Why does 5AZA matter?

[Lee]

Ruscetti has admitted that the two positive lanes in Figure 2c from the Science paper, which contained activated PBMC from CFS patients, were treated with 5AZA. He mentioned only the two CFS patient lanes, and did not say the controls were also treated. This was not mentioned in the paper. Ruscetti says it was a trivial omission, not necessary to the paper.

He is wrong, and he must know better. This is why. I'm going to start from the basics, so the story is complete.

When a retrovirus infects an animal - lets say, a human - it sometimes integrates a viral DNA sequence into the DNA of infected cells. This is a 'provirus.' The integrated provirus can ay silent, or it can express viral proteins and RNA and make new virus.

One mechanism the cell uses to try to turn the provirus off, is to methylate it - add methyl groups to the DNA. Methylation turns off expression of genes.

Sometimes a cell infected with a 'provirus' can be passed on through sperm or egg to the person's children. If that happens, the provirus sequence has now become part of the human Genome, part of our genetic heritage.

It turns out that a very large part of our human DNA consist of ancient retrovirus insertions, often from millions of years ago or more. They have mutated enough over many generation that they don't make functional virus. They are silenced by methylation. These are called endogenous retroviruses - retrovirus sequences that are not infectious and have become part of our DNA. They are called ERV's for short - and yes, this is where Abbie Smith took the name of her blog. One of her core scientific interests is endogenous retroviruses, which is one reason she is interested in this story.

ERVs are not infectious - they can't make functional virus - but many of them are capable of making virus protein if they are turned on. Including among other things, gag protein.

Some of these endogenous retroviruses are gamma-retroviruses.

So - short version - our DNA is packed with ERV genes that can make retrovirus proteins if they get turned on. They are turned off by methylation.

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If a gene - say, an ERV or an integrated provirus from a new viral infection - is turned of my methylation it can be turned back on by demethylation.

In her Ottowa presentation where JM showed 5AZA treatment of PBMCs, this is what she was talking about. She was claiming that after the initial infection, HGRVs integrate into the DNA of infected cells, and go silent She claimed that treatment with 5AZA reactivates them by demethylating them, they produce new virus, and then she can detect the virus. That's a reasonable hypothesis.

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But here's the problem Treating the PBMCs with a demethylating agent - 5AZA - can also reactivate all those silent endogenous retroviruses, which are a part of our DNA, of everyone's DNA. Take perfectly healthy cells, demethylate them, and they can start to make gag protein FROM THEIR OWN DNA. 5AZA can cause gag to be made from either ERVs or from infectious viruses.

In the Ottowa presentation, JM presented a gel from an experiment that Ruscetti did. She claimed the experiment showed that there were silenced HGRVs in infected cells, and that demethylation could make them start producing virus again. Assuming the labels on the slide she presented actually represent what was on the gel (they don't, she made that up too, but that's another story) they show patient samples that were treated with 5AZA, compared to controls that were not treated with 5AZA.

The problem is, she compared CFS-patient, 5AZA treated cells, with healthy-control cells that wereNTo treated with 5AZA, and showed that there was gag protein in the CFS samples. But from that experiment, there is no way to know if she is detecting gag from recent provirus from a fresh HGRV infection, or gag from endogenous gamma-retrovirus sequences of the human DNA.

That is, treating the patient cells with 5AZA is enough to make it produce gag from human DNA - even if it does not have a virus infection.

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Back to Figure 2c. Ruscetti has now admitted that the patient samples in Figure 2C were treated with 5AZA. That means that the gag they detected, could simply be from endogenous retrovirus sequences, including possible gamma-retrovirus - and have nothing to do with a virus infection.

This is really basic virology. I know it as an out-of-date evolutionary geneticist, because the endogenous retrovirus story has been a fundamental story in evolution. Its been a fundamental story in virology,in cell biology, in genetics. Ruscetti and Mikowits had to of known this.

Knowing that the patient samples in Figure 2c were treated with 5AZA, there is no way to tell if they are detecting XMRV, some other infections HGRV - or gag from ERVs. Just as bad, even if they are detecting an infectious HGRV, that gets turned on by 5AZA - there is no way to know if that virus might be in the controls as well, because they did not treat the controls with 5AZA.

The 5AZA treatment was a major factor in that experiment. There is NO WAY that it was a meaningless trivial detail. Ruscetti and Mikowits had to of known it. When Ruscetti said it didn't matter, every scientist I know of who has followed this, was surprised or laughed out loud.

What Figure 2C says, knowing what they did with 5AZA, is no more than - 'hey, we can find gag protein by doing things that are known to cause gag protein from healthy cells.'

This is, at best, scientific misconduct in misrepresenting the experiment they did, and what was on Figure 2c. They may not be simply making up data - maybe they do have gels showing gag in patient samples and not in controls, without 5AZA treatment, as they claim in the Science paper. But we don't know -we can't know without someone going through their lab notebooks and results - because they didn't honestly and thoroughly tell us what they did.

And at least some of what they did tell us of what they did, was not actually what they did -and they knew it when they told us.

They knew that Figure 2C had 5AZA treated patient samples, and didn't tell us. They knew that lanes labeled claimed to be from patient samples in the Ottowa presentation, were actually healthy controls.

Once a scientist has shown that they are willing to intentionally say something not true about their methods or their data, they cease to be scientists. They will no longer be trusted in science. It's a career-ender, and it makes all their work suspect, and it means that everyone who was relying on their work for their own research and research decisions cant trust the things they were relying on.

Mikowits misled us about their methods and their results, and in the case of Figure 2C and the Ottowa slide, have said they did so.

And THAT is why there is so much outrage from scientists about this.

 

[oceanblue]

If 5-aza was merely activating endogenous retroviruses - as opposed to XMRV/HGRV - would the gag proteins still be the exact same size as for XMRV/HGRV and therefore produce a band on the gel in the exact same position?

 

[asleep]

Lee,

Since you love magic tricks so much, how about this one:

Go to 1:54:32 of this video.

This is Pathak talking about Paprotka et al (the recombination paper) during a presentation. After talking about testing the early xenografts he says (transcribed by me):

In contrast, if we do RT-PCR of RNA derived from these late xenografts we can readily detect XMRV.

The results shown in the right part of the corresponding slide (entitled "XMRV-Specific PCR: Late Xenografts Do Contain XMRV") are included in their paper as the right part of Fig 2E. (This you can discern with your highly sharpened skills of matching pictures.)

Now the really interesting thing is that nowhere in their paper or their supplemental materials do they mention using RT-PCR (Reverse-Transcription PCR). Hocus Pocus!

To repeat, they include results in their paper (right part of Fig. 2E) that Pathak attributes to RT-PCR, yet they make no mention at all of this entire method in their paper.

So Lee, are they misleading the scientific community by omitting an entire method on which they based results and interpretations in their paper? Are you outraged about THIS, or do you only get outraged if Mikovits is involved?

 

[Lee]

"would the gag proteins still be the exact same size as for XMRV/HGRV and therefore produce a band on the gel in the exact same position?"

Could be. Sizes of viral proteins tend to be constrained by evolution - a virus is packing a lot of information into a very small space. Depends on what the antibody is recognizing, and if that version(s) of gag is complete and processed. Lots of variables. Point is, one can't know - which is why the 'healthy' controls are critical, and why it is critical that they told us in the first place.

 

[Lee]

asleep:

If you read the paper carefully, and the M&M in the supplement, it is clear that they are doing PCR to amplify provirus. Provirus is DNA. DNA is amplified by PCR, not RT-PCR. They did not do RT-PCR in that paper, which is why the paper does not mention RT-PCR.

In the talk, he does say RT-PCR. Once. In a line of argument in which he is talking about PCR, and it is clear that this slide is part of that discussion. He mis-spoke. Once. Virologists use RT-PCR all the time, he is used to saying it, he said RT-PCR instead of PCR.

There was no 'omission' of a description of of RT-PCR from Paprotka et al - they never used RT-PCR.

 

[asleep]

asleep:

If you read the paper carefully, and the M&M in the supplement, it is clear that they are doing PCR to amplify provirus. Provirus is DNA. DNA is amplified by PCR, not RT-PCR. They did not do RT-PCR in that paper, which is why the paper does not mention RT-PCR.

In the talk, he does say RT-PCR. Once. In a line of argument in which he is talking about PCR, and it is clear that this slide is part of that discussion. He mis-spoke. Once. Virologists use RT-PCR all the time, he is used to saying it, he said RT-PCR instead of PCR.

There was no 'omission' of a description of of RT-PCR from Paprotka et al - they never used RT-PCR.

He clearly says "...RT-PCR of RNA...". So that would be doubly misspeaking! Furthermore, how do you know that "they never used RT-PCR"? Shouldn't you, as a "skeptic" of "bad science," be up in arms about this possible egregious omission of crucial information? Certainly, the evidence points to an possible omission massive enough to warrant investigation. But all you're willing to offer is hypocritical, speculative justifications. When it's Pathak in question it's unsubstantiated excuses, but when it's Mikovits it's burn baby burn.

 

[Angela Kennedy]

**********

I think Lee has sadly underestimated his audience's understanding of the science around ME/CFS**********

 

[Lee]

asleep:

Your 'evidence' consists of a scientist saying 'RT-PCR' one time in a talk, while looking at evidence that is clearly PCR, and that is fully described in the paper as PCR.

If you're that worried about it, email him. All I see is one place in a talk where he says 'RT-PCR of RNA' instead of PCR, in a context where he clearly means PCR. There is NO EVIDENCE of having omitted anything from the paper. His methods match his results. They are detecting provirus, and then getting proviral copy numbers from Real-Time PCR - and they adequately describe the Real-Time PCR in the paper.

In the case of Mikovits and Ruscetti, they have now admitted to omitting critical experimental details, admitted to changing labels on slides to misrepresent what is in the slide.

You really think those are equivalent? Really?

 

[Lee]

Angela:

Spare the sarcasm. i said I was starting from the basics - not because I don't think a lot of people here know the basics,but because there are clearly some people here who may not know the basics in enough detail to put the entire story together. So I put the entire story there.

If you have any intention of responding with substance, I'm happy to listen. if all you can do is snark and insult, I'm not interested.

 

[oceanblue]

"would the gag proteins still be the exact same size as for XMRV/HGRV and therefore produce a band on the gel in the exact same position?"

Could be. Sizes of viral proteins tend to be constrained by evolution - a virus is packing a lot of information into a very small space. Depends on what the antibody is recognizing, and if that version(s) of gag is complete and processed. Lots of variables. Point is, one can't know - which is why the 'healthy' controls are critical, and why it is critical that they told us in the first place.

So an ERV might produce a different sized gag? The patient gag band does match that of the reference gag protein "SFFV-infected HCD-57" in lane 8, which is at least consistent with it being an XMRV/HGRV. I agree that 5-aza should have been used in controls, but ERVs could well produce bands in different positiions, or even several positions if more than one ERV is expressed.

Your theory might be right, but isn't it possible that expressed ERVs could produce quite a different WB pattern from that seen in the contentious gel?

 

[Lee]

oceanblue:
they could, yes. But again, we don't know, we cant know, because they didn't to the control and they didn't tell us they did the experiment, even though they showed us (and misrepresented) the result.

Yes, someone should look through her notes and results, and Ruscetti's, and see of she did the experiment that actually shows an interpretable result, and perhaps, if not, do the experiment to make sure one way or the other.

But JM and Ruscetti have already shown us they cant be trusted to tell us what they did, or what they got, so they cant be the ones to do it, and we cant trust anything they told us.

Again - that's why 5AZA matters. Now that we know it was used, we can't interpret their results. Any of them, really, because nearly every figure on the Science paper that has not already been retracted, can be interpreted as an artifact of 5AZA derepression - and we know that we can't rely on them to have told us whether they used 5AZA.

It is now possible that the entire Science paper is an artifact of PCR contamination and of 5AZA induction without proper controls - but we can't know, because we cant rely on them to have told us what they did.

That's why this is so egregious. All the followup work, all the time, all the money - and no one now knows whether there was actually any reason to have done any of it.

 

[Lee]

also oceanblue:
"Your theory might be right, but isn't it possible that expressed ERVs could produce quite a different WB pattern from that seen in the contentious gel?'

I think you're missing my main point. I don't have a theory - I'm not saying that gag band is from ERVs. I'm saying I don't know if that is gag from XMLV, or gag from HGRV, or gag from ERVs - and neither does anyone else.

And the reason we don't know, is that JM and Ruscetti didn't tell us what that experiment actually was - they represented it as being something else entirely.

They told us what the experiment was - activated PBMCs from patients and from healthy controls, with gag found only in patients. That experiment made sense, it was properly controlled, it was easily interpretable - patients express gag, healthy controls do not. People interpreted it that way, and designed followup experiments based on that interpretation.

But JM and Ruscetti didn't do the experiment they said they did, they did a different one and labelled it to look like the one they said they did. There is now no evidence for the experiment they said they did - we have NO EVIDENCE that they ever actually got that result.

And the experiment they actually did, now that we know what it was, doesn't tell us a damn thing.

What a waste.

 

[Lee]

asleep,

I'm about done with you - calling me a hypocrite is over the line, and I thought was a fundamental violation of the policy of this blog.

But one last time - you point out a place where a scientis says, one time - in a context where it is clear that PCR is being discussed - "RT-PCR." You accused that man of leaving out descriptions of RT-PCR from his paper - where the only evidence that it should be there is that one obvious misstatement - remember, the context of that part of his talk is detection of provirus. And in the paper, it is clear that they are detecting and determining copy number of provirus - where the technique that does that is PCR and Real-Time PCR.

I'm satisfied that it was a mis-statement. If you are not, email him.

By contrast, JM and Ruscetti have ADMITTED to failing to describe a critical part of their experiment - even though they try to say it didn't matter, the point of my post here is that it does matter - and JM has admitted to having changed labels on a gel in a way that misrepresents what is on the gel - and defended it by saying it was appropriate because it was for presentation to patients!!!.

if you honestly are willing to accuse me of being a hypocrite for not believing those two things are equivalent, I see no reason to continue talking with you.

 

[oceanblue]

also oceanblue:
"Your theory might be right, but isn't it possible that expressed ERVs could produce quite a different WB pattern from that seen in the contentious gel?'

I think you're missing my main point. I don't have a theory - I'm not saying that gag band is from ERVs. I'm saying I don't know if that is gag from XMLV, or gag from HGRV, or gag from ERVs - and neither does anyone else.

You've taken a pretty strong line in your posts here and I found what you said about ERVs very interesting (I'd thought the 5-aza was only important to unmask any XMRV/HGRV in controls) but I think it was worth pointing out that ERVs may well not give the same result as an HGRV. If you're going to critcise others, I hoped you'd be open to a little clarification on your own views. I wouldn't bother posting if I wasn't genuinely interested in what you are saying.

 

[slovokia]

Hi Lee,

Thanks for the information. I was wondering what happens when a paper like the Lombardi paper gets reviewed before publication do the reviewers assume a basic level of underlying competence in the methods/techniques used by the authors of the paper? Assuming that Mikovits et al did not lie or deliberately misinterpret data, one is left with sheer incompetence. Is it possible that reviewers would not detect that?

 

[Lee]

oceanblue:

I apologize if I wasn't clear - seems I wasn't - that yes, it is very possible that gag expression from ERVs might give a different looking result than from HGRVs - if they exist.

But they also might not. The only way to know what they would produce, is to do the control - which is why we cant use that to interpret the gel.

And, as you mention, this is not the only problem with that gel Even if that gag is from a virus, teh fact taht they only treated patient samples with 5AZA, means that we cant interpret the absence of gag from controls as meaning controls don't also have the virus. It is possible (extremely unlikely,, but possible) that they actually detected a new ubiquitous retrovirus that has nothing to do with CFS.
But again, we don't know - they didn't tell us what they did, and didn't show us the controls.

 

[Angela Kennedy]

Angela:

Spare the sarcasm. i said I was starting from the basics - not because I don't think a lot of people here know the basics,but because there are clearly some people here who may not know the basics in enough detail to put the entire story together. So I put the entire story there.

If you have any intention of responding with substance, I'm happy to listen. if all you can do is snark and insult, I'm not interested.

Lee, the Me/CFS community and their supporters have been responding with substance for many years now. You are dealing with a knowledgeable community on the whole who only want good science to progress so that they (or their loved ones) may live lives not blighted by severe physiological impairment and subject to egregious (as it's a buzz word presently) psychogenic dismissal.

This community has been subject to a 'war of attrition' - attacks on its integrity and reputation, on its safety and well-being, from people claiming scientific authority in various areas. We just do NOT get respect, nor are we listened to, no matter how substantive our concerns. We are patronised, villified, jeered at, smeared, subject to vicious ad hominem, ignored when we express vital concerns, and that's just the Bad Science crowd and Abbie Smith and her followers etc.

We also get arrogant anons coming onto our forums with monotonous regularity, basically to tell us how stupid and ignorant we are, and recently, to attack scientists who have shown integrity and scientific inquisitiveness about this illness.

I don't need any clucking noises from you, Lee. But what I have every right to do is to call you out as a mere anon with no verifiable scientific credentials, self-proclaiming scientific expertise he does not actually appear to have, and trying to promote value judgements as 'fact'.

You have NO way of backing up that "there are clearly some people here who may not know the basics". That is pure value judgement on your part, and a way of calling people stupid, likely the people disagreeing with you.

I note that my own substantive comments here are NOT being addressed by anons like you at all. So you people are clearly NOT happy to 'listen'. Your 'snark and insult' comments show this. You are attempting to impose an attack on Mikovits and colleagues. That is it. It's quite clear.

You have shown nothing but bad faith from the start. I'm not interested in your beliefs in this area because you are not bringing anything useful, but instead the same old attacks on people as detailed above. But it won't stop you spouting them, sadly. While you exhibit no integrity, I cannot take you seriously.

 

[Lee]

slovokia:
"I was wondering what happens when a paper like the Lombardi paper gets reviewed before publication do the reviewers assume a basic level of underlying competence in the methods/techniques used by the authors of the paper?"

The reviewers assume that the authors are telling the truth, as do the readers. It's a necessary assumption, and it is one of the many reasons that scientists are so adamant about NEVER EVER UNDER ANY CIRCUMSTANCE intentionally misrepresenting the methods or the data, and why intentional misrepresentation is a career killer for any scientist.

As written, that's a pretty good paper. Problem is that now, we know that at least some of what they wrote isn't what they actually did.

 

[Lee]

@Angela:
"You have NO way of backing up that "there are clearly some people here who may not know the basics". That is pure value judgement on your part, and a way of calling people stupid, likely the people disagreeing with you."

Nope, that is a response to comments I've read that say things like "I don't know the science" or "I cant follow the technical argument." I tried to lay out the science and the technical point, because I assume that the people who said things like that are intelligent and interested and will understand it if given the background.

You, on the other hand, are clearly unable or unwilling to actually address the substance of what I said, and are instead attacking me as your only response to me. Shoo.

 

[Bob]

Thanks for the info Lee. You make a very strong case for why including the info about 5AZA could have been essential to the Lombardi study. I do agree that the information that we currently have access to, is confusing and contradictory, but we don't know all of the details yet, so you are making premature conclusions based on partial information.

If all of the patient samples were treated with 5AZA, but none of the control samples were, then like you say, I think it makes the original study quite meaningless. However, it might be the case that 5AZA didn't feature in the study at all, apart from a small number of exploratory samples that weren't significant to the bulk of the study - in which case the new info doesn't change anything.

I guess that if the scenarios that you have presented correspond to actual events, then the paper may be fully retracted very soon.
But we don't yet know what the full facts are.
We'll have to wait to see what happens.

I would hope that Harvey Alter and Shing Lo would not have made similar omissions that you suggest have happened with the Lombardi paper, so I hope that they will follow up their research. And if the Lombardi et al anti-body results still stand, then I hope that that research is followed up. It is reported that Maureen Hanson has reported getting the same anti-body results, so these could potentially be used as a biomarker, and could also provide essential insights into the disease, even if they have only detected an activated ERV. I also hope that the Lipkin study goes ahead to put this subject to rest for good.