Author Topic: New sequences published  (Read 8375 times)

Daisymay

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Re: New sequences published
« Reply #45 on: May 18, 2011, 01:39:42 PM »
it looks like they are different strains but have not looked really closely. It just took me by surprise .I was looking for evidence why optimising a PCR assay using a clone is such an assenine thing to do when RNA viruses replicate using a low fidelity enzyme. I found a lot more variation than I expected because gammaretroviral reverse transcriptases are supposed to result in less mutations than HIV reverse transcriptase.

Thanks Ger

anciendaze

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Re: New sequences published
« Reply #46 on: May 18, 2011, 03:18:42 PM »
i did not expect the sequences to demonstrate such a high level of variation
My reason for expecting this level of variation includes what I've called my "entropy heuristic".  I won't claim this is defensible, only productive as an assumption.  It started out from thinking about how researchers could miss long-standing infections, and how diseases with such serious effects could avoid producing a definitive, consistent biochemical signature. 

This is not something I have predicted in detail.  I'm not trying to do that because I know cognitive impairment gives me an unacceptable chance of errors in details.  I've been looking for robust hypotheses which might still be falsifiable.  This isn't easy. 

What I noticed right away from this new data is that we are very close to overlapping HERV or MLV sequences.  Trying to exclude both of these from giving false positives in an assay is very likely to result in 0/0 outcomes.  Excluding IAP sequences may compound the problem if active retroviral infections produce similar sequences.  The problem may extend to antibodies to proteins resulting from transcription of excluded sequences. 

My guess has been that the apparent lack of variation reported in the original Lombardi/Mikovits paper was an artifact not of contamination, but of selection due to the culture test used to validate early PCR testing.  Culturing in a cell line characteristic of cancer favored a particular rapidly-replicating strain typical of aggressive prostate cancers.  The discovery that PBMCs which were not naturally activated by infection needed to be activated in the laboratory, then allowed to grow in a 'culturing' step prior to PCR, strengthened this conviction.  Extensive hypermutation, and the realization that production of enzymes causing this was turned off during mitosis of PBMCs, also fit. 

The major discovery of a gamma retrovirus in humans with ME/CFS, defined as an organic disease, was correct.  The statement published at that time about a surprising lack of variation was wrong.  Failure to publish the need for a 'culturing' step was an honest oversight.  At the time of original publication, they hadn't realized everything necessary to make their assay work reliably in practice.  There are always such oversights of important details in new research.  With publication of subsequent papers blocked, all criticism focused on those understandable missteps in the original publication. 

Instead of a vast conspiracy I see a great deal of knee-jerk reaction and predictable administrative response compounding uncertainty and confusion which typically characterizes new discoveries.  I can't rule out a half-vast conspiracy. 

Tango

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Re: New sequences published
« Reply #47 on: May 18, 2011, 03:22:41 PM »
Culturing was published in the paper.  Am I confusing what you mean anciendaze.

"I suspect there have been a number of conspiracies that never were described or leaked out. But I suspect none of the magnitude and sweep of Watergate." Woodward

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anciendaze

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Re: New sequences published
« Reply #48 on: May 18, 2011, 05:28:40 PM »
Culturing was published in the paper.  Am I confusing what you mean anciendaze.
There have been two uses of the term 'culturing':  1) the culture test for the virus using the LNCaP cell line;  2) the step of activating PBMCs and allowing them to divide several times to bring sequence numbers up to detectable levels.  The second meaning only appeared in responses after the original publication.  The 0/0 studies have consistently ignored this step, concentrating on the procedure explicitly spelled out in the original Lombardi/Mikovits Science paper. 

To head off more misunderstanding, I also want to say what I think went on in the Lo/Alter work.  That assay was developed and optimized to detect positive controls from the WPI group.  After the disagreement with the CDC group, Lo/Alter then ran tests for IAP sequences looking for contamination, in addition to the very sensitive test for mouse mtDNA.  These came back negative. 

Had the assay been developed under the restriction of avoiding these sequences, and concentrating on the few clearly identified sequences like vp62, I think it likely either the assay would not have reached the extraordinary sensitivity required, or the well-developed IAP test would have found evidence of 'contamination'.  Meeting all requirements for sensitivity and selectivity pushes you right to the edge of the state of the art.  In that sense, none of the current assays alone are satisfactory as clinical tools. 

The distinction between tests as research tools, where special care for controls is necessary, and as clinical tools to be applied by ordinary doctors, has been repeatedly glossed over.  One reason for this apparently irrational behavior is the amount of money at stake.  We are looking at a market for some millions of test kits, each probably costing over $100, at the beginning.  Once the initial market is gone, there will probably be a drop in prices as more convenient assays are developed.  Getting in at the beginning is worth considerable money.  This money can be used to bootstrap development of the second generation assays, ensuring a continuing return. 

I've seen what people will do with that kind of opportunity at stake before.  They aren't sweetly reasonable. 

SamIAm

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Re: New sequences published
« Reply #49 on: May 18, 2011, 06:52:38 PM »

hello samiam I wondered when you would show  up  these are two of them


1 gcagcactgg gggagtgtcc agcgcattgc atccaaccag tctgtggatg tcaagaagag
       61 gcgctgggtt accttctgtt ccgccgaatg gccaactttc aatgtaggat ggcctcagga
      121 tggtactttt aatttaggtg ttatctctca ggtcaagtct agagtgtttt gtcctggtcc
      181 ccacggacac ccggatcagg tcccatatat cgtcacctgg gaggcacttg cctatgaccc

      241 ccctccgtgg gtcaaaccgt ttgtctctcc taaaccccct cctttaccga cagctcccgt
      301 cctcccgccc ggtccttctg cgcaacctcc gtcccgatct gccctttacc ctgcccttac
      361 ccactc
1 agcactgggg agatgtccag cgcattgcat ccaaccagtc tgtggatgtc aagaagaggc
       61 gctgggttac cttctgttcc gccgaatggc caactttcaa tgtaggatgg cctcaggatg
      121 gtacttttaa tttaggtgtt atctctcagg tcaagtctag agtgttttgt cctggtcccc
      181 acggacaccc ggaccaggtc ccatatatcg tcacctggga ggcacttgcc tatgaccccc

      241 ctccgtgggt caaaccgttt gtctctccta aaccccctcc tttaccgaca gctcccgtcc
      301 tcccgcccgg tccttctgcg caacctccgt cccgatctgc cctttaccca aa

this makes it a bit easier to see

181 ccacggacac ccggatcagg tcccatatat cgtcacctgg gaggcacttg cctatgaccc

181 acggacaccc ggaccaggtc ccatatatcg tcacctggga ggcacttgcc tatgaccccc






That doesn't mean anything because it's not how you do sequence alignments.  You might want research this and provide the correct information.
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Gerwyn

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Re: New sequences published
« Reply #50 on: May 18, 2011, 06:55:17 PM »
 i know what shows sequence variation sami am and how a pcr based on a vp-62 clone would miss these sequences

Tango

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Re: New sequences published
« Reply #51 on: May 18, 2011, 07:00:24 PM »
That doesn't mean anything because it's not how you do sequence alignments.  You might want research this and provide the correct information.

Ha ha ha!

Tell me SamIam, how can sequences match a full length clone?  Then tell me where they match identically with that clone?  No. What a surprise.
"I suspect there have been a number of conspiracies that never were described or leaked out. But I suspect none of the magnitude and sweep of Watergate." Woodward

"I would favor any name that does not impose (or give the appearance of imposing) taxonomic preconceptions on the nomenclature." Coffin

SamIAm

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Re: New sequences published
« Reply #52 on: May 18, 2011, 07:19:53 PM »
i know what shows sequence variation sami am and how a pcr based on a vp-62 clone would miss these sequences

You can't align each sequence from 1-10 -- doesn't work like that.  I believe you think you know, but really look it up --  ;)
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Gerwyn

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Re: New sequences published
« Reply #53 on: May 18, 2011, 07:23:16 PM »
You can't align each sequence from 1-10 -- doesn't work like that.  I believe you think you know, but really look it up --  ;)

samiam you really have no idea do you . Now where does the LTR region on the 5' side end?

Tango

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Re: New sequences published
« Reply #54 on: May 18, 2011, 07:34:43 PM »
You can't align each sequence from 1-10 -- doesn't work like that.  I believe you think you know, but really look it up --  ;)

Ha ha ha you are so out of your depth there.

Go on Sam, show us what it looks like when you line them up in your special way.
"I suspect there have been a number of conspiracies that never were described or leaked out. But I suspect none of the magnitude and sweep of Watergate." Woodward

"I would favor any name that does not impose (or give the appearance of imposing) taxonomic preconceptions on the nomenclature." Coffin

Jaz

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Re: New sequences published
« Reply #55 on: May 18, 2011, 08:26:37 PM »
There have been two uses of the term 'culturing':  1) the culture test for the virus using the LNCaP cell line;  2) the step of activating PBMCs and allowing them to divide several times to bring sequence numbers up to detectable levels.  The second meaning only appeared in responses after the original publication.  The 0/0 studies have consistently ignored this step, concentrating on the procedure explicitly spelled out in the original Lombardi/Mikovits Science paper. 

To head off more misunderstanding, I also want to say what I think went on in the Lo/Alter work.  That assay was developed and optimized to detect positive controls from the WPI group.  After the disagreement with the CDC group, Lo/Alter then ran tests for IAP sequences looking for contamination, in addition to the very sensitive test for mouse mtDNA.  These came back negative. 

Had the assay been developed under the restriction of avoiding these sequences, and concentrating on the few clearly identified sequences like vp62, I think it likely either the assay would not have reached the extraordinary sensitivity required, or the well-developed IAP test would have found evidence of 'contamination'.  Meeting all requirements for sensitivity and selectivity pushes you right to the edge of the state of the art.  In that sense, none of the current assays alone are satisfactory as clinical tools. 

The distinction between tests as research tools, where special care for controls is necessary, and as clinical tools to be applied by ordinary doctors, has been repeatedly glossed over.  One reason for this apparently irrational behavior is the amount of money at stake.  We are looking at a market for some millions of test kits, each probably costing over $100, at the beginning.  Once the initial market is gone, there will probably be a drop in prices as more convenient assays are developed.  Getting in at the beginning is worth considerable money.  This money can be used to bootstrap development of the second generation assays, ensuring a continuing return. 

I've seen what people will do with that kind of opportunity at stake before.  They aren't sweetly reasonable.

ancindaze, thanks so much for explaining this.  It makes sense that this is what's likely happening. 
The goal is not to bring your adversaries to their knees but to their senses. -- Gandhi

Tango

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Re: New sequences published
« Reply #56 on: May 18, 2011, 08:34:43 PM »
There have been two uses of the term 'culturing':  1) the culture test for the virus using the LNCaP cell line;  2) the step of activating PBMCs and allowing them to divide several times to bring sequence numbers up to detectable levels.  The second meaning only appeared in responses after the original publication.  The 0/0 studies have consistently ignored this step, concentrating on the procedure explicitly spelled out in the original Lombardi/Mikovits Science paper. 

To head off more misunderstanding, I also want to say what I think went on in the Lo/Alter work.  That assay was developed and optimized to detect positive controls from the WPI group.  After the disagreement with the CDC group, Lo/Alter then ran tests for IAP sequences looking for contamination, in addition to the very sensitive test for mouse mtDNA.  These came back negative. 

Had the assay been developed under the restriction of avoiding these sequences, and concentrating on the few clearly identified sequences like vp62, I think it likely either the assay would not have reached the extraordinary sensitivity required, or the well-developed IAP test would have found evidence of 'contamination'.  Meeting all requirements for sensitivity and selectivity pushes you right to the edge of the state of the art.  In that sense, none of the current assays alone are satisfactory as clinical tools. 

The distinction between tests as research tools, where special care for controls is necessary, and as clinical tools to be applied by ordinary doctors, has been repeatedly glossed over.  One reason for this apparently irrational behavior is the amount of money at stake.  We are looking at a market for some millions of test kits, each probably costing over $100, at the beginning.  Once the initial market is gone, there will probably be a drop in prices as more convenient assays are developed.  Getting in at the beginning is worth considerable money.  This money can be used to bootstrap development of the second generation assays, ensuring a continuing return. 

I've seen what people will do with that kind of opportunity at stake before.  They aren't sweetly reasonable.

That's true, but they haven't even used the right PCR or conditions.
"I suspect there have been a number of conspiracies that never were described or leaked out. But I suspect none of the magnitude and sweep of Watergate." Woodward

"I would favor any name that does not impose (or give the appearance of imposing) taxonomic preconceptions on the nomenclature." Coffin

Jaz

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Re: New sequences published
« Reply #57 on: May 18, 2011, 08:36:33 PM »
True V. 
The goal is not to bring your adversaries to their knees but to their senses. -- Gandhi

Dr. Yes

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Re: New sequences published
« Reply #58 on: May 18, 2011, 10:29:26 PM »

Errr... Again...if anyone could point out to me which of the sequences shows the variation in question, and relative to what, I'd like to BLAST em etc... too blasted myself to go through all of them though, so any ncbi links or accession numbers for the ones you are discussing would be much appreciated.  :)
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Tango

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Re: New sequences published
« Reply #59 on: May 18, 2011, 10:35:31 PM »
These are the new partial sequences


1.
Xenotropic MuLV-related virus isolate WPI-CI-1318 gag protein (gag) gene, partial cds
348 bp linear DNA
Accession: JF907648.1 GI: 332806709
GenBank FASTA Graphics

2.
Xenotropic MuLV-related virus isolate WPI-CI-1317 gag protein (gag) gene, partial cds
343 bp linear DNA
Accession: JF907647.1 GI: 332806707
GenBank FASTA Graphics

3.
Xenotropic MuLV-related virus isolate WPI-CI-1311 gag protein (gag) gene, partial cds
340 bp linear DNA
Accession: JF907642.1 GI: 332806705
GenBank FASTA Graphics

4.
Xenotropic MuLV-related virus isolate WPI-CI-1309 gag protein (gag) gene, partial cds
349 bp linear DNA
Accession: JF907640.1 GI: 332806703
GenBank FASTA Graphics

5.
Xenotropic MuLV-related virus isolate WPI-CI-1308 gag protein (gag) gene, partial cds
325 bp linear DNA
Accession: JF907639.1 GI: 332806701
GenBank FASTA Graphics

6.
Xenotropic MuLV-related virus isolate WPI-CI-1307 gag protein (gag) gene, partial cds
374 bp linear DNA
Accession: JF907638.1 GI: 332806699
GenBank FASTA Graphics

7.
Xenotropic MuLV-related virus isolate WPI-CI-1306 gag protein (gag) gene, partial cds
340 bp linear DNA
Accession: JF907637.1 GI: 332806697
GenBank FASTA Graphics

8.
Xenotropic MuLV-related virus isolate WPI-CI-1327 gag protein (gag) gene, partial cds
372 bp linear DNA
Accession: JF907636.1 GI: 332806695
GenBank FASTA Graphics

9.
Xenotropic MuLV-related virus isolate WPI-CI-1305 gag protein (gag) gene, partial cds
325 bp linear DNA
Accession: JF907635.1 GI: 332806693
GenBank FASTA Graphics

10.
Xenotropic MuLV-related virus isolate WPI-CI-1302 gag protein (gag) gene, partial cds
352 bp linear DNA
Accession: JF907632.1 GI: 332806691
GenBank FASTA Graphics

11.
Xenotropic MuLV-related virus isolate WPI-CI-1301 gag protein (gag) gene, partial cds
366 bp linear DNA
Accession: JF907631.1 GI: 332806689
GenBank FASTA Graphics
"I suspect there have been a number of conspiracies that never were described or leaked out. But I suspect none of the magnitude and sweep of Watergate." Woodward

"I would favor any name that does not impose (or give the appearance of imposing) taxonomic preconceptions on the nomenclature." Coffin