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#211 - 🔬 Sex Differences in Neonatal Lung Injury (ft. Dr. Krithikia Lingappan)




Hello friends 👋

In this episode of the Incubator, At the bench, we talk with Dr. Krithika Lingappan who is a neonatologist and physician-scientist at the Children’s Hospital of Philadelphia and the University of Pennsylvania. Join us as we hear what inspired Dr. Lingappan to build her cutting-edge research program investigating sex-specific differences in preterm lung disease. She explains that every cell in the lung shows sex bias, including epithelial cells, endothelial cells, and immune cells. She explores the role of the X and Y chromosomes in determining susceptibility to lung diseases and the ongoing research to understand the dosage of the X chromosome and genes on the Y chromosome. Dr. Lingappan also discusses the use of multi-omics approaches, such as single-cell RNA sequencing and spatial transcriptomics, to study lung development and disease. We are fortunate to learn from her experiences including finding mentorship, building a research career that addresses a common clinical question, and establishing and then moving a research group. Dr. Lingappan also discusses her work promoting neonatology physician-scientist career development. We are thrilled to bring you this inside look into what it takes to become a neonatology physician-scientist!


Some featured manuscripts highlighting work by Dr. Lingappan:

  

Response to Therapeutic Interventions in the NICU: Role of Sex as a Biological Variable.

Albert R, Lee A, Lingappan K. Neoreviews. 2023 Dec 1;24(12):e797-e805. doi: 10.1542/neo.24-12-e797. PMID: 38036443

 

Molecular insights using spatial transcriptomics of the distal lung in congenital diaphragmatic hernia. Lingappan K, Olutoye OO 2nd, Cantu A, Cantu Gutierrez ME, Cortes-Santiago N, Hammond JD, Gilley J, Quintero JR, Li H, Polverino F, Gleghorn JP, Keswani SG.

Am J Physiol Lung Cell Mol Physiol. 2023 Oct 1;325(4):L477-L486. doi: 10.1152/ajplung.00154.2023. Epub 2023 Aug 22. PMID: 37605849

 

The Need to Address Sex as a Biological Variable in Neonatal Clinical Studies.

Lingappan K, Alur P, Eichenwald E. J Pediatr. 2023 Apr;255:17-21. doi: 10.1016/j.jpeds.2022.11.021. Epub 2022 Nov 30. PMID: 36460079

 

Remarkable sex-specific differences at single-cell resolution in neonatal hyperoxic lung injury. Cantu A, Gutierrez MC, Dong X, Leek C, Sajti E, Lingappan K. Am J Physiol Lung Cell Mol Physiol. 2023 Jan 1;324(1):L5-L31. doi: 10.1152/ajplung.00269.2022. Epub 2022 Oct 25. PMID: 36283964

 

Effect of sex chromosomes versus hormones in neonatal lung injury.

Grimm SL, Dong X, Zhang Y, Carisey AF, Arnold AP, Moorthy B, Coarfa C, Lingappan K.

JCI Insight. 2021 Jul 8;6(13):e146863. doi: 10.1172/jci.insight.146863. PMID: 34061778


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The transcript of today's episode can be found below 👇


Misty Good (00:02.188)

Welcome back to At the Bench, the Neonatology Physician-Scientist Podcast of the Incubator. My name is Misty Good and I'm the Division Chief of Neonatology at UNC Chapel Hill and I'm co-hosting today with Dr. David McCulley. David, do you want to introduce yourself?

 

David (00:18.146)

Thanks Misty. I'm David McCulley. I'm a neonatologist and physician scientist here in San Diego at the University of California in San Diego and Rady Children's Hospital. Today we are following up on our series of interviews with other neonatology physician scientists and today I'm very excited to introduce one of my good friends and one of the people who inspires me to do great work.

 

Krithika Lingapan, who's a neonatologist and physician scientist at the Children's Hospital of Philadelphia. Krithika, would you mind just introducing yourself to the audience and telling us just a little bit about the questions you're interested in and what's motivating you to do the work you've been doing.

 

Krithika Lingappan (01:01.457)

Thank you, Dave and Misty for that kind introduction. So I'm a neonatologist and associate professor at the Children's Hospital of Philadelphia. We run a basic science and translational lab and our lab is working towards finding the answers behind why girl preemies do better than boys in the NICU and what could be the biological mechanisms behind that.

 

David (01:33.022)

Excellent. So I've gotten to see you and read your work over the last couple of years. And it seems like that's the question that's been motivating you is, what are the sex-specific differences in outcomes for babies who are born prematurely, and in particular, the lung and lung function-related outcomes? Could you just talk a little bit about what motivated you to

get interested in this field of research. Was it a clinical scenario or just how did you get started?

 

Krithika Lingappan (02:09.609)

Yeah, so for me, it was absolutely the clinical question because I started out in my research journey during my fellowship. So I was an international medical graduate. I did my medical training, my medical school back in India, and then also completed my pediatrics residency there. And that's when I absolutely firmly decided that the rest of my life, I was only going to work with babies because babies are the best.

 

And in my mind, if you are able to help babies be at their best possible health, you have healthy children and then healthy adults and then healthy community and everybody's happy. So I was pretty sure that I was going to work with babies. And then when I started working in the NICU, even back in India, and then after coming here, working in the NICU as a pediatrics residence, you get hit by this so many times during rounds that if you're a girl in the NICU, you're fine. If you're a boy, you have odds stacked against you. And I would think about, you know, we accept that as a clinical truth, so to speak. But when I would ask my attending sort of even try to get behind us to, has anybody tried to find out why that is? But then what that broadened my mind towards was that this thing was not restricted just to neonatology, the role of sex as a biological variable is important in so many other diseases across the lifespan. And then that started my journey of which lab would let me ask this question or which lab was working on something similar, et cetera.

 

So my research journey was not straightforward because I initially started actually in an embryonic stem cell lab. At the end of second year of my fellowship, I ended up in a toxicologist's lab who was doing hyperoxia with adult mice. When I went into his lab, he asked me what I wanted to do. And this is what I told him, this idea. And he said, well, this is what my lab does. These are the resources available. You can go ahead and ask your question and see if it leads you anywhere.

 

That is how my research journey started. I would say that, I think I can say that there was an element of luck involved because right when I was submitting my K-award application, the NIH came out with this strong policy statement of addressing the sex as a biological variable, which I know all of the researchers initially when it came out, they were like, oh my God, yet another thing we have to put in our grant proposals.

 

Krithika Lingappan (05:03.509)

Do we have to double the number of mice that we're using, et cetera? But I think since it aligned with what I wanted to study, it somehow gave me the encouragement that maybe this is something that is worth studying. And that was basically the inspiration. And I think both of you would agree with me. The more you know, the more you don't know. So as I got into this field a little bit more, I was like, wow, there are so many gaps in knowledge right here that could be a life's worth of research and I think that I just kept going from there and I went where my data led me.

 

Misty Good (05:40.176)

I think that's terrific. I think when I was looking at your biosketch and just thinking about your training journey and knowing you and being familiar with your work, and it's really so incredible. And sex is a biological variable. Every grant that we're putting in we have to make sure that we talk about that. But you mentioned that your path wasn't exactly straight. And I know you got your PhD a little bit later, separate from your medical training, could you talk a bit about the initial motivation and then how you became interested in science? Was it truly the clinical space that then motivated you to get your PhD?

 

Krithika Lingappan (06:27.329)

Yes, so I would say that the reason behind that is probably because of the fact that I hope I still am the same person, but at least back in the day, I was a very curious person. And if I had any opportunity to learn, I would try to capitalize on that. Not just in research, even learning a new clinical skill or a new technique that would come around.

 

I would try to make time to learn it. So I did my fellowship at Texas Children's Hospital. And when I was doing my fellowship there, there was a program that the department was running called the Clinical Scientist Training Program, which was geared towards training would-be physician scientists into writing their first K proposal. So I enrolled in that. And then that particular program had a couple of tracks. It had a certificate track, a master's track, and a PhD track. And then, as would happen with all of us, with no guarantee of a job at the end of the fellowship, when I initially enrolled in the master's track, because that would end at the time when I would end my fellowship, because I was also an international medical graduate on a visa, I did not know where my next job would be. But then it just so happened that year was particularly lucky. We were five women in that group of fellows 2009 to 2012, and all five of us got recruited at the place where we did our fellowship. And four out of five of us were recruited on the research track, which in today's day and age is probably unheard of. With the protected time, but I think if I tell you how much protected time I had, you'd be like, that's not protected time, but we were told that it's protected time. And I was very happy in getting my first job, but then what that also meant was that I could go beyond the masters in the clinical scientist training program. So I approached my mentor and I said that because I'll be staying back here, can I look into pursuing a PhD at the Baylor College of Medicine graduate school. So that's how that whole process of PhD started. I do have to disclose that I didn't tell my husband until the third year of my PhD that I enrolled in the PhD program. I would just take all the courses and do all my exams secretly because I didn't know what kind of a reaction I'd get because I didn't even tell my parents.

 

Misty Good (08:56.673)

Oh my goodness.

 

Krithika Lingappan (09:09.437)

But then, towards the end of it, when everything was almost done, I was going for my defense. That's when I told them that, hey, by the way, I enrolled in this, and we're towards the end of it. And yeah, so that's, that was the story behind my PhD.

 

Misty Good (09:27.992)

That's awesome. That's such a remarkable story. That's great. I didn't know that. So that's terrific.

 

David (09:31.49)

That is amazing. I didn't either.

 

Krithika Lingappan (09:35.601)

Yeah, now my husband will know that this story is public.

 

David (09:41.838)

interesting. One of the other things that I thought was interesting that you mentioned was just the mentors that you got connected with early on. I think it's helpful for people to hear that sometimes you find mentoring where you find it and it's not necessarily obvious that the person who ends up mentoring you is going to be a helpful mentor that is studying exactly the field that you want to learn about. Sometimes it's just a good opportunity. They have the right skills or the right environment or they're doing an experiment that's at least related to what you're interested in doing and they ask good scientific questions.

 

Could you talk a little bit more about that mentoring relationship and just how it helped set you up? Because it seemed you've been remarkably productive and it's helpful for people to hear how you got established? You were asking an interesting question as well. How did you your mentoring relationship enable you to get started or make progress initially or how to can you just talk a little bit more about that?

 

Krithika Lingappan (10:47.049)

Yeah, so I would love to hear both of your thoughts about it as well, Dave and Misty, because the ideal situation is that you are in a lab with a mentor who is really geared towards your success and is exactly aligned with the field that you're doing your research in, which was not true in my case. As I said, I ended up in a toxicology lab.

 

But I think the huge advantage that I had in that lab was that my mentor, Dr. Murthy, he gave me the freedom to ask the question that I wanted to ask and was very hands-off right from the very beginning. So that kind of forced me to become a little independent from the get-go. That was the big positive.

 

But then now looking back, I would say that, a small negative of that approach was that he was not linked or hooked on to the networking that happens within the field that you're pursuing research. So that early advantages of making those connections, introductions with other research groups that other investigators who do research in your field did not happen fairly early for me. So that fell on my shoulders that, hey, that's something that I would have to do on my own. So I would say that was a small negative, but I think Misty says that so many times that the most important features or characteristics for a physician scientist is to have grit and resilience. That I knew I had plenty of. I mean, I can fall down a hundred times and I'll get up again and I'll be like, now let's go, let's go again. So.

 

I don't know how long I will have that, but back in the day I did. So I think there were pluses and minuses for that. And the reason I want to reinforce that is that for any young people who would potentially listen to this podcast who are thinking about their career track as a physician scientist, you're absolutely right, Dave.

 

The chances that you'll end up in the best mentors lab that you have in your eyes may or may not happen. But if you are a curious person and you're interested in research, you can actually find help from mentors, not just in your institution, but also outside of your institution. There's so much support and encouragement that you can get, but you just have to push yourself a little bit to out of your comfort zone to seek out that mentorship.

 

Krithika Lingappan (13:34.733)

So even though Dr. Murthy was possibly the best possible mentor that I could get at the time that I started, I also sought out mentorship from so many other mentors from outside of my educational institution who have been very, very helpful in my career.

 

David (13:50.99)

Can we follow up? Oh, go ahead, Misty, sorry. I just wanted to follow up on that, just to give, if you have specifics in mind that you could recommend to people, like did you find specific people that you thought you wanted to connect with. How did you stick yourself out there? Like, you know, how did you get engaged? Because I remember seeing you fairly early on moderating sessions at meetings that were really relevant to what you were studying. So it seemed to me that you did a really good job of doing this really early.

 

Krithika Lingappan (14:25.993)

Yeah, so I'm going to say that I would just email 25 people, cold email 25 people, saying that, dear Dr. So-and-so, I'm Krithika Lingapan. I'm an early-stage researcher interested in this particular area of research. I've read so many of your papers, learned so much from them. If there are any opportunities that you think I could contribute in a meaningful way in this particular field, you know, and attach my CV to that email. I'm sure nine times out of 10 probably that email did not get read. But, you know, there was one in 10 chance that somebody would open that email and read it. And I also did the same thing. This was hard for me to do in person. It was easy for me to send emails. But in person, I would find it really hard to walk up to someone that I did not know. And I still don't understand it. I don't understand the word networking very well. I don't know what that word means. That does not mean that I'm socially awkward. At least I don't think I'm socially awkward, but I just feel uncomfortable walking up to someone who I don't know and then just start a conversation. I think it's an art. And the other thing that I think I lack completely is humor. I don't think I have that gene. So I think people who have a tendency to incorporate humor, network better, make friends more easily than people who don't. So I don't have that. So I have to work extra hard in starting conversations. But anyway, that's what I did. Sent a lot of emails.

 

Even when I got my K08, people told me that you should now be on study sections. And when I started asking people like how does one get on study sections, and I think the answer that I got the most was, well, your mentor puts up your name or your sponsor puts up your name and says that this is a young person, they are really good at what they do, could you please consider them. I would just look up all NIH study sections that were remotely related to the kind of research that I was doing. And I would just email the SROs with my CV and say that, you know, I'm a K-funded investigator in this particular field of research. Here is my CV. Please consider me for as an ad hoc member in your study section. And I filled out the early career reviewer application. So basically finding opportunities, you know, spending some time to find opportunities, whether that be people or institutions. I think that's the way I went about it.

 

If I am a mentor now, I think I would do it a little bit differently, because you do have to spend a lot of time to do what I did, but it doesn't have to be that hard. And I think you would agree with me. You'd both agree with me.

 

Misty Good (17:30.02)

I agree. I think I did the same thing as you, in terms of the early career investigator put my name out there. I think you have mentors, you have sponsors. I always advocate for a mentorship team, you know, because I think that's important. So one mentor can't necessarily be everything to everybody. And so for all my mentees, there are certain aspects that I'm not as good at mentoring at, especially related to wellness. And so, I think being upfront about that and recognizing that not every mentor can be everything to everybody, I think is really important. But as we're talking about mentorship, at some point we all have to transition to independence. So maybe you could talk a little bit about your journey to independence and how you became an independent investigator and how has that transition from a mentorship perspective, but also launching your lab?

 

Krithika Lingappan (18:32.945)

Yeah, thank you, Misty. That is really, really important. So I actually did not, so I was very ignorant about that transition. So I think I have to be upfront about that.

 

Misty Good (18:46.86)

I think we all are right. There's no good handbook for it, I think. So don't be too hard on yourself. You're incredibly successful.

 

Krithika Lingappan (18:54.977)

So I remember when I was a K awardee, I think it was second year of my K, where I got an email from NIH actually, that NIH was running a workshop for K funded researchers. It was at DC, this particular workshop. And then when I went to that workshop, there were sessions on transitioning to independence. And literally in that workshop, like one of the things that the presenter said was that you have to think about publishing papers where your mentor is not there in the list of authors. You need to start becoming the senior author and then your K mentor needs to realize that they don't need to be on every paper that you publish. And once you start taking an independent trajectory, you need to start being assertive of your independence because that's what people want to see when you submit your R01. So I still remember after that workshop, I was standing in the metro trying to get back to the airport. And I called my mentor and I said that, you know, that I was at this NIH workshop, I'm going to submit this paper for peer review, and I don't want you to be on it. There was a pause, and then he said, yeah, that sounds great.

 

Misty Good (20:09.449)

How did that go?

 

Krithika Lingappan (20:22.157)

But I think I asked because nobody told me and I didn't know. So that's when I realized that this transition is important because I think sometimes either trainees don't know or they just do the same thing that they've been doing. And then until somebody kind of shakes them and tells them that, hey, you're gonna submit your R soon, you're going to start this transition process.

 

Krithika Lingappan (20:50.473)

Sometimes it just doesn't happen. I think that knowledge is necessary.

 

Misty Good (20:58.)

I think it doesn't always happen that easy, right? That the transition isn't always easy. Or it could be that you leave or your mentor leaves. And so, you know, but I think putting it out there for a manuscript is important. I would say probably talking about it in advance of submission is probably also ideal.

 

David (21:19.87)

Yeah, I agree that transition is not straightforward and I didn't receive a manual to help navigate it either. I think as Misty alluded to having a group of mentors is sometimes helpful just because sometimes someone in that group will recognize that this transition to independence needs to happen because as you said, Krithika, the mentee often is in the mode of like, I'm just trying to be productive. Like the quickest way for me to get published is to keep publishing with my mentor.

 

The mentor, similarly, is like, I'm mentoring. I'm going to continue to publish with my mentee. So sometimes it takes that outside perspective of someone just saying, in order for this to be successful, eventually you have to be able to publish independently. Have you talked about when you might do that or what paper you think will be independent of your mentor? Doing it ahead of time makes it a little bit easier.

 

But let's get into your research a little bit more. So, you kind of set up the clinical scenario, the sex specific differences, and I remember seeing you present early on this topic, and it just seemed so timely, and also so, like one of the things that we talk about all the time, but really just don't have a good understanding of why it happens. Can you just talk about the, maybe some of the initial experiments you did, or the approaches you were taking to try to begin to investigate the sex-specific differences, especially in lung biology?

 

Krithika Lingappan (22:50.161)

Yeah, so I think one of the main reasons that I kind of decided to start this and then continue along this path, the long term, which I hope our research would lead to that, was to find that biological pathway or pathways that either lead to female resilience or male susceptibility, and then to find either small molecules or drugs that target that.

 

To start that, we first had to show that this clinical truth that we see in our preterm neonates is able to be reproduced in mouse models that we used in basic science research. So that's how we got started. And then once we saw phenotype there, then the goal was to ask, does it happen in cells when taken out of the in vivo construct and then in vitro experiments? And I think the first cells we tested that hypothesis on were endothelial cells, which showed a very remarkable sex bias depending on whether that endothelial cell was coming from a male or a female with both HUVECs (human umbilical vein endothelial cells) and human pulmonary microvascular endothelial cells.

 

When we were doing that research, we found a particular microRNA that the female lung was making more of, microRNA 30A, and that became the focus of our first R01 that we submitted as to whether that microRNA and the gene or protein that it regulates and the upstream regulation of that microRNA expression in the female lung was the focus of the first R01. And at that point, my research was leading me towards the lung endothelial cells, because that's the data that her lab was generating. But at some point, as I was presenting my research at scientific meetings, it was categorized as “descriptive research”. I was showing that there were differences, but I was not telling people why there were differences.

 

Krithika Lingappan (25:09.793)

So that question came back to us, and understandably so, many, many times. So then we had to think about, well, we need to start answering the question, why there are differences. And I think at that point, I sought out mentors from outside of neonatology, and then I sought out Dr. Arthur Arnold, who is a scientist at UCLA, who has done a lifetime's worth in looking at sex as a biological variable, mostly in neuroscience research. But what I learned from him and his papers was that there is almost a step-wise approach when you see a sex bias in a disease. Because when you talk about sex bias in a disease, usually people start thinking about either sex hormones or sex chromosomes. So that is step one.

 

So, to answer that question, we did a study using the “four core genotype mice” (PMID: 19028515). And this was a hard mouse model for me to wrap my head around when I first learned about it. It was also very exciting because it helps you to delineate the separate effects of sex chromosomes and sex hormones on the phenotype that you are seeing. So that work was published. And then what we found in that study was that sex chromosomes predominant effect in sex hormones on the neonatal lung in the phenotype that we're seeing. So basically what we were noticing is that if the mouse had an XX chromosomal makeup, irrespective of whether the mouse had ovaries or testes, the lung was more resilient to injury compared to the mice that were XY and had either testes or ovaries. There was definitely an influence of the gonadal hormones, but sex chromosomes had a more predominant effect.

 

So, once we established that, and I'm sure it's happened to both of you too, my research has always been informed by either a manuscript reviewer who was not happy with what we had put in the manuscript or a grant review, or somebody who brings up something in a talk that I was giving. So the next thing that we got repeatedly asked was, well, which cell in the lung shows the most sex bias? So to answer that question, we did some single cell RNA-seq experiments in our lab, which then kind of led us to the result that every cell has sex bias, the epithelial cells do, the endothelial cells do, the immune cells do. Because of the very predominant sex bias that we're seeing in the lung immune cells, that is where the research is leading me now. So I am, as of last six months, reading a lot of papers on lung immunology. So that's where the research is heading right now.

 

Once we found that it was the sex chromosomes more than the sex hormones, the next question is to ask, well, is it the dosage of the X chromosome that is protecting the female? Or is it a gene on the Y chromosome that is making the male lungs susceptible? So how do you answer that question? So for that, there is another mouse model where you have mice that are XX, XO, XY, or XXY. So by doing the comparisons, you can then ask, is it the dosage of the X or the genes on the Y that are causing the effect? So that study is presently ongoing.

 

Misty Good (28:55.008)

You talked a bit about single cell RNA sequencing, but can you talk a bit, I know one of your R01s is focused on leveraging multi-omics approaches, so can you speak a little bit about the different omics approaches that you're using now, such as like spatial transcriptomics?

 

Krithika Lingappan (29:12.169)

Yeah, so I think the reason for the multi-ome in our lab was twofold. One was we very early, we did a study where we were looking at histone modifications in the newborn lung, male and female lung, when we exposed it to injury. And we were kind of blown away with the kind of differences that we saw in that result in the sense that the females were very malleable as far as histone modification was concerned after injury, whereas the male lung did not use that particular histone mark. We were studying HCK27 acetyl modification after hyperoxia exposure. But the other reason we wanted to study, so the technique that we're currently using is ataxic, looking at chromatin accessibility in the lung.

 

The other reason is there are a few genes on the X chromosomes, which are actually histone modifiers, and they undergo escape from X chromosome inactivation or XCI. So just to briefly touch upon that, because females have two X chromosomes, we don't want alleles from both of the X chromosomes to be expressed. So, all one of the X chromosomes in female cells undergoes inactivation through a process called XCI or X chromosome inactivation. However, there are a few genes that escape from XCI. They're called XCI escapees. And there are two genes in particular that have a lot of literature on them, which are KDM6A and KDM5C, both of which are histone modifiers. So that was another reason to look at the role of how the epigenome is modified and how that dictates gene transcription from a given cell. So that research is also presently ongoing.

 

The spatial data, we have not published that from the male and female lung, but that was just a new technique that our lab was really interested in when it was first launched. And with the help of a collaborator from Baylor, we did spatial transcriptomics using the NanoString platform

on lung sections in babies with congenital diaphragmatic hernia to look at which lung cell subpopulations in the distal lung are affected in babies with CDH. So that was our kind of our first foray into spatial transcriptomics.

 

Misty Good (31:53.208)

That's awesome. Thank you for sharing.

 

David (31:56.062)

It's amazing how you have learned how to use these new technologies really quickly just when they've become available and applied them to the questions that you're interested in asking. You have demonstrated that you're a neonatologist, you have this interesting clinical observation, you can try to investigate the basic mechanisms that are responsible for that difference between males and females in an extremely detailed way and be a leader in this field. And it's great to see and hear how you learned from somebody who was doing work in a parallel line of research, but a completely different scenario in sex-specific differences and sounds like neurologic disease and apply it to what we see in the NICU. So that whole story, I think, is really compelling to learn from.

 

One thing that you mentioned and that I think your long-range goal is to try to think about is how to take what you're learning as being responsible for sex-specific differences, especially in response to oxygen exposure, and then apply it to a potential treatment. So the way I think about that rescue is that it's demonstrating that the mechanism that you're investigating is really underlying the phenotype that you've observed, but it's also potentially translatable to a treatment.

 

So now that you found that the sex chromosomes seem to be really important for this phenotype and you're modulating them, how are you thinking, how are you thinking about how you could potentially translate that into a therapy? Are you still at a mechanism level or is this potentially something you're going to try to translate?

 

Krithika Lingappan (33:57.545)

Yeah, so we are thinking about that in two ways. So the first thing is, one of the things that this research rabbit hole also kind of led me to was, it's not just disease pathophysiology, but it's also response to therapy that could be different depending on whether you're a male or a female. And we know as neonatologists that there have been studies before which have shown that, like for example, the TIPP study (Trial of Indomethacin Prophylaxis in Preterm Infants) showed that the results were separate in males and females as far as the effects of Indomethacin were concerned. Lately, a few studies are showing that effects of early hydrocortisone could be different between male and female preterm neonates. So the two approaches that we're taking in the lab is... One, we are utilizing the transcriptomic profiles that we are getting from the whole lung, but also from this individual serRNA-seq experiments. And we are comparing that cellular transcriptional state to, for example, large drug databases, like the NIH LINCS database (Library of Integrated Network-based Cellular Signatures), to then identify potential drugs or small molecules that could be useful either in the female or male lung exclusively, depending on the cellular transcriptional state, or could modify the disease process in a positive way. And now there are several drug prediction algorithms, which both of you might be aware about. So we are using the single cell drug bioinformatic path pipeline to identify new drugs or small molecules to get us there.

 

But then the second approach we are also interested in asking, which hopefully we will start the study soon, is why certain drugs have differential effects in the male and female lung? What could be behind that? What makes a drug more effective in the male or female lung? What cells or what pathways are responsible for that? And the reason to do that is I think we'd all agree that for the want of something better, I think our babies get exposed to a lot of steroids, but steroids might be a big or a very strong medication for the effect that we're trying to achieve. So can we prune it down to the exact pathway or a molecule that targets the cell that it needs to in the patient that it needs to at the time that it needs to, instead of exposing babies to something like steroids, which can have effect on the brain, effect on other organ systems, and maybe not in a positive way.

 

So that would be kind of a two-pronged approach. Identify molecules that are sex-specific, but at the same time, use the drugs that we commonly use in the NICU and define how the biological mechanisms by which they act might be different in the male or the female.

 

Misty Good (37:19.488)

Yeah, it's really interesting, thinking about not just do they act differently, but also is there a dose-dependent effect that's sex-specific, adds a whole other layer of variability to the different potential therapeutic targets. So that's fantastic. Well, we really appreciate all your great work on that. We will transition a little bit.

 

You recently moved to the Children's Hospital of Philadelphia and moved your lab there. I was wondering, and hopefully you can tell us how that experience went, but it seems like it's been very successful for you. But certainly, I think our listeners would love to hear about the transition process and any challenges that you noted along the way and how you overcame them.

 

Krithika Lingappan (38:10.537)

Yeah, I think I've talked about this transition with both of you a lot. Transitions are hard. I think you have to really think through it. And I think if anybody listening to this needs advice on how to move, please reach out to any of us. We'll probably tell you the mistakes that we made that you can potentially avoid during moving a lab, et cetera.

 

Moving a lab is difficult, but I think moving as a neonatologist is also difficult because the cultures of different places are different and then aligning yourself with the culture of the place that you're moving into, letting go of the place of the practices that you are used to is also difficult. And for me, I think the transition held a mirror in front of me because what I realized was that until the physician part of me was at peace, the scientist part of me couldn't even start. So for me being comfortable as a physician in the new system at the bedside happened, I could not even focus on my research. I took it for granted in my previous institution because that's where I trained. I knew how everything worked. I knew everybody. I felt very comfortable as a physician there, which was taken away after I moved. And that took a while. So that was a learning exercise for me.

 

Misty Good (39:40.548)

How about getting your lab set up?

 

Krithika Lingappan (39:45.217)

So that was a mistake that I made. So my advice to anybody moving a lab would be to not think that you are 10 years younger because you're not. So when I moved my lab, nobody from my previous lab moved with me, but I was like, well, I've done it once, I can do it again. So, I moved my lab, my mice, everything, but when I started, it was just N equals one in my lab and that's myself. So I started the process of hiring after I moved and we all know how long hiring takes. So I felt like I was like July 1st when I started as a faculty back in 2012. It took time. And I think I should have, could have done that a little differently. And I think the mistake that I made is I should have asked for more guidance, more help, called my senior mentors, my peers to ask for guidance. But we all make mistakes. And I think I made one during that transition.

 

Misty Good (41:07.652)

Well, it all worked out for you. So even if you felt like you made a mistake, you're incredibly successful. From the outside, at least, it seems like the transition's gone really well for you.

 

David (41:19.186)

Yeah, I agree. But also hearing that you feel like it's a struggle and the idea that you said that it's hard. You're literally starting over in many cases. You may have a sense of what you need to get done and maybe you're more efficient with it the next time. But it is a really big challenge and asking people for guidance is important. But also being patient with yourself just because I think, we're used to succeeding, we're used to knowing everything and being a resource and it's hard to step back and say like, I don't know how this place works. Like I don't know how to submit a biosafety protocol. I don't know how to submit an animal protocol. Like all that stuff you have to just begin again and it's okay. And you have to be patient with yourself and know that it's going take a little while before you're back to where you were before. And may be you won’t be back to where you were before. You may be in a totally different place, but hopefully it's someplace that's exciting and interesting and allowing you to mentor people who are going be the future of our research. Can you just talk a little bit more about the environment that you're in now? Like, just curious to hear, you've been there for a couple of years, and it's such a fantastic institution. I'm just wondering about the collaborations you've found or things you think are going well, things, anything you think the audience might benefit from learning about just because moving to a new institution is a challenge, but there's also some excitement about the people that you can work with in a new place.

 

Krithika Lingappan (43:04.049)

Yes, I think I touched on this a little bit earlier about this curiosity thing in me, which can be a good thing, but can also be a bad thing. So when I moved to Penn and CHOP, you are just surrounded by the level of intellectuality that you can constantly get inspired by.

 

So there would be a talk, in some other department that I could potentially find connection with what I wanted to do. So I think the first six months that I was there, I was literally enrolling myself to watch a web, because it was during the pandemic too and everything was virtual. So I would have like three virtual talks on my calendar on a daily basis because I was just signing up to those talks because I was just so excited about. Who was giving the talk on what particular topic that could potentially help me. But I think what it has filtered down to hopefully is collaborations that are also taking our research in new directions. So we, like one example would be we are now looking at the neonatal heart and how it responds to early injury and long-term consequences with the interest in long-term cardiovascular consequences related to prematurity, and how that might be affected by sex as a biological variable. So we have an ongoing collaboration with the Penn CVRI on this particular project.

 

And as I told you, my research is leading me towards and kind of an immune focus. So we also have collaborations both within and outside CHOP to kind of help us in that area too. So I think moving to any high caliber academic institution has the potential to open up a lot of opportunities. But for me, I think I also had to tell myself to refocus, because there are too many things that can potentially excite you with ideas, concepts, diseases, people who come to give talks here. But you also need to get work done. So there was a balance needed there.

 

Misty Good (45:46.88)

It's true. I feel like every time you meet somebody else who gives a great talk, either if they're at your institution or just coming to give a talk, I think there's always the potential for collaboration. And so then making sure you stay focused, I think, is one of the things I personally struggle with. But I do think it's good advice, especially when you're in the middle of grants, for example, that you need to be productive on. And there's certainly good times to collaborate, especially when you need the help, but also recognizing that the work that you're funded to do needs to get done as well. So I can appreciate that.

 

I was going to ask you about your experience with physician scientists career development and mentoring, and specifically, both at Baylor now and at CHOP, and then also nationally through the AAP. Could you just talk a little bit about things that you've been able to pass along to your mentees or you've seen, you know, good, you've experienced good career advice along the way just for our listeners who may be early career researchers and starting out.

 

Krithika Lingappan (47:04.585)

Yeah, and I think both your work, Misty, and Dave's work has been so inspiring in these areas. You both do so much in this field, and we have a lot to be thankful to both of you in this focus area. I am just sad with the fact that, you know, the number of physician scientists in our subspecialty seems to be dwindling and dwindling fast.

 

And I just want to do anything that I can do in my power to not let that happen. When I talk to young trainees, the reasons that they give me for not engaging in research is because it's, quote unquote, too hard. Somebody told them that it's almost impossible to get a grant nowadays. You can't spend your whole life in the uncertainty of keeping your lab funded, et cetera. But then I think what people fail to highlight is the fact that being a researcher and asking new questions that nobody has ever asked before, that independence is something unique that being a physician scientist allows you to do.

 

And also the fact that you get to share your findings, you get a critique, but at the same time, you get praise, positive feedback, encouragement for your work, which kind of keeps you intellectually stimulated and going. And I think that the third biggest thing is, yes, there are no free weekends. Yes, there is no quote unquote time off for a researcher.

 

But I think as a woman, I do have to say that the flexibility that being a physician scientist gave me was unique compared to my clinician peers, who may not have that flexibility that I do. So that is an advantage that I think many a times does not get highlighted. People just dwell on the negatives of being a physician scientist and a researcher, but fail to highlight the highs that research gives you. And it does. I mean, I can't compare it to anything else but a high. When your experiment works, like if you thought it would work, or you get a finding that you did not anticipate that you'd get, or you discover something that nobody else has discovered before, I think that high is comparable to when a baby responds to a therapy that you give your patient.

 

So I think that is unique and where else would you have the ability to do that too, but in research. So I think that's what I try to at least inculcate and to not let our young trainees get disillusioned or discouraged by anything else that they might see or hear. I'm not saying research is easy.

 

Research is not easy. It is difficult. But anything that is worth anything in life is not easy. So I think if you're intellectually curious, which I think all neonatology fellows are when they enter neonatology, then I think you can be a researcher. Don't let anybody tell you that research is hard. You can do it. You think that you can do it.

 

Misty Good (50:32.256)

Yes.

 

Krithika Lingappan (50:46.441)

Yes, it will not work for everybody, but I think if you engage in research in a meaningful way, those skills will carry you for the rest of your life, whether you end up being a researcher or not a researcher. So I tried to do that at my division level, at the department level, and then whatever opportunities I get. I mean, Dave gave me this opportunity of being a co-lead for a lung biology cohort in SPR (Society for Pediatric Research).

 

And I try to do whatever I can within this section of neonatal perinatal medicine and within women in neonatology.

 

Misty Good (51:21.892)

It's incredible, so inspiring. Thank you for that.

 

David (51:25.598)

Yeah, it's really great to see you're doing this like you're leading by being a great role model, asking really good questions, taking advantage of new technologies, and talking about it in a way that people can understand and publishing regularly. You're doing all the things that we are taught to do. And you're asking a question that's really relevant to a disease that we all see in the NICU. I mean, we all see that there's this difference between males and females, especially in their pulmonary outcomes. Trying to better understand that with the idea that that's going to help tailor our treatment down the road. Otherwise, we're not going to make any new advances. I think one of the other things that, in addition to the points you mentioned about being attractive as a reason to pursue doing physician-scientist work, is also the environment that we get to work in and the people that we get to work with.

 

So just as a way to finish up our conversation here today, I was wondering, just within your group, is there something that you guys do together to maintain cohesion? I think, you know, our lab group is so much like a family. We experience our challenges together, our failures together, but also our successes. But we also like we get to do things together that are fun. Is there something that you guys like to do?

 

Krithika Lingappan (52:46.333)

Yeah, so my lab was fairly small when I was back in Texas Children's. It was a three-person lab and both of the people that I had were research staff. And I did not have any young people who are the source of most fun in any lab. So that lab was like, you know, all work.

 

When I transitioned to CHOP, we have a very vibrant lab. We have a lot of young people, so a lot of excitement in the lab. We try to cohort in the same place as much as we can. So what I mean by that is my office is down the hallway from the lab.

 

It's okay, I think I can say this. I have never gone into that office since the two years that I've been in CHOP, even though I have an office, I just always come and sit in the lab. And that's then the room that I sit in is where people have their lunches and breaks and et cetera. So inevitably social conversations happen because people are talking about what they did, where they went out for dinner and it would be a place that I've never heard of and that how conversations take off.

 

But one thing that we have now made a lab tradition is that is this really, really good bakery near my house. And if we have half a chance to celebrate anything, even an experiment working out the way that we anticipated because there are so few times that does happen, it calls for a celebration with a gourmet cake. So we have kind of made that a regular tradition. We have not yet had a big party in our lab.

 

I'm saving that for the time when my next grant gets funded. I'm kind of saving that for a big celebration. But right now, it's any chance we get to eat good cake is how our kind of lab gets together. And that is a selfie time and gets posted on the lab website.

 

Misty Good (54:55.708)

I love that. We used to have a technician that would bake cakes for like every occasion, every birthday, every paper acceptance, and I miss that. I may have to institute cake rewards. Back to the lab, I love that idea. Thank you.

 

David (55:21.646)

Okay, thank you very much, Krithika. It was really great to be able to talk with you here today. I think hearing about your work and what motivated you to do the work that you're doing will be really interesting to our audience. We are so thankful again to the Incubator podcast for giving us this opportunity to interview neonatology physician scientists. We hope that these discussions are interesting and motivating for you, and we are excited to be continuing to record interviews like this that should be coming out each month. So I would like say thank you so much again. Thank you to Misty Good and to our co-moderator, Betsy Crouch. We will talk to you again soon. Thank you so much!

 

Krithika Lingappan (56:10.049)

Thank you.

 

Misty Good (56:11.12)

Take care.

 

 

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