PONE-D-23-32313Cytotoxic activity of Ganoderma weberianum-sichuanese isolated from Lower Volta River Basin of Ghana against human prostate carcinoma (PC-3), leukemic T cell (Jurkat) and plasmacytoid dendritic cell (pDC)-derived acute leukemia (PMDC05) cell linesPLOS ONE

Dear Dr. Lombardi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Mar 09 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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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.

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We look forward to receiving your revised manuscript.

Kind regards,

Mónica L. Chávez-González, PhD

Academic Editor

PLOS ONE

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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: Partly

Reviewer #2: Yes

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

Reviewer #1: No

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: Yes

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: The article " Cytotoxic activity of Ganoderma weberianum-sichuanese isolated from Lower Volta River Basin of Ghana against human prostate carcinoma (PC-3), leukemic T cell (Jurkat) and plasmacytoid dendritic cell (pDC)-derived acute leukemia (PMDC05) cell lines" presents novel aspects on the evaluation of an extract of the fungus G. weberianum on the viability of cancer cells. However, I have some suggestions and comments to enrich the article.

1. Italicize the name Ganoderma weberianum- sichuanese in the title and in line 38.

2. Line 92, “the fruiting body was surface disinfected with 70% alcohol”.

3. Specify what type of cells are the cell lines used in the experiments.

4. How were the subfractions made? What solvent was used?

5. Was TLC performed to qualitatively differentiate the different fractions?

6. How was the calculation of the IC50 carried out, it is mentioned that through non-linear analysis, but what software was used for calculation this value?

7. In the figures it is mentioned that the mean is graphed, but are the error bars also presented?

8. It is mentioned in line 191 that there is no significant difference, but the methodology does not describe what statistical analysis was performed in each assay.

9. The effect presented is on viability (analyzed by the MTT assay), not a cytotoxic effect.

10. Improve the images, they are not properly displayed in the document due to their low quality.

11. It remains to be identified what compounds the extract contains. The introduction talks about it but its extracts lack any qualitative analysis of the presence of bioactive compounds. This is important to be able to identify the molecules responsible for the effect on the cell lines.

12. In table 2, place mL.

13. Check the mL units in the document. In some parts of the text it appears as ml.

14. Add perspectives in the conclusions, what is expected in the future with the study.

15. What was the particle size used to carry out the extraction?

16. Specify how the extract was made. What extraction technique was used?

17. I consider it more relevant to evaluate what compounds the fractions contain than to continue evaluating other cancer cells and animal models.

Reviewer #2: Some background is missing from the abstract

The authors need more to highlight the originality of the work

The authors don't explain if the extraction solvents were volatilized.

(2.3-0 36.80 μg/mL), review these data

Indicate reference to determine the selectivity index

Review the results because for the junkat cell line they report IC50 47.19 �g/mL and the graph does not show the decrease in viability for the GL-C1

Analysis is difficult due to the quality of the images

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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.

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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: No

Reviewer #2: No

**********

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PONE-D-23-32313R1Cytotoxic activity of Ganoderma weberianum-sichuanese isolated from the Lower Volta River Basin of Ghana against human prostate carcinoma PC-3, leukemic T cell Jurkat, and plasmacytoid dendritic cell pDC-derived acute leukemia PMDC05 cell lines.PLOS ONE

Dear Dr. Lombardi,

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 Sep 13 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• 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: 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,

Miquel Vall-llosera Camps

Senior Staff Editor

PLOS ONE

Additional Editor Comments:

Please address the reviewers' remaining concerns.

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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: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: I Don't Know

Reviewer #2: No

**********

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: No

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The comments above were results, however, due to the quality of the images in the previous review, comments regarding the results were not made. On this occasion, the observations made on the results are mentioned and it is recommended to address them, since the figures lack quality for publication.

Line 105: Correct “BLASTn”.

Line 135: Correct to understand what is going to be done in the fractions. “The cytotoxic activity test was carried out on the crude ethanolic extract (GL-CO1) and the seven fractions obtained”.

Line 161: Place from highest to lowest concentration: "36.80-2.30 μg/mL".

Line 223: "Chang liver cell but a moderate cytotoxic effect on Jurkat with IC50 value 224 of 47.19±4.59 μg/mL and SI =2.12 (Figure 4A and Table 1)." This information does not correspond to what appears in Table 1.

Line 314: “This finding suggests that GL-C2-C1 may have a stronger anticancer activity against PC-3 compared to curcumin and could be useful in developing ganoderma mushroom-based products for treating human prostate carcinoma.” Was a statistical analysis performed to validate this assertion?

Line 344. This could explain the results greater than 100% viability.

Why are two IC50 values for curcumin presented in each of the tables and an average is not made for the different experiments with their 3 independent experiments carried out?

A statistical analysis remains to be performed between the fractions to determine if they present a difference between the cytotoxic effects.

It remains to be mentioned whether the data is available.

The discussion needs to be improved, if the compounds present are unknown, try to address the explanation then under the extraction conditions with the different solvents and polarities used.

In the previous review, the images could not be reviewed due to low quality, so in this review, pertinent comments are made to the figures:

Figure 1. Place GL-C0 instead of GL-ethanol extract.

Figure 3. A. Remove the comma ", PMDC05". Adjust the axes, start the X axis at 0, remove the -200 (A) and -20 (B). Check the error bars in Figure 3(B). They do not present the same format as (A).

Figure 4. The information in Figure 3 is repeated here (GL-C01). Place the Figures in the same format. Same font size in the figure title. Check that the text of the axes does not overlap. The x axis in some graphs reaches up to 150 and in others up to 120 µg/mL. If the colors of the cell type lines are the same for all graphs, a single legend can be placed for all images. Figure 4 (C) Correct pDC to PMDC05.

Figure 5. Same comment in Figure 5 regarding the format and that the text does not overlap on the axes. (A) Why is there a % viability of less than 0%? Is there any control? (H) For the PMDC05 cell line, 0 is presented at all concentrations, check if it is correct. If the colors of the cell type lines are the same for all graphs, a single legend can be placed for all images. (E) Check the error bar at the concentration of 100 g/mL for PC-3. It's correct? Since it presents a lot of dispersion in the result. (F) The error bar is not presented for PMDC05.

Table 1. Check GL-CO1, it should be GL-CO according to the text.

Reviewer #2:

Include if the same solvent ratios were used for the GL-C2 subfractions.

On line 162, include if the medium with the biomass was removed??? And if a control sample without cells was used.

Include in formula format the % to determine feasibility and SI

Include reference to compare with the Chang liver cell line if not all cancer lines are of liver origin

When the decrease in viability is less than 30%, it could be referred to as a decrease, not as toxicity, according to the ISO standard….

On line 228 specify concentration

The PC3 labels are missing in figure 5

In relation to the GL-C2-C8 sufraction in Figure 5, a viability of 0 is observed in all concentrations. Is this information correct?

On line 332 discuss the results do not rewrite the same results

Include the entire statistical part in the methodology, include a reference for the description of moderate or low in terms of the decrease in viability.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: 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.

PONE-D-23-32313R2Cytotoxic activity of Ganoderma weberianum-sichuanese isolated from the Lower Volta River Basin of Ghana against human prostate carcinoma PC-3, leukemic T cell Jurkat, and plasmacytoid dendritic cell pDC-derived acute leukemia PMDC05 cell lines.PLOS ONE

Dear Dr. Lombardi,

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.

==============================

Thank you for submitting Revision 2 of your manuscript PONE-D-2332313R2. You have addressed nearly all of the reviewers’ comments, and the paper is much improved. However, before we can proceed to publication, the following points remain fundamental and must be resolved:

a)     Specify the name and version of the software used for statistical analysis in the Methods section.

b)    List all statistical tests performed and the threshold for significance (e.g., p-value).

c)     Verify and reconcile the IC₅₀ values of curcumin reported in the text against those in Table 1.

d)    Format all IC₅₀ values as, for example, “5.13 ± 0.86 μg/mL” (with spaces around “±”).

e)     Correct the spelling of the cell line “PMDC05” in the main text (it currently appears as “PMDCo5” in tables).

f)     Change all occurrences of “GL_C0” to “GL‑C0” throughout the manuscript, including Figure 1.

g)    On line 199, specify the concentration range as “500–0 μg/mL.”

h)    Clarify whether the extract alone (without cells) was included as a background control.

i)      In the Results (line 343), add the appropriate citation for the statement made in that paragraph.

Once these items have been fully addressed, your manuscript will be ready for final acceptance. Thank you for your hard work and attention to detail.

Best regards,

Karla Juarez-Moreno 

==============================

Please submit your revised manuscript by Jun 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.

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: 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,

Karla Oyuky Juarez-Moreno

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Dear Authors,

Thank you for submitting Revision 2 of your manuscript PONE-D-2332313R2. You have addressed nearly all of the reviewers’ comments, and the paper is much improved. However, before we can proceed to publication, the following points remain fundamental and must be resolved:

a) Specify the name and version of the software used for statistical analysis in the Methods section.

b) List all statistical tests performed and the threshold for significance (e.g., p-value).

c) Verify and reconcile the IC₅₀ values of curcumin reported in the text against those in Table 1.

d) Format all IC₅₀ values as, for example, “5.13 ± 0.86 μg/mL” (with spaces around “±”).

e) Correct the spelling of the cell line “PMDC05” in the main text (it currently appears as “PMDCo5” in tables).

f) Change all occurrences of “GL_C0” to “GL C0” throughout the manuscript, including Figure 1.

g) On line 199, specify the concentration range as “500–0 μg/mL.”

h) Clarify whether the extract alone (without cells) was included as a background control.

i) In the Results (line 343), add the appropriate citation for the statement made in that paragraph.

Once these items have been fully addressed, your manuscript will be ready for final acceptance. Thank you for your hard work and attention to detail.

Best regards,

Karla Juarez-Moreno

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: 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

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors made the relevant corrections, I only have a few observations.

1. Add to the methodology section the software used for statistical analysis, as well as the statistical tests performed?

2. Correct the spelling of the cell line PMDC05 in the text; in the tables it appears as PMDCo5

3. Correct the spelling of GL_C0 to GL-C0 throughout the manuscript.

4. Line 199, enter the range of 500-0.

5. Check the IC50 values of curcumin in the cell lines presented in the text since they do not agree with those presented in Table 1.

6. Adjust how the results are presented to IC50 = 5.13 ± 0.86 μg/mL, since in the text, the values are without spaces between them.

7. Correct in figure 1 GL-C0.

8. The paragraph on line 343 has no reference to what is described, add.

Reviewer #2: It is necessary to add the statistical analyses that were performed as well as the p parameter used.

The extract alone without cells was used as a background control?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: 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.

Cytotoxic activity of Ganoderma weberianum-sichuanese isolated from the Lower Volta River Basin of Ghana against human prostate carcinoma PC-3, leukemic T cell Jurkat, and plasmacytoid dendritic cell pDC-derived acute leukemia PMDC05 cell lines.

PONE-D-23-32313R3

Dear Dr. Lombardi,

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,

Huzaifa Umar

Academic Editor

PLOS ONE