Ergosterol Peroxide Isolated from Ganoderma lucidum Abolishes MicroRNA miR-378-Mediated Tumor Cells on Chemoresistance
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{"title"=>"Ergosterol Peroxide Isolated from Ganoderma lucidum Abolishes MicroRNA miR-378-Mediated Tumor Cells on Chemoresistance", "type"=>"journal", "authors"=>[{"first_name"=>"Qing Ping", "last_name"=>"Wu", "scopus_author_id"=>"14834512100"}, {"first_name"=>"Yi Zhen", "last_name"=>"Xie", "scopus_author_id"=>"8503841000"}, {"first_name"=>"Zhaoqun", "last_name"=>"Deng", "scopus_author_id"=>"13403743000"}, {"first_name"=>"Xiang Min", "last_name"=>"Li", "scopus_author_id"=>"56742836700"}, {"first_name"=>"Weining", "last_name"=>"Yang", "scopus_author_id"=>"55501123800"}, {"first_name"=>"Chun Wei", "last_name"=>"Jiao", "scopus_author_id"=>"55343249200"}, {"first_name"=>"Ling", "last_name"=>"Fang", "scopus_author_id"=>"36190570100"}, {"first_name"=>"Sen Zhu", "last_name"=>"Li", "scopus_author_id"=>"27170932200"}, {"first_name"=>"Hong Hui", "last_name"=>"Pan", "scopus_author_id"=>"55348374400"}, {"first_name"=>"Albert J.", "last_name"=>"Yee", "scopus_author_id"=>"7102354244"}, {"first_name"=>"Daniel Y.", "last_name"=>"Lee", "scopus_author_id"=>"7406664348"}, {"first_name"=>"Chong", "last_name"=>"Li", "scopus_author_id"=>"55695993500"}, {"first_name"=>"Zhi", "last_name"=>"Zhang", "scopus_author_id"=>"57192635184"}, {"first_name"=>"Jun", "last_name"=>"Guo", "scopus_author_id"=>"57199054797"}, {"first_name"=>"Burton B.", "last_name"=>"Yang", "scopus_author_id"=>"7404471996"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"365542916", "sgr"=>"84865583196", "pmid"=>"22952996", "scopus"=>"2-s2.0-84865583196", "isbn"=>"1932-6203 (Electronic) 1932-6203 (Linking)", "doi"=>"10.1371/journal.pone.0044579", "issn"=>"19326203"}, "id"=>"92516b8d-e8ce-3a6f-9545-6b294dd4e233", "abstract"=>"Due to an altered expression of oncogenic factors and tumor suppressors, aggressive cancer cells have an intrinsic or acquired resistance to chemotherapeutic agents. This typically contributes to cancer recurrence after chemotherapy. microRNAs are short non-coding RNAs that are involved in both cell self-renewal and cancer development. Here we report that tumor cells transfected with miR-378 acquired properties of aggressive cancer cells. Overexpression of miR-378 enhanced both cell survival and colony formation, and contributed to multiple drug resistance. Higher concentrations of chemotherapeutic drugs were needed to induce death of miR-378-transfected cells than to induce death of control cells. We found that the biologically active component isolated from Ganoderma lucidum could overcome the drug-resistance conferred by miR-378. We purified and identified the biologically active component of Ganoderma lucidum as ergosterol peroxide. We demonstrated that ergosterol peroxide produced greater activity in inducing death of miR-378 cells than the GFP cells. Lower concentrations of ergosterol peroxide were needed to induce death of the miR-378-transfected cells than in the control cells. With further clinical development, ergosterol peroxide represents a promising new reagent that can overcome the drug-resistance of tumor cells.", "link"=>"http://www.mendeley.com/research/ergosterol-peroxide-isolated-ganoderma-lucidum-abolishes-microrna-mir378mediated-tumor-cells-chemore", "reader_count"=>23, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Researcher"=>5, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>3, "Student > Postgraduate"=>2, "Student > Master"=>9, "Student > Bachelor"=>2}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Researcher"=>5, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>3, "Student > Postgraduate"=>2, "Student > Master"=>9, "Student > Bachelor"=>2}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>4, "Agricultural and Biological Sciences"=>11, "Medicine and Dentistry"=>1, "Pharmacology, Toxicology and Pharmaceutical Science"=>1, "Chemical Engineering"=>1, "Chemistry"=>3, "Computer Science"=>1, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Chemistry"=>{"Chemistry"=>3}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>11}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>4}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}, "Chemical Engineering"=>{"Chemical Engineering"=>1}}, "reader_count_by_country"=>{"United Kingdom"=>1}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/583588"], "description"=>"<p>(a) Upper, diagram of a construct expressing GFP, a neomycin-resistance gene, and two hairpin structures of pre-miR-378. U87 cells transfected with <i>miR-378</i> or a control vector were maintained in serum-free conditions in Petri dishes. Cell survival was monitored. Transfection with <i>miR-378</i> enhanced cell survival. Lower, in colony formation assays performed in soft agar, miR-378-expressing cells form more colonies with larger sizes. (b) U87 cells transfected with <i>miR-378</i> (obtained by flow cytometry thus containing a mix of cell population called <b>miR-378-mix or miR-378M</b>) or a control vector (<b>GFP</b>) and cells isolated from single colony of miR-378M (<b>miR-378-col</b> or <b>miR-378C</b>) were cultured in serum-free medium for 1 day. RNAs isolated from the cells were subjected to real-time PCR. miR-378C expressed highest level of miR-378. (c) miR-378M, miR-378C, and GFP cells were cultured in normal medium containing 20 µM (left) and 30 µM (right) C2-ceramide. Cells expressing <i>miR-378</i> are resistant to C2-ceramide-induced cell death.</p>", "links"=>[], "tags"=>["cells", "transfected", "c2-ceramide"], "article_id"=>254061, "categories"=>["Cancer", "Biotechnology", "Cell Biology", "Microbiology"], "users"=>["Qing-Ping Wu", "Yi-Zhen Xie", "Zhaoqun Deng", "Xiang-Min Li", "Weining Yang", "Chun-Wei Jiao", "Ling Fang", "Sen-Zhu Li", "Hong-Hui Pan", "Albert J. Yee", "Daniel Y. Lee", "Chong Li", "Zhi Zhang", "Jun Guo", "Burton B. Yang"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044579.g001", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cancer_cells_transfected_with_miR_378_are_C2_ceramide_resistant_/254061", "title"=>"Cancer cells transfected with <i>miR-378</i> are C2-ceramide resistant.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-30 01:07:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/308144", "https://ndownloader.figshare.com/files/308158", "https://ndownloader.figshare.com/files/308172", "https://ndownloader.figshare.com/files/308188", "https://ndownloader.figshare.com/files/308201", "https://ndownloader.figshare.com/files/308213", "https://ndownloader.figshare.com/files/308235", "https://ndownloader.figshare.com/files/308243", "https://ndownloader.figshare.com/files/308254", "https://ndownloader.figshare.com/files/308268"], "description"=>"<div><p>Due to an altered expression of oncogenic factors and tumor suppressors, aggressive cancer cells have an intrinsic or acquired resistance to chemotherapeutic agents. This typically contributes to cancer recurrence after chemotherapy. microRNAs are short non-coding RNAs that are involved in both cell self-renewal and cancer development. Here we report that tumor cells transfected with <em>miR-378</em> acquired properties of aggressive cancer cells. Overexpression of miR-378 enhanced both cell survival and colony formation, and contributed to multiple drug resistance. Higher concentrations of chemotherapeutic drugs were needed to induce death of miR-378-transfected cells than to induce death of control cells. We found that the biologically active component isolated from <em>Ganoderma lucidum</em> could overcome the drug-resistance conferred by miR-378. We purified and identified the biologically active component of <em>Ganoderma lucidum</em> as ergosterol peroxide. We demonstrated that ergosterol peroxide produced greater activity in inducing death of miR-378 cells than the GFP cells. Lower concentrations of ergosterol peroxide were needed to induce death of the miR-378-transfected cells than in the control cells. With further clinical development, ergosterol peroxide represents a promising new reagent that can overcome the drug-resistance of tumor cells.</p> </div>", "links"=>[], "tags"=>["ergosterol", "peroxide", "ganoderma", "lucidum", "abolishes", "microrna", "mir-378-mediated", "cells", "chemoresistance"], "article_id"=>120794, "categories"=>["Cancer", "Biotechnology", "Cell Biology", "Microbiology"], "users"=>["Qing-Ping Wu", "Yi-Zhen Xie", "Zhaoqun Deng", "Xiang-Min Li", "Weining Yang", "Chun-Wei Jiao", "Ling Fang", "Sen-Zhu Li", "Hong-Hui Pan", "Albert J. Yee", "Daniel Y. Lee", "Chong Li", "Zhi Zhang", "Jun Guo", "Burton B. Yang"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0044579.s001", "https://dx.doi.org/10.1371/journal.pone.0044579.s002", "https://dx.doi.org/10.1371/journal.pone.0044579.s003", "https://dx.doi.org/10.1371/journal.pone.0044579.s004", "https://dx.doi.org/10.1371/journal.pone.0044579.s005", "https://dx.doi.org/10.1371/journal.pone.0044579.s006", "https://dx.doi.org/10.1371/journal.pone.0044579.s007", "https://dx.doi.org/10.1371/journal.pone.0044579.s008", "https://dx.doi.org/10.1371/journal.pone.0044579.s009", "https://dx.doi.org/10.1371/journal.pone.0044579.s010"], "stats"=>{"downloads"=>1, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Ergosterol_Peroxide_Isolated_from_Ganoderma_lucidum_Abolishes_MicroRNA_miR_378_Mediated_Tumor_Cells_on_Chemoresistance/120794", "title"=>"Ergosterol Peroxide Isolated from Ganoderma lucidum Abolishes MicroRNA miR-378-Mediated Tumor Cells on Chemoresistance", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-08-30 00:13:14"}
  • {"files"=>["https://ndownloader.figshare.com/files/584244"], "description"=>"<p>(a) Ganoderma spores were subjected to oil extraction using the standard protocol, in which the spores were maintained under the standard pressure for 1–6 hours. The effects of the oil on tumor cell death were tested. (b) Ganoderma spores were digested with spore-released enzymes for different periods of time as indicated, followed by oil extraction with the standard protocol. The effects of the oil on tumor cell death were tested. (c) Ganoderma spores were subjected to oil extraction under different temperature and pressure. The effects of the oil on tumor cell death were tested. “m” means the pressure Mpa while “C” means degree of the temperature. (d) Typical results of the effects of “15m+50C” (Fraction #2) and “15m+60C” (Fraction #3) on tumor cell death are shown.</p>", "links"=>[], "tags"=>["ganoderma", "prepared", "protocols"], "article_id"=>254726, "categories"=>["Cancer", "Biotechnology", "Cell Biology", "Microbiology"], "users"=>["Qing-Ping Wu", "Yi-Zhen Xie", "Zhaoqun Deng", "Xiang-Min Li", "Weining Yang", "Chun-Wei Jiao", "Ling Fang", "Sen-Zhu Li", "Hong-Hui Pan", "Albert J. Yee", "Daniel Y. Lee", "Chong Li", "Zhi Zhang", "Jun Guo", "Burton B. Yang"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044579.g005", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effects_of_Ganoderma_oil_prepared_using_different_protocols_on_tumor_cell_death_/254726", "title"=>"Effects of Ganoderma oil prepared using different protocols on tumor cell death.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-30 01:18:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/584027"], "description"=>"<p>(a) Breast carcinoma cells (MT-1) were treated with Ganoderma oil prepared from the standard protocol (WYGO-070102), 2-year-old (maintained at room temperature) Ganoderma oil prepared from the standard protocol, and a sample of Ganoderma oil obtained from a different source (XZL-Z). Analysis of the half maximal effective concentration (EC<sub>50</sub>) indicated that the Ganoderma oil prepared from the standard protocol produced the best result in inducing tumor cell death, followed by the 2-year-old product and the sample from a different source. (b) MT-1 cells were treated double-blindly with Ganoderma oil obtained from different sources. EC50 was obtained as indicated.</p>", "links"=>[], "tags"=>["ganoderma", "sources"], "article_id"=>254510, "categories"=>["Cancer", "Biotechnology", "Cell Biology", "Microbiology"], "users"=>["Qing-Ping Wu", "Yi-Zhen Xie", "Zhaoqun Deng", "Xiang-Min Li", "Weining Yang", "Chun-Wei Jiao", "Ling Fang", "Sen-Zhu Li", "Hong-Hui Pan", "Albert J. Yee", "Daniel Y. Lee", "Chong Li", "Zhi Zhang", "Jun Guo", "Burton B. Yang"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044579.g004", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effects_of_Ganoderma_oil_from_different_sources_on_tumor_cell_death_/254510", "title"=>"Effects of Ganoderma oil from different sources on tumor cell death.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-30 01:15:10"}
  • {"files"=>["https://ndownloader.figshare.com/files/584660"], "description"=>"<p>(a) NMR analysis of the structure of the candidate. (b) Molecular structure, formula and molecular weight of ergosterol peroxide. (c) The MT-1 cells treated with or without ergosterol peroxide were fixed, and stained with the Diff-Quik-Stain kit, followed by microscopic examination and counting of cells survived. Induction of breast cancer cell death by ergosterol peroxide was concentration dependent (Upper). ** 0<0.01, n = 3. Typical vacuoles were detected in the cells treated with ergosterol peroxide (Lower).</p>", "links"=>[], "tags"=>["ergosterol", "peroxide"], "article_id"=>255133, "categories"=>["Cancer", "Biotechnology", "Cell Biology", "Microbiology"], "users"=>["Qing-Ping Wu", "Yi-Zhen Xie", "Zhaoqun Deng", "Xiang-Min Li", "Weining Yang", "Chun-Wei Jiao", "Ling Fang", "Sen-Zhu Li", "Hong-Hui Pan", "Albert J. Yee", "Daniel Y. Lee", "Chong Li", "Zhi Zhang", "Jun Guo", "Burton B. Yang"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044579.g007", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_effects_of_ergosterol_peroxide_on_tumor_cell_death_/255133", "title"=>"The effects of ergosterol peroxide on tumor cell death.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-30 01:25:33"}
  • {"files"=>["https://ndownloader.figshare.com/files/583910"], "description"=>"<p>(a-b) The GFP, miR-378M, and miR-378C cells were cultured in normal medium treated with different concentrations of Ganoderma oil (prepared form the standard protocol) as indicated for 24 hours (a) or 48 hours (b). The effects of Ganoderma oil on cell death were miR-378C > miR-378M > GFP. (c) MDA-MB-231 cells stably transfected with miR-378 or a control vector were treated with Epirubicin (2.0 µg/ml) and combined with or without Ganoderma oil 0.4 µl/ml. The miR-378-transfected cells were less sensitive to Epirubicin than the control cells. Combination with Ganoderma oil significantly increased the sensitivity of the miR-378 cells to Epirubicin. Typical photos of cell death are shown (lower panel). ** p<0.01.</p>", "links"=>[], "tags"=>["exerts", "stronger", "inducing", "cencer"], "article_id"=>254382, "categories"=>["Cancer", "Biotechnology", "Cell Biology", "Microbiology"], "users"=>["Qing-Ping Wu", "Yi-Zhen Xie", "Zhaoqun Deng", "Xiang-Min Li", "Weining Yang", "Chun-Wei Jiao", "Ling Fang", "Sen-Zhu Li", "Hong-Hui Pan", "Albert J. Yee", "Daniel Y. Lee", "Chong Li", "Zhi Zhang", "Jun Guo", "Burton B. Yang"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044579.g003", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Ganoderma_oil_exerts_stronger_effect_on_inducing_death_of_the_cencer_cells_/254382", "title"=>"Ganoderma oil exerts stronger effect on inducing death of the cencer cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-30 01:13:02"}
  • {"files"=>["https://ndownloader.figshare.com/files/584846"], "description"=>"<p>(a) GFP, miR-378M, and miR-378C cells were treated with ergosterol peroxide at the concentration of 0.23 mM for 48 hours. The chemical exhibited strongest activity on inducing death of miR-378C cells, followed by miR-378M and GFP cells. ** 0<0.01, n = 3. (b) RNAs were isolated from a number of cancer cell lines and subjected to real-time PCR analysis for miR-378 expression. The human hepatocellular carcinoma cells HepG2 express the highest levels of miR-378 among the cell lines tested. (c) Human hepatocellular carcinoma cell line HepG2 was transfected with anti-miR-378 or a control vector, followed by treatment with Ganoderma oil. Transfection with anti-miR-378 construct decreased the sensitivity of the cells to Ganoderma oil induced cell death. (d) Typical morphology of the cells treated Ganoderma oil is shown. (e) After the treatment with Ganoderma oil, the number of the cells was counted. Significant differences were seen in both groups of cells treated with different concentrations of Ganoderma oil. ** p<0.01. (f) The cells were also treated with ergosterol peroxide. Antagonizing endogenous miR-378 decreased the sensitivity of the cells to the induced cell death by Ganoderma oil. ** p<0.01.</p>", "links"=>[], "tags"=>["mir-378", "ganoderma", "ergosterol", "peroxide", "induced"], "article_id"=>255324, "categories"=>["Cancer", "Biotechnology", "Cell Biology", "Microbiology"], "users"=>["Qing-Ping Wu", "Yi-Zhen Xie", "Zhaoqun Deng", "Xiang-Min Li", "Weining Yang", "Chun-Wei Jiao", "Ling Fang", "Sen-Zhu Li", "Hong-Hui Pan", "Albert J. Yee", "Daniel Y. Lee", "Chong Li", "Zhi Zhang", "Jun Guo", "Burton B. Yang"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044579.g008", "stats"=>{"downloads"=>0, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_miR_378_on_Ganoderma_oil_and_ergosterol_peroxide_induced_cell_death_/255324", "title"=>"Effect of miR-378 on Ganoderma oil and ergosterol peroxide induced cell death.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-30 01:28:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/584457"], "description"=>"<p>(a) Biologically active fractions were eluted with different chemicals, followed by analysis of their effects on tumor cell death. The fraction eluted by methanol (Elute 4) produced the greatest activity, while Elute 1 and the sample before fractionation (Before fract) displayed the lowest activity on inducing tumor cell death. (b) The MT-1 cells treated with or without Ganoderma oil were fixed, and stained with the Diff-Quik-Stain kit, followed by microscopic examination. A great number of vacuoles were detected in the cells treated with Ganoderma oil. (c) The Ganoderma oil treated cells were subjected to electronic microscopic examination. Large vacuoles could be seen clearly.</p>", "links"=>[], "tags"=>["biologically", "components"], "article_id"=>254936, "categories"=>["Cancer", "Biotechnology", "Cell Biology", "Microbiology"], "users"=>["Qing-Ping Wu", "Yi-Zhen Xie", "Zhaoqun Deng", "Xiang-Min Li", "Weining Yang", "Chun-Wei Jiao", "Ling Fang", "Sen-Zhu Li", "Hong-Hui Pan", "Albert J. Yee", "Daniel Y. Lee", "Chong Li", "Zhi Zhang", "Jun Guo", "Burton B. Yang"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044579.g006", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_effects_of_biologically_active_components_on_tumor_cell_death_/254936", "title"=>"The effects of biologically active components on tumor cell death.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-30 01:22:16"}
  • {"files"=>["https://ndownloader.figshare.com/files/583735"], "description"=>"<p>(a) miR-378M, miR-378C, and GFP cells were cultured in normal medium containing 56 mM Cytarabine for 3 days. The number of cells was counted and analyzed statistically. The <i>miR-378</i> expressing cells exhibited higher rates of survival than the GFP cells. N = 3, ** p<0.01. (b) The cells were treated with Methotrexate at 22 mM for 6 (left) and 8 (right) days. Cells expressing <i>miR-378</i> are resistant to Methotrexate-induced cell death. (c) The cells were also treated with combination of Cytarabine and Methotrexate. The combination of both drugs was able to induce cell death at lower concentrations. (d) MDA-MB-231 cells stably transfected with miR-378 or a control vector were treated with Epirubicin (2.0 µg/ml). Cells expressing miR-378 displayed resistance to Epirubicin as compared with the vector control. Typical photos of cell death are shown (lower panel). ** p<0.01.</p>", "links"=>[], "tags"=>["cells", "transfected", "resistant", "cytarabine"], "article_id"=>254209, "categories"=>["Cancer", "Biotechnology", "Cell Biology", "Microbiology"], "users"=>["Qing-Ping Wu", "Yi-Zhen Xie", "Zhaoqun Deng", "Xiang-Min Li", "Weining Yang", "Chun-Wei Jiao", "Ling Fang", "Sen-Zhu Li", "Hong-Hui Pan", "Albert J. Yee", "Daniel Y. Lee", "Chong Li", "Zhi Zhang", "Jun Guo", "Burton B. Yang"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044579.g002", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cancer_cells_transfected_with_miR_378_are_resistant_to_Cytarabine_and_Methotrexate_/254209", "title"=>"Cancer cells transfected with <i>miR-378</i> are resistant to Cytarabine and Methotrexate.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-30 01:10:09"}

PMC Usage Stats | Further Information

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