"The something in the blood" that some scientists have made reference to, will be possible to track down, but it will take a good deal of biochemical expertise and time. This is why I am not worried we didn't hear anything about it at the Stanford symposium. EV's in general are extremely difficult to isolate. Sure, there are protocols out there, but doing it well and having a pure prep takes a lot of experimentation. Ron Davis alluded to this during his NIH talk in April 2019. My sense is they are working on it, but it will take someone a year to get good preps on 20 people, then they will go about the process of trying to identify the peptide or RNA molecule that could be triggering changes in target cells when the target cells are hydrolyzing ATP at a rate that is greater than the ATP required for just cell survival alone.
Third Annual Community Symposium on the Molecular Basis of ME/CFS Sponsored by OMF - DISCUSSION
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nandixon
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That's why they need to devote more resources to it and not be spending resources on fishing expeditions and reinventing the wheel.
I'm worried about not hearing even a single mention of what should obviously be priority number one after a whole year has passed.
They're not extremely difficult to isolate. Note that there's no need to have a pure exosome isolate initially. A crude isolate (e.g., from ultracentrifugation) is good enough for the initial purposes of (1) simply seeing if a generic isolation procedure for exosomes yields a positive result for the "something in the blood" in the nanoneedle - they should have this information already(!), and (2) fractionating the crude exosome contents using liquid chromatography methods to see if the "something in the blood" happens to be in the cargo of the exosome.
Obtaining the actual pure exosome "species" itself might only be necessary in the event that the intact exosome is needed to give a signal to the cell via an exosome surface marker (or because an active cargo component needs help entering the cell) in which case size exclusion chromatography can be used to separate the desired exosome from other extracellular vesicles.
When they fail to mention it at all after a whole year my sense is that they are not working on it.
They don't need preps from 20 people. They only need the plasma from a single ME/CFS patient who has a positive nanoneedle signal in order to begin narrowing down what the "something in the blood" is.
wigglethemouse
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Just a word of caution. We need an instrument that can scale and can produce repeatable results. Without that, it's not clear if all measured results are valid. It has been stated in the past that the nanoneedle needs a single professor to make "good" sensors and a skilled person to run and interpret the results. And that professor is now at a different University for the majority of his time. The bottleneck seems to be nanoneedle throughput - which requires an engineering effort to address along with funding, and possibly a few prototype iterations.
That's why I'm hoping commercial equipment such as the CellKey cellular impedance analyser used by Dr. Alain Moreau might be able to be used to run the salt stress tests, or a modified salt stress test such as measuring oxygen consumption rather than impedance using Agilent MitoXpress Xtra Oxygen Consumption Assay. Both instruments have multi-well plate systems that allow many experiments to be run simultaneously, including having measurement control samples which is kind of important. Commercial equipment also brings in the ability for other research groups to replicate the results.
Also, what if for a hypothetical example say 50% of diseases with immune dysfunction cause the change in impedance. Some quick tests on other disease groups should be run as well. We still don't know the answer to that.
So reading between the lines of what is said and not said, I don't think isolating the factor in the blood is so simple as we think. Fluge, Karl Morten, Bhupesh Prusty, Alain Moreau have not made much progress in isolating the factor in the blood either that they have seen in their experiments.
Karl Morten in the recent Q&A on s4me stated he couldn't repeat his plasma swap results with plasma from the UK ME biobank and questioned how could the collection and storage methods, type of tube etc, affect the experiment. He also found different cell types behaved differently and did not know what to make of that (plasma + cancer cell vs plasma + muscle cell).
Saying that, I do share your disappointment @nandixon. We all want it to go much faster.
That's why I'm hoping commercial equipment such as the CellKey cellular impedance analyser used by Dr. Alain Moreau might be able to be used to run the salt stress tests, or a modified salt stress test such as measuring oxygen consumption rather than impedance using Agilent MitoXpress Xtra Oxygen Consumption Assay. Both instruments have multi-well plate systems that allow many experiments to be run simultaneously, including having measurement control samples which is kind of important. Commercial equipment also brings in the ability for other research groups to replicate the results.
Also, what if for a hypothetical example say 50% of diseases with immune dysfunction cause the change in impedance. Some quick tests on other disease groups should be run as well. We still don't know the answer to that.
So reading between the lines of what is said and not said, I don't think isolating the factor in the blood is so simple as we think. Fluge, Karl Morten, Bhupesh Prusty, Alain Moreau have not made much progress in isolating the factor in the blood either that they have seen in their experiments.
Karl Morten in the recent Q&A on s4me stated he couldn't repeat his plasma swap results with plasma from the UK ME biobank and questioned how could the collection and storage methods, type of tube etc, affect the experiment. He also found different cell types behaved differently and did not know what to make of that (plasma + cancer cell vs plasma + muscle cell).
Saying that, I do share your disappointment @nandixon. We all want it to go much faster.
nandixon
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I think it's almost certainly going to be the case that a number of other diseases are also going to give a similar impedance signal. But that's okay because it's not important that the nanoneedle be able to be a diagnostic tool to differentiate diseases. It just needs to be used as an analytical tool to help isolate the "something in the blood" in ME/CFS patients that is causing the cells to respond to salt stress with an increased impedance signal. The other diseases are likely going to have different problems that lead to their increased impedances.
I'm afraid they're not making any attempt to try to isolate it - either because natural substances isolation is not in their repertoire or not where their scientific interest lies. I'm afraid it's also possible too that they may not fully comprehend the importance of identifying what the "something in the blood" is.
I think I understand the challenge pretty well. I probably spent at least 20% of my time as a research medicinal chemist using very similar or identical chromatographic techniques to what they'll need to use.
It may be best just to farm the isolation out to some other group that has experience doing that. (The pdf file that I attached earlier for the especially fast and simple exosome isolation procedure was from a group who was doing the same thing for sarcoidosis.)
(Note that I think the nanoneedle may be measuring mitochondrial membrane potential, either indirectly through its affect on the cellular plasma membrane potential, or some combination of both indirectly and directly because of how closely spaced the electrodes in the nanoneedle are and because of the large volume the mitochondria occupy in the cell. If that's true then there are several other methods that could be used to measure the impedance that another group could use for purposes of fractionating out and isolating the "something.")
Thanks, I recently had my 20th "anniversary" of living with this nightmare and I'm more than tired of it.
Has anyone seen the NIH grant application regarding the nano-needle? I assume that the application is for funding to develop the nano-needle as a diagnostic test - correct?
I think they should try to identify the something in the blood; I suspect the OMF group already have the capability to attempt to do that. E.g. Jonas Bergquist leads a research group with experience in protein identification and I assume that Jonas's lab. has Mass Spectrometry equipment which is at the top end of range. Also, even if there were a research group better suited to this task, OMF would be aware of that group.
Could @Ben H try to get a response i.e. whether they are working on identifying the "something in the blood"/not looking (and if so why) ---?
I'm in favour of developing the nano-needle as a diagnostic test and the identification of the "something in the blood" - until that "something" is identified I assume that it will be more difficult to access treatments e.g. re-purposed drugs.
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Talk's are in two videos (Part 1 & Part 2) here's a link to Part 1 which I think has Maureen's talk:
Part 1: Community Symposium on the Molecular Basis of ME/CFS
Check out Bupesh Prusty's talk at NIH Conference in April - he appears to have demonstrated mitocondrial fragmentation in ME (no data on incidence). Virus's can cause Mitochondrial fragmentation e.g. HHV-6; problem is Ron Davis is not finding evidence of DNA from HHV-6 etc. Possibly a search for RNA virus's might turn up something.
Hanson hypothesises that it could be a hit and run virus or a chronic virus (back to comments from Ron - no viral DNA!).
So the viral theory persists --- without any viral DNA! Possibly Bupesh's work will help to clarify.
I was eagerly waiting to watch the presentation of Jarred Younger but it did not happen. Why ? Is it that the sponsor wants to publish it in a journal or did the researech not happen as expected ? Why was his name on the list of speakers and then it did not happen ? This year's talks were more about the technology side and less of hardcore medical presentations. So I am disappointed. If funding is the issue for OMF then why have 70 + researchers on the panel ?
nandixon
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I'm just putting this out very quickly as it is quite late. There is a very interesting and potentially very exciting connection between Paul Fisher's work[1] showing an impairment in ATP synthase in ME/CFS immortalized lymphocytes and Bhupesh Prusty's work[2] showing increased fission (and decreased fusion) in mitochondria treated with ME/CFS serum, possibly due to HHV-6A (and potentially other herpes viruses like EBV and CMV).
This connection is telling us that the 'something in the blood' may be targeting ATP synthase. In particular, that it may be targeting and down-regulating a subunit of ATP synthase known as ATP5B.
It turns out that ATP5B regulates mitochondrial fission and fusion in mammalian cells.[3]
If it is the ATP5B subunit being specifically targeted then that makes the 'something in the blood' more likely to be a microRNA (perhaps being carried by an exosome) versus a peptide, for example, which might be more likely if other parts of ATP synthase were being targeted.
Prusty did find a microRNA that HHV-6A promotes the expression of and that causes mitochondria to fission.[2] This microRNA may be targeting ATP5B.
(There is at least one compound, a small molecule known as M1 that may be capable of reversing such a down-regulation of ATP5B.[4])
[1] https://forums.phoenixrising.me/thr...lized-lymphocytes-from-me-cfs-patients.77577/
[2] https://www.healthrising.org/blog/2019/04/15/nih-chronic-fatigue-lipkin-davis-prusty-oh/
[3] https://www.tandfonline.com/doi/full/10.1080/19768354.2016.1188855
[4] https://www.ncbi.nlm.nih.gov/pubmed/22907892
Edit: I emailed Dr Fisher to take a look at this possibility.
Edit 2: Hmm, I just took another look at Dr Fisher's paper and he had found ATP5B to be upregulated. Not sure if that can be reconciled or not.
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OMF has collected already almost $ 20 mill .By far more than any other ME Research group to make fair comparisons.
You might want to check out this article on Health Rising [https://www.healthrising.org/blog/2019/09/16/alzheimers-chronic-fatigue-fibromyalgia/]
Turns out a lot of Alzheimer's research may re-focus on neuro inflammation (Jarred Younger's chief area of interest I guess).
Having 70 researchers sounds good; they still haven't identified the underlying disease mechanism, so having a wide range of researchers (research areas) seems to make sense.
No idea why Jarred wasn't at the meeting/didn't present.
Thanks - you're way ahead of me.
Check out Ron's talk at the Symposium, where he refers to SS-31, apparently it changes the mitochondria from being really disorganised to being organised. So I'm wondering if this is an indication that Prusty's found something significant i.e. mitochondrial fragmentation; also, possibly there's a drug which can reverse it - SS-31.
nandixon
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Yes, I did a post a couple weeks ago on using SS-31 on the thread for Dr Fisher's ATP synthase (Complex V) impairment finding here:
https://forums.phoenixrising.me/thr...ytes-from-me-cfs-patients.77577/#post-2229686
It may be our best bet right now.
nandixon
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Here's a theoretical hierarchy of 'what may be causing what' in ME/CFS based on supporting scientific literature and the findings so far (with the associated researcher in parentheses):
Pathogen such as HHV-6A (Prusty) ---> Exosome containing microRNA (Ron Davis & Prusty, the 'something in the blood') ---> ATP synthase inhibition (Fisher) ---> Cellular impedance change due to change in mitochondrial membrane potential (Ron Davis, nanoneedle results) ---> Mitochondrial fission (Prusty)
If a pathogen has chromosomally integrated, like HHV-6A is capable of, I'm not sure where that puts us at in terms of trying to address the ultimate source of the problem. Hopefully it might work just to address ATP synthase inhibition for treatment purposes.
(Note than an actual pathogen is not the only potential cause of an inhibition of ATP synthase and subsequent mitochondrial fission.)
And again, the existence of an exosome has not been proven yet, I don't believe.
Pathogen such as HHV-6A (Prusty) ---> Exosome containing microRNA (Ron Davis & Prusty, the 'something in the blood') ---> ATP synthase inhibition (Fisher) ---> Cellular impedance change due to change in mitochondrial membrane potential (Ron Davis, nanoneedle results) ---> Mitochondrial fission (Prusty)
If a pathogen has chromosomally integrated, like HHV-6A is capable of, I'm not sure where that puts us at in terms of trying to address the ultimate source of the problem. Hopefully it might work just to address ATP synthase inhibition for treatment purposes.
(Note than an actual pathogen is not the only potential cause of an inhibition of ATP synthase and subsequent mitochondrial fission.)
And again, the existence of an exosome has not been proven yet, I don't believe.
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Thanks @nandixon - you're obviously more knowledgeable on this than I am.
I'm not sure what to make of Ron Davis's assertion that you don't find viral DNA (there's actually more in the controls) with the theory that a "hidden" virus is causing this disease. If the virus is re-activated then would viral DNA start appearing in the blood? So how do we resolve the "no viral DNA" result with the theory that there is a chromosomally integrated virus (which is relevant)?
Possibly one way to progress this is to establish how widespread mitochondrial fragmentation is e.g. do a much larger study (Bhupesh Prusty).
I'm not clear what you mean regarding exosomes. From memory Maureen Hanson has demonstrated an increase in the number of exosomes (data presented at OMF Conference?). Also, the "something in the blood", i.e. causing the change in cellular energy production, is the size of an exosome - not conclusive of course.
ljimbo423
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The type of testing Ron did was very interesting. Ron and his team used a PCR technology called "multiplex". Which is a HIGHLY sensitive PCR test and tests multiple regions on the DNA of viruses found in the blood, to assure there are no false readings.
Ron explains the method and results at 27 minutes into this video-
EDIT- If I understand the chart right. They tested 5 regions on the HHV-6 DNA they found, to make sure there were no false negatives or positives.
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nandixon
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Yes, that was partly why I did the exercise of showing how everything *might* fit together. Note in particular in the hierarchy of findings that Fisher's finding of ATP synthase impairment might be the most immediate cause of the mitochondrial fission that Prusty found. This means that even though HHV-6A can cause such fission, that it may merely be a coincidence, and that ATP synthase is impaired (and hence there is mitochondrial fission) for some other reason. That's why I wrote:
On the other hand, Davis is studying the most severely ill patients for viral DNA purposes. They're so severely ill, in fact, that some may actually be suffering from catatonia rather than ME/CFS (although both these illnesses may be on the same spectrum for all we know). Thus I'm not sure how completely applicable pathogen findings are from that cohort.
I'm just meaning that as far as I'm aware that Ron Davis just used a series of filters to narrow down the potential size of the something in the blood to being within a size range, and exosomes apparently fall within that range. But that doesn't mean the 'something' is an exosome.
Hanson finding an increase in the number of exosomes doesn't mean of course that exosomes are the something in the blood. And her p-value is weak. But if these researchers really wanted to find answers as quickly as possible they would either:
1. Courier over Hanson's exosome isolate to test in Ron's nanoneedle;
or if that's not feasible then
2. Have Hanson employ an alternative impedance testing method at her end (there are several that might possibly work as a substitute for the nanoneedle) to see if her exosomes when dissolved in healthy plasma cause healthy cells to give an increased impedance signal with salt stress.
Edited to add: Just to mention that for myself, being a former medicinal chemist researcher, that when I think of the 'something in the blood' I'm always thinking in terms of not an exosome (if one is in fact involved) but rather the chemical entity (microRNA, peptide, or surface marker) that the alleged exosome is either carrying and releasing or delivering or signaling with. Because knowing that will potentially lead to a treatment.
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I value your posts @nandixon.
Ron Davis will know severe ME because he lives with someone with very severe ME.
I may have misunderstood you @nandixon, but even if you mean by "catatonia" another disease, we shouldn't split severe ME from ME by calling it a different disease. In fact, this is what Esther Crawley does with her "Pervasive Refusal Syndrome". It is important for the perpetrators to propagate the narrative that ME is harmless and a thing like (very) severe ME does not exist. I don't think this was your intent, but it may be understood like severe ME denial.
IF catatonia is part of (very) severe ME, it is part of ME and could happen to anyone with ME, even the mildest.
We don't know for a fact who has ME and who has it not. We don't know exactly what ME is. Let's be united.
From Wikipedia:
Let's not do this. Severe ME is ME, as mild ME is ME. Mild ME can progress to severe ME. Let's stick together.
Ron Davis will know severe ME because he lives with someone with very severe ME.
I may have misunderstood you @nandixon, but even if you mean by "catatonia" another disease, we shouldn't split severe ME from ME by calling it a different disease. In fact, this is what Esther Crawley does with her "Pervasive Refusal Syndrome". It is important for the perpetrators to propagate the narrative that ME is harmless and a thing like (very) severe ME does not exist. I don't think this was your intent, but it may be understood like severe ME denial.
IF catatonia is part of (very) severe ME, it is part of ME and could happen to anyone with ME, even the mildest.
We don't know for a fact who has ME and who has it not. We don't know exactly what ME is. Let's be united.
nandixon
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@Inara, I'm pretty sure Whitney would be diagnosed with catatonia by most medical professionals familiar with that condition. Mutism, not eating and, of particular importance, a rapid response to Ativan, all of which apply to Whitney, are essentially diagnostic for catatonia. Most people think of someone who's catatonic as being perfectly rigid and completely unresponsive, but that's just a stereotype.
But catatonia is no longer considered to be just a condition associated with psychiatric diseases like schizophrenia. Rather now it's known to be associated with a wide range of medical illnesses. The Wikipedia article doesn't make that nearly clear enough. (I previously researched catatonia a fair bit because I was trying to figure out why Ativan was so helpful for some ME/CFS patients.)
So we don't know for sure whether the ultra-severe ME/CFS patients, i.e., those who may meet the catatonia criteria, are actually suffering from some number of other medical conditions which has also caused the development of catatonia in association with it (and ME/CFS may be one of those).
The idea behind the severely ill study was that by studying the most severely affected ME/CFS patients that clear markers of the disease would stand out and be more obvious. But if that group is actually not very homogeneous then that will be hard to obtain, and results like the negative viral DNA testing may not be valid for the rest of us. (I've never been too big on an ongoing infection causing my ME/CFS but I'm forced to reconsider that possibility in light of Prusty's work.)
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Haven't much time to reply - thank you for your excellent response.
I'm not familiar with Fisher's work - I'd be grateful for a link (not sure I'll understand it though). I agree that fission may be simply a consequence - but a biomarker for fission might be a diagnostic test (a plug for someone to fund more work by Bupresh Prusty)!
I think that there have been relatively large scale searches for a pathogen - I can't recall they've yielded anything! That's not to say I'm dismissing pathogen's; if you look at Alzheimer's it seems to be looking towards immune activation: "Porphyromonas gingivalis could migrate from the mouth to the brain and that a toxic protein they secrete (gingipain) destroyed brain neurons" - random quote from the web.
I agree that potentially something the same size as an exosome (but not an exosome) may be responsible. However, when you see that the numbers of this size of exosome is increased then it would require 2 coincidences i.e. size plus frequency. Hanson's P value is 0.03(?) 0.05 is often quoted - so it doesn't look too bad to me. I agree that they should throw some resources at trying to find what this is - mass spectrometry or whatever -- or explain what they've done.
I agree fully about what could be causing the signal - Bupesh Prusty lists possible candidates at the end of his NIH talk in April.
Thanks again
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