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

RogerBlack

Senior Member
Messages
902
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.
 
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A.B.

Senior Member
Messages
3,780
It's difficult to read. They did a lot of things. Help.

One interesting bit:

The pathway analysis highlighted the ‘metabolism of NADH’ as one of the most important biological functions involved in CFS. The NAD+/NADH ratio has an omnipresent role in regulating the intracellular redox status and, therefore, represents a function of the metabolic state. Some studies suggest that NADH concentrations are significantly lower in CFS patients compared with healthy controls.42, 43, 44 It is likely that high levels of MDHM and isocitrate dehydrogenase observed in CFS may be an adaptive change to deficiencies of NADH which, in turn, has a critical role in mitochondrial ATP production.

and

ATPB is the subunit beta of the ATP synthase, the key enzyme for ATP production. In addition to the increase of ATPB in CFS, we also detected significant increases of both subunits alpha and gamma using nano-LC-MS (P-value of 0.005 and 0.001 respectively; Supplementary Table S1). Tiredness and energy are tightly connected; therefore, an attractive hypothesis is that there is a metabolic dysfunction with the result that enough energy is not being produced. In this perspective the increase can be explained as an attempt to increase the ATP production.

Together with ATPB, the most promising biomarker seems to be ACON. This is the mitochondrial form of aconitase, an enzyme that catalyses the stereo-specific isomerization of citrate to isocitrate via cis-aconitate in the tricarboxylic acid cycle. ACON is commonly used as a biomarker for oxidative stress and has been suggested to serve as a sensor of redox status.47 Indeed, the reaction between ACON and superoxide generates free hydroxyl radical. It is likely that the reaction between superoxide and ACON may enhance mitochondrial oxidative damage associated with the pathophysiology of CFS.48 As a matter of fact, the excessive production of ROS and nitrogen species is usually related to CFS,16 and we found a significant upregulation of superoxide dismutase
 
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RogerBlack

Senior Member
Messages
902
It's difficult to read. They did a lot of things. Help.

One interesting bit:



and

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'.
 
Messages
2,158
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.
 
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M Paine

Senior Member
Messages
341
Location
Auckland, New Zealand
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."


tp2016184f2.jpg



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.
 
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M Paine

Senior Member
Messages
341
Location
Auckland, New Zealand
"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.

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,

Signaling pathway analysis
Proteins differentially expressed were functionally analyzed through the use of QIAGEN’s IPA (Ingenuity System, QIAGEN, Redwood City, CA, USA) to select candidates for validation.

All differentially expressed proteins with a fold variation in CFS greater than 2.0 with respect to control were included in bioinformatic analysis to identify molecular functions most strongly associated with the protein list. Supplementary Method 3 gives more details about this analysis.


From the supplementary:

Each identified protein was converted to its gene and mapped to its corresponding gene object in the IPA knowledge base. The ''Core Analysis' function, included in IPA, helps to interpret the data in the context of biological processes, pathways and networks. After the analysis, IPA provides details of the most significant biological function, associated with our proteins, that are ordered by statistical significance. The software shows which proteins are associated to the most significant functions, and how this functions are related.
 

Hutan

Senior Member
Messages
1,099
Location
New Zealand
Seems pretty exciting to me.
Yes, me too. Although I found this hilarious rather than informative
A horseradish peroxidase-conjugated goat anti-rabbit (Stressgen, Farmingdale, NY, USA) was used as a secondary antibody at 1:10000 dilution.
,
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
glyph.gif
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
 

alex3619

Senior Member
Messages
13,810
Location
Logan, Queensland, Australia
"sensitivity=93% and specificity=70%" - which is really pretty good.
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.
 

alicec

Senior Member
Messages
1,572
Location
Australia
A horseradish peroxidase-conjugated goat anti-rabbit (Stressgen, Farmingdale, NY, USA) was used as a secondary antibody at 1:10000 dilution.

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.
 

alicec

Senior Member
Messages
1,572
Location
Australia
GTP:AMP phosphotransferase AK3

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.
 

lansbergen

Senior Member
Messages
2,512
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.

Thank you.

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

M Paine

Senior Member
Messages
341
Location
Auckland, New Zealand
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.