Functional Mutation of Multiple Solvent-Exposed Loops in the Ecballium elaterium Trypsin Inhibitor-II Cystine Knot Miniprotein
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{"title"=>"Functional mutation of multiple solvent-exposed loops in the Ecballium elaterium trypsin inhibitor-II cystine knot miniprotein", "type"=>"journal", "authors"=>[{"first_name"=>"Richard H.", "last_name"=>"Kimura", "scopus_author_id"=>"12143153800"}, {"first_name"=>"Douglas S.", "last_name"=>"Jones", "scopus_author_id"=>"37009935100"}, {"first_name"=>"Lei", "last_name"=>"Jiang", "scopus_author_id"=>"56422845100"}, {"first_name"=>"Zheng", "last_name"=>"Miao", "scopus_author_id"=>"34067706900"}, {"first_name"=>"Zhen", "last_name"=>"Cheng", "scopus_author_id"=>"7401815487"}, {"first_name"=>"Jennifer R.", "last_name"=>"Cochran", "scopus_author_id"=>"7201915214"}], "year"=>2011, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"79951996425", "pmid"=>"21364742", "isbn"=>"1932-6203", "pui"=>"361327597", "issn"=>"19326203", "scopus"=>"2-s2.0-79951996425", "doi"=>"10.1371/journal.pone.0016112"}, "id"=>"059c1b48-f58f-3bdc-bc64-89cab9896ee9", "abstract"=>"BACKGROUND: The Ecballium elaterium trypsin inhibitor (EETI-II), a 28-amino acid member of the knottin family of peptides, contains three interwoven disulfide bonds that form multiple solvent-exposed loops. Previously, the trypsin binding loop of EETI-II has been engineered to confer binding to several alternative molecular targets. Here, EETI-II was further explored as a molecular scaffold for polypeptide engineering by evaluating the ability to mutate two of its structurally adjacent loops.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: Yeast surface display was used to engineer an EETI-II mutant containing two separate integrin binding epitopes. The resulting knottin peptide was comprised of 38 amino acids, and contained 11- and 10-residue loops compared to wild-type EETI-II, which naturally contains 6- and 5-residue loops, respectively. This knottin peptide bound to α(v)β(3) and α(v)β(5) integrins with affinities in the low nanomolar range, but bound weakly to the related integrins α(5)β(1) and α(iib)β(3). In addition, the engineered knottin peptide inhibited tumor cell adhesion to vitronectin, an extracellular matrix protein that binds to α(v)β(3) and α(v)β(5) integrins. A (64)Cu radiolabeled version of this knottin peptide demonstrated moderate serum stability and excellent tumor-to-muscle and tumor-to-blood ratios by positron emission tomography imaging in human tumor xenograft models. Tumor uptake was ∼3-5% injected dose per gram (%ID/g) at one hour post injection, with rapid clearance of probe through the kidneys.\\n\\nCONCLUSIONS/SIGNIFICANCE: We demonstrated that multiple loops of EETI-II can be mutated to bind with high affinity to tumor-associated integrin receptors. The resulting knottin peptide contained 21 (>50%) non-native amino acids within two mutated loops, indicating that extended loop lengths and sequence diversity were well tolerated within the EETI-II scaffold. A radiolabeled version of this knottin peptide showed promise for non-invasive imaging of integrin expression in living subjects. However, reduced serum and metabolic stability were observed compared to an engineered integrin-binding EETI-II knottin peptide containing only one mutated loop.", "link"=>"http://www.mendeley.com/research/functional-mutation-multiple-solventexposed-loops-ecballium-elaterium-trypsin-inhibitorii-cystine-kn", "reader_count"=>32, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>4, "Researcher"=>9, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>9, "Student > Master"=>3, "Other"=>2, "Student > Bachelor"=>2, "Lecturer"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>4, "Researcher"=>9, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>9, "Student > Master"=>3, "Other"=>2, "Student > Bachelor"=>2, "Lecturer"=>1}, "reader_count_by_subject_area"=>{"Engineering"=>5, "Unspecified"=>2, "Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>4, "Agricultural and Biological Sciences"=>10, "Medicine and Dentistry"=>2, "Pharmacology, Toxicology and Pharmaceutical Science"=>1, "Chemistry"=>6, "Psychology"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>5}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Chemistry"=>{"Chemistry"=>6}, "Psychology"=>{"Psychology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>10}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>4}, "Unspecified"=>{"Unspecified"=>2}, "Environmental Science"=>{"Environmental Science"=>1}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}}, "reader_count_by_country"=>{"United States"=>4, "Italy"=>1}, "group_count"=>4}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/797391"], "description"=>"<p>Biodistribution of <sup>64</sup>Cu-DOTA- knottin 3-4A in U87MG xenografts (n = 3) was measured in the tumor (T), blood (Bl), heart (H), liver (Li), lungs (Lu), muscle (M), kidneys (K), spleen (Sp), brain (Br), intestine (I), skin (Sk), stomach (St), pancreas (P), and bone (Bo). Data are presented as the %ID/g tissue ± SD (n = 3) after intravenous injection of ∼50–100 µCi probe at 1 h (black bars), 4 h (light grey bars), and 24 h (dark grey bars). To measure probe specificity, mice were injected with <sup>64</sup>Cu-DOTA- knottin 3-4A and an excess of unlabeled competitor (c(RGDyK)), and tissue biodistribution was measured after 1 h (1 h/block, white bars). Error bars represent standard deviations of experiments performed in three mice.</p>", "links"=>[], "tags"=>["u87mg"], "article_id"=>467757, "categories"=>["Biotechnology", "Biochemistry", "Biophysics"], "users"=>["Richard H. Kimura", "Douglas S. Jones", "Lei Jiang", "Zheng Miao", "Zhen Cheng", "Jennifer R. Cochran"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0016112.g006", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Biodistribution_in_U87MG_tumor_xenografts_/467757", "title"=>"Biodistribution in U87MG tumor xenografts.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-18 02:09:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/796994"], "description"=>"<p>(<b>A</b>) Vitronectin or (<b>B</b>) fibronectin coated strips were incubated with U87MG cells for 2 h with varying concentrations of echistatin (•), knottin 3-4A (⧫), and knottin 3-4C (▪). Adherent cells remaining after several wash steps were quantified with crystal violet staining by absorbance at 600 nm. Values were normalized against uncoated wells and wells containing no competing peptide. Data shown are the average of three replicates performed on different days and error bars represent standard deviations. IC<sub>50</sub> values are summarized in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016112#pone-0016112-t001\" target=\"_blank\">Table 1</a>.</p>", "links"=>[], "tags"=>["integrin-dependent"], "article_id"=>467369, "categories"=>["Biotechnology", "Biochemistry", "Biophysics"], "users"=>["Richard H. Kimura", "Douglas S. Jones", "Lei Jiang", "Zheng Miao", "Zhen Cheng", "Jennifer R. Cochran"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0016112.g003", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Inhibition_of_integrin_dependent_tumor_cell_adhesion_/467369", "title"=>"Inhibition of integrin-dependent tumor cell adhesion.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-18 02:02:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/398612", "https://ndownloader.figshare.com/files/398651", "https://ndownloader.figshare.com/files/398687", "https://ndownloader.figshare.com/files/398724", "https://ndownloader.figshare.com/files/398754", "https://ndownloader.figshare.com/files/398790"], "description"=>"<div><h3>Background</h3><p>The <em>Ecballium elaterium</em> trypsin inhibitor (EETI-II), a 28-amino acid member of the knottin family of peptides, contains three interwoven disulfide bonds that form multiple solvent-exposed loops. Previously, the trypsin binding loop of EETI-II has been engineered to confer binding to several alternative molecular targets. Here, EETI-II was further explored as a molecular scaffold for polypeptide engineering by evaluating the ability to mutate two of its structurally adjacent loops.</p> <h3>Methodology/Principal Findings</h3><p>Yeast surface display was used to engineer an EETI-II mutant containing two separate integrin binding epitopes. The resulting knottin peptide was comprised of 38 amino acids, and contained 11- and 10-residue loops compared to wild-type EETI-II, which naturally contains 6- and 5-residue loops, respectively. This knottin peptide bound to α<sub>v</sub>β<sub>3</sub> and α<sub>v</sub>β<sub>5</sub> integrins with affinities in the low nanomolar range, but bound weakly to the related integrins α<sub>5</sub>β<sub>1</sub> and α<sub>iib</sub>β<sub>3</sub>. In addition, the engineered knottin peptide inhibited tumor cell adhesion to vitronectin, an extracellular matrix protein that binds to α<sub>v</sub>β<sub>3</sub> and α<sub>v</sub>β<sub>5</sub> integrins. A <sup>64</sup>Cu radiolabeled version of this knottin peptide demonstrated moderate serum stability and excellent tumor-to-muscle and tumor-to-blood ratios by positron emission tomography imaging in human tumor xenograft models. Tumor uptake was ∼3–5% injected dose per gram (%ID/g) at one hour post injection, with rapid clearance of probe through the kidneys.</p> <h3>Conclusions/Significance</h3><p>We demonstrated that multiple loops of EETI-II can be mutated to bind with high affinity to tumor-associated integrin receptors. The resulting knottin peptide contained 21 (>50%) non-native amino acids within two mutated loops, indicating that extended loop lengths and sequence diversity were well tolerated within the EETI-II scaffold. A radiolabeled version of this knottin peptide showed promise for non-invasive imaging of integrin expression in living subjects. However, reduced serum and metabolic stability were observed compared to an engineered integrin-binding EETI-II knottin peptide containing only one mutated loop.</p> </div>", "links"=>[], "tags"=>["mutation", "solvent-exposed", "loops", "trypsin", "inhibitor-ii", "cystine", "knot", "miniprotein"], "article_id"=>138741, "categories"=>["Biotechnology", "Biochemistry", "Biophysics"], "users"=>["Richard H. Kimura", "Douglas S. Jones", "Lei Jiang", "Zheng Miao", "Zhen Cheng", "Jennifer R. Cochran"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0016112.s001", "https://dx.doi.org/10.1371/journal.pone.0016112.s002", "https://dx.doi.org/10.1371/journal.pone.0016112.s003", "https://dx.doi.org/10.1371/journal.pone.0016112.s004", "https://dx.doi.org/10.1371/journal.pone.0016112.s005", "https://dx.doi.org/10.1371/journal.pone.0016112.s006"], "stats"=>{"downloads"=>4, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Functional_Mutation_of_Multiple_Solvent_Exposed_Loops_in_the_Ecballium_elaterium_Trypsin_Inhibitor_II_Cystine_Knot_Miniprotein/138741", "title"=>"Functional Mutation of Multiple Solvent-Exposed Loops in the <em>Ecballium elaterium</em> Trypsin Inhibitor-II Cystine Knot Miniprotein", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2011-02-18 02:25:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/796812"], "description"=>"<p>(<b>A</b>) Three dimensional structure of wild-type EETI-II (pbd: 2it7, ref <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016112#pone.0016112-Heitz1\" target=\"_blank\">[13]</a>) showing Loop 1, previously engineered for high affinity α<sub>v</sub>β<sub>3</sub> integrin binding (magenta), Loop 2 (dark blue), and Loop 3 (cyan). Cysteines I to VI are labeled and shown in yellow. (<b>B</b>) Strategy for knottin engineering. The primary sequences of wild-type EETI-II and mutant 2.5D are shown with disulfide connectivities indicated in yellow. The RGD motif in Loop 1 was scrambled to <u>RDG</u> and three different Loop 3 libraries were created and screened for high affinity to α<sub>v</sub>β<sub>3</sub> integrin. After nine rounds of library screening by FACS the predominant mutant, knottin 3-4C, is shown. Three different variants 3-4A: (RGD/RGD), 3-4B: (RGD/<u>RDG</u>), and 3-4C: (<u>RDG</u>/RGD) were synthesized and folded. (<b>C</b>) FACS density dot plots showing the library sort progression for enrichment of improved α<sub>v</sub>β<sub>3</sub> integrin binders. R denotes the sort round, and the concentration of α<sub>v</sub>β<sub>3</sub> integrin is indicated. Actual sort gates are shown as polygons in the upper right quadrant.</p>", "links"=>[], "tags"=>["knottin"], "article_id"=>467182, "categories"=>["Biotechnology", "Biochemistry", "Biophysics"], "users"=>["Richard H. Kimura", "Douglas S. Jones", "Lei Jiang", "Zheng Miao", "Zhen Cheng", "Jennifer R. Cochran"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0016112.g001", "stats"=>{"downloads"=>1, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Summary_of_knottin_engineering_/467182", "title"=>"Summary of knottin engineering.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-18 01:59:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/797516"], "description"=>"<p>Urine samples or homogenized tumor, kidney, or liver tissue were analyzed by radio-HPLC and gamma counting 1 h post injection. Representative HPLC traces are shown. The intact radiotracer elutes at approximately 15 minutes, while the major hydrophilic metabolites and free copper elute with the column flow through. Only limited quantities of urine were available from the mice, resulting in higher background noise for this sample.</p>", "links"=>[], "tags"=>["vivo"], "article_id"=>467883, "categories"=>["Biotechnology", "Biochemistry", "Biophysics"], "users"=>["Richard H. Kimura", "Douglas S. Jones", "Lei Jiang", "Zheng Miao", "Zhen Cheng", "Jennifer R. Cochran"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0016112.g007", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_In_vivo_stability_of_64_Cu_DOTA_knottin_3_4A_/467883", "title"=>"In vivo stability of <sup>64</sup>Cu-DOTA-knottin 3-4A.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-18 02:11:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/797595"], "description"=>"<p>Data are reported in nM unless otherwise stated. n/d indicates not determined.</p>", "links"=>[], "tags"=>["binding", "adhesion"], "article_id"=>467961, "categories"=>["Biotechnology", "Biochemistry", "Biophysics"], "users"=>["Richard H. Kimura", "Douglas S. Jones", "Lei Jiang", "Zheng Miao", "Zhen Cheng", "Jennifer R. Cochran"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0016112.t001", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Summary_of_IC_50_values_from_competition_binding_and_cell_adhesion_assays_/467961", "title"=>"Summary of IC<sub>50</sub> values from competition binding and cell adhesion assays.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2011-02-18 02:12:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/797112"], "description"=>"<p><sup>64</sup>Cu-DOTA-knottin 3-4A was incubated with mouse serum for up to 24 h at 37°C. Representative HPLC traces are shown with radioactive signal (mV) plotted as a function of time. Intact <sup>64</sup>Cu-DOTA-knottin 3-4A (*) elutes at approximately 15 minutes. The amount of probe remaining at 1, 4, and 24 h was quantified from the area under the entire peak to be 96%, 55%, and 30%, respectively.</p>", "links"=>[], "tags"=>["biotechnology", "biophysics", "Biochemistry"], "article_id"=>467477, "categories"=>["Biotechnology", "Biochemistry", "Biophysics"], "users"=>["Richard H. Kimura", "Douglas S. Jones", "Lei Jiang", "Zheng Miao", "Zhen Cheng", "Jennifer R. Cochran"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0016112.g004", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Serum_stability_of_64_Cu_DOTA_knottin_3_4A_/467477", "title"=>"Serum stability of <sup>64</sup>Cu-DOTA-knottin 3-4A.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-18 02:04:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/796889"], "description"=>"<p>Varying concentrations of unlabeled knottin peptides or DOTA-conjugated knottin 3-4A were incubated with <sup>125</sup>I-echistatin and allowed to compete for binding to integrin receptors expressed on the surface of U87MG cells. Percent of <sup>125</sup>I-echistatin bound to the cell surface is plotted versus the concentration of unlabeled knottin (<b>A</b>) 3-4A (⧫), 3-4B (▴), 3-4C (▪), or (<b>B</b>) DOTA-knottin 3-4A (▾) and unlabeled knottin 3-4A (⧫). Echistatin (•) was used as a positive control to compare binding data from different experiments. The binding curves for the knottin peptides did not reach full inhibition because <sup>125</sup>I-echistatin binds with broad specificity to multiple integrins expressed on U87MG cells. Data shown are the average of triplicate values and error bars represent standard deviations. IC<sub>50</sub> values are summarized in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016112#pone-0016112-t001\" target=\"_blank\">Table 1</a>.</p>", "links"=>[], "tags"=>["binding", "integrin", "receptors", "u87mg"], "article_id"=>467251, "categories"=>["Biotechnology", "Biochemistry", "Biophysics"], "users"=>["Richard H. Kimura", "Douglas S. Jones", "Lei Jiang", "Zheng Miao", "Zhen Cheng", "Jennifer R. Cochran"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0016112.g002", "stats"=>{"downloads"=>2, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Competition_binding_to_integrin_receptors_expressed_on_U87MG_cells_/467251", "title"=>"Competition binding to integrin receptors expressed on U87MG cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-18 02:00:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/797264"], "description"=>"<p>(<b>A</b>) Representative microPET scans (coronal images, top; transverse images, bottom) of U87MG xenografts (n = 3) after injection of <sup>64</sup>Cu-DOTA-knottin 3-4A alone or co-injected with a large molar excess of unlabeled c(RGDyK) pentapeptide (1 h/block). The letters B, K, L, and T represent bladder, kidney, liver and tumor, respectively. (<b>B</b>) The mean %ID/g for tumor, liver, kidney, and muscle uptake were quantified on images generated 1, 2, 4, and 24 h post injection. Error bars represent standard deviations of experiments performed in three mice.</p>", "links"=>[], "tags"=>["imaging", "u87mg"], "article_id"=>467634, "categories"=>["Biotechnology", "Biochemistry", "Biophysics"], "users"=>["Richard H. Kimura", "Douglas S. Jones", "Lei Jiang", "Zheng Miao", "Zhen Cheng", "Jennifer R. Cochran"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0016112.g005", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_MicroPET_imaging_of_U87MG_tumor_xenografts_/467634", "title"=>"MicroPET imaging of U87MG tumor xenografts.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-18 02:07:14"}

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  • {"unique-ip"=>"10", "full-text"=>"7", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"1"}
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  • {"unique-ip"=>"5", "full-text"=>"5", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"13", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"6"}
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  • {"unique-ip"=>"2", "full-text"=>"2", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"10"}
  • {"unique-ip"=>"3", "full-text"=>"2", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"11"}
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  • {"unique-ip"=>"3", "full-text"=>"0", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"1", "year"=>"2017", "month"=>"1"}
  • {"unique-ip"=>"5", "full-text"=>"5", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"2"}
  • {"unique-ip"=>"3", "full-text"=>"3", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"3"}
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  • {"unique-ip"=>"1", "full-text"=>"1", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"12"}
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  • {"unique-ip"=>"5", "full-text"=>"5", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"3"}
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  • {"unique-ip"=>"2", "full-text"=>"2", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"6"}
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  • {"unique-ip"=>"4", "full-text"=>"5", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"6", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"8"}
  • {"unique-ip"=>"6", "full-text"=>"5", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"9"}
  • {"unique-ip"=>"5", "full-text"=>"5", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"10"}
  • {"unique-ip"=>"7", "full-text"=>"7", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"12"}
  • {"unique-ip"=>"10", "full-text"=>"9", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2018", "month"=>"11"}
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  • {"unique-ip"=>"1", "full-text"=>"1", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"3"}
  • {"unique-ip"=>"3", "full-text"=>"0", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"7", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"4"}
  • {"unique-ip"=>"8", "full-text"=>"7", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"4", "cited-by"=>"0", "year"=>"2019", "month"=>"5"}
  • {"unique-ip"=>"7", "full-text"=>"8", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"8"}
  • {"unique-ip"=>"5", "full-text"=>"5", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"9"}
  • {"unique-ip"=>"7", "full-text"=>"7", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"10"}
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Relative Metric

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