Smart Athlete Podcast Ep. 39 - Dr. Cheryl Keller Capone - GENOME SEQUENCING - Part 2 of 3

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JESSE: I want to transition a little bit and talk a little bit about your research. It seems like from your kind of CV background page you sent me you've done a lot of different things. I will probably have to stop you at various points and ask explanations on various pieces of jargon, as I try to work my way through what you done. So, can you start with what you worked on as your dissertation for your Ph.D. and we’ll kind of try to move forward from there or hit the high points, I guess? CHERYL: Sure. So, I did-- I will preface this by saying I did wind up doing my Ph.D. postdoc and then my current position all at Penn State University. That was not my original plan is discouraged, especially in life sciences and some other fields as well to stay in the same spot. This considered that you are not really broadening your horizons and your ability to contribute intellectually could decrease the longer you're staying at a certain place, but I stayed-- I made the decision to stay for personal reasons. You know, I met my future husband as I was finishing up my Ph.D. So, that's why I stayed and I'm fine with my choices in that respect. So, when I was in-- did my Ph.D. I studied muscle development in fruit flies. And the first thing that a lot of people are like “Oh, muscles? Fruit flies have muscles?” But they fly, they walk so yeah. And we specifically studied embryonic muscle development, so that the muscles that would form when it's a developing embryo, and that the larva would use to crawl around on the meat or whatever they're going to eat. And I studied a gene called Nautilus. And what we realized that that gene does, they’re several they called paralogs. And what that means is related genes in like vertebrates, and they're like muscle regulatory factors. And we, the gene that we studied ?? 02:11> was responsible for differentiation of muscle precursors. So, when muscles are first forming, they don't look like a muscle, they don't look like a ?? 02:22> muscle, right, it just looks like a round cell that might express some muscle proteins like myosin and actin, but when you're actually differentiating or forming into the more mature muscle type, what are the genes responsible for that process? And we found that this gene Nautilus that I worked on, was responsible for a subset of muscle differentiation in the embryo. And we used a variety of different approaches, a lot of genetics and molecular biology. I did a lot of microscopy during that time, so I really felt like it was a really good experience. I felt like I learned a lot. And I came out of there though, and kind of wanted to switch things up a little bit. Like I said, I stayed here at Penn State, so I wanted to broaden my knowledge and my experience bases as much as possible. So, I actually switched-- I did switch fields. Some people will actually stay in the same field that they get their Ph.D. in. And for me, I decided to switch and did gab receptors in mice. And gab receptors are responsible for the majority of inhibitory neurotransmission in the central nervous system. So, to kind of calm things down, right. And so that again, was-- I did a lot of work with mouse neurons, primary mouse neurons that we had to dissect out of these tiny little 14 day embryos, which I felt very bad about. But the worst thing actually was when I was doing these mouse dissections is doing these with little forceps and I had to do it under a dissecting scope. And every time we did dissections, I could not drink any caffeine that morning. That was probably the worst thing about it, right? I was like, I really want a cup of coffee, but it was so tiny that you really did not have the control to really get in there and do these dissections. JESSE: So, your dexterity would be affected by the caffeine ?? 04:30> CHERYL: Yeah, I mean, because not any noticeable. It's not like I would notice it if I was taking notes with a pencil or doing anything else. But when you're actually trying to dissect really, really small things under a microscope, it is just enough that you did you lost a little bit of that control. So, that was-- but I studied that for a couple years and I wound up studying this one, so that these gab receptors, the gamma unit, so the gap receptors are made up of a number of different proteins that together form the receptor. And one of those proteins is gamma sub unit. And I worked on trying to find various proteins that might interact with this gamma subunit to try and control-- just to try and learn more about how these gab receptors cluster in synapses. So, what you in general, right, so receptors, neuron receptors will be at synapses, where you have an axon, and then you have your dendrite, and your receptors will cluster together underneath the axon to receive input. And what we were trying to figure out is what are the mechanisms behind the clustering of these gab receptors at synapses. And so I did some screens to look for interacting proteins and I discovered the first mammalian ?? 06:02> transferees. And what that means is, it is an enzyme that attaches - group which is a fatty acid to this gamma subunit which would then affect its clustering. It would allow-- affect how the receptor gets to the cell surface and gets recycled back and so forth. So, that's sort of what I worked on as a postdoc. So, I kind of I did a couple ?? 06:31> switching model system, worked with fruit flies and I worked with mice, and I enjoyed both those things. And after that, I kind of felt like I needed a break from academia and I thought, oh, I just, I need to kind of get out here and see what else is out there. Unfortunately a year in State College, Pennsylvania, I mean, Penn State is the largest employer of science people. There are few companies around There's really not a lot of options as if there would be in a bigger city where there might be pharmaceutical companies or biotech, and we just don't really have that here. But I did work for a small company who did mitochondrial DNA forensics. So, this is back, 2004 and we tried to remember when the human genome was sequenced, I want to say 2001. JESSE: That's what I wanted to say offhand. But I would trust you more than me on that. CHERYL: Say that, around that, yeah, a little before that, but it really wasn't-- Nowadays, you can do whole genome sequencing pretty fast. In fact, that's what I do for a living now, but its DNA sequencing, but back then it was harder to sequence DNA from blood and so forth. But the other thing too is that mitochondria, so red blood cells do not have mitochondria-- Well, they don't have nuclei. So, you have if you wanted to isolate DNA from red blood cells, you could get mitochondrial DNA, but you're not going to get your nuclei gene, so genomic DNA. But anyway, we would-- So, a lot of what we would do would be to get, like samples of bone and hair in. Especially like hair samples, and a lot of it was cold cases where we would isolate mitochondrial DNA from old hair samples and a lot of it was trying to more like exclude people as being, you know, exclude people from committing the crime, right, so where they get convicted and based on some evidence. But there was not any DNA evidence and there might have been some old hair samples or blood samples or something from the case and a lot of times, we would test those and look to see whether they were a match to the person who was being accused of the crime. So, it kinda, it was interesting. I learned a lot about quality control at that point and really taking-- Not that I was not careful as a scientist, but when you're thinking that you might have somebody’s, not actual survival life on the line, but their livelihood in terms of whether or not this person is in jail or not, right, you realize you take a lot of responsibility for that and be like, wow, I am really affecting-- potentially really affect somebody's life. And I think it really kind of stepped up my awareness in terms of sample handling and quality control and things like that. So, I think that actually, that made for pretty good, for me, a good transition to my current position, which I’ll go into a minute. But I actually wound up after I left there, I took a couple years off from that. I sort of stayed at home and worked part-time when I had my son in 2005. And I never really left science. I taught a couple of classes in the evening, a couple of molecular biology classes, and I did some freelance work for some publishing companies, where I wrote some test questions for online material, studying material and things like that. Before then I got my current position as an associate research professor, and we study blood cell development. And mostly I do a lot of DNA sequencing, which is really kind of fun. JESSE: So, I want to back up a little bit. You were talking about-- No, no, you’re fine. I'll just like try to keep notes and trying to like remember okay, I want to ask about this and that. There was something about ?? 10:58> I want to ask but I just wrote that down and then I lost my question. CHERYL: Sorry. JESSE: No, no. Well, maybe I'll think of it. So, I was curious with your taking samples, you're saying you're taking, or doing like sequences of samples from cold cases. This is just a personal curiosity, but like, is there a shelf life on that kind of thing? I mean, I would think that like say you got a bone fragment for some reason. Well, the bone fragment in storage is going to be fine. Like, it's not going to decompose. But I just wondered if at the DNA level, if like there's unraveling over time, or if like the integrity of the sample can be relied on over a variable amount of time. CHERYL: That's a really good question. And there is some level of degradation of nucleic acids over time. But some of the bone fragments we worked on we're decades old and it kind of-- sometimes you would get some high-quality mitochondrial DNA from that and sometimes you wouldn't. And, excuse me, we would-- in order to sequence some of that mitochondrial DNA, we would amplify using PCR to amplify different portions of the mitochondrial genome. And we would use several different areas so that you're not just relying on one portion of the mitochondrial genome being intact. Okay. So, you may get let's say, and I don't recall honestly to be truthful, the exact number of ones we did. But let's say for each sample, you try four sites, and you may get amplification of all four or you might get amplification of just one or two depending on the age of the sample. And then you can, but you what we always do is compare it to a known, right. So, if you had a sequence from your unknown, and you got some sequence from that and then you compare it to a known individual and does that match. So, at one point, depending on the level of sites we were able to amplify r sections we could amplify and that amount of match. You know, a lot of times it was more like you would exclude somebody more than say, it's definitely this person, right? So, it was more of a system of exclusion because mitochondria are transmitted from mother to son and mother to daughter, right? So, you're always getting your mother's mitochondrial DNA. So, you would have the same mitochondrial sequence as your mother. So, you really couldn't say it was you versus her committed a crime, right? But it could exclude you from being a person from a different mother. JESSE: Right. This is essentially just another method to move forward, whether you're trying to include or exclude somebody, it's just, you're approaching the problem from two different sides, essentially, which is depending on what you have to work with, like you mentioned us sometimes it's just pieces instead of the entire sequence. You do what you can and what makes sense versus what you necessarily want to do, which is like, no, this is absolutely the-- we have the whole thing and matches perfectly. It's like no, you just work with what you can. CHERYL: And there is also no mitoc-- unless you have the hair shaft, the actual part that's stuck in the head, you don't have any genomic DNA, right? So, when you're actually just - a lot of times hair samples too, you're working with more of a little piece of the hair sample, and that does have mitochondria, but, again, unless it has the shaft, then you can't isolate genomic DNA. So, there are advantages and usefulness too still doing some mitochondrial DNA forensics. It's just that with the advantage of-- advance of genome technologies and sequencing nowadays if you have genomic material, it would be much more clear of whether there's an actual match or not. JESSE: So, I mean, it seems like no matter what project or research projects you're working on, like you seem to be focused on the gene sequences and trying to figure out what genes play a role and whatever it is. Please correct me because I'm going to get this wrong. Like when you're talking about knowledge and the development of muscles in the fly embryos, correct, you’re trying to figure out what, I want to say, what mechanism or what gene ?? 15:37> the word. What thing, I'll just say thing and you can give me the actual word here a minute was telling the muscle to create this particular muscle. Right? CHERYL: Right, right. Actually, that's a great explanation and that it's really about gene regulation, and how genes are turned on and turned off. And what are the processes involved in how genes are turned on and turned off, and what networks might be involved or other processes? And I think a lot of those will affect ?? 16:11> cell development now? And when we start with a stem cell, is it going to eventually become a white blood cell? Or is it going to become a red blood cell? And we look at how that cell fate’s decisions are made. And by learning more about those processes in a normal cell, and again, how genes are turned on and genes are turned off, and all those regulatory mechanisms; that can provide some insight into disease processes. So, you really got to kind of learn what's normal and how things work normally, and then you can ask those same questions by looking in some sort of disease state, and learn more about that, right. Which the-- again, down the road, your ultimate goal really is to improve human health and performance and so forth. JESSE: And that's kind of what the vision project’s about, right? So, trying to figure it out. So, I tried to write a summary, you provided much more detail, but basically looking at genetic traits to determine like susceptibility to disease or resistance to a particular treatment. CHERYL: Exactly. So, we-- so vision, which is sort of a bit of an acronym, stands for validated systematic integration of metaphoric epigenomes, which is a big mouthful. Really, what it really involves is that right now, we as a scientific community, who are involved in a lot of gene regulation and DNA sequencing and so forth, means the amount of data that's being generated is tremendous and data generation is no longer the limiting factor. You know, back when sequencing was first-- DNA sequencing first became more widely available it was this big rush, let's sequence everything, sequence it all. You know, and everyone was super excited but there was a cost factor in that it was very, very expensive too, both in ?? 18:22> and equipment and processing and all that. It's very expensive and time-consuming, but data generation is no longer the limiting factor. And it's more now is how do we use all that information to extract what's relevant and make hypotheses, and predictions, and then test them? Right. So, it's great to generate data, but unless you have ways to analyze it and pull out what's important, it’s not that useful, right? 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