The Microbiome-ASD Connection

All right, we're live. Hi, everyone. Thank you so much for joining us this evening. Looks like we have about 80 of you. If you can hear us in the little chat box, if you'll just let us know. Cool. We have hello from Florida, Huntington beach from Japan. Awesome.

Sweet Sedona.

Cool. Thank you guys. And everyone, can you hear us? Wonderfully. We sound checked this time pretty well, so hopefully you can hear us real clearly.

We did it ahead of time like smart people.

Okay, good. June says I can hear you well. Yes, yes. Okay, cool. So today Karan is going to talk to us about autism and the connection between autism and the gut microbiome, as most of you know. This is Karan. He is our microbiologist. He's also the chief scientific officer for us here at Microbiome Labs. All you really need to know about him is that he knows pretty much everything. So we're going to let him take it away. He's going to have about a 30 to 40 minute presentation for you guys. You can ask any questions that you have in the little chat box over here. I'm going to flag all the questions and then we'll have about a 20, 20 minute Q& A afterwards. So any question that you have, I'm going to go through. I'll ask Karan if there's anything that we don't get to. I will compile all the questions for you. We'll get them answered and then we'll send them out via email and we'll also send out the slide deck afterwards as well. So this will be recorded. You will get to watch the replay so you can pause and rewind and you'll get to have all these resources afterwards. So the. Without further ado, take it away, Karan.

Hello. Thank you, Acacia. Thank you guys all for coming on. I see a lot of people jumped on now and we have an awesome distribution of people. I saw someone from Australia, Japan, Kentucky, Boston. So hello to everyone and thank you all for coming in. We're going to talk about the connection between the microbiome and asd. As you guys know, ASD is quite a spectrum disorder. There's many different levels of asd, from mild behavioral issues to, you know, completely non social, non functioning. And one of the reasons why we want to do this because, you know, many of you may not specifically treat kids, but you treat a lot of the parents and some of those parents have kids on the spectrum. So I think you'd be able to offer help and support and clarity to those parents once you learn deeper about the kids connection between the microbiome and what the kids are going through. The other reason for it is a lot of the mechanisms that I'm going to be talking about of how what kind of disruption in the microbiome leads to ASD is actually the same, exact mechanisms and disruptions that occur in celiac patients. And we're talking about really clinically defined celiac spur, or even people that are gluten intolerant. It's the same type of dysbiosis, it's the same breakdown of mechanisms within the microbiome. So most of this information can be translated to people you're working with who have gluten intolerance, who have celiac and other inflammatory associated bowel conditions. So we'll jump in. I'm going to screen share and run through a PowerPoint. There's only about 16 or 17 slides really to get to the meat of all of this. And then after that we'll handle some questions. Let's see. Okay, Acacia, it looks good. Full screen, all that.

Yep, looks good to me.

Okay, awesome. All right, so the microbiome ASD connection, you know, and this is of course a prevalence rate of this is going up and up. And same with things like celiac disease, which is again, the same kind of mechanism. A lot of people will say, well, you know, maybe the awareness and diagnosis is increasing. That's why it seems far more prevalent these days than it did say, 20, 25 years ago. But in fact, that's actually been proven to be wrong. Many of you may not know that there's a study that was published that actually took contained serum samples of people from, I think it was in the early 80s and 70s, the samples were generated then and they've been preserved for a long period of time. And they took those serum samples, compared them to the exact same cohort, exact same type of cohort of people, meaning same sex, same age, and so on to serum samples today. And they found the prevalence rate of gluten sensitivity, for example, in today's population is way higher than it was in previous populations. And those are serum confirmed biological tests. So they've shown that it's not just about awareness. It's not about, not just about being diagnosed more often. Biologically, we are far more inflammatory, we are far more allergenic, and our guts are reacting in a much more negative way to things in our environment than they used to. And of course, the things themselves, like gluten itself, has changed quite a bit in the last 40 years or so. So keeping all those in mind, let's dive into the connection here through the microbiome. So ASD and the microbiome is now very well understood. There's further and further work that's being done. One of the challenges of studying this is it's very difficult to do clinical trials in kids. You know, we're attempting to do a couple trials right now in kids. It's very hard to get IRBs, which are the review boards that approve clinical trials, to agree to anything that may be even semi invasive in kids. Even drawing blood in kids is very hard. So a lot of these studies started in mice models of autism. And what's great about that is they've now developed really, really well established mouse models for autism, where the mice very clearly have behavioral issues that are exactly the same as spectrum disorders in kids, and they've been able to show some of the differences. Now, there are studies in kids as well, which I'll show you, but keep that in mind. The initial focus on asd, when the awareness started increasing, was to find the issue in the brain. And then, of course, when we think that the issue is coming from the brain, we always think that there's some sort of gene for this. But that was really quite unsuccessful. And that's why there's no treatments for ASD that really directly target the brain. And there's no genetic marker that's very clear for ASD either. The most updated science shows clearly that ASD is multifactorial. It's a complex condition, and there's both intrinsic and extrinsic factors that seem to play a role. For example, things like environmental chemicals. I'll talk about one of them. Diet alterations seem to drive increased risk as well. Now, a lot of the diet alterations occur as secondary factors based on the initial insults, which are the environmental exposures and the perturbations in the microbiome. Also, metabolic status can have an impact. And then, of course, the most important thing in our view is the microbiota. And hopefully you'll see the same thing when we show you the data. So, common correlations with gastrointestinal disruptions and ASD development. So, for a long time, doctors and care workers that worked with kids with ASD had clearly reported that there was a direct common correlation between having GI disturbances and having asd. The GI disturbance frequency was much higher in kids with asd. And what they were showing is that both prenatal and perinatal acetaminophen exposure, for example, during childhood had been associated with development of asd. And coincidentally, acetaminophen adenofen also has been associated to cause GI problems in kids during both prenatal and perinatal exposure. And this included acute upper GI emergencies that occur every year. There's a few thousand cases a year where you see acute upper GI emergencies associated with acetaminophen exposure. So these were starting to be reported that acetaminophen exposure during childhood seems to be associated in some cases with. With ASD as well. Also, exposure to antibiotics either early in life or during pregnancy have been linked to later development of asd. And some have suggested that early antibiotic exposure could even cause asd. So, again, another known disruptor of the microbiome, early exposure to such a disruptor has been associated with the development of asd, or at least the increased risk of developing asd. Even things like proton pump inhibitors, which are used to treat gastroesophageal reflux and which is a disaster, which is not uncommonly associated with asd, also seem to affect the ph level in the gut, which can then cause dysbiosis and can further the progression of the disease. Pesticides and heavy metals that have been linked to ASD also can cause mitochondrial dysfunction and further GI abnormalities, which then perpetuates the disease. I didn't end up putting this in here, but there was a paper that was just published about a week ago that showed real well characterized mitochondrial dysfunctions in ASD kids. And they started the studies in a rat model or in an animal model, and they were able to recreate ASD by specific mitochondrial dysfunction. So having healthy mitochondria seems to be an important aspect of progression in asd, and, of course, alleviating symptomology and starting the process of trying to reverse the disease. Now, comorbidities with GI disturbances is often observed in kids. As I mentioned, people who've been caring for ASD kids have been reporting this for some time, but they've done some actual prevalence work. And some of the published estimates show that around 70 to 90% of kids with ASD have GI disturbance comorbidities. A lot of times it's this constant bloat in the gut. There's a distension of the belly. There are severe intolerances of foods, there are severe allergies to certain foods. And of course, the feeding preferences are really, really narrow. So there's definitely a direct correlation between GI disturbances and having asd. In human studies, the presence of certain microbes have been positively associated with asd. One of them is clostridium tetani. So this was initially published back in 1998 and there's been further studies since then that have collaborated this information, but they find that it's not present in non ASD kids. And this particular Clostridia produces a lot of toxins that can really impact neurotransmitter release. Now we know that ASD is a gut bacteria, brain related conditions. So anything that affects neurotransmitter function can really affect cognitive development in kids. And then also the same mechanisms can affect adults in other kinds of disease. For example, things like anxiety, depression, mood disorders, even gambling addictions can be disrupted by the types of toxins that Tetani produces that interferes with neurotransmitter release. Clostridium bolta is also shown to be significant, significantly elevated in kids with ASD in a 2004 study. So again, the same genus of bacteria, Clostridium, in this case, a follow up study in the same group showed nine species of Clostridia that were present in the stool of ASD kids that kind of clustered. It's almost like an ASD consortium that seems to be fairly common in ASD kids. Clostridium histolyticum is another one. It's another toxin producing strain, phosphorus formed in ASD kids. And another interesting impact is the fact that non autistic siblings, so the same family born of the same mother, showed moderate levels of these same strains. And that's interesting because what it shows is that the siblings also got certain types, certain degrees of exposure to these same strains, maybe from mom, maybe from the environment that they were born into, maybe from, from close family members. You can't exactly be sure where they got exposure to these strains, but the concentration of the strains and how well they do in the child seems to dictate whether or not the disease comes on. So even though you might have gotten exposure to Clostridium tetani and histolyticum and bot, you won't necessarily develop the condition unless the exposure also was followed up by an environmental impact. When I say that, I mean like an antibiotic use or chemical exposure and so on that allowed these particular strains to take over and grow to very unhealthy levels. Right? So just exposure to the strains does not mean that the disease will come on. It also requires that environmental factor that then allows the perturbations in the microbiome and then allows the strains to actually take over and become more predominant in those subjects. So that was interesting because then it shows you there's a population aspect to it. These are some of the other strains that have also been associated with asd, but the Clostridium ones seem to be really, really prevalent. And then in addition to having high levels of clostridia, you also tend to end up with low, low levels of bifidobacteria. And in some kids, you also see high akkermansia, which is interesting, but the low levels of bifidobacteria are really key as well. So in general, the best data shows high clostridia, especially these particular strains, and low levels of bifidobacteria. And that's characteristic of an ASD child. Now, again, I want to keep reminding that this same kind of format is also characteristic of celiac disease. And from celiac, you can also evolve this same kind of characteristic in certain inflammatory bowel conditions, you know, like Crohn's and colitis and so on. That will then develop further to create more perturbations in the microbiome. In most cases, the clinical severity of the GI issues were positively correlated with severity of asd. So there is some, some data that shows that depending on how severe the GI issues are that are associated with the child with asd, the ASD itself is then subsequently worse. So there is kind of a severity dependency where very mild GI issues tends to be associated with fairly mild spectrum disorders. Very severe GI issues are more associated with severe spectrum disorders. A 2009 study showed that vancomycin treatment in the initial stages improved symptoms in children with asd. But of course, the effect was only transient once the drug was stopped. Now, that's interesting because vancomycin is a good treatment for clostridia. So the vancomycin is also the same thing they try to use to treat C. Diff when people end up with C. Diff, which is, of course, Clostridium difficile. So that further validated that the presence and the concentration of these bacteria are playing a role in causing the symptomologies, in causing the disease itself. Of course, you can't do vancomycin treatment forever. So that's not really a solution for treating kids with asd, because once you stop the drugs, the bugs come back and then the symptoms come back. But it was promising to see that the addition of vancomycin, which we know targets clostridia, actually lowered the symptomology of the disease. So we know that these clostridia seem to play a role in it. ASD kids tend to have higher levels of ammonia in stool. This is also very, very characteristics of a dysbiotic flora. So ammonia producers tend to have high levels of clostridia, tend to have high levels of certain gram negative enteric pathogens that convert more of the protein and the food coming in into ammonia. Versus someone who has healthy microbiome and has more sacralytic fermentation, they tend to have much lower levels of ammonia. But ammonia is also very characteristic of ASD kids. So keep that in mind as well. Microbiome of the mouth also could play a role. So mouth dysbiosis has been associated with severity of things like epileptic seizure. But we also know that mouth dysbiosis is directly translated to gut dysbiosis. So things that are happening in the mouth could have a severe impact on the types of microbiome within the gut. What was interesting about the epileptic seizure connection is that that's a neurological issue. So showing that dysbiosis in the mouth could actually be leading to further neurological connections which either could be a direct impact from the mouth to the brain, or from the mouth to the gut to the brain. So those things are all interesting to keep in mind. So looking at dental health and the degree of periodontal issues in kids on the spectrum would be a clue as well to see what's driving their condition. One study had suggested that the vaginal dysbiosis from maternal stress may affect offspring's neurological behavior development. I didn't put the slide here, but that was a very interesting study. So this study of course was done on a mouse model of asd. You can't do this in moms and kids. But what they did is they took two groups of pregnant mice and one group, they put the pregnant mice under a lot of stress during gestation. Another group, they had in mouse utopia, they were living a very great stress free life during gestation period. Now, the mice that, that gave birth to babies who were under the moms who were under stress ended up giving birth to baby mice that were characteristic of asd. The mice that were under low stress or no stress gave birth to normal healthy progeny, normal healthy mice. What was further interesting, so that showed some connection to gestational stress and neurological development. Now what was more interesting than that was they took the serum samples of mom, the mother mice that were raised during gestation under stress, injected it into the newborn babies from the mom that wasn't under stress. And those babies developed neurological disturbances as well. So it's something in mom's blood from Stress during gestation, that actually also can trigger the formation of ASD and other neurological symptoms as well. And we know that disruptions in the gut are absolutely associated with stress. So stress is one of the biggest drivers of leaky gut. And when mom has leaky gut because of stress, then her blood becomes much more toxic because of metabolic endotoxemia. So every time she eats food, you get a lot of LPS and a lot of other toxins, which we've talked about in many webinars before. So that's another very interesting connection as well between asd, stress, leaky gut, and of course, the whole process of giving birth. So how does dysbiosis influence asd? Again, a lot of it comes under the gut brain connection, and it comes down to the types of metabolites that are produced in the gut or should be produced in a healthy gut. For example, gaba, norepinephrine, serotonin, dopamine, even things like tryptophan metabolism, which is required for the effective production of serotonin. We do know that in ASD kids, there's the serotonin and dopamine imbalance issue. So it's all based on the gut brain connection. So digging a little bit further into that, by using the enteric nervous system and the vagus nerves, these dysbiotic microbes and their components can be directly sent to the brain. In ASD kids, they've got a dysbiotic microbiome with high levels of clostridia. And these clostridia are the particular ones that produce certain types of toxins. These toxins then are able to gain direct access to the brain through the vagus nerve and the enteric nervous system. And because of that, these toxins have the opportunity to get in and disrupt neurological development in the brain. Direct infections can manipulate vagus nerve. For example, Campylobacteria jejunum infection actually has been shown to create sudden and rapid anxiety and panic behavior. So Campylobacter is an infection that comes along typically with chickens or poultry product. And one of the characteristics of this infection is stimulation through the vagus nerve of the brain to cause anxiety behavior and cause panic attacks. Imagine you get infected by a pathogen from poultry, and the resulting infection is that you're getting panic attacks from it. This is an example of how some of these pathogens can gain direct access to your enteric nervous system and your central nervous system through. And of course, your. Your brain is part of it through the vagus nerve release of bacterial metabolites. So these can directly affect inflammation and function of the brain. We've showed previously that LPS from the gut is actually entering the brain, crossing through the blood brain barrier, entering even deep areas of the brains like the hippocampus and the amygdala, where they actually start the process of beta plaquing in the case of Alzheimer's, but in the case of asd, they get in and they start an inflammatory reaction in the brain. Even things like D, lactate and ammonia. Ammonia causes swelling in the brain, and as I mentioned previously, ammonia levels tend to be high in kids with ASD. Suppression of serotonin and dopamine. So a 2015 study showed that 95% of circulating serotonin was actually made in the gut. And of course, the dysbiosis of the gut associated with ASD causes a huge disruption in the amount of serotonin that's actually produced. And around 30% of patients with autism have very abnormal blood levels of serotonin. So there's a very clear association of serotonin dysfunction in ASD kids. Now, interestingly enough, in the 2015 study, they didn't name the specific bacteria that produce serotonin, but they said spore. Spore based organisms were responsible for stimulating something called enterochromafin cells in the gut, which are the ones that actually produce serotonin. So certain bacteria within the gut and they named spore based bacteria were some of the key ones that stimulated the intraochromafin cells to produce serotonin. Of course, micronutrient deficiencies, especially B12. Methylated B12 is produced by a healthy gut microbiome and absorbed in the ileum. Dysbiosis in the gut reduces B12 production and of course, absorption as well. Some of the key things that drive dysbiosis associated with ASD specifically target micronutrients like B12, like selenium, like molybdenum. There's a whole bunch of other minerals that are specifically chelated and disrupted in the gut. And I'll talk about what some of the biggest environmental factors are that do that. But we know that these types of disruptions in micronutrients are also associated with issues with neurological development as well. And the disruptions of these also can lead to the dysfunctional development of myelin sheets. That's the insulation that covers your neurons, which then of course gives you loss of nerve transduction. It also disrupts production of neurotransmitters and activation of genesis that can actually help repair the gut lining. Vitamin D is another example of something that can actually help with the repair. But vitamin D formation in the body can be disrupted by micronutrient deficiencies. And of course, if you're trying to take supplemental vitamin D and you have an inflamed gut, you actually don't absorb the vitamin D very well. In fact, most fat soluble vitamins absorption, it tends to be severely hindered by intestinal inflammation. So in celiac, as an example of this same kind of overall mechanism, celiac people absorb vitamin B, A, K and E at a much, much lower rate than non celiac subjects. And that actually increases the things like osteoporosis, cardiovascular disease in celiac patients, leaky gut, of course, intestinal permeability and the associated brain permeability. So leaky gut. Leaky brain also plays one of the largest roles in the risk and symptomology of asd. Those particular organisms that I talked about in asd, the Clostridia species, are actually significant drivers of leaky gut. And then that leaky gut also drives further dysbiosis. Because part of the way that your body controls the types of microbes that exist in your microbiota is by stimulating the production of antimicrobials from the intestinal epithelial cells. Well, your intestinal epithelial cells can't produce enough antimicrobials when it's in a dysbiotic state. So it's now just fighting inflammation, fighting tight junction disruption. It doesn't have the time or the capability to try to modulate the microbiome by producing and secreting out antimicrobials. Also, the stimulation of the production of antimicrobials comes from certain types of good bacteria that produce high levels of butyrate, as an example. And so that butyrate production is severely disrupted when you have high levels of clostridia and a disrupted gut. So there's mounting evidence that gut permeability starting from birth can dramatically influence the risk of developing asd. One line of evidence for this is a high fat diet, which has been shown to cause microbiome shifts and intestinal permeability. We've talked about that a lot, especially a diet very high in saturated fats. When your microbiome is not healthy, a high fat diet can actually become more disruptive to that microbiome, which leads to higher concentrations of things like endotoxins in the blood and then more neuroinflammation. This particular pathology is associated with increasing ASD risk and of course, the severity and symptomology with asd as well, going gluten free and casein free in their diet has been shown to be promising in improving ASD symptoms. So both gluten and caseins cause severe inflammation and increased gut permeability in a significant portion of the population. Which means that further reducing the exposure of things that increase permeability can actually improve the quite a bit. In a Harvard study, they showed that altered intestinal permeability was found in 43% of autistic patients, but not found in any of the control subjects, many of them the siblings of the ASD kid. And so what they were able to find is that this idea of intestinal permeability is quite prevalent in ASD kids and obviously is either a consequence of the disease which then keeps progressing the disease and maintains the condition, or it could be one of the underlying causes. The presence of toxin producing harmful bacteria like Clostridia seem to increase inflammation on the lining and of course increase then permeability as well, which is a major mechanism by which these particular species are closely associated with asd. So again, certain types of toxin producing pathogenic organisms being at high levels are also associated with high levels of intestinal permeability. And these particular organisms drive intestinal permeability by the toxins they produce, which causes an inflammatory reaction in the mucosa. That inflammatory reaction in the mucosa actually opens up the tight junctions. Researchers that were studying maternal immune activation using certain deliberate viral infections during pregnancy, showed that mice that were infected had leaky gut and gave birth to mice that demonstrated autism behaviors. Then in this case as well, they were able to extract the endotoxins from the mothers that ended up with the viral infection that ended up giving birth to ASD type of mice. When they took that blood and they injected it into the normal newborns, they were also able to develop the disease that way through the injection and transference of the infection from moms from the ones that gave birth to ASD symptomatic mice, they transferred it to non symptomatic mice and that caused ASD symptoms as well as. So one of the researchers, Patricia, which is she's well known in the ASD area and also has studied a lot on glyphosate as well. They published a seminal paper on gut hyperpermeability. So leaky gut in relation to autism. So the researchers reported that altered intestinal permeability in nearly half of ASD participants compared to the control samples. This was I think outside of the Harvard study, but they showed closer 50% prevalence rate of hyperpermeability within them. This is another study published in Scientific America where they showed that autism is primarily a disorder of the brain. But research suggests that many as 9 out of 10 individuals with the condition also suffer from GI issues, which I mentioned before, such as inflammatory bowel disease and then, of course, leaky gut. Now, again, this is part of my motivation for doing this particular webinar is that even if you're not working with anyone on asd, it's the same pathology that's associated with ibd, ibs, Crohn's colitis, and of course, celiac as well. It's important to understand the types of bacteria associated with these conditions, how they're actually affecting the microbiome, and then, you know, what are some of the things you can do about it as well. Now, all of it, of course, comes down to leaky gut, because the whole problem with having a dysbiotic flora is a dysbiotic flora leads to intestinal hyperpermeability, and that intestinal hyperpermeability feeds something called metabolic endotoxemia. And that has the inflammatory, systemic inflammatory response within the body, which then also creates inflammation in the brain. In a young kid, that typically results in autism or mood disorders or behavioral disorders in an adult, when you have this kind of endotoxemia, it leads to heart disease, diabetes, various types of cancers, and then, of course, most of these GI issues as well. Now, one of the biggest offenders as the environmental trigger that I mentioned that was important in the development of ASD is glyphosate, as most of you know it as Roundup. It's a very commonly used herbicide in the U.S. incidentally, it's been banned in many other developing countries because those governments have realized that, you know what, this stuff is way too dangerous to be spraying on our crops and having our population consume it. Of course, in the US we are largely governed by big money, so there's a lot of big money that supports the use of glyphosate. So it continues to be a prevalent issue here. I want you to recall that I mentioned a few slides back that you could have the high levels of clostridium, or you could have the presence of clostridium, not the high levels, but the presence of those clostridium species, but not develop the disease. It typically requires an environmental factor, that which is exposure to certain chemicals and the antibiotic course or two, that then allows those species to become more predominant and more prevalent. Then you end up developing the symptomology as well. And this can happen at Any time in life, if it happens in the first two and a half years of life, the risk of that overgrowth of Clostridium, because of the environmental factors, the risk of that becoming ASD is very high. But if it's happening in an adult with an already formed brain, then that adult ends up, instead of developing asd, will end up developing Crohn's colitis or celiac disease, allergies, many other things associated with the same kind of dysbiosis. Now, let me explain why glyphosate is presumed to be safe. That's not on the slide, so I'll just talk about it. Back when they were first approving glyphosate as a compound, and then also glyphosate in its various formulations. The main formulation, I think, is called bisodium lactate. Maybe it's a. No, it's a bicarbolic acid, I think. Sorry. Yeah, it's a carbolic acid formulation. So it's glyphosate. And then formulated with a carbolic acid. The carbolic acid actually increases the pervasiveness of the glyphosate into the plant tissue and so on. So that makes it absorb better into cells. That's why they formulated that way. So when they were initially looking at glyphosate to use on crops, what they had to show to prove safety is of use is that they had to show that it didn't have the general systemic toxicology that the FDA looks for in order to approve processing aids that are used in farming and food manufacturing and so on. Now, glyphosate is not toxigenic to a mammalian system the way formaldehyde would be. If you drank it, you wouldn't get an immediate toxogenic reaction. So that's what they show. There's something called 28 day feeding studies, or 90 day feeding studies, where they take mice and they give them small levels of the glyphosate. And in that 28 days or 90 days, they don't show that the mice are dying. That's why they say, okay, it's safe. They also then broke down the way glyphosate works. So glyphosate works by inhibiting a key enzyme that produces aromatic amino acids. These aromatic amino acids are really important for metabolic processes. However, this pathway, which is called the shikimate pathway, is found in plants. It's not found in mammalian cells. Their whole reasoning is this interferes with the biochemical pathway that's found in plants, but not found in humans. So it should be completely benign to humans. It shouldn't do anything. That's the basis of the approval for safety and use on crops. Now, the problem here is that glyphosate's shikimate pathway interruption does interfere with lower order plants like weeds. However, it also disrupts bacteria. So a good number of bacteria that live in your gut also depend on the shikimate pathway. And that was never really investigated. But of course, when glyphosate was initially developed, they knew nothing about the microbiome. Now that we know about the microbiome, and we know that glyphosate is in effect, an antibiotic because it kills off bacteria, it should be banned and should be prevented. And certainly you guys should avoid it as much as you can. Interestingly enough, I think it was back in 2010, Monsanto, who makes glyphosate, applied for a patent patenting the antibiotic effect of glyphosate. So they themselves have studied and shown data and applied for patents showing that glyphosate is a strong and potent antibiotic. And yet that still is not triggering any sort of regulatory issue with its use in crops in general and human consumption. And so basically, the consumption of glyphosate is like consumption consuming a particular type of antibiotic on a regular basis. Now, the thing is, glyphosate causes a very characteristic perturbation pattern in the microbiome, and one that is arguably worse than most antibiotics. So most antibiotics basically wipe down a whole bunch of bacteria within your gut. Some really strong antibiotics, like, like the Z Pak, for example, that people use for sinus infections, upper respiratory infections, those types of broad spectrum antibiotics, wipe down your entire microbiome by about 90% with a single dose, like things like Augmentin. Now, when the microbiome starts to bounce back and grow back, it causes severe perturbations. But glyphosate, this has been done with a significant number of studies in poultry, cattle and swine. The reason they've been really concerned about studying these in these animals is because these animals are constantly exposed to glyphosate. And they've actually shown huge epidemics in die off in poultry, in cattle being associated with their exposure to glyphosate. In poultry, it's clostridium infection. In cattle, I think it was like a pseudomonas infection or something like that, where there was this epidemic of cows dying off, traced it back to the exposure of these cows to glyphosate and the promotion of pathogenic organisms. So the characteristic perturbation that glyphosate does in the body that's worse than antibiotics is glyphosate seems to increase the ratio of pathogenic bacteria compared to good commensal bacteria. So it somehow specifically selects for the growth of pathogenic bacteria, in particular two kinds of Salmonella and clostridia. And the reason is Salmonella and Clostridia are highly resistant to glyphosate's negative effects on the shikimate pathway, whereas really important bacteria like Enterococcus, Bifidobacteria and Lactobacillus are very susceptible to glyphosate die off. So glyphosate is like an antibiotic that is specific for good bacteria and, and then specifically allows for the growth of bad bacteria, in particular Clostridium. And remember, we've been talking about the clostridium overgrowth is a huge issue with asd. So what's happening is you might be born with exposure to Clostridium tetani and histolyticum, but those low levels of those bacteria don't seem to cause any issue. But then if you're exposed to high levels of glyphosate in those first couple of years, then your good bacteria, like your Bifidobacteria, Lactobacillus and other bacteria are being killed off. Clostridium happens to be resistant to the glyphosate, so they're allowed to grow. And then, of course, here comes the onset of disease and symptoms. Now, again, the same thing applies to celiac disease and inflammatory bowel disease and so on. There are other pathogens that also do really well under glyphosate exposure because they carry an enzymatic capability to metabolize glyphosate. So they have an enzyme that can actually break down glyphosate. And one of those is Pseudomonas. Pseudomonas is an opportunistic pathogen. It's antibiotic resistant. It's a gram negative bacteria, which means it has a lot of LPS in it, the endotoxin that we talk about a lot and has been shown to be able to break down glyphosate because it produces a usable phosphatase that can actually negate the negative effects of glyphosate and but in turn, it'll actually turn it into formaldehyde. So not only is this a pathogenic organism that can create toxins, it actually, during the synthesis of its amino acids, it's aromatic amino acids that it pathway that it uses to bypass the shikimate pathway. One of the toxic results is it has formaldehyde as a bypass. So imagine this, that significant exposure to glyphosate allows for clostridia to overgrow, allows for pseudomonas to overgrow, and then pseudomonas, because of its ability to circumvent the shikimate pathway, and it has its own pathway of creating these aromatic amino acids, but in its own pathway, it's also producing formaldehyde. So now you've got higher formaldehyde levels in your gut. Then of course, most of these species also produce high levels of ammonia in your gut. So this is what's driving most of that dysbiosis. Here's a really interesting study that was published in 2017. It basically took two different groups of mice, male and female, and continuously fed these mice very, very low to moderate exposure levels of glyphosate. So if you look at the different exposure levels, they did 0.1 parts per billion. That's PPB. They also did 400 parts per million, and they did 5,000 parts per million. And they did this over, I believe it was a two year period. Right. And so what they were able to show, if you look at a, if you look at the two blue circles, that shows you what the control microbiome looked like in the two blue circles. And then in the red circles, you see how the microbiome has changed. Especially if you look at the far right side, the presence of all of those cream color organisms, that's denoted by the cream color on the bar. Those organisms have jumped in growth and population quite a bit after glyphosate exposure. Those organisms happen to be gram negative pathogenic bacteria, Clostridia species and so on. So this is one of the first studies that showed that in a mammalian microbiome, chronic exposure to glyphosate had huge perturbations in the microbiome and again, specifically ended up selecting for gram negative bacteria, gram negative pathogens, and also Clostridia species. Now, remember all of this stuff we talk about with metabolic endotoxemia, the migration of LPS from the gut lumen into the circulatory system. Those are all gram negative bacteria that contain that lps. Imagine if you now have an inflamed gut, you have a dysbiotic gut, you have tight junctions that are opened up and you end up having more gram negative bacteria. You're just increasing the amount of LPS in the gut that ends up leaking through. So it's all Just kind of feeding one another. Then higher levels of ammonia also cause more inflammation. Production of formaldehyde by the types of bacteria that glyphosate seems to support. I mean, it's really a profound disruption to the gut. And the thing is, studies show that there are newborn babies that are born with measurable levels of glyphosate and cord blood in their amniotic fluid and so on. So the exposure is right off the bat. So moms that are pregnant, that are thinking about getting pregnant, they need to really avoid things. Agricultural products that tend to have high glyphosate use, like soy, wheat, especially the GMO versions of those which are predominant in the US really need to look at going organic so you can try to minimize your glyphosate exposure. Because all of those things perturbs mom's gut. And then that perturbation of mom's gut increases ammonia production and so on. And then mom ends up transferring that kind of perturbation to the child itself and further increases the risk of asd because glyphosate disrupts the microbiome and increases the growth of pathogenic organisms. When you look at a plot like this, that was done by the USDA and the cdc, this is a plot on the increased use of glyphosate by each year and then the increase in deaths due to intestinal infection per 100,000 people, you see that the slopes of the plot are almost exactly identical. So if there's more glyphosate being used, there's going to be more deaths from intestinal infections. Now what's interesting, I mean, you see how well mirrored the plots are. From 2009 to 2010, there was a slight dip in the use of glyphosate. At the same time, you see that same slight dip in the reduction of intestinal infections. So it's really, really clear that those kind of environmental toxins are leading to an overgrowth of pathogens in the gut. The overgrowth of pathogens are creating severe dysbiosis. That severe dysbiosis is increasing leaky gut. You're getting more gram negative bacteria, more ammonia being produced, more than endotoxemia leaching into the system and disrupting the immune system, causing more infections and so on. Now all of this gets even deeper when you look at the plotting. And this is from the U.S. department of Education data. They plotted again the increased use of glyphosate on corn and soy with the incidence rate of ASD in kids between the ages of 6 and 21 years old, you see almost an exact mirroring of the slopes of the line. And no question about it, if you look in the early 1990s, there was virtually no use of glyphosate at that time, and the incidence rate of ASD was super low as well. It couldn't be more clear when you look at huge cohorts of data that compare the use, and this is again per 100,000 individuals. So further on, the glyphosate and microbiome, in addition to the microbial changes that you see in the microbiota, with the increase in clostridial growth, lowering of bifidobacteria, there are significant physical changes that happen to the intestinal barrier as well. Here's one study that showed in fish that were heavily exposed to glyphosate, they saw huge disruptions in the mucosal folds and significant disarray of the microvilli structures in the intestinal wall. So we're also showing that there's significant impact on the actual physical structure of the microbiome. And another deeper thing, I call it the dynamic do or deeper destruction, lots of Ds, but basically it's the relationship between glyphosate and wheat. Wheat is one of the crops that uses glyphosate the most within the U.S. and so if somebody is consuming wheat, they're consuming a lot of glyphosate, unless of course, they can find organic versions. And if somebody is getting a lot of exposure to glyphosate, they're more than likely getting good amount of exposure to wheat as well. The problem with the two is they go hand in hand in the marketplace, but they also have a scientific connection that actually makes the entire situation. Now, first of all, glyphosate, we know when consumed, is consumed heavily in wheat related products. Glyphosate, as we've shown, causes huge amounts of inflammation, causes a dysbiotic flora, leads to leaky gut, leads to disruption in the way your immune system in your gut functions, all of that. So it completely disrupts how your gut functions in general. Now, wheat contains something called gluten. As you guys know, gluten is a very antigenic peptide. So it's a peptide, it's got a certain number of amino acids in length. And gluten happens to be one of those peptides that seems to be very antigenic, meaning it can act as an antigen and stimulate an immune response in the body. However, the normal process of digestion of many of these peptides, because gluten is not the only peptide that is antigenic. There are many peptides that are antigenic that come in the diet. The thing is, once those peptides are broken down into further amino acids, or just small little dipeptides with two amino acids, their antigenic effect goes away. The antigenic effect occurs in peptides that are between eight and, I think, 15amino acids long. If the peptide is much longer than 15amino acids, typically it doesn't cause an antigenic or allergic response. If it's much shorter than 8, it typically doesn't either. So what happens to most of the peptides in food is they enter the body and then there's enzymatic proteolytic cleavage of the peptide from pancreatic enzymes and other digestive enzymes as well. So then the peptide gets broken down into amino acids. It's no longer antigenic. The problem with gluten is it's hydrophobic. Most peptides are hydrophilic, meaning they like water, they solubilize in water, and they enter the gut in the water phase of the foods that you're eating. Now, most digestive and proteolic enzymes are also hydrophilic and actually require water for their function. When gluten gets into the gut, it's actually hydrophobic, so it gets to avoid all of these digestive enzymes. What happens is gluten, it doesn't get digested through the normal process that other antigenic peptides would. So gluten, as an antigenic peptide, enters the gastrointestinal tract basically whole at high levels. And because it's entering a gastrointestinal tract that also is being exposed to glyphosate, the normal suppression of the inflammatory response of gluten doesn't exist because the gut is in disarray, it's in heavy dysbiosis. And so the combination of the two further perpetuates both problems. Because then the more you're reacting to gluten, the more immune dysfunction you have in the gut. The more leaky your gut is, the more systemic inflammation you start to see. The more leaky your gut is, the more dysbiotic your flora is and the more reactive you become. So it's a self perpetuating problem. Now, what's interesting is ASD really, I think, can be treated with probiotics quite well. We have a lot of great cases. We're still trying to figure out how to do a good study with MegaSpore and ASD kids. But it's been proposed that probiotic therapy may play a really important supportive role in the management of the condition. Now, what's interesting is there's a 2015 review paper. It's titled Microbiome Disturbance and Autism Spectrum Disorders. Now this is a review paper, which means it's a meta analysis. The researchers that published the paper looked at dozens of other studies on microbiome disturbances in ASD and basically came to a conclusion that probiotics could dramatically help asd. And if they can help asd, they also help things like celiac IBD and other inflammatory conditions in the gut. Because remember, they all have the same pathology. But the features of a probiotic that can make it useful in ASD are as follows. Now remember, this is taken directly from this published paper. It has nothing to do with us. It just so happens that the features that they have identified are really familiar to us. For one, it has to be able to alter host immunity. It has to be able to do things like increase secretory iga, lower inflammatory markers. Because without the ability to decrease or alter host immunity, people with ASD are going to continuously just react to foods that are coming into the system, also react to chemicals that are coming into the system, and that inflammation, that non stop inflammation, just further perpetuates the condition. They also said that it has to be able to restore the normal commensal flora because we know ASD is characteristic of having very high levels of clostridia and other dysbiotic flora. So a probiotic that can actually compete with those pathogenic organisms and restore the flora would be very useful in asd. They also showed that it should be able to suppress microbial pathogens. So it's got to be a probiotic with bacteria that can come in and suppress other pathogenic organisms. It also has to be able to fix and improve intestinal barrier function. Again, this is all coming directly from this published review paper. Very interestingly enough, and I was very surprised when I read this paper, they also talked about how it should promote the synthesis of antioxidants. The promotion of synthesis of antioxidants is really unique because so far in the market, there's only one strain that has been shown to promote and actually directly synthesize antioxidants. Now why did they mention this? Well, as it turns out, in ASD and other inflammatory bowel diseases, oxidative stress in the bowel has a huge issue. But then also I mentioned earlier the mitochondrial tie with asd. One of the things that has been shown most recently is that there's severe mitochondrial dysfunctions in ASD kids and that includes Mitochondrial dysfunction in neurons in the brain. These important antioxidants like zeaxanthin, astaxanthin, lutein, lycopene, alpha and beta carotene act as very potent antioxidants in cellular respiration. So they prevent mitochondrial dysfunction. They help reduce mitochondrial dysfunction at least, but in many cases they can prevent it as well. So I think that's why they mentioned that. So again, reviewing that, they're saying that it has to have the ability to alter host immunity. As you guys know, bacterial spores do just that. They improve mucosal immunity, they reduce inflammatory markers. We're doing other studies right now to show the increase in secretory iga. I think you guys have seen the publication in August on the leaky cut study where we showed 12 different inflammatory markers being reduced quite measurably in the spore treatment group, in the megaspore treatment group when you compared it to placebo treated individuals. The restoration of normal flora. We know that bacterial shifts occur with micro with the use of spore based probiotics like Megaspore. The shift in the microbiome comes from eliminating bad bacteria and helping the good bacteria flourish and also suppression of pathogens like Clostridia. Clostridia is really hardy once it overtakes the microbiome because it's a spore former itself and it forms very good biofilms. So it can hide within biofilms. So it requires spores to get in there and, and actually target and destroy other spores. And also the bacillus spores and megaspore have enzymes like alpha amylase that can break down the biofilms of other pathogenic organisms. So they seem to be able to help with that as well. We're going to be publishing a paper later this year that shows that the addition of megaspore over, I think it was a six week period, dramatically increased the alpha diversity within the microbiome. Microbiome. It of course helps suppress some of the non favorable organisms, but it also helps increase the growth of very favorable organisms like faecalin, bacteria, Prosnitzi and bifidobacteria as well. Promotes the synthesis of antioxidants. Bacillus indicus, that's in one of the flagship strains in Megaspore, is the only known probiotic that produces antioxidants in the gut in your market. So when I started studying ASD and all of the pathologies that go along with it and I found this paper, it was quite amazing to see that the type of probiotic that these researchers recommended based on an Analysis of a whole bunch of studies is exactly the type of probiotic that we have in the market. And it's the only probiotic that meets all of these criteria. So it's super exciting to know that we can help kids with asd, which also means we can help the same pathology in celiac disease and IBD and of course, maybe even C. Diff. So because of that, we've actually just signed a contract to do a C. Diff study with Cleveland Clinic. So that study is going to be underway. And then we're also working on doing a study with ASD kids, which we haven't been able to make much progress yet, but we will certainly soon. And of course, ability to fix and improve intestinal barrier. As you guys know, Megaspore is the only product that's been shown clinically to be able to actually fix intestinal barrier dysfunction or leaky gut or hyper permeability, or in our case, we measured it by metabolic endotoxemia. So it's really exciting. And you've seen, of course, this data from our study, which is really quite exciting and profound. So that's the end of the presentation part. So let me stop the screen share thing so we can chit chat and answer some questions and. Oh, there you are. Hello, Keisha.

Hi, Mac.

Yeah, so that's that. So hopefully that made sense to people and there was some interesting information that they could, you know, little kernels of information that they can grab out of there. But it, you know, becomes to me the glyphosate exposure is, is really mind boggling when you, when you think about all of the things that it does within the body. There's a couple of really, really amazing, amazingly well written publications by Stephanie Seneff that I think everybody should read. We're happy to send it out to everyone that's registered here. There are, There's a part one and part two. She's, I think she's an MD, PhD or maybe just a PhD at MIT and has done a lot of work on understanding the impact of glyphosate on the microbiome. We're going to be doing a study hopefully this year on specific impact of glyphosate on the microbiome and then trying to show that the spores hopefully will reverse that. We already know that from clinical outcomes, but we want to show specifically in a microbiome study how the spores can change the growth of Clostridium tetani and histolyticum and all of these other problematic bacteria, even Pseudomonas for example. So that's that. Yeah, hopefully. That was interesting.

I think that was mind blowing for everyone that was watching. Everyone's like, I'm cutting out. I lost a bunch of information. So don't worry, guys. We'll send out the slides, you'll get this recording, you'll be able to go back. Sorry that some of you are having technical difficulties. Difficulties. So, yeah, now we're going to go through the questions because there are a lot of questions. So since we've been talking about glyphosate exposure, what's the most effective or the most realistic thing that people can do or can recommend to their patients to either avoid glyphosate exposure or just significantly reduce that exposure?

Yeah, and unfortunately, in the United States, we probably can't avoid exposure, but we can certainly reduce it quite significantly. The biggest thing you can do is go to organic foods. Organic foods do have other herbicides on them. Some of them are kind of natural herbicides, but they don't have the kind of impact that glyphosate does. And you can't get anything organic certified if you're using glyphosate on it. So the amount of exposure is low. Now, one of the things I didn't get to mention about glyphosate is especially in agricultural products like wheat and soy, what happens is when they grow, grind the seeds and things to make flour, it actually embeds the glyphosate inside the cellular structure. So even when they wash the flowers and they clean the stalks of the plants, or they pick the soybeans and then they wash them, glyphosate is not removed from the washing process. So if it's a gmo, you know, agricultural product, it more than likely has glyphosate inside the cellular mechanisms of the plant. And then when you consume it and you break down the cellular structure of the plant from stomach acid and digestion, you're releasing the glyphosate in measurable quantities in the gut itself.

Gotcha. So then we have a few people asking if you recommend doing a humic or fulvic acid, if that would help protect from glyphosate, since we're kind of finding you can't really totally avoid it. Is that something that you would be protective against glyphosate?

You know, there's this really interesting notion that certain minerals can protect from glyphosate, like humicophobic acid. And I'll say that I love humic and fulvic acid. I think they're a really important component to a healthy overall overall microbiome. They can be overdone, which you don't want to overdo. But they are taking them in reasonable amounts are a good food source for the microbiome because they help some of the metabolic processes that the microbiome does. There's also lignites, but lignites, you know, it's in a product like restore. But there's problems with lignites if you start digging into some of the research. The lignites have are associated with kidney issues because it's their kidneys that have to get rid of it. But none of these minerals in a human study has actually been shown to have any impact on removing glyphosate. That's a misconception that I see all the time. People say, oh, you got to remove glyphosate from the body. Well, you really can't. Once it gets into the microbiome, it gets inside the cells of the bacteria and, and once it enters the bacteria, which is a cell in itself, then it already starts to disrupt the biochemical function of the bacteria. So you can't really remove it from the bacteria and then remove it from your body. It's already in there. So now it's already killing the bacteria. It's supporting the growth of the Clostridia and all that. So my view on how to help yourself from glyphosate is to do things that fix the imbalance that glyphosate is causing. That's why I wanted to really emphasize what glyphosate does in the body to show that what glyphosate is essentially doing is increasing the growth of Salmonella, increasing the growth of Clostridia, increasing the growth of Pseudomonas, totally decreasing the growth of Bifidobacteria, for example, and then increasing pathogenic gram negative bacteria as well. So having a probiotic, like the spores, for example, that can go in and specifically bring down the growth of those pathogenic organisms and then feeding the bifido and the other species good organic fiber and things like that to increase their growth so they're more competitive can also help. So it's about fixing the ecology that glyphosate is disrupting. We're not going to be able to pull glyphosate out of the body.

Got it. And so can you speak to. Because another question that we'll get a lot is oh, if that's having low Bifidobacter is present in a lot of autistic Children's guts, for example. Then why don't we just take some Bifidobacter probiotics? Like, why are the spores going to be more effective than taking a Bifidobacter probiotic?

Yeah. And, you know, and bifidobacteria do a great job of colonizing in the gut within the first six months of life. So I would say, yes, take a good, well defined Bifidobacter probiotic in the first six months. After the first six months, the gastric passage becomes too arduous for bifidobacteria. You can't recolonize your Bifidobacteria levels with outside grown strains after the first six months. Studies are showing. In fact, University of California, San Diego is doing a lot of work because they're developing a particular Bifido infantis strain that has been shown to be able to undo some of the dysbiotic issues that occur with babies who are C section born or babies that have antibiotics right after birth by adding in this Bifido infantis. But it only works in the first six months of life. After the baby's six months and older, they can't get it to get in there and regenerate, repopulate. Most kids aren't diagnosed with ASD until about two and a half years because the symptoms don't come on until over two years old. And so at that point, it's too late to try to regenerate bifido. We need to do it in a natural way. We're going to be publishing a study. I think I mentioned this when I was talking, that the spores actually increase bifido levels quite naturally. So it is a way of generating bifidobacteria anyway.

Yeah. And so how many capsules of Megaspor do you recommend for autistic children specifically?

You know, so in clinics that we work with right now that have ASD kids, we always, we always tighter them up, taper them up. Sorry. The same way we do with adults who have real inflammatory conditions. We might start with as little as half a capsule every other day. Again, you have to mix it in the food because they're not going to take a capsule. And then you taper them and you try to get them to 2 caps a day. 2 caps a day is the most effective dose. Remember, at 838 days after birth or two and a half years after birth, you've got a full adult microbiome. Even though you're a little person, your gut is essentially the same as an adult. In terms of the density of microbes in there. So you still need the same kind of dose. So weight and size is not really an issue with the dose. You just want to taper them up, try to get them two caps a day. That's the ideal amount.

Perfect. And then on that note, how long do you recommend that people take Megaspore? Is it like a one to two month thing? Is it something they have to do for life? What's your recommendation?

Yeah, so with some of the ASD kids, what we've been able to see is, you know, you get them, you taper them up over a few weeks to get them to 2 caps a day. Once you get symptomologies under control. And that includes both behavioral issues and then GI issues too, because remember, as we mentioned, the GI issues go hand in hand with the behavioral issues. So once you get all of those things under control and you're seeing measurable improvements, the beauty of the spores is because they live in the gut, they create a real functional change. You can actually go to a lower maintenance dose. You can take it two, three times a week instead of every day. You still want to keep up with the two caps at a time, but maybe you could just take it on the weekends. But it's important, important to get some continuous exposure because all of the big changes that you made during the course of taking the spores can be undone from the negative things in the environment, like again, further glyphosate exposure if you're not constantly getting some level of exposure to the probiotic.

Right, right. We have another question about Megaspor. How did you come to choose the spore specific strains that are found in Megaspore? The question was, does it recognize the possibility that there are multiple microbiomes from different geographic zones? How do we know that just these five strains of Bacillus are going to work for everyone?

Yeah, so that's a really important question because that's one of the big struggles to me with probiotics is that everybody's gut is different, everybody's gut is unique. In fact, it's really hard to tell what a healthy microbiome looks like versus a non healthy, because what may present as an unhealthy microbiome based on a microbiome analysis for one person may actually be a healthy microbiome for another person. So what we were looking at when we were looking to develop a probiotic was looking for something we call keystone strains. Scientists have been looking for keystone strains, and the reason we call them keystone strains is if you know about architecture. You know, the keystone in an architecture is the thing that brings the whole arch together. That's a final stone in the middle with a curved angle. So that keeps the entire structure together. So the idea is that we are each born with a different set of microbes from mom. Right, so your mom gave you a different set of microbes than my mom did. But even the set of microbes that my mom gave to my siblings, even though those are the same, my sibling's body and immune system created their own signature bacteria from that same cohort of bacteria that they got. Now, however, all humans after they're born are exposed to the environment almost equally, or we should be. So the idea was that in the environment, we are more than likely exposed to these keystone strains, that we all get exposure to it at equal levels. We were looking for strains that would be present in all different parts of the world, would be present in the North Pole and the South Pole, in the Sahara Desert, in the Tibetan Plateaus. And so the significant studies that show that these particular spores, and not only are they present now in every corner of the Earth, they have been present in every corner of the Earth for the last three to five million years. And they prove these by glacial ice core studies where they drill out cores from the South Pole, the North Pole, the plateaus in Tibet, and they look at the parts of the core that represent the environment 3,5 million years ago, and they find that these spores are in very high levels in all of those levels. Sorry. In all those stages of human evolution. At the end of the day, what these are, is these are keystone strains that we're all supposed to get a significant amount of exposure to during the course of being human. Being born, being exposed to dirt, being exposed to the environment, eating dirt, drinking waters out of rivers and streams. And as it turns out, humans born and developing in all corners of the Earth seem to be exposed to the same kind of strains as well. We're not saying that these are the only probiotic strains you'll ever need. That's not true. And we're also looking at developing other strains down the road. But these seem to be really important keystone strains that do significant work within the gut.

Gotcha. Wonderful answer. Let's see. Another good question is, as far as supplementing goes, what is the best all around fiber for feeding beneficial bacteria? It's probably a good lead into.

Yeah, yeah. So I'm glad you asked that. And we didn't plant this question, but, you know, we've been looking at prebiotics and fibers on the market to recommend it to people. Because we get this question all the time. And again, our whole company's philosophy is if somebody is doing it really well, there's no need for us to do it. If it's an important thing that nobody's really doing quite right, that's when we get into that. We don't have a product development plan where we just kind of do the same things that everybody else is doing and try to compete. We just look for things of significant need. Prebiotics and fibers is an area that we found a significant need. Prebiotics, I would say from a therapeutic standpoint, are more important than fibers, because a fiber is basically any sort of roughage that gets into the gut and it can or cannot be fermented. It's not that specific. Prebiotics have to specifically be shown to be fermentable by certain types of bacteria within the gut. Right. So fiber your best to get it from your diet, just eating high fiber foods, things like roots and tubers and nuts and so on. Acacia could probably give you 100 different foods that are high in soluble and insoluble fibers. She's an expert in that kind of thing, among other things. But with prebiotics, there are very specific types of prebiotics. So there's something called degrees of polymerization when it comes to prebiotics. And that basically is their complexity of the structure of the prebiotics, Especially oligos like oligosaccharides, like fructooligosaccharides, galactooligosaccharides, xyloigosaccharides, These are all really effective prebiotics. But the shorter the degree of polymerization, the more those oligosaccharides will feed bacteria in the upper gi, in the small intestine. The longer the degree of polymerization, the further down the gut they go. So we were looking for products that had a lot of these effective prebiotics, really long degrees of polymerization, so that they make it all the way to the large bowel and specifically feed good bacteria. We did not find that in the marketplace, so we're creating our own. We're very close to finishing the formulation and flavoring it and all that. So we're creating what we consider to be the market first precision prebiotic. It has three or four different sources of prebiotic in it, but we've carefully selected the degrees of polymerization that are correct for getting into the large bowel and feeding key bacteria in the large bowel. Every one of the ingredients that we have in there has been clinically shown to specifically feed certain good bacteria like bifidobacteria, faecalin bacteria, prosnitzii, in some cases akkermansia, and so on. And so we have created this product. So coming to your clinic soon, you'll have a sample of our new precision prebiotic that's coming out.

Awesome. Thank you for that. And then there are a couple questions about high fat diets. You mentioned high fat diets. So we have people asking for your opinions on the ketogenic diet. Is coconut oil good or bad? That kind of question?

Yeah. When you look at endotoxemia and there's been studies and I typically, when I do my endotoxema talk, show this slide. They studied fats that have the most endotoxic response. And as it turns out, coconut oil has the response over things like olive oil or even vegetable oils. There's a few reasons for that. One is coconut oil also has antimicrobial effects. So this endotoxic response comes from the die off of bacteria during the digestion process and the release of their lps. If the oil also has antimicrobial effects, it's going to cause more die off during the digestion process. The other thing is coconut oil is also very high in saturated fats. And what happens is when the gram negative bacteria in your gut use dietary saturated fats to make lps, they make a more toxic version of lps. So then when that LPS is released and translocates from the lumen of your intestines into your circulatory system, you have a much more toxic version of LPS than if the bacteria used, for example, N3 fatty acids or N6 omega fatty acids. So omega 3 or omega 6 fatty acids. So that's a big difference. Now, the other reason why high fat will increase endotoxemia is because your body absorbs lps, which is a lipopolysaccharide, meaning it's a fat with a big fatty acid lipid tail and a little polysaccharide head. It absorbs it like it absorbs nutritional fat. When you take a high fat diet, you release a lot of bile salts and you release a lot of things called lipid carriers that are supposed to go into the lumen and, and grab fatty acids and bring it into the body for nutritional purposes. Now, because you're increasing the number of these rafts, these lipid rafts, it's also going to go and grab LPS and bring it in at a higher level as well. Those are the reasons why the high fat diet can cause more endotoxemia. I don't think people should be on a ketogenic diet until they are somewhat sure that they've fixed their leaky gut, because I think it can make it worse. You shouldn't have to be on a ketogenic diet for long periods of time. I think it's fine if you know your leaky gut's under control. You can go keto for a short period of time, maybe three, four weeks, just to change your metabolism, just to change things up a little bit. But they've shown that continuous high fat feeding actually lowers bifidobacteria levels quite a bit, lowers fecalum bacteria levels quite a bit. So it does cause a disruption in the microbiome. And again, our ancestors, the ancestors that we come from, for the most part, or most Europeans come from, were not exposed to high fat diets during the course of evolution. So our microbiomes aren't really designed for it. In contrast, people like Inuit, Indians have a microbiome that specifically evolved to consume a high fat diet because their diets are predominantly fat, especially from seals. That's the number one part of their food. So they have a diet that's designed to be able to handle that kind of fat. We don't, and most European descendants don't either.

Good point. So, on that note, talking about lps, is there anything that we can do that can help bind LPS in the intestinal lumen to keep it from getting into circulation? And what would that be?

Yeah, so a few things. One is. Well, a couple things, really. One is something called lactoferrin. Lactoferrin is a component of colostrum. But I have to sneeze one second. But lactoferrin, I'm allergic to this question and it went away. So thank goodness. I was going to make a really loud sound in the microphone, so I was going to pull off my things real quick and turn the camera and so lactoferrin is a component in colostrum, but in typical colostrum products, it's at a very low level, so you won't get a lot of lactoferrin activity, but you can buy lactoferrin by itself. Glanbia makes a good lactoferrin product that is sold by numerous retailers. So if you look up lactoferrin, it's interesting because lactoferrin is a compound component in colostrum that actually binds LPS in the lumen. The Other thing is secretory IGA can bind and neutralize lps. That's one of its functions. So anything that increases secretory IGA production can do that. For example, colostrum in itself can increase the production of secretory iga. Very interestingly, I saw some data this last week at the naturopathic show where I was speaking, that biocidin, which is a herbal antimicrobial product, they have a study that shows that some use of it can increase secretory IGA as well. So that's another thing that might be able to help neutralize some LPs.

Awesome. Okay, let's see the next question. You talked a little bit about C. Diff. Would you mind expanding on the C. Diff protocol? How many capsules you Recommend, people taking, etc.

Yeah, so the base, the basic C. Diff protocol right now is you're basically taking two megaspore caps. Twice a day is ideal, right? So you take two in the morning, two in the evening, and then with that you're taking a restore flora cap. So you're taking one in the morning, one in the evening, and you're also taking one cap of the HU58 in the morning and the evening as well. So you're getting a very high spread out dose of the spores, especially the HU58, which is in all three of the products, because that's the main powerful spore that can competitively exclude C. Diff and other pathogens. Now, of course, you don't have to be on this forever. That's a protocol you use for six weeks, seven weeks, eight weeks, until you feel that the C. Diff is under control. Then you can go to your regular maintenance dose of just megaspore as well. And to clarify, the restore flora has the spores with a Saccharomyces boulardii. Boulardii is great because it's actually been shown in clinical trials to be able to slow down the growth or suppress the growth of clostridium and Clostridium difficile in particular. So that's another thing with ASD kids, I would say is, you know, consider even adding one cap a day of the restore flora because of the. The ability of Saccharomyces to. To slow down the growth of Clostridia as well.

Perfect. Here's another.

Someone says repeat the protocol, please. So we'll write it up in the answer section and send it to you.

Oh, yeah, yeah, we'll send that to you guys. Fear not. Okay, another question. One of your favorite questions would be what's your favorite stool test?

Oh, my God. My favorite stool test, and I do this test almost every day, is you poop in the toilet and you turn around and look at what came out. That's the, that's the only surefire stool test there is. Exactly. And if it looks and smells terrible, then you know there's something wrong with it. But, but, you know, in general, we have tried various stool tests on the the market. If you're looking for a stool test that looks at the microbiota, there just isn't one that is that accurate. I did have a very interesting conversation with Viome last week. So V I O M e there's some promise there to their stool test being a better option than what you find through eubiome, Genova and all that stuff. And, and one of the reasons is because they're looking at more RNA and they have kind of a metabolic portion of their stool test. So it's a metabolomic portion because the types of bacteria and all that in there, it's hard to tell what's dysbiotic, what's not dysbiotic, and then remind me to mention something on accuracy as well. But what I like about the volume, which we're going to test out and see if it makes sense from a clinical trial perspective, is that messenger RNA and the other RNA that they're looking at, because it looks at things like butyrate pathway, gene activation, short chain, fatty acid gene activation, things with tight junctions, fat digestion, protein digestion. So it looks at really important metabolic pathways in the gut, which shows you whether or not your gut is functioning properly or not functioning properly. Here's the bit on the accuracy. I was speaking earlier this month at the Human Microbiome Congress, which is a great show because it's really kind of the top researchers on the microbiome are there one show, it's a mix of industry and academics and they're all presenting their latest research. So you learn a lot of amazingly cool stuff. There's a company there, I believe they're called Zymo, which did a study where they took this same exact stool sample and they sent it to five different stool testing companies, including American Gut. So they sent it to American Gut and eubiome and Genova and all these things. The exact same sample came up with five different reports, with some of them being dramatically different in terms of diversity, the growth of Bifidobacter and all that. So there's no accuracy in these tests because they're not standardized in any way. What's great about what Zymo is doing is they're trying to create a standardization protocol so that these companies can test their tests to make sure that it's actually giving you the accurate data. Just the same way we calibrate equipment, we calibrate biological assays, we calibrate immunohistochemistry assays, all of those things have some sort of calibration or standardization methodology, but stool testing doesn't have it yet. So I would say that the vast majority of stool tests right now are kind of useless. Except the one where you get up, you turn around and you look in the toilet and you see what happened.

Yes. Okay, so I think we'll just do one more question and then call it a wrap because I know you guys have been with us for a long while and we really appreciate it. So just for the people who don't know the difference between Megaspore and all the other probiotics on the market, can you give a brief synopsis of the difference?

So about Megaspore and the other products? Yeah, sorry. Okay. Yeah. So Megaspore is basically a spore based probiotic. It's the first multispore based formulation. What's interesting is we've painstakingly found and picked strains that are very well studied to colonize a human gut that have no toxicology. We've done full genomic sequencing on the strains and we've looked at all the proteins that they create. So we know that they don't produce toxins, we know that they colonize really well. And we also know that they play really friendly with the rest of the microbiome. That's a really important test to do. The vast majority of companies don't test what the impact of their probiotic is on the microbiome. And that was an important thing. What we see is that what we have is a bacteria, sorry, a formulation of keystone strains that are universal colonizers, that work in virtually everybody, including other animals. We're talking about mammals and creatures that live in the sea and insects. All are colonized by these kind of similar strains. And these strains basically act as your gut police. They go through and they modulate the microbiome, they suppress the growth of unfavorable organisms, they increase the growth of favorable organisms, they increase alpha diversity, they dramatically increase things like butyrate production in the gut. One of the strains sits in your intestinal lining and produces 12 different carotenoids. Astaxanthin, zeaxanthin, lycopene, lutein, CoQ, 10. All of these things that it produces at RDA levels, they produce digestive enzymes. So they're making a complete total reconditioning of the gut floor. And these are measurable, significant changes. What's exciting about Megaspore is we've also been the first probiotic, or really any nutritional product that has been shown in a human clinical trial to be able to resolve leaky gut in as little as 30 days. And with the resolution of leaky gut, also bringing down all of these significant inflammatory markers, markers that are all associated with the onset of diabetes, various types of cancers, cardiovascular disease, and so on. So it's a very different approach of probiotics, but it's been very successful. We've got nine different clinical trials going on right now, most of which will be completed by the end of this year. I think at least five of the nine will be completed by the end of this year and the other ones will probably be completed by the end of next year. One of the trials is estimated to take around two years, but we're spending a lot of money on clinical trials, but, you know, we feel we have to do that. We can't put a mark product on the market that we don't know what it's doing in the gut.

Exactly. Well, awesome. Thank you so much for everything, Karan, and for, as usual, blowing our minds. Thank you guys all for sticking with us for so long. It's been an hour and a half and I'm so impressed. There's still 145 of you here. Well, we are going to go, everyone have a lovely evening and we'll see you all in a month.

Thank you. Bye. Bye.

Bye.