biochemistry

dna-based-bacterial-parasite-uses-completely-new-dna-editing-method

DNA-based bacterial parasite uses completely new DNA-editing method

biochemistry, Biology, DNA, gene editing, molecular biology, RNA, Science / /
Top row: individual steps in the reaction process. Bottom row: cartoon diagram of the top, showing the position of each DNA and RNA strand.

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.

DNA-based bacterial parasite uses completely new DNA-editing method Read More »

chemical-tweaks-to-a-toad-hallucinogen-turns-it-into-a-potential-drug

Chemical tweaks to a toad hallucinogen turns it into a potential drug

biochemistry, Biology, hallucinogen, health, neurobiology, psychedelic, Science, toads / /

No licking toads! —

Targets a different serotonin receptor from other popular hallucinogens.

–

Image of the face of a large toad.

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.

Nature, 2024.  DOI: 10.1038/s41586-024-07403-2

Chemical tweaks to a toad hallucinogen turns it into a potential drug Read More »

deepmind-adds-a-diffusion-engine-to-latest-protein-folding-software

DeepMind adds a diffusion engine to latest protein-folding software

AI, biochemistry, Biology, deepmind, Google, protein folding, Science / /

Added complexity —

Major under-the-hood changes let AlphaFold handle protein-DNA complexes and more.

–

image of a complicated mix of lines and ribbons arranged in a complicated 3D structure.

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.

DeepMind adds a diffusion engine to latest protein-folding software Read More »

researchers-find-a-new-organelle-evolving

Researchers find a new organelle evolving

biochemistry, Biology, endosymbiosis, evolution, nitrogen, organelle, Science / /
Image of a single celled algae.

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.

Researchers find a new organelle evolving Read More »

kamikaze-bacteria-explode-into-bursts-of-lethal-toxins

Kamikaze bacteria explode into bursts of lethal toxins

biochemistry, Biology, Genetics, infections, plague, Science, toxins / /

The needs of the many… —

If you make a big enough toxin, it’s difficult to get it out of the cells.

–

Colorized scanning electron microscope, SEM, image of Yersinia pestis bacteria

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.

Nature Microbiology, 2024. DOI: 10.1038/s41564-023-01571-z

Kamikaze bacteria explode into bursts of lethal toxins Read More »

tiny-cracks-in-rocks-may-have-concentrated-chemicals-needed-for-life

Tiny cracks in rocks may have concentrated chemicals needed for life

biochemistry, Biology, chemistry, geology, origin of life, Science / /
Cartoon of a geologically active area, showing sub-surface channels in different colors to represent various temperatures.

Enlarge / Active geology could have helped purify key chemicals needed for life.

Christof B. Mast

In some ways, the origin of life is looking much less mystifying than it was a few decades ago. Researchers have figured out how some of the fundamental molecules needed for life can form via reactions that start with extremely simple chemicals that were likely to have been present on the early Earth. (We’ve covered at least one of many examples of this sort of work.)

But that research has led to somewhat subtler but no less challenging questions. While these reactions will form key components of DNA and protein, those are often just one part of a complicated mix of reaction products. And often, to get something truly biologically relevant, they’ll have to react with some other molecules, each of which is part of its own complicated mix of reaction products. By the time these are all brought together, the key molecules may only represent a tiny fraction of the total list of chemicals present.

So, forming a more life-like chemistry still seems like a challenge. But a group of German chemists is now suggesting that the Earth itself provides a solution. Warm fluids moving through tiny fissures in rocks can potentially separate out mixes of chemicals, enriching some individual chemicals by three orders of magnitude.

Feeling the heat (and the solvent)

Even in the lab, it’s relatively rare for chemical reactions to produce just a single product. But there are lots of ways to purify out exactly what you want. Even closely related chemicals will often differ in their solubility in different solvents and in their tendency to stick to various glasses or ceramics, etc. The temperature can also influence all of those. So, chemists can use these properties as tools to fish a specific chemical out of a reaction mixture.

But, as far as the history of life is concerned, chemists are a relatively recent development—they weren’t available to purify important chemicals back before life had gotten started. Which raises the question of how the chemical building blocks of life ever reached the sorts of concentrations needed to do anything interesting.

The key insight behind this new work is that something similar to lab equipment exists naturally on Earth. Many rocks are laced with cracks, channels, and fissures that allow fluid to flow through them. In geologically active areas, that fluid is often warm, creating temperature gradients as it flows away from the heat source. And, as fluid moves through different rock types, the chemical environment changes. The walls of the fissures will have different chemical properties, and different salts may end up dissolved in the fluid.

All of that can provide conditions where some chemicals move more rapidly through the fluid, while others tend to stay where they started. And that has the potential to separate out key chemicals from the reaction mixes that produce the components of life.

But having the potential is very different from clearly working. So, the researchers decided to put the idea to the test.

Tiny cracks in rocks may have concentrated chemicals needed for life Read More »

proteins-let-cells-remember-how-well-their-last-division-went

Proteins let cells remember how well their last division went

biochemistry, Biology, cancer, DNA damage, mitosis, Science / /

Well, that went badly —

Scientists find a “mitotic stopwatch” that lets individual cells remember something.

–

Image of a stopwatch against a blue-grey background.

When we talk about memories in biology, we tend to focus on the brain and the storage of information in neurons. But there are lots of other memories that persist within our cells. Cells remember their developmental history, whether they’ve been exposed to pathogens, and so on. And that raises a question that has been challenging to answer: How does something as fundamental as a cell hold on to information across multiple divisions?

There’s no one answer, and the details are really difficult to work out in many cases. But scientists have now worked out one memory system in detail. Cells are able to remember when their parent had a difficult time dividing—a problem that’s often associated with DNA damage and cancer. And, if the problems are substantial enough, the two cells that result from a division will stop dividing themselves.

Setting a timer

In multicellular organisms, cell division is very carefully regulated. Uncontrolled division is the hallmark of cancers. But problems with the individual segments of division—things like copying DNA, repairing any damage, making sure each daughter cell gets the right number of chromosomes—can lead to mutations. So, the cell division process includes lots of checkpoints where the cell makes sure everything has worked properly.

But if a cell makes it through all the checkpoints, it’s presumably all good, right? Not entirely, as it turns out.

Mitosis is the portion of cell division where the duplicated chromosomes get separated out to each of the daughter cells. Spending a lot of time in mitosis can mean that the chromosomes have picked up damage, which may cause problems in the future. And prior research found that some cells derived from the retina will register when mitosis takes too long, and the daughter cells will stop dividing.

The new work, done by a team of researchers in Okinawa, Japan, and San Diego, started by showing that this behavior wasn’t limited to retinal cells—it seems to be a general response to a slow mitosis. Careful timing experiments showed that the longer cells spent trying to undergo mitosis, the more likely the daughter cells would be to stop dividing. The researchers term this system a “mitotic stopwatch.”

So, how does a cell operate a stopwatch? It’s not like it can ask Siri to set a timer—it’s largely stuck working with nucleic acids and proteins.

It turns out that, like many things relayed to cell division, the answer comes down to a protein named p53. It’s a protein that’s key to many pathways that detect damage to cells and stop them from dividing if there are problems. (You may recall it from our recent coverage of the development of elephant stem cells.)

A stopwatch made of proteins

The researchers found that, while mitosis was going on, p53 started showing up in a complex with two other proteins (ubiquitin-specific protease 28 and the creatively named p53-binding protein 1). If you made mutations in one of the proteins that blocked this complex from forming, the mitotic stopwatch stopped ticking. This three-protein complex only started building up to significant levels if mitosis took longer than usual, and it remained stable once it formed so that it would get passed on to the daughter cells once cell division was completed.

So, why does this complex form only when mitosis takes longer than usual? The key turned out to be a protein called a kinase, which attaches a phosphate to other proteins. The researchers screened chemicals that inhibit specific kinases that are active during mitosis and DNA repair, and found a specific one that was needed for the mitotic stopwatch. In the absence of this kinase (PLK1, for the curious), the three-protein complex doesn’t form.

So, the researchers think that the stopwatch looks like this: during mitosis, the kinase slowly attaches a phosphate to one of the proteins, allowing it to form the three-protein complex. If mitosis gets done quickly enough, the levels of this complex don’t get very high, and it has no effect on the cell. But if mitosis goes more quickly, then the complex starts building up, and it’s stable enough that it’s still around in both daughter cells. The existence of the complex helps stabilize the p53 protein, allowing it to stop future cell divisions once it’s present at high enough levels.

Consistent with this idea, all three of the proteins in the complex are tumor suppressors, meaning that mutations in them make tumor formation more likely. The researchers confirmed that the mitotic stopwatch was frequently defective in tumor samples.

So, that’s how individual cells manage to store one of their memories—the memory of problems with cell division. The mitotic stopwatch, however, is just one of the memory storage systems, with completely separate systems handling different memories. And, at the same time this is happening, a large number of other pathways also feed into the activity of p53. So, while the mitotic stopwatch may efficiently handle one specific type of problem, it’s integrated into a lot of additional, complex systems operating in the cell.

Science, 2024. DOI: 10.1126/science.add9528  (About DOIs).

Proteins let cells remember how well their last division went Read More »

new-e.-coli-strain-will-accelerate-evolution-of-the-genes-of-your-choice

New E. coli strain will accelerate evolution of the genes of your choice

biochemistry, Biology, E. coli, evolution, mutation rate, mutations, Science / /

Making mutants —

Strain eliminates the trade-offs of a high mutation rate.

–

Woman holding a plate of bacteria with clusters of bacteria on it.

Genetic mutations are essential for innovation and evolution, yet too many—or the wrong ones—can be fatal. So researchers at Cambridge established a synthetic “orthogonal” DNA replication system in E. coli that they can use as a risk-free way to generate and study such mutations. It is orthogonal because it is completely separate from the system that E. coli uses to copy its actual genome, which contains the genes E. coli needs to survive.

The genes in the orthogonal system are copied with an extraordinarily error-prone DNA replication enzyme, which spurs rapid evolution by generating many random mutations. This goes on while E. coli’s genes are replicated by its normal high-fidelity DNA copying enzyme. The two enzymes work alongside each other, each doing their own thing but not interfering with the other’s genes.

Engineering rapid mutation

Such a cool idea, right? The scientists stole it from nature. Yeast already has a system like this, with a set of genes copied by a dedicated enzyme that doesn’t replicate the rest of the genome. But E. coli is much easier to work with than yeast, and its population can double in 20 minutes, so you can get a lot of rounds of replication and evolution done fast.

The researchers generated the system by pillaging a phage—a virus that infects E. coli. They took out all of the phage genes that allow the phage to grow uncontrollably until it bursts the E. coli cell it infected open. The engineering left only a cassette containing the genes responsible for copying the phage genome. Once this cassette was inserted into the E. coli genome, it could simultaneously replicate at least three different strings of genes placed next to it in the DNA, maintaining them for over a hundred generations—all while leaving the rest of the E. coli genome to be copied by other enzymes.

The scientists then tweaked the mutation rate of the orthogonal DNA-replicating enzyme, eventually enhancing it 1,000-fold. To test if the system could be used to evolve new functions, they inserted a gene for resistance to one antibiotic and saw how long it took for that gene to mutate into one conferring resistance to a different antibiotic. Within twelve days, they got 150 times more resistance to the new antibiotic. They also inserted the gene encoding green fluorescent protein and increased its fluorescence over 1,000-fold in five days.

Evolving detoxification

Not 20 pages later, in the same issue of Science, Frances Arnold’s lab has a paper that provides evidence of how powerful this approach could be. This team directed the evolution of an enzyme the old-fashioned way: through sequential rounds of random mutagenesis and selection for the desired trait. Arnold won The Nobel Prize in Chemistry 2018 for the directed evolution of enzymes, so she knows what she’s about. In this recent work, her lab generated an enzyme that can biodegrade volatile methyl siloxanes. We make megatons of these compounds every year to stick in cleaning products, shampoos and lotions, and industrial products, but they linger in the environment. They contain carbon-silicon bonds, which were never a thing until humans made them about 80 years ago; since nature never made these bonds, there is no natural way to break them, either.

“Directed evolution with siloxane was particularly challenging,” the authors note in their introduction, for various technical reasons. “We started from an enzyme we had previously engineered for other chemistry on siloxanes—that enzyme, unlike the natural enzyme, showed a tiny bit of activity for siloxane Si-C bond cleavage. The overall project, however, from initial discovery to figuring out how to measure what we wanted, took several years,” Arnold said. And it is only the first step in possibly rendering siloxanes biodegradable. The accelerated continuous evolution that the new orthologous system allows will hopefully greatly facilitate the development of enzymes and other proteins like this that will have applications in research, medicine, and industry.

We do not (yet) have machines that can efficiently assemble long stretches of DNA or make proteins. But cells do these things extremely efficiently, and E. coli cells have long been the ones used in the lab as little factories, churning out whatever genes or proteins researchers program into them. Now E. coli can be used for one more molecular task—they can be little hotbeds of evolution.

Science, 2024.  DOI: 10.1126/science.adi5554, 10.1126/science.adk1281

New E. coli strain will accelerate evolution of the genes of your choice Read More »

gotta-go?-we’ve-finally-found-out-what-makes-urine-yellow

Gotta go? We’ve finally found out what makes urine yellow

biochemistry, Biology, Genomics, Science, urine / /

It isn’t from eating corn —

The yellow color comes from bacteria metabolizing waste from red blood cells.

–

Image of a series of scientific sample tubes filled with yellow liquids.

There are many mysteries in life that we end up shrugging off. Why is urine yellow? It just is, right? Rather than flush that 125-year-old question down the toilet, scientists sought out the answer, discovering a previously unknown microbial enzyme was to blame.

The enzyme that has eluded us for so long is now known as bilirubin reductase. It was identified by researcher and assistant professor Brantley Hall of the University of Maryland, who was part of a team based at the university and the National Institutes of Health.

Bilirubin is an orange pigment released by red blood cells after they die. Gut microbes then use bilirubin reductase to break down bilirubin into colorless urobilinogen, which degrades into yellowish urobilin, giving urine that infamous hue. While urobilin previously had an association with the color of urine, the enzyme that starts the process by producing urobilinogen was unknown until now.

“Though it was previously thought that multiple enzymes were involved in the reduction of bilirubin, our results support the finding that a single enzyme performs the reduction of bilirubin to urobilinogen,” the research team said in a study recently published in Nature Microbiology.

Gut feeling

Because some gut bacteria had been known to reduce bilirubin, Hall and his team knew where to start but wanted to fill in the unknowns by finding out which particular species actually do this—and how. This meant they had to find the gene responsible for encoding bilirubin reductase.

Previous studies had found that the species Clostridiodes difficile was capable of reducing bilirubin (though the mechanism it used was unknown). Using C. difficile as a basis for comparison, the team cultured different species of gut bacteria and exposed them to bilirubin to see whether that bacteria could produce urobilinogen, detecting its presence using a fluorescence assay.

The fluorescence assay told Hall and his colleagues that there were nine strains within the tested species that they thought were capable of reducing bilirubin, although how these bacteria were breaking it down was still unclear.  After the fluorescence assay, the genomes of the most closely related strains were analyzed,  and several turned out to share a gene that encoded an enzyme that could reduce bilirubin—bilirubin reductase.

Bacterial strains that metabolized bilirubin using bilirubin reductase all came from species that were found to belong to a single clade (the researchers informally referred to it as the bilirubin reductase clade). Within that clade, most of these species are from the class Clostridia in the phylum Firmicutes, a phylum of bacteria important to gut health.

More than … you know

The discovery of bilirubin reductase goes beyond the origin of urine color. After identifying the enzyme, the researchers found out that, while bilirubin reductase is present in healthy adults, there is a deficit in newborns and adults with inflammatory bowel disease, which could eventually influence future treatments

By sequencing infant gut genomes, Hall and his team saw that bilirubin reductase was often missing during the first few months of life. Too much bilirubin building up in the blood turns the skin and the whites of the eyes yellow, a symptom known as jaundice. Most infants have some level of jaundice, but it usually goes away on its own.

The absence of bilirubin reductase is also associated with pigmented gallstones in adults with inflammatory bowel disease (inflammatory bowel disease or IBD is a general term that can refer to several different diagnoses). Sequencing adult gut genomes showed that there was a deficit of this enzyme in most patients with Crohn’s disease or ulcerative colitis whose gut genomes were sequenced.

“With the knowledge of the species, genes, and enzymes involved in bilirubin reduction, future research can now focus on the extent to which gut microbial bilirubin metabolism affects…the role of bilirubin reduction in health and disease,” the researchers said in the same study.

There is still more research to be done on bilirubin reductase and the health implications it could have. The team thinks there may be a link between the amount of urobilin produced in the body and insulin resistance, obesity, heart disease, and even heart failure. Next to that, we finally know why urine is yellow.

Nature Microbiology, 2023. DOI: 10.1038/s41564-023-01549-x

Gotta go? We’ve finally found out what makes urine yellow Read More »

injection-of-“smart-insulin”-regulates-blood-glucose-levels-for-one-week

Injection of “smart insulin” regulates blood glucose levels for one week

biochemistry, diabetes, glucose, health, insulin, Science / /

Sugary treat treatment —

Tests in animals show the material works like the body’s own system.

–

Image of a syringe above three drug vials

Enlarge / Smart insulin has the potential to make injections far less frequent.

People with type I diabetes have to inject themselves multiple times a day with manufactured insulin to maintain healthy levels of the hormone, as their bodies do not naturally produce enough. The injections also have to be timed in response to eating and exercise, as any consumption or use of glucose has to be managed.

Research into glucose-responsive insulin, or “smart” insulin, hopes to improve the quality of life for people with type I diabetes by developing a form of insulin that needs to be injected less frequently, while providing control of blood-glucose levels over a longer period of time.

A team at Zhejiang University, China, has recently released a study documenting an improved smart insulin system in animal models—the current work doesn’t involve any human testing. Their insulin was able to regulate blood-glucose levels for a week in diabetic mice and minipigs after a single subcutaneous injection.

“Theoretically, [smart insulin is] incredibly important going forward,” said Steve Bain, clinical director of the Diabetes Research Unit in Swansea University, who was not involved in the study. “It would be a game changer.”

Polymer cage

The new smart insulin is based on a form of insulin modified with gluconic acid, which forms a complex with a polymer through chemical bonds and strong electrostatic attraction. When insulin is trapped in the polymer, its signaling function is blocked, allowing a week’s worth of insulin to be given via a single injection without a risk of overdose.

Crucial to the “glucose responsive” nature of this system is the fact that the chemical structures of glucose and gluconic acid are extremely similar, meaning the two molecules bind in very similar ways. When glucose meets the insulin-polymer complex, it can displace some of the bound insulin and form its own chemical bonds to the polymer. Glucose binding also disrupts the electrostatic attraction and further promotes insulin release.

By preferentially binding to the polymer, the glucose is able to trigger the release of insulin. And the extent of this insulin release depends on how much glucose is present: between meals, when the blood-glucose level is fairly low, only a small amount of insulin is released. This is known as basal insulin and is needed for baseline regulation of blood sugar.

But after a meal, when blood-glucose spikes, much more insulin is released. The body can now regulate the extra sugar properly, preventing abnormally high levels of glucose—known as hyperglycemia. Long-term effects of hyperglycemia in humans include nerve damage to the hands and feet and permanent damage to eyesight.

This system mimics the body’s natural process, in which insulin is also released in response to glucose.

Better regulation than standard insulin

The new smart insulin was tested in five mice and three minipigs—minipigs are often used as an animal model that’s more physiologically similar to humans. One of the three minipigs received a slightly lower dose of smart insulin, and the other two received a higher dose. The lower-dose pig showed the best response: its blood-glucose levels were tightly controlled and returned to a healthy value after meals.

During treatment, the other two pigs had glucose levels that were still above the range seen in healthy animals, although they were greatly reduced compared to pre-injection levels. The regulation of blood-glucose was also tighter compared to daily insulin injections.

It should be noted, though, that the minipig with the best response also had the lowest blood-glucose levels before treatment, which may explain why it seemed to work so well in this animal.

Crucially, these effects were all long lasting—better regulation could be seen a week after treatment. And injecting the animals with the smart insulin didn’t result in a significant immune response, which can be a common pitfall when introducing biomaterials to animals or humans.

Don’t sugarcoat it

The study is not without its limitations. Although long-term glucose regulation was seen in the mice and minipigs examined, only a few animals were involved in the study—five mice and three minipigs. And of course, there’s always the risk that the results of animal studies don’t completely track over to clinical trials in humans. “We have to accept that these are animal studies, and so going across to humans is always a bit of an issue,” said Bain.

Although more research is required before this smart insulin system can be tested in humans, this work is a promising step forward in the field.

Nature Biomedical Engineering, 2023. DOI: 10.1038/s41551-023-01138-7

Ivan Paul is a freelance writer based in the UK, finishing his PhD in cancer research. He is on X @ivan_paul_.

Injection of “smart insulin” regulates blood glucose levels for one week Read More »