One of the enzymes used in this system takes an amino acid (left) and links it to Coenzyme A. The second takes these items and links them into a polymer. Credit: Chae et. al.
Normally, PHA synthase forms links between molecules that run through an oxygen atom. But it’s also possible to form a related chemical link that instead runs through a nitrogen atom, like those found on amino acids. There were no known enzymes, however, that catalyze these reactions. So, the researchers decided to test whether any existing enzymes could be induced to do something they don’t normally do.
The researchers started with an enzyme from Clostridium that links chemicals to Coenzyme A that has a reputation for not being picky about the chemicals it interacts with. This worked reasonably well at linking amino acids to Coenzyme A. For linking the amino acids together, they used an enzyme from Pseudomonas that had four different mutations that expanded the range of molecules it would use as reaction materials. Used in a test tube, the system worked: Amino acids were linked together in a polymer.
The question was whether it would work in cells. Unfortunately, one of the two enzymes turns out to be mildly toxic to E. coli, slowing its growth. So, the researchers evolved a strain of E. coli that could tolerate the protein. With both of these two proteins, the cells produced small amounts of an amino acid polymer. If they added an excess of an amino acid to the media the cells were growing in, the polymer would be biased toward incorporating that amino acid.
Boosting polymer production
However, the yield of the polymer by weight of bacteria was fairly low. “It was reasoned that these [amino acids] might be more efficiently incorporated into the polymer if generated within the cells from a suitable carbon source,” the researchers write. So, the researchers put in extra copies of the genes needed to produce one specific amino acid (lysine). That worked, producing more polymer, with a higher percentage of the polymer being lysine.
Once his team nailed how droplets become electrically charged and how the micro-lightning phenomenon works, they recreated the Miller-Urey experiment. Only without the spark plugs.
Ingredients of life
After micro-lightnings started jumping between droplets in a mixture of gases similar to that used by Miller and Urey, the team examined their chemical composition with a mass spectrometer. They confirmed glycine, uracil, urea, cyanoethylene, and lots of other chemical compounds were made. “Micro-lightnings made all organic molecules observed previously in the Miller-Urey experiment without any external voltage applied,” Zare claims.
But does it really bring us any closer to explaining the beginnings of life? After all, Miller and Urey already demonstrated those molecules could be produced by electrical discharges in a primordial Earth’s atmosphere—does it matter all that much where those discharges came from? Zare argues that it does.
“Lightning is intermittent, so it would be hard for these molecules to concentrate. But if you look at waves crashing into rocks, you can think the spray would easily go into the crevices in these rocks,” Zare suggests. He suggests that the water in these crevices would evaporate, new spray would enter and evaporate again and again. The cyclic drying would allow the chemical precursors to build into more complex molecules. “When you go through such a dry cycle, it causes polymerization, which is how you make DNA,” Zare argues. Since sources of spray were likely common on the early Earth, Zare thinks this process could produce far more organic chemicals than potential alternatives like lightning strikes, hydrothermal vents, or impacting comets.
But even if micro-lightning really produced the basic building blocks of life on Earth, we’re still not sure how those combined into living organisms. “We did not make life. We just demonstrated a possible mechanism that gives us some chemical compounds you find in life,” Zare says. “It’s very important to have a lot of humility with this stuff.”
And it worked. Repeating the same process with an added PLACER screening step boosted the number of enzymes with catalytic activity by over three-fold.
Unfortunately, all of these enzymes stalled after a single reaction. It turns out they were much better at cleaving the ester, but they left one part of it chemically bonded to the enzyme. In other words, the enzymes acted like part of the reaction, not a catalyst. So the researchers started using PLACER to screen for structures that could adopt a key intermediate state of the reaction. This produced a much higher rate of reactive enzymes (18 percent of them cleaved the ester bond), and two—named “super” and “win”—could actually cycle through multiple rounds of reactions. The team had finally made an enzyme.
By adding additional rounds alternating between structure suggestions using RFDiffusion and screening using PLACER, the team saw the frequency of functional enzymes increase and eventually designed one that had an activity similar to some produced by actual living things. They also showed they could use the same process to design an esterase capable of digesting the bonds in PET, a common plastic.
If that sounds like a lot of work, it clearly was—designing enzymes, especially ones where we know of similar enzymes in living things, will remain a serious challenge. But at least much of it can be done on computers rather than requiring someone to order up the DNA that encodes the enzyme, getting bacteria to make it, and screening for activity. And despite the process involving references to known enzymes, the designed ones didn’t share a lot of sequences in common with them. That suggests there should be added flexibility if we want to design one that will react with esters that living things have never come across.
I’m curious about what might happen if we design an enzyme that is essential for survival, put it in bacteria, and then allow it to evolve for a while. I suspect life could find ways of improving on even our best designs.
Since these two toxicities work through entirely different mechanisms, the researchers tackled them separately.
Blocking a neurotoxin
The neurotoxic three-fingered proteins are a subgroup of the larger protein family that specializes in binding to and blocking the receptors for acetylcholine, a major neurotransmitter. Their three-dimensional structure, which is key to their ability to bind these receptors, is based on three strings of amino acids within the protein that nestle against each other (for those that have taken a sufficiently advanced biology class, these are anti-parallel beta sheets). So to interfere with these toxins, the researchers targeted these strings.
They relied on an AI package called RFdiffusion (the RF denotes its relation to the Rosetta Fold protein-folding software). RFdiffusion can be directed to design protein structures that are complements to specific chemicals; in this case, it identified new strands that could line up along the edge of the ones in the three-fingered toxins. Once those were identified, a separate AI package, called ProteinMPNN, was used to identify the amino acid sequence of a full-length protein that would form the newly identified strands.
But we’re not done with the AI tools yet. The combination of three-fingered toxins and a set of the newly designed proteins were then fed into DeepMind’s AlfaFold2 and the Rosetta protein structure software, and the strength of the interactions between them were estimated.
It’s only at this point that the researchers started making actual proteins, focusing on the candidates that the software suggested would interact the best with the three-fingered toxins. Forty-four of the computer-designed proteins were tested for their ability to interact with the three-fingered toxin, and the single protein that had the strongest interaction was used for further studies.
At this point, it was back to the AI, where RFDiffusion was used to suggest variants of this protein that might bind more effectively. About 15 percent of its suggestions did, in fact, interact more strongly with the toxin. The researchers then made both the toxin and the strongest inhibitor in bacteria and obtained the structure of their interactions. This confirmed that the software’s predictions were highly accurate.
The developers of the system call each of these potentially modifiable spots on the template an epi-bit, with the modified version corresponding to a 1 in a conventional computer bit and the unmodified version corresponding to a 0. Because no synthesis is required, multiple bits can be written simultaneously. To read the information, the scientists rigged the system so that 1s fluoresce and 0s don’t. The fluorescence, along with the sequences of bases, was read as the DNA was passed through a tiny pore.
Pictures in a meta-genome
Using this system, Zhang et al. created five DNA templates and 175 bricks to record 350 bits at a time. Using a collection of tagged template molecules, the researchers could store and read roughly 275,000 bits, including a color picture of a panda’s face and a rubbing of a tiger from the Han dynasty, which ruled China from 202 BCE to 220 CE.
They then had 60 student volunteers “with diverse academic backgrounds” store texts of their choice in epi-bits using a simple kit in a classroom. Twelve of the 15 stored texts were read successfully.
We’re not quite ready for your cat videos yet, though. There are still errors in the printing and reading steps, and since these modifications don’t survive when DNA is copied, making additional versions of the stored information may get complicated. Plus, the stability of these modifications under different storage conditions remains unknown, although the authors note that their epi-bits stayed stable at temperatures of up to 95o° C.
But once these and a few other problems are solved—and the technology is scaled up, further optimized and automated, and/or tweaked to accommodate other types of epigenetic modifications—it will be a clever and novel way to harness natural data storage methods for our needs.
Enlarge/ Zihao Ou, who helped develop this solution, holds a tube of it.
One key challenge in medical imaging is to look past skin and other tissue that are opaque to see internal organs and structures. This is the reason we need things like ultrasonography, magnetic resonance, or X-rays. There are chemical clearing agents that can make tissue transparent, like acrylamide or tetrahydrofuran, but they are almost never used in living organisms because they’re either highly toxic or can dissolve away essential biomolecules.
But now, a team of Stanford University scientists has finally found an agent that can reversibly make skin transparent without damaging it. This agent was tartrazine, a popular yellow-orange food dye called FD&C Yellow 5 that is notably used for coloring Doritos.
Playing with light
We can’t see through the skin because it is a complex tissue comprising aqueous-based components such as cell interiors and other fluids, as well as protein and lipids. The refractive index is a value that indicates how much light slows down (on average, of course) while going through a material compared to going through a vacuum. The refractive index of those aqueous components is low, while the refractive index of the proteins and lipids is high. As a result, light traveling through skin constantly bends as it endlessly crosses the boundary between high and low refractive index materials.
This scatters the light—once it penetrates the skin, it never gets back. What we see is just the light that bounces off the skin’s surface. The trick to making things transparent is mostly about making their refractive index uniform, so light, or at least some part of the spectrum, doesn’t bend all the time and doesn’t get scattered. This is exactly where the Doritos dye came in.
“The most surprising part of this study is that we usually expect dye molecules to make things less transparent,” says Guosong Hong, an assistant professor of materials science and engineering at Stanford and senior author of the paper. “For example, if you mix blue pen ink in water, the more ink you add, the less light can pass through the water. However, in our experiment, when we dissolve tartrazine in an opaque material like muscle or skin, which normally scatters light, the more tartrazine we add, the clearer the material becomes. This goes against what we typically expect with dyes.”
Transparency lotion
Hong’s team simply dissolved the dye in an aqueous solution and created a transparency-inducing lotion of sorts. It worked, because the dye reduced the difference in refractive index between water and lipids in the skin. Then the team started massaging it gently into a bit of polymer gel that emulated the light-scattering properties of tissue. From there, they moved to thinly sliced chicken breasts and to live mice.
The “transparency lotion” needed just a few minutes to start working when applied to a mouse’s skin. Massaged into a shaven scalp, it lets the scientists see the cerebral blood vessels with laser speckle contrast imaging, a technique that normally requires removal of the scalp to work. When applied to the mouse’s abdomen, it made all the internal organs, including the liver, bladder, and small intestine, visible to the naked eye. All that was needed to reverse the effect and make the skin opaque again was washing the lotion off with water.
There were some problems, though. One of them was that tartrazine absorbed most light at wavelengths around 257 and 428 nanometers, which let us see shades of violet and blue. On the other hand, it had minimal absorption above 600 nanometers, which meant that the transparent skin tinted everything red. The second issue was the depth of penetration. The lotion worked well only at spots where the skin was thin, and couldn’t penetrate deep enough where the skin was thicker.
Finally, its formulation was not universal. It relied on finding a chemical that could match the refractive index of lipids when dissolved in water, but the exact composition of the lotion was determined through trial and error. If there’s a lot of mouse-to-mouse variation, it might make it hard to come up with a one-size-fits-all solution.
Tattoos and needles
The problem of penetrating deeper into thick skin was partially solved by making the application a bit more painful. “Using microneedle patch applicators or subcutaneous injections could help deliver the molecules through thicker layers of skin,” Hong explains. The red tint issue, he suggested, might be handled by testing different dyes. “The research in my lab is currently focused on identifying molecules with sharp absorption in the near-ultraviolet region, minimizing spectral tailing into the visible range to ensure tissue transparency without the presence of a red tone,” Hong said.
“This study has only been conducted on animals. However, if the same technique could be applied to humans, it could offer a variety of benefits in biology, diagnostics, and even cosmetics,” Hong suggests. The benefits he is focusing on include evaluating deep-seated tumors without relying on biopsies, making blood tests less stressful by making locating the veins easier, and even things like improved laser tattoo removals by allowing the pigment beneath the skin to be targeted precisely.
But there is some bad news. Even though the FD&C Yellow 5 dye is widely available, replicating Hong’s results at home and making the transparency lotion on your own is not the brightest idea. “We strongly discourage attempting this on the human skin, as the toxicology of dye molecules in humans, particularly when applied topically, has not been fully evaluated,” Hong says.
And, in the end, it might not even work. “The human skin is significantly thicker than mouse skin, with the stratum corneum, the outermost layer of the epidermis, serving as a substantial barrier that prevents effective delivery of molecules into the dermis,” Hong explains
For many creatures, having a limb caught in a predator’s mouth is usually a death sentence. Not starfish, though—they can detach the limb and leave the predator something to chew on while they crawl away. But how can they pull this off?
Starfish and some other animals (including lizards and salamanders) are capable of autonomy (shedding a limb when attacked). The biology behind this phenomenon in starfish was largely unknown until now. An international team of researchers led by Maurice Elphick, professor of Animal Physiology and Neuroscience at Queen Mary University of London, have found that a neurohormone released by starfish is largely responsible for detaching limbs that end up in a predator’s jaws.
So how does this neurohormone (specifically a neuropeptide) let the starfish get away? When a starfish is under stress from a predatory attack, this hormone is secreted, stimulating a muscle at the base of the animal’s arm that allows the arm to break off.
The researchers confirmed this neuropeptide “acts as an autotomy-promoting factor in starfish and such it is the first neuropeptide to be identified as a regulator of autotomy in animals,” as they said in a study recently published in Current Biology.
Holding on
Elphick’s team studied how the neuropeptide known as ArSK/CCK1 facilitates autonomy in the European Starfish, Asterias rubens. ArSK/CCK1 is already known to inhibit feeding behavior in A. rubens by causing the stomach to contract, and muscle contraction plays a role in limb loss. The researchers found that its ability to trigger contractions goes beyond feeding.
Starfish underwent an experiment that simulated conditions where a predator’s jaw clamped down on one arm. Clamps were placed on one of three sections on a single arm, either on the end, middle, or at the site in the base where autotomy is known to occur, also known as the autotomy plane. The starfish were then suspended by these clamps above a glass bowl of seawater. During the first part of the experiment, the starfish were left to react naturally, but during the second part, they were injected with ArSK/CCK1.
Without the injection, autotomy was seen mostly in animals that had arms that were clamped closest to the autotomy plane. There was not nearly as much of a reaction from starfish when the arms were clamped in the middle or end.
In the second half of the experiment, the clamping used before was combined with an injection of ArSK/CCK1. For comparison, some were injected with the related neuropeptide ArSK/CCK2. A staggering 85 percent of ArSK/CCK1-injected animals that were clamped in the middle of the arm or closer to the autotomy plane exhibited autonomy, and some autotomized additional arms. This only happened in about 27 percent of those injected with ArSK/CCK2.
Letting go
While ArSK/CCK1 proved to be the most effective chemical trigger for autotomy, its activity in the autotomy plane depends on certain aspects of a starfish’s anatomy.
Like all echinoderms, starfish have endoskeletons built of tiny bones, or ossicles, linked by muscles and collagen fibers that allow the animals to change posture and move. Two exclusive features only found in the autotomy plane allow this structure to break. Under the skin of the autotomy plane, there is a region where bundles of collagen fibers are positioned far apart to make breakage easier. The second of these features is a band of muscle close to the region of collagen bundles. Known as the tourniquet muscle, this muscle is responsible for the constriction that allows an arm in danger to fall off.
Analyzing starfish arm tissue while it was undergoing autotomy gave the scientists a new perspective on this process. Right after a starfish has its arm seized by a predator, ArSK/CCK1 tells nerves in the tourniquet muscle to start constricting in the region right by the autonomy plane. While this is happening, the collagen in the body wall in that region softens and breaks, and so do the muscles and ligaments that hold together ossicles. It is now thought that ArSK/CCK1 is also involved in the softening of this tissue that prepares it for breakage.
After starfish autotomize a limb, that limb eventually regenerates. The same happens in other animals that can use autotomy to their advantage (such as lizards, which also grow their tails back). In the future, finding out why some animals have the ability to regenerate may tell us why we either never evolved it or some of our ancestors lost the ability. Elphick acknowledged that there might still be other unidentified factors working together with ArSK/CCK1, but further insight could someday give us a clearer picture of this process.
“Autotomy is a key adaptation for survival that has evolved in several animal taxa,” the research team said in the same study, “[and] the findings of this study provide a seminal insight into the neural mechanisms that control this remarkable biological process,”
Enlarge/ Top row: individual steps in the reaction process. Bottom row: cartoon diagram of the top, showing the position of each DNA and RNA strand.
Hiraizumi, et. al.
While CRISPR is probably the most prominent gene-editing technology, there are a variety of others, some developed before, others since. And people have been developing CRISPR variants to perform more specialized functions, like altering specific bases. In all of these cases, researchers are trying to balance a number of competing factors: convenience; flexibility; specificity and precision for the editing; low error rates; and so on.
So, having additional options for editing can be a good thing, enabling new ways of balancing those different needs. On Wednesday, a pair of papers in Nature describe a DNA-based parasite that moves itself around bacterial genomes through a mechanism that hasn’t been previously described. It’s nowhere near ready for use in humans, but it may have some distinctive features that make it worth further development.
Going mobile
Mobile genetic elements, commonly called transposons, are quite common in many species—they make up nearly half the sequences in the human genome, for example. They are indeed mobile, showing up in new locations throughout the genome, sometimes by cutting themselves out and hopping to new locations, other times by sending a copy out to a new place in the genome. For any of this to work, they need to have an enzyme that cuts DNA and specifically recognizes the right transposon sequence to insert into the cut.
The specificity of that interaction, needed to ensure the system only inserts new copies of itself, and the cutting of DNA, are features we’d like for gene editing, which places a value on better understanding these systems.
Bacterial genomes tend to have very few transposons—the extra DNA isn’t really in keeping with the bacterial reproduction approach of “copy all the DNA as quickly as possible when there’s food around.” Yet bacterial transposons do exist, and a team of scientists based in the US and Japan identified one with a rather unusual feature. As an intermediate step in moving to a new location, the two ends of the transposon (called IS110) are linked together to form a circular piece of DNA.
In its circular form, the DNA sequences at the junction act as a signal that tells the cell to make an RNA copy of nearby DNA (termed a “promoter”). When linear, each of the two bits of DNA on either side of the junction lacks the ability to act as a signal; it only works when the transposon is circular. And the researchers confirmed that there is in fact an RNA produced by the circular form, although the RNA does not encode for any proteins.
So, the research team looked at over 100 different relatives of IS110 and found that they could all produce similar non-protein-coding RNAs, all of which shared some key features. These included stretches where nearby sections of the RNA could base-pair with each other, leaving an unpaired loop of RNA in between. Two of these loops contained sequences that either base-paired with the transposon itself or at the sites in the E. coli genome where it inserted.
That suggests that the RNA produced by the circular form of the transposon helped to act as a guide, ensuring that the transposon’s DNA was specifically used and only inserted into precise locations in the genome.
Editing without precision
To confirm this was right, the researchers developed a system where the transposon would produce a fluorescent protein when it was properly inserted into the genome. They used this to show that mutations in the loop that recognized the transposon would stop it from being inserted into the genome—and that it was possible to direct it to new locations in the genome by changing the recognition sequences in the second loop.
To show this was potentially useful for gene editing, the researchers blocked the production of the transposon’s own RNA and fed it a replacement RNA that worked. So, you could potentially use this system to insert arbitrary DNA sequences into arbitrary locations in a genome. It could also be used with targeting RNAs that caused specific DNA sequences to be deleted. All of this is potentially very useful for gene editing.
Emphasis on “potentially.” The problem is that the targeting sequences in the loops are quite short, with the insertion site targeted by a recognition sequence that’s only four to seven bases long. At the short end of this range, you’d expect that a random string of bases would have an insertion site about once every 250 bases.
That relatively low specificity showed. At the high end, various experiments could see an insertion accuracy ranging from a close-to-being-useful 94 percent down to a positively threatening 50 percent. For deletion experiments, the low end of the range was a catastrophic 32 percent accuracy. So, while this has some features of an interesting gene-editing system, there’s a lot of work to do before it could fulfill that potential. It’s possible that these recognition loops could be made longer to add the sort of specificity that would be needed for editing vertebrate genomes, but we simply don’t know at this point.
Enlarge/ The Colorado River toad, also known as the Sonoran Desert Toad.
It is becoming increasingly accepted that classic psychedelics like LSD, psilocybin, ayahuasca, and mescaline can act as antidepressants and anti-anxiety treatments in addition to causing hallucinations. They act by binding to a serotonin receptor. But there are 14 known types of serotonin receptors, and most of the research into these compounds has focused on only one of them—the one these molecules like, called 5-HT2A. (5-HT, short for 5-hydroxytryptamine, is the chemical name for serotonin.)
The Colorado River toad (Incilius alvarius), also known as the Sonoran Desert toad, secretes a psychedelic compound that likes to bind to a different serotonin receptor subtype called 5-HT1A. And that difference may be the key to developing an entirely distinct class of antidepressants.
Uncovering novel biology
Like other psychedelics, the one the toad produces decreases depression and anxiety and induces meaningful and spiritually significant experiences. It has been used clinically to treat vets with post-traumatic stress disorder and is being developed as a treatment for other neurological disorders and drug abuse. 5-HT1A is a validated therapeutic target, as approved drugs, including the antidepressant Viibryd and the anti-anxiety med Buspar, bind to it. But little is known about how psychedelics engage with this receptor and which effects it mediates, so Daniel Wacker’s lab decided to look into it.
The researchers started by making chemical modifications to the frog psychedelic and noting how each of the tweaked molecules bound to both 5-HT2A and 5-HT1A. As a group, these psychedelics are known as “designer tryptamines”—that’s tryp with a “y”, mind you—because they are metabolites of the amino acid tryptophan.
The lab made 10 variants and found one that is more than 800-fold selective about sticking to 5-HT1A as compared to 5-HT2A. That makes it a great research tool for elucidating the structure-activity relationship of the 5-HT1A receptor, as well as the molecular mechanisms behind the pharmacology of the drugs on the market that bind to it. The lab used it to explore both of those avenues. However, the variant’s ultimate utility might be as a new therapeutic for psychiatric disorders, so they tested it in mice.
Improving the lives of mice
The compound did not induce hallucinations in mice, as measured by the “head-twitch response.” But it did alleviate depression, as measured by a “chronic social defeat stress model.” In this model, for 10 days in a row, the experimental mouse was introduced to an “aggressor mouse” for “10-minute defeat bouts”; essentially, it got beat up by a bully at recess for two weeks. Understandably, after this experience, the experimental mouse tended not to be that friendly with new mice, as controls usually are. But when injected with the modified toad psychedelic, the bullied mice were more likely to interact positively with new mice they met.
Depressed mice, like depressed people, also suffer from anhedonia: a reduced ability to experience pleasure. In mice, this manifests in not taking advantage of drinking sugar water when given the opportunity. But treated bullied mice regained their preference for the sweet drink. About a third of mice seem to be “stress-resilient” in this model; the bullying doesn’t seem to phase them. The drug increased the number of resilient mice.
The 5-HT2A receptor has hogged all of the research love because it mediates the hallucinogenic effects of many popular psychedelics, so people assumed that it must mediate their therapeutic effects, too. However, Wacker argues that there is little evidence supporting this assumption. Wacker’s new toad-based psychedelic variant and its preference for the 5-HT1A receptor will help elucidate the complementary roles these two receptor subtypes play in mediating the cellular and psychological effects of psychedelic molecules. And it might provide the basis for a new tryptamine-based mental health treatment as well—one without hallucinatory side effects, disappointing as that may be to some.
Enlarge/ Prediction of the structure of a coronavirus Spike protein from a virus that causes the common cold.
Google DeepMind
Most of the activities that go on inside cells—the activities that keep us living, breathing, thinking animals—are handled by proteins. They allow cells to communicate with each other, run a cell’s basic metabolism, and help convert the information stored in DNA into even more proteins. And all of that depends on the ability of the protein’s string of amino acids to fold up into a complicated yet specific three-dimensional shape that enables it to function.
Up until this decade, understanding that 3D shape meant purifying the protein and subjecting it to a time- and labor-intensive process to determine its structure. But that changed with the work of DeepMind, one of Google’s AI divisions, which released Alpha Fold in 2021, and a similar academic effort shortly afterward. The software wasn’t perfect; it struggled with larger proteins and didn’t offer high-confidence solutions for every protein. But many of its predictions turned out to be remarkably accurate.
Even so, these structures only told half of the story. To function, almost every protein has to interact with something else—other proteins, DNA, chemicals, membranes, and more. And, while the initial version of AlphaFold could handle some protein-protein interactions, the rest remained black boxes. Today, DeepMind is announcing the availability of version 3 of AlphaFold, which has seen parts of its underlying engine either heavily modified or replaced entirely. Thanks to these changes, the software now handles various additional protein interactions and modifications.
Changing parts
The original AlphaFold relied on two underlying software functions. One of those took evolutionary limits on a protein into account. By looking at the same protein in multiple species, you can get a sense for which parts are always the same, and therefore likely to be central to its function. That centrality implies that they’re always likely to be in the same location and orientation in the protein’s structure. To do this, the original AlphaFold found as many versions of a protein as it could and lined up their sequences to look for the portions that showed little variation.
Doing so, however, is computationally expensive since the more proteins you line up, the more constraints you have to resolve. In the new version, the AlphaFold team still identified multiple related proteins but switched to largely performing alignments using pairs of protein sequences from within the set of related ones. This probably isn’t as information-rich as a multi-alignment, but it’s far more computationally efficient, and the lost information doesn’t appear to be critical to figuring out protein structures.
Using these alignments, a separate software module figured out the spatial relationships among pairs of amino acids within the target protein. Those relationships were then translated into spatial coordinates for each atom by code that took into account some of the physical properties of amino acids, like which portions of an amino acid could rotate relative to others, etc.
In AlphaFold 3, the prediction of atomic positions is handled by a diffusion module, which is trained by being given both a known structure and versions of that structure where noise (in the form of shifting the positions of some atoms) has been added. This allows the diffusion module to take the inexact locations described by relative positions and convert them into exact predictions of the location of every atom in the protein. It doesn’t need to be told the physical properties of amino acids, because it can figure out what they normally do by looking at enough structures.
(DeepMind had to train on two different levels of noise to get the diffusion module to work: one in which the locations of atoms were shifted while the general structure was left intact and a second where the noise involved shifting the large-scale structure of the protein, thus affecting the location of lots of atoms.)
During training, the team found that it took about 20,000 instances of protein structures for AlphaFold 3 to get about 97 percent of a set of test structures right. By 60,000 instances, it started getting protein-protein interfaces correct at that frequency, too. And, critically, it started getting proteins complexed with other molecules right, as well.
Enlarge/ A photo of Braarudosphaera bigelowii with the nitroplast indicated by an arrowhead.
The complex cells that underlie animals and plants have a large collection of what are called organelles—compartments surrounded by membranes that perform specialized functions. Two of these were formed through a process called endosymbiosis, in which a once free-living organism is incorporated into a cell. These are the mitochondrion, where a former bacteria now handles the task of converting chemical energy into useful forms, and the chloroplast, where photosynthesis happens.
The fact that there are only a few cases of organelles that evolved through endosymbiosis suggests that it’s an extremely rare event. Yet researchers may have found a new case, in which an organelle devoted to fixing nitrogen from the atmosphere is in the process of evolving. The resulting organelle, termed a nitroplast, is still in the process of specialization.
Getting nitrogen
Nitrogen is one of the elements central to life. Every DNA base, every amino acid in a protein contains at least one, and often several, nitrogen atoms. But nitrogen is remarkably difficult for life to get ahold of. N2 molecules might be extremely abundant in our atmosphere, but they’re extremely difficult to break apart. The enzymes that can, called nitrogenases, are only found in bacteria, and they don’t work in the presence of oxygen. Other organisms have to get nitrogen from their environment, which is one of the reasons we use so much energy to supply nitrogen fertilizers to many crops.
Some plants (notably legumes), however, can obtain nitrogen via a symbiotic relationship with bacteria. These plants form specialized nodules that provide a habitat for the nitrogen-producing bacteria. This relationship is a form of endosymbiosis, where microbes take up residence inside an organism’s body or cells, with each organism typically providing chemicals that the other needs.
In more extreme cases, endosymbiosis can become obligatory. with neither organism able to survive without the other. In many insects, endosymbionts are passed on to offspring during the production of eggs, and the microbes themselves often lack key genes that would allow them to live independently.
But even states like this fall short of the situation found in mitochondria and chloroplasts. These organelles are thoroughly integrated into the cell, being duplicated and distributed when cells divide. They also have minimal genomes, with most of their proteins made by the cell and imported into the organelles. This level of integration is the product of over a billion years of evolution since the endosymbiotic relationship first started.
It’s also apparently a difficult process, based on its apparent rarity. Beyond mitochondria and chloroplasts, there’s only one confirmed example of a more recent endosymbiosis between eukaryotes and a bacterial species. (There are a number of cases where eukaryotic algae have been incorporated by other eukaryotes. Because these cells have compatible genetics, this occurs with a higher frequency.)
That’s why finding another example is such an exciting prospect.
Enlarge/ The plague bacteria, Yersina pestis, is a close relative of the toxin-producing species studied here.
Life-forms with no brain are capable of some astounding things. It might sound like sci-fi nightmare fuel, but some bacteria can wage kamikaze chemical warfare.
Pathogenic bacteria make us sick by secreting toxins. While the release of smaller toxin molecules is well understood, methods of releasing larger toxin molecules have mostly eluded us until now. Researcher Stefan Raunser, director of the Max Planck Institute of Molecular Physiology, and his team finally found out how the insect pathogen Yersinia entomophaga (which attacks beetles) releases its large-molecule toxin.
They found that designated “soldier cells” sacrifice themselves and explode to deploy the poison inside their victim. “YenTc appears to be the first example of an anti-eukaryotic toxin using this newly established type of secretion system,” the researchers said in a study recently published in Nature.
Silent and deadly
Y. entomophaga is part of the Yersinia genus, relatives of the plague bacteria, which produce what are known as Tc toxins. Their molecules are huge as far as bacterial toxins go, but, like most smaller toxin molecules, they still need to make it through the bacteria’s three cell membranes before they escape to damage the host. Raunser had already found in a previous study that Tc toxin molecules do show up outside the bacteria. What he wanted to see next was how and when they exit the bacteria that makes them.
To find out what kind of environment is ideal for Y. entomophaga to release YenTC, the bacteria were placed in acidic (PH under 7) and alkaline (PH over 7) mediums. While they did not release much in the acidic medium, the bacteria thrived in the high PH of the alkaline medium, and increasing the PH led it to release even more of the toxin. The higher PH environment in a beetle is around the mid-end of its gut, so it is now thought that most of the toxin is liberated when the bacteria reach that area.
How YenTc is released was more difficult to determine. When the research team used mass spectrometry to take a closer look at the toxin, they found that it was missing something: There was no signal sequence that indicated to the bacteria that the protein needed to be transported outside the bacterium. Signal sequences, also known as signal peptides, are kind of like built-in tags for secretion. They are in charge of connecting the proteins (toxins are proteins) to a complex at the innermost cell membrane that pushes them through. But YenTC apparently doesn’t need a signal sequence to export its toxins into the host.
About to explode
So how does this insect killer release YenTc, its most formidable toxin? The first test was a process of elimination. While YenTc has no signal sequence, the bacteria have different secretion systems for other toxins that it releases. Raunser thought that knocking out these secretion systems using gene editing could possibly reveal which one was responsible for secreting YenTc. Every secretion system in Y. entomophaga was knocked out until no more were left, yet the bacteria were still able to secrete YenTc.
The researchers then used fluorescence microscopy to observe the bacteria releasing its toxin. They inserted a gene that encodes a fluorescent protein into the toxin gene so the bacteria would glow when making the toxin. While not all Y. entomophaga cells produced YenTc, those that did (and so glowed) tended to be larger and more sluggish. To induce secretion, PH was raised to alkaline levels. Non-producing cells went about their business, but YenTc-expressing cells only took minutes to collapse and release the toxin.
This is what’s called a lytic secretion system, which involves the rupture of cell walls or membranes to release toxins.
“This prime example of self-destructive cooperation in bacteria demonstrates that YenTc release is the result of a controlled lysis strictly dedicated to toxin release rather than a typical secretion process, explaining our initially perplexing observation of atypical extracellular proteins,” the researchers said in the same study.
Yersinia also includes pathogenic bacteria that cause tuberculosis and bubonic plague, diseases that have devastated humans. Now that the secretion mechanism of one Yersinia species has been found out, Raunser wants to study more of them, along with other types of pathogens, to see if any others have kamikaze soldier cells that use the same lytic mechanism of releasing toxins.
The discovery of Y. entomophaga’s exploding cells could eventually mean human treatments that target kamikaze cells. In the meantime, we can at least be relieved we aren’t beetles.
