RNase A Does Not Translocate the Alpha-Hemolysin Pore
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{"title"=>"RNase a does not translocate the alpha-hemolysin pore", "type"=>"journal", "authors"=>[{"first_name"=>"Besnik", "last_name"=>"Krasniqi", "scopus_author_id"=>"55377387900"}, {"first_name"=>"Jeremy S.", "last_name"=>"Lee", "scopus_author_id"=>"26643581600"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"24505349", "doi"=>"10.1371/journal.pone.0088004", "sgr"=>"84895115435", "scopus"=>"2-s2.0-84895115435", "issn"=>"19326203", "pui"=>"372510252"}, "id"=>"d0eeae89-b46d-341b-afb1-a6620e2d1f73", "abstract"=>"The application of nanopore sensing utilizing the α-hemolysin pore to probe proteins at single-molecule resolution has expanded rapidly. In some studies protein translocation through the α-hemolysin has been reported. However, there is no direct evidence, as yet, that proteins can translocate the α-hemolysin pore. The biggest challenge to obtaining direct evidence is the lack of a highly sensitive assay to detect very low numbers of protein molecules. Furthermore, if an activity based assay is applied then the proteins translocating by unfolding should refold back to an active confirmation for the assay technique to work. To overcome these challenges we selected a model enzyme, ribonuclease A, that readily refolds to an active conformation even after unfolding it with denaturants. In addition we have developed a highly sensitive reverse transcription polymerase chain reaction based activity assay for ribonuclease A. Initially, ribonuclease A, a protein with a positive net charge and dimensions larger than the smallest diameter of the pore, was subjected to nanopore analysis under different experimental conditions. Surprisingly, although the protein was added to the cis chamber (grounded) and a positive potential was applied, the interaction of ribonuclease A with α-hemolysin pore induced small and large blockade events in the presence and the absence of a reducing and/or denaturing agent. Upon measuring the zeta potential, it was found that the protein undergoes a charge reversal under the experimental conditions used for nanopore sensing. From the investigation of the effect of voltage on the interaction of ribonuclease A with the α-hemolysin pore, it was impossible to conclude if the events observed were translocations. However, upon testing for ribonuclease A activity on the trans chamber it was found that ribonuclease A does not translocate the α-hemolysin pore.", "link"=>"http://www.mendeley.com/research/rnase-not-translocate-alphahemolysin-pore", "reader_count"=>21, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>3, "Student > Doctoral Student"=>2, "Researcher"=>1, "Student > Ph. D. Student"=>7, "Student > Postgraduate"=>1, "Student > Master"=>2, "Student > Bachelor"=>5}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>3, "Student > Doctoral Student"=>2, "Researcher"=>1, "Student > Ph. D. Student"=>7, "Student > Postgraduate"=>1, "Student > Master"=>2, "Student > Bachelor"=>5}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>2, "Agricultural and Biological Sciences"=>4, "Physics and Astronomy"=>6, "Chemical Engineering"=>1, "Chemistry"=>3, "Computer Science"=>1, "Engineering"=>4}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>4}, "Chemistry"=>{"Chemistry"=>3}, "Physics and Astronomy"=>{"Physics and Astronomy"=>6}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>4}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}, "Chemical Engineering"=>{"Chemical Engineering"=>1}}, "reader_count_by_country"=>{"United States"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1374528"], "description"=>"<p>Blockade current histograms obtained for (A) natively folded RNase A, (B) natively folded RNase A after being subjected to size exclusion and ion exchange chromatography, (C) reduced RNase A, (D) RNase A in presence of 1 M GdnHCl, (E) reduced RNase A in presence of 1 M GdnHCl, and (F) completely unfolded RNase A. For the analysis of completely unfolded RNase A, the protein was pre-incubated in 4 M GdnHCl and 100 mM TCEP prior to adding it to the <i>cis</i> chamber. Each event population is fitted with the Gaussian function to obtain the peak/population blockade current value. The peak blockade current values are presented in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088004#pone-0088004-t002\" target=\"_blank\">Table 2</a>. All analysis were performed at 100 mV.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "ion channels", "Transmembrane transport proteins", "biophysics", "Protein folding", "genetics", "Human genetics", "Chromosomal disorders", "translocations", "Electrochemistry", "Electrochemical cells", "electrodes", "nanotechnology", "Nanomaterials", "Particle physics", "Electric charge", "rnase"], "article_id"=>924813, "categories"=>["Physics", "Biological Sciences", "Medicine", "Chemistry"], "users"=>["Besnik Krasniqi", "Jeremy S. Lee"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0088004.g003", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Nanopore_analysis_of_RNase_A_under_different_experimental_conditions_/924813", "title"=>"Nanopore analysis of RNase A under different experimental conditions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-02-04 02:43:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/1374527"], "description"=>"<p>In (A) the absence of GdnHCl the frequency of events is lower than (B) in the presence of GdnHCl. The open pore current is higher in presence of GdnHCl as a result of higher conductivity of GdnHCl. Note the increase in frequency of the events and the change in proportion of large blockade events in the presence of GdnHCl.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "ion channels", "Transmembrane transport proteins", "biophysics", "Protein folding", "genetics", "Human genetics", "Chromosomal disorders", "translocations", "Electrochemistry", "Electrochemical cells", "electrodes", "nanotechnology", "Nanomaterials", "Particle physics", "Electric charge", "traces", "rnase", "pore", "100"], "article_id"=>924812, "categories"=>["Physics", "Biological Sciences", "Medicine", "Chemistry"], "users"=>["Besnik Krasniqi", "Jeremy S. Lee"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0088004.g002", "stats"=>{"downloads"=>0, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Segments_of_current_traces_for_the_interaction_of_RNase_A_with_the_945_hemolysin_pore_at_100_mV_/924812", "title"=>"Segments of current traces for the interaction of RNase A with the α-hemolysin pore at 100 mV.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-02-04 02:43:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/1374536"], "description"=>"<p>RNase A zeta potentials in various buffers and pHs.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "ion channels", "Transmembrane transport proteins", "biophysics", "Protein folding", "genetics", "Human genetics", "Chromosomal disorders", "translocations", "Electrochemistry", "Electrochemical cells", "electrodes", "nanotechnology", "Nanomaterials", "Particle physics", "Electric charge", "zeta", "potentials", "buffers"], "article_id"=>924821, "categories"=>["Physics", "Biological Sciences", "Medicine", "Chemistry"], "users"=>["Besnik Krasniqi", "Jeremy S. Lee"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0088004.t004", "stats"=>{"downloads"=>0, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_RNase_A_zeta_potentials_in_various_buffers_and_pHs_/924821", "title"=>"RNase A zeta potentials in various buffers and pHs.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-02-04 02:43:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/1374535"], "description"=>"<p>Lanes 1 and 2 are the solutions collected from the <i>cis</i> and <i>trans</i> chambers, respectively, before adding RNase A and while applying a potential of 100 mV. Lanes 3 and 4 are the solutions collected from cis and <i>trans</i> chambers, respectively, after adding RNase A to the <i>cis</i> chamber and under no applied voltage. Lanes 5 and 6 are similar to 3 and 4, respectively, but there was 100 mV applied. There was no lipid bilayer painted over the 150 µm aperture. Lanes 6 and 7 are positive and negative controls, respectively, for RT-PCR.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "ion channels", "Transmembrane transport proteins", "biophysics", "Protein folding", "genetics", "Human genetics", "Chromosomal disorders", "translocations", "Electrochemistry", "Electrochemical cells", "electrodes", "nanotechnology", "Nanomaterials", "Particle physics", "Electric charge", "rnase", "150"], "article_id"=>924820, "categories"=>["Physics", "Biological Sciences", "Medicine", "Chemistry"], "users"=>["Besnik Krasniqi", "Jeremy S. Lee"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0088004.g009", "stats"=>{"downloads"=>0, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Translocation_of_RNase_A_through_the_150_181_m_aperture_/924820", "title"=>"Translocation of RNase A through the 150 µm aperture.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-02-04 02:43:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/1374532"], "description"=>"<p>Lanes 1 to 8 indicate the concentrations of RNase A. Lanes 9 is the positive control for RT-PCR which contains no RNase A. The negative control for RT-PCR, lane 10, contains no RNase A or mRNA. The concentration of mRNA is the same in lanes 1 through 9.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "ion channels", "Transmembrane transport proteins", "biophysics", "Protein folding", "genetics", "Human genetics", "Chromosomal disorders", "translocations", "Electrochemistry", "Electrochemical cells", "electrodes", "nanotechnology", "Nanomaterials", "Particle physics", "Electric charge", "detection", "rt-pcr", "assay", "rnase"], "article_id"=>924817, "categories"=>["Physics", "Biological Sciences", "Medicine", "Chemistry"], "users"=>["Besnik Krasniqi", "Jeremy S. Lee"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0088004.g006", "stats"=>{"downloads"=>0, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_detection_limit_of_the_RT_PCR_based_detection_assay_for_RNase_A_/924817", "title"=>"The detection limit of the RT-PCR based detection assay for RNase A.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-02-04 02:43:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/1374531"], "description"=>"<p>Each individual population of events shown in the current blockade histograms (<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088004#pone-0088004-g004\" target=\"_blank\">Figure 4</a>) is fitted with a single exponential decay function to obtain the duration times (dwell times) for each respective population. Panels A and B show the lifetimes of the events forming the large and small blockade populations, respectively, at 50 mV. Panels C and D show the lifetimes of the events forming the large and small blockade populations, respectively, at 100 mV. Panels E and F show the lifetimes of the events forming the large and small blockade populations, respectively, at 150 mV. The duration time values for each voltage are presented in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088004#pone-0088004-t003\" target=\"_blank\">Table 3</a>.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "ion channels", "Transmembrane transport proteins", "biophysics", "Protein folding", "genetics", "Human genetics", "Chromosomal disorders", "translocations", "Electrochemistry", "Electrochemical cells", "electrodes", "nanotechnology", "Nanomaterials", "Particle physics", "Electric charge", "histograms", "rnase", "150"], "article_id"=>924816, "categories"=>["Physics", "Biological Sciences", "Medicine", "Chemistry"], "users"=>["Besnik Krasniqi", "Jeremy S. Lee"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0088004.g005", "stats"=>{"downloads"=>0, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Blockade_time_histograms_for_RNase_A_at_50_100_and_150_mV_/924816", "title"=>"Blockade time histograms for RNase A at 50, 100, and 150 mV.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-02-04 02:43:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/1374529"], "description"=>"<p>Blockade current histograms obtained for RNase A at (A) 50 mV, (B) 100 mV, and (C) 150 mV.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "ion channels", "Transmembrane transport proteins", "biophysics", "Protein folding", "genetics", "Human genetics", "Chromosomal disorders", "translocations", "Electrochemistry", "Electrochemical cells", "electrodes", "nanotechnology", "Nanomaterials", "Particle physics", "Electric charge", "voltage", "rnase"], "article_id"=>924814, "categories"=>["Physics", "Biological Sciences", "Medicine", "Chemistry"], "users"=>["Besnik Krasniqi", "Jeremy S. Lee"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0088004.g004", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_voltage_on_the_interaction_of_RNase_A_with_the_945_hemolysin_pore_/924814", "title"=>"Effect of voltage on the interaction of RNase A with the α-hemolysin pore.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-02-04 02:43:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/1374526"], "description"=>"<p>First, a nanopore experiment is conducted and at the end the solution from each chamber is collected and transferred to a microcentrifuge tube. Second, mRNA is added to the solution collected in step 1 and incubated for 24°C. Third, after incubation the solution from step 2 is used as source of template RNA for RT-PCR reaction. Fourth, RT-PCR is performed. In the fifth step, the end product from RT-PCR is run on an agarose gel. If there is RNase A present in solutions collected in step 1 then there will be a faint band or no band (depending on RNase A quantity) on the agarose gel.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "ion channels", "Transmembrane transport proteins", "biophysics", "Protein folding", "genetics", "Human genetics", "Chromosomal disorders", "translocations", "Electrochemistry", "Electrochemical cells", "electrodes", "nanotechnology", "Nanomaterials", "Particle physics", "Electric charge", "detection"], "article_id"=>924811, "categories"=>["Physics", "Biological Sciences", "Medicine", "Chemistry"], "users"=>["Besnik Krasniqi", "Jeremy S. Lee"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0088004.g001", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_RNase_A_detection_workflow_/924811", "title"=>"RNase A detection workflow.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-02-04 02:43:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/1374538"], "description"=>"a<p>I<sub>1</sub>, I<sub>2</sub>, I<sub>3</sub>, T<sub>1</sub>, T<sub>2</sub>, and T<sub>3</sub> represent the amplitudes and the durations of the current blockades of the respective event populations presented in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088004#pone-0088004-g003\" target=\"_blank\">Figure 3</a>. A<sub>1</sub>, A<sub>2</sub>, and A<sub>3</sub> are the percent of total events forming each respective population. The peaks are numbered from right to left. A dash indicates the absence of third event population.</p>b<p>The error is estimated to be ±1 pA.</p>c<p>The error is estimated to be ±10%.</p>d<p>The error is estimated to be ±1%.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "ion channels", "Transmembrane transport proteins", "biophysics", "Protein folding", "genetics", "Human genetics", "Chromosomal disorders", "translocations", "Electrochemistry", "Electrochemical cells", "electrodes", "nanotechnology", "Nanomaterials", "Particle physics", "Electric charge", "rnase", "pore"], "article_id"=>924823, "categories"=>["Physics", "Biological Sciences", "Medicine", "Chemistry"], "users"=>["Besnik Krasniqi", "Jeremy S. Lee"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0088004.t002", "stats"=>{"downloads"=>0, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Interaction_parameters_of_RNase_A_with_the_945_hemolysin_pore_under_various_experimental_conditions_/924823", "title"=>"Interaction parameters of RNase A with the α-hemolysin pore under various experimental conditions.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-02-04 02:43:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/1374539"], "description"=>"a<p>I<sub>1</sub>, I<sub>2</sub>, T<sub>1</sub>, and T<sub>2</sub>, represent the amplitudes and the durations of the current blockades of the respective event populations presented in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088004#pone-0088004-g004\" target=\"_blank\">Figure 4</a>. A<sub>1</sub>, and A<sub>2</sub> are the percent of total events forming each respective population. The peaks are numbered from right to left.</p>b<p>The error is estimated to be ±1 pA.</p>c<p>The error is estimated to be ±10%.</p>d<p>The error is estimated to be ±1%.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "ion channels", "Transmembrane transport proteins", "biophysics", "Protein folding", "genetics", "Human genetics", "Chromosomal disorders", "translocations", "Electrochemistry", "Electrochemical cells", "electrodes", "nanotechnology", "Nanomaterials", "Particle physics", "Electric charge", "voltage", "rnase"], "article_id"=>924824, "categories"=>["Physics", "Biological Sciences", "Medicine", "Chemistry"], "users"=>["Besnik Krasniqi", "Jeremy S. Lee"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0088004.t003", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_voltage_on_the_interaction_of_RNase_A_with_the_945_hemolysin_pore_/924824", "title"=>"Effect of voltage on the interaction of RNase A with the α-hemolysin pore.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-02-04 02:43:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/1374537"], "description"=>"<p>The primer sequences.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "ion channels", "Transmembrane transport proteins", "biophysics", "Protein folding", "genetics", "Human genetics", "Chromosomal disorders", "translocations", "Electrochemistry", "Electrochemical cells", "electrodes", "nanotechnology", "Nanomaterials", "Particle physics", "Electric charge", "primer"], "article_id"=>924822, "categories"=>["Physics", "Biological Sciences", "Medicine", "Chemistry"], "users"=>["Besnik Krasniqi", "Jeremy S. Lee"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0088004.t001", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_primer_sequences_/924822", "title"=>"The primer sequences.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-02-04 02:43:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/1374534"], "description"=>"<p>Lanes 1 and 2 represent the solutions collected from <i>cis</i> and <i>trans</i> chambers, respectively, before adding RNase A. Lanes 3 and 4 represent the solutions collected from cis and trans chambers, respectively, after adding RNase A to the cis chamber and conducting a nanopore experiment. The solutions used for lanes 3 and 4 were collected after the nanopore experiment and while the lipid bilayer separating the two chambers was still intact. Lane 5 represents a control for α-hemolysin solution used in the nanopore experiment where the α-hemolysin solution was tested for RNase A activity. Lanes 6 and 7 are positive and negative controls, respectively, for RT-PCR.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "ion channels", "Transmembrane transport proteins", "biophysics", "Protein folding", "genetics", "Human genetics", "Chromosomal disorders", "translocations", "Electrochemistry", "Electrochemical cells", "electrodes", "nanotechnology", "Nanomaterials", "Particle physics", "Electric charge", "detection", "rnase"], "article_id"=>924819, "categories"=>["Physics", "Biological Sciences", "Medicine", "Chemistry"], "users"=>["Besnik Krasniqi", "Jeremy S. Lee"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0088004.g008", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_RT_PCR_based_detection_of_RNase_A_in_the_trans_chamber_/924819", "title"=>"RT-PCR based detection of RNase A in the <i>trans</i> chamber.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-02-04 02:43:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/1374533"], "description"=>"<p>(A) Lanes 3 and 4 show reverse transcription of mRNA when there are Ag/AgCl electrodes immersed in the solution, whereas lanes 1 and 2 show reverse transcription of mRNA when there are no electrodes immersed in solution. (B) Lanes 1 and 2 show reverse transcription of mRNA when there are agarose salt bridges immersed in the solution instead of Ag/AgCl electrodes. Lanes 3 and 4 show the <i>cis</i> and <i>trans</i> solutions, respectively, after adding RNase A to the <i>cis</i> chamber with the lipid bilayer membrane separating the two chambers.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "ion channels", "Transmembrane transport proteins", "biophysics", "Protein folding", "genetics", "Human genetics", "Chromosomal disorders", "translocations", "Electrochemistry", "Electrochemical cells", "electrodes", "nanotechnology", "Nanomaterials", "Particle physics", "Electric charge", "rnase", "detection"], "article_id"=>924818, "categories"=>["Physics", "Biological Sciences", "Medicine", "Chemistry"], "users"=>["Besnik Krasniqi", "Jeremy S. Lee"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0088004.g007", "stats"=>{"downloads"=>0, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_Ag_AgCl_electrodes_on_the_RNase_A_detection_assay_/924818", "title"=>"Effect of Ag/AgCl electrodes on the RNase A detection assay.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-02-04 02:43:23"}

PMC Usage Stats | Further Information

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Relative Metric

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