I'm from the University of Kansas Medical Center. Today's co-chair is Dr. Ayako Suzuki, she's from Duke University. Welcome to today's session, organized by Hepatotoxin is Sick. So this is on the microbiome drug-induced liver injury. We have four excellent speakers lined up. I'm going to take advantage and get started. Took a couple of minutes. Thank you for the organizer for the invitation. Give me a chance to share my findings here. And the original title was assigned by Wenxin to me. And I actually added the second part. Well, hopefully this slide will be up. Again, I'm supposed to be the third one. Last name, Wan, W-A-N. All right. The second part of my talk is, here we go, leading drug discovery for HCC treatment. So I'm going to show you unpublished findings. Here is my disclosure. My lab has a couple patent application for using miRNA, miR22, and its inducer to treat a metabolic disease, including cancer. And a few years ago, I proposed that this regulated bioassay synthesis is a common etiological factor for liver cancer development. We know HCC is caused by diet, viral infection, and or alcohol drinking. However, we also know that elevated toxic bioassay found in obese people. I will show you detail later on. For example, deoxycholic acid, the secondary bioassay, are found in obese people's feces. And for viral infection, bioassay promote HCV replication. And HPV viral gene expression serum bioassay level actually can predict the severity of HCC and the success of interferon alpha treatment. When we drink alcohol, alcohol increase the synthesis of toxic bioassay. And alpha toxic B1 induce cholestasis. And a bioassay homeostasis is definitely disrupted in autoimmune disease, like PBC and PSC. So we are interested to find out what kind of bioassay and its associated gut microbiota actually contribute to the disease development, in part because gut microbiome generate a bioassay, mainly the secondary bioassay in the gut. We took a simple, straightforward approach. We used the Western diet and the FXR knockout mice. FXR is the bioassay receptor to study metabolic liver disease development. So when we give mice Western diet, they only induce the statosis from five months, 10 months, and up to 15 months. Definitely, the liver become very large. When we use FXR knockout mice generated by Frank and Zala's lab, it spontaneously develop statosis. And even they were on healthy control diet. And then as time goes on, it develop NASH, massive lymphocyte infiltration, eventually carcinogenesis. This has been revealed by quite a few lab, including Wendell Huang and Grace Guo's lab. And if we give FXR knockout mice, Western diet definitely statosis very apparent, massive lymphocyte infiltration, and the carcinogenesis visibly grows grossly. All right. And for female, they are definitely protected from the picture, just like a human. So we profile the bioassay as well as the gut microbiome for all those 24 experimental groups. And I will show you some sample data for 10-month-old mice. Just remember, the fourth group, which Western diet fed FXR knockout mice, has the most severe NASH. So when animal had a NASH, they had elevated beta-muricolic acid, which is the FXR antagonist, elevated the DCA, TLCA, HDCA. They also have elevated the ferrobacteria C, helicobacteria C, the sulfur fibrinol C. And you see the sexy fat is very apparent. And the female, even wild-type mice, they don't have the sulfur fibrinol C. And we also found out there's a group of bacteria whose abundance actually reduced. And when animal had a NASH, for example, erythropyelitric C and lactinospiracy, again, the sexy fat is apparent. Female mice tend to have more lactinospiracy. So we asked a question, is the statosis all the same? Western diet induces statosis. FXR knockout mice, even they were on controlled healthy diet, they also induced statosis. This is at a very early time, five months old. And so histologically, you don't see the difference. But we know FXR knockout mice, they are cancer-prone. They eventually will cause cancer spontaneously. So we would like to know at this earlier stage whether the gut microbiome are the same. The answer is no. And so the green are Western diet induced statosis. The brown were healthy diet fed FXR knockout mice. And those are the male, female. So you can use gut microbiome to predict which group of animal were actually cancer-prone. So what are the difference? The difference is mainly helicobacteria C, highly abundantly found in FXR knockout mice. Under this family, H. pylori is a risk factor for gastric cancer. The sulfofibrin C, highly abundantly found in FXR knockout mice. The sulfofibrin C producing hydrogen sulfide, which is a gut breaker. Erythropylitric C is reduced. And so this is a butary short-chain fatty acid producing bacteria. Another example somewhere here, lactinospiracy is also reduced in FXR knockout mice. And lactinospiracy also produce short-chain fatty acid. So we got interested in short-chain fatty acid. Like the previous speaker mentioned, short-chain fatty acid are produced by bacteria fermentation of fiber. And we all know we should eat our daily fiber. So we quantified one of the short-chain fatty acid producing bacteria gene called a BCOA. BCOA encoded enzyme produce butyric acid. And you're looking at the copy number per nanogram of a fecal DNA. And the BCOA gene copy number reduced by Western dye, reduced by FXR knockout, further reduced by Western dye. And so as butyric concentration in the gut, another bacteria gene we'll quantify is BAIJ. And the BAIJ is the encoded enzyme produced the acyclic acid, the secondary bio acid I mentioned early on. You can find it in obese people. And it's a carcinogen because it break the DNA. DCA increased by Western dye and reached the highest level when animal had a NASH. So the concentration of butyrate is inversely correlated with the concentration of DCA. So it looked like when the short-chain fatty acid producing bacteria expanded, then DCA producing bacteria reduced, it reached a symbiotic manner. So FXR knockout reduced the butyrate producing bacteria. And we know FXR is mainly expressed in the gut, in the liver. And a knockout FXR caused the cancer prong. We also know this FXR knockout animal is human-relevant because FXR expression is reduced in colorectal cancer, so as hepatocellular carcinoma. My lab studied gut-liver axis. So everything we study, we have to validate it in the gut and as well as the liver. So anyhow, you're looking at the normal specimen obtained adjacent to the colorectal cancer specimen. Each dot is one patient and derived from the same patient. So those are all paired, the human specimen. FXR is reduced in, again, CRC, HCC. So as short-chain fatty acid receptor, GPR 4143-109A, and short-chain fatty acid, including valerate, propionate, butyrate, they all have histone deacetylase inhibitory property. They are considered as a nature HDAC inhibitor. 109A is the butyrate-specific receptor. Another signaling we looked into is retinoic acid signaling. Why retinoic acid? We know RXR retinoid X receptor is essential for FXR to function. And so retinoic acid can activate FXR, RXR heterodimer. And retinoic acid and bio-acid actually show many redundant beneficial effects. They both metabolize lipid. They both improve insulin sensitivity. So you're looking at the expression of ALDH1A1, the retinoic acid-producing enzyme, converting retinoaldehyde into retinoic acid in the dendritic cell of the gut. And CYP26A1 is the oxidation enzyme for retinoic acid. It oxidizes retinoic acid into 4-oxo. So the synthesis is reduced so as the oxidation happens again in colon cancer as well as liver cancer. Another reason we looked at ALDH1A1 is it's epigenetically regulated. If there's a histone deacetylase inhibitory property, the ALDH1A1 get induced. So what I tried to tell you is if we eat the daily fiber, it increase your butyric acid production. And the butyric acid, in turn, will boost the retinoic acid signaling. So in consistence with the mouse data, BAIJ is increased, and the BCOA was decreased in the human specimen. So what is the commonality between all those ligands corresponding to those receptors? So we found that the ligand, retinoic acid, short-term fatty acid, which have HDAC inhibitory property. So as the SAHA very knows that the HDAC inhibitor can increase, induce a microRNA called a miR22 when retinoic acid is used in combination with a HDAC inhibitor, miR22 level father induced. And a chino-deoxycholic acid, so as the beta-cholic acid, the FXR agonist, can induce miR22. So all of those chemicals naturally found in the liver and the gut can induce miR22. What is miR22? The downstream of miR22, it inhibit potent diacetylase, including quite a few HDAC, and it's a two-in-so-a-cycling A, and we have published those data before. So we speculated miR22 should be reduced in colon cancer as well as the liver cancer. And indeed, if that's the case, I'm showing you miR22 reduction in human polyps and represent the normal adjacent specimen obtained from the same patient. miR22 reduced in colon cancer, hepatocellular carcinoma. And miR22 level can predict the HCC patient survival. miR22 high patient actually live longer. Again, miR22 silence, HDAC 1, 4, so two-in-cycling A2, we found it in liver cancer cell line, so as the colon cancer cell line. And the downstream of miR22 target correspondingly were elevated in colon rectal cancer as well as HCC. So I think I provided sufficient evidence showing you miR22 is a HCC tumor suppressor. So the next question, can miR22 used to treat HCC? And the answer is yes. And we're showing you miR22 gene therapy over expression miR22 treat HCC and prolong the mouse HCC survival time. And this is demonstrated using orthotopic HCC model generated by sleeping beauty transposal-mediated oncogene expression. This is by Razakati, and this one is a beta-catenin-positive HCC. And so I hope you can come to Ying-Hu's presentation. So the takeaway is very simple. And I made a simple story complicated. You've been known all the time we should eat our daily veggie and fiber, which generates short-term fatty acid, which through histone deacetylase activity, and it will boost the retinoic acid signaling, so as the bio-acid. They work together to activate the FXR, RXR, and they do a lot of things together. But one of them is induction of miR22, and miR22 itself, interestingly, is a histone deacetylase inhibitor. It also regulates tumor immunity found in the T cell. And again, Ying will present those data in her talk. So with that, I want to give the credit to the people who actually did the work. President Janna, Lili Shen, did the FXR work. And I want to thank Frank Gonzalez for his generosity. I want to dedicate our work and this talk to an internationally renowned liver pathologist, Dr. Samuel French, who I have collaborated for more than 30 years. And for the HCC treatment work, credit goes to Ying-Hu. And Sarah Satayashi. and I want to thank my current collaborators, Dr. Xin Liu from University Hawaii, Kit Lan for drug discovery, and definitely physician, and Dr. Samuel French's son, Dr. Samuel Willa French, for our continuous effort in obtaining human specimen to validate animal data. Thank you. Thank you, Dr. Liu. Last presenter is Dr. Christian de Totven from University Hospital, Roth Aachen. First of all, thank you very much for inviting me. So if you want to translate my name, it's familiar with wine, so I think this fits very nicely to this session, and therefore I'm very happy that I can give you the talk today, and I will cover some similar subject as the previous presenter, but I also will cover some other things which are additionally. So we also work with acetaminophen, and we know that it's a poisoning leading to ALF, and remains a condition with rapid progression, poor outcome, and high mortality. So APAP is mineralized into NAPKII-induced hepatocytes, and it uses necrosis. As a consequence, we have an activation of innate immune cells, and we showed some interesting results concerning how to block immune activation, and how to block APAP-induced liver injury. On the other side, we know that this response is especially macrophagous, and play a key role in perpetuating drug-induced liver injury in this situation, and we also know in the clinic that anesthesia in response of the highly effective treatments up to now at later phases may be difficult, and therefore we may have transplantation, which is the only treatment option. So what we used, we used another model than the previous study. We used also a model where we have two different mice. One of them is a wild-type mouse, both in the BLACK6 background, and the other mouse is an IP6 knockout mouse where we know where we have intestinal dysbiosis, and we used these two models in order to understand how translocation and how induction of immune responses between wild-type and IP6 knockout mice may alter liver injury, meaning chronic liver injury, or today I'll talk about our data in APAP-induced liver injury. So when we used these mice, we hypothesized that an IP6 mouse may have an impact on micro-liver injury due to APAP. And what we did is we made a similar model as shown before, that we have two different mice, and this time wild-types and IP6 mice in the age between seven to nine weeks, and they are male mice. We fasted them for 12 hours, and then we induced APAP-induced liver injury by injecting 500 milligrams per kilogram body weight, solved in sodium chloride. And after 12 hours, we checked how liver injury is happening, and this is a basic experimental setup how we performed our experiments. So as shown before, it's obvious that dysbiosis has an impact on APAP-induced liver injury, and in the IP6 knockout mice, you see that there is more necrosis in the liver compared to the wild-type situation, and when you validate this, there's around a one-third higher necrotic area. And this also translates into an increase in transaminases as shown here, increased ALT and also AST, again confirming the results that obviously dysbiosis in the gut microbiome might be essential to trigger liver injury in these animals. So if you look more carefully on immune activation, there's something we are interested, and you see very nicely that there's an increase in CD11B cells 12 hours after induction. And you see that if you monitor this, that specifically one population is increased, lysic A high monocyte-derived macrophages, which are strongly increased in the NIP6 group, indicating that you have a stronger influentary response after APAP-induced liver injury in these mice, and it's also shown here. So we then were interested, of course, on the microbiome, and we were interested and showed two different things. First of all, we find that the groups, as shown before in the MDS plot, that in the wild-type situation, we have a nice colloquialization of these five mice we included, and the same holds true for the NIP6 mice. But interestingly, if you now induce somehow APAP-induced liver injury, you see that in the wild-type group, you have a reduction in dysbiosis, but this is not the case in the knockout animals. And this was surprising for us, but we also confirmed this by Shannon diversity. You see very nicely that the strong difference between the wild-type and the knockout animals is no longer the case after APAP induction. However, of course, the differences are significant before and after APAP induction. So obviously, we have dysbiosis, dysbiosis changes in the wild-type situation, but it doesn't change so strongly in our NIP6 group, because maybe it's a more robust microbiome in this situation, even after inducing APAP. And these are the specific changes we found. We see that here in the control, we have a change in Firmicutes elactospiraceae, as an example. And again, here, we could confirm that in this specific situation, we have this difference between the wild-type animals and the NIP6 animals. We see the no difference after APAP induction in the NIP6 knockout animals, but we see the strong difference after APAP induction. The same holds true for Firmicutes, as shown in the lower panel. So obviously, in the NIP6, we have a very robust microbiome, even after APAP induction, which is not the case in the wild-type situation, suggesting that there is a decrease and a less of bacteria after APAP in the wild-type situation. So what we wanted to confirm is, now, if the stool is really important, and we performed, therefore, fecal microbiota transvirus experiments, as shown here, we did this in a regular path over between four- and six-week odontomels, up to seven to nine weeks. We put, in this situation, stool of the NIP6 knockout animals. The other way around is difficult, because the wild-type Fetius in NIP6 animals doesn't grow very nicely. So you can also only do it in this way, that basically, you select the wild-type animals and transfer into the wild-type animals the NIP6 stool. And then, again, the same set of experiments. After around eight beagle of age, we performed fasting for 12 hours, and then triggered APAP induced liver injury in the same dose, as shown before. And this is a set of experiments. We basically took the wild-type and NIP6 experiment. We performed FMT with either of the two stools. And this is an important group. Basically, the wild-type group transferred this NIP6 stool. And I think this is one which we'll show in a second, which is important to confirm that feedstream is essential to trigger APAP-induced liver injury in this model. And you see here, again, the experiments as shown before. You see the basic situation. You trigger APAP, and you have an increase in necrotic area. And if you now see the situation that you transfer a stool from NIP6 knockout animals into wild-type animals, you again increase in necrotic area, suggesting that the direct transfer of stool from NIP6 animals into wild-type animals increases necrotic area. And here again, the scheme, how we did it. And you also see on transaminases, you have an increase in FMT in the FMT group after transferring the NIP6 stool, which shows dysbiosis. And interestingly, this also goes along with the increase in inflammation. You see here the increase in most high-stress macrophages. And again, you see it's an increase of lysis expression macrophages, showing that there's a strong inflammatory response linked to dysbiosis after APAP induction in these animals. Importantly, we also showed that this is really dependent on the microbiome. You see, if you treat the animals with antibiotics, you can totally reverse the phenotype. You have the same amount of liver damage in wild-type mice. And I have 600 wild mice. You have an increase in transaminases to the same level with antibiotics. And on the other hand, the area of necrotic becomes larger. However, it's no more different between the two strains of animals, suggesting that obviously microbiota are essential to trigger an impact on liver injury after APAP induction. So what are the mechanisms? And here again, I want to summarize our first results. You see that in wild-type mice, you have a gut microbiota and an increase in alpha diversity. You have a decrease in alpha diversity in dysbiosis in NYP6 animals. And what happens is you have an increase in translocation. As a consequence, you can revert the phenotype to one another by NYP6 microbiome transfer, or you can leverage the two aerosols with antibiotics. And therefore, APAP seems to be very important concerning gut microbiota in this model. And at the end, you have an increase in activation of inflammation, as shown here, for Lysic-C glassy monocytes. And therefore, I think this is a very interesting model. So if you think what could be the mechanisms behind it, and there I think we have a very nice paper published of the Gonzalez groups, again, going back to these FXR knockout mice. And if you use FXR knockout mouse, you see that APAP-induced liver injury is increased. So obviously, they are more sensitive for APAP-induced liver injury. And if you look now to reverse a phenotype, you take an FXR agonist together with APAP, and you can somehow rescue the phenotype, suggesting that obviously FXR and FXR agonist might have a potential to reverse liver injury in the APAP model. So this is important also for other models. We know that there were, for example, publication concerning antibiotics. And therefore, I would like to also introduce some data from another model, again, showing that potentially we have a role of FXR and the gut liver axis in liver injury in the acute situation. We know that the cholestatic model is interesting, especially in the MDR-2 transporter, because if you knock out here specifically the MDR-2 transporter, that you don't have any more normal situation of bile. And these animals have dysbiosis of the gut, very similar as shown for PSC patients. And these mice develop biliary inflammation, peripartal fibrosis, cirrhosis, and liver cancer. So obviously a model which is interesting in the chronic situation, but maybe also in the acute situation. And what we did is we treated these animals with antibiotics. So this means you trigger antibiotics, MDR-2 mice, and you think what is happening. And interestingly, when you see in the wild type situation, nothing happens. But in this dysbiotic and disturbed animals, you have a very, very strong acute liver phenotype, suggesting you have an increase in alkaline phosphatase and a very deep increase in transaminase, suggesting that antibiotic in these models has an impact on a very strong impact of liver injury. And if you now look here again on the mechanisms of inflammation, you see that there's an increase in neutrophils, and especially also monocytes of macrophages. Again here, the lysate 6-H macrophages are increased, suggesting that also here again a similar phenotype happens, and you have a triggering of liver injury. And if you now go back to this FXR mechanism concerning bile acids, we all know that CYP7A1 is the target enzyme of bile acid production. You first have this first bile acid, which is C4, and then by B-CYP, the bile acids go into the gut, and they are metabolized and deconjugated. And it's shown here that you have a deconjugation by BSH of bacteria in the gut. And at the end, you have a feedback loop activation of FXR. FXR activates FGF15 in mice and FGF19 in humans. And this is a mechanism which I think is very important for the body concerning uptake, but also very likely concerning liver injury. And if you look now in these animals, what is happening, you see that actually FGF15 is different already in the bile types in MDR2 mice, and it's further decreased because, of course, the BSH-producing bacteria are away. And therefore, you have a downregulation of FGF15. On the other hand, as the feedback loop is changing, you have an activation of CYP7A1 in these animals, suggesting that you have more bile acid production. And this is also shown when you look on bile acids. I simplified it just as a bile acid pool, but also for C4. So obviously, C4 is increased due to CYP7A1 induction, and therefore, the bile acid pool in these animals is increased. So obviously, we have here a situation of bile acid overload, and this then triggers liver injury, as shown in the first slides. So therefore, again here, we use the same FXR agonist to somehow trigger the increase in maybe FGF15, maybe reduction in CYP7A1. And interestingly, we could find that, that actually, if you now trigger here FXR agonism, you have a downregulation of alkaline phosphatase, you have a downregulation of transaminases, again remissance to the APAP-induced liver injury. And also, you see the same on immune activation. You have a reduction on macrophages and also neutrophils, suggesting that the FXR agonist in situation does a good job. And if you look now on the bile acid pool, FGF15 goes up, the bile acid pool goes down, suggesting that there's a reduction in the situation of bile acids. And also, again, here you see a downregulation of CYP7A1 mediated by FGF15. So this means, in summary, I think that the mechanisms in acute liver injury concerning the activation of the bile acid pool is an important sensitizer of liver injury, either in the APAP model, but potentially also with other drugs, like, for example, antibiotics, when dysbiosis is already present. And therefore, I would summarize that, first of all, gut microbiota, as also shown by the previous talk, is involved in controlling APAP-induced liver injury. We know that intestinal dysbiosis increases APAP-induced liver injury, and also bile acids and isoregulation via FXR play a significant role in controlling liver injury. I would suggest also beyond APAP-induced liver injury, and at the end, we should think about if FXR agonists in certain conditions of liver injury might be useful in mice, but potentially also humans. And I would like to thank you and my collaborators who did this work mostly, and thank you for listening. Thank you. Thank you so much. Would you sit here, and the three presenters, could you come up to the stage? We're going to start a discussion. Let's open for discussion. Any question from audience? Hi, good morning. Thank you very much for this fantastic session. I'm Paul Monga from University of Pittsburgh. Good to see familiar faces up there. Just a quick question to Dr. Jiang and Dr. Trottwein. I was just wondering if you guys have ever looked at in your system if FXR analogs or if the PPA had any impact on Nrf2 activation in the liver following your acetaminophen overdose models. So if I would start, Paul, thank you very much for the question, first of all. Very nice. So what you see, of course, is that the FXR agonistic bile acids, due to the reduction in microbiota in the antibiotic mice are strongly reduced. So this means we have a change concerning reduction of BSH, reduction of deconjugation, reduction of FXR agonistic bile acids. And it at the end triggers a downregulation of FGF15 production in the terminal ileum. And therefore, the feedback loop is blocked. So did you look at Lactobacilli species at all? Because there was a study done in Emory where I think they had shown a positive impact of Lactobacilli rhamnosus, I think was the species, which had then activated Nrf2 in the context of acetaminophen overdose and shown very good protection. So I'm just wondering if the species that you are enriching using all of this is... Well, it would be great to see if exactly this species has, for example, high BSH level. And if this is the case, then of course, this is working. We have also bacteria with BSH overexpression, and we see that also those are helpful in protecting from upper induced liver injury. So I think at the end, it will depend on the bacteria, how they express these genes, which at the end might be important in this model. We still have to think that bile acids in humans and mice are different. So this means not necessarily exactly in this situation that this will also help in humans. But at the end, I would assume that at least there could be a role of FXR agonists in this situation. Great. Thank you. For PPA effects, we did the RNA-seq analysis using PPA-treated mouse sample liver tissues before acetaminophen treatment and also after, I believe, six hours after acetaminophen treatment. And we did not see any bile acid-related signaling pathway came out as a hit. So we kind of rule out the bile acid pathways based on that. Also for NRF2 pathways, we specifically looked at in in vitro system whether PPA can modulate the NRF2 signaling pathway or activates NRF2. And we did not see any effects, so we ruled that out also. And my last question is, can you speculate on what regulates CYF2E1? Is it having an impact, PPA having an impact on Wnt signaling pathway at all? Thank you for that question. I think CYF2E1 regulation, it occurs at multiple levels, transcriptional, post-translational levels. I think at least based on literature, there's some evidence that beta-catenin and HNF1-alpha are important transcription factors that's involved in CYF2E1 expression, at least at the mRNA level. Go ahead. Maybe I can have a comment. Paul, I think your question is totally justified. I would speculate, although her data didn't support propionic acid, a lot of short-term fatty acid can tighten the leaky gut. I think anti-oxidation pathway is justified, although maybe experimental data needed. For 2E1, alcohol highly-induced 2E1 is well-known, and alcoholic liver disease has a robust 2E1 induction cause oxidative stress. So I would speculate a gut fermentation maybe induced some lactobacillus, as you mentioned, may have implicated in liver injury, yeah. And in our case, our CYF2E1 upregulation was only at the protein level. So it wasn't at the transcriptional level. We did not see any difference between JAKS and taconic. So we are looking at post-translational regulatory mechanisms. Go ahead. Hi. I have a question in humans. What proportion of gut-derived acetate reaches the liver and taken up by the liver? And obviously, it's not the dominant organ which takes up the acetate. And is there a threshold at which you can see substantial effect on histone acetylation? Sorry, I didn't quite catch the first part. It was how much in humans acetate is actually taken up by the liver? Yeah. So I mean, we look primarily at mice, but I think mechanistically, acetate can be absorbed, but there's definitely a significant decrease as it goes from the luminal level as you pass into the portal level. And mammalian tissues, regardless of mice and humans, can also have acetate production as well. So I think that kind of sort of clouds the picture. Paul Watkins, University of North Carolina. So in the drug-induced liver injury network, we have over 2,500 people now who have experienced rare idiosyncratic liver toxicity. We don't have stool, but we have serum and plasma during the acute event, and most after recovery. And one of the things in our RFA to apply to renew the network very recently, I think it was last Friday, it was due. In the RFA, they talked about potentially examining the role of the gut microbiome in susceptibility. And I know Metabolon says they have several hundred, I think, metabolites they can measure that they believe are largely or exclusively of gut bacteria origin. And I'm wondering, comments you have, I mean, how worthwhile is this pursuing? Can you really infer what the actual species of bacteria are in the colon, or don't you care because it's the effect of the bacteria would be on what they release into the blood that would affect metabolism or inflammation? Comments? I would like to say that's a great idea. If the resources are available, that's probably the study we would like to do. I think looking at metabolites are, if I may say, more important than looking at specific bacteria. One of the reason being, one metabolite can be produced by so many different bacteria, per se. So many people may find certain phenotype correlated or associated with the bacteria A, bacteria B, bacteria C, finding different bacteria at the end, but they may end up making the same metabolites, and it may be the metabolite that has certain biological consequences. So I think at the end, there's a bacteria, they make something, and there's a metabolite which can have a biological effect. So if we can measure the metabolites in those samples, I think that can be very meaningful. So that the actual what's in the blood is more important than actually determining what bacterial species are in the stool, is that correct? That's my two cents. Our experience at least is that you should consider some of the metabolites which are mainly in portal blood, which you don't have, and which you have in the periphery. So at least our experience is that the metabolites in the portal blood are much more indicative than in the peripheral blood. So that's something you should at least consider. Maybe I can add my two cents. You're absolutely right, the number of metabolites we measure by NMR or GC are very limited based on the biomarker or mass you can detect. And my laboratory has recently done metabolomic. We measure the metabolites in serum, in the urine, as well as in the liver, and try to predict the development of metabolic liver, metabolic disease. And we're using AI, artificial intelligence, try to predict. We also integrated gut microbiome data. And even though the number of metabolites we detected is maybe only hundreds, we were able to find metabolites associated with aging, diet intake, as well as FXR knockout. And pretty robust data. And different metabolites from different source can predict a different thing. For example, urine metabolites can predict the reliably predicted diet intake. And the serum metabolites can predict the aging. And surprisingly, fecal microbiome can predict FX knockout. Hopefully the paper will come out soon. Thank you. We have urine as well, and we're open for collaboration. So let us know. We have a question. So I have several questions for the presentation on PPA. So since the mechanism that you propose, that PPA can actually improve acetaminophen-induced liver injury, and supposed by modulating the CP2E1 expression. So I wondered how this compound can induce, can modulate the CP2E1 expression. And also I wonder how in vitro metabolites in this compound can also modulate the acetaminophen intoxication in vitro. Because you know, in the in vitro microsystem, the CP2E1 expression cannot be changed. So there must be two aspects that this PPA can modulate the CP2E1. One is the expression, another is activity. So my question, is there any explanation? Okay, so when it comes to expression, how PPA regulates CP2E1, we are still investigating, focusing on how post-translational modification of CP2E1 may be affected by PPA. So it's still under investigation. We don't have an answer yet. And the second question is activity? Yeah, is the in vitro microsystem incubation system. I also see that PPA can modulate the acetaminophen metabolite in vitro microsystem, if I catch it correctly. We think that happens because there is decreased CP2E1 expression. So that's sort of linked to CP2E1 expression. I know, but that one is directly doing vitro incubation, so, or it is incubation from first from the microsome is isolated from the liver first, or it's from liver first, and then do acetaminophen incubation. Right, right. It's the same liver. Yeah, same liver microsystems. So another question is, because to the story of this acetaminophen presentation will focus on CP2E1 expression. So I wondered whether the other pathway are also modulated, because you know CP2E1 is the first step for acetaminophen to be metabolized into the toxic metabolite, NAPQI, which is quite reactive. So I wonder whether such as PPA or other metabolites can modulate the other process of acetaminophen induced liver injury, such as cell death, inflammation, or whatever downstream of the CP2E1. Well, at this point, we haven't really spent much time looking at the downstream, because upstream seem to have been affected by PPA, but we are looking at oxidative stress markers and other things to basically wrap up the study, but we haven't looked at them yet enough to be able to present the data yet. Okay, thank you. Hello, it's Marta from Weill Cornell, New York, and I have two small questions for Dr. Wang. So you showed that decreases of LDH1A1 and CYP26A1 in HCCs, which is known that the retinoic acid pathway is depleted in HCC. Could you also measure ALD1A2, which is more prominent in liver for the metabolism of retinoic acid? That's the first question. My memory is vague on that one. I would say likely yes. There are quite a few isozymes, and ALDH1A1, probably 1A2 as well, is the key enzyme for retinol to transform into retinoic acid. Thank you. And the other question is if you know if other retinoic acid receptors, in addition to FXR, play a role in these disorders? Retinoic acid receptor play a role in this disorder? Yeah. Again, the retinoic acid receptor, at least maybe at six, we know RAR-beta is a tumor suppressor. It's implicated in head and neck cancer. And I think RAR-beta is directly involved in regulating miR22, and Ying published that. And for RxR, alpha is predominantly expressed in the liver. My lab was one of the first knockout RxR in the liver, showing a lot of play a pivotal role in antibiotic and xenobiotic toxicity, and RxR alpha, again, dimerized with FXR. So therefore, they basically buddies. Yeah. Thank you. Thank you. We have five minutes. Let's keep the question short. Stultz, Los Angeles. I just had a question for Dr. Jiang. I hope I pronounced your name correctly. So it appeared that in the model that you used, there was less acetaminophen-induced injury. And yet, I was surprised to see that the glutathione levels had dropped to the similar amount in both sets. And then the fact that the gamma glutamyl peptide was upregulated. So I was wondering why that was that the glutathione levels were depleted to this similar extent, and yet the injury wasn't as great. I think it's a dose dependent. When we started this work, we first did the preliminary experiment to decide what dose of acetaminophen we should use. And we tried 100, 200, 300, 400 milligram per kilo. And at 200 milligram, we had a, as you know, acetaminophen toxicity shows large individual variability even in animals. So because of the variability, many of our mice showed no toxicity at all. That's why we decided on 300 milligram. And as you can see, the ALT level, it's huge as compared to other people's study. And at that dose, we were able to see the difference between Jackson and Takonik. And because at that dose, it still causes significant toxicity to the animals. I think that's why there's complete GSH depletion in both mice. So if we tweak the dose better, maybe we may be able to see that no depletion in maybe one of the groups. Question for Dr. Jiang. So I was looking at the metabolomics data that you presented and looking at the relative quant nature of it. Have you thought looking at absolute quantitative metabolomics and because absolute data can give you a look at the ability to look at the sums and ratios of metabolites, which have been shown in literature that it's sometimes more important about what is there as much as how they combine together would give us information. Have you considered looking at the sums and ratios of specific metabolites that you found? So what I hear is absolute quantification of different metabolites. Maybe I'm not really following up with the technology. Using other tools, I guess, the absolute quantification is possible for multiple metabolites? Absolutely. Yes, there's a lot of technology that can actually determine the amount of metabolites present down to the micromolar amount. And then using that, you can look at the sums and ratios and figure out if specific metabolites when they're combined together has an effect or sometimes it's a ratio of a metabolite to another. If you may enlighten me, what is the technology called? NMR-based or is it? I'm sorry? What is it used? How is it? There's several companies out there that can do that. Wendong. Okay. Thank you. And I am Wendong Huang from City of Hope Medical Center. So I have a question for Yvonne. Very nice talk. And so in the conclusion, you showed that bioassay can activate MERD-22. So do you know which bioassays can activate this microRNA? The second question is what kind of mechanism? First of all, Wendong, many thanks for your pioneer work so we can follow you. Well, it's mainly FFSA agonist. So the mechanism is receptor dependent. The reason we got into MERD-22 is because we found that this is the top one microRNA got reduced in RxR alpha knockout mice. Then later on, we found that so RxR dimerase with FXR vitamin D receptor, RAR beta. Then we found that activation of each of those pathway induced MERD-22. So MERD-22 can be induced by, again, retinoic acid, vitamin D, which I didn't talk today, bioassay, it's all receptor mediated. It's receptor dependent and require the presence of RxR alpha. Then MERD-22 got induced. Then more interestingly, once MERD-22 is induced, MERD-22 has silenced HDAC, and it go back would activate each of those receptors because all those receptors are epigenetically regulated. I hope I don't confuse anybody. Thank you. Thank you, Wendong. Hi, I'm Rachel. I'm from the University of Tulio. First, I want to say you all four did a really great presentation. I learned a lot, so thank you for that. But my question is more toward Dr. Wayne as well because I study mostly bioassays and FXR with liver injury. I'm curious, did you investigate any effects after APAP toward other nuclear receptors that are usually activated by bioassays like PXR, CAR, VDR, or even the G protein coupler receptor TGR5 because those are usually helping to detoxify bioassays like increasing those enzymes. So have you investigated those pathways as well? Yes, preferably. And you're absolutely right. There are so many bioassay receptors, PXR, VDR, S1, PR2. They are all bioassay receptors. There's a membrane receptor. There's a nuclear receptor. It's very complicated. So a single bioassay probably cannot explain all. They all work together. Some are agonists. Some are antagonists. It's like a microbiome. You have a mixture of gut microbiome, and you have a mixture of bioassays. They go up and down. It's the concentration, and it's the presence of all will determine the outcome. Thank you so much. Thank you. I think we'll close the session. Thanks so much for the speakers, wonderful talks. Thank you for all the audience.

microbiome

liver injury

hepatotoxins

short-chain fatty acids

acetaminophen-induced liver injury

gut microbiome

drug-induced liver injury

dysbiosis

hepatocellular carcinoma