Australian scientists make breakthrough in Chronic Fatigue Syndrome testing

[John Mac]

Oh, the mystery. Are we supposed to tease it out of you?

I hope we don't have to buy a book/DVD to find it out

 

[adreno]

I hope we don't have to buy a book/DVD to find it out

My guess is he is talking about H pylori. There, I saved you money!

 

[alicec]

ELISA tests can detect infections

ELISA is just a generic term for an assay technique (enzyme linked immunosorbent assay).

At its most basic, plastic wells in a 96 well plate are coated with the unknown test samples along with different amounts of the pure substance to be analysed (the analyte or antigen) to give a comparison which enables quantitation. A specific antibody to the analyte is then added. Antibody-antigen interactions are very strong and very specific, so this antibody will bind to the plate if the analyte is present in the test sample. The bound antibody is then detected by an enzyme-linked second antibody which produces a colour reaction which can be detected in a spectrophotometer.

Further refinements have been added to this basic technique to make it more sensitive.

So saying an ELISA can detect CFS is meaningless. We need to know what analyte you are planning on detecting to evaluate your claims. Is the pure substance available to provide the standard curve, do you have a specific antibody to the analyte?

 

[funkyqueen]

Published yesterday :

MicroRNAs hsa-miR-99b, hsa-miR-330, hsa-miR-126 and hsa-miR-30c: Potential Diagnostic Biomarkers in Natural Killer (NK) Cells of Patients with Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME)

Robert D. Petty , Neil E. McCarthy , Rifca Le Dieu , Jonathan R. Kerr

Published: March 11, 2016DOI: 10.1371/journal.pone.0150904

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150904

it seems this team is from UK?
I guessed they are working with Sonya M-G 's team, maybe ?

 

[BurnA]

it seems this team is from UK?

Seem to be from QMUL

 

[TiredSam]

Data Availability: All microarray data files are available from the GEO database (accession numbers: GSE70371, GSE69555).

Exemplary

 

[alex3619]

I guessed they are working with Sonya M-G 's team, maybe ?

From what I can gather at this point they are a rival team.

 

[Bob]

They have recently filed this patent (filed 10th Aug 1015 but published Feb 2016)
https://www.google.co.uk/patents/WO2016023077A1?cl=en&dq=sonya marshall-gradisnik

https://patentscope.wipo.int/search...=&sortOption=&queryString=&tab=PCTDescription

Go to [00445] and look at Table 5

Published yesterday :

MicroRNAs hsa-miR-99b, hsa-miR-330, hsa-miR-126 and hsa-miR-30c: Potential Diagnostic Biomarkers in Natural Killer (NK) Cells of Patients with Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME)

Robert D. Petty , Neil E. McCarthy , Rifca Le Dieu , Jonathan R. Kerr

Published: March 11, 2016DOI: 10.1371/journal.pone.0150904

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150904

Unfortunately neither study's main miRNA biomarker candidates match any candidates in the other study. There doesn't seem to be a match between any of the wider (less useful) selection of candidates either. It's unfortunate for there to be no matches, but I haven't carefully studied the methodological differences between the studies and that may potentially explain the differences, so it doesn't necessarily mean there's no potential for these biomarkers. I still find it interesting, and it's early stages in this research. It demonstrates why replication is necessary.

The sets of candidate miRNAs can be seen in two graphs as follows...

Australian team: See [00445] Table 5, here, or here in full context.
UK team: See Fig 1, here.

 

[worldbackwards]

Jonathan R. Kerr

Back from his travels?

 

[jimells]

Marshall-Gradisnik and her team are currently looking to partner with diagnostic companies that will be able to commercialise their new screening test and make it available to the public.

They must have a lot of confidence in this method if they are already looking for a commercial partner.

 

[Hutan]

Exemplary

CFS/ME subjects were recruited from two specialist CFS/ME centres located in Poole hospital, Dorset (N = 15) and St Helier hospital (N = 20), London following diagnosis by a clinical specialist and the exclusion of other possible sources of fatigue. All CFS/ME subjects selected for the study were diagnosed using the Fukuda clinical criteria for diagnosing CFS/ME [3]. These subjects also fulfilled the Canadian criteria for diagnosis of CFS/ME [79]. Patients with psychiatric disease were excluded from the study using the Minnesota International Neuropsychiatric Interview (MINI); all CFS/ME subjects included in this study were therefore free of major psychiatric disease and drug or alcohol abuse. Average disease duration was 6.8 yrs.

Yes, the UK team's write up is clear and sets out selection criteria nicely too.

It is indeed interesting stuff and early days.

I wonder how much impact the time since disease onset has on miRNA concentrations.

Does anyone know how stable these miRNA are? Do they change over the lifetime of a cell? Could they change following exercise?

 

[alex3619]

Does anyone know how stable these miRNA are? Do they change over the lifetime of a cell? Could they change following exercise?

My best guess is miRNA composition is dynamic and constantly changing. That could lead to a huge hurdle. However this may not be the case for every specific miRNA.

The NCNED team have been tracking very large numbers of patients over many years. So, potentially, they have a large dataset. Yet we don't have specifics of their methodology here, so its hard to compare.

It is also worth noting that the expression of these miRNA in different cell types is not the same. This may complicate things.

It is also likely that disease severity, duration of illness, comorbidities and subgroups/heterogeneity, and resting/post-activity may confound results.

 

[knackers323]

hope something comes of this

 

[Deltrus]

I'm actually looking at these TRP channels which were reported as being upregulated in this study. They specifically let mainly calcium inside, but also can let in other ions.

Calcium is a very interesting ion. The thing about calcium, is that there is regularly almost no calcium inside a cell:

The intracellular calcium level is kept relatively low with respect to the extracellular fluid, by an approximate magnitude of 12,000-fold. (https://en.wikipedia.org/wiki/Calcium_in_biology#Vertebrates)

Calcium is used as a secondary messenger in cells. It is used in things that require sensitivity. There is almost no calcium in cells, and the gradient is so steep, because the cells need radio silence. Touch, heat, pain are all integral to our survival, and need to be very sensitive. Almost no calcium in cells = no noise.

The low amount of calcium in cells is how your touch is so sensitive that you can feel stuff like a hair in your fingertips. If it was K or NA ions that regulated touch, there is already so many ions moving about, that it would create a ton of noise.


Calcium has such a steep gradient, that is requires one ATP for every calcium ion extricated using plasma membrane Ca2+ ATPase. Also on that page, you can see that even with a whole ATP, this process goes slowly. The ion really wants to flow back into the cell.

By the nature of sensitivity, ions need to both flow into a cell very fast, and there needs to be very little of the ions in a cell. This means that the gradient is super steep, and this gradient is super hard to climb. It is like forcing these calcium cells to climb up a steep mountain in order to get out.

Na+/K+-ATPase on the other hand, the transporter that uses 1/5th of a cell's energy, uses one ATP to move three Na ions and two K ions, BOTH against their ion gradient.

You can see how energy consuming excess calcium is... all because the ion gradient is a cliff.

If calcium needs to be extricated quickly, then it needs to recruit the potential energy of the ion gradient of Na, using the sodium-calcium exchanger (NCX) in order to push it out much faster.

My theory is that phenibut helps me because it keeps both the NA and ATP higher, where normally they would be used up by pumping out calcium. This means that cells operate better because they have enough NA to sustain normal function. And ATP doesn't run out either. * Turns out the NCX actually brings Na in, going from a higher concentration to a lower concentration.

My theory is that phenibut helps me because it keeps Na lower and ATP higher, where normally they would be used up by pumping out calcium. This means that cells operate better because they have enough NA to sustain normal function. And ATP doesn't run out either.


It is also becoming extremely clear why Fibromyalgia and CFS are so closely linked.

Maybe this is also why I can last almost exactly 1 hour until I run out of energy after waking up, because my cellular NA stores run out, and I'm forced to swap over from NCX to plasma membrane Ca2+ ATPase. Before 1 hour passes, I'm an extremely high functioning individual and most of my CFS symptoms aren't there. Afterwards, suddenly I'm using up a huge amount of ATP, and my NA stores are depleted Na concentration is way too high, fucking up TONS of functions in my cells.

It is like a switch, I go from super intelligent thought and suddenly just stop 1 hour in. It's like if you were talking and had a stroke, you just stop mid way, it happens instantly. I can watch my thoughts as I turn borderline retard.

EDIT: One last note, is that the TRP channels implicated in the study, are channels and not transporters. In channels, when they open, it is like sliding down a slide. With transporters, on the other hand, it is much slower. If something goes wrong with these channels, things can go really wrong.

Cavity in a carrier protein is not open simultaneously to the both environment (extracellular and intracellular). Either its inner gate is open, or outer gate is open, Carrier has binding sites, but porins and channel proteins have not. When a channel is opened, thousands to millions of ions can pass through the membrane in one time, but only one or a small amount of molecules can pass through a carrier molecule.(source)

Goodnight everyone I'l be back tomorrow with more ion channel facts. (and also more proofreading here)

 

[adreno]

@Deltrus

Did you try other calcium channel antagonists, such as magnesium, taurine or gabapentin?

BTW, calcium channel blockers are on the list of drugs to avoid in POTS, which is common in ME/CFS.

 

[alex3619]

Calcium is used as a secondary messenger in cells. It is used in things that require sensitivity. There is almost no calcium in cells, and the gradient is so steep, because the cells need radio silence.

I think this is a misinterpretation. I was really into calcium regulation about fifteen years ago, it was integral to my own model of ME.

The intracellular fluid ionized calcium concentration of cells is indeed very very low. However cells don't just use calcium influx when needed, they can induce calcium bursts from internal stores, though these are also regulated by calcium channels . Those calcium bursts were critical to my early model. So the cell stores up calcium and then releases it in large amounts when needed.

We also know that acetylcholine produces a strong and sustained response in us. This is mediated by ionized calcium.

At a personal level I benefit from Resveratrol, which alters the cAMP levels. Ca++ and cAMP operate in opposition as intracellular second messengers. Whenever you discuss Ca++ as a messenger you need to consider what is happening with cAMP. This kind of two substance control is called an axis. Very early on I considered the herb forskolin to boost cAMP but rejected it based on the risk profile. Now I wonder if that was a mistake.

 

[Deltrus]

@Deltrus

Did you try other calcium channel antagonists, such as magnesium, taurine or gabapentin?

Yes I am taking high dose magnesium, like 1.5g / day of citrate and malate forms. Taurine I've tried but haven't noticed anything. Gabapentin, I haven't tried it, but I imagine it would probably work well for me, potentially better because the lack of effect on the GABA system that phenibut has.

Keep in mind there are different types of calcium channels. I know magnesium blocks the NMDA receptor, but I don't think that channel is an issue for me. If it was, my thought would be all messed up because NMDA is so critical to thinking. I also tried memantine which blocks the NMDA receptor.

Calcium channel blockers only block one type of VDCC which in in the heart I believe.

The interesting thing about the TRP channel is that:

TRPs have important interactions with other proteins and often form signaling complexes, the exact pathways of which are unknown.(source)

The receptors are found in almost all cell types and largely localized in the cell membrane, modulating ion entry.(source)

Both phenibut and TRPs effect calcium in almost every cell in the body. But TRPs have complex signalling mechanisms that we don't understand, while VDCCs have very set activities, they fire a neuron and then stop. The only variable is receptor density.

I wonder if Complex regional pain syndrome is a brother to CFS and Fibromyalgia, except with more managable energy demands.

 

[Deltrus]

@alex3619 what dose/form of resveratrol?

Also from what I read, calcium inside the cell plasma increases 10-100 times during certain actions, which still isn't that significant. I'm almost falling over from tiredness atm, so I can't really track down any further info. Could you perhaps link the exact process you are talking about? I think I might know sort of what you mean though, calcium is pumped into a neuron during many types of action potentials, so it has to have a significant of it pumped out during certain times anyways right? Does that make the "silence" theory worse, because there's gunshots going off? Well I'l have to look into that tomorrow.

The cAMP and acetylcholine things are super interesting tho. I know acetylcholine modulates cAMP, as acetylcholine isn't directly attached to any ion channel, just has secondary messangers many of which have an effect on cAMP/calcium. Diphenhydramine, an acetylcholine receptor antagonist, also significantly helps my fatigue, just as much as phenibut actually. However it turns out that repeated use of the drug causes a much lower seizure threshold due to the H1 receptor antagonism messing with glutamine and gaba metabolism in the astrocyte immune cells.

 

[alex3619]

I knew all this stuff circa 2000, but I have forgotten most of it. I think this was related to findings in aluminum toxicity, though that could be wrong, but I no longer recall the details.

The acetylcholine work was from Vance Spence, who found vascular abnormalities and was looking into acetylcholine. I was discussing some of this with him at the time.

In looking at calcium bursts, you might also want to look up intracellular citrate metabolism. Citrate is a divalent cation chelator, and if its high in the cell, such as from a problem in the mitochondria, then the intracellular stores can release massive compensatory bursts, far beyond normal, if they are triggered. My early model was of hypercitricemia, which despite claims is still a possibility, although the mechanism that I postulated was invalidated a couple of years after I came up with it, as newer and better data was released.

 

[Deltrus]

This video and the following one in the playlist will help people make a lot of sense of what I'm talking about if you are new to the subject.

Also rewatching those videos I found out that Na actually enters the neuron when calcium exits the ion through the sodium-calcium exchanger. I thought for some reason that Na was higher in the cell, but it is actually much higher outside the cell. Thus, to power the Ca extraction, Na goes down the gradient slope into the cell.

If my "Na stores get depleted" theory was to work now, it would have to be "Na stores get too large".

EDIT next day:

So we have calcium flowing out, and Na flowing in using the NCX channel. This means that there is a great increase in intracellular positive ions. This means two things. Firstly, water will flow into the cell, creating bloating and reducing integrity of the celluluar wall. Secondly, the very leaky Tandem pore domain potassium channel would leak K out, because the inside of the cell has too many positive ions. There are much more "leaky" K channels compared to Na channels, so primarily K will leak out.



Then, the Na+/K+-ATPase kicks in, and starts using energy to remove Na, and bring K back in.


The net result would be cells that have a greater ratio of Na to K. This is the opposite of the effects of Na+/K+-ATPase, which stores potential energy in a cell by bringing Na out of a cell, against its gradient, and K into the cell, against its gradient.

Here is the original ratio:

From http://book.bionumbers.org/what-are-the-concentrations-of-different-ions-in-cells/

Normally K is trying to push itself out of the cell, going from a high concentration area to a low concentration area. The channels are always open, leaky. The reason all the K doesn't quickly push itself out of a cell, is that it is much more negative inside of a cell because of the inorganic cations, Cl, etc. This negative force pulls it back in. A greater amount of Na means that there is less negative force pulling K in, and K concentrations lower.

Something that may be correlated, is that people with CFS tend to have 10% less K than average in their cells. However total positive charge and ion concentration would have to balance out, so you would expect that lower K means more Na. Mg wouldn't increase much because there isn't much Mg in extracellular fluid.

Previously I mentioned that coxsackie b virus decreases K and increases Ca in cells. Perhaps this is the mechanism. But, according to that study, Na current stays the same. I don't really understand how voltage clamps measure this stuff so I can't say much more on the coxsackie b study.

The previous theory for why B 12 supplementation causes low potassium (hypokalemia) is that it causes increased DNA synthesis, and thus more new cells need to be filled up with ions such as K.

But I propose that B12 supplementation somehow negates the process which increases calcium in cells. Potentially it causes better DNA repair, or something like that, reducing the epigenetic changes that viruses have on Ca channels.

Doing so, K will rapidly be sucked in to the cell after its long homeostasis at a lower concentration. There is very little K stored in extracellular fluid, so you get hypokalemia.

People think that the hypokalemia is due to an increase in cell production, but to me, that doesn't make sense at all. You would get hypomagnesia too if that were the case. In addition, why would there be such a high demand for cells that they are just being constantly queued up to be formed? Increasing methylation shouldn't cause such a drastic surge in cell production.

My theory makes more sense than RichVank's original theory on B12 induced Hypokalemia, located here: http://forums.phoenixrising.me/inde...entation-needed-in-methylation-treatmt.18670/

Instead of increasing cell production, increased methylation would decrease DNA errors caused by viruses, and this would go down the line and eventually suck K ions into the cell.

Really too bad he isn't alive to discuss this with me.


EDIT: I have another theory, what if the Sodium dependent multivitamin transporter is having its functions disrupted by a less steep Na gradient, which means less energy to transport vitamins. This then lowers B vitamins in specific cells, creating a methylation block, and thus the cell cannot "turn off" DNA that viruses edit, because this is dependent on methylation/folate. This leads to long lasting epigenetic changes from the virus, such as increased calcium channel activity. The cycle perpetuates itself.