Cool kids do Basic Science

OK, so, uh, welcome everyone, um. Welcome to the first uh basic science webinar of uh YUSA. Uh, hello me too, greetings, uh, uh, also to you. Um, as you know, for over a year we've been having, uh, webinars, uh, on different clinical topics, uh, controversies, uh, and, uh. Quite some time ago, uh, Martin, uh, and I thought maybe we should also discuss basic science. We are excited about, uh, all the advancements that are happening in pediatric surgery and specifically, uh, in, with regards to basic science surgery and, uh, but we don't talk about it. So today we decided, uh, we, we are bringing to you, um, a very provocative title, Cool Kids Do Basic Science, and we're gonna discuss all topics in CDH research and for this we have uh two experts uh in the field of basic science research, uh, and I'm sure it will be a great presentation, two great presentations. Uh, but also, uh, great discussion, uh, uh, at the end. Uh, we really believe that, uh, UPSA has this uh mission also to, um, not just inform but support, uh, the, uh, basic science, um, research, the surgeon scientists, uh, we think that, uh, um, there's many members that are really interested in this. And so we wanted to um really launch a series of uh basic science uh webinars that will happen um more or less 34 times a year. Getting also some feedback from you and uh uh again we start with some cutting edge research in CDH. Martin. Yeah, thank you, um, Augusto, I think we're gonna, um, right, jump into, into it. So I will introduce the, the, the first speaker. It's a pleasure for me to introduce Dr. Richard Wagner, who I actually hired as a trainee four years ago. Um, and his research career actually started, um, at a UUSA meeting in Limassol. Uh, Richard, uh, Kaiser, maybe you remember in Cyprus 2018, you were looking for And a research fellow, he had an open position for that. And I recommended to take Richard Wagner, whom you then hired. And Richard spent, um, um, 1.5 years in your lab from 2018 to March 2019. And had a really good time in, in Winnipeg before he returned to, to Leipzig. It was a very productive time at your lab. And um since May this year, actually, he um did his second postdoc fellowship at Harvard Medical School, where he is now in the lab of Patricia Donahoe and Jing Bing Ai. Dr. Wagner is co-investigator of a Canadian Institute of Health project. Grant, which we just got, uh, which runs over 5 years, and we are part of that. Um, it is on circular RNAs in their, in their research in, um, in their role in CDH. So, with the age of 31 years, 40 years of training, he has won a couple of research grants, and he's earned over €700,000 of funding, which is quite remarkable, I think, for his young stage of his career. So, uh, for those who know him would agree that he's not a nerd kind of guy, but he has also well developed social and sportive skills like handball or wakeboarding, which I could personally witness. So he's indeed a cool kid doing basic science. And that's why Richard, we are happy to have you with us today. So welcome for this afternoon or your morning session. And we are looking forward to your talk. Yeah, um, thank you very much. I'm very honored and happy to be here today, um, to talk about that topic, uh, briefly and then later discuss. Um, as you can see, the talk is structured in 3 parts. It's cool kits, um, basic science, and CDH research. And I'm trying to participate in all three aspects of life. And, and that's why I'm super happy to share a few thoughts on basic science and CDH research today. Um, so basic science, why bother? Um, well, not only can you win a Nobel Prize, um, just like Jennifer Doudna and Emmanuel Charpentier, you can also write a book about your research. Um, and even better, you can have Walter Isaacson publish a New York Times bestseller about your research. Um, but aside from that, um, as shown with CRISPR-Cas9 gene editing, I think basic science really drives innovation. And for example, CRISPR-Cas9 is now in clinical studies for, uh, sickle cell disease or beta thalassemia, and also for cystic fibrosis. There's many more, uh, therapeutic, uh, approaches to come with novel basic science findings. So that's why I think it's, it, it makes a strong point for investigating in Uh, and doing basic science. Um, It's not advancing. Now, science can also go wrong. And especially as shown on the right side here for the surgical specialties, there has been a dramatic drop in contribution to major journals like the New England Journal of Medicine over the last 100 years, as shown in the paper by Atul Gawande. And this has also been reflected, um, in Uh, Department of Surgery funding relative to total NIH funding over a study period of 12 years. Um, this has been published in the Annals of Surgery in 2017, and they showed that there was a 23% decrease in, um, relative surgery funding to total NIH funding, and 20% drop relative to internal medicine. And I think all of us who are pediatric surgeons, scientists, we should really fight this trend and, and turn it back up again. There's many challenges that surgeons scientists face, such as showing funding it's hard to obtain, administrative duties, clinical work, uh, competition, work-life balance, and that, that you don't end up being this person, not catching a wave like a cool kid does. Um, there's a few key strategies, um, that are essential when combining basic science and clinical excellence. And we recently published this paper, um, in the Annals of Surgery, uh, together with Augusto and Richard Kaiser. And while doing the research on this and sharing thoughts about what is essential for, um, good research training in surgical residency, we found again, uh, that's been shown before that there are some key aspects of, um, good surgeon scientific pathways. And just to name a few of them, I think most important is mentors and research environment, like with the pace that basic science is going right now, um, and new innovations are coming into, into the field, we need good core facilities and really good research environment. We also need very good collaboration, dedicated time, and effective time management. And just to point out a few of these. Um, I was very fortunate to have, um, this person here as my mentors. I think many of you know, uh, Richard Kaiser, Martin Lucker, and, uh, Patricia Donahoe as outstanding pediatric surgeons, scientists, and really role models in the field. And also Shing Ben Ai, who is a Harvard, uh, lung developmental biologist and phenomenal basic researcher. Um, So these are all my mentors. And when I thought about what they share, although they come from different angles, um, I think they share inspiration and fascination for the topic. So if you have a mentor that is not fascinated for your research, then you should get another one, or have another mentor, um, finding you. This paper tried to summarize key characteristics of successful mentorship, and they name reciprocity, respect, shared values, clear expectations on the pro side, and they have poor communication, lack of commitment, conflict of interest, competition between mentor and mentee, which can happen sometimes, or lack of mentor's experience as bad for successful mentorship. Collaboration, I think, is especially important in pediatric surgical research, um, to do good basic science, and this is just a numbers game. If you are in, um, cardiovascular research or cancer research, you will have enough clinical specimen, most likely. Um, to work with, and clinical data. And if you compare that to, um, CDH where we are really in the 5 per 10,000 live births as an incidence, and also for the other surgical anomalies, we really have to team up to get enough clinical samples, um, into our, uh, cohorts to make good, uh, Um, basic science. And this is also reflected here in the 2017 study for CDH, where they labeled small CDH centers, which was the majority in that study, with a caseload less than 14 cases per year, and large centers, um, with more than 14 cases per year. And you can do the math. Even with 14 cases a year, I think you're still missing. Uh, a lot of CDH cases and good input for your studies if you don't team up. And Hm, I don't know why sometimes it's not advancing. Now, um, we really need this collaboration to get to Olympic levels in pediatric, uh, surgical research. Another very important, um, aspect is dedicated research time. There's two cool papers. One is, uh, also authored by Richard Kaiser on clinician scientists. And I think they really point out how important it is that you have protected time to sit down, write grants, supervise, uh, students in the lab, or do experiments yourself, and the importance of blocking time cannot be overemphasized. Now, to change gears a little bit and, and talk about CDH research, um, I want to point out why it is so important to really, um, proceed with good quality CDH basic science. So there's a study published in 2016 showing that the overall mortality. Uh, for CDH patients is still 45%. It will include stillbirth and terminations of pregnancy. And there has been another study in the AMSA Surgery recently that show a down trend in mortality, but it still ranges in over 30%. So that is too high, and I think we owe it to our patients. To be involved in these hot topics and CDH basic science, which I'm gonna just highlight a few of them. We're gonna talk briefly about some genetic studies, about prenatal diagnosing, uh, which is something that, uh, Richard Kaiser and myself are involved in prenatal intervention and lung maturation. And I'm sure we can discuss later a little more in detail. Um, talking about genetics, um, There's, uh, a group at Harvard Medical School here led by Patricia Donahoe, together with Wendy Chung from, uh, Columbia University in New York, and they have a large cohort of CDH patients and parents. And they perform whole exon sequencing on these patients to identify novel genetic variations within the CDH cohort. And then they can prioritize candidate genes and create genetic mouse models of CDH to study the function of the gene in lung or diaphragm development. And this is just, um, a few example genes that they identified, and then they can look into whether the gene is involved in diaphragm or lung development, or whether it's in highly pathogenic variant. And then, to give you one example of a nice study out of this, Group from McCauley and colleagues, um, they identified PBX, uh, 1 as a CDH candidate gene, and they knocked it out in the lung of, um, developing lung of mice. And what they show is that the alveoli of these knockout, uh, mice are simplified, and they also develop pulmonary hypertension, just like Human CDH patients do. So this is a very hypertrophic heart from these knockout animals after 4 weeks, uh, of age, and they show that they have, um, hypertrophic right ventricles. Uh, the left ventricle appears to be normal. Then they were also able to show that genes regulating, um, vascular smooth muscle tone. Um, are dysregulated in this knockout animal model, so genes that are constricting the vessels are increased here in black and Genes that relax the, the vascular tone are decreased in the knockout model, and then ultimately they were able to reverse that phenotype with a downstream, um, modulation of the pathway. So this is a really nice example of how you can come from clinical, uh, data, whole exome sequencing, do the knockout in an animal, show what the gene does, and then reverse the phenotype. Um, In the animal model. Um, We are very interested in prenatal biomarkers, and we recently published this in the European Respiratory Journal, um, looking at circular RNAs as potential biomarkers for CDH. Um, CDH prenatal diagnosing is still imperfect, so the sensitivity for ultrasound and MRI is around 70%, and prognostication is even harder to do, accurate. So we aim to establish biomarkers from maternal, um, body fluids to better diagnose and prognosticate CDH. And as a first step towards this, um, we screened circular RNAs in fetal CDH lungs and compared the Profiles to control to see whether there's differences between hypoplastic CDH lungs and controls. And we were able to identify a few circular RNAs that are significantly different between the two groups. Now we want to use these, um, to better prognosticate CDH in the future. This has been published this year in the New England Journal of Medicine, a great accomplishment for CDH research, um, by Jan de Prest and colleagues. And they did a randomized controlled trial for, uh, feto, um, in severe left-sided CDH. So FO is the, um, occlusion, uh, of the fetal trachea with endoscopy, and that causes the lung to mature better because lung fluid gets accumulated in the lung. And they were able to show in the randomized control trial that feto increases survival, um, significantly to discharge, but also after 6 months, um, of age. Um, so that was a great accomplishment. However, it was associated with a higher risk of preterm, um, rupture of the membranes and, and preterm birth. And They did another randomized control trial at the same time for moderate left-sided CDH, and there, they were unable to show significant increase of survival. So the fetal group had 63% survival and the control group 50%. And again, that wasn't statistically significant. So there need to be, uh, more studies in this cohort to, to improve survival of CDH patients with moderate left-sided CDH. Um, And then, of course, um, this has to be mentioned, Augusto Zani's group, uh, just published recently in Science Translational Medicine. I only want to briefly touch on this because I'm, I'm sure we can talk about it a little more in detail later with Augusto being here. But, uh, phenomenal accomplishment, um, also for CDH research, where, uh, the group uses amniotic fluid stem cell derived extracellular vesicles to promote lung growth. Um, So basically, nitrophin can mimic um CDH in a rat model, and you see that the lung is hypoplastic when you, when you put them in a, in a dish. And then by treating it with these extracellular vesicles and their content, they were able to make the lung. Grow better, they show that markers of lung differentiation, like SPC for alveolar differentiation or club cell markers are getting back to normal levels. So really, a very nice study, and I can highly recommend everybody to read it. Um, so to conclude, I think CDH research as surgeon scientists, there's many fascinating fields. I'm super happy to be part of, uh, of the group who do CDH research. Um, we have to learn so many more things on the path of biology, on prenatal treatment, prenatal diagnosis, and I think to conclude, um, is the surgeon scientist a dying breed? I hope not. Um, we as pediatric surgeons are very well positioned to do the, the best basic science possible, especially for congenital anomalies, since this is a niche that we all have to contribute to. And I think the answer, how? Uh, to make the surgeon scientist, uh, a living breed is given here. We have to decrease burdensome administrative responsibilities and protect and nurture the surgeon scientists. Um, with that, I want to thank you, and I'm looking forward to Richard's talk and to the discussion later. And Yeah, thanks. Thank you, Richard. Um, that was really very nice, uh, talk, both an overview of, um, the fundamentals of getting into basic research and the problems we are facing and the surgeon scientists is facing. Um, looking very much forward to the discussion. Um, yeah, Augusta, I think now you should proceed. And after Richard Kaiser's talk, we will go into detail. Yeah, it's my privilege and honor to introduce, uh, Richard Kaiser who's a, a colleague and a friend, and, uh, with whom we shared the, the passion for CDH research. Uh, Richard is the chief of pediatric surgery, uh, in, uh, uh, Winnipeg, uh, uh, Manitoba. Uh, he's also, uh, the inaugural Thor Larksen, uh, Chair in surgical Research and the director of research for the Department of Surgery at the University of Manitoba. As you already heard from, uh, uh, the first speaker, um, he's been driving and pushing, uh, really cutting edge research, uh, for, uh, uh, CDH to try to understand why this happens, uh, uh, why these lungs, um, um, are hypoplastic. Um, it's Richard's one of the most seminal papers, uh, in the field, uh, where, um, he came up with the two heat hypothesis more than 20 years ago. Um, and so without any further ado, I'm, uh, excited to um introduce you Richard Kaiser, uh, who will give us, uh, a little bit more of an overview of what, where, where his research is going and what to expect in the near future. Richard, OK, thank you, uh, Augusto, can you hear me OK? Very well, yes. OK, good. Well, thanks, Martin and Augusto, uh, for, uh, for inviting me, and, uh, thank you, Richard, for a great first start of the talk. Um, I'm excited to talk about it. It's near and dear to my heart. I, uh, I'll, I'll go into my personal story a little bit and then highlight some of the research we have been doing and are still doing. Uh, I, I have to declare a bit of a conflict. I, I have a patent for some of the technology described in the presentation, and together, Richard, we filed for a patent to use the circular RNA as biomarkers. Uh, so how it all began, like I went to medical school to become a doctor. I didn't even know at that point that I wanted to become a pediatric surgeon. But then during towards the end of medical school, I kind of got interested in research and, and pediatric surgery mainly. And in order to get into pediatric surgery when I was training in the Netherlands, it was very important to, to do significant research. So I went up to one of the most prolific. Uh, scientists or researchers in Rotterdam where I was training, uh, Professor Dick Tibble, and I went up to him and said, Well, can I do some chart reviews with you? And, uh, he came back to me and said, well, you know what, we don't do chart reviews here. And, and why don't you go into the lab for a couple of months? So I did that. And um I actually uh was very interested in that. So I trained with him and um I, I finished my surgical training at Erasmus in, in Rotterdam. And then moved to uh Siki to do a research fellowship with uh uh Martin Post. And I really learned how to do good science over there. I went back to the Netherlands, finished my pediatric surgery training. We lived in a small place called Mordra, which is next to Gouda from the Chiefs. Then went to Birmingham, Alabama to do some more clinical training. And then we moved from the deep South to the great white north to a place called Winnipeg. I didn't really know it well, but they offered me a great position to start my career as a surgeon scientist and to develop my own research program and at the same time have a clinical career. So as Augusto mentioned, my, my kind of claim to fame is the dual-HI hypothesis, which we developed in the, in the lab with Martin Post, and it talks about how it's not just pressure of the abdominal organs on the lungs causing abnormal lung development, but it might also be something that's inherent to the lungs while they develop. So when I moved to Winnipeg, I started my, uh, my own research program and uh we called it Miracle, microRNAs and congenital lung Anomalies. And we like to answer the question, why are these lungs abnormal, and then along the way we want to figure out how that occurs and what we need to do about it. And why I do this is because of patients like Kara Ham. She was one of my patients, uh, born with congenital diabetic hernia. She, um, she was in the intensive care unit for months actually because she had a very complicated course. Um, however, she, uh, survived. Uh, she did well and she's now growing up to be a very nice patient for follow-up, but she was lucky. She was one of the lucky ones that actually survived. Because there's too many children still dying from the abnormal lung development and congenitalma occurring. Over 400,000 patients have died since 2000, and this is unacceptable, and I think as pediatric surgeons, we need to take responsibility and own this problem with our patients and solve it. We have to do better at understanding why these patients still die of this disease in the current day. And, and as Richard mentioned, and I like to highlight that in my papers and in my grants, if you look at that paper from uh Carmen Wiesels from Stockholm. They checked like if you include the terminal, the, the, the, the terminations of pregnancies in CDH in the mortality, the mortality is not better and has not improved since the 50s. It's still close to 50%, which is a real issue. So the main problem is the abnormal lungs in my mind. As surgeons, we can close the, the diabetic defect, but these babies struggle before and after this because of their abnormal lung development. So we're trying to answer what is wrong with these lungs, and can we fix the lungs before these babies are born. So we started studying microRNAs when I started my program about 10 years ago. And the typical um dogma is that there's messenger RNA and then there's proteins, and these messenger RNA come from genes. MicroRNAs are messenger RNA and gene regulator, almost like a dimmer switch on a light, where they can influence the expression of messenger RNAs. And the translation into proteins and thereby influence the gene expression. So in 2015, we were the first to discover that certain microRNAs are upregulated in patients with congenital dimatic hernia. And in collaboration with Dr. De Prest from Leuven, we studied. Uh, tracheal fluid when these babies were plugged, so they were undergoing fetal. And when the tracheal fluid was collected at the moment, they were unplugged. So when the balloon was removed. And we found that the ones that were going to survive the fetal treatment. had much higher levels of mere 200 people. So we looked at the literature and at that moment, there wasn't a whole lot known about mere 200B. It was mainly involved in cancer, but there seemed to be a role in the balance between epithelial cells and mesenchymal cells, fibroblasts. So we went on and developed a knockout mouse for a mere 200 beat. And we found that these mice had similar lung function abnormalities as we observed in babies when they survive congenit life medicanea and grow up, and we studied their lung function when they're like early, early teenage years. So they have increased tissue damping, they have increased tissue elastence, and this means that these mice have stiffer lungs. And the mice didn't have a congenital diphhetic hernia, but their lungs looked similar to what we see in babies with congenital diphmatic hernia. So they have hypoplastic lungs, which is clear, and I don't know if you can see my arrow, but if you compare this lung on the right side of the screen, they have much thicker alveolar walls than a wall type control lung, which have normal alveolar walls. So we showed that these lungs are hypoplastic. Now, we have recently discovered that if we look more carefully at the blood vessels of these mice, they also have thicker vessel walls. So, uh, this is a normal wild type mice, and this is a normal adventitial thickness. This is a blood vessel with the blood in there, and the medial thickness. If you look at the knockout mouse, they have much thicker advantage here, and they have much thicker media. When we look functionally at that, if we do echoes, if we do echocardiographies of these mice, they actually have pulmonary hypertension. So again, even though these mice don't have a congenital diphatic hernia, they have a completely similar phenotype in their lungs as babies with congenital dihatic hernia. So they have hypoplastic lungs. And they have pulmonary hypertension. When we looked more carefully at these lungs, we found that they have disturbed endotelin signaling. And you can actually treat this endothelialtelin signaling with a drug called bosentan, which is sometimes used for pulmonary hypertension in CDH babies, so we actually did that. And we um were running these mice on a treadmill. This is a wild type. This is a knockout mouse, and this is a knockout mouse after treatment with bosentin. So this mouse was actually much more frequently falling back on the treadmill than a wild type or a mouse that was treated with bosentan. We also did a study where we looked at the microRNAs in the nitrofen model. So the nitrofen model has been used a lot for studying congenital diaphragmatic hernia in rats and in mice. And so when you treat rats with nitrofen, they have, uh, the offspring have abnormal lungs, and a lot of them have a diaphragmatic hernia. So what we did is we gave the mother rat the nitrophans. To induce abnormal lung development and uh daphatic hernia. And then we also treated them with microRNAmia 200B because we knew from our human studies that the ones that had higher levels of mere 200B were surviving more after the tracheal plugging. So we hypothesize that mere 200B needs to be up in the lungs of these uh uh rats when they're developing in order to have a better outcome. And indeed we found that. So when we treated these rats in utero with mere 200B, we found that the ones that were not treated had hypoplastic lungs, 80% had congenitalized meturnia, and they died right after birth. The ones that were treated with Mia 200B in utero. Um, had less congenital hymatic hernia, only 15%, and they actually survived. They had much better lungs. So the incidence, as I said, was in the ones that were treated with nitrofen without mere 200B over 60%. The ones that were treated with mere 200B, it came down to about 20%. Their lungs look much better. So here you have a negative control, uh, treated with nitrofen, and you can appreciate again that the lungs look much more abnormal than the ones that were treated with microRNAs at the same time that they were treated with the nitrofen. So, as I said, we're trying to find out why these lungs are abnormal, and if there's any way we can predict this better, and then eventually, if we can do fetal interventions to make the lungs better. So, one of the things that we found out while we were studying microRNAs. That circular RNAs are actually functioning as a regulator of microRNAs. They serve as microRNA sponges, so, um, they are actually one level above microRNAs in the regulation of some of the uh gene expression. So, uh, when Richard was uh with us in the lab here, he helped to study the circ RNA profile and compared the profile in CDH lungs and in control lungs. And both in mid-pregnancy as well as at end pregnancy, we were able to separate a profile from CDH in red. Over here from the profile in control. So we published that indicating that the circle RNAs are differently expressed in CDH lungs, as well as in control lungs, indicating that there might be a role for them as a biomarker. Again, in collaboration with Dr. Deret, we had amniotic fluid samples from patients that underwent fetal, again, to improve their lung development. Half of the group survived, the other half did not survive. Based on the imaging parameters of these patients that they did, they looked completely the same. The gestational age of the plaque was the same. The observed over expected lung to head ratio was the same. They had the same amount of liver herniated. They had the same amount of fetal gender, and their birth rate was the same. However, when we looked in the amniotic fluids before there were plugged, so at the moment of plugging, so before there was any influence of the veto intervention, we could distinguish the ones with who were going to survive from the ones that are not going to survive based on their circular RNA profile. So this has not been published yet. We're following up and validating these results. And we're hopefully going to be able to develop this more into a tool to better predict patients that will have more difficulty with their lung development as opposed to the ones that potentially would benefit from fetal. So this is our ultimate kind of goal. When somebody gets diagnosed with a congenital diaphragmatic hernia baby, we would be able to extract blood circular RNAs. We would screen them for how these are differently expressed, and then we would do bioinformatics and validate the circular RNAs to see if we can potentially use them as a prenatal predictor of outcome and survival. Again, I'd like to highlight why we are doing this work. It's not always easy to do basic science. A lot of it is one step forward and 5 steps backwards, and you have to be patient. You have to be in it for the long run. But I think we owe it to our patients to do better in understanding why their lung development is abnormal and why a lot of them don't survive. Kara was lucky to survive and she's growing up healthy now, but a lot of them are not healthy and are not growing up thriving. So we need to do better. And I think if we compare it to cystic fibrosis, for instance, I think as pediatric surgeons, we should own it that we've not done as well in explaining why this disease occurs. And how we can better treat it. For instance, cystic fibrosis, 1 in 3600 CDH, 1 in 3000. So it's almost as frequent. Everybody knows about cystic fibrosis. If you talk to people on the street, almost nobody has heard of congenital diabetic urnia. So we have to work together with patients to create more awareness around this. CS CF. was discovered to be caused by the CFDR gene. We still don't know exactly why congenital diabetic urnia occurs. Survival in the 50s for CF was 6 months. The first CDH patient was successfully treated in the 50s. The life expectancy of CF increased from 24 years in '82 to 48 years in 2007. The survival rate in CDH was 50% in the 80s. Now it's reported to be 80% in 2017, but if you include the prenatal termination, it's still close to 50%. And as I've said, since 2000, over 400,000 babies have died from CDH and their abnormal lung development. So we absolutely need to create more attention and get more funding to study this disease better. And as pediatric surgeons, I think we have to be involved in this. I don't want to necessarily um downplay this important finding about the roundabout receptors that were shown to be critical for forgot separation of the body wall, but this is a Morgagni hernia, and they discussed it as if it was a Bogda-like hernia. And I think this is where You could tell that these researchers were not necessarily trained in pediatric surgery, and I think it's important as pediatric surgeons that we are part of the research of our own diseases, because if we're not careful, other people will study our diseases and they might draw conclusions that are not necessarily clinically relevant. So what worked for me, uh, protected time early on in my career. I had, I was well supported by my group in Winnipeg and by my chief, BJ Hancock. I received important mentorship from Terry Clausen, uh, a very strong clinician scientist here in Winnipeg, and Andrew Haleko, a great lung researcher. Um, I, uh, I have a passion for lung research. I have a supportive environment, and I got social support from my family, um, which is super important as well. And as Steve Jobs said, people with passion can change the world for the better. So I think it's very important that pediatric surgeons engage in basic science. Even though it might be very difficult, and it's hard, but I think it's up to the leadership in pediatric surgery as well to support these people and to inspire people to do better basic science in pediatric surgery, because we owe it to our patients. I'd like to thank the people in my lab that have been working here. This is during a flag raising ceremony, and the guy in the suit with the red arrow is actually the mayor of Winnipeg. He's not uh pi padding in my lap, but he was part of the flag raising ceremony. Um. And I would acknowledge all the people that have been working in my lab and uh I'm still collaborating with and the funding, and uh I'd be happy to answer any questions. Thank you very much again. Thank you, Richard. This was, uh, excellent and thank you both Richards, uh, to, for, uh, highlighting, especially in this first basic science uh webinar, the importance of uh basic science research in pediatric surgery. Um, I, I agree with you. There's um one of my colleagues and collaborators from Brazil, uh, told me, Augusto, if we, if not us, who? The cardiologists are in, uh, have their own questions, uh, the genetics have their own questions and, and the, the, these pediatric surgical patients are sort of left alone. We are the ones that uh come from the bedside with the questions to answer and, um, we should go to the bench or to the library or uh doing also clinical uh research to then come back with uh with answers, and this is why it's very, very important that we foster this, especially for the new generations. Uh, so thank you very much for, uh, for, uh, to both of you for sharing your personal story, your passion for CDH and in general for basic science. Yeah, thank you also. Sorry, thank you also from, from, from my side. I enjoyed both talks also highlighting the, the difficulties. So, um, I, I like, for example, the point, uh, Richard, um, Kaiser, you were making, uh, regarding uh that the surgeons need to drive the research topic. I think it's, it's, it's really key to know also that families and to prenatally counsel the families, even with a very bad outcome, then to feel the pain, then go to the lab and actually get the right motivation to go into depth and to find the solution. A researcher not into the topic, not a surgeon, not knowing these patients cannot do it. And that's why I like actually your point um you made with this Morgagni hernia study. Um, that, that is not. I agree, and I, I think what's really important you mentioned too is, uh, collaborating with patients. Like, are we actually doing the research that is important for patients? I think, um, I feel we are like we need to study why this disease develops and, and how we can better intervene, but the patients that survive. Might actually have certain questions that we haven't really listened to yet. So I think there's a role for uh better listening to what's important for patients, when they survive and, and uh what they deal with in life. And we've done a study with a Hearst from disease, uh, patient organization, and, and we're currently planning on doing the same thing with CDH International. Um, yeah, we need to work together with patients here. True. Sorry, Augusto, I cut you short. You wanted to, no, no, I was just about to say that if, uh, to the audience, if you have, uh, questions, please write them uh in the, um, in the chat, uh, and I think we can start off, uh, I wanted to start off going back to, uh, actually, um, the, the, the, the topic of the circular, uh, arenas which are very, very interesting. He showed us uh um interesting data with uh uh the fetal patients, so the ones that unfortunately received fetal and died versus the ones that received fetal and survived, and I, I, I, um, heard loud and clear this is now published, so I understand it will come out at some point, but, uh, can you, um Speculate more uh on the translational aspect of that. So in the future, of course it will be very useful for families uh but can we gather more information from the circular RNAs and why some of these patients uh succumb and why instead some others, uh, is there something different in their lungs, in the ones that actually do better. Beyond the demographics that you've shown us that were pretty much the same. Yeah, uh, I can probably start with it and then Richard, you can, right? Yeah, go ahead. Well, circular RNAs are like uh fairly new to the field still. Uh, they've been regarded as, as, um, garbage in the cell, uh, because when researchers saw them for the first time 25 years ago, they were, they were not, uh, aware of what they can do. And only recently, and, and that's a, a big um thing that Richard uh brought to the field when um investigating uh these circular RNAs was that they actually have a lot of um functions in the cell. And the main function they have, uh, that has been discovered right now is they can capture microRNAs and you've seen all the implications that certain microRNAs have in lung development and CDH, um, pathobiology. So We've been looking into these circular RNAs primarily because they're highly stable in body fluids because they're like circularly shaped and they cannot be digested as good as other RNA molecules. And therefore, they're very well suited as biomarkers. But as you point out, Augusto, I think it will be crucial to understand better the exact role in lung development. And so, I, I guess our goal together, um, is to not only establish a circular RNA biomarker that can tell us which babies will benefit from feto and ultimately survive CDH. Um, but also, once we have identified exactly 1 or 5 target, uh, circular RNAs to go to animal models of lung development or animal models of CDH and study the function. Um, however, this is my point of view. Biology is so complicated, and especially circular RNAs, if you think of, they have up to hundreds of binding sites for microRNAs, microRNAs then go to mRNA and suppress gene expression, and so it's really complicated, and I think the Basic science community is not there yet to really kind of mess with uh circular RNAs on a mechanistic level, but the next step, and, and maybe Richard can, can also um tie in some thoughts on that is to really understand. Better to function during lung development. And for example, if you silence or knock out some certain circular RNAs, what happens to lung development? And can we correlate that with the, with the things we see in the amniotic fluid of these patients, um, Surviving and not surviving. Yeah, I agree. I think uh it's, uh, the, the field is very slowly developing because, uh, and I think that's one of the reasons why uh circle RNAs have been ignored so far. It's very hard still to distinguish a circle RNA from a linear messenger RNA. And there's quite a bit of overlap between the two. So it's very hard to know if you're uh influencing or dealing with a messenger RNA or a circle RNA. And like, because we were doing a certain type of study, we were able to avoid some of that difficulty. But in order to move the field forward, we would need some uh very complex uh bioinformatics and also some very complex uh molecular biology techniques that, that we're currently trying to explore in the lab and, and we're starting on that. And uh we're hoping to collaborate with you, uh, Gusteau, to do some of those studies. Yeah, no, it's um, it's fascinating and it's great that you, um, got to know about the circular RNAs that this is going to be beneficial for so many other conditions and this is what, where the collaboration comes through and there's so much unknown um in the field of pediatric surgery, neonatal surgery, fetal surgery that you can imagine to then use the same tools or the same uh Um, biomarkers potentially for other conditions, uh, once, of course, we, we know a little better. And so it's a, it's, uh, uh, fascinating and it's great that uh you have, uh, brought this into our community and, uh, and again, I think, uh, it's not just the pediatric surgeons that should, uh, uh, look into this, so we should pair up even more with the, uh, scientists, the basic scientists. Uh, who have maybe a little bit more knowledge of this, uh, advancements, uh, in, uh, in molecular biology. Yeah, I, yeah, I, I couldn't agree more. I think, uh, like, I, I don't see myself as an RNA scientist. I see myself as a pediatric surgeon that, that has heard from RNA science. And, and, but I need to rely on collaborations with people that do this as a real basic science. And that's what I want to give the audience as well. I think if you approach these people, they're super happy to work together. And they're super happy to collaborate because they are looking for a disease, diseases that they can study with their basic science discoveries. So I think I want to make a pitch for people to really try and collaborate and work together with basic scientists, um, to try and solve these problems. We should, we should collaborate and work together because You're not able to do these things on your own anymore either as a pediatric surgeon or as a basic science. It really has to be a team effort. I think that's, that's really important also to get like the right numbers. If I, if I look at your study, you, you presented uh Uh, in the European respiratory Journal, they are comparing the survivors and the not survivors. This is 3 patients versus 4 patients or something, right? But if, if you get numbers in the, in the 50s or 100s, for example, you might get a little different results or more sharp results. So this collaboration is really super key um to also get the sample numbers you need to answer the questions, right? Yeah, no, totally. And, and I think, uh, um, that's another thing I think as pediatric surgeons, we have to work on. We need to collaborate internationally because it's important to uh get a better sense of uh the, the volumes and, and of uh uh samples from patients, so that we don't have to rely on these 2 or 34 samples that are locally available or through collaboration. Well, and, and I think a good example for that is, uh, pediatric oncology, right? Almost every patient automatically, at least the, the, the countries that I, I have an overview, automatically get enrolled into a study and in a protocol, and I think Their numbers are sometimes lower, sometimes higher than congenital anomalies, but we really can, can take that example, and almost every patient with a congenital anomaly should be part of a protocol, and I think that's not happening yet, enough in the pediatric surgical field. Well, that's, that's true. I really, our fathers of oncology, they did a lot of things right, 30 or 40 years ago, at least in Europe, where, like in Germany, every patient is in a study. I don't know about the US or Canada is every oncologic patient in a study automatically. Pretty much, yeah, yeah. OK. But, uh, but congenital anomalies are still, uh, um, a big burden of, uh, mortality in pediatrics and, uh, more than that, even, uh, of morbidity and so this is a big burden for patients and families but also for the society looking at, uh, costs. So I have um a sort of a No, no, no provocative, uh, question, but a question that uh comes in my mind. I have my own answer and I want to ask you guys. Of course, we are comfortably seated in our, um, offices, um, uh, we, we are, um Richard, uh, and Richard, uh, and myself in North America where, of course, uh, uh, basic science research is, uh, um, supported Martin in Germany, which is, uh, uh, of course, uh, a country that has always been supporting the technology advancements. Uh, but there's a lot of our audience, a lot of our members that, uh, are not necessarily are in, uh, uh, countries where there is access to, uh, a lab or to pediatric surgical facilities for basic science. So how do you envisage, uh, bringing, uh, uh, This community, uh, uh, into the picture and how do you think, uh, uh, they could contribute, uh, to the bigger picture of, uh, pediatric surgery from basic science standpoint. Well, what, what I can really see is that although in, in some areas of the world, they might not have access to these, um, experimental techniques that we are having in, in, for example, Germany or the US or Canada, but I think that the question ties in well with, uh, sample size. So we should aim for, um, kind of pipelines that Whenever a CDH kit is diagnosed, even in countries that are not doing the basic science themselves, then they should be able to obtain clinical samples either from the patient or for a prenatal biomarker, ideally it would uh a blood draw from the mother, and they, they, they would be able with the necessary funding, I guess, provided from, Uh, CDH consortium kind of groups that they can ship these samples to the area where we do the experiments. And this would be a, a perfect integration of, um, researchers in, in the areas that are, that don't have access to the, to the large scale next gen sequencing or uh RNA transcriptomic analysis, which At the pace that basic science is going right now, I mean, I think we have to keep up with the state of the art in basic science, and it's clearly that we are in very privileged areas, as you say, Augusto. So, I think working together with them to get clinical samples, and if at one day we are fortunate enough to be able to, to give medical compounds prenatally to prenatally improve lung maturation, um, this would also be Good to work together with these people to have the, the, uh, sufficient sample sizes. And I want to add something to that. I, I agree. Like, I think it's through collaboration and, and, uh, uh, but also, I think what you're doing right now is inviting people to learn about this research and to uh participate in discussions around this research. I think that is important. I think if more pediatric surgeons are aware of what's happening in basic science and learn about this, that would increase. Awareness and that would increase better outcomes or support better outcomes for patients with congenital anomalies. So I think even though, as you say, and I totally agree, like it's we're we're from our offices in a privileged situation, at the same time, I think by, by working together and giving these kind of webinars, so that people can learn about this, I think that's a very important thing as well. Also, in the, I, I could not agree more. Also, Richard, in the um annual meeting of the UUSA, we have now moved the basic science session from being the last session where everybody leads to the airport already to the middle of the conference that people uh are aware that this is an integral part of what we are doing and also the responsibility of the older guys listening to it. And at least get an idea what this is about. Not totally, they probably will not understand what this is, how this works, but to raise the awareness, as you say. But can I make a pitch then, because I've sat in, uh, in, in sessions, not necessarily at EUSA, but in certain like basic science talks, and it's all too easy to fall asleep because of the way it's presented and because of the way Um, it's, it's, it's, uh, educated. I think if you are serious about this, um, we should probably organize some educational sessions. And like the talk I gave was pretty superficial and, and high level. I would be happy to at some point give a talk where I go a little bit deeper into what kind of techniques we use and why it is important to use those techniques, so that people actually learn more about it. And I think we should integrate that into the training of, of the fellows and the trainees so that they actually from the ground up, learn how to integrate some of this knowledge into their training and into the treatment of their patients. I think that's when you actually make a change. Uh, because if we're just gonna have a session where people present basic science, instead of going to the airport, they might go and have a beer on the, on the patio. Yeah, so we need to make it interesting for the public. I mean that, that raises the, the, the question, Augusto, whether we should implement a pre-congress course basic science. I think, I think we should, and I think this is important for the future generations but also for, I, uh, I, I give you my answer. I agree uh with, with Richard Wagner regarding the, um, the, the. Uh, sample pool that of course can be obviously uh beneficial for everyone, but I have to say I collaborated, uh, I was at a fork at some point and I could collaborate with um a very rich European country or with Brazil and uh for certain reasons I decided to go with Brazil. I'm so happy about that decision because then it's not just, um. Uh, exposing yourself to a different mindset. Uh, CDH, for instance, in Brazil, I got to know that is, uh, very rare because unfortunately kids don't have, mothers don't have access, uh, um, or the same type of access that we would have in other countries to antenatal. Uh, care and so there's not much of an antenatal diagnosis and so the questions are also different that come from, uh, countries that maybe have a different healthcare systems, uh, and, uh, different challenges and it's important also for our, for us scientists, uh, to, um, look at, uh, the whole picture globally. Uh, I agree, Martin, it's, uh, it, I think it's uh It would be great to make a sort of basic science for dummies, uh, section, uh, where people would actually, it would be interactive, uh, and that we would be attracting more, uh, people to, to, to the, to contributing. Uh, we're not saying people need necessarily to, uh, work in labs or, uh, but, but, uh, but even just the discussion, the questions that come, uh, come up to each and every. Pediatric surgeon around the globe, these are crucial. Yeah, and, and you know, you might need to, uh, to push people presenting when they're presenting. To also put one slide, maybe why it is clinically relevant, what they study. So to actually put it in perspective, so it's not just about the cell work that they've done, but also why it was important to study this for pediatric surgeons. Maybe we can make this a mandatory slide on, on every basic science presentation, right? And what is the, the novelty of this presentation and why this is important? I, I, I like the idea, yeah. Yeah, I, I, I think it's really uh what, what Richard Kaiser said is really um important, how you present your stuff, right? Because the way sometimes it's presented, you never know whether the, the, the person who presents it understood it, him or herself, right? Yeah. So, and then it's for the old people, it's even more hard to, to follow, and then they fall asleep, as you say, yeah. But um yeah, I, I, yeah, I, I, I have another question for Richard Wagner. You, you, you, you showed this slide with the, with the wave and the, and the surface on them with the, with the challenges of the clinician scientists and the funding administration clinic, competition and the work life. So for you personally, what, what was the What was the, the biggest um hurdle to take, or what do you think? Well, you summarized already a little bit, what are the, are the pros and, and, and cons, what you need, but, but what would you recommend for people who are in a, let's say, not the super developing country, like maybe a middle developed country and want to go into basic science? How do What, what do you think? How should they do it? Well, I, I think I can answer the, the question what worked for me. Um, it's, it, I, I don't know if I have an exact answer for, um, other type of countries. Um, I think what's really, so to answer your first question, what was really hard, I think it's, it's very hard to come out of a 24 hour shift and then write a grant at the, from 8 to 10. Um, before you do some other activity. I mean, I've been doing it a while, but I think the quality of, um, the grant writing, for example, drops if you do that right after a 24 hour shift. And so that to really combine, especially as a resident, I think when you're like on the forefront of the, the calls and everything, at least this is how it's structured in Germany, um, that has been a challenge, and Um, I think what worked for me was to take that time off, and I mean, um, I think you and, and, and Richard Kaiser gave me that opportunity to really immerse myself in basic science, and I know that Augusto did that too, and Richard has, has mentioned it in his talk, so that you really understand the fundamentals of, of basic science. And I'm super happy now to be again, uh, in a team with Shing Min Ai and Patricia Donahoe, where I realized that even though I spent 1.5 years in CDH research, I wasn't really understanding so many things of lung development. And now I start to, with another 1 or 2 years as an investment, I start to understand, um, more fundamentals of, of biology due to the great training that I'm getting here. So I think my biggest suggestion would be take off time and, um, Really spend it only on basic science because this is what I get from more experienced, um, people in like the surgeon scientist path. I think later in life, you can combine that better. You can do your cases in the OR and then afterwards check with, uh, fellows in the lab what they did. I think it's easier. As compared to, to the state status that I have right now, being, um, doing the calls and, and, and being in training in the clinic and doing supervisions in the laboratory is sometimes hard. So that's why I'm very fortunate and happy to, to take that time off. And I think the same applies basically to um. Developing countries, uh, if they have the chance, and I think everybody who's running a lab like Augusto and Richard are happy to accept people from all over the world, as long as they're motivated and fascinated for the topic, and I think, um. That would be my main take from that. Very good. Thank you. OK, well, well, I, I, in the meantime, Gaya uh posted uh our uh addresses for Instagram, Twitter, and most importantly for our YouTube channel, so we'll be, uh, these, these, like all the other webinars are then posted on the YouTube channel and they can be watched, uh, uh, at any time also for the people that, uh, could not join us. Uh, at this stage. Great. Martin, what's next? What is? We, we, we are, we have our meeting in Athens, uh, the first week of, uh, of, um, September. It's, uh, in person, uh, but the great news is that we have also a digital platform that is now available and so it will allow also the people that cannot fly to Athens or would not have, uh, flown through to Athens to, um. View the meeting, uh, participate uh to the discussion and, uh, and participate to the, to the courses. And then I think uh after that, I have one webinar on November 10th, Wednesday, November 10th, I think is a basic science with um cool, cool kids do basic science with part of the copy, if I'm correct. Yeah. We will have another one also on clinical with, on Bretria where we have uh um Uh, Professor Yamataka, who is, um, uh, laparoscopic, uh, portentrostomy, kazai portentrostomy from Japan, so there's more to come, so. Very good. We're not stopping. Even with the summer. All right. I think we're over the hour. Um, Gaia, is there anything you want to add from the beach and, um, regarding the Athens meeting or? No, uh, just, uh, we are suggesting everyone to keep an eye always on our website because we are posting them for the, um, updated information. So as soon as we have the link to the platform, we will put the information on our website and also to keep an eye on our social media because, uh, it will be now 3 weeks full of news and better is to follow us on the social and to receive all the information just in time. Thank you very much for your time. Thank you, Gaya. All right, I think we have it. Anything else to add? No, thank you, Richard, uh. And Richard, thank you, Martin. Thanks for everyone who has joined us today. See you next time. Bye bye. Bye bye. You. Bye-bye. Ciao. Bye.