Non-destructive DNA extraction for recovering mitochondrial genomes from museum grasshopper specimens

Hao Tang; Jiayi Zou; Keyao Zhang; Huateng Huang

doi:10.1371/journal.pone.0341621

Peer Review History

Original SubmissionJune 4, 2025
24 Jul 2025 Decision Letter - Marta Ciucani, Editor Dear Dr. Huang, Please submit your revised manuscript by Sep 07 2025 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. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. 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Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. [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? Reviewer #1: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: No ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: No Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: The paper by Tang et al presents a well-designed non-destructive DNA extraction protocol for museum grasshopper specimens. While the methodological advancements are clear, two major limitations should be addressed to strengthen the paper’s contribution. First, the study lacks a rigorous assessment of endogenous DNA content. Although exogenous contamination is quantified using Kraken2, the proportion of authentic grasshopper DNA in the extracts remains unverified. Given the high variability in contamination levels (50% in some specimens), this raises concerns about the protocol’s efficiency in retrieving target DNA. Incorporating negative controls during extraction and quantifying endogenous DNA would provide critical validation. Second, the title suggests a focus on mitochondrial genomes, yet the paper does not leverage these data for evolutionary or ecological insights. While the assembly of Stenocrobylus festivus mitogenomes is a valuable proof of concept, the opportunity to explore phylogenetic relationships, temporal genetic shifts, or comparative analyses with modern specimens is missed. To align the manuscript’s scope with its title, the authors should either reframe the title to emphasize methodology or include preliminary evolutionary analyses, such as a phylogeny or selection tests, to justify the current title. Reviewer #2: The manuscript titled "Mitochondrial Genomes from Museum Grasshopper Specimens via Non-Destructive DNA Extraction," which addresses a timely and relevant topic in the field of museum genomics and minimally destructive DNA extraction from historical specimens. It presents a potentially valuable approach for recovering mitogenomes from museum specimens, however, in its current form, I do not consider the manuscript suitable for publication. The study contains several methodological and interpretative issues that undermine the scientific robustness and clarity of the work. I suggest revising these points about the scientific advancements: 1) Since the manuscript is intended as a methodological paper, the experimental procedures, including design, sampling, library preparation, and data analysis, should be described in much greater detail to allow proper assessment and reproducibility. As it stands, the methodological sections, in particular library preparation or data analysis, are too sparse to support a clear methodological advancement. 2) I also suggest moving the table S1 to the main text. 3) The diagram of the DNA extraction workflow (Figure S1) is well designed and highly informative. However, I recommend that the authors ensure full consistency between Figure S1, the main text, and Supplementary Table S1 with respect to the naming or labeling of the extraction methods applied to each sample. Currently, there appear to be some ambiguities in how the methodologies are referred to across different parts of the manuscript and supplementary materials, which could make it difficult for readers to follow or reproduce the experimental design. 4) This sentence can be better explained or reformulated: l. 84 “Due to the size constraints of a 1.5 ml centrifuge tube, the hind leg of each grasshopper specimen was initially removed”. It is not clear if the leg was totally removed from the tube, or when it was placed back in the tube. 5) Statistical support: some of the reported improvements are not statistically significant (e.g., P = 0.06), and yet are described in the text as if they were. Moreover, the statistical analyses employed should be more clearly described, and the authors should avoid overinterpreting marginal or non-significant results. 6) Inappropriate or incomplete analyses: the bioinformatic approach appears suboptimal or not fully adapted to the nature of the degraded DNA typically recovered from museum specimens and probably also to the chemistry of the sequencing platform. A useful pipeline could be represented by nf-core/eager (Fellows Yates et al., 2021). More justification for the chosen tools and pipelines, as well as a discussion of potential limitations, is needed. For example the authors report the presence of polyG-rich reads or sequences. However, it is unclear whether these observations reflect technical artifacts arising from either pipeline limitations or the sequencing chemistry due to 2-channel chemistry or read length (with PE150 sequencer may read into the adapter or generate low-quality tail regions, where polyG miscalls are frequent — especially in the absence of signal toward the end of the read). 7) More about bioinformatic approach: a method to improve mappings on circular genomes is CircularMapper. Moreover, more details about the parameters selected for reconstructing the consensus sequences need to be provided. 8) Low sample size and lack of controls: the most critical limitation of the study is the relatively small and uneven sample size, especially in relation to the number of species, lysis buffers, and incubation times tested. The low number of replicates per condition significantly limits the statistical power of the analyses and the generalizability of the findings. I strongly encourage the authors to explicitly acknowledge this limitation in the manuscript and to refrain from overinterpreting marginal or non-significant results. If possible, the inclusion of additional replicates would considerably strengthen the study’s conclusions. 9) Lack of negative controls (e.g., extraction blanks): they are not mentioned in the text. They usually can assess potential contamination when working with historical material. 10) In the discussion the authors stated that “Our protocol yields 1,750–13,495 ng of DNA per specimen, surpassing values reported for grinding-based protocols (e.g., 18.31–325 ng)”. I would strongly recommend removing or rephrasing this comparison, as it lacks sufficient contextual basis. The cited values come from a different study involving samples of unclear geographic origin, age, storage conditions, preservation history, or library preparation method, which may differ substantially from those used in the present study. If the authors wish to reference previously reported yields, it would be more appropriate to do so in a descriptive or contextual manner, without implying a direct performance comparison. 11) It appears that only one mitochondrial genome has been deposited in GenBank. Could the authors clarify why the remaining mitogenomes were not submitted, and whether there are plans to make them publicly available? ****** 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: No 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. 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Revision 1
9 Oct 2025 Author Response Reviewer #1: The study lacks a rigorous assessment of endogenous DNA content. Although exogenous contamination is quantified using Kraken2, the proportion of authentic grasshopper DNA in the extracts remains unverified. Given the high variability in contamination levels (50% in some specimens), this raises concerns about the protocol’s efficiency in retrieving target DNA. Incorporating negative controls during extraction and quantifying endogenous DNA would provide critical validation. Response: We thank the reviewer for this valuable suggestion. We now include the proportion of authentic grasshopper DNA in the Sankey plots (Fig. 2), providing a direct quantification of endogenous DNA content. Besides, it is important to note that variable contamination levels are a common feature in historical DNA studies, often reflecting specimen storage conditions rather than extraction performance. Despite some samples showing high exogenous fractions, our protocol consistently yielded sufficient endogenous DNA to assemble complete mitochondrial CDS. This demonstrates that the method is effective for recovering authentic target sequences, even under less-than-ideal preservation scenarios. We did add a negative control and quantified its dsDNA amount using Nanodrop, but didn’t sequence it. We add it in our table 1. The title suggests a focus on mitochondrial genomes, yet the paper does not leverage these data for evolutionary or ecological insights. While the assembly of Stenocrobylus festivus mitogenomes is a valuable proof of concept, the opportunity to explore phylogenetic relationships, temporal genetic shifts, or comparative analyses with modern specimens is missed. To align the manuscript’s scope with its title, the authors should either reframe the title to emphasize methodology or include preliminary evolutionary analyses. Response: We agree with the reviewer’s assessment. To better reflect the methodological focus of the study, we have changed the title to:“Non-Destructive DNA Extraction for Recovering Mitochondrial Genomes from Museum Grasshopper Specimens.”This revised title avoids overstatement of evolutionary analyses while clearly emphasizing the protocol and its application. Reviewer #2: 1) Since the manuscript is intended as a methodological paper, the experimental procedures, including design, sampling, library preparation, and data analysis, should be described in much greater detail to allow proper assessment and reproducibility. As it stands, the methodological sections, in particular library preparation or data analysis, are too sparse to support a clear methodological advancement. Response: We have expanded the methodological sections to provide greater detail on the library preparation procedures and contamination analyses. This now allows for more transparent assessment and reproducibility. Besides, a clear pipeline for DNA sampling is listed in supplemental file 1. 2) I also suggest moving the table S1 to the main text. Response: We have moved the former Supplementary Table S1 into the main text as Table 1. 3) The diagram of the DNA extraction workflow (Figure S1) is well designed and highly informative. However, I recommend that the authors ensure full consistency between Figure S1, the main text, and Supplementary Table S1 with respect to the naming or labeling of the extraction methods applied to each sample. Currently, there appear to be some ambiguities in how the methodologies are referred to across different parts of the manuscript and supplementary materials, which could make it difficult for readers to follow or reproduce the experimental design. Response: We carefully revised the manuscript to ensure consistent terminology across Figure S1, the main text, and the tables. We delete those unclear characters. 4) This sentence can be better explained or reformulated: l. 84 “Due to the size constraints of a 1.5 ml centrifuge tube, the hind leg of each grasshopper specimen was initially removed”. It is not clear if the leg was totally removed from the tube, or when it was placed back in the tube. Response: We revised the sentence to clarify the step. 5) Statistical support: some of the reported improvements are not statistically significant (e.g., P = 0.06), and yet are described in the text as if they were. Moreover, the statistical analyses employed should be more clearly described, and the authors should avoid overinterpreting marginal or non-significant results. Response: We deleted the term “significantly” where the P-value was marginal (e.g., P = 0.06) and revised the statistical descriptions to avoid overinterpretation. 6) Inappropriate or incomplete analyses: the bioinformatic approach appears suboptimal or not fully adapted to the nature of the degraded DNA typically recovered from museum specimens and probably also to the chemistry of the sequencing platform. A useful pipeline could be represented by nf-core/eager (Fellows Yates et al., 2021). More justification for the chosen tools and pipelines, as well as a discussion of potential limitations, is needed. For example, the authors report the presence of polyG-rich reads or sequences. However, it is unclear whether these observations reflect technical artifacts arising from either pipeline limitations or the sequencing chemistry due to 2-channel chemistry or read length (with PE150 sequencer may read into the adapter or generate lowquality tail regions, where polyG miscalls are frequent — especially in the absence of signal toward the end of the read). Response: We have incorporated the nf-core/eager pipeline to process our data, added corresponding method and results. We compare our previous DNA damage result, and revise its with eager’s output. For poly-G, we completely understand its relation to the length of DNA insertion, thus we change its expression in the Sequence analyses section. 7) More about bioinformatic approach: a method to improve mappings on circular genomes is CircularMapper. Moreover, more details about the parameters selected for reconstructing the consensus sequences need to be provided. Response: We tested the CircularMapper (CM) tool by installing it via Conda. Specifically, we applied CM to sample 99PIT and 17STF and compared the BAM results with our previous mapping approach. Here’s our main steps to generate the BAM via CM: $java -jar /home/DataDisk/th_data/software/envs/CM/share/circularmapper-1.93.5-3/generator-1.93.5.jar -e 500 -i pi.zhengi.fasta -s "chrMT" $bwa aln -t 4 pi.zhengi_500.fasta your.merged.fastqfile -n 0.04 -l 32 -f output.sai $bwa samse pi.zhengi_500.fasta output.sai 99BTM_merge.fastq -f output_circmapper.sam $java -jar /home/DataDisk/th_data/software/envs/CM/share/circularmapper-1.93.5-3/realign-1.93.5.jar -e 500 -i output_circmapper.sam -r pi.zhengi.fasta We generate consensus sequence by Geneious prime, and map them to the reference. Although we found that in 99PIT, SNPs are fewer using CM, but appears a 196 bps gap in the sequence, and for 17STF, we find very few reads successfully mapped to the reference in 17STF using CM (We think it might cause by remoted relation between St. festivus and its reference Diabolocatantops_pinguis, for which is the only available reference of the same family). Although CM calls BWA-aln module, our sequencing strategy produce 150 bp pair end reads from historical DNA, and according to Dolenz’s research (Dolenz, 2024) BWA-mem still perform better toward 150 bp. So, we retained our original results, but further add a comment of CircularMapper. 8) Low sample size and lack of controls: the most critical limitation of the study is the relatively small and uneven sample size, especially in relation to the number of species, lysis buffers, and incubation times tested. The low number of replicates per condition significantly limits the statistical power of the analyses and the generalizability of the findings. I strongly encourage the authors to explicitly acknowledge this limitation in the manuscript and to refrain from overinterpreting marginal or non-significant results. If possible, the inclusion of additional replicates would considerably strengthen the study’s conclusions. Response: We agree and now explicitly acknowledge the limitation of sample size and replication in the revised Discussion. 9) Lack of negative controls (e.g., extraction blanks): they are not mentioned in the text. They usually can assess potential contamination when working with historical material. 10) In the discussion the authors stated that “Our protocol yields 1,750–13,495 ng of DNA per specimen, surpassing values reported for grinding-based protocols (e.g., 18.31–325 ng)”. I would strongly recommend removing or rephrasing this comparison, as it lacks sufficient contextual basis. The cited values come from a different study involving samples of unclear geographic origin, age, storage conditions, preservation history, or library preparation method, which may differ substantially from those used in the present study. If the authors wish to reference previously reported yields, it would be more appropriate to do so in a descriptive or contextual manner, without implying a direct performance comparison. Response: We did include a negative control. Although we did not sequence it, we quantified its dsDNA amount using Nanodrop. We now report this information in the revised Table 1 and discuss it in the result. Trace DNA yield in negative control shows limited contamination introduction. 11) It appears that only one mitochondrial genome has been deposited in GenBank. Could the authors clarify why the remaining mitogenomes were not submitted, and whether there are plans to make them publicly available? Response: No species of the genus Stenocrobylus had any sequences deposited in GenBank prior to our study; thus, we report it here for the first time. For the other species successfully assembled through consensus generation, corresponding sequences are already available in NCBI. We attempted to submit these as well, but received responses such as ‘Some or all of the sequences contain little similarity to other sequences in the database based on BLAST searches.’ Therefore, we now mark them as unverified, and they will be released to the public database upon publication. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0341621.r002
1 Dec 2025 Decision Letter - Marta Ciucani, Editor Dear Dr. Huang, 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 Jan 15 2026 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. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. 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We look forward to receiving your revised manuscript. Kind regards, Marta Maria Ciucani, PhD Academic Editor PLOS ONE Journal Requirements: If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 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: Based on the reviewer's feedback, I am please to inform you that the editorial decision in Minor Revision. The reviewer raised only a small number of comments that should be addressed to further improve the discussion and the manuscript. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> 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 Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #2: Yes ****** Reviewer #2: Dear Authors, Thank you for your revisions. You have addressed all my previous comments, and I appreciate the improvements made to the manuscript. I only have a few additional suggestions that may further strengthen the work. First, I recommend expanding the discussion of deamination patterns, also in light of the findings reported by Korlević et al. (2021) and Modi et al. (2021). Integrating these studies would provide useful context and help clarify how your results fit within the broader literature on ancient/historical DNA damage. Second, I would like to comment on the statement: “Although CM calls the BWA-aln module, our sequencing strategy produced 150 bp paired-end reads from historical DNA, and according to Dolenz’s research (Dolenz, 2024) BWA-MEM still performs better for 150 bp. So, we retained our original results, but further added a comment on CircularMapper.” I do not fully agree with this reasoning. Your reads are indeed 150 bp as raw output from the sequencer; however, in practice they are very likely much shorter once adapter trimming is applied. Given the short insert sizes typical of historical DNA, the opposite adapter will often be reached again, producing artificially long reads with terminal poly-G stretches. After trimming, the reads used for mapping will therefore be substantially shorter than 150 bp. For this reason, BWA-MEM may not be the most appropriate choice in this context. I suggest clarifying this potential scenario in the manuscript and discussing its implications for the mapping strategy. Overall, the manuscript is much improved, and I believe these additional clarifications would further enhance its accuracy and clarity. ****** 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: 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.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. https://doi.org/10.1371/journal.pone.0341621.r003
Revision 2
23 Dec 2025 Author Response Reviewer #2: First, I recommend expanding the discussion of deamination patterns, also in light of the findings reported by Korlević et al. (2021) and Modi et al. (2021). Integrating these studies would provide useful context and help clarify how your results fit within the broader literature on ancient/historical DNA damage. Response: We have expanded the Discussion to better describe DNA damage patterns observed in our study. We note that assessing these damage patterns helps distinguish endogenous DNA from contamination and informs mapping and assembly strategies. The manuscript has been revised accordingly. Second, I would like to comment on the statement: “Although CM calls the BWA-aln module, our sequencing strategy produced 150 bp paired-end reads from historical DNA, and according to Dolenz’s research (Dolenz, 2024) BWA-MEM still performs better for 150 bp. So, we retained our original results, but further added a comment on CircularMapper.” I do not fully agree with this reasoning. Your reads are indeed 150 bp as raw output from the sequencer; however, in practice they are very likely much shorter once adapter trimming is applied. Given the short insert sizes typical of historical DNA, the opposite adapter will often be reached again, producing artificially long reads with terminal poly-G stretches. After trimming, the reads used for mapping will therefore be substantially shorter than 150 bp. For this reason, BWA-MEM may not be the most appropriate choice in this context. I suggest clarifying this potential scenario in the manuscript and discussing its implications for the mapping strategy. Response: We agree that, despite the nominal 150 bp read length, historical DNA libraries often produce substantially shorter trimmed reads because short inserts frequently reach the opposite adapter and generate poly-G tails. This scenario may indeed reduce the suitability of BWA-MEM, which is optimized for longer reads. In response to the reviewer’s suggestion, we have added a paragraph in the Discussion acknowledging this issue and clarifying that short trimmed reads may be better handled by algorithms tailored to degraded DNA, such as BWA-aln or the internal mapping module used by CircularMapper. We also note this as an important consideration for future applications of our pipeline. The manuscript has been revised accordingly. Thank you for all your proposal. Attachments Attachment Submitted filename: Response_to_Reviewers_auresp_2.docx https://doi.org/10.1371/journal.pone.0341621.r004
11 Jan 2026 Decision Letter - Marta Ciucani, Editor Non-Destructive DNA Extraction for Recovering Mitochondrial Genomes from Museum Grasshopper Specimens PONE-D-25-30043R2 Dear Dr. Huang, 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. For questions related to billing, please contact billing support . 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, Marta Maria Ciucani, PhD Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0341621.r005
Formally Accepted
Acceptance Letter - Marta Ciucani, Editor PONE-D-25-30043R2 PLOS One Dear Dr. Huang, 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 You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. 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. Marta Maria Ciucani Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0341621.r006

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