Will It Sequence? What grows on your food?

And, what makes a tardigrade so tough?
07 June 2024
Presented by Will Tingle
Production by Will Tingle.

FOOD_MARKET

A food market with a wide array of different vegetables.

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In this episode of Naked Genetics: What the latest genetics research has to say about restless leg syndrome; I put Illumina to the test and ask, ‘what really grows on our food?’; And, what makes a tardigrade so tough?

In this episode

Legs

Restless leg syndrome study reveals new risk genes
Steven Bell, University of Cambridge

This month’s news looks into a genetic study into restless leg syndrome, or RLS, which causes those afflicted to have sudden strong urges to move their legs. This long-term neurological condition can have very detrimental effects on the sufferer’s health, but research into its risk factors is not nearly as advanced as other illnesses. But, that’s something a new study just published in Nature Genetics has changed. Co-author on the study is the University of Cambridge’s Steven Bell...

Steven - Restless leg syndrome or RLS as it's often referred to, is a neurological condition that causes a strong, often irresistible urge to move your legs, especially when you're resting or trying to sleep. And people with restless leg syndrome frequently describe it as a tingling, crawling, or creeping sensation inside their legs, similar to insects crawling under their skin. In more severe cases, it can feel like a deep aching, throbbing, or even burning, making it almost impossible to stay still. And it's thought to affect up to 1 in 10 in the European and North American population. And many people with RLS actually suffer in silence as their symptoms are frequently dismissed as fidgeting. So as you can imagine, this is incredibly frustrating and isolating, and the constant need to move one's legs disrupts their sleep, which can lead to chronic fatigue, irritability, and difficulty concentrating during the day. So it's hardly surprising that living with RLS also takes an emotional toll on people causing anxiety and depression due to relentless discomfort and the lack of understanding from others. And it's one of those conditions that also affects people's partners as well as the constant movements and need to get up will disrupt their sleep also.

Will - Had there been any research into this into the past then? Because surely if it's this detrimental, we'd have wanted to know more about it.

Steven - There have been multiple studies in the past, including genetic studies that have shown high concordance in identical twins as well as a positive family history in up to 60% of individuals who have RLS, which suggests a strong genetic component. And it's been about 20 years since the first genome wide association study of RLS was performed. And this identified three areas of the genome associated with RLS. And since then, the number has steadily trickled forward to 22 areas in total at the last count in 2020.

Will - Why did you, and people before you, decide to take a genetic approach to this particular illness?

Steven - That's a great question, and because RLS has so a few treatments available for it, one means of identifying potential avenues and routes to treatment is taking an agnostic approach via genome-wide association studies where we can identify specific areas of the genome associated with risk of RLS and see the proteins involved in this and whether these are tractable to modulate or targeting drug and therefore serve as a new treatment option for people with RLS. One of the other major differentiating factors is that our investigation was the first one to look at the genetic architecture of restless leg syndrome separately for men and women. And this is quite important as RLS affects women more frequently than men, with a reported prevalence increase of 30 to 50 percent in women. But up until now there'd been no systematic comparison of the genetic risk between sexes.

Will - The previous studies kind of tapped out at 20 risk genes, 20 genes that you'd want to look at, in order to find out whether or not you may or may not be at risk of having RLS. Did this study bump that number up?

Steven - Yes, significantly so. So we identified 161 genetic risk loci that were associated with restless leg syndrome and of these 139 were newly discovered, which represents an eightfold increase in the total number of areas of the genome that we now know to be associated with restless leg syndrome.

Will - There's a disparity between male and females. Were any of those new risk areas found on the sex chromosome?

Steven - So there were free associations on the X chromosome that we found to differ in risk of RLS overall. There were six loci that were statistically significantly different across the genome and between sex. However, these were direct concordance. That is, they were always at risk increasing in both men and women. It was just of a different magnitude and there was no consistent direction of higher risk in men always, or women always. But one of the things that were interesting about the analysis of sex were, despite the fact that we've seen a strong genetic correlation in risk of RLS across the genome in men and women, we observed that the heritability, that is the proportion of variation in restless leg syndrome that's attributed to genetic factors, was actually higher in women. So it was 32% compared to 13% for men. This emphasised the importance of studying such phenomena in genetically susceptible individuals and hopefully this will pave the way for further work to better understand precisely what these risk factors are and anything that can be done to reduce the sex disparities in the condition.

Will - Now that you've got new risk areas in the genes that might dictate whether or not you are at risk of getting RLS, what can you then do with that?

Steven - That's the million pound question really isn't it, <laugh>? And unfortunately, as practically in all studies of this kind, the short answer is there's nothing that can be done at least immediately to translate these directly to the clinic. However, a slightly more convoluted answer to that is that whilst work still needs to be done to translate our findings to the clinic, our results could drastically shorten the time taken for new treatments to be available to those suffering from more or less in the future. For example, via repurposing existing drugs to be used in the treatment of restless leg syndrome or helping to prioritise the development of entirely new therapies. Our work actually involved mapping the genes that we identified to be associated with restless leg syndrome with medications that target them. And examples that we found were two glutamate receptors. GRIA4, a target of telampanel, and GRIA1, which is a target of perampanel, both of which are anti-convulsant therapies used in the treatment of epilepsy. And as these drugs have already undergone all of the extensive safety testing and are used in existing clinical practice, the trials that would be needed to test for their efficacy in RLS would be quite drastically shorter and deliver a fraction of the time and cost. And I guess all the other genomic loci that we identified, they still can help in prioritising candidates for drugs development as only about 10% of drug development programs eventually receive approval. And an analysis published in Nature back in April showed that the probability of success for a drug that has genetic support is 2.6 times greater. And crucially, this is most pronounced in phase two and phase three clinical trials. So it's not to say that the path from obtaining a locus identified during a GWAS to a drug target is ever going to be straightforward or easy far from it because extensive studies are needed to identify the causal variance and understand their functional effects. But we've been able to provide a short list of potential candidates to be investigated further and have made our data available as well, so that further computational analysis in this area can be formed by others to support or deprioritise members of this list.

Potatoes

08:59 - What microbes actually grow on your food?

And can Illumina identify food just by its microbes?

What microbes actually grow on your food?
Trevor Ho, Illumina

I don't really know how you got in here, but welcome to my kitchen. I suppose you just caught me in the middle of preparing a delicious and very healthy meal that purely coincidentally you cannot see. Now, whilst I love preparing my fresh, very fresh and so healthy meals, let's be honest, there comes a time when the leftovers, the food that has been out for a couple of days on the side, maybe look a bit tempting. We're busy people and a fair few of us were all students once. We don't talk about what happened back then. These things can happen. And let's be honest, until you see the white puffs of disgusting random fungi emerge on the food, surely it's fair game, right? I mean, how bad for you can it really be? What really grows on your food? Now this to me is a particularly interesting question, but one that has immaculate timing because genetic sequencing company Illumina, has just handed me far more power than anyone man should ever have. I have in my possession five samples of everyday household food, food that you and I would use in any meal without thinking, except I've left them out for two to three days to see what might grow on them. I'm now going to take those samples over to Illumina and see what exactly is growing on my food. But there is a slight twist.

Will - Here's the thing. I could very easily take a bag full of swabs of old food to Illumina and ask, 'Hey, what's growing on this?' And that would be no problem at all for them to answer, but I don't think that's flexing their muscles enough, so I'm just not going to tell them what the food is. And so the question becomes twofold. One, what is growing on my food? But two, can they use their best detective intuition to work out what the food was purely based on what was growing on it. Now that sounds like more of a challenge to me. Now, if you'll excuse me, I have to go hand a bag of old food to a complete stranger.

Trevor - Hello Will, my name's Trevor. I work here at Illumina as a senior scientist. So yeah, I'm happy to grab your samples, which you're ready for me to try and sequence.

Will - Lovely. I have this bag full of rotting food. No one has ever been happy to take this from me before, but I do hope you can make something of it.

Trevor - Thanks very much. I see like a couple of tubes in front of me. I'll grab them into the lab and then I'll try and take some of the material out and then see where I can sequence them. Thank you.

Will - The bag of detritus has been successfully left at Illumina and they've asked for a couple of weeks to sequence it and see what they can find. But I'm going to give them four seconds. Okay. Let's see how they did.

Will - Now that you've received my mystery bag of strange samples of food, could you talk our audience through what sequencing you did on them?

Trevor - Sure. In general, the sequencing that we apply to those samples is called 16S sequencing, and basically 16S is often described as a housekeeping gene. Every bacteria must have one of them. But the interesting thing is that as bacteria evolve into different species, there are minute differences that accumulate on this housekeeping component of their genome over time. So by reading information from this 16S region or just like a really important essential region, then we're able to tease apart which bacteria it is. You can imagine as every bacteria have a signature region within this part of the genome. The kind of library prep or the sample prep that we apply to them is basically making copies and copies and copies of this very essential region so that we can read them accurately and with high level of confidence.

Will - You did all this and you found some interesting looking bacterial microorganisms. Was that the only tests you did?

Trevor - I'm glad I bring up Will. Actually no. So I just mentioned that we try to study the bacterial population by 16S sequencing. We actually have a very similar technology that tries to look for the fungi population, and that's called ITS sequencing. It's the same concept as before, copy paste. There's a region across all fungi that are just pretty much consistent, but surprisingly we didn't really find any. So across all the five samples, we could not identify fungi from those things. I guess that is an indication that maybe just for only three days, it's not sufficient for fungi to cause the food service and become the major source of infection. But that's also part of the reason why I was able to rule out white bread because I would've expected mould to grow on it and that I'll be able to get something out of it. But that didn't happen.

Will - Given that, you know, this food sample was prepared in perhaps not the most sterile environment being my kitchen. What were you able to tease apart from the microorganisms growing on these samples?

Trevor - So it's quite clear that the bacteria that are growing on food samples that you gave me are very diverse and probably some of them are things that we've never seen before. So it's really hard to pinpoint what's exactly in there. We can only have some glance of information from the species that we can actually identify and in fact that weren't that many that we can do. But one thing that struck me is there is one species of bacteria called Acinetobacter Johnsonii, that is very prevalent across all the five samples I received. And when I look up like when I Google and try to figure out, okay, what is this like, um, bacterial species, it lives everywhere. It lives in the sea, it lives on the soil. It is even reported to be isolated from human skins. So it's very difficult for me to guess where that bacteria actually comes from. Does it come from your hands or does it come from the air that is just floating around and then somehow sticks in the food and then they grow and then they colonise their food surface? I am not entirely sure. But what I do suspect is that that bacteria likely comes from processing because it is present in all the five swabs that you gave me.

Will - It's quite interesting though that I might have discovered a new species of bacteria in my disgusting kitchen. I mean my very sterile and clean kitchen. Yeah.

Trevor - Yeah but that also speaks to the truth of that, like, it's really important that we keep food hygiene, not just in the source of food, but rather in the processing, the cooking, the chopping and everything associated with it. There is good reason as to why people suggest actually different chopping boards for different food, to avoid cross contamination, that sort of stuff.

Will - No, absolutely. Iit more than highlights the delicate nature of having allergies as well, given how easy it's to cross contaminate in the same kitchen.

Trevor - Yeah, absolutely.

Will - So before we get onto the question of am I in any danger, obviously the samples I gave you were random samples of food and swabs of those. Could you do any detective work on them to work out what they might have been?

Trevor - I tried. I really tried, and I'm afraid I'm going to disappoint you. It's very difficult because as I said, like the majority of the population are things that we cannot identify, and then there is one single species that seems to dominate the rest. So from the rest of the population I have to make guesses based on something that looks strikingly similar. The one that stood out particularly is sample number three, which when I look at it has about 4-5% of Salmonella enterica. So salmonella is a bacteria, or a known possibly pathogenic bacteria, that can be found in paltry or other food or mostly meat. So I guess I'll make a guess that like sample three either belongs to uncooked raw beef or processed chicken. So that would be my guess. And then the other two samples, which I think I can make a bit more guess about, are sample one and sample two. These two samples, once I remove the unknowns and also like the dominating species bacteria, what's left behind are a bit of Pseudomonas, which would suggest to me that they come from something that has been in contact with soil because like that is where a lot of Pseudomonas bacteria live. So I think they are potatoes and also onions. Sample four and sample five are samples that contain, again, some salmonella, a bit of capsular oxytoca. Also these two are known pathogens, but also a bit of pseudomonas. But this is where I find it really difficult to guess what they could be. So that's my attempt and I guess like you can now tell me whether any of my guesses would land any hits.

Will - Well, I must say I'm tremendously impressed. Sample three was indeed chicken one and two are potato and onion. Yeah. And sample four was the aubergine. So you have done incredibly well there.

Trevor - Oh, so did I get three out of five? Correct. Amazing. I did not expect that.

Will - Absolutely stellar work. That is brilliant. All of that taken from one swab and some detective work.

Trevor - I was going in pretty much blind and not having a lot of confidence in anything that I can get all. So I'm amazed as well. I didn't know that there's that remaining few percent of the bacterial population that can tell me or can get me so close to the correct answer. I was really like just going blind.

Will - And the big question, should I be worried, was there anything on my food that was left out for maybe three days that I should not be ingesting?

Trevor - Absolutely. I think the general answer is yes, we should definitely be worried about, and there are two reasons. The first one is yes, we did find bacterial species which are known to be pathogens. So I mentioned Klebsiella, I also mentioned salmonella. So these two are things that could make people really sick if they get ingested. But at the same time there are also a large number of other bacteria, which they're not known to be pathogenic, but we don't know whether they are or not. But the very fact that if I eat expired food with so much bacteria that itself can upset your stomach, your guts leading to diarrhoea or general sickness or even vomiting. A lot of things consist of it. So I think in general, absolutely yes, it's not a great idea to eat food that has been left exposed to bacterial growth for a long time. And especially for people who are potentially in the compromise or they are not so good at contracting bacterial infection, getting ingesting this large amount of bacteria would pose a tremendous danger to them as well.

Will - So it comes as kind of not a surprise at all, but also a terrifying revelation that leaving food out for not actually that long in the grand scheme of things can accumulate some really nasty stuff on it.

Trevor - Yeah, really I think the best advice is to maintain good hygiene and try to consume foods that are cooked and ready to eat.

A tardigrade

What makes a tardigrade so tough?

This week, I’ve taken the decision to shrink myself down and jump into a pond. Because I want to get up close and personal with perhaps the toughest group of critters in the animal kingdom: tardigrades.

The tardigrade can grow to around half a millimetre and usually lives on a diet of algae, small invertebrates, and smaller tardigrades. A single litre of water can contain as many as 25000 of them, and they are found pretty much everywhere. These organisms, sometimes referred to as ‘Water Bears’ or ‘Moss Piglets’ were first discovered in 1773 by German scientist Johann Goeze. In 1777, Italian scientist Lazzaro Spallanzani gave them the name ‘Tardigrada,’ which translates to ‘slow-stepper.’ Me neither.

Anyway, most people haven’t heard of tardigrades because of their ‘slow steps’. They know them for being one of the toughest little guys out there.

Let’s quickly run through the accolade list, for those that aren’t aware.

Tardigrades can survive 150 degrees celsius, which is about the same temperature as the surface of Mercury.

They can survive 0.05 Kelvin, a whisker away from absolute Zero. And longer term, they can survive -20 degrees Celsius for about 30 years.

The average Tardigrade can survive pressures of 1200 atmospheres. Some can survive 6000 atmos, which is roughly 6 times the pressure of the lowest point of the Mariana Trench.

Gamma rays are produced in the most energetic parts of the universe, such as stars and supernovae. Humans have harnessed the energy of gamma rays in the medical field in order to sterilise equipment. Gamma rays are very good at killing things. A dose of 5-10 gray, the unit of absorbed ionizing radiation, could be fatal to a human. A tardigrade can withstand over 5200 of them.

Quick side note, tardigrades cannot be considered extremophiles, as an extremophile is an organism that actively chooses to live in a harsh environment. Given the choice, a tardigrade would happily curl up on a wet bog and venture nowhere near a volcanic spring. In the same way that, if needs be, I could do upwards of 5 press ups. But if you asked me to, I would jump in a volcanic spring.

Either way, this is evolutionary overkill. So, how do they do it? Well, I’m really sorry to do this to you, but we’re going to have to talk about some… chemistry.

The answer mostly comes down to retain their cell structure. The main problem with extremes of temperature or pressure or radiation is that it affects the amount of water you can keep in a cell. Water is vital to nearly all the reactions that go on inside cells, as well as retaining its structure. The cell dries out, cell functions stop, it’s bad news all round.

Intrinsically disordered proteins, named after my sleep schedule, are proteins that do not exist in one fixed three-dimensional shape. When the water concentration drops, tardigrades start to produce IDPs, or already have some ready depending on the species. As each cell desiccates, the IDPs inside them form amorphous solids. The IDPs then become a glass like structure which maintains the shape of the cell without the need for water, like those planks of wood that stop a cave from collapsing.

The tardigrade actually protects itself against drying out on two fronts. Not only does it utilise IDPs, but also employs the services of damage suppressors proteins, or DSUPs. DSUPs are exclusive to tardigrades, and prevent the breakdown of the DNA helices in cells. If a cell's DNA can remain intact, it can continue to code for IDPs and the cells can stay relatively functional, not unlike my sleep schedule.

So that’s the tardigrade: tiny, tough, and terrific. I’d wish them luck, but they don’t need it.

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