Amy Marcus: Science editors ask Mikovits to withdraw 2009 paper

[Gerwyn]

]I can’t see why it would not work.  WPI haven’t entered into this blindly and the only reason surely you would back away is if you were not confident in your own findings.  Somebody has to take the lead

if anyone is wondering why an assay that can find a low copy number of the VP-62 clone cant find wt virus the answer is relatively simple

The chemophysical properties of the DNA are different

high  levels of oxidative stress caused by hgrv infections and commonly found in studies investigating patients with ME cause oxidative modification of nucleotide bases. The most common is the addition of a hydroxyl functional group or deamination. This (for reasons involving sterochemistry and something called twisting) means that the stacking energy and hydrogen bonding which hold complimentary nucleotide bases together are dramatically reduced. This can be compensated for by using increased levels of magnesium and lowering annealing temperatures.Another strategy is to use an RNA template to begin with. mlv viruses cant replicate in resting cells so xmrv will exist in such cells as preintegrative complexes and integrated highly methylated proviruses. ROS oxidation induces a pattern of base substitution or inversion in the provirus affecting primer annealing.The presence of certain oxidised bases or even abasic sequences can stop taq polymerase "in its tracks" leading to an absence of product.Unless PCR reagent concentrations and cycling  conditions are adjusted accordingly the virus will escape detection. silverman was able to adjust his assays to find gag sequences which were already present and was fortunate enough to isolate XMRV from RNA hence largely avoiding these issues

The effect of activating PMBcs would be to allow the integration of "fresh" proviruses as the preintegrative complexes are nowhere near as prone to oxidative damage.This would also allow the creation of viral RNA

I will post the detailed version  but I wanted to post this to demonstrate what a BS decision the Science editors are making

"Science" has abandoned Science. I hope that its subscribers take note

[Nina]

The chemophysical properties of the DNA are different

high  levels of oxidative stress caused by hgrv infections and commonly found in studies investigating patients with ME cause oxidative modification of nucleotide bases. The most common is the addition of a hydroxyl functional group or deamination. This (for reasons involving sterochemistry and something called twisting) means that the stacking energy and hydrogen bonding which hold complimentary nucleotide bases together are dramatically reduced. This can be compensated for by using increased levels of magnesium and lowering annealing temperatures.Another strategy is to use an RNA template to begin with. mlv viruses cant replicate in resting cells so xmrv will exist in such cells as preintegrative complexes and integrated highly methylated proviruses. ROS oxidation induces a pattern of base substitution or inversion in the provirus affecting primer annealing.The presence of certain oxidised bases or even abasic sequences can stop taq polymerase "in its tracks" leading to an absence of product.Unless PCR reagent concentrations and cycling  conditions are adjusted accordingly the virus will escape detection. silverman was able to adjust his assays to find gag sequences which were already present and was fortunate enough to isolate XMRV from RNA hence largely avoiding these issues

You are right Gerwyn, it IS simple.

 

[Adam]

The WPI are not backing down - this business with Science is in danger of getting blown of proportion. Yes no doubt the Science editors have received communications from other parties shall we say suggesting that it might be prudent to get the WPI to retract the Oct 09 paper and just the mention of this publicly appears to add to the so-called doubt surrounding that paper, but Judy M will have been expecting this for some time. She knows her assays work, I imagine the NCI know that their assays work - not sure about the CDC - so when the BWG results are announced and as a result an agreed validated method is agreed - the WPI will be vindicated. They can then demonstrate to all concerned that this is the methodology they have been using to find XMRV in PWMECFS - some people have suggested that Judy M is naive about the politics surrounding ME/CFS - perhaps she was - at one time - I suggest she became aware quite some time ago.

Judy M remains robust and confident about her findings. Everything else is just bullshit. Money and egos are at stake here. Some scientists have gone out on a limb - some scientists might find grant monies hard to come by and some might find their reputations seriously flawed.

Chill pill time folks 

[Adam]

Nice post Gerwyn, Sir. If we stick to the facts we might get away with not beating orselves up too much. I know Judy M will stick to the facts when she receives a reply from the editors of Science - she will staet quite clearly why the combination of negative papers and papers supposedly suggesting contamination are, in fact, erroneous.

[acer2000]

Good news is coming  after the summer.

Care to elaborate? 

[jemal]

Care to elaborate? 

Yes, well all need good news...

Anyway, we still have the retrovirology conference early june, maybe we'll get some positive news from there. Or not.

[Gerwyn]

Nice post Gerwyn, Sir. If we stick to the facts we might get away with not beating orselves up too much. I know Judy M will stick to the facts when she receives a reply from the editors of Science - she will staet quite clearly why the combination of negative papers and papers supposedly suggesting contamination are, in fact, erroneous.

oxidative damage to DNA is a common feature of neurological disease

Introduction:  Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by multifocal demyelination and axonal loss. In addition to the role of the immune response in targeting oligodendrocytes and myelin in MS lesions, the extensive inflammatory infiltration is likely to lead to free radical production and oxidative damage to both oligodendrocytes/myelin and neurons. In this study we investigated the extent of free radical damage to DNA in postmortem MS spinal cord.Materials and methods:  Serial 10 µm tissue sections were cut from 26 fixed and cryoprotected spinal cord blocks from 11 MS cases (age 64 ± 4 years, pmd 13 ± 2 h) and two controls (age 70 ± 9 years, pmd 22 ± 4 h). Lesions were characterized using H&E and LFB-PAS histology and HLA-DR immunohistochemistry. DNA oxidative damage was detected using a monoclonal antibody against 8-hydroxy-2′-deoxyguanosine (8OHdG; supplied by Dr S Toyokuni, Kyoto), the most abundant modified DNA base.Results:  Quantitative analysis revealed a 150% increase in 8OHdG+ cells relative to normal appearing WM and a 400% increase relative to control WM. A positive correlation was found between the level of macrophage infiltration (HLA-DR+) and 8OHdG-expressing cells.Discussion:  These results suggest a role for oxidative damage to DNA due to free radicals in the evolution of the MS lesion.

[Adam]

Damn macrophages are supposed to help us aren't they? Been eating up my jaw bones and lost two teeth on Friday  - lost 80% depth in upper jaw - my dentist said 'not bacteria, but macrophages supposedly attacking bacteria' - abit off topic I know but I just took my Chill Pill  so I can't stress 

[Gerwyn]

This is my last post on this topic


We reasoned that genetic information encoded in such
samples may not be lost but may simply be inaccessible because the
DNA polymerases commonly used for PCR stall at sites of damage11.

[Tango]

if anyone is wondering why an assay that can find a low copy number of the VP-62 clone cant find wt virus the answer is relatively simple

The chemophysical properties of the DNA are different

high  levels of oxidative stress caused by hgrv infections and commonly found in studies investigating patients with ME cause oxidative modification of nucleotide bases. The most common is the addition of a hydroxyl functional group or deamination. This (for reasons involving sterochemistry and something called twisting) means that the stacking energy and hydrogen bonding which hold complimentary nucleotide bases together are dramatically reduced. This can be compensated for by using increased levels of magnesium and lowering annealing temperatures.Another strategy is to use an RNA template to begin with. mlv viruses cant replicate in resting cells so xmrv will exist in such cells as preintegrative complexes and integrated highly methylated proviruses. ROS oxidation induces a pattern of base substitution or inversion in the provirus affecting primer annealing.The presence of certain oxidised bases or even abasic sequences can stop taq polymerase "in its tracks" leading to an absence of product.Unless PCR reagent concentrations and cycling  conditions are adjusted accordingly the virus will escape detection. silverman was able to adjust his assays to find gag sequences which were already present and was fortunate enough to isolate XMRV from RNA hence largely avoiding these issues

The effect of activating PMBcs would be to allow the integration of "fresh" proviruses as the preintegrative complexes are nowhere near as prone to oxidative damage.This would also allow the creation of viral RNA

I will post the detailed version  but I wanted to post this to demonstrate what a BS decision the Science editors are making

"Science" has abandoned Science. I hope that its subscribers take note

Ger is this correct as a translation.

Oxygen is stopping the primers from sticking.  The way to fix this is to increased levels of magnesium and lowering annealing temperatures.  Or use an RNA template to begin with. 

[Adam]

It's not all bad.  Judy has been asked to voluntarily retract the paper.  Judy says no.  She is right.  She's not going to succumb to the pressures of the naysayers.  The study will stay unless she retracts it, that won't happen.  Judy appears to be a person who won't cave to the politics.  They obviously have no scientific basis to retract the study or they would have done it already.

This is correct and as Val said I think, Judy has the opportunity (if she wishes to make it public) to sate very clearly why she won't retract and as a result make those asking fro the retraction look foolish - it all seems abit desperate to me - especially with BWG results to come which will clearly demonstrate that the WPI can distinguish between positive and negative samples and hence validate their findings.

[Tango]

This is my last post on this topic


We reasoned that genetic information encoded in such
samples may not be lost but may simply be inaccessible because the
DNA polymerases commonly used for PCR stall at sites of damage11.

Translation

There, but we can't see it.

[Adam]

Ger is this correct as a translation.

Oxygen is stopping the primers from sticking.  The way to fix this is to increased levels of magnesium and lowering annealing temperatures.  Or use an RNA template to begin with.

I will step in for Ger for a moment and answer this one V - yes. they are cooking on the big fat burner at the back of the hob when they should beusing the itty bitty one at the front FFS 

[Tango]

 

[jemal]

The naysayers will have a field day... the P from WPI publishing a negative study in Science and Science asking the WPI to retract their study. If you haven't read all the studies in detail, you will think this is the end of it. I know I would if I was a member of the general public. There's still people commenting on the WSJ that we have a psychological disorder...