Report: The effects of topical pleurotus tuberregium (PT) aqueous extract on intraocular pressure in monkeys

Ghalib A. Akinlabi; Paul L. Kaufman; Julie A. Kiland

doi:10.1371/journal.pone.0256422

Peer Review History

Original SubmissionNovember 4, 2020
4 Nov 2020 Author Response Attachments Attachment Submitted filename: George Vousden letter to Plos one-PK-10-23-20GA.docx https://doi.org/10.1371/journal.pone.0256422.r001
6 Jan 2021 Decision Letter - Shwet Kamal, Editor PONE-D-20-33452 Report: The effects of topical Pleurotus tuber-regium (PT) aqueous extract on intraocular pressure in monkeys PLOS ONE Dear Dr. AKINLABI, 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. The study on topical use of aqueous extract of Pleurotus tuberregium mushroom on intra-ocular pressure in monkeys is a interesting study. However, there are some concerns in the study which is elaborated in the comments of the reviewers. Moreover, the authors have studied only the effect of Aqueous extracts, which contains only glucans and proteins but alkaloids, saponins, flavonoids, tannins, anthraquinnone, etc can not be extracted in water extracts. Thus the study do not represent the complete effect of PT. Please submit your revised manuscript by Feb 20 2021 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: 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'. 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Shwet Kamal, Ph.D 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 https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. In order to comply with PLOS ONE's guidelines for non-human primate experiments (http://journals.plos.org/plosone/s/submission-guidelines#loc-non-human-primates), we kindly request details regarding housing conditions, feeding regimens, environmental enrichment, and all relevant steps taken to alleviate suffering (anesthesia, analgesia, details about humane endpoints, euthanasia, etc.). Please also indicate how often animal care staff monitored the health and well-being of the animals and the criteria used to make such assessments. Lastly, please specify the disposition of animals at the end of the study (e.g. euthanasia, returned to home colony, etc.). If animals were euthanized following the study, please provide the method of sacrifice. 3. Please ensure that you refer to Figure 1 in your text as, if accepted, production will need this reference to link the reader to the figure. 4. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files. 5. 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. Additional Editor Comments (if provided): The study on topical use of aqueous extract of Pleurotus tuberregium mushroom on intra-ocular pressure in monkeys is a interesting study. However, there are some concerns in the study which is elaborated in the comments of the reviewers. Moreover, the authors have studied only the effect of Aqueous extracts, which contains only glucans and proteins but alkaloids, saponins, flavonoids, tannins, anthraquinnone, etc can not be extracted in water extracts. Thus the study do not represent the complete effect of PT. The authors are requested to re-submit a revised version in the light of the comments of the reviewers. [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: No Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A 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: 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: Dear Authors, thank you for the opportunity to read about this interesting line of research. I find this are highly relevant, however there are several major concerns regarding your study and presentation: 1. In the introduction you mention "...Scientific evidence indicates that PT is useful in treating ... ". The references provided after that are only for the in vitro and in vivo animal works. Pleas provide references to placebo-controlled clinical trials to support this statement. Otherwise it is highly misleading. 2. The scientific premise for the work is not clear. There is no suggested mechanism of action for the PT extract in lowering IOP. 3. What standardization and quality control procedures were used to normalize PT preparations and guarantee reproducibility of the studies? 4. For the IOP measurements - what time of day were they performed? Could you also describe the housing of animals and their expected circadian cycle? 5. In the discussion you make the conclusion that that the "extract has no effect on the IOP in normal eyes" and also make the statement that "there is a possibility that the study groups were small to detect any effect." These statements are contradicting - please correct, with the statistical power if needed. 6. What is the expected mechanism of action for the PT extract? 7. There are several factors that contribute to IOP regulation - could you elaborate on the actual effect of PT extract on the aqueos humour production/outflow, TM state, etc. Also, do you expect it to have differnt effect in dexamethasone-induced glaucoma and healthy eyes? Reviewer #2: Manuscript is good Few typographical or grammatical errors need to be checked Such as in abstract Line no 27. Correct the spelling of Pleurotus tuberregium Similarly line no. 54 lignocellulosic in place of lognocellulosic Correct these small errors ****** 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: 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. 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. https://doi.org/10.1371/journal.pone.0256422.r002
Revision 1
19 May 2021 Author Response RESPONSE TO REVIEWERS Thank you for favorably considering our manuscript and for the corrections pointed out therein. Below are the answers to the questions raised by the reviewers. Issues raised by academic editor The study on topical use of aqueous extract of Pleurotus tuberregium mushroom on intra-ocular pressure in monkeys is a interesting study. However, there are some concerns in the study which is elaborated in the comments of the reviewers. Moreover, the authors have studied only the effect of Aqueous extracts, which contains only glucans and proteins but alkaloids, saponins, flavonoids, tannins, anthraquinnone, etc can not be extracted in water extracts. Thus the study do not represent the complete effect of PT. Response We will study the effect of ethanolic extract on IOP. It has been reported that phytochemical analysis of ethanolic and aqueous extract of P. tuber-regium reveals that traces of polyphenols and saponins were more in ethanolic than aqueous extract, although alkaloids, glycosides, saponins, flavonoids, tannins and polyphenol, were present in both aqueous and ethanolic extract41 (line 227 - 230). Reviewer #1: 1. In the introduction you mention "...Scientific evidence indicates that PT is useful in treating ... ". The references provided after that are only for the in vitro and in vivo animal works. Please provide references to placebo-controlled clinical trials to support this statement. Otherwise it is highly misleading. Response The sentence has been changed to ‘Scientific evidence indicates that PT is useful in treating high blood pressure, diabetic hypertriglyceredemia, tumours, as well as fungal and bacterial infections in animals and humans4-15, but to our knowledge no randomized masked placebo-controlled trials have been reported.’ (line 55-58) 2. The scientific premise for the work is not clear. There is no suggested mechanism of action for the PT extract in lowering IOP. Response Mechanistic studies will be carried out later. PT was reported to contract muscarinic receptor-containing bovine iris muscle in an organ bath preparation19. However, it must be remembered that the iris has nothing to do with aqueous outflow. In live monkeys, the iris can be completely removed, and IOP, outflow facility, and the facility response to pilocarpine are unchanged23,24,25. If there is muscarinic receptor-mediated iris muscle contraction, there could also be muscarinic receptor-mediated contraction of the ciliary muscle in monkeys, and that might increase facility. Aside from that we are agnostic as to the mechanism. Assuming that PT lowers IOP in live NHP, we would identify the individual physiological parameter affected (aqueous humor formation, conventional outflow, uveoscleral outflow, Schlemm’s canal and episcleral venous pressure, etc. These parameters can each be measured 26-30. (Line 180-190). 3. What standardization and quality control procedures were used to normalize PT preparations and guarantee reproducibility of the studies? Response Mushroom crops were produced in shade houses, harvested by hand and sundried. All water used in cleaning and processing were purified with an ultra-filtration system.This super fine membrane technology filters particulate down to 0.025 microns. (Line 86-88). 4. For the IOP measurements - what time of day were they performed? Could you also describe the housing of animals and their expected circadian cycle? Response IOP measurements started at 9 am after treatment and every hour after that, for 3 hours (line 114). The monkeys were kept in standard AALAC- and USDA-approved WNPRC housing and husbandry conditions, under a 12/12hour light/dark cycle. (Line 115-117). 5. In the discussion you make the conclusion that that the "extract has no effect on the IOP in normal eyes" and also make the statement that "there is a possibility that the study groups were small to detect any effect." These statements are contradicting - please correct, with the statistical power if needed. Response The wording has been changed to: The extract had no effect on the IOP in our small group of normal eyes, but there is a possibility that the study group was too small to detect a small effect. Sample size calculation for paired organs conducted based on the mean and standard error of the current data indicated that at n equals 5 to 8, we would have sufficient power to detect a physiologic response greater than or equal to 25% of baseline for a two-sided test and 5% significance22,23. The calculation was for outflow facility; theoretically, IOP might be different, although IOP is linked to outflow facility. While our data set is small, this preliminary experiment tells us that we are not likely missing a big effect that occurs across the board in all animals. Whether a higher dose or longer duration would have mattered we cannot say, but this is not like pilocarpine, epinephrine, rho kinase inhibitors, timolol, nitric oxide donators, PGF2⍺ analogues, etc., where there is a big effect after short-term treatment in most human or monkey subjects31-40. (Line 180-190). 6. What is the expected mechanism of action for the PT extract? Response The contractile effect of PT on muscarinic receptor-containing iris muscle may explain its IOP reducing property19. If there is muscarinic receptor-mediated iris muscle contraction, there could also be muscarinic receptor-mediated contraction of the ciliary muscle in monkeys, and that might increase facility. This is only one possible mechanism; any of the other parameters affecting aqueous humor formation or outflow could be at play. One has to start somewhere……](Line 179 – 186). 7. There are several factors that contribute to IOP regulation - could you elaborate on the actual effect of PT extract on the aqueous humour production/outflow, TM state, etc. Also, do you expect it to have different effect in dexamethasone-induced glaucoma and healthy eyes? Response Earlier work shows that PT extract reduces IOP in dexamethasone-induced glaucoma in a feline model20,21. Mechanistic studies would be needed to uncover an effect of PT on aqueous humor production/outflow (trabecular meshwork/ Schlemm’s canal. EVP, uveoscleral outflow, etc.), and then to analyze morphology/ultrastructure, etc. in primates. This is far, far beyond the scope of the current exploratory study. (Line 222 – 226) Reviewer #2: Manuscript is good Few typographical or grammatical errors need to be checked Such as in abstract Line no 27. Correct the spelling of Pleurotus tuberregium Similarly line no. 54 lignocellulosic in place of lognocellulosic Correct these small errors Response The errors have been corrected (Line 27 and 54). Yours truly, G.A. Akinlabi Attachments Attachment Submitted filename: RESPONSE TO REVIEWERS 1-PKGAPKGA4-3 - PK-2GA12-3 - PKGA14-3.docx https://doi.org/10.1371/journal.pone.0256422.r003
9 Aug 2021 Decision Letter - Shwet Kamal, Editor Report: The effects of topical Pleurotus tuber-regium (PT) aqueous extract on intraocular pressure in monkeys PONE-D-20-33452R1 Dear Dr. AKINLABI, 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, Shwet Kamal, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0256422.r004
Formally Accepted
13 Aug 2021 Acceptance Letter - Shwet Kamal, Editor PONE-D-20-33452R1 Report: The effects of topical pleurotus tuberregium (PT) aqueous extract on intraocular pressure in monkeys Dear Dr. Akinlabi: 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. Shwet Kamal Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0256422.r005

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