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Decoding a key part of the cell, atom by atom

Whatever you are doing, whether it is driving a car, going for a jog, or even at your laziest, eating chips and watching TV on the couch, there is an entire…

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Whatever you are doing, whether it is driving a car, going for a jog, or even at your laziest, eating chips and watching TV on the couch, there is an entire suite of molecular machinery inside each of your cells hard at work. That machinery, far too small to see with the naked eye or even with many microscopes, creates energy for the cell, manufactures its proteins, makes copies of its DNA, and much more.

Credit: Hoelz Laboratory/Caltech Reprinted with permission from S. Petrovic et al., Science 376, eabm9798 (2022).

Whatever you are doing, whether it is driving a car, going for a jog, or even at your laziest, eating chips and watching TV on the couch, there is an entire suite of molecular machinery inside each of your cells hard at work. That machinery, far too small to see with the naked eye or even with many microscopes, creates energy for the cell, manufactures its proteins, makes copies of its DNA, and much more.

Among those pieces of machinery, and one of the most complex, is something known as the nuclear pore complex (NPC). The NPC, which is made of more than 1,000 individual proteins, is an incredibly discriminating gatekeeper for the cell’s nucleus, the membrane-bound region inside a cell that holds that cell’s genetic material. Anything going in or out of the nucleus has to pass through the NPC on its way.

The NPC’s role as a gatekeeper of the nucleus means it is vital for the operations of the cell. Within the nucleus, DNA, the cell’s permanent genetic code, is copied into RNA. That RNA is then carried out of the nucleus so it can be used to manufacture the proteins the cell needs. The NPC ensures the nucleus gets the materials it needs for synthesizing RNA, while also protecting the DNA from the harsh environment outside the nucleus and enabling the RNA to leave the nucleus after it has been made.

“It’s a little like an airplane hangar where you can repair 747s, and the door opens to let the 747 come in, but there’s a person standing there who can keep a single marble from getting out while the doors are open,” says Caltech’s André Hoelz, professor of chemistry and biochemistry and a Faculty Scholar of the Howard Hughes Medical Institute. For more than two decades, Hoelz has been studying and deciphering the structure of the NPC in relation to its function. Over the years, he has steadily chipped away at its secrets, unraveling them piece by piece by piece by piece.

The implications of this research are potentially huge. Not only is the NPC central to the operations of the cell, it is also involved in many diseases. Mutations in the NPC are responsible for some incurable cancers, for neurodegenerative and autoimmune diseases such as amyotrophic lateral sclerosis (ALS) and acute necrotizing encephalopathy, and for heart conditions including atrial fibrillation and early sudden cardiac death. Additionally, many viruses, including the one responsible for COVID-19, target and shutdown the NPC during the course of their lifecycles.

Now, in a pair of papers published in the journal Science, Hoelz and his research team describe two important breakthroughs: the determination of the structure of the outer face of the NPC and the elucidation of the mechanism by which special proteins act like a molecular glue to hold the NPC together.

A very tiny 3D jigsaw puzzle

In their paper titled “Architecture of the cytoplasmic face of the nuclear pore,” Hoelz and his research team describe how they mapped the structure of the side of the NPC that faces outward from the nucleus and into the cells’ cytoplasm. To do this, they had to solve the equivalent of a very tiny 3-D jigsaw puzzle, using imaging techniques such as electron microscopy and X-ray crystallography on each puzzle piece.

Stefan Petrovic, a graduate student in biochemistry and molecular biophysics and one of the co-first authors of the papers, says the process began with Escherichia coli bacteria (a strain of bacteria commonly used in labs) that were genetically engineered to produce the proteins that make up the human NPC.

“If you walk into the lab, you can see this giant wall of flasks in which cultures are growing,” Petrovic says. “We express each individual protein in E. coli cells, break those cells open, and chemically purify each protein component.”

Once that purification—which can require as much as 1,500 liters of bacterial culture to get enough material for a single experiment—was complete, the research team began to painstakingly test how the pieces of the NPC fit together.

George Mobbs, a senior postdoctoral scholar research associate in chemistry and another co- first author of the paper, says the assembly happened in a “stepwise” fashion; rather than pouring all the proteins together into a test tube at the same time, the researchers tested pairs of proteins to see which ones would fit together, like two puzzle pieces. If a pair was found that fit together, the researchers would then test the two now-combined proteins against a third protein until they found one that fit with that pair, and then the resulting three-piece structure was tested against other proteins, and so on. Working their way through the proteins in this way eventually produced the final result of their paper: a 16-protein wedge that is repeated eight times, like slices of a pizza, to form the face of the NPC.

“We reported the first complete structure of the entire cytoplasmic face of the human NPC, along with rigorous validation, instead of reporting a series of incremental advances of fragments or portions based on partial, incomplete, or low-resolution observation,” says Si Nie, postdoctoral scholar research associate in chemistry and also a co-first author of the paper. “We decided to patiently wait until we had acquired all necessary data, reporting a humungous amount of new information.”

Their work complemented research conducted by Martin Beck of the Max Planck Institute of Biophysics in Frankfurt, Germany, whose team used cryo-electron tomography to generate a map that provided the contours of a puzzle into which the researchers had to place the pieces. To accelerate the completion of the puzzle of the human NPC structure, Hoelz and Beck exchanged data more than two years ago and then independently built structures of the entire NPC. “The substantially improved Beck map showed much more clearly where each piece of the NPC—for which we determined the atomic structures—had to be placed, akin to a wooden frame that defines the edge of a puzzle,” Hoelz says.

The experimentally determined structures of the NPC pieces from the Hoelz group served to validate the modeling by the Beck group. “We placed the structures into the map independently, using different approaches, but the final results completely agreed. It was very satisfying to see that,” Petrovic says.

“We built a framework on which a lot of experiments can now be done,” says Christopher Bley, a senior postdoctoral scholar research associate in chemistry and also co-first author. “We have this composite structure now, and it enables and informs future experiments on NPC function, or even diseases. There are a lot of mutations in the NPC that are associated with terrible diseases, and knowing where they are in the structure and how they come together can help design the next set of experiments to try and answer the questions of what these mutations are doing.”

“This elegant arrangement of spaghetti noodles”

In the other paper, titled “Architecture of the linker-scaffold in the nuclear pore,” the research team describes how it determined the entire structure of what is known as the NPC’s linker-scaffold—the collection of proteins that help hold the NPC together while also providing it with the flexibility it needs to open and close and to adjust itself to fit the molecules that pass through.

Hoelz likens the NPC to something built out of Lego bricks that fit together without locking together and are instead lashed together by rubber bands that keep them mostly in place while still allowing them to move around a bit.

“I call these unstructured glue pieces the ‘dark matter of the pore,'” Hoelz says. “This elegant arrangement of spaghetti noodles holds everything together.”

The process for characterizing the structure of the linker-scaffold was much the same as the process used to characterize the other parts of the NPC. The team manufactured and purified large amounts of the many types linker and scaffold proteins, used a variety of biochemical experiments and imaging techniques to examine individual interactions, and tested them piece by piece to see how they fit together in the intact NPC.

To check their work, they introduced mutations into the genes that code for each of those linker proteins in a living cell. Since they knew how those mutations would change the chemical properties and shape of a specific linker protein, making it defective, they could predict what would happen to the structure of the cell’s NPCs when those defective proteins were introduced. If the cell’s NPCs were functionally and structurally defective in the way they expected, they knew they had the correct arrangement of the linker proteins.

“A cell is much more complicated than the simple system we create in a test tube, so it is necessary to verify that results obtained from in vitro experiments hold up in vivo,” Petrovic says.

The assembly of the NPC’s outer face also helped solve a longtime mystery about the nuclear envelope, the double membrane system that surrounds the nucleus. Like the membrane of the cell within which the nucleus resides, the nuclear membrane is not perfectly smooth. Rather, it is studded with molecules called integral membrane proteins (IMPs) that serve in a variety of roles, including acting as receptors and helping to catalyze biochemical reactions.

Although IMPs can be found on both the inner and outer sides of the nuclear envelope, it had been unclear how they actually traveled from one side to the other. Indeed, because IMPs are stuck inside of the membrane, they cannot just glide through the central transport channel of the NPC as do free-floating molecules.

Once Hoelz’s team understood the structure of the NPC’s linker-scaffold, they realized that it allows for the formation of little “gutters” around its outside edge that allow the IMPs to slip past the NPC from one side of the nuclear envelope to the other while always staying embedded in the membrane itself.

“It explains a lot of things that have been enigmatic in the field. I am very happy to see that the central transport channel indeed has the ability to dilate and form lateral gates for these IMPs, as we had originally proposed more than a decade ago,” Hoelz says.

Taken together, the findings of the two papers represent a leap forward in scientists’ understanding of how the human NPC is built and how it works. The team’s discoveries open the door for much more research. “Having determined its structure, we can now focus on working out the molecular bases for the NPC’s functions, such as how mRNA gets exported and the underlying causes for the many NPC-associated diseases with the goal of developing novel therapies,” Hoelz says.

The papers describing the work appear in the June 10 issue of the journal Science.

Additional co-authors of the paper, “Architecture of the cytoplasmic face of the nuclear pore,” are Anna T. Gres; now of Worldwide Clinical Trials; Xiaoyu Liu, now of UCLA; Sho Harvey, a former grad student in Hoelz’s lab; Ferdinand M. Huber, now of Odyssey Therapeutics; Daniel H. Lin, now of the Whitehead Institute for Biomedical Research; Bonnie Brown, a former research technician in Hoelz’s lab; Aaron W. Tang, a former research technician in Hoelz’s lab; Emily J. Rundlet, now of St. Jude Children’s Research Hospital and Weill Cornell Medicine; Ana R. Correia, now of Amgen; Taylor A. Stevens, graduate student in biochemistry and molecular biophysics; Claudia A. Jette, graduate student in biochemistry and molecular biophysics; Alina Patke, research assistant professor of biology; Somnath Mukherjee and Anthony A. Kossiakoff of the University of Chicago; Shane Chen, Saroj G. Regmi, and Mary Dasso of the National Institute of Child Health and Human Development; and Alexander F. Palazzo of the University of Toronto.

Additional co-authors of the paper, “Architecture of the linker-scaffold in the nuclear pore,” are Dipanjan Samanta, postdoctoral scholar fellowship trainee in chemical engineering; Thibaud Perriches, now of Care Partners; Christopher J. Bley; Karsten Thierbach; now of Odyssey Therapeutics; Bonnie Brown, Si Nie, George W. Mobbs, Taylor A. Stevens, Xiaoyu Liu, now of UCLA; Giovani Pinton Tomaleri, graduate student in biochemistry and molecular biophysics; and Lucas Schaus, graduate student in biochemistry and molecular biophysics.

Funding for the research was provided by the National Institutes of Health, the Howard Hughes Medical Institute, and the Heritage Medical Research Institute.


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Health Officials: Man Dies From Bubonic Plague In New Mexico

Health Officials: Man Dies From Bubonic Plague In New Mexico

Authored by Jack Phillips via The Epoch Times (emphasis ours),

Officials in…

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Health Officials: Man Dies From Bubonic Plague In New Mexico

Authored by Jack Phillips via The Epoch Times (emphasis ours),

Officials in New Mexico confirmed that a resident died from the plague in the United States’ first fatal case in several years.

A bubonic plague smear, prepared from a lymph removed from an adenopathic lymph node, or bubo, of a plague patient, demonstrates the presence of the Yersinia pestis bacteria that causes the plague in this undated photo. (Centers for Disease Control and Prevention/Getty Images)

The New Mexico Department of Health, in a statement, said that a man in Lincoln County “succumbed to the plague.” The man, who was not identified, was hospitalized before his death, officials said.

They further noted that it is the first human case of plague in New Mexico since 2021 and also the first death since 2020, according to the statement. No other details were provided, including how the disease spread to the man.

The agency is now doing outreach in Lincoln County, while “an environmental assessment will also be conducted in the community to look for ongoing risk,” the statement continued.

This tragic incident serves as a clear reminder of the threat posed by this ancient disease and emphasizes the need for heightened community awareness and proactive measures to prevent its spread,” the agency said.

A bacterial disease that spreads via rodents, it is generally spread to people through the bites of infected fleas. The plague, known as the black death or the bubonic plague, can spread by contact with infected animals such as rodents, pets, or wildlife.

The New Mexico Health Department statement said that pets such as dogs and cats that roam and hunt can bring infected fleas back into homes and put residents at risk.

Officials warned people in the area to “avoid sick or dead rodents and rabbits, and their nests and burrows” and to “prevent pets from roaming and hunting.”

“Talk to your veterinarian about using an appropriate flea control product on your pets as not all products are safe for cats, dogs or your children” and “have sick pets examined promptly by a veterinarian,” it added.

“See your doctor about any unexplained illness involving a sudden and severe fever, the statement continued, adding that locals should clean areas around their home that could house rodents like wood piles, junk piles, old vehicles, and brush piles.

The plague, which is spread by the bacteria Yersinia pestis, famously caused the deaths of an estimated hundreds of millions of Europeans in the 14th and 15th centuries following the Mongol invasions. In that pandemic, the bacteria spread via fleas on black rats, which historians say was not known by the people at the time.

Other outbreaks of the plague, such as the Plague of Justinian in the 6th century, are also believed to have killed about one-fifth of the population of the Byzantine Empire, according to historical records and accounts. In 2013, researchers said the Justinian plague was also caused by the Yersinia pestis bacteria.

But in the United States, it is considered a rare disease and usually occurs only in several countries worldwide. Generally, according to the Mayo Clinic, the bacteria affects only a few people in U.S. rural areas in Western states.

Recent cases have occurred mainly in Africa, Asia, and Latin America. Countries with frequent plague cases include Madagascar, the Democratic Republic of Congo, and Peru, the clinic says. There were multiple cases of plague reported in Inner Mongolia, China, in recent years, too.

Symptoms

Symptoms of a bubonic plague infection include headache, chills, fever, and weakness. Health officials say it can usually cause a painful swelling of lymph nodes in the groin, armpit, or neck areas. The swelling usually occurs within about two to eight days.

The disease can generally be treated with antibiotics, but it is usually deadly when not treated, the Mayo Clinic website says.

“Plague is considered a potential bioweapon. The U.S. government has plans and treatments in place if the disease is used as a weapon,” the website also says.

According to data from the U.S. Centers for Disease Control and Prevention, the last time that plague deaths were reported in the United States was in 2020 when two people died.

Tyler Durden Wed, 03/13/2024 - 21:40

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Riley Gaines Explains How Women’s Sports Are Rigged To Promote The Trans Agenda

Riley Gaines Explains How Women’s Sports Are Rigged To Promote The Trans Agenda

Is there a light forming when it comes to the long, dark and…

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Riley Gaines Explains How Women's Sports Are Rigged To Promote The Trans Agenda

Is there a light forming when it comes to the long, dark and bewildering tunnel of social justice cultism?  Global events have been so frenetic that many people might not remember, but only a couple years ago Big Tech companies and numerous governments were openly aligned in favor of mass censorship.  Not just to prevent the public from investigating the facts surrounding the pandemic farce, but to silence anyone questioning the validity of woke concepts like trans ideology. 

From 2020-2022 was the closest the west has come in a long time to a complete erasure of freedom of speech.  Even today there are still countries and Europe and places like Canada or Australia that are charging forward with draconian speech laws.  The phrase "radical speech" is starting to circulate within pro-censorship circles in reference to any platform where people are allowed to talk critically.  What is radical speech?  Basically, it's any discussion that runs contrary to the beliefs of the political left.

Open hatred of moderate or conservative ideals is perfectly acceptable, but don't ever shine a negative light on woke activism, or you might be a terrorist.

Riley Gaines has experienced this double standard first hand.  She was even assaulted and taken hostage at an event in 2023 at San Francisco State University when leftists protester tried to trap her in a room and demanded she "pay them to let her go."  Campus police allegedly witnessed the incident but charges were never filed and surveillance footage from the college was never released.  

It's probably the last thing a champion female swimmer ever expects, but her head-on collision with the trans movement and the institutional conspiracy to push it on the public forced her to become a counter-culture voice of reason rather than just an athlete.

For years the independent media argued that no matter how much we expose the insanity of men posing as women to compete and dominate women's sports, nothing will really change until the real female athletes speak up and fight back.  Riley Gaines and those like her represent that necessary rebellion and a desperately needed return to common sense and reason.

In a recent interview on the Joe Rogan Podcast, Gaines related some interesting information on the inner workings of the NCAA and the subversive schemes surrounding trans athletes.  Not only were women participants essentially strong-armed by colleges and officials into quietly going along with the program, there was also a concerted propaganda effort.  Competition ceremonies were rigged as vehicles for promoting trans athletes over everyone else. 

The bottom line?  The competitions didn't matter.  The real women and their achievements didn't matter.  The only thing that mattered to officials were the photo ops; dudes pretending to be chicks posing with awards for the gushing corporate media.  The agenda took precedence.

Lia Thomas, formerly known as William Thomas, was more than an activist invading female sports, he was also apparently a science project fostered and protected by the athletic establishment.  It's important to understand that the political left does not care about female athletes.  They do not care about women's sports.  They don't care about the integrity of the environments they co-opt.  Their only goal is to identify viable platforms with social impact and take control of them.  Women's sports are seen as a vehicle for public indoctrination, nothing more.

The reasons why they covet women's sports are varied, but a primary motive is the desire to assert the fallacy that men and women are "the same" psychologically as well as physically.  They want the deconstruction of biological sex and identity as nothing more than "social constructs" subject to personal preference.  If they can destroy what it means to be a man or a woman, they can destroy the very foundations of relationships, families and even procreation.  

For now it seems as though the trans agenda is hitting a wall with much of the public aware of it and less afraid to criticize it.  Social media companies might be able to silence some people, but they can't silence everyone.  However, there is still a significant threat as the movement continues to target children through the public education system and women's sports are not out of the woods yet.   

The ultimate solution is for women athletes around the world to organize and widely refuse to participate in any competitions in which biological men are allowed.  The only way to save women's sports is for women to be willing to end them, at least until institutions that put doctrine ahead of logic are made irrelevant.          

Tyler Durden Wed, 03/13/2024 - 17:20

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Congress’ failure so far to deliver on promise of tens of billions in new research spending threatens America’s long-term economic competitiveness

A deal that avoided a shutdown also slashed spending for the National Science Foundation, putting it billions below a congressional target intended to…

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Science is again on the chopping block on Capitol Hill. AP Photo/Sait Serkan Gurbuz

Federal spending on fundamental scientific research is pivotal to America’s long-term economic competitiveness and growth. But less than two years after agreeing the U.S. needed to invest tens of billions of dollars more in basic research than it had been, Congress is already seriously scaling back its plans.

A package of funding bills recently passed by Congress and signed by President Joe Biden on March 9, 2024, cuts the current fiscal year budget for the National Science Foundation, America’s premier basic science research agency, by over 8% relative to last year. That puts the NSF’s current allocation US$6.6 billion below targets Congress set in 2022.

And the president’s budget blueprint for the next fiscal year, released on March 11, doesn’t look much better. Even assuming his request for the NSF is fully funded, it would still, based on my calculations, leave the agency a total of $15 billion behind the plan Congress laid out to help the U.S. keep up with countries such as China that are rapidly increasing their science budgets.

I am a sociologist who studies how research universities contribute to the public good. I’m also the executive director of the Institute for Research on Innovation and Science, a national university consortium whose members share data that helps us understand, explain and work to amplify those benefits.

Our data shows how underfunding basic research, especially in high-priority areas, poses a real threat to the United States’ role as a leader in critical technology areas, forestalls innovation and makes it harder to recruit the skilled workers that high-tech companies need to succeed.

A promised investment

Less than two years ago, in August 2022, university researchers like me had reason to celebrate.

Congress had just passed the bipartisan CHIPS and Science Act. The science part of the law promised one of the biggest federal investments in the National Science Foundation in its 74-year history.

The CHIPS act authorized US$81 billion for the agency, promised to double its budget by 2027 and directed it to “address societal, national, and geostrategic challenges for the benefit of all Americans” by investing in research.

But there was one very big snag. The money still has to be appropriated by Congress every year. Lawmakers haven’t been good at doing that recently. As lawmakers struggle to keep the lights on, fundamental research is quickly becoming a casualty of political dysfunction.

Research’s critical impact

That’s bad because fundamental research matters in more ways than you might expect.

For instance, the basic discoveries that made the COVID-19 vaccine possible stretch back to the early 1960s. Such research investments contribute to the health, wealth and well-being of society, support jobs and regional economies and are vital to the U.S. economy and national security.

Lagging research investment will hurt U.S. leadership in critical technologies such as artificial intelligence, advanced communications, clean energy and biotechnology. Less support means less new research work gets done, fewer new researchers are trained and important new discoveries are made elsewhere.

But disrupting federal research funding also directly affects people’s jobs, lives and the economy.

Businesses nationwide thrive by selling the goods and services – everything from pipettes and biological specimens to notebooks and plane tickets – that are necessary for research. Those vendors include high-tech startups, manufacturers, contractors and even Main Street businesses like your local hardware store. They employ your neighbors and friends and contribute to the economic health of your hometown and the nation.

Nearly a third of the $10 billion in federal research funds that 26 of the universities in our consortium used in 2022 directly supported U.S. employers, including:

  • A Detroit welding shop that sells gases many labs use in experiments funded by the National Institutes of Health, National Science Foundation, Department of Defense and Department of Energy.

  • A Dallas-based construction company that is building an advanced vaccine and drug development facility paid for by the Department of Health and Human Services.

  • More than a dozen Utah businesses, including surveyors, engineers and construction and trucking companies, working on a Department of Energy project to develop breakthroughs in geothermal energy.

When Congress shortchanges basic research, it also damages businesses like these and people you might not usually associate with academic science and engineering. Construction and manufacturing companies earn more than $2 billion each year from federally funded research done by our consortium’s members.

A lag or cut in federal research funding would harm U.S. competitiveness in critical advanced technologies such as artificial intelligence and robotics. Hispanolistic/E+ via Getty Images

Jobs and innovation

Disrupting or decreasing research funding also slows the flow of STEM – science, technology, engineering and math – talent from universities to American businesses. Highly trained people are essential to corporate innovation and to U.S. leadership in key fields, such as AI, where companies depend on hiring to secure research expertise.

In 2022, federal research grants paid wages for about 122,500 people at universities that shared data with my institute. More than half of them were students or trainees. Our data shows that they go on to many types of jobs but are particularly important for leading tech companies such as Google, Amazon, Apple, Facebook and Intel.

That same data lets me estimate that over 300,000 people who worked at U.S. universities in 2022 were paid by federal research funds. Threats to federal research investments put academic jobs at risk. They also hurt private sector innovation because even the most successful companies need to hire people with expert research skills. Most people learn those skills by working on university research projects, and most of those projects are federally funded.

High stakes

If Congress doesn’t move to fund fundamental science research to meet CHIPS and Science Act targets – and make up for the $11.6 billion it’s already behind schedule – the long-term consequences for American competitiveness could be serious.

Over time, companies would see fewer skilled job candidates, and academic and corporate researchers would produce fewer discoveries. Fewer high-tech startups would mean slower economic growth. America would become less competitive in the age of AI. This would turn one of the fears that led lawmakers to pass the CHIPS and Science Act into a reality.

Ultimately, it’s up to lawmakers to decide whether to fulfill their promise to invest more in the research that supports jobs across the economy and in American innovation, competitiveness and economic growth. So far, that promise is looking pretty fragile.

This is an updated version of an article originally published on Jan. 16, 2024.

Jason Owen-Smith receives research support from the National Science Foundation, the National Institutes of Health, the Alfred P. Sloan Foundation and Wellcome Leap.

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