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Nitric oxide and its possible implication in ME/CFS (Part 1 of 2)
Andrew Gladman explores the current and historic hypotheses relating to nitric oxide problems in ME/CFS. Part 1 of a 2-part series puts nitric oxide under the microscope and explores what it is, what it does and why it is so frequently discussed in the world of ME/CFS. Part 1 focuses...
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Nitric oxide and its possible implication in ME/CFS (Part 1 of 2)

Discussion in 'Phoenix Rising Articles' started by Legendrew, Aug 25, 2014.

  1. Legendrew

    Legendrew Content team

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    Legendrew submitted a new blog post:

    Nitric oxide and its possible implication in ME/CFS (Part 1 of 2)

    Andrew Gladman explores the current and historic hypotheses relating to nitric oxide problems in ME/CFS. Part 1 of a 2-part series puts nitric oxide under the microscope and explores what it is, what it does and why it is so frequently discussed in the world of ME/CFS. Part 1 focuses on what nitric oxide is, how its produced and what exactly it does ...

    Over the years there have quite a number of proposed disease mechanisms relating to nitric oxide (NO) problems of patients suffering with ME/CFS. Studies have however, over the years, proven somewhat inconclusive in the past.

    [​IMG]
    Dr. Pall is historically one of the staunch believers in the hypothesis that ME/CFS is a result of abnormal functioning of the nitric oxide cycle and the downstream effects this can have.

    Research studies exploring nitric oxide in ME/CFS patients has however been fractured at best with some showing too much nitric oxide, while others appear to show inefficient production of the chemical messenger.

    There have also been numerous theories relating to how nitric oxide could potentially induce autoimmune disease, a hot topic in the current ME/CFS research community. Equally, there have been theories proposing that ME/CFS could indeed be a result of an autoimmune response targeting the nitric oxide cycle itself.

    It is the hypothesis relating to autoimmunity that this article focuses primarily upon. It's easy to see why this area of research has become somewhat stagnant, given the competing hypotheses proposed!

    Because of the conflicting evidence, nitric oxide and its many related hypotheses have been stuck somewhat in hypothesis limbo. However, recent studies appear to have revived this somewhat ailing line of research and could potentially point towards a novel subgroup in the heterogeneous ME/CFS cohort.

    Over the course of Part 1 and Part 2, I will aim to explore the role and function of nitric oxide, the role the endothelium plays, how the hypotheses relating to nitric oxide could explain these symptoms of ME/CFS, and the current evidence to support these hypotheses.

    What is nitric oxide and what are its functions?

    Nitric oxide (NO) has been discussed quite extensively in many chronic diseases. Only within the last decade have the numerous functions that nitric oxide plays begun to be fully understood.

    Firstly there is the role of nitric oxide as an endothelium-derived relaxing factor (EDRF) controlling the dilation of blood vessels and therefore having a major role in vascular tone. The endothelium in itself is an important part of this new hypothesis and is much more complex than it would first appear. It is therefore discussed at greater length in the following sections of this article.

    Further to this, nitric oxide acts as an intracellular messenger and neurotransmitter, as such it plays quite an important role in the nervous system, with the brain being the organ containing the highest level of nitric oxide synthase enzymes.

    Further roles of nitric oxide include functions within the immune response. Nitric oxide can be produced directly by phagocytes, acting as a toxin to pathogens through its ability as a free radical to damage DNA. Furthermore, it also acts as an activator and regulator for some cells of the immune system.

    Further, ME-relevant functions of nitric oxide include a major role within the genesis of the mitochondria. NO in this instance is hypothesised to act as a universal "trigger switch" -- triggering the initiation of mitochondrial biogenesis -- the production of new mitochondria.

    Mitochondria is an area of research that has demanded a lot of attention the field of ME in the past with numerous papers proposing dysfunctional mitochondrial, and therefore slow cellular genesis of ATP as a central mechanism with ME.

    Finally, nitric oxide has a central role in the process of sensory sensitivity of neurones within the sensory nervous system. NO activates potassium/ATP channels on the surface of the neurone and hence has direct effect on controlling the transmission of nerve impulses (action potentials). It should also be noted that this list is non-exhaustive. There are further roles of nitric oxide which are still not fully understood!

    Production of nitric oxide (NO)

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    Image showing where the ornithine cycle connects to the citrulline-NO cycle

    Nitric oxide is produced through a specialised sub-loop of a cyclical series of biochemical reactions known as the ornithine cycle (also known as the urea cycle).

    The ornithine cycle is active in hepatocytes (liver cells) and is the cycle through which the harmful ammonia produced through the breakdown of proteins is converted into urea which is then excreted through the urine.

    This side loop is known as the citrulline-NO cycle and is active in cells where production of nitric oxide is required for the functions discussed previously. This sub-cycle and the importance of these enzymes other than nitric oxide synthase is only recently being researched and understood in all its subtle complexities.

    [​IMG]

    Human inducible nitric oxide synthase enzyme (iNOS)
    By A2-33 (Own work) [CC-BY-SA-3.0], via Wikimedia Commons

    A logical hypothesis to individually make could be that an autoimmune response or perhaps damage from an infection could be targeted directly towards the enzyme nitric oxide synthase (NOS), which as the name suggests has the role of producing NO via the aforementioned cycle.
    This idea is something we will continue to loosely explore in this article. We will discuss much deeper the implications of this in the second part of this article series.

    There are 3 different types (isoforms) of nitric oxide synthase in humans: neuronal in the nervous system, inducible in phagocytes/cardiac tissue and endothelial in the endothelium, aptly abbreviated to nNOS, iNOS and eNOS respectively.

    The structural similarities of these three enzymes likely means that an autoantibody targeted at one would likely be able to bind to and hence induce the same response in any of the enzymes. This may perhaps explain the wide range of seemingly unrelated symptoms that ME patients experience.

    Because these enzymes are a specialised protein with a unique ability to catalyse reactions, their shape is incredibly important, specifically the 'active site' which is where the binding and reactions the enzyme oversees occur. The binding of an autoantibody changes this vital shape of the enzyme's active site through altering the bonding and folding within the larger structure.

    Simply put, an enzyme is like a highly specialised machine with unique parts that it needs to function. The binding of an autoantibody would serve to change the shape of these specialised parts, rendering the machine incapable of producing the required volume of nitric oxide.

    Interestingly, research has shown that iNOS appears to be functioning normally and perhaps even overproducing NO. This seems somewhat contradictory to the hypothesis that nitric oxide is being underproduced. However in general, nitric oxide synthase is grouped into two categories, inducible and constitutive. iNOS is in the inducible category while nNOS and eNOS are in the constitutive category.

    Further to this, both nNOS and eNOS are known as calcium-dependent as they require the binding of calcium to produce nitric oxide. iNOS, however, is described as being calcium-insensitive further dividing nNOS and eNOS from iNOS.

    Given this information it is easier to understand why dysfunction of NO production by nNOS and eNOS could occur while the exact opposite appears to be true for iNOS. This idea of dysregulation of nitric oxide production has been explored previously as a method of disease action in ME.

    There is quite a volume of evidence supporting that, whether directly or indirectly, the production on nitric oxide is severely hindered. However, the mechanism through which this occurs is likely more convoluted than it first appears, meaning researchers have to be a tad more crafty in their hypotheses, mechanisms and conclusions.

    Function of the endothelium

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    Diagram showing endothelial location in arteries, veins and capillaries.
    By Kelvinsong (Own work) [CC-BY-SA-3.0], via Wikimedia Commons

    Endothelial cells are incredibly important within the human body. They are primarily found lining the interiors of blood and lymphatic vessels, being present throughout the entire circulatory system, from the heart down to a single thin layer which line the capillaries.

    There are many functions provided by these cells including control of blood pressure through vasoconstriction and vasodilation, formation of new blood vessels (angiogenesis), providing a semipermeable barrier between the blood vessels and the surrounding tissue, while also aiding in the process of blood clotting.

    The large number of unique and important functions has led some researchers and scientists to regard the endothelium as an independent organ within the body.

    There are also highly specialised and differentiated endothelial cells elsewhere in the body performing important filtering tasks, examples being the blood-brain barrier and the renal glomeruli.

    The novel approach of considering the endothelium as a dynamic organ aids in the logical understanding of these cells providing a target for an autoimmune attack. The large number of functions performed by the endothelial cells mean that the cells present quite a variety of unique extracellular proteins, any of which could act as targets for an antibody mediated autoimmune response, with nitric oxide synthase being the obvious choice considering the hypothesised importance of NO in ME.

    Many of the criticisms regarding the autoimmune hypothesis as a cause of ME/CFS relate to the lack of evidence of any measurable tissue damage caused. This new hypothesis could explain why this is so. As such, a mechanism explores the potential of a non-tissue damaging and non-inflammatory autoimmune response.

    Furthermore, such a mechanism could potentially explain the wide ranging systemic symptoms. Current testing methods for endothelial dysfunction are highly specific, involving fairly complex techniques such as ultrasound flow-mediated dilation (FMD) among others, which themselves do not have complete efficiency.

    Any endothelial abnormalities would be difficult to detect using standard diagnostic medical techniques, and endothelial function is currently unlikely to be tested in ME patients.

    One of the major functions of the endothelium is the control of vascular tone, meaning the degree to which a blood vessel is dilated. This is achieved through processes known as vasoconstriction and vasodilation. The endothelium plays a dynamic role in both processes through the production and release of many different vasoactive chemicals.

    To control vasodilation, the endothelium releases endothelium-derived relaxing factors (EDRFs) such as nitric oxide, while to control vasocontriction the endothelium releases endothelium-derived contracting factors such as thromboxane and endothelin.

    These chemicals are produced and released directly by the endothelial cells, with the end result being stimulation of the surrounding smooth muscle to either relax or contract, hence dilating or contracting the blood vessel. Through this process of homeostasis, blood pressure can be tightly controlled while this mechanism is also used as an important mechanism in internal heat and temperature regulation.

    If this hypothesis proves correct, NO production is disrupted meaning that vasocontriction likely dominates. It is then logical to assume that sufferers of ME have a lowered ability to regulate heat through vascular means. This is clearly supported by the heat intolerance and symptom flares many ME patients experience in both extreme warm and cool weather.

    A potential hypothesis relating to underproduction of NO does therefore allow for the observation of quite marked dysregulation of vascular tone and endothelial function without any direct or measurable damage to the endothelium itself.

    Thankfully this means that the increased risk of atherosclerosis many other autoimmune conditions such as lupus, diabetes and rheumatic diseases unfortunately suffer with, could appear to be very minimal or non-existent in ME.

    For further discussion on the cardiovascular system in ME/CFS looking beyond nitric oxide, be sure to check out the recent article published here at Phoenix Rising.

    Stay tuned for the second article in this series where we dig deeper into nitric oxide hypotheses in ME/CFS.



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    Continue reading the Original Blog Post
     
    Last edited by a moderator: Aug 25, 2014
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  2. alex3619

    alex3619 Senior Member

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    Nitric oxide is probably important in ME and CFS. How important is what the debate is about.

    Its also connected to B12 metabolism, especially via adenosyl cobalamin.

    In 2000 Vance Spence showed that we have exaggerated activation of an enzyme called calmodullin (indirectly, by measurement of vascular response). This is a calcium dependent enzyme needed in the path to trigger NO synthesis in the endothelium.

    There is no doubt of the involvement of NO and peroxynitrate in ME. The molecular pathways are established. What we do not know, so far as I am aware, is the degree of impact this has, which could be anywhere from trivial to causative. That is why there is debate.

    NO and ONOO (perxynitrate) have profound impact on the mitochondria. NO tends to inactivate key enzymes, whereas ONOO destroys them. The ones typically targeted have an iron center. NO also increases arachidonic acid metabolism via promoting cyclooxygenase, the enzyme that is used to make series 2 and series 3 eicosanoids from arachadonic acid (for series 2 eicosanoids, not series 3). Series 2 eicosanoids are mostly (but not all of them) pro-inflammatory.

    My knowledge of this area is way out of date. Science advances, but my reading speed is still slow.

    PS Caveat: my comment on calmodullin might be due to my own interpretation, and not Vance Spence's. I have not gone back and checked the original paper.
     
    Last edited: Aug 26, 2014
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  3. Leopardtail

    Leopardtail Senior Member

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    Two points:
    Nitric Oxide Synthase can produce either Nitric Oxide : NO or Nitrogen Peroxide: ONOO or Superoxide: OO with varying levels of BH4 depletion.
    This in addition to alex's Cobamide comment above.

    NO can also be produced by 'superoxide dismutase' - hence perturbed levels may result from over production of OO, or enzymatic dysfunction.

    Nitric Oxide like ATP is also a 'co-transmitter' required for some Neurotransmitters to function. It has intracellular roles beyond vasodilation and immunity.
     
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  4. Sasha

    Sasha Fine, thank you

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    Something on NO/ONOO in Cort's blog today (on Dr Cheney's treatment protocol):

    Dr. Cheney recently noted that hydroxyB-12 was voted the single best treatment for CFS in a large survey of CFS cases (> 1000). Neither cyanoB-12 nor methylB-12 have the properties of hydroxyB-12 as far as detoxification and redox control and cyanoB-12 cannot be used at high dose as it carries cyanide.

    It is also the form of B-12 used in the famous study done by Dr. Newbold, a NYC psychiatrist, that demonstrated neuropsychiatric benefits at high doses (60 mg per day SQ/IM) over a six-month period. Newbold’s late 1990’s study showed 60mg of daily HB12 helped with a variety of mental illnesses. B-12 plays a major role in Dr. Cheney’s protocol

    The benefits Dr. Newbold saw were impressive and led him to speculate that B-12 plays a central role in all psychiatric diseases regardless of type (as well as brain trauma). B-12 also supports the methylation cycle, a significant portion of which is controlled by the redox state of the cell.

    Dr. Cheney believes proper methylation is key to good brain functioning and sleep. Hydroxy-B12 is the only form of B-12 that can control redox via its binding to nitric oxide (NO). (The term redox state is often used to describe the balance of GSH/GSSG, NAD+/NADH and NADP+/NADPH.

    It also includes processes involving free radicals). When hydroxyl-B12 binds to NO it eliminates the beginning of the NO-ONOO cycle which Martin Pall believes occurs in ME/CFS. NO binding in the presence of elevated superoxide prevents the formation of peroxynitrite (ONOO) – allegedly the most dangerous of all free radicals. It also binds to CN (Cyanide): hydroxyB-12 is the treatment of choice for cyanide poisoning.

    HydroxyB-12 binds the nitrogen residues of toxins in the blood and carries them out in the urine and skin. Cheney believes that redox control may be dose-dependent in the same way that treating cyanide poisoning is. Too little hydroxylB12 and you die of cyanide poisoning.

    This reflects Dr. Cheney’s belief that the importance of B12 in ME/CFS has nothing to do with B-12 blood levels (aka nutrition) and everything to do with scavenging toxins (i.e.. free radicals). Just as you would never measure B-12 blood levels before using it to counteract cyanide poisoning, he would never measure B-12 blood levels before using it to affect free radicals.

    HydroxyB-12 is being used to affect the redox state and therefore the methylation cycle; it has nothing to do with B-12 levels.

    Read more: The Cheney Chronicles #2: His Protocol For Chronic Fatigue Syndrome http://www.cortjohnson.org/blog/2014/08/25/cheney-chronicles-protocol-chronic-fatigue-syndrome/

    Is it just me, or does anyone else read "NO/ONOO" and see "No! Oh noooo!" :eek:
     
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  5. alex3619

    alex3619 Senior Member

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    That is not a coincidence, it was intentional. You are supposed to see that as a catchy phrase.
     
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  6. Gijs

    Gijs Senior Member

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    There are so many hypotheses. I get a little 'tired' of them. It could be true but prove it.
     
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  7. Legendrew

    Legendrew Content team

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    I feel your pain but this is the modern world of science I'm afraid. For every result there are many different hypotheses that can be drawn and only further tests can then decide whether any of these hold any water and from there the hypotheses that do hold true have to be modified, retested and so on it continues. Nothing is ever really proven, we just eventually just reach a stage where every test done agrees with the current hypothesis so it becomes the leading one but that's not to say a test in the future will not come along and blow all of that out of the water.

    Only by speculating upon hypotheses and testing these will we ever get anywhere but I do agree that with ME/CFS we perhaps deserve more answers and less hypotheses! Hopefully we're getting to that point.
     
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  8. Legendrew

    Legendrew Content team

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    The more I hear and read about nitric oxide the more and more important it seems to be for nearly every system in the body (something I believe I touch upon more in part 2/2). It seems to be one of those areas that there is a lot of buzz around in the scientific community and I can see why: research seems to be advancing at such a rate that some of this article written last month may even be out of date. This article is my attempt to give a glimpse into this world and I'm more than open to admitting that I have barely scratched the surface! This I suppose is my attempt to try and keep it reasonably simple, which I think speaks to the complexity of the processes involved and the emerging importance of NO.
     
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  9. Leopardtail

    Leopardtail Senior Member

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    It's true of many things at the intra-cellular level. Many metabolites have tens or even hundreds of functions each of which supports multiple others etc....

    It's not a co-incidence that ME is hard.
     
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  10. Leopardtail

    Leopardtail Senior Member

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    It would be nice to see your sources in these article Andrew - it would stimulate further research and analysis.
     
  11. Legendrew

    Legendrew Content team

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    You know, I did used to list them as references at the end of the text but I switched to linking in the body of the text for ease of access to internet browsers and because of this some get cut out but I think if I do an article similar to this in the future I'll go back to the old 'references' at the bottom.

    EDIT: After checking, I do have a couple of 'further reading' articles at the end of the second part from which I sourced some information. Forgive me for not remembering but this article has been on and off for quite a while from the start of this year.
     
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  12. Leopardtail

    Leopardtail Senior Member

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    Ooops, seen it now, was assuming you were using research papers.....
     
  13. Legendrew

    Legendrew Content team

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    Yeah, most of it is. Often the linked articles in turn link to a research paper. What I should do really is link to all the research articles directly for those interested in reading up themselves. As i said I try to keep these articles a little lighter to read but with something like this I can understand the desire for the sources and I'll be sure to include it in the future. I normally have a dozen or more things open at once while writing these so I often don't reference as much as I should. A problem unique to this article is that because it's been ongoing for many months a lot of the references have since been moved, deleted etc from the original link so I had to cut a lot myself due to broken links and I believe a few more were lost when in review stage for the same reason.
     
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  14. alex3619

    alex3619 Senior Member

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    That is essentially my own experience when writing blogs.
     
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  15. Aerose91

    Aerose91 Senior Member

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    One of the first things I noticed when I got M.E., the very first day, was that my veins collapsed. I used to have garden hoses for veins now it looks like indentations where they used to be.
     
  16. alex3619

    alex3619 Senior Member

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    I have had ME for probably a very long time. Yet only last year my veins all but disappeared. It took decades, but suddenly only the most experienced phlebotomists can find them. Sadly that is not most of them.

    Collapsed veins might be due to a crash in blood volume, but a failure of NO synthesis by the endothelium is another possibility.
     
    Last edited: Aug 27, 2014
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  17. Aerose91

    Aerose91 Senior Member

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    I guess I just associated it with NO because during my bodybuilding days we used to take arginine (to increase NO in the blood) and it would make me insanely vascular.
     
  18. Leopardtail

    Leopardtail Senior Member

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    Indeed the blood volume thing crossed my mind.. as did the idea that due to low blood volume the reduced NO production might be a correct physiological response in order to maintain local blood pressure. Obviously it's equally possible that overproduction of OO, ONOO in place of NO is causing the same effect. ME is one massive box of puzzles isn't it?
     
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  19. alex3619

    alex3619 Senior Member

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    Yes, nested feedback loops always have to be considered. The combinations of factors we know about, plus probably more we don't know about, make any scientific advances difficult. With so many things going on in our bodies, how do we separate causes from responses, especially when there are dynamic feedback loops?

    Advances in systems biology give us some tools. We can hope that such advances continue.
     
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  20. Leopardtail

    Leopardtail Senior Member

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    This is something I have been giving considerable thought to recently. For now I separate the major 'fly in the ointment' that way may be many illnesses under one umbrella - that has a different solution.

    Just dealing with the many factors affecting one problem - we need to test more than one variable per patient, and test each patient in multiple states (the later is harder to achieve). E.g. we know NO production depends on BH4, Cobamide, Arginine - if we are going to investigate we need to check all three along with NO, OO, ONOO then retest the later three after a litre of IV saline. We then know the biochemical picture and the impact of a very common problem. Thus we have the most immediately relevant variables all at once and can start to identify the variations between patients and get a better picture of that one problem. If we can clearly identify 30% have too much, 10% are just right, 60% have too little NO then we have our first piece of solid data. Likewise the ability to say 15% have low cobamide, 15% have low BH4, 15% have very low BH4 then we start to make tangible progress. The ability to correlate three variable each with two or three significant states gives perhaps 8 or 27 seven permutation, whereas one variable gives two or three with no ability to check cause -> effect.

    Similar things need to be done across as well as within fields of science, here I gave the example most relevant to the thread.

    well those are my thoughts at least
     

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