In Vitro Anti-Echinococcal and Metabolic Effects of Metformin Involve Activation of AMP-Activated Protein Kinase in Larval Stages of Echinococcus granulosus
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{"title"=>"In vitro anti-echinococcal and metabolic effects of metformin involve activation of AMP-activated protein kinase in larval stages of Echinococcus granulosus", "type"=>"journal", "authors"=>[{"first_name"=>"Julia A.", "last_name"=>"Loos", "scopus_author_id"=>"55240356200"}, {"first_name"=>"Andrea C.", "last_name"=>"Cumino", "scopus_author_id"=>"6602435466"}], "year"=>2015, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"604612638", "sgr"=>"84930636137", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "scopus"=>"2-s2.0-84930636137", "issn"=>"19326203", "pmid"=>"25965910", "doi"=>"10.1371/journal.pone.0126009"}, "id"=>"2942aaec-539c-306e-aaea-f2c68e333a9b", "abstract"=>"Metformin (Met) is a biguanide anti-hyperglycemic agent, which also exerts antiproliferative effects on cancer cells. This drug inhibits the complex I of the mitochondrial electron transport chain inducing a fall in the cell energy charge and leading 5'-AMP-activated protein kinase (AMPK) activation. AMPK is a highly conserved heterotrimeric complex that coordinates metabolic and growth pathways in order to maintain energy homeostasis and cell survival, mainly under nutritional stress conditions, in a Liver Kinase B1 (LKB1)-dependent manner. This work describes for the first time, the in vitro anti-echinococcal effect of Met on Echinococcus granulosus larval stages, as well as the molecular characterization of AMPK (Eg-AMPK) in this parasite of clinical importance. The drug exerted a dose-dependent effect on the viability of both larval stages. Based on this, we proceeded with the identification of the genes encoding for the different subunits of Eg-AMPK. We cloned one gene coding for the catalytic subunit (Eg-ampkɑ) and two genes coding for the regulatory subunits (Eg-ampkβ and Eg-ampkγ), all of them constitutively transcribed in E. granulosus protoscoleces and metacestodes. Their deduced amino acid sequences show all the conserved functional domains, including key amino acids involved in catalytic activity and protein-protein interactions. In protoscoleces, the drug induced the activation of AMPK (Eg-AMPKɑ-P176), possibly as a consequence of cellular energy charge depletion evidenced by assays with the fluorescent indicator JC-1. Met also led to carbohydrate starvation, it increased glucogenolysis and homolactic fermentation, and decreased transcription of intermediary metabolism genes. By in toto immunolocalization assays, we detected Eg-AMPKɑ-P176 expression, both in the nucleus and the cytoplasm of cells as in the larval tegument, the posterior bladder and the calcareous corpuscles of control and Met-treated protoscoleces. Interestingly, expression of Eg-AMPKɑ was observed in the developmental structures during the de-differentiation process from protoscoleces to microcysts. Therefore, the Eg-AMPK expression during the asexual development of E. granulosus, as well as the in vitro synergic therapeutic effects observed in presence of Met plus albendazole sulfoxide (ABZSO), suggest the importance of carrying out chemoprophylactic and clinical efficacy studies combining Met with conventional anti-echinococcal agents to test the potential use of this drug in hydatidosis therapy.", "link"=>"http://www.mendeley.com/research/vitro-antiechinococcal-metabolic-effects-metformin-involve-activation-ampactivated-protein-kinase-la", "reader_count"=>19, "reader_count_by_academic_status"=>{"Researcher"=>6, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>4, "Student > Postgraduate"=>4, "Student > Master"=>1, "Student > Bachelor"=>2, "Lecturer"=>1}, "reader_count_by_user_role"=>{"Researcher"=>6, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>4, "Student > Postgraduate"=>4, "Student > Master"=>1, "Student > Bachelor"=>2, "Lecturer"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Biochemistry, Genetics and Molecular Biology"=>2, "Medicine and Dentistry"=>4, "Agricultural and Biological Sciences"=>6, "Immunology and Microbiology"=>5}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>4}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>5}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>6}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}, "Unspecified"=>{"Unspecified"=>2}}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/2064915"], "description"=>"<p>(A) Reverse transcription-PCR analysis of the three subunits of Eg-AMPK (α, β and γ) from total RNA of protoscoleces (PTS) and metacestodes (MTC). Amplification of Eg-<i>act</i>I was used as a loading control. Molecular sizes of amplicons are indicated with arrowheads. (B) Schematic representation of <i>Homo sapiens</i> AMPKα1, AMPKβ2 and AMPKγ1, and of the predicted AMPK protein from the <i>E</i>. <i>granulosus</i> genome. Identification of kinase domain, β-subunit interaction domain (β-SID), autoinhibitory sequence (AIS), α-hook (α-H) and nuclear exportation sequence (black box) in the catalytic subunit; glycogen-binding domain (GBD) and α- and β-subunits interaction domain (αγ-SID) in the β-regulatory subunit; and divergent N-terminal domain (γNTD) and cystathionine-beta-synthase repeats (CBS 1–4) in the γ-regulatory subunit.</p>", "links"=>[], "tags"=>["genes coding", "carbohydrate starvation", "jc", "Echinococcus granulosus Metformin", "hydatidosis therapy", "antiproliferative effects", "intermediary metabolism genes", "activation", "stress conditions", "cancer cells", "energy charge depletion", "liver kinase b 1", "albendazole sulfoxide", "importance", "ampk", "expression", "ABZSO", "granulosus protoscoleces", "acid sequences show", "energy homeostasis", "Larval Stages", "asexual development", "Metabolic Effects", "LKB", "calcareous corpuscles", "Metformin Involve Activation", "Echinococcus granulosus", "immunolocalization assays", "agent", "efficacy studies", "cell energy charge", "subunit", "Genes encoding", "growth pathways", "mitochondrial electron transport chain", "gene coding", "cell survival"], "article_id"=>1412384, "categories"=>["Biological Sciences"], "users"=>["Julia A. Loos", "Andrea C. Cumino"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0126009.g004", "stats"=>{"downloads"=>1, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_and_structural_features_of_the_subunits_of_E_granulosus_AMPK_/1412384", "title"=>"Expression and structural features of the subunits of <i>E</i>. <i>granulosus</i> AMPK.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-05-12 03:46:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/2064907"], "description"=>"<p>(A) Representative confocal images showing JC-1 fluorescence in protoscoleces incubated under control conditions (a-d) or treated with 10 mM metformin (Met, e-h) for 48 h. tg: tegument; rc: rostellar cone; su: sucker; pb posterior bladder. Bars indicate 50 μm. (B) Boxplot graph showing the values of the red/green JC-1 fluorescence ratios measured in control (Co) and Met-treated protoscoleces by Image J Software. ***Statistically significant difference (<i>P</i> < 0.05) compared with control.</p>", "links"=>[], "tags"=>["genes coding", "carbohydrate starvation", "jc", "Echinococcus granulosus Metformin", "hydatidosis therapy", "antiproliferative effects", "intermediary metabolism genes", "activation", "stress conditions", "cancer cells", "energy charge depletion", "liver kinase b 1", "albendazole sulfoxide", "importance", "ampk", "expression", "ABZSO", "granulosus protoscoleces", "acid sequences show", "energy homeostasis", "Larval Stages", "asexual development", "Metabolic Effects", "LKB", "calcareous corpuscles", "Metformin Involve Activation", "Echinococcus granulosus", "immunolocalization assays", "agent", "efficacy studies", "cell energy charge", "subunit", "Genes encoding", "growth pathways", "mitochondrial electron transport chain", "gene coding", "cell survival"], "article_id"=>1412382, "categories"=>["Biological Sciences"], "users"=>["Julia A. Loos", "Andrea C. Cumino"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0126009.g003", "stats"=>{"downloads"=>4, "page_views"=>29, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Metformin_pharmacological_effect_on_mitochondrial_function_in_protoscoleces_/1412382", "title"=>"Metformin pharmacological effect on mitochondrial function in protoscoleces.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-05-12 03:46:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/2064896"], "description"=>"<p>Analysis of Eg-α-amylase (A) and Eg-lactate dehydrogenase (LDH) (B) activities in protoscoleces (PTS) and metacestodes (MTC). (C) Reverse Transcription (RT)-PCR analysis from total RNA of control (Co) or treated (Met) protoscoleces. Amplification of Eg-actin I (<i>act</i>I) was used as a loading control. Molecular sizes of amplicons are indicated with arrowheads. Eg-<i>f1</i>,<i>6bp</i>: fructose-1,6-bisphosphatase, Eg-<i>pepck</i>: phosphoenolpyruvate carboxykinase, Eg-<i>g6p</i>: glucose-6-phosphatase, Eg-<i>mdh</i><sub><i>c</i></sub>: cytoplasmic malate dehydrogenase. (D) Quantitative PCR analysis from total RNA of protoscoleces (PTS) and metacestodes (MTC) treated with Met compared to controls. Fold change expression values are plotted. Data are the mean ± S.D. of three independent experiments. ***Statistically significant difference (<i>P</i> < 0.05) compared with control. (E) Immunoblot of Eg-MDH revealed with a polyclonal antibody. Total protein extracts from control (Co) and Met-treated protoscoleces (Met) were loaded at 100 μg of total protein/lane. Actin was used as a loading control. Polypeptide size is shown.</p>", "links"=>[], "tags"=>["genes coding", "carbohydrate starvation", "jc", "Echinococcus granulosus Metformin", "hydatidosis therapy", "antiproliferative effects", "intermediary metabolism genes", "activation", "stress conditions", "cancer cells", "energy charge depletion", "liver kinase b 1", "albendazole sulfoxide", "importance", "ampk", "expression", "ABZSO", "granulosus protoscoleces", "acid sequences show", "energy homeostasis", "Larval Stages", "asexual development", "Metabolic Effects", "LKB", "calcareous corpuscles", "Metformin Involve Activation", "Echinococcus granulosus", "immunolocalization assays", "agent", "efficacy studies", "cell energy charge", "subunit", "Genes encoding", "growth pathways", "mitochondrial electron transport chain", "gene coding", "cell survival"], "article_id"=>1412371, "categories"=>["Biological Sciences"], "users"=>["Julia A. Loos", "Andrea C. Cumino"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0126009.g002", "stats"=>{"downloads"=>4, "page_views"=>35, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Metabolic_and_transcriptional_changes_induced_by_10_mM_metformin_in_parasites_treated_for_48_h_/1412371", "title"=>"Metabolic and transcriptional changes induced by 10 mM metformin in parasites treated for 48 h.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-05-12 03:46:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/2064920"], "description"=>"<p>(A) Representative immunoblots of Eg-AMPKα and Eg-AMPKα-P<sup>176</sup> revealed with heterologous antibodies against the total and phosphorylated forms of human AMPKα are shown. Total protein extracts from control (Co) and 10 mM metformin-treated protoscoleces (Met) were loaded at 100 μg of total protein/lane. Both Eg-AMPKα and Eg-AMPKα-P<sup>176</sup> were detected (see in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126009#pone.0126009.s003\" target=\"_blank\">S2A Fig</a> the epitopes recognized by each antibody). Polypeptide sizes are shown. (B) Graphs depict the fold change in the p-AMPK/AMPK. Densitometric analysis of Eg-AMPKα-P<sup>176</sup>, normalized to total AMPKα (left) and normalized to actin (right) in protoscoleces treated with 10 mM Met relative to controls (3 independent experiments with 3000 protoscoleces per sample). Values are expressed as means ± SEM (<i>p</i> < 0.05 compared to control). (C) Confocal images of <i>in toto</i> immunolocalization assays revealed with an antibody conjugated with Alexa 488—green fluorescence- and counterstained with propidium iodide—red fluorescence-. Control (a,b) and Met-treated protoscoleces (c,d) incubated with anti-AMPKα antibody (a,c) or anti-AMPKα-P antibody (b,d). Cytoplasmic expression is observed in green (arrows). Nuclear expression is observed in yellow/orange, corresponding to the merged fluorescences (arrowheads). Inset images correspond to transmission microscopy. tg: tegument; su: sucker; cc: calcareous corpuscle. Bars indicate 50 μm.</p>", "links"=>[], "tags"=>["genes coding", "carbohydrate starvation", "jc", "Echinococcus granulosus Metformin", "hydatidosis therapy", "antiproliferative effects", "intermediary metabolism genes", "activation", "stress conditions", "cancer cells", "energy charge depletion", "liver kinase b 1", "albendazole sulfoxide", "importance", "ampk", "expression", "ABZSO", "granulosus protoscoleces", "acid sequences show", "energy homeostasis", "Larval Stages", "asexual development", "Metabolic Effects", "LKB", "calcareous corpuscles", "Metformin Involve Activation", "Echinococcus granulosus", "immunolocalization assays", "agent", "efficacy studies", "cell energy charge", "subunit", "Genes encoding", "growth pathways", "mitochondrial electron transport chain", "gene coding", "cell survival"], "article_id"=>1412389, "categories"=>["Biological Sciences"], "users"=>["Julia A. Loos", "Andrea C. Cumino"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0126009.g005", "stats"=>{"downloads"=>1, "page_views"=>23, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Detection_and_immunolocalization_of_total_and_phosphorylated_Eg_AMPK_forms_from_E_granulosus_protoscoleces_/1412389", "title"=>"Detection and immunolocalization of total and phosphorylated Eg-AMPKα forms from <i>E</i>. <i>granulosus</i> protoscoleces.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-05-12 03:46:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/2064923"], "description"=>"<p>(A) Diagrammatic representation of the different mechanisms involved in cyst development during <i>in vitro</i> culture. Image reconstructed using the figure published by Rogan and Richard (1986) [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126009#pone.0126009.ref029\" target=\"_blank\">29</a>]. This image is similar but not identical to the original image, and is therefore for illustrative purposes only. (B) Transmission and confocal microscopy of a brood capsule (a,b), an everted brood capsule (c,d), protoscoleces with posterior bladders (e,f), developing cysts from posterior bladders (g-j), vesicularized protoscoleces (k-p) and pre-microcysts developed from vesicularized protoscoleces (q-x). bc: brood capsule; ebc: everted brood capsule; pb: posterior bladder; st: stalk; nu: nucleus; dh: disrupted rostellar hook; broken-lined box indicates stalk portion; solid-lined boxes indicate distribution of nuclei in vesicularized protoscoleces. Bars indicate 200 μm (b and d) and 100 μm (f, h, j, l, o, r and u).</p>", "links"=>[], "tags"=>["genes coding", "carbohydrate starvation", "jc", "Echinococcus granulosus Metformin", "hydatidosis therapy", "antiproliferative effects", "intermediary metabolism genes", "activation", "stress conditions", "cancer cells", "energy charge depletion", "liver kinase b 1", "albendazole sulfoxide", "importance", "ampk", "expression", "ABZSO", "granulosus protoscoleces", "acid sequences show", "energy homeostasis", "Larval Stages", "asexual development", "Metabolic Effects", "LKB", "calcareous corpuscles", "Metformin Involve Activation", "Echinococcus granulosus", "immunolocalization assays", "agent", "efficacy studies", "cell energy charge", "subunit", "Genes encoding", "growth pathways", "mitochondrial electron transport chain", "gene coding", "cell survival"], "article_id"=>1412392, "categories"=>["Biological Sciences"], "users"=>["Julia A. Loos", "Andrea C. Cumino"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0126009.g006", "stats"=>{"downloads"=>1, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Immunolocalization_of_Eg_AMPK_during_the_in_vitro_de_differentiation_process_of_protoscoleces_to_microcysts_/1412392", "title"=>"Immunolocalization of Eg-AMPKα during the <i>in vitro</i> de-differentiation process of protoscoleces to microcysts.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-05-12 03:46:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/2064924", "https://ndownloader.figshare.com/files/2064925", "https://ndownloader.figshare.com/files/2064926", "https://ndownloader.figshare.com/files/2064927", "https://ndownloader.figshare.com/files/2064928", "https://ndownloader.figshare.com/files/2064929"], "description"=>"<div><p>Metformin (Met) is a biguanide anti-hyperglycemic agent, which also exerts antiproliferative effects on cancer cells. This drug inhibits the complex I of the mitochondrial electron transport chain inducing a fall in the cell energy charge and leading 5'-AMP-activated protein kinase (AMPK) activation. AMPK is a highly conserved heterotrimeric complex that coordinates metabolic and growth pathways in order to maintain energy homeostasis and cell survival, mainly under nutritional stress conditions, in a Liver Kinase B1 (LKB1)-dependent manner. This work describes for the first time, the <i>in vitro</i> anti-echinococcal effect of Met on <i>Echinococcus granulosus</i> larval stages, as well as the molecular characterization of AMPK (Eg-AMPK) in this parasite of clinical importance. The drug exerted a dose-dependent effect on the viability of both larval stages. Based on this, we proceeded with the identification of the genes encoding for the different subunits of Eg-AMPK. We cloned one gene coding for the catalytic subunit (Eg-<i>ampk</i>ɑ) and two genes coding for the regulatory subunits (Eg-<i>ampk</i>β and Eg-<i>ampk</i>γ), all of them constitutively transcribed in <i>E</i>. <i>granulosus</i> protoscoleces and metacestodes. Their deduced amino acid sequences show all the conserved functional domains, including key amino acids involved in catalytic activity and protein-protein interactions. In protoscoleces, the drug induced the activation of AMPK (Eg-AMPKɑ-P<sup>176</sup>), possibly as a consequence of cellular energy charge depletion evidenced by assays with the fluorescent indicator JC-1. Met also led to carbohydrate starvation, it increased glucogenolysis and homolactic fermentation, and decreased transcription of intermediary metabolism genes. By <i>in toto</i> immunolocalization assays, we detected Eg-AMPKɑ-P<sup>176</sup> expression, both in the nucleus and the cytoplasm of cells as in the larval tegument, the posterior bladder and the calcareous corpuscles of control and Met-treated protoscoleces. Interestingly, expression of Eg-AMPKɑ was observed in the developmental structures during the de-differentiation process from protoscoleces to microcysts. Therefore, the Eg-AMPK expression during the asexual development of <i>E</i>. <i>granulosus</i>, as well as the <i>in vitro</i> synergic therapeutic effects observed in presence of Met plus albendazole sulfoxide (ABZSO), suggest the importance of carrying out chemoprophylactic and clinical efficacy studies combining Met with conventional anti-echinococcal agents to test the potential use of this drug in hydatidosis therapy.</p></div>", "links"=>[], "tags"=>["genes coding", "carbohydrate starvation", "jc", "Echinococcus granulosus Metformin", "hydatidosis therapy", "antiproliferative effects", "intermediary metabolism genes", "activation", "stress conditions", "cancer cells", "energy charge depletion", "liver kinase b 1", "albendazole sulfoxide", "importance", "ampk", "expression", "ABZSO", "granulosus protoscoleces", "acid sequences show", "energy homeostasis", "Larval Stages", "asexual development", "Metabolic Effects", "LKB", "calcareous corpuscles", "Metformin Involve Activation", "Echinococcus granulosus", "immunolocalization assays", "agent", "efficacy studies", "cell energy charge", "subunit", "Genes encoding", "growth pathways", "mitochondrial electron transport chain", "gene coding", "cell survival"], "article_id"=>1412393, "categories"=>["Biological Sciences"], "users"=>["Julia A. Loos", "Andrea C. Cumino"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0126009.s001", "https://dx.doi.org/10.1371/journal.pone.0126009.s002", "https://dx.doi.org/10.1371/journal.pone.0126009.s003", "https://dx.doi.org/10.1371/journal.pone.0126009.s004", "https://dx.doi.org/10.1371/journal.pone.0126009.s005", "https://dx.doi.org/10.1371/journal.pone.0126009.s006"], "stats"=>{"downloads"=>15, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_In_Vitro_Anti_Echinococcal_and_Metabolic_Effects_of_Metformin_Involve_Activation_of_AMP_Activated_Protein_Kinase_in_Larval_Stages_of_Echinococcus_granulosus_/1412393", "title"=>"<i>In Vitro</i> Anti-Echinococcal and Metabolic Effects of Metformin Involve Activation of AMP-Activated Protein Kinase in Larval Stages of <i>Echinococcus granulosus</i>", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-05-12 03:46:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/2064891"], "description"=>"<p>Viability of protoscoleces (A, PTS) and metacestodes (B, MTC) incubated for 10 and 4 days, respectively with 1, 5 and 10 mM of metformin alone (Met, gray bars), 15 μM albendazole sulfoxide (ABZSO, black bars) alone and 1, 5 and 10 mM Met plus 15 μM ABZSO in combination (open bars). Data are the mean ± S.D. of three independent experiments. ***Statistically significant difference (<i>P</i> < 0.05) compared with control. (C) Macroscopical damage of metacestodes treated with 10 mM Met for 4 days. Control metacestodes (Co) without morphological changes and treated metacestodes showing increased permeability (culture medium inside cysts) and collapsed germinal layer (circles). (D) Scanning electron microscopy of protoscoleces (a-d) and metacestodes (e-h) incubated with 10 mM of Met for 4 days. Control protoscolex with normal sucker and microtriches (a,b); treated protoscolex with soma region contracted and scolex region showing loss of hooks and shedding of microtriches (c,d); control murine cyst with an intact germinal layer (e,f); treated cyst with altered germinal layer (g,h). Bars indicate: 10 μm in (b, d and g-h), 20 μm in (a, c and f) and 50 μm (e).</p>", "links"=>[], "tags"=>["genes coding", "carbohydrate starvation", "jc", "Echinococcus granulosus Metformin", "hydatidosis therapy", "antiproliferative effects", "intermediary metabolism genes", "activation", "stress conditions", "cancer cells", "energy charge depletion", "liver kinase b 1", "albendazole sulfoxide", "importance", "ampk", "expression", "ABZSO", "granulosus protoscoleces", "acid sequences show", "energy homeostasis", "Larval Stages", "asexual development", "Metabolic Effects", "LKB", "calcareous corpuscles", "Metformin Involve Activation", "Echinococcus granulosus", "immunolocalization assays", "agent", "efficacy studies", "cell energy charge", "subunit", "Genes encoding", "growth pathways", "mitochondrial electron transport chain", "gene coding", "cell survival"], "article_id"=>1412366, "categories"=>["Biological Sciences"], "users"=>["Julia A. Loos", "Andrea C. Cumino"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0126009.g001", "stats"=>{"downloads"=>2, "page_views"=>32, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_metformin_and_its_combination_with_albendazole_sulfoxide_on_viability_and_ultrastructural_characteristics_of_protoscoleces_and_metacestodes_of_E_granulosus_/1412366", "title"=>"Effect of metformin and its combination with albendazole sulfoxide on viability and ultrastructural characteristics of protoscoleces and metacestodes of <i>E</i>. <i>granulosus</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-05-12 03:46:04"}

PMC Usage Stats | Further Information

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

Relative Metric

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