
Third, to investigate whether antiviral treatment during a shingles episode was associated with a reduction in the risk of dementia relative to not receiving treatment during a shingles episode, we restricted our study population to those individuals who received a diagnosis of shingles at any time after 1 January 2000 and had not received a diagnosis of dementia before 1 January 2000. Our exposure of interest in this analysis was whether or not an individual received a prescription of antiviral medication (acyclovir, famcyclovir, valacyclovir or inosine pranobex) within three months of the first shingles diagnosis. Individuals were followed up from the date of first shingles diagnosis until either the date of death, moving out of Wales, GP deregistration or end of data availability (1 March 2022). We then used a multivariable Cox proportional hazards model to regress diagnoses of dementia made after the date of the first recorded shingles episode onto whether or not the patient received an antiviral medication prescription for the first shingles episode. In a robustness check, we required that a new diagnosis of dementia must have been made at least 12 months after the date of the first shingles diagnosis. We adjusted our regressions for gender, restricted cubic splines (with three knots) of age at the first shingles infection, and the 12 variables in Supplementary Fig. 1 (excluding past breast and prostate cancer diagnoses).
Fourth, to explore whether experiencing recurrent shingles episodes was associated with a higher risk of dementia than having only a single episode, we used the same study population as in our analysis for treated versus untreated shingles. We matched individuals (via 1:1 propensity score matching) who had more than one shingles diagnosis (with the diagnosis dates having to be at least three months apart) after 1 January 2000 to individuals who only received a single shingles diagnosis after 1 January 2000. We matched individuals on proximity in the date of their first shingles diagnosis as well as the same list of baseline variables as for our analysis of treated versus untreated shingles, and forced an exact match on week of birth and gender. In each matched pair, we used the date of the second shingles diagnosis of the individual with more than one shingles diagnosis as the start date of the follow-up period. Using a Cox proportional hazards model, we then regressed new diagnoses of dementia made during the follow-up period onto whether or not the individual had received more than one shingles diagnosis. In a robustness check, we again required that a new diagnosis of dementia must have been made at least 12 months after the start date of the follow-up period.
To estimate the treatment effect heterogeneities described under this section in the main text, we fully interacted our fuzzy regression discontinuity model with a binary variable that indicates having the condition in question (for example, an autoimmune condition). Precisely, the fully interacted model was specified as:
where the subscript i indexes individuals. Yi is a binary variable equal to 1 if an individual was newly diagnosed with dementia during the follow-up period. The binary variable Vi indicates receipt of the zoster vaccine. The binary variable Di indicates eligibility for the zoster vaccine (that is, born on or after 2 September 1933). The term WOBi − c0 indicates an individual's week of birth centred around the date-of-birth eligibility threshold. The interaction term Di × (WOBi − c0) allows for the slope of the regression line to differ on either side of the date-of-birth eligibility threshold. The binary variable HETi is equal to one if an individual had the condition in question. Adding the terms (WOBi − c0) × HETi and Di × (WOBi − c0) × HETi allows the slopes to vary by this condition.
Vi and Vi × HETi are instrumented by Di and Di × HETi. Using the two-stage least-squares approach, the parameter β4 identifies the effect heterogeneity, that is, the difference in CACE on the outcome between patients with and without the condition. β1 and β1 + β4 identify the effect among compliers in the reference and comparison group, respectively. The estimates of the effects and heterogeneity are reported in absolute terms. To be consistent with our primary fuzzy regression discontinuity model (that is, without the HETi and interaction terms), we used local linear triangular kernel regressions and the MSE-optimal bandwidth from the primary model of the respective outcome.
For our analyses for autoimmune and allergic conditions, we used the 19 most common autoimmune conditions as defined previously91, and grouped the 11 least common conditions among them into a rare conditions category. We judged those conditions to be rare that had an incidence of less than 1% during the follow-up period in our cohort. These rare conditions included Addison's disease, ankylosing spondylitis, Coeliac disease, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, primary biliary cirrhosis, Sjögren's syndrome, systemic lupus erythematosus, systemic sclerosis and vitiligo. For common allergic conditions, we used those defined previously92.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
The data supporting the findings of this study are available from the SAIL Databank26. Researchers must request access to the data directly from SAIL. The authors have no permission to share the data. This paragraph describes how access to the data in the SAIL Databank can be obtained. All proposals to use SAIL data are subject to review by an independent Information Governance Review Panel (IGRP). Before any data can be accessed, approval must be given by the IGRP. The IGRP carefully considers each project to ensure the proper and appropriate use of SAIL data. When access has been granted, it is gained through a privacy-protecting trusted research environment (TRE) and remote access system referred to as the SAIL Gateway. SAIL has established an application process, which includes the payment of a fee, to be followed by anyone who would like to access data through SAIL at https://saildatabank.com/ data/apply-to-work-with-the-data/. Once approved, researchers will have to sign a data access agreement and request a gateway account. After the account is approved, researchers will be able to log into the secure SAIL Gateway remotely. Once logged in, researchers can import our SQL/R code and run the analyses by downloading our replication package (https://osf.io/cfnr6/?view_only=d3774e4fda2649e2b2031431b1234874), uploading the package (SQL and R scripts) to the SAIL Gateway through the secure file upload process, and executing the scripts in the Gateway environment.
All Read and ICD-10 codes to define variables are available in the Supplementary Codes. All statistical packages including version numbers for version control, algorithms to define variables and R analysis code are provided in an OSF repository (https://osf.io/cfnr6/?view_only=d3774e4fda2649e2b2031431b1234874)93.
Wainberg, M. et al. The viral hypothesis: how herpesviruses may contribute to Alzheimer's disease. Mol. Psychiatry 26, 5476–5480 (2021).
PubMed
PubMed Central
MATH
Google Scholar
Devanand, D. P. Viral hypothesis and antiviral treatment in Alzheimer's disease. Curr. Neurol. Neurosci. Rep. 18, 55 (2018).
PubMed
PubMed Central
MATH
Google Scholar
Moir, R. D., Lathe, R. & Tanzi, R. E. The antimicrobial protection hypothesis of Alzheimer's disease. Alzheimers Dement. 14, 1602–1614 (2018).
PubMed
MATH
Google Scholar
Itzhaki, R. F. et al. Microbes and Alzheimer's disease. J. Alzheimers Dis. 51, 979–984 (2016).
PubMed
PubMed Central
MATH
Google Scholar
Hyde, V. R. et al. Anti-herpetic tau preserves neurons via the cGAS-STING-TBK1 pathway in Alzheimer's disease. Cell Rep. 44, 115109 (2025).
Benn, C. S., Fisker, A. B., Rieckmann, A., Sørup, S. & Aaby, P. Vaccinology: time to change the paradigm? Lancet Infect. Dis. 20, e274–e283 (2020).
PubMed
Google Scholar
Benn, C. S. et al. Implications of non-specific effects for testing, approving, and regulating vaccines. Drug Saf. 46, 439–448 (2023).
PubMed
PubMed Central
MATH
Google Scholar
Aaby, P. et al. The non-specific and sex-differential effects of vaccines. Nat. Rev. Immunol. 20, 464–470 (2020).
PubMed
PubMed Central
MATH
Google Scholar
McGovern, M. E. & Canning, D. Vaccination and all-cause child mortality from 1985 to 2011: global evidence from the Demographic and Health Surveys. Am. J. Epidemiol. 182, 791–798 (2015).
PubMed
PubMed Central
MATH
Google Scholar
Michalik, F. et al. The effect of herpes zoster vaccination on the occurrence of deaths due to dementia in England and Wales. Preprint at medRxiv https://doi.org/10.1101/2023.09.08.23295225 (2023).
Wu, X. et al. Adult vaccination as a protective factor for dementia: a meta-analysis and systematic review of population-based observational studies. Front. Immunol. 13, 872542 (2022).
PubMed
PubMed Central
Google Scholar
Hernan, M. A. & Robins, J. M. Causal Inference: What If (Chapman & Hall/CRC, 2020).
Simonsen, L., Taylor, R. J., Viboud, C., Miller, M. A. & Jackson, L. A. Mortality benefits of influenza vaccination in elderly people: an ongoing controversy. Lancet Infect. Dis. 7, 658–666 (2007).
PubMed
Google Scholar
Shingles: Guidance and Vaccination Programme www.gov.uk/government/collections/shingles-vaccination-programme (UK Health Security Agency, 2021).
Harris, K. et al. The impact of routine vaccinations on Alzheimer's disease risk in persons 65 years and older: a claims-based cohort study using propensity score matching. J. Alzheimers Dis. 95, 703–718 (2023).
PubMed
PubMed Central
Google Scholar
Lophatananon, A. et al. Shingles, Zostavax vaccination and risk of developing dementia: a nested case-control study-results from the UK Biobank cohort. BMJ Open 11, e045871 (2021).
PubMed
PubMed Central
Google Scholar
Scherrer, J. F. et al. Impact of herpes zoster vaccination on incident dementia: A retrospective study in two patient cohorts. PLoS ONE 16, e0257405 (2021).
PubMed
PubMed Central
MATH
Google Scholar
Schnier, C., Janbek, J., Lathe, R. & Haas, J. Reduced dementia incidence after varicella zoster vaccination in Wales 2013–2020. Alzheimers Dement. 8, e12293 (2022).
Google Scholar
Wiemken, T. L. et al. Comparison of rates of dementia among older adult recipients of two, one, or no vaccinations. J. Am. Geriatr. Soc. 70, 1157–1168 (2022).
PubMed
MATH
Google Scholar
Lophatananon, A. et al. The association of herpes zoster and influenza vaccinations with the risk of developing dementia: a population-based cohort study within the UK Clinical Practice Research Datalink. BMC Publ. Health 23, 1903 (2023).
MATH
Google Scholar
Ukraintseva, S. et al. Associations of infections and vaccines with Alzheimer's disease point to a role of compromised immunity rather than specific pathogen in AD. Exp. Gerontol. 190, 112411 (2024).
PubMed
PubMed Central
MATH
Google Scholar
Lehrer, S. & Rheinstein, P. H. Herpes zoster vaccination reduces risk of dementia. In Vivo 35, 3271–3275 (2021).
PubMed
PubMed Central
MATH
Google Scholar
Wilkinson, T. et al. Drug prescriptions and dementia incidence: a medication-wide association study of 17000 dementia cases among half a million participants. J. Epidemiol. Community Health 76, 223–229 (2022).
PubMed
MATH
Google Scholar
Tang, E., Ray, I., Arnold, B. F. & Acharya, N. R. Recombinant zoster vaccine and the risk of dementia. Vaccine 46, 126673 (2024).
PubMed
Google Scholar
Introduction of Shingrix Vaccine for the Whole Programme and Expansion of Eligible Cohorts Letter https://www.gov.uk/government/publications/shingles-vaccination-programme-changes-from-september-2023-letter/introduction-of-shingrix-vaccine-for-the-whole-programme-and-expansion-of-eligible-cohorts-letter (UK Health Security Agency, 2023).
SAIL Databank saildatabank.com/ (Swansea University, 2025).
Attribution Data Set GP-Registered Populations Scaled to ONS Population Estimates—2011 digital.nhs.uk/data-and-information/publications/statistical/attribution-dataset-gp-registered-populations/attribution-data-set-gp-registered-populations-scaled-to-ons-population-estimates-2011 (NHS Digital, 2012).
Cattaneo, M. D., Keele, L. & Titiunik, R. A guide to regression discontinuity designs in medical applications. Stat. Med. 42, 4484–4513 (2023).
MathSciNet
PubMed
MATH
Google Scholar
Cattaneo, M. D., Idrobo, N. & Titiunik, R. A Practical Introduction to Regression Discontinuity Designs: Foundations (Cambridge Univ. Press, 2020).
Harbecke, R., Cohen, J. I. & Oxman, M. N. Herpes zoster vaccines. J. Infect. Dis. 224, S429–S442 (2021).
PubMed
PubMed Central
Google Scholar
2024 Alzheimer's disease facts and figures. Alzheimers Dement. 20, 3708–3821 (2024).
Walters, K. et al. Predicting dementia risk in primary care: development and validation of the Dementia Risk Score using routinely collected data. BMC Med. 14, 6 (2016).
PubMed
PubMed Central
MATH
Google Scholar
Global Burden of Disease Project 2019. GBD Compare vizhub.healthdata.org/gbd-compare (Institute for Health Metrics and Evaluation, 2020).
Charlson, M. E., Pompei, P., Ales, K. L. & MacKenzie, C. R. A new method of classifying prognostic comorbidity in longitudinal studies: development and validation. J. Chronic Dis. 40, 373–383 (1987).
PubMed
Google Scholar
Oxman, M. N. Zoster vaccine: current status and future prospects. Clin. Infect. Dis. 51, 197–213 (2010).
PubMed
MATH
Google Scholar
Oxman, M. N. et al. A vaccine to prevent herpes zoster and postherpetic neuralgia in older adults. N. Engl. J. Med. 352, 2271–2284 (2005).
PubMed
MATH
Google Scholar
Campling, J. et al. A review of evidence for pneumococcal vaccination in adults at increased risk of pneumococcal disease: risk group definitions and optimization of vaccination coverage in the United Kingdom. Expert Rev. Vaccines 22, 785–800 (2023).
PubMed
MATH
Google Scholar
Yun, H. et al. Risk of herpes zoster in autoimmune and inflammatory diseases: implications for vaccination. Arthritis Rheumatol. 68, 2328–2337 (2016).
PubMed
PubMed Central
MATH
Google Scholar
Marra, F., Parhar, K., Huang, B. & Vadlamudi, N. Risk factors for herpes zoster infection: a meta-analysis. Open Forum Infect. Dis. 7, ofaa005 (2020).
PubMed
PubMed Central
Google Scholar
Joesoef, R. M., Harpaz, R., Leung, J. & Bialek, S. R. Chronic medical conditions as risk factors for herpes zoster. Mayo Clin. Proc. 87, 961–967 (2012).
PubMed
PubMed Central
MATH
Google Scholar
Kawai, K. & Yawn, B. P. Risk factors for herpes zoster: a systematic review and meta-analysis. Mayo Clin. Proc. 92, 1806–1821 (2017).
PubMed
MATH
Google Scholar
McGonagle, D. & McDermott, M. F. A proposed classification of the immunological diseases. PLoS Med. 3, e297 (2006).
PubMed
PubMed Central
MATH
Google Scholar
Gandhi, N. A. et al. Targeting key proximal drivers of type 2 inflammation in disease. Nat. Rev. Drug Discov. 15, 35–50 (2016).
PubMed
MATH
Google Scholar
Shingles Vaccine Coverage (England): Report for Quarter 1 of the Financial Year 2022 to 2023 www.gov.uk/government/publications/ herpes-zoster-shingles- immunisation-programme-2022-to-2023- evaluation-reports/shingles-vaccine- coverage-england-report-for-quarter-1- of-the-financial-year-2022-to-2023 (UK Health Security Agency, 2024).
Population and Household estimates, England and Wales: Census 2021, Unrounded Data www.ons.gov.uk/peoplepopulationandcommunity/ populationandmigration/populationestimates/ bulletins/ populationandhouseholdestimatesenglandandwales/ census2021unroundeddata (ONS, 2022).
Fischinger, S., Boudreau, C. M., Butler, A. L., Streeck, H. & Alter, G. Sex differences in vaccine-induced humoral immunity. Semin. Immunopathol. 41, 239–249 (2019).
PubMed
Google Scholar
Ferretti, M. T. et al. Sex and gender differences in Alzheimer's disease: current challenges and implications for clinical practice: position paper of the Dementia and Cognitive Disorders Panel of the European Academy of Neurology. Eur. J. Neurol. 27, 928–943 (2020).
PubMed
MATH
Google Scholar
Silver, B. et al. Varicella zoster virus vasculopathy: a treatable form of rapidly progressive multi-infarct dementia after 2 years' duration. J. Neurol. Sci. 323, 245–247 (2012).
PubMed
PubMed Central
Google Scholar
Gilden, D. et al. Successful antiviral treatment after 6years of chronic progressive neurological disease attributed to VZV brain infection. J. Neurol. Sci. 368, 240–242 (2016).
PubMed
PubMed Central
Google Scholar
Bubak, A. N. et al. Targeted RNA sequencing of VZV-infected brain vascular adventitial fibroblasts indicates that amyloid may be involved in VZV vasculopathy. Neurol. Neuroimmunol. Neuroinflamm. 9, e1103 (2022).
PubMed
Google Scholar
Nagel, M. A. & Bubak, A. N. Varicella zoster virus vasculopathy. J. Infect. Dis. 218, S107–S112 (2018).
PubMed
PubMed Central
Google Scholar
Nagel, M. A. et al. The varicella zoster virus vasculopathies: clinical, CSF, imaging, and virologic features. Neurology 70, 853–860 (2008).
PubMed
MATH
Google Scholar
Nagel, M. A. et al. Varicella zoster virus vasculopathy: analysis of virus-infected arteries. Neurology 77, 364–370 (2011).
PubMed
PubMed Central
Google Scholar
Nagel, M. A. et al. Varicella-zoster virus vasculopathy: immune characteristics of virus-infected arteries. Neurology 80, 62–68 (2013).
PubMed
PubMed Central
MATH
Google Scholar
Attems, J. & Jellinger, K. A. The overlap between vascular disease and Alzheimer's disease-lessons from pathology. BMC Med. 12, 206 (2014).
PubMed
PubMed Central
Google Scholar
Boyle, P. A. et al. Cerebral amyloid angiopathy and cognitive outcomes in community-based older persons. Neurology 85, 1930–1936 (2015).
PubMed
PubMed Central
MATH
Google Scholar
Cairns, D. M., Itzhaki, R. F. & Kaplan, D. L. Potential involvement of varicella zoster virus in Alzheimer's disease via reactivation of quiescent herpes simplex virus type 1. J. Alzheimers Dis. 88, 1189–1200 (2022).
PubMed
Google Scholar
Bukhbinder, A. S., Ling, Y., Harris, K., Jiang, X. & Schulz, P. E. Do vaccinations influence the development of Alzheimer disease? Hum. Vaccin. Immunother. 19, 2216625 (2023).
PubMed
PubMed Central
Google Scholar
Anderson, M. et al. United Kingdom: health system review. Health Syst. Transit. 24, 1–194 (2022).
PubMed
MATH
Google Scholar
Ford, D. V. et al. The SAIL Databank: building a national architecture for e-health research and evaluation. BMC Health Serv. Res. 9, 157 (2009).
PubMed
PubMed Central
MATH
Google Scholar
Jones, K. H. et al. A case study of the Secure Anonymous Information Linkage (SAIL) Gateway: a privacy-protecting remote access system for health-related research and evaluation. J. Biomed. Inform. 50, 196–204 (2014).
Lyons, R. A. et al. The SAIL databank: linking multiple health and social care datasets. BMC Med. Inform. Decis. Mak. 9, 3 (2009).
Rodgers, S. E., Demmler J., Dsilva R. & Lyons R. Protecting health data privacy while using residence-based environment and demographic data. Health Place 18, 209–217 (2012).
Rodgers, S. E. et al. Residential Anonymous Linking Fields (RALFs): a novel information infrastructure to study the interaction between the environment and individuals' health. J. Public Health 31, 582–588 (2009).
Welsh Demographic Service Dataset (WDSD) web.www.healthdatagateway.org/dataset/cea328df-abe5-48fb-8bcb-c0a5b6377446# (Digital Health and Care Wales, 2022).
Welsh Longitudinal General Practice Dataset (WLGP)—Welsh Primary Care web.www.healthdatagateway.org/dataset/33fc3ffd-aa4c-4a16-a32f-0c900aaea3d2 (SAIL Databank, 2023).
Benson, T. The history of the Read Codes: the inaugural James Read Memorial Lecture 2011. Inform. Prim. Care 19, 173–182 (2011).
PubMed
MATH
Google Scholar
Patient Episode Database for Wales dhcw.nhs.wales/information-services/health-intelligence/pedw-data-online/ (Digital Health and Care Wales, 2020).
OPCS Classification of Interventions and Procedures www.datadictionary.nhs.uk/supporting_information/opcs_classification_of_interventions_and_procedures.html (NHS England, 2023).
ICD-10: International Statistical Classification of Diseases and Related Health Problems: Tenth Revision apps.who.int/iris/handle/10665/42980 (WHO, 2004).
Outpatient Database for Wales (OPDW) web.www.healthdatagateway.org/dataset/d331159b-b286-4ab9-8b36-db39123ec229 (Digital Health and Care Wales, 2022).
Welsh Cancer Intelligence and Surveillance Unit (WCISU) phw.nhs.wales/services-and-teams/welsh-cancer-intelligence-and-surveillance-unit-wcisu/ (Public Health Wales, 2023).
Annual District Death Extract (ADDE) web.www.healthdatagateway.org/dataset/15cf4241-abad-4dcc-95b0-8cd7c02be999 (Digital Health and Care Wales, 2023).
CT1303_2011 Census www.ons.gov.uk/peoplepopulationandcommunity/populationandmigration/populationestimates/adhocs/2089ct13032011census (ONS, 2024).
Bricout, H., Haugh, M., Olatunde, O. & Prieto, R. G. Herpes zoster-associated mortality in Europe: a systematic review. BMC Public Health 15, 466 (2015).
PubMed
PubMed Central
Google Scholar
Stoltenberg, E. A. Regression discontinuity design with right-censored survival data. Preprint at arxiv.org/abs/2210.02548 (2022).
Alzheimer's Association. 2022 Alzheimer's disease facts and figures. Alzheimers Dement. 18, 700–789 (2022).
MATH
Google Scholar
Boyle, P. A. et al. Attributable risk of Alzheimer's dementia attributed to age-related neuropathologies. Ann. Neurol. 85, 114–124 (2019).
PubMed
MATH
Google Scholar
Boyle, P. A. et al. Person-specific contribution of neuropathologies to cognitive loss in old age. Ann. Neurol. 83, 74–83 (2018).
PubMed
PubMed Central
MATH
Google Scholar
Villamizar-Villegas, M., Pinzon-Puerto, F. A. & Ruiz-Sanchez, M. A. A comprehensive history of regression discontinuity designs: an empirical survey of the last 60 years. J. Econ. Surv. 36, 1130–1178 (2022).
Gelman, A. & Imbens, G. Why high-order polynomials should not be used in regression discontinuity designs. J. Bus. Econ. Stat. https://doi.org/10.1080/07350015.2017.1366909 (2017).
Imbens, G. & Kalyanaraman, K. Optimal bandwidth choice for the regression discontinuity estimator. Rev. Econ. Stud. 79, 933–959 (2012).
MathSciNet
MATH
Google Scholar
Calonico, S., Cattaneo, M. D. & Titiunik, R. Robust nonparametric confidence intervals for regression-discontinuity designs. Econometrica 82, 2295–2326 (2014).
MathSciNet
MATH
Google Scholar
Imbens, G. W. & Lemieux, T. Regression discontinuity designs: a guide to practice. J. Econometr. 142, 615–635 (2008).
MathSciNet
MATH
Google Scholar
Bor, J., Moscoe, E., Mutevedzi, P., Newell, M. L. & Barnighausen, T. Regression discontinuity designs in epidemiology: causal inference without randomized trials. Epidemiology 25, 729–737 (2014).
PubMed
PubMed Central
Google Scholar
Oehlert, G. W. A note on the delta method. Am. Stat. 46, 27–29 (1992).
MathSciNet
MATH
Google Scholar
Kolesár, M. & Rothe, C. Inference in regression discontinuity designs with a discrete running variable. Am. Econ. Rev. 108, 2277–2304 (2018).
MATH
Google Scholar
Armstrong, T. B. & Kolesár, M. Simple and honest confidence intervals in nonparametric regression. Quant. Econ. 11, 1–39 (2020).
MathSciNet
MATH
Google Scholar
VanderWeele, T. J. Mediation analysis: a practitioner's guide. Ann. Rev. Publ. Health 37, 17–32 (2016).
MATH
Google Scholar
Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57, 289–300 (1995).
MathSciNet
MATH
Google Scholar
Conrad, N. et al. Autoimmune diseases and cardiovascular risk: a population-based study on 19 autoimmune diseases and 12 cardiovascular diseases in 22 million individuals in the UK. Lancet 400, 733–743 (2022).
PubMed
Google Scholar
Mukherjee, M., Wyatt, J. C., Simpson, C. R. & Sheikh, A. Usage of allergy codes in primary care electronic health records: a national evaluation in Scotland. Allergy 71, 1594–1602 (2016).
PubMed
Google Scholar
Xie, M. et al. A natural experiment on the effect of herpes zoster vaccination on dementia. Preprint at https://osf.io/cfnr6/?view_only=d3774e4fda2649e2b2031431b1234874 (2025).
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This study makes use of anonymized data held in the SAIL Databank. We thank the members of the SAIL Databank analytical services team for continuous advice and support throughout all stages of the project. We acknowledge all the data providers who made anonymized data available for research. The responsibility for the interpretation of the data supplied by SAIL is that of the authors alone. SAIL bears no responsibility for the further analysis or interpretation of their data, over and above that published by SAIL. This study was funded by grants to P.G. by The Phil & Penny Knight Initiative for Brain Resilience at the Wu Tsai Neurosciences Institute, Stanford University (KPI-003), the National Institute on Aging (R01AG084535), National Institute of Allergy and Infectious Diseases (DP2AI171011) and Chan Zuckerberg Biohub–San Francisco.
These authors contributed equally: Markus Eyting, Min Xie
Division of Primary Care and Population Health, Department of Medicine, Stanford University, Stanford, CA, USA
Markus Eyting, Min Xie, Felix Michalik, Seunghun Chung & Pascal Geldsetzer
Leibniz Institute for Financial Research SAFE, Frankfurt am Main, Germany
Markus Eyting
Faculty of Law and Economics, Johannes Gutenberg University Mainz, Mainz, Germany
Markus Eyting
Heidelberg Institute of Global Health (HIGH), Heidelberg University Hospital, Heidelberg, Germany
Min Xie & Felix Michalik
Department of Economics, Vienna University of Economics and Business, Vienna, Austria
Simon Heß
Department of Epidemiology and Population Health, Stanford University, Stanford, CA, USA
Pascal Geldsetzer
The Phil and Penny Knight Initiative for Brain Resilience at the Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA
Pascal Geldsetzer
Chan Zuckerberg Biohub – San Francisco, San Francisco, CA, USA
Pascal Geldsetzer
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M.E. and M.X. contributed equally to this work. M.E. co-conceived the study, devised the methodology, analysed and processed the data, created data visualizations, interpreted the results, wrote the Methods section of the original draft, and reviewed and edited the original draft. M.X. co-conceived the study, devised the methodology, analysed and processed the data, created data visualizations, interpreted the results, wrote the Methods section of the original draft, and reviewed and edited the original draft. F.M. interpreted the results, and reviewed and edited the original draft. S.H. devised the methodology, consulted on the data analysis, wrote the Methods section of the original draft, interpreted the results, and reviewed and edited the original draft. S.C. interpreted the results, and reviewed and edited the original draft. P.G. conceived the overall project, acquired funding, co-conceived the study, devised the methodology, was responsible for administration and supervision, interpreted the results and wrote the original draft.
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Green oasis: during a time known as the African Humid Period, it's thought that the Sahara was a lush savannah.Credit: Henrik Karlsson/Getty
The Sahara Desert has not always been the arid, inhospitable landscape we know today. Between 14,500 and 5,000 years ago, the area was unrecognizable, transformed into a lush savannah by an unusually wet interval called the African Humid Period. People roamed this green landscape for thousands of years before it was again lost to sand.
From Vikings to Beethoven: what your DNA says about your ancient relatives
From Vikings to Beethoven: what your DNA says about your ancient relatives
Ancient DNA extracted from two women who died in what is now Libya around 7,000 years ago is now helping researchers to reconstruct the origins of these early Saharans. The women's DNA profiles, described in a study published on 2 April in Nature1, represent the first full Saharan genomes from the African Humid Period — and reveal that the people were remarkably isolated from other African populations.
“The prehistory of North Africa is a big puzzle, and we only have a few pieces available,” says Rosa Fregel, a geneticist at the University of La Laguna in San Cristobal, Spain, who was not involved in the research. The work is “a significant contribution to the palaeogenomics of North Africa”, she says.
Ancient genomes from North Africa are hard to come by. Almost all palaeogenetic work is concentrated in Europe and Asia. Ancient DNA is especially rare in the Sahara, where high temperatures and strong ultraviolet light quickly degrade genetic material in remains.
The Takarkori rock shelter in Libya, where the remains were unearthed.Credit: University of Rome La Sapienza
That's why it's important to explore sites that are protected from the elements, says Nada Salem, an archaeologist at the Max Plank Institute for Evolutionary Anthropology in Leipzig, Germany.
One such site is the Takarkori rock shelter in southwestern Libya. Between 2003 and 2007, archaeologists uncovered the remains of 15 people who were buried between 8,900 and 4,800 years ago at Takarkori. Two of the corpses — both belonging to women who lived between 7,000 and 6,000 years ago — had naturally mummified.
Archaeological evidence at the site suggested that the Takarkori women belonged to a group of herders who appeared in the region around 8,000 years ago. This marked a major transition in the way of life of early Saharans, who had previously all been hunter-gatherers. Some researchers have suggested that Saharans learnt herding by intermarrying with people who were migrating into North Africa from the Levant.
To test this, Salem and her colleagues sequenced the Takarkori genomes and compared the DNA to that of around 800 modern humans and 117 ancient genomes from around Africa, southern Europe and the Middle East. The team found that the Takarkori women had only small traces of Levant ancestry — suggesting that any intermingling had happened long before the advent of herding in the region.
What's more, the analysis struggled to connect these early Saharans to any other ancient group. “This was puzzling for us. How is it that this lineage has not spread either to the east or the west or to the south?” says Salem.
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doi: https://doi.org/10.1038/d41586-025-01020-3
Read the related News & Views article: ‘Rare ancient DNA from Sahara opens a window on the region's verdant past'
Salem, N. et al. Nature https://doi.org/10.1038/s41586-025-08793-7 (2025).
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Read the paper: Ancient DNA from the Green Sahara reveals ancestral North African lineage
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Ancient DNA from the Green Sahara reveals ancestral North African lineage
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Seismic mapping of North America has revealed that an ancient slab of crust buried beneath the Midwest is causing the crust above it to "drip" and suck down rocks from across the continent.
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An ancient slab of Earth's crust buried deep beneath the Midwest is sucking huge swatches of present-day's North American crust down into the mantle, researchers say.
The slab's pull has created giant "drips" that hang from the underside of the continent down to about 400 miles (640 kilometers) deep inside the mantle, according to a new study. These drips are located beneath an area spanning from Michigan to Nebraska and Alabama, but their presence appears to be impacting the entire continent.
The dripping area looks like a large funnel, with rocks from across North America being pulled toward it horizontally before getting sucked down. As a result, large parts of North America are losing material from the underside of their crust, the researchers said.
"A very broad range is experiencing some thinning," study lead author Junlin Hua, a geoscientist who conducted the research during a postdoctoral fellowship at The University of Texas (UT) at Austin, said in a statement. "Luckily, we also got the new idea about what drives this thinning," said Hua, now a professor at the University of Science and Technology of China.
Related: Earth's crust is peeling away under California
The researchers found that the drips result from the downward dragging force of a chunk of oceanic crust that broke off from an ancient tectonic plate called the Farallon plate.
The Farallon plate and the North American plate once formed a subduction zone along the continent's west coast, with the former sliding beneath the latter and recycling its material into the mantle. The Farallon plate splintered due to the advance of the Pacific plate roughly 20 million years ago, and remnant slabs subducted beneath the North American plate slowly drifted off.
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One of these slabs currently straddles the boundary between the mantle transition zone and the lower mantle roughly 410 miles (660 km) beneath the Midwest. Dubbed the "Farallon slab" and first imaged in the 1990s, this piece of oceanic crust is responsible for a process known as "cratonic thinning," according to the new study, which was published March 28 in the journal Nature Geoscience.
Cratonic thinning refers to the wearing away of cratons, which are regions of Earth's continental crust and upper mantle that have mostly remained intact for billions of years. Despite their stability, cratons can undergo changes, but this has never been observed in action due to the huge geologic time scales involved, according to the study.
Now, for the first time, researchers have documented cratonic thinning as it occurs. The discovery was possible thanks to a wider project led by Hua to map what lies beneath North America using a high-resolution seismic imaging technique called "full-waveform inversion." This technique uses different types of seismic waves to extract all the available information about physical parameters underground.
"This sort of thing is important if we want to understand how a planet has evolved over a long time," study co-author Thorsten Becker, a distinguished chair in geophysics at UT Austin, said in the statement. "Because of the use of this full-waveform method, we have a better representation of that important zone between the deep mantle and the shallower lithosphere [crust and upper mantle]."
—Scientists discover 'sunken worlds' hidden deep within Earth's mantle that shouldn't be there
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To test their results, the researchers simulated the impact of the Farallon slab on the craton above using a computer model. A dripping area formed when the slab was present, but it disappeared when the slab was absent, confirming that — theoretically, at least — a sunken slab can drag rocks across a large area down into Earth's interior.
Dripping beneath the Midwest won't lead to changes at the surface anytime soon, the researchers said, adding that it may even stop as the Farallon slab sinks deeper into the lower mantle and its influence over the craton wanes.
The findings could help researchers piece together the enormous puzzle of how Earth came to look the way it does today. "It helps us understand how do you make continents, how do you break them, and how do you recycle them," Becker said.
Sascha is a U.K.-based staff writer at Live Science. She holds a bachelor's degree in biology from the University of Southampton in England and a master's degree in science communication from Imperial College London. Her work has appeared in The Guardian and the health website Zoe. Besides writing, she enjoys playing tennis, bread-making and browsing second-hand shops for hidden gems.
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Although it is one of the most arid regions today, the Sahara Desert was a green savannah during the African Humid Period (AHP) between 14,500 and 5,000 years before present, with water bodies promoting human occupation and the spread of pastoralism in the middle Holocene epoch1. DNA rarely preserves well in this region, limiting knowledge of the Sahara's genetic history and demographic past. Here we report ancient genomic data from the Central Sahara, obtained from two approximately 7,000-year-old Pastoral Neolithic female individuals buried in the Takarkori rock shelter in southwestern Libya. The majority of Takarkori individuals' ancestry stems from a previously unknown North African genetic lineage that diverged from sub-Saharan African lineages around the same time as present-day humans outside Africa and remained isolated throughout most of its existence. Both Takarkori individuals are closely related to ancestry first documented in 15,000-year-old foragers from Taforalt Cave, Morocco2, associated with the Iberomaurusian lithic industry and predating the AHP. Takarkori and Iberomaurusian-associated individuals are equally distantly related to sub-Saharan lineages, suggesting limited gene flow from sub-Saharan to Northern Africa during the AHP. In contrast to Taforalt individuals, who have half the Neanderthal admixture of non-Africans, Takarkori shows ten times less Neanderthal ancestry than Levantine farmers, yet significantly more than contemporary sub-Saharan genomes. Our findings suggest that pastoralism spread through cultural diffusion into a deeply divergent, isolated North African lineage that had probably been widespread in Northern Africa during the late Pleistocene epoch.
Following the last glacial period, a climatic transformation in the Sahara desert led to the AHP, which peaked around 11,000 to 5,000 years ago3,4. During this period of increased humidity, the region transformed into a ‘Green Sahara' with savanna-like landscapes, varying tree cover, permanent lakes and extensive river systems5 (Fig. 1). Evidence from ancient lake deposits, pollen samples and archaeological artifacts confirm human presence, hunting, herding and resource gathering in the currently arid desert region1,6,7. However, despite this rich history, much about the genetic history of the human population of the Green Sahara remains unclear due to limited DNA preservation under the current climatic conditions. Ancient DNA data from northwestern Africa points to a stable and isolated genetic population from at least 15,000 to 7,500 years ago2,8. This stability was disrupted by the arrival of early farming groups from southwestern Europe between 7,500 and 5,700 years ago who marked the beginning of the Neolithic in the Maghreb by introducing farming practices to the local foragers9. The earliest herders with their livestock entered Africa probably along the Sinai and the Red Sea routes, after which they rapidly spread into northeastern Africa and reached the Central Sahara around 8,300 years ago10. By 6,400 years ago, further gene flow occurred with the appearance of ancestry associated with Neolithic groups from the Levant, whose archaeological signatures are visible in the Eastern Sahara9,11,12. A previous study13 analysed mitochondrial DNA (mtDNA) from individuals recovered from the Takarkori rock shelter in the Tadrart Acacus Mountains of southwestern Libya—the same individuals examined in this study—providing the first ancient DNA from pastoralists of the Green Sahara. However, non-recombining and therefore effectively single genetic loci like mtDNA have much less statistical power to reveal population dynamics than genome-wide autosomal data. Their origins and whether the arrival of pastoralism into the Green Sahara was linked to the movement of peoples from the Levant or rather cultural diffusion remain a matter of debate10,14.
a, Timeline of climate phases and subsistence strategies during the late Pleistocene and the Holocene in North-East Africa and Central Sahara. The radiocarbon dates for both Takarkori individuals are given by the black diamond and circle. b,c, The distribution of ecozones in Northern Africa in the Green Sahara period during the early Holocene 9,000 years ago (b) and in recent times (1901–1930) (c) using the dynamic vegetation model Carbon Assimilation in the Biosphere (CARAIB). The location of the Takarkori rock shelter site is marked with a black square. The maps are adapted from refs. 20 and 60 under a Creative Commons licence CC BY 4.0.
Here we present first genome-wide data obtained from the same two approximately 7,000-year-old Saharan herders, recovered from the Takarkori rock shelter in the Central Sahara (Supplementary Figs. 1.2–1.5), a site that has yielded an exceptional wealth of data and material remains15,16,17,18,19. Our findings show that these individuals predominantly carry a previously unknown ancestral North African lineage that lacks the Neanderthal admixture typically found outside Africa and appears to have remained largely isolated, with the notable exception of small traces of Levantine admixture. These results support that pastoralism in the Sahara was established through cultural diffusion10 rather than significant human gene flow. Furthermore, the Takarkori individuals exhibit a close genetic affinity to Northwestern African foragers but no substantial ties with sub-Saharan African lineages, implying no detectable genetic exchange across the Green Sahara during the AHP from sub-Saharan to northern Africa. For a non-peer-reviewed Arabic summary of the article, see Supplementary Note 3.
The Takarkori rock shelter—situated in southwestern Libya's Tadrart Acacus Mountains—offers a remarkable glimpse into the Sahara's greener past15,20. The excavations at this archaeological site revealed a timeline of human settlement from Late Acacus hunter-gatherer-fishers from around 10,200 calibrated years before the present (cal. bp) to a long Pastoral Neolithic occupation, dated from approximately 8,300 to 4,200 cal. bp10,21. Data from these latter periods traces the sociocultural trajectories of Neolithic herding societies in Central Sahara, from early livestock introduction to the development of a full pastoral economy characterized by transhumance and the use of secondary product22,23 (Supplementary Note 1). Within the deepest recess of the rock shelter, 15 human burials were unearthed, pertaining to a timeframe between approximately 8,900 and 4,800 cal. bp22, with the majority dating back to the Early (8,300–7,300 cal. bp) and Middle (7,100–5,600 cal. bp) Pastoral period22. Strontium isotope analysis on the remains, primarily of women of reproductive age, children and juveniles, indicated a local geographical origin24. Two naturally mummified adult female individuals, attributed to the Middle Pastoral Period, were selected for our DNA analysis. These individuals were directly radiocarbon dated to 7,158–6,796 and 6,555–6,281 cal. bp (95.4% probability), respectively13 (Supplementary Note 1).
We extracted DNA from the powdered tooth root from individual TKH001 and from two fibula fragments from TKH009. Given their extremely low endogenous human DNA content (0.085–1.363%; Supplementary Table 2.1), we opted for a DNA capture approach to cost-effectively retrieve informative single-nucleotide polymorphisms (SNPs) for autosomal analysis. Sampling of other skeletal elements in the future may provide DNA extracts with higher percentage of endogenous DNA that allow for whole-genome shotgun sequencing. Using dedicated ancient DNA protocols (Methods), we prepared DNA libraries and enriched them through a DNA hybridization approach with the Twist Ancient DNA panel25 targeting 1.4 million SNPs for TKH001 and the 1240K panel26 targeting 1.2 million SNPs for TKH009. Despite challenging preservation conditions, sequencing yielded 881,765 SNPs for TKH001 and 23,317 SNPs for TKH009 (Supplementary Table 2.4). For TKH001, we conducted additional enrichment for 1.7 million SNPs, targeting sites informative about Neanderthal and Denisovan admixture (Archaic Admixture SNP capture panel)27. DNA sequences from both Takarkori individuals had a post-mortem degradation pattern typical of ancient DNA and low contamination estimates (Supplementary Figs. 2.1, 2.2 and Supplementary Table 2.3).
To visualize the variation in the genetic ancestry of the Takarkori individuals, we performed a principal component analysis (PCA) using genome-wide data from 795 present-day individuals from the whole African continent, the Near East and Southern Europe, all genotyped on the Human Origins SNP panel28,29,30,31,32,33,34,35,36,37,38 (Supplementary Data 1 and 2). We then projected the Takarkori individuals and 117 relevant published ancient genomes2,8,9,35,36,39,40,41,42,43,44 (Supplementary Data 2) onto the first two principal components (PCs). The Takarkori individuals plot broadly intermediate between West African and Near Eastern groups on PC1, albeit closer to West Africans (Extended Data Fig. 1, Supplementary Fig. 2.6 and Supplementary Data 3). To obtain a finer-scale view, we restricted the African populations to West Africa, the Sahel and East Africa, while retaining the Near Eastern and Southern European populations (Supplementary Data 3). This PCA captured the geographical distributions of these populations, whereby PC1 separates African from non-African populations and PC2 differentiates within Africa, particularly separating Sahel/West African from East African populations (Extended Data Fig. 2). Both Takarkori individuals maintained a distinct position, intermediate between Sahel/West and East African populations (Fig. 2a), and formed a tight cluster with overlapping 95% confidence interval (CI) ellipses (Supplementary Fig. 2.4). The PCA projections broadly mirror geography, including the placement of Takarkori. For subsequent population genetic analyses, we merged the low-coverage data from the TKH009 individual with the high-coverage data from the TKH001 individual. We note that many of the signals in this group analysis are probably driven by TKH001 owing to its much higher coverage.
a, PCA with projecting key ancient groups from the region (Supplementary Data 3). b, The geographical locations of ancient genomes from Africa and the Near East included in our analysis. ChL, Chalcolithic; EN, Early Neolithic; EpiPalaeo, Epipalaeolithic; IA, Iron Age; IAM, Ifri n'Amr o'Moussa; KEB, Kehf el Baroud; KTG, Kaf Taht el-Ghar; LIA, Late Iron Age; LN, Late Neolithic; MN, Middle Neolithic; OUB, Ifri Ouberrid; Palaeo, Palaeolithic; SKH, Skhirat-Rouazi.
To probe for shared genetic drift of Takarkori with other ancient and modern genomes, we computed outgroup f3 statistics30 of the form f3(Takarkori, X; South Africa 2,000 cal. bp), with X representing worldwide ancient and present-day test populations and South Africa 2,000 cal. bp as the deepest modern human outgroup lineage44. We found that the Takarkori individuals share the most genetic drift with Moroccan Palaeolithic and Early Neolithic individuals. Specifically, we observed the highest level with an Epipalaeolithic individual from Ifri Ouberrid and similarly elevated shared drift with the 15,000-year-old foragers from Taforalt and the Early Neolithic individuals from Ifri n'Amr o'Moussa (Fig. 3a, Extended Data Fig. 3 and Supplementary Fig. 2.12). Both Epipalaeolithic and Early Neolithic groups have been previously shown to maintain high genetic continuity with the much older Taforalt group8,9, explaining the similarly elevated shared drift statistics between all three groups and the Takarkori individuals.
a, Outgroup-f3 statistics f3(Takarkori, X; South Africa 2,000 cal. bp), where X represents ancient groups, mapped at their geographical positions. The colour gradient from blue to green indicates the genetic proximity to Takarkori, with the bluer colours representing closer genetic relationships. The statistics and their associated s.e. values for the top 70 signals are presented in Supplementary Fig. 2.12. b, No group shares extra affinity with Takarkori genomes compared with Taforalt, as measured by f4 statistics of the form f4(chimpanzee, X; Takarkori, Taforalt). The error bars represent 3 s.e. Group colours follow the same scheme as in Fig. 2. A more extensive list is presented in Supplementary Fig. 2.19. LSA, Late Stone Age; N, neolithic.
When restricting the comparative groups to present-day African and Near Eastern populations, we found that a group of individuals defined as FulaniA in a previous study38, which includes individuals from relatively less admixed Fulani herders from eight Sahelian countries, shows an increased genetic affinity to Takarkori, although less so to Taforalt (Extended Data Fig. 4 and Supplementary Fig. 2.16). This finding aligns with ref. 38, which observed a non-sub-Saharan ancestry component in FulaniA similar to that found in the Taforalt and Late Neolithic Moroccans. To further probe for Takarkori-like ancestry in FulaniA, we conducted an f4 analysis f4(chimpanzee, X; Masai/Datog/Iraqw, Takarkori), using Masai/Datog/Iraqw as baseline references owing to their similar amount of out-of-Africa (OoA) ancestry to Takarkori (Supplementary Fig. 2.20.1–2.20.3). The results indicated that the FulaniA have an increased affinity to Takarkori-like ancestry, as do other Sahelian and West African groups. These findings are consistent with the archaeological evidence of the southward expansion of Pastoral Neolithic groups from Central Sahara. Rock art, ceramic production and funerary practices provide detailed indications of the spread of these herders at the end of the Middle Holocene, probably driven by the progressive aridification of the Central Saharan regions45,46.
Given the high amount of shared genetic drift between the Takarkori and the ancestry first appearing in the 15,000-year-old Taforalt individuals in the outgroup f3 statistics, we subsequently investigated whether the Takarkori genomes share more alleles with other human groups compared to the Taforalt genome. For this analysis, we computed the statistics f4(chimpanzee, X; Takarkori, Taforalt), where X represents ancient and present-day groups from Africa and Eurasia. We obtained significantly positive values for Eurasian and Eurasian-admixed African groups, suggesting that the Taforalt group shares more alleles with these groups than Takarkori does. Conversely, none of the ancient or modern groups tested showed a significantly negative signal, indicating no detected closer affinity with Takarkori than Taforalt. Notably, both ancient and modern sub-Saharan groups, who are mostly unadmixed with Eurasian groups, yielded no significant value (|Z| < 3), suggesting that these groups are equally distant from both the Takarkori and Taforalt groups (Fig. 3b and Supplementary Data 4). Moreover, when running statistics f4(Chimp, Takarkori; X, Taforalt), we found significantly positive values for every population X, indicating that Takarkori shares more alleles with Taforalt than any other group (Extended Data Fig. 5 and Supplementary Data 4).
At the mtDNA level, both Takarkori individuals belong to a basal branch of haplogroup N, representing one of the deepest mtDNA lineages outside sub-Saharan Africa and predating present-day N-derived mtDNAs13. Using BEAST analysis for mtDNA dating, which included additional sequences from Upper Palaeolithic individuals and the dataset described previously13, we corroborate the previous findings of ref. 13 that the Takarkori individuals carried a basal N haplogroup lineage13, and refined the molecular split date estimate to 61,343 years old (95% highest posterior density (HPD) = 54,408–69,046) (Extended Data Fig. 6). Notably, the mtDNA lineage of the Oase 1 individual falls more basal to haplogroup N, suggesting an earlier split from the OoA lineage before the divergence of the Takarkori lineage. However, owing to incomplete lineage sorting and mtDNA representing a single lineage, the exact timing of the underlying population splits remains uncertain.
Previous work modelled the Taforalt group's ancestry as a two-way admixture of approximately 63.5% Natufian (ancient Levantine foragers) and 36.5% sub-Saharan African ancestries2. However, this model using the software qpAdm2 could not pinpoint the origin of Taforalt's African ancestry, resulting in unknown ghost ancestry only broadly linked to South, East and Central African groups2. Here we included Takarkori as a possible source of the African ancestry in Taforalt in comparison to several potential sources (namely Yoruba, Dinka, Mota, Cameroon Shum Laka, Botswana Xaro Early Iron Age (EIA) and Tanzania Zanzibar 1,300 cal. bp) through rotation-based qpAdm. We found that Saharan Takarkori provides a much better fit as an African proxy for Taforalt than the sub-Saharan groups, attaining a P value of >0.05, indicative of a much better model fit compared to the other sources (P < 2.84 × 10−34) (Extended Data Figs. 7, 8 and Supplementary Tables 2.6, 2.7). With this revised model, we estimated that the Taforalt ancestry retains a comparable 60.8% (±1.8%) contribution from Natufians, with the remaining 39.2% (±1.8%) derived from Takarkori.
We next explored the direct genetic affinity between Takarkori and the OoA ancestry found in all non-African modern humans, in contrast to African groups. For this, we computed f4 statistics of the form f4(chimpanzee, Zlatý kůň; African, Takarkori). Zlatý kůň, a 45,000-year-old Upper Palaeolithic individual from Czechia, was used as a proxy for OoA ancestry as it is probably the oldest modern human sequenced to date and represents the deepest known human lineage after the OoA lineage splits from the African lineages47. Our results showed positive values for Takarkori, indicating that it is genetically closer to Zlatý kůň than to sub-Saharan Africans, including Mota, a 4,500-year-old genome from East Africa. Nevertheless, various African populations with substantial OoA admixture were still genetically closer to Zlatý kůň than to Takarkori (Extended Data Fig. 9 and Supplementary Data 5).
These results raise the question of whether Takarkori's ancestors are closely related to OoA groups but remained in Africa, or whether they received later gene flow from OoA groups. If Takarkori experienced such later gene flow, it would carry Neanderthal admixture that is found in all OoA groups. To explore this signal, we used the data generated using the Archaic Admixture SNP panel for Takarkori and the software admixfrog48 that detects Neanderthal segments and included other ancient African and Eurasian groups as well as modern sub-Saharan African populations for comparison (Supplementary Note 2). We detected a total of 12 Neanderthal fragments that surpass 0.05 cM in length (approximately 50 kb), with the longest fragment located on chromosome 1 (Extended Data Fig. 10 and Supplementary Fig. 2.32), translating to a low level of Neanderthal ancestry in the Takarkori genome of approximately 0.15% (Fig. 4a). This percentage is less than a quarter of the Neanderthal ancestry in segments longer than 0.05 cM found in Taforalt and Neolithic Morocco individuals (0.6–0.9%), and about tenfold lower than in most OoA groups (1.4–2.36%), yet significantly higher than that in other ancient and contemporary sub-Saharan African genomes, where Neanderthal ancestry was completely absent. This pattern suggests that the Takarkori individuals have received a small amount of ancestry from OoA groups. However, the estimation of admixture dating of this OoA ancestry with DATES49 based on linkage disequilibrium (Supplementary Figs. 2.26.1–2.26.3) indicated very ancient admixture events with substantial uncertainty.
a, Detectable Neanderthal ancestry in segments longer than 0.05 cM in ancient individuals from Africa and Eurasia, along with present-day sub-Saharan African groups. The error bars represent the minimum and maximum estimates from all iterations. b, The geographical locations of groups included in the analysis. c, Admixture graph modelling of Takarkori's ancestral relationship with relevant populations.
Finally, we used the find_graphs() function from the ADMIXTOOLS 250 package to model Takarkori's ancestral relationship with other populations. We used a function for automated graph exploration with a model that includes Mota, Iran Neolithic, Natufian, Taforalt, Takarkori and the outgroup chimpanzee. The model fits with small f-statistic residuals (max |f4,expected − f4,observed| = 0.27 s.e.). The fitted graph suggests that Takarkori traces most of its ancestry (93%) to a hitherto unknown North African population, in agreement with the isolation signature obtained from the f4 statistics mentioned above (Fig. 3b). This unknown North African population is closely related to OoA populations and branches off the lineage leading to OoA later than the ancient individual from Mota Cave, Ethiopia, who represents the sub-Saharan African lineage most closely related to OoA groups identified so far. In our model, the remainder of the Takarkori individuals' ancestry (7%) is derived from a deeply ancient Levantine source. The Levantine gene flow also accounts for the Neanderthal ancestry found in Takarkori as the Levantine Neolithic genomes carry around 1.86% Neanderthal ancestry, aligning with our estimates using admixfrog. The graph also models Taforalt as a mixture of a 40% contribution from a Takarkori-related branch and 60% from a Natufian-related branch, consistent with our qpAdm results (Fig. 4c and Supplementary Data 6).
Our study introduces ancient genome-wide data from humans that inhabited the Green Sahara, providing unique insights into the genomic ancestry of populations in this region. The individuals from the Takarkori rock shelter predominantly carry an ancestral African lineage, representing an ancestry profile that has not been previously described. They share the most genetic drift with Epipalaeolithic and Early Neolithic individuals from Ifri Ouberrid and Ifri n'Amr o'Moussa, as well as the 15,000-year-old foragers from the Taforalt Cave in Morocco, suggesting a long-standing and stable population in North Africa before the AHP (14,500–5,000 bp). Plausibly, this ancestry was present in large parts of Northern Africa after the OoA event, and the Takarkori individuals inherited it from the group that inhabited the area during the final period of the Late Acacus (10,200–8,000 cal. bp). In Southwestern Libya, this period preceded the arrival of domesticates and is characterized by cultural advancements within those hunter–gatherer groups10. This included an increase in sedentism51, and the use of sophisticated material cultures such as pottery, basketry, and bone and wooden tools52,53,54.
We found that individuals from Takarkori exhibit only a marginal amount of genetic admixture from Levantine groups, suggesting that the emergence of pastoralism in the Sahara was primarily driven by the dissemination of cultural practices rather than through large-scale human migration, as suggested on archaeological basis10. Material culture at the onset of the earliest pastoral period shows both continuity and change, reflecting possibly complex assimilation dynamics during socioeconomic transitions10,19. These patterns may further suggest gradual cultural transformations rather than abrupt population replacement. This interpretation is further supported by the comparatively lower levels of Neanderthal genetic admixture found in the Takarkori individuals. Our admixture dating analysis points to events far back in time, suggesting a more heterogeneous spread of pastoralism and food production in the Sahara compared to Morocco and East Africa, where there was a noticeable increase in recent Levantine genetic admixture9,35,39,43. In addition to these findings, a run of homozygosity (ROH) analysis for TKH001 revealed no ROH segments larger than 12 cM, indicating no close-kin inbreeding (Supplementary Fig. 2.28). Several shorter ROH segments over 4 cM suggest an effective population size (Ne) of around 1,000 individuals, reflecting a moderately sized population.
Our research offers insights into the ancestry of the previously published Taforalt hunter-gatherers. While a previous study2 could not precisely ascribe the ‘sub-Saharan' component in the Taforalt genome, we now identify this ancestry as a deep North African lineage, with higher proportions found in the Saharan Takarkori individuals. This refines the earlier model, which proposed a dual admixture of Natufian and broadly sub-Saharan African ancestries. Our updated model suggests that the Taforalt ancestry is composed of a 60% contribution from a Natufian-like Levantine population, with the remaining 40% derived from a Takarkori-like ancestral North African population (Extended Data Fig. 8). Notably, both the late Pleistocene Taforalt and the mid-Holocene Takarkori individuals demonstrate equally distant relationships with sub-Saharan African lineages. This pattern suggests that no substantial genetic exchanges across the Green Sahara occurred during the AHP or other humid periods preceding the Later Pleistocene. The Sahara, spanning around 9 million km2 and housing diverse biomes, such as grasslands, wetlands, woodlands, lakes, mountains and savannas55,56, probably saw fragmented habitats impacting human gene flow. These ecological barriers, combined with social and cultural barriers, spatial structuring of populations, and the selective adoption of specific practices, may have facilitated the widespread dissemination of similar archaeological features57,58, while limiting extensive genetic admixture. This genetic discontinuity is consistent with modern data, which show substantial genetic differentiation across the Sahara beyond just a geographical gap59. Our findings suggest that sporadic Green Sahara events, particularly before pastoralism, were insufficient to allow for considerable genetic exchange, mirroring the Sahara's persistent role in limiting human genetic flow, as reflected in both ancient and modern population structures.
Our findings represent an important initial step, and future genetic studies could reveal more refined insights into human migration and gene flow across the Sahara. As sequencing costs continue to fall, whole-genome sequencing could enable more unbiased estimates regarding OoA events and other key aspects of human evolution.
The present-day communities of the Tadrart Acacus region, the Kel Tadrart pastoral group, have been actively engaged in both the excavation activities at the site (field assistance, sieving and so on) and in the decision-making processes and actions related to its conservation efforts. Other local community members contributed to the logistical management of the mission's tented camp, performing various specialized labour roles.
Although these communities maintain centuries-long connections to the territory, they do not express a specific cultural or historical affinity with the prehistoric burials uncovered there. This sentiment extends to the broader archaeological contexts and local rock art, which are viewed as expressions of ancient, pre-Islamic times, for which no direct cultural link is recognized or claimed.
Despite the lack of a perceived direct connection, ethical considerations and the implications of analysing and handling human remains have been thoroughly discussed with the Department of Antiquities (DoA) in Libya, with substantial input from M.F.M.A.-F. and M.T., both affiliated with the same institution. After the excavation concluded, and in subsequent years, the results were shared in meetings held at the local governorate office in Ghat. Unfortunately, the onset of the Libyan Revolution in 2011, shortly after the excavation activities ended, disrupted further contact. However, over the past year, coinciding with the preparation of this manuscript, online meetings with these co-authors and other DoA representatives have resumed, fostering a renewed dialogue on these issues. These inclusive approaches have ensured that both scientific research and conservation practices are aligned with local values and ethical standards, promoting a collaborative framework for heritage management and scientific collaboration.
Takarkori rock shelter counted 15 burials, varying in phases and preservation states, predominantly from the Early and Middle Pastoral periods, spanning the eighth and the seventh millennium cal. bp16,22. The burials, found immediately adjacent to the rock wall and with scarce grave goods, appear to only consist of women and children13,22,61 (Supplementary Note 1). Osteological information is reported and discussed elsewhere22, although, in brief, age estimation for subadult individuals was based on methodologies for skeletal development62,63 and dental maturation and eruption64 developed previously. For adult individuals, sex determination was based on the observation of morphological traits of the skull and the pelvis, as synthesized previously65, whereas estimation of age at death was based on the modifications of the pubic symphysis as proposed previously66, cranial sutures closure67 as well as patterns of occlusal dental wear68, which took into account the possible role of the sandy environment. Detailed layouts of the excavation areas, along with the stratigraphic and chronological insights, are depicted in Supplementary Fig 1.4. Standard stratigraphical techniques were used in the excavation of skeletal remains, with strict adherence to precautions for the sampling of biological materials. Sediments associated with the remains underwent sieving using a 2 mm mesh, and samples for laboratory-based studies were collected. The excavation and handling of human remains have been extensively discussed with the Department of Antiquities (DOA) in Tripoli, Libya (formerly the Socialist People's Libyan Arab Jamahiriya), particularly concerning ethical issues. Approval for the archaeological excavation was granted on 28 January 2004, under the reference number (translated as H98-10000 TATM 49463). Currently, the human remains are curated at the Museum of Anthropology of the University of Rome, La Sapienza.
Tooth root from individual TKH001 and fibula fragments from individual TKH009, both previously labelled as TK H1 and TK H9, respectively13, were sourced for this study. The sampling and DNA extraction procedures were carried out in a specialized ancient DNA facility at the University of Florence's Molecular Anthropology department, as outlined previously13. DNA was extracted according to the protocol described previously69. From the extracted DNA, a double-stranded DNA library was prepared in the same Florence facility, without undergoing uracil-DNA-glycosylase (UDG) treatment. An additional single-stranded DNA library, better at capturing short fragments, was prepared at the Max Planck Institute of Geoanthropology (formerly Max Planck Institute for the Science of Human History, MPI-SHH) and/or the Max Planck Institute for Evolutionary Anthropology (MPI-EVA). The prepared DNA libraries underwent shotgun sequencing to a depth of 3–5 million reads, using 75 bp single-end and/or 50 bp paired-end configurations on the Illumina HiSeq 4000 system at MPI-EVA for initial quality assessment (Supplementary Table 2.1). Both TKH001 and TKH009 libraries underwent in-solution capture targeting over a million SNPs using the Twist Ancient DNA25 and 1240k26 panels, with an additional specialized ‘archaic ancestry' panel applied to the single-stranded library from TKH001. After enrichment, all of the libraries were sequenced to 20 million reads on the Illumina HiSeq 4000 system at MPI-EVA. Read adapters were removed using AdapterRemoval70 v.2.3.0 as part of the EAGER (v.1.92.56)71, and the genome-wide captures were aligned to the human reference genome (hg19) using a mapping quality filter of 25 with BWA aligner72 v.7.12. Duplicate reads were eliminated using DeDup (v.0.12.2), which can be found at GitHub (https://github.com/apeltzer/DeDup). The contamination of the single-stranded library from TKH001 was evaluated with AuthentiCT73 v.1.0 and hapCon_ROH (https://haproh.readthedocs.io/en/latest/hapROH_with_contamination.html), and Schmutzi74 was used for the mtDNA-captured double-stranded library from TKH009 (Supplementary Table 2.3). The damage pattern was assessed with DamageProfiler75 v.1.1.
Double-stranded 1240k-captured sequences from TKH009 and single-stranded Twist-captured sequences from TKH001 were genotyped using Samtools v.1.3 and pileupCaller from SequenceTools v.1.4.0.2 (https://github.com/stschiff/sequenceTools). TKH001 has a total of 881,765 SNPs on the 1240k panel, and TKH009 libraries showed lower coverage of 22,484 SNPs. We merged the genotype data from TKH009 and TKH001. We then ran all the analyses throughout the manuscript (including PCA, f3 and f4 statistics, qpAdm, admixture graph, DATES and ADMIXTURE), except for admixfrog and hapROH, which, as individual analyses, use only the higher-coverage data from TKH001. To prevent potential artifacts from affecting the results, additional versions of the genotyped data for the TKH001 sample were generated. These included data from only the single-stranded library, data restricted to transversions only and data filtered using PMDtools (v.0.6)76 (Supplementary Table 2.4).
Using smartpca77 v.16000 from EIGENSOFT package v.8.0 with lsqmode enabled, PCAs were conducted on present-day individuals from Africa, Middle East and Southern Europe, genotyped on the Human Origins SNP panel28,29,30,31,32,33,34,35,36,37,38, alongside pertinent ancient groups from these regions2,8,9,35,36,39,40,41,42,43,44. All f3 and f4 statistics were calculated using the ADMIXTOOLS30 package v.5.1, using qp3pop v.435 for f3 statistics and qpDstat v.755 for f4 statistics. For the f3 outgroup test, the topology f3(outgroup; X, Takarkori) was used. When the outgroup was set as South Africa 2000 cal. bp, the ‘inbreed' option was enabled for qp3pop. On the other hand, when the outgroup was chimpanzee, the ‘outgroupmode' option was activated in qp3pop. This particular configuration was used to set a constant denominator of 0.01, as the inbuilt heterozygosity normalization in qp3pop does not function correctly when the outgroup is haploid, as in the case of the Chimpanzee. Furthermore, we used the Affymetrix Human Origins 1 Array, which comprises 13 unique SNP sets, to investigate potential sub-Saharan ancestry in the Takarkori and Taforalt populations. We conducted separate f4 statistics for Mbuti and Yoruba ascertained SNPs (Supplementary Figs. 2.21 and 2.22).
The Taforalt group was modelled as a two-way admixture between the Natufian population (left source) and variable African groups (right source) using qpAdm78 v.810 from ADMIXTOOLS package v.5.1. For every run, a consistent outgroup set was arranged, comprising Onge, Han, Papuan, Ust'-Ishim, Kostenki14, MA-1 and Iran_N. Every African group (Yoruba, Dinka, Mota, Cameroon Shum Laka, Botswana Xaro EIA, and Tanzania Zanzibar 1,300 cal. bp) was examined as a potential right source population in individual runs, while being omitted from the outgroup set (Supplementary Table 2.6). This methodology enabled the exploration of various admixture scenarios for the Taforalt group.
Neanderthal ancestry was inferred using admixfrog48 v.0.7.1 on single-stranded Takarkori and Taforalt libraries captured with the Archaic Admixture SNP panel27. The analysis included high-quality ancient shotgun sequencing data and present-day sub-Saharan genomes from the Allen Ancient Genome Diversity Project/John Templeton Ancient DNA Atlas (https://reich.hms.harvard.edu/ancient-genome-diversity-project) and the Human Genome Diversity Project32. Genomes were subsetted to positions in the Archaic Admixture SNP panel, with filters for mapping quality and base pair length. Libraries with insufficient SNP coverage or contamination were excluded (Supplementary Table 2.8). High-coverage genomes were subsetted to the positions covered by the Takarkori genome for direct comparison. The reference panel included three high-coverage Neanderthals79,80,81, one high-coverage Denisovan33 and the chimpanzee genome (panTro4) as the ancestral allele, along with Sub-Saharan African 1000 Genomes sequences82 for the African state.
The BEAST analysis of mitochondrial genomes involved 216 mtDNA sequences, aligned using MAFFT83 v.7.508 and adjusted by removing specific poly-C regions. The Hohlenstein–Stadel Neanderthal84 was the outgroup, and mtDNA sequences not in GenBank were processed using a specialized pipeline (mitoBench-ancientMT)85. The sequence consensus was determined using snpAD86 v.0.3.9, with haplogroups assigned by HaploGrep287 v.2.1.19. BEAST88 v.2.6.7 used these alignments, incorporating radiocarbon dates from Supplementary Table 2.9 as priors. Model suitability was assessed with bModelTest89 v.1.2.1, determining a combination of TIM(21) + I + G model, strict clock rate and ‘coalescent Bayesian skyline' tree prior as optimal. Eight independent analyses were conducted, combining results using LogCombiner and TreeAnnotator.
The ancestral relationship of the Takarkori population with others was investigated using the ‘find_graphs()' function from ADMIXTOOLS 2, which finds admixture graphs aligning with observed f statistics. Chimpanzee was set as an outgroup, and we included representatives from key relevant genetic clusters: ‘Morocco_Iberomaurusian', ‘Ethiopia_4500BP.SG', ‘Israel_Natufian_published' and ‘Iran_Ganj_Dareh_Neolithic'. Given the potential variability of ‘find_graphs()' outputs, 20 iterations were performed, using unique random graphs for each run. Graphs with a Z score of <|3| were selected (Supplementary Figs. 2.23–2.25). Moreover, Takarkori and Taforalt groups were treated as admixed populations, guided by previous knowledge from admixfrog results.
The DATES method49 v.753 was applied to the Takarkori group to detect Levantine admixture. A bin size of 0.001 and a fit range of 0.0045 to 1 in Morgan units were used. Admixture dates were estimated based on the decay of linkage disequilibrium between SNP pairs, with Levantine admixture modelled using sub-Saharan populations (Yoruba, Mbuti, Dinka, ancient groups from Ethiopia and Tanzania) and Eurasian populations (Natufian, PPNB, Ganj Dareh) (Supplementary Figs. 2.26.1–2.26.3).
The effective population size (Ne) for TKH001 was estimated using hapROH90 v.3.0. ROH segments longer than 4 cM were inferred using hapROH's recommended default settings, using the 1000 Genomes reference panel for comparison (Supplementary Fig. 2.27). This method analyses runs of homozygosity to estimate the population size and genetic background of the individual (Supplementary Fig. 2.28).
ADMIXTURE91 v.1.3.0 was used for unsupervised genetic clustering of global populations. Modern and ancient groups were subsetted from the HO-based dataset, converted to PLINK format, and transposed to pseudohaploid format to reduce artificial drift. Linkage disequilibrium pruning was performed using PLINK92 v.1.9 with a window size of 200 SNPs, a step size of 25 SNPs and an r2 threshold of 0.4. Five replicates for each k (2–9) were run, selecting the replicate with the highest log-likelihood.
Data visualization was performed in RStudio v.2022.12.0 + 353. The following R packages were used cowplot (v.1.1.2), ggplot (v.3.4.2), ggh4x (v.0.2.3), ggnewscale (v.0.4.8), janno (v.1.0.0), magrittr (v.2.0.3), maps (v.3.4.1), patchwork (v.1.1.2), purrr (v.1.0.1), RColorBrewer (v.1.1.3), readxl (v.1.4.1), tidyr (v.1.3.0) and tidyverse (v.1.3.2).
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
All newly reported ancient nuclear DNA data are archived in the European Nucleotide Archive (PRJEB84057).
Kuper, R. & Kröpelin, S. Climate-controlled Holocene occupation in the Sahara: motor of Africa's evolution. Science 313, 803–807 (2006).
PubMed
Google Scholar
van de Loosdrecht, M. et al. Pleistocene North African genomes link Near Eastern and sub-Saharan African human populations. Science 360, 548–552 (2018).
PubMed
Google Scholar
COHMAP Members. Climatic changes of the last 18,000 years: observations and model simulations. Science 241, 1043–1052 (1988).
MATH
Google Scholar
Ritchie, J. C., Eyles, C. H. & Haynes, C. V. Sediment and pollen evidence for an early to mid-Holocene humid period in the eastern Sahara. Nature 314, 352–355 (1985).
Google Scholar
Tierney, J. E., Pausata, F. S. R. & deMenocal, P. B. Rainfall regimes of the Green Sahara. Sci. Adv. 3, e1601503 (2017).
PubMed
PubMed Central
Google Scholar
Gasse, F. Hydrological changes in the African tropics since the Last Glacial Maximum. Quat. Sci. Rev. 19, 189–211 (2000).
Google Scholar
Hoelzmann, P., Keding, B., Berke, H., Kröpelin, S. & Kruse, H.-J. Environmental change and archaeology: lake evolution and human occupation in the Eastern Sahara during the Holocene. Palaeogeogr. Palaeoclimatol. Palaeoecol. 169, 193–217 (2001).
Google Scholar
Fregel, R. et al. Ancient genomes from North Africa evidence prehistoric migrations to the Maghreb from both the Levant and Europe. Proc. Natl Acad. Sci. USA 115, 6774–6779 (2018).
PubMed
PubMed Central
MATH
Google Scholar
Simões, L. G. et al. Northwest African Neolithic initiated by migrants from Iberia and Levant. Nature https://doi.org/10.1038/s41586-023-06166-6 (2023).
di Lernia, S. Earliest Herders of the Central Sahara (Tadrart Acacus Mountains, Libya): a punctuated model for the emergence of pastoralism in Africa. J. World Prehist. 34, 531–594 (2021).
Google Scholar
Smith, A. Pastoralism in Africa. Oxford Research Encyclopedia of African History https://doi.org/10.1093/acrefore/9780190277734.013.1066 (2021).
McDonald, M. M. A. The pattern of Neolithization in Dakhleh Oasis in the Eastern Sahara. Quat. Int. 410, 181–197 (2016).
MATH
Google Scholar
Vai, S. et al. Ancestral mitochondrial N lineage from the Neolithic ‘green' Sahara. Sci. Rep. 9, 3530 (2019).
PubMed
PubMed Central
MATH
Google Scholar
Manning, K. et al. Habitat fragmentation and the sporadic spread of pastoralism in the mid-Holocene Sahara. Quat. Sci. Rev. 309, 108070 (2023).
MATH
Google Scholar
Cremaschi, M. et al. Takarkori rock shelter (SW Libya): an archive of Holocene climate and environmental changes in the central Sahara. Quat. Sci. Rev. 101, 36–60 (2014).
MATH
Google Scholar
Biagetti, S. & di Lernia, S. Holocene deposits of saharan rock shelters: the case of Takarkori and other sites from the Tadrart Acacus Mountains (Southwest Libya). Afr. Archaeol. Rev. 30, 305–338 (2013).
Google Scholar
di Lernia, S., Massamba N'siala, I. & Mercuri, A. M. Saharan prehistoric basketry. Archaeological and archaeobotanical analysis of the early-middle Holocene assemblage from Takarkori (Acacus Mts., SW Libya). J. Archaeol. Sci. 39, 1837–1853 (2012).
Google Scholar
Dunne, J., Mercuri, A. M., Evershed, R. P., Bruni, S. & di Lernia, S. Earliest direct evidence of plant processing in prehistoric Saharan pottery. Nat. Plants 3, 16194 (2016).
PubMed
Google Scholar
Rotunno, R., Cavorsi, L. & di Lernia, S. A Holocene ceramic sequence in the Central Sahara: pottery traditions and social dynamics seen from the Takarkori Rockshelter (SW Libya). Afr. Archaeol. Rev. 40, 647–672 (2023).
Google Scholar
Van Neer, W. et al. Aquatic fauna from the Takarkori rock shelter reveals the Holocene central Saharan climate and palaeohydrography. PLoS ONE 15, e0228588 (2020).
PubMed
PubMed Central
Google Scholar
Cherkinsky, A. & di Lernia, S. Bayesian approach to 14C dates for estimation of long-term archaeological sequences in arid environments: the Holocene site of Takarkori rockshelter, southwest Libya. Radiocarbon 55, 771–782 (2013).
Google Scholar
Lernia, S. di, di Lernia, S. & Tafuri, M. A. Persistent deathplaces and mobile landmarks: the Holocene mortuary and isotopic record from Wadi Takarkori (SW Libya). J. Anthropological Archaeol. https://doi.org/10.1016/j.jaa.2012.07.002 (2013).
Dunne, J. et al. First dairying in green Saharan Africa in the fifth millennium BC. Nature 486, 390–394 (2012).
PubMed
MATH
Google Scholar
Tafuri, M. A., Bentley, R. A., Manzi, G. & di Lernia, S. Mobility and kinship in the prehistoric Sahara: strontium isotope analysis of Holocene human skeletons from the Acacus Mts. (southwestern Libya). J. Anthropol. Archaeol. 25, 390–402 (2006).
Google Scholar
Rohland, N. et al. Three assays for in-solution enrichment of ancient human DNA at more than a million SNPs. Genome Res. 32, 2068–2078 (2022).
PubMed
PubMed Central
MATH
Google Scholar
Mathieson, I. et al. Genome-wide patterns of selection in 230 ancient Eurasians. Nature 528, 499–503 (2015).
PubMed
PubMed Central
MATH
Google Scholar
Fu, Q. et al. An early modern human from Romania with a recent Neanderthal ancestor. Nature 524, 216–219 (2015).
PubMed
PubMed Central
MATH
Google Scholar
Lazaridis, I. et al. Ancient human genomes suggest three ancestral populations for present-day Europeans. Nature 513, 409–413 (2014).
PubMed
PubMed Central
MATH
Google Scholar
Fan, S. et al. African evolutionary history inferred from whole genome sequence data of 44 indigenous African populations. Genome Biol. 20, 82 (2019).
PubMed
PubMed Central
MATH
Google Scholar
Patterson, N. et al. Ancient admixture in human history. Genetics 192, 1065–1093 (2012).
PubMed
PubMed Central
MATH
Google Scholar
Bergström, A. et al. Insights into human genetic variation and population history from 929 diverse genomes. Science 367, eaay5012 (2020).
PubMed
PubMed Central
MATH
Google Scholar
Mallick, S. et al. The Simons Genome Diversity Project: 300 genomes from 142 diverse populations. Nature 538, 201–206 (2016).
PubMed
PubMed Central
MATH
Google Scholar
Meyer, M. et al. A high-coverage genome sequence from an archaic Denisovan individual. Science 338, 222–226 (2012).
PubMed
PubMed Central
MATH
Google Scholar
Pickrell, J. K. et al. The genetic prehistory of southern Africa. Nat. Commun. 3, 1143 (2012).
PubMed
MATH
Google Scholar
Skoglund, P. et al. Reconstructing prehistoric African population structure. Cell 171, 59–71.e21 (2017).
PubMed
PubMed Central
Google Scholar
Lazaridis, I. et al. Genomic insights into the origin of farming in the ancient Near East. Nature 536, 419–424 (2016).
PubMed
PubMed Central
MATH
Google Scholar
Pickrell, J. K. et al. Ancient west Eurasian ancestry in southern and eastern Africa. Proc. Natl Acad. Sci. USA 111, 2632–2637 (2014).
PubMed
PubMed Central
MATH
Google Scholar
D'Atanasio, E. et al. The genomic echoes of the last Green Sahara on the Fulani and Sahelian people. Curr. Biol. 33, 5495–5504 (2023).
PubMed
MATH
Google Scholar
Wang, K. et al. Ancient genomes reveal complex patterns of population movement, interaction, and replacement in sub-Saharan Africa. Sci. Adv. 6, eaaz0183 (2020).
PubMed
PubMed Central
Google Scholar
Lipson, M. et al. Ancient DNA and deep population structure in sub-Saharan African foragers. Nature 603, 290–296 (2022).
PubMed
PubMed Central
MATH
Google Scholar
Llorente, M. G. et al. Ancient Ethiopian genome reveals extensive Eurasian admixture in Eastern Africa. Science 350, 820–822 (2015).
MATH
Google Scholar
Harney, É. et al. Ancient DNA from Chalcolithic Israel reveals the role of population mixture in cultural transformation. Nat. Commun. 9, 3336 (2018).
PubMed
PubMed Central
MATH
Google Scholar
Prendergast, M. E. et al. Ancient DNA reveals a multistep spread of the first herders into sub-Saharan Africa. Science 365, eaaw6275 (2019).
PubMed
PubMed Central
MATH
Google Scholar
Schlebusch, C. M. et al. Southern African ancient genomes estimate modern human divergence to 350,000 to 260,000 years ago. Science 358, 652–655 (2017).
PubMed
Google Scholar
Paris, F. in The Origins and Development of African Livestock: Archaeology, Genetics, Linguistics, and Ethnography (ed. MacDonald, K. B. R.) 111–126 (UCL Press, 2000).
Vernet, R. in Droughts, Food and Culture 47–63 (Kluwer Academic, 2006).
Prüfer, K. et al. A genome sequence from a modern human skull over 45,000 years old from Zlatý kůň in Czechia. Nat. Ecol. Evol. 5, 820–825 (2021).
PubMed
PubMed Central
MATH
Google Scholar
Peter, B. M. 100,000 years of gene flow between Neandertals and Denisovans in the Altai mountains. Preprint at bioRxiv https://doi.org/10.1101/2020.03.13.990523 (2020).
Narasimhan, V. M. et al. The formation of human populations in South and Central Asia. Science 365, eaat7487 (2019).
PubMed
PubMed Central
MATH
Google Scholar
Maier, R. et al. On the limits of fitting complex models of population history to f-statistics. eLife 12, e85492 (2023).
PubMed
PubMed Central
MATH
Google Scholar
Garcea, E. A. A. An alternative way towards food production: the perspective from the Libyan Sahara. J. World Prehist. 18, 107–154 (2004).
MATH
Google Scholar
Mercuri, A. M., Fornaciari, R., Gallinaro, M., Vanin, S. & di Lernia, S. Plant behaviour from human imprints and the cultivation of wild cereals in Holocene Sahara. Nat. Plants 4, 71–81 (2018).
PubMed
Google Scholar
di Lernia, S. Dismantling dung: delayed use of food resources among Early Holocene foragers of the Libyan Sahara. J. Anthropol. Archaeol. 20, 408–441 (2001).
Google Scholar
Rotunno, R., Mercuri, A. M., Florenzano, A., Zerboni, A. & di Lernia, S. Coprolites from rock shelters: hunter-gatherers ‘herding' barbary sheep in the Early Holocene Sahara. J. Afr. Archaeol. 17, 76–94 (2019).
Google Scholar
Kröpelin, S. et al. Climate-driven ecosystem succession in the Sahara: the past 6000 years. Science 320, 765–768 (2008).
PubMed
MATH
Google Scholar
Hoelzmann, P. et al. in Past Climate Variability through Europe and Africa (eds Battarbee, R. W. et al.) 219–256 (Springer, 2004).
Di Lernia, S. Saharan Hunter-Gatherers: Specialization and Diversification in Holocene Southwestern Libya (Routledge, 2022).
Drake, N. A., Blench, R. M., Armitage, S. J., Bristow, C. S. & White, K. H. Ancient watercourses and biogeography of the Sahara explain the peopling of the desert. Proc. Natl Acad. Sci. USA 108, 458–462 (2011).
PubMed
Google Scholar
Peter, B. M., Petkova, D. & Novembre, J. Genetic landscapes reveal how human genetic diversity aligns with geography. Mol. Biol. Evol. 37, 943–951 (2020).
PubMed
Google Scholar
Cheddadi, R. et al. Early Holocene greening of the Sahara requires Mediterranean winter rainfall. Proc. Natl Acad. Sci. USA 118, e2024898118 (2021).
PubMed
PubMed Central
MATH
Google Scholar
Profico, A. et al. Medical imaging as a taphonomic tool: the naturally-mummified bodies from Takarkori rock shelter (Tadrart Acacus, SW Libya, 6100-5600 uncal BP). J. Cult. Herit. Manage. Sust. Dev. 10, 144–156 (2019).
Google Scholar
Stloukal, M. & Hanáková, H. Die länge der längsknochen Altslawischer bevölkerungen unter besonderer berücksichtigung von wachstumsfragen. Homo 29, 53–69 (1978).
France, D. L. Lab Manual and Workbook for Physical Anthropology (West, 1988).
Ubelaker, D. H. Human Skeletal Remains. Excavation, Analysis, Interpretation (Taraxacum, 1989)
Buikstra, J. E. & Ubelaker, D. H. Standards for Data Collection from Human Skeletal Remains (Arkansas Archaeological Survey, 1994).
Todd, T. W. Age changes in the pubic bone. I. The male white pubis. Am. J. Phys. Anthropol. 3, 285–334 (1920).
Meindl, R. S. & Lovejoy, C. O. Ectocranial suture closure: a revised method for the determination of skeletal age at death based on the lateral-anterior sutures. Am. J. Phys. Anthropol. 68, 57–66 (1985).
Lovejoy, C. O. Dental wear in the Libben population: its functional pattern and role in the determination of adult skeletal age at death. Am. J. Phys. Anthropol. 68, 47–56 (1985).
Dabney, J. et al. Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Proc. Natl Acad. Sci. USA 110, 15758–15763 (2013).
PubMed
PubMed Central
Google Scholar
Schubert, M., Lindgreen, S. & Orlando, L. AdapterRemoval v2: rapid adapter trimming, identification, and read merging. BMC Res. Notes 9, 88 (2016).
PubMed
PubMed Central
Google Scholar
Peltzer, A. et al. EAGER: efficient ancient genome reconstruction. Genome Biol. 17, 60 (2016).
PubMed
PubMed Central
Google Scholar
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
PubMed
PubMed Central
MATH
Google Scholar
Peyrégne, S. & Peter, B. M. AuthentiCT: a model of ancient DNA damage to estimate the proportion of present-day DNA contamination. Genome Biol. 21, 246 (2020).
PubMed
PubMed Central
Google Scholar
Renaud, G., Slon, V., Duggan, A. T. & Kelso, J. Schmutzi: estimation of contamination and endogenous mitochondrial consensus calling for ancient DNA. Genome Biol. 16, 224 (2015).
PubMed
PubMed Central
Google Scholar
Neukamm, J., Peltzer, A. & Nieselt, K. DamageProfiler: fast damage pattern calculation for ancient DNA. Bioinformatics 37, 3652–3653 (2021).
PubMed
MATH
Google Scholar
Skoglund, P. et al. Separating endogenous ancient DNA from modern day contamination in a Siberian Neandertal. Proc. Natl Acad. Sci. USA 111, 2229–2234 (2014).
PubMed
PubMed Central
MATH
Google Scholar
Patterson, N., Price, A. L. & Reich, D. Population structure and eigenanalysis. PLoS Genet. 2, e190 (2006).
PubMed
PubMed Central
MATH
Google Scholar
Haak, W. et al. Massive migration from the steppe was a source for Indo-European languages in Europe. Nature 522, 207–211 (2015).
PubMed
PubMed Central
MATH
Google Scholar
Prüfer, K. et al. The complete genome sequence of a Neanderthal from the Altai Mountains. Nature 505, 43–49 (2014).
PubMed
MATH
Google Scholar
Prüfer, K. et al. A high-coverage Neandertal genome from Vindija Cave in Croatia. Science 358, 655–658 (2017).
PubMed
PubMed Central
MATH
Google Scholar
Mafessoni, F. et al. A high-coverage Neandertal genome from Chagyrskaya Cave. Proc. Natl Acad. Sci. USA 117, 15132–15136 (2020).
PubMed
PubMed Central
Google Scholar
1000 Genomes Project Consortium. A global reference for human genetic variation. Nature 526, 68–74 (2015).
Google Scholar
Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).
PubMed
PubMed Central
MATH
Google Scholar
Posth, C. et al. Deeply divergent archaic mitochondrial genome provides lower time boundary for African gene flow into Neanderthals. Nat. Commun. 8, 16046 (2017).
PubMed
PubMed Central
MATH
Google Scholar
Pugach, I. et al. Ancient DNA from Guam and the peopling of the Pacific. Proc. Natl Acad. Sci. USA 118, e2022112118 (2021).
PubMed
MATH
Google Scholar
Prüfer, K. snpAD: an ancient DNA genotype caller. Bioinformatics 34, 4165–4171 (2018).
PubMed
PubMed Central
MATH
Google Scholar
Weissensteiner, H. et al. HaploGrep 2: mitochondrial haplogroup classification in the era of high-throughput sequencing. Nucleic Acids Res. 44, W58–W63 (2016).
PubMed
PubMed Central
Google Scholar
Bouckaert, R. et al. BEAST 2.5: an advanced software platform for Bayesian evolutionary analysis. PLoS Comput. Biol. 15, e1006650 (2019).
PubMed
PubMed Central
MATH
Google Scholar
Bouckaert, R. R. & Drummond, A. J. bModelTest: Bayesian phylogenetic site model averaging and model comparison. BMC Evol. Biol. 17, 42 (2017).
PubMed
PubMed Central
MATH
Google Scholar
Ringbauer, H., Novembre, J. & Steinrücken, M. Parental relatedness through time revealed by runs of homozygosity in ancient DNA. Nat. Commun. 12, 5425 (2021).
PubMed
PubMed Central
MATH
Google Scholar
Alexander, D. H., Novembre, J. & Lange, K. Fast model-based estimation of ancestry in unrelated individuals. Genome Res. 19, 1655–1664 (2009).
PubMed
PubMed Central
MATH
Google Scholar
Chang, C. C. et al. Second-generation PLINK: rising to the challenge of larger and richer datasets. Gigascience 4, 7 (2015).
PubMed
PubMed Central
MATH
Google Scholar
Download references
We thank the members of the Department of Antiquities in Tripoli for granting S.d.L. the necessary permits for the excavation, exportation, dating and analysis of human remains; M. Burri, A. Wissgott, R. Radzeviciute, M. Meyer and J. Zorn (all currently or formerly affiliated with MPI-EVA) for their substantial roles in processing the samples in the laboratory. Our research has benefited from the insights and advice of C. Jeong, E. Scerri, S. Oliveira, V. Villalba, E. Skourtanioti, S. Goldstein and M. Hajdinjak (all currently or formerly affiliated with MPI-EVA and MPI-SHH), as well as all of the members of the Population Genetics working group. The data were produced by the Ancient DNA Core Unit of the Max Planck Institute for Evolutionary Anthropology, which is funded by the Max Planck Society. We acknowledge the staff at the Multimedia Department at MPI-EVA for their expert graphical assistance in enhancing the visual aspects of our publication. This study was funded by the Max Planck Society and the Max Planck-Harvard Research Center for the Archaeoscience of the Ancient Mediterranean (MHAAM).
Open access funding provided by Max Planck Society.
Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
Nada Salem, Marieke S. van de Loosdrecht, Arev Pelin Sümer, Alexander Hübner, Benjamin Peter, Raffaela A. Bianco, Abdeljalil Bouzouggar, Kay Prüfer, Harald Ringbauer & Johannes Krause
Max Planck−Harvard Research Center for the Archaeoscience of the Ancient Mediterranean (MHAAM), Leipzig, Germany
Nada Salem & Johannes Krause
Biosystematics Group, Wageningen University, Wageningen, The Netherlands
Marieke S. van de Loosdrecht
Department of Biology, University of Florence, Florence, Italy
Stefania Vai, Martina Lari, Alessandra Modi & David Caramelli
The Department of Antiquities (DOA), Tripoli, Libya
Mohamed Faraj Mohamed Al-Faloos & Mustafa Turjman
Institut National des Sciences de l'Archéologie et du Patrimoine, Origin and Evolution of Homo sapiens Cultures, Rabat, Morocco
Abdeljalil Bouzouggar
Department of Environmental Biology, Sapienza University of Rome, Rome, Italy
Mary Anne Tafuri & Giorgio Manzi
Department of Ancient World Studies, Sapienza University of Rome, Rome, Italy
Rocco Rotunno & Savino di Lernia
School of Geography, Archaeology and Environmental Studies (GAES), University of Witwatersrand, Johannesburg, South Africa
Savino di Lernia
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J.K. and S.d.L. designed the research (Conceptualization), with N.S. leading the formal analysis assisted by M.S.v.d.L., A.P.S., A.H., H.R., B.P. and K.P.; S.d.L., D.C., M.A.T. and G.M. provided the archaeological materials and conducted excavations (resources and investigation). Laboratory work (investigation) was headed by S.V. and R.A.B. with help from R.R., M.L. and A.M. Data curation and visualization were primarily managed by N.S., with M.S.v.d.L. contributing. M.F.M.A.-F. and M.T. managed project administration and handled permit acquisitions (project administration), and A.B. contributed to embedding the findings into the broader archaeological framework (integration into archaeological framework). N.S. drafted the manuscript, with all of the authors reviewing and editing. K.P., H.R., J.K. and S.d.L. provided supervision, and J.K. secured funding. Contributions are recognized using the CRediT Taxonomy labels.
Correspondence to
Nada Salem, Harald Ringbauer, David Caramelli, Savino di Lernia or Johannes Krause.
The authors declare no competing interests.
Nature thanks Kendra Sirak and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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A) with projections of key ancient groups from these regions. B) Geographic locations of ancient genomes from Africa and Near East included in our analysis. EN: Early Neolithic, LN: Late Neolithic, ChL: Chalcolithic, LSA: Late Stone Age, IA: Iron Age.
A) with Takarkori individuals projected and highlighted in black squares. B) Geographic locations of Takarkori individuals and relevant present-day populations included in our analysis.
Within the shared drift of Takarkori group, the highest genetic drift is exhibited with Taforalt-related groups, OUB and IAM. The colour scheme for populations in the legend follows that of PCA Fig. 1. The error bars represent three standard errors.
The Taforalt and later Epipaleolithic and Neolithic groups from Morocco are included for comparison due to their highest genetic drift with Takarkori. The Takarkori group shows the second highest affinity with FulaniA after Taforalt-related groups. The colour and shape scheme for populations in the legend follows that of the PCA in Extended Data Fig. 1. The error bars represent three standard errors.
All positive f4 values underscore shared genetic drift between Takarkori and Taforalt that surpasses other potential affinities tested here. The error bars represent three standard errors.
The phylogenetic tree is constructed for the Takarkori samples TKH001 and TKH009, combined with 209 published complete genomes from ancient and modern samples. The major mitochondrial lineages and sub-lineages for the N macrohaplogroup are differentiated by distinct colours.
Takarkori exhibits a notably higher affinity to Taforalt, with an f4 value approximately 3.5 times higher than the next closest African group. The error bars represent three standard errors.
Results of modelling the Taforalt group as a two-way admixture between Natufian and various African populations. Takarkori yielded the best model fit with a P-value > 0.05, indicating the sufficiency of the two-way admixture model for Taforalt. In contrast, the P-values for the other models were all <2.84 × 10−34. The error bars represent the standard error of the ancestry proportion estimates, calculated using 5 cM block jackknifing.
The results suggest a stronger connection between Takarkori and the OoA ancestry compared to sub-Saharan Africans. The error bars represent three standard errors.
A) Detected archaic ancestry fragments in Takarkori and Taforalt inferred by admixfrog. Dark and light blue regions are homozygous and heterozygous Neanderthal fragments, respectively. Grey fragments indicate African ancestry and bar height is proportional to the posterior probability of ancestry. B) The longest archaic fragment in Takarkori is on chromosome 1 compared to the reference panel. The y-axis refers to the frequency of the alternative allele in the reference population (Africans or Neanderthals in this case) at the given position, and the x-axis stands for the SNP positions.
Supplementary Notes 1–3, including Supplementary Figs., Tables and References – see Table of Contents for details.
Supplementary Data 1–6.
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Salem, N., van de Loosdrecht, M.S., Sümer, A.P. et al. Ancient DNA from the Green Sahara reveals ancestral North African lineage.
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Temporary pacemakers are essential for the care of patients with short-lived bradycardia in post-operative and other settings1,2,3,4. Conventional devices require invasive open-heart surgery or less invasive endovascular surgery, both of which are challenging for paediatric and adult patients5,6,7,8. Other complications9,10,11 include risks of infections, lacerations and perforations of the myocardium, and of displacements of external power supplies and control systems. Here we introduce a millimetre-scale bioresorbable optoelectronic system with an onboard power supply and a wireless, optical control mechanism with generalized capabilities in electrotherapy and specific application opportunities in temporary cardiac pacing. The extremely small sizes of these devices enable minimally invasive implantation, including percutaneous injection and endovascular delivery. Experimental studies demonstrate effective pacing in mouse, rat, porcine, canine and human cardiac models at both single-site and multi-site locations. Pairing with a skin-interfaced wireless device allows autonomous, closed-loop operation upon detection of arrhythmias. Further work illustrates opportunities in combining these miniaturized devices with other medical implants, with an example of arrays of pacemakers for individual or collective use on the frames of transcatheter aortic valve replacement systems, to provide unique solutions that address risks for atrioventricular block following surgeries. This base technology can be readily adapted for a broad range of additional applications in electrotherapy, such as nerve and bone regeneration, wound therapy and pain management.
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The data supporting the results of this study are present in the paper and Supplementary Information. Source data are provided with this paper.
The code for connecting to the device via BLE, recording and analysing ECG data in real time, and configuring the pacing parameters in a closed-loop system is available on Code Ocean at https://codeocean.com/capsule/9406347/tree/v1 (ref. 49).
Choi, Y. S. et al. Fully implantable and bioresorbable cardiac pacemakers without leads or batteries. Nat. Biotechnol. 39, 1228–1238 (2021).
CAS
PubMed
PubMed Central
MATH
Google Scholar
Zhang, Y. et al. Advances in bioresorbable materials and electronics. Chem. Rev. 123, 11722–11773 (2023).
CAS
PubMed
Google Scholar
Choi, Y. S. et al. A transient, closed-loop network of wireless, body-integrated devices for autonomous electrotherapy. Science 376, 1006–1012 (2022).
ADS
CAS
PubMed
PubMed Central
MATH
Google Scholar
Lumia, F. J. & Rios, J. C. Temporary transvenous pacemaker therapy: an analysis of complications. Chest 64, 604–608 (1973).
CAS
PubMed
MATH
Google Scholar
Wood, M. A. & Ellenbogen, K. A. Cardiac pacemakers from the patient's perspective. Circulation 105, 2136–2138 (2002).
PubMed
MATH
Google Scholar
Bar-Cohen, Y. et al. Minimally invasive implantation of a micropacemaker into the pericardial space. Circ. Arrhythm. Electrophysiol. 11, e006307 (2018).
PubMed
PubMed Central
Google Scholar
Zhao, J. et al. Permanent epicardial pacing in neonates and infants less than 1 year old: 12-year experience at a single center. Transl. Pediatr. 11, 825–833 (2022).
PubMed
PubMed Central
MATH
Google Scholar
Wildbolz, M., Dave, H., Weber, R., Gass, M. & Balmer, C. Pacemaker implantation in neonates and infants: favorable outcomes with epicardial pacing systems. Pediatr. Cardiol. 41, 910–917 (2020).
PubMed
Google Scholar
Wilhelm, M. J. et al. Cardiac pacemaker infection: surgical management with and without extracorporeal circulation. Ann. Thorac. Surg. 64, 1707–1712 (1997).
CAS
PubMed
MATH
Google Scholar
Donovan, K. D. & Lee, K. Y. Indications for and complications of temporary transvenous cardiac pacing. Anaesth. Intensive Care 13, 63–70 (1985).
CAS
PubMed
Google Scholar
BRAUN, M. U. et al. Percutaneous lead implantation connected to an external device in stimulation-dependent patients with systemic infection—a prospective and controlled study. Pacing Clin. Electrophysiol. 29, 875–879 (2006).
ADS
PubMed
MATH
Google Scholar
Ouyang, H. et al. Symbiotic cardiac pacemaker. Nat. Commun. 10, 1821 (2019).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Lyu, H. et al. Synchronized biventricular heart pacing in a closed-chest porcine model based on wirelessly powered leadless pacemakers. Sci. Rep. 10, 2067 (2020).
ADS
CAS
PubMed
PubMed Central
MATH
Google Scholar
Ho, J. S. et al. Wireless power transfer to deep-tissue microimplants. Proc. Natl Acad. Sci. USA 111, 7974–7979 (2014).
ADS
CAS
PubMed
PubMed Central
MATH
Google Scholar
Wang, S. et al. A self-assembled implantable microtubular pacemaker for wireless cardiac electrotherapy. Sci. Adv. 9, eadj0540 (2023).
CAS
PubMed
PubMed Central
Google Scholar
Prominski, A. et al. Porosity-based heterojunctions enable leadless optoelectronic modulation of tissues. Nat. Mater. 21, 647–655 (2022).
ADS
CAS
PubMed
Google Scholar
Liu, Z. et al. Photoelectric cardiac pacing by flexible and degradable amorphous Si radial junction stimulators. Adv. Healthc. Mater. 9, 1901342 (2020).
CAS
Google Scholar
Wang, L. et al. A fully biodegradable and self-electrified device for neuroregenerative medicine. Sci. Adv. 6, eabc6686 (2020).
ADS
CAS
PubMed
PubMed Central
Google Scholar
Zhang, Y. et al. Self-powered, light-controlled, bioresorbable platforms for programmed drug delivery. Proc. Natl. Acad. Sci. USA 120, e2217734120 (2023).
Huang, I. et al. High performance dual-electrolyte magnesium–iodine batteries that can harmlessly resorb in the environment or in the body. Energy Environ. Sci. 15, 4095–4108 (2022).
CAS
MATH
Google Scholar
Won, S. M. et al. Natural wax for transient electronics. Adv. Funct. Mater. 28, 1801819 (2018).
MATH
Google Scholar
Choi, Y. S. et al. Biodegradable polyanhydrides as encapsulation layers for transient electronics. Adv. Funct. Mater. 30, 2000941 (2020).
CAS
Google Scholar
Song, G. Control of biodegradation of biocompatable magnesium alloys. Corros. Sci. 49, 1696–1701 (2007).
CAS
MATH
Google Scholar
Schauer, A. et al. Biocompatibility and degradation behavior of molybdenum in an in vivo rat model. Materials 14, 7776 (2021).
ADS
CAS
PubMed
PubMed Central
MATH
Google Scholar
Yin, L. et al. Dissolvable metals for transient electronics. Adv. Funct. Mater. 24, 645–658 (2014).
CAS
MATH
Google Scholar
Yu, K. J. et al. Bioresorbable silicon electronics for transient spatiotemporal mapping of electrical activity from the cerebral cortex. Nat. Mater. 15, 782–791 (2016).
ADS
CAS
PubMed
PubMed Central
MATH
Google Scholar
Kang, S.-K. et al. Bioresorbable silicon electronic sensors for the brain. Nature 530, 71–76 (2016).
ADS
CAS
PubMed
MATH
Google Scholar
Li, G. et al. Flexible transient phototransistors by use of wafer‐compatible transferred silicon nanomembranes. Small 14, e1802985 (2018).
ADS
PubMed
Google Scholar
Li, G. et al. Silicon nanomembrane phototransistor flipped with multifunctional sensors toward smart digital dust. Sci. Adv. 6, eaaz6511 (2020).
ADS
CAS
PubMed
PubMed Central
Google Scholar
López Ayerbe, J. et al. Temporary pacemakers: current use and complications. Rev. Esp. Cardiol. Engl. Ed. 57, 1045–1052 (2004).
MATH
Google Scholar
Yu, L., Nina-Paravecino, F., Kaeli, D. & Fang, Q. Scalable and massively parallel Monte Carlo photon transport simulations for heterogeneous computing platforms. J. Biomed. Opt. 23, 1 (2018).
PubMed
Google Scholar
Fang, Q. & Boas, D. A. Monte Carlo simulation of photon migration in 3D turbid media accelerated by graphics processing units. Opt. Express 17, 20178 (2009).
ADS
CAS
PubMed
Google Scholar
Taroni, P., Pifferi, A., Torricelli, A., Comelli, D. & Cubeddu, R. In vivo absorption and scattering spectroscopy of biological tissues. Photochem. Photobiol. Sci. 2, 124–129 (2003).
CAS
PubMed
MATH
Google Scholar
Khan, R., Gul, B., Khan, S., Nisar, H. & Ahmad, I. Refractive index of biological tissues: review, measurement techniques, and applications. Photodiagnosis Photodyn. Ther. 33, 102192 (2021).
CAS
PubMed
MATH
Google Scholar
Green, M. A. & Keevers, M. J. Optical properties of intrinsic silicon at 300 K. Prog. Photovoltaics Res. Appl. 3, 189–192 (1995).
CAS
Google Scholar
Firbank, M., Hiraoka, M., Essenpreis, M. & Delpy, D. T. Measurement of the optical properties of the skull in the wavelength range 650–950 nm. Phys. Med. Biol. 38, 503–510 (1993).
CAS
PubMed
Google Scholar
Rahko, P. S. Evaluation of the skin-to-heart distance in the standing adult by two-dimensional echocardiography. J. Am. Soc. Echocardiogr. 21, 761–764 (2008).
PubMed
MATH
Google Scholar
Chen, R. et al. Deep brain optogenetics without intracranial surgery. Nat. Biotechnol. 39, 161–164 (2021).
CAS
PubMed
MATH
Google Scholar
Yin, R. T. et al. Open thoracic surgical implantation of cardiac pacemakers in rats. Nat. Protoc. 18, 374–395 (2023).
CAS
PubMed
MATH
Google Scholar
Yang, Q. et al. Photocurable bioresorbable adhesives as functional interfaces between flexible bioelectronic devices and soft biological tissues. Nat. Mater. 20, 1559–1570 (2021).
ADS
CAS
PubMed
PubMed Central
MATH
Google Scholar
Shea, J. B. & Sweeney, M. O. Cardiac resynchronization therapy a patient's guide. Circulation 108, e64–e66 (2003).
Connolly, S. J., Kerr, C., Gent, M. & Yusuf, S. Dual-chamber versus ventricular pacing. Circulation 94, 578–583 (1996).
CAS
PubMed
Google Scholar
Rodés-Cabau, J., Muntané-Carol, G. & Philippon, F. Managing conduction disturbances after TAVR: toward a tailored strategy. JACC Cardiovasc. Interv. 14, 992–994 (2021).
PubMed
MATH
Google Scholar
Urena, M. & Rodés-Cabau, J. Conduction abnormalities: the true Achilles' heel of transcatheter aortic valve replacement? JACC Cardiovasc. Interv. 9, 2217–2219 (2016).
PubMed
Google Scholar
Pagnesi, M. et al. Incidence, predictors, and prognostic impact of new permanent pacemaker implantation after TAVR with self-expanding valves. JACC Cardiovasc. Interv. 16, 2004–2017 (2023).
PubMed
MATH
Google Scholar
Reiter, C. et al. Delayed total atrioventricular block after transcatheter aortic valve replacement assessed by implantable loop recorders. JACC Cardiovasc. Interv. 14, 2723–2732 (2021).
PubMed
Google Scholar
Muntané-Carol, G. et al. Ambulatory electrocardiographic monitoring following minimalist transcatheter aortic valve replacement. JACC Cardiovasc. Interv. 14, 2711–2722 (2021).
PubMed
Google Scholar
Krishnaswamy, A. et al. Feasibility and safety of same-day discharge following transfemoral transcatheter aortic valve replacement. JACC Cardiovasc. Interv. 15, 575–589 (2022).
PubMed
MATH
Google Scholar
Millimetre-scale, bioresorbable optoelectronic systems for minimally invasive electrotherapy. Code Ocean https://codeocean.com/capsule/9406347/tree/v1 (2025).
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We acknowledge support from the Querrey Simpson Institute for Bioelectronics, the Leducq Foundation grant ‘Bioelectronics for Neurocardiology' and the NIH grant (NIH R01 HL141470). Y.Z. acknowledges support from the National University of Singapore start-up grant and the AHA's Second Century Early Faculty Independence Award (grant: https://doi.org/10.58275/AHA.23SCEFIA1154076.pc.gr.173925). J. Gong and Z.M. acknowledge the support from AFOSR (grant number FA9550-21-1-0081). We thank E. Dempsey, Q. Ma, N. Ghoreishi-Haack, I. Stepien and S. Han for the help in the biocompatibility study and animal experiment. This work made use of the NUFAB facility of Northwestern University's NUANCE Center, which has received support from the SHyNE Resource (NSF ECCS-2025633), the IIN and Northwestern's MRSEC programme (NSF DMR-1720139). This work was supported by the Developmental Therapeutics Core and the Center for Advanced Molecular Imaging (RRID:SCR_021192) at Northwestern University and the Robert H. Lurie Comprehensive Cancer Center support grant (NCI P30 CA060553).
These authors contributed equally: Yamin Zhang, Eric Rytkin, Liangsong Zeng, Jong Uk Kim, Lichao Tang, Haohui Zhang
Center for Bio-Integrated Electronics, Northwestern University, Evanston, IL, USA
Yamin Zhang, Jong Uk Kim, Seung Gi Seo, Jianyu Gu, Tianyu Yang, Naijia Liu, Wei Ouyang & John A. Rogers
Querrey Simpson Institute for Bioelectronics, Northwestern University, Evanston, IL, USA
Yamin Zhang, Liangsong Zeng, Jong Uk Kim, Kaiyu Zhao, Yue Wang, Li Ding, Xinyue Lu, Elena Aprea, Gengming Jiang, Seung Gi Seo, Jin Wang, Jianyu Gu, Fei Liu, Tianyu Yang, Naijia Liu, Yinsheng Lu, Claire Hoepfner, Alex Hou, Rachel Nolander, Wei Ouyang, Igor R. Efimov & John A. Rogers
Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore, Singapore
Yamin Zhang & Lichao Tang
Department of Biomedical Engineering, Northwestern University, Evanston, IL, USA
Eric Rytkin, Yue Wang, Anastasia Lantsova, Altynai Melisova, Alex Hou, Rachel Nolander, Igor R. Efimov & John A. Rogers
Department of Mechanical Engineering, Northwestern University, Evanston, IL, USA
Liangsong Zeng, Shupeng Li, Yonggang Huang & John A. Rogers
Department of Civil and Environmental Engineering, Northwestern University, Evanston, IL, USA
Haohui Zhang & Zengyao Lv
Feinberg Cardiovascular and Renal Research Institute, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
Aleksei Mikhailov, Anna Pfenniger, Andrey Ardashev, Rishi K. Arora & Igor R. Efimov
Department of Materials Science and Engineering, Northwestern University, Evanston, IL, USA
Kaiyu Zhao, Jiayang Liu, Fei Liu, Yat Fung Larry Li, Nathan S. Purwanto, Yinsheng Lu, John M. Torkelson & John A. Rogers
Department of Cardiovascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Li Ding
The BioRobotics Institute and Department of Excellence in Robotics and AI, Scuola Superiore Sant'Anna, Pisa, Italy
Elena Aprea
Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA
Tong Wang & John M. Torkelson
Alnylam Pharmaceuticals Inc, Cambridge, MA, USA
Keith Bailey
Center for Comparative Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
Amy Burrell
Department of Electrical and Computer Engineering, Northwestern University, Evanston, IL, USA
Yue Ying
Department of Electrical and Computer Engineering, University of Wisconsin-Madison, Madison, WI, USA
Jiarui Gong & Zhenqiang Ma
Department of Electronic Engineering, Incheon National University, Incheon, Republic of Korea
Jinheon Jeong & Sung Hun Jin
Department of Chemical Engineering, Dankook University, Yongin, Republic of Korea
Junhwan Choi
Department of Applied Physical Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Wubin Bai
Thayer School of Engineering, Dartmouth College, Hanover, NH, USA
Wei Ouyang
The University of Chicago Medicine, University of Chicago, Chicago, IL, USA
Rishi K. Arora
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Y.Z. and J.A.R. initiated and conceived the self-powered, light-controlled pacing concept. Y.Z., E.R., I.R.E. and J.A.R. designed the studies and analysed the results. Y.Z., L.Z., K.Z., X.L., A.L., G.J., J.L., F.L., Y.F.L.L., Y.L., C.H., A.H. and R.N. fabricated and characterized the pacemakers. E.R., L.T., A. Mikhailov, L.D., A.B., A.P., A.A. and A. Melisova conducted animal surgeries. Y.Z., E.R., L.Z., L.T., A. Mikhailov, L.D., J.W., A.B., A.P. and W.O., performed in vivo and ex vivo cardiac pacing experiments. W.O., Y.W., J. Gu, T.Y., Y.Y. and Y.L. developed closed-loop and optical control systems. J.U.K., S.G.S., J. Gong, J.J., J.C., S.H.J. and Z.M. designed and fabricated phototransistors. H.Z., S.L., Z.L. and Y.H. performed computational simulations. E.A. and W.B. fabricated bioresorbable optical filters. T.W., N.S.P. and J.M.T. developed and synthesized the hydrogels. L.T., L.D. and K.B. evaluated the biocompatibility. Y.Z., E.R., L.Z., J.U.K., L.T., A. Mikhailov., K.Z., X.L., Y.W., H.Z., A.L., E.A., G.J., S.L., S.G.S., K.B., N.L., W.O., R.K.A., I.R.E. and J.A.R. discussed and interpreted the data. Y.Z. and J.A.R. prepared figures and wrote the paper, with input from E.R., W.O., A. Mikhailov, R.K.A. and I.R.E. In addition, J.U.K., L.Z., L.T., Y.W., H.Z., S.L. and J. Gu. assisted with the preparation of figures and text. Y.Z., L.Z., L.T., H.Z., L.D., W.O., I.R.E. and J.A.R. revised the paper. Y.Z., E.R., L.Z., J.U.K., L.T. and H.Z. contributed equally to this work.
Correspondence to
Yamin Zhang, Yonggang Huang, Wei Ouyang, Rishi K. Arora, Igor R. Efimov or John A. Rogers.
The authors declare no competing interests.
Nature thanks Gábor Duray, Hossam Haick 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, Comparisons between conventional pacemakers with leads, leadless pacemakers, bioresorbable pacemakers, and the pacemaker reported here. b, Table showing details of previously reported leadless pacemakers and the pacemaker reported here. Illustration of the pacemaker with leads in a was created with BioRender.com (https://biorender.com). Bioresorbable pacemaker in a adapted from ref. 1, Springer Nature America, Inc.
a,b, Characteristic curves of the phototransistors under various light intensities emitted from a NIR LED (850 nm, a) and a red LED (650 nm, b).
a, EIS of an agarose gel and chicken tissue. b, The output currents of the pacemaker over days. c, Output currents of the pacemaker over days under pulsed illumination.
a, Photograph showing a pacemaker placed on the surface of a mouse heart. b, ECG results before and during mouse heart pacing. c, Strength-duration curve when pacing at 480 bpm. n = 3 biologically independent animals.
a, Emission spectra for LEDs 1 and 2. b, Transmission curves for filters 1 and 2. c, Transmitted light intensities as a function of incident intensities from LEDs 1 and (2) for filters 1 and 2.
Supplementary Methods, Notes 1–11, Tables 1–3, Figs. 1–26 and References.
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Zhang, Y., Rytkin, E., Zeng, L. et al. Millimetre-scale bioresorbable optoelectronic systems for electrotherapy.
Nature 640, 77–86 (2025). https://doi.org/10.1038/s41586-025-08726-4
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Animals learn the value of foods on the basis of their postingestive effects and thereby develop aversions to foods that are toxic1,2,3,4,5,6,7,8,9,10 and preferences to those that are nutritious11,12,13. However, it remains unclear how the brain is able to assign credit to flavours experienced during a meal with postingestive feedback signals that can arise after a substantial delay. Here we reveal an unexpected role for the postingestive reactivation of neural flavour representations in this temporal credit-assignment process. To begin, we leverage the fact that mice learn to associate novel14,15, but not familiar, flavours with delayed gastrointestinal malaise signals to investigate how the brain represents flavours that support aversive postingestive learning. Analyses of brain-wide activation patterns reveal that a network of amygdala regions is unique in being preferentially activated by novel flavours across every stage of learning (consumption, delayed malaise and memory retrieval). By combining high-density recordings in the amygdala with optogenetic stimulation of malaise-coding hindbrain neurons, we show that delayed malaise signals selectively reactivate flavour representations in the amygdala from a recent meal. The degree of malaise-driven reactivation of individual neurons predicts the strengthening of flavour responses upon memory retrieval, which in turn leads to stabilization of the population-level representation of the recently consumed flavour. By contrast, flavour representations in the amygdala degrade in the absence of unexpected postingestive consequences. Thus, we demonstrate that postingestive reactivation and plasticity of neural flavour representations may support learning from delayed feedback.
Postingestive feedback signals arise from the gut as food is digested and absorbed, and animals are able to associate this delayed feedback with flavours experienced during a meal minutes or hours earlier1,2,3,4,5,6,7,8,9,10,11,12,13. This learning process is essential for survival—nutritious foods are valuable, whereas poisonous foods can be deadly—but it remains unknown how the brain is able to associate a stimulus (flavour) with a delayed reinforcement signal (postingestive feedback from the gut) that can arrive much later.
Conditioned flavour aversion (CFA) provides a classic example of this credit-assignment problem. Humans8,9,10, rodents1,2,3,4,5 and other animals6,7 develop CFAs when they experience symptoms of food poisoning (such as gastrointestinal malaise, nausea or diarrhoea), which produces a long-lasting aversion to the potentially poisonous food. Animals can develop a CFA to novel foods after a single pairing (that is, one-shot learning) even with meal-to-malaise delays of several hours16,17,18.
Previous work on CFA has focused on two primary anatomical pathways. The first begins in the mouth and sends taste signals to the gustatory insular cortex19,20, which in turn transmits these signals to the basolateral amygdala (BLA)21,22. The second begins in the gut and sends malaise signals to a genetically defined population of glutamatergic neurons in the hindbrain parabrachial nucleus (PB) called calcitonin gene-related peptide (CGRP) neurons23,24. These malaise-coding CGRP neurons then project to the central amygdala (CEA) and the bed nucleus of the stria terminalis (BST). However, it remains unclear where and how temporally separated flavour and malaise signals ultimately converge in the brain to support learning.
To gain insight into this long-standing question, we leveraged the fact that novel flavours support CFA after a single pairing with malaise, whereas familiar flavours that are already known to be safe do not14,15. Mice consumed a palatable flavour (sweetened grape Kool-Aid) that was either novel (no previous exposure before conditioning) or familiar (four daily pre-exposures before conditioning). Thirty minutes after consumption, the mice were given an intraperitoneal injection of lithium chloride (LiCl) to induce gastrointestinal malaise and related food-poisoning symptoms18,25. We then assessed learning 2 days later with a two-bottle memory-retrieval test (Fig. 1a). We used a flavour (combination of taste and odour), rather than a pure taste stimulus, in our study for ethological validity because animals rarely encounter a taste alone, and use both taste and odour to avoid foods that have made them ill26,27. Consistent with previous work14,15, mice for which the malaise-paired flavour was novel developed a strong and stable aversion, whereas mice for which the same flavour was familiar continued to prefer it to water even after the pairing with malaise (Fig. 1b).
a, Schematic of the CFA paradigm. i.p., intraperitoneal injection. b, Flavour preference across three consecutive daily retrieval tests for mice that consumed either a novel (top) or familiar (bottom; all statistical tests not significant (NS)) flavour and then were injected with either LiCl or saline on the conditioning day (n = 8 mice per group). The flavour (sweetened grape Kool-Aid) and amount consumed (1.2 ml) was the same for all groups. The group given a familiar flavour was pre-exposed to the flavour on four consecutive days before conditioning, whereas the group given the novel flavour was completely naive. c, Example FOS imaging data (100 µm maximum-intensity projection) and cell detection results from the brain-wide light-sheet microscopy imaging pipeline. Scale bars, 1 mm (left), 100 µm (bottom right) and 25 µm (top right). d, Description of the GLMM for the brain-wide FOS dataset (n = 12 mice per flavour condition per time point for e–i; see Methods and equation (2)). e, Novel – familiar ΔFOS effect distribution at each time point across all significantly modulated brain regions (n = 130 brain regions). Each point represents a single brain region. f, Hierarchical clustering of novel – familiar ΔFOS effects (see Extended Data Fig. 3d for an expanded version). g, Detail of the amygdala network (cluster 1 from f) that is preferentially activated by novel flavours at every stage of learning. h, Visualization of the difference in FOS+ cell density across flavour conditions with Allen CCF boundaries overlaid. i, Comparison of individual mice for the novel and familiar flavour conditions for the CEA at each time point. Error bars represent the mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. See Supplementary Table 2 for details of statistical tests and for exact P values. See Supplementary Table 1 for a list of brain-region abbreviations and for GLMM statistics.
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Using this experimental paradigm, we compared cellular-resolution brain-wide activation levels in response to the same flavour when it was novel versus familiar to determine where novel flavours that support learning are represented and where this representation converges with postingestive malaise signals. After each stage of CFA learning (consumption, malaise and retrieval), brain samples from experimental mice were cleared using the iDISCO+ method28 and the samples were then immunolabelled for the immediate-early gene product FOS as a proxy for neural activation. High-resolution and high-signal-to-noise whole-brain light-sheet microscopy imaging volumes were subsequently acquired. We used an automated deep-learning-assisted cell-detection pipeline (258,555 ± 14,421 (mean ± s.e.m.) FOS+ neurons per animal across the experiments in Figs. 1 and 2 ; see Methods for details) and registered the location of each FOS+ neuron to the Allen Mouse Brain Common Coordinate Framework29 (CCF) for downstream analyses (Fig. 1c).
a, Schematic of the pathway that conveys malaise signals to the amygdala. b, CGRP neurons are activated in vivo by LiCl-induced malaise (n = 5 mice). c, Top, schematic of the slice electrophysiology experiment. Bottom, traces showing strong monosynaptic connections from CGRP neurons to the CEAc and CEAl and weaker connections to the CEAm (n = 5 neurons from 3 mice per region). Dark lines represent the average and transparent lines represent individual trials for example neurons. oEPSC, optically evoked excitatory postsynaptic current; TTX, tetrodotoxin; 4AP, 4-aminopyridine. d, Top, schematic for the CGRP neuron stimulation experiment. Bottom, example image of ChR2–YFP expression and data for the retrieval test (n = 6 mice per group). e, Left, schematic of the CGRPCEA projection stimulation experiment. Middle, example image of ChRmine–mScarlet expression. Right, retrieval test data (n = 6 mice per group). f, Left, schematic of the CGRPCEA projection inhibition experiment. Middle, example image of eOPN3–mScarlet expression. Right, retrieval test data (n = 11 mice for eOPN3, 9 mice for YFP). g, Schematic of the CGRP neuron stimulation FOS experiment (n = 14 mice for novel flavour, 13 mice for familiar flavour for h–k). ITI, inter-trial interval. h, Summary of FOS+ cell counts in the CEA for individual mice. i, Correlation between average FOS+ cell count for LiCl-induced malaise versus CGRP neuron stimulation (n = 12 for the amygdala network, 117 for the other regions). j, Analogous to i, but comparing the difference between flavour conditions. k, Visualization of the difference in FOS+ cell density across flavour conditions. l, Schematic of the FISH experiment. m,n, Comparison of marker gene expression (m) and co-expression (n) (n = 6 mice for novel flavour, 7 mice for familiar flavour; 490 ± 54 Fos+ neurons per mouse (mean ± s.e.m.); all statistical tests NS). Error bars represent the mean ± s.e.m. Shaded areas in b represent the mean ± s.e.m. and in i and j represent the linear fit estimate ±95% confidence intervals. Units in j are per cent per mm3. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. See Supplementary Table 2 for details of statistical tests and for exact P values. Scale bars, 1 mm (d–f) or 100 µm (l).
Source data
We initially investigated the first stage of the CFA paradigm: consumption of flavoured water. We observed marked differences in the brain-wide activation patterns of mice that consumed a novel versus familiar flavour, even though each group had precisely the same sensory experience during FOS induction (Extended Data Fig. 1a–d and Supplementary Table 1; interactive visualization at https://www.brainsharer.org/ng/?id=872, left column). Novel flavours that support learning preferentially activated a set of sensory and amygdala structures (for example, the CEA, the BLA, the insular cortex and the piriform cortex; Extended Data Fig. 1a). These observations are consistent with previous anatomically targeted studies of immediate-early gene expression30,31 and with loss-of-function experiments19,20,21,22 that demonstrated a causal role for many of these regions in CFA. By contrast, a familiar flavour that animals had previously learned was safe primarily engaged a network of limbic regions (for example, the lateral septum (LS), the ventral hippocampus, the prefrontal cortex and the nucleus accumbens; Extended Data Fig. 1b). Unlike the novel-flavour-activated regions, most of these regions had not previously been implicated in CFA. The LS showed the strongest familiar-flavour-dependent activation, and chemogenetic activation of this region during consumption was sufficient to block CFA learning (Extended Data Fig. 2a,b) and amygdala activation by a novel flavour (Extended Data Fig. 2c–g). Together, these results validate the potential of our brain-wide FOS imaging approach to identify new regions that contribute to CFA.
We next investigated how the brain-wide activation patterns induced by a novel versus familiar flavour change during postingestive malaise and, days later, memory retrieval (interactive visualization at https://www.brainsharer.org/ng/?id=872, middle and right columns). We reasoned that preferential novel-flavour activation at these time points, respectively, may reveal where flavour and malaise signals initially converge to support CFA learning and where the CFA memory is stored and recalled. To accurately estimate the contribution of flavour novelty and experimental time point to neural activation in each brain region, we applied a generalized linear mixed model (GLMM) that accounts for variation associated with different technical batches and with the sex of the mice (Fig. 1d, Methods and Supplementary Table 1).
Although novel and familiar flavours preferentially activated an equal fraction of brain regions during consumption (Extended Data Fig. 1a,b), postingestive malaise triggered a brain-wide shift towards activation by the novel flavour (Fig. 1e and Extended Data Fig. 3a–c). This shift towards representing the novel flavour was still present when the memory was retrieved days later (Fig. 1e and Extended Data Fig. 3a–c).
To investigate which brain regions contributed to this effect, we performed hierarchical clustering on weights from the GLMM of novel-flavour versus familiar-flavour activation across experimental time points (Fig. 1f and Extended Data Fig. 3d–n). This analysis uncovered a network of amygdala regions (cluster 1) that was preferentially activated by the novel flavour at every stage of learning (Fig. 1g,h and see Extended Data Fig. 4a–d for correlation analyses within and across clusters), most notably in the CEA (Fig. 1i).
This discovery is notable for two reasons. First, the fact that the representation of a novel flavour formed during the initial consumption stage is still present 30 min later during postingestive malaise implies that this network is a site for the convergence of flavour and malaise signals. Second, the observation that this novel-flavour representation is still present upon memory retrieval suggests that the same network is also a site of storage and recall.
The idea that the amygdala could be a crucial node for the convergence of flavour representations and malaise signals is further supported by classic work establishing the amygdala as a site of associative learning32,33. Further pointing to the amygdala as a site of convergence, parabrachial CGRP neurons in the brainstem are reported to convey visceral malaise signals to the CEA23,24,34 (Fig. 2a). Indeed, we found that LiCl injection activated CGRP neurons in vivo34 (Fig. 2b). CGRP neurons formed dense monosynaptic connections in the CEA34 (Fig. 2c; latency: 5.9 ± 0.3 ms (mean ± s.e.m.)), and stimulation of CGRP neurons activated neurons throughout the amygdala in vivo (Extended Data Fig. 5a–f).
Optogenetic stimulation of CGRP neurons also recapitulated the effects of LiCl-induced malaise in mediating novel-flavour-dependent delayed CFA. Specifically, stimulation of CGRP neuron cell bodies that began 30 min after flavour consumption was sufficient to replace LiCl injection and condition an aversion to a novel but not familiar flavour (Fig. 2d). Similarly, stimulation of CGRP neuron→CEA (CGRPCEA) axon terminals 30 min after consumption of a novel flavour was also sufficient to condition a strong CFA (Fig. 2e). CGRPCEA projection inhibition during delayed LiCl-induced malaise significantly interfered with CFA acquisition (Fig. 2f), but did not fully block it. This result is consistent with previous work showing that other CGRP neuron projections, for example to the BST24, also contribute to CFA.
Given the similarities between LiCl-induced malaise and CGRP neuron stimulation, we sought to determine whether postingestive stimulation of these cells could recapitulate the effects of LiCl-induced malaise on neural activation in the amygdala and across the brain (Fig. 2g and Extended Data Fig. 6a,b). Postingestive CGRP neuron stimulation produced highly similar levels of overall neural activation (FOS+ cell counts in individual brain regions) across the entire brain compared to LiCl-induced malaise (Fig. 2i and Extended Data Fig. 6c,e). Also similar to LiCl-induced malaise, CGRP neuron stimulation induced stronger activation of the amygdala network when preceded by consumption of a novel rather than familiar flavour (Fig. 2j,k and Extended Data Fig. 6d,f). This effect was particularly prominent in the CEA (Fig. 2h) and was not observed in brain regions outside the amygdala network (Fig. 2j and Extended Data Fig. 6d,f).
We next asked whether the stronger effect of CGRP neuron stimulation on amygdala activation after consumption of a novel versus familiar flavour could be explained by recruitment of a specific CEA cell type. To address this question, we performed multiplex fluorescence in situ hybridization (FISH) in the CEA in a separate group of mice that consumed either a novel or familiar flavour followed by delayed CGRPCEA projection stimulation. We then examined co-expression of Fos mRNA with markers for several known CEA cell types35,36 (Sst, Prkcd and Calcrl; Fig. 2l). Most cells that expressed Fos also expressed Prkcd or Calcrl, and this did not depend on whether the mice had consumed a novel or familiar flavour before CGRPCEA projection stimulation (Fig. 2m,n and Extended Data Fig. 6g,h).
Taken together, these experiments show that postingestive CGRP neuron activity is necessary and sufficient to mediate delayed CFA learning and the effects of malaise on novel-flavour-dependent, amygdala-specific neural activation. This novelty-dependent activation does not seem to be instantiated by a specific CEA cell type. Instead, novel (versus familiar) flavour consumption leads to a greater number of Prkcd+ and Calcrl+ CEA neurons being activated by subsequent CGRP neuron activity.
Our brain-wide FOS measurements and CGRP neuron manipulations point to the amygdala as a unique site for the convergence of flavour representations and delayed malaise signals. However, these experiments do not resolve how these temporally separated signals are integrated at the single-cell level. One possibility is that individual novel-flavour-coding neurons may be persistently activated long after a meal in a manner that provides passive overlap with delayed CGRP neuron malaise signals (hypothesis 1 in Fig. 3a). Alternatively, CGRP neuron inputs may specifically reactivate novel-flavour-coding neurons (hypothesis 2 in Fig. 3a). Another possibility is that CGRP neuron inputs may activate a separate population of amygdala neurons that subsequently become incorporated into the novel-flavour representation during memory consolidation (hypothesis 3 in Fig. 3a). Testing these hypotheses requires tracking the activity of the same neurons across the stages of learning.
a, Hypotheses for how the amygdala associates temporally separated flavour and malaise signals. b, Schematic of the CGRP neuron-stimulation recording strategy. c, Reconstruction of recording trajectories registered to the Allen CCF. Each line represents one shank of a four-shank Neuropixels 2.0 probe (32 shanks from 8 mice). d, Average spiking of all individual neurons (n = 1,104 single units and multiunits from 8 mice). e, Average spiking of novel-flavour-preferring (n = 373), water-preferring (n = 121) and nonselective (n = 610) populations. f, Left, average spiking of individual neurons during CGRP neuron-stimulation bouts. Right, population averages (same sample sizes as e). g, Example of a multinomial logistic regression decoder session. h, Average decoder posterior time locked to CGRP neuron stimulation (mean across 6 mice). i, Average reactivation rates of novel flavour or water representations (n = 6 mice). j, PCA schematic. k, Population trajectories for novel-flavour consumption, water consumption and CGRP neuron stimulation. l, Trajectories for individual example mice. m, Schematic of the LiCl-induced-malaise recording strategy. n, Average spiking of novel-flavour-preferring (n = 280 neurons from 4 mice), water-preferring (n = 80) and nonselective (n = 218) populations. o, Average reactivation rates of novel flavour or water representations (n = 4 mice). p, Example CGRP immunoreactivity data confirming the ablation of CGRP neurons in the PB (outlined in green). Scale bar, 100 µm. q, Analogous to n, but for mice in which CGRP neurons were ablated (n = 124 novel-flavour-preferring, 20 water-preferring, 256 nonselective neurons from 4 mice; all statistical tests NS). r, Analogous to o, but for mice in which CGRP neurons were ablated (n = 4 mice). Shaded areas represent the mean ± s.e.m. Inset box plots show the 10th, 25th, 50th, 75th and 90th percentiles. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. See Supplementary Table 2 for details of statistical tests and for exact P values.
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Therefore, to distinguish these possibilities, we performed high-density recordings of individual neurons in the CEA—the core node in the amygdala network from our brain-wide FOS imaging dataset (Figs. 1 and 2)—during consumption, subsequent malaise and memory retrieval (Fig. 3b). Recordings were performed with chronically implanted four-shank Neuropixels 2.0 probes37 (Extended Data Fig. 7a). Reconstruction of individual shank trajectories confirmed that we were able to precisely target the CEA (Fig. 3c and Extended Data Fig. 7b,c; 138 ± 30 (mean ± s.e.m.) CEA neurons per animal in Fig. 3b–l). We initially trained mice to consume water at an equal rate from two port locations (Extended Data Fig. 8a and see Methods for details). On the conditioning day, we replaced one port with a novel flavour, whereas water remained in the other as an internal control. Immediately after the consumption period ended, mice were transferred to a distinct second context in which they would experience postingestive malaise. This step was performed to ensure that any neural correlates of flavour consumption that we might subsequently observe were not due to features of the original context in which consumption occurred. After a 30-min delay period in this second context, we induced postingestive malaise using one of the following methods across three groups of mice: (1) optogenetic stimulation of CGRP neuron cell bodies (Fig. 3b–l); (2) optogenetic stimulation of CGRPCEA projections (Extended Data Fig. 9a–e); or (3) injection of LiCl (Fig. 3m–r). Similar results were observed across all three groups of mice; therefore we begin by describing data from the first group.
During consumption, 34% of CEA neurons were significantly activated by the novel flavour compared with only 11% for water (Fig. 3d and Extended Data Fig. 8b) and, in a separate experiment described below, 17% for a familiar flavour. These observations are consistent with our FOS imaging data at the consumption time point (Fig. 1h,i). Almost all CEA neurons, including novel-flavour-coding neurons, were significantly less active after consumption ended (Fig. 3d,e), which suggests that persistent activation (hypothesis 1 in Fig. 3a) is not the mechanism that the amygdala uses to associate flavours with delayed malaise signals.
We next investigated how delayed CGRP neuron stimulation affects CEA neuron activity. CGRP neuron stimulation potently and selectively reactivated novel-flavour-coding CEA neurons, with only limited effects on water-coding and nonselective neurons (Fig. 3d,e; consistent only with hypothesis 2 in Fig. 3a). This reactivation was precisely time locked to individual bouts of CGRP neuron stimulation (Fig. 3f), which suggests that it is directly driven by the release of glutamate from CGRP neuron inputs34 (Fig. 2c and Extended Data Fig. 5c) rather than by a slow change in affective or physiological internal state. Similar to CGRP neuron cell-body stimulation, delayed CGRPCEA projection stimulation strongly reactivated novel-flavour-coding CEA neurons (Extended Data Fig. 9a–d).
Reactivation of novel-flavour-coding neurons was similarly present in a separate group of mice that experienced delayed LiCl-induced malaise rather than CGRP neuron stimulation (Fig. 3m,n). Moreover, genetic ablation of CGRP neurons abolished the preferential reactivation of novel-flavour-coding neurons by delayed malaise (Fig. 3p,q) and impaired learning (Extended Data Fig. 9f,g). This finding indicates that the effects of postingestive malaise on CFA—and on CEA dynamics—are mediated by CGRP neurons.
Together, these observations suggest that CEA neurons that encode a recently consumed flavour are selectively reactivated by delayed malaise signals through CGRP neuron inputs, thereby providing a potential mechanism for temporal credit assignment during CFA learning (hypothesis 2 in Fig. 3a).
Population-level analyses corroborated the conclusion that CGRP neuron activity after consumption preferentially reactivates the neural representation of the recently consumed flavour. First, we trained a multinomial logistic regression decoder using population activity during the consumption period to discriminate novel flavour or water consumption from baseline activity (Fig. 3g and Extended Data Fig. 8c,d). Cross-validated decoding accuracy was nearly perfect (Extended Data Fig. 8e). We then evaluated this decoder using population activity during the delay and CGRP neuron-stimulation periods and investigated the probability of decoding the novel flavour or water representation on a moment-by-moment basis (decoder output, P(novel flavour|population activity)). This decoding analysis showed that individual bouts of CGRP neuron stimulation reliably reactivated population-level flavour representations (Fig. 3g–i). By contrast, water representations were rarely reactivated by CGRP neuron stimulation (Fig. 3g–i). Consistent with the results from CGRP neuron cell-body stimulation, LiCl-induced malaise also strongly reactivated population-level flavour representations in the CEA (Fig. 3o) and this required functional CGRP neurons (Fig. 3r).
We next compared population-activity trajectories during consumption and during CGRP neuron stimulation by performing principal component analysis (PCA) on the pooled (across mice) trial-averaged population activity during novel-flavour and water consumption. Neural activity during consumption was low-dimensional, with the first two principal components (PCs) explaining >70% of variance in trial-averaged population activity (Fig. 3j). Plotting neural trajectories during novel-flavour and water consumption on the PC1–PC2 axis revealed that PC2 perfectly discriminated these two flavours (Fig. 3k). Using these PCA loadings to project population activity during CGRP neuron stimulation onto the same PC1–PC2 axis showed that this experience closely mirrored the neural trajectory of novel-flavour consumption (Fig. 3k). This strong effect was also apparent in the population activity of individual mice (Fig. 3l) and in the population activity of mice that received delayed CGRPCEA projection stimulation (Extended Data Fig. 9e). Thus, population-level analyses confirmed that CGRP neuron activity specifically reactivates flavour representations in the CEA during delayed postingestive malaise (hypothesis 2 in Fig. 3a).
Tracking neural activity across flavour consumption and malaise revealed that malaise signals preferentially reactivate flavour representations in the amygdala (Fig. 3), thereby providing a potential mechanism for the brain to link flavours experienced during a meal with delayed postingestive feedback. If this mechanism contributes to learning, then postingestive CGRP neuron activity would be expected to trigger functional plasticity in flavour representations in the amygdala that could underlie the CFA memory. To test this hypothesis, we examined whether postingestive reactivation of novel-flavour-coding CEA neurons is predictive of stronger flavour responses when the CFA memory is retrieved. To accomplish this task, we took advantage of the high stability of our chronic recordings to track the same CEA neurons across days and analysed their responses to flavour consumption before (conditioning day) and after (retrieval day) pairing with CGRP neuron stimulation (Fig. 4a,b and see Methods for details).
a, Spike waveforms, autocorrelograms (Autocorr.) and flavour response rasters for an example neuron tracked across conditioning and retrieval days. b, Average spiking of all tracked neurons during the consumption, delay and CGRP neuron-stimulation periods on the conditioning day and during consumption on the retrieval day (n = 939 neurons from 8 mice). c, Left, average spiking of the novel-flavour-preferring population (n = 265 neurons) during flavour consumption on conditioning and retrieval days. Middle and right, average spiking of novel-flavour-preferring neurons with the highest 10% CGRP response magnitudes and of the remaining novel-flavour-preferring neurons. d, Correlation between the change (retrieval – conditioning) in flavour response or selectivity for each neuron during consumption to its average response during the CGRP neuron-stimulation period. The novel-flavour-preferring (n = 265 neurons), water-preferring (n = 123 neurons) and nonselective (n = 551 neurons) populations are shown separately. e, Analogous to d, but for mice with CGRPCEA projection stimulation (n = 286 novel-flavour-preferring neurons from 8 mice). f, Left, schematic of the flavour-familiarization experiment. Right, average spiking of the initially flavour-preferring population (n = 201 neurons from 7 mice; classified on novel-flavour day) during flavour consumption on the novel day and the familiar day. g, Illustration of the neural mechanism for learning from delayed postingestive feedback using malaise-driven reactivation and stabilization of flavour representations in the amygdala. Shaded areas in c and f represent the mean ± s.e.m. and in d and e represent the linear fit estimate ±95% confidence intervals. Inset box plot shows the 10th, 25th, 50th, 75th and 90th percentiles. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001. See Supplementary Table 2 for details of statistical tests and for exact P values.
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The trial-averaged response of novel-flavour-coding CEA neurons (classified on the conditioning day) was largely stable across days (Fig. 4c). However, sorting these neurons on the basis of the magnitude of their response to CGRP neuron stimulation revealed a notable effect. Specifically, novel-flavour-coding CEA neurons with the greatest CGRP neuron input responded more strongly to the flavour during memory retrieval, whereas the responses of novel-flavour-coding neurons with weak or no CGRP neuron input remained relatively unchanged (Fig. 4c,d). By contrast, we did not observe a similar correlation for water-coding or nonselective CEA neurons (Fig. 4d), which further suggests that postingestive malaise does not recruit additional neurons into the initial flavour representation (as in hypothesis 3 in Fig. 3a). Together, these observations indicate that CGRP neurons induce functional plasticity that stabilizes the response of the amygdala to the conditioned flavour after learning. Consistent with this conclusion, the population-level flavour representation, as visualized using PCA, was highly stable across conditioning and retrieval days (Extended Data Fig. 10b,l).
CGRPCEA projection stimulation (Fig. 4e and Extended Data Fig. 10c–e) and LiCl-induced malaise (Extended Data Fig. 10f) had similar stabilizing effects on novel-flavour-coding neuron responses during memory retrieval compared to CGRP neuron cell-body stimulation. By contrast, the responses of novel-flavour-coding neurons significantly decreased during memory retrieval in mice lacking CGRP neurons (Extended Data Fig. 10g). Thus, CGRP neuron activity is necessary and sufficient for both the reactivation and stabilization of flavour representations in the amygdala by delayed malaise signals.
For comparison, we next asked how amygdala activity evolves after familiarization (that is, experience with a flavour without any aversive postingestive consequences, as in the ‘Familiar' condition in Figs. 1 and 2). Consistent with our initial FOS imaging data comparing activation patterns between novel and familiar flavours (Fig. 1h,i), tracking CEA neuron activity before and after familiarization revealed that the proportion of flavour-coding neurons significantly decreased after familiarization (Extended Data Fig. 10h,i). Similarly, the trial-averaged response of individual flavour-coding neurons (classified on the novel-flavour day) significantly decreased after familiarization (Fig. 4f). This result was in contrast to the stability we observed following conditioning with CGRP neuron stimulation (Fig. 4c, left) and LiCl-induced malaise (Extended Data Fig. 10f). Furthermore, initially water-preferring neurons increased their response to the flavour after it became familiar (Extended Data Fig. 10j). Together, these observations suggest that familiarization degrades flavour representations in the amygdala such that the representation of a flavour moves closer to the representation of pure water. Consistent with this conclusion, the modulation of population-level activity along the PC2 dimension that discriminates flavour from water was almost completely abolished following familiarization (Extended Data Fig. 10k,l).
Thus, CGRP neurons convey malaise signals that preferentially reactivate flavour representations in the amygdala, which may enable the brain to bridge the delay between a meal and postingestive feedback during CFA learning. Moreover, these postingestive signals induce plasticity to stabilize or strengthen flavour representations after conditioning, whereas flavour representations rapidly degrade in the absence of malaise signals as flavours become familiar and safe (Fig. 4g).
So far, we have focused on the role of neural activity in supporting CFA. Previous work in the field has taken a complementary approach to examine the role of biochemical signals38,39. For example, many studies have shown an important role in the amygdala for cAMP response element-binding protein (CREB)40,41,42, a transcription factor that regulates neural excitability, and protein kinase A (PKA)43,44, which phosphorylates and activates CREB. CREB activity levels at the time of conditioning are thought to bias neurons towards allocation to the CFA ‘memory engram'45,46: the ensemble of cells that are activated during retrieval of the CFA memory (Fig. 5a).
a, Simplified schematic of the biochemical pathway that has been proposed to allocate a subset of amygdala neurons to the CFA memory-retrieval ensemble (or ‘memory engram')40,41,42,43,44,45,46. b, Schematic of the strategy for recording PKA activity in the CEA across flavour-familiarization using the AKAR2 sensor47. F, familiar; N, novel. c, Example image of AKAR2 expression in the CEA. Scale bar, 1 mm. d, PKA activity in the CEA in response to consumption of a novel or familiar flavour (port A) and to water (port B) across four consecutive days (n = 13 mice). Points and error bars at the top of each plot indicate the timing of the next reward consumption. e, PKA activity for individual mice in response to a novel or familiar flavour and to water consumption. Mice were sorted by novel-flavour response (day 1). f, Summary of PKA activity in response to a novel or familiar flavour and to water consumption (n = 13 mice). g, PKA activity in response to novel flavour (left) and water (right) consumption on day 1 for individual mice aligned to the Allen CCF (n = 13 mice). h, Schematic of a putative hypothesis linking biochemistry with neural activity during CFA, with novelty-dependent increases in PKA in novel-flavour-coding amygdala neurons leading to increased reactivation of these cells by delayed CGRP neuron inputs and recruitment to the CFA memory-retrieval ensemble. Error bars in d represent the 25th, 50th and 75th percentiles and in f represent the mean ± s.e.m. Shaded areas represent the mean ± s.e.m. *P ≤ 0.05, ****P ≤ 0.0001. See Supplementary Table 2 for details of statistical tests and for exact P values.
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We therefore investigated how the PKA→CREB pathway might relate to the malaise-driven reactivation and stabilization of flavour representations that we describe here. One possibility is that this biochemical pathway could be preferentially triggered in the amygdala by novel-flavour consumption, which may in turn contribute to increased excitability or responsiveness of novel-flavour-coding neurons to CGRP neuron inputs during malaise.
To test the first part of this hypothesis—that the PKA→CREB pathway is preferentially activated by novel flavours—we recorded in vivo PKA activity in the CEA through fibre photometry measurements of the AKAR2 sensor47 (Fig. 5b,c). These recordings showed that novel flavours drive a strong increase in PKA activity in the CEA, whereas familiar flavours have little impact on PKA activity (Fig. 5d–g). This increase in PKA activity (tens of seconds) was substantially longer in duration than the increase in spiking for each bout of consumption (Fig. 3d). Downstream effects on CREB, gene expression and neural excitability are presumably far longer-lasting. Thus, novelty-dependent gating of PKA could serve as a biochemical eligibility trace that increases the responsiveness of novel-flavour-coding neurons to delayed malaise signals, thereby permitting the selective reactivation and plasticity of novel-flavour representations in the amygdala (Fig. 5h).
The major reason that learning is challenging is because of delays between a stimulus or action and its outcome. This raises the question of how the brain assigns credit to the correct previous event. Most work on credit assignment has been limited to examining learning in the case of relatively short delays (on the order of seconds)48,49,50,51. Postingestive learning paradigms, such as CFA, provide an opportunity to study how the brain assigns credit across much longer delays.
Here we described a neural mechanism that may contribute to solving the credit-assignment problem inherent to CFA. That is, postingestive malaise signals selectively reactivate the neural representations of flavours experienced during a recent meal, and this reactivation serves to stabilize or strengthen the flavour representation upon memory retrieval.
Although previous work, mostly in the hippocampus and cortex, has suggested a role for neural reactivations in learning and memory52,53, our study advances this idea in multiple important ways. First, we applied this concept to a new paradigm: postingestive learning and CFA. Second, we discovered a role for the outcome signal (unconditioned stimulus) in directly triggering reactivations and demonstrated that a cell-type-specific malaise pathway mediates this effect. Third, we discovered a relationship between outcome-driven flavour reactivations and strengthened flavour representations during memory retrieval.
Our entry point into this problem was the fact that novel flavours more easily support postingestive learning than familiar flavours14,15. By comparing brain-wide neural-activation patterns in animals that consumed the same flavour when it was novel versus familiar, we identified an amygdala network that was unique in preferentially responding to novel flavours across every stage of learning. Although we focused here on how novel flavours become associated with aversive postingestive feedback in this amygdala network, it is possible that these same ideas generalize to the processes of familiarization or postingestive nutrient learning. Specifically, a parallel and mechanistically similar process may be at work when learning that a food is safe or nutritious. In that case, safety (or reward11,12,13) signals may reactivate recently consumed flavour representations, in contrast to the aversive CGRP neuron-mediated reactivations we report here. This may enable the weakening of flavour representations in the amygdala (Fig. 4f) and/or the strengthening of flavour representations in the LS and other limbic regions (Extended Data Fig. 1b).
The degree of specificity of malaise-driven reactivations for a recently consumed novel flavour (versus other flavours), and what mechanisms may contribute to such specificity, remain open questions. One possibility is that preferential activation of the PKA→CREB pathway by consumption of a particular novel flavour (Fig. 5) may provide a biochemical eligibility trace to facilitate the selective reactivation and strengthening of the neural representation of that flavour (versus other flavours) by delayed outcome signals.
Previous recording experiments during CFA have concentrated on the role of the gustatory insular cortex54,55,56,57,58,59. A consistent finding is that CFA amplifies the cortical representation of the conditioned tastant and shifts it to be more similar to innately aversive tastants. Recent work has further shown that homeostatic synaptic plasticity in the insula contributes to a transition, over the course of hours or days, from the initial formation of a more generalized taste aversion to a tastant-specific CFA memory60. How the malaise-driven reactivation of flavour representations in the amygdala we report here relates to such mechanisms in the insula requires further study. One possibility is that the reactivation of flavour representations by delayed malaise signals contributes to the formation of an initial memory, which is then further refined through homeostatic mechanisms in the insula and its reciprocal connections with the amygdala.
Overall, our results reveal how dedicated novelty-detection circuitry and built-in priors (preferential reactivation of recent flavour representations by postingestive malaise) work together to enable the brain to correctly link stimuli and outcomes despite long delays.
All experimental procedures were approved by the Princeton University Institutional Animal Care and Use Committee following the NIH Guide for the Care and Use of Laboratory Animals. Wild-type mice (JAX 000664) and Calcacre mice34 (JAX 033168) were obtained from the Jackson Laboratory. Adult mice (>8 weeks old) of both sexes were used for all experiments. Mice were housed under a 12-h light–dark cycle, and experiments were conducted during the dark cycle. Ambient temperature was maintained at 21–26 °C and humidity at 30–70%. Stereotaxic surgeries were performed under isoflurane anaesthesia (3–4% for induction, 0.75–1.5% for maintenance). Mice received pre-operative antibiotics (5 mg kg–1 Baytril subcutaneous (s.c.)) and pre-operative and post-operative analgesia (10 mg kg–1 Ketofen s.c.; 3 daily injections). Post-operative health (evidence of pain, incision healing, activity and posture) was monitored for at least 5 days. For all CFA experiments, mice were water-restricted and maintained at >80% body weight for the duration of the experiment.
For CGRP neuron cell-body stimulation experiments (Figs. 2–4), we bilaterally injected 400 nl of AAV5-EF1a-DIO-hChR2(H134R)-eYFP (titre, 1.2 × 1013 genome copies (GC) per ml; manufacturer, Princeton Neuroscience Institute (PNI) Viral Core Facility)61,62 at –5.00 mm anterior–posterior (AP), ±1.40 mm medial–lateral (ML) and –3.50 mm dorsal–ventral (DV) into Calcacre mice. We used these stereotaxic coordinates to target the PB in all subsequent experiments. For CGRP neuron fibre photometry experiments (Fig. 2b), we unilaterally injected 400 nl of AAV9-hSyn-FLEX-GCaMP6s (titre, 1.0 × 1013 GC per ml; manufacturer, PNI Viral Core Facility)63 into the PB of Calcacre mice. For CGRPCEA projection stimulation experiments (Figs. 2 and 4 and Extended Data Fig. 9), we bilaterally injected 350 nl of AAV5-EF1a-DIO-hChR2(H134R)-eYFP (titre, 1.2 × 13 GC per ml; manufacturer, PNI Viral Core Facility; RNAscope FISH experiment)61,62, AAV5-EF1a-DIO-ChRmine-mScarlet (titre, 9.0 × 1012 GC per ml; manufacturer, PNI Viral Core Facility; all other experiments)64 or AAV5-EF1a-DIO-eYFP (titre, 1.5 × 1013 GC per ml; manufacturer, PNI Viral Core Facility) into the PB of Calcacre mice. For CGRPCEA projection inhibition experiments (Fig. 2f), we bilaterally injected 350 nl of AAV5-hSyn-SIO-eOPN3-mScarlet (titre, 9.0 × 1012 GC per ml; manufacturer, Addgene)65 or AAV5-EF1a-DIO-eYFP (titre, 1.5 × 1013 GC per ml; manufacturer, PNI Viral Core Facility) into the PB of Calcacre mice. For CGRP neuron ablation experiments (Fig. 3p–r and Extended Data Fig. 9g,h), we bilaterally injected 350 nl of AAV5-EF1a-FLEX-taCasp3-TEVp (titre, 1.6 × 1013 GC per ml; manufacturer, Addgene)66 into the PB of Calcacre mice. For control LiCl conditioning and Neuropixels implantation experiments (Fig. 3n,o), we bilaterally injected 350 nl of AAV5-Camk2a-eYFP (titre, 7.5 × 1011 GC per ml; manufacturer, University of North Carolina (UNC) Vector Core) into the PB of wild-type mice. For CEA PKA recording experiments (Fig. 5), we unilaterally injected 300 nl of AAV5-hSyn-ExRai-AKAR2 (titre, 2.4 × 1013 GC per ml; manufacturer, PNI Viral Core Facility)47 at –1.15 mm AP, –2.65 mm ML and –4.85 mm DV into wild-type mice. For LS activation experiments (Extended Data Fig. 2), we bilaterally injected AAV5-hSyn-hM3D(Gq)-mCherry (titre, 3.8 × 1012 GC per ml; manufacturer, Addgene)67 or AAV5-Camk2a-eYFP (titre, 7.5 × 1011 GC per ml; manufacturer, UNC Vector Core) at one (500 nl at +0.55 mm AP, ±0.35 mm ML and –4.00 mm DV) or two (150 nl each at +0.85 mm or +0.25 mm AP, ±0.60 mm ML and –3.75 mm DV) coordinates into wild-type mice. Virus was infused at 100 nl min–1. Coordinates are given relative to bregma. We allowed 3 weeks for AKAR2 and GCaMP expression, at least 4 weeks for ChR2 and hM3D expression, 5 weeks for CGRP neuron ablation by taCasp3-TEVp and 8 weeks for CGRPCEA terminal expression of ChRmine, ChR2 and eOPN3.
Optical fibres encased in stainless-steel ferrules were implanted into the brain for optogenetic and fibre photometry experiments. For bilateral optogenetic stimulation of CGRP neurons (Fig. 2), we implanted 300 µm core diameter, 0.39 NA fibres (Thorlabs, FT300EMT) above the PB at a 10° angle, with the fibre tips terminating 300–400 µm above the viral injection coordinate. For unilateral stimulation of CGRP neurons (Figs. 3 and 4), we implanted a 300 µm core diameter, 0.39 NA fibre above the left PB at a 25–30° angle, with the fibre tip terminating 300–400 µm above the viral injection coordinate. For bilateral optogenetic manipulation of CGRPCEA projections (Fig. 2), we implanted 300 µm core diameter, 0.39 NA fibres above the CEA, with the fibre tips terminating at –1.15 mm AP, ±2.85 mm ML and –4.25 mm DV. For unilateral optogenetic stimulation of CGRPCEA projections (Fig. 4 and Extended Data Figs. 9 and 10), we implanted a 300 µm core diameter, 0.37 NA fibre (Doric, MFC_300/360-0.37_10mm_MF2.5_FLT) above the left CEA at a +55° angle, with the fibre tip terminating at –1.20 mm AP, +2.25 mm ML and –3.55 mm DV. For fibre photometry recording of CGRP neurons (Fig. 2b), we implanted a 400 µm core diameter, 0.48 NA fibre (Doric, MFC_400/430-0.48_5.0mm_MF2.5_FLT) above the left PB at a –10° to –30° angle, with the fibre tip terminating approximately at the viral injection coordinate. For fibre photometry recording of CEA PKA activity (Fig. 5), we implanted a 400 µm core diameter, 0.48 NA fibre (Doric, MFC_400/430-0.48_6.0mm_MF2.5_FLT) above the left CEA, with the fibre tip terminating approximately at the viral injection coordinate. Optical fibres were affixed to the skull with Metabond (Parkell, S380), which was then covered in acrylic dental cement.
We used four-shank Neuropixels 2.0 probes37 (test-phase; Imec), as they were miniaturized to make them more suitable for chronic implantation in mice. To avoid directly cementing the probes to the skull (that is, so that the probes could be reused), we designed a chronic implant assembly (Extended Data Fig. 7a) based on the design for Neuropixels 1.0 probes previously validated in rats68. Similar to that design, the assembly was printed on Formlabs SLA 3D printers and consisted of four discrete parts: (1) a dovetail adapter permanently glued to the probe base; (2) an internal holder that mated with the dovetail adapter and facilitated stereotaxic manipulation of the probe; and (3–4) an external chassis, printed in two separated parts, that encased and protected the entire assembly. The external chassis and internal holder were attached using screws that could be removed at the end of the experiment to enable explantation and reuse. The external chassis of the final implant assembly was coated with Metabond before implantation. After explantation, probes were cleaned with consecutive overnight washes in enzyme-active detergent (Alconox Tergazyme) and silicone cleaning solvent (Dowsil, DS-2025) before reuse. The dimensions of the Neuropixels 2.0 implant assembly were significantly smaller than the Neuropixels 1.0 implant assembly68, primarily because of the smaller size of the probe and headstage. The maximum dimensions were 24.7 mm (height), 12.2 mm (width) and 11.2 mm (depth), with a weight of 1.5 g (not including the headstage). Space was made for the headstage to be permanently housed in the implant, as opposed to the previous design in which the headstage was connected only during recording and was secured to a tether attached to the animal. This made connecting the animal to the assembly for a recording significantly easier and obviated the need for a bulky tether that limits the movements of the animal. This change was made possible owing to improvements in Neuropixels cable design, which required fewer cables per probe and less reinforcement of the cables during free movement. Design files and instructions for printing and assembling the chronic Neuropixels 2.0 implant are available from GitHub (https://github.com/agbondy/neuropixels_2.0_implant_assembly).
Surgery was performed 3–4 weeks after AAV injection to allow time for viral expression and behavioural training. First, three craniotomies were drilled: one small craniotomy (500 µm diameter) above the left PB (approached at a –10° to –30° angle) or the left CEA (approached at a +55° angle) for the optical fibre, another small craniotomy above the cerebellum for the ground wire, and one large craniotomy (1 × 2 mm) above the left CEA for the Neuropixels probe. Next, a single optical fibre was placed above the left PB or left CEA as described above. At this point, the optical fibre was affixed to the skull with Metabond and the exposed skull was covered with Metabond. Next, a prefabricated chronic Neuropixels assembly was lowered at 2.5 µm s–1 into the CEA using an ultraprecise micromanipulator (Sensapex µMp). The probe shanks were aligned with the AP axis of the skull, with the most anterior shank tip terminating at –0.95 mm AP, –2.95 mm ML and –6.50 mm DV. Once the probe was fully lowered, the stainless-steel ground wire was inserted 1–2 mm into the cerebellum and affixed with Metabond. The CEA craniotomy and probe shanks were then covered with medical-grade petroleum jelly, and Dentin (Parkell, S301) was used to affix the chronic Neuropixels assembly to the Metabond on the skull. The optical fibres and CEA Neuropixels probe were both placed in the left hemisphere because CGRP neuron projections are primarily ipsilateral24.
As shown in Figs. 1 and 2, we used a one-reward CFA paradigm that used either a novel or familiar flavour. Experiments were performed in operant boxes (Med Associates) using MedPC software (https://med-associates.com/product/med-pc; v.IV). Operant boxes were situated in sound-attenuating chambers and equipped with a single nosepoke port and light. The nosepoke port contained a reward-delivery tube that was calibrated to deliver 20 µl of reward through a solenoid valve (Lee Technologies, LHDA2433315H). Every behavioural session (training and conditioning) had the following basic structure. First, the mouse was allowed to acclimate to the chamber for 5 min. Then, the consumption period began and the port light turned on to indicate that rewards were available. During this period, each nosepoke, detected by an infrared beam break with a 1 s time-out period, triggered the delivery of a single reward, and the period ended when 1.2 ml of reward was consumed or 10 min had passed. Then, the delay period began and lasted until 30 min after the beginning of the consumption period. During training sessions, mice were returned to the home cage after the end of the delay period.
Mice assigned to the novel-flavour condition first received four training days as described above with water as the reward and no LiCl or CGRP neuron stimulation. On the conditioning day, sweetened grape Kool-Aid (0.06% grape and 0.3% saccharin sodium salt; Sigma, S1002) was the reward. Mice assigned the familiar-flavour condition had sweetened grape Kool-Aid as the reward for all four training days as well as on the conditioning day.
On the LiCl conditioning day (Fig. 1), mice received an i.p. injection of LiCl (125 mg kg–1; Fisher Scientific, L121) or normal saline after the 30-min delay after the end of the consumption period. For the CGRP neuron cell-body stimulation (Fig. 2d) and CGRPCEA projection stimulation (Fig. 2e) experiments, mice then received 45 min of intermittent stimulation beginning after the 30 min of delay. Blue light was generated using a 447 nm laser for ChR2 experiments. Green light was generated using a 532 nm laser for ChRmine experiments. The light was split through a rotary joint and delivered to the animal using 200 µm diameter core patch cables. Light power was calibrated to approximately 10 mW at the patch cable tip for ChR2 experiments and 3 mW for ChRmine experiments. During the experiment, the laser was controlled with a Pulse Pal signal generator (Sanworks, 1102) programmed to deliver 5 ms laser pulses at 10 Hz. For the duration of the stimulation period, the laser was pulsed for 1.5–15 s intervals (randomly chosen from a uniform distribution with 1.5 s step size) and then off for 1–10 s intervals (randomly chosen from a uniform distribution with 1 s step size). For the eOPN3 experiment (Fig. 2f), photoinhibition began 1 min before the LiCl injection and then continued for 90 min (532 nm laser, 10 mW power, 500 ms laser pulses at 0.4 Hz). Mice were then returned to the home cage. For the LS activation experiments (Extended Data Fig. 2), mice received an i.p. injection of 3 mg kg–1 clozapine N-oxide (CNO; Hellobio, 6149) 45 min before the experiment began.
We assessed learning using a two-bottle memory retrieval test. Two bottles were affixed to the side of a mouse cage (Animal Care Systems Optimice) such that the sipper tube openings were located approximately 1 cm apart. One day after conditioning, mice were given 30 min of access with water in both bottles. We calculated a preference for each mouse for this session and then counterbalanced the location of the test bottle for the retrieval test such that the average water day preference for the two bottle locations was as close to 50% as possible for each group. The next day, mice were given 30 min of access with water in one bottle and sweetened grape Kool-Aid in the other bottle. Flavour preference was then calculated using the weight consumed from each bottle during this retrieval test: flavour/(flavour + water).
To initially characterize behaviour in our CFA paradigm (Fig. 1b), retrieval tests were conducted on three consecutive days with the flavour bottle in the same location each day for each mouse. We then fit a GLMM to this dataset using the R package glmmTMB69 (https://github.com/glmmTMB/glmmTMB; v.1.17) with a Gaussian link function and the formula:
where Preference is the retrieval test result, Novel (novel, familiar), Injection (LiCl, saline), Day (day 1, day 2, day 3) and Sex (female, male) are fixed-effect categorical variables, (1|Subject) is a random effect for each mouse, the asterisk represents the main effects and interactions, and the tilde means ‘distributed as'. This GLMM showed a strong novel–injection interaction effect (P = 2.22 × 10–6, coefficient estimate z test, n = 32 mice) and a weak effect of novel alone (P = 0.025), but no effect of sex (P = 0.137) or injection (P = 0.574) alone or for any other effects. Using the coefficients from this GLMM, we then used the R package marginaleffects70 (https://github.com/vincentarelbundock/marginaleffects; version 0.12.0) to calculate the marginal effect of the flavour condition (novel – familiar) on each day independently for each injection group. We used the marginal effect estimates and s.e. values to calculate a P value for each injection–day combination with a z test, and then corrected for multiple comparisons in each injection group using the Hochberg–Bonferroni step-up procedure71.
For subsequent experiments (Fig. 2d–f and Extended Data Fig. 2b), we performed a single retrieval test per animal and tested for significant differences across groups using Wilcoxon rank-sum tests.
We visualized mCherry, mScarlet and YFP signals to validate transgene expression in our LS chemogenetics (Extended Data Fig. 2a,b) and CGRP neuron optogenetics (Fig. 2d–f) experiments. Mice were deeply anaesthetized (2 mg kg–1 Euthasol i.p.) and then transcardially perfused with PBS followed by 4% paraformaldehyde (PFA) in PBS. Brains were then extracted and post-fixed overnight in 4% PFA at 4 °C and then cryoprotected overnight in 30% sucrose in PBS at 4 °C. Free-floating sections (40 μm) were prepared with a cryostat (Leica Microsystems, CM3050S), mounted with DAPI Fluoromount-G (Southern Biotech, 0100) and imaged with a slide scanner (Hamamatsu, NanoZoomer S60) using NDP Scan software (https://www.hamamatsu.com; v.3.4).
To visualize ExRai–AKAR2 signals (Fig. 5c), we stained for GFP immunoreactivity in the CEA. To validate CGRP neuron ablation following taCasp3-TEVp injection (Fig. 3p and Extended Data Fig. 9f), we stained for CGRP immunoreactivity in the PB. In brief, sections were washed, blocked (3% normal donkey serum (NDS) and 0.3% Triton-X in PBS for 90 min) and then incubated with primary antibody (rabbit anti-GFP, Novus, NB600-308, 1:1,000; mouse anti-CGRP, Abcam, ab81887, 1:250) in blocking buffer overnight at 4 °C. Sections were then washed, incubated with secondary antibody (Alexa Fluor 647 donkey anti-rabbit, Invitrogen, A31573, 1:500; Alexa Fluor 568 donkey anti-mouse, Life Technologies, A10037, 1:500) in blocking buffer for 90 min at room temperature, washed again, mounted with DAPI Fluoromount-G (Southern Biotech, 0100) and imaged with a slide scanner (Hamamatsu, NanoZoomer S60) using NDP Scan software (https://www.hamamatsu.com; v.3.4).
Basic image processing, such as brightness and contrast adjustment, was performed using Fiji72 (https://fiji.sc; v.1.52).
The reference atlas we used is based on the 25 μm resolution Allen Mouse Brain CCF v.3 (https://atlas.brain-map.org)29. For FOS imaging experiments, we considered every brain region in the atlas that met the following criteria: (1) total volume ≥0.1 mm3; (2) lowest level of its branch of the ontology tree (cortical layers or zones not included). We made two modifications to the standard atlas for this study.
First, we reassigned brain region identifiers to increase the clarity of our FOS visualizations that incorporate the atlas and to accurately represent the full functional extent of the CEA. We merged several small regional subdivisions together into the larger LG, PVH, PV, MRN, PRN and SPV regions (see Supplementary Table 1 for a list of brain-region abbreviations). We reassigned all cortical layers and zones to their immediate parent regions (for example, ‘Gustatory areas, layers 1–6b' (111–117) were reassigned to ‘Gustatory areas' (110)). We merged all unassigned regions (tagged with the ‘-un' suffix in the Allen CCF) into relevant parent regions (for example, ‘HPF-un' (563) was reassigned to ‘Hippocampal formation' (462)). We reassigned the voxels immediately surrounding the CEA that were assigned to the ‘Striatum' (581) to the ‘Central amygdalar nucleus' (605), because we found that cells localized to these CEA-adjacent voxels had highly similar FOS and Neuropixels responses compared with cells localized strictly in the CEA. These atlas changes were used throughout the paper (FOS imaging experiments and Neuropixels experiments). Summaries across the entire CEA (for example, Figs. 1i, 2h, 3 and 4) included all atlas voxels assigned to the parent CEA region (605) and to the CEAc (606), CEAl (607) and CEAm (608) subdivisions. The summary across the entire LS (Extended Data Fig. 2d) included all atlas voxels assigned to the parent ‘Lateral septal complex' region (594) and to the LS subdivision (595).
Second, we made the left and right hemispheres symmetric to facilitate the pooling of data from both hemispheres for our FOS visualizations. To ensure that the hemispheres of the Allen CCF were perfectly symmetric, we replaced the left hemisphere with a mirrored version of the right hemisphere. This atlas change was used only for the FOS experiments.
All mice used for the FOS experiments (Figs. 1 and 2 and Extended Data Figs. 1–4 and 6) were trained in the one-reward CFA paradigm as described above. For the consumption time point (Fig. 1), mice were euthanized 60 min after the end of the consumption period on the conditioning day (no LiCl injection was given). For the malaise time point (Fig. 1), mice were euthanized 60 min after the LiCl injection on the conditioning day. For the retrieval time point (Fig. 1), mice received the LiCl conditioning described above and then were returned to the operant box 2 days later for another consumption of the paired flavour using the same task structure as described above. Mice were euthanized 60 min after the end of the consumption period of the retrieval session (no LiCl was given during the retrieval session). For the CGRP neuron stimulation time point (Fig. 2), mice were euthanized 60 min after the onset of CGRP neuron stimulation on the conditioning day and stimulation continued for the full 60 min. For the LS activation time point (Extended Data Fig. 2), mice received an i.p. injection of 3 mg kg–1 CNO 45 min before consumption and were then euthanized 60 min after the LiCl injection on the conditioning day.
Mice were deeply anaesthetized (2 mg kg–1 Euthasol i.p.) and then transcardially perfused with ice-cold PBS and heparin (20 U ml–1; Sigma, H3149) followed by ice-cold 4% PFA in PBS. Brains were then extracted and post-fixed overnight in 4% PFA at 4 °C.
Brain samples were cleared and immunolabelled using an iDISCO+ protocol as previously described28,73. All incubations were performed at room temperature unless otherwise noted.
Brain samples were serially dehydrated in increasing concentrations of methanol (Carolina Biological Supply, 874195; 20%, 40%, 60%, 80% and 100% in doubly distilled water (ddH2O); 45 min–1 h each), bleached in 5% hydrogen peroxide (Sigma, H1009) in methanol overnight and then serially rehydrated in decreasing concentrations of methanol (100%, 80%, 60%, 40% and 20% in ddH2O; 45 min–1 h each).
Brain samples were washed in 0.2% Triton X-100 (Sigma, T8787) in PBS, followed by 20% DMSO (Fisher Scientific, D128), 0.3 M glycine (Sigma, 410225) and 0.2% Triton X-100 in PBS at 37 °C for 2 days. Brains were then washed in 10% DMSO and 6% NDS (EMD Millipore S30) and 0.2% Triton X-100 in PBS at 37 °C for 2–3 days to block nonspecific antibody binding. Brains were then washed twice for 1 h at 37 °C in 0.2% Tween-20 (Sigma P9416) and 10 mg ml–1 heparin in PBS (PTwH solution) followed by incubation with primary antibody solution (rabbit anti-FOS, 1:1,000; Synaptic Systems, 226008) in 5% DMSO, 3% NDS and PTwH at 37 °C for 7 days. Brains were then washed in PTwH 6 times for increasing durations (10 min, 15 min, 30 min, 1 h, 2 h and overnight) followed by incubation with secondary antibody solution (Alexa Fluor 647 donkey anti-rabbit, 1:200; Abcam, ab150075) in 3% NDS and PTwH at 37 °C for 7 days. Brains were then washed in PTwH 6 times for increasing durations again (10 min, 15 min, 30 min, 1 h, 2 h, overnight).
CGRP neuron stimulation time point samples also received primary (chicken anti-GFP, 1:500; Aves, GFP-1020) and secondary (Alexa Fluor 594 donkey anti-chicken, 1:500; Jackson Immunoresearch, 703-585-155) antibodies for ChR2–YFP immunolabelling during the above protocol.
Brain samples were serially dehydrated in increasing concentrations of methanol (20%, 40%, 60%, 80% and 100% in ddH2O; 45 min–1 h each), then incubated in a 2:1 solution of dichloromethane (Sigma, 270997) and methanol for 3 h then washed twice for 15 min in 100% dichloromethane. Before imaging, brains were stored in the refractive-index-matching solution dibenzyl ether (Sigma, 108014).
Cleared and immunolabelled brain samples were glued (Loctite, 234796) ventral side-down to a 3D-printed holder and imaged in dibenzyl ether using a dynamic axial-sweeping light-sheet fluorescence microscope74 (Life Canvas Technologies, SmartSPIM) using SmartSPIM acquisition software (https://lifecanvastech.com/products/smartspim; v.5.6). Images were acquired using a ×3.6, 0.2 NA objective with a 3,650 × 3,650 µm field of view onto a 2,048 × 2,048 pixel sCMOS camera (pixel size, 1.78 × 1.78 µm) with a spacing of 2 µm between horizontal planes (nominal axial point spread function, 3.2–4.0 µm). Imaging of the entire brain required 4 × 6 tiling across the horizontal plane and 3,300–3,900 total horizontal planes. Autofluorescence channel images were acquired using 488 nm excitation light at 20% power (maximum output, 150 mW) and 2 ms of exposure time, and FOS channel images were acquired using 639 nm excitation light at 90% power (maximum output, 160 mW) and 2 ms of exposure time. For CGRP neuron stimulation time point samples, a bilateral volume encompassing both PB regions was imaged separately using 561 nm excitation light at 20% power (maximum output, 150 mW) and 2 ms of exposure time to confirm ChR2–YFP expression.
After acquisition, tiled images for the FOS channel were first stitched into a single imaging volume using the TeraStitcher C++ package75 (https://github.com/abria/TeraStitcher; v.1.11.10). These stitching parameters were then directly applied to the tiled autofluorescence channel images, which produced two aligned 3D imaging volumes with the same final dimensions. After tile stitching, striping artefacts were removed from each channel using the Python package Pystripe76 (https://github.com/chunglabmit/pystripe; v.0.2.0).
We registered the final FOS imaging volume to the Allen CCF using the autofluorescence imaging volume as an intermediary73. We first downsampled both imaging volumes by a factor of five for computational efficiency. Autofluorescence→atlas alignment was done by applying an affine transformation to obtain general alignment using only translation, rotation, shearing and scaling, followed by applying a b-spline transformation to account for local nonlinear variability among individual brains. FOS→autofluorescence alignment was done by applying only affine transformations to account for brain movement during imaging and wavelength-dependent aberrations. Alignment transformations were computed using the Elastix C++ package77,78 (https://github.com/SuperElastix/elastix; v.4.8). These transformations enabled us to transform FOS+ cell coordinates first from their native space to the autofluorescence space and then to Allen CCF space. In rare cases when this two-step alignment strategy failed, we directly registered the FOS imaging volume to the Allen CCF by applying both affine and b-spline transformations.
We first use standard machine-vision approaches to identify candidate FOS+ cells based on peak intensity and then use a convolutional neural network to remove artefacts. Our pipeline builds on the Python package ClearMap28,79 (https://github.com/ChristophKirst/ClearMap2; v.2.0) for identifying candidate cells and the Python package Cellfinder80 (https://github.com/brainglobe/cellfinder; v.0.4.20) for artefact removal.
ClearMap operates through a series of simple image-processing steps. First, the FOS imaging volume was background-subtracted using a morphological opening (disk size, 21 pixels). Second, potential cell centres were found as local maxima in the background-subtracted imaging volume (structural element shape, 11 pixels). Third, the cell size was determined for each potential cell centre using a watershed algorithm (see below for details on the watershed-detection threshold). Fourth, a final list of candidate cells was generated by removing all potential cells that were smaller than a preset size (size threshold, 350 pixels). We confirmed that our findings were consistent across a wide range of potential size thresholds.
We implemented three changes to the standard ClearMap algorithm. First, we de-noised the FOS imaging volume using a median filter (function, scipy.ndimage.median_filter; size, 3 pixels) before the background-subtraction step. Second, we dynamically adjusted the watershed-detection threshold for each sample based on its fluorescence intensity. This step was important for achieving consistent cell-detection performance despite changes in the background and signal intensity across batches and samples owing to technical variations in clearing, immunolabelling and imaging. In brief, we selected a 1,000 × 1,000 × 200 pixel subvolume at the centre of each sample's FOS imaging volume. We then median-filtered and background-subtracted this subvolume as described above. We then used sigma clipping (function, astropy.stats.sigma_clipped_stats; sigma=3.0, maxiters=10, cenfunc=‘median', stdfunc=‘mad_std') to estimate the mean background signal level for this subvolume, µbg, and set the watershed-detection threshold for each sample to 10*µbg. Third, we removed from further analyses all cell candidates that were located outside the brain, in the anterior olfactory areas or cerebellum (which were often damaged during dissection), or in the ventricles, fibre tracts and grooves following registration to the Allen CCF.
One limitation of the watershed algorithm implemented by ClearMap is that it identifies any high-contrast feature as a candidate cell, including exterior and ventricle brain edges, tissue tears, bubbles and other aberrations. To overcome this limitation, we re-trained the 50-layer ResNet81 implemented in Keras (https://keras.io; v.2.8.0) for TensorFlow (https://www.tensorflow.org; v.2.8.0) from the Python package Cellfinder80 to classify candidate FOS+ cells in our high-resolution light-sheet microscopy imaging dataset as true FOS+ cells or artefacts. This network uses both the autofluorescence and FOS channels during classification because the autofluorescence channel has significant information about high-contrast anatomical features and imaging aberrations. We first manually annotated 2,000 true FOS+ cells and 1,000 artefacts from each of four brain samples across two technical batches using the Cellfinder Napari plugin, which produced a total training dataset of 12,000 examples. We then re-trained the Cellfinder network (which had already been trained on approximately 100,000 examples from serial two-photon images of GFP-labelled neurons) over 100 epochs with a learning rate of 0.0001 and 1,200 examples (10% of the training dataset) held out for validation. Re-training took 4 days 16 min 41 s on a high-performance computing cluster using 1 GPU and 12 CPU threads. We achieved a final validation accuracy of 98.33%. Across all samples in our main brain-wide FOS dataset, our trained convolutional neural network removed 15.99 ± 0.58% (mean ± s.e.m.; range, 2.96–32.71%; n = 99 brains across the experiments in Figs. 1 and 2) of cell candidates from ClearMap as artefacts.
We used the ClearMap interface with Elastix to transform the coordinates of each true FOS+ cell to the Allen CCF space using the transformations described above. We then used these coordinates to assign each FOS+ cell to an Allen CCF brain region. For each sample, we generated a final data structure that contained the Allen CCF coordinates (x,y,z), size and brain region for each true FOS+ cell.
We generated 3D maps of FOS+ cell density by applying a Gaussian kernel-density estimate (KDE) (function, scipy.stats.gaussian_kde) in Python to all FOS+ cells across all animals in a given experimental condition (for example, novel flavour + consumption time point). These maps are visualized in Figs. 1h and 2k and Extended Data Figs. 1c–h, 2g and 6e,f.
We first generated a table containing the Allen CCF coordinates (x,y,z) for every FOS+ cell in every animal in an experimental condition. At this stage, we listed each cell twice (once with its original coordinates and once with its ML (z) coordinate flipped to the opposite hemisphere) to pool data from both hemispheres. We then assigned each cell a weight equal to the inverse of the total number of FOS+ cells in that animal to ensure that each animal in an experimental condition would be equally weighted. We then fit a 3D Gaussian KDE for each experimental condition using the scipy.stats.gaussian_kde function and manually set the kernel bandwidth for every experimental condition to be equal at 0.04. We then evaluated this KDE at every voxel in the Allen CCF (excluding voxels outside the brain or in anterior olfactory areas, cerebellum, ventricles, fibre tracts and grooves) to obtain a 3D map of FOS+ density for each condition. Last, we normalized the KDE for each experimental condition by dividing by its sum as well as the voxel volume of the atlas to generate a final 3D map with units of ‘per cent FOS+ cells per mm3'. For the CGRP neuron stimulation time point, we assigned each cell a weight equal to the inverse of the number of FOS+ cells in the PB of that animal, rather than the total number FOS+ cells, to account for variations in ChR2 expression across mice and flavour conditions.
To examine the difference in FOS+ cell density across flavour conditions (for example, in Extended Data Fig. 1d for the consumption time point) we simply subtracted the 3D KDE volumes for the two conditions, novel – familiar, and then plotted coronal sections through this subtracted volume with Allen CCF boundaries overlaid. The colour bar limits for all novel – familiar ΔFOS KDE figures are ±0.5% FOS+ cells per mm3 and for all average FOS KDE, figures are 0–1% FOS+ cells per mm3.
We used the WebGL-based Neuroglancer to generate interactive 3D visualizations of the FOS+ cell density maps for each experimental time point (https://www.brainsharer.org/ng/?id=872). To achieve this, we used the Python package cloudvolume (https://github.com/seung-lab/cloud-volume; v.8.5.1) to convert our 3D KDE volumes from the numpy format to precomputed layers compatible with Neuroglancer and then loaded these layers into the Brainsharer web portal to create the final visualization.
We adopted a GLMM to analyse the brain-wide FOS data (Figs. 1 and 2). This process enabled us to model the contribution of flavour and experimental time point to neural activation in each brain region while also accounting for the overdispersed, discrete nature of the data by using a negative binomial link function, the contribution of batch-to-batch technical variation in tissue clearing, immunolabelling and imaging by modelling this as a random effect, and the potential contribution of sex as a biological variable by modelling this as a fixed effect.
The first step was to determine whether there was any effect of novel or familiar flavour, experimental time point or their interaction for each brain region while controlling the false discovery rate (FDR) across all regions. To accomplish this, we fit a full GLMM for each brain region using the R package glmmTMB69 (https://github.com/glmmTMB/glmmTMB; v.1.1.7) with a negative binomial link function (nbinom2) and the formula:
where FOS counts is the number of FOS+ cells in a brain region, Novel (novel, familiar), Time point (consumption, malaise, retrieval) and Sex (female, male) are fixed-effect categorical variables, (1|Batch) is a random effect for each technical batch (that is, each set of samples that underwent tissue clearing, immunolabelling and light-sheet microscopy imaging together), ln(Total counts) is an offset term for the total number of FOS+ cells in each sample and the asterisk represents all possible main effects and interactions (Fig. 1d). We then fit a reduced GLMM for each brain region, which was the same as the full model (equation (2)) but with the Novel*Time point terms (that is, all main effects and interactions related to flavour novelty and experimental time point) removed. We compared these two models for each brain region using likelihood-ratio χ2-tests and then adjusted the resultant P values using the Benjamini–Krieger–Yekutieli two-step procedure82 to permit a 10% FDR across all brain regions. The 10% FDR threshold used here is standard for brain-wide FOS studies83,84,85. Of the 200 brain regions tested, 130 met this criterion and were included for downstream analyses.
We next specifically tested the effect of flavour novelty on FOS counts separately at each experimental time point for the 130 brain regions that passed the above-defined FDR threshold. To calculate the marginal effect of the flavour condition (novel – familiar) at each time point for each brain region, we used the R package marginaleffects70 (https://github.com/vincentarelbundock/marginaleffects; v.0.12.0) to do post hoc testing of the full GLMM. We used the marginal effect estimates and s.e. values to calculate a P value for each time point with a z test and then corrected for multiple comparisons across time points in each brain region using the Hochberg–Bonferroni procedure71. We also used the ratio of these marginal effect estimates and s.e. values to compute the standardized average difference in FOS+ cell counts across flavour conditions for each brain region at each time point (Z = estimate/s.e.; Fig. 1e–g and Supplementary Table 1). The advantage of this metric is that it explicitly accounts for variation within and across groups, for effects of sex and technical batch, and is independent of brain region size.
When displaying FOS+ cell counts for individual samples (Fig. 1i and Extended Data Figs. 1a,b and 6b), we divided the number of FOS+ cells for each animal or brain region by the total number of FOS+ cells in that animal and by the Allen CCF volume of that brain region, so that the data for each region are presented as ‘per cent FOS+ cells per mm3'. We used the P values from the GLMM marginal effect z tests described above to assess significance.
To examine the brain-wide shift in novel – familiar coding across time points (Fig. 1e and Extended Data Fig. 3a–c), we used the Matlab package Violinplot (https://github.com/bastibe/Violinplot-Matlab) to plot the distribution of standardized average difference Z values at each time point for the brain regions that passed our FDR threshold and then used Kolmogorov–Smirnov tests to assess whether these distributions were significantly different from each other, correcting for multiple comparisons across time points using the Hochberg–Bonferroni procedure71.
To identify structure in novel – familiar coding across time points (Fig. 1f and Extended Data Fig. 3d–n), we used the built-in Matlab linkage function (method=‘ward', metric=‘chebychev') to create a hierarchical tree using the standardized average difference Z values at each time point for the brain regions that passed our FDR threshold. The input matrix was 130 brain regions × 3 time points. We then used the built-in Matlab dendrogram function to plot this hierarchical tree and used a distance threshold of 4.7 for clustering.
We followed an analogous procedure to analyse brain-wide FOS data for the CGRP neuron stimulation time point (Fig. 2 and Extended Data Fig. 6). To account for variations in ChR2 expression across mice and flavour conditions, we weighted FOS+ cell counts by the number of FOS+ cells in the PB in these analyses. Specifically, to compare the effects of CGRP neuron stimulation and LiCl-induced malaise on overall FOS levels (Extended Data Fig. 6c), we fit a GLMM for each brain region with the negative binomial link function and the formula:
where FOS counts is the number of FOS+ cells in a brain region, Time point (consumption, malaise, retrieval, CGRP neuron stimulation) and Sex (female, male) are fixed-effect categorical variables, (1|Batch) is a random effect for each technical batch, ln(PB counts) is an offset term for the total number of FOS+ cells in the PB of each sample and the asterisk represents all possible main effects and interactions. For this model, we did not include any terms related to flavour novelty because we were specifically investigating changes in overall FOS levels. We then plotted the coefficient estimate Z values from this GLMM (Extended Data Fig. 6c). To compare the effects of CGRP neuron stimulation and LiCl-induced malaise on FOS levels in the novel versus familiar flavour condition (Extended Data Fig. 6d), we fit a GLMM for each brain region with the formula:
where FOS counts is the number of FOS+ cells in a brain region, Novel (novel, familiar), Time point (malaise, CGRP neuron stimulation) and Sex (female, male) are fixed-effect categorical variables, (1|Batch) is a random effect for each technical batch, ln(PB counts) is an offset term for the total number of FOS+ cells in the PB of each sample and the asterisk represents all possible main effects and interactions. For this model, we only included the experimental time points in which CGRP neurons were activated either optogenetically (CGRP neuron stimulation) or pharmacologically (malaise); see Extended Data Fig. 6b for the quantification of PB activation. We then calculated and plotted the marginal effect of the flavour condition (novel – familiar) separately for each time point and brain region (Extended Data Fig. 6d). We also used the marginal effect from the GLMM in equation (4) to calculate the P value for Fig. 2h. When displaying FOS+ cell counts for individual animals (Fig. 2h; CGRP neuron stimulation time point) or brain regions (Fig. 2i,j; malaise and CGRP neuron stimulation time points), we first divided the number of FOS+ cells for each brain region in each animal by the number of FOS+ cells in the PB for that animal and by the Allen CCF volume of that brain region. We then divided this number by the average ratio of total FOS+ cells to PB FOS+ cells across every sample in that time point (malaise or CGRP neuron stimulation), which produced a final measure of FOS+ cells of each animal or region as a percentage of the entire brain's FOS+ cells weighted by the relative count of PB FOS+ cells for that animal. We obtained consistent results by instead subsampling the animals in the CGRP neuron stimulation time point to have approximately equal FOS+ cell counts in both flavour conditions and then weighting by the total FOS+ cell count of each animal.
To quantify FOS correlations across individual mice (Extended Data Fig. 4), we considered each experimental time point (consumption, malaise, retrieval) separately. We first assembled the relative FOS+ cell counts (per cent per mm3) for every brain region that passed our FDR threshold and then sorted these regions using the hierarchical tree fit described above, which resulted in a 130 brain region × 24 animal input matrix for each experimental time point. We then used the built-in Matlab corr function to calculate and visualize pairwise correlations among all brain regions (Extended Data Fig. 4a). To estimate the correlation among individual brain regions in the amygdala cluster at each time point (Extended Data Fig. 4b), we averaged pairwise correlations for each brain region with all other amygdala cluster regions in the correlation matrices described above. We tested whether the correlation among individual amygdala cluster brain regions was significant at each time point using Wilcoxon signed-rank tests, correcting for multiple comparisons across time points using the Hochberg–Bonferroni procedure71. To estimate the correlation between the amygdala cluster and every other cluster at each time point (Extended Data Fig. 4c), we averaged the pairwise correlations for all brain region pairs across the two clusters.
To compare the effects of LS activation on FOS levels in the LS and the CEA (Extended Data Fig. 2d,e), we calculated P values for these two regions using a GLMM with the formula:
where FOS counts is the number of FOS+ cells in a brain region, hM3D (hM3D, YFP) and Sex (female, male) are fixed-effect categorical variables, (1|Batch) is a random effect for each technical batch, ln(Total counts) is an offset term for the total number of FOS+ cells in each sample and the asterisk represents all possible main effects and interactions. The P values in the figure are from the hM3D coefficient estimates.
To compare the effects of LS activation of FOS levels across the brain (Extended Data Fig. 2f), we plotted the average FOS level (per cent FOS+ cells per mm3) across all mice in each condition (hM3D, YFP) separately for three groups of brain regions: the amygdala network, the septal complex and other regions. We then used the built-in Matlab aoctool function to fit a one-way analysis of covariance model for these three groups of brain regions and the built-in Matlab multcompare function to test whether the estimated slopes were significantly different, correcting for multiple comparisons using the Bonferroni procedure.
We sliced 18–25-µm-thick sections from perfused brain samples. Multiplex FISH (Fig. 2l–n and Extended Data Fig. 6g,h) was performed using an RNAscope86 Multiplex Fluorescent Assay v2 (ACD 323120) with the following probes: Mm-Calcrl (452281), Mm-Sst-C2 (404631-C2, 1:50 dilution in C1 solution), Mm-Prkcd-C3 (441791-C3, 1:50 dilution in C1 solution) and Mm-Fos-C4 (316921-C4, 1:50 dilution in C1 solution). The Calcrl, Sst, Prkcd and Fos probes were linked to Opal 690, Opal 520, Opal 620 and Opal 570 fluorophores, respectively (Akoya Biosciences). All fluorophores were reconstituted in DMSO according to instructions from the manufacturer and diluted 1:1,200 in tyramide signal amplification buffer included in the RNAscope kit. After in situ hybridization, slides were coverslipped using DAPI Fluoromount-G (Southern Biotech, 0100).
We obtained ×20 z stacks from the CEA with a confocal microscope (Leica TCS SP8 X) using Leica Application Suite X software (https://www.leica-microsystems.com; v.1.8). We then converted these z stacks into maximum-intensity projections for each labelled RNA. We trained a Cellpose87,88 (https://cellpose.readthedocs.io; v.3.0.8) model to identify Fos+ cells in the maximum-intensity projections using eight manually corrected examples, and then used this model to identify Fos+ cells in the remaining images. We then manually classified whether every Fos+ cell identified by Cellpose also expressed Sst, Prkcd and/or Calcrl. We used the full z stacks for each labelled RNA for this process to ensure that potentially overlapping cells were labelled separately. We also imaged each tissue section with a slide scanner (Hamamatsu, Nanozoomer S60) using NDP Scan software (https://www.hamamatsu.com; v.3.4) and then registered them to the Allen CCF using ABBA89 (https://abba-documentation.readthedocs.io; v.0.8.0). To remove Fos+ cells outside the CEA from analysis, we manually aligned the confocal and slide scanner images using the Fos channel in each image as a guide and then manually transferred the CEA boundaries to the confocal images. Manual cell classifications and basic image processing tasks were performed using Fiji72 (https://fiji.sc; v.1.52).
All slice electrophysiology recordings (Fig. 2c) were performed on brain slices collected at approximately the same time of day. Calcacre mice were first injected with 400 nl of AAV5-EF1a-DIO-hChR2(H134R)-eYFP (titre, 1.2 × 1013 GC per ml; manufacturer, PNI Viral Core Facility) bilaterally into the PB 6 weeks or more before the experiment. On the day of the recordings, mice were anaesthetized with isoflurane and decapitated to remove the brain. After extraction, the brain was immersed in ice-cold NMDG ACSF (92 mM NMDG, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 2 mM thiourea, 5 mM sodium ascorbate, 3 mM sodium pyruvate, 0.5 mM CaCl2·4H2O, 10 mM MgSO4·7H2O and 12 mM N-acetyl-l-cysteine; pH adjusted to 7.3–7.4) for 2 min. Afterwards, coronal slices (300 μm) were sectioned using a vibratome (Leica VT1200s) and then incubated in NMDG ACSF at 34 °C for approximately 15 min. Slices were then transferred to a holding solution of HEPES ACSF (92 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 2 mM thiourea, 5 mM sodium ascorbate, 3 mM sodium pyruvate, 2 mM CaCl2·4H2O, 2 mM MgSO4·7H2O and 12 mM N-acetyl-l-cysteine, bubbled at room temperature with 95% O2 and 5% CO2) for at least 60 min until recordings were performed.
Whole-cell recordings were performed using a Molecular Devices Multiclamp 700B amplifier and Digidata 1440A low-noise data acquisition system. Recording pipettes had a resistance of 4–7 MΩ and were filled with an internal solution containing 120 mM potassium gluconate, 0.2 mM EGTA, 10 mM HEPES, 5 mM NaCl, 1 mM MgCl2, 2 mM Mg-ATP and 0.3 mM NA-GTP, with the pH adjusted to 7.2 with KOH and the osmolarity adjusted to approximately 289 mmol kg−1 with sucrose. During recordings, slices were perfused with a recording ACSF solution (100 μM picrotoxin, 120 mM NaCl, 3.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 1.3 mM MgCl2, 2 mM CaCl2 and 11 mM D-(+)-glucose) with 1 µM TTX and 100 µM 4AP that was continuously bubbled with 95% O2 and 5% CO2. Infrared differential interference contrast-enhanced visual guidance was used to select neurons that were 3–4 cell layers below the surface of the slices. All CEAc and CEAl recordings were made for which eYFP-expressing CGRP neuron axons were visible, and all CEAm recordings were made more medial to this location using the Allen CCF as a guide The recording solution was delivered to slices through superfusion driven by a peristaltic pump (flow rate of 4–5 ml min–1) and was held at room temperature. The neurons were held at −70 mV (voltage clamp), and the pipette series resistance was monitored throughout the experiments by hyperpolarizing steps of 1 mV with each sweep. If the series resistance changed by >20% during the recording, the data were discarded. Whole-cell currents were low-pass filtered at 4 kHz online and digitized and stored at 10 kHz using Clampex software (https://www.moleculardevices.com; v.10.7). Currents were then filtered at 1 kHz offline before analysis. During the experiment, we measured light-evoked oEPSCs every 30 s with light stimulation (0.074 mW mm–2) delivered for a duration of 5 ms. Twenty repetitions of the stimulation protocol were recorded per cell after stable oEPSCs were achieved. All experiments were completed within 4 h after slices were made to maximize cell viability and consistency.
Traces from example CEAc, CEAl and CEAm neurons are shown in Fig. 2c. Across all monosynaptically connected neurons, the amplitude of CGRP neuron→CEAc or CEAl oEPSCs was –327.0 ± 136.3 pA (mean ± s.e.m.; n = 5 out of 5 connected neurons from 3 mice) and of CGRP neuron→CEAm oEPSCs was –15.6 ± 6.4 pA (mean ± s.e.m.; n = 4 out of 5 connected neurons from 3 mice).
For Neuropixels recording experiments (Figs. 3 and 4), we used a two-reward CFA paradigm (rather than the one-reward paradigm used for the FOS experiments). This enabled us to compare neural correlates of the novel-flavour reward, which was delivered from one port, with responses related to a control port that delivered water. Experiments were performed in an operant box (Med Associates) using MedPC software (https://med-associates.com/product/med-pc; v.IV). The operant box was situated in a sound-attenuating chamber and equipped with a single speaker and with two custom 3D-printed nosepoke ports with built-in lights and infrared beam breaks in each port. The nosepoke ports each contained a reward delivery tube that was calibrated to deliver 20 µl of reward through a solenoid valve (Lee Technologies LHDA2433315H). The ports were located on either side of the same wall of the operant box.
Mice first underwent a basic task procedure to train them to drink from two reward ports in a cued manner (Extended Data Fig. 8a). Each behavioural session had the following structure. First, the mouse was allowed to acclimate to the chamber for 5 min. Then, the consumption period began and rewards were made available in a trial-based manner that forced mice to drink from the two ports at a relatively equal rate throughout the session. At the beginning of each trial, one port was randomly selected and made available to the mouse. This was cued through the port light turning on and a distinct tone (2.5 kHz or 7.5 kHz; 70 dB) playing. The mouse had 10 s to enter the port and receive a reward, which was detected by the infrared beam break. The end of the 10 s reward availability period or entering either port ended the trial; at this point, the cueing light and sound were terminated and an inter-trial interval was initiated (randomly selected from a uniform distribution of 10–20 s; 1 s step size). At the end of the inter-trial interval, a new trial would begin as long as the mouse had not entered either reward port in the previous 2 s; otherwise, the next trial was delayed until this criterion was satisfied. To ensure that mice drank from the two ports at a relatively equal rate, we required that each consecutive block of ten successful rewarded trials must be evenly split between the two ports. The consumption period ended when 1.2 ml (60 rewards) was consumed. Mice learned to perform this task nearly perfectly (<5 unsuccessful trials per session) in approximately 1 week. During initial training, both ports delivered water. After mice were trained in the task, they underwent the chronic Neuropixels surgery described above and were allowed to recover for at least 5 days. Mice were then returned to daily training, with the addition of a delay period following the consumption period. At the beginning of the delay period (immediately after the final reward), mice were transferred to a distinct second context, which was triangular in shape with smooth white acrylic walls. Mice remained in this second context for at least 30 min before returning to the home cage. Mice were acclimated to the delay period and second context, and to tethering of the Neuropixels assembly and optical fibre, for at least 4 days before proceeding to the conditioning experiments. Variations to this basic task structure for specific experiments that were used following training and surgery are described below.
For these experiments (Figs. 3 and 4), on the conditioning day, the same behavioural session structure was followed, but now one port delivered water and the other port delivered sweetened grape Kool-Aid (0.06% grape and 0.3% saccharin sodium salt). The novel-flavour port was counterbalanced across mice. Mice (n = 8) were run in two separate groups separated by approximately 2 months. After the 30-min delay period in the second context, the CGRP neuron stimulation period began and lasted for 45 min in the same second context. Blue light was generated using a 447 nm laser and delivered to the animal using a 200 µm diameter patch cable. Light power was calibrated to approximately 10 mW at the patch cable tip. The laser was controlled with a Pulse Pal signal generator programmed to deliver 5 ms laser pulses at 10 Hz. For the duration of the CGRP neuron stimulation period, the laser was pulsed for 3 s bouts and then off for random intervals chosen from an exponential distribution (minimum, 1 s; mean, 3 s; maximum, 7.8 s). Following the 45-min neuron stimulation period, mice were returned to the home cage overnight. The following day, mice underwent a forced retrieval session that followed the same trial structure as previous sessions, and the flavour and water were delivered from the same ports as on the conditioning day.
These experiments (Fig. 4e and Extended Data Figs. 9a–e and 10c–e) were performed using the same strategy as the cell-body stimulation experiment described above in a separate group of mice (n = 8) with the following changes. Green light was generated using a 532-nm laser and calibrated to approximately 3 mW at the patch cable tip. To minimize potential photoelectric artefacts in our recordings, we positioned the tip of the optical fibre 1.5 mm from the Neuropixels shanks in the CEA for an irradiance at the electrodes of approximately 0.1 mW mm–2 and reduced the laser pulse width to 2 ms. These stimulation parameters were sufficient to activate the ultrasensitive opsin ChRmine64.
This experiment (Fig. 3m–r and Extended Data Figs. 9f,g and 10f,g) was performed in a separate group of mice (n = 4 control mice and 4 CGRP neuron ablation mice). It followed the same structure as above except that LiCl (125 mg kg–1 i.p.) was injected to induce gastrointestinal malaise after 30 min in the second context (delay period) instead of CGRP neuron stimulation. For behavioural validation of CGRP neuron ablation (Extended Data Fig. 9g), we included five mice that were not used for recordings but either received taCasp3 virus (n = 2 ablation mice) or did not undergo surgery (n = 3 control mice).
For these experiments (Fig. 4f and Extended Data Fig. 10h–k), a different flavour, sweetened cherry Kool-Aid (0.06% cherry and 0.3% saccharin sodium salt), was used. The experiment followed the same basic task structure as during initial training for the two-reward CFA paradigm, without any aversive conditioning experiences (LiCl injection or CGRP neuron stimulation). The experiment was run on three consecutive days. On the first day (novel day), one port contained the novel sweetened cherry Kool-Aid flavour and the other port contained water. On the second day, the port locations were switched. On the third day (familiar day), the port locations were switched again (that is, back to the initial locations from novel day).
This experiment (Extended Data Fig. 9h–j) did not involve rewards or use the task structure described above. First, mice were allowed to acclimate to the operant box recording chamber for 5 min. Then, CGRP neurons were photostimulated using the same protocol as for the acute Neuropixels recording experiment described below. In brief, mice received 1 s of 10 Hz CGRP neuron stimulation followed by a 9-s inter-trial interval for a total of 10 min per 60 trains. After a 5-min recovery period, LiCl (125 mg kg–1 i.p.) was then injected to induce gastrointestinal malaise. Mice remained in the recording chamber for at least 15 min before being returned to the home cage.
We used 27 mice for chronic Neuropixels recording experiments (Extended Data Fig. 7c). Animals 1–4 were used for the CGRP neuron cell-body stimulation conditioning experiment. Animals 5–8 were used for multiple experiments with the following timeline: (1) CGRP neuron cell-body stimulation conditioning experiment, (2) familiarization experiment, (3) CGRP neuron stimulation→LiCl injection experiment. Animals 9–12 were control mice used for the LiCl conditioning experiment. Animals 13–16 were CGRP neuron ablation mice used for the LiCl conditioning experiment. Animals 17–24 were used for the CGRPCEA projection stimulation conditioning experiment. Animals 25–27 were used for the familiarization experiment.
Before beginning experiments, we performed a series of test recordings for each mouse to identify the recording sites along each Neuropixels shank that were located in the CEA. We recorded for approximately 10 min from the bottom 384 recording sites of each shank. We found that recording sites properly targeted to the CEA could be identified by a dense band of single-unit and multiunit activity (see Extended Data Fig. 7b for examples). This process enabled us to design custom Imec readout tables (recording site maps; https://billkarsh.github.io/SpikeGLX/help/imroTables) for each mouse that maximized the yield of CEA neurons during subsequent experiments.
We recorded 384 Neuropixels channels per session at 30 kHz using National Instruments PXI hardware and SpikeGLX software (https://billkarsh.github.io/SpikeGLX; v.3.0). Experimental TTL signals (representing reward cues, port entries, reward deliveries and laser pulses) were recorded simultaneously using the same system.
We used CatGT (https://billkarsh.github.io/SpikeGLX; v.3.3) to apply global common average referencing (-gblcar) and to isolate the action potential frequency band (-apfilter=butter,12,300,9000). We then used the International Brain Laboratory's (IBL) Python Kilosort 2.5 implementation90,91 (https://github.com/int-brain-lab/pykilosort) to correct for sample drift along the length of the probe, to detect and remove failing channels and to apply a spatial de-striping filter.
We also used the IBL's Python Kilosort 2.5 implementation91,92,93 (https://github.com/int-brain-lab/pykilosort) for spike sorting. We then used the Python package Phy (https://github.com/cortex-lab/phy; v.2.0) for interactive visualization and manual curation of spike sorting output. We used Phy to classify clusters from Kilosort as single-unit (good) or multiunit (MUA) clusters and to remove noise. We relied on waveform shape, autocorrelogram shape, spike amplitude time course and cluster separation for classification. Following curation with Phy, we used Matlab to compute three statistics for each cluster. First, we calculated the median amplitude of each cluster using the template scaling amplitudes from Kilosort (stored in amplitudes.npy), converted from bits to µV using the gain factor 2.34375. These template-scaling amplitudes were calculated after whitening the data and were significantly smaller than the equivalent raw spike amplitudes (µV). Second, we calculated the estimated false-positive rate of each cluster based on 2 ms refractory period violations94. Third, we calculated the firing rate (sp s–1) of each cluster. For experiments with multiple epochs (for example, consumption and CGRP neuron stimulation in Fig. 3), we calculated these metrics separately for each epoch and then kept the minimum median template-scaling amplitude, the maximum estimated false-positive rate and the minimum firing rate across epochs for each cluster. We removed clusters with median template-scaling amplitude values of <20 µV, estimated false-positive rates of >100% or firing rates <0.05 sp s–1 as noise. We classified the remaining clusters that were labelled ‘good' in Phy and had an estimated false-positive rate <10% as single units and the rest as multiunits. We included both single-unit and multiunit clusters throughout the article. We confirmed that our findings were consistent across a range of amplitude thresholds and for only single-unit clusters. Finally, we binned the spikes for each included neuron into 10 ms bins for downstream analyses.
All probes were coated in CellTracker CM-DiI (Invitrogen C7000) before implantation. After the conclusion of experiments, animals were euthanized and the brains cleared with an abbreviated version of the iDISCO+ protocol described above without immunostaining. The cleared brains were then imaged on a light-sheet microscope (LaVision Ultramicroscope II) using LaVision BioTec ImSpector software (https://www.lavisionbiotec.com; v.7.0). Images were acquired using 488 nm (autofluorescence channel) and 561 nm (CM-DiI channel) excitation light with 10 µm between horizontal planes and 5.91 µm per pixel resolution. Atlas alignment then followed the IBL's pipeline95 (https://github.com/int-brain-lab/iblapps/wiki). The autofluorescence volume was registered to the atlas using the Python package Brainreg96 (https://github.com/brainglobe/brainreg; v.0.4.0), and these transformations were then directly applied to the CM-DiI volume. Individual Neuropixels shank trajectories were then manually annotated in the atlas-registered CM-DiI volume using the Brainreg-segment Napari module (https://github.com/brainglobe/brainreg-segment; v.0.2.16). Every recording site was then localized to an Allen CCF coordinate (x,y,z) and brain region using the IBL's alignment GUI. The alignment process was performed separately for each Neuropixels probe shank. For all analyses, we only included neurons from recording sites that were localized to the CEA.
We made two changes to our processing pipeline to track units across two recording sessions (for example, the conditioning and retrieval sessions in Fig. 4b–e and Extended Data Fig. 10a–g, and the novel and familiar sessions in Fig. 4f and Extended Data Fig. 10h–k). First, we concatenated the two recording sessions using CatGT after applying global common average referencing and isolating the action potential frequency band. We then performed spike sorting using the IBL's Python Kilosort 2.5 implementation as described above. Second, during manual curation in Phy, we removed clusters with obvious discontinuities or irregularities across days as noise. We then evaluated our quality metrics in Matlab as described above. To improve Kilosort's ability to track units in multiday recordings, pairs of sessions were separated by only 1 day (conditioning and retrieval) or 2 days (novel and familiar). We then used the Matlab package Spikes (https://github.com/cortex-lab/spikes) to extract spike waveforms and to generate autocorrelograms for each recording session (Fig. 4a).
For experiments involving reward delivery, we classified all neurons as novel-flavour-preferring, water-preferring or nonselective (for example, in the heatmaps in Figs. 3d and 4b). We first z scored each neuron's 10-ms binned spiking across the entire consumption period. We then calculated the average neural activity in the 10 s following every reward delivery, which was triggered by the animal entering the port. We distinguished nonselective neurons from reward-selective neurons using a Wilcoxon rank-sum test on these average responses for novel flavour and water trials while permitting a 5% FDR across all recorded neurons (pooled across mice within each experiment) with the Benjamini–Krieger–Yekutieli procedure82. We then classified reward-selective neurons as novel-flavour-preferring if their average neural activity in the 10 s following reward delivery was greater for novel-flavour trials than for water trials; the remaining neurons were classified as water-preferring. When tracking neurons across days and examining the change in their flavour response or selectivity (Fig. 4 and Extended Data Fig. 10), we classified neurons as novel-flavour-preferring or water-preferring based on their responses during consumption on the first day (novel or conditioning) only. We defined ‘novel flavour response' as the average neural activity in the 10 s following novel flavour delivery and ‘novel flavour selectivity' as the average neural activity in the 10 s following novel flavour delivery minus the average neural activity in the 10 s following water delivery. When correlating CGRP response to the change in flavour response and selectivity across days (Fig. 4d,e), we subtracted the baseline activity (–10 s to –5 s before reward delivery) from each trial when calculating reward responses to account for potential changes in baseline firing rate across days.
We generated peri-event time histograms (PETHs) surrounding reward delivery (–5 s to +10 s) using the z scored traces calculated above and averaging across all novel-flavour or water reward deliveries. When generating reward PETHs for the second day of multiday recordings (retrieval day, familiar day), we used the mean and s.d. calculated while z scoring the consumption period trace for the first day to ensure that units were comparable across days. We generated PETHs surrounding CGRP neuron stimulation or CGRPCEA projection stimulation trains (–1 s to +4 s) using the mean and s.d. calculated while z scoring that day's consumption period trace, and then subtracted the baseline (–1 s to 0 s) mean of each neuron's PETH. For plotting reward delivery PETHs as heatmaps and traces, we convolved each neuron's PETH with a causal half-Gaussian filter with 100-ms s.d.
We generated delay→CGRP neuron stimulation PETHs (Figs. 3d,e and 4b, c), delay→CGRPCEA projection stimulation PETHs (Extended Data Figs. 9b,c and 10c) and delay→LiCl PETHs (Fig. 3n,q) using the 10-ms binned spiking from 30 min before to 45 min after the onset of CGRP neuron stimulation or CGRPCEA projection stimulation LiCl injection. We then z scored these traces using the mean and s.d. from the final 20 min of the delay period and downsampled the final normalized PETHs to 1 sample per min for plotting. We defined ‘CGRP response' as the average neural activity across the entire 45-min CGRP neuron stimulation or CGRPCEA projection stimulation period in the PETHs described above (Figs. 3e and 4c–e and Extended Data Fig. 9c) and ‘LiCl response' as the average neural activity from 5–15 min after LiCl injection in the PETHs described above (Fig. 3n,q). We generated whole-experiment PETHs (Fig. 3e,n,q and Extended Data Fig. 9c) by concatenating the 10-ms binned spiking from the final 15-min of the consumption period, the first 30-min of the delay period and the first 45-min of the CGRP neuron stimulation period or CGRPCEA projection stimulation period or LiCl-induced malaise period. We then z scored these traces using the mean and s.d. of the delay period calculated above and downsampled the final normalized PETHs to 1 sample per min for plotting. When comparing the CGRP response (Fig. 3e,f and Extended Data Fig. 9c,d) or LiCl response (Fig. 3n,q) of novel flavour-preferring, water-preferring and nonselective neurons, we corrected for multiple comparisons using the Hochberg–Bonferroni procedure71.
For the CGRP neuron stimulation→LiCl injection experiment (Extended Data Fig. 9h–j), we first classified each neuron's CGRP response type using the strategy from the acute recording experiment described below. We first z scored each neuron's 10-ms binned spiking across the entire 10-min CGRP neuron stimulation period and generated baseline-subtracted PETHs surrounding CGRP neuron stimulation trains (–1 s to +2 s). We then applied the Gaussian mixture model (GMM) fit on the acute recording data (Extended Data Fig. 5c) to these PETHs to determine each neuron's CGRP response type and to identify CGRP-activated neurons (Extended Data Fig. 9i). We then generated LiCl injection PETHs using the 10-ms binned spiking from 5 min before to 15 min after the LiCl i.p. injection. We then z scored these traces using the mean and s.d. from the 5-min acclimatization period before CGRP neuron stimulation began and baseline-subtracted the normalized PETHs using the mean activity during the period between CGRP neuron stimulation and LiCl i.p. injection (–5 min to –1 min before LiCl). We downsampled the final normalized PETHs to 1 sample per min for plotting and then plotted average LiCl injection PETHs separately for CGRP-activated neurons and for other neurons (Extended Data Fig. 9j). We defined ‘LiCl response' as the average neural activity from 5 to 15 min after LiCl injection in the PETHs described above (Extended Data Fig. 9j).
To identify reactivations of neural flavour representations (Fig. 3g–i,o,r), we trained a multinomial logistic regression decoder using the LogisticRegression class from the Python package scikit-learn97 (https://scikit-learn.org; v.1.0.2) separately for each mouse using all CEA neurons during the consumption period on the conditioning day and then evaluated this decoder across the entire conditioning session. We included all mice with >75 simultaneously recorded CEA neurons for the decoding analysis (6 out of 8 CGRP neuron stimulation mice in Fig. 4h–i; 8 out of 8 LiCl injection mice in Fig. 4o,r). The decoder was trained to discriminate behavioural states during the consumption period across three categories: novel-flavour consumption (represented by normalized spike counts within 1 s after novel-flavour delivery; normalization procedure described below); water consumption (normalized spike counts within 1 s after water delivery); and baseline (normalized spike counts within 1 s before each cue onset). We trained the decoder using Lasso regularization and tested λ from 10–4 to 104 (nine logarithmically spaced values; Extended Data Fig. 8c). We chose λ = 1 for the final decoder because it provided a high level of regularization without decreasing log-likelihood in the held-out data during tenfold cross-validation. Cross-validation also verified that the decoder correctly identified the animal's behavioural state (novel flavour, water, baseline) during the consumption period (Extended Data Fig. 8e).
We normalized spike counts separately for each neuron and task period. For the consumption period (decoder training), we calculated each neuron's average spike counts within 1 s before cue onsets and subtracted it from the binned spike counts. We then divided these baseline-subtracted spike counts by the s.d. during the consumption period. For the delay and CGRP neuron stimulation periods (decoder evaluation), we z scored each neuron's spike counts based on its mean and s.d. during the delay period.
We then evaluated the decoder using neural activities across the session. We first used a 1-s sliding window with 150 ms steps to bin the spikes across the start to the end of the session. After obtaining the n neuron × n time bin normalized spike counts, we used the decoder to classify the behavioural state for each time bin based on the corresponding normalized spike counts. To visualize the decoder's performance (Fig. 3g), we plotted the decoder output along with the simultaneously recorded neural activity (spike trains convolved with a causal half-Gaussian filter with 25 ms s.d.) grouped by novel flavour or water preference, defined using criteria described above. For clarity, we only display a subset of recorded neurons (50 out of 90) in the example raster in Fig. 3g: all novel flavour-preferring and water-preferring neurons, along with 15 randomly chosen nonselective neurons. We focused our analysis on the comparison between the decoded probabilities for the novel flavour and water categories (Fig. 3g, top, and Fig. 3h). We detected peaks (local maxima with values > 0.5) of the decoder output as reactivation events, and counted the number of novel flavour and water reactivations with a sliding window of 1 min width and 30 s step size (Fig. 3i,o,r).
We used the built-in Matlab pca function (Fig. 3j). We began by taking the novel flavour delivery, water delivery and CGRP neuron stimulation PETHs described above, all convolved with a causal half-Gaussian filter with 100 ms s.d., for all reward-selective neurons (n = 494 pooled across all mice). We baseline-subtracted each neuron's reward-delivery PETHs using the mean baseline activity (–5 s to –4 s before reward delivery) averaged across both reward types and then peak-normalized each neuron's PETHs using the maximum absolute value across both reward types. We baseline-subtracted each neuron's CGRP neuron stimulation PETH using the mean baseline activity (–1 s to 0 s) before laser onset and then peak-normalized each neuron's PETH using the maximum absolute value of the CGRP neuron stimulation PETH. To identify PC loadings and to calculate the variance explained, we concatenated each neuron's novel flavour and water reward PETHs (0 s to +5 s from reward delivery), which produced a final input matrix that was 494 neurons × 1,000 time bins for PCA. We centred every column of this matrix before performing PCA along the neuron dimension.
To plot neural trajectories during novel flavour consumption and water consumption (Fig. 3k), we used the PC loadings defined above to calculate PC1 and PC2 values for the entire population at each time bin of the PETH (–5 s to +10 s from reward delivery). We followed an analogous procedure to plot neural trajectories during CGRP neuron stimulation. In both cases, we centred every column (time bin).
We repeated this entire analysis using only the neurons from individual mice (Fig. 3l) or using all neurons from a separate group of mice that received CGRPCEA projection stimulation (Extended Data Fig. 9e).
When analysing changes in PC trajectories across days (retrieval in Extended Data Fig. 10b and familiarization in Extended Data Fig. 10k), we followed basically the same procedure as above. For these analyses, we identified PC loadings using only the first day's (conditioning day or novel day) reward delivery PETHs and then used this set of PC loadings when plotting the PC trajectories for both days. Similarly, we baseline-subtracted and peak-normalized the second day's PETHs using values that were calculated using only the first day's PETHs. These measures ensured that PC trajectories were comparable across multiday recordings.
Calcacre mice were first injected with 400 nl of AAV5-EF1a-DIO-hChR2(H134R)-eYFP (titre, 1.2 × 1013 GC per ml; manufacturer, PNI Viral Core Facility) into the left PB. Four weeks later, in a second surgery, an optical fibre (300 µm diameter core, 0.39 NA) was implanted at a –30° angle above the injection site (see the section ‘Viral injections and optical fibre implantations' for details), a steel headbar (approximately 1 g) was implanted at AP +1.25 mm, and a ground pin (Newark Electronics) was placed above the right hemisphere of the cerebellum. Finally, a 2 mm2 recording chamber was built with Dentin (Parkell S301) above the left hemisphere extending from AP 0 mm to AP –2.0 mm and ML –2.5 mm to ML –3.5 mm. The exposed skull was removed and the brain covered with a silicone elastomer (Kwik-Cast, World Precision Instruments).
Mice were habituated to head fixation (3× 30-min sessions). On the recording day, mice were head-fixed in a custom-built recording rig91, the silicone elastomer removed and the exposed brain briefly cleaned with normal saline. Neuropixels 1.0 probes98 had a soldered connection to short ground to external reference, which was also connected to the mouse's ground pin during recording. Immediately before the start of the recording session, the probe was coated in CellTracker CM-DiI (Invitrogen C7000). A single probe was lowered (approximately 10 μm s–1) with an ultraprecise micromanipulator (Sensapex µMp) into the amygdala. To prevent drying, the exposed brain and probe shank were covered with a viscous silicone polymer (Dow-Sil, Corning). After reaching the targeted location, the brain tissue was allowed to settle for 15 min before starting the recording. Recordings were acquired at 30 kHz using National Instruments PXI hardware and SpikeGLX software (https://billkarsh.github.io/SpikeGLX; v.3.3). Six recording locations were targeted in each animal. During the recording, mice received 1 s CGRP neuron stimulation followed by a 9 s inter-trial interval for a total of 10 min. Blue light was generated using a 447 nm laser and delivered to the animal using a 200 µm diameter core patch cable. Light power was calibrated to approximately 8 mW at the fibre tip. The laser was controlled with a Pulse Pal signal generator (Sanworks, 1102) programmed to deliver 5 ms laser pulses at 10 Hz.
Spike sorting, manual curation and atlas alignment were performed as described above for chronic Neuropixels recordings. To precisely map our electrophysiological data to the anatomical subdivisions of the amygdala in this experiment, we used the IBL's electrophysiology alignment GUI (https://github.com/int-brain-lab/iblapps/wiki) to manually tune the alignment of each recording to the Allen CCF using electrophysiological landmarks. We then z scored each neuron's 10-ms binned spiking across the entire 10-min CGRP neuron stimulation period. We then generated PETHs surrounding CGRP neuron stimulation trains (–1 s to +2 s), and subtracted the baseline (–1 s to 0 s) mean of each neuron's PETH (Extended Data Fig. 5c). We then used the built-in Matlab fitgmdist function (CovarianceType=‘diagonal', RegularizationValue=1e-5, SharedCovariance=false, Replicates=100) to fit a GMM with four response types to this dataset. Specifically, we used the time bins during stimulation (0 s to +1 s) from all amygdala neurons to generate a 3,524 neuron × 100 time bin input matrix for GMM fitting. This GMM revealed two CGRP neuron stimulation-activated response types (shown in green in Extended Data Fig. 5c, left), one CGRP neuron stimulation-inhibited response type (shown in purple) and one unmodulated response type (shown in grey). We then plotted average CGRP neuron stimulation PETHs for each response type separately (Extended Data Fig. 5c, right) and analysed the distribution of CGRP neuron stimulation-activated neurons across amygdala regions (Extended Data Fig. 5d,e).
We recorded CGRP neuron GCaMP signals (Fig. 2b) with standard fibre photometry acquisition hardware99,100. Excitation light was supplied at two wavelengths—isosbestic 405 nm (intensity at patch cable tip, 5–10 µW; sinusoidal frequency modulation, 531 Hz) and activity-dependent 488 nm (intensity, 15–25 µW; sinusoidal frequency modulation, 211 Hz)—using an LED driver (Thorlabs, DC4104) coupled to a low-autofluorescence patch cable (Doric, MFP_400/430/1100-0.57_ 0.45m_FCM-MF2.5_LAF). Emission light was collected through the same patch cable using a low-light photoreceiver (Newport Femtowatt 215) and then digitized using a base processor (Tucker Davis Technologies, RZ5D) that served both as an analog-to-digital converter and a lock-in amplifier. We then low-pass filtered (2 Hz) and downsampled (100 Hz) the isosbestic 405 nm and activity-dependent 488 nm signals. To control for photobleaching, we applied a linear fit to the isosbestic signal to align it to the activity-dependent signal and then subtracted this fitted isosbestic signal from the activity-dependent signal to obtain the final de-bleached activity-dependent GCaMP signal.
To assess changes in CGRP neuron activity due to LiCl-induced malaise, we generated PETHs using the 10 min before and 30 min after LiCl injection (125 mg kg–1 i.p.). We z scored the entire PETH for each mouse using the mean and s.d. of the full 10 min before LiCl injection and then downsampled (1 Hz) and smoothed (1-min centred moving average) the final normalized PETHs for plotting.
We recorded CEA AKAR2 signals (Fig. 5) using the same acquisition system described above for GCaMP recordings. We low-pass filtered (1 Hz) and downsampled (100 Hz) both the 405 nm and 488 nm signals. Because AKAR2 is a ratiometric indicator of PKA activity47, we divided the 488 nm signal by the 405 nm signal to obtain the final de-bleached activity-dependent PKA signal.
This experiment was run on four consecutive days using a version of the two-reward familiarization paradigm described above (Fig. 5b). On day 0 (water day), both ports contained water. On day 1 (novel day), one port (port A) contained the novel sweetened grape Kool-Aid flavour and the other port (port B) contained water. On days 2 and 3 (familiar days), the same ports contained the same sweetened grape Kool-Aid flavour and water as on day 1. The flavour port was counterbalanced across mice.
We generated PETHs surrounding reward delivery (–10 s to +30 s) using the final PKA signal described above. We z scored these PETHs separately for each mouse and day using the following procedure. First, we centred each individual reward event PETH by subtracting the mean baseline signal (–5 to –1 s before reward delivery). Then we concatenated this baseline epoch from all individual reward events from both ports for that mouse per day and calculated the s.d. of this vector. Then we divided each individual reward event PETH for that mouse per day by the calculated s.d. Last, we averaged across all rewards of each type (port A, port B) for that mouse per day (Fig. 5d,e).
To quantify PKA activity for statistical analysis, we calculated the average response from +5 to +15 s after reward delivery for each mouse per day per port using the final averaged PETHs calculated above. We then fit a GLMM using the R package glmmTMB69 (https://github.com/glmmTMB/glmmTMB; v.1.1.7) with a Gaussian link function and the formula:
where PKA activity is as described above, Port (port A, port B) and Day (day 0, day 1, day 2, day 3) are fixed-effect categorical variables, (1|Subject) is a random effect for each mouse and the asterisk represents the main effects and interactions. Using the coefficients from this GLMM, we used the R package marginaleffects70 (https://github.com/vincentarelbundock/marginaleffects; v.0.12.0) to calculate the marginal effect of port (port A – port B) on each day. We used the marginal effect estimates and s.e. values to calculate a P value for each day with a z test, and then corrected for multiple comparisons across days using the Hochberg–Bonferroni procedure71 (Fig. 5f).
The recording location for each animal was determined using the same procedure as for our Neuropixels recordings described above. In brief, we manually annotated the tip of the optical fibre lesion for each animal in the light-sheet microscopy imaging data after registration to the Allen CCF and visualized these recording locations on the Allen CCF (Fig. 5g) as 100 µm circles centred on the fibre tip location for each animal.
We excluded data in three instances. First, we excluded one mouse with no hM3D(Gq)–mCherry expression and one mouse with no YFP expression in the LS from Extended Data Fig. 2b. Second, we excluded one mouse with no eOPN3–mScarlet expression in the CEA from Fig. 2f. Third, we excluded confocal images with poor FISH labelling (defined as <35 total Fos+ CEA cells, or <25% of Fos+ cells also Sst+, or <25% of Fos+ cells also Prkcd+, or <25% of Fos+ cells also Calcrl+) from Fig. 2m,n (22 out of 109 images).
No statistical methods were used to predetermine sample sizes. Sample sizes were chosen based on previous studies investigating CTA and CGRP neurons (for example, refs. 23,24,34) and on the availability of animals. All attempts at replication were successful. Most experiments were replicated in multiple independent groups of animals with all experimental groups present in each cohort. We used multiple independent experimental approaches, and multiple independent analyses within each experiment, to confirm our findings whenever possible. For experiments with multiple groups, individual animals or entire cages were randomly assigned to a group either at the time of surgery or at the beginning of behavioural testing (for animals that did not require surgery) with the constraint of balancing sex across groups. Automated analyses and automated experimental hardware and software, without manual intervention, were used whenever possible. Experimenters were not blinded to the group assignments of the animals. Statistical tests and data analyses were performed in Matlab (R2021a), Python (3.8.10) and R (4.2.1) as described above. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 throughout the article. Individual data points are shown when practical (and always for n ≤ 10), and box plots show the data distribution for larger sample sizes. Sample sizes, statistical tests, multiple comparisons corrections, exact P values, error bars, shaded areas and box plots are defined in the figure captions, Methods and Supplementary Table 2.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data used in this paper are publicly available from Figshare (https://doi.org/10.6084/m9.figshare.28327118)101. Source data are provided with this paper.
Code used in this paper is publicly available from GitHub (https://github.com/cazimmerman/cta and https://github.com/bichanw/cta).
Garcia, J., Kimeldorf, D. J. & Koelling, R. A. Conditioned aversion to saccharin resulting from exposure to gamma radiation. Science 122, 157–158 (1955).
ADS
PubMed
Google Scholar
Garcia, J. & Koelling, R. A. Relation of cue to consequence in avoidance learning. Psychon. Sci. 4, 123–124 (1966).
MATH
Google Scholar
Garcia, J., McGowan, B. K., Ervin, F. R. & Koelling, R. A. Cues: their relative effectiveness as a function of the reinforcer. Science 160, 794–795 (1968).
ADS
PubMed
Google Scholar
Garcia, J., Hankins, W. G. & Rusiniak, K. W. Behavioral regulation of the milieu interne in man and rat. Science 185, 824–831 (1974).
ADS
PubMed
Google Scholar
Adams, C. D. & Dickinson, A. Instrumental responding following reinforcer devaluation. Q. J. Exp. Psychol. 33, 109–121 (1981).
Google Scholar
Wilcoxon, H. C., Dragoin, W. B. & Kral, P. A. Illness-induced aversions in rat and quail: relative salience of visual and gustatory cues. Science 171, 826–828 (1971).
ADS
PubMed
Google Scholar
Gustavson, C. R., Garcia, J., Hankins, W. G. & Rusiniak, K. W. Coyote predation control by aversive conditioning. Science 184, 581–583 (1974).
ADS
PubMed
Google Scholar
Garb, J. L. & Stunkard, A. J. Taste aversions in man. Am. J. Psychiatry 131, 1204–1207 (1974).
PubMed
Google Scholar
Bernstein, I. L. Learned taste aversions in children receiving chemotherapy. Science 200, 1302–1303 (1978).
ADS
PubMed
MATH
Google Scholar
Bernstein, I. L. & Webster, M. M. Learned taste aversions in humans. Physiol. Behav. 25, 363–366 (1980).
PubMed
MATH
Google Scholar
Holman, G. L. Intragastric reinforcement effect. J. Comp. Physiol. Psychol. 69, 432–441 (1968).
MATH
Google Scholar
Sclafani, A. Conditioned food preferences. Bull. Psychon. Soc. 29, 256–260 (1991).
MATH
Google Scholar
de Araujo, I. E., Schatzker, M. & Small, D. M. Rethinking food reward. Annu. Rev. Psychol. 71, 139–164 (2020).
PubMed
Google Scholar
Revusky, S. H. & Bedarf, E. W. Association of illness with prior ingestion of novel foods. Science 155, 219–220 (1967).
ADS
PubMed
Google Scholar
Kalat, J. W. & Rozin, P. ‘Learned safety' as a mechanism in long-delay taste-aversion learning in rats. J. Comp. Physiol. Psychol. 83, 198–207 (1973).
PubMed
MATH
Google Scholar
Garcia, J., Ervin, F. R. & Koelling, R. A. Learning with prolonged delay of reinforcement. Psychon. Sci. 5, 121–122 (1966).
MATH
Google Scholar
Smith, J. C. & Roll, D. L. Trace conditioning with X-rays as an aversive stimulus. Psychon. Sci. 9, 11–12 (1967).
MATH
Google Scholar
Nachman, M. Learned taste and temperature aversions due to lithium chloride sickness after temporal delays. J. Comp. Physiol. Psychol. 73, 22–30 (1970).
PubMed
MATH
Google Scholar
Braun, J. J., Slick, T. B. & Lorden, J. F. Involvement of gustatory neocortex in the learning of taste aversions. Physiol. Behav. 9, 637–641 (1972).
PubMed
Google Scholar
Rosenblum, K., Meiri, N. & Dudai, Y. Taste memory: the role of protein synthesis in gustatory cortex. Behav. Neural Biol. 59, 49–56 (1993).
PubMed
Google Scholar
Nachman, M. & Ashe, J. H. Effects of basolateral amygdala lesions on neophobia, learned taste aversions, and sodium appetite in rats. J. Comp. Physiol. Psychol. 87, 622–643 (1974).
PubMed
Google Scholar
Kayyal, H. et al. Activity of insula to basolateral amygdala projecting neurons is necessary and sufficient for taste valence representation. J. Neurosci. 39, 9369–9382 (2019).
PubMed
PubMed Central
MATH
Google Scholar
Carter, M. E., Han, S. & Palmiter, R. D. Parabrachial calcitonin gene-related peptide neurons mediate conditioned taste aversion. J. Neurosci. 35, 4582–4586 (2015).
PubMed
PubMed Central
Google Scholar
Chen, J. Y., Campos, C. A., Jarvie, B. C. & Palmiter, R. D. Parabrachial CGRP neurons establish and sustain aversive taste memories. Neuron 100, 891–899 (2018).
PubMed
PubMed Central
Google Scholar
Nachman, M. & Ashe, J. H. Learned taste aversions in rats as a function of dosage, concentration, and route of administration of LiCl. Physiol. Behav. 10, 73–78 (1973).
PubMed
Google Scholar
Rusiniak, K. W., Hankins, W. G., Garcia, J. & Brett, L. P. Flavor-illness aversions: potentiation of odor by taste in rats. Behav. Neural Biol. 25, 1–17 (1979).
PubMed
MATH
Google Scholar
Palmerino, C. C., Rusiniak, K. W. & Garcia, J. Flavor-illness aversions: the peculiar roles of odor and taste in memory for poison. Science 208, 753–755 (1980).
ADS
PubMed
Google Scholar
Renier, N. et al. Mapping of brain activity by automated volume analysis of immediate early genes. Cell 165, 1789–1802 (2016).
PubMed
PubMed Central
MATH
Google Scholar
Wang, Q. et al. The Allen Mouse Brain Common Coordinate Framework: a 3D reference atlas. Cell 181, 936–953 (2020).
PubMed
PubMed Central
MATH
Google Scholar
Koh, M. T., Wilkins, E. E. & Bernstein, I. L. Novel tastes elevate c-fos expression in the central amygdala and insular cortex: implication for taste aversion learning. Behav. Neurosci. 117, 1416–1422 (2003).
PubMed
Google Scholar
Barot, S. K., Kyono, Y., Clark, E. W. & Bernstein, I. L. Visualizing stimulus convergence in amygdala neurons during associative learning. Proc. Natl Acad. Sci. USA 105, 20959–20963 (2008).
ADS
PubMed
PubMed Central
Google Scholar
Phelps, E. A. & LeDoux, J. E. Contributions of the amygdala to emotion processing: from animal models to human behavior. Neuron 48, 175–187 (2005).
PubMed
MATH
Google Scholar
Janak, P. H. & Tye, K. M. From circuits to behaviour in the amygdala. Nature 517, 284–292 (2015).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Carter, M. E., Soden, M. E., Zweifel, L. S. & Palmiter, R. D. Genetic identification of a neural circuit that suppresses appetite. Nature 503, 111–114 (2013).
ADS
PubMed
PubMed Central
Google Scholar
O'Leary, T. P. et al. Neuronal cell types, projections, and spatial organization of the central amygdala. iScience 25, 105497 (2022).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Wang, Y. et al. Multimodal mapping of cell types and projections in the central nucleus of the amygdala. eLife 12, e84262 (2023).
PubMed
PubMed Central
Google Scholar
Steinmetz, N. A. et al. Neuropixels 2.0: a miniaturized high-density probe for stable, long-term brain recordings. Science 372, eabf4588 (2021).
PubMed
PubMed Central
MATH
Google Scholar
Bermúdez-Rattoni, F. Molecular mechanisms of taste-recognition memory. Nat. Rev. Neurosci. 5, 209–217 (2004).
PubMed
MATH
Google Scholar
Gal-Ben-Ari, S. & Rosenblum, K. Molecular mechanisms underlying memory consolidation of taste information in the cortex. Front. Behav. Neurosci. 5, 87 (2011).
PubMed
MATH
Google Scholar
Lamprecht, R., Hazvi, S. & Dudai, Y. cAMP response element-binding protein in the amygdala is required for long- but not short-term conditioned taste aversion memory. J. Neurosci. 17, 8443–8450 (1997).
PubMed
PubMed Central
Google Scholar
Josselyn, S. A., Kida, S. & Silva, A. J. Inducible repression of CREB function disrupts amygdala-dependent memory. Neurobiol. Learn. Mem. 82, 159–163 (2004).
PubMed
Google Scholar
Zhou, Y. et al. CREB regulates excitability and the allocation of memory to subsets of neurons in the amygdala. Nat. Neurosci. 12, 1438–1443 (2009).
PubMed
PubMed Central
MATH
Google Scholar
Koh, M. T., Thiele, T. E. & Bernstein, I. L. Inhibition of protein kinase A activity interferes with long-term, but not short-term, memory of conditioned taste aversions. Behav. Neurosci. 116, 1070–1074 (2002).
PubMed
Google Scholar
Koh, M. T., Clarke, S. N. D. A., Spray, K. J., Thiele, T. E. & Bernstein, I. L. Conditioned taste aversion memory and c-Fos induction are disrupted in RIIβ-protein kinase A mutant mice. Behav. Brain Res. 143, 57–63 (2003).
PubMed
Google Scholar
Josselyn, S. A. & Frankland, P. W. Memory allocation: mechanisms and function. Annu. Rev. Neurosci. 41, 389–413 (2018).
PubMed
PubMed Central
MATH
Google Scholar
Josselyn, S. A. & Tonegawa, S. Memory engrams: recalling the past and imagining the future. Science 367, eaaw4325 (2020).
PubMed
PubMed Central
MATH
Google Scholar
Zhang, J.-F. et al. An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice. Nat. Chem. Biol. 17, 39–46 (2021).
ADS
PubMed
MATH
Google Scholar
Ahmed, M. S. et al. Hippocampal network reorganization underlies the formation of a temporal association memory. Neuron 107, 283–291 (2020).
PubMed
PubMed Central
MATH
Google Scholar
Hamid, A. A., Frank, M. J. & Moore, C. I. Wave-like dopamine dynamics as a mechanism for spatiotemporal credit assignment. Cell 184, 2733–2749 (2021).
PubMed
PubMed Central
MATH
Google Scholar
Parker, N. F. et al. Choice-selective sequences dominate in cortical relative to thalamic inputs to NAc to support reinforcement learning. Cell Rep. 39, 110756 (2022).
PubMed
PubMed Central
MATH
Google Scholar
Krausz, T. A. et al. Dual credit assignment processes underlie dopamine signals in a complex spatial environment. Neuron 111, 3465–3478 (2023).
PubMed
PubMed Central
MATH
Google Scholar
Foster, D. J. Replay comes of age. Annu. Rev. Neurosci. 40, 581–602 (2017).
PubMed
MATH
Google Scholar
Klinzing, J. G., Niethard, N. & Born, J. Mechanisms of systems memory consolidation during sleep. Nat. Neurosci. 22, 1598–1610 (2019).
PubMed
Google Scholar
Yamamoto, T., Matsuo, R., Kiyomitsu, Y. & Kitamura, R. Taste responses of cortical neurons in freely ingesting rats. J. Neurophysiol. 61, 1244–1258 (1989).
PubMed
Google Scholar
Yasoshima, Y. & Yamamoto, T. Short-term and long-term excitability changes of the insular cortical neurons after the acquisition of taste aversion learning in behaving rats. Neuroscience 84, 1–5 (1998).
PubMed
MATH
Google Scholar
Accolla, R. & Carleton, A. Internal body state influences topographical plasticity of sensory representations in the rat gustatory cortex. Proc. Natl Acad. Sci. USA 105, 4010–4015 (2008).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Grossman, S. E., Fontanini, A., Wieskopf, J. S. & Katz, D. B. Learning-related plasticity of temporal coding in simultaneously recorded amygdala–cortical ensembles. J. Neurosci. 28, 2864–2873 (2008).
PubMed
PubMed Central
Google Scholar
Moran, A. & Katz, D. B. Sensory cortical population dynamics uniquely track behavior across learning and extinction. J. Neurosci. 34, 1248–1257 (2014).
PubMed
PubMed Central
MATH
Google Scholar
Lavi, K., Jacobson, G. A., Rosenblum, K. & Lüthi, A. Encoding of conditioned taste aversion in cortico-amygdala circuits. Cell Rep. 24, 278–283 (2018).
PubMed
MATH
Google Scholar
Wu, C.-H., Ramos, R., Katz, D. B. & Turrigiano, G. G. Homeostatic synaptic scaling establishes the specificity of an associative memory. Curr. Biol. 31, 2274–2285 (2021).
PubMed
PubMed Central
MATH
Google Scholar
Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8, 1263–1268 (2005).
PubMed
Google Scholar
Nagel, G. et al. Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr. Biol. 15, 2279–2284 (2005).
PubMed
MATH
Google Scholar
Chen, T.-W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Marshel, J. H. et al. Cortical layer–specific critical dynamics triggering perception. Science 365, eaaw5202 (2019).
PubMed
PubMed Central
Google Scholar
Mahn, M. et al. Efficient optogenetic silencing of neurotransmitter release with a mosquito rhodopsin. Neuron 109, 1621–1635 (2021).
PubMed
PubMed Central
MATH
Google Scholar
Yang, C. F. et al. Sexually dimorphic neurons in the ventromedial hypothalamus govern mating in both sexes and aggression in males. Cell 153, 896–909 (2013).
PubMed
PubMed Central
MATH
Google Scholar
Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S. & Roth, B. L. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc. Natl Acad. Sci. USA 104, 5163–5168 (2007).
ADS
PubMed
PubMed Central
Google Scholar
Luo, T. Z. et al. An approach for long-term, multi-probe Neuropixels recordings in unrestrained rats. eLife 9, e59716 (2020).
PubMed
PubMed Central
Google Scholar
Brooks, M. E. et al. glmmTMB balances speed and flexibility among packages for zero-inflated generalized linear mixed modeling. R J. 9, 378–400 (2017).
MATH
Google Scholar
Arel-Bundock, V., Greifer, N. & Heiss, A. How to interpret statistical models using marginaleffects for R and Python. J. Stat. Softw. 111, 1–32 (2024).
MATH
Google Scholar
Hochberg, Y. A sharper Bonferroni procedure for multiple tests of significance. Biometrika 75, 800–802 (1988).
MathSciNet
MATH
Google Scholar
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
PubMed
MATH
Google Scholar
Renier, N. et al. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159, 896–910 (2014).
PubMed
MATH
Google Scholar
Dean, K. M., Roudot, P., Welf, E. S., Danuser, G. & Fiolka, R. Deconvolution-free subcellular imaging with axially swept light sheet microscopy. Biophys. J. 108, 2807–2815 (2015).
ADS
PubMed
PubMed Central
Google Scholar
Bria, A. & Iannello, G. TeraStitcher—a tool for fast automatic 3D-stitching of teravoxel-sized microscopy images. BMC Bioinformatics 13, 316 (2012).
PubMed
PubMed Central
MATH
Google Scholar
Swaney, J. et al. Scalable image processing techniques for quantitative analysis of volumetric biological images from light-sheet microscopy. Preprint at bioRxiv https://doi.org/10.1101/576595 (2019).
Klein, S., Staring, M., Murphy, K., Viergever, M. A. & Pluim, J. P. W. elastix: a toolbox for intensity-based medical image registration. IEEE Trans. Med. Imaging 29, 196–205 (2010).
PubMed
MATH
Google Scholar
Shamonin, D. P. et al. Fast parallel image registration on CPU and GPU for diagnostic classification of Alzheimer's disease. Front. Neuroinform. 7, 50 (2013).
PubMed
MATH
Google Scholar
Kirst, C. et al. Mapping the fine-scale organization and plasticity of the brain vasculature. Cell 180, 780–795 (2020).
PubMed
MATH
Google Scholar
Tyson, A. L. et al. A deep learning algorithm for 3D cell detection in whole mouse brain image datasets. PLoS Comput. Biol. 17, e1009074 (2021).
PubMed
PubMed Central
MATH
Google Scholar
He, K., Zhang, X., Ren, S. & Sun, J. Deep residual learning for image recognition. In Proc. IEEE Conference on Computer Vision and Pattern Recognition 770–778 (IEEE, 2016).
Benjamini, Y., Krieger, A. M. & Yekutieli, D. Adaptive linear step-up procedures that control the false discovery rate. Biometrika 93, 491–507 (2006).
MathSciNet
MATH
Google Scholar
DeNardo, L. A. et al. Temporal evolution of cortical ensembles promoting remote memory retrieval. Nat. Neurosci. 22, 460–469 (2019).
PubMed
PubMed Central
MATH
Google Scholar
Madangopal, R. et al. Incubation of palatable food craving is associated with brain-wide neuronal activation in mice. Proc. Natl Acad. Sci. USA 119, e2209382119 (2022).
PubMed
PubMed Central
Google Scholar
Hsueh, B. et al. Cardiogenic control of affective behavioural state. Nature 615, 292–299 (2023).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Wang, F. et al. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J. Mol. Diagn. 14, 22–29 (2012).
PubMed
PubMed Central
Google Scholar
Stringer, C., Wang, T., Michaelos, M. & Pachitariu, M. Cellpose: a generalist algorithm for cellular segmentation. Nat. Methods 18, 100–106 (2021).
PubMed
Google Scholar
Pachitariu, M. & Stringer, C. Cellpose 2.0: how to train your own model. Nat. Methods 19, 1634–1641 (2022).
PubMed
PubMed Central
MATH
Google Scholar
Chiaruttini, N. et al. ABBA, a novel tool for whole-brain mapping, reveals brain-wide differences in immediate early genes induction following learning. Preprint at bioRxiv https://doi.org/10.1101/2024.09.06.611625 (2024).
International Brain Laboratory. Spike sorting pipeline for the International Brain Laboratory. Figshare https://doi.org/10.6084/m9.figshare.19705522 (2022).
International Brain Laboratory et al. Reproducibility of in vivo electrophysiological measurements in mice. eLife 13, RP100840 (2024).
Google Scholar
Pachitariu, M., Steinmetz, N., Kadir, S., Carandini, M. & Harris, K. D. Kilosort: realtime spike-sorting for extracellular electrophysiology with hundreds of channels. Preprint at bioRxiv https://doi.org/10.1101/061481 (2016).
Pachitariu, M., Sridhar, S., Pennington, J. & Stringer, C. Spike sorting with Kilosort4. Nat. Methods 21, 914–921 (2024).
PubMed
PubMed Central
Google Scholar
Hill, D. N., Mehta, S. B. & Kleinfeld, D. Quality metrics to accompany spike sorting of extracellular signals. J. Neurosci. 31, 8699–8705 (2011).
PubMed
PubMed Central
MATH
Google Scholar
Liu, L. D. et al. Accurate localization of linear probe electrode arrays across multiple brains. eNeuro https://doi.org/10.1523/ENEURO.0241-21.2021 (2021).
Tyson, A. L. et al. Accurate determination of marker location within whole-brain microscopy images. Sci. Rep. 12, 867 (2022).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Pedregosa, F. et al. Scikit-learn: machine learning in Python. J. Machine Learn. Res. 12, 2825–2830 (2011).
MathSciNet
MATH
Google Scholar
Jun, J. J. et al. Fully integrated silicon probes for high-density recording of neural activity. Nature 551, 232–236 (2017).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Gunaydin, L. A. et al. Natural neural projection dynamics underlying social behavior. Cell 157, 1535–1551 (2014).
PubMed
PubMed Central
MATH
Google Scholar
Lerner, T. N. et al. Intact-brain analyses reveal distinct information carried by SNc dopamine subcircuits. Cell 162, 635–647 (2015).
PubMed
PubMed Central
MATH
Google Scholar
Zimmerman, C. A. Data for “A neural mechanism for learning from delayed postingestive feedback”. Figshare https://doi.org/10.6084/m9.figshare.28327118 (2025).
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We thank T. Harris and W. Sun for the design, manufacture and attachment of Neuropixels probe dovetail adapters; T. Luo for help troubleshooting Neuropixels recordings; E. Engel, O. Huang, A. Chan and staff at the PNI Viral Core Facility for AAV production; J. Stirman and staff at Life Canvas Technologies for light-sheet microscopy imaging support; members of the D. Kleinfeld and S. Wang laboratories for collaboration on the Brainsharer web portal; J. Pillow for advice on the decoding analysis; K. Deisseroth, D. Kim, R. Palmiter, B. Roth, N. Shah, J. Wells, O. Yizhar, J. Zhang and staff at the HHMI Janelia GENIE Project for sharing reagents; A. Sirko and staff at the Princeton Laboratory Animal Resources for help with animal husbandry; A. Kalmbach and M. Magos for technical assistance; and P. Dayan, Z. Knight, B. Jarvie, W. Fleming and members of the Witten laboratory for feedback on the manuscript. Funding was provided by the Helen Hay Whitney Foundation (to C.A.Z.), the Brain Research Foundation (to I.B.W.), the Simons Collaboration on the Global Brain (to I.B.W.), the Howard Hughes Medical Institute (to I.B.W.) and the National Institutes of Health (K99-DA059957 to C.A.Z.; P50-MH136296, U19-NS123716 and DP1-MH136573 to I.B.W.; U19-NS104648 to I.B.W. and S.S.-H.W.; and RF1-MH128776 to S.S.-H.W.).
Princeton Neuroscience Institute, Princeton University, Princeton, NJ, USA
Christopher A. Zimmerman, Scott S. Bolkan, Alejandro Pan-Vazquez, Bichan Wu, Emma F. Keppler, Jordan B. Meares-Garcia, Eartha Mae Guthman, Robert N. Fetcho, Brenna McMannon, Junuk Lee, Austin T. Hoag, Laura A. Lynch, Sanjeev R. Janarthanan, Juan F. López Luna, Adrian G. Bondy, Annegret L. Falkner, Samuel S.-H. Wang & Ilana B. Witten
Howard Hughes Medical Institute, Princeton University, Princeton, NJ, USA
Ilana B. Witten
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C.A.Z. conceived the project with input from I.B.W. C.A.Z. and I.B.W. designed and interpreted the experiments. I.B.W. supervised all aspects of the project. C.A.Z. performed the experiments, analysed the data and generated the figures with contributions from all the authors as described below. A.P.-V., with support from C.A.Z., R.N.F. and B.M., performed the acute Neuropixels recordings. B.W., with support from C.A.Z., performed the decoding analyses. E.F.K. performed stereotaxic surgeries. E.F.K. and J.B.M.-G. performed histology. S.S.B. and J.B.M.-G., with support from C.A.Z., performed the RNAscope FISH experiments. E.M.G., with support from J.L. and A.L.F., performed and analysed the slice electrophysiology recordings. C.A.Z., with support from A.T.H., developed the deep-learning-assisted cell-detection pipeline. L.A.L., S.R.J. and C.A.Z., with support from S.S.-H.W., performed tissue clearing and light-sheet microscopy imaging. A.P.-V. and S.S.B. provided support for the chronic Neuropixels recordings. J.F.L.L. designed and manufactured behavioural equipment. A.G.B. designed the chronic Neuropixels 2.0 implant assembly. C.A.Z. and I.B.W. wrote the paper with input from all the authors.
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Christopher A. Zimmerman or Ilana B. Witten.
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a, Comparison of individual familiar and novel flavour condition mice for every brain region that was significantly novel flavour-activated during consumption (n = 12 mice per flavour condition). b, Analogous to a, but for brain regions that were significantly familiar flavour-activated during consumption (n = 12 mice per flavour condition). c, Map of average FOS+ cell density across all mice for the consumption time point (n = 24 mice). The Allen CCF is overlaid. Coronal sections are spaced by 0.5 mm, the section corresponding to Bregma is marked with a *, and key brain regions are labeled. d, Map of the difference in average FOS+ cell density across novel versus familiar flavour condition mice for the consumption time point (n = 12 mice per flavour condition). e, Map of average FOS+ cell density across all mice for the malaise time point (n = 24 mice). f, Map of the difference in average FOS+ cell density across novel versus familiar flavour condition mice for the malaise time point (n = 12 mice per flavour condition). g, Map of average FOS+ cell density across all mice for the retrieval time point (n = 24 mice). h, Map of the difference in average FOS+ cell density across novel versus familiar flavour condition mice for the retrieval time point (n = 12 mice per flavour condition). An interactive visualization of these FOS+ cell density maps is available at https://www.brainsharer.org/ng/?id=872. Error bars represent mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. See Supplementary Table 2 for details of statistical tests and for exact P values. See Supplementary Table 1 for list of brain region abbreviations.
Source data
a, Schematic and example hM3D-mCherry expression data for the bilateral chemogenetic LS activation experiment. CNO was delivered 45-min before the experiment began to ensure that the LS was activated throughout consumption. b, Retrieval test flavour preference for the experiment described in a (n = 18 hM3D mice, 12 YFP mice). c, Schematic of the LS activation FOS time point (n = 12 mice per group for d–g). As in a, CNO was delivered 45-min before the experiment began, and the flavour was novel for both groups. d, Comparison of LS FOS (including the entire ‘Lateral septal complex' in the Allen CCF) for individual YFP and hM3D mice, confirming strong activation by hM3D. e, Comparison of CEA FOS for individual YFP and hM3D mice, showing reduced malaise-driven activation in hM3D mice. f, Correlation between the average FOS+ cell count of each brain region for hM3D versus YFP mice. The amygdala network (from Fig. 1f, g; n = 12 regions), septal complex (n = 4 regions), and all other regions (n = 114 regions) are shown separately. g, Visualization of the difference in FOS+ cell density across YFP versus hM3D mice with Allen CCF boundaries overlaid. Error bars represent mean ± s.e.m. Shaded areas represent linear fit estimate ± 95% confidence interval. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. See Supplementary Table 2 for details of statistical tests and for exact P values. See Supplementary Table 1 for list of brain region abbreviations.
Source data
Panels a–c show that the brain-wide shift towards activation by the novel flavour is primarily localized to subcortical regions; outlines represent kernel-density estimates of the empirical distributions. a, Novel – familiar ΔFOS effect distribution of all cortical regions (cerebral cortex in the Allen CCF) at each time point (n = 38 brain regions; all statistical tests not significant). b, Novel – familiar ΔFOS effect distribution of all subcortical forebrain regions (cerebral nuclei, thalamus and hypothalamus in the Allen CCF) at each time point (n = 54 brain regions). c, Novel – familiar ΔFOS effect distribution of all midbrain and hindbrain regions (midbrain, pons and medulla in the Allen CCF) at each time point (n = 38 brain regions). d, Hierarchical clustering of novel – familiar ΔFOS effects. This is an expanded version Fig. 1f showing all brain region names. e–n, Left, Illustration of the brain regions comprising each cluster from the hierarchical clustering analysis. Right, Summary of the novel – familiar ΔFOS effect for each cluster at each time point, showing each brain region as an individual point. Error bars represent mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. See Supplementary Table 2 for details of statistical tests and for exact P values. No statistical tests were performed for e–n. See Supplementary Table 1 for list of brain region abbreviations and for GLMM statistics.
Source data
a, Correlation matrices showing the animal-by-animal pairwise FOS correlation for every pair of brain regions during consumption (left), delayed malaise (middle), or memory retrieval (right). Brain regions are sorted using the hierarchical clustermap obtained from the novel – familiar ΔFOS effects in Fig. 1f. b, Summary of the average within-cluster FOS correlation for individual amygdala network regions (Cluster 1 from Fig. 1f, g) by time point (n = 12 regions). The high animal-by-animal correlation among all of the regions in cluster 1 suggest that these regions form a functional network. Panels c,d show that activation of other clusters of brain regions is more correlated with amygdala network activation at experimental time points when those clusters are more strongly novel flavour-selective, including for clusters that were specifically engaged during the initial flavour consumption (comprising sensory cortices; cluster 2) or during retrieval (including the BST; cluster 6). c, Summary of the average across-cluster FOS correlation between the amygdala network and every other cluster at each time point as a function of the other cluster's standardized novel – familiar effect at that time point (n = 9 clusters × 3 time points). d, Scatter plots showing the pairwise correlation between AIp (top; example cluster 2 region) or BST (bottom; example cluster 6 region) and the CEA (n = 24 mice per time point) at each experimental time point. Error bars represent mean ± s.e.m. Shaded areas represent linear fit estimate ± 95% confidence interval. NS, not significant, ***P ≤ 0.001, ****P ≤ 0.0001. See Supplementary Table 2 for details of statistical tests and for exact P values. See Supplementary Table 1 for list of brain region abbreviations.
Source data
a, Schematic of the acute Neuropixels recording experiment (created using BioRender.com). b, Reconstruction of recording trajectories registered to the Allen CCF. Each line represents one insertion of a single-shank Neuropixels 1.0 probe (n = 24 insertions from 4 mice). c, Left, PETHs of neural activity time-locked to CGRP neuron stimulation trains (n = 3,524 amygdala neurons from 24 insertions). Neurons were divided into four response types using a GMM (see Methods): two CGRP neuron stimulation-activated response types (7.3% strongly activated, dark green; 22.4% weakly activated, light green), one CGRP neuron stimulation-inhibited response type (24.6%, purple), and one unmodulated response type (45.8%, gray). Right, Average PETHs for each GMM response type. d, Percentage of recorded neurons that were CGRP neuron stimulation-activated based on the GMM across amygdala subregions (n = 339 CEAc, 272 CEAl, 717 CEAm, 526 BMAa, 129 COAa, 133 IA, 41 BLAp, 30 PAA, 182 MEA, 354 BLAa, 54 Other (AAA, LA, PA), 44 BLAv, 250 BMAp, and 58 COAp neurons). Regions in the CFA amygdala network (cluster 1 from Fig. 1f, g) are shown in red, and other amygdala regions are shown in black. e, Anatomical distribution of all CGRP neuron stimulation-activated (green), CGRP neuron stimulation-inhibited (purple), and unmodulated (gray) neurons projected onto a single coronal or sagittal section of the Allen CCF. f, Left, Light-sheet microscopy data for each animal showing Neuropixels probe trajectories aligned to the Allen CCF with amygdala subregions overlaid. Right, Reconstructions of recording trajectories. For each animal, a single sagittal section corresponding to the center-of-mass of the active recording sites is shown. The colormap for amygdala regions in b is also used in e,f. See Supplementary Table 1 for list of brain region abbreviations.
Source data
a, Example brain-wide FOS imaging data (200-µm maximum-intensity projections) for four example CGRP neuron stimulation animals. b, Summary of FOS+ cell counts in the PB at each time point (n = 24 consumption, 24 malaise, 27 CGRP neuron stimulation, 24 retrieval mice). c, Analysis analogous to Fig. 2i but using a GLMM, showing the correlation among the standardized coefficients for the main LiCl-induced malaise and CGRP neuron stimulation effects from Equation 3 (n = 12 amygdala, 117 other regions). d, Analysis analogous to Fig. 2j but using a GLMM, showing the correlation among the average marginal effects of flavour from Equation 4 (n = 12 amygdala, 117 other regions). e, Map of average FOS+ cell density across all mice for the CGRP neuron stimulation time point (n = 27 mice). f, Map of the difference in average FOS+ cell density across novel versus familiar flavour condition mice for the CGRP neuron stimulation time point (n = 14 novel flavour mice, 13 familiar flavour mice). g, Top, Schematic of the CGRPCEA projection stimulation RNAscope FISH experiment. Bottom, Example slide scanner image of FOS expression with the Allen CCF overlaid. h, Example confocal image showing Fos, Sst, Prkcd, and Calcrl expression. This is an expanded version of Fig. 2l. The top row shows the full field-of-view, and the bottom row is magnified with Fos+ cell outlines overlaid in black. Error bars represent mean ± s.e.m. Shaded areas represent linear fit estimate ± 95% confidence interval. NS, not significant, *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. See Supplementary Table 2 for details of statistical tests and for exact P values. No statistical tests were performed for b. See Supplementary Table 1 for list of brain region abbreviations and for GLMM statistics.
Source data
a, Left, Schematic illustration of the chronic Neuropixels 2.0 implant assembly at progressive stages of construction from top left to bottom right. Right, Schematic illustration of the chronic Neuropixels 1.0 implant assembly68 upon which the 2.0 implant design is based, shown for size comparison. b, Test recordings used to select recording sites (black bars) properly targeting the CEA (red bars, based on postmortem reconstruction) for two example animals. The shanks are arranged from anterior (1, left) to posterior (4, right) and span 750-µm total. We found that we could distinguish the CEA from nearby brain regions along the vertical axis of the probe shanks as a band of dense neural activity. c, Left, Light-sheet microscopy data for each animal showing Neuropixels shank trajectories aligned to the Allen CCF with CEA subregions overlaid. Right, Reconstruction of trajectories in the CEA. For each animal, a single sagittal section corresponding to the center-of-mass of all active recording sites is shown. See Methods for a description of which animals were included in each experiment.
a, Cumulative intake of the novel flavour and water in the two-reward CFA paradigm in Fig. 3b (n = 8 mice). We used a randomized, trial-based structure to ensure that mice consumed the two options at an equal rate (see Methods). b, Left, PETHs to novel flavour delivery for the novel flavour-preferring (n = 373 neurons from 8 mice), water-preferring (n = 121 neurons), and nonselective (n = 610 neurons) CEA neurons in Fig. 3c–l. Middle, PETHs to water delivery for the same novel flavour-preferring, water-preferring, and nonselective CEA neurons. Right, Pie chart visualizing the proportion of novel flavour-preferring, water-preferring, and nonselective CEA neurons. Panels c–e provide additional characterization of the multinomial logistic regression decoder using CEA population activity in Fig. 4g–i. c, Cross-validated log-likelihood for the decoder classifying periods of novel flavour consumption versus water consumption versus baseline activity across a range of regularization parameter (λ) values (n = 6 mice). d, Decoder output time-locked to novel flavour delivery (top) and water delivery (bottom) in the initial consumption period (mean across 6 mice). The decoder's predicted probability for novel flavour consumption and water consumption are both shown. e, Confusion matrix summarizing the cross-validated decoder performance across all mice (n = 6 mice). The overall misclassification rate was 0.56% (4 out of 720). Error bars and shaded areas represent mean ± s.e.m. No statistical tests were performed for c.
Panels a–e show that CGRPCEA projection stimulation reactivates flavour representations in the amygdala. a, Reconstruction of recording trajectories registered to the Allen CCF for mice with CGRPCEA projection stimulation (n = 32 shanks from 8 mice). b, Average spiking of individual neurons during the CGRPCEA projection stimulation conditioning experiment (n = 1,221 neurons from 8 mice). c, Average spiking of the novel flavour-preferring (n = 354 neurons), water-preferring (n = 129 neurons), and nonselective (n = 738 neurons) populations across the entire experiment. d, Average spiking of the novel flavour-preferring, water-preferring, and nonselective populations during individual bouts of CGRPCEA projection stimulation (same sample sizes as c). e, Neural trajectories in PC-space for novel flavour consumption, water consumption, and CGRPCEA projection stimulation. Panels f,g show that genetic ablation of CGRP neurons by taCasp3-TEVp impairs delayed CFA learning. f, Example CGRP immunoreactivity data confirming genetic ablation of CGRP neurons by taCasp3-TEVp. This is an expanded version of Fig. 3p. g, CGRP neuron ablation mice show significantly higher acceptance of the conditioned flavour following LiCl-induced CFA when compared to wild type controls (n = 6 taCasp3 mice, 7 control mice). Panels h–j show that CGRP neuron stimulation-activated CEA neurons are also activated by LiCl injection. h, Schematic. i, Average spiking during CGRP neuron stimulation and then during LiCl-induced malaise (n = 821 neurons from 4 mice). j, Average spiking of the CGRP neuron stimulation-activated neurons (n = 189 neurons) and other neurons (n = 632 neurons) during LiCl-induced malaise. Shaded areas represent mean ± s.e.m. Inset box plots show the 10th, 25th, 50th, 75th, and 90th percentiles. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. See Supplementary Table 2 for details of statistical tests and for exact P values.
Source data
Panels a,b relate to Fig. 4b–d. a, Proportion of flavour-preferring neurons classified separately on conditioning or retrieval day (n = 8 mice). b, Population trajectories for flavour consumption, water consumption, and CGRP neuron stimulation. Panels c–e relate to Fig. 4e. c, Average spiking of all individual neurons for the CGRPCEA projection stimulation experiment (n = 1,042 neurons from 8 mice). d, Analogous to a, but for mice with CGRPCEA projection stimulation (n = 8 mice). e, Average spiking of the novel flavour-preferring neurons with the highest 10% CGRPCEA response magnitudes and of the remaining novel flavour-preferring neurons. Panels f,g show that LiCl-induced malaise stabilizes the flavour representation upon retrieval, and that this is impaired by CGRP neuron ablation. f, For control mice, average spiking of the novel flavour-preferring population (n = 279 neurons from 4 mice). g, Analogous to f, but for mice with CGRP neuron ablation (n = 109 neurons from 4 mice). Panels h–k relate to Fig. 4f. h, Average spiking of all individual neurons (n = 924 neurons from 7 mice). i, Proportion of flavour-preferring neurons classified separately on novel or familiar day (n = 7 mice). j, Average spiking of the initially water-preferring population (n = 160 neurons from 7 mice; classified on novel day) during flavour consumption. k, Population trajectories for flavour and water consumption. l, Time-courses along the PC2 axis during consumption following CGRP neuron stimulation conditioning (from b) and familiarization (from k). Error bars and shaded areas represent mean ± s.e.m. Inset box plots show the 10th, 25th, 50th, 75th, and 90th percentiles. NS, not significant, *P ≤ 0.05, **P ≤ 0.01. See Supplementary Table 2 for details of statistical tests and for exact P values.
Source data
Supplementary Table 1 contains FOS GLMM statistics for individual brain regions, including a list of abbreviations. Supplementary Table 2 contains a summary of statistical tests, including exact P values.
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Zimmerman, C.A., Bolkan, S.S., Pan-Vazquez, A. et al. A neural mechanism for learning from delayed postingestive feedback.
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Bian, X. et al. Regulation of gene expression by glycolytic and gluconeogenic enzymes. Trends Cell Biol. 32, 786–799 (2022).
CAS
PubMed
Google Scholar
Lunt, S. Y. & Vander Heiden, M. G. Aerobic glycolysis: meeting the metabolic requirements of cell proliferation. Annu. Rev. Cell Dev. Biol. 27, 441–464 (2011).
CAS
PubMed
Google Scholar
Xu, D. et al. The evolving landscape of noncanonical functions of metabolic enzymes in cancer and other pathologies. Cell Metab. 33, 33–50 (2021).
CAS
PubMed
Google Scholar
Warburg, O., Wind, F. & Negelein, E. The metabolism of tumors in the body. J. Gen. Physiol. 8, 519–530 (1927).
CAS
PubMed
PubMed Central
Google Scholar
Thompson, C. B. et al. A century of the Warburg effect. Nat. Metab. 5, 1840–1843 (2023).
PubMed
Google Scholar
Ashton, T. M., McKenna, W. G., Kunz-Schughart, L. A. & Higgins, G. S. Oxidative phosphorylation as an emerging target in cancer therapy. Clin. Cancer Res. 24, 2482–2490 (2018).
CAS
PubMed
Google Scholar
El-Botty, R. et al. Oxidative phosphorylation is a metabolic vulnerability of endocrine therapy and palbociclib resistant metastatic breast cancers. Nat. Commun. 14, 4221 (2023).
CAS
PubMed
PubMed Central
Google Scholar
Menk, A. V. et al. Early TCR signaling induces rapid aerobic glycolysis enabling distinct acute T cell effector functions. Cell Rep. 22, 1509–1521 (2018).
CAS
PubMed
PubMed Central
Google Scholar
DeBerardinis, R. J., Lum, J. J., Hatzivassiliou, G. & Thompson, C. B. The biology of cancer: metabolic reprogramming fuels cell growth and proliferation. Cell Metab. 7, 11–20 (2008). This study reveals the coordinated regulation of glycolysis and mitochondrial activity by the protein kinase activity of phosphoglycerate kinase 1 to drive the Warburg effect.
CAS
PubMed
Google Scholar
Li, X. et al. Mitochondria-translocated PGK1 functions as a protein kinase to coordinate glycolysis and the TCA cycle in tumorigenesis. Mol. Cell 61, 705–719 (2016).
CAS
PubMed
PubMed Central
Google Scholar
Claps, G. et al. The multiple roles of LDH in cancer. Nat. Rev. Clin. Oncol. 19, 749–762 (2022).
PubMed
Google Scholar
Miao, P., Sheng, S., Sun, X., Liu, J. & Huang, G. Lactate dehydrogenase A in cancer: a promising target for diagnosis and therapy. IUBMB Life 65, 904–910 (2013).
CAS
PubMed
Google Scholar
Li, X., Egervari, G., Wang, Y., Berger, S. L. & Lu, Z. Regulation of chromatin and gene expression by metabolic enzymes and metabolites. Nat. Rev. Mol. Cell Biol. 19, 563–578 (2018).
CAS
PubMed
PubMed Central
Google Scholar
Lu, Z. & Hunter, T. Metabolic kinases moonlighting as protein kinases. Trends Biochem. Sci. 43, 301–310 (2018).
CAS
PubMed
PubMed Central
Google Scholar
Dai, J. et al. Primary prostate cancer educates bone stroma through exosomal pyruvate kinase M2 to promote bone metastasis. J. Exp. Med. 216, 2883–2899 (2019).
CAS
PubMed
PubMed Central
Google Scholar
Wang, Y., Xia, Y. & Lu, Z. Metabolic features of cancer cells. Cancer Commun. 38, 65 (2018).
CAS
Google Scholar
Tessarz, P. & Kouzarides, T. Histone core modifications regulating nucleosome structure and dynamics. Nat. Rev. Mol. Cell Biol. 15, 703–708 (2014).
CAS
PubMed
Google Scholar
Zhang, D. et al. Metabolic regulation of gene expression by histone lactylation. Nature 574, 575–580 (2019). This study reveals that lactylation occurs on histones, thereby regulating gene expression.
CAS
PubMed
PubMed Central
Google Scholar
Yang, W. et al. EGFR-induced and PKCepsilon monoubiquitylation-dependent NF-kappaB activation upregulates PKM2 expression and promotes tumorigenesis. Mol. Cell 48, 771–784 (2012).
CAS
PubMed
PubMed Central
Google Scholar
Yang, W. et al. ERK1/2-dependent phosphorylation and nuclear translocation of PKM2 promotes the Warburg effect. Nat. Cell Biol. 14, 1295–1304 (2012). This study reveals that nuclear pyruvate kinase M2 activates MYC to promote glycolytic gene expression, demonstrating that the Warburg effect is regulated by a nuclear glycolytic enzyme upon receptor tyrosine kinase activation.
CAS
PubMed
PubMed Central
Google Scholar
Lu, Z. & Hunter, T. Prolyl isomerase Pin1 in cancer. Cell Res. 24, 1033–1049 (2014).
CAS
PubMed
PubMed Central
Google Scholar
Lv, L. et al. Mitogenic and oncogenic stimulation of K433 acetylation promotes PKM2 protein kinase activity and nuclear localization. Mol. Cell 52, 340–352 (2013).
CAS
PubMed
PubMed Central
Google Scholar
Wang, H. J. et al. JMJD5 regulates PKM2 nuclear translocation and reprograms HIF-1alpha-mediated glucose metabolism. Proc. Natl Acad. Sci. USA 111, 279–284 (2014).
CAS
PubMed
Google Scholar
Yang, W. et al. PKM2 phosphorylates histone H3 and promotes gene transcription and tumorigenesis. Cell 150, 685–696 (2012). This study reveals that pyruvate kinase M2 can function as a protein kinase and phosphorylate histone H3.
CAS
PubMed
PubMed Central
Google Scholar
Yang, W. et al. Nuclear PKM2 regulates beta-catenin transactivation upon EGFR activation. Nature 480, 118–122 (2011). This study reveals that receptor tyrosine kinase activation confers a nuclear function on pyruvate kinase M2, allowing it to regulate β-catenin and promote tumour cell proliferation.
CAS
PubMed
PubMed Central
Google Scholar
Hosios, A. M., Fiske, B. P., Gui, D. Y. & Vander Heiden, M. G. Lack of evidence for PKM2 protein kinase activity. Mol. Cell 59, 850–857 (2015).
CAS
PubMed
PubMed Central
Google Scholar
Yang, W. & Lu, Z. Nuclear PKM2 regulates the Warburg effect. Cell Cycle 12, 3154–3158 (2013).
CAS
PubMed
PubMed Central
Google Scholar
Wang, X. et al. PKM2-induced the phosphorylation of histone H3 contributes to EGF-mediated PD-L1 transcription in HCC. Front. Pharmacol. 11, 577108 (2020).
CAS
PubMed
PubMed Central
Google Scholar
Li, S. et al. Serine and SAM responsive complex SESAME regulates histone modification crosstalk by sensing cellular metabolism. Mol. Cell 60, 408–421 (2015).
CAS
PubMed
Google Scholar
Yu, Q. et al. Regulation of SESAME-mediated H3T11 phosphorylation by glycolytic enzymes and metabolites. PLoS ONE 12, e0175576 (2017).
PubMed
PubMed Central
Google Scholar
Hu, P. et al. Nuclear-localized pyruvate kinases control phosphorylation of histone H3 on threonine 11. Nat. Plants 10, 1682–1697 (2024).
CAS
PubMed
Google Scholar
Zhang, Y. et al. Metabolic switch regulates lineage plasticity and induces synthetic lethality in triple-negative breast cancer. Cell Metab. 36, 193–208.e8 (2024).
CAS
PubMed
Google Scholar
Jing, Y. Y. et al. Epigenetic regulation of the Warburg effect by H2B monoubiquitination. Cell Death Differ. 27, 1660–1676 (2020).
CAS
PubMed
Google Scholar
Liu, Y. et al. Nuclear lactate dehydrogenase A senses ROS to produce alpha-hydroxybutyrate for HPV-induced cervical tumor growth. Nat. Commun. 9, 4429 (2018).
PubMed
PubMed Central
Google Scholar
Kornberg, M. D. et al. GAPDH mediates nitrosylation of nuclear proteins. Nat. Cell Biol. 12, 1094–1100 (2010).
CAS
PubMed
PubMed Central
Google Scholar
Yin, C. et al. ALDOB/KAT2A interactions epigenetically modulate TGF-beta expression and T cell functions in hepatocellular carcinogenesis. Hepatology 81, 77–93 (2023).
PubMed
Google Scholar
Shi, R. et al. Fructose-1,6-bisphosphatase 1 suppresses PPARalpha-mediated gene transcription and non-small-cell lung cancer progression. Am. J. Cancer Res. 13, 4742–4754 (2023).
CAS
PubMed
PubMed Central
Google Scholar
Wang, Z. et al. Fructose-1,6-bisphosphatase 1 functions as a protein phosphatase to dephosphorylate histone H3 and suppresses PPARalpha-regulated gene transcription and tumour growth. Nat. Cell Biol. 24, 1655–1665 (2022). This study is the first to report a metabolic enzyme functioning as a protein phosphatase.
CAS
PubMed
Google Scholar
Li, B. et al. Fructose-1,6-bisphosphatase opposes renal carcinoma progression. Nature 513, 251–255 (2014).
CAS
PubMed
PubMed Central
Google Scholar
Moreno-Yruela, C. et al. Class I histone deacetylases (HDAC1-3) are histone lysine delactylases. Sci. Adv. 8, eabi6696 (2022).
CAS
PubMed
PubMed Central
Google Scholar
Torrini, C. et al. Lactate is an epigenetic metabolite that drives survival in model systems of glioblastoma. Mol. Cell 82, 3061–3076.e6 (2022).
CAS
PubMed
PubMed Central
Google Scholar
Latham, T. et al. Lactate, a product of glycolytic metabolism, inhibits histone deacetylase activity and promotes changes in gene expression. Nucleic Acids Res. 40, 4794–4803 (2012).
CAS
PubMed
PubMed Central
Google Scholar
Takata, N. et al. Lactate-dependent transcriptional regulation controls mammalian eye morphogenesis. Nat. Commun. 14, 4129 (2023).
CAS
PubMed
PubMed Central
Google Scholar
Heim, C. E. et al. Lactate production by Staphylococcus aureus biofilm inhibits HDAC11 to reprogramme the host immune response during persistent infection. Nat. Microbiol. 5, 1271–1284 (2020).
CAS
PubMed
PubMed Central
Google Scholar
Zhu, R. et al. ACSS2 acts as a lactyl-CoA synthetase and couples KAT2A to function as a lactyltransferase for histone lactylation and tumor immune evasion. Cell Metab. 37, 361–376 (2024). This study is the first to identify a mammalian lactyl-coenzyme A synthetase.
Google Scholar
Liu, R. et al. Nuclear GTPSCS functions as a lactyl-CoA synthetase to promote histone lactylation and gliomagenesis. Cell Metab. 37, 377–394 (2024).
PubMed
Google Scholar
Wang, S. et al. Lactate reprograms glioblastoma immunity through CBX3-regulated histone lactylation. J. Clin. Investig. 134, e176851 (2024).
CAS
PubMed
PubMed Central
Google Scholar
Huang, Z. W. et al. STAT5 promotes PD-L1 expression by facilitating histone lactylation to drive immunosuppression in acute myeloid leukemia. Signal. Transduct. Target. Ther. 8, 391 (2023).
CAS
PubMed
PubMed Central
Google Scholar
Li, W. et al. Tumor-derived lactate promotes resistance to bevacizumab treatment by facilitating autophagy enhancer protein RUBCNL expression through histone H3 lysine 18 lactylation (H3K18la) in colorectal cancer. Autophagy 20, 114–130 (2024).
CAS
PubMed
Google Scholar
He, Y. et al. Numb/Parkin-directed mitochondrial fitness governs cancer cell fate via metabolic regulation of histone lactylation. Cell Rep. 42, 112033 (2023).
CAS
PubMed
Google Scholar
Li, X. M. et al. Histone lactylation inhibits RARgamma expression in macrophages to promote colorectal tumorigenesis through activation of TRAF6-IL-6-STAT3 signaling. Cell Rep. 43, 113688 (2024).
CAS
PubMed
Google Scholar
Noe, J. T. et al. Lactate supports a metabolic–epigenetic link in macrophage polarization. Sci. Adv. 7, eabi8602 (2021).
CAS
PubMed
PubMed Central
Google Scholar
Ippolito, L. et al. Lactate rewires lipid metabolism and sustains a metabolic–epigenetic axis in prostate cancer. Cancer Res. 82, 1267–1282 (2022).
CAS
PubMed
PubMed Central
Google Scholar
Gao, X., Wang, H., Yang, J. J., Liu, X. & Liu, Z. R. Pyruvate kinase M2 regulates gene transcription by acting as a protein kinase. Mol. Cell 45, 598–609 (2012).
CAS
PubMed
PubMed Central
Google Scholar
Damasceno, L. E. A. et al. PKM2 promotes Th17 cell differentiation and autoimmune inflammation by fine-tuning STAT3 activation. J. Exp. Med. 217, e20190613 (2020).
CAS
PubMed
PubMed Central
Google Scholar
Angiari, S. et al. Pharmacological activation of pyruvate kinase M2 inhibits CD4(+) T cell pathogenicity and suppresses autoimmunity. Cell Metab. 31, 391–405.e8 (2020).
CAS
PubMed
PubMed Central
Google Scholar
Zhao, X. et al. Pyruvate kinase M2 interacts with nuclear sterol regulatory element-binding protein 1a and thereby activates lipogenesis and cell proliferation in hepatocellular carcinoma. J. Biol. Chem. 293, 6623–6634 (2018).
CAS
PubMed
PubMed Central
Google Scholar
Yu, P. et al. PKM2-c-Myc-survivin cascade regulates the cell proliferation, migration, and tamoxifen resistance in breast cancer. Front. Pharmacol. 11, 550469 (2020).
CAS
PubMed
PubMed Central
Google Scholar
Luo, W. et al. Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1. Cell 145, 732–744 (2011).
CAS
PubMed
PubMed Central
Google Scholar
Zheng, F. et al. The HIF-1alpha antisense long non-coding RNA drives a positive feedback loop of HIF-1alpha mediated transactivation and glycolysis. Nat. Commun. 12, 1341 (2021).
CAS
PubMed
PubMed Central
Google Scholar
Palsson-McDermott, E. M. et al. Pyruvate kinase M2 regulates Hif-1alpha activity and IL-1beta induction and is a critical determinant of the Warburg effect in LPS-activated macrophages. Cell Metab. 21, 347 (2015).
CAS
PubMed
Google Scholar
Yang, L. et al. PKM2 regulates the Warburg effect and promotes HMGB1 release in sepsis. Nat. Commun. 5, 4436 (2014).
CAS
PubMed
Google Scholar
Hamabe, A. et al. Role of pyruvate kinase M2 in transcriptional regulation leading to epithelial–mesenchymal transition. Proc. Natl Acad. Sci. USA 111, 15526–15531 (2014).
CAS
PubMed
PubMed Central
Google Scholar
Morfouace, M. et al. Control of glioma cell death and differentiation by PKM2–Oct4 interaction. Cell Death Dis. 5, e1036 (2014).
CAS
PubMed
PubMed Central
Google Scholar
Jin, X. et al. Pyruvate kinase M2 promotes the activation of dendritic cells by enhancing IL-12p35 expression. Cell Rep. 31, 107690 (2020).
CAS
PubMed
Google Scholar
Guo, D., Meng, Y., Jiang, X. & Lu, Z. Hexokinases in cancer and other pathologies. Cell Insight 2, 100077 (2023).
PubMed
PubMed Central
Google Scholar
Guo, D. et al. Aerobic glycolysis promotes tumor immune evasion by hexokinase2-mediated phosphorylation of IκBα. Cell Metab. 34, 1312–1324.e6 (2022). This study reveals that the Warburg effect can directly regulate tumour cell immune evasion through the protein kinase activity of hexokinase 2.
CAS
PubMed
Google Scholar
He, H., Xiao, L., Wang, J., Guo, D. & Lu, Z. Aerobic glycolysis promotes tumor immune evasion and tumor cell stemness through the noncanonical function of hexokinase 2. Cancer Commun. 43, 387–390 (2023).
Google Scholar
Thomas, G. E. et al. The metabolic enzyme hexokinase 2 localizes to the nucleus in AML and normal haematopoietic stem and progenitor cells to maintain stemness. Nat. Cell Biol. 24, 872–884 (2022).
CAS
PubMed
PubMed Central
Google Scholar
Dasgupta, S. et al. Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer. Nature 556, 249–254 (2018).
CAS
PubMed
PubMed Central
Google Scholar
Meng, J., Chen, X. & Han, Z. PFKFB4 promotes lung adenocarcinoma progression via phosphorylating and activating transcriptional coactivator SRC-2. BMC Pulm. Med. 21, 60 (2021).
CAS
PubMed
PubMed Central
Google Scholar
Xu, D. et al. The gluconeogenic enzyme PCK1 phosphorylates INSIG1/2 for lipogenesis. Nature 580, 530–535 (2020). This study reveals an integrated regulation of glucose metabolism and lipogenesis by the protein kinase activity of phosphoenolpyruvate carboxykinase 1.
CAS
PubMed
Google Scholar
Jiang, H., Zhu, L., Xu, D. & Lu, Z. A newly discovered role of metabolic enzyme PCK1 as a protein kinase to promote cancer lipogenesis. Cancer Commun. 40, 389–394 (2020).
Google Scholar
Shao, F. et al. Association of phosphoenolpyruvate carboxykinase 1 protein kinase activity-dependent sterol regulatory element-binding protein 1 activation with prognosis of oesophageal carcinoma. Eur. J. Cancer 142, 123–131 (2020).
PubMed
Google Scholar
Liao, K. et al. A feedback circuitry between polycomb signaling and fructose-1,6-bisphosphatase enables hepatic and renal tumorigenesis. Cancer Res. 80, 675–688 (2020).
CAS
PubMed
Google Scholar
Huangyang, P. et al. Fructose-1,6-bisphosphatase 2 inhibits sarcoma progression by restraining mitochondrial biogenesis. Cell Metab. 31, 174–188.e7 (2020).
CAS
PubMed
Google Scholar
De Rosa, V. et al. Glycolysis controls the induction of human regulatory T cells by modulating the expression of FOXP3 exon 2 splicing variants. Nat. Immunol. 16, 1174–1184 (2015).
PubMed
PubMed Central
Google Scholar
Liu, Q. et al. Glycogen accumulation and phase separation drives liver tumor initiation. Cell 184, 5559–5576.e19 (2021).
CAS
PubMed
Google Scholar
Zheng, L., Roeder, R. G. & Luo, Y. S phase activation of the histone H2B promoter by OCA-S, a coactivator complex that contains GAPDH as a key component. Cell 114, 255–266 (2003).
CAS
PubMed
Google Scholar
Gao, X. et al. NleB, a bacterial effector with glycosyltransferase activity, targets GAPDH function to inhibit NF-kappaB activation. Cell Host Microbe 13, 87–99 (2013).
CAS
PubMed
PubMed Central
Google Scholar
Mondragon, L. et al. GAPDH overexpression in the T cell lineage promotes angioimmunoblastic T cell lymphoma through an NF-kappaB-dependent mechanism. Cancer Cell 36, 268–287.e10 (2019).
CAS
PubMed
Google Scholar
Lee, J. H. et al. EGFR-phosphorylated platelet isoform of phosphofructokinase 1 promotes PI3K activation. Mol. Cell 70, 197–210.e7 (2018). This study reveals that phosphofructokinase 1, a rate-limiting glycolytic enzyme, functions as a signalling molecule essential for receptor tyrosine kinase-mediated PI3K–AKT activation.
PubMed
PubMed Central
Google Scholar
Lee, J. H. et al. Stabilization of phosphofructokinase 1 platelet isoform by AKT promotes tumorigenesis. Nat. Commun. 8, 949 (2017).
PubMed
PubMed Central
Google Scholar
Lee, J. H. et al. Phosphofructokinase 1 platelet isoform promotes beta-catenin transactivation for tumor development. Front. Oncol. 10, 211 (2020).
PubMed
PubMed Central
Google Scholar
Enzo, E. et al. Aerobic glycolysis tunes YAP/TAZ transcriptional activity. EMBO J. 34, 1349–1370 (2015).
CAS
PubMed
PubMed Central
Google Scholar
Gao, X. et al. Nuclear PFKP promotes CXCR4-dependent infiltration by T cell acute lymphoblastic leukemia. J. Clin. Invest. 131, e143119 (2021).
CAS
PubMed
PubMed Central
Google Scholar
Song, J. et al. Aldolase A accelerates cancer progression by modulating mRNA translation and protein biosynthesis via noncanonical mechanisms. Adv. Sci. 10, e2302425 (2023).
Google Scholar
Singh, R. & Green, M. R. Sequence-specific binding of transfer RNA by glyceraldehyde-3-phosphate dehydrogenase. Science 259, 365–368 (1993).
CAS
PubMed
Google Scholar
Ding, R. et al. Lactate modulates RNA splicing to promote CTLA-4 expression in tumor-infiltrating regulatory T cells. Immunity 57, 528–540 (2024).
CAS
PubMed
Google Scholar
Sun, L. et al. Lactylation of METTL16 promotes cuproptosis via m(6)A-modification on FDX1 mRNA in gastric cancer. Nat. Commun. 14, 6523 (2023).
CAS
PubMed
PubMed Central
Google Scholar
Sizemore, S. T. et al. Pyruvate kinase M2 regulates homologous recombination-mediated DNA double-strand break repair. Cell Res. 28, 1090–1102 (2018).
CAS
PubMed
PubMed Central
Google Scholar
Qu, J. et al. Phosphoglycerate mutase 1 regulates dNTP pool and promotes homologous recombination repair in cancer cells. J. Cell Biol. 216, 409–424 (2017).
CAS
PubMed
PubMed Central
Google Scholar
Gustafsson, N. M. S. et al. Targeting PFKFB3 radiosensitizes cancer cells and suppresses homologous recombination. Nat. Commun. 9, 3872 (2018).
PubMed
PubMed Central
Google Scholar
Shi, M. et al. GAPDH facilitates homologous recombination repair by stabilizing RAD51 in an HDAC1-dependent manner. EMBO Rep. 24, e56437 (2023).
CAS
PubMed
PubMed Central
Google Scholar
Ci, S. et al. Src-mediated phosphorylation of GAPDH regulates its nuclear localization and cellular response to DNA damage. FASEB J. 34, 10443–10461 (2020).
CAS
PubMed
Google Scholar
Sobanski, T. et al. The fructose-bisphosphate, Aldolase A (ALDOA), facilitates DNA-PKcs and ATM kinase activity to regulate DNA double-strand break repair. Sci. Rep. 13, 15171 (2023).
CAS
PubMed
PubMed Central
Google Scholar
Wu, S. et al. Pyruvate facilitates FACT-mediated gammaH2AX loading to chromatin and promotes the radiation resistance of glioblastoma. Adv. Sci. 9, e2104055 (2022).
Google Scholar
Qian, X. et al. Phosphoglycerate kinase 1 phosphorylates Beclin1 to induce autophagy. Mol. Cell 65, 917–931 (2017).
CAS
PubMed
PubMed Central
Google Scholar
Roberts, D. J., Tan-Sah, V. P., Ding, E. Y., Smith, J. M. & Miyamoto, S. Hexokinase-II positively regulates glucose starvation-induced autophagy through TORC1 inhibition. Mol. Cell 53, 521–533 (2014).
CAS
PubMed
PubMed Central
Google Scholar
Qian, X., Li, X. & Lu, Z. Protein kinase activity of the glycolytic enzyme PGK1 regulates autophagy to promote tumorigenesis. Autophagy 13, 1246–1247 (2017).
CAS
PubMed
PubMed Central
Google Scholar
He, C. L. et al. Pyruvate kinase M2 activates mTORC1 by phosphorylating AKT1S1. Sci. Rep. 6, 21524 (2016).
CAS
PubMed
PubMed Central
Google Scholar
Lee, M. N. et al. Glycolytic flux signals to mTOR through glyceraldehyde-3-phosphate dehydrogenase-mediated regulation of Rheb. Mol. Cell. Biol. 29, 3991–4001 (2009).
CAS
PubMed
PubMed Central
Google Scholar
Chang, C. et al. AMPK-dependent phosphorylation of GAPDH triggers Sirt1 activation and is necessary for autophagy upon glucose starvation. Mol. Cell 60, 930–940 (2015).
CAS
PubMed
Google Scholar
Iqbal, I. K., Bajeli, S., Sahu, S., Bhat, S. A. & Kumar, A. Hydrogen sulfide-induced GAPDH sulfhydration disrupts the CCAR2–SIRT1 interaction to initiate autophagy. Autophagy 17, 3511–3529 (2021).
CAS
PubMed
PubMed Central
Google Scholar
Huang, R. et al. Deacetylation of nuclear LC3 drives autophagy initiation under starvation. Mol. Cell 57, 456–466 (2015).
CAS
PubMed
Google Scholar
Li, M. et al. Fructose-1,6-bisphosphatase 1 dephosphorylates and inhibits TERT for tumor suppression. Nat. Chem. Biol. 20, 1505–1513 (2024). This study reveals that fructose-1,6-bisphosphatase 1 functions as a protein phosphatase, negatively regulating TERT and telomeres to govern immortality and ageing.
CAS
PubMed
Google Scholar
Zhu, W. et al. Fructose-1,6-bisphosphatase 1 dephosphorylates IκBα and suppresses colorectal tumorigenesis. Cell Res. 33, 245–257 (2023).
CAS
PubMed
PubMed Central
Google Scholar
Zhang, G., Tao, J., Lin, L., Qiu, W. & Lu, Z. Repurposing FBP1: dephosphorylating IκBα to suppress NFκB. Cell Res. 33, 419–420 (2023).
CAS
PubMed
PubMed Central
Google Scholar
Yang, J. S. et al. GAPDH inhibits intracellular pathways during starvation for cellular energy homeostasis. Nature 561, 263–267 (2018).
CAS
PubMed
PubMed Central
Google Scholar
Jacquin, M. A. et al. GAPDH binds to active Akt, leading to Bcl-xL increase and escape from caspase-independent cell death. Cell Death Differ. 20, 1043–1054 (2013).
CAS
PubMed
PubMed Central
Google Scholar
Jang, M. et al. Glyceraldehyde-3-phosphate, a glycolytic intermediate, plays a key role in controlling cell fate via inhibition of caspase activity. Mol. Cell 28, 559–563 (2009).
CAS
Google Scholar
Majewski, N. et al. Hexokinase–mitochondria interaction mediated by Akt is required to inhibit apoptosis in the presence or absence of Bax and Bak. Mol. Cell 16, 819–830 (2004).
CAS
PubMed
Google Scholar
Qi, H. et al. Succinylation-dependent mitochondrial translocation of PKM2 promotes cell survival in response to nutritional stress. Cell Death Dis. 10, 170 (2019).
PubMed
PubMed Central
Google Scholar
Liang, J. et al. Mitochondrial PKM2 regulates oxidative stress-induced apoptosis by stabilizing Bcl2. Cell Res. 27, 329–351 (2017).
CAS
PubMed
Google Scholar
Huang, C. et al. Lactate promotes resistance to glucose starvation via upregulation of Bcl-2 mediated by mTOR activation. Oncol. Rep. 33, 875–884 (2015).
CAS
PubMed
Google Scholar
Hsu, C. C. et al. Inositol serves as a natural inhibitor of mitochondrial fission by directly targeting AMPK. Mol. Cell 81, 3803–3819.e7 (2021).
CAS
PubMed
PubMed Central
Google Scholar
Meng, Y. et al. Glycolytic enzyme PFKL governs lipolysis by promoting lipid droplet-mitochondria tethering to enhance beta-oxidation and tumor cell proliferation. Nat. Metab. 6, 1092–1107 (2024). This study reveals that phosphofructokinase, liver-type coordinately regulates glycolysis and lipolysis through its protein kinase activity.
CAS
PubMed
Google Scholar
Chen, J. et al. PFKP alleviates glucose starvation-induced metabolic stress in lung cancer cells via AMPK-ACC2 dependent fatty acid oxidation. Cell Discov. 8, 52 (2022).
CAS
PubMed
PubMed Central
Google Scholar
Zhang, T. et al. G6PD maintains the VSMC synthetic phenotype and accelerates vascular neointimal hyperplasia by inhibiting the VDAC1-Bax-mediated mitochondrial apoptosis pathway. Cell Mol. Biol. Lett. 29, 47 (2024).
CAS
PubMed
PubMed Central
Google Scholar
Zhong, B. et al. Glucose-6-phosphate dehydrogenase neutralizes stresses by supporting reductive glutamine metabolism and AMPK activation. Signal. Transduct. Target. Ther. 6, 46 (2021).
CAS
PubMed
PubMed Central
Google Scholar
Li, X. et al. Nuclear PGK1 alleviates ADP-dependent inhibition of CDC7 to promote DNA replication. Mol. Cell 72, 650–660 (2018).
CAS
PubMed
Google Scholar
Jiang, Y. et al. PKM2 regulates chromosome segregation and mitosis progression of tumor cells. Mol. Cell 53, 75–87 (2014). This study reveals that pyruvate kinase M2 functions as a protein kinase and directly regulates mitosis progression.
CAS
PubMed
Google Scholar
Jiang, Y. et al. PKM2 phosphorylates MLC2 and regulates cytokinesis of tumour cells. Nat. Commun. 5, 5566 (2014).
CAS
PubMed
Google Scholar
Liu, W. et al. Lactate regulates cell cycle by remodelling the anaphase promoting complex. Nature 616, 790–797 (2023). This study reveals that lactate functions as an anaphase-promoting complex/cyclosome modulator to control mitotic exit.
CAS
PubMed
Google Scholar
Orozco, J. M. et al. Dihydroxyacetone phosphate signals glucose availability to mTORC1. Nat. Metab. 2, 893–901 (2020).
CAS
PubMed
PubMed Central
Google Scholar
Liu, J. et al. Metabolic enzyme LDHA activates Rac1 GTPase as a noncanonical mechanism to promote cancer. Nat. Metab. 4, 1830–1846 (2022).
CAS
PubMed
PubMed Central
Google Scholar
Qian, X. et al. PTEN suppresses glycolysis by dephosphorylating and inhibiting autophosphorylated PGK1. Mol. Cell 76, 516–527.e7 (2019).
CAS
PubMed
Google Scholar
Liu, F. et al. PKM2 methylation by CARM1 activates aerobic glycolysis to promote tumorigenesis. Nat. Cell Biol. 19, 1358–1370 (2017).
CAS
PubMed
PubMed Central
Google Scholar
Yang, Y. C. et al. Cytosolic PKM2 stabilizes mutant EGFR protein expression through regulating HSP90–EGFR association. Oncogene 36, 4234 (2017).
CAS
PubMed
Google Scholar
Keller, K. E., Doctor, Z. M., Dwyer, Z. W. & Lee, Y. S. SAICAR induces protein kinase activity of PKM2 that is necessary for sustained proliferative signaling of cancer cells. Mol. Cell 53, 700–709 (2014).
CAS
PubMed
PubMed Central
Google Scholar
Cheng, T. Y. et al. Pyruvate kinase M2 promotes pancreatic ductal adenocarcinoma invasion and metastasis through phosphorylation and stabilization of PAK2 protein. Oncogene 37, 1730–1742 (2018).
CAS
PubMed
Google Scholar
Zhang, D. et al. Phosphoglycerate mutase 1 promotes cancer cell migration independent of its metabolic activity. Oncogene 36, 2900–2909 (2017).
CAS
PubMed
Google Scholar
Blaha, C. S. et al. A non-catalytic scaffolding activity of hexokinase 2 contributes to EMT and metastasis. Nat. Commun. 13, 899 (2022).
CAS
PubMed
PubMed Central
Google Scholar
Li, M. et al. Aldolase B suppresses hepatocellular carcinogenesis by inhibiting G6PD and pentose phosphate pathways. Nat. Cancer 1, 735–747 (2020).
CAS
PubMed
Google Scholar
He, X. et al. Loss of hepatic aldolase B activates Akt and promotes hepatocellular carcinogenesis by destabilizing the Aldob/Akt/PP2A protein complex. PLoS Biol. 18, e3000803 (2020).
CAS
PubMed
PubMed Central
Google Scholar
Gu, L. et al. Fructose-1,6-bisphosphatase is a nonenzymatic safety valve that curtails AKT activation to prevent insulin hyperresponsiveness. Cell Metab. 35, 1009–1021.e1009 (2023).
CAS
PubMed
PubMed Central
Google Scholar
Wang, J. et al. A non-metabolic function of hexokinase 2 in small cell lung cancer: promotes cancer cell stemness by increasing USP11-mediated CD133 stability. Cancer Commun. 42, 1008–1027 (2022).
Google Scholar
Ma, Q. et al. The moonlighting function of glycolytic enzyme enolase-1 promotes choline phospholipid metabolism and tumor cell proliferation. Proc. Natl Acad. Sci. USA 120, e2209435120 (2023).
CAS
PubMed
PubMed Central
Google Scholar
Yang, W. & Lu, Z. Pyruvate kinase M2 at a glance. J. Cell Sci. 128, 1655–1660 (2015).
CAS
PubMed
PubMed Central
Google Scholar
Oslund, R. C. et al. Bisphosphoglycerate mutase controls serine pathway flux via 3-phosphoglycerate. Nat. Chem. Biol. 13, 1081–1087 (2017).
CAS
PubMed
PubMed Central
Google Scholar
Hitosugi, T. et al. Phosphoglycerate mutase 1 coordinates glycolysis and biosynthesis to promote tumor growth. Cancer Cell 22, 585–600 (2012).
CAS
PubMed
PubMed Central
Google Scholar
Cai, X. et al. Lactate activates the mitochondrial electron transport chain independently of its metabolism. Mol. Cell 83, 3904–3920.e7 (2023).
CAS
PubMed
PubMed Central
Google Scholar
Li, F. et al. FBP1 loss disrupts liver metabolism and promotes tumorigenesis through a hepatic stellate cell senescence secretome. Nat. Cell Biol. 22, 728–739 (2020).
PubMed
PubMed Central
Google Scholar
Dou, X. et al. PDK4-dependent hypercatabolism and lactate production of senescent cells promotes cancer malignancy. Nat. Metab. 5, 1887–1910 (2023).
CAS
PubMed
PubMed Central
Google Scholar
Wei, Y. et al. Pyruvate kinase type M2 promotes tumour cell exosome release via phosphorylating synaptosome-associated protein 23. Nat. Commun. 8, 14041 (2017).
CAS
PubMed
PubMed Central
Google Scholar
Hou, P. P. et al. Ectosomal PKM2 promotes HCC by inducing macrophage differentiation and remodeling the tumor microenvironment. Mol. Cell 78, 1192–1206.e10 (2020).
CAS
PubMed
Google Scholar
Funasaka, T., Hogan, V. & Raz, A. Phosphoglucose isomerase/autocrine motility factor mediates epithelial and mesenchymal phenotype conversions in breast cancer. Cancer Res. 69, 5349–5356 (2009).
CAS
PubMed
PubMed Central
Google Scholar
Han, J. et al. GPI is a prognostic biomarker and correlates with immune infiltrates in lung adenocarcinoma. Front. Oncol. 11, 752642 (2021).
CAS
PubMed
PubMed Central
Google Scholar
Tsutsumi, S., Hogan, V., Nabi, I. R. & Raz, A. Overexpression of the autocrine motility factor/phosphoglucose isomerase induces transformation and survival of NIH-3T3 fibroblasts. Cancer Res. 63, 242–249 (2003).
CAS
PubMed
Google Scholar
Lu, Z., Ghosh, S., Wang, Z. & Hunter, T. Downregulation of caveolin-1 function by EGF leads to the loss of E-cadherin, increased transcriptional activity of beta-catenin, and enhanced tumor cell invasion. Cancer Cell 4, 499–515 (2003).
CAS
PubMed
Google Scholar
Lay, A. J. et al. Phosphoglycerate kinase acts in tumour angiogenesis as a disulphide reductase. Nature 408, 869–873 (2000).
CAS
PubMed
Google Scholar
Colbert, L. E. et al. Tumor-resident Lactobacillus iners confer chemoradiation resistance through lactate-induced metabolic rewiring. Cancer Cell 41, 1945–1962.e11 (2023). This study reveals that tumour-resident probiotic bacterial species produce lactate, thereby regulating tumour metabolism and gene expression.
CAS
PubMed
PubMed Central
Google Scholar
Wolf, A. J. et al. Hexokinase is an innate immune receptor for the detection of bacterial peptidoglycan. Cell 166, 624–636 (2016).
CAS
PubMed
PubMed Central
Google Scholar
Jia, J. et al. Protection of extraribosomal RPL13a by GAPDH and dysregulation by S-nitrosylation. Mol. Cell 47, 656–663 (2012).
CAS
PubMed
PubMed Central
Google Scholar
Wang, X. et al. A GAPDH serotonylation system couples CD8(+) T cell glycolytic metabolism to antitumor immunity. Mol. Cell 84, 760–775.e7 (2024).
CAS
PubMed
Google Scholar
Khan, F. et al. Lactate dehydrogenase A regulates tumor-macrophage symbiosis to promote glioblastoma progression. Nat. Commun. 15, 1987 (2024).
CAS
PubMed
PubMed Central
Google Scholar
Terrasse, R. et al. Human and pneumococcal cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins are both ligands of human C1q protein. J. Biol. Chem. 287, 42620–42633 (2012).
CAS
PubMed
PubMed Central
Google Scholar
Chen, T. et al. NSUN2 is a glucose sensor suppressing cGAS/STING to maintain tumorigenesis and immunotherapy resistance. Cell Metab. 35, 1782–1798.e8 (2023).
CAS
PubMed
PubMed Central
Google Scholar
Huang, T. Y. et al. Phosphoenolpyruvate regulates the Th17 transcriptional program and inhibits autoimmunity. Cell Rep. 42, 112205 (2023).
CAS
PubMed
Google Scholar
Ho, P. C. et al. Phosphoenolpyruvate is a metabolic checkpoint of anti-tumor T cell responses. Cell 162, 1217–1228 (2015). This study reveals that phosphoenolpyruvate promotes the antitumour functions of CD4+ and CD8+ T cells by regulating calcium signalling and gene expression.
CAS
PubMed
PubMed Central
Google Scholar
Zhang, W. et al. Lactate is a natural suppressor of RLR signaling by targeting MAVS. Cell 178, 176–189.e15 (2019).
CAS
PubMed
PubMed Central
Google Scholar
Feng, T. et al. Adipocyte-derived lactate is a signalling metabolite that potentiates adipose macrophage inflammation via targeting PHD2. Nat. Commun. 13, 5208 (2022).
CAS
PubMed
PubMed Central
Google Scholar
Goswami, S., Zhang, Q., Celik, C. E., Reich, E. M. & Yilmaz, O. H. Dietary fat and lipid metabolism in the tumor microenvironment. Biochim. Biophys. Acta Rev. Cancer 1878, 188984 (2023).
CAS
PubMed
PubMed Central
Google Scholar
Lee, Y. S. et al. Increased adipocyte O2 consumption triggers HIF-1alpha, causing inflammation and insulin resistance in obesity. Cell 157, 1339–1352 (2014).
CAS
PubMed
PubMed Central
Google Scholar
Chen, Y. D., Varasteh, B. B. & Reaven, G. M. Plasma lactate concentration in obesity and type 2 diabetes. Diabete Metab. 19, 348–354 (1993).
CAS
PubMed
Google Scholar
Stine, Z. E., Schug, Z. T., Salvino, J. M. & Dang, C. V. Targeting cancer metabolism in the era of precision oncology. Nat. Rev. Drug Discov. 21, 141–162 (2022).
CAS
PubMed
Google Scholar
Mellinghoff, I. K. et al. Vorasidenib in IDH1- or IDH2-mutant low-grade glioma. N. Engl. J. Med. 389, 589–601 (2023).
CAS
PubMed
PubMed Central
Google Scholar
Li, H. et al. FBP1 regulates proliferation, metastasis, and chemoresistance by participating in C-MYC/STAT3 signaling axis in ovarian cancer. Oncogene 40, 5938–5949 (2021).
CAS
PubMed
PubMed Central
Google Scholar
Wenes, M. et al. The mitochondrial pyruvate carrier regulates memory T cell differentiation and antitumor function. Cell Metab. 34, 731–746.e9 (2022).
CAS
PubMed
PubMed Central
Google Scholar
Cao, Z. et al. Lactate oxidase nanocapsules boost T cell immunity and efficacy of cancer immunotherapy. Sci. Transl. Med. 15, eadd2712 (2023). This study presents a potential strategy to modulate lactate levels in the tumour microenvironment to enhance T cell function for more effective cancer immunotherapy.
CAS
PubMed
PubMed Central
Google Scholar
Download references
This study was supported by grants from the National Natural Science Foundation of China (82188102 and 82030074 to Z.L.; 82372816 to D.G.; 82203553 to Y.M.; 82002445 to G.Z.), the Ministry of Science and Technology of the People's Republic of China (2020YFA0803300 to Z.L.) and the National Center of Technology Innovation for Biopharmaceuticals (NCTIB2022HS02006 to Z.L.). Z.L. is the Kuancheng Wang Distinguished Chair.
These authors contributed equally: Dong Guo, Ying Meng, Gaoxiang Zhao.
Zhejiang Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, Zhejiang Key Laboratory of Frontier Medical Research on Cancer Metabolism, and Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China
Dong Guo, Ying Meng, Qingang Wu & Zhimin Lu
Institute of Fundamental and Transdisciplinary Research, Cancer Center, Zhejiang University, Hangzhou, China
Dong Guo, Ying Meng, Qingang Wu & Zhimin Lu
Department of Oncology, Cancer Institute of The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao Cancer Institute, Qingdao, China
Gaoxiang Zhao
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D.G., Y.M., G.Z. and Z.L. researched data for the article. D.G., Y.M. and G.Z. contributed substantially to the discussion of the content. Z.L. wrote the article. D.G., Y.M., Q.W. and Z.L. reviewed and/or edited the manuscript before submission.
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Zhimin Lu.
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Nature Reviews Cancer thanks Sara Sdelci and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Precipitation over East Asia and the western United States is projected to increase as a result of global warming, although substantial uncertainties persist regarding the magnitude. A key factor driving these uncertainties is the tropical surface warming pattern, yet the mechanisms behind both this warming pattern and the resulting regional precipitation changes remain elusive. Here we use a set of climate model experiments to argue that these changes are partly driven by global teleconnection from the Southern Ocean, which rapidly absorbs anthropogenic heat but releases it with a delay of decades to a century. We show that the delayed Southern Ocean warming contributes to broad tropical ocean warming with an El Niño-like pattern, enhancing precipitation during summer in East Asia and winter in the western United States. The atmospheric teleconnections from the tropical ocean link the Southern Ocean warming to the Northern Hemisphere regional wetting. Southern Hemisphere low clouds are a key regulator of this teleconnection, partly explaining the projected uncertainty of regional precipitation. The documented teleconnection has practical implications: even if climate mitigation reduces carbon dioxide levels, the delayed Southern Ocean warming will sustain a wetter East Asia and western United States for decades to centuries.
Effective adaptation to climate change hinges on accurate regional precipitation projections. This is particularly true for East Asia and the western United States—highly populated regions that experience substantial precipitation variability and associated socioeconomic impacts from flood and droughts1,2,3. State-of-the-art climate models, however, show a high uncertainty of future precipitation changes in these regions4. A factor contributing to this uncertainty is inter-model differences in sea surface temperature (SST) projections over the tropics5,6,7,8,9. For example, climate models projecting a stronger El Niño-like warming pattern—stronger warming in the eastern equatorial Pacific compared to the western part—tend to exhibit a more pronounced precipitation increase in summer over East Asia10 and winter over the western United States11.
However, the underlying drivers of projection uncertainty in tropical SSTs, and thus regional precipitation, remain elusive. A key insight emerges from the common slow timescale shared by the tropical Pacific SST and regional precipitation responses. When CO2 concentration is abruptly quadrupled in climate models, the eastern equatorial Pacific warming is initially muted but gradually evolves to an El Niño-like warming pattern12 (Extended Data Fig. 1d). The regional precipitation over East Asia and the western United States also slowly increases following the slow timescale of the El Niño-like warming13 (Extended Data Fig. 1e,f). Notably, the slowly emerging processes are the primary contributors to the multi-model mean and inter-model spread of the long-term responses (Extended Data Fig. 1g–i). This raises a critical question: what process in the slow timescale governs the uncertainty of the tropical Pacific warming and, consequently, the regional precipitation enhancements?
Building on these insights, we hypothesize that delayed warming of the Southern Ocean (SO) is a key slow process. Initially, ocean circulation over the SO mitigates SST warming by absorbing heat into the deep ocean. This ocean heat uptake gradually slows, and manifests as the delayed SO warming14. Meanwhile, the anomalous SO warming causes the tropical Pacific to warm, particularly in the eastern basin, inducing the El Niño-like warming pattern, as revealed by recent idealized model experiments15,16,17. In this Article, through a series of climate model experiments, we demonstrate that tropical ocean warming, including with an El Niño-like pattern, is partly attributable to the delayed SO warming. This SO warming, in turn, increases precipitation during summer in East Asia and winter in the western United States, through teleconnections from the tropical SST changes. We show that Southern Hemisphere low cloud feedback modulates the entire teleconnection from the SO, partially explaining the inter-model spread of the regional precipitation projections over East Asia and the western Unites States.
The teleconnection from the delayed SO warming emerges in the slow response. In this study, we define the slow response as the difference between the last and first 30 years of the 150-year CMIP6 abrupt-4xCO2 simulation, in which CO2 is abruptly quadrupled from pre-industrial levels18 (Methods). In this section we will focus on the remote impact the delayed SO warming has on the tropical SSTs, before investigating the impact on regional precipitation.
With abrupt CO2 increases, the SO initially absorbs heat from the atmosphere, but gradually releases it around 45° S (Fig. 1a). Consequently, in the CMIP6 slow response, the SST warming is pronounced between 60° S and 40° S, indicating delayed SO warming (Fig. 1b). Meanwhile, the tropical Pacific warms in a triangular shape from the South Pacific (Fig. 1b, purple triangle), corresponding to El Niño-like warming (Supplementary Fig. 1). To isolate the effect of the delayed SO warming, we conducted a numerical experiment with the CESM1-CAM4 fully coupled model, wherein a regional thermal forcing is applied between 40° S and 60° S (Methods). The prescribed SO heating induces a response that resembles the CMIP6 slow response (compare Fig. 1c and 1b), with a high pattern correlation, 0.76 over the tropical Pacific between 30° S and 30° N and 120° E and 300° E. In other words, an anomalous zonally uniform heating to the SO drives global-scale teleconnections, including a warming of the eastern Pacific. Therefore, the delayed SO warming partially contributes to the tropical Pacific SST pattern of the slow response.
a, Muti-model and zonal mean ocean heat uptake (OHU) response of abrupt-4xCO2 relative to piControl, shown for 15-year periods. OHU corresponds to a downward positive net surface heat flux. b,c, Annual mean SST anomaly from the multi-model mean CMIP6 slow response to CO2 quadrupling (b) and the CESM1-CAM4 SO warming experiment (c) (Methods). Note that the SST averaged over the tropics between 20° S and 20° N is used as the centre of the colour scale to emphasize the tropical SST pattern. Stippling in b indicates where >70% of 35 models agree on the sign of the pattern change. Unhatched shading in c indicates statistically significant signals at the 95% confidence using a t-test, compared to the inter-annual variability of the tropical SST pattern in the control climate.
Previous studies have revealed the physical mechanisms by which zonally uniform SO heating induces El Niño-like warming over the tropical Pacific15,16,19,20,21. Specifically, SO warming is advected equatorward by the climatological southeasterlies west of South America15. The warm anomalies weaken these winds, leading to a reduction in evaporative cooling and coastal upwelling, which further amplifies the warming response15,16,22. The resulting Southeast Pacific SST warming reduces low cloud cover west of South America, and this positive low cloud feedback is the key that mediates the SO-driven teleconnection15,16. Once the warming reaches the eastern equatorial Pacific, the El Niño-like warming pattern is sustained by Bjerknes feedback.
The SO warming also propagates towards the tropical Indian Ocean and the tropical Atlantic Ocean (Fig. 1b,c). The west of Australia and South Africa exhibit climate states similar to those west of South America, with prevalent southeasterlies and stratocumulus clouds, thus exhibiting a similar teleconnection mechanism15. However, over the Indian Ocean, tropical warming is not evident in the SO warming experiment compared to the CMIP6 slow response. We suspect that this muted teleconnection towards the tropical Indian Ocean is due to the erroneously negative low cloud feedback west of Australia in CESM1-CAM4 (Supplementary Fig. 2 and Methods). Regardless of such tropical pattern differences, the delayed SO warming partially contributes to the enhanced equatorial warming—the stronger SST warming near the equator compared to the tropical ocean7. The enhanced equatorial warming is dominantly shaped by the tropical Pacific heating (Fig. 1b,c and Supplementary Fig. 1).
The delayed SO warming further contributes to the regional precipitation increases in the Northern Hemisphere, via tropical SST anomalies and subsequent atmospheric teleconnections. In this section we first focus on the precipitation over East Asia (25° N–40° N and 110° E–145° E) during the boreal summer months of June, July and August (JJA). This geographic domain includes a densely populated coastal region subject to flood risks, as well as portions of ocean, to capture the large-scale precipitation signal.
The East Asia summer precipitation increases in both the CMIP6 slow response and the response to the SO warming alone (Fig. 2a,b, green boxes). Thus, the slow increase in the East Asia summer precipitation can be partly attributed to the delayed SO warming. The atmospheric teleconnection occurring in the summer following El Niño in the winter illustrates the mechanism, which relies on enhanced equatorial warming increasing the East Asia summer precipitation by shifting the Asian jet southward6,7,8,23.
a,b, Precipitation (shading) and 200-hPa zonal wind (contours) anomalies from the multi-model mean CMIP6 slow response (a) and the CESM1-CAM4 SO warming experiment (b). The solid (dashed) grey contours represent positive (negative) anomalies (interval = 0.8 m s−1), where the thick line specifies zero. c–f, The multi-model mean CMIP6 slow response related to the southward Asian jet shift. c, Response in the vertical temperature profile averaged over the Asian sector (60° E–120° E). d, Response (shading) and climatology (contours; interval = 6 m s−1) of the vertical profile of the zonal wind (U) over the Asian sector (60° E–120° E), with the solid (dashed) contours indicating westerly (easterly) winds. e, Vertical profile of the meridional wind (V) response (110° E–130° E). f, 500-hPa zonal wind changes (shading) and 500-hPa temperature climatology (contours; interval = 2 K from 265 K). The target East Asia domain is indicated by the green box in a, b and f, and the thick green line in e indicates the latitudes of the target East Asia domain. The thick green lines in f denote longitudes east of the Tibetan Plateau corresponding to e. Stippled and unhatched regions indicate statistical significance as in Fig. 1b,c. All variables are averaged for JJA.
First, the enhanced equatorial warming, driven by the delayed SO warming, leads to tropical upper-level warming following the moist adiabat (Fig. 2c). The resultant southward shift in the maximum meridional temperature gradient leads to the southward displacement of the Asian summer jet23 (Fig. 2d). As the Asian jet shifts southward, the atmospheric column it passes through becomes shallower due to the elevated topography (Fig. 2d). Potential vorticity conservation implies a reduction in relative vorticity24, and the subsequent anticyclonic flow triggered along the topography entails a northerly wind downstream of the Tibetan Plateau8 (Fig. 2e). Northerly anomalies result in moisture convergence within East Asia and thereby lead to the precipitation increase6. In addition, the southward-shifted jet favours warm advection to East Asia in the mid-troposphere, as after the jet shift, the westerlies blow from the climatologically warmer regions over the Tibetan Plateau25 (Fig. 2f). To balance the mid-tropospheric energy budget, the enhanced warm advection requires compensatory adiabatic cooling by anomalous upward motion (Supplementary Fig. 3). The anomalous upward motion enhances East Asia precipitation26.
Indeed, the East Asia summer moistening is tightly linked to the southward jet shift across models, which is associated with the different degree of enhanced equatorial warming, hence the tropical Pacific warming (Extended Data Fig. 2 and Supplementary Fig. 1). The aforementioned dynamics in the CMIP6 slow response can be consistently identified in the SO warming experiment (compare Fig. 2c–f to Extended Data Fig. 3). In other words, the delayed SO warming causes the enhanced equatorial warming, shifting the Asian jet southward, resulting in the anomalous northerly and mid-tropospheric warm advection east of the Tibetan Plateau, thereby increasing the East Asia precipitation.
Although dynamical mechanisms related to jet shift are consistent between the CMIP6 slow response and the SO warming experiment, the precipitation decreases south of East Asia only in the CMIP6 slow response (Fig. 2a,b). We attribute this difference to the muted warming over the tropical Indian Ocean in CESM1-CAM4 (Fig. 1b,c). In previous studies, the equatorial warming over the Indian Ocean relative to the western Pacific is shown to trigger a subtropical high-pressure anomaly over the western North Pacific9,10. The resulting anticyclonic flow enhances East Asia precipitation by supplying additional moisture, and the descent motion associated with the anticyclone decreases the precipitation south of the East Asia9,27. Indeed, the western North Pacific subtropical high-pressure anomaly is developed only in the CMIP6 slow response (Extended Data Fig. 4a,b). Similarly, the teleconnection mechanisms related to the subtropical high-pressure anomaly exist in the slow response but can be negligible in models in which the relative Indian Ocean warming is muted (Extended Data Fig. 4e–g).
We now examine the slow CO2-forced precipitation response in the western United States (30° N–45° N, 215° E–245° E) during the boreal winter months (December, January, February, DJF). Note that the selected region contains the highly populated coastal cities. The western United States precipitation increases in the CMIP6 slow response as well as in the SO warming experiment (Fig. 3a,b, green rectangles). Hence, the slow increase in the western United States winter precipitation is partly attributed to the delayed SO warming.
a,b, Precipitation (shading) and 200-hPa geopotential height pattern (contours) anomalies from the multi-model mean CMIP6 slow response (a) and the CESM1-CAM4 SO warming experiment (b). The geopotential height pattern is calculated by subtracting the Northern Hemisphere average (0°–40° N) from the original value (Methods). The solid (dashed) grey contours represent negative (positive) anomalies (interval = 10 m). c,d, The multi-model mean CMIP6 slow response related to the Pacific/North America pattern. c, The 850-hPa geopotential height pattern (shading) and 850-hPa horizontal wind (vectors). The vectors are shown in the region where >70% of 35 models agree on the sign of either the zonal or meridional wind changes. d, The changes (shading) and climatology (contours) of the 200-hPa zonal wind. Only positive contours larger than 12 m s−1 are shown; the contour interval is 8 m s−1. The green rectangle (parallelogram) corresponds to regions of western United States (southeastern United States) precipitation enhancement. Stippled and unhatched regions indicate statistical significance as in Fig. 1a,b. All variables are averaged for DJF.
The wetting in the western United States can be induced by an El Niño-like teleconnection, starting from the delayed SO warming. Previous research has revealed that tropical Pacific warming induces low geopotential height anomalies reminiscent of the Pacific/North America (PNA) pattern via Rossby wave responses, thereby enhancing winter precipitation over the western United States5,11. Indeed, in response to the delayed SO warming, the 200-hPa geopotential height anomalies exhibit minima over the northeastern Pacific (Fig. 3a,b, grey contours), which resemble the responses in a typical El Niño year28. The resulting low height anomalies generate southwesterly winds directed towards the western United States (Fig. 3c), which in turn supply moisture and enhance precipitation. In addition, the low height anomalies in the northeastern Pacific correspond to the eastward extension of the Pacific jet, which would otherwise be concentrated in the western Pacific (Fig. 3d). Given that climatological storm track activity decreases from the Pacific to the western United States29, this eastward jet extension steers more storms towards the western United States, thereby enhancing precipitation30.
Accordingly, models with stronger tropical Pacific warmings tend to have lower height anomalies, modulating the western United States precipitation response among models (Extended Data Fig. 5a–c). In addition, the dynamic mechanisms behind the western United States moistening are similar in the CMIP6 slow response and the SO warming experiment (compare Fig. 3c,d to Extended Data Fig. 6a,b). Therefore, the delayed SO warming induces a tropical Pacific warming, triggering a low geopotential height anomaly over the northeastern Pacific, increasing moisture supply and steering more storms to the western United States, thereby intensifying precipitation there.
The slow precipitation increase in the southeastern United States is also partly explained by the SO-driven teleconnection. Precipitation during winter increases with the warming from the SO (Fig. 3a,b, green parallelograms). The tropical Pacific warming induces low height anomalies over the southeastern United States (Fig. 3a,b, grey contours), thereby increasing the rainfall in the southeastern United States as in El Niño years31. This El Niño-like teleconnection is further confirmed by the inter-model correlation between the tropical Pacific warming, low height anomaly and southeastern United States precipitation increase (Extended Data Fig. 5d–f).
Likewise, the SO-driven teleconnections affect not only the tropical SSTs but also regional precipitation over East Asia, the western United States and the southeastern United States. Previous studies have identified Southern Hemisphere low cloud feedback as a key determinant of the teleconnection from the SO to the tropics15,16. Here we examine whether variations in low cloud feedback can explain the inter-model differences not only in tropical SST anomalies, but also in regional precipitation changes.
We first quantified the Southern Hemisphere low cloud feedback (CFSH) as the sensitivity of the shortwave (SW) cloud radiative effect to underlying SSTs averaged over Southern Hemisphere regions with climatological low clouds32, indicated by black boxes in Fig. 4a (Methods and Supplementary Fig. 4). We then regressed the CMIP6 slow response against CFSH across different climate models (Fig. 4a–c). The models with a stronger CFSH exhibit more warming in the SO and tropical oceans. To be specific, the triangular warming over the South Pacific becomes stronger, with a significant inter-model correlation of 0.74 (Fig. 4g). Furthermore, the models with a larger CFSH tend to have a more pronounced increase in precipitation over East Asia and the western United States (Fig. 4b,c), with significant correlation coefficients (Fig. 4h,i). Therefore, the Southern Hemisphere low cloud feedback may amplify the SO-driven teleconnection, regulating the slow response of the tropical SST pattern as well as regional precipitation over the Northern Hemisphere.
a–c, Regression across models of the CMIP6 slow response onto CFSH for the annual mean tropical SST pattern (a), precipitation (shading) and 200-hPa zonal wind (contours; interval = 0.8 m s−1) during summer (JJA; b) and precipitation (shading) and 200-hPa geopotential height pattern (contours; interval = 10 m) during winter (DJF; c). d–f, Same variables as in a–c but for the CFSH-induced responses estimated with regional cloud-locking experiments (Methods). Unhatched shading indicates statistically significant signals at the 95% confidence level using a t-test. g–i, CFSH versus the CMIP6 slow responses for the tropical Pacific triangular warming (g); precipitation during the East Asian (EA) summer (h) and western United States (WUS) winter (i). Annotations indicate inter-model correlation coefficients. The symbols are colour-coded from blue to red in ascending order of the CFSH. The black symbols indicate the multi-model mean, and open (filled) green rectangles indicate the CESM1-CAM5 experiment in which the clouds are regionally locked (globally interactive). In i, the western United States winter is defined as November, December, January for the CMIP6 slow response and as January, February, March for the cloud-locking experiment, when each signal is maximized.
We further quantified the role of CFSH by means of a regional cloud-locking experiment. The abrupt-4xCO2 simulation with the default CESM1-CAM5 was compared with a simulation in which the cloud radiative feedbacks were disabled regionally over the climatological low cloud regions in the Southern Hemisphere (Methods and Supplementary Fig. 5). The differences in the slow response between the cloud-interactive and regional cloud-locked configurations represent the impact of CFSH (Fig. 4d–f). The tropical Pacific warming and the regional precipitation increases were amplified with interactive CFSH relative to the cloud-locked experiments (Fig. 4g–i, green rectangles). Therefore, it is evident that CFSH regulates the global teleconnections from the SO in the models, as suggested by the similarity between the regional cloud-locking experiment and the inter-model regression map (Fig. 4a–f).
The atmospheric teleconnections discussed in previous sections propagate the uncertainty of the diverging CFSH among models to regional precipitation change. For the East Asia precipitation increase, the southward jet shift and associated interaction with the Tibetan Plateau (Fig. 2c–f) explain the model uncertainty associated with CFSH, shown by the consistent signals both in the inter-model regression (Extended Data Fig. 7) and the regional cloud-locking experiment (Extended Data Fig. 8). The stronger CFSH also favours a stronger high-pressure anomaly over the western North Pacific subtropical regions (Extended Data Fig. 4c,d), explaining the reduced precipitation south of East Asia (Fig. 4b,e). For the western United States and southeastern United States precipitation enhancement, the Rossby wave responses and subsequent PNA pattern (Fig. 3c,d) are also amplified with a stronger CFSH (Extended Data Fig. 6c–f).
Our analysis has concentrated on the slow climate response to abrupt CO2 quadrupling, specifically the gradual evolution of tropical Pacific warming and related changes in regional precipitation. In fact, the Southern Hemisphere low cloud feedback explains the projection uncertainties not only for the slow responses, but also for the long-term responses (Extended Data Fig. 9). The inter-model correlation is dominated by four CESM2 variant models, but the cloud-locking experiment aligns with the inter-model relationship (compare the black regression lines and green dashed lines in Extended Data Fig. 9). Given that the cloud-locking experiment provides actual causal evidence, whereas the inter-model correlation does not, we argue that the CFSH is partly responsible for the inter-model spread, even in the non-robust inter-model correlation. Thus, under the scenario with CO2 enhancements, the misrepresentation of low clouds in the Southern Hemisphere will contribute to the projection uncertainties of tropical SSTs and regional precipitation changes over East Asia and the southeastern United States.
Here we reveal a teleconnection in which the delayed SO warming expected under anthropogenic climate change contributes to enhanced equatorial warming with an El Niño-like pattern, thereby enhancing regional precipitation over East Asia, the western Unites States and the southeastern United States, as summarized in Fig. 5. The Southern Hemisphere low cloud feedback regulates the strength of the SO-driven teleconnection and partly explains the inter-model uncertainties of the regional precipitation projections. In fact, the highly model-dependent feedback from the Southern Hemisphere low clouds has been identified as a major source of uncertainty in global mean temperature increase and thus climate sensitivity33,34. Therefore, recent field campaigns focusing on the Southern Hemisphere low cloud35,36 will prove valuable to improve not only climate sensitivity estimates but also regional precipitation projections.
The delayed SO warming propagates equatorward, preferentially west of the continents, following the climatological southeasterlies. This equatorward teleconnection is further promoted by positive low cloud feedback. As the warming signal reaches the equator, it is further amplified by Bjerknes feedback, leading to the El Niño-like warming pattern. In summer, the enhanced equatorial warming heats the tropical troposphere following the moist adiabat, shifting the Asian jet southward and intensifying precipitation over East Asia (EA) due to strengthened interactions between the jet and the Tibetan Plateau. In winter, the El Niño-like warming induces the Rossby wave responses, producing a PNA circulation pattern with corresponding low (L) and high (H) pressure anomalies, which in turn enhances precipitation across the western United States (WUS) and southeastern United States (SEUS). The multi-model mean of the slow response in abrupt-4xCO2 and piControl climatology across the CMIP6 ensemble is used for the schematic.
In response to global warming, the slow teleconnections we describe here occur on centennial timescales as the SO slowly absorbs and releases heat, implying less impact on the near-future transient climate. However, the teleconnection impact will be more evident as humans reduce greenhouse gases (GHGs)—SO warming will persist while other regions will cool or equilibrate faster due to the differing heat capacities37. Indeed, in recent climate model experiments in which CO2 is removed after transient quadrupling38, SO warming becomes pronounced when CO2 is removed (Fig. 6a–d). The SO warming sustains the enhanced warming over the tropical oceans, inducing dynamical changes identical to this study, thereby sustaining a precipitation increase over East Asia, the western United States and the southeastern United States (Fig. 6e–h). The sustained warming and wetting are consistent with recent studies with similar model experiments27,39. Eventually, long-term adaptation policies need to reflect these regional climate changes induced by SO warming, which will remain even with CO2 reductions.
a, Lists of CMIP6 models participating in the Carbon Dioxide Removal Model Intercomparison Project (CDRMIP)38. b, Transient CO2 quadrupling and subsequent reduction used as the forcing in CDRMIP. The long-term persisting climate changes are measured by the difference between years 191–230 and 51–90 when CO2 concentrations are identical. c,d, SST responses in summer (c) and winter (d). e, Vertical profile of zonal wind changes in summer (shading), with the corresponding climatology (contours; interval = 6 m s−1). f, Responses in the 200-hPa geopotential height pattern during winter. g,h, Precipitation responses in summer (g) and winter (h). Stippling indicates where >6 of 8 models agree on the sign of the change. Green boxes are regions with precipitation enhancements investigated in this study.
Generally, there is growing evidence indicating that the SO is a global climate pacemaker in recent trends, producing remote impacts such as those highlighted in this study40,41,42. Specifically, in the process of developing decadal climate predictions, higher spatial resolution has been shown to improve the prediction skill of surface temperature over the SO. This SO skill improvement extends to a better hindcast of the tropical Pacific SST as well as the western United States and southeastern United States precipitation during winter42. Therefore, an accurate representation of recent SO cooling trends may play a crucial role in resolving model–observation discrepancies of the recent tropical Pacific cooling as well as the western United States drying. The mechanisms revealed in this study lend further support to this hypothesis. These cumulative findings could help alleviate model errors in simulating recent regional precipitation trends and enable more trustworthy future projections.
We use monthly mean outputs of two fully coupled CMIP618 experiments: piControl and abrupt-4xCO2. piControl mimics the climate with a pre-industrial CO2 concentration (280 ppm), and abrupt-4xCO2 simulates the climate with quadrupled CO2. We calculated the total response to CO2 quadrupling as the difference between the last 30 years of abrupt-4xCO2 and the last 100 years of piControl. We calculated the slow response as the last minus first 30 years of abrupt-4xCO2, in which the effects of ocean dynamics and slowly evolving SSTs dominate. Although previous studies used the first ten years to capture the slow evolution of the SST, here we use the first 30 years to minimize the high internal variability in regional precipitation. This definition of the slow response clearly characterizes the delayed SO warming and associated global impacts (Supplementary Fig. 6). All CMIP6 data are interpolated to 1° × 1° horizontal resolution, and one ensemble per model was used for the analysis. A total of 35 models were selected based on their data availability (model lists are provided in Supplementary Table 1).
To confirm the SO-driven teleconnection impact, a SO warming experiment was conducted using the fully coupled CESM1-CAM443. We first equilibrate to the pre-industrial climate and then added the SO warming to run a forced simulation for 150 years. The target warming was added to the longwave heat flux term in the ocean coupler code. We added a sinusoidal heat flux between 40° S and 60° S, with a maximum of 20 W m−2. The averaged difference between the last 50 years of forced climate and last 100 years of pre-industrial climate was analysed. The imposed heat flux was eventually released towards the atmosphere (dashed–dotted blue line in Supplementary Fig. 6), affecting the global climate through teleconnections. Note that the tropical Pacific response in this experiment is weaker than in the CMIP6 slow response, even if the forcing magnitude is larger (Fig. 1b,c). The weaker response can be attributed to the weak positive low cloud feedback in CESM1-CAM4, which might dampen the cloud radiative anomalies and thus the teleconnection in this particular model (Supplementary Fig. 2). To be specific, the negative low cloud feedback west of Australia might hinder the equatorward propagation of the SO warming in CESM1-CAM4, muting the tropical Indian Ocean warming (Fig. 1c). Note that the SST response in the North Atlantic and North Pacific in the SO warming experiment differs from the CMIP6 slow response. In the SO warming experiment, the stronger North Atlantic warming and displacement of the North Pacific cooling maxima to the northwest modulate the jet streams, which could result in weaker precipitation signals over East Asia and the western United States. However, the overall teleconnection impacts from the tropical SST anomalies are consistent.
In this study we aim to find the cause of the inter-model spread in the SW cloud radiative effect (SWCRE) responses over the Southern Hemisphere low cloud regions. We decompose the SWCRE response into the product of the forced SST response and the SWCRE sensitivity to SST, a relationship that holds, particularly for low cloud regions33. The SWCRE sensitivity at each grid point is the estimated strength of the local SW cloud feedback, which will be intrinsic to a model's parameterizations.
We calculated the local SW cloud feedback strength by regressing the monthly SWCRE at the top of atmosphere (TOA) onto the monthly SST at each model grid point, using the de-seasonalized and de-trended deviations of 100-year piControl simulations. The local SW cloud feedback was then averaged over the Southern Hemisphere low cloud regions, where the inter-model correlation between the SWCRE changes and the tropical Pacific triangular warming is large (Supplementary Fig. 4a,b, green and blue boxes). The averaged feedback is what we refer to as the Southern Hemisphere low cloud feedback (CFSH). This low cloud feedback can explain the inter-model spread of SWCRE changes both in the subtropical and SO domains, even without considering SST changes (Supplementary Fig. 4d,e). This indicates that the difference in low cloud parameterization is the dominant cause of the inter-model spread in the SWCRE responses. We estimated the observational CFSH with the same method, using the CERES-EBAF44 for SWCRE and OISSTv545 for SST from March 2000 to February 2020.
Here, the Southern Hemisphere low cloud includes the cloud over the subtropics and SO, and the subtropical and SO cloud feedbacks are correlated among climate models (r = 0.63; Supplementary Fig. 4c). In addition, note that we directly link the low cloud and SWCRE changes to the SST. In previous studies using cloud-controlling factor analysis, estimated inversion strength is another important factor for explaining SWCRE changes33,46,47. However, for the Southern Hemisphere low cloud regions, the anticorrelation between the time series of SST and estimated inversion strength is strong, so the univariate regression with SST is sufficient to capture the inter-model spread of the SWCRE responses.
To elucidate the role of CFSH, we conducted regional cloud-locking experiments48 using the fully coupled CESM1-CAM549. First, the eight cloud parameters were extracted every 2 h from a randomly chosen year of the equilibrated pre-industrial simulation. Then, in the regional cloud-locking experiments, the 2-h cloud parameters were prescribed repetitively every year in the radiative transfer code for the target region. The target regions we focused on were the SO and off the west coast of all major Southern Hemisphere continents where CFSH is defined. One abrupt-4xCO2 simulation was integrated for 150 years with the interactive clouds and locked clouds. The effect was that the SW cloud radiative forcing in the interactive cloud experiment was muted in the regionally locked experiment (Supplementary Fig. 5). The difference between the interactive and locked cloud experiments demonstrates the role of CFSH. The slow response was used for the analysis as in CMIP. Note that the CESM1-CAM5 has relatively realistic SW cloud feedback strength over the SH low cloud regions when compared to other models (Supplementary Fig. 2d), confirming the fidelity of the cloud-locking experiment.
The tropical SST pattern was calculated as the SST from which the tropical (20° S–20° N) mean has been subtracted. For the 200-hPa and 850-hPa geopotential height patterns, the deviation from the Northern Hemisphere (0°–40° N) average value was used to capture the circulation responses under global warming, following a previous study50.
The shift of the Asian summer jet was quantified as the change in meridional asymmetry of the 200-hPa zonal wind as follows. First, we took the 200-hPa zonal wind during the summer (JJA) over the Asian continent (60° E–120° E). Next, we calculated the climatological Asian jet position as the latitude of the maximum zonally averaged zonal wind. To assess the meridional asymmetry of the zonal wind (U200asy), the meridionally averaged zonal wind between the climatological jet position and 20° south of the jet was subtracted from that between the climatological jet and 20° north of the jet. Negative changes in U200asy denote a southward shift in the Asian summer jet.
CMIP6 data are available from the ESGF data portals (https://esgf-node.llnl.gov/projects/esgf-llnl/). The post-processed CESM1-CAM4 Southern Ocean warming experiment and CESM1-CAM5 regional cloud-locking experiment are available at https://doi.org/10.5281/zenodo.14797112. The observational SST (OISST v2) can be downloaded from the Physical Sciences Laboratory website (https://psl.noaa.gov/data/gridded/data.noaa.oisst.v2.html). The CERES-EBAF v4.1 TOA radiation data are available from https://ceres.larc.nasa.gov/data/. The post-processed data for reproducing figures are available at https://doi.org/10.5281/zenodo.14797112.
The raw data were first averaged by NetCDF Operator (NCO; https://nco.sourceforge.net/). All calculations, analysis and visualizations were then carried out using MATLAB. The MATLAB codes are available at https://doi.org/10.5281/zenodo.14797112.
Zhang, W. et al. Increasing precipitation variability on daily-to-multiyear time scales in a warmer world. Sci. Adv. 7, eabf8021 (2021).
Article
Google Scholar
Donatti, C. I. et al. Global hotspots of climate-related disasters. Int. J. Disaster Risk Reduct. 108, 104488 (2024).
Article
Google Scholar
Howitt, R., MacEwan, D., Medellín-Azuara, J., Lund, J. & Sumner, D. Economic Analysis of the 2015 Drought for California Agriculture (UC Davis Center for Watershed Sciences, ERA Economics, UC Agricultural Issues Center, 2015).
Douville, H. et al. in Climate Change 2021: The Physical Science Basis (eds Masson-Delmotte, V. et al.) Ch. 8 (IPCC, Cambridge Univ. Press, 2023).
Allen, R. J. & Luptowitz, R. El Niño-like teleconnection increases California precipitation in response to warming. Nat. Commun. 8, 16055 (2017).
Article
CAS
Google Scholar
Chiang, J. C. H., Fischer, J., Kong, W. & Herman, M. J. Intensification of the Pre-Meiyu Rainband in the Late 21st Century. Geophys. Res. Lett. 46, 7536–7545 (2019).
Article
Google Scholar
Zhou, W., Xie, S.-P. & Yang, D. Enhanced equatorial warming causes deep-tropical contraction and subtropical monsoon shift. Nat. Clim. Change 9, 834–839 (2019).
Kong, W. & Chiang, J. C. H. Southward shift of westerlies intensifies the East Asian early summer rainband following El Niño. Geophys. Res. Lett. 47, e2020GL088631 (2020).
Article
Google Scholar
Xie, S.-P. et al. Indian Ocean capacitor effect on Indo-western Pacific climate during the summer following El Niño. J. Clim. 22, 730–747 (2009).
Article
Google Scholar
He, C. & Zhou, T. Responses of the western North Pacific subtropical high to global warming under RCP4.5 and RCP8.5 scenarios projected by 33 CMIP5 models: the dominance of tropical Indian Ocean-tropical western Pacific SST gradient. J. Clim. 28, 365–380 (2015).
Article
Google Scholar
Dong, L., Leung, L. R., Song, F. & Lu, J. Uncertainty in El Niño-like warming and California precipitation changes linked by the Interdecadal Pacific Oscillation. Nat. Commun. 12, 6484 (2021).
Article
CAS
Google Scholar
Heede, U. K. & Fedorov, A. V. Eastern equatorial Pacific warming delayed by aerosols and thermostat response to CO2 increase. Nat. Clim. Change 11, 696–703 (2021).
Zappa, G., Ceppi, P. & Shepherd, T. G. Time-evolving sea-surface warming patterns modulate the climate change response of subtropical precipitation over land. Proc. Natl Acad. Sci. USA 117, 4539–4545 (2020).
Article
CAS
Google Scholar
Armour, K. C., Marshall, J., Scott, J. R., Donohoe, A. & Newsom, E. R. Southern Ocean warming delayed by circumpolar upwelling and equatorward transport. Nat. Geosci. 9, 549–554 (2016).
Article
CAS
Google Scholar
Kim, H., Kang, S. M., Kay, J. E. & Xie, S.-P. Subtropical clouds key to Southern Ocean teleconnections to the tropical Pacific. Proc. Natl Acad. Sci. USA 119, e2200514119 (2022).
Article
CAS
Google Scholar
Mechoso, C. R. et al. Can reducing the incoming energy flux over the Southern Ocean in a CGCM improve its simulation of tropical climate?: Southern Ocean-Tropics link in a CGCM. Geophys. Res. Lett. 43, 11057–11063 (2016).
Article
Google Scholar
Xiang, B., Zhao, M., Ming, Y., Yu, W. & Kang, S. M. Contrasting impacts of radiative forcing in the Southern Ocean versus southern Tropics on ITCZ position and energy transport in one GFDL climate model. J. Clim. 31, 5609–5628 (2018).
Article
Google Scholar
Eyring, V. et al. Overview of the Coupled Model Intercomparison Project Phase 6 (CMIP6) experimental design and organization. Geosci. Model Dev. 9, 1937–1958 (2016).
Article
Google Scholar
England, M. R., Polvani, L. M., Sun, L. & Deser, C. Tropical climate responses to projected Arctic and Antarctic sea-ice loss. Nat. Geosci. 13, 275–281 (2020).
Article
CAS
Google Scholar
Kang, S. M. et al. Walker circulation response to extratropical radiative forcing. Sci. Adv. 6, eabd3021 (2020).
Article
Google Scholar
Shin, Y. et al. Evolution of the tropical response to periodic extratropical thermal forcing. J. Clim. 34, 6335–6353 (2021).
Google Scholar
Zhang, H., Clement, A. & Di Nezio, P. The South Pacific meridional mode: a mechanism for ENSO-like variability. J. Clim. 27, 769–783 (2014).
Article
Google Scholar
Zhou, W., Leung, L. R. & Lu, J. Seasonally and regionally dependent shifts of the atmospheric westerly jets under global warming. J. Clim. 35, 5433–5447 (2022).
Article
Google Scholar
Holton, J. R. An Introduction to Dynamic Meteorology (Elsevier, 2004).
Boos, W. R. & Kuang, Z. Dominant control of the South Asian monsoon by orographic insulation versus plateau heating. Nature 463, 218–222 (2010).
Article
CAS
Google Scholar
Sampe, T. & Xie, S.-P. Large-scale dynamics of the Meiyu-Baiu Rainband: environmental forcing by the westerly jet. J. Clim. 23, 113–134 (2010).
Article
Google Scholar
Song, S.-Y. et al. Asymmetrical response of summer rainfall in East Asia to CO2 forcing. Sci. Bull. 67, 213–222 (2022).
Article
CAS
Google Scholar
Horel, J. D. & Wallace, J. M. Planetary-scale atmospheric phenomena associated with the southern oscillation. Mon. Weather Rev. 109, 813–829 (1981).
Article
Google Scholar
Chang, E. K. M., Zheng, C., Lanigan, P., Yau, A. M. W. & Neelin, J. D. Significant modulation of variability and projected change in California winter precipitation by extratropical cyclone activity. Geophys. Res. Lett. 42, 5983–5991 (2015).
Article
Google Scholar
Neelin, J. D., Langenbrunner, B., Meyerson, J. E., Hall, A. & Berg, N. California winter precipitation change under global warming in the Coupled Model Intercomparison Project Phase 5 ensemble. J. Clim. 26, 6238–6256 (2013).
Article
Google Scholar
Ropelewski, C. F. & Halpert, M. S. North American precipitation and temperature patterns associated with the El Niño/Southern Oscillation (ENSO). Mon. Weather Rev. 114, 2352–2362 (1986).
Article
Google Scholar
Myers, T. A. et al. Observational constraints on low cloud feedback reduce uncertainty of climate sensitivity. Nat. Clim. Change 11, 501–507 (2021).
Article
Google Scholar
Brient, F. & Schneider, T. Constraints on climate sensitivity from space-based measurements of low-cloud reflection. J. Clim. 29, 5821–5835 (2016).
Article
Google Scholar
Tan, I., Storelvmo, T. & Zelinka, M. D. Observational constraints on mixed-phase clouds imply higher climate sensitivity. Science 352, 224–227 (2016).
Article
CAS
Google Scholar
McFarquhar, G. M. et al. Observations of clouds, aerosols, precipitation, and surface radiation over the Southern Ocean: an overview of CAPRICORN, MARCUS, MICRE, and SOCRATES. Bull. Am. Meteorol. Soc. 102, E894–E928 (2021).
Mechoso, C. R. et al. Ocean-cloud-atmosphere-land interactions in the southeastern Pacific: the VOCALS program. Bull. Am. Meteorol. Soc. 95, 357–375 (2014).
Article
Google Scholar
Kim, S.-K. et al. Widespread irreversible changes in surface temperature and precipitation in response to CO2 forcing. Nat Clim. Change 12, 834–840 (2022).
Article
CAS
Google Scholar
Keller, D. P. et al. The Carbon Dioxide Removal Model Intercomparison Project (CDRMIP): rationale and experimental protocol for CMIP6. Geosci. Model Dev. 11, 1133–1160 (2018).
Article
CAS
Google Scholar
Kug, J.-S. et al. Hysteresis of the intertropical convergence zone to CO2 forcing. Nat. Clim. Change 12, 47–53 (2022).
Article
CAS
Google Scholar
Kang, S. M., Ceppi, P., Yu, Y. & Kang, I.-S. Recent global climate feedback controlled by Southern Ocean cooling. Nat. Geosci. 16, 775–780 (2023).
Article
CAS
Google Scholar
Kang, S. M. et al. Global impacts of recent Southern Ocean cooling. Proc. Natl Acad. Sci. USA 120, e2300881120 (2023).
Article
CAS
Google Scholar
Yeager, S. G. et al. Reduced Southern Ocean warming enhances global skill and signal-to-noise in an eddy-resolving decadal prediction system. NPJ Clim. Atmos. Sci 6, 107 (2023).
Article
Google Scholar
Gent, P. R. et al. The community climate system model version 4. J. Clim. 24, 4973–4991 (2011).
Article
Google Scholar
Loeb, N. G. et al. Clouds and the Earth's Radiant Energy System (CERES) Energy Balanced and Filled (EBAF) Top-of-Atmosphere (TOA) Edition-4.0 Data Product. J. Clim. 31, 895–918 (2018).
Article
Google Scholar
Reynolds, R. W., Rayner, N. A., Smith, T. M., Stokes, D. C. & Wang, W. An improved in situ and satellite SST analysis for climate. J. Clim. 15, 1609–1625 (2002).
Article
Google Scholar
Qu, X., Hall, A., Klein, S. A. & Caldwell, P. M. On the spread of changes in marine low cloud cover in climate model simulations of the 21st century. Clim. Dyn. 42, 2603–2626 (2014).
Article
Google Scholar
Ceppi, P. & Nowack, P. Observational evidence that cloud feedback amplifies global warming. Proc. Natl Acad. Sci. USA 118, e2026290118 (2021).
Article
CAS
Google Scholar
Middlemas, E. A., Kay, J. E. & Medeiros, B. M. Quantifying the influence of cloud radiative feedbacks on Arctic surface warming using cloud locking in an Earth system model. Geophys. Res. Lett. 47, e2020GL089207 (2020).
Article
Google Scholar
Hurrell, J. W. et al. The Community Earth System Model: a framework for collaborative research. Bull. Am. Meteorol. Soc. 94, 1339–1360 (2013).
Article
Google Scholar
He, C. et al. Enhanced or weakened western North Pacific subtropical high under global warming? Sci. Rep. 5, 16771 (2015).
Article
CAS
Google Scholar
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H.K. and F.L. are supported by NOAA MAPP award no. NA21OAR4310349. H.K. and S.M.K. have been supported by the research programme for the carbon cycle between oceans, land and atmosphere of the National Research Foundation (NRF) funded by the Ministry of Science and ICT (NRF-2022M3I6A1090965). A.G.P. and F.L. acknowledge support from the US Department of Energy, Office of Science, Office of Biological and Environmental Research, Regional and Global Model Analysis (RGMA) component of the Earth and Environmental System Modeling Program under award no. DE-SC0022070 and National Science Foundation IA 1947282. P.C. is supported by UK Research and Innovation (UKRI) grants NE/V012045/1, NE/T006250/1 and EP/Y036123/1. The National Center for Atmospheric Research is sponsored by the National Science Foundation. We thank the developer of the Synda Transfer Module, which was used for downloading CMIP data (https://espri-mod.github.io/synda/index.html). S.-W.Y. is supported by the Korea Environment Industry and Technology Institute (KEITI) through Climate Change R&D Project for New Climate Regime funded by Korea Ministry of Environment (MOE) (2022003560001). Y.S. is supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2024-00334637). We deeply appreciate Y. Kamae and the other anonymous reviewers for their contributions to improving the paper.
Department of Earth and Atmospheric Sciences, Cornell University, Ithaca, NY, USA
Hanjun Kim, Angeline G. Pendergrass & Flavio Lehner
Max Planck Institute for Meteorology, Hamburg, Germany
Sarah M. Kang
Climate and Global Dynamics Laboratory, National Center for Atmospheric Research, Boulder, CO, USA
Angeline G. Pendergrass & Flavio Lehner
Polar Bears International, Bozeman, MT, USA
Flavio Lehner
School of Earth and Environmental Sciences, Seoul National University, Seoul, South Korea
Yechul Shin
Department of Physics, Imperial College London, London, UK
Paulo Ceppi
Department of Marine Science and Convergence Engineering, Hanyang University, ERICA, Ansan, South Korea
Sang-Wook Yeh
Earth Research Institute, University of California, Santa Barbara, CA, USA
Se-Yong Song
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H.K. and S.M.K. conceived the study and designed the experiments. H.K. carried out the analysis and wrote a first draft. H.K., Y.S. and S.-Y.S. performed modelling experiments. H.K., A.G.P. and F.L. analysed the teleconnection mechanisms in detail. All authors contributed to discussions of the results and revisions of the paper.
Correspondence to
Hanjun Kim or Sarah M. Kang.
The authors declare no competing interests.
Nature Geoscience thanks Youichi Kamae and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary Handling Editor: James Super, in collaboration with the Nature Geoscience team.
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Total response is difference between last 30 years of abrupt-4xCO2 and last 100 years of pre-industrial control simulation, while slow response is difference between last 30 and first 10 years of abrupt-4xCO2 simulation. Fast response is subtraction of the slow from the total response. a–c, Multi-model mean total response in annual-mean SST (a), summer precipitation (b), and winter precipitation (c). d–f, Decadal averaged timeseries of the response in equatorial Pacific SST gradient (d), precipitation over East Asia (e) and the Western United States (f). The zonal SST gradient and regional precipitations are calculated over the black boxes in (a-c). Black lines represent multi-model mean and shading indicate \(\pm\)1 inter-model standard deviation. g–i, Total responses versus slow/fast responses for equatorial Pacific SST gradient (g), precipitation over East Asia (h) and the Western United States (i). Red (Blue) circles indicate the slow (fast) responses, and the open circles indicate the multi-model mean. For slow/fast response, inter-model correlation coefficients to the total response are inserted as text. Note that the definition of slow response—which uses first 10 years—is specific to this figure; in main analysis we use first 30 years to minimize the internal variability of the regional precipitation (Methods).
a–c, Relationship between the Asian jet shift and the East Asian (EA) precipitation changes (a), the tropical Pacific triangular warming and the Asian jet shift (b), and the triangular warming and the EA precipitation changes (c). The Asian jet shift is measured by changes in meridional asymmetry of 200-hPa zonal wind, in which negative sign indicates southward shift (Methods). The precipitation anomaly is averaged over EA between 25°N-40°N and 110°E-145°E (green boxes in Fig. 2a,b). Circle symbols are CMIP6 slow responses, black is multi-model mean. Orange triangles indicate the CESM1-CAM4 SO warming experiment. The inter-model correlation coefficients are inserted as text. All variables are averaged for JJA.
a–d, Same to Fig. 2c–f but for the CESM1-CAM4 SO warming experiment. Unhatched regions indicate statistically significant responses at the 95% confidence level using a t test, compared to the inter-annual variability in control climate.
a–d, 850-hPa geopotential height pattern (shading) and 850-hPa horizontal wind (vectors) from multi-model mean CMIP6 slow response (a), CESM1-CAM4 SO warming experiment (b), inter-model regression of CMIP6 slow response onto the CFSH (c), and CESM1-CAM5 regional cloud-locking experiment (d). e-g, Relationship between the tropical Pacific triangular warming and the relative warming over the tropical Indian ocean between 10°S-10°N and 50°E-100°E, compared to the tropical western Pacific between 10°S-10°N and 150°E-180°E (e), the relative tropical Indian ocean warming and the western North Pacific subtropical high (WNPSH) height anomaly (f), the height anomaly and the East Asia summer precipitation enhancement (g). The WNPSH height anomaly is calculated as the averaged 850-hPa geopotential height pattern (Methods) over the western North Pacific between 10°N-25°N and 110°E-145°E (black boxes in (a-d)). Annotations in (e-g) follow those in Extended Data Fig. 2. All variables are averaged for JJA.
a–c, Relationship between the tropical Pacific triangular warming and the northeastern Pacific 200-hPa geopotential height pattern anomaly (a), the height anomaly and WUS precipitation enhancement (b), and the triangular warming and WUS precipitation enhancement (c). The height anomaly is averaged over the northeastern Pacific between 30°N–55°N and 185°E–235°E, and the precipitation anomaly is averaged over WUS between 30°N-45°N and 215°E-245°E (green boxes in Fig. 3a,b). d–f, Same relationship as in a–c except for the height anomalies over the southeastern United States (30°N–55°N and 185°E–235°E) and the SEUS precipitations averaged over the green parallelogram in Fig. 3a,b. The symbols and texts follow those in Extended Data Fig. 2. All variables are averaged for DJF.
a,c,e, The 850-hPa geopotential height pattern (shading) and 850-hPa horizontal wind (vectors) from the CESM1-CAM4 SO warming experiment (a), the inter-model regression of CMIP6 slow response onto the CFSH (c), and CESM1-CAM5 regional cloud-locking experiment (e). The vectors are shown if either the zonal or meridional component are statistically significant at the 95% confidence interval using a t test. b,d,f, Same as a, c and e, but for the changes (shading) and climatology (contours) of 200-hPa zonal wind. Only the positive contours larger than 12 m s−1 are shown; the contour interval is 8 m s−1. Green boxes indicate the Western and Southeastern United States, where precipitation increases. All variables are averaged for DJF.
a–d, Same to Fig. 2c–f but for the inter-model regression of CMIP6 slow response onto the CFSH. Unhatched regions indicate statistically significant regression coefficients at the 95% confidence level using a t test.
a–d, Same to Fig. 2c–f but for the CESM1-CAM5 regional cloud-locking experiment. Unhatched regions indicate statistically significant regression coefficients at the 95% confidence level using a t test.
a–d, CFSH versus the tropical Pacific triangular warming (a), the precipitation responses during the EA summer (b), WUS winter (c), and SEUS winter (d). Circle (cross) symbols indicate CMIP6 slow (total) response. The inserted texts indicate inter-model correlation coefficients, where the values in parentheses are calculated after excluding four CESM2 variant models with the highest CFSH (Supplementary Table 1). The symbols are color-coded from blue to red in ascending order of the CFSH. Black symbol indicates muti-model mean and empty (filled) green rectangle indicates the CESM1-CAM5 experiment in which the clouds are regionally locked (globally interactive). The vertical black line indicates the observational estimate of CFSH (Methods). Note that WUS winter is defined as November, December, January for CMIP6 slow response and as January, February, March for the cloud-locking experiment, when each signal is maximized.
Supplementary Table 1 and Figs. 1–6.
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Kim, H., Kang, S.M., Pendergrass, A.G. et al. Higher precipitation in East Asia and western United States expected with future Southern Ocean warming.
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The solute carrier protein SLC19A1 is crucial for transporting folate nutrients, antifolate chemotherapeutics, and more recently cyclic dinucleotides (CDNs) immune transmitters, influencing various physiological and pathological processes. While the inward-open state of human SLC19A1 (hSLC19A1) has been previously described, key aspects regarding its conformational dynamics, substrate selectivity, and precise mechanisms underlying CDNs transport remain elusive. Using an antibody-facilitated conformation screening strategy, we present cryo-electron microscopy structures of hSLC19A1 in its outward-open state with and without bound substrates, revealing detailed mechanisms of substrate recognition and conformational changes during transport. We identify both general and specific features for folate/antifolate recognition, including an SLC19A1-specific pocket for accommodating γ-carboxylate-modified antifolates. Intriguingly, CDNs bind as monomers within the canonical pocket of outward-open hSLC19A1, contrasting with dimeric binding in inward-open structures. Together with functional assays, these findings provide a framework for developing antifolate drugs and CDN-targeted therapies, advancing our understanding of SLC19A1's physiological and therapeutic functions.
Folate, an essential nutrient for mammalian growth and development, acts as a crucial one-carbon unit donor in fundamental metabolic processes and plays pivotal roles in synthesizing key biomolecules such as thymidine, purine nucleotides, serine, and methionine1,2. The solute carrier protein SLC19A1, also known as the reduced folate carrier, has been extensively studied for its role in folate transport over several decades3,4,5. Additionally, SLC19A1 facilitates the transport of antifolate drugs, such as methotrexate and pemetrexed, which are widely used in the treatment of cancer, rheumatoid arthritis, and psoriasis6. Interestingly, recent studies have unveiled a novel function of SLC19A1 in the transport of cyclic dinucleotides (CDNs)7,8. CDNs serve as pivotal immune signaling molecules capable of eliciting broad downstream immune responses by binding to the stimulator of interferon genes (STING)9,10,11,12,13. SLC19A1 transports not only mammalian endogenous CDN: 2'3'-cGAMP synthesized by cyclic GMP–AMP synthase (cGAS)14,15,16, but also bacterial CDN and synthetic CDN-type drugs11,17,18,19. The dual role of SLC19A1 in folate/antifolate and CDN transport underscores its significance in fundamental metabolism, infection, immunopathology, cancer chemotherapy, and immunotherapy4,5,20,21,22.
In line with the major facilitator superfamily (MFS) fold, SLC19A1 comprises 12 transmembrane (TM) helixes, with TM1-6 forming the N-terminal domain (NTD) and TM7-12 constituting the C-terminal domain (CTD)23. The transmembrane transport of substrates by MFS protein normally occurs through a rocker-switch rotation model, wherein the two domains undergo conformational changes from an outward-open to an inward-open state24. Previous cryo-electron microscopy (cryo-EM) studies have provided insights into the substrate binding details of human SLC19A1 (hSLC19A1) in its inward-open state, with folate and antifolate binding as monomers within a canonical pocket25,26,27 and CDNs forming a compact dimeric unit within a non-canonical cavity25. However, all the reported structures exhibit the same inward-open conformation, leaving unresolved questions about how hSLC19A1 changes conformation and recognizes substrates during the transport cycle. Moreover, it is still unclear whether the captured CDN dimer in the inward-open state represents the actual form in which CDNs are transported by hSLC19A1 or just a potential substrate binding mode, since CDNs are not located in the typical pocket of SLC proteins25. Therefore, whether CDNs are transported in the dimeric form remains an open question25. Further structural studies of hSLC19A1 in complex with different substrates at the outward-open conformation are warranted to fully elucidate the transport cycle and diverse substrate recognition mechanisms of hSLC19A1, thereby advancing understanding of its physiological roles and potential therapeutic implications.
Previously reported hSLC19A1 structures all exhibit the inward-open conformation, suggesting that purified hSLC19A1 may have a preference for this state in vitro. To capture hSLC19A1 in other conformations, we sought to systematically introduce mutations at the NTD-CTD interface aimed at destabilizing the inward-open state without affecting the substrate recognition tunnel. By taking advantage of our previously isolated inward-open specific antibody to hSLC19A1 (mAb70#)25, we were able to efficiently distinguish the conformation of each mutant in cells using flow cytometry, rather than going through tedious purification and structure determination procedures for each sample (Fig. 1a). In addition, we also generated two conformation-unspecific antibodies recognizing linear epitopes as control antibodies, mAb5# and mAb6#, targeting the disordered N-terminal tail and intracellular loop between TM6 and TM7 (IL6-7), respectively (Supplementary Fig. 1a). Subsequent flow cytometry results show that, among multiple mutants tested, the G307F mutation completely disrupts the binding between hSLC19A1 and mAb70# while leaving interactions with mAb5# and mAb6# unaffected (Supplementary Fig. 1b, c). Size-exclusion chromatography experiments with purified hSLC19A1WT and hSLC19A1G307F further confirm these results (Supplementary Fig. 1d, e). Consistently, the 3H-labelled antifolate uptake of hSLC19A1G307F is abolished in the cellular uptake assay (Fig. 1b and Supplementary Fig. 2), highlighting that the G307F mutation may have trapped hSLC19A1 in a conformation other than the inward-open state.
a Schematic diagram of the antibody-facilitated strategy for identifying conformationally altered hSLC19A1 mutants. b [3H]-MTX uptake assays using SLC19A1−/− HeLa cells stably expressing either WT hSLC19A1 or G307F mutant. Data were normalized to WT and are mean ± s.e.m. of n = 4 biologically independent experiments. Statistical analysis was performed using two-tailed unpaired Student's t-tests. EV, empty vector; WT, wild type. c Cryo-EM density map of outward-open hSLC19A1. d The overall structure of outward-open hSLC19A1. The N- and C-terminal domains (NTD and CTD) are coloured in blue and yellow, respectively. e Cut-open side view of the electrostatic potential surface of the hSLC19A1 outward-open structure. f Domain-wise alignments between outward- (blue and yellow) and inward-open (grey) hSLC19A1. g Rotation of representative TM segments between outward- (blue and yellow) and inward-open (grey) hSLC19A1 relative to the membrane norm. h Cytosolic bottom view of the intracellular gate in the outward-open conformation. Key residues are shown as sticks. i Conformational changes of Phe141 (left), Gln150 (middle) and Arg157 (right) on the basis of NTD-centered structural superimposition of outward- (blue and yellow) and inward-open hSLC19A1 (grey). Key residues are shown as sticks and hydrogen bonds are represented as dashed lines. j [3H]-MTX uptake assays using SLC19A1−/− HeLa cells stably expressing either WT hSLC19A1 or intracellular-gate residue mutants. Data were normalized to WT and are mean ± s.e.m. of n = 4 biologically independent experiments. Statistical analysis was performed using two-tailed unpaired Student's t-tests. ***P ≤ 0.001, ****P ≤ 0.0001. Source data are provided as a Source Data file.
hSLC19A1 is a relatively small membrane protein and lacks rigid soluble domains, making it challenging for high-resolution structure determination. To facilitate cryo-EM reconstructions, we designed a hSLC19A1G307F construct carrying an N-terminal BRIL domain and a C-terminal specific nanobody (termed hSLC19A1EM-G307F; Supplementary Fig. 3a), both of which can specifically bind to a widely used anti-BRIL Fab fragment. In this way, the subsequent addition of anti-BRIL Fab may bridge the N- and C-terminus of hSLC19A1 together, thereby enhancing sample stability and facilitating cryo-EM particle alignment. It should be noted that the N- and C-terminal fusions to the wild-type protein still largely preserve the substrate cellular uptake activity (Supplementary Figs. 2 and 3b), suggesting that the fusion strategy per se does not significantly affect hSLC19A1 function.
To verify the conformational details of the engineered protein, we reconstituted the complex of hSLC19A1EM-G307F and anti-BRIL Fab (Supplementary Fig. 3c, d) and successfully determined its cryo-EM structure at a resolution of 2.94 Å (Fig. 1c-e, Supplementary Fig. 3e–i and Supplementary Table 1). The density is of sufficient quality to trace the transmembrane regions of hSLC19A1 from residues 24 to 457 (Supplementary Fig. 4a). Some parts of the intracellular loop between TM6 and TM7 (IL6-7, residues 213-248) and the extracellular loop between TM7 and TM8 (EL7-8, residues 295-302) were not observed in the cryo-EM map, possibly due to the dynamic nature of these regions (Fig. 1d). Interestingly, in contrast to previously reported structures, the engineered hSLC19A1 adopts an outward-open conformation (Fig. 1d). In this conformation, TM1 and TM2 tilt away from TM7 and TM8, exposing the entrance to a large solvent-accessible and highly conserved cavity that extends more than halfway across the cell membrane (Fig. 1d and Supplementary Fig. 4b). Characterized by a narrow extracellular entrance, the upper portion of the cavity extends toward the NTD and presents an overall negative electrostatic surface (Fig. 1e). Conversely, the lower half of the cavity contracts and shows an overall positive charge, consistent with the anionic properties of CDN and folate (Fig. 1e). On the intracellular side, TM4 and TM5, along with TM10 and TM11, snugly interlock to seal the cavity from the cytoplasm (Supplementary Fig. 4c). Additionally, IL2-3, IL4-5, IL6-7 and IL10-11 closely associate to further stabilize the outward-open conformation (Supplementary Fig. 4c).
Domain-wise alignments between outward- and inward-open structures of hSLC19A1 show that the alternate access is achieved through a rocker-switch motion of the NTD and CTD domains. Individual NTD and CTD of outward-open hSLC19A1 align well with those in inward-open state, with root-mean-square deviation (RMSD) values of 0.69 Å over 168 Cα atoms and 0.71 Å over 155 Cα atoms, respectively (Fig. 1f). Both domains of hSLC19A1 rotate relative to an axis perpendicular to the membrane plane by approximately 18°, allowing the transmembrane regions to remain embedded within the membrane (Fig. 1g). The highly conserved and functionally important N-terminal region of IL6-728 (aa203-aa212), which is tightly embedded in a surface groove formed by the intracellular ends of TM2, TM3, TM4 and TM11, also undergoes relative motion to accommodate the movement of CTD in the outward-open state (Supplementary Fig. 4d). In the current outward-open conformation, three pairs of hydrogen bonds (Gln150-Ala324/Val327 and Arg157-Gln377) and a set of hydrophobic interactions (Phe141-Ala388-Phe400), presumably contribute to the closure of the intracellular gate (Fig. 1h, i). Interestingly, structural superimposition with the inward-open structure reveals dramatic conformational changes in Phe141, Gln150 and Arg157, which would disrupt the intracellular gate and trigger the closure of the extracellular gate as well as the release of substrates (Fig. 1i and Supplementary Fig. 4e). To validate these structural observations, we substituted these gate residues and assessed the cellular uptake of 3H-labelled antifolate in SLC19A1−/− HeLa cells stably expressing either wild-type or mutant hSLC19A1. The F141S and R157A substitutions nearly abolish antifolate uptake, Q150A, A388S and F400S reduce antifolate uptake by about 70–80%, and Q377A decreases the activity by around 25% (Fig. 1j, Supplementary Figs. 2 and 4f).
Our structure also provides a detailed explanation of why the G307F single point mutation drives conformational changes (Supplementary Fig. 1b, c). In the inward-open hSLC19A1 structure, Gly307 on TM8 forms a hydrogen bond with the carbonyl group of Gln172 on TM5 (Supplementary Fig. 4g). However, by wedging into the lateral interface between NTD and CTD, Phe307 disrupts the hydrogen bond and stacks with Gln172 to lock the outward-open conformation (Supplementary Fig. 4g). To check whether substitutions of Gly307 with residues of different side chain sizes can enable hSLC19A1 in alternative intermediate states of the transport cycle, we further generated G307W, G307S, and G307A mutations. Cryo-EM analysis shows that G307W and G307S both adopt the same outward-open structures as observed with G307F, while a smaller side chain mutation G307A maintains the inward-open conformation similar to the wild-type protein (Supplementary Fig. 4h). These results suggest that the outward- and inward-open conformations likely represent two relatively stable states of the SLC19A1 transport cycle.
While previous studies have provided insights into the interaction pattern of inward-open hSLC19A1 with folate and antifolate, a comprehensive understanding of the transport cycle of these substrates necessitates further structural elucidation. Therefore, using the aforementioned engineering strategy, we determined the cryo-EM structures of outward-open hSLC19A1 in complex with the reduced folate 5-methyltetrahydrofolate (5-MTHF) and the antifolate methotrexate (MTX) at resolutions of 3.43 Å and 3.44 Å, respectively (Fig. 2a–f, Supplementary Fig. 5 and Supplementary Table 1).
a, d Cryo-EM density maps of outward-open hSLC19A1 bound to 5-MTHF (a) and MTX (d). b, e The overall structures of outward-open hSLC19A1 bound to 5-MTHF (b) and MTX (e). The NTD and CTD are coloured in blue and yellow, respectively. 5-MTHF and MTX are shown as a space-filling representation and are coloured in cyan and wheat, respectively. c, f A cut-open side view of the electrostatic potential surface of outward-open hSLC19A1 bound to 5-MTHF (c) and MTX (f). g Different conformations of the bound 5-MTHF in the outward- (cyan) and inward-open state (grey) of hSLC19A1 on the basis of NTD-centered structural superimposition. h Hydrogen bonding interactions between 5-MTHF (sticks; cyan) and outward-open hSLC19A1 (grey). Key residues are shown as sticks and hydrogen bonds are represented as dashed lines. i Hydrophobic interactions between 5-MTHF (sticks; cyan) and outward-open hSLC19A1 (grey). Key residues are shown as sticks and dots. j Different conformations of the MTX in the outward-open (wheat) and inward-open state (grey) on the basis of NTD-centered structural superimposition. k [3H]-MTX uptake assays using SLC19A1−/− HeLa cells stably expressing either WT hSLC19A1 or folate-binding pocket mutants. Data were normalized to WT and are mean ± s.e.m. of n = 4 biologically independent experiments. Statistical analysis was performed using two-tailed unpaired Student's t-tests. ***P ≤ 0.001, ****P ≤ 0.0001. Source data are provided as a Source Data file.
5-MTHF is a predominant natural folate in diet and blood and plays a crucial role in normal metabolism and development. The well-resolved density map reveals that 5-MTHF is situated at the polar cavity in an approximately upright conformation, with its long axis aligned parallel to the transmembrane helices (Fig. 2a–c, g and Supplementary Fig. 6a, b). Anchored by hydrogen bonds with Thr49 of TM1 and Glu123 of TM4 (Fig. 2h), the pterin moiety of 5-MTHF inserts diagonally into the upper negatively charged pocket within the NTD (Fig. 2c). The pterin ring and methyl group establish extensive hydrophobic contacts with Glu45/Ile48 of TM1, Leu72 of TM2 and Tyr126 of TM4 (Fig. 2i). Meanwhile, the benzoyl moiety extends downward perpendicular to the plane of the pterin ring, forming a hydrogen bond with Arg133 of TM4, along with hydrophobic interactions with Tyr126/Met130 of TM4 and Tyr286 of TM7 (Fig. 2h, i). In addition, the glutamate moiety faces the intracellular side and reaches the positively charged region (Fig. 2c, h). The β-carboxylate group faces the CTD and interacts with Tyr281 of TM7, Arg373 and Gln377 of TM10, while the γ-carboxylate group turns back towards the NTD and interacts with Arg133 of TM4 (Fig. 2c, h).
It is noteworthy that SLC19A1 shows a pronounced preference for reduced folate over folic acid4. Compared to folic acid, 5-MTHF contains an additional methyl group at position N5 (Supplementary Fig. 6a). Our previous inward-open structure reveals that this extra methyl group is embedded in a hydrophobic pocket formed by Glu45, Ile48 and Tyr126, thereby enhancing the affinity of 5-MTHF for hSLC19A125. Although the overall binding site and interaction mode between 5-MTHF and the current outward-open hSLC19A1 are similar to those of the inward-open structure, the detailed conformations of the pterin ring, benzoyl and glutamate moieties of 5-MTHF exhibit notable differences (Fig. 2g). Specifically, the pterin ring of 5-MTHF rotates ~180° with the N5 methyl group serving as the pivot, maintaining the methyl group within the hydrophobic pocket (Fig. 2g, i). The benzoyl and glutamate moieties also undergo appropriate adjustments accordingly (Fig. 2g). These findings highlight the contribution of the N5 methyl group to the reduced folate selectivity preference of SLC19A1 and indicate that 5-MTHF likely possesses some degree of flexibility to accommodate the transport cycle.
MTX is a widely used antifolate drug for the treatment of cancer and autoimmune diseases. Consistent with the 5-MTHF-bound structure, MTX also adopts an upright conformation and binds within the central cavity, with the pteridine ring occupying the electronegative pocket at NTD and the glutamate moiety extending toward the electropositive region (Fig. 2d–f and Supplementary Fig. 6c). The interactions between MTX and the outward-open hSLC19A1 are similar to those of 5-MTHF (Supplementary Fig. 6d, e). Interestingly, in the previously reported inward-open hSLC19A1 structure bound to an N-hydroxysuccinimide (NHS)-conjugated MTX26, the glutamate moiety of MTX was covalently linked to Lys411 of TM11 via NHS-mediated crosslinking, resulting in a 180° rotation of the glutamate and benzoyl groups when aligning the NTD of SLC19A1 of the two structures (Fig. 2j). These results indicate that the glutamate moiety of antifolate exhibits high dynamics within the electropositive cavity to accommodate the binding and dissociation with hSLC19A1.
The residues lining the binding sites for 5-MTHF and MTX are highly conserved among vertebrates (Supplementary Fig. 7). To investigate the contribution of the observed SLC19A1-substrate interactions, we introduced mutations into the corresponding residues of hSLC19A1 (Supplementary Fig. 2) and examined the effects on cellular 3H-labelled antifolate uptake. The representative substitutions lead to a substantial reduction in antifolate uptake, with E123A, R133A and R373A mutations almost abolishing the activity (Fig. 2k). Furthermore, the initial-rate analysis confirms the critical roles of representative residues for folate/antifolate recognition (Supplementary Fig. 4f).
Tumor cells tend to use different types of folate transporters to sustain the elevated folate levels necessary for their rapid growth. Studies have shown that SLC19A1 primarily functions in hematologic malignancies and solid tumors, including acute lymphoblastic leukemia and osteosarcoma3,29,30. In contrast, SLC46A1 (also known as proton-coupled folate transporter, PCFT), another crucial pathway for folate and antifolate drug delivery, is prominently expressed in solid tumors, particularly in breast, lung and ovarian tumor cell lines4,31,32,33. The widely used antifolate drugs, including MTX and pemetrexed (PMX), can be absorbed by healthy cells through both SLC19A1 and SLC46A1, resulting in significant cytotoxicity34. Therefore, selective targeting of SLC19A1 or SLC46A1 through designer drugs would facilitate more effective treatments while mitigating cytotoxic effects35.
The γ-carboxylate groups of folates and antifolates have been demonstrated to be important for their uptake by SLC19A1 and SLC46A125,33. However, PT523, an atypical antifolate featuring a substituted γ-carboxylate (Supplementary Fig. 8a), emerges as an even more favorable substrate for SLC19A1 while exhibiting minimal transport by SLC46A14,36,37. To elucidate the SLC19A1-specific recognition mechanism of PT523, we determined the cryo-EM structure of outward-open hSLC19A1 in complex with PT523 at a resolution of 3.25 Å (Fig. 3a–c, Supplementary Fig. 8b–g and Supplementary Table 1). The cryo-EM density is of high quality and corresponds well to the molecular shape of PT523 (Supplementary Fig. 8a, g). An additional smeared density was also observed near PT523 in the pocket, likely due to non-specific attachment, which diminishes with decreasing PT523 concentration during cryo-EM sample preparation (Supplementary Fig. 9a, b). Similar to 5-MTHF, PT523 is tightly embedded in the central polar cavity with its long axis aligning parallel to the transmembrane helices (Fig. 3b, c and Supplementary Fig. 9c). The pteridine ring of PT523 occupies the electronegative pocket of the NTD, forming interactions with Glu45/Ile48/Thr49 of TM1, Leu72 of TM2 and Glu123 of TM4 (Fig. 3d, e). The benzoyl moiety forms hydrophobic interactions with Tyr126/Met130 of TM4 and the carbonyl is hydrogen bonded by Arg133 of TM4 (Fig. 3d, e). The β-carboxylate group faces the CTD and engages in hydrogen bonds with Tyr281 of TM7 and Arg373/Gln377 of TM10 (Fig. 3c, d). Remarkably, the γ-substituted hemiphthaloyl group delves into a distinct pocket within the NTD at the bottom of the central cavity (Fig. 3c, f, g and Supplementary Fig. 9d). The aromatic ring is supported from below by Tyr136 of TM4 and is flanked on both sides by hydrophobic interactions with the side chains of Arg133 of TM4 and Val160 of TM5 (Fig. 3f, g). Furthermore, the carbonyl and carboxyl are further stabilized by Arg133/Ser137 of TM4 and Arg157 of TM5 through hydrogen bonds (Fig. 3f, g). Thus, these additional contacts appear to contribute to the high transport activity of PT523. In addition, the existence of the specific and conserved pocket (Supplementary Fig. 7), lined by Arg133, Tyr136, Ser137, Arg157 and Val160, provides structural insights into the preference of SLC19A1 for antifolates with γ-carboxylate modifications over those with β-carboxylate modifications38.
a Cryo-EM density map of outward-open hSLC19A1 bound to PT523. b The overall structure of outward-open hSLC19A1 bound to PT523. The NTD and CTD are coloured in blue and yellow, respectively. PT523 is shown as a space-filling representation and is coloured in salmon. c A cut-open side view of the electrostatic potential surface of outward-open hSLC19A1 bound to PT523. d, f Hydrogen bonding interactions between PT523 (sticks; salmon) and outward-open hSLC19A1 (grey). Key residues are shown as sticks and hydrogen bonds are represented as dashed lines. e, g Hydrophobic interactions between PT523 (sticks; salmon) and outward-open hSLC19A1 (grey). Key residues are shown as sticks and dots.
In contrast, in the recently solved SLC46A1-PMX outward-open structure, PMX binds at the base of the polar cavity in an orientation nearly parallel to the membrane plane33. The pyrimidine moiety of SLC46A1-bound PMX resides in a negatively charged cavity within the CTD, while the glutamate group extends toward the NTD with the γ-carboxylate group nestled in a positively charged pocket (Supplementary Fig. 9e). Consequently, SLC46A1 ligands must establish an appropriate distance between the ring and the γ-carboxylate group to adapt to the minimal distance (~16 Å) of the functional groups within the binding site33,35. The length of the long axis of PT523 (~23 Å) exceeds the capacity of the substrate pocket of SLC46A1, thus the γ-substituted hemiphthaloyl group will apparently create significant steric hindrance with the positively charged pocket within the NTD (Supplementary Fig. 9c, e). Taken together, the additional interactions of PT523's hemiphthaloyl group with SLC19A1, coupled with its steric hindrance with the positively charged pocket of SLC46A1, may contribute to the high affinity and substrate selectivity preference of PT523 for SLC19A1. These results provide new clues for the design of next-generation antifolate drugs with enhanced transporter specificity and reduced side effects. Moreover, besides serving as delivery systems for antifolate drugs, SLC19A1 and SLC46A1 also represent attractive targets for modulating folate uptake in tumor cells. A comparative analysis of substrate binding modes between SLC19A1 and SLC46A1, both in their outward-open conformation, will likely provide a framework for the rational design of specific exofacial inhibitors aimed at blocking folate uptake in different tumor types, as the substrate binding pocket of the outward-open conformation is readily accessible on the extracellular surface39,40.
In our previously reported hSLC19A1-CDN inward-open structures, CDNs were observed to bind as a compact dual-molecule unit near the intracellular side of hSLC19A1, even at low CDN:transporter molar ratios25. However, the compact CDN dimer does not bind within the canonical substrate cavity of SLC proteins and is likely to hinder closure of the intracellular gate of hSLC19A1 due to its numerous interactions with gate residues (Supplementary Fig. 11a). Therefore, the CDN dimer may only represent a potential substrate binding mode, and it remains unclear whether CDNs are transported by SLC19A1 in such a dimeric form. To further understand the precise transport mechanism of CDNs by SLC19A1, we determined the structure of outward-open hSLC19A1 bound to a representative CDN-type drug, 2′3′-CDAS, at a resolution of 3.34 Å (Fig. 4a–c, Supplementary Fig. 10 and Supplementary Table 1).
a Cryo-EM density map of outward-open hSLC19A1 bound to 2′3′-CDAS. b, The overall structure of outward-open hSLC19A1 bound to 2′3′-CDAS. The NTD and CTD are coloured in blue and yellow, respectively. 2′3′-CDAS is shown as a space-filling representation and is coloured in green. c, A cut-open side view of the electrostatic potential surface of outward-open hSLC19A1 bound to 2′3′-CDAS. d Hydrogen bonding and hydrophobic interactions between 2′3′-CDAS (sticks; green) and outward-open hSLC19A1 (grey). Key residues are shown as sticks and dots, and hydrogen bonds are represented as dashed lines. e SLC19A1−/− THP-1 cells stably expressing either WT hSLC19A1 or CDN-binding-pocket mutants were exposed to 2′3′-cGAMP. The induction of IFNB1 mRNA was measured by quantitative PCR with reverse transcription (RT-qPCR). Data are mean ± s.e.m. of n = 4 biological replicates. Statistical analysis was performed using two-tailed unpaired Student's t-tests. f, h 2′3′-cGAMP (f) and [3H]-MTX (h) uptake assays using SLC19A1−/− HeLa cells stably expressing either WT hSLC19A1 or mutants of folate-binding and CDN-binding pocket. Data were normalized to WT and are mean ± s.e.m. of n = 4 biologically independent experiments. Statistical analysis was performed using two-tailed unpaired Student's t-tests. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Source data are provided as a Source Data file. g Structural superimposition of 5-MTHF (cyan) and 2′3′-CDAS (green) bound in the canonical substrate pocket of the outward-open hSLC19A1.
2′3′-CDAS represents one of the most extensively evaluated synthetic CDN-type drugs which consists of two AMP moieties connected by 2′-5′/3′-5′ mixed linkages with bisphosphothioate substitutions (Supplementary Fig. 10f). The cryo-EM density is of good quality to distinguish 2′-5′ and 3′-5′ phosphodiester bonds and different chemical groups (Supplementary Fig. 10g). Intriguingly, in contrast to inward-open structures, only one 2′3′-CDAS molecule binds within the canonical substrate cavity of hSLC19A1 (Fig. 4a–d). Positioned within the central canonical cavity of hSLC19A1, 2′3′-CDAS monomer adopts a U-type conformation (Fig. 4a–d). The sugar-phosphate ring faces the intracellular side and wedges diagonally into the positively charged region of the cavity, with the 3′-5′ thiophosphate interacting with Lys411 of TM11 and the 2′-5′ thiophosphate interacting with Tyr281 of TM7 (Fig. 4d and Supplementary Fig. 11b). Moreover, the free 3′-OH is held in place by a hydrogen bond with Arg133 of TM4 (Fig. 4d and Supplementary Fig. 11b). Consistent with the orientation of the sugar-phosphate ring, the 2′-5′ side adenine base occupies a lower position and is tightly packed to the NTD by hydrophobic interactions with Leu72 of TM2 as well as Tyr126/Met130 of TM4 (Fig. 4d). On the other hand, the 3′-5′ side adenine base is located at a higher position by interacting with His289 of TM7 in the CTD and is almost perpendicular to the 2′-5′ side adenine base (Fig. 4d). The backbones of 2′3′-CDAS-bound hSLC19A1 align well with those in substrate-free and folate-bound structures, with the side chains within the cavity exhibiting reasonable adjustments to adapt different type of substrates (Supplementary Fig. 11c, d).
To verify the importance of residues involved in CDN recognition, we assessed the effect of hSLC19A1 mutants on extracellular CDN-mediated STING pathway activation by cellular assays (Fig. 4e and Supplementary Fig. 2). In SLC19A1−/− THP-1 cells stably expressing WT or mutant hSLC19A1, IFNB1 mRNA production levels induced by extracellular 2′3′-cGAMP are reduced to varying extents by representative mutations (Fig. 4e). In particular, substitutions of Arg133, His289 and Lys411 to alanine nearly abolish STING pathway activation (Fig. 4e). Direct measurement of intracellular 2′3′-cGAMP accumulation41 using the cGAMP ELISA Kit further confirms the importance of Arg133, His289 and Lys411 in CDN recognition and cellular uptake (Fig. 4f).
Although both folate/antifolate and CDN occupy the canonical substrate cavity of the outward-open hSLC19A1, their interaction details differ significantly (Fig. 4g). The pterin moiety of 5-MTHF occupies the upper negatively charged pocket within the NTD, and the glutamate moiety extends to the intracellular side and reaches the positively charged region (Fig. 4g). Despite sharing partial structural similarities with the pterin group of 5-MTHF (Supplementary Figs. 6a and 10f), the adenine bases of 2′3′-CDAS occupy lower positions and establish contacts with both domains instead of fitting into the negatively charged pocket of the NTD as folate does (Fig. 4g). Structural superimposition reveals certain overlap between the 2′-5′ side adenine base of 2′3′-CDAS and the benzoyl moiety of 5-MTHF (Fig. 4g), and therefore, not surprisingly, the two types of substrates may compete with each other in cellular uptake processes. Mutations of residues involved in the interaction of hSLC19A1 with both CDN and folate (e.g., Arg133) markedly reduce the uptake of both types of substrates (Fig. 4f, h, Supplementary Figs. 2 and 4f). However, mutations targeting CDN pocket-specific residues (e.g., His289 and Lys411) almost abolish CDN uptake while only mildly affecting folate uptake, highlighting the specific role of these residues in CDN recognition (Fig. 4f, h, Supplementary Figs. 2 and 4f). These findings lay the groundwork for the development of SLC19A1-specific inhibitors that selectively block CDN uptake without significantly affecting folate, which will be constructive in the treatment of autoimmune diseases.
As an antiporter, SLC19A1 mediates the import of folates or CDNs by coupling the export of organic anions such as ATP, ADP, AMP, 5-aminoimidazole-4-carboxamide ribotide monophosphate (ZMP), and glucose 6-phosphate (G6P)4,5. Among these, thiamine pyrophosphate (TPP) stands out as one of the preferred coupled substrates of SLC19A1, binding to inward-open SLC19A1 in a manner similar to folates (Supplementary Fig. 11e)27. To understand how SLC19A1 recognizes the exported organic anion in its outward-open conformation, we further determined the cryo-EM structure of outward-open hSLC19A1 in complex with TPP at 3.74 Å (Supplementary Fig. 11f, g and Supplementary Table 1). The pyrimidine ring of TPP aligns well with the density at the folate pocket and its interactions with SLC19A1 are similar to those of pterin ring in 5-MTHF (Supplementary Fig. 11h, i and Fig. 2h, i). However, the thiazolium ring and the pyrophosphate moiety remain unresolved due to the weak densities (Supplementary Fig. 11h). After the intracellular release of substrates in the inward-open conformation, organic anions bind to the inward-open SLC19A1, triggering the transporter to switch to its outward-open conformation, which drives the transport of folates and CDNs (Fig. 5). It is possible that the affinity of TPP for the outward-open cavity becomes weaker, facilitating the extracellular release of TPP. Interestingly, our cellular results indicate that the mutation in the folate-specific pocket (e.g., E123A) significantly reduces the uptake of both folates and CDNs (Fig. 4f, h and Supplementary Fig. 2), probably because the mutation affects the recognition of not only folates but also the coupled organic anions.
Schematic diagram of the alternating-access model for SLC19A1-mediated folate/antifolate and CDN transport. Alternating access of SLC19A1 is achieved through conformational changes alternately expose central substrate binding sites to each side of the membrane. Key residues involved in substrate recognition are highlighted.
Originally recognized as a transporter for folate and antifolate, and recently identified as an immune transmitter transporter, SLC19A1 plays a pivotal role in basal metabolism, antitumor therapy, infection and immunopathology. Although previous works have established a structural framework for understanding the interaction modes of folate/antifolate and CDN in the inward-open conformation, further elucidation of the conformational change, substrate selectivity, and precise transport mechanism of CDN remain necessary. In the current work, using an antibody-facilitated screening strategy, we successfully identified a mutation capable of fixing SLC19A1 in the outward-open state and determined the structures of SLC19A1 in the substrate-free state and in complex with different types of substrates. This work also demonstrates the potential of an antibody-facilitated mutant screening strategy in elucidating the transport cycle of other SLC family proteins.
In the outward-open conformation of SLC19A1, TM1 and TM2, as well as TM7 and TM8, diverge to reveal the extracellular entrance to the hydrophilic cavity, facilitating substrate access and binding. Hydrogen bonds of Gln150-Ala324/Val327 and Arg157-Gln377, along with hydrophobic interactions of Phe141-Ala388-Phe400, form the intracellular gates, sealing off the cavity from the intracellular side. Notably, structural superimposition reveals that Phe141, Gln150 and Arg157 undergo dramatic conformational changes during the transport cycle, which would open the intracellular gate and trigger the intracellular release of substrates. In addition to the conformational changes during the transport cycle, the 5-MTHF-, MTX- and PT523-bound structures also shed light on the molecular mechanisms underlying substrate preference for SLC19A1 (Fig. 5). Remarkably, SLC19A1 NTD forms a specific pocket at the bottom of the cavity, lined by Arg133, Tyr136, Ser137, Arg157 and Val160, to accommodate the γ-substituted hemiphthaloyl group of PT523. However, comparison with the recently determined outward-open structure of SLC46A133, another folate transporter, reveals a potentially strong steric hindrance between the cavity of SLC46A1 and the γ-carboxylate modifications of PT523. Given the different expression levels of SLC19A1 and SLC46A1 in different tumor types, the integration of current molecular insights may accelerate the development of novel antifolates targeting specific transporters with minimized side effects.
Different from inward-open structures, the 2′3′-CDAS-bound outward-open structure shows that only one CDN molecule binds within the canonical cavity of SLC19A1 in a U-shaped conformation. The detailed binding pattern of CDN differs significantly from that of folate/antifolate, despite of sharing some similarities in chemical structure (Fig. 5). Specifically, CDN is sandwiched between the two domains of SLC19A1 at the positively charged position, whereas folate and antifolate interact more prominently with the NTD, with the pterin moiety extending into the upper negatively charged pocket. Through systematic mutant analysis, we identified that mutations targeting CDN-specific residues (e.g., H289A and K411A) nearly abolish SLC19A1-mediated CDN uptake while only slightly affecting folate/antifolate transport. These results highlight the distinctive recognition strategies of SLC19A1 for different substrate types, and provide critical insights for the development of potential small molecule drugs with selective regulatory activity for CDN and folate uptake processes. Inspired by the CDN-bound outward-open cryo-EM structure, we performed molecular docking and found that CDN may also be able to bind as a monomer to the canonical cavity of inward-open SLC19A1 (Supplementary Fig. 11j). The calculated binding free energy suggests that CDN binds to this pocket with low affinity (Supplementary Fig. 11k), which probably facilitates the release of substrates into cytoplasm and also makes it challenging to capture the inward-open structure with CDN monomer bound within the canonical pocket. Taken together, the structural, cellular and computational results indicate that CDN can be transported in its monomeric form by SLC19A1 via a canonical mechanism of SLC family proteins (Fig. 5).
Considering that in our previously reported SLC19A1-CDN inward-open structures25, the compact CDN dimer binds in a non-canonical pocket near the intracellular side and probably hinders the efficient intracellular release of CDN due to stronger interactions, the CDN dimer captured in these structures may not represent an effective transport unit of SLC19A1. On the other hand, given that CDN dimer or oligomer exhibits dynamic behavior in solution and plays important roles in other biological processes42,43,44, and that CDN can bind as a dimer even at low CDN:SLC19A1 molar ratios, it is reasonable to speculate that the dimer-bound conformation may represent a substrate binding mode of SLC19A1 with potential regulatory functions. Further studies will be required to determine whether this dimeric binding state functions in different immune activation or suppression scenarios. In addition, the non-canonical dimeric binding mode may also provide an unexpected avenue and strategy for the design of new classes of SLC19A1-specific drugs (Fig. 5).
In summary, the data elucidated in this study reveal the molecular basis for the transport mechanism and substrate preference of SLC19A1, establishing the framework for the development of antineoplastic and anti-autoimmune drugs.
Monoclonal antibodies targeting human SLC19A1 (hSLC19A1) were screened using single-cell B cell receptor (BCR) sequencing of antigen-specific cells, following a previously established protocol25. Approximately 10,000 antigen specific B cells (hSLC19A1-bound CD3-B220+CD19+CD38−GL7+ B cells) were isolated from the spleens and lymph nodes of immunized mice using the BD FACSAria III cell sorter. These cells were barcoded through the 10x Chromium Single Cell platform (10x Genomics). The libraries were sequenced on the Illumina Novaseq X Plus platform in a 150 bp pair-ended manner (Berry Genomics Corporation). A total of 54 VH and VL sequences were selected, synthesized, and cloned into mammalian expression vectors with constant regions of mouse IgG2a heavy chain and κ light chain respectively. The recombinant antibodies were then transiently expressed in HEK-293T cells using lipo2000 (Thermo Fisher Scientific) according to the manufacturer's instruction. Culture supernatants were collected 24 hours post transfection. HEK-293T cells overexpressing hSLC19A1, mouse SLC19A1 (mSLC19A1) and chimeras of hSLC19A1 with specific regions replaced by mSLC19A1 were stained with the culture supernatants as the primary antibodies and PE goat anti-mouse IgG (BioLegend) as the secondary antibody. Epitope mapping of the recombinant antibodies was performed using western blot and flow cytometry on an Attune NxT Flow Cytometer (Thermo Fisher Scientific). The monoclonal antibodies (mAb70#, mAb5#, and mAb6# targeting different binding epitopes) were produced in HEK-293F Human Embryonic Kidney cells and purified using a protein A agarose prepacked column.
The DNA sequences encoding engineered mutants of hSLC19A1, designed to be locked in different conformations, were cloned into a modified pTT3 vector containing a GFP reporter gene linked with an IRES sequence. HEK-293T cells were transfected with these constructs using lipo2000 (Thermo Fisher Scientific). Following transfection, the medium was replaced with fresh DMEM supplemented with 10% (v/v) fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) after 6 hours. After 24 hours, the transfected cells were collected, labeled with hSLC19A1-specific antibodies targeting different epitopes (mAb70#, mAb5#, and mAb6#) as the primary antibodies and PE goat anti-mouse IgG (BioLegend) as the secondary antibody. Flow cytometry screening for outward-open proteins was conducted using an Attune NxT Flow Cytometer (Thermo Fisher Scientific).
The cDNA of hSLC19A1 (Uniport ID: P41440) was cloned into a modified PTT5 vector with a C-terminal 10×His tag using the ClonExpress II One Step Cloning Kit (Vazyme) for overexpressing in HEK-293F. To facilitate cryo-EM structures determination, the gene encoding BRIL fragment was fused to the N-terminus of TM1 of hSLC19A1 and the gene encoding anti-Fab nanobody was fused into the C-terminus of TM12 by a helical linker (AEEEKRKAEEEKRK). All mutants were generated with a standard PCR-based strategy. To produce hSLC19A1 protein, 0.5 mg hSLC19A1 plasmids were pre-incubated with 1.5 mg PEI in 50 mL Opti-MEM medium at 37 °C. After 20 mins, the mixture was added drop by drop into 500 mL HEK-293F cells at a density of 2 ~ 2.5 × 106 cells/mL. The transfected cells were cultured for 72 hours before harvesting by centrifugation. Harvested HEK-293F cells were resuspended in lysis buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10 mM imidazole, 5% glycerin and 1 mM PMSF. The cells were disrupted by sonication, and 1% (w/v) n-dodecyl-b-D-maltoside (DDM, Anatrace) and 0.1% (w/v) cholesteryl hemisuccinate (Anatrace) were added for protein solubilizing at 4 °C for 2 hours. After centrifugation (30,000 × g, 30 mins, 4 °C), the supernatant was incubated with nickel affinity resin at 4 °C for 1 hour, and then the resin was washed with the washing buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 60 mM imidazole, 0.05% (w/v) n-dodecyl-b-D-maltoside (DDM, Anatrace) and 0.005% (w/v) cholesteryl hemisuccinate (Anatrace) and 5% glycerin. The protein was eluted with 50 mM HEPES pH 7.5, 300 mM NaCl, 400 mM imidazole, 0.05% (w/v) n-dodecyl-b-D-maltoside (DDM, Anatrace) and 0.005% (w/v) cholesteryl hemisuccinate (Anatrace) and 5% glycerin. The eluted protein was further purified through a Superdex 200 Increase 10/300 GL column (GE Healthcare) in the SEC buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, 0.01% (w/v) GDN. Peak fractions were collected and concentrated to ~2 mg/mL.
For complex assembly, the purified hSLC19A1EM and anti-BRIL Fab were mixed on ice at a molar ratio of 1:1.1. After incubation for 20 mins, the mixture was subjected to a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated with SEC buffer (20 mM HEPES pH 7.5, 150 mM NaCl and 0.01% GDN) to remove the excess anti-Bril Fab. For nanodisc reconstitution, hSLC19A1EM, MSP1D1, POPG and anti-BRIL Fab were mixed at a molar ratio of 1:2.1:84:1.1. Detergents were removed by 100 mg/mL Bio-beads with gentle agitation overnight. Subsequently, the Bio-beads were removed and the nanodisc reconstitution mixture was loaded onto a Superdex 200 Increase 10/300 GL column equilibrated with 20 mM HEPES pH 7.5 and 150 mM NaCl. Peak fractions were analyzed by SDS-PAGE and then concentrated for cryo-EM sample preparation.
For apo hSLC19A1 samples, the peak fractions of hSLC19A1EM-Fab in GDN (20 mM HEPES pH 7.5, 150 mM NaCl and 0.01% GDN) were concentrated to 8.4 mg/mL. For substrate-bound samples, the peak fractions of the SLC19A1EM-Fab in nanodisc (20 mM HEPES pH 7.5 and 150 mM NaCl) were concentrated to 10.7 mg/mL and separately incubated with 5 mM 5-MTHF, 8 mM MTX, 5 mM PT523, 3 mM 2'3'-CDAS and 5 mM TPP for 3 h. 3 μL of the protein sample was deposited onto the glow-discharged holey carbon, 300 mesh R1.2/1.3 Au grids (Quantifoil). Subsequently, the grids were blotted for 2.5 s and plunged into liquid ethane for quick freezing using the Vitrobot Mark IV (Thermo Fisher Scientific). All cryo-EM datasets were collected under the dose rate of 9.2 e− Å−2 s−1 and dose-fractioned into 32 frames, yielding a total dose of 60 e− Å−2 by SerialEM45 on the Talos Arctica 200 kV FEG (Thermo Fisher Scientific) with a K2 summit direct electron elector (Gatan) and a GIF quantum energy filter (Gatan). The pixel size was calibrated at 0.5 Å (×130,000) under super-resolution mode, with the defocus ranging from −0.8 to −1.5 μm. Images were recorded using beam–image shift data collection methods46.
3301 micrographs were collected for outward-open apo-hSLC19A1 structure dataset. Beam-induced motion correction and dose weighting were performed with MotionCor247. Corrected micrographs were then imported into cryoSPARC48 for contrast transfer function (CTF) estimation with CTFFIND449. A total of 2,001,272 particles were picked and extracted. After two rounds of 2D classification, 854,517 particles with clear features were picked and subjected to the Ab initio reconstruction. The following two rounds of heterogeneous refinement yielded 345,903 good particles which resulted in a reconstruction with an overall resolution of 3.26 Å. The particles were then imported into RELION50 for Bayesian polishing, followed by non-uniform refinement and local refinement in cryoSPARC, which yielded a final reconstruction to 2.94 Å resolution. 5-MTHF-bound (6,961 micrographs; 220,166 particles), MTX-bound (2,333 micrographs; 97,106 particles), PT523-bound (2,082 micrographs; 94,583 particles), 2′3′-CDAS-bound (5,257 micrographs; 301,880 particles) and TPP-bound (1,389 micrographs; 50,812 particles) datasets were processed using a similar procedure and yielded reconstructions at 3.43 Å, 3.44 Å, 3.25 Å, 3.34 Å and 3.74 Å respectively. The local resolution estimation was performed to determine the local resolution of the final map in cryoSPARC.
The inward-open hSLC19A1 (PDB: 7XPZ)25 was used as initial template for model building. The NTD and CTD were roughly fitted into the hSLC19A1 outward-open maps by UCSF Chimera51. After manually adjustment by Coot52, the resulting model was further refined in PHENIX53. The atomic model of ligand-bound hSLC19A1 was generated by several rounds of real space refinement in COOT and PHENIX with the apo outward-open hSLC19A1 as the initial model. 5-MTHF, MTX, PT523, 2′3′-CDAS or TPP was fitted into the density using COOT, then the resulting model was manually rebuilt in COOT and further refined in PHENIX. The models were validated through evaluation of the Clash scores, MolProbity scores and statistics of the Ramachandran plots by PHENIX. All the structural figures were prepared by PyMOL54 and UCSF ChimeraX55.
The experiments were carried out as previously described in ref. 25. In brief, the DNA sequence encoding human SLC19A1 or its mutants was cloned into a lentiviral plasmid containing a GFP reporter gene connected by a 2 A sequence. The mutant-carrying lentiviruses were produced in HEK-293T cells and used to infect SLC19A1−/− Hela or SLC19A1−/− THP-1 cells in the presence of 8 μg/ml polybrene (Sigma-Aldrich). After 72 hours of culture, lentiviral-infected cells showing similar levels of GFP expression were isolated using a BD FACSAria III cell sorter (BD Biosciences).
The cellular [3H]-labelled antifolate uptake assay was performed under published protocols25,56. The cell lines were generated as mentioned above. For uptake assays, 2 × 105 WT or SLC19A1 mutant cells per well were seeded into 24-well plates. After 12 h growth, the cells were washed twice with 500 µL HBSS buffer at pH 7.4 (Pricella). The buffer contains 140 mg/L CaCl2, 100 mg/L MgCl2•6H2O, 100 mg/L MgSO4•7H2O, 350 mg/L NaHCO3 and 1000 mg/L D-glucose. The cells were then incubated with 200 µL HBSS buffer supplemented with 25 nM [3H]-labelled methotrexate (American Radiolabeled Chemicals) for 10 mins. The uptake was terminated and washed twice by 500 µL ice-cold HBSS buffer. Lysed by 200 µL 0.2 M NaOH, the amount of accumulated [3H]-labelled methotrexate was calculated by scintillation counting in Ultima Gold (Perkin Elmer). For measurement of the initial uptake rates, the amount of accumulated [3H]-labelled methotrexate was calculated at 0.5 min, 1 min, 1.5 min and 2 min respectively.
The experiments were according to established protocols25. Briefly, SLC19A1−/− THP-1 cells stably expressing either wild-type or mutant hSLC19A1 were seeded in a 24-well plate at a density of 3 × 105 cells per well in 400 µL medium. The cells were then stimulated with 30 μM 2′3′-cGAMP for 3 hours. Cells were collected and total RNAs were extracted using TRIzol reagent (Thermo Fisher Scientific). Genomic DNA was removed by DNase I treatment (RQ1, Promega). Reverse transcription was carried out using M-MLV Reverse Transcriptase (Promega). Real-time PCR was performed with 1x PowerUp™ SYBR™ Green Master Mix (Applied Biosystems) according to the manufacturer's instructions. The primers used were as follows: β-actin, 5'-ACCGAGCGCGGCTACAG-3' and 5'-CTTAATGTCACGCACGATTTCC-3'; IFNB1: 5'-GTCTCCTCCAAATTGCTCTC-3' and 5'-ACAGGAGCTTCTGACACTGA-3'. Samples were analyzed on a QuantStudio Q7 (Applied Biosystems).
SLC19A1−/− Hela cells stably expressing either wild-type or mutant hSLC19A1 were seeded into 24-well plates at a density of 3 × 105 cells per well and cultured for 12 hours. After being washed twice with PBS, the cells were exposed to fresh DMEM complete medium containing 50 μM cGAMP for 4 hours. Following this, the cells were washed five times with PBS, lysed in 100 μL of RIPA buffer (150 mM NaCl, 1% Triton-X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0) on ice for 15 mins, and then centrifuged to remove insoluble material before cGAMP quantification. The intracellular cGAMP levels were measured using the Direct 2'3'-Cyclic GAMP ELISA Kit (Arbor Assays).
The binding modes between hSLC19A1 and 2'3'-cGAMP were determined by using molecular docking method. Structural models of inward-open (PDB: 7XPZ) and outward-open (PDB: 9JOZ) hSLC19A1 were set to be rigid in the docking process. The ligand 2'3'-cGAMP was set to be flexible. The docking process was accomplished by using the AutoDock Vina software and performed within 25 Å × 25 Å × 25 Å cubes centered on the center points of the chosen canonical substrate binding pocket with exhaustiveness set to 32. The results were ranked based on the lowest binding energy. Structural drawings were generated using PyMol.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
The data that support this study are available from the corresponding authors upon request. The cryo-EM maps have been deposited into the Electron Microscopy Data Bank under accession numbers EMD-61690 (outward-open hSLC19A1), EMD-61756 (outward-open hSLC19A1 + 5-MTHF), EMD-61758 (outward-open hSLC19A1+MTX), EMD-61759 (outward-open hSLC19A1 + PT523), EMD-61760 (outward-open hSLC19A1 + 2'3'-CDAS) and EMD-61761 (outward-open hSLC19A1+TPP). The coordinates have been deposited into the Protein Data Bank under accession numbers PDB: 9JOZ (outward-open hSLC19A1), PDB: 9JRI (outward-open hSLC19A1 + 5-MTHF), PDB: 9JRK (outward-open hSLC19A1+MTX), PDB: 9JRL (outward-open hSLC19A1 + PT523), PDD: 9JRM (outward-open hSLC19A1 + 2'3'-CDAS) and PDB: 9JRN (outward-open hSLC19A1+TPP). A source Data file is included with this manuscript. Source data are provided with this paper.
Zheng, Y. & Cantley, L. C. Toward a better understanding of folate metabolism in health and disease. J. Exp. Med. 216, 253–266 (2019).
PubMed
PubMed Central
MATH
Google Scholar
Clare, C. E., Brassington, A. H., Kwong, W. Y. & Sinclair, K. D. One-carbon metabolism: linking nutritional biochemistry to epigenetic programming of long-term development. Annu. Rev. Anim. Biosci. 7, 263–287 (2019).
PubMed
Google Scholar
Matherly, L. H., Hou, Z. & Deng, Y. Human reduced folate carrier: translation of basic biology to cancer etiology and therapy. Cancer metastasis Rev. 26, 111–128 (2007).
PubMed
MATH
Google Scholar
Hou, Z. & Matherly, L. H. Biology of the major facilitative folate transporters SLC19A1 and SLC46A1. Curr. Top. Membr. 73, 175–204 (2014).
PubMed
PubMed Central
Google Scholar
Matherly, L. H. & Hou, Z. Structure and function of the reduced folate carrier: a paradigm of a major facilitator superfamily mammalian nutrient transporter. Vitam. hormones 79, 145–184 (2008).
Google Scholar
Goldman, I. D., Chattopadhyay, S., Zhao, R. & Moran, R. The antifolates: evolution, new agents in the clinic, and how targeting delivery via specific membrane transporters is driving the development of a next generation of folate analogs. Current opinion in investigational drugs (London, England: 2000) 11, 1409–1423 (2010).
Luteijn, R. D. et al. SLC19A1 transports immunoreactive cyclic dinucleotides. Nature 573, 434–438 (2019).
ADS
PubMed
PubMed Central
Google Scholar
Ritchie, C., Cordova, A. F., Hess, G. T., Bassik, M. C. & Li, L. SLC19A1 is an importer of the immunotransmitter cGAMP. Mol. cell 75, 372–381. e375 (2019).
PubMed
PubMed Central
Google Scholar
Danilchanka, O. & Mekalanos, J. J. Cyclic dinucleotides and the innate immune response. Cell 154, 962–970 (2013).
PubMed
PubMed Central
MATH
Google Scholar
Zaver, S. A. & Woodward, J. J. Cyclic dinucleotides at the forefront of innate immunity. Curr. Opin. cell Biol. 63, 49–56 (2020).
PubMed
PubMed Central
MATH
Google Scholar
Zhang, X. et al. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol. cell 51, 226–235 (2013).
PubMed
MATH
Google Scholar
Gao, P. et al. Structure-function analysis of STING activation by c [G (2′, 5′) pA (3′, 5′) p] and targeting by antiviral DMXAA. Cell 154, 748–762 (2013).
PubMed
PubMed Central
MATH
Google Scholar
Shang, G., Zhang, C., Chen, Z. J., Bai, X.-C. & Zhang, X. Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP–AMP. Nature 567, 389–393 (2019).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Wu, J. et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339, 826–830 (2013).
ADS
PubMed
MATH
Google Scholar
Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z. J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786–791 (2013).
ADS
PubMed
MATH
Google Scholar
Gao, P. et al. Cyclic [G (2′, 5′) pA (3′, 5′) p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase. Cell 153, 1094–1107 (2013).
PubMed
PubMed Central
MATH
Google Scholar
Burdette, D. L. et al. STING is a direct innate immune sensor of cyclic di-GMP. Nature 478, 515–518 (2011).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Diner, E. J. et al. The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING. Cell Rep. 3, 1355–1361 (2013).
PubMed
PubMed Central
MATH
Google Scholar
Fu, J. et al. STING agonist formulated cancer vaccines can cure established tumors resistant to PD-1 blockade. Sci. Transl. Med. 7, 283ra252–283ra252 (2015).
Google Scholar
Mekers, V. E., Kho, V. M., Ansems, M. & Adema, G. J. cGAS/cGAMP/STING signal propagation in the tumor microenvironment: key role for myeloid cells in antitumor immunity. Radiother. Oncol. 174, 158–167 (2022).
Xie, W. & Patel, D. J. Structure-based mechanisms of 2' 3'-cGAMP intercellular transport in the cGAS–STING immune pathway. Trends Immunol. 44, 50–467 (2023).
Matherly, L. H. & Hou, Z. Folate transporter offers clues for anticancer drugs. Nature 612, 39–41 (2022).
ADS
PubMed
PubMed Central
Google Scholar
Hou, Z., Ye, J., Haska, C. L. & Matherly, L. H. Transmembrane domains 4, 5, 7, 8, and 10 of the human reduced folate carrier are important structural or functional components of the transmembrane channel for folate substrates. J. Biol. Chem. 281, 33588–33596 (2006).
PubMed
Google Scholar
Quistgaard, E. M., Löw, C., Guettou, F. & Nordlund, P. Understanding transport by the major facilitator superfamily (MFS): structures pave the way. Nat. Rev. Mol. Cell Biol. 17, 123–132 (2016).
PubMed
Google Scholar
Zhang, Q. et al. Recognition of cyclic dinucleotides and folates by human SLC19A1. Nature 612, 170–176 (2022).
ADS
PubMed
MATH
Google Scholar
Wright, N. J. et al. Methotrexate recognition by the human reduced folate carrier SLC19A1. Nature 609, 1056–1062 (2022).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Dang, Y. et al. Molecular mechanism of substrate recognition by folate transporter SLC19A1. Cell Discov. 8, 141 (2022).
PubMed
PubMed Central
MATH
Google Scholar
Liu, X. Y., Witt, T. L. & Matherly, L. H. Restoration of high-level transport activity by human reduced folate carrier/ThTr1 thiamine transporter chimaeras: role of the transmembrane domain 6/7 linker region in reduced folate carrier function. Biochem. J. 369, 31–37 (2003).
PubMed
PubMed Central
Google Scholar
Liu, M. et al. Transcriptional regulation of the human reduced folate carrier in childhood acute lymphoblastic leukemia cells. Clin. cancer Res. 12, 608–616 (2006).
PubMed
MATH
Google Scholar
Ifergan, I., Meller, I., Issakov, J. & Assaraf, Y. G. Reduced folate carrier protein expression in osteosarcoma: implications for the prediction of tumor chemosensitivity. Cancer 98, 1958–1966 (2003).
PubMed
MATH
Google Scholar
Qiu, A. et al. Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption. Cell 127, 917–928 (2006).
PubMed
MATH
Google Scholar
Zhao, R., Aluri, S. & Goldman, I. D. The proton-coupled folate transporter (PCFT-SLC46A1) and the syndrome of systemic and cerebral folate deficiency of infancy: hereditary folate malabsorption. Mol. Asp. Med. 53, 57–72 (2017).
Google Scholar
Parker, J. L. et al. Structural basis of antifolate recognition and transport by PCFT. Nature 595, 130–134 (2021).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Zhao, R. et al. The proton-coupled folate transporter: impact on pemetrexed transport and on antifolates activities compared with the reduced folate carrier. Mol. Pharmacol. 74, 854–862 (2008).
PubMed
MATH
Google Scholar
Newstead, S. Structural basis for recognition and transport of folic acid in mammalian cells. Curr. Opin. Struct. Biol. 74, 102353 (2022).
PubMed
PubMed Central
MATH
Google Scholar
Rhee, M. S., Galivan, J., Wright, J. E. & Rosowsky, A. Biochemical studies on PT523, a potent nonpolyglutamatable antifolate, in cultured cells. Mol. Pharmacol. 45, 783–791 (1994).
PubMed
Google Scholar
Rosowsky, A., Wright, J. E., Vaidya, C. M. & Forsch, R. A. The effect of side-chain, para-aminobenzoyl region, and B-ring modifications on dihydrofolate reductase binding, influx via the reduced folate carrier, and cytotoxicity of the potent nonpolyglutamatable antifolate Nα-(4-amino-4-deoxypteroyl)-Nδ-hemiphthaloyl-l-ornithine. Pharmacol. Therapeutics 85, 191–205 (2000).
Google Scholar
Deng, Y. et al. Role of lysine 411 in substrate carboxyl group binding to the human reduced folate carrier, as determined by site-directed mutagenesis and affinity inhibition. Mol. Pharmacol. 73, 1274–1281 (2008).
PubMed
MATH
Google Scholar
Wang, J. et al. Glucose transporter inhibitor-conjugated insulin mitigates hypoglycemia. Proc. Natl Acad. Sci. 116, 10744–10748 (2019).
ADS
PubMed
PubMed Central
Google Scholar
Wang, N. et al. Molecular basis for inhibiting human glucose transporters by exofacial inhibitors. Nat. Commun. 13, 2632 (2022).
ADS
PubMed
PubMed Central
MATH
Google Scholar
Maltbaek, J. H., Cambier, S., Snyder, J. M. & Stetson, D. B. ABCC1 transporter exports the immunostimulatory cyclic dinucleotide cGAMP. Immunity 55, 1799–1812.e1794 (2022).
PubMed
PubMed Central
Google Scholar
Zhang, Z., Kim, S., Gaffney, B. L. & Jones, R. A. Polymorphism of the signaling molecule c-di-GMP. J. Am. Chem. Soc. 128, 7015–7024 (2006).
PubMed
PubMed Central
MATH
Google Scholar
Chan, C. et al. Structural basis of activity and allosteric control of diguanylate cyclase. Proc. Natl Acad. Sci. 101, 17084–17089 (2004).
ADS
PubMed
PubMed Central
Google Scholar
Tschowri, N. et al. Tetrameric c-di-GMP mediates effective transcription factor dimerization to control Streptomyces development. Cell 158, 1136–1147 (2014).
PubMed
PubMed Central
MATH
Google Scholar
Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).
PubMed
MATH
Google Scholar
Wu, C., Huang, X., Cheng, J., Zhu, D. & Zhang, X. High-quality, high-throughput cryo-electron microscopy data collection via beam tilt and astigmatism-free beam-image shift. J. Struct. Biol. 208, 107396 (2019).
PubMed
Google Scholar
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. methods 14, 331–332 (2017).
PubMed
PubMed Central
MATH
Google Scholar
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. methods 14, 290–296 (2017).
PubMed
Google Scholar
Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
PubMed
PubMed Central
MATH
Google Scholar
Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. elife 7, e42166 (2018).
PubMed
PubMed Central
MATH
Google Scholar
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Computational Chem. 25, 1605–1612 (2004).
MATH
Google Scholar
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. Sect. D: Biol. Crystallogr. 66, 486–501 (2010).
ADS
MATH
Google Scholar
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. Sect. D: Biol. Crystallogr. 66, 213–221 (2010).
ADS
MATH
Google Scholar
Schrödinger, L. The PyMOL Molecular Graphics System, Version 1.8. (No Title) (2015).
Goddard, T. D. et al. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).
PubMed
MATH
Google Scholar
Visentin, M., Zhao, R. & Goldman, I. D. Augmentation of reduced folate carrier-mediated folate/antifolate transport through an antiport mechanism with 5-aminoimidazole-4-carboxamide riboside monophosphate. Mol. Pharmacol. 82, 209–216 (2012).
PubMed
PubMed Central
Google Scholar
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Cryo-EM data collection was carried out at the Center for Biological Imaging, Core Facilities for Protein Science at the Institute of Biophysics, Chinese Academy of Sciences. The computation work was performed using High-performance computing resources, the Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Science. All radioactivity experiments were performed at the radioactive isotope laboratory (Institute of Biophysics, CAS), with guidance from H.J. Zhang in handling radioactive materials. The plasmid expressing membrane scaffold protein 1D1 (MSP1D1) was a gift from Dr. Z. Liu's group. We also thank Cryo-Electron Microscopy Platform of Medical Science and Technology Innovation Center of Shandong First Medical University for the support of Cryo-EM data collection. This work was supported by grants from National Natural Science Foundation of China (32325028 & 32130057 to P.G., 32171219 to A.G., 32401005 to Q.Z., 82371853 to X.Z.), National Key R&D Program of China (2024YFA1307400 to P.G.), Beijing Natural Science Foundation (Z220018 to P.G.), Basic Research Program Based on Major Scientific Infrastructures-CAS (JZHKYPT-2021-05 to P.G.), CAS Project for Young Scientists in Basic Research (YSBR-074 to P.G.), and China Postdoctoral Science Foundation (2023M740257 & BX20230468 to Q.Z.).
These authors contributed equally: Qixiang Zhang, Xuyuan Zhang, Kexin Liu.
Key Laboratory of Molecular Medicine and Biotherapy, Aerospace Center Hospital, School of Life Science, Beijing Institute of Technology, Beijing, China
Qixiang Zhang, Yalan Zhu & Ang Gao
National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
Xuyuan Zhang, Kexin Liu, Xiaohua Nie, Junxiao Ma, Panpan Sun, Zhaolong Li, Yina Gao, Songqing Liu, Liguo Zhang & Pu Gao
University of Chinese Academy of Sciences, Beijing, China
Kexin Liu, Junxiao Ma, Panpan Sun, Zhaolong Li, Liguo Zhang & Pu Gao
Science and Technology Innovation Center, Shandong First Medical University, Shandong Academy of Medical Sciences, Jinan, China
Ang Gao & Pu Gao
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Q.Z. and K.L. purified proteins, prepared cryo-EM samples, collected and processed cryo-EM data, and reconstructed density maps. Y.G. assisted with cryo-EM data processing. Q.Z. and P.G. built and refined models. X.Z. and Q.Z. performed antibody screening and validation. X.Z., Q.Z. and K.L. performed cellular assays. Y.Z., X.N., and J.M. assisted with antibody screening. P.S. and S.L. assisted with cell culture and protein expression. Z.L. performed molecular docking assay. P.G., L.Z., and A.G. initiated the project and directed the research. P.G. and Q.Z. wrote the manuscript with help of all authors.
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Ang Gao, Liguo Zhang or Pu Gao.
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Zhang, Q., Zhang, X., Liu, K. et al. Molecular basis of SLC19A1-mediated folate and cyclic dinucleotide transport.
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There is active crosstalk between tumor cells and the tumor microenvironment during metastatic progression, a process that is significantly affected by obesity, particularly in breast cancer. Here we analyze the impact of a high fat diet (HFD) on metastasis, focusing on the role of platelets in the formation of premetastatic niches (PMNs). We find that a HFD provokes pre-activation of platelets and endothelial cells, promoting the formation of PMNs in the lung. These niches are characterized by increased vascular leakiness, platelet activation and overexpression of fibronectin in both platelets and endothelial cells. A HFD promotes interactions between platelets, tumor cells and endothelial cells within PMNs, enhancing tumor cell homing and metastasis. Importantly, therapeutic interventions like anti-platelet antibody administration or a dietary switch reduce metastatic cell homing and outgrowth. Moreover, blocking fibronectin reduces the interaction of tumor cells with endothelial cells. Importantly, when coagulation parameters prior to neoadjuvant treatment are considered, triple negative breast cancer (TNBC) female patients with reduced Partial Thromboplastin time (aPTT) had a significantly shorter time to relapse. These findings highlight how diet and platelet activation in pre-metastatic niches affect tumor cell homing and metastasis, suggesting potential therapeutic interventions and prognostic markers for TNBC patients.
Metastasis is a complex process in which cancer cells must adapt to the diverse microenvironments they encounter as they disseminate to distant tissues1,2. In recent decades, it has become clear that tumor cell metastasis depends both on intrinsic alterations to tumor cells and extrinsic effects of the tumor microenvironment (TME)3. For example, it is known that obesity contributes to the progression of several cancer types, including endometrial, ovarian, pancreatic, colon and breast cancers (BCs)4,5. In addition, obesity is correlated to poor outcomes of BC in postmenopausal women6 and in triple-negative breast cancer (TNBC) patients7. As well as affecting primary BC tumor growth, obesity enhances the metastasis of these cells to the lungs in a manner that is dependent on neutrophils8,9, involving vascular dysfunction and increased endothelial transmigration of the tumor cells10. Moreover, obesity also induces chronic inflammation, while enhancing pro-thrombotic signaling in both platelets and endothelial cells11,12, and promoting a state of hypercoagulability in cancer patients13. Notably, exacerbated coagulation is related to worse patient survival in a variety of tumor types, underscoring the pro-metastatic role of platelets14.
Platelets have been implicated in tumor progression through several mechanisms, as they can interact with different cell types like leukocytes, or stromal, endothelial and cancer cells15,16. Indeed, platelets surrounding circulating tumor cells play an important role in tumor cell survival and metastasis, impeding their recognition by immune cells17,18,19. Hence, defining the precise mechanisms involved in the tumor-platelet-endothelial cell interactions is of particular interest. Platelets have also been implicated in the early metastatic dissemination of colon cancer and melanoma20,21. Upon activation, platelets release growth factors, cytokines and extracellular vesicles that help remodel the extracellular matrix (ECM) and that are involved in the recruitment of other cells to the metastatic organs20,21. However, the precise influence of extrinsic factors like a high fat diet (HFD) on platelet activation and the formation of pre-metastatic niches (PMNs)22 remains unclear and warrants further investigation.
Here we show that a HFD promotes the generation of PMNs in distal organs, aiding metastatic cell homing. Our data demonstrate that a HFD contributes to platelet activation, and that it enhances vascular permeability and fibronectin (FN) overexpression in the lungs. Moreover, we find that adipose tissue in HFD-fed mice secretes high levels of TNF-α, which leads to increased FN levels and its subsequent accumulation in platelets. Notably, platelets from HFD-fed mice exhibit greater interactions with tumor cells within PMNs, reinforcing the tumor-endothelial-platelet cell axis. Furthermore, a HFD exacerbates tumor cell homing and metastasis, yet only in highly-metastatic BC models, suggesting that both intrinsic and extrinsic factors are crucial for metastatic success. Our results suggest that therapeutic interventions, such as anti-platelet antibody administration or dietary modifications, reduce metastatic cell homing and outgrowth, underscoring the potential of dietary and platelet-modulating strategies in influencing disease outcomes. Additionally, blocking FN reduces platelet-mediated interactions between tumor cells and endothelial cells, highlighting its pivotal role in this process. Finally, in triple-negative breast cancer (TNBC) patients, we observe a correlation between the body mass index (BMI) and higher pro-coagulant activity. Patients with a shorter activated Partial Thromboplastin time (aPTT) show increased risk and lower survival rates over three years post-treatment, suggesting a short aPTT as a risk factor in TNBC prior to surgery. Collectively, our findings reveal the impact of HFD on platelet activation, PMN formation, and metastasis in TNBC, offering potential therapeutic targets and prognostic markers for cancer management.
In this study, a diet-induced obesity (DIO) model was chosen to assess how a HFD might affect PMN formation in pre-metastatic lungs (Suppl. Figure 1A), initially verifying that the HFD produced a weight gain in these mice over the 12week study period (Suppl. Figure 1B-E). The impact of the HFD on platelet activation was assessed and since platelet activation results in dense granule secretion and a conformational change of integrin αIIbβ323,24, these parameters were analyzed in mice by flow cytometry using anti-GPIIb/IIIa (JON/A) and anti-P-selectin (CD62P) antibodies (Fig. 1A). Interestingly, the platelets from HFD mice were more susceptible to activation by exposure to thrombin for 5 or 15 minutes relative to those from ND mice (Fig. 1A, B). In terms of dense granule secretion in response to thrombin stimulation, ATP luminometry revealed a significant increase in such release by platelets from HFD mice relative to those from ND mice (Fig. 1C). Since fibrinogen (Fg) binding to platelets promotes their spreading, an important step in the amplification process25, we analyzed this phenomenon in mice. Enhanced Fg adhesion was evident in platelets from HFD mice (Fig. 1D), coupled to significantly accelerated spreading (Fig. 1E-G). There was a particular enrichment in the filopodial (Fp, red bars, Fig. 1G) and spread phenotypes (Sp, green bars, Fig. 1G), for which cytoskeletal reorganization is crucial. Moreover, the HFD exacerbated platelet aggregation (Fig. 1 H), demonstrating that a HFD promotes both platelet activation and aggregation.
A Analysis of platelet activation by flow cytometry, measuring the expression of activated αIIbβ3 integrin and CD62P in both the presence (Activated platelets) and absence (Resting platelets) of thrombin (0.05 IU/ml). B Graph showing the quantification of the experiment shown in (A), at the times indicated. 5 independent experiments: ND n = 4, HFD n = 5 platelet samples per group. C Platelets from ND or HFD mice were incubated with the luciferase-luciferin reagent and ATP release was measured in a luminometer upon thrombin stimulation. 2 independent experiments: n = 3 platelet samples per group. D Quantitative analysis of the relative platelet adhesion to fibrinogen (%) over time. 4 independent experiments: n = 4 platelet samples per group. E Spreading of platelets on fibrinogen. Platelets were stained with α-tubulin (red) and phalloidin (green). Bar: 5 μm. F Quantification of platelet morphology after thrombin activation on fibrinogen for 60 minutes. Representative images of platelet morphology are shown: discoid (Ds), filopodial (Fp), pseudopodial (Pp), lamellipodia (Lp), and spread (Sp). Bar: 5 μm. The plot below indicates the proportion of cells in each category ( > 150 cells per experiment), showing the mean ± SEM of each morphology. G Detail of the proportion of platelets with a Fp (red bars) and Sp morphology (green bars). 2 independent experiments: n = 9 images per group. H Analysis of platelet aggregation in ND and HFD mice, assessing their response to thrombin (0.01 IU/ml). 3 independent experiments: ND n = 9, HFD n = 10 mice. I-M) Western blots of (I) phospho Src/Src, J phospho PKC/Actin, K phospho AKT/AKT, L phospho FAK/FAK and (M) phospho Talin/Talin in platelets isolated from ND (Upper panels) or HFD (Lower panels) mice, untreated (0) or activated with thrombin for the times indicated. The data corresponds to 3 independent experiments: ND n = 9, and HFD n = 10. All the data represent the mean ± s.d. and the P values were calculated two-way ANOVA (C, D, H) or with a two-tailed Student's t-test (B, G).
To gain further insights into the mechanisms driving platelet activation and aggregation in response to thrombin, signaling activation in platelets from ND and HFD mice was analyzed at different time points after treatment (Fig. 1I–M, Suppl. Figures 1F–J). Platelets from HFD mice displayed sustained Src phosphorylation (Fig. 1I, Suppl. Figure 1F), which is essential to initiate and propagate signals from αIIbβ3 through the “inside-out” signaling pathway26,27. A similar effect was observed on protein kinase C (PKC: Fig. 1J, Suppl. Figure 1G), which has been shown to play a major role in inducing platelet granule secretion28,29. Moreover, at later times there was enhanced AKT phosphorylation ((Fig. 1K, Suppl. Figure 1H), previously implicated in α-granule secretion30), FAK (Fig. 1L, Suppl. Figure 1I) and Talin (Fig. 1M, Suppl. Figure 1J), both Src substrates involved in cell adhesion and cytoskeletal reorganization in platelets31.
In the light of all these data, our results suggest that a HFD promotes the activation of integrin αIIbβ3 through the ‘inside-out' signaling pathway, followed by ‘outside-in' downstream signaling32,33 that leads to platelet activation, cytoskeletal reorganization and platelet spreading.
Platelets have been implicated in early metastasis in mice20, yet the impact of platelets and a HFD on PMN formation has been little explored. To assess the effect of HFD on PMN formation, platelet recruitment to the lungs of mice was analyzed. Interestingly, significantly enhanced P-selectin expression was evident in the lungs of HFD mice relative to the controls (Fig. 2A, B), showing a higher presence of platelets in the metastatic organs of HFD mice. Since obesity-associated inflammation is involved in endothelial cell disfunction34, we also studied the impact of a HFD on lung endothelial cell leakiness. Vascular leakiness was assessed by injecting fluorescent dextran into HFD or ND mice, which underwent greater extravasation in the former (Fig. 2C, D). Whether this leakiness was due to high adiposity, or the diet itself was explored in ob/ob mice35,36. These ob/ob mice also exhibited significantly enhanced vascular permeability in the lungs relative to wild type (WT) mice (Suppl. Figures 2A, 2B), demonstrating the increase in vascular leakiness regardless of the model assessed.
A Representative images of P-Selectin staining in the lungs of ND and HFD-fed mice. Bottom panels are magnifications of the boxed area. Bar: 50 μm (B) Quantitative analysis of P-Selectin staining in the lungs of ND and HFD mice. 2 independent experiments: ND n = 9 fields, HFD n = 10 fields per group. C Representative images of the vascular permeability in the lungs of ND and HFD mice visualized with NIR-Dextran. D Quantification of (C). 3 independent experiments: n = 11 mice per group. E Heatmap of genes in endothelial cells from ND and HFD mice based on RNA-seq data: n = 3 mice per group. F Correlation between the RNA-seq data and the proteomic data from ND and HFD mice (FDR < 0.05) and fold change. G Representative images of FN1 staining in the lungs of ND and HFD mice. Bottom panels are magnifications of the boxed area. Bar: 100 μm. H Quantification of G. 2 independent experiments: n = 14 fields per group. I Representative Western blot of FN1 in whole lung extracts comparing non-perfused and perfused lungs from both ND and HFD mice. J Quantification of the experiments in I. Data correspond to 2 independent experiments ND (n = 8 perfused and n = 8 non-perfused) and HFD (n = 8 perfused and n = 7 non-perfused). Representative Western blot of FN1 in plasma (K) and platelets (L) isolated from ND and HFD mice. M, N Quantification of the experiments in K and L respectively. 2 independent experiments, M (ND n = 7, HFD n = 8), N (ND n = 7, HFD n = 6) platelet samples per group. O) Analysis of platelet fibronectin uptake by flow cytometry expressed as total counts (P) or MFI (Q). The data corresponds to 4 independent experiments: n = 3 platelet samples per group. All the data represent the mean ± s.d., and the P values were calculated with a two-tailed Student's t-test (B, D, H, M, N, P, Q) or Mann-Whitney test (J). Values are shown in box‐and‐whisker plots where the line in the box corresponds to the mean. Boxes extend from the minimum to the maximum value.
We next explored the impact of the HFD on lung endothelial cells by analyzing gene expression in CD45−CD31+ lung endothelial cells isolated from the mice by RNA-sequencing (RNA-seq) (Suppl. Figure 2C, Suppl. Data 1). A gene set enrichment analysis (GSEA) identified signatures of enrichment related to the inflammatory response, cell adhesion and hemostasis, amongst other gene pathways (Suppl. Figure 2D, Suppl. Data 1). We found that three of the top ten pathways were enriched in genes related to cell adhesion (Jam2, Ocln, Dsp, Panx1), inflammatory responses (Ikbkb, Tlr3, Cmip) and hemostasis (Plaur, Plat, Fn1) (Suppl. Figure 2D, Table 1). To identify common pathways affected in both platelets and endothelial cells isolated from HFD mice we integrated the RNA expression data from endothelial cells and the proteomic analyzes of platelets-derived from mice fed with either diet (Fig. 2F). Out of 2916 candidate genes modulated, we identified 6 that were upregulated in both endothelial cells and platelets from HFD mice, with a FDR < 0.05 (Fig. 2F). Among these, fibronectin (FN) attracted our attention since it was previously associated with lung PMN formation37.
Immunohistochemical staining revealed FN to be significantly overexpressed in the lungs of HFD mice (Fig. 2G, H). To determine the origin of this FN expression, the lungs of mice perfused intracardially with PBS and non-perfused lungs were analyzed in Western blots (Fig. 2I). A significantly enhanced FN expression was detected in non-perfused whole lung tissues (Fig. 2J), suggesting an increase in blood circulation. We next analyzed FN expression in plasma and platelets isolated from the blood of ND and HFD mice (Fig. 2K, L) and while there were no differences in the FN in plasma (Fig. 2M), a significant increase in FN was evident in platelets reflecting its upregulation (Fig. 2N).
To assess the expression of FN in HFD-induced PMNs, we examined lung sections from our DIO model by confocal immunofluorescence microscopy (Suppl. Figure 2E). We analyzed FN co-expression with P-selectin (platelets) and CD31 (endothelial cells). We found that its expression was elevated in platelet aggregates (Suppl. Figure 2F), exhibiting a higher fluorescence intensity within the lungs of HFD mice (Suppl. Figure 2G). Conversely, there was negligible co-expression in the lungs of ND mice and as expected, there were almost no platelet aggregates in the lungs of these mice (Suppl. Figure 2E), indicative of specific co-expression and deposition of FN in platelet aggregates (P-selectin+ areas) in HFD mice. Furthermore, there was only a marginal and non-significant increase in FN staining of endothelial cells (CD31+, Suppl. Figure 2H), demonstrating that platelets are the primary source of FN in the lungs of HFD mice reflecting their activation at PMNs.
Since thrombus formation has been proposed to facilitate fibrinogen and fibronectin internalization in other contexts38, we investigated whether platelets from ND- and HFD-fed mice differ in their ability to bind and internalize FN. To do this, platelets isolated from both groups were incubated with FITC-FN and then fixed before and after the addition of trypan blue to distinguish between FN binding and internalization, respectively. Our results revealed that platelets from HFD-fed mice displayed a increassed capacity to both bind and internalize FN (Fig. 2O, P, Q) compared with those from ND-fed mice.
To identify potential factors involved in this mechanism, we analyzed the expression of cytokines in adipose tissue and platelets from HFD-fed mice compared to ND-fed mice. Our analysis revealed that several factors, including TNF-α, CCL3, Thrombopoietin, Angiopoietin-1, TIM-1, IL-22, Leptin, among others were significantly elevated in both conditions (Suppl. Figure 2I). Given the crucial role of TNF-α in obesity39, we further investigated its effect on FN production by platelets. Interestingly, we observed that the treatment significantly increased FN levels in platelets, reaching levels comparable to those observed in platelets from HFD-fed mice (Suppl. Figure 2J, K).
To evaluate the impact of a HFD on spontaneous BC metastasis, EO771 cells were injected into the mammary fat pad of DIO mice and analyzed 22 days after injection. Primary tumor growth increased in the HFD mice (Fig. 3A) and when tumor cells were stained for p5340, a 3.17-fold increase in lung metastasis was detected (Fig. 3B, C). Moreover, among these metastases 69.3% existed as individual cells (Fig. 3C). To mitigate the potential impact of variations in primary tumor size on spontaneous metastasis, we compared the metastases in mice with similar tumor sizes on day 14 (Fig. 3D) when there were 4.7-fold more metastases in the lung of HFD mice (Fig. 3E, F). Interestingly, among these metastases 64.9% were individual cells (Fig. 3F), suggesting that the HFD specifically enhanced tumor cell homing and early adhesion.
A Primary tumor growth of EO771 cells injected into the mammary fat pad of ND or HFD-fed. 3 independent experiments: ND n = 16 mice, HFD n = 17 mice. B Representative images of p53 staining in the lungs of ND and HFD mice on day 22 post-tumor cell injection (p.i.) Bar: 200 μm. C Quantification of the total number of metastatic foci in the experiment shown in (A). Lesions were binned into four size categories based on the number of tumor cells: 1-2 cells, 3-10 cells, 11-49 cells, and ≥50 cells. 3 independent experiments: ND n = 15, HFD n = 17 mice. D Representative images of tumors from ND and HFD mice on day 14 p.i. (no significant differences in tumor size). E Representative images of p53 stained lungs from ND and HFD mice on day 14 p.i. Bar: 200 μm. F Quantification of the total number of metastatic foci in the lungs of ND and HFD mice on day 14 p.i. Lesions were binned and analyzed as in (C). 4 independent experiments: ND n = 21, HFD n = 28 mice. G Scheme illustrating the EO771 cell homing assay corresponding to (H). H Representative images of the 1-hour homing assay with EO771 -GFP-luciferase tumor cells injected into the tail vein of ND and HFD mice. I Quantification of experiment in (H), 3 independent experiments, n = 12 mice per group. J Representative pictures of p53 staining in the lungs of ND and HFD mice at day 14 p.i. Bar: 100 μm. K Quantification of the total number of metastatic foci in the lungs of ND and HFD mice at day 14 p.i. by tail vein injection. Lesions were binned and analyzed as in (C), corresponding to 3 independent experiments: n = 15 mice per group. All data represent the mean ± s.d., and the P values were calculated by two-way ANOVA in (A) and with a two-tailed Student's t-test in C, F, I, and Mann-Whitney test in (K). Values are shown in box‐and‐whisker plots where the line in the box corresponds to the mean. Boxes extend from the minimum to the maximum value.
To assess how a HFD influences tumor homing, EO771-GFP-luciferase cells were injected into the tail vein of ND and HFD mice and mice were sacrificed after one hour (See Fig. 3G for a scheme of tumor cell homing assays). Remarkably, the HFD significantly enhanced tumor cell homing to the lungs (Fig. 3H-I). These findings were consistent when three additional murine BC models were assessed (Py230-Luc, Py8119-Luc and AT3-Luc: Suppl. Figure 3A–C). Tumor cell homing was also analyzed in ob/ob mice to determine whether this effect was influenced by high adiposity or the diet, and as no significant differences in tumor cell homing were observed in the lungs (Suppl. Figure 3D), it appears that this phenomenon is only affected by the HFD.
The impact of HFD on tumor cell metastasis was further investigated in the highly metastatic EO771 model, analyzing lung metastasis at early (12 days post injection –p.i.) and late (3 weeks p.i.) time points. The HFD enhanced metastasis at the early time point (Fig. 3J, Supp. Figure 3E), when the metastases were predominantly detected as single cells (2.2-fold change), followed by small metastases (2.8-fold change), micrometastases (1.5-fold change) and macrometastases (1.5-fold change: Fig. 3K). This effect was particularly prominent at the later time points (Suppl. Figure 3F–H). Similarly, with the Py8119-luc model an 11-fold increase in experimental metastasis was evident in the lungs of HFD compared to ND mice (Suppl. Figure 3I). By contrast, the HFD did not increase metastasis when the low metastatic AT3 model was assessed (Suppl. Figure 3J), suggesting that both intrinsic and extrinsic changes influence metastatic spread.
The pro-metastatic effects of platelets have traditionally been attributed to their ability to promote tumor cell adhesion or survival21, leading us to study the influence of a HFD on platelet and tumor cell interactions with endothelial cells. Platelets derived from HFD models adhered more to EO771 tumor cells than platelets derived from ND mice (Fig. 4A, B), as they also did to the low metastatic model AT3 cells (Suppl. Figure 4A, B). We next examined whether platelets from HFD-fed and ND-fed mice differ in their shape upon contact with tumor cells, as platelet morphology provides valuable insights into their activation potential. Our findings revealed that activated platelets from HFD-fed mice displayed an increased number of aggregating platelets, characterized by numerous membrane extensions and the formation of larger aggregates as demonstrated by scanning electron microscopy (Fig. 4C, D).
A Representative images showing EO771 tumor and platelets. Bar: 15 μm. B Quantification of (A), 2 independent experiments: ND n = 18, HFD n = 17 fields per group. C Representative SEM images showing EO771 tumor cells and platelets. Bar, 20 μm. D Quantification of C. ( > 150 platelets per experiment), showing the mean ± SEM of each morphology. 2 independent experiments: n = 12 images per group. E Representative images of EO771 tumor cell adhesion to endothelial cell monolayers stimulated by platelets. Bar: 50 μm. F Quantification of the endothelial cell surface area covered by platelets. 2 independent experiments, n = 16 fields per group. G Quantification of the number of EO771-GFP cells (#) adhered to endothelial cells incubated with platelets, 3 independent experiments: n = 16 fields per group. H Representative images by intravital microscopy of EO771-GFP cells and platelets. Bar, 30 μm (I) Quantification of H, n = 4 mice per group, 16 independent positions. J Representative images of immunofluorescence staining for FN1 and CD41 in HUVEC cells incubated with platelets for 16 h. Bar, 100 μm. K Quantification of cortical FN1 staining from the experiment in (J) 2 independent experiments: n = 15 fields per group. L Representative Western blot images in HUVEC cells after a 16 h incubation with platelets. N = 3 independent experiments. M Representative images of EO771-GFP cells, platelets and HUVEC cells incubated for 16 h with platelets from HFD mice in the presence or absence of the anti-FN1 antibody. Bar, 100 μm. N Quantification of (M). 2 independent experiments: ND n = 15, HFD n = 16 fields. O Representative images of the homing assay with EO771 and EO771-KOα5 tumor cells injected tail vein of HFD mice. P Quantification of (O), 2 independent experiments: n = 11 mice per group. Data represent the mean ± s.d., and P values were calculated by Mann-Whitney test in (F, I, K) and with a two-tailed Student's t-test in (B, D, G, N and P). Values are shown in box‐and‐whisker plots where the line in the box corresponds to the mean. Boxes extend from the minimum to the maximum value.
To determine if the enhanced tumor-platelet interaction also influences their interaction with endothelial cells, tumor-platelet-endothelial interactions were assayed in chambers coated with a monolayer of HUVECs (Human Umbilical Vein Endothelial Cells: Fig. 4E). Platelets from HFD mice adhered more to endothelial cells (Fig. 4F) and tumor cells (Fig. 4G), and interestingly, transwell trans-endothelial migration (TEM) assays revealed that platelets from obese mice migrated more efficiently than those from lean mice when injected with Py230 or EO771 cells (Suppl. Figure 4C).
To study the effect of a HFD on platelet-tumor interactions in vivo, high-resolution intravital imaging of EO771GFP tumor cells injected into the tail vein was carried out on the lung of HFD or ND mice. The area covered by platelets surrounding the tumor cells was measured in vivo (see Suppl. Figure 4D–E for a scheme), indicating that platelets were recruited around tumor cells in the lungs of mice as early as 1 minute after tumor cell injection in both ND and HFD mice (Suppl. Figure 4F–G and Video 1). Tumor-platelet aggregation 30 minutes after injection reflected how platelets from HFD mice interacted more efficiently with tumor cells in PMNs than those from ND mice (Fig. 4H–I).
The enhanced FN expression by platelets and endothelial cells induced by a HFD suggests that FN might modulate tumor-endothelial-platelet interactions. Hence, FN expression by endothelial cells exposed to platelets from ND and HFD mice was assessed by immunofluorescence, with FN and CD41 staining highlighting the upregulation of FN expression (Fig. 4J, green channel) and the enhanced interaction of platelets from HFD mice with endothelial cells (Fig. 4J, red channel). Platelets from HFD mice induced stronger FN expression in fibers surrounding endothelial cells (Fig. 4J panel inset, lower panels), whereas no fibers were observed with platelets from ND mice, when FN was poorly organized (Fig. 4J panel inset, upper panels) a significant increase in cortical FN was evident in endothelial cells treated with platelets from HFD mice relative to those from ND mice (Fig. 4K).
Since FN is known to be involved in intracellular signaling in endothelial cells41,42,43, we assessed the activity of the main downstream pathways implicated in regulating cell adhesion, cytoskeletal reorganization and endothelial permeability, through the effectors Src and Talin. Talin and Src appeared to be activated in endothelial cells after their interaction with platelets from HFD mice44 (Fig. 4L). Whether the effect on endothelial cell adhesion was dependent on HFD-induced FN was assessed in a tumor-endothelial adhesion assay using platelets from HFD-fed mice in the presence or absence of FN-neutralizing antibodies (Fig. 4M). Adhesion of EO771 cells to HUVECs in the presence of blocking antibodies was reduced by 61% relative to that of HUVECs preconditioned with platelets from HFD mice (Figs. 4M, N), suggesting that FN is involved in the adhesion of tumor cells to endothelial cells promoted by platelets from HFD-fed mice.
It is known that integrin α5β1 is the primary FN receptor responsible for cell migration and adhesion45,46. Integrin α5β1-FN interactions are of particular interest since they contribute to cancer metastasis, as witnessed by its upregulation in invasive cancer cells47,48,49. Both subunits of this receptor were expressed in our BC cell models, with particularly high levels in the highly metastatic EO771 cells (Suppl. Figure 5A). To demonstrate the relevance of this integrin interaction, we blocked integrin α5 expression in EO771 cells (Suppl. Figure 5B) and performed tumor homing assays (Fig. 4O). The absence of integrin α5 significantly dampened metastatic cell homing (Fig. 4P), highlighting the significance of this integrin in tumor cell homing to PMNs in HFD mice.
We next investigated whether the effects of a HFD were reversible by subjecting mice to a “diet resting” protocol (S.Diet) in which mice were fed for 12 weeks with a HFD and then switched them to a ND (Switch-diet) for 7 days (Suppl. Figure 5C). We first verified that mice experienced a significant average weight loss of 10% after switching diet (Suppl. Figure 5D) and subsequently, this switch was seen to dampen the activation of platelets, as witnessed by the weaker expression of activated integrin αIIbβ3 and P-Selectin in platelets from the S.Diet mice (Fig. 5A, B). Consistent with the earlier observations, platelet aggregation was also significantly reduced after switching diet (Fig. 5C). The expression of FN in the plasma and platelets of all the mice was assessed (Fig. 5D–F) and interestingly, FN expression returned to normal in platelets (Fig. 5F) after the resting period (S.Diet) while that in the plasma remained relatively constant (Fig. 5E). We also analyzed the impact of the S.Diet on the adhesive properties of tumor cells to platelet-activated endothelium in vitro (Fig. 5G). As observed previously, the HFD heightened the platelet-endothelial interaction, while in mice that underwent S.Diet, the platelet endothelial interaction was dampened relative to HFD mice (Fig. 5H). Importantly, the normalization of these interactions by switching diet also significantly reduced the adhesion of EO771 tumor cells to endothelial cells (Fig. 5I). Overall, the S.Diet positively influenced mice by reducing platelet activation/aggregation, restoring the FN levels in platelets and normalizing the interaction with endothelial cells, reducing their adhesion to tumor cells.
A Flow cytometry analysis of platelet activation, measuring the expression of activated αIIbβ3 (JON/A) and P-Selectin (CD62P) in the absence (Resting platelets) and presence (Activated platelets) of thrombin (0.05 IU/m) from the mice following a HFD or S.Diet. B Graph of the quantification of the experimental data shown in A. 5 independent experiments: n = 3 platelet samples per group. C Aggregation of platelets obtained from both HFD and S.Diet mice, and their response to thrombin (0.01 IU/m). The data correspond to 3 independent experiments: ND n = 10, and HFD n = 8 platelet samples. D Representative Western blots analyzing FN1 expression in plasma (top panels) and platelets (bottom panels) from ND, HFD and S.Diet mice. E, F Quantification of FN1 expression in the plasma (E) and platelets (F) from the data shown in D, corresponding to 2 independent experiments: n = 6 platelet samples per group. G Representative images of EO771 cell adhesion (green) to endothelial cell monolayers stimulated with platelets (red) from ND, HFD and S.Diet mice. Bar: 100 μm. H Quantification of the endothelial cell surface area covered by platelets obtained from ND, HFD and S.Diet mice (shown in G). I Quantification of the number of EO771-GFP cells (#) adhered to endothelial cells stimulated with platelets from ND, HFD and S.Diet mice (shown in G). 2 independent experiments: n = 20 fields analyzed per group. J Representative Western blots detecting FN1, p-Src, Src, p-Talin, Talin, and Hsp90 expression in HUVEC cells after a 16 h incubation with platelets from ND, HFD and S.Diet mice. K–M Graphs of the quantification of the experimental data shown in (J), n = 5 independent experiments. All the data represent mean ± s.d., and P values were calculated by two-tailed Student's t-test in (B, K, L, M) one-way ANOVA in (E, F, H, I,) and two-way ANOVA in (C). Values are shown in box‐and‐whisker plots where the line in the box corresponds to the mean. Boxes extend from the minimum to the maximum value.
The dietary switch might modify the signaling pathways downstream of FN in endothelial cells, specifically those involving Src and Talin41,50. Indeed, the S.Diet significantly dampened FN expression in endothelial cells (Fig. 5J, K), in conjunction with a reduction in Src activation (Fig. 5J, L). We also observed weaker talin activation, albeit not significantly (Fig. 5J, M). Based on these findings, diet appears to modulate both the activation and aggregation of platelets, changes concomitant with a normalization of FN expression in platelets, limiting the interaction of these cells with tumor and endothelial cells, and the ensuing downregulation of downstream signaling pathways.
We next investigated whether a dietary switch could affect tumor cell homing 1 hour after injection of tumor cells. We observed tumor cell homing was enhanced in the HFD mice (Fig. 6A, B), although it was significantly reversed after a dietary switch (Fig. 6A, B). We also examined the impact of the S.Diet on lung vascular leakiness and while the HFD significantly increased vascular leakiness (Fig. 6C, D), dextran leakiness tended to revert in the S.Diet mice, albeit not significantly (Fig. 6C, D). Hence, while diet may modulate platelet activation and tumor cell homing, endothelial cell function did not appear to be completely restored by dietary switching. This data suggests that tumor cell homing is mainly affected by platelet activation rather than vascular permeability. In addition, we analyzed the effect of diet switching on experimental metastasis by examining lung metastases. Notably, the dietary change led to a significant reduction in tumor cell metastasis within one week (Fig. 6E, F), indicating that diet switch dampens platelet activation and thereby affects both tumor cell homing and metastasis.
A Representative bioluminescence images 1 hour after EO771-Luc tumor cell tail vein injection in ND, HFD and S.Diet mice. B Quantification of the average radiance per mice in the experiment shown in (A). 3 independent experiments: ND n = 11, HFD n = 12, and S.Diet n = 13 mice. C Representative images of vascular permeability in the lungs of ND, HFD and S.Diet mice using NIR-Dextran. D Quantification of NIR-Dextran fluorescence from the experiments in (C). 2 independent experiments: ND n = 8, HFD n = 8, and S.Diet n = 9 mice. E Representative bioluminescence images from the metastasis assay with EO771 tumor cells injected into the tail vein of HFD and S.Diet mice. F Quantification of the average radiance per mice from the experiment shown in (E), corresponding to 2 independent experiments: ND n = 6, HFD n = 9, and S.Diet n = 10 mice. G Representative bioluminescence images of EO771-Luc cells injected into the tail vein 48 hours after R300 antibody treatment of ND and HFD mice during metastatic homing assays. H Quantification of the average radiance per mice from the experiment shown in (G). 2 independent experiments: n = 5 mice per group. I Representative bioluminescence images of EO771-Luc cells injected into the tail vein 48 hours after R300 antibody treatment of ND and HFD mice in metastasis assays. The metastases were analyzed 21 days p.i. J Quantification of the average radiance per mice from the experiment shown in (I). 2 independent experiments: ND n = 9, ND + R300 n = 9, HFD n = 8, ND + R300 n = 8. All the data represent the mean ± s.d. and the P values were calculated by One way ANOVA. Values are shown in box‐and‐whisker plots where the line in the box corresponds to the mean. Boxes extend from the minimum to the maximum value.
To analyze the relevance of platelets in tumor cell homing after HFD-induced PMN formation, we depleted platelets in HFD mice prior to the injection of tumor cells using anti-GPIbα (R300) antibodies51 (Suppl. Figure 5E). Mice received a single R300 injection 48 hours before that of the tumor cells, inducing a window of platelet depletion just when tumor cells home to the PMNs. After verifying platelet depletion, tumor cells were injected into the tail vein and tumor cell homing was analyzed 1 hour later (Supp. Figure 5E). Platelet depletion promoted a significant decrease in tumor cell homing in HFD and in ND mice (Fig. 6G, H), although the reduction was not significant in the latter. The effect of platelet depletion 48 hours before tumor cell injection on experimental metastasis assays in HFD mice was also analyzed in the lungs after 21 days (Fig. 6I–J). Importantly, the R300 antibody not only dampened tumor cell homing but also metastasis in HFD mice, yet not in the ND mice (Fig. 6J). Hence, platelets appear to be crucial to promote tumor cell interactions at PMNs. The activation of platelets in these niches by a HFD can be reversed by a dietary switch or platelet depletion during PMN formation, offering potential therapeutic strategies to reduce tumor cell homing to PMNs.
Since a HFD appears to promote platelet activation and aggregation, a HFD and increased BMI might also affect the coagulation profile in this model. Thus, the blood coagulation profile was analyzed in ND and HFD mice, measuring the Prothrombin Time (PT: Fig. 7A), Partial Thromboplastin time (aPTT:Fig. 7B), Thrombin Time (TT: Fig. 7C) and Fibrinogen (Fg:Fig. 7D) in the DIO model. Of all the parameters analyzed, the aPTT was significantly reduced in HFD mice (Fig. 7B).
A PT values measured in ND, and HFD mice. 10 independent experiments: ND n = 36, HFD n = 37 mice. B aPTT values measured in ND, and HFD mice. 9 independent experiments: ND n = 29, HFD n = 29 mice. C TT values measured in ND, and HFD mice. 5 independent experiments: ND n = 14, HFD n = 20 mice. D Fibrinogen values measured in ND, and HFD mice. 6 independent experiments: ND n = 28, HFD n = 29 mice. All data is expressed as the mean ± s.d and P values were calculated with two-tailed Student's t-test in (A, B, C and Mann-Whitney test in D). E Plot of BMI vs aPTT values among TNBC patients. Patients with BMI < 25 were categorized as normal weight and patients with BMI > 25 were categorized as overweight or obese. BMI > 25 is a positively correlated with a lower aPTT (correlation coefficient r = 0.08824, **p = 0.0083- analyzed by simple linear regression test). The red solid line is the fitted regression line, and the green shaded area corresponds to a range of standard aPTT values. The Kaplan–Meier survivor functions according to (F) aPTT, G PT, H INR and I Fibrinogen levels in patients. Log-rank tests between a normal aPTT and a shorter aPTT, PT and INR are shown (lowest quartile), and for fibrinogen the log-rank test is between normal and higher values (highest quartile are shown).
Based on our hypothesis and the association between a high BMI, pro-thrombotic risk and cancer4, we analyzed the potential association of BMI and coagulation parameters with disease outcomes in patients with TNBC. TNBC has an elevated risk of metastatic disease development and lower survival rates than hormone-positive BC52. Moreover, being overweight is associated with a shorter disease-free and overall survival time among TNBC patients7. Hence, the correlation of BMI and different coagulation parameters before surgery with disease outcome was analyzed in a cohort of 82 early TNBC patients treated, with neoadjuvant and/or adjuvant chemotherapy. The PT, aPTT, INR (International Normalized Ratio), Fg and platelet counts were evaluated in TNBC patients prior to surgery, examining the correlation between these parameters and the BMI. The BMI was inversely correlated with a reduced aPTT, albeit moderately (Fig. 7E), whereas no significant correlation was observed between the BMI and platelet count, platelet volume, total Fg or the INR.
Further analysis of these parameters relative to disease outcome demonstrated that patients with the shortest aPTT (1st Quartile - Q1) experienced a significantly shorter time to relapse (estimated mean time to relapse of 8.5 years) than other patients (11.9 years, P = 0.0014: Fig. 7F). However, a multivariate analysis did not reveal any further correlations of BMI with disease outcome or among the other parameters that were related to higher coagulation profile (Fig. 7G–I): PT, p = 0.568; INR, p = 0.789; or Fg, p = 0.135. Overall, these data suggest that although BMI was inversely correlated with a shorter aPTT, only a shorter aPTT could be considered as an independent additional risk factor of disease progression in TNBC patients prior to neoadjuvant treatment and surgery.
There is considerable experimental evidence that platelets influence metastasis17,18,19,21,53 and early metastatic dissemination20,21, therefore this study set out to assess the influence of additional systemic cancer risk factors in platelet-driven BC metastasis, in particular that of a HFD. The data obtained suggest that a HFD increases the activation, adhesion and the aggregation of platelets in circulation, and in the lungs of mice, leading to hypercoagulation.
Obesity is known to promote prothrombotic signaling in relation to platelet activation and hypercoagulability54,55. Our findings reveal that Src and Talin are activated in platelets obtained from HFD mice, and in endothelial cells exposed to platelets from these mice. Interestingly, the activation of Src is involved in the activation of platelets56 and endothelial cells57, promoting the recruitment of several proteins like Talin and integrins58, the latter serving as receptors for ECM molecules like FN59. Moreover, it is worth noting that prolonged Src activation has been implicated in disrupting barrier permeability57. Hence, these findings support a model in which a HFD induces Src hyperactivation and Talin expression in both platelets and endothelial cells, leading to increased vascular permeability and platelet activation, and enhancing tumor cell interactions through the upregulation of FN. Furthermore, our data also suggests that platelets from HFD mice exhibited prolonged phosphorylation, both inside-out and outside-in, producing elevated levels of pSrc and pPKC. By contrast, Src is partially inactivated in the inside-out pathway in platelets from ND mice, and activated again in the outside-in pathway, whereas Src exhibited constant activation in platelets from HFD mice. Src phosphorylation can induce filopodia and lamellipodia formation during platelet spreading by mediating calcium signaling and PKC activity60, while PKC and AKT are important regulators of platelet granule secretion28,29,30,61. The data obtained here suggests the pre-activation of inside-out signaling pathways in HFD mice, and more rapid outside-in signaling upon stimulation. It is worth noting that this type of activation has been associated with adhesion to the vascular wall and platelet aggregation62.
In addition to platelet activation and aggregation, the HFD increased vascular leakiness, modulating the expression of genes associated with cell adhesion and platelet activation, and endothelial cell damage63,64. These results are consistent with studies indicating that vascular injury leads to a shift in the endothelium from an anticoagulant to a procoagulant phenotype, resulting in the exposure of fibers and other endothelial matrix proteins65,66. Indeed, studies have shown that obesity enhances vascular leakiness, thereby promoting BC extravasation67. Our data suggests that systemic changes induced by HFD could affect the generation of PMNs, changes that influence vascular leakiness, platelet activation and ECM remodeling, favoring tumor cell homing and metastasis. Interestingly, similar changes have been proposed as hallmarks of PMN formation by primary tumors22, suggesting that they may be shared between these microenvironments.
We identified FN as a key molecule upregulated in endothelial cells and activated platelets in the lungs, both in vitro and in vivo. There are two forms of FN, plasma FN (pFN) that lacks extra EDA and EDB segments, and tissue cellular FN (cFN) that is synthesized by many cell types (e.g., fibroblasts, endothelial cells, platelets and monocytes) and that bears varying proportions of EDA and EDB segments68. Both forms of FN can be incorporated into the fibrillar network of the ECM68. Of these, pFN supports hemostasis and regulates thrombosis69, while cFN is implicated in efficient adhesion, activation and aggregation of platelets, promoting inflammation and thrombosis70,71,72. We found that the overexpression of FN in platelets induced by a HFD is crucial in regulating the interaction between platelets, endothelial and BC cells.
Moreover, FN plays a critical role in PMN formation37 and lung metastasis73. Interestingly, cFN levels are significantly elevated in the plasma of patients with thromboembolism or diabetes74 and they are positively correlated with the BMI75. The results here support the notion that platelets play a crucial role for successful vascular retention and metastatic outgrowth, consistent with an effect of HFD on platelets that enriches FN, influencing tumor cell homing and metastasis. These observations are supported by previous studies on FN-EDA KO mice administered a HFD, which exhibit reduced thromboembolism relative to WT mice on the same diet. Hence, FN appears to promote thromboembolism in the context of DIO75. Interestingly, an ECM rich in FN primes endothelial cells for activation in response to oxidized low-density lipoprotein76 and hyperglycemia77. Furthermore, atheroprone shear stress has been implicated in the upregulation of FN expression creating a positive feedback loop that sustains endothelial cell inflammation78. A HFD appears to promote platelet activation and aggregation at PMNs, concomitant with FN overexpression and deposition at their surface, that interacts with integrin α5β1 receptors on tumor cells. The involvement of integrin α5β1 in tumor cell migration, invasion and metastatic tumor cell homing makes it an attractive therapeutic target47,48,49,79. However, given the complexity of obesity-associated inflammation and BC metastasis8,9,10,80, and the multifaceted role of platelets in PMNs81, the mechanisms driving FN overexpression at PMNs are likely to be multifactorial54. Consistent with previous studies39, we observed that adipose tissue from HFD-fed mice secretes elevated levels of TNF-α. Notably, this factor promoted FN expression and accumulation in platelets, suggesting an additional mechanism underlying FN expression in circulating platelets from HFD-fed mice. Furthermore, given the crucial role of FN in platelet aggregation and activation70,82, our findings support a model in which HFD-induced signaling enhances platelet FN uptake and retention, potentially contributing to the prothrombotic state associated with HFD-fed mice.
It is widely accepted that obesity is associated with a higher incidence of BC, and with increased mortality and metastasis6,7. While several studies have shown that obesity is associated with BC metastasis8,9,80, the impact of platelets in this process has not been thoroughly assessed. Our data demonstrates a distinct role for platelets in the obese microenvironment, primarily influencing tumor cell homing and subsequently impacting later stages of metastasis. We found that the tumor cells in HFD mice have a greater capacity to extravasate and successfully survive as solitary cells in the lung parenchyma when compared to those in ND mice. We also found that platelets from HFD mice adhere more efficiently to both tumor cells and endothelial cells, rapidly binding to these upon their homing to metastatic organs and resulting in the formation of significantly larger aggregates. One possible explanation could be that after they home to the target organ, tumor cells are protected from the action of immune cells (e.g., NK cells) by the surrounding platelets, as suggested elsewhere18. Nevertheless, we did not analyze the implication of immune cells in this system, and the mechanism by which a HFD may lead to platelets favoring individual cell metastasis remains unclear.
Importantly, our findings indicate that the platelets derived from HFD mice interact more strongly with cells from both high and low metastatic TNBC models, although a HFD specifically reinforces single cell homing only in highly metastatic models. Hence, both extrinsic and intrinsic characteristics of the cancer cells appear to play an important role in this process. Notably, platelets engage with cancer cells and form stable aggregates primarily through the expression of P-selectin83,84. Indeed, platelets derived from HFD mice not only exhibit enhanced P-selectin expression but also, their interactions with tumor cells are heightened both in vitro and in vivo. In addition, we observed that platelets from HFD-fed mice, aside from being more activated, also exhibited an enhanced spreading phenotype when interacting with tumor cells compared to platelets from ND-fed mice. These findings align with previous studies showing that platelet shape is modified upon contact with tumor cells85 and further demonstrate that platelets from HFD-fed mice display a heightened activation status while on the surface of tumor cells.
Once cancer cells have disseminated, their ability to home to a supportive niche becomes crucial for successful metastasis22. While a HFD appears to influence tumor cell interactions with endothelial cells in mice, the data presented here suggest that dietary intervention can reverse or mitigate certain consequences of a HFD. However, vascular damage appears to be more persistent and may require additional time to recover completely, as indicated elsewhere10. These findings imply that the impact on cell dissemination in HFD mice is probably due to the diet itself rather than solely being a result of high adiposity. Strikingly, the data from the ob/ob mouse model supports the notion that high adiposity induces increased vascular leakiness, regardless of diet.
While several studies suggest that weight loss is associated with improved BC outcomes86, the impact of diet on coagulation and metastasis has been little studied. Remarkably, the dietary switch restored FN levels concomitant with a decrease in lung metastasis. However, it is noteworthy that vascular injury persisted even after weight loss over the period analyzed here, probably due to the systemic effects of inflamed adipose tissue. These data highlight the complex interaction between diet and metastasis, such that vascular leakiness alone is not sufficient to affect tumor cell homing and that further factors are also required, for example platelet activation. It would be interesting to analyze coagulation parameters and clinical outcomes in ongoing clinical studies on dietary intervention in BC as an additional clinical factor.
From a clinical perspective, our study demonstrates that TNBC patients with stronger hypercoagulation have a worse prognosis. Although no direct association with BMI was seen, HFD mice and patients with a higher BMI had shorter aPTTs. Given the role of platelets in hematogenous metastasis, interest has grown in developing drugs that target platelets for anti-tumor therapy. Most of these drugs focus on inhibiting platelet activity and thus, they have the drawback that other platelet functions may be affected87. Our study demonstrates that a dietary switch or depleting platelets reduces metastasis in the lungs of mice on a HFD. Platelet depletion was particularly successful in reducing metastasis that developed on a background of a HFD right before tumor cell homing. Interestingly, aspirin treatment during tumor cell homing, yet not after tumor cell extravasation, reduces lung metastasis by inhibiting the platelet-derived COX-1/thromboxane A2 axis88. It would be interesting to test other anti-platelet drugs, such as inhibitors of αIIbβ3 and selectins in this context since their effect on cancer progression has yet to be evaluated89. Our findings support the effectiveness of anti-platelet therapies in blocking tumor cell homing, particularly in HFD-induced microenvironments. It should be recognized that other anti-coagulant treatments may affect later stages of metastasis, as previously suggested elsewhere75. Indeed, it was recently reported that targeting platelet-specific receptors like glycoprotein VI (GPVI) in mouse models reduces the growth of established metastases85. However, evidence of the effective clinical use of anticoagulant and anti-platelet drugs in obese patients and in the premetastatic setting is limited.
The human studies performed here were all approved previously by the ethics committees from the Hospital Universitario de Fuenlabrada (protocol approval number CEI: 11/37). The study included one cohort of 82 patients diagnosed with early TNBC between 2012 and 2015, and who were eligible for neoadjuvant and/or adjuvant chemotherapy. The diagnosis was based on negative expression of ER/PR (less than 10%) by immunohistochemistry (IHC) and HER2, either IHC 0-1 or fluorescent in situ hybridization (FISH)-negative if ≥ 2 on IHC (see Suppl. Table 2 for the clinical characteristics of the patients). The PA clinical study protocol was approved to gather their clinical, demographic and blood test data, and all the participating patients signed an informed consent form. The study was carried out in accordance with the Helsinki Declaration and approved by the Institutional Review Board. TNBC was defined as negative ( < 1%) for ER and PR expression, and no amplification of HER2 (result of 0+ or 1+ in an Herceptest or a result of 2+ with negative FISH).
There were no restrictions regarding gender or ethnicity in our protocol; however, the recruited population was primarily Caucasian and Hispanic due to the local demographics.
All the experimentation with mice was first approved by the Institutional Ethics Committees of the CNIO (IACUC-015-2017, IACUC-020-2018, IACUC-017-2022), the Instituto de Salud Carlos III (CBA-19_2017, CBA 01_2019v4, CBA-04_2023v2) and the Comunidad Autónoma de Madrid (PROEX 227/17, PROEX091/19, PROEX77.1/23). The maximum tumor size (1000 mm3) and burden permitted by our ethics committee were not exceeded in the subcutaneous and metastasis models. All the experiments on animals were performed in accordance with the guidelines for ethical conduct in the care and use of animals, as stated in the International Guiding Principles for Biomedical Research involving Animals and devised by the Council for International Organizations of Medical Sciences.
C57BL/6JolaHsd male and female mice and C57BL/6-Tg (CAG-EGFP) 10 sb/J male (8–12 weeks old: Envigo and Jackson Laboratory) were used in these studies, and they were housed at 21 ± 2 °C with 50–60% humidity, on a13 h light and 11 h dark cycle. For studies on male mice, 8-week-old mice were maintained on either a high-fat (HFD, 60% kcal, Research Diet D12492) or standard (ND, 6.2% Kcal, Envigo 2018S) diet for 12 weeks. For studies on female mice, 8-week-old mice were maintained on either a high-fat (HFD, 60% kcal, Research Diet D12492) or low-fat (LFD, 4% kcal, Envigo 2014) for 20 weeks. The key experiments (i.e., vascular leakiness, tumor cell homing, experimental metastasis, platelet parameter analysis, platelet aggregation analysis) were performed on both sexes of the DIO model and we did not find differences between sexes. Weight was monitored throughout the experiments. At the end-point, animals were either euthanized for coagulation analysis or injected with NIR-dextran (see Permeability assay with NIR-Dextran) or tumor cells (see sections on primary tumor growth and spontaneous metastasis, and experimental metastasis assay). For the dietary switch (HFD/ND-LFD), 8-week-old male or female BL6 mice were fed with a HFD diet until the end of DIO protocol and then switched to the ND or LFD for 1 week prior to sacrifice.
To analyze the effects of adipose tissue, 4-week-old female B6.Cg-Lepob (ob/ob: Jackson Laboratory) mice were maintained on a normal rodent diet (ND: Envigo 2018S). Their weight was monitored over time from 5 weeks of age and the mice were sacrificed when their weight exceeded 40 g, a period that was significantly shorter (6 weeks) than that of the DIO model (12 weeks). After 6 weeks, the mice were injected with NIR-dextran or tumor cells to perform 1 h homing assays.
To prepare NIR fluorescent dextran, 6.5 mg of dextran was diluted in 1.8 ml of 0.1 M NaHCO3, and 190 µl of DMSO and 10 µl of cyanine 7.5 NHS ester (25.6 mM) was then added to the solution. The mixture was kept in the dark and stirred at room temperature (RT) for 4 hours, after which the reaction was purified with 50 kDa Amicon filters (filtration 14,000 rpm, 10 min; recovery 7500 rpm, 5 min). The tail vein of male and female DIO mice was injected with 1 mg g−1 dextran and lung permeability was analyzed 1 h after dextran injection by bioluminescent imaging (Xenogen IVIS-200 Optical In Vivo Imaging System).
EO771 cells were purchased from CH3, the Py230 cell line was purchased from the ATCC and HUVECs were obtained from Lonza. Py8119 cells were kindly provided by Dr Cyrus Ghajar (Fred Hutchinson Cancer Center), MC38 cells by Dr Luuke Hawinkels (Leiden University Medical Center) and AT3 cells by Dr Inge Verbrugge (The Netherlands Cancer Institute). The EO771 and AT3 tumor cell lines were grown in high-glucose DMEM (Lonza) supplemented with 10% fetal bovine serum (FBS: Hyclone), 2 mM glutamine and 20 μg ml−1 gentamicin. MC38, Py230 and Py8119 cells were grown in Ham's F12 (Lonza) supplemented with 10% FBS (Hyclone), 2 mM glutamine and 20 μg ml−1 gentamicin. The HUVEC line (Clonetics, Lonza, Verviers, Belgium) was cultured in EGM-2 medium containing growth factors (Clonetics), 20 μg ml−1 gentamicin and 10% (v/v) FBS. Culture flasks were pre-coated with gelatin 0.2% in ultra-purified water for 15 min and all cells were grown at 37 °C in a humidified 5% CO2/95% air atmosphere.
For Itga5 gene inactivation, sgRNAs were designed using the Benchling CRISPR sgRNA tool (http://www.benchling.com). Specific sgRNAs were tested against the Itga5 gene (NM_010577, exons 3 and 4), and also a non-targeting (NT) guide was used as a control. These sequences were cloned into the lentiCRISPRv2 vector (plasmid #52961, Addgene) and verified by Sanger sequencing. Recombinant lentiviruses were produced by transient plasmid transfection into HEK293T cells using the calcium phosphate method. Cells were transfected using second-generation packaging plasmids (psPAX2 and pMD.2 G, #12260 and #12259, respectively, Addgene) and the appropriate transfer plasmid (pLV CRISPR sgItga5 or pLV CRISPR sgNT). The medium was collected after 48 hours, cleared by low-speed centrifugation, and filtered through 0.45 μm pore-size PVDF filters (Millipore). Viral titers were calculated by qPCR and values ranged from 107 to 108 TU/ml.
To investigate the effects of a HFD on breast cancer (BC) primary tumor growth, 1×106 BC cells (EO771) were injected into the right thoracic mammary fat pad of 20-week-old female DIO mice. Cells were injected in 1:1 serum-free DMEM/growth factor-reduced Matrigel (BD Biosciences). The tumor volume was measured with a digital caliper and calculated as V = (L × W2)/2. Key time points throughout the trial were: 0 d, prior to tumor cell injection; 14 d, when primary tumors presented no statistical differences between the groups; 22 d, when primary tumors presented statistical differences between the groups. To assess spontaneous metastasis to the lungs, mice were euthanized on day 14 or 22, and the lungs were fixed with 10% formalin, paraffin-embedded, and hematoxylin and eosin (H&E) stained. For EO771 cells, tissue sections (10 μm) were stained with p53 and metastases were counted using a Leica Aperio digital pathology slide scanner and software.
To investigate the effects of a HFD on experimental metastasis to the lungs, 1×105 EO771 BC cells labeled with GFP and luciferase (GFP-Luc) were injected into the tail vein of 22-week-old male and female DIO mice. Cells were resuspended in calcium- and magnesium-free PBS, and filtered through a 40 μm mesh immediately prior to injection. Animals were injected with luciferin (3 mg: MB102, syd labs), and lung metastases were then analyzed by bioluminescent imaging (Xenogen IVIS-200 Optical In Vivo Imaging System) 1 h, 24 h, 2 or 5 weeks after tumor cell injection using Living Image 4.7.2 software (PerkinElmer).
The effect of platelet depletion was analyzed in male DIO mice. Platelet depletion was achieved by injecting a single intravenous dose of an anti-Gp1bα antibody (R300, 4 μg g−1: Emfret Analytics) or rat IgG2a isotype control (C301, Emfret Analytics) 48 h before tumor cells (1 × 105 EO771 GFP-Luc cells) were injected intravenously. Tumor cell homing was analyzed 1 h after injection by bioluminescent imaging (BLI: Xenogen IVIS-200 Optical In Vivo Imaging System). To analyze the effect of platelet depletion on experimental metastasis, platelet depletion was performed as indicated above 48 h before injecting the EO771 tumor cells (1 × 105 cells). Lung metastases were analyzed over 3 weeks after injection by BLI, confirming the effectiveness of the anti-platelet antibody in each experimental setting by counting platelets prior to and during the homing assay.
Platelets were isolated as described previously90. Briefly, female and male DIO mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg), and whole blood was drawn by cardiac puncture using a syringe containing acid citrate dextrose (1 volume of anticoagulant/9 volumes of blood). Blood was mixed with 1 volume of modified HEPES-Tyrode's buffer (140 mM NaCl, 2 mM KCl, 12 mM NaHCO3, 0.3 mM NaH2PO4, 1 mM MgCl2, 5.5 mM glucose, 5 mM HEPES, 2 mM EGTA, and 0.035% BSA, pH 6.7) and it was centrifuged at 150 g for 2 minutes to obtain platelets or platelet-rich plasma (PRP). Prostaglandin E1 (PGE1, 5 nM: Millipore Sigma) was then added and the platelets were pelleted by centrifugation at 1500 g for 4 minutes at 37 °C. Platelet pellets were finally suspended in modified HEPES-Tyrode's buffer without EGTA and BSA (pH 7.4) but in the presence of 0.02 U/ml apyrase (grade VII: Millipore Sigma). Platelets were counted and pooled at the density necessary for each experiment.
For murine platelet aggregation studies, washed platelets isolated from female and male mice, and female DIO mice (250 μl of 5 × 108 platelets/ml) were used. Optical platelet aggregation experiments were monitored by turbidimetry using an aggregometer (Chrono-log 490 Optical Aggregometer) with continuous stirring at 1200 rpm at 37 °C. A final concentration of 0.5 IU/ml thrombin and 5 μg/ml collagen (Chrono-log Corporation) was used as an agonist for the aggregation studies.
To analyze αIIbβ3 integrin activation (GPIIb/IIIa, CD41/CD61) in murine platelets isolated from male mice after DIO, washed platelets (100 μl of 1 × 106 platelets/ml) were activated with 0.05 IU/ml thrombin or left untreated, and analyzed by flow cytometry after incubating them with PE-labeled anti- GPIIb/IIIa (Emfret, clone JON/A) and anti-CD62-P FITC antibodies (Emfret, clone Wug.E9) for 15 minutes at RT. Reactions were stopped by adding 400 μl PBS and the samples were analyzed within 30 minutes on a FACS CANTO II (BD, San Jose CA) and using FloJo v10 (BD). All antibody details and dilutions are available in Suppl. Table 1. Pools of platelets from 3 mice per diet were used in each condition. Ex vivo fibronectin binding and uptake assays were performed following the protocol described previously for fibrinogen adapted with some modifications91, briefly washed platelets (1.0 × 107/mL) were incubated with FITC-FN (0.15 mg/mL, Cytoskeleton, Inc.) at 37 °C for the indicated times. The platelets were fixed with 2% paraformaldehyde and analyzed by FACS. To study fibronectin uptake, platelets were fixed with 2% paraformaldehyde and analyzed by flow cytometry after addition of 0.04% trypan blue (Sigma). Pools of platelets from 3 mice per diet were used in each condition.
Washed platelets isolated from male DIO mice (250 μl of 5 × 108 platelets/ml) were activated with 0.05 IU/ml thrombin (Chrono-log Corporation) and incubated at 37 °C for different times: untreated, 0 min; activated 15 and 30 seconds, and 3, 7 or 15 min. Subsequently, 250 μl 2× RIPA lysis buffer was added: 100 mM Tris-HCl [pH 8.0], 0.2% SDS, 2% NP-40, 1% sodium deoxycholate, supplemented with a mixture of protease and phosphatase inhibitors (PMSF 2 mM, Leupeptine 10 μg/ml, NaF 50 mM, Aprotinin 5 μg/ml, Sodium Orthovanadate 0.3 mM). Signaling pathways were analyzed in immunoblots of total protein extracts from washed platelets probed with the antibodies indicated (Suppl. Table 1). Pools of platelets from 3 mice per diet were used in each condition. When indicated, platelets were pretreated with TNFα (1ug/ml, Peprotech) for 5, 15 and 30 minutes at 37 °C before activation.
The release of ATP stored in platelet dense granules was measured as light output generated following an ATP-luciferin-luciferase reaction. The assay was carried out as described previously92. Briefly, washed platelets isolated from male DIO mice (5 × 108/mL, 180 μL) were incubated in a white Corning Costar flat bottom 96-well plate for 5 minutes at 37 °C on an orbital shake. Platelet agonizts (Thrombin 0.01 IU/ml) and CaCcl2 (2 mM) were then added, and platelets were activated for 30 seconds (s) at 37 °C on an orbital shaker. ATP standards were assayed in the same plate. Finally, 50 μL of the Chronolume detection reagent (Chrono-Log Corporation, Havertown, PA, USA) was added to each well and the corresponding luminescence emitted was measured (in arbitrary units) for 3 s in a multiple-well Reader (ClarioStarPlus. Luminiscence) following a 1 s delay. Pools of platelets from 3 mice per diet were used in each condition. The assay was performed in triplicate on at least two independent experiments.
Spreading assays were performed as described previously91. In brief, Lab-Tek Chamber Slides (Nunc) were coated overnight at 4 °C with 100 μg/ml human fibrinogen (Millipore Sigma) in PBS [pH 8.0] and after washing with PBS, they were blocked for 2 hours at RT with heat-denatured BSA (5 mg/ml) and then washed again. Platelets isolated from male DIO mice (1 × 108 platelets/ml) were incubated on the fibrinogen-coated chambers and allowed to spread for 5, 15, 30 and 60 minutes at 37 °C in the presence of 0.01 IU/ml thrombin. Pools of platelets from 3 mice per diet were used in each condition. The chambers were then washed and the adhered platelets were fixed with paraformaldehyde (PFA, 4%) for 10 minutes. After washing again, the chambers were incubated with 100 mM glycine for 5 minutes, washed and then permeabilized with 0.2% Triton X-100 for 5 minutes. The plates were washed further, blocked for 30 minutes with 10% normal goat serum (NGS: Millipore Sigma) and then probed with an anti–α-tubulin primary antibody, followed by the Alexa Fluor 488 goat anti-mouse secondary antibody. Phalloidin–Alexa Fluor 564 (A22283, ThermoFisher) was used to stain F-actin (T9026, Sigma) and images were acquired with a TCS SP5 X confocal microscope (Leica-Microsystems) equipped with AOBS, a HCX PLAN APO CS 63x/1.4 Oil Immersion, a HCX PLAN APO CS 100×/1.4 Oil Immersion objective and LAS AF v2.7.5 software. To quantify platelet morphology, at least 150 platelets per sample were measured using Fiji v2.1software.
Adhesion assays were performed as described previously93. In brief, microplate wells coated with fibrinogen (Millipore Sigma) were blocked for 1 h at RT and washed with PBS before adding the platelets isolated from male DIO mice. The platelets (50 ul of 1 × 108 platelets/ml) were unactivated, or activated by exposing to thrombin (0.05 U/ml, final activity) for 20 to 60 min at RT, and had been washed four times with PBS before they were diluted in the phosphatase substrate solution (5 mM p-nitrophenyl phosphate). After a 1 hour incubation, 2 M NaOH was added and the resulting formation of p-nitrophenol was measured spectrophotometrically in Multi-well Clario Star Plus Reader at λ = 405 nm absorbance. The assay was performed in triplicate for at least two independent experiments.
EO771 (1.5 × 105 tumor cells) were washed in serum free media prior incubation with platelet suspensions (1.5 × 106 platelets) at indicated concentrations. Purified platelets were incubated with EO771 tumor cells for 1 h at room temperature then fixed with 2.5% glutaraldehyde and 4% paraformaldehyde prepared in Milloning phosphate buffer for 4 h. Pools of platelets from 3 mice per diet were used in each condition. After washing in distilled water, samples were dehydrated in an ethanol series (30, 50, 70, 80, 90, 95 and 100% for 10 min each). The samples were dried in a critical point dryer (Leica EM CPD300; Leica Microsystems). A 10 nm gold layer was sputtered onto the surface of the samples. Image acquisition was performed at high resolution (20 kV) and ×3000–6000 magnification under an JEOL JSM 6400 scanning electron microscope.
A commercially available antibody array kit (Proteome Profiler Mouse XL Cytokine Array, R & D Systems, ARY028) which was coated with 111 different cytokine antibodies in duplicate on a nitrocellulose membrane (dot blot) was used, following the manufacturer's instructions. This cytokine antibody array was used to determine the effects of platelets exposure on cytokine synthesis by EO771 cells compared with the analysis of adipose tissue-secreted soluble factors from ND and HFD-fed mice. The assay required 500 μL of cell culture supernatants (unstimulated and Platelet stimulated (1 × 106). Pools of platelets from 3 mice per diet were used in each condition. Membrane images were quantified, processing and analyzed using Fiji v2.1. software. Levels of cytokines were expressed and normalized against of the reference spots and relativized to the control condition (EO771 or ND secreted factors). Only the cytokines that showed similar expression patterns in both between the HFD conditions are shown.
HUVECs were grown to form a monolayer on gelatin-coated 6-well plates and activated by adding 100 μl of PKH26-labeled-platelets from male DIO mice (1×108 platelets/ml) for 45 min. Pools of platelets from 3 mice per diet were used in each condition. Platelets were washed and 1x105 EO771 cells were added to each well and allowed to adhere for 45 min. The cells were gently washed twice with medium and fixed in 4% PFA. When indicated, HUVECs were treated with an anti-FN1 blocking antibody (100 mg/ml) for 16 h (IST9, Abcam, ab6328). Images were acquired on a Thunder Imaging System (Leica-Microsystems) equipped with a 5X/0.15 Air Objective, a 8-LED light source, a DFC9000GTC camera and LAS-X v3.9.1 software. Random fields were obtained and analyzed with Fiji v.2.1 software.
TEM assays were set up as described previously10. Briefly, endothelial cells (HUVEC or HMEC) were seeded in Transwell chambers (8 μm, 24-well format) at a density 100,000 cells/well and allowed to form monolayers. Endothelial monolayers were pre-treated with platelets isolated from female DIO mice (500,000 platelets/well, 45 min exposure). Pools of platelets from 3 mice per diet were used in each condition. BC cells were labeled with the Vybrant CFDA SE Cell Tracer (Invitrogen) to differentiate them from endothelial cells, they were seeded in the upper chamber (4000 cells/well, Py230 or EO771 cells) and then allowed to migrate across the endothelial monolayer to the underside of the Transwell chamber over 24 h along a 0-2% FBS gradient. The cells that migrated to the underside of the Transwell chamber were quantified under a fluorescent microscope using a 10x objective.
Western blots were performed on 30-60 μg of total protein extract from murine platelets and RIPA whole cell lysates, resolved by SDS-PAGE. Membranes were blocked for 1 hour at RT with 5% (w/v) non-fat milk in TBS-T (50 mM Tris-Cl, 100 mM NaCl, 0.1% Tween-20, pH 7.4) and probed overnight at 4 °C with the antibodies indicated (Suppl. Table 1). The membranes were then rinsed 3 times with TBS-T and incubated with the secondary conjugated antibodies. Signals were detected using the ECL Western Blotting Substrate kit (GE Healthcare) or the Odyssey CLX system (Licor).
Tissue samples were fixed in 10% neutral buffered formalin (4% formaldehyde in solution), paraffin-embedded, and 3 μm sections were mounted on TOMO®slides and dried overnight. For different staining methods, slides were deparaffinized in xylene and re-hydrated through a series of graded ethanols until they reached water. Consecutive sections were H&E stained and different immunohistochemistry reactions were carried out in an automated immunostaining platform (Discovery XT-ULTRA, Ventana-Roche). Antigen retrieval was performed with the appropriate pH buffer (CC1: Ventana, Roche) and endogenous peroxidases were blocked with hydrogen peroxide (3%). The slides were then incubated with the appropriate primary antibody (anti-P53, anti-fibronectin or anti-P-selectin: Suppl. Table 1) and then with the corresponding horseradish peroxidase conjugated secondary antibodies: anti-rat (Vector) and OmniMap anti-Rabbit visualization systems (Ventana, Roche). The immunohistochemical reaction was developed using 3, 30-diaminobenzidine tetrahydrochloride (DAB: ChromoMap DAB, Ventana, Roche) and the nuclei were counterstained with hematoxylin. Finally, the slides were dehydrated, cleared and mounted in a permanent mounting medium for microscopy. Positive control sections known to be positive for the primary antibody were included in each staining run. The images from the whole slides were acquired on a slide scanner (AxioScan Z1, Zeiss) and captured with the Zen Blue Software (V3.1 Zeiss).
Cells were blocked with 4% PFA for 20 min and after three washes with PBS, the sections were incubated for 15 min with PBS containing 0.2% Triton X-100. Samples were blocked for 1 h at RT with PBS containing 1% BSA, 5% donkey serum and 0.05% Triton X-100. After three washes, the tissues were probed with the primary antibodies at 4 °C overnight (see Suppl. Table 1) and after another three washes, they were incubated for 1 h with the Alexa Fluor secondary antibodies (diluted 1:500: Molecular Probes) and washed again. The samples were mounted with Prolong DAPI (Thermo), images were obtained using a TCS-SP8 STED 3X confocal microscope (Leica-Microsystems) equiped with a 63X/NA1.4 oil immersion objective, AFC, AOBS, a tunable white laser and LAS-X software v3.7.5. Images were analyzed with Fiji v2.1 software.
Mouse lung endothelial cells (MLECs) were obtained and cultured as described previously94,95. Briefly, lungs from male DIO mice were excised, disaggregated and digested in 0.1% collagenase (Gibco) for 1 h at 37 °C. The cell suspensions was seeded onto plates coated with 10 µg/ml fibronectin (Sigma), 10 µg/ml collagen I (PureCol) and 0.1% gelatin (Sigma). After attachment, the cells were negatively selected with an anti-CD16/CD32 mAb (BD Biosciences, 553141) coupled to magnetic beads (Dynal, Invitrogen, 11035) and then positively selected with anti-ICAM-2 (BD Biosciences, 553325) coupled to magnetic beads. The cells recovered were seeded onto plates coated with 10 µg/ml collagen I and left to reach confluency after a further 7 days. The cell suspension was stained with CD45-PB (Invitrogen, MCD4528), CD31-APC (BD Biosciences, 551262) and 7-AAD Viability Dye (Invitrogen, A1310). Doublets and dead cells were gated prior to FACS sorting in a FACS ARIA IIu (BD), and viable CD45– CD31+ ECs were sorted into collecting medium containing 10% FBS (Thermo Fisher Scientific) and immediately frozen to isolate RNA (all the details on the antibodies and their dilutions are available in Suppl. Table 1).
Total RNA was extracted from the cells using the TRIzol Reagent (Invitrogen). After recovering the aqueous phase using chloroform, DNase treatment and further purification was performed with the RNeasy Micro kit (Qiagen), following the manufacturer's instructions. Subsequently, 500 cells per sample were processed with the “NEBNext Single Cell/Low Input RNA Library Prep” kit (NEB #E6420), according to the manufacturer's instructions. Briefly, extracts obtained by suspending the sample in cell lysis buffer were subjected to oligo(dT) primed reverse transcription in a template switching reaction. Double stranded cDNA was then produced by limited-cycle PCR, and sequencing libraries were completed with the “NEBNext Ultra II FS DNA Library Prep Kit for Illumina” (NEB #E7805) and analyzed on an Illumina NextSeq 550 (with v2.5 reagent kits). Single-end 85 bp reads were obtained, their clontech adapters were removed with Cutadapt (https://cutadapt.readthedocs.io/en/stable/), subsequently removing the last 15 bp from the reads, and they were then analyzed in the nextpresso pipeline96: sequencing quality was checked with FastQC v0.11.7 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/); reads were aligned to the mouse reference genome (GRCm38) with TopHat-2.0.1097, using Bowtie 1.0.098 and Samtools 0.1.1999, and allowing three mismatches and twenty multi-hits; read counts were obtained with HTSeq-count v0.6.1100, using the mouse gene annotation from GENCODE (encode.vM20.GRCm38.Ensembl95)101; differential expression was established with DESeq2102, using a 0.05 FDR; GSEAPreranked103 was used to perform gene set enrichment for several gene signatures on a pre-ranked gene list, setting 1,000 gene set permutations. Only those gene sets with significant enrichment (FDR q-value < 0.25) were considered.
Platelets from male DIO mice were lysed in 7 M urea and 2 M thiourea, digested with trypsin using the standard FASP protocol and labeled using the iTRAQ® reagent 8-plex. Pools of platelets from 3 mice per diet were used in each condition For whole proteome analysis, peptides were fractionated using an online HpH reverse-phase HPLC system and then, concatenated into 15 fractions. Samples were analyzed by LC-MS/MS using an Impact Mass Spectrometer (Bruker) and the Raw files obtained were analyzed with MaxQuant against a mouse protein database. Statistical analysis was achieved with Prostar. To integrate the RNA-seq performed on CD45-, CD31+ MLECS and the proteomic profile of platelets isolated from ND (N = 3) and HFD (N = 3) male mice, 2916 genes in common RNA–protein pairs were considered. Co-regulated pairs were FDR < 0.05 (colored), 5 above the FC cut-off (in solid color) applying a protein fold change >0.25 or <−0.25.
Intravital microscopy of the lung was performed as reported elsewhere104. Briefly, male DIO mice were anesthetized and ventilated mechanically through the trachea using a small-animal ventilator (model 687: Harvard Apparatus). A right lateral thoracotomy was performed and the lung was positioned under the window of a custom-built fixation device, applying a mild vacuum to hold the lung in position during microscopy. To visualize platelets, anti-CD41-PE was injected intravenously (1 μg/mouse), and the lungs were then analyzed 10 min after the mice were injected with an anti-CD41-PE-antibody and 1x105 EO771-GFP cells. Kinetic parameters were quantified, such as the Tumor-Platelet Aggregation index (Fig. 4G), quantifying the platelets in the tumor cell area and those in the surrounding region. A ratio between the interacting platelets (closer) and the total platelets in the area (both interacting and non-interacting) was then calculated (see Suppl. Figure 3E for more details). We calculated the platelet accumulation around tumor cells and the same area measured in a different region (random location) without green cells to verify the specificity of the interactions. Intravital experiments were performed on a TCS SP5 confocal microscope (Leica-Microsystems) equipped with a 20x/NA 0.7 dry objective, an AOBS and a temperature controller driven by the LAS AF v2.6 software, and Z-stack time lapse videos were obtained and analyzed using an ImageJ macro105. The algorithm developed involved: first finding the tumor cell in each frame and its segmentation into the green channel; this region of interest is dilated to obtain a new area around the cell (surrounding region); subsequently, the platelet area is segmented into the red channel; and finally, the total area is measured and the number of particles in each region of interest was assessed (tumor cells and surrounding region). In addition, other measures were calculated, like the segmented area of the platelets in the full frame and the size of the tumor cell. These measures were studied and analyzed using SPSS and Microsoft Excel to obtain the results shown here.
The sample size for in vitro and in vivo experiments was selected based on pilot studies. Animals were randomly allocated for dietary studies and for studies involving tumor cell injection. For all other experiments, no specific randomization method was followed as this was irrelevant for the in vitro assays. Researchers were blind to the final outcome assessment in the mouse experiments but not to the allocation or treatment of the mice in the experiments. For the remaining in vitro experiments, researchers were not blinded but the experiments were performed by different individuals. Error bars in the graphical data represent the mean ± s.d. unless otherwise specified. For in vivo and in vitro assays, the number of independent experiments and biological replicates are indicated in the figure legends. Statistical significance was determined using GraphPad Prism software v.9.1.0, applying a two-tailed unpaired or paired Student's t-tests, one-way or two-way ANOVA, or non-parametric tests as appropriate. For human studies, Pearson's correlation was assessed for bivariate correlations between the BMI and each coagulation test parameter. Survival analyzes were carried out with Kaplan-Meier estimates and LogRank tests. Multivariate analyzes were performed according to the Cox's proportionate hazards methods. All tests were two-sided and a P-value cut-off of 0.05 was deemed statistically significant. All these tests were performed with the SPSS V.11 software package.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Raw data from RNA sequencing and mass spectrometry analysis are publicly available. The RNA analysis data has been deposited in the GEO database: accession No. GSE237839. Mass spectrometry raw data raw data is deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD051176. All the data and materials referred to in the main text or the supplementary materials are available upon request. Source data are provided with this paper.
Massague, J. & Ganesh, K. Metastasis-initiating cells and ecosystems. Cancer Discov. 11, 971–994 (2021).
Article
CAS
PubMed
PubMed Central
MATH
Google Scholar
Lambert, A. W., Pattabiraman, D. R. & Weinberg, R. A. Emerging biological principles of metastasis. Cell 168, 670–691 (2017).
Article
CAS
PubMed
PubMed Central
MATH
Google Scholar
de Visser, K. E. & Joyce, J. A. The evolving tumor microenvironment: from cancer initiation to metastatic outgrowth. Cancer Cell 41, 374–403 (2023).
Article
PubMed
Google Scholar
Rubio-Jurado, B. et al. Obesity, thrombotic risk, and inflammation in cancer. Adv. Clin. Chem. 85, 71–89 (2018).
Article
CAS
PubMed
MATH
Google Scholar
Calle, E. E., Rodriguez, C., Walker-Thurmond, K. & Thun, M. J. Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults. N. Engl. J. Med 348, 1625–1638 (2003).
Article
PubMed
Google Scholar
Petrelli, F. et al. Association of obesity with survival outcomes in patients with cancer: a systematic review and meta-analysis. JAMA Netw. Open 4, e213520 (2021).
Article
PubMed
PubMed Central
MATH
Google Scholar
Harborg, S. et al. Overweight and prognosis in triple-negative breast cancer patients: a systematic review and meta-analysis. NPJ Breast Cancer 7, 119 (2021).
Article
PubMed
PubMed Central
Google Scholar
McDowell S. A. C., et al. Obesity alters monocyte developmental trajectories to enhance metastasis. J Exp Med 220, e20220509 (2023).
Quail, D. F. et al. Obesity alters the lung myeloid cell landscape to enhance breast cancer metastasis through IL5 and GM-CSF. Nat. Cell Biol. 19, 974–987 (2017).
Article
CAS
PubMed
PubMed Central
Google Scholar
McDowell, S. A. C. et al. Neutrophil oxidative stress mediates obesity-associated vascular dysfunction and metastatic transmigration. Nat. Cancer 2, 545–562 (2021).
Article
CAS
PubMed
Google Scholar
Ellulu, M. S., Patimah, I., Khaza'ai, H., Rahmat, A. & Abed, Y. Obesity and inflammation: the linking mechanism and the complications. Arch. Med Sci. 13, 851–863 (2017).
Article
CAS
PubMed
Google Scholar
Henning, R. J. Obesity and obesity-induced inflammatory disease contribute to atherosclerosis: a review of the pathophysiology and treatment of obesity. Am. J. Cardiovasc Dis. 11, 504–529 (2021).
CAS
PubMed
PubMed Central
MATH
Google Scholar
Falanga, A., Russo, L., Milesi, V. & Vignoli, A. Mechanisms and risk factors of thrombosis in cancer. Crit. Rev. Oncol. Hematol. 118, 79–83 (2017).
Article
PubMed
Google Scholar
Erpenbeck, L. & Schon, M. P. Deadly allies: the fatal interplay between platelets and metastasizing cancer cells. Blood 115, 3427–3436 (2010).
Article
CAS
PubMed
PubMed Central
MATH
Google Scholar
Braun, A., Anders, H. J., Gudermann, T. & Mammadova-Bach, E. Platelet-cancer interplay: molecular mechanisms and new therapeutic avenues. Front Oncol. 11, 665534 (2021).
Article
CAS
PubMed
PubMed Central
Google Scholar
Xu, X. R. et al. Platelets are versatile cells: New discoveries in hemostasis, thrombosis, immune responses, tumor metastasis and beyond. Crit. Rev. Clin. Lab Sci. 53, 409–430 (2016).
Article
CAS
PubMed
MATH
Google Scholar
Nieswandt, B., Hafner, M., Echtenacher, B. & Mannel, D. N. Lysis of tumor cells by natural killer cells in mice is impeded by platelets. Cancer Res 59, 1295–1300 (1999).
CAS
PubMed
Google Scholar
Palumbo, J. S. et al. Platelets and fibrin(ogen) increase metastatic potential by impeding natural killer cell-mediated elimination of tumor cells. Blood 105, 178–185 (2005).
Article
CAS
PubMed
MATH
Google Scholar
Gay, L. J. & Felding-Habermann, B. Contribution of platelets to tumour metastasis. Nat. Rev. Cancer 11, 123–134 (2011).
Article
CAS
PubMed
PubMed Central
Google Scholar
Labelle, M., Begum, S. & Hynes, R. O. Platelets guide the formation of early metastatic niches. Proc. Natl Acad. Sci. USA 111, E3053–E3061 (2014).
Article
ADS
CAS
PubMed
PubMed Central
MATH
Google Scholar
Labelle, M. & Hynes, R. O. The initial hours of metastasis: the importance of cooperative host-tumor cell interactions during hematogenous dissemination. Cancer Discov. 2, 1091–1099 (2012).
Article
CAS
PubMed
PubMed Central
MATH
Google Scholar
Peinado, H. et al. Pre-metastatic niches: organ-specific homes for metastases. Nat. Rev. Cancer 17, 302–317 (2017).
Article
CAS
PubMed
MATH
Google Scholar
Coller, B. S. & Shattil, S. J. The GPIIb/IIIa (integrin alphaIIbbeta3) odyssey: a technology-driven saga of a receptor with twists, turns, and even a bend. Blood 112, 3011–3025 (2008).
Article
CAS
PubMed
PubMed Central
Google Scholar
Shattil, S. J., Kim, C. & Ginsberg, M. H. The final steps of integrin activation: the end game. Nat. Rev. Mol. Cell Biol. 11, 288–300 (2010).
Article
CAS
PubMed
PubMed Central
MATH
Google Scholar
Shattil, S. J. & Newman, P. J. Integrins: dynamic scaffolds for adhesion and signaling in platelets. Blood 104, 1606–1615 (2004).
Article
CAS
PubMed
MATH
Google Scholar
Wu, Y. et al. The Tyrosine Kinase c-Src Specifically Binds to the Active Integrin alphaIIbbeta3 to Initiate Outside-in Signaling in Platelets. J. Biol. Chem. 290, 15825–15834 (2015).
Article
CAS
PubMed
PubMed Central
MATH
Google Scholar
Shen, B. et al. A directional switch of integrin signalling and a new anti-thrombotic strategy. Nature 503, 131–135 (2013).
Article
ADS
CAS
PubMed
PubMed Central
MATH
Google Scholar
Konopatskaya, O. et al. PKCalpha regulates platelet granule secretion and thrombus formation in mice. J. Clin. Invest 119, 399–407 (2009).
CAS
PubMed
PubMed Central
Google Scholar
Yoshioka, A. et al. Identification of protein kinase Calpha as an essential, but not sufficient, cytosolic factor for Ca2+-induced alpha- and dense-core granule secretion in platelets. J. Biol. Chem. 276, 39379–39385 (2001).
Article
CAS
PubMed
MATH
Google Scholar
Chen, J. et al. Impaired platelet responses to thrombin and collagen in AKT-1-deficient mice. Blood 104, 1703–1710 (2004).
Article
CAS
PubMed
MATH
Google Scholar
Mazharian, A., Thomas, S. G., Dhanjal, T. S., Buckley, C. D. & Watson, S. P. Critical role of Src-Syk-PLCgamma2 signaling in megakaryocyte migration and thrombopoiesis. Blood 116, 793–800 (2010).
Article
CAS
PubMed
Google Scholar
Levy-Toledano, S. Platelet signal transduction pathways: could we organize them into a ‘hierarchy'? Haemostasis 29, 4–15 (1999).
CAS
PubMed
Google Scholar
Levy-Toledano, S. et al. Phosphorylation and dephosphorylation mechanisms in platelet function: a tightly regulated balance. Thromb. Haemost. 78, 226–233 (1997).
Article
CAS
PubMed
MATH
Google Scholar
Kwaifa I. K., Bahari H., Yong Y. K., Noor S. M. Endothelial dysfunction in obesity-induced inflammation: molecular mechanisms and clinical implications. Biomolecules 10, 291 (2020).
Ingalls, A. M., Dickie, M. M. & Snell, G. D. Obese, a new mutation in the house mouse. J. Hered. 41, 317–318 (1950).
Article
CAS
PubMed
Google Scholar
Coleman, D. L. & Hummel, K. P. The influence of genetic background on the expression of the obese (Ob) gene in the mouse. Diabetologia 9, 287–293 (1973).
Article
CAS
PubMed
MATH
Google Scholar
Kaplan, R. N. et al. VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature 438, 820–827 (2005).
Article
ADS
CAS
PubMed
PubMed Central
Google Scholar
Ni, H., Papalia, J. M., Degen, J. L. & Wagner, D. D. Control of thrombus embolization and fibronectin internalization by integrin alpha IIb beta 3 engagement of the fibrinogen gamma chain. Blood 102, 3609–3614 (2003).
Article
CAS
PubMed
Google Scholar
Hotamisligil, G. S., Shargill, N. S. & Spiegelman, B. M. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 259, 87–91 (1993).
Article
ADS
CAS
PubMed
MATH
Google Scholar
Johnstone, C. N. et al. Functional and molecular characterisation of EO771.LMB tumours, a new C57BL/6-mouse-derived model of spontaneously metastatic mammary cancer. Dis. Model Mech. 8, 237–251 (2015).
CAS
PubMed
PubMed Central
MATH
Google Scholar
Zou, L., Cao, S., Kang, N., Huebert, R. C. & Shah, V. H. Fibronectin induces endothelial cell migration through beta1 integrin and Src-dependent phosphorylation of fibroblast growth factor receptor-1 at tyrosines 653/654 and 766. J. Biol. Chem. 287, 7190–7202 (2012).
Article
CAS
PubMed
PubMed Central
Google Scholar
Pulous, F. E., Grimsley-Myers, C. M., Kansal, S., Kowalczyk, A. P. & Petrich, B. G. Talin-dependent integrin activation regulates ve-cadherin localization and endothelial cell barrier function. Circ. Res 124, 891–903 (2019).
Article
CAS
PubMed
PubMed Central
Google Scholar
Hu, G., Place, A. T. & Minshall, R. D. Regulation of endothelial permeability by Src kinase signaling: vascular leakage versus transcellular transport of drugs and macromolecules. Chem. Biol. Interact. 171, 177–189 (2008).
Article
CAS
PubMed
MATH
Google Scholar
Li, Z., Delaney, M. K., O'Brien, K. A. & Du, X. Signaling during platelet adhesion and activation. Arterioscler Thromb. Vasc. Biol. 30, 2341–2349 (2010).
Article
CAS
PubMed
PubMed Central
MATH
Google Scholar
Sechler, J. L., Corbett, S. A. & Schwarzbauer, J. E. Modulatory roles for integrin activation and the synergy site of fibronectin during matrix assembly. Mol. Biol. Cell 8, 2563–2573 (1997).
Article
CAS
PubMed
PubMed Central
Google Scholar
Schumacher S., et al. Structural insights into integrin alpha(5)beta(1) opening by fibronectin ligand. Sci. Adv. 7, eabe9716 (2021).
Ju, J. A. et al. Hypoxia Selectively enhances integrin alpha(5)beta(1) receptor expression in breast cancer to promote metastasis. Mol. Cancer Res 15, 723–734 (2017).
Article
CAS
PubMed
PubMed Central
MATH
Google Scholar
Varner, J. A., Emerson, D. A. & Juliano, R. L. Integrin alpha 5 beta 1 expression negatively regulates cell growth: reversal by attachment to fibronectin. Mol. Biol. Cell 6, 725–740 (1995).
Article
CAS
PubMed
PubMed Central
MATH
Google Scholar
Pantano, F. et al. Integrin alpha5 in human breast cancer is a mediator of bone metastasis and a therapeutic target for the treatment of osteolytic lesions. Oncogene 40, 1284–1299 (2021).
Article
CAS
PubMed
PubMed Central
MATH
Google Scholar
Schimmel L., et al. c-Src controls stability of sprouting blood vessels in the developing retina independently of cell-cell adhesion through focal adhesion assembly. Development 147, dev185405 (2020).
Bergmeier, W., Rackebrandt, K., Schroder, W., Zirngibl, H. & Nieswandt, B. Structural and functional characterization of the mouse von Willebrand factor receptor GPIb-IX with novel monoclonal antibodies. Blood 95, 886–893 (2000).
Article
CAS
PubMed
Google Scholar
Dent, R. et al. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin. Cancer Res 13, 4429–4434 (2007).
Article
PubMed
MATH
Google Scholar
Menter, D. G. et al. Platelets and cancer: a casual or causal relationship: revisited. Cancer Metastasis Rev. 33, 231–269 (2014).
Article
PubMed
PubMed Central
MATH
Google Scholar
Blokhin, I. O. & Lentz, S. R. Mechanisms of thrombosis in obesity. Curr. Opin. Hematol. 20, 437–444 (2013).
Article
CAS
PubMed
PubMed Central
MATH
Google Scholar
Badimon, L., Hernandez Vera, R. & Vilahur, G. Atherothrombotic risk in obesity. Hamostaseologie 33, 259–268 (2013).
Article
CAS
PubMed
MATH
Google Scholar
Senis, Y. A., Mazharian, A. & Mori, J. Src family kinases: at the forefront of platelet activation. Blood 124, 2013–2024 (2014).
Article
CAS
PubMed
PubMed Central
Google Scholar
Kim, M. P., Park, S. I., Kopetz, S. & Gallick, G. E. Src family kinases as mediators of endothelial permeability: effects on inflammation and metastasis. Cell Tissue Res 335, 249–259 (2009).
Article
CAS
PubMed
Google Scholar
Petrich, B. G. Talin-dependent integrin signalling in vivo. Thromb. Haemost. 101, 1020–1024 (2009).
Article
CAS
PubMed
MATH
Google Scholar
Hynes, R. O. Integrins: a family of cell surface receptors. Cell 48, 549–554 (1987).
Article
CAS
PubMed
MATH
Google Scholar
De Kock L., Freson K. The (Patho)biology of SRC kinase in platelets and megakaryocytes. Medicina (Kaunas) 56, 633 (2020).
Harper, M. T. & Poole, A. W. Diverse functions of protein kinase C isoforms in platelet activation and thrombus formation. J. Thromb. Haemost. 8, 454–462 (2010).
Article
CAS
PubMed
MATH
Google Scholar
Murphy J. M., Jeong K., Lim S. S. FAK family kinases in vascular diseases. Int. J. Mol. Sci. 21, 3630 (2020).
Yau, J. W., Teoh, H. & Verma, S. Endothelial cell control of thrombosis. BMC Cardiovasc Disord. 15, 130 (2015).
Article
PubMed
PubMed Central
MATH
Google Scholar
Bondareva, O. et al. Single-cell profiling of vascular endothelial cells reveals progressive organ-specific vulnerabilities during obesity. Nat. Metab. 4, 1591–1610 (2022).
Article
CAS
PubMed
PubMed Central
MATH
Google Scholar
Neubauer, K. & Zieger, B. Endothelial cells and coagulation. Cell Tissue Res 387, 391–398 (2022).
Article
CAS
PubMed
MATH
Google Scholar
Bergmeier W., Hynes R. O. Extracellular matrix proteins in hemostasis and thrombosis. Cold Spring Harb. Perspect Biol. 4, a005132 (2012).
Cloutier, N. et al. Platelets can enhance vascular permeability. Blood 120, 1334–1343 (2012).
Article
CAS
PubMed
MATH
Google Scholar
To, W. S. & Midwood, K. S. Plasma and cellular fibronectin: distinct and independent functions during tissue repair. Fibrogenes. Tissue Repair 4, 21 (2011).
Article
CAS
MATH
Google Scholar
Wang, Y. et al. Plasma fibronectin supports hemostasis and regulates thrombosis. J. Clin. Invest 124, 4281–4293 (2014).
Article
PubMed
PubMed Central
MATH
Google Scholar
Maurer, E. et al. Fibrillar cellular fibronectin supports efficient platelet aggregation and procoagulant activity. Thromb. Haemost. 114, 1175–1188 (2015).
Article
PubMed
MATH
Google Scholar
Cho, J. & Mosher, D. F. Enhancement of thrombogenesis by plasma fibronectin cross-linked to fibrin and assembled in platelet thrombi. Blood 107, 3555–3563 (2006).
Article
CAS
PubMed
PubMed Central
MATH
Google Scholar
Prakash, P., Kulkarni, P. P., Lentz, S. R. & Chauhan, A. K. Cellular fibronectin containing extra domain A promotes arterial thrombosis in mice through platelet Toll-like receptor 4. Blood 125, 3164–3172 (2015).
Article
CAS
PubMed
PubMed Central
Google Scholar
Malik, G. et al. Plasma fibronectin promotes lung metastasis by contributions to fibrin clots and tumor cell invasion. Cancer Res 70, 4327–4334 (2010).
Article
CAS
PubMed
PubMed Central
MATH
Google Scholar
Kanters, S. D. et al. Plasma levels of cellular fibronectin in diabetes. Diab. Care 24, 323–327 (2001).
Article
CAS
Google Scholar
Dhanesha, N., Jain, M., Doddapattar, P., Undas, A. & Chauhan, A. K. Cellular fibronectin promotes deep vein thrombosis in diet-induced obese mice. J. Thromb. Haemost. 19, 814–821 (2021).
Article
CAS
PubMed
Google Scholar
Yurdagul, A. Jr. et al. alpha5beta1 integrin signaling mediates oxidized low-density lipoprotein-induced inflammation and early atherosclerosis. Arterioscler Thromb. Vasc. Biol. 34, 1362–1373 (2014).
Article
CAS
PubMed
PubMed Central
MATH
Google Scholar
Green, J., Yurdagul, A. Jr., McInnis, M. C., Albert, P. & Orr, A. W. Flow patterns regulate hyperglycemia-induced subendothelial matrix remodeling during early atherogenesis. Atherosclerosis 232, 277–284 (2014).
Article
CAS
PubMed
Google Scholar
Feaver, R. E., Gelfand, B. D., Wang, C., Schwartz, M. A. & Blackman, B. R. Atheroprone hemodynamics regulate fibronectin deposition to create positive feedback that sustains endothelial inflammation. Circ. Res 106, 1703–1711 (2010).
Article
CAS
PubMed
PubMed Central
Google Scholar
Schaffner, F., Ray, A. M. & Dontenwill, M. Integrin alpha5beta1, the fibronectin receptor, as a pertinent therapeutic target in solid tumors. Cancers (Basel) 5, 27–47 (2013).
Article
CAS
PubMed
Google Scholar
Bousquenaud, M., Fico, F., Solinas, G., Ruegg, C. & Santamaria-Martinez, A. Obesity promotes the expansion of metastasis-initiating cells in breast cancer. Breast Cancer Res 20, 104 (2018).
Article
PubMed
PubMed Central
Google Scholar
Foss, A., Munoz-Sagredo, L., Sleeman, J. & Thiele, W. The contribution of platelets to intravascular arrest, extravasation, and outgrowth of disseminated tumor cells. Clin. Exp. Metastasis 37, 47–67 (2020).
Article
PubMed
Google Scholar
Lickert, S. et al. Platelets drive fibronectin fibrillogenesis using integrin alphaIIbbeta3. Sci. Adv. 8, eabj8331 (2022).
Article
CAS
PubMed
PubMed Central
Google Scholar
Kim, Y. J., Borsig, L., Varki, N. M. & Varki, A. P-selectin deficiency attenuates tumor growth and metastasis. Proc. Natl Acad. Sci. USA 95, 9325–9330 (1998).
Article
ADS
CAS
PubMed
PubMed Central
MATH
Google Scholar
Borsig, L. et al. Heparin and cancer revisited: mechanistic connections involving platelets, P-selectin, carcinoma mucins, and tumor metastasis. Proc. Natl Acad. Sci. USA 98, 3352–3357 (2001).
Article
ADS
CAS
PubMed
PubMed Central
MATH
Google Scholar
Garcia-Leon, M. J. et al. Platelets favor the outgrowth of established metastases. Nat. Commun. 15, 3297 (2024).
Article
ADS
CAS
PubMed
PubMed Central
MATH
Google Scholar
Lake, B., Damery, S. & Jolly, K. Effectiveness of weight loss interventions in breast cancer survivors: a systematic review of reviews. BMJ Open 12, e062288 (2022).
Article
PubMed
PubMed Central
MATH
Google Scholar
Bian, X. et al. Roles of platelets in tumor invasion and metastasis: a review. Heliyon 8, e12072 (2022).
Article
CAS
PubMed
PubMed Central
MATH
Google Scholar
Lucotti, S. et al. Aspirin blocks formation of metastatic intravascular niches by inhibiting platelet-derived COX-1/thromboxane A2. J. Clin. Invest 129, 1845–1862 (2019).
Article
PubMed
PubMed Central
MATH
Google Scholar
Lucotti, S. & Muschel, R. J. Platelets and metastasis: new implications of an old interplay. Front Oncol. 10, 1350 (2020).
Article
PubMed
PubMed Central
MATH
Google Scholar
Prevost, N., Kato, H., Bodin, L. & Shattil, S. J. Platelet integrin adhesive functions and signaling. Methods Enzymol. 426, 103–115 (2007).
Article
CAS
PubMed
MATH
Google Scholar
Hurtado, B. et al. Thrombocytopenia-associated mutations in Ser/Thr kinase MASTL deregulate actin cytoskeletal dynamics in platelets. J. Clin. Invest 128, 5351–5367 (2018).
Article
PubMed
PubMed Central
MATH
Google Scholar
Mitrugno, A., Williams, D., Kerrigan, S. W. & Moran, N. A novel and essential role for FcgammaRIIa in cancer cell-induced platelet activation. Blood 123, 249–260 (2014).
Article
CAS
PubMed
Google Scholar
Bellavite, P. et al. A colorimetric method for the measurement of platelet adhesion in microtiter plates. Anal. Biochem 216, 444–450 (1994).
Article
CAS
PubMed
Google Scholar
Oblander, S. A. et al. Distinctive functions of membrane type 1 matrix-metalloprotease (MT1-MMP or MMP-14) in lung and submandibular gland development are independent of its role in pro-MMP-2 activation. Dev. Biol. 277, 255–269 (2005).
Article
CAS
PubMed
Google Scholar
Esteban, S. et al. Endothelial MT1-MMP targeting limits intussusceptive angiogenesis and colitis via TSP1/nitric oxide axis. EMBO Mol. Med 12, e10862 (2020).
Article
CAS
PubMed
Google Scholar
Graña, O., Rubio-Camarillo, M., Fdez-Riverola, F., Pisano, D. & Glez-Peña, D. Nextpresso: next generation sequencing expression analysis pipeline. Curr. Bioinforma. 13, 583–591 (2018).
Article
MATH
Google Scholar
Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).
Article
CAS
PubMed
PubMed Central
MATH
Google Scholar
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Article
PubMed
PubMed Central
Google Scholar
Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Article
PubMed
PubMed Central
MATH
Google Scholar
Frankish, A. et al. Gencode 2021. Nucleic Acids Res 49, D916–D923 (2021).
Article
CAS
PubMed
Google Scholar
Anders, S., Pyl, P. T. & Huber, W. HTSeq–a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).
Article
CAS
PubMed
MATH
Google Scholar
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Article
PubMed
PubMed Central
MATH
Google Scholar
Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).
Article
ADS
CAS
PubMed
PubMed Central
Google Scholar
Adrover, J. M. et al. Programmed ‘disarming' of the neutrophil proteome reduces the magnitude of inflammation. Nat. Immunol. 21, 135–144 (2020).
Article
CAS
PubMed
PubMed Central
MATH
Google Scholar
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).
Article
CAS
PubMed
PubMed Central
Google Scholar
Download references
This work was supported by the Worldwide Cancer Research UK (24-0197, 16-1244), Agencia Estatal de Investigación/Ministerio de Ciencia e Innovación (AEI/MCIN: PID2020-118558RB-I00/AEI/10.13039/501100011033), WHRI-ACADEMY, a COFUND of Marie Curie action (WHRI-309), Fundación Bancaria “la Caixa” (HR18-00256) granted to HP. This work was also supported by grants from the Spanish National Research and Development Plan, Instituto de Salud Carlos III, and FEDER (PI20/01837 and PI23/01932 to S.R-P.); and AECC (AECC_Lab 2020) to S.R-P and ISCIII/FEDER (PI21/01641) to R.T-R. The CNIO is a certified Severo Ochoa Center of Excellence, supported by the Spanish Government through the Instituto de Salud Carlos III (ISCIII). We thank Drs. Cyrus Ghajar, Luuke Hawinkels and Inge Verbrugge for providing cell lines, and we are grateful to Beatriz Salinas (Hospital General Universitario Gregorio Marañon) for synthetizing the NIR-IF dextran. We also thank the CNIO Histopathology Unit for their technical support and Alicia Garcia Arroyo (Centro de Investigaciones Biologicas) for her help in isolating MLECs. Figures were partially created with Servier Medical Art (https://smart.servier.com/) and Adobe Illustrator 24.1.
Microenvironment and Metastasis Laboratory, Molecular Oncology Programme, Spanish National Cancer Research Center (CNIO), Madrid, Spain
Marta Hergueta-Redondo, Sara Sánchez-Redondo, Vanesa Santos, Marina S. Mazariegos & Héctor Peinado
Cancer Cell Cycle Group, Preclinical & Translational Research Department, Vall d'Hebron Institute of Oncology (VHIO), Barcelona, Spain
Begoña Hurtado
Confocal Microscopy Unit, Biotechnology Programme, Spanish National Cancer Research Center (CNIO), Madrid, Spain
Manuel Pérez-Martínez, Gadea Mata & Diego Megias
Proteomics Unit, Biotechnology Programme, Spanish National Cancer Research Center (CNIO), Madrid, Spain
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Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, Quebec, Canada
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Lund Stem Cell Center (SCC), Lund University, Lund, Sweden
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Molecular Cytogenetics Unit, Human Cancer Genetics Programme, Spanish National Cancer Research Center (CNIO), Madrid, Spain
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Division of Hematopoietic Innovative Therapies, Biomedical Innovation Unit, Centro de Investigaciones Energéticas, Medioambientales y Tecnologicas (CIEMAT), Madrid, Spain
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Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain
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Flow Cytometry Core Unit, Biotechnology Programme, Spanish National Cancer Research Center (CNIO), Madrid, Spain
Lola Martínez
Bioinformatics Unit, Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
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Departamento de Ciencias Médicas Básicas, Instituto de Medicina Molecular Aplicada (IMMA-Nemesio Díez), Facultad de Medicina, Universidad San Pablo-CEU, CEU Universities, 28925, Alcorcón, Spain
Osvaldo Graña-Castro
Advanced Optical Microscopy – ISCIII Madrid, Madrid, Spain
Diego Megias
Breast Cancer Clinical Research Unit, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain
Miguel Quintela-Fandino
Medical Oncology, Hospital de Fuenlabrada, Madrid, Spain
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M.H.-R., S.S.-R., V.S., M.P.-M., P.X.-E., S.A.C.M., M.S.M., R.T.-R., performed the experiments. M.H.-R., B.H., P.X., M.S.M., M.P.-M., G.M., O.G.-C., D.M. designed the experiments and analyzed the data. H.P., S.R.-P., L.M., D.M., D.Q., M.Q.-F. supervised the experiments. H.P. and M.H.-R. conceived the hypothesis, oversaw the project and wrote the paper. H.P. supervised all the work and obtained funding. All authors have read the final version of the manuscript and agree to its publication.
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April 02, 2025
Preliminary results from a new study showed that the risk for sudden cardiac death (SCD) was more than twice as high in individuals who used antidepressants for 6 or more years compared with that in those not taking the medications.
While the findings suggest a possible association between long-term antidepressant use and SCD, researchers caution that the results do not imply these medications are inherently dangerous or should be discontinued.
“I hope doctors don't think they need to discontinue this treatment because it might pose a risk of sudden cardiac death,” study investigator Jasmin Mujkanovic, MD, PhD student, Copenhagen University Hospital, Copenhagen, Denmark, told Medscape Medical News.
Mujkanovic emphasized that depression itself is a known risk factor for SCD and that effective treatment can improve quality of life and support healthier lifestyle choices, which may in turn help reduce heart-related risks.
The findings were presented on March 30 at the European Heart Rhythm Association 2025, a scientific congress of the European Society of Cardiology.
Previous research has shown a link between long-term antidepressant use and poor cardiac health. Antipsychotics have also been tied to an increase risk for cardiovascular disease in older adults with dementia.
While earlier studies suggested that patients with depressive disorders have an increased risk for SCD, the effect of antidepressant exposure on SCD risk was unclear.
Researchers reviewed all death certificates and autopsy reports from 2010 among the 4.3 million residents of Denmark aged 18-90 years. The year 2010 was chosen because some of Mujkanovic's colleagues had already assessed every death certificate for that year and entered pertinent information into a database.
The researchers defined antidepressant exposure as having filled at least two prescriptions for the medication within a single year during the 12 years leading up to 2010, the start of the follow-up period. Exposure was categorized as either 1-5 years or 6 years or more.
Based on available data, deaths were categorized as SCD or non-SCD. There were 6002 deaths due to SCD, which included 1981 in the antidepressant cohort and 4021 in the unexposed population.
Investigators found that the risk for SCD was significantly higher in individuals exposed to antidepressants than in the general population across all age groups — except those aged 18-29 years, where the association was not statistically significant. Mujkanovic suggested that younger patients may not have used antidepressants long enough to be classified as having prolonged exposure.
After adjusting for age, sex, and comorbidities, including ischemic heart disease, chronic obstructive pulmonary disease, diabetes and arrhythmias, the risk for SCD was 56% higher in those with 1-5 years of exposure to antidepressants (hazard ratio [HR], 1.56; P < .001) and more than double for those with 6 or more years of exposure (HR, 2.17; P < .001).
The risk for SCD among individuals taking antidepressants, compared with the unexposed general population, varied slightly across age groups. However, among those aged 40-79 years, the incidence of SCD was significantly higher in individuals with 6 or more years of antidepressant use than in those with 1-5 years of exposure.
When comparing longer to shorter durations of antidepressant use, the differences in risk were significant in the following age groups: 40-49 years (incidence rate ratio [IRR], 1.7; P = .03), 50-59 years (IRR, 2.0; P < .001), and 60-69 years (IRR, 1.4; P < .001).
The differences in risk between antidepressant exposure durations were not statistically significant among the younger participants (age, 18-39 years) and the oldest group (age, 80-90 years).
Due to the study's design, it's challenging to determine whether the increased risk for SCD is linked to the underlying condition of depression or to the antidepressant medications themselves, Mujkanovic said. However, if the medications do contribute to the risk, it may be because some can alter the heart's electrical activity, he noted.
Mujkanovic also pointed out that certain antidepressants are associated with significant weight gain and QT interval prolongation, which can lead to metabolic syndrome — a known risk factor for atherosclerosis.
“It's like a chain reaction — atherosclerosis is a known risk factor for ischemic heart disease, and ischemic heart disease increases the risk for myocardial infarction, which increases the risks for cardiac death,” Mujkanovic said.
The study did not distinguish between different classes of antidepressants — such as selective serotonin reuptake inhibitors and tricyclics — and participants may have switched between classes during the study period.
It also did not analyze men and women separately, though Mujkanovic expressed interest in exploring sex-specific differences in future research.
Several experts weighed in on the findings in a statement from the UK-based independent nonprofit Science Media Centre (SMC).
Paul Keedwell, MB, PhD, consultant psychiatrist and fellow of the Royal College of Psychiatrists in London, the United Kingdom, agreed with the investigators' caution against discontinuing antidepressants based on the study's findings.
“The results should be treated with caution because the study was unable to separate the risks of antidepressant treatment from the risk of having depression per se,” Keedwell said.
Depression is linked to a 60% higher risk for heart disease, including SCD, a 50%-90% increased risk for life-threatening arrhythmias, and approximately double the risk for heart attack, Keedwell noted.
He added that individuals with depression tend to die earlier than those in the general population — with men dying up to 14 years earlier and women dying up to 10 years earlier.
While suicide contributes to this excess mortality, Keedwell emphasized that the leading cause is poor physical health, likely driven by an unhealthy lifestyle.
“More research is needed to directly compare the life expectancy in treated and untreated depression, but as things stand, the weight of evidence supports the conclusion that the risk of early death is much higher when depression is left untreated than when it is treated,” he said. “Therefore, people should not stop their antidepressant treatment based on this study.”
Also commenting for the SMC, Charles Pearman, MB, PhD, consultant cardiologist and electrophysiologist at Manchester University NHS Foundation Trust, noted that previous studies have identified a link between antipsychotic use and an increased risk for SCD.
He added that antidepressants may not directly cause SCD but could instead serve as markers for other underlying health issues or risk factors — such as obesity, hypertension, diabetes, smoking, or physical inactivity.
“The investigators tried to account for this possibility, but it is unclear which risk factors they considered,” Pearman said.
Pearman emphasized that the overall risk remains low and, like the study authors, advised against patients abruptly discontinuing their antidepressant treatment. Overall, while there was an increased risk from taking antidepressants, the risk remains small, he noted.
“People who are concerned about their risks should speak to their GP [general practitioner] rather than stopping their medicines abruptly,” he said.
The study received no external funding. Mujkanovic and Keedwell reported n o related conflicts of interest. Pearman disclosed no relevant conflicts.
Send comments and news tips to news@medscape.net.
Carlo Ancelotti appeared in court on Wednesday to face charges over unpaid taxes that could land the Real Madrid coach more than four years in prison.
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The soccer analyst believes that the Norwich City forward needs to find the right balance between his performances for club and country
Chelsea star Sam Kerr could return before the end of the season after an ACL injury, but her international call-up does not hint at an early comeback.
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Granit Xhaka was spotted furiously arguing with Bayer Leverkusen fans after the team's humiliating exit from DFB Pokal semi-final.
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Tottenham have confirmed that 18-year-old central midfielder Callum Olusesi has signed a new four-year contract with the north London club.
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Antonio Cassano slammed under-fire AC Milan forward Rafael Leao, claiming he's "half the player" that his teammate Christian Pulisic is.
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Birmingham City minority owner Tom Brady has been backed by Tim Sherwood to oversee back-to-back promotions for the club.
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Ousmane Dembele starred for PSG against Dunkerque and fans are calling for him to win the Ballon d'Or as reward for his stellar 2024-25 season.
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Check out our football expert's Liverpool vs Everton predictions and betting tips, prior to Wednesday's 15:00 ET Premier League clash (04/02).
All odds are courtesy of bet365, correct at the time of publishing and subject to change.
Our Liverpool vs Everton predictions indicate things couldn't have gone much worse for Liverpool before the international break. One minute they were competing for a treble, the next they were out of the Champions League and League Cup. However, they are unbeaten in 25 Premier League games and have one hand on the trophy.
Everton, meanwhile, have put together a streak of their own under David Moyes. They're without defeat in nine league matches and find themselves in 15th place. While top-flight safety isn't mathematically confirmed yet, it's not far away, and they've proven hard to beat of late.
Liverpool Expected Lineup: Alisson, Quansah, Konate, Van Dijk, Robertson, Gravenberch, Mac Allister, Salah, Szoboszlai, Gakpo, Nunez
Everton Expected Lineup: Pickford, O'Brien, Tarkowski, Branthwaite, Mykolenko, Gueye, Garner, Harrison, Doucoure, Alcaraz, Beto
Arne Slot's men may have faced setbacks outside the Premier League, but they remain strong in the league. They've won four in a row at Anfield, scoring 11, and haven't lost at home since September. They'll be desperate to continue that form against their bitter rivals this week.
The Toffees haven't picked up many wins recently, but they also haven't lost many. You have to go back to mid-January to find their last league defeat, and they've scored in every game since. If the Reds are still reeling from their CL and cup defeats, Moyes' side will sense an opportunity.
It's only a few weeks since these two sides last faced off, and that game ended with four goals between them. However, potential injuries to Alisson, Ryan Gravenberch and Vitali Mykolenko are worth considering, and a tight game is expected.
The Reds are deservedly favourites for this one, but Everton will look for any weaknesses in their defence. Moyes' side have scored in nine consecutive Premier League games and got 17 goals along the way. They've had eight different scorers in that time period.
A total of 25 Premier League matches have gone by since Liverpool failed to score in one. Therefore, a clean sheet for the Toffees would come as a surprise, but they'll feel confident of breaking down their rivals. Their chances of doing so increase if Iliman Ndiaye and Dwight McNeil can get back in the mix.
Both players were back in training last week, but it remains to see how much - or even if - they will feature at Anfield. Both teams have scored in five of Liverpool's last nine across all competitions - for Everton, it's seven in 10.
Not many teams will be eager to play against Mo Salah right now, but Everton have extra reason to fear him. In his 12 games against the Toffees so far, Salah has scored eight and assisted two - including one of each the last time they met.
The Egyptian has only failed to score in three of his 11 league outings since the turn of the year. He may not have been able to get things going against PSG or Newcastle United, but Everton should fear him. Slot's side will be out for revenge given the nature of their draw in February, and Salah is their destroyer-in-chief.
There's been a winner in eight of the last 12 Merseyside derbies, and Liverpool are desperate to put March behind them with three big points.
Bayern Munich star Harry Kane has been backed to return to the Premier League and "have a go" at Alan Shearer's goalscoring record.
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A furious Phil Parkinson accused referee Thomas Parsons of "trying to make a name for himself" after Wrexham's damaging draw with Cambridge.
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Endrick opened up on his future after scoring a vital goal for Real Madrid in their Copa del Rey semi-final thriller against Real Sociedad.
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Enzo Maresca has issued an injury update on Cole Palmer ahead of Chelsea's crunch clash with bitter rivals Tottenham.
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Atletico Madrid are eager to bring in Cristian Romero from Tottenham before the Club World Cup and the defender is tempted by a move.
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Anne Hathaway sent a special message to Bukayo Saka as the Hollywood star donned an Arsenal shirt to celebrate his goal on his return from injury.
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The Rojiblancos' campaign would effectively be over if they were to lose to Barcelona in the second leg of their Copa del Rey semi-final on Wednesday
As Antonio Rudiger raced away to celebrate with his team-mates after converting the decisive spot-kick in Real Madrid's shootout win over city rivals Atletico, Diego Simeone made a beeline for his disconsolate players. The message was clear: keep your heads up, you've done yourselves and your club proud. Then, the coach turned to fans and urged them to applaud a side that had given its all but once again come up agonisingly short in a Champions League tie against their neighbours from across the Spanish capital.
This was a particularly bitter pill for Atleti to swallow, though, as an incredibly controversial call had ultimately proved decisive, with the Video Assistant Referee (VAR) ruling out Julian Alvarez's converted penalty in the shootout for a 'double touch' that was neither deliberate nor obvious even in the multiple video replays that followed. Consequently, despite Simeone's best efforts to lift his players, centre-back Clement Lenglet admitted that they were all still in a "state of shock" in the dressing room.
"We produced a massive performance from start to finish," he told Movistar Plus after Atleti had won 1-0 on the night to draw 2-2 on aggregate. "I think we deserved [to win] as a team, a club, for the fans and their support, which helped us a lot. But we will handle the blow to our morale in the days ahead. We have a lot of work left to do, we have important things to play for, the season doesn't end today."
It might on Wednesday, though, as it appears as if Atletico still haven't come to terms with their desperately unfortunate Champions League elimination on March 12...
The 23-year-old shares her best travel tips, favorite jet lag hack and more in our exclusive chat.ByStephanie LivaudaisPublished Apr 02, 2025 copy_link
Published Apr 02, 2025
© 2023 Chris Smith
After back-to-back weeks across North America, Emma Navarro is finally in the home stretch. Born in New York but raised in South Carolina, the 23-year-old gearing up for her home tournament, the Credit One Charleston Open.But first, she has to make a detour.“Go see my family and my dogs. They're always my first stop when I get home to Charleston," Navarro tells TENNIS.com. "It's something that I always look forward to.”Read More: The rise of mold-breaking Emma Navarro - Peter BodoNavarro is soaking up as much quality family time as possible before the WTA Tour moves on to Europe. The American also is seeking a reset after mixed success in recent weeks, having won her first title of the season in Merida but departing Indian Wells and Miami with just one match win.The clay-court season kicks off in Charleston, a city with deep family connections for Navarro. She went to school at Ashley Hall in nearby downtown, while her father, Ben Navarro, is not only the WTA tournament's owner but also the owner of Credit One Bank, the event's title sponsor.
But first, she has to make a detour.“Go see my family and my dogs. They're always my first stop when I get home to Charleston," Navarro tells TENNIS.com. "It's something that I always look forward to.”Read More: The rise of mold-breaking Emma Navarro - Peter BodoNavarro is soaking up as much quality family time as possible before the WTA Tour moves on to Europe. The American also is seeking a reset after mixed success in recent weeks, having won her first title of the season in Merida but departing Indian Wells and Miami with just one match win.The clay-court season kicks off in Charleston, a city with deep family connections for Navarro. She went to school at Ashley Hall in nearby downtown, while her father, Ben Navarro, is not only the WTA tournament's owner but also the owner of Credit One Bank, the event's title sponsor.
“Go see my family and my dogs. They're always my first stop when I get home to Charleston," Navarro tells TENNIS.com. "It's something that I always look forward to.”Read More: The rise of mold-breaking Emma Navarro - Peter BodoNavarro is soaking up as much quality family time as possible before the WTA Tour moves on to Europe. The American also is seeking a reset after mixed success in recent weeks, having won her first title of the season in Merida but departing Indian Wells and Miami with just one match win.The clay-court season kicks off in Charleston, a city with deep family connections for Navarro. She went to school at Ashley Hall in nearby downtown, while her father, Ben Navarro, is not only the WTA tournament's owner but also the owner of Credit One Bank, the event's title sponsor.
Read More: The rise of mold-breaking Emma Navarro - Peter BodoNavarro is soaking up as much quality family time as possible before the WTA Tour moves on to Europe. The American also is seeking a reset after mixed success in recent weeks, having won her first title of the season in Merida but departing Indian Wells and Miami with just one match win.The clay-court season kicks off in Charleston, a city with deep family connections for Navarro. She went to school at Ashley Hall in nearby downtown, while her father, Ben Navarro, is not only the WTA tournament's owner but also the owner of Credit One Bank, the event's title sponsor.
Navarro is soaking up as much quality family time as possible before the WTA Tour moves on to Europe. The American also is seeking a reset after mixed success in recent weeks, having won her first title of the season in Merida but departing Indian Wells and Miami with just one match win.The clay-court season kicks off in Charleston, a city with deep family connections for Navarro. She went to school at Ashley Hall in nearby downtown, while her father, Ben Navarro, is not only the WTA tournament's owner but also the owner of Credit One Bank, the event's title sponsor.
The clay-court season kicks off in Charleston, a city with deep family connections for Navarro. She went to school at Ashley Hall in nearby downtown, while her father, Ben Navarro, is not only the WTA tournament's owner but also the owner of Credit One Bank, the event's title sponsor.
"My family and my dogs... They're always my first stop when I get home" Navarro tells TENNIS.com. © Instagram @emma_navarro48
© Instagram @emma_navarro48
It was here where she made her 2019 WTA debut as an 18-year-old wildcard, falling in straight sets to Laura Siegemund. Navarro then opted to go to college, and would go on to play tennis for University of Virginia and make a name for herself in becoming the 2022 NCAA champion before joining the pro tour.Read More: “I can be playful, and that comes through in my jewelry”: Emma Navarro becomes Mejuri brand ambassadorNow the 11th ranked player in the world, Navarro says her rapid rise has been a ‘learning experience'—and the lessons have been a bit unexpected.“So many super niche tennis lessons, like physical things,” she explained. “Just the things I'm learning about myself and how I have to play, and how other players play…“Off the court, or in a more philosophical sense, I've ‘learned how to lose,' for sure. I've learned how to win, as well, and how to manage stress, travel, and the ups and downs that come with tour life.“I think I've also learned that there's so much more to learn. I'm buckled up for a lot more lessons along the way.”
Read More: “I can be playful, and that comes through in my jewelry”: Emma Navarro becomes Mejuri brand ambassadorNow the 11th ranked player in the world, Navarro says her rapid rise has been a ‘learning experience'—and the lessons have been a bit unexpected.“So many super niche tennis lessons, like physical things,” she explained. “Just the things I'm learning about myself and how I have to play, and how other players play…“Off the court, or in a more philosophical sense, I've ‘learned how to lose,' for sure. I've learned how to win, as well, and how to manage stress, travel, and the ups and downs that come with tour life.“I think I've also learned that there's so much more to learn. I'm buckled up for a lot more lessons along the way.”
Now the 11th ranked player in the world, Navarro says her rapid rise has been a ‘learning experience'—and the lessons have been a bit unexpected.“So many super niche tennis lessons, like physical things,” she explained. “Just the things I'm learning about myself and how I have to play, and how other players play…“Off the court, or in a more philosophical sense, I've ‘learned how to lose,' for sure. I've learned how to win, as well, and how to manage stress, travel, and the ups and downs that come with tour life.“I think I've also learned that there's so much more to learn. I'm buckled up for a lot more lessons along the way.”
“So many super niche tennis lessons, like physical things,” she explained. “Just the things I'm learning about myself and how I have to play, and how other players play…“Off the court, or in a more philosophical sense, I've ‘learned how to lose,' for sure. I've learned how to win, as well, and how to manage stress, travel, and the ups and downs that come with tour life.“I think I've also learned that there's so much more to learn. I'm buckled up for a lot more lessons along the way.”
“Off the court, or in a more philosophical sense, I've ‘learned how to lose,' for sure. I've learned how to win, as well, and how to manage stress, travel, and the ups and downs that come with tour life.“I think I've also learned that there's so much more to learn. I'm buckled up for a lot more lessons along the way.”
“I think I've also learned that there's so much more to learn. I'm buckled up for a lot more lessons along the way.”
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Navarro, who has been ranked as high as world No. 8 and owns two WTA titles to her name, is still seeking a strong result in Charleston. She owns a 2-4 record on the green clay, including one round-of-16 appearance.While the American will be applying all her lessons learned on court at her home tournament, off the court she's indulging her wanderlust as a way to unwind from the WTA grind.“On my days off, I prioritize rest for sure. But I also like to get out there and see some things,” Navarro tells me. "I love to walk around different cities and neighborhoods especially, just see how the people live or see the different architecture."Things like that make me feel like I'm a little bit in touch with the local people and my surroundings.”
While the American will be applying all her lessons learned on court at her home tournament, off the court she's indulging her wanderlust as a way to unwind from the WTA grind.“On my days off, I prioritize rest for sure. But I also like to get out there and see some things,” Navarro tells me. "I love to walk around different cities and neighborhoods especially, just see how the people live or see the different architecture."Things like that make me feel like I'm a little bit in touch with the local people and my surroundings.”
“On my days off, I prioritize rest for sure. But I also like to get out there and see some things,” Navarro tells me. "I love to walk around different cities and neighborhoods especially, just see how the people live or see the different architecture."Things like that make me feel like I'm a little bit in touch with the local people and my surroundings.”
"Things like that make me feel like I'm a little bit in touch with the local people and my surroundings.”
"I love to walk around different cities and neighborhoods especially, just see how the people live or see the different architecture."© Instagram @emma_navarro48
© Instagram @emma_navarro48
Navarro will start her Charleston campaign on Wednesday at Credit One Stadium, where she will face No. 90 Hailey Baptiste in the second round. Baptiste, a fellow American, overcame Olivia Gadecki 6-2, 6-4 to advance.In the meantime, the No. 4 seed shared a few more "super niche tennis lessons"—including how she fights jetlag—with TENNIS.com:Travel tips from Emma Navarro“Hydration is key. You get super dehydrated when you're flying, so stay on top of your hydration.”“If you wake up in the middle of the night, or at like two or three a.m. or whatever, don't fight it. Just accept that you're awake. You're probably not going back to sleep. So just relax, but don't go on your phone and look at screens! Just lay there.”“Get out and exercise, or even just be outside and get some sunlight on your skin. That helps a bunch.”
In the meantime, the No. 4 seed shared a few more "super niche tennis lessons"—including how she fights jetlag—with TENNIS.com:Travel tips from Emma Navarro“Hydration is key. You get super dehydrated when you're flying, so stay on top of your hydration.”“If you wake up in the middle of the night, or at like two or three a.m. or whatever, don't fight it. Just accept that you're awake. You're probably not going back to sleep. So just relax, but don't go on your phone and look at screens! Just lay there.”“Get out and exercise, or even just be outside and get some sunlight on your skin. That helps a bunch.”
Ethan Quinn's first clay-court match on the ATP Tour was a memorable one.
The 21-year-old defeated World No. 35 Jordan Thompson 4-6, 6-4, 6-3 on Tuesday, earning the highest-ranked win of his career at the Fayez Sarofim & Co. U.S. Men's Clay Court Championship.
“That was a unique experience, my first one on clay. Not too comfortable out there. There are a few slips and a few almost falls,” said Quinn, who is competing in Houston at a career-high No. 130 in the PIF ATP Rankings.
“To get through that one, not on the floor, but also against a really tough opponent in Jordan — he beat me when I was a little bit younger just on Tour at a Challenger. To get revenge a little bit felt really good, but also to get one on clay and start the clay-court season.”
The wild card saved nine of the 13 break chances he faced, according to Infosys ATP Stats, and will next play American qualifier Colton Smith, a standout senior at the University of Arizona. Smith defeated Quinn earlier this season at an ATP Challenger Tour event in Cleveland, overcoming the 2023 NCAA singles champion 1-6, 6-1, 6-4 in the first round. Smith went on to win the title.
The 22-year-old Smith, who is also competing at a career high (No. 200) this week, cruised past Australian James Duckworth 6-2, 6-2 in opening-round action at the ATP 250.In other Houston action, Kei Nishikori claimed his eighth tour-level win of the season by racing past American Mitchell Krueger 6-4, 6-2 in a dominant display under the lights."I served really well today, I think that was the key to winning like this today," said Nishikori, who won 77 per cent of his first-serve points. "Overall, I played pretty well. I think I'm pretty happy with every shot, every moment."
Nishikori is making his third appearance at the clay-court event and first since reaching the final in 2011. One win shy of his 450th tour-level victory, the Japanese star will next face Christopher Eubanks.
The 28-year-old Eubanks and fellow American Mackenzie McDonald advanced with three-set wins. Eubanks defeated Rinky Hijikata 6-2, 4-6, 6-3 in a rematch of their first-round clash last year at the same event. Eubanks and Hijikata are now level at 1-1 in their Lexus ATP Head2Head series.
McDonald outlasted Daniel Elahi Galan 6-2, 6-7(3), 6-4 to set a second-round clash against fourth seed Brandon Nakashima. The 29-year-old McDonald will test his perfect 4-0 record against Nakashima.Watch Extended Highlights from Tuesday's action in Houston:
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The wild card saved nine of the 13 break chances he faced, according to Infosys ATP Stats, and will next play American qualifier Colton Smith, a standout senior at the University of Arizona. Smith defeated Quinn earlier this season at an ATP Challenger Tour event in Cleveland, overcoming the 2023 NCAA singles champion 1-6, 6-1, 6-4 in the first round. Smith went on to win the title.
The 22-year-old Smith, who is also competing at a career high (No. 200) this week, cruised past Australian James Duckworth 6-2, 6-2 in opening-round action at the ATP 250.
In other Houston action, Kei Nishikori claimed his eighth tour-level win of the season by racing past American Mitchell Krueger 6-4, 6-2 in a dominant display under the lights.
"I served really well today, I think that was the key to winning like this today," said Nishikori, who won 77 per cent of his first-serve points. "Overall, I played pretty well. I think I'm pretty happy with every shot, every moment."
Nishikori is making his third appearance at the clay-court event and first since reaching the final in 2011. One win shy of his 450th tour-level victory, the Japanese star will next face Christopher Eubanks.
The 28-year-old Eubanks and fellow American Mackenzie McDonald advanced with three-set wins. Eubanks defeated Rinky Hijikata 6-2, 4-6, 6-3 in a rematch of their first-round clash last year at the same event. Eubanks and Hijikata are now level at 1-1 in their Lexus ATP Head2Head series.
McDonald outlasted Daniel Elahi Galan 6-2, 6-7(3), 6-4 to set a second-round clash against fourth seed Brandon Nakashima. The 29-year-old McDonald will test his perfect 4-0 record against Nakashima.
Watch Extended Highlights from Tuesday's action in Houston:
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2025 Charleston
WTA Staff
Three Americans were the first players to reach the WTA 500 Credit One Charleston Open Round of 16, with Madison Keys, Amanda Anisimova and defending champion Danielle Collins all booking third-round spots on Tuesday evening.
Charleston: Draws | Scores | Order of play
No. 2 seed Keys had the trickiest path of the three into the Round of 16. The reigning Australian Open champion needed eight match points before she could close out her fellow American Caroline Dolehide 6-3, 7-6(4).
Keys held her first four match points while up a set and a break at 6-3, 5-4. However, Dolehide erased those chances, and two more in the following game, battling back to level footing in the second set of the power-hitting clash.
Currently ranked a career-high World No. 5, Keys needed to hold firm in the tiebreak before she at last defeated her compatriot, earning her 20th match-win of the year.
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"I think [Dolehide] started playing some of her best tennis at the end, and I think I got a little bit passive, and then all of a sudden, seven match points later..." Keys trailed off, shaking her head in her on-court interview. "The drama is escalating, and everyone just kinda wants to go home.
"But I got it done, and I get to come back out and play another match here."
The stage is still set for Keys to pick up her second Charleston title. She won the event in 2019, defeating Caroline Wozniacki to capture her first clay-court title, in a city where she has the best vibes.
"I've been coming here for more than the last decade, I love it here," Keys said. "[Bjorn Fratangelo and I] got married here, I always feel like this is one of my favorite places in the entire world, and I'm so happy to be back playing here."
Defending champ cruises: No. 7 seed Collins followed on Credit One Stadium, and she kicked off her title defense by besting fellow American Robin Montgomery 6-3, 6-1 in 1 hour and 9 minutes. Collins collected her seventh straight victory in Charleston.
Collins' title run last year was the middle of a 15-match winning streak on tour, which pushed her back into the Top 10 before the year was through. Collins won her first WTA 500 title here last year -- one week after she won her first WTA 1000 title at the 2024 Miami Open.
The determination to keep her Charleston streak going was evident from Collins on Tuesday, as she powered past 20-year-old Montgomery. Collins won 80 percent of her first-serve points and denied World No. 105 Montgomery any break points all evening.
"I'm most happy with my mindset, and just being relentless," Collins said on court, after her win. "Going after my shots, not getting too down on myself after mistakes, and just giving myself room to breathe."
Anisimova advances: The night on Credit One Stadium ended with No. 8 seed Anisimova's 6-2, 6-2 victory over the 2021 Charleston champion, Veronika Kudermetova. Anisimova was 0-2 against Kudermetova coming into Tuesday's match, but she finally got her first win over the former Top 10 player.
"It's always nice to be back, the first clay tournament of the year," Anisimova said on court after her victory. "Veronika's a tough opponent, so I'm happy to be into the next round."
Anisimova reached the semifinals here in 2022, which is her top showing at any WTA 500 event. She will hope to better that this week, during a stretch where she is arguably playing her career-best tennis. She won her biggest career title at WTA 1000 Doha in February, and yesterday she set a new career-high ranking of No. 16.
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Plus, why Graydon Carter's ghost writer should get a raise and Marvel's extremely weird musical chair stunt breaks livestream records.
By
Benjamin Svetkey and Julian Sancton, Editors
Yes, politics indeed makes strange bedfellows — in Gavin Newsom‘s case, his ex-wife. Turns out Kimberly Guilfoyle, who was married to the governor when he was mayor of San Francisco in the early 2000s — and who after the divorce became a Fox News star and then, for a time, Donald Trump Jr.'s girlfriend as well as a major MAGA player — has been helping Newsom with his recent political pivot. According to various reports, Guilfoyle, 56, was the one who persuaded right-wingers Steve Bannon and Charlie Kirk to appear on her ex-husband's new extremist-friendly podcast, This Is Gavin Newsom, where the potential 2028 Democratic presidential candidate is, in his own words, “exploring the other side” to figure out why Republicans are “kicking our ass.” Not surprisingly, Newsom has been taking some serious heat over the dialogues and for his apparent about-face on issues like trans women in sports (“Deeply unfair,” Newsom called it during his chat with Kirk), and not just from his indigo-blue base. “What the hell is going on with Gavin Newsom?” CNN anchor Erin Burnett wondered out loud during a recent segment about the podcasts. According to insiders, though, the least shocking part of Newsom's seeming right turn is Guilfoyle's participation. Newsom, who has been married since 2008 to documentary filmmaker Jennifer Siebel Newsom, has been friendly with Guilfoyle for years despite her palling around with a president who frequently refers to her ex-husband as “Newscum.” Says one insider: “This doesn't surprise me at all. Gavin has had a decent professional connection — or maybe transactional is the word — with Kimberly before she started dating Donald Trump Jr. He wasn't fazed by her political transformation, and he'd talk to her every once in a while. As far as I know, there wasn't a year that they didn't talk.” — ADDITIONAL REPORTING BY PETER KIEFER
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Graydon Carter became a rich and famous magazine editor by publishing juicy gossip about the rich and famous — so, in honor of his just-published memoir, When the Going Was Good, let's do some of that for a while. Rumor has it Penguin Press ponied up something close to $900,000 for Carter's book, in which he details his rise from a Canadian railroad lineman (no joke) to a Time Inc. staff writer to a founding co-editor of the late, great Spy magazine and, finally, to his long tenure as editor-in-chief of Vanity Fair, the pinnacle of glossy publishing during the late 20th century. Nearly a million dollars may sound like a lot by today's standards, but back during VF‘s heyday in the late 1990s, Carter, now 75, would sometimes offer mere magazine writers $500,000 contracts to pen a measly three articles a year. More intriguing is how little Carter's ghostwriter — if ghost is even the term that applies — got paid for the memoir. Sources say James Fox, author of the 1984 best-seller White Mischief as well as Keith Richards' 2010 memoir, Life, was given a stipend of $10,000 a month over the course of a year, earning about $120,000. Aside from the unusual monthly arrangement — ghostwriters are typically paid an advance upfront and the rest of their pay upon delivery and publication — Fox's fee seems notably anemic. “U.K. writers tend to be paid less, but I'd be surprised if Fox were only paid that much,” offers a well-placed source in the ghostwriting industry. “Also, we don't know if Fox did a complete write or whether he just edited what Graydon had written.” No, we don't, and neither Carter's publishers nor Fox responded to Rambling's request for clarification. But Carter recently hinted at how critical Fox had been in getting his memoir between covers. “I'm a writer, so it wasn't the same as when he worked with Keith Richards,” Carter told The Guardian. “But he was instrumental, showing me how to shape it. Not everything happens in a linear fashion. Nobody wants to read a book that begins in Toronto General Hospital.”
What were you doing between 8 a.m. and 1:27 p.m. on Wednesday, March 26? Because several million otherwise rational people were glued to their computer screens — for five hours and 27 minutes — watching a camera panning slowly, so very slowly, across a stage filled only with empty director chairs. This was how Marvel Studios unveiled the cast of its next superhero tentpole, Avengers: Doomsday, with a livestream that played out more like an experimental Andy Warhol movie than a pumped-up marketing stunt. While the names inscribed on the 27 director chairs shown on the feed were certainly impressive — Pedro Pascal as Reed Richards, Vanessa Kirby as Sue Storm, Joseph Quinn as Johnny Storm, plus a bunch of old-timey X-Men (Patrick Stewart, Ian McKellen and Rebecca Romijn) and assorted other MCU regulars (Chris Hemsworth, Paul Rudd and Anthony Mackie) — none of these stars turned up for the event. Or at least almost none. If you stuck around for the full 316 minutes of chair viewing (sometimes accompanied by a trippy, humming, ambient soundtack), you were treated at the end to a very brief appearance by Iron Man actor Robert Downey Jr., who sat in his chair and silently mugged for the camera for a moment. The weirdest, most inexplicable part of the bizarre spectacle? Viewers loved it. Marvel's chair-a-palooza racked up an astonishing 275 million views, making it the biggest livestream in Marvel history. “It was a roaring success,” crows a Marvel source. “Like, very roaring.”
This story appeared in the April 2 issue of The Hollywood Reporter magazine. Click here to subscribe.
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Days after the viral moment, the singer has listed T-shirts and trucker hats with the phrase for $45 on his website.
By
Ethan Millman
Morgan Wallen seems to be leaning into his controversial viral moment from Saturday Night Live, as the country superstar is now selling “Get Me to God's Country” merchandise on his website.
Wallen caused a stir over the weekend as he walked off stage during the closing moments of the show, then posted a picture on his Instagram story of an airplane along with the caption “Get Me to God's Country.” T-shirts and trucker hats with the phrase are listed for $45 each.
A source familiar with the matter says it wasn't a pre-planned moment, and that discussions about the merch had begun between Wallen and Bravado — Universal Music Group's merchandise arm — as early as Sunday when the singer's post had turned into a viral moment. Bravado had the design done by Monday, and the merch hit his website Tuesday afternoon.
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The “Last Night” singer's abrupt dip and Instagram post have caused speculation for days on what happened, and Wallen himself hasn't addressed the situation. A source told The Hollywood Reporter on Tuesday that Wallen also did the walk-off during dress rehearsal. Wallen was also asked to participate in the “Big Dumb Line” musical sketch that aired Saturday, but a source said he wasn't available, and Joe Jonas did the part instead.
Wallen has courted controversy in the past, most notably when he was caught on video using the N-word back in 2021, putting a temporary setback on his career. He's got some history with SNL, with his initial debut canceled back in 2020 after a video surfaced of him breaking social distancing protocols at the height of the pandemic. He would make his official debut on the show two months later.
Kenan Thompson weighed in on the move in an interview with Entertainment Weekly earlier this week.
“I don't know what goes through people's minds when they decide to do stuff like that,” Thompson said. “I don't know if he understood the assignment or not or if he was really feeling a certain kind of way.”
SNL writer Josh Patten, who says he's a fan of Wallen, poked fun at the situation, posting the “God's Country” phrase to highlight a Krispy Kreme truck.
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The company behind the box office phenomenon ‘Sound of Freedom' confirmed during its CinemaCon presentation that it has set a release date for Aug. 6 for ‘Sketch,' which debuted to raves at TIFF.
By
Chris Gardner
Angel Studios has drawn up a deal for Seth Worley's Sketch.
The company behind the box office phenomenon Sound of Freedom confirmed during its CinemaCon presentation at Caesars Palace on Wednesday morning that it has acquired the film for distribution and set a release date for Aug. 6. Wonder Project and UTA's Independent Film Group negotiated the deal.
Written and directed by Worley, Sketch stars Tony Hale, D'Arcy Carden, Bianca Belle and Kue Lawrence in the story of a single dad who is swept up in an insane adventure when his daughter's comically dark drawings come to life and terrorize their small town. Hale also produced with Steve Taylor and Dusty Brown.
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Sketch debuted at last September's Toronto International Film Festival to critical raves. “If you were to take the wide-eyed wonder of a Steven Spielberg, the impish mischief of a Joe Dante, plus the vibrant visuals of prime Pixar and somehow blitz them together in a Magic Bullet blender, the resulting concoction might well resemble Sketch, an audaciously gonzo first feature by Seth Worley,” wrote The Hollywood Reporter's film critic Michael Rechtshaffen, who praised it as “dazzlingly inventive.”
Worley, who has a long list of short films to his credit, released a director's statement about the film, revealing that Sketch is a passion project that took seven years to make. “I've made a movie that is 100 percent me, and I still can't figure out how to describe it to people,” he wrote. “Executives, producers, and financiers would read this movie and either interpret it as The Babadook if it were ‘inappropriately funny and warm' or ‘Goosebumps if it were too good and weirdly dark.' But to me, it's Inside Out meets Jurassic Park. It challenges stigmas surrounding grief and explores childhood emotions, the darker places they can go, and how we live our lives in the wake of trauma.”
He continued: “This movie is my childhood. This movie is my kids. If I only get to make one movie in my life, this is the one. It's weird, it's thrilling, it's funny, it's deeply emotional, and hopefully, I've put enough of myself into it for you to find yourself in it, too.”
CinemaCon, the annual gathering of cinema owners and Hollywood studios, is hosted in Las Vegas by the newly rebranded Cinema United, which for decades was known as the National Association of Theatre Owners. This year's edition runs from March 31 to April 3.
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The Boygenius star also posted that the pop star's praise has her "shaking."
By
Hannah Dailey
Lucy Dacus just had a magical moment as a Lady Gaga fan. After the Boygenius star covered the pop icon's Mayhem single “Abracadabra” on BBC Radio 1's Live Lounge, Gaga left Dacus stunned by showering the rendition with praise on TikTok.
Commenting on a video of the “Ankles” singer's performance on Wednesday (April 2), Mother Monster wrote, “I LOVE this so much,” adding a crying emoji. “wow,” she added. “captures the purity of the song.”
On X shortly afterward, Dacus posted a screenshot of Gaga's message and simply wrote, “I'm shaking.”
BBC first shared the cover the day prior. In the video, Dacus substituted the intense, flashy pop of “Abracadabra” with soft vocals, mellow piano and finger-picked acoustic guitar, bringing out an entirely different side of the track. “With a haunting dance, now you're both in a trance/ It's time to cast your spell on the night/ Abracadabra/ Amor oo na na/ Abra ca da bra,” Dacus sang pensively during the performance.
Gaga dropped “Abracadabra” in February ahead of Mayhem, which debuted at No. 1 on the Billboard 200 last month. So far, the single has peaked at No. 13 on the Billboard Hot 100.
Dacus also shared a new LP in March: Forever Is a Feeling, her first album since Boygenius' Grammy-winning The Record and the Virginia native's own Home Video album from 2021. In a recent interview with Billboard about the project, Dacus — who recently confirmed her romance with Boygenius bandmate Julien Baker — opened up about writing specificity into the love songs on her new record.
“Once you focus on one thing and one person, it actually recontextualizes everything else,” she said. “And you realize that every detail is its own universe.”
See Gaga's comment and Dacus' reaction below.
@lucy dacus covers @ladygaga ‘Abracadabra' on the new music show 🪄🖤 #abracadabra #ladygaga #lucydacus
I'm shaking pic.twitter.com/GAryx8u5Hf
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The Weeknd has sent at least one song to the Hot 100's tier each year since 2014. Plus, who has the record on the Billboard 200 albums chart?
By
Gary Trust
Submit questions about Billboard charts, as well as general music musings, to askbb@billboard.com. Please include your first and last name, as well as your city, state and country, if outside the United States.
Or, reach out on Bluesky.
Let's open the latest mailbag.
Hi Gary,
Remember when you, myself and another Billboard reader went over music acts with the longest streaks of gaining a new Billboard Hot 100 top 10 year after year? We had come to the consensus that, with 12 years apiece, Mariah Carey (1990-2001) and Prince (1983-94) were the two front-runners in that club. Well, now we have a third.
Thanks to his “Rather Lie,” with Playboi Carti, which debuted on the March 29 chart, The Weeknd has now put a new song in the Hot 100's top 10 for a 12th consecutive year.
Here's a rundown of all of his top 10s, in chronological order of their peaks:
Who else to tie such a historic streak than The Weeknd, right? Someone who happens to be: A, one of my favorite popular music acts of all time, and B, known for citing Prince as an influence.
Regards,
Jake RiveraMashpee, Mass.
Hi Jake,
Thanks for pointing out the update, and congrats to The Weeknd on his record-tying streak of Hot 100 top 10s in 12 consecutive years (or more than 600 weekends).
Notably, another act has joined the mix for potentially matching the mark: Drake is now up to an active streak of 11 years in a row with new Hot 100 top 10s, from 2015 (“Hotline Bling”) through 2025 (“Gimme a Hug” and “Nokia”). He could, thus, tie the record next year — or The Weeknd could claim the honor all to himself with at least one new top 10 in 2026.
Meanwhile, what about the same feat on the Billboard 200 albums chart? On first thought, a lengthy streak of annual new top 10s might seem less likely there, as, compared to singles, acts for the most part don't release as many as albums, and somewhat rarely every year historically.
Let's count down the artists, from The Beatles to Taylor Swift, Drake and more, with the most consecutive years of sending at least one new album to the Billboard 200's top 10 (dating to Aug. 17, 1963, when the chart began combining mono and stereo releases into one ranking). The act atop the list might seem surprising, although perhaps less so once looking into why.
Seven consecutive years with new Billboard 200 top 10s:
Eight consecutive years:
Nine consecutive years:
And, the act with the longest such streak overall …
12 consecutive years (the same as the Hot 100 record):
The leading group – of rotating members – tallied all 24 of its Billboard 200 top 10s from Kidz Bop 7 through Kidz Bop 32. (In that run, only Kidz Bop 17 and Kidz Bop 30 missed the tier, both reaching No. 12; meanwhile, the collective has hit a No. 2 best with five releases.)
The act scored its record run of consistency in the Billboard 200's top 10 thanks to its steady stream of all-ages covers of big pop hits. Kidz Bop Kids additionally earned 101 entries, including 42 top 10s, on the Kid Digital Song Sales chart, both bests in the list's history. Four reached No. 1, led by their family-friendlier take on Meghan Trainor's former Hot 100 No. 1 “All About That Bass,” which led for six weeks in 2015.
In 2014, Victor Zaraya, then an executive for the ensemble, mused about its win-win nature. “It's favorable to have your song being sung,” he said. “Maybe a kid heard the Kidz Bop cover of an artist's song before they heard the actual version. Will they remember it as a Kidz Bop song? Maybe. Will they remember it with the original artist? Maybe. But it's only furthering that artist's song.”
Beyond remakes of familiar songs, Zaraya noted that the act's singers contributed to the enduring appeal of Kidz Bop, which in 2025 celebrates its 25th year, including with tour dates. To date, the troupe has sold 18.7 million albums and drawn 8.1 billion official streams for its songs in the U.S., according to Luminate.
“We want to let kids know that [the Kidz Bop Kids] are real — they sing, dance and perform,” Zaraya said. “They can be brand ambassadors for us. They have personalities. They are stars.”
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Los Alegres del Barranco will not be able to tour the U.S. as planned.
By
Natalia Cano
The U.S. State Department has canceled the work and tourist visas of the members of Mexican corrido group Los Alegres del Barranco after they displayed images of the leader of the Jalisco New Generation Cartel (CJNG), Nemesio Oseguera Cervantes, also known as “El Mencho,” during a concert on Saturday (March 29) at an auditorium at the University of Guadalajara.
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The news was confirmed on Tuesday (April 1) by Christopher Landau, the Deputy Secretary of State, in a post on X.
I'm a firm believer in freedom of expression, but that doesn't mean that expression should be free of consequences. A Mexican band, “Los Alegres del Barranco,” portrayed images glorifying drug kingpin “El Mencho” — head of the grotesquely violent CJNG cartel — at a recent concert… pic.twitter.com/neSIib7EC4
“I'm a firm believer in freedom of expression, but that doesn't mean that expression should be free of consequences,” wrote Landau in his post. “A Mexican band, Los Alegres del Barranco, portrayed images glorifying drug kingpin “El Mencho” — head of the grotesquely violent CJNG cartel — at a recent concert in Mexico. I'm pleased to announce that the State Department has revoked the band members' work and tourism visas. In the Trump Administration, we take seriously our responsibility over foreigners' access to our country. The last thing we need is a welcome mat for people who extol criminals and terrorists.”
The State Department, through Secretary of State Marco Rubio, announced on Feb. 20 the designation of eight cartels and transnational organizations — including the Jalisco New Generation Cartel — as Foreign Terrorist Organizations (FTO) and Specially Designated Global Terrorists (SDGT).
Prior to Landau's announcement, the concert had caused significant controversy and outrage in Mexico, which has long tried to curb the glorification of drug lords in popular Mexican music and narcocorridos.
The concert was condemned on Monday (March 31) by Mexican President Claudia Sheinbaum. It prompted the Jalisco State Prosecutor's Office to launch an investigation for “glorification of crime.”
📢 Tras la proyección de imágenes en un concierto en Zapopan, que presuntamente hacían alusión a un personaje vinculado a un grupo criminal, la Fiscalía del Estado inició una carpeta de investigación. (1-3) pic.twitter.com/OU4R8EYr6q
On Tuesday (April 1), the governor of Jalisco, Pablo Lemus, wrote in a post on X that his government supports the measures adopted by the University of Guadalajara to prevent criminal acts from being glorified at concerts, as occurred over the weekend at the Telmex Auditorium. The local leader said he signed an executive order to ensure that no singer or group with a history of endorsing criminal activity will perform at events linked to his government.
“Next week, I will introduce an initiative to ensure that, in any legally sanctioned public event, producers and performers are held accountable for what happens during their shows, and no one can wash their hands of responsibility,” Lemus announced.
Los Alegres del Barranco were scheduled to play shows in several U.S. cities, where the band was announced as part of the lineup for the Bésame Mucho festival April 5 in Austin, Texas. In a TikTok livestream on Tuesday, Pavel Morales, a member of the Sinaloan group, stated that the majority of their audience supports them and referred to their critics as “confused.”
#polemica #paratiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii #losalegresdelbarranco #pavelmoreno
Billboard Español reached out for comment to the band's reps, but hasn't received a reply by press time. Meanwhile, authorities from the municipality of Pedro Escobedo, in the Mexican state of Querétaro, confirmed on Tuesday that the group's scheduled performance for April 19 was canceled because “it does not meet the necessary municipal permits for its realization,” the local government said in a statement on Tuesday.
The projection of the controversial images took place during a concert titled “Los Señores del Corrido” at the Telmex Auditorium, where Los Alegres del Barranco performed the song “El Dueño del Palenque” (The Owner of the Palenque) and displayed on screen photos of the cartel leader, as well as other images created by AI.
The images appeared on multiple videos on social media. They include the moments in which fans burst into cheers when the images of the cartel leader were shown, adding to the controversy.
In a statement, Auditorio Telmex Adistanced itself from the events, arguing that the venue “has no influence on the selection of the repertoire, speeches, or audiovisual material that artists decide to share with their audiences.” However, it acknowledged that the images of the kingpin could be considered an “exultation of crime.”
The controversy over the alleged tribute to the drug trafficker arises after information has surfaced over how the cartel uses clandestine ranches to recruit people through deceptive job offers, according to federal authorities and media reports. This followed the recent discovery of Izaguirre Ranch in early March in the municipality of Teuchitlán, where acts of torture and murder were allegedly committed, according to the Guerreros Buscadores collective.
🚨#AlertaADN¡Se cancela! El municipio de Pedro Escobedo, Querétaro, suspendió la presentación de "Los Alegres del Barranco", prevista para el 19 de abril, tras la controversia por un homenaje a "El Mencho" en un concierto en Jalisco pic.twitter.com/ChxD61VNps
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The rapper joined Cabello on her single "He Knows" last summer.
By
Gil Kaufman
Lil Nas X feels lucky to have Camila Cabello in his friend orbit. The rapper who has been sprinkling singles all over the place in the lead-up to the release of his as-yet-unscheduled Dreamboy album, told Paper magazine that the “I Luv It” singer was really there for him when he was having a crisis of faith in his personal and professional lives.
“We did a great job at uplifting each other and showing each other how we view the situation and how we view each other from outside of ourselves,” he said of his bestie, who he teamed up with last summer for her “He Knows” single. “Once you made up your mind: ‘Oh, the world doesn't see me as something great or feels like I've done anything amazing.‘ To hear [affirmation] from somebody who you greatly respect and you love what they've done, it feels great, especially because you can feel like it comes from a genuine place, and not like someone trying to get something from you.”
Lil Nas said he had fun hanging out with Cabello at a time when he was just getting comfortable “going out places. It was just genuine fun. It didn't feel like work. Camila made me feel so good, she made me feel so great,” he said. “And that was the beginning of me getting my confidence back. To finally be at this place, where I'm like, ‘Okay, I know who the f–k I am. I know where I'm going. I don't care what none of these b–ches say. I didn't care at first. Why do I care now?'”
The rapper also said he's taking a completely different approach on the new album than he did with his 2021 full-length debut album, Montero. Whereas last time he said he brought the world to him, this time “I'm bringing myself to the world,” describing Dreamboy as a collection of “everything and anything I've ever loved or hated… it's just me going out there and doing me in whichever form I feel.”
So far, Lil Nas has released eight songs that could be featured on the album, including “Hotbox,” “Light Again,” “Need Dat Boy,” “Dreamboy,” “Big Dummy!,” “Swish,” “Right There!” and “Lean on My Body,” which he recently collected on the Days Before Dreamboy EP.
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"He was a good friend when I needed one," Springsteen wrote.
By
Jessica Lynch
Bruce Springsteen has paid tribute to Joe DePugh, the New Jersey pitcher who inspired his hit song “Glory Days,” following news of DePugh's death this week at the age of 75.
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“Just a moment to mark the passing of Freehold native and ballplayer Joe DePugh,” Springsteen wrote in an Instagram post on March 30. “He was a good friend when I needed one. ‘He could throw that speedball by you, make you look like a fool' …. Glory Days my friend.”
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DePugh and Springsteen grew up together in Freehold, N.J., and played baseball in the same youth league. Their now-legendary chance encounter at a bar in 1973 served as the real-life basis for one of the most iconic verses on Springsteen's Born in the U.S.A. album.
DePugh later confirmed the moment in interviews, recalling how the two reconnected outside the Headliner in Neptune, then spent hours catching up inside.
“Whenever we're together, it's the same dynamic: I'm the star and he's the guy at the end of the bench,' said DePugh to the Palm Beach Post in 2011. “That's who he has always been to me, my right fielder.”
” … Once I saw Bruce we went back in and closed the place. He had a little entourage with him. They all sat in a booth, but it was just me and him at the bar. All of a sudden, it's 1:30 (a.m.) and they started blinking the lights.”
“Glory Days” reached No. 5 on the Billboard Hot 100 in 1985, and DePugh told the Palm Beach Post that he was “tickled pink I would even get into the song.”
“When I first heard the song, I thought the song said ‘and all we kept talking about was glory days,' “ said DePugh. “And years later, I finally saw the lyrics and saw ‘all he kept talking about was glory days.' And I thought, ‘Huh, (he) took a little shot at me!'
DePugh and Springsteen remained friends throughout their lives, occasionally crossing paths in Palm Beach County, where Springsteen owns a home and DePugh lived in Lake Worth.
DePugh died after a battle with cancer. He is remembered fondly by friends, including longtime Freehold teacher and coach Rich Kane, who said: “All he wanted to do was raise his brothers, play baseball, play basketball and just hang in Freehold Borough. This one hurt. Joe and I were very close.”
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The New Jersey senator's epic floor session drew many cues from Hollywood, and may teach it a few things too.
By
Steven Zeitchik
Senior Editor, Technology and Politics
Was it a little like William Wallace's freedom speech in Braveheart?
Did it contain the drama of Don Larsen working toward that World Series perfect game?
Or maybe the vibe was more Spalding Gray in Swimming to Cambodia, the monologist as circus master.
For those steeped in screen entertainment, the analogies came and fast and furious as Cory Booker took the Senate floor Monday night and Tuesday. Fortunately, he gave us a lot of time to come up with them.
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You may not have watched anything but the last hour, or few minutes, or even a second of the Democratic senator's 25-hour, one-sitting (or standing) opus on YouTube or C-SPAN. Trust me then when I tell you the whole thing was the kind of spectacle that should be eligible for an Emmy, so subtle were its layers and so ambitious the performance.
On its face, Booker's speech building an elaborate case against the policies of Donald Trump and Elon Musk was pure political theater — if theater involved a prize for never relying on a chair, food or the bathroom. As he broke Strom Thurmond's 24-hour, 18-minute mark for longest Senate-floor speech in history, the New Jersey lawmaker spoke from giant loose-leaf binders of facts and read anecdotes off index cards; he thanked the Parliamentarian and gave at least his vocal cords a break by deferring to extended questions (that were more of a comment) from other senators. Booker balanced rousing constitutional ideals with basic economic litmus tests, reappropriating Ronald Reagan's famous 1980 debate line of, “Are you better off now than you were four years ago?” as (fittingly for these lightning times), “Are you better off now than you were 71 days ago?”
But what Booker was doing was nothing less than creating a cinematic spectacle, a binge-worthy awards contender in which all 25 hours happened to drop at once. And while the consequences are deadly serious, the techniques came from some of our most popular entertainment. Focus on different through lines of Booker's performance and you'd experience different arcs; come in at different moments and you'd infer different genres.
For stretches, the speech had a kind of relaxed podcast vibe, as Booker kidded with other senators or even did quasi-comedy bits (his re-enactment of Elon Musk trying to describe to Joe Rogan why Social Security was a Ponzi scheme, complete with Muskian verbal tics, was especially Laugh Factory-ish.)
He created a series of supporting characters, including his offscreen mother, the Las Vegas-dwelling standby who had a surrogate here in Jacky Rosen, the Nevada senator.
Like all good heroes, Booker had a catch phrase. “I yield for a question while retaining the floor,” he said, over and over again, each time causing viewers to clench up just a little; should he mix up the words and yield the floor, the game would be up.
At times the speech had an air of televangelism, with Booker's booming oration about the “moral moment” or, more literally, when he tossed to Delaware Sen. Chris Coons, a Yale Divinity School graduate who proceeded to cite Isaiah and talk about the Bible's attitude toward the poor. When it wasn't God-heavy, the speech had the vibe of secular religion and its High Priest John Lewis, whose story of Civil Rights-era martyrdom and tagline of “good trouble” became a leitmotif for Booker.
Research was doubtless important — with so many substantive facts and policies on everything from Social Security-office closures to changes in veterans benefits to the details of the Federalist Papers, this was a feat as much of preparation as anything else. Sam Rockwell has his acting coach; Cory Booker has his Hill staffers. But for all the planning, Booker also had to make it look like he wasn't straining. “An actor has to burn inside with an outer ease,” the performance guru Michael Chekhov once said, and the senator seemed to embody that, joking about the length of time he was up there, how much he generally loved the mic and how he wanted not to gaffe his way into giving up the floor, making fun of himself in a way that put us on his side. Far from someone trying to convince us he could break the record, Booker was right there with us sharing our doubts.
The whole will-he-get-there narrative kept recalling to me the 1990s cult documentary Hands on a Hardbody, in which a series of humble strivers try to win a truck by simply outlasting their competitors in keeping their hands on a Nissan. Only this time, the prize was historical supremacy, a turn Booker was happy to lean into. Thurmond made his record-setting speech in 1957 breathing segregationist fury railing against the Civil Rights Act, and Booker reminded us just often enough that Thurmond could now be upstaged by a man with the kind of background he dedicated his career to keeping down.
Perhaps the secret ingredient — the unknown spice in the must-see TV casserole — is how Booker let us in on the social workings of the Senate, making it seem like just a few friends who were not that different from your own workplace buddies. Booker would pepper his speech with references to the times he and Connecticut Sen. Chris Murphy texted; he referenced Ted Cruz's mid-speech kibbitz about pulling a fire alarm; he even, upon getting a request from Chuck Schumer to ask a question, smiled wanly and said it's the only time he'll ever tell him no, like the boss you push back on but still secretly worry about.
In this regard, Booker may owe his biggest debt to The Kardashians, The Simple Life and other proto-celebrity reality shows, dangling the forbidden glamor only to take us behind the curtain to show how the people there jive like everyone else. Even when Booker said he had a story about another senator but wasn't going to tell it now — a line he used several times — it drew us nearer, like the uncle who says he'll finally tell us the secret if we can just wait till Thanksgiving.
As the speech went on the audience grew, Booker's YouTube audience going from just 10,000 people Monday night to several times that Tuesday morning as people woke up and realized he was still going. By the time Booker neared the record Tuesday evening, the channel's viewers had swelled to 140,000. (Millions more would watch him on television and in clips on X and Instagram.) What they saw was grand speechmaking that recalled Mr. Smith Goes Washington. And while no one would confuse Booker with a political acolyte, his Jersey accent and references to his college football days instantly undercut any talk about Democratic elites.
In fact, Frank Capra's presence hovered in multiple ways. At the very moments Booker was returning to his villainous “the world's most powerful man and the world's richest man,” Elon Musk was seen on various news channels presenting cartoon-sized checks to buy votes in Wisconsin, casting in this It's a Wonderful Filibuster film a ready-made Henry Potter character.
Booker also understood what Oscar contenders have grasped, from Robert De Niro in Raging Bull to the climbers in Free Solo — audiences love physical commitment. Whether gaining weight for a role or clinging to the side of a mountain with your fingertips, if you show that you've defied some odds of the natural world, you'll immediately endear yourself to a crowd. “I am going to go for as long as I am physically able to go,” Booker said in a social video post Monday before heading out to the floor, a double-barreled statement that suggested both his commitment to the part and his uncertainty of whether he could play it.
That gave the proceeding a level of drama no congressional speech has the right to have, the record both there and not there during the entire run, like the announcers never mentioning a no-hitter even as the linescore shows zeros. As the Bluesky influencer Ben Collins noted, “MSNBC and CNN should be airing this Cory Booker thing in the same way you take over programming if there's a no-hitter or a basketball player with 60 points in three quarters.” MSNBC, at least, eventually did, letting host Ari Melber lead his show with the performance as a clock kept score in the corner.
The length also pulled meaningfully against our viral-clip era. The speech might have aired on YouTube and TikTok, but it played against their ethos, almost delighting its audience, Brutalist-style, with its longform counterprogramming. Not for nothing did cheering comments flash dizzyingly through the YouTube live-chat or did 400 million (!) like the speech on TikTok.
Implicit in the endurance feat was also the man Booker was speaking against, who prides himself on treating podiums like ultramarathons. Just a few weeks ago, Trump set a record for longest address to a joint session of Congress with a 100-minute oration — or as Booker calls it, a quick digression. Next to Booker, Joe Rogan seems like a newsflash.
And of course came the real own, one that right-wing media is unlikely to cop to but that practically vibrated from left-wing quarters: Booker was mastering the medium that gave Trump the presidency in the first place.
None of this should surprise those who've been paying attention to Booker. The senator is hardly a stranger to screen drama, having dated Rosario Dawson for four years ending in 2022; surely Ahsoka Tano taught Grogu a few tricks. And let's not forget that Booker came to national prominence on film, as the charismatic and at times controversial star of Street Fight, Marshall Curry's documentary about the bid by Booker, then a Newark city councilman, to win the city's mayoralty that premiered at the Tribeca Film Festival 20 years ago this month. He is both aware, and the beneficiary, of powerful cinematic images.
In a time when addictive-watching is down, Booker reminded us — and maybe reminded streamers and studios too — of the reasons we liked it in the first place: the unknown finale, the lovable side characters, the sense that we are part of something that matters.
And in a time when liberal morale is down, Booker was there to pick it up. What the Democrats do from here is the obvious next question, as pundits tried to quantify what this brief momentum-burst meant. “A cool glass of water in the desert,” MSNBC pundit and former Missouri Sen. Claire McCaskill said after the speech ended. To judge by the live-chat comments, many Democrats felt a sense of pride and hope that has been sparse since the election, and talk quickly turned to a series of rallies planned for this weekend, a Democratic win in that Wisconsin Supreme Court race and other mobilizations.
The size of the speech's halo, of course, can't be predicted. But any actor will tell you the hardest thing is first getting a distracted audience's attention. Booker did that. Now the weight is on the rest of the cast to finish the story.
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The much-loved actor died on Tuesday from pneumonia after battling throat cancer for several years.
By
Abid Rahman
International Editor, Digital
Michael Mann, Francis Ford Coppola, Nicolas Cage and Josh Brolin were among the many entertainment industry figures who paid tribute to Val Kilmer after news of his death was announced.
Kilmer died of pneumonia in Los Angeles, his daughter, actress Mercedes Kilmer, told The New York Times. He was 65. The actor was diagnosed with throat cancer in 2015.
His death was announced as the actor was meant to be arriving for a rare red carpet appearance at the Beverly Hills Film Festival on Tuesday night. “It was a shock to find this out. We had just confirmed Val to attend the West Coast premiere of American Badass: A Michael Madsen Retrospective this past weekend,” Nino Simone, founder and president of the Beverly Hills Film Festival, said on Wednesday.
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The much-loved and incredibly versatile actor had a diverse filmography, excelling both as a leading man (Batman Forever, The Saint, Top Secret!, Willow, The Doors, Real Genius) and as a scene-stealing supporting player (Tombstone, Top Gun, Kiss Kiss Bang Bang, Heat) in a variety of genres. Kilmer was also the subject of the 2021 documentary Val, which is an intimate portrait of the actor that explores his battle with throat cancer, his artistic passions and his reflections on his career.
In a statement to The Hollywood Reporter, filmmaker Michael Mann said, “While working with Val on Heat I always marveled at the range, the brilliant variability within the powerful current of Val's possessing and expressing character. After so many years of Val battling disease and maintaining his spirit, this is tremendously sad news.”
Nicolas Cage said in a statement to THR, “I always liked Val and am sad to hear of his passing. I thought he was a genius actor. I enjoyed working with him on Bad Lieutenant and I admired his commitment and sense of humor. He should have won the Oscar for The Doors.”
Francis Ford Coppola said on Instagram: “Val Kilmer was the most talented actor when in his High School, and that talent only grew greater throughout his life. He was a wonderful person to work with and a joy to know — I will always remember him.”
Josh Brolin wrote on Instagram, “See ya, pal. I'm going to miss you. You were a smart, challenging, brave, uber-creative firecracker. There's not a lot left of those. I hope to see you up there in the heavens when I eventually get there. Until then, amazing memories, lovely thoughts. ❤️💔❤️💔❤️💔💎 #valkilmer #marktwain,” The second hashtag a reference to Kilmer's lifelong passion for Mark Twain, which culminated in the actor portraying the famed writer and humorist twice onscreen and in the theater production Citizen Twain.
Also on Instagram, actor Josh Gad wrote, “RIP Val Kilmer. Thank you for defining so many of the movies of my childhood. You truly were an icon.”
White Lotus star Michelle Monaghan, his co-star in Kiss Kiss Bang Bang, shared to Instagram: “A kind, curious, committed, rebellious, and radical gent. I learned from one of the greats. An artist through and through. I treasured my time with you.❤️ Godspeed buddy.”
Matthew Modine tweeted, “RIP Val Kilmer. If it wasn't for our chance encounter at the Source in 1985, I may never have been cast in FULL METAL JACKET. Thanks, Val. 🙏☮️”
On Bluesky, Emmy-nominated TV writer and comedian Mike Drucker wrote, “Val Kilmer felt like an actor who, no matter what the material, always understood the assignment.”
Brian Lynch, the screenwriter behind Minions and Secret Life of Pets, wrote on Bluesky, “The Doors. Top Secret! Tombstone. Top Gun. Real Genius. Willow. Batman Forever. Kiss Kiss Bang Bang. True Romance. Macgruber. Heat. There was nobody like Val Kilmer. May he Rest in Peace.”
On X, Jennifer Tilly offered her memory of Kilmer auditioning for The Doors. “A long time ago, I was auditioning for the movie ‘The Doors' It was kind of a cattle call,” she wrote. “They paired together potential Jims with potential Pamela‘s. And they were running behind so we were spilling out of the casting office, sitting on the porch, the lawn, and the driveway. All of a sudden, a sixties convertible came screeching up, blaring Doors Music at top volume. And a guy jumped out and strode inside: He had wild hair and he was barefoot, shirtless, and wearing nothing but a pair of tight leather pants.”
Tilly added, “We all looked at each other like … Who is this guy? We were more than a little shook by the sheer audacity of his entrance. Well of course it was Val Kilmer and from that minute on, nobody else stood a chance. Rip King.”
Cher, who dated Kilmer in the '80s, wrote on X: “VALUS Will miss u, U Were Funny, crazy, pain in the ass, GREAT FRIEND, kids 💜 U, BRILLIANT as Mark Twain, BRAVE here during ur sickness.”
Lucasfilm executive Pablo Hidalgo wrote on Bluesky, “Oh man, not Val Kilmer. His comedic chops were top notch but he had a fascinating storm roiling beneath the surface. Madmartigan was an icon.”
Famed movie critic Richard Roeper tweeted, “Val Kilmer should have been nominated for Best Supporting Actor for Tombstone and for Heat. He was a brilliant presence in some of the most enduring films of his generation. Rest well. Thank you for the incredible work.”
Writer-director Dylan Park-Pettiford tweeted, “Listen, Val Kilmer had me wanting to fly fighter jets, be Batman, rob banks, and hunt lions as a kid. They don't make too many movie stars like him anymore. Generational.”
Ringer owner Bill Simmons tweeted, “There wasn't anyone quite like Val Kilmer. Really enjoyed his work. RIP.”
On X, the official Top Gun movie account tweeted, “Remembering Val Kilmer, whose indelible cinematic mark spanned genres and generations. RIP Iceman.”
Like many other users on social media, Indonesian filmmaker Timo Tjahjanto (The Shadow Strays, Nobody 2), posted a famous scene from Heat, where Kilmer's character interacts with Ashley Judd's character for the final time. Tjahjanto wrote, “A masterpiece exercise in saying nothing and expressing everything. Rest in Peace truly.”
Actress Sasha Grey tweeted on X, “Rest In Peace Val Kilmer, an absolute legend. He played some of my favorite characters in some of my favorite movies of all time. Condolences to his family 🫶 He was 65.”
On Instagram, Harry Potter star David Thewlis wrote about co-starring with Kilmer in The Island of Dr. Moreau (1996): “I spent the most bizarre 5 months of my entire life with Val Kilmer, out in the Australian rainforest, on the ill-fated Island of Dr Moreau. It was so spectacularly bleak and awful it was almost wonderful. Look it up sometime. As Val wrote in his final mail to me: ‘What an incredible story we lived, you and I. One of the greatest.' Bittersweet to be back here in Australia and hear the heartbreaking news. He was one of the most extraordinary people I have ever met. Proud to have called him a friend and co-conspirator.”
Actor Joe Manganiello shared a tribute on Instagram: “He was one of the main reasons I wanted to become an actor and why I felt it was so important for me to seek out classical training. He could do it all… drama, comedy, classical… He was great with a sword, a gun, a fighter jet, or a shot cup. Then on top of all of that, he could sing… case in point: for most of my life when I tried to picture Jim Morrison, I could only see him. His performances were brave and intelligent, and he showed me that you didn't always have to be the star in order to make the most impact. If the situation presented itself, you could take a tiny, nothing part on paper and elevate it into a scene stealer. Oh, and he got to be Batman. RIP to one of the all time greats…”
The account for Film at Lincoln Center posted on X, “Rest in peace to our former campus mate, the great Val Kilmer (1959-2025), who at 17 was the youngest drama student ever admitted to The Julliard School's Drama Division.”
This is a developing story.
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The Hives are returning with "a new record so full of energy, joy, anger and life that you will be questioning reality as you have known it."
By
Tyler Jenke
Incendiary Swedish rockers The Hives are readying themselves for a big year, with a new album announced alongside a global run of tour dates.
The veteran quintet – who celebrated their 30th anniversary in 2023 – shared the news of their seventh album in a press release on Tuesday (April 1), with the announcement taking the form of an open letter formally addressed to “whom it may concern,” but clarified to mean “everyone.”
“This message is to inform you that International Rock Sensation The Hives, the best live band on the planet and still your new favourite band, lauded on all continents for their masterful skill and reckless abandon in the rock music field, have once again, sooner than you expected, created a new body of work the likes of which have never been heard or indeed probably will again,” the message began.
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“A new record so full of energy, joy, anger and life that you will be questioning reality as you have known it,” it continued. “They finally did it. Every single song a single, every single single a hit, every hit a direct hit in the face of the man.”
The record in question is titled The Hives Forever Forever The Hives and will be released via Play It Again Sam on Aug. 29. According to the press release, the 13-track album was “crafted with commitment, abandon and skill in Sweden in cohorts with the esteemed producers Pelle Gunnerfeldt and Mike D of Beastie Boys.”
The group have also unveiled “Enough Is Enough” as the album's lead single, with a video directed by filmmaker Eik Kockum capturing the group in Bucharest, Romania.
“Who in their right mind would start a song like this? No one but The Hives,” the group explained. “They are here again sooner than you expected and they have had enough of everyone at this point. Hence the title. Dig? Dig.”
The Hives will celebrate the release of their forthcoming album with a world tour which launches in Australia in July, before visiting North America, Europe, and the U.K. throughout the rest of the year.
The Hives Forever Forever The Hives arrives as the band's seventh album, and follows on from the release of 2023's The Death of Randy Fitzsimmons – ending an eleven-year gap between releases for the band.
The group initially formed in 1993, though largely avoided widespread recognition until the release of the 2001 compilation Your New Favourite Band. The compilation resulted in the re-release of their single “Hate to Say I Told You So,” which peaked at No. 86 on the Hot 100, and saw their following album – 2004's Tyrannosaurus Hives – reach a career high of No. 33 on the Billboard 200.
The Hives – World Tour 2025
July 17 – Metropolis Freo, Perth, AUJuly 19 – Forum, Melbourne, AUJuly 23 – Enmore Theatre, Sydney, AUJuly 24 – Fortitude Music Hall, Brisbane, AUSept. 8 – Stubb's Waller Creek Ampitheater, Austin, TXSept. 9 – House of Blues, Houston, TXSept. 10 – House of Blues, Dallas, TXSept. 12 – Ogden Theatre, Denver, COSept. 13 – The Union Event Center, Salt Lake City, UTSept. 15 – Showbox SoDo, Seattle, WASept. 16 – The Commodore Ballroom, Vancouver, BCSept. 17 – Revolution Hall, Portland, ORSept. 19 – Ace of Spades, Sacramento, CASept. 20 – The Warfield, San Francisco, CASept. 22 – The Sound, Del Mar, CASept. 25 – Hollywood Palladium, Los Angeles, CAOct. 17 – Sentrume Scene, Oslo, NOOct. 18 – KB Hall, Copenhagen, DKOct. 21 – Colombia Halle, Berlin, DEOct. 24 – Zenith, Munich, DEOct. 25 – Haus Auensee, Leipzig, DEOct. 26 – Gasometer, Wien, ATOct. 28 – Xtra, Zurich, CHOct. 29 – Alcatraz, Milan, ITNov. 1 – Sant Jordi Club, Barcelona, ESNov. 2 – Wizink, Madrid, ESNov. 4 – Sagres Campo Pequeno, Lisbon, PTNov. 19 – Forest National, Brussels, BENov. 20 – Le Zenith, Paris, FRNov. 22 – AFAS, Amsterdam, NLNov. 24 – Utilitia Arena, Cardiff, UKNov. 26 – Ovo Hydro, Glasgow, UKNov. 28 – Aviva Studios, Manchester, UKNov. 29 – Alexandra Palace, London, UKDec. 1 – Palladium, Cologne, DEDec. 2 – Jahrhunderthalle, Frankfurt, DEDec. 3 – Sporthalle, Hamburg, DEDec. 6 – Avicii Arena, Stockholm, SE
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Charismatic and versatile, he also won praise for memorable roles as Iceman in 'Top Gun' and Doc Holliday in 'Tombstone,' but his unpredictable behavior ruffled some feathers in Hollywood.
By
Mike Barnes
Senior Editor
Val Kilmer, the charisma-oozing leading man who lost himself portraying such tormented, self-loathing characters as Jim Morrison, gunslinger Doc Holliday and Batman during his all-too-brief career, died Tuesday. He was 65.
Kilmer, who came to fame for playing the competitive naval aviator Tom “Iceman” Kazansky alongside Tom Cruise in Tony Scott's 1986 mega box-office hit Top Gun, died of pneumonia in Los Angeles, his daughter, actress Mercedes Kilmer, told The New York Times.
Kilmer was diagnosed with throat cancer in 2015, and Val, a stirring documentary about his life that premiered at Cannes in July 2021, showed him needing a breathing tube.
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His death was announced as the actor was meant to be arriving for a rare red carpet appearance at the Beverly Hills Film Festival on Tuesday night. “It was a shock to find this out. We had just confirmed Val to attend the west coast premiere of American Badass: A Michael Madsen Retrospective this past weekend,” Nino Simone, founder and president of the Beverly Hills Film Festival, said on Wednesday.
Raised in the San Fernando Valley in the shadow of Hollywood, Kilmer also was known for his meaty performances as Robert De Niro's nasty henchman in Michael Mann's Heat (1995); as Marlon Brando's insane assistant in John Frankenheimer's The Island of Dr. Moreau (1996); as the suave crook Simon Templar in Phillip Noyce's The Saint (1997); and as the homosexual detective Gay Perry in Shane Black's tribute to film noir, Kiss Kiss Bang Bang (2005).
Kilmer also impressively channeled Elvis Presley in Scott's True Romance (1993), written by Quentin Tarantino, and porn star/cocaine addict John Holmes in Wonderland (2003).
He was married to British actress Joanne Whalley from 1988 until their divorce in 1996. They met while working together on Willow and wed months later.
In Oliver Stone‘s The Doors (1991), Kilmer, with long brown hair and skintight black leathers, was eerily realistic as Morrison, the L.A. band's iconic frontman who succumbed to drugs in 1971 at age 27. The actor took months to prepare for the role, and he recorded his baritone voice against the backing of original Doors master tapes for the film.
The soundtrack “combines Morrison's original vocals and new vocals by Val Kilmer so seamlessly that there is never, not even for a moment, the sensation that Kilmer is not singing everything we hear,” Roger Ebert wrote in his review.
“That illusion is strengthened by Kilmer's appearance. He looks so uncannily like Jim Morrison that we feel this is not a case of casting, but of possession. The performance is the best thing in the movie — and since nearly every scene centers on Morrison, that is not small praise. Val Kilmer has always had a remarkable talent, which until now has been largely overlooked.”
Kilmer also was quite compelling with his scene-stealing turn as the doomed Holliday, a sickly alcoholic who's quick on the draw, in the modern Western classic Tombstone (1993).
“He works harder than most actors to make it look believable,” Tombstone director George Cosmatos told the Los Angeles Times in 1993. “He's in the ranks of the great actors in America like [Al] Pacino or De Niro.”
Kilmer then took the cowl vacated by Michael Keaton to star as the moody Caped Crusader in Joel Schumacher's Batman Forever (1995). The film raked in $336 million at the global box office, and only Toy Story grossed more that year.
“For me, Val Kilmer was the best Batman,” Schumacher once said, even though they reportedly clashed on the set. (Frankenheimer also didn't get along with Kilmer; after Dr. Moreau, he said that the two things he would never do again were “climb Mount Everest and work with Val Kilmer again.”)
On Batman Forever, “Everything was different about this job than I'd experienced before,” the actor told Entertainment Tonight in 1995. “The size of the character and how strange it was that Michael Keaton had decided not to do it — I just said yes, without reading the script.”
When he and Warner Bros. couldn't agree to terms, Kilmer was one and done as Batman, opting not to return for Schumacher's Batman & Robin (1997) as George Clooney stepped in.
Val Edward Kilmer, part Cherokee, Irish, German and Swedish, was born on New Year's Eve 1959 in the L.A. suburb of Chatsworth. His father was an aerospace engineer and real estate developer and his mother a housewife — they would end up divorcing when he was 9 — and he had one older brother, one younger.
Wesley, his younger sibling, suffered an epileptic seizure and drowned in a swimming pool at the family home that his father had bought from Western movie legends Roy Rogers and Dale Evans. At the time, Kilmer was about to leave to study acting at Juilliard in New York; he was 17 and the youngest person to be admitted to the school's drama division.
“It was quite an emotional time for me, and in a way, the extremely high standards and the activity of the school I'm sure were good for me, because I was forced to really challenge myself about my very life, you know — what I believe about life and death,” he said in a 2005 interview.
At Juilliard, he co-wrote How It All Began, a play based on the true story of a West German radical, and it wound up being directed by Des McAnuff and produced by Joseph Papp for The Public Theater. He made his Broadway debut in 1983's Slab Boys, also featuring Sean Penn and Kevin Bacon.
In his first film, Kilmer starred as rockabilly teen idol Nick Rivers in the daffy spy spoof Top Secret! (1984) from Jim Abrahams and the Zucker brothers. (He also was dating Cher around this time.)
He made an ABC Afterschool Special called One Too Many, in which he played a teenage alcoholic alongside Mare Winningham, then portrayed a lazy laser-technology whiz kid in Real Genius (1985), from director Martha Coolidge.
When Scott approached him for Top Gun, Kilmer said he wasn't interested. “I told Tony at the meeting, ‘Frankly, I don't like this.' I loved what I'd seen of his work, but I just didn't want to do that movie,” he recalled in the Times interview. “He said, ‘Don't worry, your hair will look great.' He thought that would make a difference. He was infectious that way.”
For many fans, Iceman was his signature role: “People talk about it pretty much every time I go to an airport,” he said.
Kilmer returned for the 2022 sequel, and his health issues were evident. His brief scenes in the movie, David Rooney wrote in THR‘s review, generated “resonant pathos. There's reciprocal warmth, even love, in a scene between Iceman and [Cruise's] Maverick that acknowledges the characters' hard-won bond as well as the rivalry that preceded it, with gentle humor.”
Kilmer also provided the voice of K.I.T.T. in a new version of TV's Knight Rider in 2008-09; played opposite Nicolas Cage in Werner Herzog's Bad Lieutenant: Port of Call New Orleans (2009); portrayed the bad guy Cunth for laughs in MacGruber (2010); starred for Francis Ford Coppola in Twixt (2011); was a creepy building superintendent in The Super (2018); and directed, wrote and starred as Mark Twain in Citizen Twain, a one-man show that he brought to stages around the country and then to the big screen.
In 2011, Kilmer sold off most of his 6,000-acre ranch outside Santa Fe, New Mexico, where he had lived for decades. He told THR in December 2017 that his faith as a Christian Scientist helped him deal with his cancer ordeal.
Survivors include his son, Jack, an actor as well.
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Wallen's early exit from Saturday Night Live has been turned into a shirt and hat design.
By
Tyler Jenke
Fresh off the back of his early exit from Saturday Night Live over the weekend, Morgan Wallen has launched a new line of “Get Me to God's Country” merchandise.
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Launched on Tuesday (April 1), the new merch options are rather simple, consisting of a white shirt and a hat, with the latter available in two different color variants. All the items feature the same design, however, with the Coors logo worked into a design which bears the words “Get Me to God's Country.”
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Despite the timing of the merch drop, the Club Wallen Instagram account has assured fans it's not part of an April Fools' Day joke.
The nascent run of merch comes just days after the phrase rose to prominence following Wallen's early walk-off from Saturday Night Live. After performing a pair of songs from his forthcoming album, I'm the Problem, Wallen briefly joined the SNL cast at the end of the show, as is customary on the long-running sketch comedy series. He then whispered something into host Mikey Madison's ear, gave her a hug and abruptly walked off the stage at Studio 8H.
Shortly after the incident, Wallen shared a photo on his Instagram Stories from his private plane, with the caption “Get me to God's country” written over an image of the runway.
Given the somewhat mysterious nature of the phrase and the swift launch of the merchandise line, it's currently unclear whether the slogan is part of Wallen's new album, or something else entirely. The forthcoming record, I'm the Problem, is scheduled to be released on May 16 and features a total of 37 tracks, though only a handful of their titles have been released to date.
In an interview with Entertainment Weekly following Wallen's walk-off, longtime cast member Kenan Thompson said the incident was “definitely a spike in the norm.”
“We're so used to everybody just turning around and high-fiving us, everybody's saying, ‘Good job, good job, good job.' So when there's a departure from that, it's like, hmm, I wonder what that's about?” Thompson added, noting that Prince had previously done the same thing during his appearance on the show.
“I'm not saying Morgan Wallen is Prince, but we weren't surprised because Prince was notoriously kind of standoffish. It's just how he was. So we just thought like, ‘Okay, now he's gone back into fantasyland.'”
Wallen is yet to publicly comment on the incident or provide information as to the meaning behind the phrase.
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"Sidney Poitier said, 'You are not Denzel [Washington], and you are not Morgan [Freeman]. You are a breath of fresh air, and don't f*** with that!'" Murphy recalls.
By
Carly Thomas
Associate Editor
Eddie Murphy is opening up about some advice Sidney Poitier once gave him that left him surprised.
In Apple TV+'s new documentary, Number One on the Call Sheet: Black Leading Men in Hollywood, the Beverly Hills Cop actor shared that the Oscar winner told him not to star in 1992's Malcom X. At the time, the film was initially being helmed by Norman Jewison, Poitier's In the Heat of the Night director, who cast Denzel Washington in the lead role before Spike Lee took over as director.
“They were talking about doing Malcolm X,” Murphy recalled. “Norman Jewison was putting it together. They were gonna use The Autobiography of Malcolm X by Alex Haley. And they approached me about playing Alex Haley. Around that same time, I bumped into Sidney Poitier at something, and I asked him, ‘Yeah, I'm thinking about playing Alex Haley!' And Sidney Poitier said, ‘You are not Denzel [Washington], and you are not Morgan [Freeman]. You are a breath of fresh air, and don't fuck with that!'”
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Murphy admitted he “didn't know” if Poitier's advice “was an insult or a compliment,” but was just shocked to be compared to Washington and Freeman. “I was like, ‘What?'” he added.
Elsewhere in Black Leading Men in Hollywood, Murphy speculated as to why Poitier didn't put him in the same group as Washington and Freeman.
“I was in uncharted waters. For Sidney and all those guys, when I showed up, it was something kinda new,” the Dreamgirls actor said. “They didn't have a reference for me, they couldn't give me advice, 'cause I was 20, 21 years old, and my audience was the mainstream — all of everywhere. My movies [were] all around the world, and they had never had that with a young Black person. So nobody could give me advice, really. Everything broke really big and really fast.”
While Lee's Malcom X didn't include Haley as a character, the film ended up being a huge critical success, earning two Oscar nominations: best actor for Washington and best costume design for Ruth E. Carter.
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It's fourth leader for Gomez and first for blanco.
By
Keith Caulfield
Selena Gomez and benny blanco's first collaborative album, I Said I Love You First, debuts at No. 1 on Billboard's Top Album Sales chart (dated April 5). It's the fourth leader for Gomez and first for blanco. The set sold 71,000 copies in the U.S. in the week ending March 27, according to Luminate. It's the best sales week for Gomez since 2015 and blanco's best sales week ever.
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Of the 71,000 sold, vinyl purchases comprise 21,000 – the biggest week on vinyl for either artist. The title – the first album pairing from the real-life couple – also enters at No. 1 on the Vinyl Albums chart.
In total, I Said I Love You First is Gomez's eighth top 10 on Top Album Sales, and the first for blanco.
Also debuting in the top 10 on the latest Top Album Sales chart: Japanese Breakfast's For Melancholy Brunettes (& Sad Women) and My Morning Jacket's is.
Billboard's Top Album Sales chart ranks the top-selling albums of the week based only on traditional album sales. The chart's history dates back to May 25, 1991, the first week Billboard began tabulating charts with electronically monitored piece count information from SoundScan, now Luminate. Pure album sales were the sole measurement utilized by the Billboard 200 albums chart through the list dated Dec. 6, 2014, after which that chart switched to a methodology that blends album sales with track equivalent album (TEA) units and streaming equivalent album (SEA) units.
Of the 71,000 copies sold of I Said I Love You First, physical sales comprise 38,000 (21,000 on vinyl and 17,000 on CD) and digital download sales comprise 33,000.
The opening-week sales of I Said I Love You First were bolstered by its availability across seven vinyl variants (different color editions, some with alternate covers; including a signed version), three CD versions (a standard CD, a signed edition, and a zine/CD version with expanded packaging), a deluxe box set containing branded merch and a CD, and 10 different digital download iterations.
Of the download editions, firstly, there was a widely available standard set at digital retail. Then, through the album's opening week, nine additional download variants were issued, all initially exclusively available through Gomez's webstore, and each sold for $5. All of the variants included the standard album's 14 songs, plus bonus material. Five of the variants each had one bonus track (“Stained,” “Talk,” “That's What I'll Care [Seven Heavens Version],” “Scared of Loving You [Live From Vevo]” and “How Does It Feel To Be Forgotten [Live From Vevo],” respectively) and one contained two bonus cuts (an acoustic version and extended version of the album single “Call Me When You Break Up”). There was also an Explained: Narrated by Selena Gomez edition (with 14 bonus tracks with Gomez providing commentary on each of the set's 14 songs), a Slowed & Reverbed edition (with 14 bonus slowed and reverbed versions of the album's songs) and an Instrumentals edition (with 14 bonus instrumental versions of the tracklist).
All nine of the variants became available in the iTunes Store on Wednesday (March 26). The variants were only sold in the iTunes Store through March 27, the final day they were also sold in Gomez's store.
Lady Gaga's chart-topping MAYHEM is a non-mover at No. 2 on Top Album Sales with a little over 13,000 sold (down 44%).
Japanese Breakfast lands its best sales week ever and highest charting title on Top Album Sales as For Melancholy Brunettes (& Sad Women) debuts at No. 3 with 13,000 sold. (It's the second top 10 for the act.) The album's sales were aided by its availability across eight vinyl variants (including a signed version), along with a standard download, CD and cassette edition. Vinyl sales tallied up to just over 10,000 – the act's best week on vinyl, and it yields at No. 2 debut on the Vinyl Albums chart.
Kendrick Lamar's former No. 1 GNX is steady at No. 4 on Top Album Sales (a little more than 11,000; down 9%) while The Weeknd's chart-topping Hurry Up Tomorrow jumps 15-5 (11,000; up 92% following the release of a deluxe CD boxed set edition of the album sold in the artist's webstore).
My Morning Jacket's latest studio album, titled is, debuts at No. 6 on Top Album Sales with nearly 10,000 sold – with 7,000 of that sum on vinyl. It's the fifth top 10-charting effort for the act. It also launches at No. 4 on the Vinyl Albums chart. The set was available across five vinyl variants, along with a standard CD and download edition.
Rounding out the rest of the top 10 on Top Album Sales: Sabrina Carpenter's former leader Short n' Sweet slips 6-7 (nearly 10,000; down 5%), LE SSERAFIM's HOT falls 1-8 in its second week (8,000; down 80%), Playboi Carti's MUSIC drops 3-9 (just over 7,000; down 51%) and Chappell Roan's chart-topping The Rise and Fall of a Midwest Princess dips 9-10 (7,000; down 13%).
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"If the fact that I think Donald Trump is the worst president in the history of our great country could stop me from coming back, what does that say for Freedom?" Young asked.
By
Tyler Jenke
Veteran Canadian rocker Neil Young has shared his fears over a potential ban from the U.S. that may await him upon his return from Europe.
The musician – who is a dual citizen of Canada and the U.S. – has not been shy in regard to his criticism of President Donald Trump in the past. Previously, Young has gone so far as to call Trump “a disgrace to my country,” and most recently, claim that “the US has lost its standing” on the world stage under the President's leadership.
However, with an upcoming European tour set to be followed by a run of dates in the U.S., Young has taken to his Archives website to ruminate on the notion that he too may be barred from entering the country for sharing his critical thoughts on Trump.
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“When I go to play music in Europe, if I talk about Donald J. Trump, I may be one of those returning to America who is barred or put in jail to sleep on a cement floor with an aluminum blanket,” Young wrote on Tuesday (April 1). “If I come back from Europe and am barred, can't play my USA tour, all of the folks who bought tickets will not be able to come to a concert by me.
“If the fact that I think Donald Trump is the worst president in the history of our great country could stop me from coming back, what does that say for Freedom? I love America and its people and its music and its culture.”
As Young continued, he reflected on the constitutional right to freedom of speech within the country, likely referring to recent news stories such as the arrest and orders to deport Syrian-born permanent resident and pro-Palestinian activist Mahmoud Khalil.
“By these latest actions of our US government, it seems that those who speak out freely with their own opinions are now vulnerable to a non-existent Trump law,” Young wrote. “Then it seems to me that if you voted for Kamala Harris over Trump, that makes it possible for you to go to jail or be detained, punished in some way for not showing allegiance to what? How spineless is that? Trump is not be able to stand up to anyone who does not agree with his ideas?
“Remember, all months have 30 days,” he concluded. “One country, indivisible, with Liberty and Freedom for all. Remember that? I do.”
Young is currently scheduled to launch his forthcoming tour with the Chrome Hearts in Rättvik, Sweden on June 18, with North American dates set to begin in Charlotte, NC on Aug. 8. The rocker's previously-announced plans for a free concert in Ukraine to launch the tour were recently cancelled, with Young citing safety concerns as the reason for the decision.
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Warner Bros. recently confirmed that it would be pushing the release date on Gyllenhaal's Frankenstein pic ‘The Bride' from Sept. 26, 2025, to March 6, 2026.
By
Chris Gardner
Maggie Gyllenhaal took the veil off The Bride in Las Vegas.
The filmmaker, joined by title star Jessie Buckley, took to the Colosseum stage inside Caesars Palace on Tuesday afternoon during Warner Bros.‘ studio presentation to offer the first look at her anticipated sophomore feature. They were joined by WB bosses Michael De Luca and Pam Abdy.
The Bride finds Gyllenhaal putting her own spin on Mary Shelley's story, setting it in 1930s Chicago and following a lonesome Frankenstein's monster who enlists the help of Dr. Euphronios to create a companion for himself. They revive a murdered woman, creating the titular bride. Buckley stars opposite Christian Bale, Penélope Cruz, Annette Bening, John Magaro and Julianne Hough. It's also a bit of a family affair, as Gyllenhaal cast her younger brother, Jake Gyllenhaal, and husband, Peter Sarsgaard.
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Gyllenhaal said that after her directorial debut, The Lost Daughter, she was looking for a follow-up that was “pop and big” and “radical” at the same time. Then she went to a party where she encountered a man with a tattoo of the bride of Frankenstein on his arm. “It hooked me and I went home and watched the movie,” she continued. “The bride is in the movie for about three minutes, and she doesn't speak, which could not be more different than our bride.”
So Gyllenhaal conceived of a bride who is beyond Frankenstein's wildest imagination, a mate who doesn't really fit in a box. She added that she's also interested in monsters. “All of us have a little aspect of something monstrous in us, which is why we love monster movies, probably,” the filmmaker said. “You can run from it your entire life, or you can shake hands with it.”
As for Buckley, she said when she read Gyllenhaal's script, it felt as if she were being plugged into an electrical current. “It was so wild and so unique. I love her, she's an amazing, amazing woman. She could've asked me to be a sheep, and I would've said yes.”
Asked to describe The Bride's relationship with Frankenstein, Buckley called it “the punkest love that's ever existed,” with traces of Bonnie and Clyde and Wild at Heart. “Our one has some petrol in its skin and we're holding a match.”
Gyllenhaal noted that those who have seen the film report back that “they haven't really seen anything like it before.” Gyllenhaal, who also spoke about shooting the film for Imax, enthusiastically addressed theater owners during her comments. “This is a cinematic movie,” he said. “Frankenstein is a lover of movies in our movie. His closest relationship before The Bride is with a movie star. I wanted to make this punk, monstrous love story in a classic cinematic way that really respected these characters.”
She then introduced the footage that showed scenes of a burial, followed by Buckley's character being revived by the monster, who says in a voiceover, “There's nothing left to do now than live.” The following scenes show the pair making their way through various locations and encountering a cast of characters that show an epic journey through vivid landscapes. In a scene reminiscent of Joker and Harley Quinn, Frankenstein is seen violently murdering someone in a street. Across the screen flashed a tagline, “Here Comes the Mother Fucking Bride.” To close it out, Buckley's bride is seen asking, “What's my name? I can't remember.”
A post shared by Maggie Gyllenhaal (@mgyllenhaal)
Warner Bros. also recently confirmed that it would be pushing the release date on The Bride from Sept. 26, 2025, to March 6, 2026. Gyllenhaal made her directorial debut on Netflix's The Lost Daughter after a long career in front of the camera.
CinemaCon, the annual gathering of cinema owners and Hollywood studios, is hosted in Las Vegas by the newly rebranded Cinema United, which for decades was known as the National Association of Theatre Owners. This year's edition runs from March 31 to April 3.
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New Line's video game adaptation shared clips at CinemaCon, including Karl Urban as the sunglasses enthusiast.
By Aaron Couch, Ryan Gajewski
April 1, 2025 5:30pm
Mortal Kombat 2 kicked its way into the Colosseum at Caesar's Palace Tuesday, where New Line chief Richard Brenner introduced the first footage from the film.
A sizzle reel teased the video game adaptation's key characters, including Karl Urban as Johnny Cage. “I am Johnny fucking Cage,” Urban says in the clip. “It's showtime.”
Brenner said of Mortal Kombat 2, “Expect amazing fights, epic battles and a few fatalities.” It hits theaters Oct. 24.
The feature is based on the popular video game franchise that dates back to the 1990s. It arrives four years after the previous Mortal Kombat movie hit amid the pandemic and hails from returning director Simon McQuoid. This time, Urban headlines as fan-favorite character Johnny Cage, with Tati Gabrielle joining the franchise as Jade and Adeline Rudolph joining as Kitana. Returning stars include Ludi Lin (Liu Kang), Mehcad Brooks as Jax, Lewis Tan as Cole Young, Jessica McNamee as Sonya Blade, Hiroyuki Sanada as Scorpion, Tadanobu Asano as Lord Raiden and Joe Taslim as Sub-Zero.
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The sequel centers on the group of ragtag fighters who must defend Earth from the evil forces of Outworld. The films come decades after a pair of Mortal Kombat features made in the 1990s. The first, 1995's Mortal Kombat, was profitable and is still fondly remembered. 1997's Mortal Kombat: Annihilation came and went without making a splash.
The new feature is produce by Atomic Monster's James Wan and Michael Clear, and Broken Road Productions' Todd Garner. McQuoid and E. Bennett Walsh are also producing.
A previous version of this story misstated the name of the 1997 Mortal Kombat movie.
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The country music star, who a source says walked away from the dress-rehearsal goodnights much like the live show, declined.
By
Tony Maglio
Apparently, the way “to God's country” has a shortcut right through the heart of 30 Rock's famed Studio 8H.
Country music star Morgan Wallen exited stage dead center midway during Saturday Night Live's “goodnights” late Saturday night/early Sunday morning. The abrupt departure stirred up the internet when, post-show, Wallen posted an Instagram story depicting a jet and the caption: “Get me to God's country.” Wallen hails from Tennessee.
Wallen has not directly addressed his exodus, but a source now tells The Hollywood Reporter that Wallen did the same thing during dress rehearsal, which is taped. On the dress tape, Wallen could be seen onstage starting the process with everyone else — but after a quick cutaway, he was gone. Show host Mikey Madison (Anora) and others carried on with the hugs and the waves, just as they did for the real deal. SNL's dress rehearsal has a live studio audience (albeit a different one), just like the live show. Dress rehearsal is two hours long versus the live show's 90-minute runtime.
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For the live show, with the director staying centerstage, Wallen's exit at least looked more dramatic. The steady camera angle perfectly and fully captured the route Wallen used to exit. En route to God's country, Wallen walked right off the Studio 8H stage and into your living room, basically.
Still, at the time, no one really thought much of it, a source recalls. Wallen's team was “so nice” throughout the entire week, the person who spoke with THR on the condition of anonymity, adds. It was the internet that made this a thing, which is what the internet does best.
Longtime castmember Kenan Thompson says he “definitely” noticed Wallen dip.
“You see somebody before you get a chance to say hi or say good job or anything like that, they just dipping,” Thompson told Entertainment Weekly. “I thought maybe he had to go to the potty or something.
“I don't know what goes through people's minds when they decide to do stuff like that,” Thompson said. “I don't know if he understood the assignment or not, or if he was really feeling a certain kind of way.”
On occasion, especially when a sketch gets cut, an upset performer may not end up joining everyone else. No one forces them up on the stage, the source said.
But this was so much more noticeable, and some amateur internet sleuths believe it was a purposefully visible protest. Wallen appeals to a huge number of conservatives, and SNL skews very liberal.
Wallen has a bit of a checkered history with Saturday Night Live. In October 2020, SNL canceled Wallen's scheduled debut as SNL musical guest after videos of the singer brazenly violating CDC-recommended social-distancing guidelines went viral. He ultimately performed on the show just two months later and participated in a parody of the incident and its fallout.
Sketch-comedy conspiracy theorists are now also locking on to the fact that Wallen did not appear in a pretaped sketch, which typically are filmed on a Friday (sometimes a Thursday). That's also not unusual, the source close to the show said. Sometimes the show's musical guests are particularly busy away from the SNL sets — or maybe they just want focus on their musical performance. Sometimes they just don't like the proposed sketch.
Joe Jonas popped up on Saturday's SNL to sing the bridge on Big Dumb Line, a pretaped musical number about waiting in long New York City lines for viral foods, which grow increasingly stranger in nature. Wallen was asked to the do part, a source says, but he “wasn't available.”
Wallen performed a pair of songs from his upcoming album, I'm the Problem, on the final SNL of March 2025. And then he was out.
“I had a wonderful time, thank you all,” Madison said, as the house band played, and the closing credits began to roll. Wallen then gave Madison a hug and said something in her ear.
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SPHEREx's first images — containing roughly 100,000 points of light stars, galaxies and nebulae — have confirmed that the telescope is working according to its design.
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A new NASA space telescope has turned on its detectors for the first time, capturing its first light in images that contain tens of thousands of galaxies and stars.
The Spectro-Photometer for the History of the Universe, Epoch of Reionization, and Ices Explorer (SPHEREx) arrived in orbit atop a SpaceX Falcon 9 rocket on March 11.
The six released images, collected by the space telescope on March 27, were each snapped by three different detectors. The top three images span the telescope's complete field of view, and are captured again in the bottom three which are colored differently to represent varying ranges of infrared wavelengths.
Within each image's full field of view — an area roughly 20 times wider than the full moon — roughly 100,000 light sources from stars, galaxies, and nebulas can be glimpsed.
"Our spacecraft has opened its eyes on the universe," Olivier Doré, a SPHEREx project scientist at Caltech and NASA's Jet Propulsion Laboratory, said in a statement. "It's performing just as it was designed to."
Related: Euclid space telescope: ESA's groundbreaking mission to study dark matter and dark energy
Costing a total of $488 million to build and launch, the new telescope has been in development for roughly a decade, and is set to map the universe by observing both optical and infrared light. It will orbit Earth 14.5 times a day, completing 11,000 orbits during its lifetime to filter infrared light from distant gas and dust clouds using a technique called spectroscopy.
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Once it is fully online in April, SPHEREX will scan the entire night sky a total of four times using 102 separate infrared color sensors, enabling it to collect data from more than 450 million galaxies during its planned two-year operation. This amounts to roughly 600 exposures a day, according to NASA.
This dataset will give scientists key insights into some of the biggest questions in cosmology, enabling astronomers to study galaxies at various stages in their evolution; trace the ice floating in empty space to see how life may have begun; and even understand the period of rapid inflation the universe underwent immediately after the Big Bang.
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SPHEREx's wide panorama view makes it the perfect complement for the James Webb Space Telescope, flagging regions of interest for the latter to study with greater depth and resolution.
After lofting it to space, NASA scientists and engineers have performed a nail-biting series of checks on the new telescope. This includes ensuring that its sensitive infrared equipment is cooling down to its final temperature of around minus 350 degrees Fahrenheit (minus 210 degrees Celsius) and that the telescope is set to the right focus — something that cannot be adjusted in space.
Based on these stunning preliminary images, it appears that everything has worked out.
"This is the high point of spacecraft checkout; it's the thing we wait for," Beth Fabinsky, SPHEREx deputy project manager at JPL, said in the statement. "There's still work to do, but this is the big payoff. And wow! Just wow!"
Ben Turner is a U.K. based staff writer at Live Science. He covers physics and astronomy, among other topics like tech and climate change. He graduated from University College London with a degree in particle physics before training as a journalist. When he's not writing, Ben enjoys reading literature, playing the guitar and embarrassing himself with chess.
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Surprising results from hypersonic air flow simulations could help design stronger, faster and more durable supersonic vehicles.
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A close look at air flow around high-speed shapes reveals surprising turbulence, according to a new study. The findings, published March 7 in the journal Physical Review Fluids, could inform the design of future high-speed vehicles.
In the study, researchers used three-dimensional simulations to reveal unexpected disturbances around fast-moving cones.
At hypersonic speeds — above Mach 5, or more than 5 times the speed of sound (3,836 mph or 6,174 kilometers per hour) — the flow of air around a vehicle's surface becomes complex and bumpy. Most simulations assume that the flow is symmetrical around the whole cone, but until recently, studies of the transition from streamlined to turbulent were only possible in two dimensions so we couldn't be sure that there weren't any asymmetries in flow around a three-dimensional structure.
The findings could help engineers design stronger, faster vehicles able to withstand the extreme temperatures, pressures and vibrations felt during hypersonic flight.
"Transitioning flows are 3D and unsteady in nature, regardless of the flow geometry," study co-author Irmak Taylan Karpuzcu, an aerospace engineer at the University of Illinois Urbana-Champaign, said in a statement. "Experiments were conducted in 3D in the early 2000s [but they] didn't provide enough data to determine any 3D effects or unsteadiness because there weren't enough sensors all around the cone-shaped model. It wasn't wrong. It was just all that was possible then."
Using the Frontera supercomputer at the Texas Advanced Computing Center, Karpuzcu and aerospace engineer Deborah Levin simulated how air flow around a cone-shaped object — often used as a simplified model for hypersonic vehicles — changes in three dimensions at high speed. They studied both a single cone and a double cone, which helps scientists study how multiple shock waves interact with each other.
"Normally, you would expect the flow around the cone to be concentric ribbons, but we noticed breaks in the flow within shock layers both in the single and double cone shapes," Karpuzcu said.
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These breaks were particularly prevalent around the tip of the cone. At high speeds, the shock wave lies closer to the cone, squeezing air molecules into unstable layers and amplifying instabilities in the airflow. The team confirmed their findings by running a program that tracks each simulated air molecule and captures how collisions between the molecules affect air flow.
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The disturbances also seem to develop at high speeds. "As you increase the Mach number, the shock gets closer to the surface and promotes these instabilities. It would be too expensive to run the simulation at every speed, but we did run it at Mach 6 and did not see a break in the flow," Karpuzcu said.
The breaks could affect design considerations for hypersonic vehicles, which could be used for shipping, weapons and transportation, Karpuzcu said, as engineers will need to account for the newly observed discontinuities.
Skyler Ware is a freelance science journalist covering chemistry, biology, paleontology and Earth science. She was a 2023 AAAS Mass Media Science and Engineering Fellow at Science News. Her work has also appeared in Science News Explores, ZME Science and Chembites, among others. Skyler has a Ph.D. in chemistry from Caltech.
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Seismic mapping of North America has revealed that an ancient slab of crust buried beneath the Midwest is causing the crust above it to "drip" and suck down rocks from across the continent.
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An ancient slab of Earth's crust buried deep beneath the Midwest is sucking huge swatches of present-day's North American crust down into the mantle, researchers say.
The slab's pull has created giant "drips" that hang from the underside of the continent down to about 400 miles (640 kilometers) deep inside the mantle, according to a new study. These drips are located beneath an area spanning from Michigan to Nebraska and Alabama, but their presence appears to be impacting the entire continent.
The dripping area looks like a large funnel, with rocks from across North America being pulled toward it horizontally before getting sucked down. As a result, large parts of North America are losing material from the underside of their crust, the researchers said.
"A very broad range is experiencing some thinning," study lead author Junlin Hua, a geoscientist who conducted the research during a postdoctoral fellowship at The University of Texas (UT) at Austin, said in a statement. "Luckily, we also got the new idea about what drives this thinning," said Hua, now a professor at the University of Science and Technology of China.
Related: Earth's crust is peeling away under California
The researchers found that the drips result from the downward dragging force of a chunk of oceanic crust that broke off from an ancient tectonic plate called the Farallon plate.
The Farallon plate and the North American plate once formed a subduction zone along the continent's west coast, with the former sliding beneath the latter and recycling its material into the mantle. The Farallon plate splintered due to the advance of the Pacific plate roughly 20 million years ago, and remnant slabs subducted beneath the North American plate slowly drifted off.
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One of these slabs currently straddles the boundary between the mantle transition zone and the lower mantle roughly 410 miles (660 km) beneath the Midwest. Dubbed the "Farallon slab" and first imaged in the 1990s, this piece of oceanic crust is responsible for a process known as "cratonic thinning," according to the new study, which was published March 28 in the journal Nature Geoscience.
Cratonic thinning refers to the wearing away of cratons, which are regions of Earth's continental crust and upper mantle that have mostly remained intact for billions of years. Despite their stability, cratons can undergo changes, but this has never been observed in action due to the huge geologic time scales involved, according to the study.
Now, for the first time, researchers have documented cratonic thinning as it occurs. The discovery was possible thanks to a wider project led by Hua to map what lies beneath North America using a high-resolution seismic imaging technique called "full-waveform inversion." This technique uses different types of seismic waves to extract all the available information about physical parameters underground.
"This sort of thing is important if we want to understand how a planet has evolved over a long time," study co-author Thorsten Becker, a distinguished chair in geophysics at UT Austin, said in the statement. "Because of the use of this full-waveform method, we have a better representation of that important zone between the deep mantle and the shallower lithosphere [crust and upper mantle]."
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To test their results, the researchers simulated the impact of the Farallon slab on the craton above using a computer model. A dripping area formed when the slab was present, but it disappeared when the slab was absent, confirming that — theoretically, at least — a sunken slab can drag rocks across a large area down into Earth's interior.
Dripping beneath the Midwest won't lead to changes at the surface anytime soon, the researchers said, adding that it may even stop as the Farallon slab sinks deeper into the lower mantle and its influence over the craton wanes.
The findings could help researchers piece together the enormous puzzle of how Earth came to look the way it does today. "It helps us understand how do you make continents, how do you break them, and how do you recycle them," Becker said.
Sascha is a U.K.-based staff writer at Live Science. She holds a bachelor's degree in biology from the University of Southampton in England and a master's degree in science communication from Imperial College London. Her work has appeared in The Guardian and the health website Zoe. Besides writing, she enjoys playing tennis, bread-making and browsing second-hand shops for hidden gems.
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Potentially Misleading TitleNASA has taken down the Raw Image of the Tiny Mars Tic-Tac Object (self.UFOs)
submitted 14 hours ago * by tcom2222
UPDATE:
It has been restored around 12pm est!
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Please first see the original Tiny Mars Tic-Tac Object post, link in OP Comment Post so this doesn't get deleted.
Case Update:
Originally there was a panorama that had been put together and a raw photo of the Mars Tic-Tac Object which could be found at mars. nasa . gov however, the panorama was soon taken down, but it was saved by the person who had originally found the object (link and pic in the OP). That being said there was still the raw image itself of the object at their site. I purposefully took a screenshot of this raw image open in my browser, and included the picture in the OP. I was just notified however that, the raw image is now no longer accessible either. "403 Forbidden. Access Denied" If you check out the OP and OP comment post you'll still find the image, the link, and the metadata. Of note, the raw image of the different timeframe where the object is NOT there IS still available to view (link in OP).
NASA, are you new here? Why not just make an age old comment like, "hey we see that the public took great interest in this object, we don't know what it is for sure, and likely will not without having more data, sorry, however of course we think its swamp gas/natural phenomenon/just a rock of course..." why take it down and have that be the latest in updates to this case fueling even more speculation? There should be no reason not to leave these images open for public availability, analysis, and scrutiny, especially if its just a perfectly smooth oddly shaped reflective rock mistaken to be flying in one image and gone in another from a different time, “for the widest practicable and appropriate dissemination of information concerning its [NASA's] activities and the results thereof.”
Note* Instead of refraining from making a comment on size since its just estimation, I've added "tiny" so this doesn't get tagged as misleading. I've included no pictures so this doesn't get deleted. I've included no links so this doesn't get deleted, and below is the mandated DTG&L of the sighting so this doesn't get deleted. Below is a comment post, that will host the links so this doesn't get deleted.
Time: Sol 2692 3 March 2020 (2020-03-03 02:32:29 UTC )
Location: Mars Longitude: 137.38077432° Latitude: -4.73673265°
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NASA has taken down the Raw Image of the Tiny Mars Tic-Tac Object ()
submitted 5 hours ago by quantify-it to r/AliensRHere
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