Exciting Advantages to Single-Cell Sequencing of Parasites: Researchers Walzer and Chi Explain

Richard Jacobs: This is Richard Jacobs with the Finding Genius Project. I have 2 people today. Dr. Jen-Tsan Ashley Chi And Katelyn Ann Walzer, Dr. Jen-Tsan is from Taiwan. He obtained a Ph.D. from Stanford University; postdoctoral work at Stanford and he is working with such genomic analysis and gene expression to look at the influences of the tumor, microenvironment stresses, cancer, and the heterogeneity tumors. Katelyn here is now a postdoc at the University of Pennsylvania and she’s looking at actual single cell type analysis. So we’ll get into that. I want to welcome both of you. Thanks for coming

Katelyn Ann Walzer: Thank you for having us.

Richard Jacobs: So, Katelyn, if it’s alright, we’ll be focusing on your work, probably the most fast-moving and interesting just for the sake of this podcast. Please tell me about what you’re working on right now and I’ll ask my questions around that.

Katelyn Ann Walzer: So, for over 10 years, actually, I’ve been working on apicomplexan parasites. I started out on Toxoplasma gondii, which actually is the easiest one to work on and I progressively worked on parasites that are more difficult to handle. So I currently work on a parasite called cryptosporidium parvum that causes diarrheal diseases in humans. It’s also a big agricultural pathogen. So I did my Ph.D. with Ashley in the genetics and genomics program at Duke and I’ve learned a lot of genomic technologies by being in his lab and through my graduate program. So I took the knowledge of those genomic technologies and have done a lot of sequencing of parasites. So currently we did single-cell sequencing, RNA sequencing of cryptosporidium, parasites, and previously we did not know the genes that were expressed throughout what’s called the asexual cycle and then also the sexual cycle.

So note that what we know so far in parasites is they also have male and female. So there are two mating types. I’ve been able to figure out exactly which genes are expressed in each of those mating types through single-cell sequencing, both in Plasmodium falciparum, but now in cryptosporidium parvum.

Richard Jacobs: A bunch of questions here. So cryptosporidium, what are its hosts? How does it get into people? Let’s just give a little bit of broad background on that.

Katelyn Ann Walzer: So cryptosporidium is primarily an agricultural pathogen. So actually, it’s at the oasis stage is what’s transmissible. So we could actually get oasis from cows and then those oases are very environmentally resilient. So they can just hang out, we get the oasis through feces and then those can stay in the environment for long periods of time and then a human can pick that up through multiple ways, for instance, by eating it. So then you can end up getting cryptosporidium that way. Again, the cycle just continues. One thing we are trying to figure out right now is, are the oasis so they can exit the host. But then we also believe that some of the oasis can reactivate and that actually causes chronic infection and so why this is such a huge concern is in many countries, particularly in Africa, you can have children who already have or are susceptible to this diarrheal disease, but then they keep wasting away in terms of having this pathogen chronically infect them and that’s really why it’s such a big problem.

Richard Jacobs: How novel is it that you’ve been able to sequence single cells. From what I hear, that’s just really a very new technology. For some reason, I don’t know why it is difficult. Can you talk about that?

Katelyn Ann Walzer: So RNA sequencing only became popular probably in the 2000s, and we they quickly moved from being able to do RNA sequencing, Ashley, my advisor here, had done microarray, which previous to RNA sequencing, and he did that early 2000s. So in the span of even just 10 years, we’ve gone from being able to do just sequencing and then taking that to a single cell. It didn’t take too long to move the technology forward very quickly and then I was within the first wave of scientists to do this in the malaria-causing species, Plasmodium, and it did involve some optimization. But there’s been multiple groups now and it’s kind of taken off that we’ve been able to analyze single cells. In the beginning, it was difficult because the first couple of times I did this in graduate school, I didn’t give very many reasons for my sequencing.

It was very difficult to decipher anything from it. But as technology has improved, we’ve been able to garner more and more from this data. So in graduate school, I focused on the fluid on technology and the smart sequencing, which with what I did there, we only focused on tens to hundreds of cells. But now I actually did 10X genomics for cryptosporidium, and we were able to get up to like 5000 cells pretty easily. So the technology has vastly improved over a short period of time.

Dr. Jen-Tsan Ashley Chi: So, early days, we basically; most people analyzed as a parasite the bulk population where we get hundreds of millions of cells. We all, like took data and analyzed the expression. So what Katey here has done is powered by different technology now actually picks the individual parasite and analyzes the expression. So you can understand when you analyze a population, maybe on the individual difference are kind of masked. You actually never see that. So the interior actually started to analyze individual parasites, so then you actually see it in part in terms of the individual; we call it shady correlating generic. Basically this difference in a bunch of parasites wasn’t apparent when they actually sequenced bulk and when you sequenced the individual, that all reveals itself. It’s actually it’s a very big deal.

I think that’s in the early days when I was doing my Ph.D., people would try to do a single cell because a T cell receptor, each T cell has a different T cell receptor. People use repeat drops and sometimes use that try to pick the individual cell themselves. So after that, it was an instrument that Katey was using the micro fully device-based fluid on that was able to get hundreds of cells. But now, actually, she’s doing this next generation, this emulsion repeat droplet-based technology that typically will get a couple of cells into the cell individual sequence. So that’s become very, very powerful.

Richard Jacobs:  Questions about cryptosporidium. When it’s inside a person, is it just one cryptosporidium or does it make millions and billions of them?

Katelyn Ann Walzer: It makes more. So actually, I think that in order to get infected, you only need up to 10 oases. So it’s just very little and has a lot. So, yes, it does amplify very much once it goes to your intestines.

Richard Jacobs:  So in someone that is showing symptoms, they’re actively sick. What’s your guess on how much cryptosporidium they have in them versus someone that seems fine?

Katelyn Ann Walzer: I wouldn’t know the answer off the top of my head.

Richard Jacobs: The reason I ask is you’re doing this single-cell level analysis now, which is great. But, from what I’ve heard, parasites are incredibly diverse, certainly in the sexual reproduction stage. Does that happen in people? And if so, if you have a million different variations of the parasite, what’s the point of sequencing just one of them?

Katelyn Ann Walzer: Sex does take place in order to create a bunch of different oases. So far, my work has focused on doing in vitro culture, which isn’t so diverse. Actually, what we found, I’m in Boris Streetman’s lab and I’m one of the graduate students who determine that in culture fertilization is actually blocked. So with that, I’ve done so far, we don’t even have some of the in vivo stages but we work on the mouse model and through there we can figure out, and then this is that this is an experiment that I had wanted to do right before we ended up going into quarantine. So we are patiently waiting to kind of get the lab started up again so I can do this experiment but we wanted to do single-cell sequencing in vivo to see what the sexual stages looked like and then how did the parasites then change?

We can see that then after they have sex if there are differences in transcription. But again, another thing that we would like to do in the future is to do not just single-cell RNA sequencing but the new DNA sequencing that we haven’t got to do.

Dr. Jen-Tsan Ashley Chi: Katey, you only actually talked about your Ph.D. work where you are actually using single-cell to figure out male and female mitosis in malaria. I think that’s actually, the sexual stage is very relevant.

Katelyn Ann Walzer: Right and I think that was very relevant too because previously it was focused on a bulk population and you would have all sexual parasites together and people also did flow sorting. We never had a good sense of the developmental progression. So in falciparum, the maturation from asexual to sexual takes up to two weeks, which is a very long time. So through my data set, we were able to decipher earlier on what the male and female differences are. I think that’s very useful, not just for our science interest, but in terms of looking at even with markers or field isolates. A lot of the markers that are used in flu patients are actually only female-specific. So P25 is a big one, is a big marker gene in plasmodium falciparum but its only female specifics, so we don’t have a good idea of what the gametocyte carriage is for patients. So I feel like that’s one big advantage of my work, is that we were able to find a bunch of different genes that are male and female-specific and you can’t find that.

Richard Jacobs: I guess you could also find two in a given population where they do reproduce sexually. Do they produce more males and females? Do they have different roles in the host? But again, if you’re not seeing the females or you’re not seeing the males, what does that tell you or misinform you about, etc.?

Katelyn Ann Walzer:  I read papers that said that there are differences. So actually, there’s a different ratio. So in falciparum, crypto too, it’s a 5 to 1 ratio of females to males. So there are so many more females in these species than males. I’ve read that there are differences in the male ratio, sometimes in patient isolates, at least for falciparum. So that can make a difference and it’s something to look for in terms of how virulent these parasites are. I think the one thing that really got me interested in doing parasite work is understanding how virulence factors arise and these often occur through sex. So it’s something I’ve always been interested in following up in my own research.

Dr. Jen-Tsan Ashley Chi: Yeah, maybe you could also you mention that; I think also the answer is actually this basically identify this male population is really the power of single-cell because they are so rare. That is very rare, in the bulk cell population it is very, very hard to find a male. So Katey was able to find this very, very rare population, especially today, we talk about diversity and individual person, when we are using the single-cell approach, we can magnify a very rare cell with a very special property like I think the parasites are very rare. If it’s a male, even more so because there are so few and it’s very rare.

Katelyn Ann Walzer:  Yeah. So we’ve had the same issue in cryptosporidium. So actually a paper that we published from our lab before I arrived is in our Nature microbiology paper. That’s mainly a female population that was sexual. But now we’ve been able to figure out male by doing single cell and we couldn’t do that before. So doing a single cell has been very, very helpful, and really quickly moving the field forward.

Richard Jacobs:  Do you notice there’s a cycling of the ratios of males, females at different stages? Does it correspond with virulence or remission etc.?

Katelyn Ann Walzer:  I wouldn’t say it’s so many different stages. It seems to be different strains. So I’ve noticed at least when I did this in falciparum that there are a couple of different parasite strains that we use. It’s not just one strain that infects for falciparum. You have a couple of different ones and there are differences in male and female ratios for those.

Dr. Jen-Tsan Ashley Chi: Also, remember that our result was the male actually is even more under-represented than what’s commonly perceived, right? So Katey does an animation about a specific number to give Jacob a sense.

Katelyn Ann Walzer:  Oh I think like one in 10 were male.

Dr. Jen-Tsan Ashley Chi: Yeah. So basically, I think the traditional conventional wisdom was basically one male versus three females. So your data is actually one male among 10 female and also this radical is important because this a male and female chromocyte is the only cell that can transmit to another person in plasmodium, in Malaria because it basically more so the parasite, a body attached to a dorsal sac in the red blood cell. But when they get sucked into that mosquito, they just don’t just typically digest it and die. But only it is a male and female, they must mate in the gut of the mosquito and they actually form the next generation of parasite for inter-personal transmission. So, to actually figure out this transmission and their mating is a big deal because that’s one block. Right.

Richard Jacobs:  So what are you trying to figure out now about cryptosporidium in particular? Have you identified between males and females, the genes as well, or is that the malaria parasite? What’s your current work about?

Katelyn Ann Walzer:  So my current work is I’ve always been really interested in transcriptional regulators. So, by doing those single-cell sequencing, we now have a bunch of different candidates for different stages of the life cycle. So my next goal is to look at which regulators are involved in asexual progression and also the switch to a sexual progression, as well as male and female. So that’ll be the focus of my; I’ve been on my postdoc for a year now. So we kind of got all the genomics stuff done and out of the way. So now the next step is to take that to a functional study in particular in parasites for AP Complex, the AP2 family is very important for stage-specific differences and so on cryptosporidium, there are 16 of those and I’ve kind of been able to hone in on those and see which ones might be important for the different stage-specific differences. So that’s my next step.

The other part of it, too, though, is what actually is the advantage of cryptosporidium compared to falciparum. Plasmodium species infect red blood cells, and they can also infect liver cells, too, but cryptosporidium infects intestinal epithelial cells. So something else we’re interested in following up on is the host immune response on a single cell level and to see if there are differences in the host immune response, that’s going to be another aspect of my project.

Richard Jacobs:  What do you mean? What aspects of the host immune response on a single cell level would you look for?

Katelyn Ann Walzer:  So I think its interferon responses important at certain. So cryptosporidium what we think is a 12-hour lifecycle. So in looking at different immune genes that are up-regulated at different times, we’re still trying to figure out exactly, different parts of the parasite progression, what the immune genes are. But just taking the first look recently at the human sequences in RNA compared to the parasite across time progression, we do see some differences. What we’re just trying to figure out exactly what those are and what those mean.

Dr. Jen-Tsan Ashley Chi: So one thing I just want to actually chop in a little bit. So, basically the idea is all these parasites are intercellular parasites or intercellular pathogens. So their house, their brain works for a house, so they are laying low, which is in plasmodium, it’s basically is a red blood cell in an intestinal epithelial cell. So what’s tedious thinking about where you can actually do a single cell on a sequencing post in the host, as well as in the parasite and comparing them together. Is that what you’re thinking about, Katey?

Katelyn Ann Walzer:  That’s what we are doing.

Dr. Jen-Tsan Ashley Chi: Yes, so that’s something we’re talking about doing in graduate school. We are actually doing a red blood cell together with some malaria parasites.

Richard Jacobs:  So these parasites are actually inside the cells, they’re not in between them. Living inside.

Katelyn Ann Walzer:  Yeah. So when I did the sequencing for 10X, we were able to get; so if you capture the bead and within the same bead, you get a host cell and you get a parasite cell. So you can kind of match those up with barcodes and figure out, okay, these are the host transcripts and these are the parasite transcripts and then you can see what

Richard Jacobs:  How do these parasites get inside these cells?

Katelyn Ann Walzer:  So we’re investigating; my lab’s investigating that. But I can talk about Red Cell then crypto. But I’m trying to say it’s very quick. I know that because we actually did work with Nic Boschler’s Lab at Duke. He’s at NC State now. But we were investigating how the parasites burst out and then how they end up going to the red blood cell but they attach the red blood cell and then they quickly invade.

Richard Jacobs:  So do they enter like a virus? Do they bind to a receptor and enter? Has this ever been visualized? Are they large enough to do that like microscopy?

Katelyn Ann Walzer:  Yeah. Yeah.

Richard Jacobs:  So what’s been seen? How does it does that happen at least in some cases?

Katelyn Ann Walzer:   I’m not sure if I can comment in detail on that.

Richard Jacobs:  But it’s interesting. I just don’t know if it’s. Is it I guess it’s purely adversarial. The parasite says I’m coming in. That’s it. It comes in. It doesn’t destroy the cell and it’s not like the cell says, all right, you can come in and allows it.

Katelyn Ann Walzer:   So the parasite forms, I guess I can’t comment on the nitty-gritty details, but the parasite does form a parasitophorous vacuole. Anything complex, they do that. So they just they come in and they form their own little vacuole as they’re coming in. So they have minimal damage than to the host membrane and are able to then kind of burrow into the host cell and form their own little home. That’s the easiest way to put it for a general audience.

Richard Jacobs:  Okay, yeah, I just never thought about that. So what have you been able to figure out by pairing an individual infected cell with its own little parasite? What’s seen that wasn’t seen before?

Katelyn Ann Walzer:  So, an individual parasite, you mean in relation to host or just in the general single parasite.

Richard Jacobs:  Yeah, like the individual cell host to parasite relationship.

Katelyn Ann Walzer:  So I’d say it’s a work in progress.

Dr. Jen-Tsan Ashley Chi: For example, one of the things we talked about is whether it’s possible that parasite only invades a subset of cell that even though they all look the same scene as a red cell or intestinal epithelial cell but it’s possible parasite only invaded a small percentage of the cell. We didn’t know, right? Morphologically it looked like red blood cell or epithelial cell but they may actually exist with either a particularly severe receptor or resistant factor that can determine where certain cells were invaded or not. So, it’s similar to the recent COVID-19. We all can talk about where there’s some genetic component of either the severity of transmission.

So we can actually do in a single cell level where you actually compare all the cell, individual whole-cell whether they were infected or not infected. That’s one way. Another way to look at it is possible. You can also see the host, an individual host’s response to the invasion. So maybe the host abdicates the invasion, it triggered interferer response and all the other things. So there’s a very rich area where you can actually figure out both.

Richard Jacobs:  Yeah, it’s amazing, for one person to study even one parasite and know everything about it is like, no way. I know it’s the same.

Katelyn Ann Walzer:  Especially with crypto, there’s so much we don’t know and then recently so I had a couple of publications from my PhD but there are a number of other groups working on Plasmodium single cell and so recently there’s been they were able to do single-cell and Plasmodium buybacks, which is actually not able to be cultured in the lab. So, now you’re able to find beyond just doing things lab-based but you can look at patient and clinical samples, which I think is highly relevant and highly interesting.

Richard Jacobs:  Is there a cyst stooge for Crypto in people? in turn.

Katelyn Ann Walzer:  So there’s an oasis stage in terms of the cyst, not that I know of.

Richard Jacobs:  So, what’s an oasis?

Katelyn Ann Walzer:  So I’m thinking in terms of cysts, so Toxoplasma has this because Toxoplasma can form brain cysts. So that’s kind of the chronic stage of Toxoplasma. But I don’t believe the crypto has the same thing. So the oasis is just the sexual stage after fertilization. Does that make sense?

Richard Jacobs:  I mean, once it’s in you, it doesn’t go into a dormant state where it’s very difficult to get it out?

Katelyn Ann Walzer:  So, the oasis stage, so that’s kind of one thing that we’re going to try and figure out is, yes, there is an oasis dormant stage. But like I mentioned earlier, we know that there’s a chronic infection in humans and so some of those oases might not be dormant. So if they’re single cells, we can see if there are differences in oasis because some papers have mentioned that there are a thin-walled and thick-walled oasis. But we have no idea how those are different and on a single cell, level is how we can figure out or do they have differential transcription? So that’s one goal of my postdoc, is to see if that’s actually the case.

Richard Jacobs:  Right. But there’s nothing that jumps attitude, there’s no, like, really thick-walled morphologically different type crypto that you are like, alright, that’s it’s a dormant stage.  

Katelyn Ann Walzer:  We think that the thick-walled is the dormant stage that would be resilient, exit the host, and then be able to be picked up by other humans or by cattle for greater or more infection and another host. Does that make sense?

Dr. Jen-Tsan Ashley Chi: Yeah. I think also in Katey’s Ph.D. work, I think one of the things she does is beyond we get a single-cell sequencing. She actually also is using in situ hybridization as well as morphological examination to answer the question you asked, where a certain cyst that she found at the separate different stage actually associate was a particular morphological stage on the individual cell level, as you can see, because you can actually, using the fluorescent and many different techniques to visualizing the individual cell and match was on expression to exactly answer the question you talked about.

Richard Jacobs:  How easy is it to look at the transcription? Is it quick? I guess there’s this tradeoff. You can do a single cell, but if it takes a long time or a lot of effort or money to do it, then it’s like ugh. But if you can do it really quickly and easily, then even though you do single so you can load up thousands.

Katelyn Ann Walzer:  So in my postdoc, in the last year I’ve been here, I’ve learned how to do the computational analysis for a single cell. So in the beginning, if you don’t know how to do it, it can be difficult. But once you know how to do it, it’s very quick and I’m actually very happy with 10X in particular because their protocol can be done literally in a day. So I went ahead and I infected cells. It was a 48-hour time point or so and then you can load those onto a 10x and you can do all of this within like two days and then you put on the sequencer and you have your data. I have to let the things go really quickly and pen for that. So I  can have my data within five days. Since I know how to analyze it now, it’s very quick. The only thing is it’s a little bit pricey, but I think it’s worth it.

Richard Jacobs:  But this is data for one cell. So, I mean, can you load up?

Katelyn Ann Walzer:  Oh, no, this is like; you can do thousands at once, because what it does is each individual or at least for 10x in particular, as I think about this a lot now because it’s what I’m doing in the meantime. But each bead that captures a cell has an individual barcode. So you have an individual bar code for each cell. But then in addition to that, you have an individual what’s called a unique molecular identifier for each individual transcript and this is why you’re able to even take the host and the parasite together and you can put those all into that individual cell and you can put a bunch it; like you can do up to ten thousand cells for 10X in just one sample. But then each chip actually has eight wells, you could actually do eighty thousand at once. It’s crazy.

Richard Jacobs:  You can apply a big data approach while still looking at the individual cell level.

Katelyn Ann Walzer:  Oh yeah.

Dr. Jen-Tsan Ashley Chi: Yeah. What is beautiful about 10X is that they basically make it, we are getting a machine as well. So they are basically, each cell becomes put in a liquid emulsion or a droplet. So doing a reverse cDNA synthesis. They put a small back hole and after that they just like the whole liquid. So they actually put everything together through sequence together and solve that cell together. So, really the per cell basis is actually much.

Richard Jacobs:  Yeah. I mean, there’s a real basic question with a crypto parasite. Is it a single-celled organism or are you taking one of its cells?

Katelyn Ann Walzer:  It’s a single-celled organism. But when we’re doing single-celled, we’re actually doing the single parasite and then the human cell with it. So you have kind of have two cells in there. But yeah, it’s a single cell.

Richard Jacobs:  Yeah. I just wonder if, have you found, when you do the genomics, was there any DNA there that wasn’t accounted for? What if there’s a virus that infects crypto? What if it has its own micro bio?

Katelyn Ann Walzer:  Yeah. It’s like phages infect bacteria. I haven’t found anything, but I’m sure the information is there if I need to look for it. So far, we’ve just kind of looked at aligning the known that the transcripts to the transcripts that we know already so that it’s finding what the differential gene expression is. That’s kind of been my main focus. But, down the line is to kind of find out what are the unique genes that we didn’t know about before? By looking at unique genes and you could probably find something like that.

Dr. Jen-Tsan Ashley Chi: So a typical case where you’re talking about is like a TCTA tumor where the sequence or genetic contents are tumors. So after they figure out a mutation, now, many groups study to look at the virus sequence and how they related to that. So even though their intention was never to look at the virus, but a virus data is still there, so people can actually go back to that.

Richard Jacobs:  So far, in looking at the single parasite, what have you learned in looking at its transcripts? Is there anything that jumps out at you so far or the change between male and female is great but anything else yet?

Katelyn Ann Walzer:  So we’ve found the differences between males and females. But another paper from my graduate work in post genetics. We looked at the asexual cycle and what we saw is a couple of things. We noticed that there is gene expression. So in Plasmodium on a blood cell level, it was found, concluded very widely throughout the field that there is a Just-In-Time expression that each gene is expressed once during the life cycle and it’s expressed in a smooth transition and through my single-cell analysis, we found that that’s not always the case. So in later stages of asexual, which is what I focused on in my paper, we found that there are distinct transitions and this was validated through the RNA and C2 hybridization data.

Then we also saw that there was gene expression in particular of a gene called EXP2, which is an exported protein that was actually expressed at multiple times throughout the life cycle and what’s interesting is another paper had shown that EXP2 actually has a dual function, so that actually makes sense that’s expressed multiple times. But really, on a single cell level is how you can see this differential expression across time, and then finally, one of the other things that we notice on a single cell level was that there is a wide variety in the gene expression levels. So, for instance, there are genes called rotaries and these are important for invasion and these invasion genes show low expression in some cells, and in other cells, they’re very high.

So while maybe that could be due to some stage-specific differences, a more interesting conclusion is that potentially maybe some parasites have a higher propensity to invade than others do and what if they do have some different sorts of roles and different functions and there is very much diversity in these group of parasites. We think that they’re all the same, but what if they’re not? So we did see on a transcripts level, variance in their amount of transcription.

Richard Jacobs:  So what’s ahead for you once you get back in the lab? You mentioned it briefly, but if you could just recap.

Katelyn Ann Walzer:  So this is my first week back. We were actually supposed to go back last week. We are back this week now. So we actually just got the lab started up this week and we’re training our new lab manager who just started. But the next step really is to do more of the single cells. So we have the in vitro work but to do more of the stages and then to figure out, I already have candidates to follow up functionally, but there are more that we want to follow up on based on additional data. I think a key thing, at least for crypto, is there’s only one treatment available and that treatment is actually not very useful for people who are immuno-compromised. So I hope that my work can lead to additional candidates for treatments for cryptosporidium and that’s really one of the reasons why I got into doing science, is because I really want to be able to my work to have an effect on helping people who have these diseases and who are infected by these parasites. So those are the main next steps and as I mentioned before, also looking at the host immune system is going to be another big thing in our lab.

Richard Jacobs:  Last data point, how many people worldwide on average are affected by cryptosporidium?

Katelyn Ann Walzer:  So I think in terms of infections, it’s millions. But in terms of deaths, it’s not as high. So malaria is much more deadly. I know that malaria is over 400,000 deaths per year, which is very high. I think about a lot in terms of even COVID now because hundreds of thousands of people die, but even every year still 400000 people die of malaria. I guess crypto is less than that. It’s only in I think it’s 40000. But this issue with Crypto more so though is it is deadly, but it leads to the not as high quality of life for many children who are affected by it because these are lifelong effects. So they have stunted growth. They don’t have; once they’re infected as young children, it’s a tough road back.

Richard Jacobs:  Well, very good. Ashley and Katelyn, thanks to both of you to come. I appreciate it. I was trying to hang on by the seat of my pants because it’s complicated stuff. But you and Katelyn explained it very well. So thank you both for coming. What’s the best way for people to find out more about Katelyn ‘s work and then perhaps yours actually as well?

Dr. Jen-Tsan Ashley Chi: So actually, I’m going to give you a copy, a link to two of Katey’s paper. I think that in her assistant’s work there’s also been Open Access Journal. So any of your audience can just go and look in for itself, there is the paper freely available and we’re really happy to answer any question in the future.

Richard Jacobs:  Very good. Thank you both for coming. I appreciate it.

Katelyn Ann Walzer:  Thank you.

Dr. Jen-Tsan Ashley Chi: Thank you so much. Thank you.