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New WPI and CDC XMRV sequences in genbank

ukxmrv

Senior Member
Messages
4,413
Location
London
Todd,

Dr Mikovits had a look of respect on her face for Dr Coffin and whatever this paper is. She didn't have a look of being threatened by or scathing of whatever it is Dr Coffin is due to publish.

I've seen her now at two IiME conferences and Dr Mikovits has always spoken in supportive/glowing/respectful tones of other researchers papers and findings (even when they do not match hers). Dr Mikovits was also talking to and mixing with Dr Peterson and others there freely and on good terms.
 

currer

Senior Member
Messages
1,409
UKXMRV is right. Dr Mikovits simply mentioned Dr Coffins paper in passing, quite respectfully and professionally. She did not give the impression that there were any issues there.
 

toddm1960

Senior Member
Messages
155
Location
Rochester, New York
That's good to hear, I was afraid she knew of more piling on of contamination studies. As long as it's nothing more than he's said to this point.......it's good news.
 

insearchof

Senior Member
Messages
598
Hi insearchof,

From wikipedia: http://en.wikipedia.org/wiki/Polymerase_chain_reaction_optimization#Magnesium_concentration

Magnesium concentration

Magnesium is required as a co-factor for thermostable DNA polymerase. Taq polymerase is a magnesium-dependent enzyme and determining the optimum concentration to use is critical to the success of the PCR reaction.[3] Some of the components of the reaction mixture such as template concentration, dNTPs and the presence of chelating agents (EDTA) or proteins can reduce the amount of free magnesium present thus reducing the activity of the enzyme.[4] Primers which bind to incorrect template sites are stabilized in the presence of excessive magnesium concentrations and so results in decreased specificity of the reaction. Excessive magnesium concentrations also stabilize double stranded DNA and prevent complete denaturation of the DNA during PCR reducing the product yield.[3][4] Inadequate thawing of MgCl2 may result in the formation of concentration gradients within the magnesium chloride solution supplied with the DNA polymerase and also contribute to many failed experiments.[4]


Back to me again - what does this mean? The polymerase used is stable at very high temperatures (it is derived from an organism that lives in extremely hot water), and those temperatures alter the physical characteristics of the DNA, speparating the twin strands, which makes PCR possible. The polymerase enzyme is magnesium dependent, it can't copy DNA without magnesium. Too little magnesium means the polymerase will not work, too much protects the DNA even so that it is stable at high temperatures - it remains double stranded. Double stranded DNA cannot be copied without first separating the strands.

I gather that optimization of magnesium concentrations is dependent on the target DNA. I do not know enough about this process to comment further on this point, I would only be speculating.


http://www.caister.com/molecular-biology-blog/2008/11/pcr-troubleshooting-mg-concentration.html

This site discusses Mg troubleshooting for PCR briefly. When certain other substances are used in the reaction, they can alter magnesium ion concentrations and so the magnesium level has to be adjusted. There are entire manuals on PCR optimization online, and a number of books.

I have never used a PCR machine, although I was involved in labwork in which PCR was used, but the machine was handled by an experience operator. My knowledge is more theoretical, and now old - there are many new types of PCR reactions than I studied when I was at university.

Of interest in the XMRV debate is that reverse transcription (RT-PCR) is often using MuLV reverse transcriptase, which is one reason that contamination testing is required for all reactions I suppose. RT-PCR is for amplifying RNA rather than DNA - and XMRV is an RNA virus. So if you are looking for the virus, and not its integrated DNA form, you need to look for RNA.

As to when and how it is added, I can only speculate. It is probably added at the very beginning with the rest of the reaction mixture. I doubt any is added later, if for no other reason than this would increase risk of contamination. However, this is an engineering issue, and different machines may or may not allow this - I do not know.

Bye
Alex

Hi Alex

Thank you so much for this information. It just goes to show how involved these scientific processes are and how easy it is for these experiments to fail.

So, is magnesium used to amplify RNA or is it used for other purposes?
 

Bob

Senior Member
Messages
16,455
Location
England (south coast)
Hi Alex

Thank you so much for this information. It just goes to show how involved these scientific processes are and how easy it is for these experiments to fail.

So, is magnesium used to amplify RNA or is it used for other purposes?

Hi insearchof,

From reading that helpful wiki entry, it seems that magnesium concentrations optimise the efficiency of the enzyme (polymerase) that is used to detect DNA/RNA in the PCR test.

The article doesn't specifically mention RNA, but I assume that it works in a similar way when searching for RNA, but there are probably other steps in the process involved.

If too much magnesium is used, then the enzyme (polymerase) will become more sensitive but less specific, so there will be more positive readings but there might also be a higher proportion of false positive readings. (i.e. more sensitive but less accurate.)

And if too little magnesium is used then the enzyme will be less sensitive but more specific, so it will detect less of what you are looking for, but there would be a lower proportion of false positive readings. (i.e. less sensitive but more accurate.)

So the magnesium doesn't amplify the RNA directly, but it can have the result of increasing the detection rates of RNA sequences, due to acting on the enzyme (polymerase) that is used to detect the DNA/RNA sequences.

That's my understanding of it anyway, but I may have misinterpreted the article.

I might be wrong about this, but I believe that the WPI use a less specific test, which detects a wider range of DNA/RNA sequences, but then they add another step to the procedure, which analyses the sequence that they have detected in order to determine if it is an XMRV/PMRV sequence, or not. So they only confirm that it is an XMRV RNA sequence once they have sequenced it. (Again, I might be wrong about this, but this is what I understand.) But I don't know if they work like that all of the time, for all of their studies.
 

alex3619

Senior Member
Messages
13,810
Location
Logan, Queensland, Australia
Hi Alex

Thank you so much for this information. It just goes to show how involved these scientific processes are and how easy it is for these experiments to fail.

So, is magnesium used to amplify RNA or is it used for other purposes?

DNA polymerase is the enzyme that actually copies single DNA it binds to. It copies that strand and turns it into another strand. Primers are small segments of DNA that can bind to recognized sequences and start this copying.

The heat of the process is what separates the strands for potential copying.

DNA polymerase needs magnesium to work. Too much magnesium can lead to either the DNA becoming more stable and not separating (so it cannot be copied) which can cause the reaction to fail, or as Bob commented it can alter the reaction so that it becomes easy to copy the wrong thing - magnesium concentration is critical. I do not fully understand the topic of magnesium optimization. My guess is that more than a few PhDs were earned working that out alone.

DNA polymerase does not copy RNA. To amplify an RNA virus, it first has to be changed to a DNA strand, and this requires reverse transcriptase, an enzyme found in retroviruses.

Bye
Alex
 

alex3619

Senior Member
Messages
13,810
Location
Logan, Queensland, Australia
OK so we have a variable retrovirus in prostate cancer, and one that isnt varying enough in ME/CFS!

I am going to the Invest in ME Conference in London tomorrow, J. Mikovits will be there, giving a talk along with other researchers.
I can ask her about this.
Any questions anyone? I'll put them forward for you.

I agree, at the moment things dont look good for XMRV and CFS.

Hi currer, something I have been thinking about is this: what is a big difference between a CFS-wise asymptomatic XMRV positive prostate cancer patient and those with ME or CFS? Our immune systems are fighting like crazy! Maybe sequence diversity will always be higher in prostate cancer patients due to ME/CFS patients fighting and destroying the virus more often. It could be that we kill and deactivate more virus, and slow its replication even further. This is just speculation, but I thought it was interesting.

Bye
Alex
 

currer

Senior Member
Messages
1,409
Hi Alex,

I'm just coming round after the IiME conference - it has taken me almost a week to get back to normal. Sorry I could not post much earlier.
I agree with you that we are fighting the virus, and as JM speculates, this may be protective against some of the associated cancers.

However, when JM was discussing her research, the slide she showed had the "family tree" of all the viruses they had isolated, and how they relate to each other.
XMRV was on the bottom branch and above were many PMLV branches, showing all the PMLV variants they had isolated so far.

So I think that JM is simply suffering from the difficulty of getting her research published. They really are finding as many variants as they would expect for this type of virus.
She said that we should not use an HIV model when thinking about XMRV. The true parallel is HTLV1, which does not vary much. The same virus can be found in patients forty years on, HTLV1 does not mutate much. Remember HIV has a highly abnormal replication rate - it is astronomical! (Check out the Wiki page) This is why it mutates so much. XMRV is much more usual in its replication.

She did say that they had looked at their antibody tests. Sometimes you get an antibody to a gag protein showing up clearly, but no antibody to the expected env (this is just from memory). She interpreted this to mean that the env had mutated so that the antibody test could not pick it up. They were looking at these results in more detail as this was a sign that there was a new PMLV variant in this patient. (clever, huh!)

This is why they are behind on some research looking for straight XMRV, because they want to be sure and isolate all the PMLV examples they find in their blood samples.

She also quite definitely stated that active infection correlates with the biomarkers and cytokines. As I remember she said that they had found a specific cytokine profile in infection which was different to the Klimas one.
 

heapsreal

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I also wonder if we have an immune defiency first and second comes all the different infections. In japan cfs is known as Nk dysfunction syndrome, or something like that. Although not 100% perfect, but the only real known cure for cfs is ampligen and it works through strengthening our immune system, it doesnt fight any particular virus or infection but strengthens our immune system as a whole to fight these different infections.

Maybe we/they should be looking at ways to strengthen our immune system and or nk function as this is the most common immune abnormality. Also instead of trying to resurrect ampligen, maybe they should be looking for an alternative, one that is easier to use and administer then twice weekly ampligen IV's. Secondly go after co-infections with antivirals, antibiotics etc but the immune system should be first. Maybe then we wouldnt being arguing about 'the' infection causing cfs.

cheers!!!
 

alex3619

Senior Member
Messages
13,810
Location
Logan, Queensland, Australia
Hi, I wonder if we shouldn't be more active in promoting HTLV as a model. This has been a better model for a long time, but so many have never heard of it but most know of HIV. Like HTLV, many carriers seem to be asymptomatic. Indeed, if the prevalence of XMRV carriers is around 7% and the prevalence of symptomatic carriers is like 1 or 2% of the population, this would look a lot like HTLV.

currer, it is good to know that the range of virus being found includes full PMLVs.

heapsreal, fegarding ampligen, I was not aware that anyone was cured. I do know that many have periods of significant remission in symptoms, but these return. If anyone knows of someone who was cured, do you a link or reference? This would be an important step. It is however a drug with a long history of successful treatment, no other highly successful drug has been studied for as long.

Bye
Alex
 

heapsreal

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hi alex,
probably in my dodgy wording of cure , not 100% perfect but should have said the closest we have come to a cure and i would take a significant remission of symptoms while on the drug as close enough. I would grab it with both hands. Im thinking like HIV patients being almost cured on arv's and they also go backwards without their meds. HIV people have alot better quality of life and longevity on arv's, so im refering to a cure like this.

But what Im getting at is going after fixing the immune system first, which they have had the best success with ampligen. I just think that ampligen has been around along time, surely someone out there has made improvements on this type of treatment by now. Then maybe marrying up this type of treatment with going after the infections we know we have will get us over the line. I think this is something that should be happening now, narrowing down the exact bug maybe secondary. I get the impression this is maybe apart of what montoya and stanford are doing by studying different infections in cfs.

cheers!!!
 

currer

Senior Member
Messages
1,409
Hi Alex,
Yes Ive added this to my last post #68 but JM is spending a lot of her time now looking for the PMLV variants in blood. This has made her behind on some of the work just to do with the XMRV virus.
 

Bob

Senior Member
Messages
16,455
Location
England (south coast)
There are further XMRV sequences submitted to genbank by the WPI.
I've added them to the opening post:

Whittemore Peterson Institute
Lombardi,V.C. and Khaiboullina,S.F.
21st May 2011


Xenotropic MuLV-related virus isolate WPI-CI-1303 gag protein (gag) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/JF907633.1

Xenotropic MuLV-related virus isolate WPI-CI-1304 gag protein (gag) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/JF907634.1

Xenotropic MuLV-related virus isolate WPI-CI-1310 gag protein (gag) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/JF907641.1

Xenotropic MuLV-related virus isolate WPI-CI-1313 gag protein (gag) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/JF907643.1

Xenotropic MuLV-related virus isolate WPI-CI-1314T gag protein (gag) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/JF907644.1

Xenotropic MuLV-related virus isolate WPI-CI-1315 gag protein (gag) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/JF907645.1

Xenotropic MuLV-related virus isolate WPI-CI-1316 gag protein (gag) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/JF907646.1
 

eric_s

Senior Member
Messages
1,925
Location
Switzerland/Spain (Valencia)
Great, i hope some of them will sink Coffin's hypothesis (in case the ones already uploaded were not enough to prove this hypothesis wrong).

I'm really looking forward to the day when this crazy controversy is over and we can just focus on getting help for the people affected with ME/CFS.
 

Jemal

Senior Member
Messages
1,031
I'm really looking forward to the day when this crazy controversy is over and we can just focus on getting help for the people affected with ME/CFS.

Same here. I have the feeling though the controversy will probably last for some time yet... I am not a generally positive person though, so I hope I am wrong.
 

eric_s

Senior Member
Messages
1,925
Location
Switzerland/Spain (Valencia)
I also think we might have to wait a bit more, but nevertheless there are interesting new results coming out every now and then.

I think we can use that time to strengthen our community, make new connections, try to improve the strucutes etc. so that we will be ready to shower the good reserach with more money, gain more influence with politicians/officials, get more awareness in the public and so on.
 

joshualevy

Senior Member
Messages
158
TWIV #136 has a discussion of the sequences in genbank. (I'm not sure about all of them, but certainly the first batch.) The summary is simple:
  • The tiny differences between the different sequences provide strong evidence that what was found was contaminant.
  • Far from helping the WPI cause, they provide direct evidence that the XMRV found was a contaminant.

I must say I have never heard any Virologist who did not work for WPI, say anything positive about those gene sequences. Everyone who is knowledgeable in the field says the same thing: they are evidence of contamination.

BTW, This Week in Virology #136 is well worth listening too.

Joshua (not Jay!) Levy
 

Bob

Senior Member
Messages
16,455
Location
England (south coast)
TWIV #136 has a discussion of the sequences in genbank. (I'm not sure about all of them, but certainly the first batch.) The summary is simple:
  • The tiny differences between the different sequences provide strong evidence that what was found was contaminant.
  • Far from helping the WPI cause, they provide direct evidence that the XMRV found was a contaminant.

I must say I have never heard any Virologist who did not work for WPI, say anything positive about those gene sequences. Everyone who is knowledgeable in the field says the same thing: they are evidence of contamination.

BTW, This Week in Virology #136 is well worth listening too.

Joshua (not Jay!) Levy

The Coffin paper has been challenged by the new sequences in genbank - Both Switzer's and the WPI's newly published sequences.
Many people probably are not aware of the new sequences, because a high profile paper hasn't been published about them.
 

joshualevy

Senior Member
Messages
158
The Coffin paper has been challenged by the new sequences in genbank - Both Switzer's and the WPI's newly published sequences.
Many people probably are not aware of the new sequences, because a high profile paper hasn't been published about them.

The TWIV #136 comments were in June, and the new sequences were in May, so I think their comments covered those sequences. Are you saying there were sequences added in June?

Joshua (not Jay!) Levy
 

Bob

Senior Member
Messages
16,455
Location
England (south coast)
The TWIV #136 comments were in June, and the new sequences were in May, so I think their comments covered those sequences. Are you saying there were sequences added in June?

Joshua (not Jay!) Levy

I'm saying that most people are only aware of the 3 sequences that the WPI originally published.
The latest sequences were published on 21st May.
A professional analysis has only been carried out, and peer reviewed, in relation to the original WPI sequences, and not the recent sequences, as far as I am aware.
The information and analysis, that I have seen, suggests that the new sequences do have a greater variety than the original sequences.