How evolution tamed a deadly virus and why we should still worry

The story of the rise and fall of western equine encephalitis as a lethal disease offers essential lessons about how a pathogen can gain or lose its ability to jump from animals to humans.

That story is captured in newly published research from Harvard Medical School that identifies the mechanisms the western equine encephalitis virus used to infect humans and matches changes in that ability over time to a decline in illness and deaths caused by the pathogen.

The study results, published July 24 in Nature, offer important lessons for public health experts looking to prepare for future outbreaks, the researchers said.

The work took many unexpected turns, the researchers said. The findings challenge some of the basic assumptions that scientists have relied on in their attempts to understand how viruses interact with human cells and what causes outbreaks to ebb and flow, such as the notion that any given virus targets one host receptor to gain entry and infect cells.

"This was a real scientific detective story," said study senior author Jonathan Abraham, associate professor of microbiology in the Blavatnik Institute at Harvard Medical School. "The virus kept surprising us and taught us some important lessons about how to study viruses."

The researchers identified the specific proteins expressed on host cells that different strains of the virus have used to infect a variety of animals, including horses, humans, and birds over the last century. Their findings tied differences in the virus's ability to sicken humans and horses to changes in the viral genome that left the virus unable to target proteins found in humans and horses, while leaving intact the virus's ability to infect birds and reptiles that serve as reservoirs for the virus.

The surprising diversity and variability in the virus's ability to infect host cells highlights the importance of studying viruses broadly across time, space, and host species to track potential outbreaks and monitor for emerging and re-emerging viruses.

A virus changes

The protagonist in the story is the western equine encephalitis virus (WEEV), a member of a viral family known as alphaviruses.

One key to understanding how a virus interacts with a host is identifying the precise path it takes to enter cells and cause infection.

WEEV and others in the alphavirus family typically attach a spike protein to a compatible protein -- the receptor -- on the surface of a host cell. Once attached to the host receptor, the virus enters the cell. Once inside the cell, the virus hijacks the cells' armamentarium to enable its own replication, spread, and survival.

The researchers made harmless replicas of various viral strains collected from different times and places and tested their ability to infect host cells in lab dishes. They also tested some of the strains in mice.

Several deadly strains of WEEV are known to cause severe brain inflammation in both horses and humans. Some years, thousands of horses were killed and hundreds of humans were sickened. Case fatality rates for people were as high as 15 percent in North America in the early and middle decades of the 20th century.

Abraham's group found that some of these early strains could stick their spike proteins to several different types of receptors to enter animal cells. That was an unexpected finding because the prevailing dogma in virology thus far has been that viruses typically attack by targeting only one type of host cell receptor.

The team observed that the strains circulating during the years of frequent outbreaks could use multiple receptors that are expressed on brain cells of humans and horses, including proteins known as PCDH10 and VLDLR.

Although the virus still circulates between birds, mosquitoes, and other animals, the most recent outbreak in the United States in humans was in 1987, according to the Centers for Disease Control and Prevention. Since then, there have been only five cases identified in the United States.

By contrast, when the researchers tested more recently isolated strains recovered from mosquitos in California in 2005, they found that the viral spike protein failed to recognize the human receptors, but could still interact with similar proteins found in birds.

Based on these findings, the researchers hypothesize that the virus had evolved, perhaps because horses can be vaccinated and are no longer prevalent enough in the agriculture or transportation industries to serve as effective amplifiers for the virus. Alternatively, the researchers note, the virus may have evolved through simple antigenic drifting, a process by which random mutations cause a series of small changes to a viral genome that, over time, may end up changing the way a virus interacts with its host. Whatever the reason, the researchers said, subtle shifts in the shape of the viral spike proteins changed the cellular receptors with which the virus could connect.

This change in targetable host receptors is likely the central reason why the virus "submerged" as a human pathogen in North America, the research team said. This newly gleaned appreciation of the dynamic complexity of viral receptors is an essential tool for understanding how this virus or others like it might one day re-emerge, the scientists said.

"We need to understand what happens to viruses when they submerge, to better prepare for when they re-emerge," said first author Wanyu Li, a Harvard Kenneth C. Griffin Graduate School of Arts and Sciences doctoral student in the virology program in the Harvard Division of Medical Sciences at HMS.

For example, knowing whether dangerous versions of the pathogen persist in isolated populations of insects, or if the virus has gained the ability to infect other animals, could provide important early warning signs for potential resurgences of illnesses that are thought to have disappeared.

A virus's complex behavior

Through their experiments, the researchers discovered that certain old WEEV strains behaved differently than expected.

The team used eastern equine encephalitis virus -- a deadlier cousin of WEEV -- as a control in some experiments. In one test, the team found that an old strain of WEEV could use the same receptor as the eastern virus, which is something that newer WEEV strains could not do. They also found different strains of WEEV that used different receptors. Some strains could stick to avian versions of the receptor protein but not those expressed in human or equine cells.

The findings serve as an important reminder that viruses are part of a dynamic system and that the viruses themselves are dynamic, with subtle but significant differences across time and geography -- a notion that was powerfully underscored by the rapidly shapeshifting SARS-CoV-2 virus that fueled the COVID-19 pandemic, the researchers said.

"It was a wake-up call," Abraham said. "It's telling us that we can't just study one strain of a virus and assume we know the whole story. Viruses seem simple, but they're quite complex, and they're constantly changing."

Applying lessons to pandemic preparedness

In standard virology, researchers often only check a limited number of viral strains. These new findings show that that's not enough to truly understand the virus.

"There's so much more biology to be learned by exploring the diversity of these complex systems," Abraham said. He also noted that it's necessary to explore as much of that viral diversity as possible in order to prepare for possible outbreaks.

Many viruses circulate in insects and animals that live around us, Abraham said. Some, like the tick-borne infection Powassan, which is endemic in New England, occasionally flare up to cause deadly or debilitating disease.

There could be many reasons for the flare-ups, Abraham said. Are there different strains of Powassan that carry different levels of risk? Is it an environmental change or an evolutionary shift in the pathogen itself that causes new outbreaks? Looking at all these aspects and the breadth of viral diversity will help researchers predict and protect against outbreaks.

In another twist, as Abraham and his team conducted their experiments, a new outbreak of WEEV occurred in South America, which had also seen steep declines in the disease in recent years. The viral populations in South and North America appear to be genetically distinct, and the South American strain of the virus doesn't remain viable long enough for migrating birds to transfer it from one continent to the other regularly. Still, Abraham noted, the new outbreak in South America emphasizes the importance of vigilance and of improving scientific understanding of these volatile, shapeshifting viruses.

"WEEV's return caught everyone by surprise," Li said. "Now with its cellular host receptors known, we have the tools to understand the molecular aspects of WEEV's re-emergence."

Abraham and collaborators are now investigating the strains associated with recent outbreak in South America.

"One small shift in the viral genome, in the intensity of a rainy season that allows mosquitos to proliferate, or in the place humans live or work, could trigger an outbreak," Abraham said. "The more we know, the better we'll be able to protect ourselves."

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Researchers grow embryonic humanized kidneys inside pigs for 28 days

Guangzhou Institutes of Biomedicine and Health researchers have successfully created chimeric embryos containing a combination of human and pig cells. When transferred into surrogate pig mothers, the developing humanized kidneys had normal structure and tubule formation after 28 days. This is the first time that scientists have been able to grow a solid humanized organ inside another species, though previous studies have used similar methods to generate human tissues such as blood or skeletal muscle in pigs. The work appears September 7 in the journal Cell Stem Cell.

The researchers focused on kidneys because they are one of the first organs to develop, and they're also the most commonly transplanted organ in human medicine.

"Rat organs have been produced in mice, and mouse organs have been produced in rats, but previous attempts to grow human organs in pigs have not succeeded," says senior author Liangxue of the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, and Wuyi University. "Our approach improves the integration of human cells into recipient tissues and allows us to grow human organs in pigs."

Integrating human stem cells into pig embryos has been a challenge because pig cells outcompete human cells and pig and human cells have different physiological needs. "We have been working on mechanisms to overcome the extremely low efficiency in interspecies chimera," says senior author Guangjin Pan of the Guangzhou Institutes of Biomedicine and Health. "We identified a couple of critical factors that enhance the formation of interspecies chimera by facilitating cell competition."

The team's technique depends on three key components:
 

• First, they created a niche within the pig embryo so that the human cells would not have to compete with pig cells by using CRISPR to genetically engineer a single-cell pig embryo so that it was missing two genes that are needed for kidney development.
• Second, the researchers engineered human pluripotent stem cells -- cells that have the potential to develop into any cell type -- to make them more amenable to integration and less likely to self-destruct by temporarily shutting down apoptosis. Then, they converted these cells into "naïve" cells resembling early human embryonic cells by culturing them in a special medium.
• Third, before implanting the developing embryos in surrogate sows, the researchers grew the chimeras in conditions that were optimized to provide unique nutrients and signals to both the human and pig cells, since these cells usually have disparate needs.

Altogether, the researchers transferred 1,820 embryos to 13 surrogate mothers. After either 25 or 28 days, they terminated gestation and extracted the embryos to assess whether the chimeras had successfully produced humanized kidneys.

The researchers collected five chimeric embryos for analysis (two at 25 days and three at 28 days post-implantation) and found that they had structurally normal kidneys for their stage of development and were composed of 50-60% human cells. At 25-28 days, the kidneys were in the mesonephros stage (the second stage of kidney development); they had formed tubules and buds of cells that would eventually become ureters connecting the kidney to the bladder.

The team also investigated whether human cells were contributing to other tissues throughout the embryos, which could have ethical implications, especially if abundant human cells were found in neural or germline tissues and the pigs were brought to term. They showed that human cells were mostly localized to the kidneys, whereas the remainder of the embryo was composed of pig cells.

"We found that if you create a niche in the pig embryo, then the human cells naturally go into these spaces," says senior author Zhen Dai of Guangzhou Institutes of Biomedicine and Health. "We saw only very few human neural cells in the brain and spinal cord and no human cells in the genital ridge, indicating that the human pluripotent stem cells did not differentiate into germ cells." This may be further prevented by knocking out further genes in the human pluripotent stem cells, which can be tested in future studies, the researchers say.

Now that they have optimized conditions for growing humanized kidneys in human-pig chimeras, the team wants to allow the kidneys to develop for a longer duration. They're also working to generate other human organs in pigs, including the heart and pancreas.

The long-term goal is to optimize this technology for human organ transplantation, but the researchers acknowledge the work will be complex and could take many years. Growing a fully functional humanized organ in a pig would require some additional steps because organs are composed of multiple types of cells and tissues. In this study, the researchers created a niche for only one subset of cells, which meant that the kidneys had pig-derived vascular cells, and this could cause organ rejection if they were used in a transplant scenario.

"Because organs are not composed of just one cell lineage, in order to have an organ where everything comes from the human, we would probably need to engineer the pigs in a much more complex way and that also brings some additional challenges," says senior author Miguel A. Esteban of Guangzhou Institutes of Biomedicine and Health.

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Human cells help researchers understand squid camouflage

Squids and octopuses are masters of camouflage, blending into their environment to evade predators or surprise prey. Some aspects of how these cephalopods become reversibly transparent are still "unclear," largely because researchers can't culture cephalopod skin cells in the lab. Today, however, researchers report that they have replicated the tunable transparency of some squid skin cells in mammalian cells, which can be cultured. The work could not only shed light on basic squid biology, but also lead to better ways to image many cell types.

The researchers will present their results at the spring meeting of the American Chemical Society (ACS).

For many years, Alon Gorodetsky, Ph.D., and his research group have been working on materials inspired by squid. In past work, they developed "invisibility stickers," which consisted of bacterially produced squid reflectin proteins that were adhered onto sticky tape. "So then, we had this crazy idea to see whether we could capture some aspect of the ability of squid skin tissues to change transparency within human cell cultures," says Gorodetsky, who is the principal investigator on the project.

The team at the University of California, Irvine focused their efforts on cephalopod cells called leucophores, which have particulate-like nanostructures composed of reflectin proteins that scatter light. Typically, reflectins clump together and form the nanoparticles, so light isn't absorbed or directly transmitted; instead, the light scatters or bounces off of them, making the leucophores appear bright white.

"We wanted to engineer mammalian cells to stably, instead of temporarily, form reflectin nanostructures for which we could better control the scattering of light," says Gorodetsky. That's because if cells allow light through with little scattering, they'll seem more transparent. Alternatively, by scattering a lot more light, cells will become opaque and more apparent. "Then, at a cellular level, or even the culture level, we thought that we could predictably alter the cells' transparency relative to the surroundings or background," he says.

To change how light interacts with cultured cells, Georgii Bogdanov, a graduate student in Gorodetsky's lab who is presenting the results, introduced squid-derived genes that encoded for reflectin into human cells, which then used the DNA to produce the protein. "A key advance in our experiments was getting the cells to stably produce reflectin and form light-scattering nanostructures with relatively high refractive indices, which also allowed us to better image the cells in three dimensions," says Bogdanov.

In experiments, the team added salt to the cells' culture media and observed the reflectin proteins clumping together into nanostructures. By systematically increasing the salt concentration, Bogdanov got detailed, time-lapse 3D images of the nanostructures' properties. As the nanoparticles became larger, the amount of light that bounced off the cells increased, consequently tuning their opacity.

Then, the COVID-19 pandemic hit, leaving the researchers to wonder what they could do to advance their investigation without being physically in the lab. So, Bogdanov spent his time at home developing computational models that could predict a cell's expected light scattering and transparency before an experiment was even run. "It's a beautiful loop between theory and experiments, where you feed in design parameters for the reflectin nanostructures, get out specific predicted optical properties and then engineer the cells more efficiently -- for whatever light-scattering properties you might be interested in," explains Gorodetsky.

On a basic level, Gorodetsky suggests that these results will help scientists better understand squid skin cells, which haven't been successfully cultured in a laboratory setting. For example, previous researchers postulated that reflectin nanoparticles disassemble and reassemble to change the transparency of tunable squid leucophores. And now Gorodetsky's team has shown that similar rearrangements occurred in their stable engineered mammalian cells with simple changes in salt concentration, a mechanism that appears analogous to what has been observed in the tunable squid cells.

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'We're not all that different': Study IDs bacterial weapons that could be harnessed to treat human disease

When it comes to fighting off invaders, bacteria operate in a remarkably similar way to human cells, possessing the same core machinery required to switch immune pathways on and off, according to new University of Colorado Boulder research.

The study, published Feb. 8 in the journal Nature, also sheds light on how that shared, ancient machinery -- a cluster of enzymes known as ubiquitin transferases -- works.

Better understanding, and potentially reprogramming this machine, could ultimately pave the way to novel approaches for treating a host of human diseases, from autoimmune disorders like Rheumatoid arthritis and Crohn's disease to neurodegenerative diseases like Parkinson's disease, the authors said.

"This study demonstrates that we're not all that different from bacteria," said senior author Aaron Whiteley, an assistant professor in the Department of Biochemistry. "We can learn a lot about how the human body works by studying these bacterial processes."

The next CRISPR?

The study is not the first to showcase the lessons bacteria can teach humans.

Mounting evidence suggests that portions of the human immune system may have originated in bacteria, with evolution yielding more complex iterations of bacterial virus-fighting tools across plant and animal kingdoms.

In 2020, University of California Berkeley biochemist Jennifer Doudna won the Nobel Prize for CRISPR, a gene-editing tool that repurposes another obscure system bacteria use to fight off their own viruses, known as phages.

The buzz around CRISPR ignited renewed scientific interest in the role proteins and enzymes play in anti-phage immune response.

"Over the past three to five years people have realized it doesn't end with CRISPR. The potential is so much bigger," said Whiteley.

Missing link in evolutionary history

For the study, Whiteley and co-first author Hannah Ledvina, a Jane Coffin Childs Postdoctoral Fellow in the department, collaborated with University of California San Diego biochemists to learn more about a protein called cGAS (cyclic GMP-AMP synthase), previously shown to be present in both humans and, in a simpler form, bacteria.

In bacteria and in humans, cGAS is critical for mounting a downstream defense when the cell senses a viral invader. But what regulates this process in bacteria was previously unknown.

Using an ultra-high-resolution technique called cryo-electron microscopy alongside other genetic and biochemical experiments, Whiteley's team took an up-close look at the structure of cGAS's evolutionary predecessor in bacteria and discovered additional proteins that bacteria use to help cGAS defend the cell from viral attack.

Specifically, they discovered that bacteria modify their cGAS using a streamlined "all-in-one version" of ubiquitin transferase, a complex collection of enzymes that in humans control immune signaling and other critical cellular processes.

Because bacteria are easier to genetically manipulate and study than human cells, this discovery opens a new world of opportunity for research, said Ledvina.

"The ubiquitin transferases in bacteria are a missing link in our understanding of the evolutionary history of these proteins."

Editing proteins

The study also revealed just how this machine works, identifying two key components -- proteins called Cap2 and Cap3 (CD-NTase-associated protein 2 and 3) -- which serve, respectively, as on and off switches for the cGAS response.

Whiteley explained that in addition to playing a key role in immune response, ubiquitin in humans can serve as a sort of marker for cellular garbage, directing excess or old proteins to be broken down and destroyed. When that system misfires due to mutations in the machine, proteins can build up and diseases, such as Parkinson's, can occur.

The authors stress that far more research is needed but the discovery opens exciting scientific doors. Just as scientists adapted the ancient bacterial defense system CRISPR into scissor-like biotechnology that can snip mutations out of DNA, Whiteley believes pieces of the bacterial ubiquitin transferase machine -- namely Cap3, the "off switch" -- could ultimately be programmed to edit out problem proteins and treat disease in humans.

He and his team, with the help of Venture Partners at CU Boulder, have already filed for intellectual property protection, and they're moving forward with more research.

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How some gut microbes awaken 'zombie' viruses in their neighbors

Some gut bacteria have a spooky superpower: they can reanimate dormant viruses lurking within other microbes.

This viral awakening unleashes full-blown infections that destroy the virus-carrying cells, Howard Hughes Medical Institute Investigator Emily Balskus's lab first published as a preprint on bioRxiv and later in the journal Nature on February 23, 2022. A cryptic molecule called colibactin can summon the killer viruses from their slumber, they found.

Microbes often generate noxious compounds to attack one another within the cramped quarters of the gut. But among these chemical weapons, colibactin appears unusual, says Balskus, a chemical biologist at Harvard University. "It doesn't directly kill the target organisms, which is what we normally think of bacterial toxins doing within microbial communities." Instead, colibactin tweaks microbial cells just so, activating latent -- and lethal -- viruses tucked away in some bacteria's genomes.

Humans have long sought out the potent compounds that microbes produce. "We know a lot about their chemical properties, we purify them in the lab, and we use them as medicine, including antibiotics," says Breck Duerkop, who studies bacterial viruses at the University of Colorado School of Medicine.

But why bacteria make these compounds and what effects they have on neighboring organisms are open-ended questions, says Duerkop, who was not involved in this research. He calls Balskus's teams new work "one step in the right direction."

Chemical dark matter

Scientists have known for years that colibactin can wreak havoc on human cells. Research by Balskus and many others has shown that the compound damages DNA, which can lead to colorectal cancer. But establishing a connection between this compound and disease proved particularly formidable.

In 2006, a French team reported that mammalian cells that encountered the gut bacteria E. coli suffered fatal damage to their DNA. The researchers linked this damage to a cluster of E. coli genes encoding machinery for building a complex molecule. Dubbed colibactin, the molecule was extraordinarily difficult to study. After many tries, researchers simply couldn't isolate it from the E. coli making it.

Colibactin is one of many ephemeral compounds that scientists suspect microbes make. Like invisible particles of dark matter in space, this "chemical dark matter" requires creative means to study. As part of her exploration of the gut's microbial chemistry, Balskus uses indirect approaches to examine these elusive molecules.

Over the past 10 years, her team has probed colibactin by studying the microbial machinery that manufactures it. She and her colleagues have pieced together colibactin's structure and determined that it damages DNA by forming errant connections within the double helix.

Building off this work, scientists elsewhere uncovered a definitive link to cancer: the molecule's distinctive fingerprints appear in genes known to drive colorectal tumor growth.

A role for viruses

Balskus's most recent colibactin study got its start with another disease: COVID-19. Like many other labs, hers had to rearrange things to reduce physical contact among researchers. As part of the reshuffling, postdoc Justin Silpe and graduate student Joel Wong ended up working near one another for the first time. Their conversations led them and Balskus to wonder how colibactin affected other microbes in a crowded gut.

Early on, they found that exposing colibactin-producing bacteria to non-producers had little effect, suggesting that, on its own, the molecule isn't particularly deadly. Silpe and Wong weren't sure if colibactin, a large, unstable molecule, could even enter bacterial cells to damage their DNA. They then wondered if a third party -- bacteria-infecting viruses -- might be involved. Hardly more than bits of genetic information, these viruses can slip into bacteria's DNA and lie quietly in wait. Then, once triggered, they cause an infection that blows up the cell like a landmine.

When the researchers grew colibactin producers alongside bacteria carrying such latent viruses, they saw the number of viral particles spike, and the growth of many virus-containing bacteria drop. That suggested the molecule sparked a surge in active, cell-killing infections. Colibactin does indeed enter bacteria and damage DNA, the team showed. That damage sounds a cellular wake-up bell that rouses the viruses.

Many microbes appeared equipped to protect themselves against colibactin. Balskus's lab identified a resistance gene encoding a protein that neutralizes the compound in a wide variety of bacteria.

Though colibactin clearly has a dangerous side, it may serve as more than just a lethal weapon, Balskus says. For example, both DNA damage and awakened viruses can also induce genetic changes, rather than death, in neighboring bacteria, potentially benefiting colibactin producers.

Balskus's team's discoveries suggest that cancer may be collateral damage caused by whatever else colibactin-producing bacteria are doing. "We always suspected that bacteria made this toxin to target other bacteria in some way," she says. "It didn't make sense from an evolutionary perspective that they acquired it to target human cells."

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Research advances knowledge of the battle between viruses and human cells

In the long-term battle between a herpesvirus and its human host, a University of Massachusetts virologist and her team of students have identified some human RNA able to resist the viral takeover -- and the mechanism by which that occurs.

This discovery, described in a paper published Feb. 15 in Proceedings of the National Academy of Sciences, represents an important step in the effort to develop anti-viral drugs to fight off infections.

"This paper is about trying to understand the mechanism that makes these RNA escape degradation," says senior author Mandy Muller, assistant professor of microbiology. "The next step is to figure out if we can manipulate this to our advantage."

In the Muller Lab, student researchers work with Muller studying how Kaposi sarcoma-associated herpesvirus (KSHV) hides for years inside the human body before seeking to gain control over human gene expression to complete the viral infection. At that point, people with a weakened immune system may develop Kaposi sarcoma cancer lesions in the mouth, skin or other organs.

The researchers use genome-wide sequencing, post-transcriptional sequencing and molecular biology to examine how the human cell or the virus knows how to prevent degradation.

"Viruses are very smart, that's what I love to say," Muller says. "They have lots of strategies to stick around, and they don't do a lot of damage for a very long time, because that's one way to hide from the immune system.

"But then, at some point -- many, many years later -- they reactivate. The way they do this is by triggering a massive RNA degradation event where the virus will wipe out the mRNA from the cell. That means the human system can no longer express the proteins that it needs to express, and that means also that a lot of resources are suddenly available for the virus."

How and why some RNA are able to escape the viral degradation are questions Muller's team -- including lead author and graduate student Daniel Macveigh-Fierro and co-authors and undergraduates Angelina Cicerchia, Ashley Cadorette and Vasudha Sharma -- has been investigating.

"We show that RNA that escape have a chemical tag on them -- a post-transcriptional modification -- that makes them different from the others," Muller explains. "By having this tag, M6A, they can recruit proteins that protect them from degradation."

Muller has been studying KSHV since she was an undergraduate in her native France, and her mission continues.

"We know you need this protein to protect the RNA from degradation, but we still don't know how that physically stops the degradation, so that's what we're going to look at now," she says.

Ultimately, understanding the mechanisms and pathways involved in KSHV infection may lead to the development of RNA therapeutics to treat viral diseases.

"By identifying the determinants of what makes an mRNA either resistant or susceptible to viral-induced decay, we could use those findings to our advantage to better design anti-viral drugs and reshape the outcome of infection," Muller says.

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Long-range four-stranded DNA structures found to play a role in rare aging disease

A special form of four-stranded DNA, recently seen in human cells, has been found to interact with a gene that causes Cockayne Syndrome when faulty.

As well as the classic double-helix, researchers have recently discovered a whole host of other DNA strand configurations, including quadruple-helix DNA, which forms knot-like structures called G-quadruplexes.

While many of these new DNA configurations have only been observed in cells in dishes, G-quadruplexes have recently been observed in living human cells. However, their possible functions in cells have not been discovered.

Now, researchers from the Molecular Science Research Hub at Imperial College London have observed a protein called Cockayne Syndrome B (CSB) preferentially interacting with one specific type of G-quadruplex. These special G-quadruplexes arise when distant parts of DNA interact, something that researchers thought was impossible to form within cells.

Normally functioning CSB proteins do not cause any ill effects, but mutations of the gene that produce CSB protein can cause the fatal premature ageing disorder Cockayne Syndrome, which kills many sufferers before adulthood.

The team found that CSB proteins with mutations that cause Cockayne Syndrome are no longer able to interact with the long-range G-quadruplexes. While we don't yet know why this might be, the team's results, published today in theJournal of the American Chemical Society, suggest that these long-range DNA G-quadruplexes are specifically linked with the functional role of CSB.

Lead researcher Dr Marco Di Antonio, from the Department of Chemistry at Imperial, said: "Our genomic DNA is more than two metres long, but is compressed into a space only a few microns in diameter. It shouldn't therefore be a surprise that there are ways the long-range looped structures are leveraged to compress DNA in more complex interactions than we imagined.

"There is still so much we don't know about DNA, but our results show that how and where G-quadruplex structures form affects their function, making them more important biologically than previously thought."

DNA strands are incredibly long and are wound in tight structures to fit inside our cells. Previously, researchers had assumed that G-quadruplexes form only from regions of DNA that sit next to each other. However, the team discovered G-quadruplexes that are formed from parts of the DNA strand that are spatially distant one from the other.

It's these G-quadruplexes that specifically interact with the CSB protein. The team shows that CSB could potentially use the G-quadruplexes to link together distant portions of the DNA.

Exactly what the interaction results in is yet to be determined, but previous independent research found that cells without CSB have difficulty processing the DNA around sequences with the potential to form G-quadruplexes.

The Imperial team have now found that the mutated form of CSB that causes Cockayne Syndrome is specifically attracted to G-quadruplexes that link distant DNA portions. This could mean further study of the mutated CSB gene might reveal the specific biological function of these long-range DNA structures.

Next, the researchers want to image the G-quadruplexes and the functional CSB gene bound together to determine exactly what the relationship does: whether the CSB helps the G-quadruplex hold the two distant regions of the DNA together, or whether CSB actually initiates the break-up of G-quadruplexes once they have completed their function, or a combination of both.

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Ultrashort-pulse lasers kill bacterial superbugs, spores

Life-threatening bacteria are becoming ever more resistant to antibiotics, making the search for alternatives to antibiotics an increasingly urgent challenge. For certain applications, one alternative may be a special type of laser.

Researchers at Washington University School of Medicine in St. Louis have found that lasers that emit ultrashort pulses of light can kill multidrug-resistant bacteria and hardy bacterial spores. The findings, available online in the Journal of Biophotonics, open up the possibility of using such lasers to destroy bacteria that are hard to kill by other means. The researchers previously have shown that such lasers don't damage human cells, making it possible to envision using the lasers to sterilize wounds or disinfect blood products.

"The ultrashort-pulse laser technology uniquely inactivates pathogens while preserving human proteins and cells," said first author Shaw-Wei (David) Tsen, MD, PhD, an instructor of radiology at Washington University's Mallinckrodt Institute of Radiology (MIR). "Imagine if, prior to closing a surgical wound, we could scan a laser beam across the site and further reduce the chances of infection. I can see this technology being used soon to disinfect biological products in vitro, and even to treat bloodstream infections in the future by putting patients on dialysis and passing the blood through a laser treatment device."

Tsen and senior author Samuel Achilefu, PhD, the Michel M. Ter-Pogossian Professor of Radiology and director of MIR's Biophotonics Research Center, have been exploring the germicidal properties of ultrashort-pulse lasers for years. They have shown that such lasers can inactivate viruses and ordinary bacteria without harming human cells. In the new study, conducted in collaboration with Shelley Haydel, PhD, a professor of microbiology at Arizona State University, they extended their exploration to antibiotic-resistant bacteria and bacterial spores.

The researchers trained their lasers on multidrug-resistant Staphylococcus aureus (MRSA), which causes infections of the skin, lungs and other organs, and extended spectrum beta-lactamase-producing Escherichia coli (E. coli), which cause urinary tract infections, diarrhea and wound infections. Apart from their shared ability to make people miserable, MRSA and E. coli are very different types of bacteria, representing two distant branches of the bacterial kingdom. The researchers also looked at spores of the bacterium Bacillus cereus, which causes food poisoning and food spoilage. Bacillus spores can withstand boiling and cooking.

In all cases, the lasers killed more than 99.9% of the target organisms, reducing their numbers by more than 1,000 times.

Viruses and bacteria contain densely packed protein structures that can be excited by an ultrashort-pulse laser. The laser kills by causing these protein structures to vibrate until some of their molecular bonds break. The broken ends quickly reattach to whatever they can find, which in many cases is not what they had been attached to before. The result is a mess of incorrect linkages inside and between proteins, and that mess causes normal protein function in microorganisms to grind to a halt.

"We previously published a paper in which we showed that the laser power matters," Tsen said. "At a certain laser power, we're inactivating viruses. As you increase the power, you start inactivating bacteria. But it takes even higher power than that, and we're talking orders of magnitude, to start killing human cells. So there is a therapeutic window where we can tune the laser parameters such that we can kill pathogens without affecting the human cells."

Heat, radiation and chemicals such as bleach are effective at sterilizing objects, but most are too damaging to be used on people or biological products. By inactivating all kinds of bacteria and viruses without damaging cells, ultrashort-pulse lasers could provide a new approach to making blood products and other biological products safer.

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Study reveals existing drugs that kill SARS-CoV2 in cells

Since the beginning of the pandemic, researchers worldwide have been looking for ways to treat COVID-19. And while the COVID-19 vaccines represent the best measure to prevent the disease, therapies for those who do get infected remain in short supply. A new groundbreaking study from U-M reveals several drug contenders already in use for other purposes -- including one dietary supplement -- that have been shown to block or reduce SARS-CoV2 infection in cells.

The study, published recently in the Proceedings of the National Academy of Science, uses artificial intelligence-powered image analysis of human cell lines during infection with the novel coronavirus. The cells were treated with more than 1,400 individual FDA-approved drugs and compounds, either before or after viral infection, and screened, resulting in 17 potential hits. Ten of those hits were newly recognized, with seven identified in previous drug repurposing studies, including remdesivir, which is one of the few FDA-approved therapies for COVID-17 in hospitalized patients.

"Traditionally, the drug development process takes a decade -- and we just don't have a decade," said Jonathan Sexton, Ph.D., Assistant Professor of Internal Medicine at the U-M Medical School and one of the senior authors on the paper. "The therapies we discovered are well positioned for phase 2 clinical trials because their safety has already been established."

The team validated the 17 candidate compounds in several types of cells, including stem-cell derived human lung cells in an effort to mimic SARS-CoV2 infection of the respiratory tract. Nine showed anti-viral activity at reasonable doses, including lactoferrin, a protein found in human breastmilk that is also available over the counter as a dietary supplement derived from cow's milk.

"We found lactoferrin had remarkable efficacy for preventing infection, working better than anything else we observed," Sexton said. He adds that early data suggest this efficacy extends even to newer variants of SARS-CoV2, including the highly transmissible Delta variant.

The team is soon launching clinical trials of the compound to examine its ability to reduce viral loads and inflammation in patients with SARS-CoV2 infection.

The trials are adding to the list of ongoing studies of promising repurposed drugs. Sexton noted that over the course of the pandemic, other drug repurposing studies have identified different compounds with potential efficacy against SARS-CoV2. "The results seem to be dependent on what cell system is used," he said.

"But there is an emerging consensus around a subset of drugs and those are the ones that have the highest priority for clinical translation. We fully expect that the majority of these won't work in human beings, but we anticipate there are some that will."

A surprising finding about certain drugs and COVID

Remarkably, the U-M study also identified a class of compounds called MEK-inhibitors, typically prescribed to treat cancer, that appear to worsen SARS-CoV2 infection. The finding sheds light on how the virus spreads among cells.

"People going in for chemotherapy are at risk already due to a lowered immune response. We need to investigate whether some of these drugs worsen disease progression," said Sexton.

The next step, he noted, is to use electronic health records to see whether patients on these drugs have worse COVID-19 outcomes.

The work is one of the first major discoveries to come out of the new U-M Center for Drug Repurposing (CDR), which was established in November 2019, just as the pandemic began. The Michigan Institute for Clinical & Health Research (MICHR), with partners across campus, launched the Center with the goal of finding potential therapeutics for the thousands of human diseases for which there is no treatment.

"Repurposing existing therapeutic interventions in the clinical setting has many advantages that result in significantly less time from discovery to clinical use, including documented safety profiles, reduced regulatory burden, and substantial cost savings," said George A. Mashour, MD, PhD, co-director of MICHR and founder/executive sponsor of the CDR.

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New discovery shows human cells can write RNA sequences into DNA

Cells contain machinery that duplicates DNA into a new set that goes into a newly formed cell. That same class of machines, called polymerases, also build RNA messages, which are like notes copied from the central DNA repository of recipes, so they can be read more efficiently into proteins. But polymerases were thought to only work in one direction DNA into DNA or RNA. This prevents RNA messages from being rewritten back into the master recipe book of genomic DNA. Now, Thomas Jefferson University researchers provide the first evidence that RNA segments can be written back into DNA, which potentially challenges the central dogma in biology and could have wide implications affecting many fields of biology.

"This work opens the door to many other studies that will help us understand the significance of having a mechanism for converting RNA messages into DNA in our own cells," says Richard Pomerantz, PhD, associate professor of biochemistry and molecular biology at Thomas Jefferson University. "The reality that a human polymerase can do this with high efficiency, raises many questions." For example, this finding suggests that RNA messages can be used as templates for repairing or re-writing genomic DNA.

The work was published June 11th in the journal Science Advances.

Together with first author Gurushankar Chandramouly and other collaborators, Dr. Pomerantz's team started by investigating one very unusual polymerase, called polymerase theta. Of the 14 DNA polymerases in mammalian cells, only three do the bulk of the work of duplicating the entire genome to prepare for cell division. The remaining 11 are mostly involved in detecting and making repairs when there's a break or error in the DNA strands. Polymerase theta repairs DNA, but is very error-prone and makes many errors or mutations. The researchers therefore noticed that some of polymerase theta's "bad" qualities were ones it shared with another cellular machine, albeit one more common in viruses -- the reverse transcriptase. Like Pol theta, HIV reverse transcriptase acts as a DNA polymerase, but can also bind RNA and read RNA back into a DNA strand.

In a series of elegant experiments, the researchers tested polymerase theta against the reverse transcriptase from HIV, which is one of the best studied of its kind. They showed that polymerase theta was capable of converting RNA messages into DNA, which it did as well as HIV reverse transcriptase, and that it actually did a better job than when duplicating DNA to DNA. Polymerase theta was more efficient and introduced fewer errors when using an RNA template to write new DNA messages, than when duplicating DNA into DNA, suggesting that this function could be its primary purpose in the cell.

The group collaborated with Dr. Xiaojiang S. Chen's lab at USC and used x-ray crystallography to define the structure and found that this molecule was able to change shape in order to accommodate the more bulky RNA molecule -- a feat unique among polymerases.

"Our research suggests that polymerase theta's main function is to act as a reverse transcriptase," says Dr. Pomerantz. "In healthy cells, the purpose of this molecule may be toward RNA-mediated DNA repair. In unhealthy cells, such as cancer cells, polymerase theta is highly expressed and promotes cancer cell growth and drug resistance. It will be exciting to further understand how polymerase theta's activity on RNA contributes to DNA repair and cancer-cell proliferation."

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A rift in the retina may help repair the optic nerve

 In experiments in mouse tissues and human cells, Johns Hopkins Medicine researchers say they have found that removing a membrane that lines the back of the eye may improve the success rate for regrowing nerve cells damaged by blinding diseases. The findings are specifically aimed at discovering new ways to reverse vision loss caused by glaucoma and other diseases that affect the optic nerve, the information highway from the eye to the brain.

"The idea of restoring vision to someone who has lost it from optic nerve disease has been considered science fiction for decades. But in the last five years, stem cell biology has reached a point where it's feasible," says Thomas Johnson, M.D., Ph.D., assistant professor of ophthalmology at the Wilmer Eye Institute at the Johns Hopkins University School of Medicine.

The research was published Jan. 12 in the journal Stem Cell Reports.

A human eye has more than 1 million small nerve cells, called retinal ganglion cells, that transmit signals from light-collecting cells called photoreceptors in the back of the eye to the brain. Retinal ganglion cells send out long arms, or axons, that bundle together with other retinal ganglion cell projections, forming the optic nerve that leads to the brain.

When the eye is subjected to high pressure, as occurs in glaucoma, it damages and eventually kills retinal ganglion cells. In other conditions, inflammation, blocked blood vessels, or tumors can kill retinal ganglion cells. Once they die, retinal ganglion cells don't regenerate.

"That's why it is so important to detect glaucoma early," says Johnson. "We know a lot about how to treat glaucoma and help nerve cells survive an injury, but once the cells die off, the damage to someone's vision becomes permanent."

Johnson is a member of a team of researchers at the Johns Hopkins Wilmer Eye Institute looking for ways scientists can repair or replace lost optic neurons by growing new cells.

In the current study, Johnson and his team grew mouse retinas in a laboratory dish and tracked what happens when they added human retinal ganglion cells, derived from human embryonic stem cells, to the surface of the mouse retinas. They found that most of the transplanted human cells were unable to integrate into the retinal tissue, which contains several layers of cells.

"The transplanted cells clumped together rather than dispersing from one another like on a living retina," says Johnson.

However, the researchers found that a small number of transplanted retinal cells were able to settle uniformly into certain areas of the mouse retina. Looking more closely, the areas where the transplanted cells integrated well aligned with locations where the researchers had to make incisions into the mouse retinas to get them to lie flat in the culture dish. At these incision points, some of the transplanted cells were able to crawl into the retina and integrate themselves in the proper place within the tissue.

"This suggested that there was some type of barrier that had been broken by these incisions," Johnson says. "If we could find a way to remove it, we may have more success with transplantation."

It turns out that the barrier is a well-known anatomical structure of the retina, called the internal limiting membrane. It's a translucent connective tissue created by the retina's cells to separate the fluid of the eye from the retina.

After using an enzyme to loosen the connective fibers of the internal limiting membrane, the researchers removed the membrane and applied the transplanted human cells to the retinas. They found that most of the transplanted retinal ganglion cells grew in a more normal pattern, integrating themselves more fully. The transplanted cells also showed signs of establishing new nerve connections to the rest of the retinal structure when compared with retinas that had intact membranes.

"These findings suggest that altering the internal limiting membrane may be a necessary step in our aim to regrow new cells in damaged retinas," says Johnson.

The researchers plan to continue investigating the development of transplanted retinal ganglion cells to determine the factors they need to function once integrated into the retina.

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