The Bioinformatics CRO Podcast

Transcript of Episode 37: Jason Arnold

Disclaimer: Transcripts may contain errors.

Grace Ratley: [00:00:00] Welcome to The Bioinformatics CRO Podcast. My name is Grace Ratley, and today I’m joined by Dr. Jason Arnold, who is the technical director of the Microbiome Core at UNC Chapel Hill. Welcome, Jason.

Jason Arnold: [00:00:12] Thanks. It’s great to be here.

Grace Ratley: [00:00:13] So, Jason, tell us a little bit about your research and your role as technical director of the Microbiome Core.

Jason Arnold: [00:00:20] A lot of universities don’t have these sorts of facilities. I know where I did my PhD. We had nothing like this. It was a relatively small I don’t want to call it a liberal arts college. It was science based, but the research infrastructure was limited. So anytime we needed to do any sequencing, we would have to outsource that sequencing to either somebody else in our department who had the facilities to do so, which was very limited, or we would have to send it to a company and have it done there. So at UNC, we have all sorts of different core facilities and these core facilities are accessible to anybody at UNC as well as anybody worldwide really to be able to come to us and say simply, Look, I have these samples. I’m not a microbiologist. I don’t know how to do this, Can you help us? And we provide the expertise in terms of development of their experiment, designing which direction it should go and what they would really need to do, as well as the technology. So we have access to all of these fairly high end machines for automated DNA extractions and library preparations and sequencing and all of this stuff. So we’re able to utilize our infrastructure to help others inside the university and outside the university to reach their research goals and to be able to accomplish what they’re trying to do.

[00:01:33] And it’s not academic exclusive either. I actually have a friend who’s part of a startup company and he often comes to us and we discuss like, Well, what can we work on together? R&D for things that he works with. And this all goes through UNC systems and we’re able to really work collaboratively with industry as well as with academics. And it’s a really great experience because it gives me the exposure to all sorts of different things that I would never, in my own research ever run into. So if my focus is on aging or if my focus is on a specific bacteria, I’m never going to be thinking about veterinary medicine or microbial interactions in primate nests in Africa. Like you don’t think about these things, but these are the projects that come in and we then get the opportunity to work alongside these other researchers and ensure that they get the best results they can get. And hopefully that data gets published. A lot of times it does and it’s affordable as well as opposed to having to buy sequencing machines and all of this stuff, they can just bring it here, use the infrastructure we have in most cases, just the cost of reagents.

Grace Ratley: [00:02:38] It’s very similar to the business model of The Bioinformatics CRO. We’re kind of like a core facility for genomics.

Jason Arnold: [00:02:46] So my personal research, which I do at the same time as I work with a lot of these collaborators, I focus on the impact of the gut microbiome on host physiology in the context of aging. So what I look at is we use animal models for aging and we look at the physiology of the gut and what happens as these animals start to get older. We start to see physiological defects and associated with those physiological defects. We see distinct changes in the composition and function of the microbial community. So what we’re interested in is seeing how we can control that microbial community and modulate the composition of what’s there to be able to reverse some of the physiological defects associated with aging. One of the big ones that we’ve focused on in our research is intestinal permeability. So as you get older, the barrier of the gut starts to fail, starts to fall apart, and you get more small molecules and invasive bacteria being able to pass through into the system. But with specialized prebiotic compounds and modulation of the gut microbiome, we’re able to actually reverse some of that defective barrier, allowing for the hosts to remain healthier for longer.

Grace Ratley: [00:03:55] Yeah. So tell us a little bit about these compounds. What kinds of prebiotics are you using?

Jason Arnold: [00:04:00] So the prebiotic we focus on most is galacto-oligosaccharides. So what these compounds are is they’re basically a galactose attached to a lactose molecule and you have a chain of galactose is attached to that. Now these are compounds that are commonly found in mammalian breast milk, and they’re often thought of as beneficial to infants as the infant is growing. It helps with the early development and colonization, with the initial microbiome these animals have. These compounds interestingly are not digested by the host. So if you, for example, were to eat a bunch of these galacto-oligosaccharides, if you didn’t have a gut microbiome, they’d pass right through you and have very little impact. We don’t know for sure that it’s no impact. We’re actually doing some work now to understand if there is a host prebiotic interaction without the presence of the microbiome. But generally speaking, these compounds have to be broken down by the microbiome or by members of the microbiome in order to have an impact on the host.

[00:05:02] So one of the things we like to look at is the specific genes present within bacteria that are able to break some of these compounds down. So galacto-oligosaccharides are generally metabolized by Galactosidase, β-Galactosidase, α-Galactosidase. And what happens is the bacteria will either internalize the prebiotic compound, break it up into bits that it can use and then secrete what it can’t or the bacteria will secrete these enzymes that are able to interact with compounds in their environment, break those compounds down into components that they have binding receptors for. They can bind to that component that they can use, internalize that and use that. So interestingly, there’s this network within the community where you have a primary bacteria that’s able to take this prebiotic compound, break it down into whatever components come out from that bacteria. Those components, those byproducts are then able to be used by other bacteria in the community. So you form this cross feeding network where, let’s say lactobacillus, for example, will internalize a galactooligosaccharide, break it down into lactate, release that lactate, taking the small simple sugars off that it can use for itself. That lactate now goes out into the community where another bacteria like let’s say Roseburia, can internalize that lactate and potentially convert it to something else, adding residues to it, taking residues off. Ultimately, what we are interested in looking at is how these compounds are broken down and repurposed into short chain fatty acids. Those short chain fatty acids then directly interact with the host providing either benefits or growth factors or well, the ability to grow. So it’s a very complex system. There’s a lot we don’t know yet and there’s a lot we’re working on. So yeah, that’s the small molecules we use.

Grace Ratley: [00:06:48] Yeah, you bring up a lot of really interesting topics to discuss here, but just for our listeners, could you give us a little description of what Prebiotics are and how they compare to things like probiotics or synbiotics?

Jason Arnold: [00:07:00] That’s right. So a probiotic is a live microorganism that when you consume a bunch of them and you have enough. It’s supposed to have a positive impact on the host. Now, not all probiotics do, many do. That’s a whole different conversation for a whole different day. Prebiotics are indigestible carbohydrate compounds, usually fibers or complex sugars of some variety that the host can’t break down. They can’t digest it, but the microorganisms can. And what these compounds generally do is they promote the growth of beneficial microorganisms hence being called prebiotics. Synbiotics are a combination of a probiotic and a prebiotic. So think about it as you’re eating yogurt and your yogurt has lactobacillus and bifidobacterium in it. Beneficial microorganisms with enough of them in there that you’re eating it, it’ll have some potentially beneficial properties. Now if that yogurt was engineered in such a way that it consisted of lactobacillus bifidobacteria and indigestible carbohydrates that those bacteria can utilize to produce additional small molecules that are potentially beneficial to the host, That would be what would be considered a synbiotic because you have a mixture of a prebiotic and a probiotic, and the idea is that there’s synergy, hence symbiotic between these two compounds. Now there’s been a lot of studies done that suggest that the specificity of the prebiotic and the probiotic in order to actually function in a symbiotic setting is extraordinary. It’s very difficult to find a prebiotic probiotic pair that actually does produce an effect that’s greater than just the sum of the parts. So the idea of probiotics and prebiotics is if you’re consuming these things, you’re doing so because you’ve expected it’s going to provide a benefit to you in some way.

[00:08:49] However, it depends so much on what’s already in your microbiome, what bacteria are already there that you may or may not see an effect, and you’re going to have differences between individuals. There’s going to be host genetic factors that play a role. So there’s a lot in terms of the context of what’s really there in the host. Now when a lot of diseases you end up having a dysbiotic microbial community, which means that it’s imbalanced in some way. You have bacteria that are there in higher abundance than they should normally be, or that they would be in a healthy person. And sometimes those imbalances will cause drastic differences in a person’s response to a therapeutic or to a probiotic or to a prebiotic. So one of the things we’re interested in and that we try to do a lot of work with is understanding how how these dysbiotic communities come about. For example, in aging, we know as an animal gets older, these communities start to shift and you start to get this dysbiosis, which makes it harder for certain drugs to impact the host. We’re trying to understand a little bit about how that happens. Is it because diet is changing or is it because all of a sudden people are starting to get older and they’re taking more antibiotics and that tends to skew the community’s dynamics a bit. There’s a huge amount of information out there, but there’s still a lot that needs to be done to be able to really understand what’s going on.

Grace Ratley: [00:10:10] Yeah, the translate ability is really interesting to me. So what topics within microbiota science are you really excited about or do you think we’ll see the most advances in the next five years?

Jason Arnold: [00:10:23] I think in terms of what isn’t currently easily accessible and available is the idea of personalized medicine. We’re moving more toward systems where we’re able to understand the host’s genetic background and how the host’s genetic background interacts with the microbial community. Historically, we haven’t had technology that allows us to do that. Now, with the advances in cell culture techniques and with the advances in next generation sequencing, we’re able to actually start designing models and experimental systems where we can, even from an individual host in the hospital, take a biopsy from intestine, grow stem cells from that biopsy and be able to actually generate host specific organoids, for example, is a system that we’ve done a lot of work in, and those organoids, they’re like micro organs. It’s very hard to describe without showing a picture of them. Basically what happens is you take a subset of primary tissue from the host, which could be a biopsy, it could be a tissue section from the surgery, grow them in specialized growth conditions that allows for those stem cells to separate from everything else that’s there and differentiate to form a what we like to think of as a miniaturized organ. So you’re going to have all the different cell types that are in the small intestine or large intestine, depending on where those stem cells come from. And you’re going to have that host’s genetic background. It’s just ingrained into the tissue because you’re using it from a host.

[00:11:47] Now what we can do is we can actually take complex communities, individual microorganisms, individual small molecules, whatever we want to look at, put those inside this this three dimensional organoid and look directly at host gene expression and host response to that compound or that microbial community or individual microorganism. And that provides us a wonderful tool to be able to start to think about personalized medicine, because these aren’t very expensive to run these sorts of experiments now that the technology exists. So I think that’s one direction the microbiome field is going. Another direction that is actually really exciting to me is historically when we’re thinking about the microbiome and the microbial community and what’s all there in the gut, I’m thinking intestine specifically, you’re sequencing everything that’s there and you’re going to get a combination of bacteria derived from food bacteria that are alive, actively growing in the mucosal layer. You’re going to have bacteria that are just in the lumen and you’re going to have just DNA from bacteria that may or may not even be alive. So you get this big picture of everything that’s there. But in terms of being able to translate that to health, it’s very difficult to do because we have no good way of saying we had no good way of saying, well, these are the bacteria that are actively alive in doing something versus everything that’s there. Now, we have been working on developing and we finally to the point where we’re able to use this technology where we can actually differentiate in sequence data what bacteria are alive versus what bacteria are not alive or DNA. That’s just free DNA.

[00:13:23] So we can take everything that’s dead or excluded from the live bacteria section out and then start to get a picture of what bacteria are actually there. So we ran a study recently in mice, actually, because we were finding that in a lot of the mice that we were using in some of our experiments, we had a huge amount of lactococcus and we did some experiments where we were like, Well, is the Lactococcus there alive? Is the Lactococcus in the food? Like, where is this coming from? And by differentiating live bacteria from non live bacteria, we were able to identify that a lot of this lactococcus signal that was coming up in our sequence data was actually derived from the food. It was not even bacteria. It was just free DNA because portions of the diet were run through a fermenter, a big chemical vat of sorts that produces a large amounts of this food for the animal diet. And there was just lactococcus in there. And we had just lactococcus DNA. It was nothing alive. But in some cases it consisted of like 20 to 30% of the overall microbial community. When we came back with our sequencing data, when we actually looked at what was alive, it was zero. It wasn’t there at all.

Grace Ratley: [00:14:29] I think that would be extremely useful, especially for things like gnotobiotic studies where you’re trying to take a known source of microbes and colonize some sort of mouse with them, seeing how well those microbes colonize within the gut, that could be really useful in eliminating some of the variability that you see in studies, especially with things like fecal microbiota transplants.

Jason Arnold: [00:14:56] Yeah. Also in production of probiotics. This is something that we’re actually thinking about because a lot of times you go to a store and you buy your probiotic and it says there’s ten to the seventh bacteria. Is it alive? So using something like this can help in development of these products. So the people who are actually producing and selling these to other individuals in the hopes that it is going to be some therapeutic benefit, you actually know for sure that these are in fact, alive and you can do tests then. I mean, aside from culturing now, culturing for most probiotics is sufficient, but this is going to be a much higher throughput. When you use this method in conjunction with the PCR, you’ll be able to actually get an exact number of how many bacteria are there in a matter of a couple of hours as opposed to having to wait a couple of days for culture plates to grow. And then you have to take into consideration, well, the plates desiccate too much. And not every colony grew to the point where it has to. Am I miscounting? Is the system I’m using not working right? Being able to do this in a molecular approach, it just eliminates a lot of the the background, so to speak, and a lot of the human error. It will allow for development of much more, I guess, precision in terms of the development of probiotic mixtures.

Grace Ratley: [00:16:08] Well, that’s a really exciting technology. I hadn’t heard of that before. That’s something that you’re working on.

Jason Arnold: [00:16:13] Yeah. So there were some work done years ago that discussed this sort of thing, like basically in environmental samples being able to exclude contaminating DNA. So we repurposed this DNA contaminant exclusion system for sequencing applications. So it’s been done before in, like I said, for like identifying DNA contaminants and for viability of bacteria using PCR based methods. But it had never been done before in a sequencing platform. So yes, we in the Microbiome Core are actually working on getting all of that developed to a point where we have people actually coming in now and using that technology here where it’s actually accessible for people who want to do that kind of research.

Grace Ratley: [00:16:58] So I am very interested in your reptile business. Can you tell us a little bit about that?

Jason Arnold: [00:17:05] Oh, yeah, yeah. So I guess I should preface this by saying I grew up in New York, Western New York on a farm, didn’t have much in terms of technology out that way. I mean, we did, but I wasn’t big into that sort of thing. But I was extremely allergic to most of the animals we had on the farm. So as a kid, I’m like, We have horses and goats and chickens and dogs and everything and I’m allergic to all of these. I’m like, This is horrible. I love the animals. I love being around them, but my eyes are watering all the time. So I must have been like eight years old, nine years old, and I wanted a pet. And somehow ill advised, I convinced my mother to get me an iguana. And I’m like, Now that’s a terrible idea. Never have an iguana as a pet. They’re bad. But little kid Jason was like, Yeah, this is a great idea and it was just out of control from that point on. So I realized, Hey, these are animals that I can interact with and work with and not be allergic. So years later, I after high school, I’m starting my undergrad. I started keeping more reptiles. So I started having some snakes and some other types of lizards. And I started working with other breeders and collectors and zoos and conservationists in the area up in New York.

[00:18:21] And I got to learn a lot about the genetics of these animals and the husbandry and how these animals are taken care of and how exactly you can keep large numbers of them. And as I started graduate school, I started a business where I was breeding a variety of different reptiles for genetic mutations, for colors and patterns, and I was providing them to zoos and to other collectors and breeders. And actually I had produced the first of a lot of different species. My bloodlines are in all different countries, all over the world from years and years ago. So I did this. I also did an educational demo program that I worked with others in the community. So in New York, there were a lot of really strict licensing requirements to be able to have anything venomous or anything potentially dangerous. So I had all those licenses and I had acquired them over the years, having worked with a lot of these people and working in conjunction with high tier rescues and organizations that do a lot of work with this. And I had started a business where I was doing educational demos for schools and for small outreach programs where I would be able to show pretty much every native animal in the United States, like all the different native venomous snakes.

[00:19:34]

So we had like copperheads and water moccasins and all of these different types of venomous snakes and we had crocodilians and we had all of this, you’d think it would like a zoo and we would go from school and we’d show people these things and we’d educate them and teach them best practices in terms of like, if you were to encounter something like this in the wild, how would you interact with it in a way that is safe. And in most cases, it’s just don’t even go near it. Just stay away. It’ll stay away from you. But it was really a great experience. And I learned a lot about the animals themselves, the behavior of the animals. And then after finishing graduate school, I dialed it back a little. I still have a very large collection of a variety of different snakes and gecko species. My breeding projects have all been turned off because I’m focusing on other bigger things. But yeah, I actually still have some eggs incubating right now from some of my geckos. I had a couple of baby geckos hatch last week. It’s a great hobby. I enjoy it as a hobby. The way I thought of it is, I started it as a hobby and then it transitioned into a business.

[00:20:36] And then when I started feeling too much like a business, I was like, Huh, I’m going to dial it back and it’ll be a hobby and I’ll just do this as I enjoy doing it. It overlaps a little bit with my research because there’s a lot of very interesting metabolic potential in reptiles that we don’t often think about. For example, I have snakes in my collection that will go a year at a time without eating. Well they will eat one meal and they will sit for a year. They won’t lose any weight. They won’t behave any differently, but they won’t eat. So this led me to believe like, well the metabolism. Something is absolutely fascinating about their metabolism. And if we can somehow repurpose that microbial community or the function of that microbial community, there’s a lot of potential in terms of what can be done to increase yields in agriculture or even in some cases, help with a variety of different human diseases. If it’s something that we could use to control metabolism, slow metabolism down, if it has to be slowed down, there’s a lot that can be learned from these animals that I think a lot of people don’t take into consideration.

Grace Ratley: [00:21:38] Yeah, certainly. I was just about to ask if you regularly sequenced their microbiomes.

Jason Arnold: [00:21:44] I’ve wanted to. I haven’t gotten funding for that yet. Presumably at some point I will actually do it. I have enough animals in my collection now to generate pretty robust data from it, but what I would probably do is I would network with others and other organizations that I’ve worked with, get samples from a variety of different housing sources and make sure that we have a wide variety to be able to get some decent statistics out of it at least.

Grace Ratley: [00:22:09] That’s so fascinating. So is that how you got into science through reptiles?

Jason Arnold: [00:22:16] Well, it’s a tough question. I’m going to go with No. I think I’m more so got into reptiles because I was interested in science. Some of the earliest things I remember when I was a kid is I would have picture books of animals that live in specific areas. And I would always tell my mom and my family, I want to be a marine biologist. I’m going to be a marine biologist when I grow up. We lived nowhere near the ocean. Western New York, there isn’t an ocean near there. So needless to say, that didn’t happen. The life in the ocean fascinates me, like biology in general has always fascinated me. My undergraduate was actually predominantly in chemical engineering, so I went for studying chemical engineering for three and a half years. I actually had one semester left before I transitioned out into genetics, and it was because I took a genetics course and that was when I was starting to think about reptile breeding. And so I guess it overlaps a little bit. I wouldn’t say that the reptiles got me into science or that necessarily the science got me into reptiles, but they go hand in hand very well. So yeah, after taking one genetics course, I was like, All right, I’m going to put all that money that I could be making as a chemical engineer aside, and I’m going to do science. So that’s how we ended up a biologist.

Grace Ratley: [00:23:31] So then how did you move from genetics to microbiome science?

Jason Arnold: [00:23:36] Then in my undergrad, I was studying genetics and cell biology, and I took a couple of microbiology courses just because they were required. And I thought it would be interesting to understand I mean the smaller side of things. After taking one of those courses, I enrolled in an undergraduate research program, and this is the first time I had done research. And it was this professor who worked on fungal development. So I worked with him for a summer and I realized that A, I really like this, and B, I was pretty good at it. So I was like, okay, this is good. This is a good fit. But I had never even considered graduate school at that point. I was thinking, okay, I’ll finish a bachelor’s degree and I’ll go be a lab tech or do whatever it is I want to do. After doing research for one semester and one summer, I was like, No, no, no, I’m going to have to go to graduate school because this is just what I want to do. So I joined the graduate program, and the program that I was in required us to do rotations in multiple different labs. So I was thinking to myself, Well, I’m just going to stay in this lab doing what I’m doing in fungal biology. This is great. But I was forced to go rotate another lab. So one of the labs I rotated with was a biochemist, like an old school biochemist. It was all the polyacrylamide gels for gel shift assays and all of this stuff from ages past that we don’t really do very often now.

[00:24:54] And that professor was starting a small research project where they were interested in understanding how bacteriophages these viruses that infect bacteria allow for the production of toxins that cause disease in humans. I was very interested in that. And one of the big questions he was asking, because the idea was that he wanted to study the evolution of these toxins. And the thought process was that these toxins were evolving as not so much as a mechanism to make people sick, but as a defense for the bacteria against either environmental factors or environmental predators. So during my rotation project, I had started with this guy and he had told me, All I want you to do is just get a picture, a microscope image of an amoeba eating a bacteria. That’s all I want. And it seemed pretty straightforward and it seemed easy enough. But I kept getting pictures of dead amoeba and I was like, I don’t understand why they keep dying. This doesn’t make any sense. So I met up with my advisor and I was like, Look, I’m using this bacteria that the other, the graduate student gave me and he said, This is the bacteria that he’s working on. It should be fine. The amoeba should eat it, what’s going on? And he was like, Oh, that’s interesting. He gave you the wrong strain. He was supposed to give you a strain that was a knockout for the toxin gene in that strains not but the literature shows that amoeba are immune to this toxin. What’s going on? So we switched the strains immediately, got beautiful pictures of amoeba eating bacteria. No problem whatsoever. Switched back. All the amoeba are dying again.

[00:26:29] So my entire PhD thesis was built around the concept that this paper from the 70s was wrong. And this publication suggested that the toxin that this bacteria was producing, so it was Shiga toxin. It had shown amoeba is immune to it. And they did all of these complex assays where they took pure toxin and they incubated amoeba with it and the amoeba would live no problem at all. And we ended up finding out that, yes, amoeba are immune to the toxin in its pure form. However, if the bacteria is producing the toxin from inside the amoeba, the amoeba will die. So it was very interesting to find that out. And what we ended up doing is we did a lot of molecular research and we did the actual molecular mechanism of what was happening in the amoeba. Unlike mammalian cells, lack the cell surface receptor that’s responsible for binding to the toxin subunit that allows it to get into the cell. So pure toxin is just going to bounce off the amoeba as if nothing is there. However, when the bacteria is already internalized and it’s in a vacuum inside the amoeba and that toxin is released from inside that vacuole, it passes right through the vacuole wall into the cytoplasm and kills the amoeba.

[00:27:36] So we did a whole bunch of experiments on that. And then we actually went a step further and even identified the mechanism by which the amoeba were identifying what bacteria is, food and what isn’t, which was very interesting. And it led to my thesis, which focused on the evolution of bacteria in the context of antipredator response. And that to me was fascinating. So we have this polymicrobial interaction, and I was thinking about that as I was getting close to graduating and I’m like, Yeah, polymicrobial interactions are really interesting. What about very complex communities? What about the gut microbiome? And this is around the time where next gen sequencing was emerging at a point where it was accessible and it was affordable, and we could actually do high throughput sequencing studies and metagenomics was just coming up the conference. I was at my final year of graduate school was, I mean, all metagenomics [] the new thing it was the awesome fun thing that everybody was doing. And all of this data for complex communities was being released. And I said, You know, that’s interesting. I was on the toxin producing pathogen side. Let’s take a walk over to the other side and look at how these beneficial microorganisms interact with the microbial community to help benefit hosts. So that’s how I ended up in that.

Grace Ratley: [00:28:52] That’s an awesome journey and Amoeba is just mind boggling. It makes me wonder a little bit about within the microbiome. Obviously the most commonly studied microorganism is bacteria, but you also have the microbiome, which is the fungus that live in our gut and the virome, the viruses in the gut. Have you done any work with these polymicrobial interactions in your microbiome research?

Jason Arnold: [00:29:20] Yes, I have an active collaboration now. We’re working on finishing our first publication, looking at Protozoal pathogens in the gut in human cohorts, and how these pathogens not only cause disease, because in some cases the disease itself isn’t really noticeable or isn’t really even measurable, but how they interact with the microbiome in such a way that I don’t want to call it symbiotic because it’s the opposite of that for the host. But how these interactions between protozoal pathogen and microbial community lead to more severe disease in the host. So we have some studies ongoing there. And then parallel to that, we have some studies looking at the same protozoal pathogens and how probiotics can be used to combat infection and how specific bacteria colonizing specific niche space within the large and small intestine can displace these pathogens and help clear the infection more rapidly than in regular, conventionally raised animals without probiotic treatment. So we are doing some work there. In addition to that, with technology increasing and the new technologies emerging and becoming more affordable, most microbiome studies historically have been focused on sequencing one specific region of a bacterial gene that’s conserved across all bacteria, the 16S ribosomal gene. So a lot of sequencing studies do is they amplify that region and they sequence it and that’ll give you a snapshot of all the bacteria that are in databases and in that community. So as we advance and as sequencing technologies get better and more affordable, we’re able to get much higher depth.

[00:31:04] And instead of just simply sequencing one gene or one fragment of a gene, we can sequence the whole genome of not only the bacteria, but everything that’s in the community at once. So a lot of the studies that have been coming through recently, within the past 2 or 3 years or so, it’s transitioning more toward this whole genome shotgun sequencing approach where you’re sequencing every bit of DNA that’s there. You’re going to get some host, you’re going to get some bacteria, but you’re also going to get all of the DNA viruses and protozoal pathogens and other any other eukaryote that’s there. Right now, the limitation to a lot of those studies is that the databases just simply aren’t good enough. They don’t have enough information there, especially from the protozoa. That’s one that the databases for protozoa pathogens are so lacking that even from whole genome shotgun data were in many cases unable to even identify the presence of many protozoal pathogens. Viruses are the same thing because viruses are so diverse that there’s not really a well curated database for viral DNA sequences. And unfortunately, we’re not at a point yet where we can well, I mean we can, but not affordably be able to sequence both all of the DNA and all of the RNA and decipher like what RNA viruses are there. So virome studies within the gut microbiome, we’re not there yet. We’re on our way. But hopefully within the next couple of years we’ll be able to actually have technologies that work for that.

Grace Ratley: [00:32:31] What other ways can we improve microbiota science? Like how can we improve the rigor of the studies? Or what needs do you think are unmet in the microbiome science?

Jason Arnold: [00:32:46] There are quite a few. So rigor and reproducibility, at least for the NIH and for most people who work in the microbiome are key factors to keep in mind whenever you’re developing a microbiome study. So one of the big things that is difficult in microbiome work is being able to take your results and replicate those results in somebody else’s lab because tiny things that seem like they won’t make a difference, the impact that those tiny things actually have on the overall output of the data that comes out is overwhelming, diet being a big thing. Like if you have a big human study, you can’t really control for diet in a large human study. So it makes it very difficult to reproduce a human study because the diet of your cohort may not be the same as the diet of the cohort from the original study. In mice, that’s a little easier because you could use defined diets. But things as simple as how the mice are housed can completely change the outcome of a microbiome study. Let’s say you have a study with ten mice and you have the mice housed individually in ten cages, and all ten of those mice are being fed the same diet. And then you run the exact same experiment, but you don’t have the housing available for ten cages. So we’re just going to house them in pairs. You can get completely different results because something so simple as the stress that the animal is under being housed singly versus being housed in a pair or in a group that could result in the production of of stress hormone factors that modulate the microbiome and change what happens in the community. The batch of food that you can use can be different.

[00:34:19] The animal’s genetic background could be different the way they were housed originally. It’s endless. The number of different things that could go wrong, not even necessarily go wrong, but could result in a different result than you were expecting. One of the big things we try to focus on is standardization of our methods. So whenever we start an animal study, we standardize all of the animals we co-housed everything in groups for a couple of weeks prior, all being fed the same diet. Then we split everything. So you have this homogenized microbiome before you start. Now the homogenized microbiome in our study may end up being different than the homogenized microbiome in a different study, but at least starting at a normalized point will give you an idea of what changes are occurring. So those will allow for comparisons on that front. Even though you may see dramatic differences over the course of the entire study, we always try to make sure that the genetic background is clearly recorded and that we know exactly what we’re working with. And when we’re doing an animal study, when it comes to human studies, the only thing you can really do is just increase your sample size, get to a point where your sample size is large enough that any confounding factor that gets introduced is just going to be lost in the background. That’s often very hard to do because a lot of these studies are not cheap and trying to get to a point where you have 12,000, 15,000 humans, it’s very difficult to do.

[00:35:37] So yeah, there’s a lot that needs to be taken into consideration in terms of reproducibility, also randomization of samples when you’re preparing DNA isolation. So you have your samples, they all come in, you’re ready to start your extractions for the sequencing and you’re going to have to do it in three batches. All right, the first 40 samples here, next 40 samples, next 40 samples, run them all. You’re going to have batch effects. So if you don’t randomize all of your samples first, you may see differences between sample groups that are in different extraction subgroups. And those differences could potentially give you false positive results or false negative results. In addition to that, you want to do the same thing again when sequencing. So sequencing batch effects are a real thing. So if you have one sequencing run on an OVA sequence and you have a second sequencing run on a different NovaSeq, even if it’s the exact same samples, the results are going to be slightly different. So what you want to do again is make sure that when you’re loading your samples that you have biological replicates all randomized within all of the different sequencing runs. You have to run library preparation matters. That’s another thing that people often don’t think about.

[00:36:48] The kits that you use or the reagents you use are going to differ from different lots, even from the same company. So if your reactions are being all prepared from a variety of different lots, you’re going to want to make sure that you have all of your biological replicates separated across all the different lots that you have. Otherwise you may introduce biases. And the last thing that we think about when we’re preparing samples for clients or for ourselves is the hands of the person doing the experiment matter. So me making a library versus you making a library, it may be the exact same protocol, exact same reagents, but they’re different. And it’s just a matter of a person’s technical handling of the materials. And those differences all matter when it comes to reproducing a result in a microbiome study. So we try to we think about it in the Core as the fewer cooks in the kitchen, the better. So if you have one person who takes the project and it’s their project and they’re going to run through the whole thing, nobody else is going to be involved because then you’re not introducing all these additional biases as you’re going through the process. Standard operating protocols are always very important as well, making sure that everybody is using the same protocol all the time, but for the big things that we think about.

Grace Ratley: [00:38:06] Yeah. And then microbiota science has become extremely popular within the general public. People are very interested in improving their health using microbiome science. What misconceptions do people have about microbiota science, or what ways can people get reliable information about microbiome science?

Jason Arnold: [00:38:29] So, yeah, this is the age of misinformation, after all it seems in some cases. So it isn’t easy. And because this is emerging as a new popular topic, you have a lot of mixed reviews on what’s real and what’s not and what’s good and what’s bad. And it’s very hard to really know. Now scientific literature helps, but scientific literature is limited. And not everybody has full access to scientific literature. People may be able to get access to abstracts and be able to read it over. Oh, lactobacillus, such and such is beneficial for colitis. Great. But what are the results actually show? Because sometimes people are publishing things and the publication and the title and the abstract don’t necessarily match exactly with what the results are showing. So there is a bit of scientific rigor that has to go into understanding really what’s happening now. I think for the most part, news outlets, they don’t go too deep into the science often. Maybe that’s good, maybe that’s bad. It’s really hard to say. Unfortunately, the information that the general public has easy access to isn’t always complete. So generally speaking, eat a healthy diet. That’s a good thing. There’s no downside, even if it isn’t scientifically proven to change the abundance of lactobacillus and whatever. There isn’t a downside to eating less fast food and eating more healthy fresh food.

[00:39:54] Things like that are good. There’s no real downside to most probiotics. Most probiotics are not going to cure cancer. They’re not going to make your hair grow back and they’re not going to do all these magical things that some of the manufacturers want them to. But adding beneficial microorganisms to your gut won’t hurt. So that’s the way I generally think about it. And I try to think more along the lines of instead of what is the magic bullet, what’s going to make everybody healthy. It’s more along the lines of trial and error for yourself, because personalized medicine isn’t really to the stage where we can say yet. So if it works for you, great. But it doesn’t mean it’s going to work for everyone. So FDA doesn’t work on probiotics and prebiotics at this point. They don’t do any approvals. So any claims that are being made by these companies that produce these mixtures or compounds, it’s unfounded in many cases. I mean, there may be scientific literature that backs it up, but nothing is federally regulated. And that’s something hopefully in the future we move toward thinking about regulations because you can make a claim and have it be based on nothing. That’s kind of problematic.

Grace Ratley: [00:41:02] Do you cook a lot of fermented type foods or drink kombucha type of things?

Jason Arnold: [00:41:07] So you would think I would. I don’t like the taste of it, so I don’t. My fiance does. My diet is predominantly pasta. It’s really unfortunate because you would think with all of this background in microbiome and understanding like what diets you really need to have. I eat a lot of fresh fruits and vegetables, I fresh make my food every day. I don’t eat out very often, but it’s not like eating gallons of yogurt and drinking kombucha or any other random fermented stuff just because I don’t like it. It’s not because I don’t think it’s good for me. I’m sure it is. But it’s just a taste preference and I just can’t get over that for myself, even though I know it’s probably the right thing to do.

Grace Ratley: [00:41:49] It’s totally valid. I’m a fan of Kombucha, but I don’t eat a ton of other fermented type foods.

Jason Arnold: [00:41:56] So on that note, we actually did do fermentation of ginger beers for a while. Like a bunch of us in our group, we thought it would be fun to just make our own ginger beers. So we were actually making our own. We got a starter culture from a friend of ours who was doing this for a long time and we made by modulating the sugar that went in. We fermented different like I made a melon beer and I made just a variety of these different ginger beers and they were delicious. So just like as a hobby. And I wouldn’t like ferment foods full time, but it was all right.

Grace Ratley: [00:42:29] Yeah, I see. I love that aspect of the science. I mean, yeah, there’s a lot of misinformation out there and microbiome friendly hand soaps, which are kind of ridiculous.

Jason Arnold: [00:42:39] Sure, yeah, it is a little silly.

Grace Ratley: [00:42:41] But I do think it’s interesting because it gets people interested in science. It gets them interested in making things or eating healthier. And I do appreciate that about it being popularized. So I guess to wrap up our episode, I normally ask if you had advice to give to people who were trying to go into your career path, people maybe who are at a postdoc stage or early career scientists. What advice would you give to these people?

Jason Arnold: [00:43:13] There’s a lot. I guess the first thing that I would say is probably my biggest regret is, I never learned how to be incredibly proficient at coding. If you have the opportunity to learn how to code in R and Python or whatever, do it, it will benefit you beyond your imagination down the line. I have a very limited experience with it and like I said, that’s one of my biggest regrets is not really going harder into that. It allows you to do a lot more. It unlocks a lot of tools that you may not have access to otherwise. As far as early career scientists like postdocs, graduate students, I guess I would recommend, the biggest thing that I think I learned throughout my PhD was accepting what you don’t know. That’s always something I tell all my students. If you don’t know the answer to something, that’s not a bad thing. It just shows you what you could learn. I remember at my thesis defense, actually, one of my committee members was asking questions that he knew I didn’t know the answer to because he wanted to hear me say, I don’t know. And he basically just asked a question. I was like, I don’t know. But here’s where you can find that information or this is how I would go about finding that information.

[00:44:29] And it forces you to think about not only you don’t want to have that idea that you know everything because nobody does, and being able to acknowledge what you don’t know, it gives you the opportunity to grow as a scientist. And that’s a big thing that I took out of my training and my years of doing this is the more you don’t know, the better in some cases because it gives you more to learn. And I don’t know about everybody out there, but I like to learn. That’s one of the things that I enjoy about what I do. And being able to acknowledge that you don’t know something is the best way to be able to learn what you can learn. Aside from that, mentorship is important. You want to make sure that you work with somebody who’s able to provide you with the feedback you need and the support that you need to make the next steps in the career. It’s not easy. It’s a very competitive in academia especially. I mean it’s competitive in industry as well. But in academia especially, it’s extremely, extremely difficult. So without that kind of guidance and support by somebody who has the experience, it becomes a lot harder.

Grace Ratley: [00:45:29] Well, thank you so much for coming on the podcast, Jason. It was wonderful to hear your perspective about all the interesting microbiome science and also reptile science.

Jason Arnold: [00:45:36] Yeah, absolutely.