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of genes / QTLs, which is already represented in column 1) and add the most striking phenotypic data, when possible and relevant for discussion.
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The ms is the result of an intensive and years-long work of breeding, that eventually pyramidized several resistance genes and QTLs for abiotic traits into an indian elite rice variety.
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plants11050622_makarova
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The new text does not conceptually link PTMs with PGRMC1 protein stability, which is the only reason they should be mentioned.
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What characterisation was performed to confirm the identity of cells as ESCs?
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reprodmed3020015_makarova
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SEM is a function of how many measurements were made, and does not reflect sample variability.
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It is important to determine whether these mechanisms are both operating.
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reprodmed3020015_makarova
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Line 207: “The miRNA target prediction database, miRDB (http://www.mirdb.org), was screened to identify miRNAs with potential to interact with PGRMC1.” miR-98 is already known to regulate PGRMC1.
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The western blot (also Fig.3) result must show the entire molecular weight range (e.g.
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reprodmed3020015_makarova
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If siRNA-resistant PGRMC1 levels are able to inhibit decidualization it would provide convincing evidence in favour of the hypothesis that lowered PGRMC1 is required for decidualization, and is perhaps even a trigger.
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Suggest change to “In the present study, we showed that microRNA-mediated the regulation of PGRMC1 abundance during the process of decidualization.” (if this is what is meant) Lines 271-76.
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reprodmed3020015_makarova
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This is a critical point because the very title claims that PGRMC1 downregulation is required for decidualization.
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If further mechanism exploration is carried out, the research will be more complete and persuasive.
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reprodmed3020015_makarova
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Translational level changes can be due to changed levels of transcription, to altered mRNA stability (such as that proposed here for miR-98), or to altered efficiency of translation of a steady state mRNA pool.
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The conclusion has been verified by other 2-3 articles, and the design of this study is not rigorous enough.
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reflects variability between replicate measurements.
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The conclusion has been verified by other 2-3 articles, and the design of this study is not rigorous enough.
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reprodmed3020015_makarova
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4) Lines 263-267: “Because the effect of AG-205 and PGRMC1 knock-down on decidualization was common in the present study, these findings further support for a role for PGRMC1 downregulation in promoting ESC decidualization during the secretory phase.” The reviewer does not understand the sentence.
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This is a critical point because the very title claims that PGRMC1 downregulation is required for decidualization.
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reprodmed3020015_makarova
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In general, there is very little detail on these primary cultured lines.
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At the very least this mechanism merits detailed discussion in that section.
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reprodmed3020015_makarova
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The authors have addressed most of my concerns.
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Line 207: “The miRNA target prediction database, miRDB (http://www.mirdb.org), was screened to identify miRNAs with potential to interact with PGRMC1.” miR-98 is already known to regulate PGRMC1.
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reprodmed3020015_makarova
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Appropriate human ethics approvals were obtained.
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The new text does not conceptually link PTMs with PGRMC1 protein stability, which is the only reason they should be mentioned.
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reprodmed3020015_makarova
0
It is important to determine whether these mechanisms are both operating.
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What characterisation was performed to confirm the identity of cells as ESCs?
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reprodmed3020015_makarova
0
Line 207: “The miRNA target prediction database, miRDB (http://www.mirdb.org), was screened to identify miRNAs with potential to interact with PGRMC1.” miR-98 is already known to regulate PGRMC1.
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We are told in the methods that primary cell cultures were obtained from 3 patients (see lines 88-89).
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reprodmed3020015_makarova
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Author Response Responses to Reviewer 2 We appreciate your review of our manuscript and constructive comments and suggestions.
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The manuscript explored the mechanisms of PGRMC1 regulation during the decidualization of human ESCs.
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reprodmed3020015_makarova
0
And please provide basic information of the patient.
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The authors must refer to the fact that AG-205 is not specific for PGRMC1, contrary to many citations in the literature (such as would be the case here, if uncorrected).
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reprodmed3020015_makarova
0
Such changes may occur at the translational or post-translational level.
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However, the effects of P4 in the absence of db-cAMP would also require control in cells without db-cAMP, +/- P4.
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reprodmed3020015_makarova
0
This is a critical point because the very title claims that PGRMC1 downregulation is required for decidualization.
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In fact, proteasomal degradation could be much more important that miRNA reduction of the transcript in the reduction of PGRMC1 levels during decidualization.
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reprodmed3020015_makarova
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It would be possible to introduce an expression cassette via a lentiviral vector at MOI 1.
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Line 261. mechanisms HAVE not (plural mechanisms) Lines 261-262.
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reprodmed3020015_makarova
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Cite PMID: 34944026, PMID: 34680104, PMID: 32924377.
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The authors mention technical difficulties in establishing ESCs that express miRNA-resistant PGRMC1.
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reprodmed3020015_makarova
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This is a critical point because the very title claims that PGRMC1 downregulation is required for decidualization.
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The paragraph describes a result showing that the results are independent of P4.
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reprodmed3020015_makarova
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Appropriate human ethics approvals were obtained.
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Aspiration to clarify this in future papers does not merit publication of the present paper.
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reprodmed3020015_makarova
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Downregulation of PGRMC1 During ESC in vitro Decidualization.” This seems to be the next section heading, in which case it should follow a line break, and say: “3.2.
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It would be possible to introduce an expression cassette via a lentiviral vector at MOI 1.
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reprodmed3020015_makarova
0
SEM is a function of how many measurements were made, and does not reflect sample variability.
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The new text does not conceptually link PTMs with PGRMC1 protein stability, which is the only reason they should be mentioned.
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reprodmed3020015_makarova
0
We are told in the methods that primary cell cultures were obtained from 3 patients (see lines 88-89).
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In response the authors have made minimal changes to the manuscript, effectively restricted to rewording the title, and several minor text changes.
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reprodmed3020015_makarova
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Assuming binomial distribution, a two-way ANOVA is required for these experiments.
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PGRMC1 (Hpr6.6) increased the rate of cell death (in a non-apoptotic mechanism) in MCF-7 cancer cells in response to H2O2 (PMID: 14523988).
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reprodmed3020015_makarova
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It is suggested to add cell lines or normal endometrial cells for verification.
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Critically, these figures compare the effects of two variables each, and therefor one way ANOVA is not the appropriate test.
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reprodmed3020015_makarova
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4) Lines 263-267: “Because the effect of AG-205 and PGRMC1 knock-down on decidualization was common in the present study, these findings further support for a role for PGRMC1 downregulation in promoting ESC decidualization during the secretory phase.” The reviewer does not understand the sentence.
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Critically, these figures compare the effects of two variables each, and therefor one way ANOVA is not the appropriate test.
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reprodmed3020015_makarova
0
However, as described above AG-205 is not a specific inhibitor of PGRMC1.
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If higher PGRMC1 levels are indeed critical for the suppression of decidualization, as is reasoned by the authors, then it should be possible to maintain PGRMC1 levels artificially to inhibit decidualization, as discussed below.
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reprodmed3020015_makarova
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Statistical sample sizes were unfavourably small.
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It is important that the involvement of the ubiquitin pathway contribution be either quantified or discarded in well controlled experiments to justify the title of the paper.
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reprodmed3020015_makarova
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Comparison of the miR-98/PGRMC1 abundance in proliferative and secretory phases of endometrial biopsy may contribute to the diagnostic prediction of uterine receptivity for embryo implantation.
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Therefore, a resubmission after major revision is recommended.
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reprodmed3020015_makarova
0
We are told in the methods that primary cell cultures were obtained from 3 patients (see lines 88-89).
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The western blot (also Fig.3) result must show the entire molecular weight range (e.g.
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reprodmed3020015_makarova
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PGRMC1 protein instability could be the dominant effect involved, with miRNA transcript regulation playing only a minor part.
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Figure 2 seems to depict just one cell culture (which is not even named), with 4 replicates of each measurement.
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reprodmed3020015_makarova
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The research has a certain degree of scientific significance.
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And please provide basic information of the patient.
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reprodmed3020015_makarova
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The fact that PGRMC1 levels fall does not mean that low PGRMC1 levels are required.
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That new experimental result is what elevated the required changes to a major revision.
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reprodmed3020015_makarova
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Give the catalogue number of the secondary antibody.
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If higher PGRMC1 levels are indeed critical for the suppression of decidualization, as is reasoned by the authors, then it should be possible to maintain PGRMC1 levels artificially to inhibit decidualization, as discussed below.
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reprodmed3020015_makarova
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Were primary cultures used directly, or were they stored cryogenically?
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What percentage acrylamide were the SDS PAGE gels.
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reprodmed3020015_makarova
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It is also suggested throughout to use “AG-205” (the correct name for the reagent: https://www.sigmaaldrich.com cat#: a1487) rather than AG205 (often incorrectly used in the literature).
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Which ImageJ plug-in was used for quantification?
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reprodmed3020015_makarova
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Author Response We appreciate your review of our revised manuscript, constructive comments, and suggestions, which have helped us to improve the manuscript.
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This could be accomplished by making an expression plasmid encoding a codon-altered PGRMC1 gene, and cotransfecting this in a controlled matrix design with siRNA.
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reprodmed3020015_makarova
0
It is also suggested throughout to use “AG-205” (the correct name for the reagent: https://www.sigmaaldrich.com cat#: a1487) rather than AG205 (often incorrectly used in the literature).
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Author Response We appreciate your review of our revised manuscript, constructive comments, and suggestions, which have helped us to improve the manuscript.
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reprodmed3020015_makarova
0
This new text should be discussing the possibility of the latter also contributing to the observed effects, as well as the effects of mRNA level that the paper pursues.
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The manuscript explored the mechanisms of PGRMC1 regulation during the decidualization of human ESCs.
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reprodmed3020015_makarova
0
“Data of cell culture are expressed as the mean ± SD from three independent experiments with at least repeated two times.” Change to: Data of cell culture are expressed as the mean ± SD from three independent experiments.
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Sensitivity of a phenomenon to AG-205 is consistent with possible PGRMC1 involvement, but does not demonstrate PGRMC1 involvement.
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reprodmed3020015_makarova
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The entire set of experiments needs to be replicated to demonstrate reproducibility before it can be considered to publish these results.
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It would be possible to introduce an expression cassette via a lentiviral vector at MOI 1.
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reprodmed3020015_perova
0
And please provide basic information of the patient.
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Assuming binomial distribution, a two-way ANOVA is required for these experiments.
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reprodmed3020015_perova
0
The discussion must qualify the results of Salsano et al.
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PGRMC1 (Hpr6.6) increased the rate of cell death (in a non-apoptotic mechanism) in MCF-7 cancer cells in response to H2O2 (PMID: 14523988).
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reprodmed3020015_perova
0
Unfortunately, the quality of experimental design and results processing are somewhat problematic.
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Individual mutation of these residues could stabilise PGRMC1 levels to reduce decidualization if miR-98 is only partially responsible for reducing PGRMC1 levels.
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reprodmed3020015_perova
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Author Response Responses to Reviewer 1 We appreciate your review of our manuscript and constructive comments and suggestions.
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Comparison of the miR-98/PGRMC1 abundance in proliferative and secretory phases of endometrial biopsy may contribute to the diagnostic prediction of uterine receptivity for embryo implantation.
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reprodmed3020015_perova
0
Individual mutation of these residues could stabilise PGRMC1 levels to reduce decidualization if miR-98 is only partially responsible for reducing PGRMC1 levels.
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Our responses to your comments are as follows.
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reprodmed3020015_perova
0
“In the present study, we showed that microRNA-mediated PGRMC1 regulation during the process of decidualization.” This is confusingly worded.
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This new text should be discussing the possibility of the latter also contributing to the observed effects, as well as the effects of mRNA level that the paper pursues.
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reprodmed3020015_perova
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Were primary cultures used directly, or were they stored cryogenically?
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This is a critical point because the very title claims that PGRMC1 downregulation is required for decidualisation.
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reprodmed3020015_perova
0
Which ImageJ plug-in was used for quantification?
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It is much preferable to refer to protein abundance, and changes in abundance.
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reprodmed3020015_perova
0
However, the effects of P4 in the absence of db-cAMP would also require control in cells without db-cAMP, +/- P4.
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Individual mutation of these residues could stabilise PGRMC1 levels to reduce decidualization if miR-98 is only partially responsible for reducing PGRMC1 levels.
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reprodmed3020015_perova
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I am uncertain because the methods do not explain how the experiment was performed.
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At the very least this mechanism merits detailed discussion in that section.
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reprodmed3020015_perova
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Such vectors typically include fluorescent protein reporters, enabling discrimination between infected and non-infected cells in one plate, or population separation by FACS.
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The simplest direct comparison would be db-cAMP/P4 vs. db-cAMP to permit the argument the authors are presenting.
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reprodmed3020015_perova
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Since that is by no means certain, the study should not be published in its present form.
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The introduction succinctly provides background relevant for the following research description.
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reprodmed3020015_perova
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Therefore, a resubmission after major revision is recommended.
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Appropriate human ethics approvals were obtained.
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reprodmed3020015_perova
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The authors go on to show that expogenous expression of miR-98 lowers PGRMC1 abundance levels (a more apt description than the fuzzy description of “PGRMC1 downregulation”, which could be by a number of mechanisms as discussed below).
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Such changes may occur at the translational or post-translational level.
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reprodmed3020015_perova
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The experiemtnal design for this conclusion is problematic, as described below.
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Translational level changes can be due to changed levels of transcription, to altered mRNA stability (such as that proposed here for miR-98), or to altered efficiency of translation of a steady state mRNA pool.
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reprodmed3020015_perova
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The latter could well contribute to alterations in PGRMC1 abundance cited by this study.
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The latter could well contribute to alterations in PGRMC1 abundance cited by this study.
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reprodmed3020015_perova
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In fact, proteasomal degradation could be much more important that miRNA reduction of the transcript in the reduction of PGRMC1 levels during decidualization.
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It is much preferable to refer to protein abundance, and changes in abundance.
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reprodmed3020015_perova
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Suggest change to; “Further study is required to determine the relationship between miR-98-mediatd PGRMC1 regulation and pregnancy.
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Which ImageJ plug-in was used for quantification?
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reprodmed3020015_perova
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PGRMC1 levels are also controlled by proteolysis, mediated by the ubiquitin proteasome system as described above.
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Before such a claim is published in the scientific literature, it must be supported by convincing evidence.
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reprodmed3020015_perova
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It is important that the involvement of the ubiquitin pathway contribution be either quantified or discarded in well controlled experiments to justify the title of the paper.
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Suggest change to; “Further study is required to determine the relationship between miR-98-mediatd PGRMC1 regulation and pregnancy.
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reprodmed3020015_perova
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PGRMC1-independe (add t) Lines 263-267: “Because, the effect of AG-205 and PGRMC1 knock-down on decidualization was common in the present study, these findings further support for a role for PGRMC1 downregulation in promoting ESC decidualization during the secretory phase.” The reviewer does not understand the sentence.
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Immunocytochemistry (ICC) of vimentin and cytokeratin in isolated human ESCs.
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reprodmed3020015_perova
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Downregulation of PGRMC1 During ESC in vitro Decidualization.” All subsequent section header numbers in results should be modified to accommodate the new section.
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An inducible vector may be advisable, such as tetracycline, IPTG, or other available systems.
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reprodmed3020015_perova
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What characterisation was performed to confirm the identity of cells as ESCs?
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Suggest change to “In the present study, we showed that microRNA-mediated the regulation of PGRMC1 abundance during the process of decidualization.” (if this is what is meant) Lines 271-76.
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reprodmed3020015_perova
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Suggest change to; “Further study is required to determine the relationship between miR-98-mediatd PGRMC1 regulation and pregnancy.
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However, in the first instance the manuscript cannot be published as it stands, and should therefore be rejected now with the stated option of subsequent resubmission only if new evidence is obtained.
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reprodmed3020015_perova
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If PGRMC1 down-regulation is critical, it should be possible to perturb decidualization by expression of an exogenous PGRMC1 protein.
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Such changes may occur at the translational or post-translational level.
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reprodmed3020015_perova
0
It is also suggested throughout to use “AG-205” (the correct name for the reagent: https://www.sigmaaldrich.com cat#: a1487) rather than AG205 (often incorrectly used in the literature).
null
null
Suggest change to “In the present study, we showed that microRNA-mediated the regulation of PGRMC1 abundance during the process of decidualization.” (if this is what is meant) Lines 271-76.
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reprodmed3020015_perova
0
However, the effects of P4 in the absence of db-cAMP would also require control in cells without db-cAMP, +/- P4.
null
null
The authors go on to show that expogenous expression of miR-98 lowers PGRMC1 abundance levels (a more apt description than the fuzzy description of “PGRMC1 downregulation”, which could be by a number of mechanisms as discussed below).
null
null
reprodmed3020015_perova
0
An inducible vector may be advisable, such as tetracycline, IPTG, or other available systems.
null
null
Our responses to your comments are as follows.
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reprodmed3020015_perova
0
Individual mutation of these residues could stabilise PGRMC1 levels to reduce decidualization if miR-98 is only partially responsible for reducing PGRMC1 levels.
null
null
It is important that the involvement of the ubiquitin pathway contribution be either quantified or discarded in well controlled experiments to justify the title of the paper.
null
null
reprodmed3020015_perova
0
Our responses to your comments are as follows.
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Appropriate human ethics approvals were obtained.
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reprodmed3020015_perova
0
The methods could describe that all subjects were ethnically Japanese/Asian (or otherwise).
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Suggest observations that may be able to discriminate between different possibilities.
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reprodmed3020015_perova
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In fact, proteasomal degradation could be much more important that miRNA reduction of the transcript in the reduction of PGRMC1 levels during decidualization.
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demonstrate that PGRMC1 abundance levels are higher in proliferative phase versus secretory phase endometrial patient biopsies (fig.1).
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reprodmed3020015_perova
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Our responses to your comments are as follows.
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Statistical sample sizes were unfavourably small.
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reprodmed3020015_perova
0
The entire set of experiments needs to be replicated to demonstrate reproducibility before it can be considered to publish these results.
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demonstrate that PGRMC1 abundance levels are higher in proliferative phase versus secretory phase endometrial patient biopsies (fig.1).
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reprodmed3020015_perova
0
This is a critical point because the very title claims that PGRMC1 downregulation is required for decidualisation.
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Author Response We appreciate your review of our revised manuscript, constructive comments, and suggestions, which have helped us to improve the manuscript.
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reprodmed3020015_perova
0
Unfortunately, the authors have been unable to demonstrate the direct involvement of PGRMC1 downregulation in decidualization.
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And please provide basic information of the patient.
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reprodmed3020015_perova
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The manuscript has been modified accordingly.
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The salient point concerning the ubiquitination events is that they are correlated for other proteins with proteasome-mediated degradation.
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reprodmed3020015_perova
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In fact, proteasomal degradation could be much more important that miRNA reduction of the transcript in the reduction of PGRMC1 levels during decidualization.
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Suggest observations that may be able to discriminate between different possibilities.
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reprodmed3020015_perova
0
It has improved the readability, clarity, and quality of our manuscript.
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Thus, the refined GGMs provide better local quasi-geoid results.
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rs14061437_makarova
0
Response to Reviewer 1 Comments: We thank you for your constructive and detailed comments.
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It has improved the readability, clarity, and quality of our manuscript.
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rs14061437_makarova
0
If you have any information, please don’t hesitate to let us know.
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Please comment on that question quantitatifely and qualitatively.
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rs14061437_makarova
0
The point-by-point response can be found from attachment.
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Models Max Min Mean STD EIGEN-6C4 1.007 -1.696 0.048 0.187 GECO 1.579 -1.703 0.041 0.223 SGG-UGM-1 1.003 -1.671 0.052 0.194 SGG-UGM-2 1.003 -1.704 0.051 0.191 XGM2016 1.016 -1.757 -0.020 0.214 XGM2019 1.705 -1.737 0.081 0.213 Point 6: Your method for combining the satellite-only GFM with EGM2008 (sec.
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rs14061437_makarova
0
Email: [email protected] 9 4 National Geomatics Center of China, Beijing 100830, China; [email protected]; [email protected] 10 * Correspondence: [email protected];Tel.
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Author Response We thank you for your constructive and detailed comments.
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rs14061437_makarova
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Introduction 33 With the emergence of Global Navigation Satellite System (GNSS), users can obtain 34 consistent ellipsoidal height at global scale.
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Please comment on that question quantitatifely and qualitatively.
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rs14061437_makarova
0
3.3) seems quite simple for me and probably not optimal.
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I have a few minor points (marked in the attached PDF) and two general points, which I like you to address.
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rs14061437_makarova
0
Importantly, even though all local height datums are re- 42 lated to the MSL, the vertical offsets between them may be up to 2 m at global scale [1].
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The point-by-point response can be found from attachment, please see attachment.
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rs14061437_makarova
0
The point-by-point response can be found from attachment.
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The ellipsoid height relative to a given geo- 35 centric ellipsoid can be obtained quickly and accurately by using GNSS.
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rs14061437_makarova
0
I have a few minor points (marked in the attached PDF) and two general points, which I like you to address.
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Why do you think, that a sharp cut-off SH degree between the satellite-only GFM and EGM2008 is better than a rigorous combination by applying some sophisticated weighting approach?
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rs14061437_perova
0
I have a few minor points (marked in the attached PDF) and two general points, which I like you to address.
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Author Response We thank you for your constructive and detailed comments.
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rs14061437_perova
0
Why do you think, that a sharp cut-off SH degree between the satellite-only GFM and EGM2008 is better than a rigorous combination by applying some sophisticated weighting approach?
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Dear authors, thank you for this interesting and well written study.
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rs14061437_perova
0
Email: [email protected] 9 4 National Geomatics Center of China, Beijing 100830, China; [email protected]; [email protected] 10 * Correspondence: [email protected];Tel.
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The goal of this paper is to derive 13 the geopotential value for the Chinese height datum (CNHD) in order to realize the height datum 14 unification in China.
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rs14061437_perova
0
29 Keywords: Chinese height datum; GRACE/GOCE; residual terrain model; spectral expansion ap- 30 proach; height datum geopotential 31 32 1.
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Point 5: What is the main difference (except of the terrestrial data over China) to other combined GFMs, such as XGM2016 or XGM2019?
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rs14061437_perova
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3.3) seems quite simple for me and probably not optimal.
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Author Response We thank you for your constructive and detailed comments.
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rs14061437_perova
0
We have added descriptions and contents in the revised manuscript.
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8 3 China University of Mining and Technology-Beijing, Beijing 100083, China.
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rs14061437_perova
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If you have any information, please don’t hesitate to let us know.
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However, the 36 ellipsoidal height is not related to the Earth's gravity field.
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rs14061437_perova
0
3.3) seems quite simple for me and probably not optimal.
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: +86-0278-6752107 11 Abstract: A unified height datum is essential for global geographic information resource construc- 12 tion, ecological environment protection and scientific research.
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rs14061437_perova
0
However, I would like to make a few comments.
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- I suggest presenting the paper's aim as the last idea in the introduction to make the manuscript easier to read.
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s22072682_makarova
0