Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, Manuscript ID: cells-4295868

The inhibitory effect of myricetin was evaluated against T. gondii. The study was well-conducted, and the results recorded are significant and encouraging for further investigation. However, the defect in this study lies in the lack of evidence to prove the selectivity of the compound by assessment its effect on catalytic activity on the enzyme analog (DHO) in mammals. In addition, positive control was not used to validate the experimental procedure by ensuring it could produce the expected positive result confirming that the reagents, equipment, and methods were working correctly. There are also several minor observations:

1. The introduction includes a brief comparison between the enzyme in the gondii and its analog in mammals.
2. In section 2.2, specify the incubation time for the enzyme with its substrate, and the source of the enzyme used.
3. In section 2.5, which parasite strains were used?
4. In section 2.8, how was it confirmed that the infection in the mice had occurred successfully?
5. Appendix A.1.A and S1. A, write the unit of measurement used.

 

Author Response

Comment 1: The introduction includes a brief comparison between the enzyme in the gondii and its analog in mammals.

Response: We thank the reviewer for this comment. According to your suggestion, we have added a comparison between the enzyme in T. gondii and its mammalian homolog in lines 74-78.

 

Comment 2: In section 2.2, specify the incubation time for the enzyme with its substrate, and the source of the enzyme used.

Response: We thank the reviewer for this comment. We have already added the reaction time and the source of the enzyme in section 2.2.

 

Comment 3: In section 2.5, which parasite strains were used?

Response: We thank the reviewer for this comment. We have supplemented the information on the T. gondii strain used in section 2.5.

 

Comment 4: In section 2.8, how was it confirmed that the infection in the mice had occurred successfully?

Response: We thank the reviewer for this comment. We have supplemented the detailed procedures and observational criteria used to confirm successful infection of mice with T. gondii.

 

Comment 5: Appendix A.1.A and S1. A, write the unit of measurement used.

Response: We are grateful to the reviewer for this remark. We have added the units of measurement used

Reviewer 2 Report

Comments and Suggestions for Authors

In this paper from Ge et al, the authors tested the effect of Myr on Toxoplasma gondii chronic stage. Here are my comments to the paper:

Could the authors explain why they used RH and/or Pru for different experiments? Is there a special reason?

In the result session, lane 161-172 must be moved to the introduction or to the methods. The recombinant DHO protein was already produced and the activity has been validated in a previous study so this is not a result. The authors should improve the explanation of the rationale of the experiments.

The authors should explain why citotoxicity was tested only on RH parasites.

Figures 4E, 4F, 5E and 5F are not mentioned in the text.

If possible, I suggest that it is indicated the exact p-value in the experiments

Comments on the Quality of English Language

English is fine but there are typos

Author Response

Comment 1: Could the authors explain why they used RH and/or Pru for different experiments? Is there a special reason?

Response: We thank the reviewer for raising this point. We have added the characteristics and applications of the RH and Pru strains at line 89.

 

Comment 2: In the result session, lane 161-172 must be moved to the introduction or to the methods. The recombinant DHO protein was already produced and the activity has been validated in a previous study so this is not a result. The authors should improve the explanation of the rationale of the experiments.

Response: We thank the reviewer for this comment. We have moved the content originally in lines 161–170 to the Introduction section, and have clarified the rationale of the experimental design in the Introduction as well.

 

Comment 3: The authors should explain why citotoxicity was tested only on RH parasites.

Response: First, it should be noted that the cytotoxicity assay measured the toxicity of the compounds (MYR and PLU) to host cells. For T. gondii, this study determined the concentration of each compound required to inhibit 50% of the growth of the RH strain (the half-maximal inhibitory concentration, IC₅₀). As for why the IC₅₀ was only measured against the RH strain, the reason is that the IC₅₀ reflects the inhibitory effect of the compounds on extracellular tachyzoites in vitro, whereas the Pru strain (a low-virulence type II genotype) used in this study was primarily employed to evaluate the effects of the drugs on bradyzoites and chronic infection stages, and was not involved in the determination of the IC₅₀ against tachyzoites.

 

Comment 4: Figures 4E, 4F, 5E and 5F are not mentioned in the text.

Response: We thank the reviewer for this comment. The descriptions corresponding to Figures 4E, 4F, 5E, and 5F have been corrected.

 

Comment 5: If possible, I suggest that it is indicated the exact p-value in the experiments

Response: We thank the reviewer for this comment. The exact p-values have been indicated.

Reviewer 3 Report

Comments and Suggestions for Authors

In the current study, the authors demonstrated that MYR inhibits TgDHO enzyme activity, exerts significant anti-Toxoplasma effects in vitro and in vivo, and induces morphological abnormalities in brain cysts. These findings provide new insights and experimental evidence supporting the development of therapeutic strategies for both acute and chronic toxoplasmosis.

Some suggestions:

1. Lines 53-54: please add what are myricetin and plumbagin from a structural point of view. 

2. Line 68-69: Only DMSO was used to prepare the stock solutions?

3.Line 70 – please add how many mice were used in the study.

4. Lines 85-86 you wrote “with varying concentrations of the substrate L-dihydroorotate (L-D) and in the presence or absence of MYR or PLU”. What amount of L-dihydroorotate and MYR or PLU  where used? Please add.

5.Lines 91-92: add please some details about  how the “The cytotoxicity of the drugs was determined in HFF cells with a CellTiter 96® AQueous One Solution Cell Proliferation Assay”.

6.Line 104 – what kind of microscope was used? Please specify.

7.Lines 141-143 you wrote “At 24-hour post-infection, each 141 mouse received a daily intraperitoneal injection of either MYR (dissolved in sterile saline  containing 5% DMSO) or the vehicle control (sterile saline containing 5% DMSO). Isn’t 5% DMSO a bit much? It is toxic.

8.Under what conditions were the mice kept in cages during the experiment? Please add.

9. How were the mice euthanized and how were the tissues collected/preserved until their analysis? Please add. 

10. Please specify what you consider to be the limitations of the study.

 

 

 

Author Response

Comment 1: Lines 53-54: please add what are myricetin and plumbagin from a structural point of view.

Response: We thank the reviewer for this constructive suggestion. Accordingly, We have added the structural description of myricetin and plumbagin at line 56 of the revised manuscript.

We believe this addition improves the clarity for readers. The changes have been highlighted in the revised manuscript.

 

Comment 2: Line 68-69: Only DMSO was used to prepare the stock solutions?

Response: We thank the reviewer for this comment. Yes, only DMSO was used to prepare the stock solutions due to the poor water solubility of the compounds. The stock solutions were then diluted with culture medium to the desired working concentrations. The final DMSO concentration in all treatment groups (including controls) was kept below 0.1% (v/v) to avoid any solvent-induced cytotoxicity. The control group received an equal volume of DMSO (diluted accordingly) as the vehicle control.

 

Comment 3: Line 70-please add how many mice were used in the study.

Response: We thank the reviewer for this comment. We have supplemented the number of mice used in this study.

 

Comment 4: Lines 85-86 you wrote “with varying concentrations of the substrate L-dihydroorotate (L-D) and in the presence or absence of MYR or PLU”. What amount of L-dihydroorotate and MYR or PLU where used? Please add.

Response: We thank the reviewer for this comment. We have supplemented the compound dosages at line 89.

 

Comment 5: Lines 91-92: add please some details about how the “The cytotoxicity of the drugs was determined in HFF cells with a CellTiter 96® AQueous One Solution Cell Proliferation Assay”.

Response: We thank the reviewer for this suggestion. We have now added the following details regarding the cytotoxicity assay in the revised manuscript (Lines 91–94):

“HFF cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and allowed to adhere overnight. The cells were then treated with various concentrations of MYR or PLU (ranging from 0.1 to 10 µM) for 48 hours. Subsequently, 20 µL of CellTiter 96® AQueous One Solution reagent was added to each well, and the plates were incubated for an additional 2 hours at 37°C. The absorbance was measured at 490 nm using a microplate reader. Cell viability was calculated as the percentage of absorbance relative to the DMSO-treated control group (0.1% DMSO, v/v).”

 

Comment 6: Line 104 – what kind of microscope was used? Please specify.

Response: We thank the reviewer for this comment. We have now specified the microscope used in the revised manuscript.

 

Comment 7: Lines 141-143 you wrote “At 24-hour post-infection, each 141 mouse received a daily intraperitoneal injection of either MYR (dissolved in sterile saline containing 5% DMSO) or the vehicle control (sterile saline containing 5% DMSO). Isn’t 5% DMSO a bit much? It is toxic.

Response: Thank you for highlighting this important point regarding the DMSO concentration. We acknowledge that 5% DMSO is at the upper limit of the recommended range for intraperitoneal injection in mice. However, based on our preliminary experiments and a literature review, the vehicle control group (5% DMSO in saline) showed no signs of distress, abnormal behavior, or weight loss, and tolerated the entire treatment course comparably to the treatment groups. Given the poor water solubility of myricetin and plumbagin, we reduced the total volume per injection to 50 μL to minimize the impact on the mice.

 

Comment 8: Under what conditions were the mice kept in cages during the experiment? Please add.

Response: We thank the reviewer for this suggestion. We have now added the detailed housing conditions of the mice in the revised manuscript. Briefly, the mice were kept under standard conditions: temperature 22 ± 2°C, relative humidity 50 ± 10%, 12 h light/dark cycle, with free access to food and water. Each cage housed up to 5 mice.

 

Comment 9: How were the mice euthanized and how were the tissues collected/preserved until their analysis? Please add.

Response: We thank the reviewer for this important question. Accordingly, we have added the following details in the revised manuscript (Lines XX–XX):

“At the end of the experiment, mice were euthanized by carbon dioxide (CO₂) inhalation followed by cervical dislocation. Brain tissues were immediately harvested, and each whole brain was homogenized individually in 1 mL of phosphate-buffered saline (PBS).”

 

Comment 10: Please specify what you consider to be the limitations of the study.

Response: We thank the reviewer for this comment. We have added the limitations of this study at line 150, as follows:

1. Myricetin exhibits good inhibitory effects against T. gondii both in vitro and in vivo; however, its selectivity index is suboptimal, and the toxicity issue cannot be overlooked.
2. The inhibitory effect of myricetin on TgDHO is not specific; therefore, no specific targeted inhibitor of TgDHO has been identified in this study.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

All comments have been addressed. This version is suitable for publication.

Author Response

Comment: All comments have been addressed. This version is suitable for publication.

Response: We sincerely thank the reviewer for the positive evaluation and for the time and effort dedicated to our manuscript. We are glad to know that the reviewer finds our revision satisfactory. 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

In figure 5, the legend should be corrected (first D)

Comments on the Quality of English Language

English is fine 

Author Response

Comment: In figure 5, the legend should be corrected (first D).

Response: We thank the reviewer for pointing out this error. We have carefully checked Figure 5 and corrected its legend as suggested.