Where No Botulinum Toxin Has Gone Before


Toxin found for first time in Enterococci bacteria

Enterococci bacteria.

Enterococci are hardy microbes that thrive in the gastrointestinal tracts of nearly all land animals, including our own, and generally cause no harm. But their ruggedness has lately made them leading causes of multi-drug-resistant infections, especially in settings like hospitals where antibiotic use disrupts the natural balance of intestinal microbes.

So the discovery of a new toxin in a strain of Enterococcus is raising scientific eyebrows. Isolated from cow feces sampled at a South Carolina farm, the bug was unexpectedly found to carry a toxin resembling the toxin that causes botulism. The finding was reported in Cell Host and Microbe.

“This is the first time a botulinum neurotoxin has been found outside of Clostridium botulinum—and not just the toxin, but an entire unit containing the toxin and associated proteins that prevent the toxin from being degraded in the GI tract,” said Min Dong, Harvard Medical School assistant professor of surgery at Boston Children’s Hospital and one of the world’s experts on botulinum toxins.

The toxin, dubbed BoNT/En, is the ninth botulinum toxin to be described. Last August, Dong and colleagues reported the eighth, BoNT/X, made by C. botulinum—the first new botulinum toxin to be found in close to 50 years.

Should we be scared?

No. At least, not yet, said Sicai Zhang, a postdoctoral fellow in Dong’s lab and one of three co-first authors on the new report.

“The enterococcal isolate carrying the toxin luckily remains susceptible to key antibiotics,” said Zhang. “It was found only once from a single animal, and no signs of botulism disease were observed.”

When Sicai and his colleague Jie Zhang tested the toxin in rodents in the lab, it had little or no effect. Only when they manipulated the toxin to better target mouse and rat neurons did it become potent, shutting down nerve function and causing paralysis.

Dong’s lab is now testing BoNT/En in cultured human neurons to find out whether it’s toxic to humans.

Making the leap

How could this botulinum toxin jump from one bacterial species to another? Teams led by Dong’s collaborators, Michael Gilmore, the HMS Sir William Osler Professor of Ophthalmology at Massachusetts Eye and Ear, and Andrew Doxey, a bioinformatician at the University of Waterloo, found that the BoNT/En botulinum toxin genes were carried by a plasmid.

Plasmids are mobile structures that contain DNA independently of chromosomes and can be swapped from one bacterium to another. Plasmids are quite common in enterococci. In fact, they have been associated with the acquisition of resistance to vancomycin, a last-resort antibiotic, and transfer of resistance to the fearsome Staphylococcus aureus.

Down on the farm

Gilmore’s lab sequenced the toxin producing E. faecium strain as part of a much wider search for the origins of enterococcal antibiotic resistance and disease-causing ability.

“We were not looking for a neurotoxin in E. faecium,” said Francois Lebreton, HMS instructor of ophthalmology at Mass. Eye and Ear and another co-first author on the paper, who specializes in examining the genome sequences of these microbes. “There was no reason to suspect its existence.”

“This is a unique discovery of a botulinum neurotoxin in a bacterium that is both ubiquitous in animals and a serious problem in human health.” —Francois Lebreton

Lebreton has been investigating the evolution of enterococci from their commensal Paleozoic origins to their rise as a hospital threat.

“In intensive agriculture, antibiotics are administered to farm animals to promote weight gain in often crowded facilities. We believe that this creates an environment in the animal gut that allows antibiotic-resistant enterococci to thrive and come into contact with humans,” he explained. “We know that the highly antibiotic-resistant E. faecium strain we fight in the hospital is very closely related to strains found in the GI tracts of farm-raised animals.”

From farm to computer

Doxey’s lab specializes in mining genome data to discover new toxins and virulence genes. Analyzing the newly sequenced E. faecium genome, the lab’s computer programs quickly spotted the genetic sequence for the novel botulinum toxin.

“The way that we discovered this toxin using computational methods is different from how toxins used to be identified in the past, and may become a standard approach in biomonitoring,” Doxey said. “It represents scientific collaboration and data sharing at its best.”

A perfect storm?

The newly discovered toxin does raise some concern that botulinum toxin could turn up in antibiotic-resistant enterococci, perhaps stemming from gene transfer in the gut of an animal harboring both C. botulinum and Enterococcus.

“This is a unique discovery of a botulinum neurotoxin in a bacterium that is both ubiquitous in animals and a serious problem in human health,” said Lebreton. “E. faecium is in the gut of nearly every human; it is extremely tough and survives a lot of stresses, often including efforts to disinfect hospital surfaces. A hospital-adapted, antibiotic-resistant, hard-to-kill bug carrying a neurotoxin would be a worst-case scenario.”

Exactly what animal this ninth botulinum toxin is meant to target remains unknown. Gilmore’s lab continues to expand and study its collection of enterococcal isolates.

“Most of what we know about Enterococcus comes from the few strains circulating in the hospital,” said Lebreton. “It’s possible that BoNT/En, or even other novel toxins, will turn up in other enterococci isolated from the wild. We just never looked for those before.”

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Nature, Meet Nurture


Single-cell analysis reveals dramatic landscape of genetic changes in the brain after visual stimulation

A “brainbow” of cerebral cortex neurons labeled with different colors.

“Nature and nurture is a convenient jingle of words, for it separates under two distinct heads the innumerable elements of which personality is composed. Nature is all that a man brings with himself into the world; nurture is every influence from without that affects him after his birth.” – Francis Galton, cousin of Charles Darwin, 1874.

Is it nature or nurture that ultimately shapes a human? Are actions and behaviors a result of genes or environment?

Variations of these questions have been explored by countless philosophers and scientists across millennia.

Yet, as biologists continue to better understand the mechanisms that underlie brain function, it is increasingly apparent that this long-debated dichotomy may be no dichotomy at all.

In a study published in Nature Neuroscience on Jan. 21, neuroscientists and systems biologists from Harvard Medical School reveal just how inexorably interwoven nature and nurture are.

Using novel technologies developed at HMS, the team looked at how a single sensory experience affects gene expression in the brain by analyzing more than 114,000 individual cells in the mouse visual cortex before and after exposure to light.

Their findings revealed a dramatic and diverse landscape of gene expression changes across all cell types, involving 611 different genes, many linked to neural connectivity and the brain’s ability to rewire itself to learn and adapt.

The results offer insights into how bursts of neuronal activity that last only milliseconds trigger lasting changes in the brain, and open new fields of exploration for efforts to understand how the brain works.

“What we found is, in a sense, amazing. In response to visual stimulation, virtually every cell in the visual cortex is responding in a different way,” said co-senior author Michael Greenberg, the Nathan Marsh Pusey Professor of Neurobiology and chair of the Department of Neurobiology at HMS.

“This in essence addresses the long-asked question about nature and nurture: Is it genes or environment? It’s both, and this is how they come together,” he said.

One out of many

Neuroscientists have known that stimuli—sensory experiences such as touch or sound, metabolic changes, injury and other environmental experiences—can trigger the activation of genetic programs within the brain.

Composed of a vast array of different cells, the brain depends on a complex orchestra of cellular functions to carry out its tasks. Scientists have long sought to understand how individual cells respond to various stimuli. However, due to technological limitations, previous genetic studies largely focused on mixed populations of cells, obscuring critical nuances in cellular behavior.

To build a more comprehensive picture, Greenberg teamed with co-corresponding author Bernardo Sabatini, the Alice and Rodman W. Moorhead III Professor of Neurobiology at HMS, and Allon Klein, assistant professor of systems biology at HMS.

Spearheaded by co-lead authors Sinisa Hrvatin, a postdoctoral fellow in the Greenberg lab, Daniel Hochbaum, a postdoctoral fellow in the Sabatini lab and M. Aurel Nagy, an MD-PhD student in the Greenberg lab, the researchers first housed mice in complete darkness to quiet the visual cortex, the area of the brain that controls vision.

They then exposed the mice to light and studied how it affected genes within the brain. Using technology developed by the Klein lab known as inDrops, they tracked which genes got turned on or off in tens of thousands of individual cells before and after light exposure.

The team found significant changes in gene expression after light exposure in all cell types in the visual cortex—both neurons and, unexpectedly, non-neuronal cells such as astrocytes, macrophages and muscle cells that line blood vessels in the brain.

Roughly 50 to 70 percent of excitatory neurons, for example, exhibited changes regardless of their location or function. Remarkably, the authors said, a large proportion of non-neuronal cells—almost half of all astrocytes, for example—also exhibited changes.

The team identified thousands of genes with altered expression patterns after light exposure, and 611 genes that had at least two-fold increases or decreases.

Many of these genes have been previously linked to structural remodeling in the brain, suggesting that virtually the entire visual cortex, including the vasculature and muscle cell types, may undergo genetically controlled rewiring in response to a sensory experience.

There has been some controversy among neuroscientists over whether gene expression could functionally control plasticity or connectivity between neurons.

“I think our study strongly suggests that this is the case, and that each cell has a unique genetic program that’s tailored to the function of a given cell within a neural circuit,” Greenberg said.

Goldmine of questions

These findings open a wide range of avenues for further study, the authors said. For example, how genetic programs affect the function of specific cell types, how they vary early or later in life and how dysfunction in these programs might contribute to disease, all of which could help scientists learn more about the fundamental workings of the brain.

“Experience and environmental stimuli appear to almost constantly affect gene expression and function throughout the brain. This may help us to understand how processes such as learning and memory formation, which require long-term changes in the brain, arise from the short bursts of electrical activity through which neurons signal to each other,” Greenberg said.

One especially interesting area of inquiry, according to Greenberg, includes the regulatory elements that control the expression of genes in response to sensory experience. In a paper published earlier this year in Molecular Cell, he and his team explored the activity of the FOS/JUN protein complex, which is expressed across many different cell types in the brain but appears to regulate unique programs in each different cell type.

Identifying the regulatory elements that control gene expression is critical because they may account for differences in brain function from one human to another, and may also underlie disorders such as autism, schizophrenia and bipolar disease, the researchers said.

“We’re sitting on a goldmine of questions that can help us better understand how the brain works,” Greenberg said. “And there is a whole field of exploration waiting to be tapped.”

No Llamas Required


Researchers develop alternate method to uncover protein structures, design new drugs

https://player.vimeo.com/video/254936336?color=456d86&title=0&byline=0&portrait=0

Detouring around a major research roadblock, researchers have found a new way to create valuable antibodies without needing … llamas?

It is a little-known fact that llamas, alpacas, camels and other members of the camelid family make a unique class of antibodies that allow scientists to determine the structures of otherwise impossible-to-study proteins in the body, helping them to understand how those proteins malfunction in disease and how to design new drugs that act on them.

As one might imagine, there are downsides to taking advantage of this evolutionary happenstance.

First, not all researchers who need camelid antibodies for their experiments have access to llama (or alpaca or camel) facilities. Second, while the animals are not harmed, vaccinating them to generate the desired antibodies is expensive, takes as long as six months per attempt and often doesn’t work.

So, biochemists Andrew Kruse at Harvard Medical School and Aashish Manglik at the University of California, San Francisco, teamed up to create a llama-free solution: vials of specially engineered yeast.

The yeast method, described Feb. 12 in Nature Structural and Molecular Biology, can be done in a test tube in a researcher’s own lab. It has a higher success rate and faster turnaround time than both llama vaccination and previous attempts to circumvent camelids, the authors say.

It also marks the first time a camelid-bypass system has been made freely available for nonprofit use.

“There’s a real need for something like this,” said Kruse. “It’s low-tech, it’s a low time investment and it has a high likelihood of success for most proteins.”

“People who have struggled to nail down their protein structures for years with llamas are getting them now,” he said.

Lock and key

The active segments of camelid antibodies are often called nanobodies because they can be much smaller than regular antibodies. A llama nanobody might bind only to a particular conformation—for example, “open” or “closed”—of a particular protein. Nanobodies also can bind to challenging proteins, such as receptors that work in oily cell membranes.

Structural biologists like Kruse and Manglik want to find the exact nanobody that matches their protein of interest so they can lock the protein in one position and run tests to figure out its atomic structure. Learning the structure allows them to study how the protein works and provides a blueprint for designing drugs that target it.

Nanobodies have opened long-locked doors in biomedical science. For example, they have allowed researchers to see for the first time how neurotransmitters such as adrenaline and opioids bind to receptors in the brain.

Scientists just needed an easier way to find the keys.

Glowing success

Right now, a scientist who wants to study a difficult membrane protein has to laboriously generate several milligrams of it, inoculate a llama with it—usually done through a third-party service—and hope the animal’s immune system responds. Only then can she search for antibodies in a blood sample and hope there are enough to work with.

By contrast, Kruse and Manglik’s research team, led by first author Conor McMahon, a postdoctoral researcher in the Kruse lab, created a library of 500 million camelid antibodies using yeast cells.

Each yeast cell has a slightly different nanobody tethered to its surface, made by a slightly different piece of synthetic DNA.

The researchers mixed all the yeasts together and froze them for safekeeping. Anytime they want to run an experiment, they simply defrost a test tube’s worth: a miniature llama immune system. (The tube contains 10 to 20 times the amount needed to ensure that at least one of each unique antibody is included.)

The team developed a method where, instead of injecting a llama, scientists can now label their protein of interest with a fluorescent molecule and add it to the test tube. Yeast with surface nanobodies that recognize the protein will glow.

The researchers then use fluorescence-activated cell sorting, or FACS, to separate the glowing yeast from the rest.

They sequence the DNA of those glowing yeast cells to learn what the nanobodies are. They can then use E. coli bacteria to grow as many of those nanobodies as they need.

The whole process takes three to six weeks instead of three to six months.

Money for nothing, and yeast for free

The team tested its yeast system on two proteins: the beta-2 adrenergic receptor, linked to asthma, and the adenosine receptor, which is a gateway for caffeine to deliver its buzz. In both cases, the nanobody bound to the desired receptor, bound only to that receptor, and bound to it only when it was “on.”

“We found that the yeast-derived nanobodies can do everything llama-derived antibodies can,” said Kruse.

The team is now offering vials of the yeast mix and usage instructions free of charge to any nonprofit labs that want them. Commercial companies can license the yeast. “We made a big batch,” said McMahon, so there’s plenty to go around.

More than 40 labs requested vials before the paper was even published.

“Nanobodies are making it possible to develop drugs for biological targets that antibodies were simply too big to hit,” said Manglik. “By making nanobody discovery quick and easy, we hope our platform will dramatically accelerate the potential applications of this exciting technology.”

“I think we’ll see things that blow the animal immune system away,” added McMahon. “This is new technology. It’s only going to get better. Hopefully it will work as well or better so we won’t need llamas anymore.”

“Walking Fish” Discovery Scraps Evolutionary Theory of Human Locomotion


Walking is a lot more complicated than putting one foot in front of the other. For that to happen, motor neurons in the brain and spinal cord must instantaneously coordinate the muscles you need to move forward, then manage the limbs, lungs, and brain to work in harmony to get you where you need to go. The origins of this elaborate organizational strategy are murky: Until recently, the most accepted theory is the one you’ve seen drawn out on high school biology posters, showing that the ability to walk evolved as vertebrates transitioned from sea to land.

 But new research, released Thursday, revises that theory in a counterintuitive way. In the journal Cell, an international team of scientists report that the ability of spinal cord nerves to articulate muscles for walking emerged millions of years ago in the sea.

“We have learned that some of the things we generally think evolved in more ‘advanced’ animal species, such as the nerve cells controlling walking, are actually much more ancient than previously thought,” co-author and New York University neuroscientist Jeremy Dasen, Ph.D., tells Inverse.

This means that the first creatures that developed the ability to walk — the common ancestor linking fish and humans — stayed underwater. Some of their descendants eventually became walking invertebrates on land, while others remain on the ocean floor today, still walking.

skates
Skates use large pectoral fins for swimming and small pelvic fins for walking.

One of these seafloor dwellers, the little skate, was the focus of this new study. Skates, which look similar to rays, are cartilaginous fish that haven’t changed much in the hundreds of millions of years that they’ve existed. And they “walk,” but you probably couldn’t tell by looking. Previous research showed that they wave their smaller pelvic fins in alternating left-right motions to creep along the ocean floor — which would hardly be noticeable to a scuba diver floating over them in the western Atlantic Ocean.

“One of the most surprising findings was how similar the movement of the skates’ pelvic fins are to the way we use our legs during walking,” says Dasen. “We could only appreciate this from taking videos from underneath skates while they are walking. This showed that many of the basic elements of walking, like the alternation between left and right legs, leg extensions and flexion, were present in skates.”

Dasen and his team began studying a group of skates as they developed in their egg cases. In a skate embryo, the tail is the strongest thing that pushes its locomotion, but after it hatches, the tail eventually regresses — presumably because locomotion through pelvic fins is poised to dominate.

A follow-up experiment on the skates used RNA sequencing to assess what genes were expressed in the skate’s motor neurons and compared them to genes linked to mammal locomotion. This showed that skates and mammals actually have a lot in common, including molecules expressed in the motor neurons of land vertebrates, molecular switches that control muscles, and interneurons that control locomotion.

evolution
Skates and tetrapods like mice have a common ancestor.

“Many of the genes we studied in skates were known to be very important for the function of motor neurons that control walking in mammals,” says Dasen. “Some of these genes produce proteins that are known to function as ‘genetic switches’, which turn genes on or off. Our study shows that these same switches are used in both skates and mammals to help wire the nerve circuits essential for walking.”

Taken together, the observations indicate that the circuits involved in limb control began with a vertebrate ancestor millions of years before anything walked on land. By the time our ancestors wiggled onto the sand with their primordial limbs, the processes that generated their movement had long been established. With this in mind, Dasen and his team will continue to study the little skates to understand how exactly their motor neurons connect, with the hope that one day this knowledge can help people with serious spinal injuries.

“We actually know very little about how the nerve cells in the brain and spinal cord communicate with the motor neurons that control walking,” says Dasen.

“We hope we can take advantage of the relative simplicity of the skate fin to figure out some of the important nerve connections that make walking possible, and eventually test whether these same connections are important for mammals.”

Weird Clutch of Eggs Found in One of the Most Inhospitable Places on Earth


Strange conditions in the murkiest depths of the ocean have led to the evolution of weird-looking animals that sometimes look like dicks but are, impressively, able to thrive in uniquely severe environments. One of the oddest and most inhospitable of these habitats is the area surrounding underwater volcanoes known as black smokers, which spew forth hot jets of chemicals from the Earth’s mantle that can reach temperatures of 650º-750º Fahrenheit. Scientists know that some hardy organisms rely on these chemical feasts to thrive, but they only recently realized that the searing heat from those vents might be crucial to their survival as well.

In a paper published Thursday in the journal Scientific Reports, an international team of scientists led by Pelayo Salinas-de-León, Ph.D. report an especially unusual discovery: that Pacific white skates (Bathyraja spinosissima), relatives of sharks that grow to have a wingspan of up to five feet, lay their eggs around hydrothermal vents in the Iguanas-Pinguinos vent field, about one mile below the sea just north of the Galápagos Islands.

pacific white skate deep sea hydrothermal vents
Pacific white skates can live up to 10,000 feet below the surface of the sea.

While the fossil record has shown that dinosaurs laid eggs in volcanic soil, just like the still-living megapode, a ground-dwelling bird that lays its eggs in mounds of heat-generating, decomposing matter in Asia and Australia, this report marks the first time anyone has observed this behavior in a marine animal.

Scientists found egg cases between 30 feet and 450 feet from hydrothermal vents.
Scientists found egg cases between three feet and 450 feet from hydrothermal vents.

Pacific white skates are wide, flat fish that can live up to 10,000 feet below the surface of the ocean. What makes this species especially unique is that their eggs take a really long time to hatch: Researchers estimate that these eggs incubate for 1,500 days — more than four years. Laying the eggs around these deep-sea vents, the researchers hypothesize, could help shorten the time it takes for the eggs to hatch.

This image, taken from the ROV's POV, shows skate eggs around a hydrothermal chimney.
This image, taken from the ROV’s POV, shows skate eggs around a hydrothermal chimney.

Since the hydrothermal vents of the Galápagos Marine Reserve are so deep underwater, the researchers had to use a remotely operated underwater vehicle (ROV) to explore what life could thrived there. Using the camera on the ROV, as well as a robotic arm that can gently manipulate objects, they observed the scene and collected four samples to confirm the species. In total, they found 157 egg cases, each about four inches long. Many of the cases were fresh, suggesting that the site was an active incubation area.

Deep-sea hydrothermal vents, known as black smokers, host a wide range of life, including skate eggs.
Deep-sea hydrothermal vents, known as black smokers, host a wide range of life, including skate eggs.

With the ROV’s camera, they also observed a bunch of older egg cases, indicating that skates had been laying their eggs around these vents for quite some time.

Even with the help of the hydrothermal vents, the water is still quite cold way down there — only a few degrees above freezing. So it makes sense that the skates are taking advantage of this environmental freebie.

Researchers collected four egg cases to confirm the species.
Researchers collected four egg cases to confirm the species.

Aside from being a startlingly strange find, documenting this phenomenon could assist conservation efforts in the future, as these deep-sea hydrothermal vents could soon be under threat. While it seems like something a mile under the ocean should be safe from human hinderance, they’ve recently become a target for mining companies hoping to extract methane or harvest the geothermal energy.

“We hardly know anything about the deep sea, and we are fishing, and mining, before we even get a chance to even document what species live down there and what unique behaviors [they] could reveal [to] us,” Salinas-de-León told National Geographic in an interview this month. Perhaps learning that these vents not only host the crabs and worms that we already knew about but also serve as nurseries for these strange and beautiful skates will teach us to be a little more hesitant to decimate these habitats for our own gain.

Abstract: The discovery of deep-sea hydrothermal vents in 1977 challenged our views of ecosystem functioning and yet, the research conducted at these extreme and logistically challenging environments still continues to reveal unique biological processes. Here, we report for the first time, a unique behavior where the deep-sea skate, Bathyraja spinosissima, appears to be actively using the elevated temperature of a hydrothermal vent environment to naturally “incubate” developing egg-cases. We hypothesize that this behavior is directly targeted to accelerate embryo development time given that deep-sea skates have some of the longest egg incubation times reported for the animal kingdom. Similar egg incubating behavior, where eggs are incubated in volcanically heated nesting grounds, have been recorded in Cretaceous sauropod dinosaurs and the rare avian megapode. To our knowledge, this is the first time incubating behavior using a volcanic source is recorded for the marine environment.

Genetic Flip Helped Organisms Go From One Cell to Many


Clockwise from top left: microscopic views of glands in frog skin, a sheep’s hoof, a tamarin’s skin and fish scales. 

Narwhals and newts, eagles and eagle rays — the diversity of animal forms never ceases to amaze. At the root of this spectacular diversity is the fact that all animals are made up of many cells — in our case, about 37 trillion of them. As an animal develops from a fertilized egg, its cells may diversify into a seemingly limitless range of types and tissues, from tusks to feathers to brains.

The transition from our single-celled ancestors to the first multicellular animals occurred about 800 million years ago, but scientists aren’t sure how it happened. In a study published in the journal eLife, a team of researchers tackles this mystery in a new way.

The researchers resurrected ancient molecules that once helped single-celled organisms thrive, then recreated the mutations that helped them build multicellular bodies.

The authors of the new study focused on a single molecule called GK-PID, which animals depend on for growing different kinds of tissues. Without GK-PID, cells don’t develop into coherent structures, instead growing into a disorganized mess and sometimes even turning cancerous.

GK-PID’s job, scientists have found, is to link proteins so cells can divide properly. “I think of it as a molecular carabiner,” said Joseph W. Thornton, an evolutionary biologist at the University of Chicago and a co-author of the new study

When a cell divides, it first has to make an extra copy of its chromosomes, and then each set of chromosomes must be moved into the two new cells. GK-PID latches onto proteins that drag the chromosomes, then attaches to anchor proteins on the inner wall of the cell membrane. Once those proteins are joined by GK-PID, the dragging proteins pull the chromosomes in the correct directions.

Bad things happen if the chromosomes head the wrong way. Skin cells, for example, form a stack of horizontal layers. New cells needs to grow in the same direction so skin can continue to act as a barrier. If GK-PID doesn’t ensure that the chromosomes move horizontally, the cells end up in a jumble, like bricks randomly set at different angles.

Previous studies have offered clues to how this important molecule might have evolved in the ancestors of animals. All animals (ourselves included) carry a gene sequence that’s very similar to the one producing GK-PID. But that gene encodes a different molecule with a different job: an enzyme that helps build DNA. The enzyme can be found even in other organisms, like fungi to bacteria.

Dr. Thornton and his colleagues wondered whether that enzyme and its cousin GK-PID shared some kind of evolutionary history.

First, they made a careful study of the different forms of GK-PID and the DNA-building enzyme in about 200 species. Then they worked out how the genes for these molecules must have mutated over the millenniums.

That analysis allowed the scientists to figure out the DNA sequence for GK-PID in the single-celled ancestors of animals — a gene that hasn’t been seen in hundreds of millions of years. Then Dr. Thornton and his colleagues did something even more amazing: They recreated those ancient molecules to see how they once functioned.

The ancestral version of GK-PID wasn’t a carabiner, the scientists found. Instead, it behaved like a DNA-building enzyme. That finding suggests that in the ancestors of animals, the gene for the enzyme was accidentally duplicated. Later on, mutations in one copy of the gene turned it into a carabiner.

But how many mutations did it take to transform the molecule? That’s the most remarkable part of the new study. The scientists altered the gene for the ancestral enzyme with the earliest mutations that evolved in it. They found it took a single mutation to flip GK-PID from an enzyme to a carabiner.

“Genetically, it was much easier than we thought possible,” Dr. Thornton said. “You don’t need some elaborate series of thousands of mutations in just the right order.”

The evolution of a molecular carabiner did not by itself give rise to the animal kingdom, of course. Other adaptations were needed to grow multicellular bodies. Dr. Thornton said that it might be possible to resurrect other ancestral molecules to figure out how those adaptations evolved, as well.

And if GK-PID is any guide, Dr. Thornton said, their evolution may have been surprisingly simple. A single mutation might have been enough to switch a molecule from one job to another.

Antonis Rokas, an evolutionary biologist at Vanderbilt University who was not involved in the study, agreed. “One of evolution’s most striking major innovations may be the end-product of a series of many minor innovations,” he said.

Researchers See New Importance in Y Chromosome


The male, or Y, chromosome in humans, right, is much smaller than the X, left. 

There is new reason to respect the diminutive male Y chromosome.

Besides its long-known role of reversing the default state of being female, the Y chromosome includes genes required for the general operation of the genome, according to two new surveys of its evolutionary history. These genes may represent a fundamental difference in how the cells in men’s and women’s bodies read off the information in their genomes.

When researchers were first able to analyze the genetic content of the Y chromosome, they found it had shed hundreds of genes over time, explaining why it was so much shorter than its partner, the X chromosome. All cells in a man’s body have an X and a Y chromosome; women’s have two X chromosomes.

The finding created considerable consternation. The Y had so few genes left that it seemed the loss of a few more could tip it into extinction.

But an analysis in 2012 showed that the rhesus monkey’s Y chromosome had essentially the same number of genes as the human Y. This suggested that the Y had stabilized and ceased to lose genes for the last 25 million years, the interval since the two species diverged from a common ancestor.

Two new surveys have now reconstructed the full history of the Y chromosome back to its evolutionary origin. One research group was led by Daniel W. Bellott and David C. Page of the Whitehead Institute in Cambridge, Mass., and the other by Diego Cortez and Henrik Kaessmann of the University of Lausanne in Switzerland. Their findings were reported on Wednesday in the journal Nature.

In the past 12 years, with the help of the genome sequencing centers at Washington University in St. Louis and the Baylor College of Medicine in Houston, Dr. Page’s group has decoded the DNA sequence of the Y chromosome of eight mammals, including the rhesus monkey and humans. The Y chromosome is so hard to decode that many early versions of the human genome sequence just omitted it. Dr. Kaessmann’s group, on the other hand, devised a quick method of fishing out Y chromosome genes by simply comparing the X and Y DNA of various species and assuming that any genetic sequences that did not match to the X must come from the Y.

Dr. Kaessmann calculates that the Y chromosome originated 181 million years ago, after the duck-billed platypus split off from other mammals but before the marsupials did so.

In some reptiles, sex is determined by the temperature at which the egg incubates. Genetic control over sex probably began when a gene on one of the X chromosomes called SOX3 became converted to SRY, the gene that determines maleness, and thus the Y chromosome came into being.

Until this time, the predecessors of the X and the Y had been an equal pair of chromosomes just like any of the others. Humans have 23 pairs of chromosomes, with one member of every pair being inherited from each parent. People with an XX pair among their 23 are female; those with an XY pair are male.

Before generating eggs and sperm, the 23 pairs of chromosomes line up and each chromosome exchanges chunks of DNA with its partner, a process known as recombination. But recombination between the X and Y had to be banned, except at their very tips, lest the male-determining SRY gene slip across to the X and wreak havoc.

Recombination creates novel arrays of DNA that keep genes adapted to the environment; without recombination they decay and are shed from the genome.

The reconstructions by the Page and Kaessmann groups show that most such genes were shed almost immediately and that the few genes remaining on the Y have been stable for millions of years.

One of these genes is SRY, and others are involved in sperm production. A third category of genes is unusual in being switched on not just in the testis but in tissues all over the body. These active genes, of which there are 12 in humans, all have high-level roles in controlling the state of the genome and the activation of other genes.

The 12 regulatory genes have counterpart genes on the X with which they used to recombine millions of years ago. They escaped the usual decay caused by lack of recombination, presumably being kept functional by purifying selection, a geneticists’ term meaning that any mutations were lethal to the owner. They have, however, become somewhat different from their 12 counterpart genes on the X.

This means that female, or XX, cells have a slightly different set of these powerful genes from male or XY cells, since the X and Y genes are producing slightly different proteins. In females, usually one X chromosome is inactivated in each cell, but the 12 genes are so important that they escape inactivation, and XX cells, like XY cells, receive a double dose of the gene’s products.

“Throughout human bodies, the cells of males and females are biochemically different,” Dr. Page said. The genome may be controlled slightly differently because of this variation in the 12 regulatory genes, which he thinks could contribute to the differing incidence of many diseases in men and women.

Differences between male and female tissues are often attributed to the powerful influence of sex hormones. But now that the 12 regulatory genes are known to be active throughout the body, there is clearly an intrinsic difference in male and female cells even before the sex hormones are brought into play.

“We are only beginning to understand the full extent of the differences in molecular biology of males and females,” Andrew Clark, a geneticist at Cornell University, wrote in a commentary in Nature on the two reports.

A Gene Mystery: How Are Rats With No Y Chromosome Born Male?


An Amami spiny rat. Cells of the rat, which is from Japan, are sexually flexible and capable of adapting to either ovaries or testes. 

In most mammals, us included, biological sex is determined by a lottery between two letters: X and Y, the sex chromosomes. Inherit one X each from mom and dad, and develop ovaries, a womb and a vagina. Inherit an X from mom and a Y from dad, and develop testes and a penis.

But there are rare, mysterious exceptions. A small number of rodents have no Y chromosomes, yet are born as either females or males, not hermaphrodites. Now, scientists may be one step closer to figuring out how sex determination works in one of these rodents.

In a study published in Science Advances on Friday, Japanese scientists suggested that cells of the endangered Amami spiny rat, from Japan, are sexually flexible and capable of adapting to either ovaries or testes. When the researchers injected stem cells derived from a female rat into male embryos of laboratory mice, the cells developed into and survived as sperm precursors in adult males. The result was surprising since scientists have never been able to generate mature sperm from female stem cells, largely because sperm production normally requires the Y chromosome.

Found only in the subtropical forests of an island in Japan called Amami Oshima, Amami spiny rats are threatened by habitat destruction, competition with black rats not native to the island and predation by mongooses and feral cats and dogs. Their range has been reduced to less than 300 square miles, an area smaller than New York City.

Both female and male Amami spiny rats have only one X chromosome, an arrangement only known to occur in a handful of rodents among mammals. Arata Honda, associate professor at the University of Miyazaki and the lead author of the paper, said in an email that he was partly motivated to study Amami spiny rats in the hope that learning about them might reduce their risk of extinction.

No one knows how or why, but at some point the rats lost their Y chromosome and, along with it, an important gene called SRY that’s considered the “master switch” of male anatomical development in most mammals.

A chimera containing genes from an Amami spiny rat and a mouse. 

It’s possible that a new gene that wasn’t linked to the Y chromosome took over the role of SRY in these rats, said Monika Ward, a professor and expert on the Y chromosome at the University of Hawaii in Honolulu who was not involved in this study. In addition, research has shown that other genes involved in male sexual differentiation were not lost, but rather transferred from the Y chromosome to other parts of the rat’s genome, including to the X chromosome.

Because the rats are endangered, scientists cannot directly do experiments on them. To get around this, Dr. Honda and his colleagues converted skin cells from the tail tip of a female Amami spiny rat into special stem cells called induced pluripotent stem cells (also called iPS cells), which can multiply indefinitely and become any other cell type in the body. The scientists injected the stem cells into mice embryos and transplanted the embryos into female mice, which birthed 13 so-called chimeras.

After the chimeras reached adulthood, the researchers located the spiny rat iPS cells within their bodies. They were surprised to find some iPS cells appeared in the ovary as immature egg cells, and others in the testis as immature sperm cells.

This study shows that the spiny rat’s sex cells have “astounding” fluidity, said Diana Laird, an associate professor in reproductive sciences at the University of California, San Francisco, who was not involved in the study. The cells were able to sense whether they were in an ovary or testis, and “not only hear but obey the signals” coming from that foreign environment, she added.

Dr. Ward emphasized that these results are not universal — if you were to take iPS cells from a normal female mouse and put them in a male embryo, the few cells that became sperm precursors would die very quickly. The female stem cells in this study were able to approach mature sperm development because of the Amami spiny rat’s unique biology, she said.

The study is also significant because the researchers managed to create chimeras from an endangered species, said Marisa Korody, a postdoctoral associate at the San Diego Zoo Institute for Conservation Research who researches how iPS cells might be used to protect endangered animals.

“One of the lofty goals we have for using stem cells,” she said, is “to differentiate them into egg and sperm and hopefully create embryos that can be transplanted into a surrogate.”

But there are limits to the findings because the researchers have not yet shown that the spiny rat’s stem cells can fully develop into mature eggs and sperm. “That’s the million dollar question,” Dr. Laird said.

Somehow, This Fish Fathered a Near Clone of Itself


Squalius alburnoides and Squalius pyrenaicus inside an artificial pond where researchers studied them.

A female and male get together. One thing leads to another, and they have sex. His sperm fuses with her egg, half of his DNA combining with half of her DNA to form an embryo.

As humans, this is how we tend to think of reproduction.

But there are many other bizarre ways reproduction can take place. For instance, scientists have discovered a fish carrying genes only from its father in the nucleus of its cells. Found in a type of fish called Squalius alburnoides, which normally inhabits rivers in Portugal or Spain, this is the first documented instance in vertebrates of a father producing a near clone of itself through sexual reproduction — a rare phenomenon called androgenesis — the researchers reported in the journal Royal Society Open Science on Wednesday.

The possibility of androgenesis is just one of many mysteries about Squalius alburnoides. It’s not a species in the usual sense, but rather something called a hybrid complex, a group of organisms with multiple parental combinations that can mate with one another.

The group is thought to have arisen from hybridization between females of one species, Squalius pyrenaicus, and males of another species, now extinct, that belonged to a group of fish called Anaecypris. To sustain its population, Squalius alburnoides mates with several other closely related species belonging to the Squalius lineage.

That it can reproduce at all is unusual enough. Most hybrids, like mules, are sterile because the chromosomes from their parents of different species have trouble combining, swapping DNA and dividing — steps required for egg or sperm production.

Squalius alburnoides males circumvent this problem by producing sperm cells that do not divide, and therefore contain more than one chromosome set. This is important because most animals, Squalius alburnoides included, need at least two chromosome sets to survive.

Mostly, animals have sex cells containing only one chromosome set, which means the egg and sperm are both required for reproduction. But in Squalius alburnoides, sperm with multiple chromosome sets can provide all the nuclear genetic material needed for a viable offspring — paving the way for androgenesis.

A Squalius alburnoides fish, which researchers discovered had reproduced through androgenesis. 

The details in Squalius alburnoides are still unknown, but in general androgenesis is thought to occur in a couple of ways, said Miguel Morgado-Santos, a graduate student at the University of Lisbon and an author of the study: Sperm could fertilize an egg that contains no chromosomes, or it could destroy the genetic content from the nucleus of the egg after fertilization.

Mr. Morgado-Santos’s group found this instance of androgenesis by accident, while studying the mating patterns of Squalius alburnoides. The researchers put male and female Squalius alburnoides with males and females of another Squalius species in an artificial pond, let the fish reproduce and then genetically analyzed 100 randomly selected offspring. One of these offspring had only paternal chromosomes.

“We weren’t expecting to find that,” Mr. Morgado-Santos said, adding that at first he and his collaborators thought they had made a mistake.

Though he acknowledges one in 100 fish is a rare occurrence, Mr. Morgado-Santos thinks this instance of androgenesis could represent a “snapshot” of a population moving toward becoming its own species. Put another way, androgenesis may help this fish become independent from the other Squalius species it relies on to reproduce.

If androgenesis turns out to be a regular feature in this population, Mr. Morgado-Santos’s group might be catching the ”very early stages” of a new reproductive mode for the fish, which would be exciting, said Tanja Schwander, a professor of ecology and evolution at the University of Lausanne in Switzerland who did not participate in this research.

But for now, she added, the researchers cannot rule out the possibility that this one instance is a random exception (perhaps the fish’s mother accidentally produced an egg that contained no chromosomes, for instance).

Accident or not, it happened, and shows that reproduction can vary in all sorts of “weird and wonderful” ways across the natural world, said Benjamin Oldroyd, a professor of genetics at the University of Sydney who was not involved in this study.

“It may not add up to a hill of beans other than realizing that the world is much more complicated than we assume,” he said. “But it’s part of what life is, as a curious human, to understand these things.”

This Mutant Crayfish Clones Itself, and It’s Taking Over Europe


The marbled crayfish is a mutant species that clones itself, scientists report. The population is exploding in Europe, but the species appears to have originated only about 25 years ago. 

Frank Lyko, a biologist at the German Cancer Research Center, studies the six-inch-long marbled crayfish. Finding specimens is easy: Dr. Lyko can buy the crayfish at pet stores in Germany, or he can head with colleagues to a nearby lake.

Wait till dark, switch on head lamps, and wander into the shallows. The marbled crayfish will emerge from hiding and begin swarming around your ankles.

“It’s extremely impressive,” said Dr. Lyko. “Three of us once caught 150 animals within one hour, just with our hands.”

Over the past five years, Dr. Lyko and his colleagues have sequenced the genomes of marbled crayfish. In a study published on Monday, the researchers demonstrate that the marble crayfish, while common, is one of the most remarkable species known to science.

Before about 25 years ago, the species simply did not exist. A single drastic mutation in a single crayfish produced the marbled crayfish in an instant

The mutation made it possible for the creature to clone itself, and now it has spread across much of Europe and gained a toehold on other continents. In Madagascar, where it arrived about 2007, it now numbers in the millions and threatens native crayfish.

“We may never have caught the genome of a species so soon after it became a species,” said Zen Faulkes, a biologist at the University of Texas Rio Grande Valley, who was not involved in the new study.

The marbled crayfish became popular among German aquarium hobbyists in the late 1990s. The earliest report of the creature comes from a hobbyist who told Dr. Lyko he bought what were described to him as “Texas crayfish” in 1995.

The hobbyist — whom Dr. Lyko declined to identify — was struck by the large size of the crayfish and its enormous batches of eggs. A single marbled crayfish can produce hundreds of eggs at a time.

Soon the hobbyist was giving away the crayfish to his friends. And not long afterward, so-called marmorkrebs were showing up in pet stores in Germany and beyond.

As marmorkrebs became more popular, owners grew increasingly puzzled. The crayfish seemed to be laying eggs without mating. The progeny were all female, and each one grew up ready to reproduce.

In 2003, scientists confirmed that the marbled crayfish were indeed making clones of themselves. They sequenced small bits of DNA from the animals, which bore a striking similarity to a group of crayfish species called Procambarus, native to North America and Central America.

Ten years later, Dr. Lyko and his colleagues set out to determine the entire genome of the marbled crayfish. By then, it was no longer just an aquarium oddity.

For nearly two decades, marbled crayfish have been multiplying like Tribbles on the legendary “Star Trek” episode. “People would start out with a single animal, and a year later they would have a couple hundred,” said Dr. Lyko.

Many owners apparently drove to nearby lakes and dumped their marmorkrebs. And it turned out that the marbled crayfish didn’t need to be pampered to thrive. Marmorkrebs established growing populations in the wild, sometimes walking hundreds of yards to reach new lakes and streams. Feral populations started turning up in the Czech Republic, Hungary, Croatia and Ukraine in Europe, and later in Japan and Madagascar.

Sequencing the genome of this animal was not easy: No one had sequenced the genome of a crayfish. In fact, no one had ever sequenced any close relative of crayfish.

Dr. Lyko and his colleagues struggled for years to piece together fragments of DNA into a single map of its genome. Once they succeeded, they sequenced the genomes of 15 other specimens, including marbled crayfish living in German lakes and those belonging to other species.

The rich genetic detail gave the scientists a much clearer look at the freakish origins of the marbled crayfish.

It apparently evolved from a species known as the slough crayfish, Procambarus fallax, which lives only in the tributaries of the Satilla River in Florida and Georgia.

The scientists concluded that the new species got its start when two slough crayfish mated. One of them had a mutation in a sex cell — whether it was an egg or sperm, the scientists can’t tell.

Normal sex cells contain a single copy of each chromosome. But the mutant crayfish sex cell had two.

Somehow the two sex cells fused and produced a female crayfish embryo with three copies of each chromosome instead of the normal two. Somehow, too, the new crayfish didn’t suffer any deformities as a result of all that extra DNA.

It grew and thrived. But instead of reproducing sexually, the first marbled crayfish was able to induce her own eggs to start dividing into embryos. The offspring, all females, inherited identical copies of her three sets of chromosomes. They were clones.

Now that their chromosomes were mismatched with those of slough crayfish, they could no longer produce viable offspring. Male slough crayfish will readily mate with the marbled crayfish, but they never father any of the offspring.

In December, Dr. Lyko and his colleagues officially declared the marbled crayfish to be a species of its own, which they named Procambarus virginalis. The scientists can’t say for sure where the species began. There are no wild populations of marble crayfish in the United States, so it’s conceivable that the new species arose in a German aquarium.

[READ: A Gene Mystery: How Are Rats With No Y Chromosome Born Male?]

All the marbled crayfish Dr. Lyko’s team studied were almost genetically identical to one another. Yet that single genome has allowed the clones to thrive in all manner of habitats — from abandoned coal fields in Germany to rice paddies in Madagascar.

In their new study, published in the journal Nature Ecology and Evolution, the researchers show that the marbled crayfish has spread across Madagascar at an astonishing pace, across an area the size of Indiana in about a decade.

Thanks to the young age of the species, marbled crayfish could shed light on one of the big mysteries about the animal kingdom: why so many animals have sex.

Only about 1 in 10,000 species comprise cloning females. Many studies suggest that sex-free species are rare because they don’t last long.

In one such study, Abraham E. Tucker of Southern Arkansas University and his colleagues studied 11 asexual species of water fleas, a tiny kind of invertebrate. Their DNA indicates that the species only evolved about 1,250 years ago.

There are a lot of clear advantages to being a clone. Marbled crayfish produce nothing but fertile offspring, allowing their populations to explode. “Asexuality is a fantastic short-term strategy,” said Dr. Tucker.

In the long term, however, there are benefits to sex. Sexually reproducing animals may be better at fighting off diseases, for example.

If a pathogen evolves a way to attack one clone, its strategy will succeed on every clone. Sexually reproducing species mix their genes together into new combinations, increasing their odds of developing a defense.

The marbled crayfish offers scientists a chance to watch this drama play out practically from the beginning. In its first couple decades, it’s doing extremely well. But sooner or later, the marbled crayfish’s fortunes may well turn.

“Maybe they just survive for 100,000 years,” Dr. Lyko speculated. “That would be a long time for me personally, but in evolution it would just be a blip on the radar.”

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