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Analyses of Soft Tissue from Tyrannosaurus rex Suggest the Presence of Protein

  • Beth Israel Deaconess Medical Center / Harvard Medical School

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We performed multiple analyses of Tyrannosaurus rex (specimen MOR 1125) fibrous cortical and medullary tissues remaining after demineralization. The results indicate that collagen I, the main organic component of bone, has been preserved in low concentrations in these tissues. The findings were independently confirmed by mass spectrometry. We propose a possible chemical pathway that may contribute to this preservation. The presence of endogenous protein in dinosaur bone may validate hypotheses about evolutionary relationships, rates, and patterns of molecular change and degradation, as well as the chemical stability of molecules over time.
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DOI: 10.1126/science.1138709
, 277 (2007); 316Science
et al.Mary Higby Schweitzer,
Suggest the Presence of Protein
Tyrannosaurus rexAnalyses of Soft Tissue from (this information is current as of April 18, 2007 ):
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17. The Doppler broadening of the radar echo due to rotation
of the target is B =(4pD/lP) sin a, where B is the limb-
to-limb bandwidth of the echo, D is the target diameter
producing the Doppler shift at the current viewing
geometry and rotation phase, l is the radar wavelength,
P is the spin period of the target, and a is the inclination
of the spin axis to the line of sight.
18. S. J. Ostro, Rev. Mod. Phys. 65, 1235 (1993).
19. Resolution in time delay, and equivalently range, is
achieved by transmitting a time-dependent signal and
analyzing the received signal according to arrival time.
Thetimeincrementt used in the transmitted signal yields
arangeresolutionct/2, where c is the speed of light.
20. We typically define the limb-to-limb bandwidth as the
full width of the radar echo at the level of twice the root
mean square (RMS) of the off-DC, off-target noise. The
exception is the strong 2004 Arecibo data, for which we
use 10 times the RMS as the threshold to avoid
contributions from frequency sidelobes.
21. This assumes PH5 is a principal axis (PA) rotator where
the spin axis remains fixed in inertial space and aligned
with the axis of maximum moment of inertia. The spin
axis of PH5 must then be oriented such that the angles it
makes with the lines of sight satisfy the observed
bandwidths (17). The damping time scale (28)toPA
rotation for PH5 is of order 0.1 million years.
22. Materials and methods are available as supporting
material on Science Online.
23. The spin state solution is also validated by the phase
agreement of infrared lightcurves from the Spitzer Space
Telescope with synthetic lightcurves produced with our
shape (27).
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in Hazards Due to Comets and Asteroids, T. Gehrels,
M. S. Matthews, A. M. Schumann, Eds. (Univ. of Arizona
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(2006), p. 95.
28. I. Sharma, J. A. Burns, C.-Y. Hui, Mon. Not. R. Astron.
Soc. 359, 79 (2005).
29. We thank the staffs of the Arecibo Observatory and the
Goldstone Solar System Radar for their support in
performing this research. The Arecibo Observatory is part
of the National Astronomy and Ionosphere Center, which
is operated by Cornell University under a cooperative
agreement with NSF. Some of this work was performed at
the Jet Propulsion Laboratory, California Institute of
Technology, under contract with NASA. This material is
based in part on work supported by NASA under the
Science Mission Directorate Research and Analysis
Programs. P.A.T. and J.L.M. were partially supported by
NASA grant NNG04GN31G. The work of P.P. and D.V. was
supported by the Grant Agency of the Czech Republic.
D.J.S. acknowledges support from the NASA Planetary
Geology and Geophysics Program. S.C.L. and A.F.
acknowledge support from the Leverhulme Trust and
PPARC, respectively. C.M. was partially supported by
NSF grant AST-0205975. The International Astronomical
Union has approved the name YORP for asteroid (54509)
2000 PH5.
Supporting Online Material
Figs. S1 to S5
Tables S1 and S2
19 December 2006; accepted 21 February 2007
Published online 8 March 2007;
Include this information when citing this paper.
Analyses of Soft Tissue from
Tyrannosaurus rex Suggest the
Presence of Protein
Mary Higby Schweitzer,
* Zhiyong Suo,
Recep Avci,
John M. Asara,
Mark A. Allen,
Fernando Teran Arce,
John R. Horner
We performed multiple analyses of Tyrannosaurus rex (specimen MOR 1125) fibrous cortical and
medullary tissues remaining after demineralization. The results indicate that collagen I, the main
organic component of bone, has been preserved in low concentrations in these tissues. The findings
were independently confirmed by mass spectrometry. We propose a possible chemical pathway that
may contribute to this preservation. The presence of endogenous protein in dinosaur bone may
validate hypotheses about evolutionary relationships, rates, and patterns of molecular change and
degradation, as well as the chemical stability of molecules over time.
t has long been assumed that the process
of fossilization results in the destruction of
virtually all original organic components
of an organism, and it has been hypothesized
that original molecules will be either lost or
altered to the point of nonrecognition over
relatively short time spans (well under a mil-
lion years) (17). However, the discovery of
intact structures retaining original transparency,
flexibility, and other characteristics in speci-
mens dating at least to the Cretaceous (8, 9)
suggested that, under certain conditions, rem-
nant organic constituents may persist across
geological time.
The skull, vertebrae, both femora and tibiae,
and other elements of an exceptionally well-
preserved Tyrannosaurus r ex [MOR 1125 (8)]
were recovered from the base of the Hell Creek
Formation in eastern Montana (USA), buried
within at least 1000 m
of medium-grained,
loosely consolidated sandstone interfingered with
fine-grained muds, interpreted as stream channel
sediments. Demineralization of femur and tibia
fragments revealed the preservation of fibrous,
flexible, and apparently original tissues, as well
as apparent cells and blood vessels (8), but the
endogeneity and composition of these structures
could not be ascertained without further analyses.
We present molecular and chemical (10)
analyses of tissues remaining after partial de-
mineralization (11) of the left and right femora
and associated medullary bone (12) that would,
in extant bone, represent the extracellular matrix
(osteoid) dominated by collagen I (13). Because
of its ordered structure as a triple helix (14, 15),
collagen I has unique characteristics that are
highly conserved across taxa, making validation
of its presence relatively straightforward. The
molecular composition of collagen incorporates
glycine, the smallest amino acid, at every helical
turn. Therefore, an amino acid profile of colla-
gen results in ~33% glycine content (14). This
molecular structure also results in packing of
microfibrils with a banded repeat of ~70 nm
(15, 16). Collagen also shows posttranslational
hydroxylation of about half of all proline and
some lysine residues; thus, the detection of
hydroxyproline and hydroxylysine in extracts of
organic material is viewed as strong evidence for
the presence of collagen (17, 18). Finally, colla-
gen is identified by polyclonal or monoclonal
antibody reactivity that can distinguish between
collagen types (19). We focused on identifying
collagen-like compounds because in addition to
being abundant and easily identified by multiple
Department of Marine, Earth and Atmospheric Sciences, North
Carolina State University, Raleigh, NC 27695, USA.
Carolina Museum of Natural Sciences, Raleigh, NC 27601,
Museum of the Rockies, Montana State University,
Bozeman, MT 59717, USA.
Image and Chemical Analysis
Laboratory Facility, Department of Physics, Montana State
University, Bozeman, MT 59717, USA.
Division of Signal
Transduction, Beth Israel Deaconess Medical Center, Boston,
MA 02115, USA.
Department of Pathology, Harvard
Medical School, Boston, MA 02115, USA.
Department of
Chemistry and Biochemistry, Montana State University,
Bozeman, MT 59717, USA.
Center for Nanomedicine,
Pulmonary and Critical Care Medicine, Department of
Medicine, University of Chicago, Chicago, IL 60637, USA.
*To whom correspondence should be addressed. E-mail: SCIENCE VOL 316 13 APRIL 2007 277
on April 18, 2007 www.sciencemag.orgDownloaded from
and independent methods, this protein is durable
(20, 21) and resistant to degradation.
The fibrous nature of demineralized dinosaur
tissues was demonstrated by optical (8) and elec-
tron (fig. S1) microscopy. Furthermore, regions
of dinosaur cortical and medullary (12) bone
demonstrated a repeat pattern with periodicity of
copy (AFM) (Fig. 1, A to D), consistent with
collagen in extant bone (Fig. 1, E and F) and
similar to that previously observed in fragments of
demineralized Cretaceous avian bone (22). How-
ever, this periodic pattern was rarely observed in
ultrathin sections of MOR 1 125 demineralized
bone by transmission electron microscopy (TEM)
(fig. S1). This may be a methodological problem,
or the periodic features we observe (Fig. 1, A to
D) may be due to surface features generated when
demineralization removed most of the apatite
crystals emplaced during biomineralization, when
collagen acted as a template. Thus, the banded
features may represent a type of natural molecular
imprinting (23), because banded fibers have been
observed by TEM for other dinosaur tissues (24).
TEM studies confirm that, unlike extant bone,
dinosaur bone did not completely demineralize
after p rolonged incubation i n EDTA (11).
Selected-area electron diffraction (SAED) of the
tissues (fig. S1D, inset) showed that this retained
mineral is biogenic hydroxylapatite (25). It is not
possible to determine this conclusively because of
the similarity in structure between hydroxylapatite
and fluorapatite; however, the observed diffrac-
tion circle intensities are most consistent with hy-
droxylapatite. This finding suggests that the bone
mineral is virtually unchanged from the living
state and has undergone little if any alteration.
Force curve measurements of demineralized
dinosaur medullary and cortical bone indicate
that the elasticity of dinosaur tissues was similar
to that of demineralized extant bone. We mea-
sured both embedded sections (fig. S2A) and
unembedded whole mounts (fig. S2B) of demin-
eralized bone in both air and liquid (11). The
demineralized bone surface softened after expo-
sure to buffer, allowing the AFM tip to penetrate
deeper into the tissues with less resistance. Thus,
the modulus of elasticity (fig. S2C) was reduced
in liquid by more than three orders of magnitude
(fig. S2B). Although ~2000 nN of force was
required to penetrate ~40 nm into MOR 112 5
bone matrix in air , only ~15 nN of force was
required to depress the tip ~75 nm into the same
matrix when hydrated (fig. S2B, inset).
MOR 1 125 cortical and medullary whole-
bone extracts showed reactivity to antibodies
raised against chicken collagen I (11)whenmea-
sured by enzyme-linked immunosorbent assay
(ELISA), although the degree of binding varied
widely. Reactivity was greatly reduced in dinosaur
extracts relative to extant samples (fig. S3), but
still at least twice that observed in negative con-
Fig. 1. AFM images of partially demineralized bones of MOR 1125 (A to D)andemu(E and F). (A) Phase
image of MOR 1125 cortical bone imaged in air; (B) deflection image of MOR cortical bone imaged in
phosphate-buffered saline; (C) amplitude image of embedded and sectioned MOR medullary bone
imaged in air; (D) phase image of MOR 1125 medullary bone imaged in air (note longitudinal and cross-
sectional orientation of fiber-like structures at right angles to each other); (E) amplitude image of emu
cortical bone imaged in air; (F) amplitude image of emu medullary bone imaged in air.
Fig. 2. In situ immunochemistry on
300-nm sections of demineralized
MOR 1125 cortical bone (A to D)and
medullary bone (E to H). (A) and (E),
no primary antibodies added (negative
control); (B) and (F), antibodies to
avian collagen I; (C) and (G), anti-
bodies to actin protein (nonrelevant,
negative control); (D) and (H), anti-
bodies to avian collagen I, inhibited by
incubating with purified chicken colla-
gen before exposing to dinosaur
tissues. All data were collected using
the same parameters at 122-ms inte-
gration. (I to K) MOR 1125 cortical
tissue exposed to (I) no primary, (J) antibodies to avian collagen I, or
(K) collagenase digestion followed by antibodies to avian collagen I,
as described (11). Data in (I), (J), and (K) were collected at 149-ms
on April 18, 2007 www.sciencemag.orgDownloaded from
trols of coextracted sediments and buffer without
sample, similarly treated.
We confirmed the antibody reactivity data by
in situ immunohistochemistryinaseriesof
experiments. W e exposed thin (0.3 to 0.5 mm)
sections of demineralized cortical (Fig. 2, A to D
and I to K) and medullary (Fig. 2, E to H) dinosaur
bone to antibodies raised against avian collagen I,
both before (Fig. 2, B and F) and after inhibition of
antibodies with chicken collagen (Fig. 2, D and H)
(11). Additionally , antibody reactivity (Fig. 2J) was
significantly decreased after we digested dinosaur
tissues with collagenase (Fig. 2K), although this
enzyme effect was not consistently observed. Re-
activity to antibodies, measured by fluorescence,
was significantly greater than in negative controls
(Fig. 2, A, C, E, G, and I) and was localized to
tissues. We also observed some binding of os-
teocalcin antibodies to dinosaur tissues (fig. S4).
These patterns were similar to those observed with
extant emu cortical and medullary bone (fig. S5).
Immunoreactivity in dinosaur tissues was greatly
reduced from that observed in extant bone, as illus-
trated by longer integration times and fainter signal,
but was greater than in negative controls. Immuno-
histochemistry performed on sediments was nega-
tive for binding. These results imply that the
concentration of reactive epitopes in the dinosaur
tissues is very low, consistent with the ELISA re-
sults. That antibody reactivity was more consistent-
ly observed in situ than in ELISA could be due to
greater alteration and/or loss of organic compounds
during extraction procedures, or to reduced binding
of degraded antigen to ELISA plate polymers.
The presence of collagen-derived epitopes in
demineralized tissues is supported by mass spec-
trometry data. T ime-of-flight secondary ion mass
spectrometry (TOF-SIMS) detects surface ions
associated with molecular fragmentation with
high mass resolution, and can localize signal to
whole samples without subjecting them to chem-
ical extraction. In situ TOF-SIMS analyses were
performed to unambiguously detect amino acid
residues consistent with the presence of protein in
demineralized MOR 1125 tissues (Fig. 2 and fig.
S6). We obtained ratios of glycine (Gly), the most
abundant amino acid in collagen [~33% (14)], and
alanine (Ala), which constitutes about 10% of
collagenous amino acids, to support the presence
of the specific collagen a1type1proteininthese
tissues. Small peaks representin g proline (Pro)
at mass/charge ratio (m/z) 70 (Fig. 3C), lysine
(Lys) at m/z 84 (fig. S6A), and leucine or
isoleucine at m/z 86 (fig. S6B) were also detected.
TOF-SIMS is highly matrix dependent, and de-
sorption and ionization of some amino acid res-
idues, especially modified residues such as
hydroxylated Pro, are less efficient than for other
residues (26). These modified residues were not
detected by this method but were readily identified
by other mass spectrometry methods (10).
The Gly:Ala ratio for published chicken
collagen a1 type 1 sequence (27)is2.5:1.The
TOF-SIMS results show that the Gly:Ala ratio in
medullary bone of MOR 1125 is 2.6:1 (Fig. 3, A
and B). Sandstones entombing the dinosaur , sub-
jected to TOF-SIMS as a control, showed little or
no evidence for these amino acids (Fig. 3, D and E).
We identified a variety of nitrogen-cont ain ing
speciesincluding an alkyl amine group, C
located at 130 amu (fig. S6C)in all dinosaur
samples tested, but not in any surrounding sedi-
ments. We also observed a number of Fe-C-H
associated with the dinosaur matrix (fig. S7) but
not seen in extant material. Similar compounds
were observed in the sediments surrounding the
dinosaur . These may be microbial products, as
sequences from iron-containing microbial en-
zymes were identified by mass spectrometry
(10). W e interpret these fragments as evidence that
iron may help preserve soft tissue through initiation
of intra- and intermolecular cross-links (9).
Dinosaur protein sequence, including collagen,
should be most similar to that of birds among
extant taxa, according to other phy logenetic
information (28). The hypothesis that molecular
fragments of original proteins are preserved in the
mineralized matrix of bony elements of MOR
1 125 is supported by peptide sequences recovered
from dinosaur extracts, some of which align
uniquely with chicken collagen a1type1(10).
The amount of protein or protein-like com-
ponents in MOR 1 125 is minimal. The percent
yield afte r extraction and lyophilization was
~0.62% for cortical bone and 1.3% for medullary
bone. Protein-derived material is only a small per-
centage of the lyophilate relative to other material
coextracted from bone, as assessed by comparison
of immunoreactivity with extant samples. This is
verified by mass spectrometry , which identifies
only femtomole amounts of sequenceable mate-
rial (10) in a heterogeneous mixture of extracted
Microenvironments within a single bone vary
greatly, and not every fragment of bone examined
yielded positive results. There was a high degree
of variability between extractions, and we have
also noted progressive reduction of signal in more
recent extractions, indicating bone degradation in
modern environments (29). Therefore, each of the
analyses we report has been repeated numerous
times, and we have set a minimum of three rep-
etitions with similar results before reporting an
assay as positive. Additionally , experiments have
been conducted independently in at least three
different labs and by numerous investigators, and
the results strongly support the endogeneity of
collagen-like protein molecules.
We hypothesize that these molecular frag-
ments are preserved because reactive sites on the
Fig. 3. TOF-SIMS spectra of demineralized MOR 1125 medullary bone (A to C) and entombing
sedimentary matrix (D to F). The imonium ions for Gly (m/z = 30), Ala (m/z = 44), and Pro (m/z =
70) can be unambiguously identified for MOR 1125; no signal was observed in sediment controls
that corresponded to these amino acids. See text for discussion. SCIENCE VOL 316 13 APRIL 2007
on April 18, 2007 www.sciencemag.orgDownloaded from
original protein molecules became irreversibly
cross-linked, both to similar molecules and to
mineral or exogenous organic components. These
cross-linking reactions may have been initiated by
unstable metal ions that formed free radicals
(30, 31),whichinturnreactedwithorganicmol-
ecules to form polymers (6, 7, 9, 32). W e propose
that the unstable metal ions were derived from the
post mortem degradation of iron-containing di-
nosaur biomolecules such as hemoglobin, myo-
globin, and possibly cytochromes (9, 31). Once
stabilized by these cross-linking reactions, the
molecules were no longer available as substrates
for further degrad ative reactio ns.
The intimate relationship between apatite and
the organic phase of bone also contributes to the
preservation of organic matter (16, 3338), but we
propose that the mineral phase may be stabilized
by this relationship as well. The presence of bio-
genic apatite in these 68-million-year-old bones
can only be rationalized by protection from an
intact organic phase, which in turn is only satisfied
by a synergistic relationship between collagen and
mineral phases. Whereas extant bone retains no
detectable calcium after days to weeks of de-
mineralization, dinosaur bone retains a fraction of
recognizable apatite crystals after months of
treatment (fig. S1). Another contributing factor
in the retention of original mineral may be that
apatite is stabilized in the presence of calcite (33).
Sandstones surrounding MOR 1125 contain
abundant calcite cements.
The depositional environments may affect
organic preservation in other ways. Comparison
of fossils from a variety of enviro nments indicates
that those derived from sandstones are more likely
to retain soft tissues and/or cells (9). W e hypoth-
esize that the porosity of sandstones may facilitate
draining of enzymes of decay and suppurating
fluids as the organism degrades, whereas organ-
isms buried in nonporous mudstones or clays may
be exposed to these longer and therefore may be
more completely degraded.
Our findings indicate the need for optimizing
methods of extraction and handling of fossil
material. In particular, the decrease in signal we
observed over time supports the need to establish
field collection and storage of fossils according to
protocols that allow future analytical studies (29).
The data presented here illustrate the value of a
multidisciplinary approach to the characterization
of very old fossil material and validate sequence
data reported elsewhere (10). The inclusion of
fossil-derived molecular sequences into existing
phylogenies may provide greater resolution and
may allow reconstruction of character evolution
beyond what is currently possible. Elucidating
modifications to ancient molecules may shed light
on patterns of degradation and diagenesis. The
presence of original molecular components is not
predicted for fossils older than a million years
(17), and the discovery of collagen in this well-
preserved dinosaur supports the use of actualistic
conditions to formulate molecular degradation
rates and models, rather than relying on theoretical
or experimental extrapolations derived from
conditions that do not occur in nature.
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1125, B. rex. Supported by NSF grants EAR-0541744
and EAR-0548847 and the David and Lucile Packard
Foundation (M.H.S.), NASA Experimental Program to
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Supporting Online Material
Materials and Methods
Figs. S1 to S7
11 December 2006; accepted 19 March 2007
Protein Sequences from Mastodon
and Tyrannosaurus Rex Revealed by
Mass Spectrometry
John M. Asara,
Lisa M. Freimark,
Matthew Phillips,
Lewis C. Cantley
Fossilized bones from extinct taxa harbor the potential for obtaining protein or DNA sequences that
could reveal evolutionary links to extant species. We used mass spectrometry to obtain protein
sequences from bones of a 160,000- to 600,000-year-old extinct mastodon (Mammut americanum)
and a 68-million-year-old dinosaur (Tyrannosaurus rex). The presence of T. rex sequences indicates
that their peptide bonds were remarkably stable. Mass spectrometry can thus be used to determine
unique sequences from ancient organisms from peptide fragmentation patterns, a valuable tool to
study the evolution and adaptation of ancient taxa from which genomic sequences are unlikely to
be obtained.
btaining genome sequences from a
number of taxa has dramatically en-
hanced our abilities to study the evolu-
tion and adaptation of organisms. However ,
difficulties in the acquisition of DNA or RNA
from ancient extinct taxa limit the abi lity to
examine molecular evolution. Recent advances
in mass spectrometry (MS) technologies have
on April 18, 2007 www.sciencemag.orgDownloaded from
... As for the sedimentary environments, there are different geological configurations formed from sediments (e.g., fluvial deposits, sandstones, karst deposits, claystone, and marine deposits). However, research has shown that, at least for the recovery of nonmineralized biomaterials (e.g., blood vessels, matrix tissues), rapid burial in a fluvial or sandstone depositional environment seems preferable over to other environments (Schweitzer et al., 2007(Schweitzer et al., , 2009. ...
... As Timothy P. Cleland, a molecular paleobiologist, at the Smithsonian Museum Conservation Institute, states, "Fossils found in sandstone appear to have a better preservation than those that come from shale or clay" (Morton, 2017, p. 40, 42). The hypothesis raised is that the phenomenon is due to the porous nature of the sand, which would allow suppurating fluids, rich in microorganisms and loaded with enzymes, to disperse more quickly, providing a drier environment and thus delaying the decomposition of materials (Schweitzer et al., 2007). ...
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Este trabalho é fruto de uma pesquisa exploratória e descritiva, de natureza quantitativa de dados, que teve por objetivo investigar o conhecimento de estudantes do Ensino Médio do sul do Brasil sobre aspectos relacionados à Paleontologia em geral e, mais especificamente, aos conceitos básicos da Paleontologia Molecular. O grupo amostral foi composto por 52 estudantes da 2ª série do Ensino Médio, de duas escolas privadas, localizadas nos municípios de Araucária (PR) e Palhoça (SC), região sul do Brasil. Os dados foram obtidos através de questionário estruturado on-line, elaborado via plataforma Google Forms, composto de 21 questões objetivas de múltipla escolha. A análise dos questionamentos foi feita utilizando-se de estatística descritiva para exame da frequência de respostas distribuídas nas três categorias de análise: 1) características socioeconômicas, 2) conhecimentos gerais sobre Paleontologia e 3) conhecimentos específicos sobre Paleontologia Molecular. Os resultados demonstram que as respostas dos estudantes sobre conceitos básicos da Paleontologia Molecular encontram-se coerentes com os dados disponíveis na literatura científica, o que demonstra que os alunos do Ensino Médio são capazes de adquirir e aplicar conteúdos complexos vinculados à biologia molecular, fósseis e evolução, em ambiente escolar. Uma vez que são raros os estudos que buscaram investigar a compreensão de alunos da educação básica a respeito de assuntos da Paleogenômica e Paleoproteômica, percebe-se que os dados aqui apresentados poderão suprir uma lacuna do conhecimento existente em nosso país.
... Results from these studies showed exceptional micromorphological preservation extending to the cellular level (eg. osteocytes, possible endothelial cells; ( [17,18,[22][23][24][25], Schweitzer et al. in prep) as well as several biochemical signatures of bone-specific proteins (e.g., collagen; [17][18][19]23]). Additionally, these studies confirmed the observed molecular signatures differed from various diagenetically-induced morphologies/chemistries (e.g., biofilms; [14]) and were comparable to similar morphologies/chemistries of extant archosaurs. ...
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Biomolecules preserved in deep time have potential to shed light on major evolutionary questions, driving the search for new and more rigorous methods to detect them. Despite the increasing body of evidence from a wide variety of new, high resolution/high sensitivity analytical techniques, this research is commonly met with skepticism, as the long standing dogma persists that such preservation in very deep time (>1 Ma) is unlikely. The Late Cretaceous dinosaur Tyrannosaurus rex (MOR 1125) has been shown, through multiple biochemical studies, to preserve original bone chemistry. Here, we provide additional, independent support that deep time bimolecular preservation is possible. We use synchrotron X-ray fluorescence imaging (XRF) and X-ray absorption spectroscopy (XAS) to investigate a section from the femur of this dinosaur, and demonstrate preservation of elements (S, Ca, and Zn) associated with bone remodeling and redeposition. We then compare these data to the bone of an extant dinosaur (bird), as well as a second non-avian dinosaur, Tenontosaurus tilletti (OMNH 34784) that did not preserve any sign of original biochemistry. Our data indicate that MOR 1125 bone cortices have similar bone elemental distributions to that of an extant bird, which supports preservation of original endogenous chemistry in this specimen.
... Based on the historical order of their discovery, 28 types of collagens-type I through type XXVIII-have been identified and described up to the current day [39]. The oldest collagen identified to date was found in the soft tissue of a fossilized Tyrannosaurus rex bone that dates back 68 million years [40,41]. ...
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Fish collagen garnered significant academic and commercial focus in the last decades featuring prospective applications in a variety of health-related industries, including food, medicine, pharmaceutics, and cosmetics. Due to its distinct advantages over mammalian-based collagen, including the reduced zoonosis transmission risk, the absence of cultural-religious limitations, the cost- effectiveness of manufacturing process, and its superior bioavailability, the use of collagen derived from fish wastes (i.e., skin, scales) quickly expanded. Moreover, by-products are low cost and the need to minimize fish industry waste’s environmental impact paved the way for the use of discards in the development of collagen-based products with remarkable added value. This review summarizes the recent advances in the valorization of fish industry wastes for the extraction of collagen used in several applications. Issues related to processing and characterization of collagen were presented. Moreover, an overview of the most relevant applications in food industry, nutraceutical, cosmetics, tissue engineering, and food packaging of the last three years was introduced. Lastly, the fish- collagen market and the open technological challenges to a reliable recovery and exploitation of this biopolymer were discussed.
... Given the above conceptual models and mechanisms of postburial protein decay, it is not surprising that reports of collagen preservation in Mesozoic fossil bones (e.g., Schweitzer et al., 1997Schweitzer et al., , 2007 have been met with skepticism, citing the lack of certainty in determining whether the protein signals are endogenous due to the high potential for cross-contamination (Buckley et al., 2008(Buckley et al., , 2017Saitta et al., 2018), although additional studies were published which further support the endogeneity of the proteins in question (Schroeter et al., 2017;Schweitzer et al., 2013). Several disparate explanations for the preservation of proteins in fossils from deep time have been proposed such as rapid mineralization leading to the incorporation of organic material into the mineral matrix (Schweitzer et al., 2005) stabilization resulting from iron chelation (Schweitzer et al., 2014) and the existence of a suitable microenvironment conducive to decay inhibition (Dhiman et al., 2021;Dutta et al., 2020). ...
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The mechanism of protein degradation has remained a topic of debate (specifically concerning their preservation in deep time), which has recently been invigorated due to multiple published reports of preservation ranging from Miocene to the Triassic that potentially challenge the convention that protein preservation beyond the Cenozoic is extremely uncommon or is expected to be absent altogether, and thus have attracted skepticism. In this paper, we analyze fossil fish scales from the Cretaceous, Jurassic, and Triassic using comprehensive pyrolysis gas chromatography coupled with time-of-flight mass spectrometry and compare the pyrolytic products so obtained with a well-preserved fish scale from Late Pliocene, in an attempt to better understand the effects of diagenesis on protein degradation at the molecular level through deep time. We find that the Pliocene fish scale displays a large number of N-bearing pyrolytic products, including abundant substituted cyclic 2,5-diketopiperazines (2,5-DKPs) which are diagnostic products of peptide and amino acid pyrolysis. We identify N-bearing compounds in the Mesozoic fish scales—however, among the 2,5-DKPs that were identified in the Pliocene scale, only diketodipyrrole (or cyclo (Pyr-Pyr)) is present in the Mesozoic scales. We discuss the implications of N-bearing pyrolytic products with emphasis on 2,5-DKPs in geological samples and conclude that the discrepancy in abundance and variety of N-bearing products between Pliocene and Mesozoic scales indicates that the protein component in the latter has been extensively diagenetically altered, while a suite of DKPs such as in the former would imply stronger evidence to indicate preservation of protein. We conclude that analytical pyrolysis is an effective tool for detecting preservation of intact proteins, as well as for providing insights into their degradation mechanisms, and can potentially be utilized to assign proteinaceous origin to a fossil sample of unknown affinity.
... The specific types of ions detected via this process vary depending upon specimen chemical makeup; this allows the unique histological structures of a specimen to be targeted so that chemical makeup can be connected to morphology (Sodhi, 2004;Thiel & Sjövall, 2011;Touboul & Brunelle, 2016). A few studies have employed ToF-SIMS to analyze ancient specimens (Lindgren et al., 2012(Lindgren et al., , 2015(Lindgren et al., , 2017(Lindgren et al., , 2018McNamara et al., 2016;Orlando et al., 2013;Schweitzer, Suo, et al., 2007;Surmik et al., 2016). One recent publication used the method to analyze the biomolecular histology of demineralized epidermis from an exceptionally preserved Jurassic ichthyosaur (Lindgren et al., 2018). ...
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Researchers' ability to accurately screen fossil and subfossil 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 nominal use for specimens of similar depositional environments. Cell and tissue biomolecular histology is thus proposed as a novel proxy for determining sequence preservation potential of ancient specimens with improved accuracy. Biomolecular histology as a proxy is hypothesized to elucidate why fossils/subfossils of some depositional environments preserve sequences while others do not and to facilitate selection of ancient specimens for use in molecular studies. Researchers' ability to accurately screen fossil and subfossil specimens for preservation of DNA and protein sequences remains limited. Cell and tissue biomolecular histology is thus proposed as a novel proxy for determining sequence preservation potential of ancient specimens with improved accuracy. Biomolecular histology as a proxy is hypothesized to elucidate why fossils/subfossils of some depositional environments preserve sequences while others do not and to facilitate selection of ancient specimens for use in molecular studies.
... Material detected in dinosaur fossils, notably Tyrannosaurus rex (MOR 1125, 66 Ma), Brachylophosaurus canadensis (MOR 2598, 80 Ma), and Lufengosaurus (CXPM Z4644, 195 Ma), is very suggestive of collagen I based on multiple methods of analysis. Analysis of the amino acid sequences suggests that there is a strong evolutionary relationship between dinosaurs and both avian and crocodilian species [31][32][33][34]. More specifically, amino acid sequencing of fragments of collagen α1(I) and α2(I) from T. rex has demonstrated a strong evolutionary relationship to modern Gallus gallus and Struthio camelus (the common chicken and the common ostrich, respectively) with a 0.90 confidence in Bayesian analysis in the generated phylogenetic tree [35]. ...
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The multidisciplinary fields of tissue engineering and regenerative medicine have the potential to revolutionize the practise of medicine through the abilities to repair, regenerate, or replace tissues and organs with functional engineered constructs. To this end, tissue engineering combines scaffolding materials with cells and biologically active molecules into constructs with the appropriate structures and properties for tissue/organ regeneration, where scaffolding materials and biomolecules are the keys to mimic the native extracellular matrix (ECM). For this, one emerging way is to decellularize the native ECM into the materials suitable for, directly or in combination with other materials, creating functional constructs. Over the past decade, decellularized ECM (or dECM) has greatly facilitated the advance of tissue engineering and regenerative medicine, while being challenged in many ways. This article reviews the recent development of dECM for tissue engineering and regenerative medicine, with a focus on the preparation of dECM along with its influence on cell culture, the modification of dECM for use as a scaffolding material, and the novel techniques and emerging trends in processing dECM into functional constructs. We highlight the success of dECM and constructs in the in vitro, in vivo, and clinical applications and further identify the key issues and challenges involved, along with a discussion of future research directions.
Dinosaurs have attracted varying degrees of scientific and public interest since their initial description in 1824. Interest has steadily increased, however, since the late 1960s when the Dinosaur Renaissance began, and when the Canadian Journal of Earth Sciences started to publish. Since then, there has been a feedback system (international in scope) promoting increased scientific activity and ever-increasing public attention. This has led to ever more dinosaur discoveries internationally; increased numbers of museums and parks displaying dinosaurs; more publications, blogs, and other media on dinosaurs; and (most importantly) increased numbers of people and institutions doing research on dinosaurs. About 30 new species of dinosaurs are now being described every year, adding to the more than 1000 species already known. Furthermore, it is now acknowledged by most biologists and palaeontologists that modern birds are the direct descendants of dinosaurs, and that they are classified as part of the Dinosauria. Recognizing that there are more than 11 000 species of living dinosaurs has given us a better understanding of many aspects of the biology of nonavian dinosaurs. Along with technological improvements, this has revealed new—and often surprising—facts about their anatomy (bones, soft tissues, and even colours), interrelationships, biomechanics, growth and variation, ecology, physiology, behaviour, and extinction. In spite of the intensity of research over the last six decades, there is no indication that the discovery of new species and new facts about their biology is slowing down. It is quite clear that there is still a lot to be learned!
Knowledge of evolutionary history is based extensively on relatively rare fossils that preserve soft tissues. These fossils record a much greater proportion of anatomy than would be known solely from mineralized remains and provide key data for testing evolutionary hypotheses in deep time. Ironically, however, exceptionally preserved fossils are often among the most contentious because they are difficult to interpret. This is because their morphology has invariably been affected by the processes of decay and diagenesis, meaning that it is often difficult to distinguish preserved biology from artifacts introduced by these processes. Here we describe how a range of analytical techniques can be used to tease apart mineralization that preserves biological structures from unrelated geological mineralization phases. This approach involves using a series of X-ray, ion, electron and laser beam techniques to characterize the texture and chemistry of the different phases so that they can be differentiated in material that is difficult to interpret. This approach is demonstrated using a case study of its application to the study of fossils from the Ediacaran Doushantuo Biota.
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 molecular histology is thus proposed as a proxy for determining sequence preservation potential of ancient specimens with improved accuracy. Molecular 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.
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The differing patterns of molecular abundances in organisms are fundamental to the understanding of the biomolecular palaeontological record. All organisms contain DNA, RNA, protein, polysaccharides and lipid components, together with glycolipids, lipopolysaccharides and other complex molecules. Certain biopolymers, however, are restricted in their distributions; for example, lignin, cutin and sporopollenin are found only in terrestrial plants. The detailed chemical structures, namely the bond types present and their precise intramolecular environments, determine resistance to degradation. Observations of biomolecular preservation are compared with predictions based on chemical structure and on conditions encountered during decay.
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▪ Abstract The term bone refers to a family of materials, all of which are built up of mineralized collagen fibrils. They have highly complex structures, described in terms of up to 7 hierarchical levels of organization. These materials have evolved to fulfill a variety of mechanical functions, for which the structures are presumably fine-tuned. Matching structure to function is a challenge. Here we review the structure-mechanical relations at each of the hierarchical levels of organization, highlighting wherever possible both underlying strategies and gaps in our knowledge. The insights gained from the study of these fascinating materials are not only important biologically, but may well provide novel ideas that can be applied to the design of synthetic materials.
Amino acid analyses of undecalcified samples of fossil crocodile and rhinoceros enamel and dentin from mature teeth revealed that the total protein content of these mineralized fossil tissues varied from ~0.01–0.007% by weight. Except in one instance, amino acid analyses of the enamel proteins revealed them to be free of collagen and to have an amino acid composition similar to the proteins obtained from the enamel of mature modern vertebrates. Molecular sieving of the acid soluble enamel proteins demonstrated that the components consisted principally of small peptides and free amino acids, as in the enamel of modern vertebrates. Based on the presence of hydroxyproline (hyp) and hydroxylysine (hyl), collagen was detected in undecalcified mature dentin of fossil rhinoceros, but not in undecalcified crocodile dentin. It was only by sequential extraction procedures that the presence of collagen in the dentin of fossil crocodile was established, emphasizing the utility and importance of analyzing the soluble components in specific extracts of the fossil. Based on these data and the concentrations of hyp and hyl in the material solubilized by the various solvents, the dentin of fossil rhinoceros contained considerably more collagen per weight and as a percentage of the total protein in the dentin than did the dentin of fossil crocodile. As with modern dentin, EDTA and dilute acid solubilize the noncollagenous proteins and peptides found in fossil enamel and dentin, some of which contain O-phosphoserine [Ser(P)], an amino acid unique to mineralized connective tissues. Similar to recent reported findings from fossil bone, less of the original content of the noncollagenous proteins, including those phosphorylated, is degraded and removed from the enamel and dentin during fossilization than the percentage of dentinal collagen and the nonphosphorylated domains of the enamel proteins which are removed. This selective resistance to degradation and removal of the noncollagenous proteins, including phosphoproteins, may reflect the strong interaction of these proteins with the mineral phase of fossilized tissues. The amount and close packing of the inorganic crystals may also inhibit the interaction of the proteins in the interior of the enamel and dentin with the geochemical environment.
Histological examination of a growth series of humeri of Lapparentosaurus demonstrates the bone-tissue changes occurring during growth. Those changes are quantified by computerized image analysis. The speed of primary bone deposition was very high during early life then tapered somewhat towards still high values during later ontogeny, with a somewhat cyclical pattern of deposition suggesting yearly cycles of growth. Details of epiphyseal structures show precise adaptations to active longitudinal growth, even at large body size, and to the support of heavy strains among adults. Adult condition may have been reached at an age of two decades but ultimate longevity could not be assessed. On the other hand, observation of demineralized sections with the TEM has shown clear evidence of well preserved collagen fibrils in bone, and probably also in cartilage. Overall, the data are accordant with the current interpretation of Sauropod biology, namely one of land dwelling tetrapods with an active, indefinite growth and specialized thermo-metabolical physiology. -from English summary
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.
Type I collagen is the major protein in bones. The mineral matrix protects collagen from denaturation, thus permitting the recovery of large collagen peptides from fossil bones thousands or millions of years old. Collagen peptides are more or less denatured in fossil bones, with diagenetic alteration being the major cause of such denaturation. Classical extraction methods alter the remaining large collagen peptides by extensive solubilization. A method is described here that used shorter collagen solubilization times. Resulting collagen yields are favourably compared with classical methods. The size of the large peptide (>10kDa) fraction improves considerably. Combined with a particular concentration step, the use of this shorter solubilization technique should be useful for collagen analyses that necessitate large peptides, as in the case of palaeoimmunology.
After a synthesis on some recent data on the biology of bone tissues senso lato, this paper focuses on the limits of known variability of bone regarding its amount of mineralisation (maximal and minimal) and on the underlying control mechanisms. Two models of hypermineralised bones are reviewed among cetaceans: the rostrum of the ‘beaked whale’ Mesoplodon and the tympanic bulla of the dolphin Delphinus. In the first case, hypermineralisation is reached lately through an ultrastructural specialization of the secondary osteons. In the second case, hypermineralisation starts at once, during foetal life, through a specialization of the primary osteons. In both cases, ultrastructural peculiarities of collagen fibrillogenesis are demonstrated. Diameter and spacing of the fibrils leave an exceptional empty space available for mineral deposition. Conversely, hypomineralisation is observed on the model of the Teleost scale. The basal plate of the scale is made of a regular ‘biological plywood’ of densely packed collageneous fibres. The mineral is laid down mostly in the interfibrillary spaces. All such tissue modulations seem to be rigorously controlled by details of the osteoblast biosynthetic activities. In a given situation, osteoblasts may express only a subset of molecules from their potential complete biosynthetic repertoire. The debate now centres on how osteoblasts acquire positional information to express this subset. To cite this article: L. Zylberberg, C. R. Palevol 3 (2004).
The portion of organic matrix in bones that is present within fused aggregates of hydroxyapatite crystals was isolated by oxidizing the rest of the bone organic matter with sodium hypochlorite. The aggregate organic matter from a variety of modern and prehistoric bones was subjected to elemental and stable C and N isotopic analysis. For comparison, collagen from modern bones and the fraction from prehistoric bones with the same solubility characteristics as collagen (referred to herein as "collagen") were subjected to the same analytical procedures. Collagen and aggregate organic matter in modern bones have similar 15 N values but dissimilar 13 C values. The difference may be caused by the presence of non-collagenous proteins (NCPs) in the aggregate organic matter, because the NCPs have 13 C values different from those of collagen from the same bone. The organic matter in aggregates is not subject to the same diagenetic processes that can alter the isotope ratios of collagen, and appears to retain an in vivo isotope signal even in cases in which that of "collagen" has been altered. These conclusions apply to samples that were burned prehistorically as well as to those that suffered postmortem alteration only in the depositional environment. The organic matter in aggregates represents a new substrate that should prove useful for stable isotopic studies and possibly for radiocarbon and other biogeochemical analyses of bone.
Authenticity is all in research on ancient DNA. Experience has taught us that even the most exciting claims of the retrieval of ancient DNA are not worth much if they cannot be independently reproduced. Hence the importance of a paper on page 490of this issue, in which Ovchinnikov et al.1 describe the extraction, amplification and sequencing of DNA from 29,000-year-old archaeological bone material of a Neanderthal recovered from the Mezmaiskaya Cave in the northern Caucasus (see map on page 490). This is the second time that such a claim has been made, the first being in 1997 (ref. 2). The paper by Ovchinnikov et al. is probably the more important of the two, for it provides invaluable corroboration for the authenticity of Neanderthal DNA sequences. Moreover, sequences of the DNA from a second Neanderthal offer more detailed insight into the contentious evolutionary relationship between Neanderthals and modern humans.
Bone mineral solubility is an important parameter for understanding the preservation of bones in the archaeological and palaeontological records. In this study we have measured the solubility of the carbonated hydroxyl apatite of sub-recent and fossil bones, as well as synthetic hydroxyl apatite in deionized water and in pH-buffered solutions. The solutions were open to the atmosphere and the pH values were around neutral; measurement conditions that are relevant to bone mineral preservation in nature, but that were absent from most previous studies. We obtained internally consistent results from both the water and the buffered experiments supporting the notion that we are measuring an inherent property of the mineral phase. We found that bone mineral is much more soluble than synthetic hydroxyl apatite. We measured the ionic activity products at “steady state” conditions and we identify a recrystallization window between pH 7.6 and 8.1, which defines the conditions under which bone crystals dissolve and reprecipitate as a more insoluble form of carbonated hydroxyl apatite. As these conditions are common in nature, most fossil bones will not maintain their original crystals with time. We also found that bones that contained small amounts of calcite did not dissolve at all during our experiments. These results provide a basis for better understanding the conditions in sediments under which bones are preserved and the relative states of preservation of bone. They also have important implications for the selection of the most appropriate bone samples for paleoenvironmental and paleodiet analyses and dating.