Field of Science

My Letter to Science

(If you're looking for the long post I wrote on Saturday, the one that started the controversy about the Wolfe-Simon arsenic bacteria paper by describing all the problems I found, it's here.)

Below is the text of my formal Letter to Science about the Wolfe-Simon paper.  Letter submissions are supposed to be limited to 300 words (this is a bit over at 371), so I'm only bringing up the issues concerning contamination.  This is an improved version that incorporates many of the suggestions provided in the comments below.  So some of the comments (in the first 10-15) won't make sense any more.

Because this paper has LOTS of other problems, it would be great if many other researchers could also submit Letters and Technical Comments (limit of 1000 words, peer-reviewed).  Here's a link to the Instructions for Authors entry page.  I won't mind if your Letter gets accepted and mine doesn't.

Here's mine:


Wolfe-Simon et al. (1) meticulously eliminated contamination of the reagents and equipment used in their elemental analyses, but they made much less effort to eliminate contamination in their biological samples.

The reagents used for the culture media were not pure.  The 3.1 µM PO4 contaminating the As+/P- medium provided enough P for all of the cell growth seen in this medium, using the authors’ estimate of 7.5x106 atoms of P per genome and the generous assumption that phosphate-starved cells use 90% of their P for molecules other than DNA (2).  This calculation (not done by the authors) obviates their hypothesis that the cells could only grow by replacing P with As.

An independent contamination problem is the omission of standard DNA purification steps when testing for As in DNA (2).  Contamination is typical in DNA/RNA pellets produced by ethanol precipitation of the aqueous phases from phenol:chloroform extractions.  This is partly because this fraction contains most of the small molecules from the cytoplasm (contrary to the authors’ assertion), which are often less soluble in 70% ethanol than in water.  Pellets are also typically contaminated with small amounts of the ethanol supernatant.  Yet the usual step of washing the pellets was omitted, and the dried pellets were simply resuspended in water and loaded on an agarose gel.

Most surprisingly, the chromosomal DNA fractions (boxed in Fig. 2A) were not purified from the gel slices (a standard ten-minute procedure).  Instead the authors simply dried the gel slices and assayed them.  Not only does this bring in any contaminants present in the gel, but since each gel slice would have contained at least 1 mg of agarose (100 mg of 1% agarose gel), and each DNA band no more than 1 µg of DNA, at least 99.9% of the carbon in these samples would have come from the agarose, not the DNA.  No correction can be made for the agarose-derived C because the actual amounts of DNA and agarose are not known.  Omission of the gel-removal step for these critical samples is surprising because the authors did use it in preparing the rDNA fragments they sequenced for their phylogenetic analysis.

1.      Wolfe-Simon F, Blum JS, Kulp TR, Gordon GW, Hoeft SE, Pett-Ridge J, Stolz JF, Webb SM, Weber PK, Davies PC, Anbar AD, & Oremland RS (2010). Science Express. PMID: 21127214
2.      W. Makino, J. Cotner, R. Sterner, J. Elser, Funct Ecol 17,121 (2003).
3.      J. Sambrook, D. W. Russell.  Molecular Cloning, A Laboratory Manual. 3rd Ed.  Cold Spring Harbor Press, New York 2001.

51 comments:

  1. as a suggestion, I would remove the comment on the cost of the kit. If it is 2 dollars or 200, they still should have done it, and it just sounds "envy" in a way.
    Good luck,

    c.

    ReplyDelete
  2. Why a letter and not a technical comment?

    ReplyDelete
  3. Hi Marty,
    I understand the reasons for many of your points, but have you taken into consideration that DNA with As integrated into the backbone would be highly unstable when solubilized in dH2O. Perhaps the authors were limiting seemingly unnecessary resuspension/washing steps to maintain the integrity of the DNA. Are there other methods that could have been done to stabilize the As-DNA backbone? Are your suggestions taking this problem into account?

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  4. Science's response will be: please outline the *experiments* you did to confirm your statements. Only experimental evidence can refute experimental evidence.

    ReplyDelete
  5. Sorry Rosie. Used Marty's name because he tweeted your blog post.

    ReplyDelete
  6. *"Wolfe-Simon et al. meticulously eliminated contamination of the reagents and materials used in their elemental analyses, but they made much less effort to eliminate contamination in the materials they were assaying."

    I'd reword this opening paragraph. It's sort of confusing because you say they eliminated contamination in their materials but not in their assayed materials.

    *"This simple calculation..."

    They might find it overly prickish to refer to it "simple". Though it is. (Also, is it weird you open the paragraph with "First" but there is never a "Second" anywhere?)

    *"And because the actual amounts of DNA and agarose are not known, no correction can be made for this."

    Should reword this sentence so it doesn't start with "And because". No correction can be made for the agarose contribution to the total carbon because the levels of DNA and agarose in the gel slice are unknown yaddaydaddayadda.

    ReplyDelete
  7. and also:

    *"Most surprisingly, the gel slices containing the chromosomal DNA fractions (boxed in Fig. 2A) were not purified."

    the sentence suggests it's the gel slices that must be purified- i'd technically reword it to be The chromosomal DNA was not purified from the agarose gel slices, but instead dried yaddayaddayadda.

    *and overall you might have to word things drier. Make it all science and less emotion. Get rid of the "simple", "simply", "generous assumption", "most surprisingly", "particularly surprising" (in the same paragraph no less) type opinion comments that really change the tone of the comment. Let the science speak for itself.

    ReplyDelete
  8. I love blogging - this is all good advice!

    ReplyDelete
  9. @Jamie: I'm just doing a Letter because it's much easier and faster, and I barely have room to say the thinks I do. In any case, the authors may raise your points in response.But they did dissolve their DNA in pure H2O, they just didn't wash away the contaminants first.

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  10. *and seriously- I said this in the last post comments too but the Occam's Razor answer to "how can these bacteria survive in this environment" ISN'T that they can use the arsenate (which would require lots of enzymes/biological pathways to completely overhaul how they work at every step) but that they are simply better at NOT incorporating arsenate in their pathways (which would require more stringent binding for just the enzymes at the beginning of the pathways yaddayaddayadda)

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  11. There seems to be a lack of references to back up a number of your statements in this letter. Perhaps this concern reflects my considerable lack of expertise in the subject and the things you mention are so standard and obvious that they don't require further support. But on face value at least, I think your statements require the extra support provided by proper referencing.

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  12. *They used a qiagen kit for cleaning out agarose for their PCR products (albeit one with a cutoff of 10kb... maybe this thing has a huge genome?), but to be honest I don't understand the exact nature of the contaminant you are worried about.
    *The presence of agarose only matters if it was greatly different in one sample vs. the other, right?
    And if you can't tell how much agarose is in there, how can you do the math to figure out how many arsenic atoms should be represented?

    *Arsenate (in arsenate form) should easily be cleaned off via electrophoresis. It being nicely charged and tiny. Or maybe it would hydrolyze? Unless I'm missing something, or it's chelated to something that isn't cleaned by agarose and sticks to DNA.

    *Personally, I'm unsettled by the differences in the bands. The ribosomal RNA is so different the arsenic-fed cells must have been pretty sick.
    And *maybe* the arsenic DNA *is* fragmented more... if that were reliable, it might even mean that the DNA tends to fragment more with arsenic in it (if you'd asked me before this paper that would have seemed plausible).

    ReplyDelete
  13. Again, this is evidence that the scientists involved in this disclosure are living up to a fraternal responsibility to the foundation(s) that brought them to their place in the world.

    The timeline in our culture demands that: Too much; to soon would reek havoc.

    It seems that scientists could, during the scope of an investigation, come across human like feces behind "Little Red Riding Hoods" bush- but overlook it until funds were appropriated for that search.

    ReplyDelete
  14. Again, this is evidence that the scientists involved in this disclosure are living up to a fraternal responsibility to the foundation(s) that brought them to their place in the world.

    The timeline in our culture demands that: Too much; to soon would reek havoc.

    It seems that scientists could, during the scope of an investigation, come across human like feces behind "Little Red Riding Hoods" bush- but overlook it until funds were appropriated for that search.


    I don't recommend including this in your letter.

    ReplyDelete
  15. My 2 cents:

    >This is a critique of one of several issues associated with research published by Wolfe-Simon et al.
    Although Wolfe-Simon et al. seem to have eliminated contamination in reagents and materials used in their elemental analyses, much less effort was made to eliminate contamination in the materials they were assaying. The reagents used for the culture media were insufficiently pure. The 3.1 µM PO4 in the As+/P- medium provided enough P for all of the cell growth seen in this medium, using the authors’ estimate of 7.5x106 atoms of P per genome and the assumption that phosphate-starved cells use 90% of their P for molecules other than DNA (Makino ref.). This calculation (not done by the authors) would obviate their hypothesis that the cells replaced P by As in some molecules. An independent contamination problem is the omission of standard DNA purification steps when assaying for As in DNA. Contamination is typical in DNA/RNA pellets produced by ethanol precipitation of the aqueous phases from phenol:chloroform extractions, partly because this fraction contains most of the small molecules from the cytoplasm (contrary to the authors’ assertion),[NEED REFERENCE HERE] which are often less soluble in 70% ethanol than in water. Pellets are also typically contaminated with small amounts of the ethanol supernatant. Yet the standard step of washing the pellets was omitted, and the dried pellets were simply resuspended in water and loaded on an agarose gel. In addition,the gel slices containing the chromosomal DNA fractions (boxed in Fig. 2A) were not purified. Instead of dissolving the agarose and washing it and other contaminants away (a standard ten-minute procedure), the authors simply dried the gel slices and assayed them. Not only does this include contaminants present in the gel, but since each gel slice would have contained at least 1 mg of agarose (100 mg of 1% agarose gel), and each DNA band no more than 1 µg of DNA, at least 99.9% of the carbon in these samples would have come from the agarose, not the DNA. Further, since the actual amounts of DNA and agarose are not known, no correction can be made for this. Omission of the gel-removal step for these critical samples is particularly surprising because the authors did use it in preparing the rDNA fragments they sequenced for their phylogenetic analysis. We hope to obtain a response to these issues from the authors in order that the remarkable results of their research can be understood, validated and hopefully reproduced.<

    ReplyDelete
  16. My 2 cents:

    >This is a critique of one of several issues associated with research published by Wolfe-Simon et al.
    Although Wolfe-Simon et al. seem to have eliminated contamination in reagents and materials used in their elemental analyses, much less effort was made to eliminate contamination in the materials they were assaying. The reagents used for the culture media were insufficiently pure. The 3.1 µM PO4 in the As+/P- medium provided enough P for all of the cell growth seen in this medium, using the authors’ estimate of 7.5x106 atoms of P per genome and the assumption that phosphate-starved cells use 90% of their P for molecules other than DNA (Makino ref.). This calculation (not done by the authors) would obviate their hypothesis that the cells replaced P by As in some molecules. An independent contamination problem is the omission of standard DNA purification steps when assaying for As in DNA. Contamination is typical in DNA/RNA pellets produced by ethanol precipitation of the aqueous phases from phenol:chloroform extractions, partly because this fraction contains most of the small molecules from the cytoplasm (contrary to the authors’ assertion),[NEED REFERENCE HERE] which are often less soluble in 70% ethanol than in water. Pellets are also typically contaminated with small amounts of the ethanol supernatant. Yet the standard step of washing the pellets was omitted, and the dried pellets were simply resuspended in water and loaded on an agarose gel. In addition,the gel slices containing the chromosomal DNA fractions (boxed in Fig. 2A) were not purified. Instead of dissolving the agarose and washing it and other contaminants away (a standard ten-minute procedure), the authors simply dried the gel slices and assayed them. Not only does this include contaminants present in the gel, but since each gel slice would have contained at least 1 mg of agarose (100 mg of 1% agarose gel), and each DNA band no more than 1 µg of DNA, at least 99.9% of the carbon in these samples would have come from the agarose, not the DNA. Further, since the actual amounts of DNA and agarose are not known, no correction can be made for this. Omission of the gel-removal step for these critical samples is particularly surprising because the authors did use it in preparing the rDNA fragments they sequenced for their phylogenetic analysis. We hope to obtain a response to these issues from the authors in order that the remarkable results of their research can be understood, validated and hopefully reproduced.<

    ReplyDelete
  17. My 2 cents:

    >This is a critique of one of several issues associated with research published by Wolfe-Simon et al.
    Although Wolfe-Simon et al. seem to have eliminated contamination in reagents and materials used in their elemental analyses, much less effort was made to eliminate contamination in the materials they were assaying. The reagents used for the culture media were insufficiently pure. The 3.1 µM PO4 in the As+/P- medium provided enough P for all of the cell growth seen in this medium, using the authors’ estimate of 7.5x106 atoms of P per genome and the assumption that phosphate-starved cells use 90% of their P for molecules other than DNA (Makino ref.). This calculation (not done by the authors) would obviate their hypothesis that the cells replaced P by As in some molecules. An independent contamination problem is the omission of standard DNA purification steps when assaying for As in DNA. Contamination is typical in DNA/RNA pellets produced by ethanol precipitation of the aqueous phases from phenol:chloroform extractions, partly because this fraction contains most of the small molecules from the cytoplasm (contrary to the authors’ assertion),[NEED REFERENCE HERE] which are often less soluble in 70% ethanol than in water. Pellets are also typically contaminated with small amounts of the ethanol supernatant. Yet the standard step of washing the pellets was omitted, and the dried pellets were simply resuspended in water and loaded on an agarose gel. In addition,the gel slices containing the chromosomal DNA fractions (boxed in Fig. 2A) were not purified. Instead of dissolving the agarose and washing it and other contaminants away (a standard ten-minute procedure), the authors simply dried the gel slices and assayed them. Not only does this include contaminants present in the gel, but since each gel slice would have contained at least 1 mg of agarose (100 mg of 1% agarose gel), and each DNA band no more than 1 µg of DNA, at least 99.9% of the carbon in these samples would have come from the agarose, not the DNA. Further, since the actual amounts of DNA and agarose are not known, no correction can be made for this. Omission of the gel-removal step for these critical samples is particularly surprising because the authors did use it in preparing the rDNA fragments they sequenced for their phylogenetic analysis. We hope to obtain a response to these issues from the authors in order that the remarkable results of their research can be understood, validated and hopefully reproduced.<

    ReplyDelete
  18. My thoughts, FWIW.
    Pre Strunk & White ("Omit needless words.")

    Strike:

    meticulously;
    "they made much less effort to" (change to "did not" or "failed to");

    "First, the reagents used for the culture media were insufficiently pure." and "This simple calculation (not done by the authors) obviates their hypothesis that the cells replaced P by As in some molecules." (change to "Despite their efforts, insufficiently pure reagents used for the culture media obviates their hypothesis that the cells replaced P with As in some molecules." as topic sentence);

    "An independent contamination problem is the omission of standard DNA purification steps when testing for As in DNA." (do you need this intro for some reason? not sure);

    "ten-minute";

    "critical";

    "particularly";

    "it" (change to "the standard procedure").

    Just my two cents on the language; no comment on the technical aspects.

    /an interested former science student-turned-lawyer

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  19. Good for you. Found your blog via CJR: http://www.cjr.org/the_observatory/the_right_place_for_scientific.php

    ReplyDelete
  20. 1) Arseno-DNA is mildly plausible.

    2) Arseno-RNA has serious hydrolytic kinetics problems given anchimeric assistance from the additional sugar hydroxyl. Even this is not the boojum.

    3) Phosphoric polyanhydrides persist in aqueous solution re ATP and Calgon. Arsenic polyanhydrides would hydrolyze almost immediately. If the bacterium can substitute arsenic for phosphorus, what does it use for its energy metabolism to replace ATP?

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  21. Apart from the Makino reference about phosphorus content (also cited by the authors, but for well-fed cells), I'll cite a basic manual of molecular biology methods. But this is so basic it's not in papers.

    ReplyDelete
  22. I have been following this blog during the last two days. Much has been said about doing scientific research and the way it should be published, reviewed and criticized.
    What I perceived is that a load of valuable ideas, suggestions and knowledge finds its way to this blog and within 2 days this complex piece of work is thoroughly analyzed, erroneous information is corrected within an hour by specialized people (+ review), new insight is born and supplied with feedback and it leads to a scientific "report" which is immediately reviewed on contents and language.
    And I think: wouldn't it be perfect if this process would become the standard for scientific research? The knowledge for everything is somewhere and every somewhere is connected to the internet. I'm afraid that the old fashioned way with the magazines, at least for the millions of "half way publications", is not suitable anymore.

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  23. you have never addressed the fact that their cells DID NOT GROW in the -As/-P condition that contained just as much P contaminant at the +As condition. Why was 3 uM P enough with As present but not when As wasn't added? The answer: It isn't enough, the cells needed the As (or extra P in the +P condition) to be added. They grew with (using) the As.

    ReplyDelete
  24. No comment on the letter as I'm not in a position to judge. I hope though that this will spur Science to move with the times. Demanding a published letter or technical comment is too old-fashioned - the minimum should be an online (open access) comments section for each article. It would benefit the authors as well, who by declining to comment on the various blogs have allowed them to multiply and in some cases become abusive.

    I have some sympathy with the authors not wanting to respond to blogs as they appear. Clearly in this case there are questions to be answered which have been raised by serious scientists - but this will not always be the case and blogs can be used for many reasons, sometimes misguided. This though is a problem for Science and the other journals to solve (many journals already do have comments sections, the British Medical Journal has had an active section for many years, predating in fact the rapid expansion of the blogosphere).

    ReplyDelete
  25. I would also ask the same question as anonymous above. Why did the cells grow so little with only neither As nor P present, but seem to start growing when arsenic is added? That seems like a pretty important question. Also, I don't know essentially anything about DNA hydrolysis, but I would have guessed that a rather massive kinetic chelate effect would keep a DNA molecule together based on the enhanced activity of the nucleophile remaining nearby after bond hydrolysis. Maybe this doesn't come up in biochemistry that often, but it seems at least a reasonable explanation for why the DNA wouldn't fall apart at such an alarming rate in vivo. Well as a semiconductor photochemist, I will defer to your expertise, but I would like to know how you would respond to the above commments.

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  26. I agree that the if chromosomal dna was to be gel purified then washing the pellet with extra 70% would have achieved nothing as small molecule electrolytes will run well ahead of the dna band. That is why we call running dna on a gel 'purifying' it, particularly large chromosomal dna.

    As to your criticism of the analysis of the dna in the gel, as others have said, so long as they are drawing inferences from a comparison with control dna also in gel your criticism is invalid.

    ReplyDelete
  27. In fact on reflection you should limit your comment to first part on the presence of phosphate in the culture medium. That is a good point.

    The gel criticism is ill informed unless you can specify what 'contaminants' you think are not separated from the dna by the gel. Any free ions or ionic molecules will run far ahead of the dna (and probably ended up in the bottom tank). Proteins not removed by the phenol/chloroform extraction and the ethanol precipitation will be excluded from the gel (probably), so what do you think the 'contamination' might be?

    ReplyDelete
  28. Does publishing on Faculty of 1000 probably fit into the scope of NASA's "acceptable" rebuttals?

    Laura Landweber just did it:
    http://f1000.com/6854956?key=y11r1klww5vkfxh

    ReplyDelete
  29. Hi

    I agree with other comments that the strong point is the phospate contents in the medium. Also, I think that you make a good point with the discrepancies between the OD600 values and Cell numbers.

    Good Luck

    ReplyDelete
  30. "Why did the cells grow so little with only neither As nor P present, but seem to start growing when arsenic is added?"

    A 2004 paper from Oremland (coauther of the Science paper) et al. already pointed out that arsenic can be used "either as an electron acceptor for anaerobic respiration, or as an electron donor to support chemoautotrophic fixation of CO2 into cell carbon." It is not surprising at all that bacteria grow slower when no P and As present.

    "The microbial arsenic cycle in Mono Lake, California"
    Oremland, Stolz, Hollibaugh
    FEMS Microbiology Ecology 48 (2004) pp.15–27

    ReplyDelete
  31. these papers weren't reducing As(V), no As(III) production occured and how could they get energy that way anyhow this was an aerobic experiment and dissimilatory As(V) reduction is an anaerobic process.

    ReplyDelete
  32. sorry, meant to say the bugs in the paper weren't reducing As(V)...

    ReplyDelete
  33. I've read your previous post and the letter and I think that the concerns you raise are quite thought-provoking. I would encourage you to consider, however, whether or not this blog post constitutes "prior publication" as outlined by Science's publishing policies.

    http://www.sciencemag.org/site/feature/contribinfo/prep/gen_info.xhtml#prior

    With controversial studies, and especially criticisms of them, the journal may be reluctant to accept such critique, using any grounds they might find. I found your blog from a link on Science's site itself, so the journal obviously knows about your thoughts.

    For what its worth, I think you could make an even stronger argument beyond simply the contamination issue with many of the other points you raised in your first post. The math was quite detailed, as well as the glaring issue with Fig. 1. Let the debate continue!

    ReplyDelete
  34. Regarding no growth in As-/P- and growth in As+/P-. If these cells are similar to their close relative (E. coli) they will survive very well in the starvation media, no growth no death. When you add arsenic maybe cells are dying and releasing phosphate for the other cells to grow on. This will explain the rise in OD.

    ReplyDelete
  35. That math doesn't work, if P is coming from dying cells you should reach a steady state, as cells die others may scavage P but there is no extra P being added to the system so how can the total population of cells increase as much as it does? Its not just OD they have cell counts that show population increase. A no amendment control is a pretty standard method in microbiology so I don't know why people have such a hard time accepting this one.

    ReplyDelete
  36. There was 33 comments before this.

    Have you considered the rise angle of the spirals.
    The double helix in dna's "A" form is ~33.3 degrees.

    This is a tetrahedral number.
    shorthand for the sine of the angle 19.471 basically.

    When crick/watson discovered the winding stairway of dna...there are three known forms:
    A,B and Z dna.

    Since phospher has a "mimic in arsenic"

    and it's number is 33=As

    The angle of the corkscrew is ~33 degrees.

    Perhaps the fact that the triple point of water may solve certain 'hydrolize' scenarios.


    Water has three phases.
    These Phases are gas/liquid/solid or vice versa.


    That is One Molecule with 3(Three) identities as an elemental entity.

    1 / 3 = .333 wich is the Sine of the Terahedral 19.471.

    Ice is PROVEN to be terahedral.
    Water is still a scientific ANOMALY believe it or not and the majority of all scientists do not understand it in its liquid phase.

    If Phosphorous has a tetrahedral underpinning like water and DAN do...then we are dealing with GEOMETRY and not onl chemistry.


    If the tetrahedrals are not considered then you have ZERO letter to even begin to write to the editors any form of critique.


    Obviously,Sheer and pure Geometry is overlooked and may be the key to the mystery...in my opine.

    ReplyDelete
  37. Basically,RR...


    You better Edit your letter the the Edfitor even further as not one respose has dealt with the chiral spiral nor the sine/.333 that may be viral around the biochemists and astrobiologists in near future.


    33 may be the numer for AS.

    But it's more likely ~33.3 will attenuate the signature as the geometry is viewed in its finer resolution.


    Water is tetrahedral.
    DNA's backbone is terahedral.


    is Phosphorus Tetrahedral???
    if it is...then so is the Masked Heirophant that plays the part in the same act :Agent/code-named--- AS-33 arsenic. lol.


    But siriusly,study the geometry before you get an inkling that if you are putting Pen to paper or "Inking Quill to Vellum" let alone typing on keyboard to cyberspace...get your geometry properly demonstrated or it will never convince any real specialists.

    Since this is rather "NEW" science.
    You need to Do the geometry...please and thank you.

    Before you can truly assess the situation at these punitive almost Quantum nano-measures.

    Understand at least that if there is a "church of biology."

    The Spires of the Cathedrals are all tetrahedral.Like DNA and water.

    ReplyDelete
  38. ah, yes. Now we are starting to see the value of "peer review by blog." That geometry stuff is really compelling (like most of the arguments on here) put that in your letter for sure.

    ReplyDelete
  39. No geometry...
    No Backbone.

    No Backbone.
    No base pairs as stairs.
    No information can ascend nor descend the code.

    As analogy or metaphor...
    even angels can't climb jacob's ladder.

    If you don't consider ~33.3 degrees...
    I consider any skepticism as lacking in basic fundamental properties.

    address the geometry...take certain careful steps and arrive at the conclusion:
    As = 33 = Cowan.

    ReplyDelete
  40. yes, oh geometric one, tell us how one person's perception can overcome empirical data in the age of the almighty BLOG!!!!! and how science is better off if our scientific process becomes like our political discourse! only those scientific findings that meet the "commonness" filter will be recognized!!! No fancy schmanchy government PhD's need apply! Any research group more well known than my own must be WRONG! At this age I realize that I'm not going to light the world on fire with my own discoveries so I might as well tear down other people.

    ReplyDelete
  41. Sorry, sensible readers, but the five comments above this one appear to be from a geometry crank. In the spirit of openness I won't delete them.

    ReplyDelete
  42. "That math doesn't work, if P is coming from dying cells you should reach a steady state, as cells die others may scavage P but there is no extra P being added to the system so how can the total population of cells increase as much as it does? Its not just OD they have cell counts that show population increase."

    What is your math, mentioned but not observed? The P amount is claimed to be limiting, there is an extra source that may not be observed and the live cells can grow and grow in number. [Disclaimer: I'm not a biologist.]

    The As+/P- cell population reach the steady state you ask for according to both OD and stain cell count. (I take it the used stain is a poor one from perusing the web, seeing dead & live cells alike, but nevertheless correlating with live cells if proper care is taken). I seem to remember comments [so many!] concerned that the time for this particular experiment was too small, IIRC, it seems long enough for your stated purpose.

    While I'm commenting on that, as it relates to my previous layman comments, I do believe fig 1 is wrong on axis. (Not "the glaring issue", I take it, but an error nevertheless.) But the easier explanation is simply mislabeling and then curves being comparable in the sense I just used.

    ReplyDelete
  43. Mission accomplished

    When you landing on the aircraft carrier with the banner and all...

    Let's see this put UBC on the map for all the wrong reasons

    ReplyDelete
  44. Sorry, sensible readers, but the five comments above this one appear to be from a geometry crank. In the spirit of openness I won't delete them.

    actually...oly 3 of 5 are from this 'author' though ALL are labled anonymous.

    I label that near lible 3/5

    water is tetrahedral.
    phosphorus is tetrahedral.

    water or 'hydrolating' arsenic bonds has a half-life of approx. 10 minutes.therefore the bonds should be completely broken near 19.5 -20 minutes of sexigismal/and tertrahedral timing based on 360 degrees and hour,minute,second intervals like a clockface in essence.

    IF you consider a 'crank' then even a geometrical one has got a handle on it.

    you have'nt a clue what water can do,and water never broke the bonds of arsenic so no comprende' amigos.

    if you do't understand h2o i liquid form good luck with the rest of the story...

    anonymous =a.k.a.=C.S.I.

    ReplyDelete
  45. Let's try to leave this blog troll-free, shall we?

    ReplyDelete
  46. Torbjorn, a microbiologist will recognize that the steady state you mention as the "stationary phase" that any bacterial culture reaches after exponential growth, which is what you call that "upward slanty" part of the line that preceeds it. Now, if you start with say, 1000 cells and it grows to say 10,000,000 cells before leveling off, how do you think there was enough P in the original 1000 to support 10,000,000 by scavaging from dead cells? Its a closed system, no new P coming in. Do I really need to show that math?

    ReplyDelete
  47. The paper of Fritz Widdel and Norbert Pfennig cited in the Supplementary material in the context of vitamin stock preparation does not have a protocol, but instead, the reference to the paper of same authors in the same journal (Arch Microbiol) published earlier. If you look at that original paper you'll see that amongst vitamins there is the B12 which is ...bingo... phosphate-containing.

    sorry if the same was posted before

    ReplyDelete
  48. P content in the media was measured and reported in the paper after amendment with vitamins. Any P that came from anywhere was accounted for, be it from vitamins or salts. The authors did not overlook or neglect to report this. Stuff grew in media with vitamins and As (or P added) but not with vitamins and no As or extra P. The contribution from vitamins would be the same in each condition.

    ReplyDelete
  49. Thanks for taking time to deal with that drivel. I guess most molbiologists once they had a look at that very Arse-nium paper just rolled their eyes and went back to pipetting or pymoling. I know we did. But if we all be that cynical (I know I am going to stick this paper to Science editors next time we get some crap about structure resolution), these oboi-playing scuba-diving dilettantes will take over the world...

    ReplyDelete

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