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Nature-Translational Psychiatry: Different protein expression in saliva of people with/ without CFS.

Discussion in 'Latest ME/CFS Research' started by RogerBlack, Sep 28, 2016.

  1. RogerBlack

    RogerBlack Senior Member

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    http://www.nature.com/tp/journal/v6/n9/full/tp2016184a.html

    "Bottom-up proteomics suggests an association between differential expression of mitochondrial proteins and chronic fatigue syndrome."
    Is potentially interesting.
    They took two twins, with and without CFS, and analysed the proteins found in the mitochondria.
    This is different from the DNA - the proteins tell you what the genes are actually doing.
    They analysed the differences and found three candidate marker proteins that are found in the mitochondria of platelet cells in blood - based on differences between the two twins.
    They then went on to measure these three proteins in a cohort of 50 people with and 50 without CFS (Fuduka criteria). The test in this group was done on saliva.

    "The selected proteins were as follows: aconitate hydratase (ACON), ATP synthase subunit beta (ATPB) and malate dehydrogenase (MDHM)."

    I stalled out partway through the analysis section, but my understanding is that this is an interesting test - which is great for 'proving it's a real disease' - but seems not to find all CFS patients.
    For the FACIT criteria - "sensitivity=93% and specificity=70%" - which is really pretty good.
    As I understand it, the three proteins chosen were chosen from a subset of the ones they could have looked at, as these three proteins were thought likely to be an issue.
    it may be that adding more proteins would help in discrimination, or finding disparate patient populations with variants of cfs.
     
    Last edited: Sep 28, 2016
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  2. Sidereal

    Sidereal Senior Member

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    That's not Nature though. The journal is Translational Psychiatry.
     
  3. RogerBlack

    RogerBlack Senior Member

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    I meant to edit it and say Translational Psychiatry - one of Natures open journals, but realised after clicking submit that I hadn't gotten around to that.
    Oops.
     
  4. A.B.

    A.B. Senior Member

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    You can edit the title. Under the title, on the right, in Thread Tools.
     
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  5. A.B.

    A.B. Senior Member

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    It's difficult to read. They did a lot of things. Help.

    One interesting bit:

    and

     
    Last edited: Sep 28, 2016
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  6. RogerBlack

    RogerBlack Senior Member

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    I am not sure the above can be more usefully summed up than:
    The two proteins we found elevated in a large fraction of CFS patients are both involved in energy production. These may both be increased as an attempt to boost energy production in the cell which is damaged by some unidentified mechanism. This increase and its side-effects may cause some of the symptoms of CFS, adding to the underlying defecit in energy production.

    The test I would want to try immediately next is to take the same 45 CFS patients, and get them to spit into a sample bottle and freeze it on a very good day, an average day, just after doing something that will cause a crash, and at the worst point of a crash.
    Then look at these proteins again.

    However, I have not looked into the half-life of these proteins in the body, if it's weeks, then the above would not be useful.

    It may also be interesting to look at subsets of these patients which have strong signals in this for which bits of the genome are 'turned on'.
     
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  7. trishrhymes

    trishrhymes Save PR. Sack the President of the Board.

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    I've had a go at reading the paper, skimming over the bits I don't understand. As I see it, they have potential biomarkers in saliva which can be used to identify at least a significant subgroup of ME patients, and to distinguish between more and less severely affected patients.

    The particular chemicals found are fascinating, though here my rather limited knowledge of mitochondrial biochemistry limits me.

    The picture I get is of a problem of low amounts of the transporter proteins in the mitochondrial membrane that transport the ADP (low energy) into the mitochondria where it is changed to ATP (high energy) and transporting the ATP back out into the cytoplasm were it can be used for energy by the cell.

    The mitochondria produce more of the proteins that carry out the conversion of ADP to ATP in attempt to overcome this problem, but this doesn't help because not enough ADP is reaching them.

    I think this is fascinating, and hope this will lead to more detailed understanding of why this is happening and to finding a way to switch the ADP/ATP transporter proteins back on again.

    Please feel free to correct me if I've got this wrong.

    It seems, again with my limited understanding to fit very well with the Naviaux study which also found problems with mitochondria not functioning properly.

    Seems pretty exciting to me.
     
    Last edited: Sep 28, 2016
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  8. M Paine

    M Paine Senior Member

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    I thought the part which involved comparing CFS patients to the Ill Twin was interesting. It makes me unsure about how to interpret the data when they compare the CFS twin to the two groups of CFS patients, split by illness severity.

    "Then, we classified patients according to their clinical features from questionnaires, that is, we examined the following: FACIT, FIQ, VAS_pain, VAS_fatigue and VAS_sleep. CFS patients were split into two groups considering as reference the twin suffering from CFS. One group with the clinical features whose values were in common with (or greater than) those of the ill twin (group A), and the second with the patients with lower values (group B). Intensities were higher for each of the three markers in group A with respect to group B (Figure 2). Details are given in SR3."


    [​IMG]


    With the recent metabolomics papers, there seem to be pronounced and decinct differences between men and women. It might have been nice to see them gender match the twin to the CFS Groups, (which was 25 females, 20 males. The Twins are male). Or perhaps to add a control group.



    "It is noteworthy that the increase of ATPB in CFS correlated positively with the increase in IFN-γ and TNF-α. Immunological dysregulation has been always proposed as a significant component of the CFS, and CFS patients had usually high levels of pro-inflammatory cytokines, such as IFN-γ and TNF-α.14, 51, 52 In addition, MDHM correlated positively with TNF-α. These correlations are instructive, as all these variables can be thought as markers. It is interesting to point out that ACON levels correlated negatively with IL-2, in line with the fact that the twin with CFS had lower levels of IL-2 with respect to his healthy twin brother"

    It's nice to see some direct correlation between these cytokines and the drop in protein expression.
     
    Last edited: Sep 28, 2016
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  9. M Paine

    M Paine Senior Member

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    I think you're right, that other proteins could be identified. Another set of monozygotic twins, discordant for CFS would be nice for that purpose. Especially if they are in a different subgroup.


    From the paper,


    From the supplementary:

     
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  10. Hutan

    Hutan Senior Member

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    Yes, me too. Although I found this hilarious rather than informative
    ,
    so, what would I know?

    The idea of comparing proteins/metabolites in monozygotic twins with and without CFS is a great one. It is relatively cheap as the sample is so small but a whole lot of possible confounding factors are controlled.

    I was impressed with the detail they have given about their methodology - I think it would help anyone who does this type of analysis to evaluate and potentially replicate what they have done.

    Does anyone know the researchers?

    This table lists the mitochondrial proteins that differed between the twins (one with CFS and one without).
    http://www.nature.com/tp/journal/v6/n9/fig_tab/tp2016184t2.html#figure-title
    Table 2. List of mitochondrial proteins found differentially expressed by nano-LC-MS analysis, and with a fold of change [​IMG]2, in mitochondria from platelets of the twins discordant for CFS

    Figure 1 is copied below. The three proteins that they were most interested in are circled in red in Fig 1a. Note that levels of these proteins were increased in the CFS twin (Fig 1b) (and two of the three proteins were increased in the larger CFS group Fig 1c).

    horseradish.jpg
     
  11. lansbergen

    lansbergen Senior Member

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    Does it say wich one?
     
  12. Hutan

    Hutan Senior Member

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    From Table 2 - Superoxide dismutase (Mn)
    Manganese co-factor
     
  13. lansbergen

    lansbergen Senior Member

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    Did they find that in the mitos or in the cytosol?
     
  14. RogerBlack

    RogerBlack Senior Member

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    They separate out (as much as they can) the mitochondria by centrifugation.
     
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  15. lansbergen

    lansbergen Senior Member

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    Can somebody explain this?

     
  16. alex3619

    alex3619 Senior Member

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    From old reading its generally accepted that 95% for both sensitivity and specificity is the standard for a good test. However there are tests with much lower figures, I guess because sometimes a poor test is better than no test at all. This is early days for the research, and I have not read the full paper yet, but it looks promising and may fit with the hypometabolism hypothesis. They also used a second method to check the first method's accuracy.
     
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  17. alicec

    alicec Senior Member

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    That's just a reagent that's used in immunoassays to visualise and quantitate the binding of specific antibody (the first antibody) to the analyte.

    So in this case, the first or specific antibody to whatever substance they are assaying (the analyte or antigen) was raised in a rabbit. In a common immunoassay procedure, the test samples, along with a standard curve of purified substance for comparison, would be bound to the wells of an ELISA plate. Specific antibody would then be added which would bind to the plate in proportion to the amount of specific antigen already bound there.

    In order to detect the bound antibody, a generic anti-rabbit antibody (in this case raised in a goat) to which an enzyme has been conjugated (in this case horseradish peroxidase) is then added. This second antibody can be detected by adding the substrate for the enzyme (various ones are used) which results in a colour reaction which can be quantitated in a spectrophotometer.

    Amplification of signal is achieved at each step so that, ultimately, small but unknown amounts of analyte/antigen in the original test sample can be detected and quantitated by comparison of the colour reaction with known amounts in the standard curve.
     
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  18. alicec

    alicec Senior Member

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    That is an enzyme, coded for by the AK3 gene, whose function is to maintain nucleotide homeostasis in the cell. It does this by catalysing the transfer of phosphate groups from one nucleoside to another.

    The generic reaction transfers a phosphate from a nucleotide triphosphate (for this enzyme either GTP or ITP) to a nucleotide monophosphate (AMP in both cases), producing a nucleotide diphosphate and ADP.
     
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  19. lansbergen

    lansbergen Senior Member

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    Thank you.

    Does the 2.2 fold mean that 2.2 times more phosphate is transfered from GTP/ITP to AMP in the patient?
     
  20. M Paine

    M Paine Senior Member

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    In these kinds of systems, it's sort of like a mass production facility. Think of the protein as a machine in a factory. Putting a label on a bottle for example. If you had enough labels, enough power, enough bottles, enough labour, enough space, enough waste management, and efficient transportation for your labels and bottles to get from and to your machine,... Than sure, twice as fast.
     
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