The effect of autophagy and mitochondrial fission on Harderian gland is greater than apoptosis in male hamsters during different photoperiods

Jin-Hui Xu; Zhe Wang; Jun-Jie Mou; Xiang-Yu Zhao; Xiao-Cui Geng; Ming Wu; Hui-Liang Xue; Lei Chen; Lai-Xiang Xu

doi:10.1371/journal.pone.0241561

Peer Review History

Original SubmissionJune 7, 2020
14 Jul 2020 Decision Letter - Hemachandra Reddy, Editor PONE-D-20-17311 The effect of autophagy and mitochondrial fission on Harderian gland is greater than apoptosis in male hamsters during different photoperiods PLOS ONE Dear Dr. Xu, 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 by both the reviewers. PLEASE ADDRESS CONCERNS CAREFULLY - clarity of writeup, experimental design and presentation of data. If applicable, we recommend that you deposit your laboratory protocols in protocols to 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. We look forward to receiving your revised manuscript. 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Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No 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: No 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: No Reviewer #2: Yes ****** 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 “The effect of autophagy and mitochondrial fission on Harderian gland is greater than apoptosis in male hamsters during different photoperiods” done by Zhe Wang et al., represents many questionable things to readers and how reliable this research will draw with scientific conclusions. While the manuscript has been reading, the approaches remain unclear, the data do not support the interpretations reached, and the lack of experimentation makes this manuscript not of priority for publication. The writing of the manuscript and the author communications does not reach the level needed for adequate communications. This manuscript does not provide significant autophagy and mitochondrial fission properties experimental validation of the phenomena described. The writing part is poor from abstract write-up is not up to mark, first time description need to be expand the abbreviations. Results presentation in abstract is vaguely represented. However, there are some major issues identified the manuscript. 1. Poor write up the complete manuscript without proper understanding autophagy and mitochondrial fission mechanisms. 2. The methodology and experimental design is in adequate like why only the selected Autophagy proteins chosen by the authors like LC3-I, LC3-II is questionable and these are not LC3 and P62 proteins are the only players influences the Autophagy and mitophagy..? 3. Changes in mitochondrial function involve mitochondrial fission and fusion involve lot of proteins why only the Dynamin related protein 1 (DRP1) and focused the authors?. 4. This manuscript taking about the effect of autophagy disappointed without mentioning conserved genes Autophagy-related genes (Atg) and their validation which is key for the macroautophagy development. 5. This is no statistical validation or signicance “p” values across all western blot data. The manuscript complete lack of scientific novelty and methods for the autophagy and mitochondrial dynamics and biogenesis. In particular, we emphasize on the viability of the Autophagy-related genes (Atg) validation in mitochondrial apoptosis is important. Without checking significant autophagy and mitophagy genes lead to wrong statistical inferences and consequently wrong scientific conclusions. I feel that this manuscript not to deserve to plos one journal Reviewer #2: 1. What is the rationale of selecting only male adult hamsters in this study? 2. What is the melatonin level in the blood of hamsters in three photoperiodic groups? 3. Please provide the morphological structure of the Harderian gland (HG). 4. Have you checked the key enzymes that are involved in the day/night rhythmic production of melatonin? 5. Have you done the mitochondrial functional assay? 6. Have you analyzed the mitochondrial number and length? 7. In Fig 1B, I did not see any intact cristae structure. Please provide a clear picture. 8. In Fig4, 5, the blots (bax, LC3I, LC3II) is not matching with the respective graphs. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Revision 1
29 Sep 2020 Author Response Dear Editor, Thank you very much for giving us the opportunity to revise our manuscript entitled, “Effects of autophagy and mitochondrial fission on Harderian gland are greater than that of apoptosis in male striped dwarf hamsters (Cricetulus barabensis) under different photoperiods”, (Manuscript ID: PONE-D-20-17311). We greatly appreciate the positive and constructive comments from you and the reviewers. We have devoted effort and careful review to revising the manuscript based on the comments and recommendations. In addition, we have asked a native English speaker with a science background to help us review and polish the manuscript. The revised content is presented in blue in our response to the reviewers’ comments and within the manuscript. We believe the revised manuscript to be much improved and hope it has reached the standards required for publication in PLOS ONE. We thank you for your consideration and look forward to your response. Yours sincerely, Prof Lai-Xiang Xu, Ph.D. (On behalf of all co-authors) College of Life Sciences, Qufu Normal University 57# Jingxuanxilu Road, Qufu, Shandong 273165, China Mobile: 86-13853709058 Email: xulx@qfnu.edu.cn Reviewer 1: This manuscript does not provide significant autophagy and mitochondrial fission properties experimental validation of the phenomena described. The writing part is poor from abstract write-up is not up to mark, first time description need to be expand the abbreviations. Results presentation in abstract is vaguely represented. However, there are some major issues identified the manuscript. Response: Thank you very much for your guidance. Firstly, we used preserved paraffin-embedded Harderian gland (HG) tissue for electron microscopy, added clearer figures of mitochondrial morphology, and determined the mitochondrial cross-sectional area (CSA). Results showed that there was no significant change in the CSA of mitochondria among the three groups. However, because HGs are secretory glands and contain a large number of lipid droplets, autophagic vesicles are difficult to observe, so we are unable to provide a more appropriate figure of autophagy. Secondly, we used samples previously preserved in liquid nitrogen for quantitative protein expression analysis of beclin1 (BECN1), as beclin1 is a key autophagy initiator (Schaaf et al., 2016). Please see details in 81-83, 183-187, 226-227, 273-276, 307-309, 375-379. Finally, we have rewritten the abstract, introduction, and discussion sections of the manuscript, rearranged the results, and carefully examined details in the manuscript, such as the use of acronyms. Schaaf, M. B., Keulers, T. G., Vooijs, M. A., & Rouschop, K. M. (2016). LC3/GABARAP family proteins: autophagy-(un)related functions. Faseb j, 30(12), 3961-3978. doi:10.1096/fj.201600698R 1. Poor write up the complete manuscript without proper understanding autophagy and mitochondrial fission mechanisms. Response: Thank you very much for your question, and for highlighting the shortcomings of our experiment. According to your suggestion, we studied the mechanism of autophagy and mitochondrial division, examined a substantial amount of data, and determined the indicators for in-depth study of the mechanism of autophagy and mitochondrial division. However, due to the COVID-19 pandemic, all experiments based on wild animals are now strictly limited. In addition, because of the small amount of HG tissue (0.02~0.03 g), we were unable to complete all suggested experiments. Instead, we used tissue we had previously preserved in liquid nitrogen to quantify protein expression of beclin1, as beclin1 is a key factor for the initiation of autophagy (Schaaf et al., 2016). We will investigate other indicators in our next study and further explore the underlying mechanism of autophagy and changes in mitochondrial function. We have modified the results and discussion sections of the revised manuscript, please see details in line 81-83,307-309,375-379. Schaaf, M. B., Keulers, T. G., Vooijs, M. A., & Rouschop, K. M. (2016). LC3/GABARAP family proteins: autophagy-(un)related functions. Faseb j, 30(12), 3961-3978. doi:10.1096/fj.201600698R 2. The methodology and experimental design is in adequate like why only the selected Autophagy proteins chosen by the authors like LC3-I, LC3-II is questionable and these are not LC3 and P62 proteins are the only players influences the Autophagy and mitophagy? Response: Thank you very much. According to your suggestion, we used samples previously preserved in liquid nitrogen for protein expression quantification of beclin1, a key initiator of autophagy. Results showed that the protein expression level of beclin1 was highest in the MP group but decreased under the SP and LP groups. However, as the amount of HG tissue was small (0.02~0.03 g), after the quantitative beclin1 experiments, we did not have enough samples to study other ATG family-related proteins. Furthermore, due to the COVID-19 pandemic, all experiments based on wild animals are now strictly limited. Therefore, we had no choice but to temporarily abandon more in-depth study of autophagy. We will undertake further studies in the future. In the revised manuscript, we have described and discussed the autophagy results more rigorously. Please see details in line 81-83, 121-124, 367-370, 371-379,423-433, 525-530. 3. Changes in mitochondrial function involve mitochondrial fission and fusion involve lot of proteins why only the Dynamin related protein 1 (DRP1) and focused the authors? Response: Thank you for highlighting some of the issues regarding our experiment. According to your suggestion, we have read many references, which state that changes in mitochondrial function are related to the processes of mitochondrial division and fusion. Drp1 is the most important factor in mitochondrial division and Mff promotes activity of Drp1 variation (Fekkes et al., 2000), while Fis1 inhibits mitochondrial division (Liu & Chan, 2015). Mitochondrial genome maintenance 1 (Mgm1) is a key component of mitochondrial membrane fusion, which is necessary to maintain the dynamics and morphology of mitochondria (Rujiviphat et al., 2009). Mitofusins 1 and 2 (Mfn1, Mfn2) are integral proteins of the outer mitochondrial membrane (Santel & Fuller, 2001) and act in trans in either homo- or heterotypic interactions to tether apposing mitochondria as the initial step of fusion (Koshiba et al., 2004). However, due to the COVID-19 pandemic, we were not able to recapture and re-treat hamsters. In addition, we only had sufficient HG tissue, which was previously preserved in liquid nitrogen, for the quantitative study of beclin1. However, we will further explore the underlying mechanism of mitochondrial functional changes in the future. Fekkes, P., Shepard, K. A., & Yaffe, M. P. (2000). Gag3p, an outer membrane protein required for fission of mitochondrial tubules. J Cell Biol, 151(2), 333-340. Liu, R., & Chan, D. C. (2015). The mitochondrial fission receptor Mff selectively recruits oligomerized Drp1. Mol Biol Cell, 26(24), 4466-4477. doi:10.1091/mbc.E15-08-0591 Rujiviphat, J., Meglei, G., Rubinstein, J. L., & McQuibban, G. A. (2009). Phospholipid Association Is Essential for Dynamin-related Protein Mgm1 to Function in Mitochondrial Membrane Fusion. Journal of Biological Chemistry, 284(42), 28682-28686. doi:10.1074/jbc.M109.044933 Santel, A., & Fuller, M. T. (2001). Control of mitochondrial morphology by a human mitofusin. J Cell Sci, 114(Pt 5), 867-874. Koshiba, T., Detmer, S. A., Kaiser, J. T., Chen, H., McCaffery, J. M., & Chan, D. C. (2004). Structural basis of mitochondrial tethering by mitofusin complexes. Science (New York, N.Y.), 305(5685), 858-862. doi:10.1126/science.1099793 4. This manuscript taking about the effect of autophagy disappointed without mentioning conserved genes Autophagy-related genes (Atg) and their validation which is key for the macroautophagy development. Response: Thank you very much. According to your suggestion, we have read numerous papers on autophagy-related factor beclin1, a key initiator of autophagy (Schaaf et al., 2016). The transformation ratio of LC3-I to LC3-II represents the formation of autophagy lysosome membranes (Mariño et al., 2014). ATG4 can couple LC3 with phospholipid ethanolamine on the autophagy membrane, which is a key step in autophagy biogenesis and circulation (Tanida et al., 2004). At the same time, ATG5 and ATG9 are essential for initiating the formation of autophagosomes (He et al., 2013; Koyama-Honda et al., 2013). However, given the HG tissue restrictions (0.02~0.03 g) and strict limits on wild animal research due to the COVID-19 pandemic, we did not have enough wild hamsters to repeat the experiment. We could only choose one of the most critical factors (beclin1) for further experimentation. However, we will explore the mechanism of autophagy in future study. We have made relevant changes in the results and discussion sections in the revised manuscript. Please see detail in line 81-83, 121-124, 367-370, 371-379, 423-433, 525-530. Schaaf, M. B., Keulers, T. G., Vooijs, M. A., & Rouschop, K. M. (2016). LC3/GABARAP family proteins: autophagy-(un)related functions. Faseb j, 30(12), 3961-3978. doi:10.1096/fj.201600698R Mariño, G., Niso-Santano, M., Baehrecke, E. H., & Kroemer, G. (2014). Self-consumption: the interplay of autophagy and apoptosis. Nature reviews. Molecular cell biology, 15(2), 81-94. doi:10.1038/nrm3735 Tanida, I., Sou, Y.-s., Ezaki, J., Minematsu-Ikeguchi, N., Ueno, T., & Kominami, E. (2004). HsAtg4B/HsApg4B/autophagin-1 cleaves the carboxyl termini of three human Atg8 homologues and delipidates microtubule-associated protein light chain 3- and GABAA receptor-associated protein-phospholipid conjugates. J Biol Chem, 279(35), 36268-36276. doi:10.1074/jbc.M401461200 Koyama-Honda, I., Itakura, E., Fujiwara, T. K., & Mizushima, N. (2013). Temporal analysis of recruitment of mammalian ATG proteins to the autophagosome formation site. Autophagy, 9(10), 1491-1499. doi:10.4161/auto.25529 He, Z., Liu, H., Agostini, M., Yousefi, S., Perren, A., Tschan, M. P., . . . Simon, H. U. (2013). p73 regulates autophagy and hepatocellular lipid metabolism through a transcriptional activation of the ATG5 gene. Cell death and differentiation, 20(10), 1415-1424. doi:10.1038/cdd.2013.104 5. This is no statistical validation or signicance “p” values across all western blot data. The manuscript complete lack of scientific novelty and methods for the autophagy and mitochondrial dynamics and biogenesis. In particular, we emphasize on the viability of the Autophagy-related genes (Atg) validation in mitochondrial apoptosis is important. Without checking significant autophagy and mitophagy genes lead to wrong statistical inferences and consequently wrong scientific conclusions. I feel that this manuscript not to deserve to plos one journal Response: Thank you very much for your question. According to your guidance, we re-examined the original data and statistics in the manuscript and replaced the inappropriate representative western blot bands. We also consulted the references carefully. Autophagy-related factor beclin1 is a key initiator of autophagy (Schaaf et al., 2016). The transformation ratio of LC31 to LC32 represents the formation of autophagy lysosome membranes (Mariño et al., 2014). ATG4 can couple LC3 with phospholipid ethanolamine on the autophagy membrane, which is a key step in autophagy biogenesis and circulation (Tanida et al., 2004). At the same time, ATG5 and ATG9 are essential for initiating the formation of autophagosomes (He et al., 2013; Koyama-Honda et al., 2013). However, given the HG tissue restrictions (0.02~0.03 g) and strict limits on wild animal research due to the COVID-19 pandemic, we did not have enough wild hamsters to repeat the experiment. We could only choose one of the most critical factors (beclin1) for protein quantitative research using samples previously stored in liquid nitrogen. Therefore, the mechanism of autophagy in HGs under different photoperiods needs further study. This deficiency has been added to the study limitations in the revised manuscript. Schaaf, M. B., Keulers, T. G., Vooijs, M. A., & Rouschop, K. M. (2016). LC3/GABARAP family proteins: autophagy-(un)related functions. Faseb j, 30(12), 3961-3978. doi:10.1096/fj.201600698R Mariño, G., Niso-Santano, M., Baehrecke, E. H., & Kroemer, G. (2014). Self-consumption: the interplay of autophagy and apoptosis. Nature reviews. Molecular cell biology, 15(2), 81-94. doi:10.1038/nrm3735 Tanida, I., Sou, Y.-s., Ezaki, J., Minematsu-Ikeguchi, N., Ueno, T., & Kominami, E. (2004). HsAtg4B/HsApg4B/autophagin-1 cleaves the carboxyl termini of three human Atg8 homologues and delipidates microtubule-associated protein light chain 3- and GABAA receptor-associated protein-phospholipid conjugates. J Biol Chem, 279(35), 36268-36276. doi:10.1074/jbc.M401461200 Koyama-Honda, I., Itakura, E., Fujiwara, T. K., & Mizushima, N. (2013). Temporal analysis of recruitment of mammalian ATG proteins to the autophagosome formation site. Autophagy, 9(10), 1491-1499. doi:10.4161/auto.25529 He, Z., Liu, H., Agostini, M., Yousefi, S., Perren, A., Tschan, M. P., . . . Simon, H. U. (2013). p73 regulates autophagy and hepatocellular lipid metabolism through a transcriptional activation of the ATG5 gene. Cell death and differentiation, 20(10), 1415-1424. doi:10.1038/cdd.2013.104 Reviewer #2: 1. What is the rationale of selecting only male adult hamsters in this study? Response: Thank you very much for your question. Our research group has previously reported on the effects of photoperiod on the HG in female striped dwarf hamsters (Wang et al., 2020). As an organ with uncertain function, the HG may play an important role in photosensitivity. In addition, HGs show marked sexually dimorphism (Buzzell, 1996; Chieffi et al., 1996). Therefore, studies on HGs in male hamsters are helpful for understanding the function of HGs. In the revised manuscript, we added comparison of HGs between the sexes. Please see details in line 16-19, 47-55, 261-268, 328-336, 413-418, 480-484. Wang Zhe, Xu Jin-Hui, Mou Jun-Jie, et al. Photoperiod affects Harderian gland morphology and secretion in female Cricetulus barabensis: Autophagy, apoptosis, and mitochondria. Frontiers in Physiology, 2020, 11:408. DOI=10.3389/fphys.2020.00408. Buzzell, G. R. (1996). The Harderian gland: perspectives. Microscopy research and technique, 34(1), 2-5. doi:10.1002/(sici)1097-0029(19960501)34:1<2::Aid-jemt2>3.0.Co;2-w Chieffi, G., Baccari, G. C., Di Matteo, L., d'Istria, M., Minucci, S., & Varriale, B. (1996). Cell biology of the harderian gland. International review of cytology, 168, 1-80. doi:10.1016/s0074-7696(08)60882-7 2. What is the melatonin level in the blood of hamsters in three photoperiodic groups? Response: Thank you very much for your question. According to your suggestion, we used previously preserved serum to determine the concentration of melatonin, with results showing the order SP > MP > LP (300 pg/ml~500 pg/ml). We have added the relevant description in the revised manuscript. Please see details in the methods, results, and discussion sections and in Fig 1. 3. Please provide the morphological structure of the Harderian gland (HG). Response: Thank you very much. According to your suggestion, we used H&E-staining on paraffin-embedded tissue to observe the morphology of the HG in male striped dwarf hamsters. Please see details in Fig2. 4. Have you checked the key enzymes that are involved in the day/night rhythmic production of melatonin? Response: Thank you very much. According to your suggestion, we used previously preserved protein extracts to supplement quantitative detection of proteins in two key enzymes of melatonin synthesis, i.e., arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT). Results showed that there were no significant differences in the protein expression of AANAT among the three groups, but the expression of HIOMT in the SP group was significantly higher than that in the other groups. This may be one of the reasons for the increase in serum melatonin level in the SP group. We added a description of this in the revised draft. Please see details in line 22-25, 47-55, 120-124, 293-296, 525-530. 5. Have you done the mitochondrial functional assay? Response: Thank you very much for your question. As mitochondria are mainly responsible for ATP supply and aerobic oxidation, we detected the enzyme activity of two key factors, i.e., ATP synthase and citrate synthase. Results showed that the activity of ATP in the MP group was significantly higher than that in the other two groups, but there was no significant difference in CS activity among the three groups. This suggests that, compared with the MP control group, mitochondrial function may be weakened to some extent in the SP and LP groups. In the revised draft, we have emphasized the results and significance of this part of the study. Please see details in line 206-213, 388-390, 397-402, 525-530 6. Have you analyzed the mitochondrial number and length? Response: Thank you very much. According to your suggestion, we used preserved paraffin-embedded tissue to re-observe the mitochondria of the HG in striped dwarf hamsters, analyzed electron microscope pictures, and calculated the CSA of a single mitochondrion. Results showed that there were no significant changes in the CSA of mitochondria among the three groups. This has been discussed in the results section. Please see details in line183-187, 273-276, 480-484. 7. In Fig 1B, I did not see any intact cristae structure. Please provide a clear picture. Response: We apologize for the unclear picture. According to your suggestion, we used preserved paraffin-embedded tissue to re-observe the mitochondria of the HG of striped dwarf hamsters. Results showed that the morphology of the mitochondria in the three groups was basically intact and the mitochondrial crista structure could be seen. We have rewritten the description as “There were no significant differences in the mitochondrial structures of the three groups, with intact membranes and ridge structures and no degeneration, such as vacuolation or sparse ridges, observed”. Please see details in Fig3. 8. In Fig4, 5, the blots (bax, LC3I, LC3II) is not matching with the respective graphs. Response: We apologize for providing inappropriate pictures. We have reselected appropriate representative pictures of the western blots. Please see details in Fig9. https://doi.org/10.1371/journal.pone.0241561.r002
19 Oct 2020 Decision Letter - Hemachandra Reddy, Editor The effect of autophagy and mitochondrial fission on Harderian gland is greater than apoptosis in male hamsters during different photoperiods PONE-D-20-17311R1 Dear Dr. Xu, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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, Hemachandra Reddy 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: All concerns raised by the reviewers have been addressed by the authors and please accept the manuscript as it is. ****** 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: No https://doi.org/10.1371/journal.pone.0241561.r003
Formally Accepted
13 Nov 2020 Acceptance Letter - Hemachandra Reddy, Editor PONE-D-20-17311R1 The effect of autophagy and mitochondrial fission on Harderian gland is greater than apoptosis in male hamsters during different photoperiods Dear Dr. Xu: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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. Hemachandra Reddy Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0241561.r004

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