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The persistence of original soft tissues in Mesozoic fossil bone is not explained by current chemical degradation models. We identified iron particles (goethite-αFeO(OH)) associated with soft tissues recovered from two Mesozoic dinosaurs, using transmission electron microscopy, electron energy loss spectroscopy, micro-X-ray diffraction and Fe micro-X-ray absorption near-edge structure. Iron chelators increased fossil tissue immunoreactivity to multiple antibodies dramatically, suggesting a role for iron in both preserving and masking proteins in fossil tissues. Haemoglobin (HB) increased tissue stability more than 200-fold, from approximately 3 days to more than two years at room temperature (25°C) in an ostrich blood vessel model developed to test post-mortem 'tissue fixation' by cross-linking or peroxidation. HB-induced solution hypoxia coupled with iron chelation enhances preservation as follows: HB + O2 > HB - O2 > -O2 ≫ +O2. The well-known O2/haeme interactions in the chemistry of life, such as respiration and bioenergetics, are complemented by O2/haeme interactions in the preservation of fossil soft tissues.
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, 20132741, published 27 November 2013281 2014 Proc. R. Soc. B
Elizabeth Theil, Matthew A. Marcus and Sirine C. Fakra
Mary H. Schweitzer, Wenxia Zheng, Timothy P. Cleland, Mark B. Goodwin, Elizabeth Boatman,
tissues, cells and molecules from deep time
A role for iron and oxygen chemistry in preserving soft
Supplementary data
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http://rspb.royalsocietypublishing.org/content/suppl/2013/11/22/rspb.2013.2741.DC1.h
"Data Supplement"
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Research
Cite this article: Schweitzer MH, Zheng W,
Cleland TP, Goodwin MB, Boatman E, Theil E,
Marcus MA, Fakra SC. 2014 A role for iron and
oxygen chemistry in preserving soft tissues,
cells and molecules from deep time.
Proc. R. Soc. B 281: 20132741.
http://dx.doi.org/10.1098/rspb.2013.2741
Received: 21 October 2013
Accepted: 29 October 2013
Subject Areas:
biochemistry, palaeontology, cellular biology
Keywords:
soft tissue preservation, haemoglobin,
iron, goethite, Fenton chemistry,
protein cross-linking
Author for correspondence:
Mary H. Schweitzer
e-mail: mhschwei@ncsu.edu
Present address: The Department of
Biomedical Engineering, Rensselaer Polytechnic
Institute, Troy, NY 12182, USA.
Present address: American Association for the
Advancement of Science, Arlington, VA, USA.
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rspb.2013.2741 or
via http://rspb.royalsocietypublishing.org.
A role for iron and oxygen chemistry
in preserving soft tissues, cells and
molecules from deep time
Mary H. Schweitzer1,2, Wenxia Zheng1, Timothy P. Cleland1,†,
Mark B. Goodwin3, Elizabeth Boatman4,‡, Elizabeth Theil5,6,
Matthew A. Marcus7and Sirine C. Fakra7
1
Marine, Earth, and Atmospheric Sciences, North Carolina State University, Campus Box 8208, Raleigh,
NC 27695, USA
2
North Carolina Museum of Natural Sciences, 11 West Jones Street, Raleigh, NC 27601, USA
3
Museum of Paleontology, and
4
Department of Material Sciences and Engineering, University of California,
Berkeley, CA 94720, USA
5
CHORI (Children’s Hospital Oakland Research Institute), 5700 Martin Luther King, Jr. Way, Oakland,
CA 94609, USA
6
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh,
NC 27695-7622, USA
7
Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
The persistence of original soft tissues in Mesozoic fossil bone is not explai-
ned by current chemical degradation models. We identified iron particles
(goethite-aFeO(OH)) associated with soft tissues recovered from two Meso-
zoic dinosaurs, using transmission electron microscopy, electron energy loss
spectroscopy, micro-X-ray diffraction and Fe micro-X-ray absorption near-
edge structure. Iron chelators increased fossil tissue immunoreactivity to
multiple antibodies dramatically, suggesting a role for iron in both preserving
and masking proteins in fossil tissues. Haemoglobin (HB) increased tissue
stability more than 200-fold, from approximately 3 days to more than two
years at room temperature (258C) in an ostrich blood vessel model developed
to test post-mortem ‘tissue fixation’ by cross-linking or peroxidation.
HB-induced solution hypoxia coupled with iron chelation enhances pre-
servation as follows: HB þO
2
.HB 2O
2
.2O
2
þO
2
. The well-known
O
2
/haeme interactions in the chemistry of life, such as respiration and bioener-
getics, are complemented by O
2
/haeme interactions in the preservation of
fossil soft tissues.
1. Introduction
Preservation of structures in fossils that were not originally mineralized in the
living organisms is uncommon, but is represented in microbes, plants and ani-
mals in disparate environments throughout the fossil record (e.g. [1] and
references therein). Soft tissue structures retaining some aspects of original
material, and thus not completely replaced replicas, have been described in Meso-
zoic fossil bone as early as the 1960s [2– 5]. This ‘exceptional preservation’ has
been observed for decades, but is not addressed by models of fossilization pro-
cesses wherein an organism is buried and degraded, and spaces left by
degrading organics are subsequently filled by precipitation of exogenous min-
erals. Modes of preservation to explain the persistence of these secondarily
mineralized, but originally soft tissues include microbially mediated stabilization
[6,7], early diagenetic mineralization or authigenic replacement [8– 10], ‘sulfuriza-
tion’ [11,12] and others (reviewed in [6,13,14]), but few of these preservation
modes have been experimentally tested.
Recently, still-soft biomaterials have been identified in bones of multiple taxa
from the Cretaceous to the Recent, with morphological and molecular character-
istics consistent with an endogenous source [15– 20]. An alternative hypothesis,
that these structures result from microbial biofilms [21], is eliminated by several
&2013 The Author(s) Published by the Royal Society. All rights reserved.
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lines of evidence, including but not limited to: (i) immunologi-
cal reactivity (multiple antibodies binding both with chemical
extracts and in situ, independent of contributions from, and not
reactive to, biofilms [17,22,23]); (ii) peptide sequence data from
proteins not found in microbes [17,22– 25]; and (iii) identifi-
cation of histones—nuclear, chromosomal proteins that are
eukaryote-specific—by both amino acid sequence and anti-
body localization [22]. Multiple lines of evidence support the
endogeneity of these recovered molecules in Cretaceous speci-
mens, despite hypothesized temporal limits on molecular
preservation of less than 1 Myr for proteins and approximately
100 000 years for DNA [26–30] (but see [31]) that are based
upon degradation proxies of heat and/or pH [28,32], theore-
tical models of breakdown kinetics [33,34], and, recently,
extrapolation from a select and time-limited set of fossils [35].
For soft tissues and the proteins comprising them to persist
beyond these limits, a mode of preservation sufficiently rapid
to outpace decay is required [6]. Here, we propose a chemi-
cal explanation for molecular and tissue ‘fixation’ over time
involving iron-catalysed free-radical reactions.
Redox-active iron, abundant in living cells and tissues, is
stabilized in haeme proteins (e.g. haemoglobin (HB), myoglo-
bin, cytochromes [3639]), non-haeme iron proteins (e.g.
ribonucleotide reductase, fatty acid desaturase [40]) and ferri-
tin, a protein which synthesizes iron oxyhydroxide mineral
nanoparticles [41,42]. These proteins control the rapid gener-
ation of oxygen-free radicals by environmental dioxygen (O
2
)
[42– 44]. Although approximately 85% of iron in animals
resides in HB [45],thousands of ironatoms are also sequestered
in life in a single ferritin molecule [46]. When iron–protein
binding is disrupted through death or disease [47–49], iron-
induced Fenton-type reactions occur, producing insoluble (K
s
approx. 10
218
M) mineralized iron/rust and highly reactive
hydroxyl radicals [37,42,50,51]. Ferritin is a complex pro-
tein that synthesizes iron biominerals, the form of which is
environmentally dependent. In its antioxidant mode, ferritin
scavenges cytoplasmic iron (II) and sequesters it as protein-
caged, iron biomineral [41]. Nevertheless, some iron escapes,
contributing to formation of oxy radicals that amplify peroxi-
dation of membrane lipids [43,50,52,53]. Oxy radicals also
facilitate protein cross-linking [54] in a manner analogous to
the actions of tissue fixatives (e.g. formaldehyde), thus increas-
ing resistance of these ‘fixed’ biomolecules to enzymatic or
microbial digestion [55,56]. Lipid peroxidation and protein
condensation reactions are harmful to living tissues [52,54],
but could act to preserve tissues and biomolecules after death.
Here, we show data from both fossil and extant organic
material to support the hypothesis that iron contributes to
preservation of soft tissues and molecules. We present
direct evidence that iron is closely associated with still-soft
tissues (e.g. semi-transparent, pliable ‘vessels’, osteocyte-like
microstructures and associated contents) recovered from fos-
sils using our aseptic protocols [17,22]; and that treatment of
these materials with the iron chelators pyridoxal isonicotinic
hydrazide (PIH [57]), salicylaldehyde isonicotinic hydrazide
(SIH [58,59]) or polyethylene glycol 600 (PEG600 [60])
increased antibody recognition in situ, with PIH the most
efficient and least damaging to tissues.
When extant, post-mortem ostrich blood vessels were incu-
bated in a red blood cell lysate rich in solubilized HB, iron
deposits formed quickly and these materials have resisted
tissue degradation for many months at room temperature
with no further treatment (see the electronic supplementary
material). We also compared vessels in aerobic or hypoxic con-
ditions and found that tissues incubated in HB in the presence
of dioxygen displayed the greatest stability and longevity, to
date more than 2 years.
2. Material and methods
For details of actualistic experiments and additional figures (S1– S6),
see the electronic supplementary material.
3. Results
Transmission electron microscopy (TEM) shows iron intimately
associated with vessels recovered from demineralized dinosaur
tissues (figure 1). Both isolated Tyrannosaurus rex (MOR 1125)
vessels (figure 1a,c,e)andBrachylophosaurus canadensis (MOR
2598) vessels (figure 1b,d) show iron-rich nanoparticles, often
embedded in an amorphous, apparently ‘organic’ layer that is
sometimes almost completely obscured by electron-dense iron
particles (figure 1b). Figure 1cshows a structure protruding
into the lumen of the dinosaur vessel that is similar in mor-
phology to nuclei of the endothelial cells (EN) that comprise
extant ostrich vessel walls (figure 1f). Higher magnification of
the boxed region in figure 1cshows the intimate relationship
between the organic layer and iron (figure 1e). Lower magnifi-
cation of an ostrich vessel in cross section (figure 1f)shows
distinct EN-containing chromatin protruding into the lumen
of the vessel and a tight junction (TJ) uniting two cell mem-
branes. The tapering nature of the endothelial cell (EC)
cytoplasm is clearly visible, and is consistent with structures
seen under higher magnification of T. rex vessels (figure 1c).
Figure S1 in the electronic supplementary material
shows backscatter TEM images of isolated osteocytes [22]
of T. rex (electronic supplementary material, figure S1A) and
B. canadensis (electronic supplementary material, figure S1C).
Electron energy loss spectroscopy (EELS) elemental maps
show iron localized to cells and intracellular contents of both
dinosaurs (electronic supplementary material, figure S1B,D).
When these cells were treated to chelate iron, EELS shows
that iron signal is greatly decreased and more diffuse(electronic
supplementary material, figure S2). This supports the intimate
association of iron to these preserved, still-soft structures.
Synchrotron microprobe techniques were used to investi-
gate dinosaur and ostrich vessels, intravascular material
and chemical speciation of associated iron. Micro-X-ray fluor-
escence (m-XRF) distribution maps of iron at 3 mm resolution
(red pixel intensity corresponds to iron concentration) are
shown in extended regions of HB-incubated ostrich (figure 2a),
B. canadensis (figure 2b)andT. rex (figure 2c) vessel samples,
which had been air-dried on an Si
3
N
4
window (Silson). In all
cases, iron was found in intimate association with the vessel
structures. Micro-X-ray absorption near-edge structure (m-
XANES) spectroscopy was used to determine the chemical spe-
ciation of iron at multiple locations on each sample (white
numerical labels), with representative plots of the correspond-
ing spectra shown in figure 2d–f for HB-incubated ostrich,
B. canadensis and T. rex, respectively. In each Fe m-XANES
plot, the experimental spectrum is in black, the corresponding
least-square linear combination fit is displayed in red, and
green shows the residuals. The chemical speciation of iron
in the HB-incubated ostrich tissue was a combination of oxyhae-
moglobin and a disordered Fe oxyhydroxide, which was
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represented in fits by biogenic Fe oxide [61] and which will be
referred to hereafter as ‘biogenic-like oxide’ (BLO). Both
dinosaur tissues were found to contain a combination of
goethite (a-FeO(OH)) and biogenic iron oxyhydroxide.
Optical microscopy was used to identify intravascular
material in the vessels of B. canadensis (figure 2g) and T. rex
(figure 2k). m-XRF mapping revealed high concentrations of
iron in these intravascular structures (figure 2h,l, respectively).
Micro-X-ray diffraction (m-XRD) analysis of intravascular
structures in the dinosaur vessels identified these features as
crystalline goethite (figure 2iat location 1 and figure 2mat
location 13). Iron m-XANES performed at the same locations
showed that, in addition to crystalline goethite, these loca-
tions also contained highly disordered amorphous (i.e.
poorly diffracting) iron oxyhydroxides best matching a bio-
genic iron oxyhydroxide standard. Similar accumulations
EN??
Fe particles
Fe particles
Fe particles
amorphous
organic
layer
amorphous
organic
layer
amorphous
organic
layer
iron layer
EC
cytoplasm
EN
TJ
4.0 Kx
1µm
2µm2µm
2µm m
m
(b)
(a)
(c)(d)
(e)(f)
Figure 1. TEM images of (a,c,e)T. rex (MOR 1125) vessels, (b,d)B. canadensis (MOR 2598) vessels and ( f) ostrich vessels. Tyrannosaurus rex vessels show iron
particles infiltrating a relatively amorphous ‘organic’ layer (arrows, a,c). Higher magnification (c) shows a structure similar in morphology to an endothelial cell
nucleus seen in ostrich vessel (EN, f), protruding into the lumen of an isolated vessel. No chromatin or nuclear membrane is visible in the dinosaur structures,
but these features are visible in the ostrich. (e) Higher magnification of the area within the box in (c) shows variation in texture within the ‘organic’ layer. B.
candensis vessels are more completely infiltrated with iron (b), but in some views (d), an organic layer is still visible. The ostrich vessel (f) shows nuclear membrane
(EN) within the endothelial cell (EC). Cytoplasmic extensions make up the bulk of the vessel wall, and a TJ uniting two endothelial cells. Scale bar, 2 mm for (a–d),
1mm for (e,f).
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1
1.5
1.0
0.5
0
2000 1.5
1.0
0.5
0
1.0
0.5
0
1500
1000
500
0
10 15 20 25 30 35 40 7050 7100 7150 7200 7250 7300 7350 7400
7100 7200
47% oxyhemoglobin 85% goethite
15% biogenic Fe oxyhydroxide
70% goethite
30% biogenic Fe oxyhydroxide
75% goethite
25% biogenic Fe oxyhydroxide
72% goethite
28% biogenic Fe oxyhydroxide
53% biogenic Fe oxyhydroxide
experimental
fit
(200)
(200)
(101)
(201)
(301)
(210) (111)
(212)
(511)
(112)
(410)
(401)
(131)
(211)
(201)
(101)
(111)
(301)
(210)
(211)
(401)
(131)
(410)
(112)
(212)
(312) (511)
(020)
residual
experimental
fit
residual
experimental
fit
residual
spot 13
fit
residual
experimental
fit
residual
photon energy (eV) photon energy (eV)
photon energy (eV)
Q (nm–1)
10 15 20 25 30 35 40 7050 7100 7150 7200 7250 7300 7350 7400
photon energy (eV)
Q (nm–1)
photon energy (eV)
7300 7400 7100 7200 7300 7400 7050 7100 7150 7200 72507300 73507400
3
2
67
9
10
1
1
2
3
1
2
3
14
13
14
13
2
34
5
8
1
2
50 µm
50 µm
20 µm
50 µm 50 µm
normalized absorbance
intensity (arb.units)
1000
800
600
400
200
0
intensity (arb.units)
normalized absorbance
normalized absorbance
(b)
(a)
(g)
(k)(l)
(h)
(c)
(d)(e)
(i)
(m)(n)
(j)
(f)
Figure 2. (a–c)m-XRF maps of HB-incubated ostrich, B. canadensis (MOR 2598) and T. rex (MOR 1125) vessel tissues at 3 mm resolution, with locations of analysis identified
by white numerical labels, illustrating the intimate association of Fe with each vessel tissue. (d–f)m-XANES analysis of iron chemical speciation at representative locations,
respectively, for each vessel tissue, where the experimental data are plotted in black and the least-square linear combination fits are in red. HB-incubated ostrich tissue was
found to contain iron in the form of oxy-HB and biogenic (disordered non-crystalline) iron oxyhydroxide; by contrast, dinosaur vessels contained goethite (a-FeO(OH)) in
addition to disordered biogenic-like oxide (BLO) iron oxyhydroxide. Green curves are residuals and indicate good fits for all three samples. (g,h) Optical microscopy, m-XRF
analysis of intravascular structures in B. canadensis. (i,j)m-XRD and iron m-XANES chemical analysis of intravascular structure (location 1) in B. canadensis identifies these
features as crystalline goethite with an additional fraction of biogenic iron oxyhydroxide, which is amorphous (i.e. poorly diffracting). (k–n) Investigation of intravascular
structures in T. rex revealed similar findings (i.e. location 13 was identified as a combination of goethite and biogenic Fe oxyhydroxide). Whole pattern m-XRD insets of (i,m)
exhibit thin, continuous rings for both B. canadensis and T. rex, indicating that the goethite in both samples is nanocrystalline. Labels associated with peaks (e.g. 200, 101)
represent h,k,lMiller indices of diffracting planes. XRD was used to identify the nature of the crystalline iron oxyhydroxides and XANES spectroscopy was used to probe the
poorly crystalline or amorphous (i.e. poorly diffracting) phase(s) using least-square linear combination fitting and a set of iron standards. (Online version in colour.)
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of iron were observed in HB-treated ostrich vessels, as shown in
figure 2a, locations 1 and 2, which is in contrast to the amor-
phous pattern of the ostrich vessel tissue observed at regions
of low iron concentration (e.g. location 3 in figure 2a; electronic
supplementary material, figure S3). However, all analysed
regions of B. canadensis and T. rex vessels exhibited finely
crystalline character (thin, continuous rings in whole pattern
insets of figure 2i,m), suggesting that these vessel tissues are
associated with nanocrystalline goethite.
Iron chelation increased immunoreactivity of proteins in
osteocytes and vessels of dinosaurs [17,22,23]. Dinosaur osteo-
cytes had minimal response to anti-actin antibodies before
chelation (figure 3a,b,e,f), but responses increased dramatically
after iron chelation (figure 3c,d,g,h). When vessels from both
dinosaurs were exposed to elastin antibodies, a highly con-
served protein found in vessel walls of all extant vertebrates
[62] (figure 3i–p), chelators also enhanced binding over
untreated tissues. Both SIH and PIH resulted in increased
signal in vessels recovered from B. canadensis (figure 3il)
and T. rex (figure 3m–p), but PIH was most effective, and
only data from this chelation treatment are presented.
An ostrich blood vessel model was used to determine
post-mortem conditions, possibly contributing to preservation
of tissues, as observed in the dinosaur samples. Ostrich vessels
were incubated in a concentrated solution of red blood
cell lysate (see the electronic supplementary material) to
approximate post-mortem erythrocyte lysis. Control tissues
were prepared identically, then incubated in either sterile dis-
tilled water or phosphate buffered saline (PBS). Haemoglobin
was chosen to test its preservation properties for four reasons:
(i) HB is in known to be bacteriostatic [63,64]; (ii) in the presence
of dioxygen, HB produces free radicals [65]; (iii) blood vessels
fill with large amounts of HB after death as red cells begin
to die and lyse, thus it is naturally present in large vertebra-
tes [45]; and (iv) haeme released from HB, when degraded,
will release iron, possibly accounting for the iron particles
associated with preserved soft tissues [42,66] (figure 1).
HB-treated vessels have remained intact for more than
2 years at room temperature with virtually no change, while
control tissues were significantly degraded within 3 days. Indi-
cators of tissue stability include thick vessel walls (figure 4a,b,
black arrows) and visible surface structures consistent with
endothelial nuclei (figure 4a,b, white arrowheads). In many
cases, material could be seen inside the vessel lumen, appear-
ing most often as structureless masses (figure 4a,b, asterisk).
There was no difference between tissues incubated in HB/
hypoxy and HB/oxyconditions (see the electronic supplemen-
tary material), including the presence of the intravascular
material, except that distinct red blood cells were also present
in the HB/oxy condition (figure 4c,d, asterisk). By contrast,
the absence of HB resulted in extensive tissue degradation,
indicated by bacilliform bacteria (figure 4h, asterisk), fungal
(b)
(i)
(m)(n)(o)(p)
(j)(k)(l)
(a)
(g)(h)
(c)(d)
(e)(f)
Figure 3. Overlay and fluorescence microscopy images of dinosaur ‘osteocytes’ and vessels after exposure to polyclonal antibodies. (a–h) Cells exposed to antibodies
against actin protein; (ip) vessels exposed to antibodies raised against elastin protein, a component of vessel walls. (a,b)Brachylophosaurus canadensis and (e,f)T.
rex osteocytes show minimal reactivity to antibodies before treatment with PIH; treatment with the iron chelator PIH result in increase in binding to actin antibodies
in both B. canadensis (c,d) and T. rex (g,h) isolated osteocytes. Similar increase in antibody binding is visible in dinosaur vessels. (i,j) isolated vessel from
B. canadensis exposed to elastin antibodies without PIH; binding is greatly increased after chelation (k,l). A similar pattern is seen for T. rex vessels before
(m,n) and after chelation (o,p). All data collection parameters are identical for each condition. Scale bar is 50 mm for each image. (Online version in colour.)
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50 µm
*
*
*
**
*
**
*
50 µm 50 µm
50 µm
50 µm
50 µm
50 µm
50 µm
(b)
(a)
(c)(d)
(e)
(g)(h)
(f)
Figure 4. Ostrich vessels isolated from extant bone. (a,b) Vessels incubated in HB in deoxygenated condition; (c,d) vessels incubated in HB, but exposed to oxygen. (e,f)
Deoxygenated vessels incubated in distilled water; (g,h) vessels incubated in distilled waterand oxygenated. Black arrows show endothelial layeris much thickerand better
preserved in HB-soaked vessels. Other details are seen in HB vessels, for example possible endothelial nuclei on vessel surfaces (white arrowhead). White asterisks indicate
red blood cells within vessel of only HB-oxygenated condition; black asterisks show microbial invasion. Scale bar as indicated. (Online version in colour.)
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invasion, vessel wall thinning and collapse (figure 4e), loss of
vessel contents, and complete loss of tissue integrity. Incu-
bation in water or PBS without HB resulted in rapid
degradation (approx. 3 days), but when dioxygen was
removed with an argon purge, tissue degradation was some-
what delayed. More importantly, ostrich vessels incubated
with HB in the presence of oxygen were bright red (light
microscopy) (figure 4c,d), while those in the argon-purged
(hypoxic) solution were darker (figure 4a,b), indicating that a
haeme– oxygen complex formed and coincided with enhanced
tissue stability. The range of tissue stabilities observed with dif-
fering combinations of HB and O
2
were: HB þO
2
.HB –
O
2
¼PBS – O
2
.. PBS þO
2
, emphasizing the importance of
both HB and oxygen to tissue stabilization.
Ostrich blood vessel structure was stabilized when HB
was present, but in the presence of oxygen, degradation
was further inhibited relative to the hypoxic state. Scanning
electron microscopy (SEM) shows that HB-incubated vessels
in the absence of oxygen contain minimal areas of collapse
(electronic supplementary material, figure S4A,B, black
arrows) and degradation (electronic supplementary material,
figure S3C, white arrows) in otherwise intact vessel walls,
and endothelial nuclei (white arrowheads) were still visible
(electronic supplementary material, figure S4C). When both
HB and oxygen were present, the blood vessels were fully
inflated and there were no areas of collapse, suggesting
intact elastin proteins (electronic supplementary material,
figure S4D). At higher magnification (electronic supple-
mentary material, figure S4E,F), no regions of breakdown
were detected, and surface texture was intact (electronic
supplementary material, figure S4F).
However, in vessels incubated in PBS/water, tissue degra-
dation was much greater; vessel collapse was less frequently
observed in the absence of oxygen (black arrows, electronic
supplementary material, figure S4G,I) than in its presence
(electronic supplementary material, figure S4J–L). This was
further evidenced at the end of one month, where microbial
contamination was still not observed in either HB condition
(electronic supplementary material, figure S5A,B), but almost
completely consumed vessels in both control conditions (elec-
tronic supplementary material, figure S5C,D) after days to
weeks. No microbial influence is detected in either HB con-
dition even after storage at room temperature for six months
to 2 years.
When ostrich blood vessels were soaked in HB, rinsed and
exposed to antibodies against a synthetic HB peptide (NH2-
TSLWGKVNVADCGAEALAR-OH) [67] in in situ antibody
assays, with or without iron chelation, patterns similar to the
dinosaur vessels were observed. The ostrich vessels treated
with PIH to remove HB-derived iron (electronic supplementary
material, figure S6B,D) showed significantly more antibody bind-
ing than those vessels incubated in HB without PIH chelator
(electronic supplementary material, figure S6A,C).
4. Discussion
The HB –oxygen interactions investigated here explain both the
association of iron with many exceptionally preserved fossils
and the enhanced preservation of tissues, cells and molecules
over deep time. Iron and oxygen chemistry, at the centre of
bioenergetics and terrestrial life [41], are now seen to play
key roles in the preservation of biomaterials after death.
The hypothesis that iron contributes to preservation in
deep time, perhaps by both free-radical-mediated fixation and
anti-microbial activity, is supported by data presented herein.
Although the exact mechanism of microbial inhibition by HB
is not known, it has been noted in earlier works [63,64]. The
iron may be directly protecting proteins by blocking active
sites recognized by enzymes of degradation (supported by
the increase in antibody signal after treatment with iron chela-
tor), or it may be providing protection indirectly by binding
to oxygen, and thus preventing oxidative damage [68,69] or
outcompeting bacterial mechanisms, similar to ferritins [45].
Here, we observe the intimate association between iron
(goethite) particles and soft tissues recovered from dinosaurs.
In life, blood cells rich in iron-containing HB flow through
vessels, and have access to bone osteocytes through the
lacuna-canalicular network [70,71]; after death, HB could
cause localized, haeme-based radical cross-linking in dinosaur
tissues. Moreover, HB-derived haeme, previously identified in
dinosaur bone [72], has recently been identified in Miocene
mosquitoes, supporting the durability of this prosthetic unit
[73]. But are these reactions sufficient to result in long-term
preservation?
In our test model, incubation in HB increased ostrich vessel
stability more than 240-fold, or more than 24000% over control
conditions. The greatest effect was in the presence of dioxygen,
but significant stabilization by HB also occurred when oxygen
was absent (figure 4; electronic supplementary material, figure
S5). Without HB treatment, blood vessels were more stable in
the absence of oxygen, whereas the most rapid degradation
occurred with oxygen present and HB absent. Two possible
explanations for the HB/O
2
effect on stabilizing blood vessel
tissues are based on earlier observations in different environ-
ments: (i) enhanced tissue fixation by free radicals, initiated
by haeme–oxygen interactions [65]; or (ii) inhibition of
microbial growth by free radicals [63,64]. Ironically, haeme, a
molecule thought to have contributed to the formation of life
[41,74], may contribute to preservation after death.
Goethite-like iron particles similar to those observed in
these fossil soft tissues have been identified in modern tissues
and are possibly derived from HB through formation of ferritin
protein-caged iron biominerals [44,75–79] during degradation.
Ferritins are stable proteins that retain activity post-mortem.
They are capable of scavenging iron released from less stable
proteins and converting it to biominerals such as goethite,
depositing it as crystals of relatively uniform size, in surround-
ing tissues. These iron nanoparticles may have stabilized cell
architecture and may even be responsible for preserving intra-
cellular components chemically consistent with DNA [22]
through iron-mediated DNA– protein cross-links [80].
However, just as iron contributes to reduction of anti-
body reactivity (figure 3; electronic supplementary material,
figure S5), it may also confound efforts to sequence bio-
molecules, by diminishing signals in mass spectrometry via
ion suppression or by inhibiting enzymes required for DNA
sequencing [81,82]. Iron chelation in soft tissue analysis is a
technical advance in analysing biomaterials from fossil
bone because chelation reduces signal inhibition in many
fossil analyses, thus broadening the range of specimens
from which molecular data may be obtained.
Biomolecules recovered from fossils have great potential
to reveal aspects of the biology and environments of extinct
organisms by: (i) independently testing and/or resolving
evolutionary relationships determined by morphological
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characters; (ii) combining morphological and molecular data
from both fossil and extant taxa to generate more robust phy-
logenies; (iii) illuminating acquisition of physiological
strategies not discernible from morphology alone (e.g. cold-
adapted HB in Mammuthus primigenius [83]); and (iv) provid-
ing, independently, estimates on rate and direction of
molecular evolution. In addition, molecules derived from fos-
sils can elucidate molecular mechanisms for organismal
survival during prolonged periods of global climate change.
Finally, studying molecular diversity of organisms in the
fossil record before major ‘bottleneck’ events illuminates
population structures and may suggest mechanisms to miti-
gate the current decline in diversity in some extant lineages.
However, despite this potential, fossils older than
approximately 1 Ma have not been targeted for molecular
studies, because proposed limits on preservation of organic
components in bone [28,30,33,34] obscured the possibility of
molecular survival. These models/proxies predict degra-
dation of tissues on even shorter time scales. Therefore, the
apparent recovery of structures in Cretaceous bone consistent
with an endogenous origin that share identical location, tex-
ture, morphology, translucency, molecular characteristics
and immunoreactivity with extant osteocytes and blood
vessels [17,22,24,84] has remained controversial. Here, our
data support a naturally occurring mechanism that results in
stabilization of these presumably transient components over
geological time. Because we observed iron particles in associ-
ation with soft tissues in these fossils (figure 1), and earlier
studies localized iron to the vessels of bone, not the bone
matrix or surrounding sediments [72,85,86], we focused our
attention on identifying a protein source for iron after death.
Redox reactions of iron are modulated by insertion of iron
into porphyrins bound to specific proteins (HB, myoglobin
and cytochromes), by integration in iron–sulfur clusters
[47,48], or used to synthesize and sequester iron biominerals
by ferritins. Multiple cellular repair mechanisms exist to com-
pensate for free-radical-induced damage caused by errant
iron (or dioxygen) [55]. After death, iron released from
these proteins becomes available for free-radical chemistry
with oxygen, leading to protein and lipid cross-linking,
tissue fixation and resistance to enzymatic/bacterial degra-
dation [55,56], and also forms particles in situ in tissues, as
our data demonstrate. Thus, damaging reactions in life can
be preserving reactions after death. Stabilization of cellular
and vascular components by HB iron in solution and/or
anoxia in the ostrich vessel model suggests that iron observed
in extant and dinosaur tissues is derived from HB degra-
dation. However, other metals also contribute to hydroxyl
radical formation; iron may be only one of many metals play-
ing a role in exceptional fossil preservation. Whatever the
exact mechanism, iron removal by chelation may increase
the number of fossil samples amenable to molecular analyses.
(Note: during the course of review of this manuscript, a
paper was published in PNAS [73] that directly relates to
our conclusion that iron influences preservation of biomole-
cules across geological time and speaks of the longevity of
some iron-containing biomolecules.)
Acknowledgements. We thank J. Horner, R. Harmon (Museum of the
Rockies, Montana State University) and the rest of the palaeontology
crew for access to specimens; S. Brumfeld and N. Equall (Montana
StateUniversity) for TEMand SEM data,respectively;J. Fountainfor geo-
chemical discussions and input; North Carolina State University and the
NC Museumof Natural Sciences forcontinued support of ourefforts;and
colleagues who contributed seed ideasthrough multiple discussions.
Funding statement. This research was financially supported by the
National Science Foundation (DGE-0750733 to T.P.C. and EAR
0541744 to M.H.S.) and the David and Lucile Packard Foundation
to M.H.S. The operations of the Advanced Light Source at Lawrence
Berkeley National Laboratory are supported by the Director, Office of
Science, Office of Basic Energy Sciences, US Department of Energy
under contract number DE-AC02-05CH11231.
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rspb.royalsocietypublishing.org Proc. R. Soc. B 281: 20132741
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... However, this observation only holds in a general sense and to a relative degree. Other variables affecting protein and DNA preservation include sediment composition (Briggs, 2003;Collins et al., 2002;Collins, Riley, Child, & Turner-Walker, 1995;Gupta, 2014;Kendall et al., 2018;Lindahl, 1993;Schweitzer, Schroeter, Cleland, & Zheng, 2019;Schweitzer et al., 2014), moisture content (Briggs, 2003;Collins et al., 2002;Gupta, 2014;Hedges & Millard, 1995;Kendall et al., 2018;Lennartz, Hamilton, & Weaver, 2020;Lindahl, 1993;Nielsen-Marsh et al., 2000;Schweitzer et al., 2019;Trueman, Behrensmeyer, Tuross, & Weiner, 2004), and oxygen content (Briggs, 2003;Collins et al., 2002;Gupta, 2014;Kendall et al., 2018;Lennartz et al., 2020;Lindahl, 1993;Schweitzer et al., 2019;Wiemann, Crawford, & Briggs, 2020;Wiemann et al., 2018), among others. Consequently, multiple studies have reported differing degrees of sequence preservation even for specimens sharing similar thermal histories and/or geologic ages (Fortes et al., 2016;C. ...
... Reported sequence preservation varied widely between specimens and was unattributable to any specific variables (Mackie et al., 2017). These differences in preservation likely result from a combination of other variables including differences in composition (Briggs, 2003;Collins et al., 2002;Collins et al., 1995;Gupta, 2014;Kendall et al., 2018;Lindahl, 1993;Schweitzer et al., 2019;Schweitzer et al., 2014), moisture content (Briggs, 2003;Collins et al., 2002;Gupta, 2014;Kendall et al., 2018;Lennartz et al., 2020;Lindahl, 1993;Nielsen-Marsh et al., 2000;Schweitzer et al., 2019;Trueman et al., 2004), and oxygen content (Briggs, 2003;Collins et al., 2002;Gupta, 2014;Kendall et al., 2018;Lennartz et al., 2020;Lindahl, 1993;Schweitzer et al., 2019;Wiemann et al., 2020;Wiemann et al., 2018) of burial sediments, among others . The complex range of variables potentially affecting sequence preservation supports that factors beyond geologic age and thermal history are responsible for specimens demonstrating exceptional sequence preservation. ...
... The presence of such exogenous minerals supports that this tibia had undergone substantial chemical alteration. Both mineral precipitants are consistent with observations from older tertiary (Boskovic et al., 2021;Cadena, 2016Cadena, , 2020 and even Mesozoic specimens (Armitage & Anderson, 2013;Boatman et al., 2019;Schweitzer et al., 2013;Schweitzer et al., 2014;Surmik et al., 2016), and their presence likely precludes it from being considered a "sub-fossil". Despite the apparent chemical alteration to its biomolecular histology, the tibia still preserved collagen sequences identifiable via mass spectrometry (Rybczynski et al., 2013). ...
Preprint
Researcher ability to accurately screen fossil and sub-fossil specimens for preservation of DNA and protein sequences remains limited. Thermal exposure and geologic age are usable proxies for sequence preservation on a broad scale but are of limited use for specimens of similar depositional environments and/or ages. Cell and tissue biomolecular histology is thus proposed as a proxy for determining sequence preservation potential of ancient specimens with improved accuracy. Biomolecular histology as a proxy is hypothesized to elucidate why fossil/sub-fossils of some depositional environments and or geologic ages preserve sequences while others do not and to facilitate selection of ancient specimens for use in molecular studies.
... Furthermore, hemoglobin from invertebrates has also become a significant subject of research. Thus, potential of extracellular hemoglobin as an additive for organ and tissue preservation [72,73] and as a growth stimulator of mesenchymal stromal cells (MSC), which maintain "stemness" in vitro [74], have been described. Therefore, one should be aware that under the same term "hemoglobin", the studies on extracellular hemoglobin functions imply both vertebrate and evolutionary distant invertebrate hemoglobin. ...
... Furthermore, hemoglobin from invertebrates has also become a significant subject of research. Thus, potential of extracellular hemoglobin as an additive for organ and tissue preservation [72,73] and as a growth stimulator of mesenchymal stromal cells (MSC), which maintain ''stemness" in vitro [74], have been described. Therefore, one should be aware that under the same term "hemoglobin", the studies on extracellular hemoglobin functions imply both vertebrate and evolutionary distant invertebrate hemoglobin. ...
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Hemoglobin is essential for maintaining cellular bioenergetic homeostasis through its ability to bind and transport oxygen to the tissues. Besides its ability to transport oxygen, hemoglobin within erythrocytes plays an important role in cellular signaling and modulation of the inflammatory response either directly by binding gas molecules (NO, CO, and CO2) or indirectly by acting as their source. Once hemoglobin reaches the extracellular environment, it acquires several secondary functions affecting surrounding cells and tissues. By modulating the cell functions, this macromolecule becomes involved in the etiology and pathophysiology of various diseases. The up-to-date results disclose the impact of extracellular hemoglobin on (i) redox status, (ii) inflammatory state of cells, (iii) proliferation and chemotaxis, (iv) mitochondrial dynamic, (v) chemoresistance and (vi) differentiation. This review pays special attention to applied biomedical research and the use of non-vertebrate and vertebrate extracellular hemoglobin as a promising candidate for hemoglobin-based oxygen carriers, as well as cell culture medium additive. Although recent experimental settings have some limitations, they provide additional insight into the modulatory activity of extracellular hemoglobin in various cellular microenvironments, such as stem or tumor cells niches.
... Though the acidic pH that would have temporarily accompanied reducing conditions within the medullary cavity of this specimen may seem (at face value) preclusive to molecular preservation, weak acidity has been implicated in the rapid nucleation of inert, protective, microcrystalline goethite crystals within 'osteocytes' and 'blood vessels' recovered from fossil bones [4,53]. For this reason, initial redox conditions in the immediatẽ 48 h after death may ultimately be the most critical, as it has been demonstrated that inter-and intra-molecular crosslinking (i.e., stabilization) reactions can operate in this brief timeframe, promoting equilibration with the early-diagenetic environment which may then persist through fossilization and late diagenesis [53]. ...
... This fact indicates that processes which stabilized diagenetiforms within this specimen in the initial hours to days postmortem imparted remarkable long-term resiliency. Novel experiments by Schwietzer et al. [4] and Boatman et al. [53] have begun to shed light on how this may occur, but testing of more fossils and further actualistic studies are needed to fully resolve the endurance of diagenetiforms under the wide array of physicochemical/thermodynamic regimes of natural diagenetic environments. ...
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Recent recoveries of peptide sequences from two Cretaceous dinosaur bones require paleontologists to rethink traditional notions about how fossilization occurs. As part of this shifting paradigm, several research groups have recently begun attempting to characterize biomolecular decay and stabilization pathways in diverse paleoenvironmental and diagenetic settings. To advance these efforts, we assessed the taphonomic and geochemical history of Brachylophosaurus canadensis specimen MOR 2598, the left femur of which was previously found to retain endogenous cells, tissues, and structural proteins. Combined stratigraphic and trace element data show that after brief fluvial transport, this articulated hind limb was buried in a sandy, likely-brackish, estuarine channel. During early diagenesis, percolating groundwaters stagnated within the bones, forming reducing internal microenvironments. Recent exposure and weathering also caused the surficial leaching of trace elements from the specimen. Despite these shifting redox regimes, proteins within the bones were able to survive through diagenesis, attesting to their remarkable resiliency over geologic time. Synthesizing our findings with other recent studies reveals that oxidizing conditions in the initial ~48 h postmortem likely promote molecular stabilization reactions and that the retention of early-diagenetic trace element signatures may be a useful proxy for molecular recovery potential.
... In spite of the diverse, growing literature that original, endogenous organic material can preserve for millions of years, these reports are often regarded with skepticism (e.g., [15]), in no small part, because the geochemical mechanisms that allow for such preservation are not completely understood. Although hypotheses exist as to geochemical factors that may positively influence preservation (e.g., involvement of iron [16,17], microbial activity ( [18]), and/or condensation reactions [19][20][21][22]), such studies often examine specific cases in isolation, making it difficult to infer larger scale relationships between the geochemical environment and preservation. As a result, the comprehensive depositional and geochemical data that could aid in building robust, multi-faceted hypotheses about molecular preservation in deep time are lacking from most paleomolecular reports [23,24]. ...
... If 'normal' bone fossilization processes do not reduce molecular preservation potential to zero, then the pool of fossil specimens that may yield biomolecular material is drastically larger than previously thought (indeed, if this is the case, molecular preservation might not actually be 'exceptional'). Although recrystallization and permineralization have each been hypothesized to possibly promote molecular preservation in fossil bones (via mineral encapsulation [16,101,[125][126][127] and hindrance of microbial infiltration [18,19,128], respectively), it remains premature to claim that 'average' fossil bones constitute favorable paleomolecular samples because this outlook remains based on a sample size of one: Dreadnoughtus humerus MPM-PV 1156-49. All other protein-bearing, pre-Cenozoic fossil bones whose trace element inventories have been characterized to date exhibit less REE enrichment [49,100], and the REE content of all other specimens documented to yield original molecules (e.g., those analyzed by Tuross [125] and Schweitzer et al. [13]) remain unknown. ...
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Evidence that organic material preserves in deep time (>1 Ma) has been reported using a wide variety of analytical techniques. However, the comprehensive geochemical data that could aid in building robust hypotheses for how soft-tissues persist over millions of years are lacking from most paleomolecular reports. Here, we analyze the molecular preservation and taphonomic history of the Dreadnougtus schrani holotype (MPM-PV 1156) at both macroscopic and microscopic levels. We review the stratigraphy, depositional setting, and physical taphonomy of the D. schrani skeletal assemblage, and extensively characterize the preservation and taphonomic history of the humerus at a micro-scale via: (1) histological analysis (structural integrity) and X-ray diffraction (exogenous mineral content); (2) laser ablation-inductively coupled plasma mass spectrometry (analyses of rare earth element content throughout cortex); (3) demineralization and optical microscopy (soft-tissue microstructures); (4) in situ and in-solution immunological assays (presence of endogenous protein). Our data show the D. schrani holotype preserves soft-tissue microstructures and remnants of endogenous bone protein. Further, it was exposed to LREE-enriched groundwaters and weakly-oxidizing conditions after burial, but experienced negligible further chemical alteration after early-diagenetic fossilization. These findings support previous hypotheses that fossils that display low trace element uptake are favorable targets for paleomolecular analyses.
... We considered the Hornerstown Formation as a lithosome potentially favorable for the preservation of endogenous organics because glauconite is rich in iron, which has been suggested to aid in molecular preservation. Specifically, it has been hypothesized that dissolved iron in diagenetic pore fluids reacts with peroxides sourced from decaying lipids to form iron free radicals, which in turn can induce chemical chain reactions resulting in crosslinking of biomolecules, their decay products, metal cations in solution, and dissolved humics [3,32,33]. Therefore, we examined twelve specimens of five taxa from the MFL and higher within the Formation for soft tissue and biomolecular preservation. This included analyzing one specimen of the marine crocodile Thoracosaurus for the preservation of the primary structural protein collagen I using molecular assays. ...
... Thus, our findings corroborate other recent studies [3,6,10,16,35] which refute the traditional hypothesis that marine paleoenvironments are inconducive to biomolecular preservation due to hydrolysis caused by constant exposure to water [25,26]. As iron is hypothesized to aid in molecular preservation in many cases [3,32,33], it is possible that the high concentration of glauconite, a mineral rich in iron, aided in the preservation of these endogenous organics, (e.g., via iron free radical-induced molecular crosslinking [33]). It is also possible that the presence of abundant dissolved iron was responsible for some of the analytical challenges encountered herein (see below). ...
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Endogenous biomolecules and soft tissues are known to persist in the fossil record. To date, these discoveries derive from a limited number of preservational environments, (e.g., fluvial channels and floodplains), and fossils from less common depositional environments have been largely unexplored. We conducted paleomolecular analyses of shallow marine vertebrate fossils from the Cretaceous–Paleogene Hornerstown Formation, an 80–90% glauconitic greensand from Jean and Ric Edelman Fossil Park in Mantua Township, NJ. Twelve samples were demineralized and found to yield products morphologically consistent with vertebrate osteocytes, blood vessels, and bone matrix. Specimens from these deposits that are dark in color exhibit excellent histological preservation and yielded a greater recovery of cells and soft tissues, whereas lighter-colored specimens exhibit poor histology and few to no cells/soft tissues. Additionally, a well-preserved femur of the marine crocodilian Thoracosaurus was found to have retained endogenous collagen I by immunofluorescence and enzyme-linked immunosorbent assays. Our results thus not only corroborate previous findings that soft tissue and biomolecular recovery from fossils preserved in marine environments are possible but also expand the range of depositional environments documented to preserve endogenous biomolecules, thus broadening the suite of geologic strata that may be fruitful to examine in future paleomolecular studies.
... The presence of such exogenous minerals supports that this tibia had undergone substantial chemical alteration. Both mineral precipitants are consistent with observations from older tertiary [44][45][46] and even Mesozoic specimens 23,42,43,47,48 , and certainly precludes it from being considered a "sub-fossil". Despite the apparent chemical alteration to its biomolecular histology, the tibia still preserved collagen sequences identifiable via mass spectrometry 2 . ...
Preprint
Researcher ability to accurately screen fossil and sub-fossil specimens for preservation of DNA and protein sequences remains limited. Thermal exposure and geologic age are usable proxies for sequence preservation on a broad scale but are of limited use for specimens of similar depositional environments and/or ages. Cell and tissue biomolecular histology is thus proposed as a proxy for determining sequence preservation potential of ancient specimens with improved accuracy. Biomolecular histology as a proxy is hypothesized to elucidate why fossil/sub-fossils of some depositional environments and or geologic ages preserve sequences while others do not and to facilitate selection of ancient specimens for use in molecular studies.
... The presence of such exogenous minerals supports that this tibia had undergone substantial chemical alteration. Both mineral precipitants are consistent with observations from older tertiary [44][45][46] and even Mesozoic specimens 23,42,43,47,48 , and certainly precludes it from being considered a "sub-fossil". Despite the apparent chemical alteration to its biomolecular histology, the tibia still preserved collagen sequences identifiable via mass spectrometry 2 . ...
Preprint
Researcher ability to accurately screen fossil and sub-fossil specimens for preservation of DNA and protein sequences remains limited. Thermal exposure and geologic age are usable proxies for sequence preservation on a broad scale but are of limited use for specimens of similar depositional environments and/or ages. Cell and tissue biomolecular histology is thus proposed as a proxy for determining sequence preservation potential of ancient specimens with improved accuracy. Biomolecular histology as a proxy is hypothesized to elucidate why fossil/sub-fossils of some depositional environments and or geologic ages preserve sequences while others do not and to facilitate selection of ancient specimens for use in molecular studies.
... Eumelanin is brown to black in color and contains repeating units of 5,6-dihydroxyindole (6) and 5,6-dihydroxyindole-2-carboxylic acid (7). In its biosynthesis, it is derived from the amino acid tyrosine (8), which, upon action of tyrosinase, or by oxidation, is converted to DOPA-quinone (9), which is then cyclized and decarboxylated to form 5,6-dihydroxyindole (6) through the intermediate compounds leucodopachrome (10) and dopachrome (11) [25,85,86] (for structures see Figure 2). Some indole units may randomly undergo partial oxidative cleavage via formation of an ortho-benzoquinone leading to pyrrole-di-carboxylic acid derivatives, which are incorporated into the polymeric structure of eumelanin [89,90]. ...
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This review provides an overview of organic compounds detected in non-avian dinosaur fossils to date. This was enabled by the development of sensitive analytical techniques. Non-destructive methods and procedures restricted to the sample surface, e.g., light and electron microscopy, infrared (IR) and Raman spectroscopy, as well as more invasive approaches including liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), time-of-flight secondary ion mass spectrometry, and immunological methods were employed. Organic compounds detected in samples of dinosaur fossils include pigments (heme, biliverdin, protoporphyrin IX, melanin), and proteins, such as collagens and keratins. The origin and nature of the observed protein signals is, however, in some cases, controversially discussed. Molecular taphonomy approaches can support the development of suitable analytical methods to confirm reported findings and to identify further organic compounds in dinosaur and other fossils in the future. The chemical properties of the various organic compounds detected in dinosaurs, and the techniques utilized for the identification and analysis of each of the compounds will be discussed.
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Chapter
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Significance Fossils, in addition to documenting the existence of extinct species, can often provide information on the behavior of ancient organisms. The present study describes the fossil of a blood-engorged mosquito in oil shale from northwestern Montana. The existence of this rare specimen extends the existence of blood-feeding behavior in this family of insects 46 million years into the past. Heme, the oxygen-carrying group of hemoglobin in the host’s blood, was identified in the abdomen of the fossil mosquito by nondestructive mass-spectrometry analysis. Although large and fragile molecules such as DNA cannot survive fossilization, other complex organic molecules, in this case iron-stabilized heme, can survive intact and provide information relative to the mechanisms of the fossilization process.
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Fossil deposits that preserve soft-bodied organisms provide critical evidence of the history of life. Usually, only more decay resistant materials, e.g., cuticles, survive as organic remains as a result of selective preservation and subsequent diagenesis to more resistant biopolymers. Permineralization, the permeation of tissues by mineralizing fluids, may preserve remarkable detail, particularly of plants. However, evidence of more labile tissues, e.g., muscle, normally requires the replication of their morphology by rapid in situ growth of minerals, i.e., authigenic mineralization. This process relies on the steep geochemical gradients generated by decay microbes. The minerals involved, and the level of detail preserved (which may be subcellular), depend on a number of factors, including the nature of microbial activity and amount of decay, availability of ions, and the type of organism that is fossilized. Understanding these controls is essential to determining the conditions that favor exceptional preservation.
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The ability of iron to accept or donate electrons, coupled with the ability of oxygen to act as an electron acceptor, renders both elements essential to normal cellular biology. However, these same chemical properties allow free iron in solution to generate toxic free radicals, particularly in combination with oxygen. Thus, closely interwoven homeostatic mechanisms have evolved to regulate both iron and oxygen concentrations at the systemic and the cellular levels. Systemically, iron levels are regulated through hepcidin-mediated uptake of iron in the duodenum, whereas intracellular free-iron levels are controlled through iron-regulatory proteins (IRPs). Cardiorespiratory changes increase systemic oxygen delivery, whereas at a cellular level, many responses to altered oxygen levels are coordinated by hypoxia-inducible factor (HIF). However, the mechanisms of iron homeostasis also are regulated by oxygen availability, with alterations in both hepcidin and IRP activity. In addition, many genes involved in iron homeostasis are direct targets of HIF. Furthermore, HIF activation is modulated by intracellular iron, through regulation of hydroxylase activity, which requires iron as a cofactor. In addition, HIF-2alpha translation is controlled by IRP activity, providing another level of interdependence between iron and oxygen homeostasis.
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