PONE-D-23-18419Characterization of genetic and molecular tools for studying the endogenous expression of Lactate dehydrogenase in Drosophila melanogasterPLOS ONE

Dear Dr. Tennessen,

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.

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“We thank the Bloomington Drosophila Stock Center (NIH P40OD018537) for providing fly stocks, the Drosophila Genomics Resource Center (NIH 2P40OD010949) for genomic reagents, Flybase (NIH 5U41HG000739), and the Indiana University Light Microscopy Imaging Center. Targeted GC-MS analysis was conducted using instruments housed in the Indiana University Mass Spectrometry Facility, which is supported, in part, by NSF MRI Award 1726633. G.C is supported by the DBT/Wellcome Trust India Alliance Fellowship/Grant [grant number IA/I(S)/17/1/503085]. J.M.T. is supported by the National Institute of General Medical Sciences of the National Institutes of Health under a R35 Maximizing Investigators’ Research Award (MIRA; 1R35GM119557).”

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J.M.T. is supported by the National Institute of General Medical Sciences of the National Institutes of Health under a R35 Maximizing Investigators’ Research Award (MIRA; 1R35GM119557). https://nigms.nih.gov. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

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Additional Editor Comments:

The reviewers have suggested minor revisions to the manuscript. Need for additional statistical analyses, references, and images for supplementary data has been raised. Certain aspects of the materials and methods section also need detailing.

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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: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

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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: No

Reviewer #2: Yes

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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: Yes

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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: In this manuscript Rai et. al. demonstrate where Ldh is localized in Drosophila larva across developmental stages and tissue types. The authors provide validation for valuable tools to examine expression of this enzyme that is highly relevant to development and many other physiological processes. Moreover, their discussion is clear and concise. The primary strengths of this study are the number of tissues measured and the direct comparison of similar tools in equivalent contexts. The weaknesses of this study are a lack of quantification for the fluorescence images across larval development, lack of any comment on individual variation, and absence of raw values for quantitative data with statistical analyses. Nonetheless, this study provides insightful findings that are suitable for publication in this journal. I recommend the editors accept this manuscript with revisions.

Here are my recommended revisions in order of importance:

1. Quantitative data provided in this manuscript is presented exclusively in Figure 1B-D. In the figure legend of Figure 1 the authors state that this data was analyzed using “one-way ANOVA followed by a post hoc Tukey HSD.” On page 9 the authors write in the methods section that for metabolite “Data were compared using a Kruskal–Walli’s test followed by a Dunn’s multiple comparison test.” The raw values and statistical analyses for all quantitative data (including the viability assay) were not provided. I recommend that the authors provide the raw values and statistical analyses (including test statistics and p value for all tests) for all quantitative data in the manuscript and clarify in the methods section which data were analyzed by ANOVA and Tukey or Kruskal-Wallis and Dunn’s.

2. In Figure 1 and S1, the authors provide qualitative data depicting detection of change in Ldh level across larval development using two different lines, Ldh-GFPGenomic and Ldh-mCherryGenomic. To strengthen these findings, I recommend the authors provide a quantification of the fluorescence depicted in these images in order to highlight the magnitude of changes that were detected and to compare more clearly to previous findings.

3. Throughout the manuscript, the authors provide images from a single representative individual animal fore every result. Although this may be sufficient for a binary measure whereby the outcome is only either positive or negative, instances whereby patterns are evaluated should be examined in more than a single animal. I recommend the authors provide supplementary images from additional animals for at least Figure 1, S1, and 2. At a minimum, even if additional images are not provided, I recommend that the authors discuss in the body text whether they observed any individual variation in their results from each tool they used, including Ldh-mCherry.

4. The authors highlight the discovery of new functions of Ldh on page 3. This description of Ldh seems to conflate the function of lactate to the function of Ldh. I recommend clarification of the distinction between functions attributed to Ldh and the substrates of Ldh, lactate and pyruvate, of which Ldh reversibly interconverts. Furthermore, a particularly important newly discovered function of lactate which warrants citing because it may have a significant impact on development is the ability of lactate to regulate cell cycle through zinc chelation (Liu, W., Wang, Y., Bozi, L.H.M., Fischer, P., Jedrychowski, M.P., Xiao, H., Wu, T., Darabedian, N., He, X., Mills, E.L., et al. (2023). Lactate regulates cell cycle by remodeling the anaphase promoting complex. Nature 616, 1–2. DOI:10.1038/s41586-023-05939-3 PMID:36921622.)

5. The authors state on page 3 “Ldh expression and activity are very high during this growth phase when compared to other phases of fly life-cycle [19]”. This citation only measures Ldh activity and not expression. I recommend adding a citation that provides evidence supporting the claimed changes in expression, for example (Graveley, B., Brooks, A., Carlson, J. et al. The developmental transcriptome of Drosophila melanogaster. Nature 471, 473–479 (2011). DOI: 10.1038/nature09715)

6. The authors cite three studies related to the connection between lactate metabolism activity and “neuronal health and aging [24-27]”. I recommend four additional critical citations (1. Hunt, L.C., and Demontis, F. (2021). Age-Related Increase in Lactate Dehydrogenase Activity in Skeletal Muscle Reduces Lifespan in Drosophila. J. Gerontol. A. Biol. Sci. Med. Sci. XX, 1–32. DOI:10.1093/gerona/glab260 PMID:34477202. 2. Frame, A.K., Robinson, J.W., Mahmoudzadeh, N.H., Tennessen, J.M., Simon, A.F., and Cumming, R.C. (2023). Aging and memory are altered by genetically manipulating lactate dehydrogenase in the neurons or glia of flies. Aging (Albany. NY). 10, 1–53. DOI:10.18632/aging.204565. 3. Long, D.M., Frame, A.K., Reardon, P.N., Cumming, R.C., Hendrix, D.A., Kretzschmar, D., and Giebultowicz, J.M. (2020). Lactate dehydrogenase expression modulates longevity and neurodegeneration in Drosophila melanogaster. Aging (Albany. NY). 12, 10041–10058. DOI:10.18632/aging.103373. 4. Lee, J.E., Oney, M., Frizzell, K., Phadnis, N., and Hollien, J. (2015). Drosophila melanogaster Activating Transcription Factor 4 Regulates Glycolysis During Endoplasmic Reticulum Stress. G3 Genes|Genomes|Genetics 5, 667–675. DOI:10.1534/g3.115.017269 PMID:25681259.

7. On page 6 the authors describe the production of Ldh-mCherryGenomic with reference to a past study which has used this resource and having produced this resource in a similar manner to Ldh-GFPGenomic. Citation 28 does not provide an explanation for the method used to generate Ldh-mCherryGenomic and citation 32 describes how Ldh-GFPGenomic was generated using a PCR based method and plasmid injection by Rainbow Transgenics. I recommend the authors clarify here whether the methods used for generating Ldh-mCherryGenomic were identical and cite Rainbow Transgenics if their services were used.

8. In the methods section the authors do not provide an explanation for how Ldh-mCherryGenomic was imaged. Was an antibody against mCherry required? I recommend the authors provide this information.

9. On page 8 the authors describe their confocal microscopy method. I recommend the authors provide the excitation and emission wavelengths used for detection of each fluorophore imaged, the method used for combining Z-stacks (e.g. maximum intensity projection vs average intensity projection), and the objectives used.

10. The authors describe the results from Figure one on page 10 without reference to Figure 1A or 1B. I recommend that the authors refer to those figures where they have described them.

11. On page 10 the authors acknowledge a slight decrease in lactate and 2HG in their Ldh-GFPGenomic rescue compared to control without reference to any statistical test in the figure or results. I recommend the authors provide the results of a statistical test comparing the rescue with control. Moreover, I recommend the authors add discussion of potential reasons for differences in fusion protein activity compared to endogenous Ldh. For example, protein metabolite interaction (Hicks, K.G., Cluntun, A.A., Schubert, H.L., Hackett, S.R., Berg, J.A., Leonard, P.G., Ajalla Aleixo, M.A., Zhou, Y., Bott, A.J., Salvatore, S.R., et al. (2023). Protein-metabolite interactomics of carbohydrate metabolism reveal regulation of lactate dehydrogenase. Science (80-. ). 379, 996–1003. DOI:10.1126/science.abm3452 PMID:36893255.), post-translational modification (Storey, K.B. (2016). Comparative enzymology—new insights from studies of an “old” enzyme, lactate dehydrogenase. Comp. Biochem. Physiol. Part - B Biochem. Mol. Biol. 199, 13–20. DOI:10.1016/j.cbpb.2015.12.004 PMID:26688543.), or post-transcriptional regulation (Jungmann, R.A., Huang, D., and Tian, D. (1998). Regulation of LDH-A gene expression by transcriptional and posttranscriptional signal transduction mechanisms. J. Exp. Zool. 282, 188–195. DOI:10.1002/(SICI)1097-010X(199809/10)282:1/2<188::AID-JEZ21>3.0.CO;2-P PMID:9723176.).

12. In Figure 2 the image for anti-Ldh staining without the entire ventral nerve cord visible. For fair comparison, I recommend addition of an image with anti-Ldh antibody with the ventral nerve chord entirely in frame like those larval nervous systems depicted for the GFP genomic and enhancer lines.

13. On page 13 the authors summarize their findings. For clarity and to highlight differences between the methods, I recommend the authors provide a summary table comparing the methodology and Ldh characteristics identified for the different methods they recommend. Moreover, I recommend the authors comment on the utility (or lack thereof) of the Ldh-mCherry line despite having characterized it less than the other methods. If the Ldh-mCherry line does not require any antibody staining to detect the signal above autofluorescence, then this may be an advantage for studies utilizing this line outside of the context whereby the authors have identified discrepant patterns of expression (pupal development).

14. In the legend for Figure 2, 3, 4, and 5 the authors state “For Ldh-GFPGenomic and Ldh-GFPenhancer expression analysis, the CNS was fixed and stained with an anti-GFP antibody as described in the methods.” Reference to the Ldh-GFPenhancer line in Figure 3 seems to be irrelevant because the authors also state in that figure legend that only Ldh-GFPgenomic was used. In addition, having fixation and staining procedures in the methods section makes mention of this method in the figure legends redundant unless they differ from the description in the methods section. I recommend that the authors remove all statements “For Ldh-GFPGenomic and Ldh-GFPenhancer expression analysis, the CNS was fixed and stained with an anti-GFP antibody as described in the methods.“ from the figure legend unless there is a reason to distinguish a method in any figure from the method described in the methods section.

15. For all Figures, the scale bars and labels should be formatted consistently. I recommend that the authors label staining for GFP in Ldh-GFPGenomic and Ldh-GFPEnhancer in Figure 2, 3, 4, 5, and 6 consistently as either only αGFP or only LDH. I recommend the authors make all labels denoting the stain used in images below consistently displayed in a font color matching the pseudocolor in the image below, as they have done in Figure 3. I recommend that the authors remove all labels describing the length of scale bars from within the image and keep this description of scale bar length restricted to the Figure legend, as they have done in Figure 5.

16. In the legend for Figure 3, the authors state refer to (I-K) whereby they seem to be referring to (I-L). I recommend the authors change this to (I-L).

17. The label above Figure 5F and L is (αGFP). This seems to be a mistake. If this is incorrect, I recommend the authors change this to αLDH as they have done in Figure 4.

18. In the legend for Figure S2 the authors state that the brightness was increased in panel L. I recommend the authors provide the percentage the brightness was increased compared to the other images in this figure.

19. On page 13 the statement “Previous studies of the Ldh enzyme activity suggest that Ldh expression levels are relatively low in the larval salivary glands and fat body” is not cited. I recommend the authors add a citation supporting this claim.

20. The authors state “(see methods)” on page 5 where reiterating the catalog number and supplier of the antibody here would provide more clarity to the reader: (Bosterbio; DZ41222)

Reviewer #2: Dear Editor,

In the manuscript "Characterization of genetic and molecular tools for studying the endogenous expression of Lactate dehydrogenase in Drosophila melanogaster", Ray et al. provide a detailed and accurate characterization of D. melanogaster LDH reporter lines and a new anti-LDH antibody.

Although the information provided in this manuscript may be limited to a specialized audience, the experiments reported here provide an important set of data to validate l reagents previously used in several publications and now available to the Drosophila community.

I have a few minor comments below that I think should be addressed prior to publication, but overall I consider the manuscript to be of high quality and have no major comments that would preclude its publication after this minor corrections.

Sincerely

Minor comments:

- Figure 1C: The figure legend indicates that the Ldh-GFP fusion protein is able to restore steady-state levels of (C) lactate and (D) 2-hydroxyglutarate (2HG).

Although lactate and 2HG levels are significantly increased in p{Ldh-GFP} compared to Ldh null mutants, the statistical difference in metabolite levels between Ldh and p{Ldh-GFP} was not tested. The phrase "restore steady-state levels" is ambiguous in this context. The authors should either perform an additional statistical test and retain the sentence only in case of lack of statistical differences or rephrase this sentence to more accurately describe the observed results, as is already the case in the Results section.

- Figure 3C: References justifying the choice of different markers (REPO, PROS, MIRA) could be added to the text.

- The term "AMP cluster" should be defined.

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Reviewer #1: Yes: Ariel K. Frame

Reviewer #2: No

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PONE-D-23-18419R1Characterization of genetic and molecular tools for studying the endogenous expression of Lactate dehydrogenase in Drosophila melanogasterPLOS ONE

Dear Dr. Tennessen,

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. A reviewer has suggested minor revision that includes consistent labeling of figures.

Please submit your revised manuscript by Jan 26 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.

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• 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'.

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,

Jyotshna Kanungo, Ph.D.

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.

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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: All comments have been addressed

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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: Yes

**********

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

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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

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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: All comments were adequately addressed. There are a few very minor changes I recommend before final publication:

1) I agree that Abu-Shumays et al. is sufficient evidence for the claim that Ldh expression is relatively high during larval developement. But, it seems this citation was added to the sentence prior to this claim (Line 79). I recommend moving it to the sentence discussion Ldh expression (Line 80).

2) The method for combining Z-stacks is still not provided (e.g. maximum intensity projection / average intensity projection). I recommend this is added to the sentence on Line 196.

3) Figure 4 and 5 have the green channel legend labeled αLDH whereas figure 2 and everywhere else in the body text and figure legends denotes this without all uppercase as αLdh. I recommend changing figure 4 and 5 to match everywhere else (i.e. αLdh).

Reviewer #2: I consider that the authors have responded adequately to all my comments. I recommend the publication of this study.

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Reviewer #1: Yes: Ariel K. Frame

Reviewer #2: No

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Characterization of genetic and molecular tools for studying the endogenous expression of Lactate dehydrogenase in Drosophila melanogaster

PONE-D-23-18419R2

Dear Dr. Tennessen,

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.

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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,

Jyotshna Kanungo, 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 #1: 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 #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: 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

**********

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

**********

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)

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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: Ariel K. Frame

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