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The authors have addressed most of the concerns raised by the reviewers.
[# PeerJ Staff Note - this decision was reviewed and approved by Glynn Gould, a PeerJ Section Editor covering this Section #]
No comment
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The review process is now complete, and two thorough reviews from highly qualified referees are included at the bottom of this letter. Although there is merit in your paper, we identified several concerns that must be considered in your resubmission.
1-Please, consider reviewing the abstract aiming to emphasize, in a direct manner, the importance and novelty of the study. Restructure the abstract topics according to their relevance and aims, and avoid misplaced information such as conclusions in MM and Results. Include information related to the Material and Methods used and a pertinent Conclusion. In the same way, the sections of the manuscript must be reorganized to avoid unnecessary repetitions.
2-The English language needs to be improved to make the manuscript clear for readers.
[# PeerJ Staff Note: Please ensure that all review and editorial comments are addressed in a response letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. #]
[# PeerJ Staff Note: The Academic Editor has identified that the English language must be improved. PeerJ can provide language editing services - please contact us at copyediting@peerj.com for pricing (be sure to provide your manuscript number and title) #]
Article is well written. Sufficient literature is provided.
Study is well designed. Please provide justification for use of 100% DMSO as solvent for curcumin preparation. DMSO can interfere with study results, as reported literature indicate:
- DMSO is reported to induce apoptosis at concentrations >10% (v/v)
- DMSO stimulate prompt Acanthamoeba differentiation into a rounded cyst-like stage with a single envelope
- DMSO increases penetration of drugs into parasite cysts
- DMSO is cytotoxic
Findings are reported nicely. Please provide suitable justification to clarify that results obtained in this study are because of curcumin use only and there is no interference in study results with the use of DMSO and if DMSO interferes with study data, then please include role of DMSO also in discussion and conclusion.
The manuscript describes the effect of sub-lethal doses of curcumin on Acanthamoeba encystation, by investigating morphological parameters and expression of genes related to encystation. In its essence, the study proposal is sound because encystation inhibition is considered an auxiliary strategy for treating infections by Acanthamoeba. The article is well structured scientifically with adequate figures, the author presented consistent supplementary material and raw data. A robust set of references are presented, but some of them are dispensable. English writing makes understanding difficult in some sections. The text is wordy and an overall revision of the English language must be done. I strongly recommend the assistance of a colleague who is proficient in English or from a professional editing service.
The experimental approach involves the comparison of trophozoites treated or not with sublethal doses of curcumin, in starvation or nutrient-rich conditions, regarding morphological parameters and expression of encystation-related genes. The idea is original and based on previous evidence of the curcumin effect on Acanthamoeba inactivation. An English language revision would improve the overall quality of the text to make clear the main question. Some specific issues in methodology with impact even in the title should be addressed (see specific comments)
The data supported the conclusion that curcumin inhibits Acanthamoeba encystation, but the link with Atg genes was not confirmed. The conclusion description should be more assertive and concise in this direction.
Specific comments and questions are the following:
Main issues:
1. Abstract: In the Background subsection, the term “surviving amoebas” does not make sense if one does not know they were treated. Consider including “amoebas treated with sublethal doses of curcumin”. The aim of the study is not clear and assertive, I suggest rewriting mainly the last two phrases in the Background subsection.
2. Introduction: What do the authors consider the focus of the work? The inhibition of encystation by curcumin or the Atg genes expression? As written, the introduction section makes the central aim of the work ambiguous and does not properly connect these two topics. To my understanding, the curcumin effect on encystation is the central point, and the investigation of Atg and other genes expression will give insights into the encystation mechanisms. Therefore, the second paragraph's idea should be before the third one, and the connection of both should be improved.
3. Material and Methods: The encystation inductor medium (PAS+5% glucose) was based on Aqeel et al (2003) as the authors said. However, Aqeel et al used PBS + 10% glucose + 50mM MgCl2. If a modification was inserted, it must be described, as well as the reason for the modification.
4. Material & Methods: Does the term “starvation” adequate, considering that glucose is a nutrient able to support trophozoites activity for the period used in the assays (24h)? Besides, it is clear in the work of Aqeel et al (2003) that high osmolarity is the trigger for encystation. Thus, it seems conceptually incorrect to indicate starvation as the only inducer of encystation. Several recent and old works described encystation saline (no nutrients) to induce cyst formation by starvation as the Neff's encystation medium or other. (Examples: Rolland et al 2020, doi: 10.3390/pathogens9050321; Coulon et al. 2010, doi:10.1128/JCM.00309-10; Bowers & Korn 1969, doi:10.1083/jcb.41.3.786). Why don´t you use them instead of a glucose medium? This conceptual issue demands correction in the paper and in the title (I wonder if distinct types of encystation stimuli could influence the effect of curcumin and even the Atg genes expression, an approach for future investigations.)
5. Material & Methods: What do you mean by "irregular trophozoites"? Are they pleomorphic, presenting pseudopodia, acanthopodia, and vacuoles as active and healthy forms? If so, the term “irregular” can be misinterpreted.
6. Discussion: This section should be shortened to address some issues more objectively. Examples are descriptions on lines 383 to 392 and 516 to 541. Removing references and text restructuring will provide greater conciseness.
7. Conclusions: Pieces of information previously described in Introduction and Results are unproperly repeated here. I recommend rewriting to indicate the main findings according to the aim and the significance. It must be concise.
Additional minor comments:
8. Abstract: The methods should be described more technically.
9. Abstract: In the Results subsection, the description of the results should follow the same sequence as the main text.
10. Abstract: In the Results subsection, line 83: “Altogether, the data reveals that curcumin stress does not induce cysts formation…” Consider replacing by “curcumin stress inhibits cyst formation”
11. Introduction: A revision of the English language is needed. Several phrases can be improved in the style (Example on lines 78-85). What is “cidal”?? (line 129).
12. Material and Methods: The subsection “A. triangularis cultivation” should be the first.
13. Results: Tables S1, S2 and S3 do not indicate the evaluated parameter (percentage viability)
14. Results: Asterisks indicative of p-value significance are lacking in Atg 16 graphic. Include that transcriptional expression of other encystation-related genes is in response to curcumin in the caption of Figure 4.
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