Soft tissue in Cretaceous dino bone. Still controversial.

The T-rex collagen (Schweitzer et al. 2016) controversy is back, this time on PlosOne.

From the abstract
“Recovery of still-soft tissue structures, including blood vessels and osteocytes, from dinosaur bone after demineralization was reported in 2005 and in subsequent publications. Despite multiple lines of evidence supporting an endogenous source, it was proposed that these structures arose from contamination from biofilm-forming organisms. To test the hypothesis that soft tissue structures result from microbial invasion of the fossil bone, we used two different biofilm-forming microorganisms to inoculate modern bone fragments from which organic components had been removed. We show fundamental morphological, chemical and textural differences between the resultant biofilm structures and those derived from dinosaur bone. The data do not support the hypothesis that biofilm-forming microorganisms are the source of these structures.”

From the comments
Tom Kaye writes:We are delighted to see that seven years after publication, our biofilm hypothesis still has enough merit to generate a manuscript to test it experimentally. Our comments below.

1. The two week growth period was arbitrary.
2. No details of the nutrient supply over time.
3. Biofilms mineralize as is well known by plaque on teeth. Ultra-pure water means no minerals to modify the biofilm.
4. Iron is the basis for Dr. Schweitzer?s claims of extraordinary preservation. No iron is available in this experiment, but the resulting biofilms are compared to dinosaur ?vessels? with iron. 
5. The experiment DID produce tubular branching structures fully consistent with the claims of Kaye et al 2008.

The design of the experiment with limited inputs insured only limited outcomes which were then compared to a natural process that included factors outside the range of the experiment. Mineralized or iron reinforced biofilms could never be expected in the results of this experiment by design. 

We look forward to further experiments that mimic the natural process of biofilm formation.” 

Schweitzer’s Reply to the Kaye Comments:
“Kaye’s original comments, and our responses below each .

1. The two week growth period was arbitrary.
Mr Kay misunderstand the point of the current paper. Our intent in undertaking the current project was to test the hypothesis that biofilm would 1. invade fossil bone under naturally occurring conditions that might reasonably exist in dinosaur bone; and 2. take on the shape and character of vessels (uniform wall diameter, coherency and retention of branching patterns, and retention of an open lumen). We also tested the hypothesis that specific antibodies can differentiate source and composition of materials retained in fossils. We did accomplish these goals with a growth period of 2 weeks (also see below). There is much in the literature about optimal phases of microbial growth, and there is precedent for this growth period in the literature, cited below. Additionally, much is already known about the conditions of growing biofilm; that was not the point of this manuscript. Conditions used in labs to generate optimal biofilm growth (ie, continual flow of water, additional nutrients, agitation, etc as employed by Kaye et al 2008) would not occur in dinosaur bone exposed in the Montana badlands. That was what we tried to imitate.
Note also that Tom does not address our chemical/molecular data at any point in these comments, only our approach for growing biofilm. We were able to show biofilm growth in bone under highly regulated conditions, which was our goal, thus how we might have done it differently is irrelevant to our central hypothesis (see below). Furthermore, duration of growth period, once the biofilm invaded bone, was not pertinent to our central questions. It could grow forever and not produce the vertebrate proteins recognized by our antibodies. Chemically, the biofilm hypothesis is not supported. Neither is it supported morphologically.

2. No details of the nutrient supply over time.  
See below, and our inoculation details in the methods provided in the paper.

3. Biofilms mineralize as is well known by plaque on teeth. Ultra-pure water means no minerals to modify the biofilm.  
There are adequate minerals present in the bone “framework” in which we conducted our experiments, and microbes are perfectly capable of mobilizing these as a mineral source. Yes, biofilms do mineralize, and in that capacity no doubt play a role in preserving some “soft” materials in the rock record through consolidation. Biofilm can overgrow flat materials, like skin or feathers, in a thin layer which, when mineralized, will preserve aspects of the underlying structure (but obviously not the chemistry of the original material unless it is also preserved)… But NO data, including that presented by Kaye et al. 2008, support the hypothesis that mineralized biofilm will result in solid-walled three-dimensional structures with a lumen. Additionally, we have shown here that when minerals are REMOVED, any shape that was initially present is immediately lost. Thus, mineralized biofilm is not supported as a source for our solid walled, easily manipulated, lumen-possessing structures consistent with vertebrate vessels.

4. Iron is the basis for Dr. Schweitzer?s claims of extraordinary preservation. No iron is available in this experiment, but the resulting biofilms are compared to dinosaur ?vessels? with iron. 
Iron is quite obviously not the only means of preservation, just one we have tested and demonstrated experimentally. We proposed that iron-generated reactive oxygen species were responsible for chemical crosslinking that acted as a fixative to stabilize the vessels before decay. The biofilms used in our experiment were chemically fixed, thus an adequate model with which to compare “iron-fixed” dinosaur vessels.

5. The experiment DID produce tubular branching structures fully consistent with the claims of Kaye et al 2008.
Absolutely *NOT*. A ‘tubular structure’ by definition contains a lumen. We never observed a lumen in these biofilm structures (as we stated in the text); thus they were NOT tubular structures. The vessels, consistent with all vertebrate blood vessels, always demonstrate a lumen, in all analyses, from SEM to TEM sections and in sectioning for LM/fluorescence. Furthermore, as we state in the paper, once the bone was removed through demineralization the biofilms were disrupted with the slightest agitation and did not hold any shape even when fixed—that is why sections of these materials differ so completely from dinosaur vessels also figured. The vessels were physically removed from the chelating buffers used to demineralize the bone, washed multiple times, collected into embedding bullets, and sectioned, and still retained both structure and lumen. This was impossible to accomplish with the biofilm, even when fixed.

6. The design of the experiment with limited inputs insured only limited outcomes which were then compared to a natural process that included factors outside the range of the experiment. Mineralized or iron reinforced biofilms could never be expected in the results of this experiment by design. 
This is simply not true. In Kaye’s original paper, he grew biofilms on a flat surface under conditions of controlled recirculated water flow and nutrients added, which are not “natural” conditions occurring inside a fossil bone, buried in or exposed on the surface of sediments; thus his original paper also included factors “outside the range” of natural processes. As stated above, minerals can be (and often are) mobilized from bone itself and most certainly would have been required deep in dinosaur cortical bone in the arid, isolated badlands of Montana, where access to minerals and nutrients were limited primarily to the dinosaur bone itself and access to water was limited and sporadic. The process of microbial mobilization of bone mineral and organic matrix leaves characteristic alterations in bone microstructure, which were not observed in our specimens. For the experiments in the current paper, our goal was to attempt to mimic what might occur in nature, under natural conditions, in dinosaur cortical bone. To preserve any fossil material requires that the materials are stabilized before they can decay. Thus, the initial stages of the decay/stabilization process is capable of being approximated in the lab in relatively short time spans. Our previous experiments showed that without some kind of fixative, blood vessels isolated from bone degrade almost to completion in less than a week. Because stabilization must occur, or at least begin, within this range, a two week growth period overlapped this value. Kaye’s own contention (2008) is that structures he observed in isolated pieces of ‘float’ bone fragments, mostly from turtle plastron/carapace, resulted from modern biofilm invasions. Thus, our experiment tested these conditions rigorously.

Furthermore, conventional wisdom states that fossil bone does not retain original organics. Precisely because of this conventional and widely held belief, we designed our experiment to account for this presumed lack of organics, and this step of removing organics from bone was vital to our initial hypothesis. Although we have shown using multiple methods in multiple fossils that organics most likely persist in bone, we tested, again, this starting and widely held assumption. On the other hand, it is well known that microbes will grow almost anywhere there is organic material available to them, so to work with untreated bone still retaining organics would provide no new information. Thus, our experimental design required removing the organics from bone, to better approximate what is believed by many to best represent the composition of fossil material. This step was important to testing our hypothesis, which we state clearly in the paper. When Kaye argues that “No details were provided as to nutrient level or other parameters of the biofilm”, our response is that these details were not pertinent to our central hypothesis. We simply show that when additional nutrients are supplied, biofilm *will* grow in “naked” bone. There is precedent for this, cited in Neu et al 2003, where it is stated “a study is described in which a river inoculum was used as a sole source of nutrients (and inoculated only once, similar to our study) and the after 4 weeks the biofilm plateaued and no longer grew”. Based on these findings, then, we chose to halt the experiment half way, when we could observe biofilm growth but before sloughing of cells occurred, thus optimizing molecular response if it were present, as well as optimizing the chance of recovering coherent intact biofilm.” 

7. We look forward to further experiments that mimic the natural process of biofilm formation. 
We are satisfied that we have ruled out biofilm as a source of our vessels with abundant and varied data and will not pursue this. We will continue to base our future research on the well-supported hypothesis that these structures are endogenous. Multiple lines of evidence support this hypothesis, including recovery of sequences of multiple vertebrate proteins commonly associated with blood vessels and other bone organic components, but which are not generated by or associated with biofilm-producing organisms, the presence of which cannot therefore be explained by a biofilm origin. The data we present here, rigorously testing this alternative hypothesis, further eliminate the possibility that these arise from biofilm. Biofilms do not contain vertebrate proteins. Biofilms do not cross react with antibodies to vertebrate proteins. Dinosaur blood vessels do not respond to antibodies against bacterial proteins. Biofilms are morphologically distinct from blood vessels, are not cylindrical, and do not contain a lumen. There is no evidence to support a biofilm origin for the vessel structures recovered from these and other dinosaur materials.”

Once again,
I can’t weigh in on this argument. Both sides are steadfast. Only one can be correct. I know how both of them feel. A lot of work on both sides has come to loggerheads. Full disclosure: I have seen and tugged on the rubbery biofilms in the Tom Kaye lab.

References
Kaye TG, Gaugler G, Sawlowicz Z. Stepanova A. 2008. Dinosaurian soft tissues interpreted as bacterial biofilms. PLoS One. 2008;3(7):e2808. doi: 10.1371/journal.pone.0002808. pmid:18665236
Schweitzer MH, Moyer AE and Zheng W-X 2016. Testing the Hypothesis of Biofilm as a Source for Soft Tissue and Cell-Like Structures Preserved in Dinosaur Bone. DOI: 10.1371/journal.pone.0150238

M. Schweitzer on 60 minutes here

Kaye pictures here:
http://scienceblogs.com/grrlscientist/2008/07/30/a-closer-look-at-dinosaur-soft/

Advertisements

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s