https://orcid.org/0000-0001-8877-4200
Peer Review History
| Original SubmissionSeptember 13, 2023 |
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PONE-D-23-29797Leishmania Ribosomal Protein (RP) paralogous genes compensate each other’s expression maintaining protein native levelsPLOS ONE Dear Dr. Cruz, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== Your paper has been evaluated by two reviewers. Please revise your paper according to the reviewers' comments. Please also respond to the reviewers' comments in a point-by-point manner that is easy for the reviewers to understand. ============================== Please submit your revised manuscript by Nov 16 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript:
If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Keisuke Hitachi Academic Editor PLOS ONE Journal requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Partly ********** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ********** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ********** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: No ********** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The manuscript entitled "Leishmania Ribosomal Protein (RP) paralogous genes compensate each other’s expression maintaining protein native levels", is a very compact and elegant work that sheds light on the mechanisms of gene expression in trypanomatids, particularly in Leishmania. However, the manuscript can be improved in its presentation. Some suggestions are: 1. Although the authors state that the study focuses on the role of different UTRs in regulation (lines 255-259), rather than function, since approximations are made to the possible non-canonical functions of the ribosomal genes under study (nutritional stress assays), it is suggested: - Point out the a.a. that change between copies of RPL13a in the Introduction. - To perform in silico models of the three-dimensional structure of RPL13a in order to discard possible structural changes affecting the protein function. 2. Avoid repeating the information provided to the reader in the Introduction in the Discussion. 3. The results concerning the Cis elements found in the UTRs are very important and, in fact, are included in the abstract. therefore, I suggest including them in Materials and methods and results and including the figure in the body of the manuscript. 4. Lines 328 to 331 correspond more to the discussion than to the results. 5. Clarify the stage of the parasites in Figure 3C. 6. RT-qPCR results of expression of RPS16 80 and 90 paralogs in mutant vs. wild-type parasites are not shown. 7. As for the discussion, I suggest changing the order. I mean, after presenting the two possible reasons for the parasite to have the same gene with two equal UTRs (lines 465 to 468), continue with the regulation issues, then the functionality, to finish with the compensatory effect. 8. Regardless of the stage, procyclic or metacyclic promastigotes, there is one copy of both genes that is expressed at the protein level more than the other. Could the length of the corresponding UTRs have something to do with it? 9.I wonder what the authors think about what would happen in the amastigote stage in relation to their research questions. I suggest including in the discussion. 10. It would be very interesting for the authors to discuss the possible effect of mutations at the level of promastigote replication, metacyclogenesis, infective capacity and amastigote replication. 11. Revise figures and supplementary material carefully as there are changes in gene or protein names, double legends, etc. 12. Include the database to which readers can refer to look up the raw data from the study, especially the pull-down findings. Reviewer #2: Many ribosomal protein (RP) genes are duplicated in Leishmania. Dr Angela Cruz and coworkers have investigated the expression of the paralogous pairs of RP RPS16 and RPL13a during the transition from normal growth to starvation conditions. They report that the deletion of one of the two paralogous genes leads to increased protein expression from the other, though the corresponding change in mRNA levels is not observed. This agrees with the current model that most regulation of gene expression is post-transcriptional in Leishmania. Interestingly, microscopic experiments indicate that cytoplasmic r-proteins are distributed in several foci, which do not change during the transition from active growth to starvation. Finally, they analyze the ribosome organization through polysome gradient experiments. A peak sedimenting a little faster than the 80S is interpreted to represent a non-ribosomal RP-mRNA complex. The problem is interesting, but several aspects of the work need further investigation prior to publication. Major issues • Please mention the names of the RPs in the universal nomenclature (http://dx.doi.org/10.1016/j.sbi.2014.01.002) in order to facilitate comparison to results with other species. • There are several problems with the polysome experiment (Figure 6) o Show the OD trace from active growth and starvation conditions on comparative scales to allow a (semi-)quantitative comparison of the peaks. o The peak moving slightly faster than the 80S particles runs similarly to the half-mers found in several other species. The authors should do a northern analysis of the fractions, at least in the 80S-disome range. If the authors’ interpretation is correct, no rRNA peak corresponding to the OD trace should be found between the 80S and the disome. • The analysis of proteins binding to the 3’ORF should be continued with washes of incremental salt concentration to determine the hierarchy of affinities. • Line 44: the statement is too general. The are many scries without such gene duplication, e.g., K. lactis and C albicans. • The writing style should be tightened. The current version is quite conversational, at places it goes on extensive excursions for which there is little rigorous evidence (e.g., lines 598-603). • Lines 197-198: samples did not enter the separation gel? How were the proteins then fractionated? Minor issues • The English language is generally good, but a review of the use of articles is suggested. • Recommend more comparisons to yeast expression of paralogous RP. ********** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: Concepcion J Puerta Reviewer #2: No ********** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. |
| Revision 1 |
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PONE-D-23-29797R1Leishmania Ribosomal Protein (RP) paralogous genes compensate each other’s expression maintaining protein native levelsPLOS ONE Dear Dr. Cruz, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== Please respond so that reviewer 2's concerns can be addressed. ============================== Please submit your revised manuscript by Jan 12 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript:
If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Keisuke Hitachi Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: (No Response) ********** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: No ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ********** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ********** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ********** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) Reviewer #2: The authors have addressed some of my issues, but the most serious criticism (Figure 5) stands. I cannot recommend publication until this is resolved. 1. Thank you for referencing the universal nomenclature. This facilitates the comparison to articles about other species. 2. I appreciate that the authors inserted scales for the A254 profiles of the sucrose gradients (Figure 5). However, my comment was not directed towards the comparison of strains, but rather the comparison of the profiles for M199 grown cultures (blue trace) with starved culture (red trace). The former peaks go off the scale and the patterns cannot be compared to the latter. Since the Materials and Methods indicate that 5x108 cells were harvested from both growing and starved cultures the very significant differences in signal intensity of the sucrose gradient A254 traces suggest that much more A254 material from the M199 cultures was loaded on the gradients as compared to the starved cultures. That is, the yield of A254 material was much greater from the M199 cultures than from the starved cultures. Why? Is the ribosome concentration decimated during starvation? A certain loss of ribosome density is expected but the extent indicated by the comparison of the blue and red traces is surprising. Is the lysis of the starved cultures inefficient? This issue should be further investigated by a. tabulating the A254 yield of the cleared lysates and by quantitative northern analysis of rRNA in the lysates b. rerunning the gradients with smaller amounts of material from the M199 cultures. It is essential to see how that trace changes during the starvation procedure. The pattern of the 40S-80S region does not look normal, even for the parental strain. Furthermore, the comparisons of peak heights in the revised manuscript is not understandable at all c. running quantitative northern analysis on samples of all sucrose gradient fractions in order to identify peaks of the individual rRNA species. 3. The authors fail to address the issue of the peak running slightly ahead of the 80S. The peak does certainly not show the expected sedimentation rate of a disome peak (said on the basis of evaluating theoudsans of sucrose gradients over the years). I agree with the authors that the separation of the 80S and named peak is not complete, but running northern analysis of samples from each fraction would show if there is an rRNA peak that co-sediments with the named peak. If the authors are correct about a peak of L13 with mRNA, this should not be the case. The authors spend a lot of the text discussing potential extra-ribosomal functions of ribosomal proteins. Thus, it is essential to settle whether the experiments presented support this idea or not. Other issues 1. Explain abbreviations such as E-64, MTT, TKM, and others throughout. 2. Line 44: human ribosomes have 47 60S ribosomal proteins. 3. Line 253: buffer lysis > lysis buffer. 4. Line 254: something missing after the end of the parenthesis. 5. Line 254: agitation in a pipette; add the type of pipette and how many times, which could affect lysis efficiency. 6. Figure 1: Define asterisks. 7. Line 298: are the insertions also confirmed by sequencing? 8. Quantify blots throughout. 9. Indicate the base value of the log base in Figure 3B. 10. Line 395: I suggest that the text be changed to “which were completely reversed” 11. Figure 4D and Figure 6: What are the units on the histogram scale? 12. Line 431: The 60S peak is already reduced relative to the 40S peak in the pT007 culture. 13. Line 458 and in Figure 5: Free polysome (FP)> polysome free (PF). 14. Figure 5C: Puromicyn> Puromycin. 15. Line 516: not up-to-date; see e.g. papers from Maria Barnes’ group 16. Line 572: The authors should mention the effect of extra-ribosomal proteins on p53 stability. ********** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: Concepción J Puerta Reviewer #2: Yes: Lasse Lindahl ********** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.
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| Revision 2 |
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PONE-D-23-29797R2Leishmania Ribosomal Protein (RP) paralogous genes compensate each other’s expression maintaining protein native levelsPLOS ONE Dear Dr. Cruz, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== Although this is after two revisions, just a few more minor revisions are required. Please respond appropriately before publishing.============================== Please submit your revised manuscript by Feb 03 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript:
If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Keisuke Hitachi Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: (No Response) ********** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes ********** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #2: Yes ********** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: Yes ********** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: The authors have responded to my critique of Figure 5 by eliminating the results of the starvation experiment. I applaud his decision since it will take some time to complete this investigation and the starvation experiment is not essential for interpreting the expression of the L16a and L13 decision alleles. See however below. The authors also addressed many of my comments under “Minor Points” but a few points need to be addressed. 1. Were the myc insertions sequenced? 2. Some blots, e.g. Figure 2c, should be quantified. 3. The estimates of the peak heights in Figure 5 are not done correctly. The baselines for the 40S and 80S peaks is not at the bottom of the “valleys” between peaks, because the different peaks overlap. The best way to compare is to overlay the A254 tracings, line up the corresponding peaks, and graphically adjust the 40S peaks to have equal height. The authors arrive at the correct conclusion, but the procedure in not correct. Here is an example: (See attachment for figure) 4. Line 264-265: Absolute OD is not a proper term, since OD, most correctly called A (absorption), is defined as the log10 of the ratio between the light transmission in a control solution relative to the experimental solution. ********** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: Yes: Lasse Lindahl ********** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.
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| Revision 3 |
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PONE-D-23-29797R3Leishmania Ribosomal Protein (RP) paralogous genes compensate each other’s expression maintaining protein native levelsPLOS ONE Dear Dr. Cruz, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Feb 16 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript:
If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Keisuke Hitachi Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: Thank you for your efforts addressing the revisions. However, the reviewers' requirements have not yet been completely met. Please respond fully to the reviewers' comments before accepting the manuscript. I wish you a Happy New Year. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: (No Response) ********** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: (No Response) ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: (No Response) ********** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #2: (No Response) ********** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: (No Response) ********** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: Thank you for your responses to R2. However, I see two outstanding issues: 1. Please define the calculation and scale for "band skew. I brought this is in my comments on version R1. 2. The myc insertions have not been sequenced. I think this should be done. ********** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: Yes: Lasse Lindahl ********** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. |
| Revision 4 |
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PONE-D-23-29797R4Leishmania Ribosomal Protein (RP) paralogous genes compensate each other’s expression maintaining protein native levelsPLOS ONE Dear Dr. Cruz, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Mar 07 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript:
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If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: As per the reviewer's comments, please correctly describe the scale. Also, please add the addition to Methods, as noted in the comments. The reviewer's comment about sequencing is valid, and I ask that you respond to it. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: (No Response) ********** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Partly ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes ********** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #2: Yes ********** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: (No Response) ********** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: Critique of PONE-D-23-29797_R3. Reviewer #2: Thank you for your responses to R2. However, I see two outstanding issues: 1. Please define the calculation and scale for "band skew”. I brought this is in my comments on version R1. We apologize for the laconic explanation given on R1. The band intensity on western blots was determined by ImageJ using “ROI manager” (ROI = Regions of Interest); the tool allows quantification of all bands, based on their size and intensity. Inside the ImageJ software, the array can be segmented into ROIs of identical size, where each ROI corresponds to one protein, certifying that the same area was considered for future comparisons between the samples. Thus, band skew shows how much the band intensity decreased comparing the RP expression in the presence or absence of its paralogue (Nat Protoc. 2016 August; 11(8): 1508–1530. doi:10.1038/nprot.2016.089). Reviewer’s reply and comment to the authors answers to R3 comments I know the standard procedure. However, "Band Skew" is, to my knowledge, not a standard term (I did not know it and I have read a lot of papers and worked in the field for a long time). As such Band Skew should be defined in Materials and Methods. And the scale should be defined (log2 or log10 change of expression). Note that I twice specifically requested that the scale be defined. 2. The myc insertions have not been sequenced. I think this should be done. We will ask reviewer #2 to reconsider this specific demand for the reasons below. We agree that sequencing knockouts, tagging or any genome editing is the most robust form of confirming the expected modification, without any further undesirable event, or off-target insertion. Nevertheless, I want to raise some points that might convince Reviewer #2 that, in this case specifically, we have robust evidence that both knockout and myc tags occurred as aimed. First aspect to consider is that genome editing in Leishmania has been first achieved in 1990, using the classical method of gene replacement that had been previously employed for Saccharomyces cerevisiae. We (Cruz and Beverley, 1990) pioneered the field of Leishmania genetic manipulation when we showed that “Following introduction of a construct containing dihydrofolate reductase–thymidylate synthase (dhfr-ts) flanking sequences fused to neomycin phosphotransferase, 45% of the colonies contained the planned homologous replacement; this frequency rose to nearly 100% in transfections using low amounts of DNA. Integrative transfection in Leishmania thus resembles that of Saccharomyces cerevisae in giving predominantly homologous events.” (http://dx.doi.org/10.1038/348171a0). This pioneer work demonstrated that the parasite has a highly efficient machinery of homologous recombination, which largely differs from most eukaryotes; insertion does not occur randomly and even when we exceeded in the amount of DNA to be transfected, what we observed was multiple insertions of the fragment in the homologous sequence/aimed site (same article above). The establishment of the CRISPR-Cas9 in these parasites (doi: 10.1098/rsos.170095) came to facilitate the previously labor-intensive and time-consuming approach, and represents a significant advance in the field. Even after 6 years of CRISPR/Cas9 use in Leishmania, we find no publications reporting off-target editing in Leishmania, and there are authors that report complete sequencing of the genome of transfected parasite, with no off-target events, this is probably partially due to the intrinsic genetic features of the organism. In addition to the abovementioned, we must consider that the results we report now compose a robust indication that myc insertion occurred in the expected genomic site. The tag insertion in the RP genes studied in our work was also confirmed based on immune detection, as we used S16 antibody; the subcellular distribution of the tagged proteins using α-myc antibody was undistinguishable from the distribution of RPS16 protein detected by a specific α-S16 antibody (see supplementary material SF2). We have also used S16 polyclonal antibody in WB and the pattern was the same (not shown). The compensatory effect that maintains protein levels at similar levels comparing KO with wild type parasites was coherent, for all the paralogues (S16 and L13), showing that we are dealing with the protein we aimed to tag. For these reasons, we believe that the conventional PCR was an adequate tool in this study and confirmed the high efficiency of homologous recombination in Leishmania parasites, for both knockout and tagged parasites generation. Reviewer reply I am fully convinced of the authors’ experience in manipulating the Leishmania genome. However, my concern is not whether insertions and tags were successfully placed in the S16 genes. Rather, in spite of the efficiency of CRISPR, it seems possible that the target sequence is not copied accurately into the genome, which could lead to a change of the amino acid sequence of the tag, and thus a change in the antibody affinity for the tag. This should not affect the relative expression of a specific paralog but may affect the estimate of the relative expression of the two paralogs. Can the authors conclusively refute this scenario? ********** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: Yes: Lasse Lindahl ********** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. 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| Revision 5 |
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Leishmania Ribosomal Protein (RP) paralogous genes compensate each other’s expression maintaining protein native levels PONE-D-23-29797R5 Dear Dr. Cruz, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Alexander F. Palazzo, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: All comments have been addressed ********** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes ********** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #2: Yes ********** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: Yes ********** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: Thank you for explaining band skew and the accuracy of the myc tag in your revision. I accept your response to my comments. ********** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: Yes: Lasse Lindahl ********** |
| Formally Accepted |
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PONE-D-23-29797R5 PLOS ONE Dear Dr. Cruz, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Alexander F. Palazzo Academic Editor PLOS ONE |
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