Publications by authors named "Danilo Gomes Moriel"

14 Publications

  • Page 1 of 1

Rational Design of a Glycoconjugate Vaccine against Group A .

Int J Mol Sci 2020 Nov 13;21(22). Epub 2020 Nov 13.

GSK Vaccines Institute for Global Health (GVGH), Via Fiorentina 1, 53100 Siena, Italy.

No commercial vaccine is yet available against Group A (GAS), major cause of pharyngitis and impetigo, with a high frequency of serious sequelae in low- and middle-income countries. Group A Carbohydrate (GAC), conjugated to an appropriate carrier protein, has been proposed as an attractive vaccine candidate. Here, we explored the possibility to use GAS Streptolysin O (SLO), SpyCEP and SpyAD protein antigens with dual role of antigen and carrier, to enhance the efficacy of the final vaccine and reduce its complexity. All protein antigens resulted good carrier for GAC, inducing similar anti-GAC IgG response to the more traditional CRM conjugate in mice. However, conjugation to the polysaccharide had a negative impact on the anti-protein responses, especially in terms of functionality as evaluated by an IL-8 cleavage assay for SpyCEP and a hemolysis assay for SLO. After selecting CRM as carrier, optimal conditions for its conjugation to GAC were identified through a Design of Experiment approach, improving process robustness and yield This work supports the development of a vaccine against GAS and shows how novel statistical tools and recent advancements in the field of conjugation can lead to improved design of glycoconjugate vaccines.
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http://dx.doi.org/10.3390/ijms21228558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696035PMC
November 2020

Structure, dynamics and immunogenicity of a catalytically inactive CC chemokine-degrading protease SpyCEP from .

Comput Struct Biotechnol J 2020 13;18:650-660. Epub 2020 Mar 13.

Department of Life Sciences, Imperial College London, South Kensington Campus, SW7 2AZ, UK.

Over 18 million disease cases and half a million deaths worldwide are estimated to be caused annually by Group A Streptococcus. A vaccine to prevent GAS disease is urgently needed. SpyCEP (Streptococcus Cell-Envelope Proteinase) is a surface-exposed serine protease that inactivates chemokines, impairing neutrophil recruitment and bacterial clearance, and has shown promising immunogenicity in preclinical models. Although SpyCEP structure has been partially characterized, a more complete and higher resolution understanding of its antigenic features would be desirable prior to large scale manufacturing. To address these gaps and facilitate development of this globally important vaccine, we performed immunogenicity studies with a safety-engineered SpyCEP mutant, and comprehensively characterized its structure by combining X-ray crystallography, NMR spectroscopy and molecular dynamics simulations. We found that the catalytically-inactive SpyCEP antigen conferred protection similar to wild-type SpyCEP in a mouse infection model. Further, a new higher-resolution crystal structure of the inactive SpyCEP mutant provided new insights into this large chemokine protease comprising nine domains derived from two non-covalently linked fragments. NMR spectroscopy and molecular simulation analyses revealed conformational flexibility that is likely important for optimal substrate recognition and overall function. These combined immunogenicity and structural data demonstrate that the full-length SpyCEP inactive mutant is a strong candidate human vaccine antigen. These findings show how a multi-disciplinary study was used to overcome obstacles in the development of a GAS vaccine, an approach applicable to other future vaccine programs. Moreover, the information provided may also facilitate the structure-based discovery of small-molecule therapeutics targeting SpyCEP protease inhibition.
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http://dx.doi.org/10.1016/j.csbj.2020.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113628PMC
March 2020

The Intimin-Like Protein FdeC Is Regulated by H-NS and Temperature in Enterohemorrhagic Escherichia coli.

Appl Environ Microbiol 2014 Dec 19;80(23):7337-47. Epub 2014 Sep 19.

Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Queensland, Australia

Enterohemorrhagic Escherichia coli (EHEC) is a Shiga-toxigenic pathogen capable of inducing severe forms of enteritis (e.g., hemorrhagic colitis) and extraintestinal sequelae (e.g., hemolytic-uremic syndrome). The molecular basis of colonization of human and animal hosts by EHEC is not yet completely understood, and an improved understanding of EHEC mucosal adherence may lead to the development of interventions that could disrupt host colonization. FdeC, also referred to by its IHE3034 locus tag ECOK1_0290, is an intimin-like protein that was recently shown to contribute to kidney colonization in a mouse urinary tract infection model. The expression of FdeC is tightly regulated in vitro, and FdeC shows promise as a vaccine candidate against extraintestinal E. coli strains. In this study, we characterized the prevalence, regulation, and function of fdeC in EHEC. We showed that the fdeC gene is conserved in both O157 and non-O157 EHEC and encodes a protein that is expressed at the cell surface and promotes biofilm formation under continuous-flow conditions in a recombinant E. coli strain background. We also identified culture conditions under which FdeC is expressed and showed that minor alterations of these conditions, such as changes in temperature, can significantly alter the level of FdeC expression. Additionally, we demonstrated that the transcription of the fdeC gene is repressed by the global regulator H-NS. Taken together, our data suggest a role for FdeC in EHEC when it grows at temperatures above 37°C, a condition relevant to its specialized niche at the rectoanal junctions of cattle.
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http://dx.doi.org/10.1128/AEM.02114-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249167PMC
December 2014

Global dissemination of a multidrug resistant Escherichia coli clone.

Proc Natl Acad Sci U S A 2014 Apr 31;111(15):5694-9. Epub 2014 Mar 31.

Australian Infectious Diseases Research Centre, and School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia.

Escherichia coli sequence type 131 (ST131) is a globally disseminated, multidrug resistant (MDR) clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with several factors, including resistance to fluoroquinolones, high virulence gene content, the possession of the type 1 fimbriae FimH30 allele, and the production of the CTX-M-15 extended spectrum β-lactamase (ESBL). Here, we used genome sequencing to examine the molecular epidemiology of a collection of E. coli ST131 strains isolated from six distinct geographical locations across the world spanning 2000-2011. The global phylogeny of E. coli ST131, determined from whole-genome sequence data, revealed a single lineage of E. coli ST131 distinct from other extraintestinal E. coli strains within the B2 phylogroup. Three closely related E. coli ST131 sublineages were identified, with little association to geographic origin. The majority of single-nucleotide variants associated with each of the sublineages were due to recombination in regions adjacent to mobile genetic elements (MGEs). The most prevalent sublineage of ST131 strains was characterized by fluoroquinolone resistance, and a distinct virulence factor and MGE profile. Four different variants of the CTX-M ESBL-resistance gene were identified in our ST131 strains, with acquisition of CTX-M-15 representing a defining feature of a discrete but geographically dispersed ST131 sublineage. This study confirms the global dispersal of a single E. coli ST131 clone and demonstrates the role of MGEs and recombination in the evolution of this important MDR pathogen.
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http://dx.doi.org/10.1073/pnas.1322678111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3992628PMC
April 2014

The serum resistome of a globally disseminated multidrug resistant uropathogenic Escherichia coli clone.

PLoS Genet 2013 3;9(10):e1003834. Epub 2013 Oct 3.

Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland, Australia ; School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia.

Escherichia coli ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with antibiotic resistance; however, this phenotype alone is unlikely to explain its dominance amongst multidrug resistant uropathogens circulating worldwide in hospitals and the community. Thus, a greater understanding of the molecular mechanisms that underpin the fitness of E. coli ST131 is required. In this study, we employed hyper-saturated transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genes required for in vitro growth and the serum resistome (i.e. genes required for resistance to human serum) of E. coli EC958, a representative of the predominant E. coli ST131 clonal lineage. We identified 315 essential genes in E. coli EC958, 231 (73%) of which were also essential in E. coli K-12. The serum resistome comprised 56 genes, the majority of which encode membrane proteins or factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis confirmed a role in serum resistance for 46 (82%) of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, were most strongly associated with serum resistance. While LPS was the main resistance mechanism defined for E. coli EC958 in serum, the enterobacterial common antigen and colanic acid also impacted on this phenotype. Our analysis also identified a novel function for two genes, hyxA and hyxR, as minor regulators of O-antigen chain length. This study offers novel insight into the genetic make-up of E. coli ST131, and provides a framework for future research on E. coli and other Gram-negative pathogens to define their essential gene repertoire and to dissect the molecular mechanisms that enable them to survive in the bloodstream and cause disease.
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http://dx.doi.org/10.1371/journal.pgen.1003834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789825PMC
March 2014

EsiB, a novel pathogenic Escherichia coli secretory immunoglobulin A-binding protein impairing neutrophil activation.

mBio 2013 Jul 23;4(4). Epub 2013 Jul 23.

Novartis Vaccines and Diagnostics Srl, Siena, Italy.

Unlabelled: In this study, we have characterized the functional properties of a novel Escherichia coli antigen named EsiB (E. coli secretory immunoglobulin A-binding protein), recently reported to protect mice from sepsis. Gene distribution analysis of a panel of 267 strains representative of different E. coli pathotypes revealed that esiB is preferentially associated with extraintestinal strains, while the gene is rarely found in either intestinal or nonpathogenic strains. These findings were supported by the presence of anti-EsiB antibodies in the sera of patients affected by urinary tract infections (UTIs). By solving its crystal structure, we observed that EsiB adopts a superhelical fold composed of Sel1-like repeats (SLRs), a feature often associated with bacterial proteins possessing immunomodulatory functions. Indeed, we found that EsiB interacts with secretory immunoglobulin A (SIgA) through a specific motif identified by an immunocapturing approach. Functional assays showed that EsiB binding to SIgA is likely to interfere with productive FcαRI signaling, by inhibiting both SIgA-induced neutrophil chemotaxis and respiratory burst. Indeed, EsiB hampers SIgA-mediated signaling events by reducing the phosphorylation status of key signal-transducer cytosolic proteins, including mitogen-activated kinases. We propose that the interference with such immune events could contribute to the capacity of the bacterium to avoid clearance by neutrophils, as well as reducing the recruitment of immune cells to the infection site.

Importance: Pathogenic Escherichia coli infections have recently been exacerbated by increasing antibiotic resistance and the number of recurrent contagions. Attempts to develop preventive strategies against E. coli have not been successful, mainly due to the large antigenic and genetic variability of virulence factors, but also due to the complexity of the mechanisms used by the pathogen to evade the immune system. In this work, we elucidated the function of a recently discovered protective antigen, named EsiB, and described its capacity to interact with secretory immunoglobulin A (SIgA) and impair effector functions. This work unravels a novel strategy used by E. coli to subvert the host immune response and avoid neutrophil-dependent clearance.
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http://dx.doi.org/10.1128/mBio.00206-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3735183PMC
July 2013

Functional heterogeneity of the UpaH autotransporter protein from uropathogenic Escherichia coli.

J Bacteriol 2012 Nov 17;194(21):5769-82. Epub 2012 Aug 17.

Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia.

Uropathogenic Escherichia coli (UPEC) is responsible for the majority of urinary tract infections (UTI). To cause a UTI, UPEC must adhere to the epithelial cells of the urinary tract and overcome the shear flow forces of urine. This function is mediated primarily by fimbrial adhesins, which mediate specific attachment to host cell receptors. Another group of adhesins that contributes to UPEC-mediated UTI is autotransporter (AT) proteins. AT proteins possess a range of virulence properties, such as adherence, aggregation, invasion, and biofilm formation. One recently characterized AT protein of UPEC is UpaH, a large adhesin-involved-in-diffuse-adherence (AIDA-I)-type AT protein that contributes to biofilm formation and bladder colonization. In this study we characterized a series of naturally occurring variants of UpaH. We demonstrate that extensive sequence variation exists within the passenger-encoding domain of UpaH variants from different UPEC strains. This sequence variation is associated with functional heterogeneity with respect to the ability of UpaH to mediate biofilm formation. In contrast, all of the UpaH variants examined retained a conserved ability to mediate binding to extracellular matrix (ECM) proteins. Bioinformatic analysis of the UpaH passenger domain identified a conserved region (UpaH(CR)) and a hydrophobic region (UpaH(HR)). Deletion of these domains reduced biofilm formation but not the binding to ECM proteins. Despite variation in the upaH sequence, the transcription of upaH was repressed by a conserved mechanism involving the global regulator H-NS, and mutation of the hns gene relieved this repression. Overall, our findings shed new light on the regulation and functions of the UpaH AT protein.
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http://dx.doi.org/10.1128/JB.01264-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486098PMC
November 2012

Uropathogenic Escherichia coli mediated urinary tract infection.

Curr Drug Targets 2012 Oct;13(11):1386-99

Australian Infectiuos Diseases Research Centre, School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane QLD 4072, Australia.

Urinary tract infection (UTI) is among the most common infectious diseases of humans and is the most common nosocomial infection in the developed world. They cause significant morbidity and mortality, with approximately 150 million cases globally per year. It is estimated that 40-50% of women and 5% of men will develop a UTI in their lifetime, and UTI accounts for more than 1 million hospitalizations and $1.6 billion in medical expenses each year in the USA. Uropathogenic E. coli (UPEC) is the primary cause of UTI. This review presents an overview of the primary virulence factors of UPEC, the major host responses to infection of the urinary tract, the emergence of specific multidrug resistant clones of UPEC, antibiotic treatment options for UPEC-mediated UTI and the current state of vaccine strategies as well as other novel anti-adhesive and prophylactic approaches to prevent UTI. New and emerging themes in UPEC research are also discussed in the context of future outlooks.
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http://dx.doi.org/10.2174/138945012803530206DOI Listing
October 2012

FdeC, a novel broadly conserved Escherichia coli adhesin eliciting protection against urinary tract infections.

mBio 2012 10;3(2). Epub 2012 Apr 10.

Novartis Vaccines and Diagnostics Srl, Siena, Italy.

Unlabelled: The increasing antibiotic resistance of pathogenic Escherichia coli species and the absence of a pan-protective vaccine pose major health concerns. We recently identified, by subtractive reverse vaccinology, nine Escherichia coli antigens that protect mice from sepsis. In this study, we characterized one of them, ECOK1_0290, named FdeC (factor adherence E. coli) for its ability to mediate E. coli adhesion to mammalian cells and extracellular matrix. This adhesive propensity was consistent with the X-ray structure of one of the FdeC domains that shows a striking structural homology to Yersinia pseudotuberculosis invasin and enteropathogenic E. coli intimin. Confocal imaging analysis revealed that expression of FdeC on the bacterial surface is triggered by interaction of E. coli with host cells. This phenotype was also observed in bladder tissue sections derived from mice infected with an extraintestinal strain. Indeed, we observed that FdeC contributes to colonization of the bladder and kidney, with the wild-type strain outcompeting the fdeC mutant in cochallenge experiments. Finally, intranasal mucosal immunization with recombinant FdeC significantly reduced kidney colonization in mice challenged transurethrally with uropathogenic E. coli, supporting a role for FdeC in urinary tract infections.

Importance: Pathogenic Escherichia coli strains are involved in a diverse spectrum of diseases, including intestinal and extraintestinal infections (urinary tract infections and sepsis). The absence of a broadly protective vaccine against all these E. coli strains is a major problem for modern society due to high costs to health care systems. Here, we describe the structural and functional properties of a recently reported protective antigen, named FdeC, and elucidated its putative role during extraintestinal pathogenic E. coli infection by using both in vitro and in vivo infection models. The conservation of FdeC among strains of different E. coli pathotypes highlights its potential as a component of a broadly protective vaccine against extraintestinal and intestinal E. coli infections.
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http://dx.doi.org/10.1128/mBio.00010-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3324786PMC
June 2012

Genome-based vaccine development: a short cut for the future.

Hum Vaccin 2008 May-Jun;4(3):184-8. Epub 2010 May 15.

Novartis Vaccines, Siena, Italy.

Bacterial infectious diseases remain a major cause of deaths and disabilities in the world. Although conventional vaccinology approaches were successful in conferring protection against several diseases, they failed in providing efficient vaccines against many others. Together to the sequencing of the first genome, a new chapter in the vaccinology history started to be written. Reverse vaccinology changed the way to think about vaccine development, using the information provided by the microorganisms' genome against themselves. Since then, reverse vaccinology has evolved and helped researchers to overcome the limits of the conventional vaccinology approaches and led to the discovery and development of novel vaccines concerning emerging diseases, like Neisseria meningitidis B and Streptococcus agalactiae. A lot of work must be done, but deciphering the information provided by genome sequences and using it to better understand the host-pathogen interactions has proved to be the key for protection.
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http://dx.doi.org/10.4161/hv.4.3.6313DOI Listing
November 2010

Identification of protective and broadly conserved vaccine antigens from the genome of extraintestinal pathogenic Escherichia coli.

Proc Natl Acad Sci U S A 2010 May 3;107(20):9072-7. Epub 2010 May 3.

Novartis Vaccines and Diagnostics, 53100 Siena, Italy.

Extraintestinal pathogenic Escherichia coli (ExPEC) are a common cause of disease in both mammals and birds. A vaccine to prevent such infections would be desirable given the increasing antibiotic resistance of these bacteria. We have determined the genome sequence of ExPEC IHE3034 (ST95) isolated from a case of neonatal meningitis and compared this to available genome sequences of other ExPEC strains and a few nonpathogenic E. coli. We found 19 genomic islands present in the genome of IHE3034, which are absent in the nonpathogenic E. coli isolates. By using subtractive reverse vaccinology we identified 230 antigens present in ExPEC but absent (or present with low similarity) in nonpathogenic strains. Nine antigens were protective in a mouse challenge model. Some of them were also present in other pathogenic non-ExPEC strains, suggesting that a broadly protective E. coli vaccine may be possible. The gene encoding the most protective antigen was detected in most of the E. coli isolates, highly conserved in sequence and found to be exported by a type II secretion system which seems to be nonfunctional in nonpathogenic strains.
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http://dx.doi.org/10.1073/pnas.0915077107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2889118PMC
May 2010

Towards a vaccine against Escherichia coli-associated urinary tract infections.

Future Microbiol 2010 Mar;5(3):351-4

Novartis Vaccines & Diagnostics, Research Centre, Via Fiorentina 1, 53100 Siena, Italy.

Evaluation of: Alteri CJ, Hagan EC, Sivick KE, Smith SN, Mobley HLT: Mucosal immunization with iron receptor antigens protects against urinary tract infections. PLoS Pathog. 5(9), E1000586 (2009). Urinary tract infection is one of the most common infections in humans. The eradication of uropathogenic Escherichia coli-mediated urinary tract infections has still not been achieved and no effective licensed vaccines are currently available. To overcome the limitations of previous approaches in developing an efficacious vaccine, Alteri et al., through a functional genomic approach, identified six novel vaccine candidates shown to be protective against urinary tract infection in a mouse model. The six proteins all belong to the class of outer membrane iron receptors, are upregulated in iron-restricted conditions and were demonstrated to induce, upon mucosal vaccination, antigen-specific antibodies and cytokine responses, which correlated with protection in a mouse model of urinary tract infection. Therefore, for the first time, antigens that were previously recognized as necessary for bacterial pathogenesis, being involved in iron acquisition in an iron-limited environment such as the urinary tract, are now proposed as potential candidates for the development of a vaccine against uropathogenic strain-associated urinary tract infections.
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http://dx.doi.org/10.2217/fmb.10.6DOI Listing
March 2010

Genome-based vaccine development: a short cut for the future.

Adv Exp Med Biol 2009 ;655:81-9

Novartis Vaccines, Via Fiorentina 1, 53100, Siena, Italy.

Bacterial infectious diseases remain a major cause of deaths and disabilities in the world. Although conventional vaccinology approaches were successful in conferring protection against several diseases, they failed in providing efficient vaccines against many others. Together to the sequencing of the first genome, a new chapter in the vaccinology history started to be written. Reverse vaccinology changed the way to think about vaccine development, using the information provided by the microorganisms' genome against themselves. Since then, reverse vaccinology has evolved and helped researchers to overcome the limits of the conventional vaccinology approaches and led to the discovery and development of novel vaccines concerning emerging diseases, like Neisseria meningitidis B and Streptococcus agalactiae. A lot of work must be done, but deciphering the information provided by genome sequences and using it to better understand the host-pathogen interactions has proved to be the key for protection.
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http://dx.doi.org/10.1007/978-1-4419-1132-2_8DOI Listing
August 2010

Proteomics characterization of outer membrane vesicles from the extraintestinal pathogenic Escherichia coli DeltatolR IHE3034 mutant.

Mol Cell Proteomics 2008 Mar 2;7(3):473-85. Epub 2007 Nov 2.

Biochemistry and Molecular Biology Unit, Novartis Vaccines and Diagnostics, 53100 Siena, Italy.

Extraintestinal pathogenic Escherichia coli are the cause of a diverse spectrum of invasive infections in humans and animals, leading to urinary tract infections, meningitis, or septicemia. In this study, we focused our attention on the identification of the outer membrane proteins of the pathogen in consideration of their important biological role and of their use as potential targets for prophylactic and therapeutic interventions. To this aim, we generated a DeltatolR mutant of the pathogenic IHE3034 strain that spontaneously released a large quantity of outer membrane vesicles in the culture supernatant. The vesicles were analyzed by two-dimensional electrophoresis coupled to mass spectrometry. The analysis led to the identification of 100 proteins, most of which are localized to the outer membrane and periplasmic compartments. Interestingly based on the genome sequences available in the current public database, seven of the identified proteins appear to be specific for pathogenic E. coli and enteric bacteria and therefore are potential targets for vaccine and drug development. Finally we demonstrated that the cytolethal distending toxin, a toxin exclusively produced by pathogenic bacteria, is released in association with the vesicles, supporting the recently proposed role of bacterial vesicles in toxin delivery to host cells. Overall, our data demonstrated that outer membrane vesicles represent an ideal tool to study Gram-negative periplasm and outer membrane compartments and to shed light on new mechanisms of bacterial pathogenesis.
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http://dx.doi.org/10.1074/mcp.M700295-MCP200DOI Listing
March 2008