Thank you to the organizers for allowing me the opportunity to share data from our ongoing work regarding novel therapeutic targets for Wilson's disease. I have no financial relationships to disclose. And here's the QR code if you would like to provide virtual feedback or questions. So as we all know, nuclear receptors are a group of transcription factors that contain a highly conserved DNA binding domain that require zinc in the zinc finger. Studies to examine whether or not different transition metals could impair the ability of the DNA binding domain of the estrogen receptor with an oligonucleotide containing an ERE showed that cadmium and cobalt did not impair the ability of the estrogen receptor DNA binding domain to form a complex with its response element, however, copper and nickel did. Subsequent studies with copper showed that copper incorporation into the DNA binding domain resulted in a disordered structure that was unable to coordinate with EREs. This was interesting from the standpoint of there being a metal that could displace zinc and impede upon nuclear receptor activity, which is probably relevant to other nuclear receptors considering the highly conserved nature of the DNA binding domains of all nuclear receptors. So this had applicability to the nuclear receptor field in broad general terms. So copper is an essential trace element that is absorbed in the duodenum and is transported into the hepatocyte via the CTR1 transporter. It is then transported to the ATP7B transporter on the trans-Golgi network, which then incorporates copper into the secretory pathway where it is bound to a ferroxidase called ceruloplasmin and secreted into circulation. Excess copper is eliminated via biliary copper excretion. This and the ATP7B transporter result in an autosomal recessive disorder called Wilson's disease. Wilson's disease is a rare disorder with an occurrence of one in 30,000 and is fatal if not treated, but is usually treated with chelation and or zinc therapy. This excess copper that accumulates in the liver in patients with Wilson's disease causes lots of cellular stress and redox stress as well as mitochondrial toxicity and inflammation. We hypothesized that this state of chronic excess copper exposure in the hepatocyte could likely impair nuclear receptor function and contribute to the pathology seen in Wilson's disease. As we know, nuclear receptors are key in regulating many metabolic homeostatic processes from bile acid regulation of bile acid synthesis, as well as lipid and cholesterol metabolism. We hypothesized that excess copper could, like the estrogen receptor, impair the ability of these nuclear receptors to coordinate with their DNA response elements. For these studies, we used a Wilson's disease animal model. This animal model is very similar to, has a mutation in the ATP7B transporter that results in excess copper concentration similar to those seen clinically, as well as increased markers of liver stress such as ALT and bilirubin. You can see that in the panel at the end of the slide that on a cellular level, these cells are damaged. There's enlarged hepatocytes with pleomorphic nuclei and microsteatosis, but it takes a while for this to develop. It's a chronic process that usually you don't see pathology until the animals are about 5 months of age, and later they develop nodular formation. Studies with pre-symptomatic animals from the lab of Svetlana Lutsenko at Johns Hopkins showed, interestingly, that serum cholesterol levels were lower in ATP7B knockout animals. This corresponded with an increased abundance in differentially expressed genes from lipid metabolism pathways. Again, these are pre-symptomatic animals. They're not sick yet, but they have this profound change in cholesterol and lipid metabolism. This supports the idea that nuclear receptors are somehow impaired in this animal model because we know nuclear receptors such as FXR and PPAR-alpha are key in regulating many homeostatic processes in the liver, as well as there's a coordination of nuclear receptor feedback from the gut to the liver as well. Our studies initially looked at the effect of copper in vitro in hep G2 cells. We found that hep G2 cells treated with those naturally occurring FXR log in CdCA, that this induction of B-sep expression was strongly attenuated in the presence of copper, but was restored when zinc was added to the media with copper and CdCA. This improvement on FXR binding to its response element was shown with an IMSA analysis. Those studies were supported by further studies in the ATP7B knockout mice, which showed that recruitment of LRH1 to the LRH1 response elements was decreased, significantly decreased, as well as FXR recruitment to the FXREs of B-sep and SHIP, supporting the concept that nuclear receptor activity was impaired. I want to point out that it's not completely obliterated, that the nuclear receptors maintain some ability to bind to their response elements. We next did in vivo studies with the ATP7B knockout mice that were similar in concept to those in hep G2 cells. We gave the animals child diets supplemented with zinc to see if we could improve liver outcomes as well as restore or improve nuclear receptor activity. Markers of liver stress, such as ALT, bilirubin, and bile acid serum levels were all improved in the knockout animals given a child diet supplemented with zinc. We can see that the FXR target gene B-sep, which I should point out is essential for bile acid secretion from the liver and preventing toxicity from hydrophobic bile acids. You can see that gene expression is improved as well as FXR and RXR recruitment to the promoter. Gene and our transcriptomic analysis from this data set revealed interesting differences between animals fed, knockout animals fed the child diet or the zinc diet. So as you can see, the red circle shows that knockout animals on child diet versus wild type animals on child diet had, there were a lot of differentially expressed genes with some overlap here, but the animals fed, knockout animals fed a zinc diet relative to the wild type animals fed a child diet did not have as much differential, did not have as many differentially expressed genes, indicating that it's a likelihood that the zinc enriched diet normalized gene expression in the knockout animals. We looked more globally at the ability of FXR to coordinate with FXREs across the genome and the panel in brown shows that there was an abundance of peaks in the, that were able to form from wild type animals but were lost in the knockout animals. These peaks within the genome represented genes that were most abundant in metabolic processes, predominantly bile acid metabolism. There were, there was a cohort of peaks that overlapped between wild type and knockout mice, but the abundance of these peaks that were, the abundance of the peaks that were formed in the knockout animals were decreased in amplitude relative to the wild type mice. And again, the pathways represented with, of genes within, that contain these peaks were metabolic, represented metabolic pathways. Genes that had enriched or more binding of FXR to elements within the genome relative to wild type reflected more of a stress response rather than a metabolic response. Similarly, we looked at PPAR-alpha recruitment across the genome and found similar trends. There were a lot of peaks that were missing in the knockout animals but present in the wild type animals. And again, like the FXR ChIP-seq, we see that they're mostly, the genes represented with, in these peaks are mostly metabolic genes in metabolic pathways. Looking at the transcriptomic profiles of these animals, knockout animals, and again, I want to point out these are on chow, they're not on a supplemented diet. Again, in support of this metabolic defective signature, we see that most of the differentially expressed genes on transcriptomic analysis are in the metabolic pathways pathway. And you see a lot of other metabolic processes including biosecretion, PPAR-alpha signaling, as well as protein metabolism with some other pathways being represented, chemical carcinogen, such as chemical carcinogenesis and ACM receptor interaction being what we would consider to be a possible response to the toxic levels of copper. We overlapped the transcriptomic profiles with the ChIP-seq peaks that were either missing or decreased in the ATP7B knockout. So we took the genes that did not have, that had this reduced binding of FXR or PPAR-alpha and looked at gene expression, and you can see in yellow, most of the, most of these genes actually have correlating decreased messenger RNA expression. There are groups of genes in each pathway that do have an opposite, an opposite pattern, but mostly this, the gene expression corresponds with the nuclear receptor binding. And on this side, on the right side of the panel, you can see examples of this. So ATP11A, for example, in the wild-type animals, you can see that there are peaks in the wild-type animal that are completely missing in the knockout or almost completely gone. Similarly with PPAR-alpha, you see that there are a lot of enriched regions in the G6, in the glucose 6-phosphatase gene that are reduced in amplitude in the knockout. When we looked at metabolic profiles in these animals, we see similarly that there is a metabolic signature that seems to overlap this decreased nuclear receptor activity that we see. So in the top, these are metabolites, and this, I should say, this was not a global, this was not an untargeted approach. This was a targeted approach. So we looked at glycolytic TCA cycle amino acid as well as bile acid metabolites, and we see in the top panel the reduced metabolites, which are interestingly pyruvate lactate, glucose, and Cetyl-CoA. But a lot of these metabolites are actually increased in the knockout animals relative to the wild-type animals. And we think, we're not sure why this is, but we think it may be due to issues with metabolic flux. About the time these studies were being done, clinical groups were, had published results from Wilson's disease patients that had showed that Wilson's disease patients, in fact, have an altered metabolic profile as well. So they had increased colic acid. They had the human counterpoint increased glycolic acid, which in the mouse is tauricolic acid, and we see is increased in the mice as well, as well as other altered metabolites, indicating that the mouse and human likely have overlap in their defective nuclear receptor signatures. Animals given a Western diet challenge revealed some interesting results that supported this aberrant metabolism in the knockout animals. In the top panel, in the red, the red line are wild-type animals on a Western diet. You can see a nice steady increase in weight, as expected, relative to wild-type animals on chow, the white line. But in blue and yellow, you can see these are knockout animals on either chow or Western diet. The knockout animals have slightly different weights than their counterpart, than the wild-type cohort, and you can also see that there is no weight gain in the knockout animals on Western diet. This was quite a striking finding. And this overlapped a lack of, they don't gain fat, so there's less fat, and they don't gain fat on Western, their fat mass doesn't increase on Western diet, and correlates with epidermal white adipose tissue weight, as well as decreased liver weight. They also had very impressive differences in insulin sensitivity. You can see on chow diet, in the blue line, these are the knockouts on Western diets, and on Western diet, the red line, you can see that they have an apparent improvement in insulin sensitivity that's quite striking, and that correlates with that increased and improved glucose homeostatic status, as well. Overlapping this was decreased gluconeogenesis, as found by pyruvate tolerance testing, decreased hepatic glycogen, and decreased hepatic lipid, which was in support of previous data from Svetlana Lutsenko's lab, as well. So even though this appeared to be a metabolic improvement, we didn't really consider this to be an improvement, because these, the livers in these animals are sick, and they, and it's likely due to a defective, a defective functionality of the nuclear receptors, rather than a true metabolic improvement. So in conclusion, FXR and PPAR-alpha are decreased in vivo and in vitro in response to elevated copper. Excess zinc supplementation reduces copper dysregulation of hepatic gene expression, and ATP7B knockout mice have metabolic homeostasis that correlates with nuclear receptor dysfunction. So what are our next steps? So as I said in the previous slide, we know that FXR is activated in the gut by increased bile acids. So when bile acids increase in the gut, that activates FXR, which turns on expression of a protein, of the FGF15-19 protein, which many people in this audience probably appreciate, negatively regulates bile acid synthesis in the hepatocyte. So we wanted to know, this study was done quite a while ago, but we wanted to know if the gut had a similar dysfunction as the liver, because we also knew that copper homeostasis in the intestine is not impaired in Wilson's disease, because differences in transporter expression. So we found that there was, basally, there was a decrease in AT, in FGF15 expression in the terminal ileums of knockout mice. However, it responded to the FXR login very strongly, GW4064. So this indicated to us that there was a good chance that we could target nuclear receptors in the gut, and in turn, improve the metabolic outcomes of the liver. And so, in addition to that, so in addition to the possibility of targeting nuclear receptors in the gut via specific activation of FXR in the gut, and in turn, negatively regulating bile acid synthesis to hopefully prevent, to normalize the levels of toricolate that we had found in the livers of these mice, which should reduce some of the toxicity that's seen in the liver, it also, we also could use specific logins to target FXR, PPAR alpha, among others. So these, we actually have, we have data, and these studies were ongoing, and the pandemic happened right as we were starting to really work out the dose curves on those. Because it's tricky, because these mice, they already have liver disease, so giving them logins is tricky, because that's an added stress to the liver. Also, we're going to look more closely at how zinc is actually, you know, what's the mechanism of zinc protection for nuclear receptor function, and the health of the liver. So we actually think that this zinc-mediated effect, so clinically, it's well known that zinc outcompetes, can outcompete copper absorption in the gut when added in much excess, and that's actually considered to be the protective mechanism of zinc. We, you know, our studies, our findings showed that zinc had a specific liver effect as well, but we think, you know, in terms of holistically, that it's likely that there is some coordination between the gut and the liver, the hepatocyte, in terms of the protective effect of zinc. So it's not just a gut mechanism, there's definitely a liver mechanism, and whether or not it's, you know, limited to the improvement of nuclear receptors or not is yet to be seen. And in terms of specific login dosing, I think, you know, that is a promising approach too, but it's going to be a little bit tricky because, again, we have to find the right ligand at the right dose and given at the right time in order to prevent a lot of toxicity in the liver. So my acknowledgments, this work was initiated in the laboratory of David Moore before he moved to Berkeley and when I was a postdoctoral fellow in his lab. Allie Antar and Yong Zhu from Baylor College of Medicine were essential in performing the studies with the animals, the metabolic studies with the animals on Western diet. Micah Grusak, who's now, I think, in North Dakota. He's a USDA ARS scientist, measured copper metals for us, as well as my technician and others at Baylor College of Medicine. Thank you. That was an excellent talk. I guess I'll start. So 30% of all proteins are metal-binding proteins. And have you performed proteomics to look at the impact on the proteome? Yes, so we have with older animals. So the older animals actually develop nodules that we think are mostly regenerative. Can people hear me? That are mostly regenerative. It's been on a very small scale, but there is an enrichment in this, I think, ketone keg pathway and metal stress pathways, which we sort of casually dismissed, because that's what an expected result. But I think it's worth probing, because that's very critical to the homeostatic processes of the liver as well, and even bile acid secretion. Nice talk, Ruth. Carolyn Cummins, University of Toronto. I was wondering, you might have mentioned this, are there treatments for Wilson's disease? Is zinc considered? I'm sorry? Are there treatments for Wilson's disease? Yeah, so the standard of care is chelation therapy first. So that helps to neutralize all of the free copper in the liver. Zinc, I think, is kind of a second line approach. So either patients who have been on chelation therapy for a long time and are well-maintained, they sometimes switch them to zinc, because it's an easier therapeutic approach, but it's not as clinicians debate over which one to do. It's hard to give them at the same time, because you give the chelator in the morning, give the zinc in the afternoon, or something like that. But it's tricky, because these therapies, although they can essentially cure a patient in some ways, it's not long-term, not a well-tolerated system. The chelation therapy is especially not well-tolerated. And there's a whole different area of Wilson's disease, neurological Wilson's disease, that I didn't talk about. And those patients don't handle chelation therapy very well. They tend to do better on zinc therapy. And it's not known. It's a big unknown. So we actually are going to start looking at some of the neurological stuff, aspects of this with Yongzhu at the CNRC. But yeah, but zinc is, and there are, I think in general, that there are certain foods that they want people with Wilson's disease to avoid, because they have more copper than others. I think like red meats and things like that. OK, thanks. Nice talk, Ruth. I just have two couple questions, quick questions. I can't remember, of course, being from David's lab, I have to ask, what's CAR levels in these mice? Are they activated because high bile acid? And the second question is, you nicely showed CA and UDCA are upregulated or high in your animals. But we know MCA would be the primary bile acid and an antagonist for FXR. So what's MCA levels in these? Great question, Sah. So CAR, that's a great question. So I actually looked at, so I tried generating the CAR 7B double knockout, because there's a lot of xenobiotic metabolism lights up, and a lot of CAR PXR targets are increased in these animals. However, we don't have evidence that CAR is really, it's not clear. But when we, some very early studies, when we gave CAR-like TC compound, they actually didn't, they had an attenuated response to CAR, it wasn't completely gone. But interestingly, when we tried to generate the 7B CAR double knockouts, they don't survive, or very few survive. So it probably is playing some role. Regarding, so in the patients, so patients have the increased UDCA, CA. Our animals have increased CA. Muricolic acids are actually decreased. So I didn't, I think that was in the small, like in the upper panel, it was hard to see. But yeah, so muricolic acid is decreased, which you would expect in a cholestatic condition and a toxic liver condition. Great questions, yeah. I have another question, wondering about the metabolic phenotype, so that they were, they had an improved metabolic phenotype. Wondering, is that also seen in patients? That's a great question, and it's not clear. So there are, so it's, because it's a rare disorder, there's not a lot of data. There are some reports that these, so there are clinical reports that say these persons have increased steatosis, fatty liver. There are other reports that say they're anemic and undernourished and thin. We actually are in a different project. We are looking at GDF-15, because in our mice, they have elevated GDF-15. So we are interested in trying to sort out whether or not that's relevant to patients as well. Thank you. Okay, thank you. Thank you. Okay, our next speaker is Dr. Laura Salt from the newly rebranded University of Florida Scripps Research Institute. And today, she's gonna talk to us about chemical modulators to dissect ROR function. Great, okay, can everyone hear me? Thank you to the organizers for inviting me to this, and thank you for those who came out for the 8 a.m. session. Those who are talking, appreciate it. So today, I'm gonna talk to you about ROR-alpha. We're gonna go back to immunology a little bit and look at what's going on in Th17 cells. And since this session is talking about the guts, it's gonna focus more on IBD. So I have no financial disclosures. These are the slides that I needed to do. Okay, anyway, so autoimmune and chronic inflammatory diseases are on the rise, and some of the ones shown on this particular screen, on this screen right here, are the ones that are probably most well-known to people and the most talked about in the literature. However, there's more than 100 different autoimmune and chronic inflammatory diseases, and about 30 million people in the United States suffer from these diseases, which drives about $100 billion annually in treatment costs. So to me, that's a lot of money. I'm sure to the people who are suffering from this, this is a lot of money. But basically, one of the problems with the treatments for a lot of these is that for some diseases, there's really no treatments. For some of the other ones, the treatments are more global immune suppressants, which is not ideal because it can leave the patient susceptible to opportunistic infections or cancer, or for some of the therapies, they start working, but then they lose efficacy over time. So what this basically means to me is that we need to develop a better understanding of those things that drive disease and see if we can make more focused therapeutics to help patients. So David gave a really nice intro yesterday morning, but for those of you who weren't here, I'm just gonna go into inflammatory bowel disease again. So it's chronic inflammation of the GI tract, and it affects about three million people in the U.S. You present with abdominal pain, bleeding, and severe diarrhea. While it's called IBD, there's two different diseases. There's ulcerative colitis, which basically the inflammation's stuck to the colon. Then there's Crohn's disease, where you can get inflammation in any part of the GI tract. There are existing medications that dampen the immune system, and some are very effective, and so I put anti-TNF here, because that's a biologic that's often used. However, this is one of the ones that either a subset of patients don't react to this, or they lose efficacy over time. Failure to basically keep this under control means that a lot of patients have to undergo surgery to basically dissect out those sections. You can get colorectal cancer, and there's just lifelong disability with these patients. And so many autoimmune and chronic inflammatory diseases are driven by CD4 positive T cells. So again, back to immunology, there's usually two types of T cells, CD4 and CD8. We're focusing on the CD4s. And naive CD4 T cells that have not seen any antigen, once they do see their accommodate antigen, they can differentiate into any flavor of T helper cell populations shown here, depending upon the different cytokines in the milieu. Now all these populations have their own specific function. So we have TH1s that are required to, or help fight off intracellular pathogens and viruses. TH2 that fights off, basically helps expel parasites. And TH17s, which are important to basically, for immunity against extracellular bacteria and fungi. Now here you have these T regulatory cells. So these are not pro-inflammatory, unlike these. These are anti-inflammatory. And they're basically help down-regulate the pro-inflammatory immune responses. Now we're interested in TH17 cells, which secrete the cytokines IL-17A and F, largely because they've been shown to drive various diseases. But TH17 cells can actually acquire a TH1-like phenotype during chronic inflammation. So in the presence of things like IL-23, they up-regulate not only, they actually start to up-regulate interferon gamma, as well as IL-17A. And it's these double-producing cells that are thought to be the mediators of pathogenesis. So, like I said, TH17 cells have been implicated in the pathogenesis of various autoimmune diseases. And I just told you that under normal conditions, it's important for mucosal immunity. But they can become apparently activated and drive autoimmune and chronic inflammatory diseases, like multiple sclerosis, psoriasis, IBD, they're implicated in. And the reason that, you know, several, actually over a decade ago, when ROR gamma T was identified as the lineage-defining transcription factor, it was really exciting, because as a nuclear receptor, it meant that we could target cells and be a little bit more specific with our therapeutics. And so that's actually what the question was broached probably about 10 years ago. Oh, that's ROR, okay. So since that time, there's been a lot of work put into understanding, you know, how ROR gamma T is regulated by both endogenous and synthetic ligands. So some of the synthetic ligands for ROR gamma T are cholesterol derivatives. And RORs are thought to, for the most part, be constitutively active. And in the presence of ligands, it's gonna down-regulate its transcriptional activity. However, because gamma T was important for TH17 cells, a lot of groups and a lot of pharmaceutical companies put in a ton of effort trying to develop ROR gamma modulators for the clinical use. And these are just some of the early ones that were identified, and these are some of the ones we've identified in-house. So since that time, there's been about 20 different ROR gamma modulators that have gone into clinical trials. Unfortunately, most of them have either been withdrawn or suspended due to safety issues. Some of them are known, some of them are unknown. But one of the things that is known about ROR gamma T, which isn't exactly ideal, is that not only is it important for TH17 cell development, it's also important for T cell development in the thymus. And so this is something we published several years ago, and many other groups have shown this. But basically, this is what a normal thymus should look like, normal CD8 and CD4 expression. When you knock out gamma and T cells, this obviously looks nothing like this. And that's because gamma T is important to drive anti-apoptotic factors. So in its absence, you get increased thymic apoptosis, leads to decreased total T cells in the thymus. And what happens to these mice over time is a lot of them succumb to lymphomas. And what people have found is that long-term treatment with gamma modulators starts to phenocopy what they see genetically. So that isn't exactly ideal. So because of this, people started asking the question, actually, we started asking the question too, are there alternative targets in TH17 cells that might be therapeutically tractable? And the answer in a nutshell is yes, because several years ago, Chen Dong's lab published that ROR alpha is also expressed in TH17 cells. So this is a close family member. And this is basically just showing that ROR alpha is upregulated with kinetics similar to ROR gamma at both the mRNA and protein levels. But what people fail to realize is that even though ROR gamma is considered the lineage-defining transcription factor, it's not the be-all, end-all in TH17 cells. If you see here, when you knock out ROR gamma, there's still a pretty decent amount of IL-17 expression coming from these cells. That's about 20%, despite no ROR gamma expression coming from these particular cells. So what was found is that if you knock out both alpha and gamma, you ablate IL-17 expression. So basically, you needed both transcription factors. But the problem was, so as you're probably aware in the nuclear receptor field, if there isn't an obvious phenotype to something, people think that the receptor's redundant to something else. And that's basically what people thought, like alpha's redundant to gamma. I don't think nature makes redundant proteins. I just think about all the effort that went into this evolutionarily to make these proteins. So why would you do something that's just a backup? So with that in mind, we started to dive a little bit into what ROR alpha was doing in TH17 cells and how it might be inducing pathogenicity. And so what we showed is that if you overexpress and knock out ROR alpha, it modulates TH17 cells. So overexpression of ROR alpha, you get increased TH17s. And here we knocked out ROR alpha in T cells, and you can see decreased IL-17 expression, despite there being equal amounts of ROR gamma T protein expression between the wild type and knockout mice. So ROR alpha is basically critical. But what was really interesting to us, and oh, I'm sorry, this is supposed to have colors to it. Anyway, so we did RNA-seq of alpha knockout TH17 cells, and then we compared that data to the transcriptome of ROR gamma knockout TH17 cells. And there's really very little overlap in the transcriptional signatures between alpha and gamma knockout TH17 cells. The overlap is arising in the core TH17 genes. But there's a lot that ROR alpha is regulating by itself, independent of gamma, suggesting that this really isn't redundant. And if you can see here, the KEGG pathway analysis, one of the top hits is inflammatory bowel disease, which TH17s are thought to play a role in. So with that in mind, knowing this in vitro phenotype, we wanted to determine how alpha was affecting pathogenicity. And so we run various mouse models of autoimmunity and chronic inflammation in the lab. But since this is a gut-mediated session, I'm gonna focus on our models of colitis. So this was introduced yesterday, but just in case. So you can induce colitis by transferring naive T cells that you sort, and transferring them into RAG1 or RAG2 deficient hosts. Now, these animals don't have T or B cells. And so what happens is they develop a wasting disease over time, which is consistent with what you see with colitis. So after eight to 12 weeks, you basically, actually you monitor the mice for eight to 12 weeks. And then what we found was that mice receiving alpha knockout T cells were largely protected from wasting relative to the wild type, excuse me, the animals receiving wild type T cells. And when we looked in the colons, just for global immune, excuse me, pro-inflammatory cytokine expression, we saw that in the absence of alpha, there's significantly less pro-inflammatory cytokine expression shown here, which is consistent with, you know, the mice being protected from disease. Now, what we wanted to do next was look at a more, actually a shorter time point at weeks five to six, because this is more indicative of when the cells actually are starting to home into the colon and induce disease. And so when we looked in the animals after five to six weeks post-transfer, we saw that there's significantly less T cells in the colon relative to those mice receiving wild type T cells. And that was because we found that there was decreased expression of certain chemokine receptors, like alpha-4 beta-7 and CCR6. So alpha-4 beta-7 is a key chemokine that helps T cells, or immune cells in general, home into the colon. And there's actually biologics out there right now that target alpha-4 beta-7 for IBD. So this, and CCR6 is another chemokine receptor, which basically is important for T cell homing into sites of inflammation. And it's been shown, at least in mouse models of multiple sclerosis, that this is key for the Th17 cells to home into the CNS. So this decreased number of cells in the colon is partly indicative to decreased cell ability to home into the colon. And so we also looked at, obviously, the phenotype of the T cells. And interestingly, unlike in vitro, we saw that in the absence of alpha, there was decreased ROR gamma-T positive cells, which suggests that alpha is important for stabilizing the Th17 phenotype and ROR gamma-T expression. But of those ROR gamma-T positive cells, we saw consistently decreased IL-17 expression, and again, decreased expression of cells expressing both alpha and interfering gamma. So like I said, those double producers are the ones that are thought to drive disease pathogenesis. And there's also this, at least for immunology, this dichotomy between Th17 cells and Tregs. And so when you get one, you get maybe less of the other. So considering the fact there's less gamma-T here, it wasn't exactly surprising to us that we saw increased T regulatory cells. So these are the anti-inflammatory cells, which could also help be accounting for the lack of disease severity. So there's a lot of things going on here. And so we also wanted to make sure that this was a cell-intrinsic effect and not a cell-extrinsic effect. And so what we did was we did another transfer of colitis where we basically did a one-to-one transfer of wild-type and knockout T cells, which you can differentiate between the surface marker CD45.1 versus CD45.2. And after five weeks, we just looked at the phenotype of these cells. And consistent with what we saw in colitis, there's decreased homing of the cells to the lymph nodes and the colon or the mesenteric lymph nodes in the colon with not as much effect on the spleen. So there seems to be this issue of the cells homing to the colon and sites of inflammation, which again, we saw a decreased expression of CCR6, gamma-T, and IL-17A expression, which is consistent with what we saw in all our other in vivo models. So alpha seems to be important for Th17 cells, particularly in IBD. And we did some, as immunologists, we like to make these fancy-schmancy, complicated reporter mice. And that's what we did here. But basically what we wanted to do was look at Th17 cells, different populations of Th17 cells in the gut. So these mice, IL-17 is marked by GFP, FOXP3 is marked by RFP, and any cell that ever expressed IL-17 was marked with human CD2. So this is a fate reporter mouse. So we basically did a transfer and we sorted these different cells and we did single-cell RNA sequencing. And it might be hard to see, but basically what we see here is that there's not as much gamma expression in these Th17 cells, unlike ROR alpha, which is kind of consistent with alpha being pretty important in Th17 cells. And this keys into what I'm going to talk about on the next slide. So to summarize the genetic data, alpha is important for the regulation of TH17 cells. It's required for full pathogenicity in both colitis and what I haven't shown you, but mouse models of multiple sclerosis. And several months after we published our data, this paper came out from Chen Dong's lab, which basically corroborated our data in colitis. And in fact, another group, Dan Littman's group has something on bio archives, which is about ROR alpha and TH17 cells, which is very similar. But what was really interesting about the cell reports paper was that in this paper, they took transcriptomic analysis from different actually patients with IBD. And what they found was that ROR alpha was highly expressed in active anti-TNF resistant UC patients. So if you remember at the beginning of the talk, I said that there's a lot of patients with IBD who they'll take anti-TNF and it loses efficacy over time, or they don't respond to it in general in the first place. So this was really interesting to us and we wanted to know whether we could actually target ROR alpha for TH17 driven diseases. And so we dug into our arsenal of actually gamma modulators that we had from a program a long time ago, but several years ago, we published that 3,3,3,5 was an alpha selective ligand. So we differentiated TH17 cells and treated them with 3,3,3,5. And you can see that IL-17 was inhibited in a dose dependent manner. And that is supposed to be micromolar, so I apologize, that's a conversion issue. Anyway, inhibition of IL-17 in a dose dependent manner, there's no effects on viability. And we actually did, oops, sorry. Oh, I didn't put this on here. Anyway, but we did treat TH1s, TH2s, Tregs, and alpha knockout TH17 cells with 3,3,3,5, and we saw no effects. So this was largely selective to alpha and TH17 cells. So everything we'd done so far had been in murine models and mouse TH17 cells. So we wanted to see whether this effect translated to human T cells, because if it didn't, then this was kind of a moot point, because we want something that's translational. And so we took advantage of this in vitro TH17 differentiation system that was published by the McGeeky Lab, where basically you only use anti-CD3, IL-1 beta, and IL-23, and it gives you robust IL-17 expression in T cells. And so we used this, and we saw that there was a, again, with 3,3,3,5, a dose dependent inhibition of IL-17 without affecting the viability. Now these treatments that I just showed you in vitro, they were all prophylactic. And so we really wanted to next ask, what about memory cells? So these are the T cells that are already activated in the periphery, floating around and will become reactivated when they experience antigen again, and will be driving disease. So with the help of my colleague, Mark Sundred, we sorted on human TH17 and TH17.1 cells from PBMCs, and TH17.1 expressed IL-17A and interferon gamma, just an FYI. So we sorted on those populations and basically treated them with 3,3,3,5. And if I just direct your attention here, you can see that 3,3,3,5 inhibited IL-17 from these different populations. And we also treated other T helper populations with 3,3,3,5, and we didn't see effects on those. So again, it was largely selective to alpha and TH17 cells. And actually, we found this really interesting because TNF is a pro-inflammatory cytokine that's often greatly overexpressed in the gut of IBD patients. And actually, 3,3,3,5 seemed to be inhibiting TNF in these populations, which is actually a good thing. And we didn't see this in any other T helper populations. But we also wanted to look at gene expression from these memory T cells. So we did nanostring analysis, and we saw that basically 3,3,3,5 was largely phenocopying our mouse alpha knockout TH17 cell RNA-seq data. Oh, and we also, with the help of Mark Sundred, we got PBMCs from UC patients, and we found that 3,3,3,5 inhibited IL-17 from UC patients as well. And so to see whether this worked in vivo, we actually, we did several mouse models. So we showed that 3,3,5 was very effective in several mouse models of multiple sclerosis. But again, since this is a gut session, I focused on colitis. We also looked at active colitis. So we went back to our transfer model here, and basically, we transferred naive CD4 T cells. And after three weeks, just when the cells are just starting to go into the colon, we started treatment with 3,3,3,5 for another two weeks, and then we sacrificed some mice to look at the phenotype. And we didn't see changes in body weight, which is good, because we didn't want the drug treatment to induce body weight. But what we saw is, this is pretty interesting. So when mice start to get sick, their colons start to shrink in length. And so we saw protection from this relative to the vehicle controls. There was also decreased cells homing into the colon. And again, consistent with what we saw in our genetic models, this was due to decreased expression of CCR6 and decreased ROR gamma T expression as well. So this seemed to be phenocopying our alpha knockout genetic data, and it also suggested that we could target TH17 cells for colitis. Now, if you remember, at the beginning of the talk, I also told you that there was a lot of gamma modulators out there, and they had safety issues, and one of them was thymic apoptosis. So we wanted to evaluate whether, oops, sorry, there was also decreased expression of this with the 3-3-3-5 treatment. So we also wanted to evaluate whether alpha knockouts or targeting alpha gave you the same phenotype as what a gamma modulator would do. And so to test this, this is actually a really short, quick experiment that was published several years ago by Dan Quah's lab, where you just take normal B6 mice, and you can treat them for three days, and then collect the thymus and do fax analysis looking at just T cell development. And so we treated mice with 3-3-3-5 and an in-house gamma inverse agonist, and 2211 did what it does and what we expected it to do. There was increased thymic apoptosis and decreased T cells in general in the thymus. 3-3-3-5 treatment basically didn't seem to have this profound effect relative to gamma modulators. And what I'm not showing you, but we also looked genetically at this, and there's no effect on thymic development, T cell development in the thymus and the alpha knockout mice. So, oh, and some of the things that we're doing now is just trying to understand how ROR-alpha is being regulated with different co-regulators. And so we're taking advantage of the system that one of my other colleagues has, Matthew Pipkin, where he has a SH RNA library, various different co-factors. And so what we're trying to do here is if you overexpress ROR-alpha and knock down different co-factors, you should see if they're required, they're gonna be affecting IL-17 expression. And we see that SRC-1, excuse me, SRC-2 seems to be affecting IL-17 relative to SRC-1 or SRC-3, which is also interesting because gamma seems to require SRC-1 and SRC-3. So alpha seems to be more selectively requiring SRC-2 for IL-17 in these animals. So to summarize everything that I just basically told you is that alpha is required for TH17 cell development and pathogenicity, both in colitis and in mouse models of multiple sclerosis. We can inhibit ROR-alpha pharmacologically, and this seems to be effective in inhibiting TH17 cells without the same phenotype that you see with gamma modulators. So targeting ROR-alpha may not have the same safety liabilities as targeting ROR-gamma. So with that, I'd like to thank the people in my lab. And so this was obviously taken during COVID. And so I like to put the smiley faces on here because I can assure you everyone's smiling under the masks. But past postdocs, Ran Wang and Amir helped with a lot of this, as well as Sarah Mosher did a lot of, very talented graduate student to a lot of the bioinformatic analysis, as well as my technician, Sean Campbell. Like I mentioned, the Sundred lab helped with a lot of the human T cell work, the Kmenica lab for med chem support, and the Cameron lab for DMPK support. So with that, I'd like to, oops, maybe that's not the right thing. Anyway, I like, thank you for your attention and I'd be happy to take any questions. So Laura, fantastic talk, and I have to apologize for not acknowledging you yesterday when I was talking about IBD. It was okay. I was wondering, you focused a lot on the inflammatory signals and things like that, which makes a lot of sense, but what about the anti-inflammatory signals like IL-10 and things like that? Is there any response to that? Yeah, so that's interesting. It's been shown that alpha drives IL-10 expression in another T helper population called TR1 cells. So we actually see decreased IL-10, which isn't exactly ideal. And so we're trying to figure out how alpha might be regulating IL-10, and yeah, so we're seeing increased Tregs and Tregs secrete IL-10, but we see decreased IL-10 expression. So we have to figure out how effective and functional those Tregs are that we are getting increases of in vivo. That answers your question. Hi, beautiful work. I just, it's more of a comment. When I was at the AEI meeting, Vijay Kuchu was talking about two populations of TH17 cells, and I haven't seen the publication yet, but it really just reminded me a lot of your talk. And you were saying there's one population that circulates between the spleen and the gut, and they're kind of anti-inflammatory, and then the other population circulates to the CNS and back, and they're more pro-inflammatory. So I'm wondering if somehow the signals are getting crossed in your knockout mice, and it'd be something to keep in mind, but I haven't seen the paper yet, so hopefully. Yeah, no, actually, yeah, it's pretty known, well, yes, it's known that they circulate, and there's actually, you can actually differentiate non-pathogenic TH17 cells in culture versus pathogenic TH17 cells. And we've done all that genetic, we've done all of that with the knockouts and the drug treatment, and it affects them all equally. But yes, you're right, I mean, we need to look at how it's affecting the non-pathogenic cells versus those that are apparently activated, which are gonna be driving disease. Very nice work, thank you. Thanks. Really nice talk, so a naive question. You said the benefits, which you beautifully showed, is that RR-alpha may not be affecting your thymus. So have you done any long-term, because you saw a TNF show up in the long-term efficacy study, so have you done a long-term with the RR-alpha agonist? No, we haven't done that, and it's something that we definitely need to do, but unfortunately, on the priority list, that was not one of them. But I mean, that's, when we do talk to drug companies, that's one of the things that they're asking. Have you done long-term treatment? Not yet. If you pay me money, I'll be happy to do it, so, yeah. Thank you, nice work. Thank you. Any other questions? Thank you. Our next speaker is Dr. Hendrik Kraus from University of Toronto. He's gonna talk about new gut-derived lipids to modulate all the 48 nuclear receptor. I'm excited to hear all about it. Hendrik? Thank you very much. I should start by thanking the organizers, you guys, for giving me the opportunity to talk here. And I'd also like to start with a small apology for detouring you a little bit away from the gut for a moment at the beginning. This project actually started as an attempt to find new nuclear receptor-targeted signals that could affect signaling between the gut and the brain. And so, the first ligand that I'm gonna tell you about that we found was found with brain. But the approach we're using applies to all of them. And in the future, we're heading mostly down microbiome route where the money that we seem to be getting access to is present. So, nothing to say. Why the gut and brain? I think everybody knows that there's a lot of signaling that goes on back and forth. We know quite a bit about that, but I think there's still a lot more to learn. Nuclear receptors and their hormonal-like lipids can play a major role that is yet to be discovered. There are highly understudied areas of nuclear receptor activity historically. And yet, basically, almost all of the receptors are present in the brain and around the gut. The brain has very different metabolism, uses very different energy sources, has full of very different lipids. So, lots of opportunities for finding different kinds of ligands and roles. And then the gut, I think we all know now, is full of trillions of different microbes that produce hundreds of thousands of metabolites, many of which are small polar lipids that have excellent potential to be nuclear receptor ligands. Okay, so this is the approach that we developed to find new ligands. It's deceptively very simple. Basically, we're using His-tagged nuclear receptor ligand binding domains. We make extracts from various different tissues, the papillic extracts. Part of the trick is making them in a way that we can actually use them and they're stable. And then, let's see, we take those extracts and we simply mix them with a His-tagged ligand binding domain of one of the nuclear receptors that is in a lysate, an E. coli lysate that was used to induce it. So, it's a very messy mixture. We do the incubation for a short period of time and then using nickel beads, we fish out only the molecules that are tightly bound and we extract them with methanol and do MS to find what they are. So, there were a lot of finesses that had to go into this. Our collaborator, Hui Peng, was instrumental in this. He's a chemist, a small molecule mass spectrometry person. And basically, with my post-doc, they worked out how to do this in 96-well plates in a fairly efficient process. So, essentially, here's what we do. We take the ligand binding domains of each of the 48 human nuclear receptors, put in a 6S-tag, and we did a trick devised by the Lesburg lab for PXR. They used the LXXLL motif of SRC1 to help stabilize PXR, which was giving them solubility problems. And we found that for about a third of the receptors that we were expressing, they did have solubility issues. And those are the 16 or so on the end there. So, here's an example of what that does. This is for PPAR-beta. This is without the peptide, and then this is with the peptide. Increasing amounts of known ligand and effects on thermostability. And so, you can see the peptide made a nice big change in the thermal stability half-life. And importantly, the more stable protein is still very receptive to ligand, perhaps even more so. So, here's a proof of concept. I'll show you a couple of these before diving into the data. This is the PPAR-gamma receptor. And into this, we've tossed in nanomolar amounts of rosiglitazone, a known high-affinity target. And you can see that after the pull-down, we get a very nice enrichment out of the metabolites over 10 to the fourth fold. And we also do these in competition with another nuclear receptor that's related. And you can see that on that scale, it's also highly selected. This is another similar experiment where we basically took a pool of known ligands for each of these receptors down here. We mixed them all up, put them in our extracts, and then pulled them down one by one with different receptors. And you can see that they were all very selective. For example, PPAR-gamma was the only one that could pull down ROSI, PPAR-alpha, this GW compound, FXR. I can't read it from here. Basically, anywhere you see more than one line, those are known cross-reactions. So exactly what you would expect. So this is working quite nicely. Okay, so we've tried, this is very early days for us, we've tried six TAG receptors. You can see here on the outside here. We've pulled out 14 highly selective masses that you can see here. A lot of these are really interesting. Most of them are in early days of being characterized. One day, I wanna talk about these CAR ligands. Basically, they're all related to one another. And if you look closely, you can see each one is about 14 Daltons, bigger than the other one. But today, these stories aren't ready for prime time. I'm gonna talk about three different PPAR ligands that we found. One from brain and the others from human gut and mouse gut. And those are identified there. So, first with the brain. We started with brain and PPAR-alpha because PPAR-alpha is expressed throughout the brain, all brain neurons, essentially. And it's been shown to play roles in neuroprotective activity and positive activity. Possibly with neuronal function. So, this shows when we do our pull-down with PPAR-alpha. We started with hippocampus and then we went to cortex and we get more or less a single mass. And there are quite a few PPAR-alpha previously published ligands in brain. And in our pull-down, we didn't get any of those. We found some of those in other tissues, but not in the brain. We only got this one mass. What is it? Turns out it's a modified hydroxyl form of DHA, which is one of the most abundant omega-3 fatty acids and forms much of the membranes of neurons in the brain and so on. But this is a hydroxyl version of it. And Zhabao was able to show that the hydroxyl was at the seven position in this molecule. Basically, if you look at these two metabolites, one with the hydroxyl and one without in the MS2 ionized form, you can see them over here. I think one's 113 and one's 141. And this is what we pulled down and this is purchased seven HDHA. So that was nicely validated. This is roughly what it looks like compared to DHA. As you can see, it's got the omega-3 at this third position here. That's how they're defined. And 7-HDHA has the OH at the seven position relative to the carboxyl. And I just reminded you some points about DHA. It's been applied not just in having structural roles, but also having roles in cognition. It's put in formula that's fed to babies. It's thought to have brain-developing and functional assays. But the mechanisms are still very much up in the air. Okay. So this slide basically shows the relative affinities for the different PPARs. It can bind to PPAR gamma, but at least a tenfold higher or lower affinity. And we think these levels are probably too low to act through PPAR gamma. And it doesn't bind to delta-beta at all. And the KD, the binding affinity, is roughly around one micromolar. But the hydroxyl in the middle position with a double bond allows different isoforms, stereo isoforms of this molecule to exist. And that can make a big difference in terms of which way the hydroxyl group is pointing relative to the rest of the chain. And so with help from one of Carolyn Cummins' colleagues, who's a structural or organic chemist at York University, and he made us pure forms of the R and S isoforms of 7-HDHA, and we tested these, and we found it's the 7S form, which seems to have by far the highest affinity. And it was the S form which we were pulling down with our receptor from the brain extracts. And so we wanted to know what kind of difference this might make, so we aided, got the help of a structural proteomics guy at U of T, Janati Pota, and he basically did some predictive programming, and his data suggested that this hydroxyl would be interacting with the cysteine-276 in the pocket. So to test this, my postdoc mutated cysteine-276 to alanine, and then he also did the same for an adjacent cysteine, 275 to alanine, and then looked to see what they did to various activities of the protein. And I apologize here, I think the numbers got shifted, but basically this is wild-type unmutated, and you can see that the orangey curve is when you add 7S DHA, basically this is a melting curve, and that stabilizes, or increases the stability of the LBD. And the same was true when the 275, that's 275, the control cysteine was mutated, it didn't affect the thermal stability, and it could still respond to the ligand. But when this cysteine was changed to an alanine, it was basically, there was no longer any response to 7S DHA. And then we went to another colleague, available to us in the Donnelly Center where I work at U of T. This is Samir, a graduate student in Lilliana Adesano's lab, and they were able to take brain cortical neurons and make primary neurons with these, and then treat them with our 7H DHA. And at very low concentration, they were able to see pretty dramatic effects, very dramatic effects. So what you can kind of see is that these neurons usually have one long neurite, probably an axon, and usually only one neurite, that's a dendrite. But when you add the ligand, you now typically get about four different dendrites, and they tend to get longer than the normal dendrite. We got a similar effect with the GW control, and then no effect with the antagonist. And all of these were dependent on PPAR alpha. If you knock it out, those effects go away. And if you kind of look over here, if you look at the dark shading, basically these are numbers of neurons with more than three to four dendrites. And you can kind of see here that the 7H DHA had the strongest effect, even compared to the positive GW control. And all of these effects are gone away with SI knockout. And then over here, we're looking at a number of target genes, PPAR alpha target genes, that are known to be involved in synaptic plasticity regulation. And all of those were upregulated in a PPAR alpha dependent fashion. So we think that this molecule is a good candidate for treating diseases that have to do with memory or cognition. And maybe one of the active, if not the active, components of DHA, which itself is a very low affinity agonist for PPAR alpha, and mainly stuck in membranes. Okay, so quickly, basically we've found that this is a new ligand for PPAR alpha found in the brain. It's the only one that we could find using this pull-down approach. It has much higher affinity than any other published natural ligand for PPAR alpha that's out there. And it seems to be a strong potentiator of neuron, brain neuron, arboration, and potentially plasticity. And in terms of where it comes from, it can be made enzymatically from DHA. There are enzymes in the brain that can do that. We also think it's possible that it can come from the diet. It's highly abundant in omega-3 supplements, and it's really abundant in fish, especially fish brains and eyes. So now on to the microbiome. So again, we're starting with PPAR alpha here. And in this initial case, we used a mixture of human microbes that were pooled together to basically comprise the most abundant microbes in the human gut. And we did our first pull-downs with that. And the first thing we got was a derivative of stearic acid. And again, it was hydroxylated. And we did, again, MS to figure out where the hydroxyl group was. And it turns out it was at the 10 position. So here is the previous molecule that we were working with. Obviously not a saturated fatty acid. 10-HSA is. The hydroxyl group is in a similar place, but not exactly the same place. And we're working out to see what that does to, in terms of specific interaction and stabilizing effects. And I'll also point out here that we subsequently found a related molecule to this in the same pull-downs, 10-oxo, in which the OH is turned into a double bond oxygen. And it turns out that this one is even higher in affinity and activity. So I think all of you know that stearic acid is probably the most abundant lipid in animal fat. So there's plenty of it out there. But these derivatives are unique. Okay, what I wanted to point out here is that these are made in the gut by conversion of oleic acid with an enzyme that's found predominantly in lactobacilli into 10-HSA. And they also have an enzyme that can convert that into 10-oxo-SA. And oleic acid can also be derived indirectly from stearic acid, but stearic acid can produce a lot of other things, many of which are not great. So oleic acid in the diet has been considered good. On the right, we work together with another colleague of mine at U of T, William Navar, and his graduate student, and my post-doc. And basically they looked at the levels of the metabolites that are produced by each of these different strains of lactobacilli when fed oleic acid. And these ones down here are obviously the more interesting ones to us. And especially these two ruteri strains, which are very close cousins to each other, one seems to produce the oxo and the other one not. So they'll be interesting to test in probiotic-type experiments going forward. Here's some work that was done by a student in Carolyn's lab, Chidam Sahin. And basically what you can see here is that here's 10-HSA in a FRET assay binding, displacing a competitor in comparison to previously identified ligands for PPAR. Often you can see that it functions much better than any of these previously discovered ones. And its KD more or less is in the nanomolar range together with the 7S-HDHA. And then the 10-oxo is another 10-fold higher almost. So we are really interested in testing these guys for different effects. And as many of the others here, we're kind of gravitating towards IBD in terms of what these might do. So we naively thought that if we looked at patients with IBD, that if these microbes are producing these helpful ligands that would act positively on PPAR-alpha, which has been shown to have anti-inflammatory properties, that we might see in people with IBD that if we looked at their stool samples, that there would be lower levels there. And so that would have led to the increased inflammation. But instead we found that the healthy individuals had very tightly clustered levels, not very high, of 10-HSA and 10-oxo. And the patients that were unhealthy, their levels were all over the map, most cases higher. So this is pretty preliminary stuff. Right now we're working under the assumption that what's happening here is that these guys actually are not getting absorbed into the intestines and not getting to PPAR-alpha. And PPAR-alpha is therefore not having a positive effect. And that could lead to a big positive-negative feed loop pathway. And so we're currently looking at biopsies from these same patients to see what the levels of the metabolites are and the PPAR-alpha target gene expression levels. So fortunately I have to leave that. That's where, it's very preliminary data. So just to summarize all this. So we've gotten some, oops, new unusually high affinity inactive ligands that are produced by microbes in the gut for PPAR-alpha. These two new ones are even better than 7-HDSA. And we think that potentially as natural products, either the metabolites or the microbes themselves could be used to deal with IBD-type issues and disorders. And yeah, finish there. I'll just say that about 80% of what I showed you was done by an amazing post-doc in the lab, Zhai Baolu. He also made a major role in recruiting a lot of the rest of the people that have helped out. Don't look at me. Hui is our MS chemist who's really been super important, super helpful, and Carolyn and her people have done almost all of the cell culture experiments, the transfections, and some of the mouse work. And Liliana's lab did the mouse cortical neuron stuff. And yeah, we're starting to look at IBD together with Dr. Sridhar Mani's group at Einstein. So I'll stop there. Thank you. I think I have 48 questions, but I'll ask only one. Something that has always puzzled me is the PC binding to PPR alpha from liver that was described some time ago in cells, you probably know, right? So do you see any evidence for that? Because that seemed at the time like it was the best example of, you know, technologies like this, and it gave such an unexpected result, and it seems like it's an outlier. So what can you say about endogenous ligands or gamma, especially in the liver, or anywhere, actually? So in terms of our pulldowns with alpha, we never saw it with any tissue. So I would think that either the affinity is not sufficiently high or relative concentration is not sufficient. Specific question is whether the PC species would actually be endogenous. Yes. We looked at almost everything that's been shown to bind previously, and they're all there more abundant than the 7-HDHA. The only thing we pulled out of any other tissue that's anywhere in the ballpark is 13 HODE, and I think 9, but 9 tends to be a very poor agonist. But everything else is orders of magnitude worse. I mean, one of the issues I think here, you know, that people have been batting around a lot are what are the levels of these things in tissues, and I think maybe we should be more sophisticated about that because there are transporters and fatty acid binding proteins and things that can really change the subcellular concentrations of these molecules in specific cell types and so on. So I think looking at an overall tissue and the overall levels of abundance of a metabolite is a pretty crude way of assessing whether or not they have potential to be active. That was very interesting. I really, really enjoyed that. So actually I have two questions. So with the IBD, were all the patients being treated or were they not? I'm just wondering if part of the reason that you're seeing such a diverse outcome is because of, you know, different treatments which may or may not be affecting the microbiome themselves. And then I'm wondering if you can maybe correlate which treatment to, you know, which dot would be helpful. Yeah, that's a great question. I know that a lot of these patients are in various different stages of the disease. Some of them are even in remission and some have different severity. Some have been treated and others not or with different things. So we're starting to try to see if there's any correlation between any of that, and there might be. But, you know, that's a good question and a complicated process to unravel. Yeah, unfortunately, yes, that's the case. And so my other question was you did these pull-outs with the SRC peptide bound to? Am I understanding that correctly? The SRC peptide with the LBD? Yeah. Do you think that if you would change that peptide, it might have, like, any profound effects on the different metabolites that you're pulling out? Yeah, it's something that we wondered about. What I can say so far, I mean, it seems to have a very general effect. And with all of the different fusions that we've tried, they seem to still bound the known ligands that normally interact and nothing new. So it doesn't seem to be having an allosteric effect on the pocket availability, but it is a possibility that it could affect something. And there are some molecules that also interact with that surface that could influence things. So it's something that we're keeping in mind. Okay, thank you. Really fascinating talk. I really enjoyed it. So I was thinking two things went ding, ding in my head. As an Indian, we eat a lot of yogurt, so lactobacillus, right? So is there a way you can compare? I know I'm going off of Laura's question, like the diet or something of these patients. And also if you have access to patients who have antibiotic treatments, which seems to be, like, the norm, then, you know, look at these. Yeah, I think there was—I read something about antibiotic treatment with these patients. I think, if I remember correctly, that they were not being actively treated at the point of these samples being obtained. But, yeah, good question. And, yeah, I don't know what they're eating, if they're all eating the same thing or different things. They're eating yogurt. It's hard to control. Yeah, they should be eating more yogurt. I would advocate—sorry. I have one quick question. Your neurite outgrowth experiments, did you use—I didn't see DHA on your slide. Did you try DHA? Did we do DHA? I think we did go back. We didn't initially. And I think we did go back and did try and didn't see anything. One thing we did do was treat DHA to rats for a month or more, and the levels of 7-DHA went up substantially. But when we treat DHA with the protein in cells or in vitro, the binding affinity is so low that we never really saw anything. Okay. I was thinking for your—if it had worked, you know, lipoxygenase inhibitors and alpha-K-lox to figure out whether you're generating it. It could be converted to, yeah. Thank you very much. Thanks to all our speakers today. This was a really excellent session. Thank you.

ROR-alpha

TH17 cells

therapeutic target

inflammatory bowel disease

IL-17 expression

ROR-gamma

colitis

pro-inflammatory cytokine

phenotype stability

nuclear receptor ligands

gut-brain signaling

PPAR-alpha