WASF3 disrupts mitochondrial respiration and may mediate exercise intolerance in myalgic encephalomyelitis/chronic fatigue syndrome, 2023 Hwang et al

I've done the densitometry myself and quantified relative to GAPDH as they would presumably have done (a "housekeeping" gene there to normalise for total protein levels). Bear in mind I'm working with the snippets of the blot membranes they are showing in the paper and not the membrane itself, and it is split across different figures. Hopefully the contrast has been maintained between the different crops they've done. This also made it tricky to get a good read of the background signal which you need to normalise properly. That being said:


P-values (two-tailed unpaired student's t test) and effect sizes (cohen's D):
Gene: WASF3
P-value: 0.0859249
Cohen's d: 0.7596534

Gene: MTCO1
P-value: 0.0007785032
Cohen's d: 2.143763

Gene: COX17
P-value: 0.00237593
Cohen's d: 1.633564

Gene: PERK
P-value: 0.02902655
Cohen's d: 0.9279234

Gene: BiP
P-value: 0.004006675
Cohen's d: 1.298791

Weirdly WASF3 doesn't actually make the 0.05 threshold in my attempt, but it is close so I can see how it might have squeezed past the threshold when they did it. Some of the others though scream through with tiny p values and enormous effect sizes.

 

From what I understood PERK and BiP are supposed to go up together during ER stress but in ME/CFS PERK is high and BiP low. This sounds a bit like the "blunted/absent physiological response to exercise" finding in some recent studies.

Figure 6 shows immunoblots of patient S1 fibroblasts treated with ER stress inhibitors TUDCA and salubrinal. Sadly the effect of TUDCA is modest. It would have been nice if it worked better because it's already available.

The effect of salubrinal on oxygen consumption of patient S1's fibroblasts seems meaningful.

PS: some are going to find fault with this paper but I love it. We've never had anything like this published before. In particular a mouse model that appears to replicate at least some of the reduced stamina that patient suffer from. They have shown that interesting discoveries can be made. Even if it's not going to be THE explanation it's still valuable. Now we have to figure out what's going on with the ER stress response.

 

Did you check the supplemental materials for some of the info you're looking for? You could also write Paul Hwang and ask for it.

 

Yes, he's looking at Relyzrio, approved as an ALS drug a few years ago.

Edit: He was also looking at TUDCA + another supplement but it sounded like he wanted to start with an already FDA-approved drug.

 

Interesting, Relyvrio is a combination of 3g sodium phenylbutyrate and 1g TUDCA.

 

https://en.wikipedia.org/wiki/Sodium_phenylbutyrate/ursodoxicoltaurine

 

History on relyvrio..
https://www.newyorker.com/magazine/2023/06/26/relyvrio-als-fda-approval

 

@chillier thanks for asking for detail and holding my statements accountable. Should actually happen more.

Density (contact inhibition): I don't know about published seahorse studies off the top of my head but I have seen it firsthand in the lab with seahorse, and others have demonstrated it with other methods eg: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1000514

Trypsinisation: seahorse incubation times can vary between 30 minutes to 24 hours. Since this is not described in the paper, a shorter incubation time would mean that the treatment and circumstances of the cells prior to seeding in seahorse plate would likely matter. This may not be an issue with the study if they use a longer time, but since the method is not described, who knows? I have asked in an email to Hwang. I expect that it was probably a longer time to allow for adherence. We will see!

I would say this is sensible, my view is just that it opens up possibilities for new variables to influence the result, which is counter to the purpose of normalisation. Even if it was a discrepancy of only 0.5% protein content between samples, why do it? Especially when it's adding another step + technique to the assay, and using a different instrument, which opens up more chances for sources of variation or error to creep in. One can measure average cell size while counting if cell size is a concern. It won't add another step or any manual handling, most counting machines do this already.

It's also coming after a seahorse assay which is not only involving biological changes due to inhibition but also mechanical manipulation by mixing and probing. It's quite the ordeal. What's coming out by the very end is not matching what went in.

Everything I have done over literally tens of thousands of seahorse samples (including microscopy assessment of wells after each run), suggests that careful manual handling and a sufficient number of replicates produces reliable data that is not confounded by loss of cells during handling.

There is one possibility which is that overnight incubation is employed and that variable rate of proliferation of cells between samples is intended to be controlled for. But I'm not aware of a way to mess with them with a protein assay in the seahorse well before the seahorse assay that wouldn't be problematic. What one could do there is prepare a growth curve in a previous experiment and calculate an effect based on incubation time. Or do some quick bright-field imaging-based well scans to quantify cell surface area per well.

GAPDH is a commonly used housekeeping gene that is considered indicative of total protein levels within a cell. Proteins of interest can move up or down with total protein even if they are not dysregulated so measuring them on their own is a problem, you have to measure total protein alongside your protein of interest in some way (this is all meant in the context of westerns).

This may raise alarm bells as a housekeeping gene if glycolysis stands out as a potential problem area in some people's theories, but my whole-cell proteomics shows only a 2.5% average difference (ie: nothing) between healthy and pwME so I would say it's a safe pick. (Housekeeping genes used in a disease context should be known to be unaffected. People use the same old housekeeping genes every time without checking for effect of disease, GAPDH is one of them, but I think this paper dodged this bullet.)

Hey, yeah I did, but unfortunately it's not in there. I have written to him . Make no mistake, this is not negativity on my part. There is a lot of work in this paper with an impressive approach and study design, and the parts unaffected by my comments are saying very interesting things. I just cannot conclude much from some components of the cell-based work without knowing the full context of how the experiments were performed.

In the past I have been guilty of multiple of the presentational things you have outlined in the comment (albeit inadvertently, just doing things by what I thought was convention as a student). Especially this part about scatter plots. So just saying that I hear you loud and clear and will improve my efforts.

 

Just trivial sleuthing again, but noticing that NIAID Director 'liked' this today and hopefully is following closely too with NHLBI/NINDS.

 

This article gave a great explanation of the study for a wider audience:

Science Alert This Protein Could be Responsible For The Exhaustion in Chronic Fatigue Syndrome

Quote:

Baffling perhaps to medical doctors who have long dismissed ME/CFS, but not so much to researchers who have steadily been building a picture of what triggers this debilitating illness, nor to those who live with its unrelenting exhaustion every day.


Inside every cell are energy-making machines, the mitochondria, which power our cells, replenish our brains, and keep our muscles moving.


Now a new study from a team of US researchers adds evidence to a growing theory that malfunctioning mitochondria might be one potential cause of energy-limiting illnesses such as ME/CFS and long COVID.

 

article
This Protein Could Be Responsible For The Exhaustion in Chronic Fatigue Syndrome

https://www.sciencealert.com/this-p...or-the-exhaustion-in-chronic-fatigue-syndrome

 

Don’t want to sound too negative but I am not optimistic about Relyvrio. Also not cheap!

Price of New A.L.S. Treatment Will Be $158,000 Per Year, Maker Says

https://www.nytimes.com/2022/09/30/...ejxwju7P-KjeRv310JMKX9iO9uJU0T&smid=url-share

Also don’t see much in the pipeline for this pathway.

 

Just curious -- when you say you don't see much in the pipeline for this, do you mean in terms of potential treatments? (Just wanting to better understand

 

Thread on Hacker News.

 

From a quick scan there seems to be little to no discussion of the study itself.

 

Paul Hwang sent me a couple of lovely responses, seems like a really nice guy. I didn't get the full picture because he's out of the office and my questions are very technically detailed. I may get more info later from another author. When I have concrete info I may share here if appropriate - (while preserving the intrinsic assumed confidentiality of email). Would only be very brief dot points pertaining to relevant topics from the paper. I want the body of work to be as meaningful as it can possibly be!

 

Paul Hwang also replied to my email which was very nice given the fact that he is out of office . He said he will follow up with any questions.

 

TUDCA is licensed in Italy as Tudcabil for the treatment of bile production disorders. 20 250mg tablets cost about €11.

An affordable self-administered drug like this for ME/CFS would be amazing.