 Great good afternoon, and thanks much Cindy and also many thanks to Lita and the organizing committee for the opportunity to participate in this meeting It really has been fantastic By way of introduction my background is in nutrition So I'm going to take of a more of a nutrition-centric view of our discussion about relationships between the gut microbial community and diet and also before I start I'd also like to acknowledge my colleague Meredith Huller who's a microbial ecologist who's been working with us for a number of years now who really has helped in Our group beginning to think about some of the interactions and the issues with regards to these relationships So from a nutritional standpoint when we think about Health and risk of disease often this is the very broad and general Paradigm is that Aspects of diet are influencing risk of disease and in many cases we focus primarily on a number of chronic diseases that we're faced with From a public health standpoint and we can think about this not only from the standpoint of Fuel availability which certainly in the last Number of years has come to the forefront with our concerns over adiposity and the implications of our increase in obesity in the Western world, but also the aspect of other dietary constituents whether they be Recognized essential nutrients or a whole host of other components of diet that can influence various aspects of disease risk in the context of our discussion today then really thinking about the gut microbial community as a Potential modifier of effective diet certainly we've heard a lot of discussion about how various microbes can have direct effects on tissues and on the Human system, but also the potential for the gut microbial community to influence fuel availability and modulation of Signaling that affects energy balance But also the impact of the gut microbes on various other dietary constituents, so I'll really focus on this piece today Certainly, we've also heard in a number of presentations the capacity for diet to directly affect the gut microbial community So there's always that back-and-forth connection So as far as Discussion today like to address a couple of questions in a very broad sense And I think this is really more to bring us back to thinking about the complexity of studying gut microbial community and the various other aspects of exposures in humans or other Animals and how this may impact on the community structure. So first of all, what are the gut microbes doing with our food? What is the effect of the gut microbiome on host dietary exposures particularly in the context of a lot of Population-based studies where we often rely heavily on a food frequency questionnaire to try to identify Dietary exposure, but to what extent is that really Capturing what's going on in vivo and how might this influence disease risk in the Cases where we have some examples of this. So I really certainly this is very broad. It's also Capturing a couple of examples by no means No means comprehensive and many of you can bring up additional examples that we can Discuss to and then I'll finish with gaps needs and challenges So there's no question about it. The human diet is extremely complex Which means when we come to think about it We're not only dealing with exposure to thousands of compounds on a daily basis But also how we handle food whether it be processing in a factory whether it be how we are Preparing a meal in the kitchen, but various aspects of this can affect structure and particle size bioavailability of compounds to the host and With reduced biobility of compounds to the host what you're actually likely seeing is increased bioavailability Bioavailability of substrates to the microbiome I Tend to think about this and with regards to gut microbial metabolism designed to make the most of the situation and in the context of flow of food Through the gastrointestinal tract really first line of handling is through human digestion and often we go back to the old nutrition and Medical textbooks. We're really focusing only on What we can handle in the way of enzymatic reactions that allow for availability of nutrients and energy sources for the human But there are a whole host of components One that could consider the indigestibles that is the dietary fiber. We've heard about resistance starch. That's not easily For men are metabolized by humans as well as a number of other components what we might call the leftovers which are Other bits that we cannot handle either because from a structural standpoint, they are encased in non-digestible Coating as for example in seeds some nuts Depending how well you chew your large chunks of meat availability of protein all these things that then become part of the bacterial substrate mix and really a lot of the Reactions that we come to consider in relation to gut microbial metabolism are designed to handle these fermentation. We've heard about Reduction of nitrate and sulfates Stereofication aromatic fission, etc. That come into play When we look at this in relation to the Contribution of genes to That are related to metabolic pathways good majority of these are focused on carbohydrate and amino acid metabolism as well as xenobiotic degradation which Pete talked a little bit about in relation will quite a bit in relation to drugs But when we think about xenobiotics from a dietary standpoint We're also talking about a whole host of other compounds as such as many of the plant compounds that for from a human Standpoint we also view as xenobiotic But are also major substrates for gut microbes as well as many Pyrolysis products that come with how we prepare food by cooking So we've heard a lot already About production of short chain fatty acids in relation to carbohydrate fermentation So I'm really not going to spend much time on this beyond to just remind ourselves that Acetate-appropriate and butyrate besides serving as Energy sources both for the colonic epithelium in the case of butyrate as well as capacity for lipogenesis and gluconeogenesis For acetate and appropriate But the also the importance of these compounds as signaling molecules butyrate acting as a histone deacetylase inhibitor in the colonic epithelium and therefore having effects on epigenetic modulation of apoptosis and proliferation as well as signaling Down further systemically as we heard From Gary and potential to have even an impact in the lungs So I think this is an area that of that will continue to hear a lot about Looking at microbial metabolism of proteins and amino acids again. This is a range of different types of Compounds from the standpoint of substrates and it really depending on the type of amino acid one is generating a variety of different Products the or the bacteria are certainly in the context of the aromatic amino acids a range of phenols and indols Sulphur amino acids produce a whole host of different sulfur compounds. I'll talk a little bit more about that in a minute and The rest of the other amino acids can be deaminated or decarboxylated to contribute to ammonia production Amines as well as can also be Deaminated and fermented Additionally to produce short chain fatty acids and in the case of the branch chain amino acids to produce branch chain Short-chain branch chain mean branch chain fatty acids, excuse me So by way of example in the context of the aromatic Amino acid metabolism l tryptophan can be converted to indole and pyruvate and poster number 18 has also another nice example of an additional conversion of tryptophan to Indole pyruvate which is of concern in relation to Metabolite that's been identified with autism if you're interested in some more metabolic pieces of that But in the case of production of indole has received some attention both from the standpoint that it's Present in both the human and rodent gut lumen at low millimolar concentrations and has been shown to modulate expression of both pro and anti-inflammatory genes typically showing increases in the anti-inflammatory genes and down regulation of the pro inflammatory genes as well as strengthening some of the epithelial cell Barrier and decreasing pathogen colonization. So I think I think what this is just one example, but it really points out The importance of so many of these small molecules many of which we probably don't even begin to understand their potential to To impact on the host Another amino acid Product of amino acid metabolism in the case of sulfur amino acids is generation of hydrogen sulfide This has been shown to be toxic to colonocytes both an in vitro and in vivo models and can contribute to inflammation and therefore is of concern in relation to ulcerative colitis and colon cancer and From the standpoint of handling by a gut bacteria Hydrogen sulfide can be generated both by fermentation of sulfur-containing amino acids, but also the action of the Sulfate reducing bacteria on inorganic sulfate. So sulfate and sulfite many of which are used as preservatives in food and also just present In foods in general. So again another source there Just by way of an example people have been saying today. That's often really hard to do studies in humans But one can do dietary interventions and although we aren't necessarily looking at who gets ulcerative colitis at the end This is an example of a controlled feeding study in which case Volunteers were sequestered in a metabolic ward Participated in five different treatments of Ten days each of meat Supplementation and in a controlled diet and they were looking at fecal sulfide concentrations as they also looked at urinary sulfate excretion In relation to these treatments and as you can see as you increase the amount of meat in the diet You see increases in fecal sulfide concentration. So the human studies such as these are small But they can often add important Connecting bits of information in relation to the impact of these compounds in human health and exposure to them Another story that's received quite a bit of attention and last couple of years is the production of Trimethylamine oxide which Comes from the bacterial conversion of choline to trimethylamine Choline is derived from phosphatidylcholine or lethacin that is a good part of the diet particularly if you include Meat products, and you've been eating the hard-boiled eggs on the last couple of mornings at breakfast you've been receiving your phosphatidylcholine challenge and Choline is released upon lipolysis in the gut it undergoes metabolism by gut microbes to trimethylamine is absorbed further metabolized by Hepatic-flavine mono oxygenases and it's converted to trimethylamine oxide, which has been shown in animal models to be atherosclerotic and I'll show you a little data in a minute about potential risk in humans Study a lot of work has been done in animal models already, but I'm in the context of again a human study with dosing of 40 individuals with This phosphatidylcholine challenge both as an eggs as egg source as well as a Deuterated phosphatidylcholine they could show you see increases in TMAO as a result of the intervention Provide a broad-sprec antibiotic could show decreases as a result and then a reappearance of the TMAO upon Withdrawal of the adiabiotic again showing the the relationship between Bacterial production of the trimethylamine and subsequent TMAO as part of this Study they also evaluated prospectively The relationship between plasma TMAO concentrations in 4,000 individuals who were then followed for three years for cardiovascular events and showed significant differences among quartiles of Plasma TMAO concentrations in relation to risk of myocardial infarction stroke and death With certainly the highest risk among those individuals in the highest quartile again showing this relationship of production of a Plasma component in relation to disease outcome so the other piece of Metabolism that is Little near and dear to my heart is in the area of phytochemicals and there's an estimate We are exposed to some 20,000 different phytochemicals and over the span of about 70 different plant foods and many of these compounds are also handled by gut microbes Certainly a lot of the phenolics terpenoids which include the carotenoids various sulfur containing compounds and the Glucosinolates and indoles also so what I'll do in the next few minutes is Focus as a by way of example on the glucosinolates in the isoflavones But certainly there are many other examples of the impact of gut microbes on exposure to these compounds into their metabolites First example is in relation to production of isothiocyanates from glucosinolates in the area of Cancer prevention cruciferous vegetable that is the broccoli containing our broccoli family cabbage family veggies are Major source of these in the human diet and as they exist in the plants. They are have a glucose moiety bound to them and aptly named glucosinolates and These are not bioactive in this form, but their Release of the glucose moiety to production of the isothiocyanate is production of the bioactive component that's been Shown to be important with regards to reduce carcinogenesis and animal models as well as associated with lower risk of several common cancers in human epidemiologic studies now as As the compound exists there is a enzyme in Cruciferous vegetables that can cleave this glucose moiety to release the isothiocyanate and the thought is there that the primary reason the plant does this is in the face of A threat or not a threat but having been chomped on or chewed that it actually releases the isothiocyanate which has a bitter and pungent Feel in the mouth and will cause the insect or animal to stop consuming it In the in most cases in humans We actually don't consume cruciferous vegetables raw and that means that the myrosinase is denatured as a result of cooking But our gut bacteria Many of which contain thioglucosidases can also carry out this reaction and allow for release of isothiocyanates and I think this is a nice example of the difference in exposure to isothiocyanates Across a group of individuals. This is work of Tom Kensler's from a number of years ago But those they were conducting an intervention trial with glucosinolate supplement Glucoraphane in which is the glucosinolate precursor of sulforaphane and We're looking at the impact on reducing aflatox and DNA addyx in a population in China As you can see here the urinary isothiocyanate recovery Ranged from 1 to 45 percent of the dose administered when they measured The metabolites in urine Suggesting that the availability of these compounds varies considerably across individuals in this as you increase Recovery in the urine you see a decrease in DNA addict Formation suggesting that those exposed to more may be benefited more from the impact of the isothiocyanate We were interested in whether to what extent we could understand the Relationship between these urinary measures of phenotype as far as recovery of isothiocyanates in relation to gut microbial activity and Faily in our group recruited a group of individuals challenged them with a dose of cooked broccoli so ie glucosinolate rich, but know my rosinase to cleave the isothiocyanate and Could recover between 1 to 28 percent of the isothiocyanate in urine you can see there's quite a range in and the distribution And then took the high and low urinary ITC excretors and incubated fecal bacteria from their set of stool samples with gluoraphanin and looked at rate of decrease in relation of gluoraphanin in relation to being either a high excreter in yellow or a low excreter in blue and Suggesting that those individuals who are Excreting more isothiocyanates are typically more likely to break down the gluoraphanin Which allows for release and absorption? Another example of Phytochemicals that are heavily Metabolized by gut microbes are the soy isoflavones These have been of interest for a number of decades now in relation to their weak estrogenic properties Capacited bind to the estrogen receptors both alpha and beta and elicit either weak estrogenic responses or to Have anti estrogenic effects in the context of this story The soy isoflavone dietzen is handled by gut microbes to convert to major metabolites Equal anodezmethylene-golensin or ODMA what we find in most populations that Most individuals have gut microbes that can carry out this reaction and if you measure urinary ODMA levels in a Population 80 to 90 percent of individuals will have substantial ODMA excretion and Whereas the equal Really varies one across populations and certainly is a lot lower in production And it's not necessarily that either you're an ODMA producer or an equal producer typically ODMA producers can be equal producers or vice versa This just shows that if you give individuals a soy challenge Containing dietzen you can see a range of excretion. This is 24-hour urine excretion But this is a tenfold jump between those individuals who really aren't excreting any in this case What we're measuring here is likely what's coming from? Other animal sources of equal coming from milk and meat sources of ruminants compared to What you seek produced by the humans? The reason people have been interested in this is that in a number of the soy intervention studies there had been identified differences in response between those with an equal producing capacity and many of these relate to sex hormone effects either in relation to menstrual cycle or steroid hormone concentrations bone mineral density as well as some differential gene expression that is Estrogen receptor driven in peripheral lymphocytes and then also similarly some observational studies that show differences, but these are certainly not always Shown in all studies there are certainly are in all studies too, but So what gut microbes are producing equal there have been a number of attempts to identify these and there's a number of specific bacteria that have been shown to be able to convert dietzing to equal Listed here but often when you go looking by QPCR for some of these specifically in Human stool samples you don't necessarily find them There are also some suggestions that it may rely Some individuals may rely on a consortium of bacteria some that can carry out the first part of the reaction and some that carry out The second so that I think this sort of is an indication of the importance of being able to Come to a more functional approach to be able to characterize some of these activities because We're going to find for many of these reactions that there are a whole host of bacteria that can do this But and they're not necessarily all within the same group So that I think that's an important piece. So just to summarize presentation I think hope I've shown that I'm gut microbial metabolism can modify a variety of different dietary components That the differences in the gut microbial community capacity to handle substrates can be Detected using metabolic phenotypes even on a more gross level of monitoring Urinary metabolites and certainly as we move into using the metabolomics approaches can get a wide range of ways to evaluate this I think one of the things that is always a good reminder is that diet as Consumed not is not necessarily That experienced by the host so from the standpoint of the components The metabolites produced by the bacteria these may be in some cases quite different from those that went into your mouth and lastly I Think the gut microbiome really needs to be considered in the context of the host diet in order for us to best understand its impact on both host metabolism as well as its own and on disease risk in relation to some of the gaps needs and challenges Coming at this from a very Sort of the nutritional angle and it's interesting to see how these are sort of Recapitulating across many of our presentations in the last two days I think one of our big challenges certainly is trying to test the causality of gut microbiome and Contribution to health and disease and certainly in the context of diet and in that regard I think We have a strong need for a number of different approaches to this both from the standpoint of the prospect of cohorts that allow us to follow individuals for extended periods of time with good measure of Exposure whether it be diet or otherwise as well as capacity to sample and characterize the gut microbiome I think there's a place for well-controlled dietary interventions to try to get at some of the relationships for inner individual variation and finding ways to Harness these and make the most of them. I think is important and certainly the model systems are going to be critical As there's so many things that we certainly can't do in humans And lastly on a more broader level I think to Reiterate Vince's comment this morning really trying to facilitate Transdisciplinary research to allow for the breadth and depth of knowledge that we need in this area Not only from a standpoint of a funding situation But certainly from the support of our institutions to be able to allow for this cross fertilization and Big plug for methods for assessing composite functionality of the Got microbial community and being able to integrate the structure and functional relationships and Lastly having the computational methods to integrate Multiple omics platforms, and I think we've heard that one before so lastly just want to Thank the members of my group as well as collaborators at the Hutch and University of Washington Texas A&M University with collaborations Rob Chapkin on Project and long-term collaborations and lastly to thank NCI for the support over the years. Thank you We have time for a few questions Everybody's just ready for dinner Well, if people aren't rushing for questions now, it'll probably give us the opportunity to someone's I do have one question You mentioned lecithin and you were looking at meat and egg sources. I was wondering have you looked at all at soy Lecithin, I know it's very commonly and almost ubiquitously put into processed foods. I Would expect it probably is going to be a similar I mean as Phosphatidylcholine it's going to be similarly Broken down and the choline is going to be available as a substrate for the bacteria So whether or not it's a pure source or whether it's coming from different food products likely to have an effect Well, please join me in showing appreciation for all the speakers in this session And we'll now start the discussion session for this afternoon