Microbiome and Cardiovascular Disease Biomarkers – Stanley Hazen

Microbiome and Cardiovascular Disease Biomarkers – Stanley Hazen


Stanley Hazen:
I’m going to start with the end, and then show you the data that supports the overall
scheme. And that is, what I’m going to try to convince you is that intestinal microbes
play a contributory role to the development of cardiovascular disease. What our group
has found is that diets that were familiar — or food sources that were familiar with
increased risk of cardiovascular disease, such as those abundant in fat and cholesterol,
and illustrated on the left there, are also associated with increased levels of trimethylamine-containing
nutrients, such as phosphatidylcholine or choline, shown in the bottom left, and also
carnitine shown in the mid-left. And what I’m going to show you is that gut flora contain
enzymes that will actually cleave the trimethylamine moiety off of the nutrient generating TMA,
or trimethylamine. This is a gas at body temperatures. We have within our livers a cluster of enzymes
called flavin monooxygenases, which are capable of then very efficiently oxidizing TMA into
what we, or the field, used to think of as purely a nitrogenous waste product, TMAO,
or trimethylamine oxide, but what I hope to show you is that this actual compound has
biological activity, and can actually alter cholesterol and sterile metabolism at multiple
different compartments. And not only is it associated with cardiovascular disease outcomes
in humans, but in animal models, will actually facilitate or accelerate the development of
atherosclerosis, suggesting that gut flora, through this axis, can actually perturb the
development of cardiovascular disease. So there are also a couple other take-home
messages that I’d like to try and discuss today, and that is, the first is that if you
think about things coming from either genetics or environment, how we perceive that environment
is influenced through the filters that are there. And our largest environmental exposure
is what we eat. We literally consume kilogram quantities of foreign material, if you think
of food as a foreign object, and it is through this filter of the intestinal microbiome and
if you take different compositions, and you’re making slighting different metabolites at
different levels from one person to another, the filters can actually influence phenotype
in that way. The second thing that I’d like to also suggest
is that we should think of the intestinal microbiome as an endocrine organ. An endocrine
organ is, of course, something that makes a hormone, and if a hormone, by definition,
is a biologically-active compound that will diffuse in the circulation and act at a distant
site, I’m going to give you examples of how compounds are being generated in the intestines,
and diffusing and then acting at the site of, let’s say, a macrophage in the artery
wall, or hepatocyte in the liver, or an enterocyte in the intestine, and influencing the biology.
So it’s a quite plastic organ, to be sure, and it changes depending upon what the chronic
dietary exposure is, and what the acute dietary intake is, but nonetheless, it’s functioning
like an endocrine organ. And then lastly, while I’m not going to show
you data on this, I think what is exciting about this whole field is that this is a drugable
target. If we start to understand the chemical Braille of the different pathways of what
the microbes are generating and how they’re biologically active, then these becomes targets
for pharmacologic intervention through inhibitors or agonists, not an antibiotic but an inhibitor.
And so perhaps in our future we’ll look at our medicine cabinet and see, let’s say, a
statin, and say, oh, that’s our homo sapien enzyme inhibitor for blocking cholesterol
synthesis, and right next to it will be a tablet for blocking a specific enzyme in bacteria
that doesn’t kill it but actually prevents the formation of compounds that might be linked
to cardiometabolic diseases, or obesity, or whatever, or maybe even our behavior, as we
just recently heard. Okay, so how did we actually get into this?
I am not a microbiologist, and I am not — actually, my history has not been working on a microbiome.
Instead, I’m actually a chemist and run the mass spectrometry facility at my institution,
and we stated with an unbiased metabolomic screening study. We’re using a cohort for
which I’m the PI, and it’s called GenBank, has over 10,000 subjects for whom we’ve collected
their blood and then followed longitudinally over time. And so from this cohort we identified
a small subset who went on and experienced a heart attack, stroke, and death, in age-
and gender-matched control subjects, and then within each person’s plasma in the small case
control cohort, we asked, can we find small molecules that are associated with cardiovascular
disease risk? So we did this in a learning set, and we did
it in a validation cohort. In the end we came up with a small subset of compounds which
actually seem to reproducibly associate with cardiovascular disease. Well, the surprising
finding was, is that some of these compounds fell into a common pathway, and that was involved
in phosphatidylcholine metabolism. Now, the three main candidates in our first paper were
choline, betaine, which is a oxidation product of choline, and then this small molecule that
I call TMAO, or trimethylamine N-oxide, which is shown in the structures at the bottom.
And so what this association study suggested is that there at least was an association
between multiple metabolites and phosphatidylcholine metabolism and cardiovascular disease risk. So we first wanted to actually look at mechanistic
studies to see was there a causal link. So, oh, I’m sorry, I’m switching the order of
my slides here. So the first thing we actually wanted to also confirm was, if one looks at
phosphatidylcholine metabolism, the classic pathway that — how all of us digest phosphatidylcholine
is we have lipases that cleave off the fatty acids, and then glycerol phosphatidylcholine
and the fatty acids are absorbed in the intestines. However, there was a suggestion that TMA and
TMAO might be ultimately generated via gut microbes because certain bacteria could cleave
choline in forming TMA. But this really hadn’t been shown, especially for phosphatidylcholine,
to involve intestinal microbes. So we received germ-free animals from Taconic,
and then did the experiment where we took the germ-free animals, and immediately upon
opening the microisolators, gavaged them with isotope-labeled phosphatidylcholine, or egg
yolk phosphatidylcholine, and then monitored, in the plasma, the appearance of the different
isotopologues of the metabolites of phosphatidylcholine, such as choline, betaine, and TMAO. And as
can be seen on the left, what was found is that in the germ-free animals, following ingestion
of phosphatidylcholine, no TMAO was generated or appeared in the plasma over time. But in
contrast, if you the same germ-free animals and now we put them in conventional cages,
and two weeks later they’ve now assumed intestinal microbial communities, they’ve become conventionalized,
and now when we gavage them with phosphatidylcholine, TMAO rapidly appeared in the plasma following
ingestion of the phosphatidylcholine, showing that TMAO was indeed formed in an obligatory
way, or it required the role of gut flora to be generated, or gut microbiota. Now you’ve seen this slide as shown on — I
guess throughout this — just earlier today, but more recently, Wilson Tang in our group
kind of tried to extend these studies to humans. And what we looked at was using egg yolk,
or two hard-boiled eggs, as the dietary source of phosphatidylcholine in the subjects, and
at baseline, we would give them the hardboiled eggs, and then monitor for the appearance
of this metabolite TMAO in the plasma and in the urine We also gave them a capsule of synthetic isotope-labeled
phosphatidylcholine where the end-methyl [spelled phonetically] hydrogens were now deuterons,
so we could trace the — and show for certain the metabolite we were measuring, TMAO, came
from phosphatidylcholine, because there’s many things inside a hardboiled egg besides
phosphatidylcholine. And what we were able to show is that, at baseline, you could readily
detect TMAO of — this is six hours following ingestion of two hardboiled eggs. These are
healthy volunteers who are placed on a cocktail of oral antibiotics, and then came back five
or six days later and repeated the challenge. The formation of the TMAO was virtually completely
eliminated, and so none was detected in both the plasma and in the urine. And then after
cessation of the antibiotics, and going home and coming back a month later, repeated the
challenge, and now you see it again. So this showed an obligatory role for intestinal microbes
in the formation of this metabolite that we’re measuring in the blood. Now, why do we think that’s important? So,
first of all, if we actually measured the metabolite in the plasma of subjects, we saw
that it was strikingly associated with cardiovascular risk. What its shown here is an independent
study of over 1,800 subjects where we have the baseline plasma level, and we’re looking
at cardiovascular disease. This is — the risk is shown on the Y axis, the odds of having
cardiovascular disease after adjustment for traditional risk factors such as those in
the Framingham risk factor or formula: age, gender, diabetes, hypertension, smoking, LDL,
HDL. It also has the addition of other labs that are currently used for evaluating cardiac
risk, such as triglyceride and CRP, and an estimate of renal function. And if you focus
your attention on the center graph, what you can see is there’s a striking distribution
or association between the plasma level of TMAO and the likelihood of risk of having
cardiovascular disease in subjects. So the line in the middle is the odds ratio, and
the dotted lines are the 95 percent confidence interval, and there’s quite a steep association.
And if one were to look at LDL cholesterol, for example, it’d be a much more horizontal
line in this cohort. So this is still just associative data, but
nonetheless it’s compelling because that was independent and above, on top of traditional
risk factors. More recently we’ve extended this to an alternative independent cohort
for whom we’ve followed over time who went on to develop heart attack, stroke, or death.
And what we see is that baseline levels are predicting future risks of myocardial infarction,
stroke, and death. If one looks at the composite of this, of major adverse cardiac events that’s
in the bottom left-hand corner, the forth quartile, so the top 1,000 subjects compared
to the bottom 1,000, the first quartile is the index group for comparison, they have
about a fourfold increase likelihood of experiencing an event in the next three-year period, and
the line represents the 95 percent confidence interval. And shown on the right are Kaplan-Meier
survival plot, showing that your plasma TMAO level is a good independent predictor of prospective
survival. So to take this into a more mechanistic realm,
we started doing animal model studies. And at first, we actually fed mice phosphatidylcholine,
and actually saw that they got accelerated atherosclerosis. But they also gained more
weight, they were getting more calories, and there was the whole issue of the fatty acid
composition; maybe that was contributing to the disease. So we instead started just giving
choline in the diet. Now choline, for animals, for mammals, has no calories. We don’t catabolize
choline and generate calories from it. And we also don’t see changes in either the cholesterol,
or the lipoprotein, or glucose or insulin levels in the animals that are on the choline
diet. But nonetheless what we found is that augmenting
an atherosclerosis-prone animal’s diet with choline led to accelerated atherosclerosis.
So shown in the open bars were the wild-type conventionalized animals on a normal chow
diet on the control side, and the choline represents a high-choline diet, which is approximately
what would be a very high-fat Western diet in terms of its choline content. Shown in
the black bars are animals in which the intestinal microbial community was suppressed by a cocktail
of broad-spectrum antibiotics. The TMAO levels in the plasma, which are shown below in red,
were suppressed to near zero levels throughout the course of the study, which is about a
20-week duration. And what was seen is that the increase in atherosclerotic plaque, here
measured by macrophage, the major cell type in the– in the aorta, but also this has been
done by cholesterol content, or oil red O [spelled phonetically] staining, that the
diet-induced atherosclerosis was inhibited in the animals in which the gut flora was
suppressed and TMAO was not generated. Now importantly, what was also found, and I’m
sorry I don’t have it here, what was also found is that if we fed TMAO directly to the
animals and bypassed the microbes, that alone were sufficient to increase and augment accelerated
atherosclerosis in the mouse model. Now working out the mechanism, we started
looking at a variety of different places, and if — what was found as that the macrophage
was accumulating cholesterol, so we focused on cholesterol metabolism. And so for the
macrophage kind of shown in that ugly cell on the right in purple is a black box, and
you see it’s accumulating cholesterol. You can either have enhanced pathways for delivering
cholesterol into the cell, more cholesterol being synthesized in the cell, or decreased
removal of cholesterol from the cell. You know, increase flux in, decrease flux out,
or more synthesis in the cell. And so we kind of looked at it in this simplified black-box
approach and looked at candidates involved in cholesterol metabolism to try to work out
the details of how is cholesterol accumulating in cells of the artery wall. It turned out to be a little bit of a complex
mechanism, and we still don’t know precisely all of the pathways that are involved, but
what was found was that there was both an enhanced forward cholesterol transport, and
in particular, on the macrophage, there was an upregulation of genes involved in recognition
of modified forms of LDL, such as scavenger receptor SRA1 and CD36. These were effects
of, directly, of TMAO when fed to animals, as well as when cultured with — when incubated
with cultured macrophages. What was also observed is that there were
changes in cholesterol and bile acid metabolism at the level of the hepatocyte and also the
enterocyte. So there’s a substantial reduction in bile acid pool size and composition, changes
in the bile acid transport pathways, such as CYP27A and CYP7A1, and then also changes
in enterocyte cholesterol and bile acid transporters as well. The net effect is shown on the top
right-hand corner. There’s enhanced forward cholesterol transport, and if one actually
directly measured the reverse cholesterol transport pathway using methods that were
first developed by Dan Rader and colleagues at University of Pennsylvania, we saw that
there was actually about a 30 percent reduction in reverse cholesterol transport that was
mediated by either TMAO directly in animals that were fed the TMAO, or in animals that
were fed the precursor choline when they had intact flora. But if you suppressed the flora
and blocked the TMAO formation, you no longer saw the changes in cholesterol and sterol
metabolism, or in bile acid synthesis that was being observed. Now I, so far, kind of showed you data that
was mostly done with choline, but more recently, we’ve actually started expanding these studies
to alternative dietary nutrients that are similarly trimethylamine containing. And so,
like, carnitine was the one we were focusing on. Now the reason we were focusing on carnitine
is because there’s substantial epidemiology data that argues that red meat is associated
with increased cardiovascular risks. And in particular, for example, this recent study
that came out by the Harvard Nutrition Group, looking at both the Health Profession Follow-Up
Study and the Nurses Health Study, combined to have over almost 3 million follow-up years
of information with almost 24,000 deaths, followed with an average follow-up period
of over 20 years. And what was seen is that for each one portion increase per day in red
meat amongst the individuals that were followed in these studies, it accounts for somewhere
between a 13 to a 20 percent increase in mortality over the course of the duration of follow-up,
which is, as I said, either 20- or 28-year period, depending on the two different studies. And, by the way, a portion size by the nutritionists,
it always strikes me as a little bit funny that they’re so small. Its only three ounces,
is considered one portion of red meat. I don’t know about you, but that seems to me like
the snack that you eat before you have dinner as opposed to the real portion size. [laughter] So why the interest in red meat? Well, carnitine
is a nutrient that’s almost exclusively found in red meat, certainly in meat. And the structure
is shown here, and it has that same trimethylamine moiety on it. And it actually derives its
name from the Latin root word where carnivore comes from, meaning flesh. And that’s because
the chemist who first discovered carnitine structure over a century ago found that the
food substances that it was in were essentially of flesh, and red meat in particular has a
high level. And if you’re wondering what red meat has the highest level, kangaroo, by the
way, has a 50-fold higher level of carnitine than beef, so don’t go out and eat kangaroo
patties if you want to try and cut down on your carnitine ingestion. But anyway, carnitine plays a role normally
in fatty acid transport into mitochondria. We make all the carnitine we eat from out
diet. It’s not an essential amino acid. It is made by lysine after — and lysine being
the single most abundant amino acid in both plant and animals proteins. Most individuals,
unless you have a genetic defect, do not have carnitine deficiency, or it’s a very rare
phenotype. So we were interested in this same kind of
story: Could carnitine generate TMAO and accelerate atherosclerosis? Just to speed up, because
I want to leave time for questions, we used the germ-free animals, and were able to show
that when you ingest, or when animals ingest carnitine, and they are germ-free, they do
not make TMAO. But when they’re conventionalized, they do. We then wanted to translate this
to human clinical studies. Bob Koeth, a post doc — or, I’m sorry, is
the MSTP in my lab, this was his thesis research, the carnitine story. He did this study where
actually we generated — we used, literally, a grill that had that on the box cover, and
used steak as our source of — natural source of carnitine, isotope-labeled carnitine in
a pill, and did the same kind of experiment, where at visit one, they’d get a carnitine
challenge, and over time, measure the appearance and disappearance of metabolites, go on antibiotics,
suppress the intestinal flora, et cetera. So doing that same kind of experience as we
did with the phosphatidylcholine, what we saw is that while individuals have intact
flora, they readily generate TMAO from ingestion of carnitine. But following suppression of
the intestinal flora, no TMAO is generated by ingesting carnitine, suggesting that carnitine
formation of TMAO has an obligatory requirement for gut flora as well. So taking this to atherosclerosis animal models,
we used the apoE mouse model. What we saw is there was about a two-fold increase in
aorta-accrued atherosclerosis in animals that were on a carnitine diet, despite no changes
in their cholesterol levels, their weights, or triglyceride levels. If you suppress the
intestinal flora, and bring the TMA and TMAO levels down to near zero, over the course
of the duration of the study, we saw — I’m looking at the right-hand panel — no diet-dependent
increase in atherosclerosis. Now one of the intriguing findings that we
saw during the course of these studies was that the animals that were on the chronic
carnitine diet showed a 10-fold increase in their synthetic capacity to make TMAO compared
to normal chow mice. This suggested that the chronic dietary exposure to carnitine had
actually shifted the intestinal microbial composition to such that the microbes were
now — those that preferred carnitine as a substrate had become — had a selected advantage
and had grown more, and now carnitine-dependent conversion into TMA and TMAO was occurring
more readily because of this shift in intestinal microbes. And actually, through looking at
the 16S ribosomal DNA of the feces, or — and also of the cecal contents, we saw that this
had actually, in fact, happened. In studies that we actually performed in collaboration
with our U Penn colleagues, such as Rick Bushman and Gary Wu, who are here, we actually looked
at omnivores versus vegetarians and vegans as well to see if this naturally-occurring
diversity in carnitine ingestion, that is, vegetarians and vegans have a very low carnitine
ingestion rate compared to omnivores, could we see a microbial shift, if you will, in
the formation of TMA production in the subjects? And this is data just from one omnivore who
was characteristic or exemplary, and one vegan who was an intrepid vegan who agreed to eat
a steak, as well as take the capsule with the methylated — the deuterated carnitine.
And what was seen is that following ingestion of carnitine, the vegan had an exceedingly
low synthetic capacity to make TMAO compared to the omnivore. And this was true with both
the natural abundance as well as the isotope-labeled precursor carnitine. Shown here are data with larger numbers that,
when we compared a larger number of vegetarians to omnivores, that the omnivores had a higher
level of TMAO in general. And then shown on the right are for those who went through the
full isotope-labeled carnitine challenge, there’s a very dramatic difference in TMAO
synthetic capacity or production rate following ingestion of carnitine seen in the vegans
compared to the omnivores. The vegans, as was seen with the other vegan who ate only
the steak and capsule, those who just had the capsule were very, very poor at generating
TMAO. Now when doing the microbial composition analysis
of the — from feces analysis, what was seen is that there were multiple different taxonomic
groupings of microbes that associated not only with the dietary pattern but also with
the plasma TMAO level. And shown in the red box here is an example of two different patterns.
The top one is illustrated as a lower proportion in vegetarian and vegans, this particular
tax, and associated with lower TMAO levels. And in contrast, for example, in the lachnospira,
that’s a proportion of a specific genus that’s higher in the vegetarian and vegan, and associated
with lower TMAO. And so the chronic dietary pattern had actually shifted the intestinal
microbial composition of the subjects, and this was associated with the altered TMAO
level. Now to bring this back to humans and cardiovascular
disease, which is my main research interest, we wanted to know how could we do this. And
it turns out that there have been many studies that suggest that individuals who eat more
carnitine have higher plasma levels of carnitine. And so we actually, then, went ahead and measured
carnitine in over 2,500 subjects, and asked, does it track with cardiac risks. And the
answer was it did, actually quite well; if you adjust it to traditional risk factors,
it worked as good or better as LDL cholesterol, or blood sugar, or any of the current diagnostic
tests that we use for — and are associated with cardiac risk, carnitine was independent
and better, if you will. But what we also found is that in individuals,
if you then stratify by their TMAO level, only those subjects who had a concurrent high
TMAO level — this is above and below the medium analysis, so you needed to have a high
carnitine and a high TMAO level to have increased cardiac risk. If you have a high carnitine
level but a low TMAO level, that is, let’s say your microbiota composition was healthier,
you didn’t — you weren’t a TMAO producer, you’re at low risk, even though you had the
high carnitine level. Presumably these were high carnitine ingesting subjects. We don’t
know the dietary patterns of these individuals, unfortunately. This was a study of over 2,500
subjects. So to summarize, I’d like to suggest that,
in addition to foods high in cholesterol and saturated fat, there are other dietary nutrients
that, through the action of gut microbes, that is like choline and carnitine, can generate
small-molecule metabolites that are biologically active, acting at a distant site, and influencing
cholesterol and sterile metabolism in the artery wall, such as in the macrophage in
the liver, such as in the synthesis of bile acids, and in the enterocyte in terms of cholesterol
absorption and cholesterol in bile acid metabolism. And collectively, it’s serving like a rheostat
on the light switch. You need cholesterol to have atherosclerosis, just like you need
electricity to have a light bulb turn on. But you can have a rheostat, and if you have
a high TMAO level, the dimmer switch is bright, and at any given cholesterol level, you have
a more — bigger chance of getting atherosclerosis. If the dimmer switch is down, and you have
a lower TMAO level, you have a less chance of getting atherosclerosis. Now I’m going to end just by saying, where
else do we get carnitine? Well, in addition to these foods, it is a very common recent
dietary supplement, and not only in things that are shown here, but also, you know, some
of these energy drinks have an extraordinary amount of carnitine within them. If you’ll
notice, the carnitine content in Monster in particular is in front of the glucose, so
it’s the most abundant additive that they have. And it’s — one portion of a steak is
approximately 160 to 180 milligrams, so we’re talking a lot of carnitine in a can. So we were interested in asking — this is
the last piece of data; this is unpublished — if you’re on a chronic vegan diet, are
you protected if you start taking carnitine supplements because, actually, many vegetarians
and vegans, to try to augment their carnitine, will be on a daily carnitine supplement, thinking
that they’re missing it because they’re on a vegetarian or vegan diet. And what’s shown
on the left are the carnitine challenges that were done at baseline, or after one month
or two months of daily carnitine supplementation, and the vegans that we’ve done so far shown
on the left, the omnivores on the top and the omnivores on the bottom, and what can
be seen — and then on the right-hand side is just fasting plasma levels, and it tells
a story. If you have chronic carnitine supplementation, regardless of your diet, you know, we are
walking tissue culture dishes, and if you give a nutrient that the microbes like, those
that have the selective — they get a selective advantage, and now they’re going to be more
populous and grow. And so, actually, chronic diet carnitine exposure is changing the intestinal
microbial composition and making the subjects more prone to making TMAO, which we think
is a good proatherogenic phenotype. So it suggests, or makes us wonder, if chronic carnitine
exposure in things even in like energy drinks might be a long-term adverse thing that certainly
needs further studies in the future. I’ll just end by thanking you for your attention,
and point out four main individuals in particular. The four major papers that I’ve discussed
were each first authors by individuals in my group: Zeneng Wang, Bob Koeth, and Wilson
Tang; Brian Bennett working with — at UCLA with Jake Lusis was the author of the cell
metab paper. I also want to point out our colleagues at U Penn, Rick Bushman, Dr. Chen,
and Gary Wu as well. Thank you very much. [applause] Male Speaker:
Okay, maybe one question while we’re transitioning between speakers here. We’re running a little
behind. Male Speaker:
Well, here’s one. The — over here. The — since a number of lipids can be a source of trimethylamine
with the choline head group, why is it that carnitine sticks out as being a substrate,
or is that the conclusion that you have? Or is that not the case? In other words, is it
that making the trimethylamine is one thing, but then there’s some other ecology or interactive
process related to L. carnitine which goes with the cardiovascular risk? Stanley Hazen:
Well, I think that any of the trimethylamine nutrients, whether it’s carnitine, or phosphatidylcholine,
or choline, possibly betaine, acetylcarnitine, all of these can be converted into TMA and
TMAO, and so far, in an animal model level, all of them are associating with accelerated
atherosclerosis. So I don’t think there’s anything unique about carnitine; I think we
studied carnitine after our first study on choline. So carnitine just has that trimethylamine
group, and is — yet, the gut flora can actually cleave it off, and it has the same conserved
group. That’s why we started looking at that carnitine as a — but the two most abundant
trimethylamines in a Western diet are going to be carnitine and phosphatidylcholine, lesser
than [spelled phonetically]. Male Speaker:
Okay, do you have a quick question? Male Speaker:
Excellent talk. You mentioned that carnitine can be bad if TMO levels are high, but carnitine
has a lot of benefit in certain diseases of interest. There are diseases with low carnitine;
autism, the work we’re doing in working in. Are there efforts to look at TMO levels in
other disorders where carnitine metabolism is involved? And you made an excellent point,
which — for supplements, what might be good for some disorder may be very bad for another. Stanley Hazen:
We haven’t looked at autism. We actually — after our paper came out, I got a landslide of emails
asking about acetylcarnitine because, apparently, it’s a common nutrient or supplement that’s
used with reported benefits for cognition, and in various neurodegenerative disorders
or dementia. We have been studying it. All I can say is that we’re looking at it from
the cardiovascular standpoint, not the dementia standpoint, and I can say that you get accelerated
— or you get enhanced TMA and TMAO development by ingestion of acetylcarnitine. Whether that
leads to accelerated atherosclerosis in an animal model, we don’t know the answer yet. Male Speaker:
But it’s a good example that, you know, for some disorders it shows promise in specific
studies as opposed to being a carte blanche thing that everyone needs. It’s an excellent
study. Stanley Hazen:
Thank you. Male Speaker:
Okay, thank you. I think we need to move on. [applause] Okay, the next speaker is Christian Jobin
from the University of Florida School of Medicine, and he’s going to be talking about the gut
microbiome in colorectal cancer.