SciCafe: How "Paleo" is Your Diet?

SciCafe: How "Paleo" is Your Diet? - Transcript

Christina Warinner (Professor of Anthropology, University of Oklahoma):

Thank you so much for coming. I'm so excited to be here and to share with you some of the really exciting findings that we have been pursuing and some of the new research that we've been doing.

Focusing on humans, focusing on our microbes, one thing that's always really fascinated me is that we don't know very much about the evolution of our own microbes. And that's something that I've been trying really hard to address. So, the human species evolved more than 100,000 years ago, but the context in which we evolved is extremely different from the one in which we live today in these urban and industrialized environments. And, in fact, many of the largest changes that have occurred have occurred over a very short time period in the grand scheme of human evolution. So, for example, if we think about just our diet alone, we went from eating out of ecosystems like this to this in fewer than 500 generations. That's a massive amount of change. And it's been argued that this disconnect between our ancient bodies and these modern, urban, industrialized environments that we have created for ourselves are the basis of today's so-called diseases of civilization. And this idea is the premise behind things like the fad diet the Paleo Diet and the ideas that if we could just eat more like our Paleolithic ancestors many of these modern problems would go away.

However, this is a huge challenge for us because it is almost impossible to eat like our Paleolithic ancestors. If you go to a supermarket, there are no Paleolithic foods that are present. They're almost all the product of modern agriculture. They've been radically changed through selective breeding over thousands of years. The only really foraged food that you can even find in a supermarket are a few species of perhaps fish. And even if we could forage for all of our food, the earth simply cannot support 7 billion hunters and gatherers. That would be impossible. So, the question is: Is it really agriculture that's the source of our problems, or are there other aspects of our modern lives that are causing some of these problems? And so we can kind of work around this. Now, one way to address this is to study the evolution and changing ecology of our human microbiome. It's becoming increasingly clear that no study of human evolution is complete without also considering the trillions of microorganisms that live in and on our bodies and then contribute to our basic biology.

It was only in 2001 that the molecular biologist Joshua Lederberg coined the term microbiome, which he defined to signify the ecological community of commensal, symbiotic and pathogenic microorganisms that literally share our body space and have been all but ignored as determinants of health and disease. And so while we have made great strides in understanding and revealing the diversity variation in evolution of the human genome, we know surprisingly little about the origins and evolution of this microbial portion of ourselves; our microbiome. And this is really remarkable because these communities perform major essential functions within their host bodies. Here's just a few in digestion, vitamin production, metabolism, education of immune system and defense of pathogens.

So, digestion is the most obvious one. These microbes help us digest our diets. Now, this is a fantastic graph. This is work done by Ruth Ley, who's a really amazing researcher. So, each point of light is a different microbe, and each microbe is connected to 60 different animal species that have that microbe in their bodies. And if we color-code it—so, for example, if we color-code the lines, so each line that connects an herbivore to its microbe a really amazing pattern emerges where all the herbivores kind of group together because they have similar types of gut microbes because they have a similar diet and they require similar bacteria to break it down. Omnivores clustered together there in blue, and carnivores clustered together in red. Now, because I'm interested in humans, I'm curious about where primates would fall in here. And so I've added primates here, and they fall in this intersection between omnivores and herbivores. This makes a lot of sense. The primates that are primarily folivores like gorillas, they fall closer to the herbivores than primates that eat a more diverse diet or a more varied diet like bonobos and chimps. They fall right at that boundary.We have three there that fall right squarely in the omnivore group, and those are spider monkeys and marmosets. They primarily eat plants, but they also supplement their diet with small animals and eggs and insects.

And we can put humans on this graph and ask where do they fall. Humans fall squarely within the omnivores. It's clear from this and from our gut microbiota that we are omnivores We've been omnivores for a really long period of time. This is a clue towards our ancient diets. And so looking at all this information, at the University of Oklahoma my colleagues and I were really curious in applying this information to humans and human evolution. And specifically we wanted to look at subsistent strategies; really different subsistent strategies that people have had over time. How does this affect human microbiota? Most of the studies that have been done on humans have focused on North American and European populations. They focus on urban industrialized populations. And what we wanted to know is what if we look beyond that. What if we look at hunter-gatherers? What if we look at subsistence farmers? Will we see a greater amount of diversity there? Will we see patterns?

And so we focused on three different groups. One was the Matses hunter-gatherers of the Amazonian jungle. They primarily eat a diet of sloths. They eat fish. They eat wild tubers and various fruits. We also looked at the Tunapuco of Highland Peru. They're potato farmers. They're subsistence farmers. They grow the food that they eat. And they live at high elevation. And we also looked at people living in cities in Oklahoma. I may have participated in this study.

Anyways, we looked at these three groups and we wanted to know to what degree did they differ. This is a principle coordinates graph, and what you can see here is each point actually represents the gut microbiome of one individual in the study. And how close or far away they are indicates how similar they are in terms of their microbial community. And from even just a small number of individuals, we can start to see this pattern where we see that people following a more traditional diet, whether that's hunting and gathering or traditional agriculture, cluster away. There's some differentiation there, but they overlap. But they cluster away from these urban industrial populations. We thought this was really interesting.

So, we scoured the literature for every other study ever published on hunter-gatherer or traditional farmer or people living in urban industrialized environments, and we added them to this graph. And we add these additional populations, which include people now from Africa, from Europe, from North America and from South America. We see a stark pattern. And we see a very strong differentiation between people who eat traditional diets and people who eat out of an urban industrialized food chain. What really struck me about these results is that unlike what might have been predicted, it's not agriculture that structures the gut microbiome that changes it. It appears to be industrialization because what we see is that the people that follow a subsistence agricultural diet and people that are hunter-gatherers overlap to a large degree in their gut microbiome. But it's these urban industrial populations that are so different.

In addition to helping us with digestion, the microbiome also produces vitamins for us. And one of the reasons you've never had to take a vitamin K supplement is because your gut microbes produce it for you. They also produce a number of B vitamins. They also metabolize drugs, and that's part of the reason why there's some variation in how much dosage a person might need of a particular drug. The third thing that the microbiome does is it plays a really critical role in educating your immune system, especially in the first three years of your life. At this point, your immune system is maturing. It's developing. It's differentiating. And these native bacteria of the human body go to great lengths to teach and educate your immune system what to react to like pathogens and what not to react to like pollen and dust mites and things like that. And, finally, the microbiome plays a major role is just defending you from pathogens. They occupy space. They occupy space on your skin and keep pathogens away. And they prevent the growth of other bacteria. This is perhaps most clearly seen the vaginal microbiome where native populations of lactobacilli create a high acid environment that prevents the growth of, for example, STDs.

So, but in addition to the many beneficial things the microbiome does, it can also cause disease. The oral microbiome is actually the reservoir for many opportunistic pathogens that cause respiratory disease. For example, the microbe streptococcus pneumoniae is quite widespread. The carriage rate's actually quite high. Possibly as high as 30 percent of you carry this microbe in your mouth. Now, you're quite healthy. You have a strong immune system. It doesn't cause problems, but if you become immunocompromised it can leave your oral cavity, descend into your lungs and cause pneumonia. And there are many, many opportunistic pathogens in the mouth that can do this. So, the microbiome clearly plays an important role in human biology and in human evolution. But we don't know very much about how it's changed through time. So, this is something that's really fascinated me because I am a molecular anthropologist, and I focus on ancient DNA. And I wanted to use these techniques and see if I can go into the archaeological past and actually reconstruct the microbiome at different points to help us understand how it's changed through time and to confirm some of these hypothesis that we've been formulating about major changes that might have occurred in the archaeological record and at what point they occurred and what stimuli caused them.

Archaeologists and people who focus on ancient DNA primarily study bones and teeth. And there's a very good reason for that, and that's because mainly that's what we have in the archaeological record. That's the primary form of human remains that we have. And we've learned an awful lot from bones and teeth. We've been able to take these and reconstruct the genomes of extinct animals, the genomes of archaic humans. And we've even been able to reconstruct pathogens that infected humans and plants long ago. But how are we going to get to ancient microbiota?

As we said earlier, microbes don't typically fossilize. How do you find them? The human body itself rapidly decomposes after death. And it's true that in some cases we get mummification, but mummification's actually quite rare. And in the vast majority of cases, what we're really left with is just a skeleton. A skeleton itself does not have a microbiome. But there are a couple of substrates that do preserve in the archaeological record. One is paleofeces or coprolites. Sometimes it is not common, and it only occurs in some cases, but feces can preserve, especially if they are dried out very rapidly in the archaeological record. And we do sometimes find paleofeces in archeological deposits in dry caves or in salt mines; prehistoric salt mines. But like I said, they're quite rare. One thing I've been working on much more recently that seems to be extremely promising is something called dental calculus. Now, you probably know it by its other name, which is tartar. It's that stuff that you pay dentists to take off of your teeth when you go into your dental visits. What it effectively is is calcified dental plaque. And what's really amazing about it is that this calcification process— actually the mineralogic component of it is the same as in your bones and teeth. It fossilizes just like the rest of the skeleton and can preserve for millions of years. And this gives us ready access to the oral microbiome.

So, I'm going to talk about the work that we've done so far on these two substrates. I'm going to begin with coprolites. Now, most of you probably don't know what coprolites look like, what paleofeces look like. So, I'm going to give you a couple of pictures. I've looked at a lot of coprolites now, and I can tell you that they're very diverse. So, human poop in the past looks—can look like a lot of things. They're different colors. They're different shapes. They're different sizes. One thing that is very common is they're often very fibrous, I have learned, by looking at them. The human diet in the past was a lot more fibrous than today. We often find whole pieces of plant preserved inside, whole seeds. These are coprolites from three different continents. So, on the top, this is a coprolite from Mexico. The one on the left is a coprolite from Austria, and the one on the right is a coprolite from Iran. But one of the problems we face immediate with coprolites is, as you might imagine, that feces decay and decompose really quickly. So, just because it looks well preserved to the naked eye, how well preserved is it really at a molecular level, at a DNA level?

Well, a really clever man named Dan Knights came up with this program, which is fantastic. It uses Bayesian statistics to estimate or to quantify the preservation of things like coprolites. So, we can say, well, we think this coprolite might be a mixture of bacteria from the gut. And lots of people have studied the gut, so we have many, many sequences of DNA and bacteria from the gut in public databases.

And we think it might also have some skin contamination; maybe some skin microbiome contamination from the archaeologists. So, we'll add into this. We'll add that as a possible source. We can also take things like soil. Lots of people study soil. There's millions and millions of DNA sequences from soil that have been deposited in databases. We can add that. And we can use this program. And it tries to infer how much of each of those communities contributed to our coprolite. We can use this to authenticate it. So, these are two real coprolites from a real study that I'm doing right now. One's from Europe. One's from the Middle East. When we apply this method, what we find is actually it's incredibly well preserved. And that's because they're from salt mines, which turns out to be a really great preservational environment for coprolites. What we find here is that more than 50 percent of the bacteria that we can identify within these paleofeces are typical of the human gut microbiome. And so we now have a really good source of an ancient human gut microbiome. They date to the Iron Age. They're a little bit more than 1,500 years old.

But as I mentioned before, the human gut microbiome is not monolithic. We see this big difference between traditional guts and industrialized guts. And so what we can actually do is we can split that gut microbiome component; that source component, and we can now ask what proportion of these ancient gut microbiomes— they're, like I said, about 1,500 years old. So, not all the way back to the Paleolithic. More recent than that. Do they look like these industrialized or traditional guts? When we apply that—what we find actually is they look like traditional gut. And this is really interesting to me because these people were not subsistence farmers and they weren't hunter-gatherers. They come from the Celtic Empire. They come from the Sassanid Empire. These were miners in an ancient salt mine. And yet they look like these traditional guts. These ancient Europeans here look more like hunter-gatherers and subsistence farmers do today than they do like living Europeans in Europe right now. And that implies that there's been a tremendous change in the gut microbiome ecology over a very, very short time scale.

So, many of the genera that are found in the human gut microbiome are universal. We all have them. And here's what I call my A-list of fecal celebrities. These are the top fecal genera. And they're fantastic. We have Clostridium, Prevotella, Faecali, Coprococcus, Blautia, Roseburia. I guarantee every single one of you in this room has these bacteria in your gut right now digesting your dinner. These are the workhorses of the gut microbiome. But when we first got these results, there was one that really puzzled us because we weren't really sure how to interpret it. And that's this one here, which is Treponema. Now, most of you have probably never heard of Treponema. But if you have, what you've probably heard of is Treponema pallidum. That's the causative agent of syphilis. And Treponema denticola is the causative agent of periodontal disease. Treponema have a bad rap. There's a lot of pathogens in Treponema. There's spirochetes, spiral-shaped bacteria. And so we were really puzzled when we saw them in the human gut, and wondering what they were doing there. Now, this isn't syphilis. This isn't denticola. This isn't causing periodontal disease. It's a different type of Treponema, but what is it doing in the human gut? Because the Treponema is not thought to be part of the human gut microbiome.

But then we realized that's because we were comparing to databases made up of sequences deposited from European and North American samples. And so we started to look a little more broadly. What we discovered is that Treponema is found in the gut microbiome of the great apes. It's also found in the gut microbiota of hunter-gatherers. And it's also found in the gut microbiome of traditional subsistence farmers. But where it is not found is in modern-day Europe or North America. So, this seems to be a group of bacteria that's been systematically lost from industrialized populations. We've been able to take the data that we've obtained from this metagenomics soup that we obtained from fecal samples, and we've been able to determine there's at least 5 species of Treponemas found in humans that are no longer found in industrialized populations. We can analyze their genomes. We've actually used a computer algorithms to stitch these sequences back into reconstructed genomes. And we can tell that they're probably fiber digesters. They probably specialize in fiber digestion, and that's actually extremely interesting. So, bacteria that digests fiber, produce fatty acids as a by-product. And some of those fatty acids are very important.

So, one fatty acid that's produced by some fiber digesters is called butyrate. And butyrates, it's actually the primary nutrient source for your colonocytes, the cells that line your colon. So, unlike many of your other cells, which are fed by nutrients that are traveling in your bloodstream, your colonocytes, your colon lining cells, they're actually fed directly from your gut from things like butyrate. And so these fiber-digesting bacteria are extremely important in feeding those cells. And so here we have what seems to be an entire clade of fiber-digesting bacteria that's been systematically lost from industrialized populations. And so we're following this up now, like I said.

Okay, next I'm going to talk about dental calculus. I think it's an incredibly interesting and promising new area of research. Dental plaque was never so cool. Again, you probably don't know what it looks like, so here's a couple of images. Here's a good close up. This is a mandible. This is about 1,000 years old. It comes from a Medieval German cemetery. We can take a closer look at this tooth here. We're going to look at that dental calculus deposit. This is just a scanning electron microscopy image. We can look at it in cross section. I'm just going to zoom in on that calculus deposit right there. So, there it is again. We're going to zoom in again, and here we have it in cross section. And there's a number of really interesting things about it. First of all, you'll notice that it's layered. It seems to have this laminar structure. And that's because of how it forms. You first form a layer of dental plaque. It calcifies, and then you form another layer of dental plaque on top of it and so forth. And so it really builds up like tree rings or layers of an onion. And what's extraordinary about that—what's really exciting is that means this is actually a temporal record of this person's life. So, the interior layers are when they were younger. And this exterior layer was formed just before this particular woman died. And inside we have an ordered record of her life history fossilized in place. Incidentally, dental calculus is the only part of your body that actually fossilizes while you're still alive. And this is really important because it means it's protected from these post-mortem decomposition processes. Because it calcifies while you're alive, it doesn't actually decompose after you die. It's very resistant to decomposition, and so preserves for extraordinary amounts of time.

Now, the oldest dental calculus that I know to exist comes from a fossil primate, and is 12 million years old. We can zoom in even closer, and here it becomes really apparent where this really shines. So, unlike a coprolites, which like I said are open systems, they can decompose. Calculus is so different. What we see here, you can see individual microbial cells. These are the original oral microbial bacteria that have just been completely calcified. This white material here is calcium phosphate ions. It's been completely fossilized in place. All these bacteria are exactly where they were during life. And we can now decalcify this using a chemical that's the same chemical that's in your shampoo that gets rid of hard water. We use it on the teeth, and it removes the calcium and we're able to liberate this DNA.

Now, one thing that's really extraordinary, too, about calculus, which I didn't know. This is something we discovered and was really exciting, is when we work with ancient samples they typically have very little DNA. And it makes it really hard to work with them. So, to put this in perspective, if we take dentin, for example, which is just the root of the tooth and you extract DNA from it, we get very little. So, the scale here, it's logarithmic. It's base 2. And it's the number of nanograms of DNA that we get per milligram of tissue that we analyze. And for something like dentin, it's really low. It's less than one nanogram of DNA per milligram. Coprolites have more DNA. They have on average about five nanograms of DNA per milligram of tissue. But calculus has an enormous quantity of DNA, in part because it's preserved so well. It has on average 40 nanograms of DNA per milligram of tissue that we can analyze. And the highest I've ever measured comes from a medieval sample in the U.K. It's this one on the far right. It had 506 nanograms of DNA per milligram of tissue. It was amazing. We saw messages on our machines we didn't know existed. Too much DNA, cannot measure. All right. So, if you're going to—please pardon my pun here, but I think we're only beginning to scratch the surface of what we can learn from dental calculus. But I'm going to show you some of the really exciting findings that we have so far.

One thing that is really, really exciting—and I mentioned before that the trouble with coprolites is that they rarely preserve and we can't really predict where they're going to preserve or when. That's not true for calculus. So, calculus is found in every known living human population today. It's also found in every known archaeological population. We find it on Neanderthal teeth. We find it on Australopithecus teeth. And even chimpanzees make it quite abundant. So, here we have a record of the oral microbiome that spans the entire period of human evolution. And what can we find when we analyze calculus? Well, first of all, it's mostly bacteria. About 99 percent of it is bacterial. That makes sense. It's made of dental plaque. But about half a percent is eukaryotic. When we look at that, what we find is that is a combination of human DNA and dietary DNA. And I'll come back to that in a few minutes.

We also find an abundance of proteins preserved within dental calculus. About 80 percent of them are bacterial, and about 20 percent of those are human. If we take those bacterial DNA sequences, though, that we've analyzed and we ask what we have, we have more than 2,000 different species we've identified. Most of them—about 85 percent—resolve to about 100 really common species. And some of them are extremely interesting. So, I mentioned before that the oral cavity is the natural reservoir for a large number of opportunistic pathogens. And so one thing we wanted to know was is that only true today, or what that also true in the past? And so we tested these medieval German samples for this. And what we found was a quite wide array of these opportunistic pathogens. Now, again, that doesn't mean they were necessarily infected with all of these diseases, but they carried the pathogens that have the potential to cause these diseases.

We identified a large number of them. Just a few highlights are Streptococcus pnemoniae, which can cause pneumonia, Streptococcus pyogenes; that causes strep throat, Haemophilus influenza, that cause a variety of respiratory infections and Neisseria meningitides is one of the leading causes of meningitis. We also found bacteria associated with periodontal disease. There's three major ones that are associated with periodontal disease today. That's Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola. We found all three in these ancient samples, confirming that periodontal disease has the same etiology in the past as it does today. We then compared them to the frequency of these bacteria that we find healthy people today, and can confirm that our ancient individuals had very high levels of these bacteria.

In one, the Tannerella forsythia, we actually had so many sequences from this that we were able to reconstruct its full genome. This was pretty exciting, so this is a full ancient human microbiome member that we were able to reconstruct. But one thing that really troubled us initially—we didn't quite know what to make of it— is we had this huge gap in our assembly. And this really bothered me because I felt like why is that DNA missing? Why can't we find that DNA? It's a large gap. It's 48,000 consecutive bases. It's 53 consecutive genes. And I really scratched my head over this on why we didn't have it until I realized what this is. It's something called a conjugative transposon.It is a mobile piece of DNA. It can move from species to species. And it encodes antibiotic resistance. And so what we found was that our ancient sample lacks it. It doesn't have it at all. This is something that is more recent. And that was really interesting. We also—although, we didn't find antibiotic resistance in this particular species— in this particular reconstruction—we did find many genes in this large metagenomics soup that have putative antibiotic resistance function. This is really interesting. This actually plays into how we understand how antibiotic resistance evolves. So, although we don't think that any of these ancient bacteria had a functional antibiotic resistance, many of the genes that are associated with antibiotic resistance today actually originally had different functions that had been repurposed for antibiotic resistance. And what this taught us is that the capacity for antibiotic resistance is very deep within the human microbiome. And so even if we could wipe out every strain today of functionally antibiotic resistant bacteria, they have the genetic machinery to regenerate it anew. And this goes very, very deep.

One thing that's super cool and really exciting about some of these ancient microbiomes is that in addition to learning about the microbiome itself, in addition to learning about these bacteria, calculus and coprolites also act as a kind of sink for other really interesting host and dietary information. So, I'm going to focus on the host. So, I mentioned before that we sometimes find human DNA— we find it in both calculus and we find it in coprolites. And so one thing we wanted to know was is it enough to do something interesting with? And so we started playing around with it, and we found that we can actually now reconstruct full mitochondrial genomes just from the DNA that got left over inside dental calculus and coprolites. This is fantastic because we can use mitochondrial genomes to then infer ancestry and also large migration events. We find a lot of human proteins in dental calculus. This is just a protein interaction where each dot is a different protein. I've color-coded it by what the protein does, what basic function is. And red is the innate immune system. We saw a lot of immune proteins in calculus. What we're actually seeing—this is amazing. We're not only seeing pathogens that are in there, periodontal pathogens that are causing disease virulence factors, we can actually see the host immune response to that ancient infection. We have a complete infectious reaction that we can see entrapped in these ancient calculus samples.

We also have really interesting information about diet. One thing we find is we sometimes find little bits—little micro fossils that are visible under the microscope of people's diets. So, as you know, sometimes food gets stuck in your teeth.And that happened in the past as well. And thousands of years later we can use a microscope and identify it. So, this particular Medieval German had a variety of things stuck in their teeth. They had a little bit of connective tissue from some meat that they had been eating. They had some plant [unintelligible]. And they had a couple of starch granules, which we see here on the bottom. This goes to the fact that plaque can sometimes calcify so quickly that even little starch granules that you've eaten in your diet can become entrapped and preserved over thousands of years. This one on the left is consistent with wheat. And the one on the right is very similar to what we see in peas. We can also take DNA out of these samples and look for specific dietary DNA signatures. And from this sample, we were able to identify four different food stuffs. We were able to identify sheep, pigs, cabbage and wheat. Not particularly surprising for a Medieval German diet, but a great proof of principle that we were on the right track. But what's also interesting is that we can start to get at another question.

So, I'm very interested in the origins of diary. As many of you know, one of the tenets of the Paleo Diet is that you shouldn't eat diary. And diary is a really odd thing. We are the only mammals on earth that continue to drink milk long after our infancy. And that is very strange. But diary on a global scale is big business. The global milk production today is 697 million metric tons per year of milk. That is huge. So, 697 million metric tons is the same as 278,800 Olympic-sized swimming pools, or 217 Walden Ponds of milk are produced every year on this planet. This is enough for every single person on earth every day to consume a quarter liter of milk. And, yet, the vast majority of the world's population is lactose intolerant. So, how did we come to this position where we have— where diary is so widespread and, yet, most people can't consume milk as adults? And it turns out that dental calculus can help us understand this. Because one of the proteins that we started finding over and over again in these ancient samples was something called beta lactoglobulin. Beta lactoglobulin is a protein that is specific to milk. What's also interesting is humans don't make it. We actually have a defect in this gene, so we don't produce it. But it's produced by all of the diary livestock that we use today. And so what we can do is we can go to dental calculus of particular populations in the past, we can measure their calculus.

We can recover this protein, and we can infer who was drinking milk at a personal level throughout prehistory. And so we've done this. So, we went and we got 100 dental calculus samples from across Europe, spanning more than 4,000 years, and we were able to type who was drinking milk where and when. And using this information, we were able to say, for example, that Bronze Age northern Italians liked goat milk because there are mutations that actually indicate the species. And we could also say that people living in Rome and Britain preferred sheep and cattle milk. The Norse Vikings really liked their cattle milk. And so we can start to go back and reconstruct this history of diary. We've now expanded this project, and we're focusing now on the Neolithic and trying to push it back even further in the near East and in Europe and trying to track the spread of milk across the continent.

This is exciting to me because milk is otherwise extremely difficult to study, as you might imagine. It does not preserve well in the archeological record. We don't actually know when people started dairy. And so this is a new area of research for us. And so I hope I've convinced you that studying the microbiome is incredibly interesting and also valuable and that we can do this by both studying diverse people today and also by directing going into the past and studying these preserved microbiome substrates. By putting all this information together, we can start to gain a much better appreciation of our complete selves, so not only our own evolution, but the evolution of the trillions of microbes that call us home.

Thank you very much. And I want to acknowledge all of these wonderful people who helped with this research.

[Applause]

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