Construction of a highly flexible and comprehensive gene collection representing the ORFeome of the human pathogen Chlamydia pneumoniae

Background The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strat...

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Published inBMC genomics Vol. 13; no. 1; p. 632
Main Authors Maier, Christina J, Maier, Richard H, Virok, Dezso Peter, Maass, Matthias, Hintner, Helmut, Bauer, Johann W, Önder, Kamil
Format Journal Article
LanguageEnglish
Published London BioMed Central 16.11.2012
BioMed Central Ltd
BMC
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ISSN1471-2164
1471-2164
DOI10.1186/1471-2164-13-632

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Abstract Background The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis. Results Here we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn ( Chlamydia pneumoniae strain CWL029) in Gateway® ‘entry’ vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway® entry clones (83% coverage) and an additional 168 PCR-verified ‘pooled’ entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in Escherichia coli and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of E. coli harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates. Conclusion This first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host–pathogen analysis. The high yield of recombinant proteins and the promising interactors for Cpn virulence factors described here demonstrate the possibilities for proteome-wide studies.
AbstractList The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis.BACKGROUNDThe Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis.Here we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn (Chlamydia pneumoniae strain CWL029) in Gateway(®) 'entry' vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway(®) entry clones (83% coverage) and an additional 168 PCR-verified 'pooled' entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in Escherichia coli and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of E. coli harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates.RESULTSHere we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn (Chlamydia pneumoniae strain CWL029) in Gateway(®) 'entry' vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway(®) entry clones (83% coverage) and an additional 168 PCR-verified 'pooled' entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in Escherichia coli and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of E. coli harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates.This first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host-pathogen analysis. The high yield of recombinant proteins and the promising interactors for Cpn virulence factors described here demonstrate the possibilities for proteome-wide studies.CONCLUSIONThis first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host-pathogen analysis. The high yield of recombinant proteins and the promising interactors for Cpn virulence factors described here demonstrate the possibilities for proteome-wide studies.
The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis. Here we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn (Chlamydia pneumoniae strain CWL029) in Gateway[R] 'entry' vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway[R] entry clones (83% coverage) and an additional 168 PCR-verified 'pooled' entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in Escherichia coli and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of E. coli harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates. This first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host-pathogen analysis. The high yield of recombinant proteins and the promising interactors for Cpn virulence factors described here demonstrate the possibilities for proteome-wide studies.
Doc number: 632 Abstract Background: The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis. Results: Here we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn (Chlamydia pneumoniae strain CWL029) in Gateway® 'entry' vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway® entry clones (83% coverage) and an additional 168 PCR-verified 'pooled' entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in Escherichia coli and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of E. coli harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates. Conclusion: This first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host-pathogen analysis. The high yield of recombinant proteins and the promising interactors for Cpn virulence factors described here demonstrate the possibilities for proteome-wide studies.
The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis. Here we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn (Chlamydia pneumoniae strain CWL029) in Gateway(®) 'entry' vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway(®) entry clones (83% coverage) and an additional 168 PCR-verified 'pooled' entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in Escherichia coli and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of E. coli harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates. This first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host-pathogen analysis. The high yield of recombinant proteins and the promising interactors for Cpn virulence factors described here demonstrate the possibilities for proteome-wide studies.
Background: The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis. Results: Here we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn (Chlamydia pneumoniae strain CWL029) in Gateway registered 'entry' vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway registered entry clones (83% coverage) and an additional 168 PCR-verified 'pooled' entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in Escherichia coli and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of E. coli harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates. Conclusion: This first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host-pathogen analysis. The high yield of recombinant proteins and the promising interactors for Cpn virulence factors described here demonstrate the possibilities for proteome-wide studies.
Abstract Background The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis. Results Here we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn (Chlamydia pneumoniae strain CWL029) in Gateway® ‘entry’ vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway® entry clones (83% coverage) and an additional 168 PCR-verified ‘pooled’ entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in Escherichia coli and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of E. coli harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates. Conclusion This first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host–pathogen analysis. The high yield of recombinant proteins and the promising interactors for Cpn virulence factors described here demonstrate the possibilities for proteome-wide studies.
Background The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis. Results Here we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn ( Chlamydia pneumoniae strain CWL029) in Gateway® ‘entry’ vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway® entry clones (83% coverage) and an additional 168 PCR-verified ‘pooled’ entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in Escherichia coli and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of E. coli harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates. Conclusion This first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host–pathogen analysis. The high yield of recombinant proteins and the promising interactors for Cpn virulence factors described here demonstrate the possibilities for proteome-wide studies.
Background The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis. Results Here we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn (Chlamydia pneumoniae strain CWL029) in Gateway[R] 'entry' vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway[R] entry clones (83% coverage) and an additional 168 PCR-verified 'pooled' entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in Escherichia coli and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of E. coli harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates. Conclusion This first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host-pathogen analysis. The high yield of recombinant proteins and the promising interactors for Cpn virulence factors described here demonstrate the possibilities for proteome-wide studies. Keywords: ORFeome, Chlamydia pneumoniae, Omics, Pathogen, Systems biology, Infectious diseases
ArticleNumber 632
Audience Academic
Author Maier, Richard H
Maier, Christina J
Virok, Dezso Peter
Maass, Matthias
Bauer, Johann W
Önder, Kamil
Hintner, Helmut
AuthorAffiliation 1 Department of Dermatology, Paracelsus Medical University, Salzburg, Austria
2 Institute of Clinical Microbiology, University of Szeged, Szeged, Hungary
3 Department of Microbiology, Paracelsus Medical University, Salzburg, Austria
AuthorAffiliation_xml – name: 1 Department of Dermatology, Paracelsus Medical University, Salzburg, Austria
– name: 2 Institute of Clinical Microbiology, University of Szeged, Szeged, Hungary
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  surname: Maier
  fullname: Maier, Richard H
  organization: Department of Dermatology, Paracelsus Medical University
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  givenname: Dezso Peter
  surname: Virok
  fullname: Virok, Dezso Peter
  organization: Institute of Clinical Microbiology, University of Szeged
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BackLink https://www.ncbi.nlm.nih.gov/pubmed/23157390$$D View this record in MEDLINE/PubMed
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COPYRIGHT 2012 BioMed Central Ltd.
2012 Maier et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Copyright ©2012 Maier et al.; licensee BioMed Central Ltd. 2012 Maier et al.; licensee BioMed Central Ltd.
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– notice: COPYRIGHT 2012 BioMed Central Ltd.
– notice: 2012 Maier et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Issue 1
Keywords Chlamydia pneumoniae
Infectious diseases
Pathogen
Systems biology
ORFeome
Omics
Language English
License This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Snippet Background The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as...
The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and...
Background The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as...
Doc number: 632 Abstract Background: The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for...
Background: The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as...
Abstract Background The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections...
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StartPage 632
SubjectTerms Animal Genetics and Genomics
Bacterial genetics
Bacterial pneumonia
Biomedical and Life Sciences
Bronchitis
Chlamydia
Chlamydia pneumoniae
Chlamydophila pneumoniae
Chlamydophila pneumoniae - genetics
Chlamydophila pneumoniae - pathogenicity
Cloning, Molecular
Construction
Deoxyribonucleic acid
DNA
DNA sequencing
DNA, Bacterial
Drug development
E coli
Electrophoresis
Electrophoresis, Polyacrylamide Gel
Escherichia coli
Escherichia coli - genetics
Gene Library
Genes
Genetic aspects
Genetic research
Genome, Bacterial
Genomes
Genomics
Health aspects
Humans
Infection
Infectious diseases
Life Sciences
Microarrays
Microbial Genetics and Genomics
Microbiology
Nucleotide sequencing
Omics
Open reading frames
Open Reading Frames - genetics
ORFeome
Pathogen
Pathogens
Physiological aspects
Plant Genetics and Genomics
Plasmids
Pneumonia
Prokaryote microbial genomics
Protein interaction
Proteins
Proteomics
Recombinant proteins
Recombinant Proteins - genetics
Research Article
Sequence Analysis, DNA
Sexually transmitted diseases
STD
Systems biology
Two-Hybrid System Techniques
Vectors
Virulence
virulence factors
Virulence Factors - genetics
Yeasts
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Title Construction of a highly flexible and comprehensive gene collection representing the ORFeome of the human pathogen Chlamydia pneumoniae
URI https://link.springer.com/article/10.1186/1471-2164-13-632
https://www.ncbi.nlm.nih.gov/pubmed/23157390
https://www.proquest.com/docview/1265818232
https://www.proquest.com/docview/1272714048
https://www.proquest.com/docview/1273118461
https://pubmed.ncbi.nlm.nih.gov/PMC3534531
https://doaj.org/article/2c3a6540c65443ba909c10972e9288f1
Volume 13
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