Huge opportunities exist in overseas markets for SMEs in pharma sector: Pharmexcil

Peethaambaran Kunnathoor, Chennai Saturday, June 11, 2011, 08:00 Hrs [IST]

Huge potential exists for the pharmaceutical manufacturing units in the SME sector to venture into overseas markets and they can avail of various export incentive schemes offered the Union government. The government is extending financial assistances to the aspiring exporters through export promotion councils for the pharma industry under MDA and MAI schemes, said Abhay Kumar Sinha, assistant regional director, Pharmexcil, New Delhi. He was delivering a lecture in a seminar organised for the manufacturers of West Bengal by Pharmaceutical Export Promotion Council in Kolkata last week. The objectives of the Market Development Assistance (MDA) Scheme is basically to assist the SMEs to promote exports of their products and to explore new markets. The companies which intend to market their products abroad have to follow certain norms and procedures. Those who apply for MDA shall not be under investigation or charged for any offence or prosecuted for any violation or debarred due to malpractice or black listed under the Foreign Trade Policy of India or under any other law relating to export and import business. The maximum MDA assistance is inclusive of assistances received from all Government bodies, EPCs, FIEO, ITPO etc, he said. Exporting companies with an FOB value of exports of up to Rs.15 crore in the preceding year are eligible for MDA scheme. The member exporters of EPCs would also be eligible for MDA assistance for participation in events organized by ITPO abroad. Their applications or claims can be routed or reimbursed through the concerned EPC. A maximum of 3 participations in a particular trade fair or exhibition would be entertained for the assistance, he told the manufacturers present there. There is other assistance available for participation through Council sponsored activities. This is permissible to one regular employee or director or partner or proprietor of the company. Exporter of foreign nationality or holding foreign passport is not eligible. Applications for this assistance are received in any of the offices of Pharmexcil with a minimum of 14 days advance notice excluding the date of receipt of application and the date of departure from the Country. Whereas, the Market Access Initiatives (MAI) Scheme, Abhay Kumar said, is an export promotion scheme envisaged to act as a catalyst to promote India’s export on a sustained basis. This scheme is formulated on ‘focus product – focus country’ approach to evolve specific market and specific product through market studies and surveys. Through this scheme assistance is provided to EPCs or TPOs for enhancement of export through accessing new markets or through increasing the share in the existing markets. He explained that Indian domestic pharmaceutical market size was estimated at US$ 12.2 billion (Rs.55,454 crore) in the year 2008. The country’s healthcare expenditure during the period was estimated at US$ 68.6 billion. He said that medicine purchases forms 17.78 per cent share of health care expenditure and 0.8 per cent in GDP. He said that India is the 15th largest exporter of bulk drugs & formulations in value terms with a share of 1.44 per cent during 2008. The country ranked 14th in value terms and 3rd in volume terms. It is the 9th largest exporter of APIs/Bulk Drugs and the 8th in terms of export volumes. Exports estimated for the year 2009-10 were US$ 8.88 billion. Pharmaceutical exports occupied a share of around 5 per cent in India’s total exports over the last 5 years, he said. With regard to rising export opportunities, India has very good scope. He said in the case of Generic Opportunity, 450 molecules worth US$ 350 billion are going off patent in the coming years. The contract manufacturing opportunity of prescription drugs is estimated to increase from a value of $26.2 billion to $43.9 billion. in the next 5 years. Besides, there are many important categories such as monoclonal antibodies, peptides, biotech, etc, which are not filed by India, but cater global opportunity of approximately US$ 90 billion. Several technologies such as sustained release/controlled /modified release lyophilized pharmaceuticals, bio-pharmaceuticals, specialty generics, drug Intermediates, NIP based APIs/Formulations, regulatory compliant infrastructure, are not attempted in India. There are more than 285 drugs in this category with an opportunity of US$ 50 billion. Further, in the drug research value chain, there exists an opportunity to capture the market share in global clinical R&D market such as clinical trials, data management, testing, etc. By 2012, nearly half of all newly approved prescription drugs will be biologics. By the end of 2010, patents will expire on brand biologics with $15 billion in annual sales. The Indian manufacturers, especially those from the SME sector, can utilize all these opportunities with the help of Pharmexcil, Aabhay Kumar told the manufacturers of West Bengal. He said with the aim to promote pharma exports, Pharmexcil provides several services to the Indian pharmaceutical companies such as dissemination of trade enquiries, organizing trade delegations from India to overseas markets and also reverse delegations, organizing buyers-sellers meets, organizing participation in international trade fairs, providing policy inputs to the government of India on India’s bilateral trade in pharmaceuticals, dissemination of India’s pharmaceutical trade statistics, preparing technical publications, exporters’ directory, resolving grievances of pharmaceutical importers and exporters, organizing national & international seminars on areas related to pharmaceutical industry and certificate of origin.

Proteins & Peptides Soon, alternative drugs to battle antibiotic-immune pathogens!

India Soon, alternative drugs to battle antibiotic-immune pathogens! Washington | June 09, 2011 12:01:13 AM IST Krishna announces a slew of benefits for Haj pilgrims The indiscriminate use of antibiotics has allowed an increasing number of pathogens to develop immunity to penicillin and other antibiotics. The threat is evident from the World Health Organization warning that if measures are not taken quickly, it may soon not be possible to treat many frequently occurring infections. But research scientists at the Fraunhofer Institute for Cell Therapy and Immunology IZI say they've found an alternative in the nick of time - antimicrobial peptides. "We have already identified 20 of these short chains of amino acids which kill numerous microbes, including enterococci, yeasts and molds, as well as human pathogenic bacteria such as Streptococcus mutans, which is found in the human oral cavity and causes tooth decay," said Dr. Andreas Schubert, group manager at Fraunhofer IZI.. Even the multi-resistant hospital bug Staphylococcus aureus is not immune, and in our tests its growth was considerably inhibited," Schubert, said. From familiar fungicidal and bactericidal peptides the research scientists produced sequence variations and tested them in vitro on various microbes. They then compared the survivability of the pathogens with an untreated control. Antibiotic peptides unblock their microbicidal effect within a few minutes, stated Schubert, and work at a concentration of less than 1uM. Conventional antibiotics require a concentration of 10uM. "The spectrum of efficacy of the tested peptides includes not only bacteria and molds but also lipid-enveloped viruses," Schubert added. "Another key factor is that the peptides identified in our tests do not harm healthy body cells." (ANI)

Peptides & Proteins ~ Antibiotic Quotes

Antibiotic Quotes

It's no one's fault, and there's not much that could be done. That's the hardest thing for family members and friends to see. It makes you feel like, 'Could my child die tomorrow?' We're not used to that in the United States. [Meningitis] is not common, but it's one of the few things remaining that takes relatively healthy people.SOURCE: SOUTH FLORIDA SUN-SENTINEL 2011-06-14 00:01:00

Most of these people who die just seem to be completely fine and then extremely ill the next day.SOURCE: SOUTH FLORIDA SUN-SENTINEL 2011-06-14 00:01:00

Henry will truly be missed by those who had the honor to know him or to even meet him.SOURCE: MIAMI HERALD 2011-06-13 23:39:00

It seems impossible that I will not see your beautiful smiling face again.SOURCE: MIAMI HERALD 2011-06-13 23:39:00

The community support has been absolutely amazing.SOURCE: MIAMI HERALD 2011-06-13 23:39:00

It is a sporadic disease that happens from time to time.SOURCE: MIAMI HERALD 2011-06-13 23:39:00

Everyone did everything they could..SOURCE: MIAMI HERALD 2011-06-13 23:39:00

He was an extremely happy, healthy, active 7-year-old ... We have lost a very special boy..SOURCE: MIAMI HERALD 2011-06-13 23:39:00

Perhaps the most disgraceful aspect of our agricultural system — I say this as an Oregon farmboy who once raised sheep, cattle and hogs — is the way antibiotics are recklessly stuffed into healthy animals to make them grow faster..SOURCE: MONDAY MORNING 2011-06-13 21:47:00

A series of simple fecal microbe tests (as reliable as current standard tests) that do not require local people administering them to have advanced training in environmental microbiology or water quality analysis..SOURCE: MONDAY MORNING 2011-06-13 21:47:00

Flooding associated with natural disasters can cause widespread fecal contamination of both surface and groundwater supplies. This contamination poses microbial risks to human health, as waterborne disease outbreaks have been linked to fecal contamination from flood waters….SOURCE: MONDAY MORNING 2011-06-13 21:47:00

The single state of North Carolina uses more antibiotics for livestock than the entire United States uses for humans..SOURCE: MONDAY MORNING 2011-06-13 21:47:00

The Food and Drug Administration reported recently that 80 percent of antibiotics in the United States go to livestock, not humans. And 90 percent of the livestock antibiotics are administered in their food or water, typically to healthy animals to keep them from getting sick when they are confined in squalid and crowded conditions..SOURCE: MONDAY MORNING 2011-06-13 21:47:00

Perhaps the most disgraceful aspect of our agricultural system — I say this as an Oregon farmboy who once raised sheep, cattle and hogs — is the way antibiotics are recklessly stuffed into healthy animals to make them grow faster..SOURCE: MONDAY MORNING 2011-06-13 21:47:00

We’re doing everything that we are supposed to be doing.SOURCE: ORANGEVILLE.COM 2011-06-13 21:23:00

We’re certainly meeting with physicians and talking about antibiotics..SOURCE: ORANGEVILLE.COM 2011-06-13 21:23:00

We have increased vigilance around hand hygiene, and we are increasing cleaning of the rooms.SOURCE: ORANGEVILLE.COM 2011-06-13 21:23:00

There are certain antibiotics that can cause C. difficile ... This isn’t unique to Headwaters, it happens at other hospitals. People do not have to be concerned at all..SOURCE: ORANGEVILLE.COM 2011-06-13 21:23:00

This is not due to faulty testing. This is an outbreak ... Any test now that is not strongly positive we would send off to Sunnybrook..SOURCE: ORANGEVILLE.COM 2011-06-13 21:23:00

The community does not need to be concerned about coming to Headwaters for a scheduled test, procedure or emergency visit ... Good hand hygiene practices by patients, visitors and hospital staff remain the best protection in preventing the spread of C. difficile or introducing it into the hospital from the community..SOURCE: ORANGEVILLE.COM 2011-06-13 21:23:00

Tell your doctor right away if you experience unexplained muscle pain, tenderness or weakness.SOURCE: DESERET NEWS 2011-06-13 17:29:00

Muscle problems, including muscle breakdown, can be serious in some people and rarely cause kidney damage that can lead to death..SOURCE: DESERET NEWS 2011-06-13 17:29:00

There are countless bacteria out there capable of mutating into equally dangerous counterparts to the EHEC bacterium. For example, 94 per cent of the chicken in our supermarkets is infected with ESBL, which is also resistant to antibiotics..SOURCE: IRISH TIMES 2011-06-12 23:35:00

It has already been tested using the ESBL bacterium, but it is also applicable against the EHEC bacterium..SOURCE: IRISH TIMES 2011-06-12 23:35:00

It appears to be a valuable alternative to antibiotics, and can achieve the same effects as antibiotics.SOURCE: IRISH TIMES 2011-06-12 23:35:00

The doctors said they were talking about a matter of hours or a day or two before risking sepsis and both of them might die ... Obviously, if it was a choice of whether both Karen and the child are going to die or just the child is going to die, I mean it’s a pretty easy call..SOURCE: CROOKS AND LIARS 2011-06-12 20:27:00

intends to convene a task force within the United Nations system, to study the findings and recommendations made by the Independent Panel of Experts to ensure prompt and appropriate follow-up.... On behalf of the UN family, the Secretary-General reiterates his deepest sympathies to the victims of the epidemic and their loved ones..SOURCE: INNER CITY PRESS 2011-06-11 19:55:00

We need to broaden the fight against bacteria to include a better understanding of how they cause disease; the discovery of new classes of antibiotics; strategies to slow the growth of antibiotic resistance; faster diagnosis of infection and better ways to screen food..SOURCE: BIG THINK 2011-06-11 13:18:00

I think our people got on it pretty early and took care of it. So hopefully we got it early enough for it not to be worse than what it could be..SOURCE: MSNBC SPORTS 2011-06-10 23:29:00

On Tuesday he's getting treated for a ... raspberry and then just that quickly that thing turned from just being a raspberry to being a pretty serious infection.SOURCE: MSNBC SPORTS 2011-06-10 23:29:00

Peptides & Proteins ~ Nanoparticle therapeutics might help people suffering from hearing disorders

Nanoparticle therapeutics might help people suffering from hearing disorders

Abstract: For millions of people hearing disorders make a negative impact on their lives. Scientists are looking into new ways of treating hearing disorders, by using different sorts of nanoparticles as original inner ear delivery devices. Their hope is that nanoparticles will be able to deliver drugs that can improve or restore hearing. Nanoparticle therapeutics might help people suffering from hearing disorders Italy | Posted on June 13th, 2011 Due to different anatomic and physiological barriers it is hard to treat hearing disorders by using conventional systemic drug delivery. Therefore scientists are investigating different ways to locally apply drugs using nanoparticles. Researchers at Guangdong Pharmaceutical University in China highlight that several nanoparticles have a combination of properties, such as target specificity, stability in vivo, biocompatibility and capacity to incorporate the encapsulated drugs into the cell, making it possible to use smaller doses and reduce the side effects of therapeutic agents. They anticipate that nanoparticles will play an extremely important role in the development of drug delivery systems, which can find the desired target sites and release the drugs in a controlled way within the cell. In the European Commission-funded project NANOEAR, scientists from several countries in Europe are studying where different nanoparticles go within the inner ear and if these nanoparticles could be harmful or useful in therapeutics. They are currently testing eight nanoparticle classes, for example, biodegradable liposomes, micelles and lipid-core nanocapsules, regarding their delivery of genes, peptides, corticosteroids, siRNA and shRNA. Since nanoparticles also have properties that separate them, the researchers need to find the best carrier for each drug. The project‚s researchers are working on nanoparticles targeted towards hair cells, neurons and supporting cells. They have identified fundamental peptides helping the nanoparticles to avoid being trapped and dysfunctionalized by the lysosomal enzymes in the cell‚s endosome system, so the nanoparticles are able to deliver the therapeutic drugs to their targets. Nanoparticles can be modified with coatings to escape immune responses. Three of NANOEAR‚s partners are focusing on safety and they have not observed any adverse reactions. Within the consortium, researchers at the Medical University of Innsbruck in Austria studied nanoparticles conjugated with a neurotrophin-derived peptide ligand. They found that these nanoparticles selectively targeted the mouse inner ear cells and can potentially be used as inner ear drug delivery devices. Many research projects are in the early stage development. There will be rigorous controls and take years until patients may be helped by the innovations. However, if the researchers manage to make these nanoparticles do exactly what they are made to do, nanoparticle therapeutics will radically change the way hearing disorders are treated. #### For more information, please click here Contacts: Elisabeth Schmid Phone: +39 02 70002 572 Fax: +39 02 70002 540 E-mail: elisabeth.schmid@youris.com

Peptides Proteins ~Dinosaur Peptides Suggest Mechanisms of Protein Survival

Dinosaur Peptides Suggest Mechanisms of Protein Survival

James D. San Antonio^1*, Mary H. Schweitzer^2,3,4, Shane T. Jensen⁵, Raghu Kalluri^6,7, Michael Buckley^8,9, Joseph P. R. O. Orgel^10*

1 Operations, Orthovita, Inc., Malvern, Pennsylvania, United States of America, 2 Department of Marine, Earth and Atmospheric Sciences, North Carolina State University, Raleigh, North Carolina, United States of America,3 North Carolina Museum of Natural Sciences, Raleigh, North Carolina, United States of America, 4 Museum of the Rockies, Montana State University, Bozeman, Montana, United States of America, 5 Department of Statistics, The Wharton School, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 6 Division of Matrix Biology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, United States of America, 7 Department of Biological Chemistry and Molecular Pharmacology, Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology, Boston, Massachusetts, United States of America, 8 Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom, 9 Department of Archaeology, The University of York, York, United Kingdom, 10 Department of Biology, Pritzker Institute of Biomedical Science and Engineering, Illinois Institute of Technology, Chicago, Illinois, United States of America

Abstract Top

Eleven collagen peptide sequences recovered from chemical extracts of dinosaur bones were mapped onto molecular models of the vertebrate collagen fibril derived from extant taxa. The dinosaur peptides localized to fibril regions protected by the close packing of collagen molecules, and contained few acidic amino acids. Four peptides mapped to collagen regions crucial for cell-collagen interactions and tissue development. Dinosaur peptides were not represented in more exposed parts of the collagen fibril or regions mediating intermolecular cross-linking. Thus functionally significant regions of collagen fibrils that are physically shielded within the fibril may be preferentially preserved in fossils. These results show empirically that structure-function relationships at the molecular level could contribute to selective preservation in fossilized vertebrate remains across geological time, suggest a ‘preservation motif’, and bolster current concepts linking collagen structure to biological function. This non-random distribution supports the hypothesis that the peptides are produced by the extinct organisms and suggests a chemical mechanism for survival.

Citation: San Antonio JD, Schweitzer MH, Jensen ST, Kalluri R, Buckley M, et al. (2011) Dinosaur Peptides Suggest Mechanisms of Protein Survival. PLoS ONE 6(6): e20381. doi:10.1371/journal.pone.0020381

Editor: Hendrik W. van Veen, University of Cambridge, United Kingdom

Received: February 2, 2011; Accepted: May 1, 2011; Published: June 8, 2011

Copyright: © 2011 San Antonio et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This research was funded by NIH RR 08630 and an NSF Career Award 0644015 to JPROO; NIH DK 55001 grant to RK; and grants from NSF and The David and Lucile Packard Foundation to MHS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The lead author is an employee of private industry, Orthovita, Inc. However, this is an academic study that the company is allowing to be researched and published, and with which they have no financial or proprietary interest. The last author, J. Orgel, is a Biochemistry Section Editor of PLOS ONE. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

* E-mail: jsanantonio@orthovita.com (JDSA); orgel@iit.edu (JPROO)

Introduction Top

While it is widely accepted that proteins have the potential to survive significantly longer periods of time than DNA [1], persistence of original bone proteins in fossils at least 68 million years old is controversial [2], [3], despite multiple lines of evidence supporting this hypothesis [4], [5], [6], [7], [8],[9]. Current temporal limits for survival of original biomaterials [10], [11] are based upon theoretical kinetics and laboratory experiments designed to simulate protein diagenesis through exposure to harsh conditions (e.g. low pH and high temperature [10], [12]) and predict complete degradation of measurable biomolecules in well under a million years if degradation proceeds at simulated rates. Modeled degradation of DNA [13] places temporal limits of ~100,000 years (at a constant 10°C), whereas models of protein degradation (e.g. [1], [14]) extend this to a few million years (at a constant 10°C). However, these predictions have been surpassed (e.g. [15]), supporting the suggestion that current models may not be appropriate, in part because they do not consider the molecules in their native state (i.e., folded, closely-packed, cross-linked or, in the case of bone, stabilized by association with the mineral phase [16]). Recovery of what appear to be cells, blood vessels and tissues from multiple fossils from varying ages and depositional settings [4], and protein sequence data from two dinosaurs [5], [6], [7], [9], also suggests that these models may be incomplete. Examining endogenous biomolecules other than DNA avoids synthetic amplification and reduces contamination issues that significantly impeded early ancient DNA research. Technological improvements in recent years, including soft ionization mass spectrometry, allow increased detection of minute traces of biomolecules that may persist for extended periods of time via crystal encapsulation [17], [18], even in the presence of exogenous contamination that precluded earlier forms of analysis such as amino acid composition analyses and stable isotope analyses [13].

The possibility of using information contained in ancient molecules to address contemporary questions of basic biology and ecology is intriguing, and has unexpected potential beyond paleontology. For example, identifying the elements of the collagen fibril most resistant to degradation in fossils may lead to the rational design of collagenous scaffolds with enhanced in vivo longevities to support tendon or bone regeneration in humans. Similarly, identifying naturally occurring modifications on these molecules that contribute to preservation may also shed light on molecular-based disease processes. We show here that molecular preservation is linked to protein function, and discuss how sequences of ancient peptides can test models of molecular function in extant organisms. In addition, we show how models of extant protein function suggest a mechanism for the survival of proteins in exceptionally well preserved fossils.

Results and Discussion Top

Type I collagen peptides were extracted and sequenced from ~ 68 million years old fossils ofTyrannosaurus rex (Museum of the Rockies [MOR] 1125) [5], [7], (Fig. 1). However, despite multiple lines of evidence to support the presence of collagen, including in situ antibody binding, the endogeneity of MOR 1125 peptides was disputed, and the sequences instead were suggested to arise from either microbial invasion [19], extant collagens introduced in laboratory experiments [2], or even statistical artifact [3]. Collagen peptide sequences were subsequently derived from a second dinosaur, Brachylophosauraus canadensis (MOR 2598) [9], and included many of the earlier lines of supporting evidence as well as independent replication of data in multiple labs.

Figure 1. Tyrannosaurus rex femur (MOR 1125) from which demineralized matrix (insets; bars, 20 µm) and peptides were obtained.

Courtesy Museum of the Rockies.

doi:10.1371/journal.pone.0020381.g001

Surprisingly, advances in collagen biology also support the authenticity of the fossil peptides. The molecular structure of collagen favors preservation. The triple-helical arrangement and intra- and intermolecular cross-links confer stability upon this ubiquitous structural molecule [20], [21], [22], [23],[24], [25]. Additionally, when collagen is surrounded by or adsorbed to mineral surfaces, as in bone, its preservation potential is greatly enhanced (e.g. [18], [26], [27], [28], [29], [30]). In fibrillar collagens, individual triple-helical molecules aggregate, forming a fibril with a characteristic 67 nm banding pattern that is readily recognized by electron microscopy (Fig. 2) [31], [32]. Within each 67 nm wide D-period, segments of neighboring molecules are referred to as monomers 1–5 (Fig. 2), and specific functional regions have been mapped to each monomer using a variety of experimental approaches [33], [34], [35].

Figure 2. The collagen fibril (A) is composed of triple-helical monomers that polymerize in an overlapping fashion (B), and are derived from proteolysis of the soluble procollagen precursor (C).

Fibrils appear as periodic banded structures by electron microscopy; one D-period (expanded two-dimensional view of 67 nm segment of microfibril, box) contains the complete collagen sequence from elements of five monomers and includes an overlap and gap zone; arrow, left border of overlap zone. Image of the X-ray diffraction-derived fibril subunit structure: the microfibril (D) shows aggregates of five triple-helical, rope-like monomers; magnified view shows triple helix containing three peptide chains (two α1 and one α2 chains) (E). Many thousands of microfibrils polymerize and cross-link to form cable-like collagen fibrils of vertebrates. Modified from original research [33].

doi:10.1371/journal.pone.0020381.g002

The stability and unique function conferred by the triple-helical structure of collagen has been known for over forty years, but just how molecules assemble into microfibrils to form the massive cable-like fibrils in tissues has been less well understood. However, recent advances in technology have allowed molecular resolution images of type I collagen microfibrils and fibrils [35], [36]. This new information, coupled with non-random distribution of collagen functional sequences and mutations [33], has led to the formation of a testable model linking structure to function in this massive protein assemblage. Discrete cell- and matrix- interaction domains have been identified, and collagen-binding ligands that cooperatively carry out fibril functions have been recognized.

We reasoned that particular functional molecular regions may contribute to their preferential resistance to biological degradation throughout the lifetime of an individual organism. This property not only needs to remain highly conserved through species but also may render those regions resistant to degradation in the burial environment. Thus, molecular models for differential functions of collagen fibril domains or sequences may provide a chemical or structural rationale for preservation. We mapped eleven fossil-derived peptide sequences from two dinosaurs, Tyrannosaurus rex andBrachylophosauraus canadensis [7], [9], [37] on molecular models of extant human and rat collagens[33], [34] (Table 1, Figs. 3 and 4). These peptides represent eight sequences which localize to seven regions of the monomer, and comprise less than fifteen percent of the length of the collagen triple helix. They were non-randomly distributed in several respects (Fig. 3 and Statistical Analyses [seeMaterials and Methods]). In particular, fossil sequences mapped to regions of the protein partly shielded by tight molecular packing (Fig 4) [34], which may physically stabilize and protect them from enzymatic degradation, thus contributing to their preservation. Comparing the amino acid compositions of fossil peptides with sequences of the entire human protein for predicted properties such as hydrophobicity, polarity and charge revealed that most fossil peptides were from regions of collagen which contain relatively few acidic residues [38], and eight of the peptides (five sequences) lacked such residues altogether, which would limit their solubility and propensity for proteolytic degradation (Table 1). Also, five peptides mapped to a uniquely hydrophobic fibril region [39]. The results imply that the most stable regions of the protein are those with a more hydrophobic, less acidic nature. That the more exposed, charged regions of collagen with high densities of trypsin cleavage sites yielded fewer fossil peptides suggested their susceptibility to proteolysis in early diagenesis, and supports non-random degradation and preservation patterns for the diverse type I collagen sequence set in fossil bone. It is also interesting to note that perhaps the least stable region, the hydroxyproline deficient thermally-labile domain located towards the C-terminal end of the molecule[40], is not represented by any of the fossil peptides.

Figure 3. Dinosaur peptide sequence positions were mapped on the two dimensional human collagen fibril D-period schematic³³.

doi:10.1371/journal.pone.0020381.g003

Figure 4. X-ray diffraction model of the rat collagen microfibril in situ; Integrins, predominant cell-binding site; MMP, matrix metalloproteinase cleavage site; FN, fibronectin binding site; decoron, decorin proteoglycan core protein binding sites; putative cell and matrix interaction domains³⁵.

doi:10.1371/journal.pone.0020381.g004

Table 1. Number of Acidic Residues.

doi:10.1371/journal.pone.0020381.t001

All fossil-derived peptides mapped to monomers 2, 3, and 4 on the extant collagen models. The remaining monomers, 1 and 5, are joined across microfibrillar layers by intermolecular cross-links that, while stabilizing the molecule and protecting from enzymatic attack, may also hinder peptide extraction. In fact, the only position where alpha 1 chain peptides (Peptides 3 and 8) co-localize with an alpha 2 chain peptide (Peptide 11) mapped to the integrin binding site that promotes cell-collagen interactions, angiogenesis, and osteoblast differentiation; its fibril location and association with severe mutations also suggest its crucial nature [33] and hence strong selective pressure for conservation of sequence. One peptide (Peptide 4) mapped to the Matrix Metalloproteinase-1 (MMP-1) cleavage domain crucial for collagen remodeling, and a site for fibronectin binding. In living tissues, the integrin binding site and MMP-1 cleavage/fibronectin binding sequences are somewhat buried under the surface of the collagen fibril, thus fibril proteolysis or injury may be needed to render them available for cell-collagen interactions and tissue regeneration [35]. The molecularly “sheltered” environment required to protect crucial biological function may also account for enhanced survival of those protein regions in fossils. Although the majority of the dinosaur peptides are from highly conserved regions of the molecule, both of the alpha 2 chain peptides are highly variable [41], [42]. That they are not exclusively from sequences with a high similarity to residues in public databases, suggests that the peptides were not identified solely because they derive from highly conserved sequences; thus, the gaps in our model are not simply due to the lack of peptide identification due to divergence from known organisms. Additional preservation potential may be conferred by association with biomineral, especially if some regions of the collagen molecule are more intimately associated with mineral than others. Conversely, the absence of peptide matches elsewhere in the molecule may be due to lack of response to trypsin resulting from unusual post-mortem modifications which may also confer resistance to proteolytic degradation and contribute to preservation over time [20]. Additional collagen sequences may have survived over time, but because of chemical modification or lack of representation in current databases, may not have been recognized by existing search algorithms and therefore not identified in original analyses.

Our results add to the evidence provided by sequence data [5], [7], [9], [37], molecular phylogenetic analyses [8], [9], microstructure [4], [6], [9], [43] and immunoreactivity to anti-collagen antibodies [6],[9], [43], that supports persistence of elements of native collagen fibril structure across geological time in some fossils. Most of the peptide sequences aligned perpendicularly with one or more other sequences on the fibril model, implying that neighboring triple-helical segments, or fragments thereof, may have been preserved en bloc. If supported by further peptide recovery and mapping, this observation would validate current models of collagen monomer arrangement in the fibril [35], [44].

Mapping the distribution of fossil collagen peptides observed using mass spectrometry to models of collagen function demonstrates that preservation of fossil-derived collagen sequences concurs with current concepts of collagen biology, and provides a molecular mechanism for the preservation of this protein in fossil bone. Moreover, these findings support the endogeneous source and longevity of fossil-derived peptides, because peptides arising from recent contamination are expected to be more concentrated and random in distribution. They would not be expected to be over-represented in regions that so well reflect collagen fibril structure/function relationships in native vertebrate tissue[33], [34].

Finally, by showing that functionally crucial protein regions are more stable than others over geologic time, we provide insight into selective pressures constraining the molecular structure, function, and hence sequence, of collagen. Paleoproteomics therefore not only holds significant promise for elucidating evolutionary relationships between extinct and extant organisms, but is potentially useful for enhancing our understanding of protein function in living animals. Also, elucidating molecular functions of extant proteins may help predict proteins or protein regions most likely to preserve in fossils, as has also been shown for the highly-conserved and structurally sheltered mineral-binding mid-region of the bone protein osteocalcin [45]. As technologies continue to improve in both sensitivity and resolution, the recovery of additional protein sequences from fossils will be enhanced. The understanding of preferential preservation driven by molecular function may be used to adapt search algorithms to optimize studies of ancient molecules recovered from multiple extinct taxa. The recovery of additional sequences, allowed by these advances, may shed further light on the biology of extracellular matrix superstructures of living organisms.

Materials and Methods Top

Peptide sequences

Eleven peptides representing eight sequences recovered from the bones of Tyrannosaurus rex (MOR 1125) and Brachylophosauraus canadensis (MOR 2598 were obtained from previous publications [5],[6], [7], [9].

Peptide mapping on collagen models

Human microfibril.

The two dimensional expanded schematic of the human collagen fibril D-period used here was as presented previously [33]. Positions of select binding sites and functional domains from the D-period ligand binding and mutation map [33] are indicated by symbols placed next to the relevant sequences on the schematic, and the positions of dinosaur peptide sequences were mapped to homologous human sequences according to their linear distance from the N-terminus of the collagen triple helix.

Rat microfibril.

The three dimensional collagen microfibril model used in this study was composed from the packing structure of rat tendon type I collagen molecules in situ [35]–[36]. This molecular model was constructed based on the primary sequences of the α1 and α2 chains of rat collagen, and the superhelical parameters were established from crystallographic structure determinations of collagen-like peptides constrained within the lower resolution fiber diffraction molecular envelope [35]. To map the position of the dinosaur peptide sequences on the three-dimensional rat microfibril, solvent-accessible surface calculation and rendering was performed using SPOCK [46] with the default probe size of 0.14 nm to compose a molecular outline. The Cα “worm” traces of relevant portions of individual triple helices were marked (see Fig. 4 for color key) to indicate the positions of peptide sequences from either Tyrannosaurus rex or Brachylophosauraus canadensis, or both (where they co-localized on the collagen molecule). The significant homology between vertebrate collagen protein sequences justifies the approach of localizing functional domains of human type I collagen on the rat type I collagen microfibril.

Statistical Analysis of Peptide Distributions on Collagen

We show the alignment of the eleven dinosaur peptides with homologous sequences on the human collagen map (Fig. 3). By visual inspection, the peptide locations appear to be non-random in several ways. For example, there appears to be co-localization between peptides from the two species on the collagen monomer at three positions. The most interesting finding is that at one of these positions, the alpha 1 chain peptide also co-localizes with its matching alpha 2 chain peptide which occurs at the integrin binding site. Also, all peptides map to Monomers 2, 3, and 4, but not to Monomers 1 and 5. We evaluated the statistical significance of these and other seemingly non-random features through their comparison to a null hypothesis of completely random alignment of the peptides to the collagen map. The null distribution of random alignment was calculated via simulation: a large number (m = 100,000) of simulated maps were generated where the eleven peptides were randomly placed. Each map was generated by sampling eleven random numbers from a discrete uniform distribution (with replacement) among all possible map locations. The uniqueness of a given feature of the peptide alignment to the collagen map was evaluated by calculating the proportion of random maps sharing that feature. We refer to this proportion as the randomization p-value, and deem features with an exceedingly small p-value to be significant (i.e. very few random maps share that feature). We calculated the randomization p-value for nine features of the peptide alignment to the human collagen map. In calculating our threshold for declaring significance, we must account for the fact that we are performing multiple tests (for nine different features). We use the conservative Bonferroni correction to determine our significance threshold, which divides the nominal significance level of 0.05 by the number of tests performed. Thus, our p-value threshold for declaring significance was 0.05/9 = 0.0056. As detailed below, two of the nine features were found to be significantly non-random by this criterion and seven were found to not be significant:

Significant Features

Significant Feature #1.

Localization to the integrin (cell) binding site: p-value = 0.0024

Details: Three of eleven peptides (two unique sequences) were observed to overlap with the integrin binding site of the fibril which we define as comprising residues 502–510.

Significant Feature #2.

Co-localization between the two species: p-value = 0.0034

Details: Three pairs of peptides (three unique sequences) from the two species co-localized on the collagen monomer.

Non-Significant Features

Non-Significant Feature #1.

Overlap zone vs. gap zone: p-value = 0.022

Details: Ten of eleven peptides (seven unique sequences) localized to the overlap zone.

Non-Significant Feature #2.

Cell interaction domain: p-value = 0.212

Details: Three of eleven peptides (two unique sequences) localized to the cell interaction domain.

Non-Significant Feature #3.

Monomers 2, 3, and 4: p-value = 0.016

Details: All peptides (eight unique sequences) mapped to monomers 2, 3, and 4, and none to monomers 1 and 5.

Non-Significant Feature #4.

Co-localization of peptides: p-value = 0.036

Details: Four of the eleven peptides (four unique sequences) did not overlap with any other peptides.

Non-Significant Feature #5.

Overlap with cross-links: p-value = 0.097

Details: Five of the eleven peptides (three unique sequences) overlapped with the intermolecular cross-links.

Non-Significant Feature #6.

Overlap with any functional domain: p-value = 0.014

Details: Eight out of eleven peptides (five unique sequences) co-localized with at least one of the following functional domains: the central integrin binding site; MMP-1-cleavage site; decoron ligation sequences; and overlapping of the intermolecular crosslinks, or aligning with them across the fibril.

Non-Significant Feature #7.

Overlap with the master control region: p-value = 0.018

Details: Ten of eleven peptides (seven unique sequences) occupied the master control region, a fibril zone where most of the collagen fibrils crucial functional sequences are located.

Acknowledgments Top

We gratefully acknowledge Jack Horner for access to dinosaur specimens, Wenxia Zheng for sample preparation and John Asara and Chris Organ for production and analyses of sequences published previously.

Author Contributions Top

Conceived and designed the experiments: JSA MS JO SJ. Performed the experiments: JSA MS JO SJ MB. Analyzed the data: JSA MS JO SJ MB RK. Contributed reagents/materials/analysis tools: JSA MS JO SJ MB. Wrote the paper: JSA MS JO SJ MB RK.

References Top

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