Scalable Production in Human Cells and Biochemical Characterization of Full-Length Normal and Mutant Huntingtin
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{"title"=>"Scalable production in human cells and biochemical characterization of full-length normal and mutant huntingtin", "type"=>"journal", "authors"=>[{"first_name"=>"Bin", "last_name"=>"Huang", "scopus_author_id"=>"55321772000"}, {"first_name"=>"Tanja", "last_name"=>"Lucas", "scopus_author_id"=>"37100188000"}, {"first_name"=>"Claudia", "last_name"=>"Kueppers", "scopus_author_id"=>"55315510300"}, {"first_name"=>"Xiaomin", "last_name"=>"Dong", "scopus_author_id"=>"55322090000"}, {"first_name"=>"Maike", "last_name"=>"Krause", "scopus_author_id"=>"55873744900"}, {"first_name"=>"Alexander", "last_name"=>"Bepperling", "scopus_author_id"=>"35344606200"}, {"first_name"=>"Johannes", "last_name"=>"Buchner", "scopus_author_id"=>"7005734918"}, {"first_name"=>"Hans", "last_name"=>"Voshol", "scopus_author_id"=>"6602667664"}, {"first_name"=>"Andreas", "last_name"=>"Weiss", "scopus_author_id"=>"15021604600"}, {"first_name"=>"Bertran", "last_name"=>"Gerrits", "scopus_author_id"=>"16028477200"}, {"first_name"=>"Stefan", "last_name"=>"Kochanek", "scopus_author_id"=>"17035235000"}], "year"=>2015, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "scopus"=>"2-s2.0-84925728810", "pui"=>"603282290", "doi"=>"10.1371/journal.pone.0121055", "isbn"=>"1932-6203 (Electronic)\r1932-6203 (Linking)", "sgr"=>"84925728810", "pmid"=>"25799558"}, "id"=>"40abc4ca-4c43-34aa-8078-7c890e8ec1b6", "abstract"=>"Huntingtin (Htt) is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington's disease (HD) is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ) expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q) and mutant (46Q and 128Q) Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on \"gutless\" adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs) were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.", "link"=>"http://www.mendeley.com/research/scalable-production-human-cells-biochemical-characterization-fulllength-normal-mutant-huntingtin", "reader_count"=>24, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Researcher"=>7, "Student > Ph. D. Student"=>7, "Student > Postgraduate"=>1, "Other"=>1, "Student > Master"=>2, "Student > Bachelor"=>4, "Lecturer"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Researcher"=>7, "Student > Ph. D. Student"=>7, "Student > Postgraduate"=>1, "Other"=>1, "Student > Master"=>2, "Student > Bachelor"=>4, "Lecturer"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>8, "Medicine and Dentistry"=>2, "Agricultural and Biological Sciences"=>8, "Neuroscience"=>3, "Design"=>1, "Psychology"=>1}, "reader_count_by_subdiscipline"=>{"Design"=>{"Design"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Neuroscience"=>{"Neuroscience"=>3}, "Psychology"=>{"Psychology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>8}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>8}, "Unspecified"=>{"Unspecified"=>1}}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1981692"], "description"=>"<p>Alpha-solenoid/Alpha-rod regions (αS1, αS2 and αS3) in Htt were predicted by the neural network-based ARD2 application in its most recent update ([<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121055#pone.0121055.ref060\" target=\"_blank\">60</a>], <a href=\"http://cbdm.mdc-berlin.de/~ard2\" target=\"_blank\">http://cbdm.mdc-berlin.de/~ard2</a>) for predicting HEAT repeats and related sequences. The phosphorylation sites were indicated with respect to the predicted alpha-solenoid domain structure of Htt. Phosphorylated amino acids detected in this study and not previously described are indicated in bold. Phosphorylated amino acids detected in this study and confirming previously described phosphorylation are indicated as bold/underlined. Previously described phosphorylation sites not covered by this study are indicated in italic. Previously described phosphorylation sites covered but not found phosphorylated in this study are indicated in regular letters. Known calpain cleavage sites (arrows) and known caspase 2/3, caspase 3 and caspase 6 cleavage sites (triangles) between alpha-solenoid regions 1 and 2 are indicated. As can be seen is this scheme, the phosphorylation sites mainly cluster in regions outside of predicted alpha-solenoid domains.</p>", "links"=>[], "tags"=>["Gene transfer", "phosphorylation sites", "milligram quantities", "Scalable Production", "exon 1", "CAG triplet amplification", "Htt function", "Cell lines", "hd", "ptm", "scalable system", "Human Cells", "form dimers", "posttranslational modifications", "Mutant Huntingtin Huntingtin", "350 kD intracellular protein", "Biochemical Characterization", "oligomer formation", "128 Q", "order oligomers"], "article_id"=>1350389, "categories"=>["Biological Sciences"], "users"=>["Bin Huang", "Tanja Lucas", "Claudia Kueppers", "Xiaomin Dong", "Maike Krause", "Alexander Bepperling", "Johannes Buchner", "Hans Voshol", "Andreas Weiss", "Bertran Gerrits", "Stefan Kochanek"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0121055.g003", "stats"=>{"downloads"=>0, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Schematic_representation_of_Htt_phosphorylation_sites_/1350389", "title"=>"Schematic representation of Htt phosphorylation sites.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-03-23 04:18:35"}
  • {"files"=>["https://ndownloader.figshare.com/files/1981686"], "description"=>"<p>(A) Constructs for expression of Htt17 in B1.21 cells and for expression of Htt46 in C2.6 cells. Htt17 and Htt46 are c-terminally fused to a FLAG-His affinity tag and are expressed under the control of a Dox-inducible and bidirectional minimal CMV promoter tetracycline responsive element (CMV-TRE-CMV). (B) Adenovirus vector constructs for expression of Htt17 (HC-Ad-Htt17Q-FH) and of Htt128 (HC-Ad-Htt128Q-FH). Htt17 and Htt128 are c-terminally fused to a FLAG-His tag and are expressed under control of the CMV promoter. HC-Ad-Htt16Q-FH and HC-Ad-Htt128Q-FH vectors also contain stuffer DNAs (hprt and c346) from human genomic DNA, the inverted terminal repeats (ITR) of hAd5 and the packaging signal of hAd5 (Ψ). (C) The expression of Htt17 and Htt46 in B1.21 and C2.6 cells was analyzed 48 hours after induction with Dox by SDS-PAGE and Coomassie Blue staining (C,S.) and was further confirmed by Western Blot (W.B.) analysis. Compared to un-induced cells (-), an additional band of about 350 kD size was clearly visible in cells 48 hours after induction (+). 2166 Ab: antibody recognizing Htt; Flag Ab: antibody recognizing the FLAG tag. (D) Expression of Htt17 and Htt128 following transduction of 293 cell-based 116 cells with HC-Ad-Htt17Q-FH and HC-Ad-Htt128Q-FH vector was analyzed 48 hours after gene transfer by SDS-PAGE and Coomassie Blue staining and was further confirmed by Western Blot analysis. Compared with un-transduced cells (-), additional bands of about 350 kD size and reacting with anti-Htt and anti-FLAG antibodies are visible following HC-Ad vector mediated transduction (+).</p>", "links"=>[], "tags"=>["Gene transfer", "phosphorylation sites", "milligram quantities", "Scalable Production", "exon 1", "CAG triplet amplification", "Htt function", "Cell lines", "hd", "ptm", "scalable system", "Human Cells", "form dimers", "posttranslational modifications", "Mutant Huntingtin Huntingtin", "350 kD intracellular protein", "Biochemical Characterization", "oligomer formation", "128 Q", "order oligomers"], "article_id"=>1350383, "categories"=>["Biological Sciences"], "users"=>["Bin Huang", "Tanja Lucas", "Claudia Kueppers", "Xiaomin Dong", "Maike Krause", "Alexander Bepperling", "Johannes Buchner", "Hans Voshol", "Andreas Weiss", "Bertran Gerrits", "Stefan Kochanek"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0121055.g001", "stats"=>{"downloads"=>2, "page_views"=>57, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Production_of_full_length_normal_and_mutant_Htt_in_stable_cell_lines_and_following_adenovirus_mediated_gene_transfer_/1350383", "title"=>"Production of full-length normal and mutant Htt in stable cell lines and following adenovirus-mediated gene transfer.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-03-23 04:18:35"}
  • {"files"=>["https://ndownloader.figshare.com/files/1981694"], "description"=>"<p>(A) CD spectra of Htt17, Htt46 and Htt128. (B) Based on the determined CD spectra, the secondary structures of Htt17, Htt46 and Htt128 were analyzed with Selcon3. Htt is not a purely helical protein (helix >50% and strand <5%), but rather a helix-rich protein (40% helix) mixed with strand (10%), which is consistent with the results of Htt secondary prediction by the PHD neuro-network. Significant differences in the secondary structure of Htt17, Htt46 and Htt128 were not observed.</p>", "links"=>[], "tags"=>["Gene transfer", "phosphorylation sites", "milligram quantities", "Scalable Production", "exon 1", "CAG triplet amplification", "Htt function", "Cell lines", "hd", "ptm", "scalable system", "Human Cells", "form dimers", "posttranslational modifications", "Mutant Huntingtin Huntingtin", "350 kD intracellular protein", "Biochemical Characterization", "oligomer formation", "128 Q", "order oligomers"], "article_id"=>1350391, "categories"=>["Biological Sciences"], "users"=>["Bin Huang", "Tanja Lucas", "Claudia Kueppers", "Xiaomin Dong", "Maike Krause", "Alexander Bepperling", "Johannes Buchner", "Hans Voshol", "Andreas Weiss", "Bertran Gerrits", "Stefan Kochanek"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0121055.g005", "stats"=>{"downloads"=>1, "page_views"=>24, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_CD_analysis_of_Htt17_Htt46_and_Htt128_/1350391", "title"=>"CD analysis of Htt17, Htt46 and Htt128.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-03-23 04:18:35"}
  • {"files"=>["https://ndownloader.figshare.com/files/1981695"], "description"=>"<p>Htt is a molecule with elongated shape and mutant Htt128 is more extended than Htt17 and Htt46. (A) Htt17, Htt46 and Htt128 were analyzed by SEC with a Superdex-200 10/300 GL column to estimate the molecular weight (MW) and stoke radius (Rs). Htt oligomers eluted at peak 1 corresponding to the void volume of this column and Htt monomers eluted at peak 2. Htt17 and Htt46 eluted at retention volume of about 9.5 ml, while Htt128 eluted at a retention volume of about 9.18 ml. (B) There were no significant differences of the retention volumes between Htt16 and Htt46, while Htt128 eluted significantly earlier than Htt17 and Htt46. (C) From the SEC results and considering a calibration curve the MW of Htt would be calculated to have a MW of about 600 kD, which is significantly larger than its actual 350 kD MW, indicating that Htt is not a globular protein. The friction ratio f/fm represents the total shape asymmetry of molecules. The f/fm of 1.6 predicts that Htt has an elongated shape. The larger f/fm value of Htt128 than Htt17 and Htt46 indicated that the shape of Htt128 was more extended than the shapes of Htt17 and Htt46.</p>", "links"=>[], "tags"=>["Gene transfer", "phosphorylation sites", "milligram quantities", "Scalable Production", "exon 1", "CAG triplet amplification", "Htt function", "Cell lines", "hd", "ptm", "scalable system", "Human Cells", "form dimers", "posttranslational modifications", "Mutant Huntingtin Huntingtin", "350 kD intracellular protein", "Biochemical Characterization", "oligomer formation", "128 Q", "order oligomers"], "article_id"=>1350392, "categories"=>["Biological Sciences"], "users"=>["Bin Huang", "Tanja Lucas", "Claudia Kueppers", "Xiaomin Dong", "Maike Krause", "Alexander Bepperling", "Johannes Buchner", "Hans Voshol", "Andreas Weiss", "Bertran Gerrits", "Stefan Kochanek"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0121055.g006", "stats"=>{"downloads"=>1, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Analytical_size_exclusion_chromatography_/1350392", "title"=>"Analytical size exclusion chromatography.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-03-23 04:18:35"}
  • {"files"=>["https://ndownloader.figshare.com/files/1981693"], "description"=>"<p>(A) c(s) distributions of Htt17 (black curve) and Htt128 (grey curve) obtained by Sedfit analysis, indicating one major peak at 10 S for the momoneric species. Dimers and trimers are observed around 15 S and 20 S, respectively. At s-values higher than 22 S oligomers of higher order and aggregates are detectable. (B, C) Summary of the sedimentation viscosity (SV) analysis of Htt17 and Htt128. The main species in both samples is the monomeric form (52.1% and 35.6%). Dimers have a fraction size from around 20% and trimers are higher populated in the Htt128 sample (17.3% vs. 8.8%). The amount of visible higher oligomers and aggregates is more pronounced in Htt128. Note: the AUC analyses of Htt17 and Htt128 were performed in two independent analyses likely explaining the smaller s<sub>20,W</sub>-value of Htt128 in this experiment. (D) Htt17 and Htt128 were analyzed with HR-native PAGE and Coomassie Blue staining with a result that was consistent with the SV analysis. M: Htt monomer; D: Htt dimer; T: Htt trimer.</p>", "links"=>[], "tags"=>["Gene transfer", "phosphorylation sites", "milligram quantities", "Scalable Production", "exon 1", "CAG triplet amplification", "Htt function", "Cell lines", "hd", "ptm", "scalable system", "Human Cells", "form dimers", "posttranslational modifications", "Mutant Huntingtin Huntingtin", "350 kD intracellular protein", "Biochemical Characterization", "oligomer formation", "128 Q", "order oligomers"], "article_id"=>1350390, "categories"=>["Biological Sciences"], "users"=>["Bin Huang", "Tanja Lucas", "Claudia Kueppers", "Xiaomin Dong", "Maike Krause", "Alexander Bepperling", "Johannes Buchner", "Hans Voshol", "Andreas Weiss", "Bertran Gerrits", "Stefan Kochanek"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0121055.g004", "stats"=>{"downloads"=>1, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Polyglutamine_expansion_correlates_with_an_enhanced_formation_of_oligomers_and_aggregates_/1350390", "title"=>"Polyglutamine expansion correlates with an enhanced formation of oligomers and aggregates.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-03-23 04:18:35"}
  • {"files"=>["https://ndownloader.figshare.com/files/1981690"], "description"=>"<p>(A) FLAG affinity purification of Htt17 from B1.21 cells. B1.21 cells were harvested 72 hours after induction and lysed by freeze/thawing. The cell lysate was cleared by centrifugation and filtration. Htt17 in the cleared cell lysate was purified by anti-FLAG beads. The cleared cell lysate (lane 1), the cell lysate after incubation with the anti-FLAG beads (lane 2) and Htt17 eluted from the anti-FLAG beads (lane 3) were analyzed by SDS-PAGE and Coomassie Blue staining. (B) Htt17, after elution from the anti-FLAG beads was further purified by SEC using a Superose 6 column in 20 mM Tris, 500 mM NaCl, 0.4% CHAPS, 5 mM DTT and 5% Glycerol. Htt eluted in a major peak [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121055#pone.0121055.ref002\" target=\"_blank\">2</a>] and a shoulder [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121055#pone.0121055.ref001\" target=\"_blank\">1</a>]. (C) Purified Htt17 was analyzed by SDS-PAGE and Coomassie Blue staining. 2 μg of FLAG-purified Htt (lane 1) and FLAG-SEC purified Htt (peak 2 from SEC, lane 2) was evaluated by 6% and 10% SDS-PAGE. Minor contaminating proteins in the FLAG-only purified Htt17 are indicated with an asterisk. Contaminating proteins were absent in FLAG-SEC purified Htt17. (D) The integrity of the FLAG-SEC purified Htt17 was confirmed by Western Blot analysis with the 2B4 anti-Htt antibody (reacting with the N-terminus of Htt) and Anti-FLAG antibody (detecting the C-terminus of Htt). Lane 1: unloaded; Lane 2: FLAG-SEC purified Htt17. (E) Analysis of Htt46 and Htt128 after FLAG-SEC purification. 2 μg FLAG-SEC purified Htt46 and Htt128 were evaluated by SDS-PAGE and Coomassie Blue staining. Contaminating proteins were not detected.</p>", "links"=>[], "tags"=>["Gene transfer", "phosphorylation sites", "milligram quantities", "Scalable Production", "exon 1", "CAG triplet amplification", "Htt function", "Cell lines", "hd", "ptm", "scalable system", "Human Cells", "form dimers", "posttranslational modifications", "Mutant Huntingtin Huntingtin", "350 kD intracellular protein", "Biochemical Characterization", "oligomer formation", "128 Q", "order oligomers"], "article_id"=>1350387, "categories"=>["Biological Sciences"], "users"=>["Bin Huang", "Tanja Lucas", "Claudia Kueppers", "Xiaomin Dong", "Maike Krause", "Alexander Bepperling", "Johannes Buchner", "Hans Voshol", "Andreas Weiss", "Bertran Gerrits", "Stefan Kochanek"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0121055.g002", "stats"=>{"downloads"=>5, "page_views"=>104, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Htt_purification_using_FLAG_affinity_beads_followed_by_size_exclusion_chromatography_SEC_/1350387", "title"=>"Htt purification using FLAG-affinity beads followed by size exclusion chromatography (SEC).", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-03-23 04:18:35"}
  • {"files"=>["https://ndownloader.figshare.com/files/1981712", "https://ndownloader.figshare.com/files/1981713", "https://ndownloader.figshare.com/files/1981714", "https://ndownloader.figshare.com/files/1981715", "https://ndownloader.figshare.com/files/1981716", "https://ndownloader.figshare.com/files/1981717", "https://ndownloader.figshare.com/files/1981718", "https://ndownloader.figshare.com/files/1981719", "https://ndownloader.figshare.com/files/1981720"], "description"=>"<div><p>Huntingtin (Htt) is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington’s disease (HD) is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ) expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q) and mutant (46Q and 128Q) Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on “gutless” adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs) were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.</p></div>", "links"=>[], "tags"=>["Gene transfer", "phosphorylation sites", "milligram quantities", "Scalable Production", "exon 1", "CAG triplet amplification", "Htt function", "Cell lines", "hd", "ptm", "scalable system", "Human Cells", "form dimers", "posttranslational modifications", "Mutant Huntingtin Huntingtin", "350 kD intracellular protein", "Biochemical Characterization", "oligomer formation", "128 Q", "order oligomers"], "article_id"=>1350404, "categories"=>["Biological Sciences"], "users"=>["Bin Huang", "Tanja Lucas", "Claudia Kueppers", "Xiaomin Dong", "Maike Krause", "Alexander Bepperling", "Johannes Buchner", "Hans Voshol", "Andreas Weiss", "Bertran Gerrits", "Stefan Kochanek"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0121055.s001", "https://dx.doi.org/10.1371/journal.pone.0121055.s002", "https://dx.doi.org/10.1371/journal.pone.0121055.s003", "https://dx.doi.org/10.1371/journal.pone.0121055.s004", "https://dx.doi.org/10.1371/journal.pone.0121055.s005", "https://dx.doi.org/10.1371/journal.pone.0121055.s006", "https://dx.doi.org/10.1371/journal.pone.0121055.s007", "https://dx.doi.org/10.1371/journal.pone.0121055.s008", "https://dx.doi.org/10.1371/journal.pone.0121055.s009"], "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Scalable_Production_in_Human_Cells_and_Biochemical_Characterization_of_Full_Length_Normal_and_Mutant_Huntingtin_/1350404", "title"=>"Scalable Production in Human Cells and Biochemical Characterization of Full-Length Normal and Mutant Huntingtin", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-03-23 04:18:35"}

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  • {"unique-ip"=>"5", "full-text"=>"5", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"11"}
  • {"unique-ip"=>"11", "full-text"=>"15", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"6", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"9"}
  • {"unique-ip"=>"11", "full-text"=>"12", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"2"}
  • {"unique-ip"=>"11", "full-text"=>"12", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"3"}
  • {"unique-ip"=>"18", "full-text"=>"16", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"6", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"4"}
  • {"unique-ip"=>"16", "full-text"=>"16", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"5"}
  • {"unique-ip"=>"13", "full-text"=>"17", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"8"}
  • {"unique-ip"=>"16", "full-text"=>"14", "pdf"=>"7", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"9"}
  • {"unique-ip"=>"10", "full-text"=>"10", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"10"}
  • {"unique-ip"=>"10", "full-text"=>"13", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"12"}
  • {"unique-ip"=>"13", "full-text"=>"12", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"4", "cited-by"=>"0", "year"=>"2020", "month"=>"2"}
  • {"unique-ip"=>"13", "full-text"=>"15", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"31", "cited-by"=>"0", "year"=>"2020", "month"=>"3"}
  • {"unique-ip"=>"10", "full-text"=>"9", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2020", "month"=>"4"}
  • {"unique-ip"=>"13", "full-text"=>"14", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2020", "month"=>"5"}
  • {"unique-ip"=>"14", "full-text"=>"19", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2020", "month"=>"6"}
  • {"unique-ip"=>"12", "full-text"=>"13", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2020", "month"=>"7"}
  • {"unique-ip"=>"15", "full-text"=>"15", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"4", "cited-by"=>"0", "year"=>"2020", "month"=>"8"}
  • {"unique-ip"=>"17", "full-text"=>"18", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2020", "month"=>"9"}

Relative Metric

{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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