Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ACHIEVING PHYSICAL EXAMINATION COMPETENCE THROUGH OPTIMIZING HANDS-ON PRACTICE CYCLES: A PROSPECTIVE COHORT COMPARATIVE STUDY OF MEDICAL STUDENTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Thank you for giving me the opportunity to review this interesting manuscript that tackles an important undergraduate medical education topic. After revisions, I believe the manuscript now is much clearer in enlisting aims and goals, and language and structure are more fluent and easier to follow. It does help when unnecessary information and data are removed or abbreviated to give the reader a more concise and direct view of the flow of the study and its results, and I believe this what happened here. All the data were appropriately shared and demonstrated in simple figures and tables. It was quite clear why authors wanted to do the study. Finding an optimal practice frequency for one of the important physical exam (PE) skills - abdominal examination - needed for students without prior clinical experience to achieve a pre-defined competence is crucial for their development as competent future physicians. It also assists in developing PE curricula during medical school training.
Experimental design: Deliberate practice (DP), as outlined in the manuscript, was found to be associated with better skills acquisition in medical simulation in general, for example during surgical and procedural training. PE can be looked at as a 'bedside medical procedure' where DP can be applied to reach acceptable level of professionalism. This manuscript directly addresses the question of how many practical sessions, on average, a medical student without clinical experience will need to achieve an agreed upon competence level in performing abdominal examination. Although the number of students involved is relatively small but still the two studies used to answer the question (the learning curve study and the comparative study) were appropriate and help formulating an answer to that question. Applying similar methods with larger number of students, in different learning environment and institutions, with other physical examination domains will be even more informative and educationally useful. Standardizing of assessment was established with excellent k-value and that is very important in this kind of studies.
Validity of the findings: Based on the methods described and the settings in which the study was performed, the findings make sense and reasonable. It will be interesting to know if the number of practices needed to achieve competency will decrease when the ratio of instructors to students is less than the ratio used in the study (between 1:12 and 1:14). Smaller ratio is expected to give more feedback time for the instructors and that might, at least theoretically, enhance the learning curve. Also, doing the final test on a standardized patient with actual findings on abdominal exam (e.g., splenomegaly) would have made the study more pragmatic and powerful.
Additional comments: Overall, I think this is a useful and informative study that can add to body of literature related to physical examination training in undergradulate medical education. I encourage the authors to do similar studies in other aspects of physical exam and consider points mentioned above to make these studies even more robust.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ACHIEVING PHYSICAL EXAMINATION COMPETENCE THROUGH OPTIMIZING HANDS-ON PRACTICE CYCLES: A PROSPECTIVE COHORT COMPARATIVE STUDY OF MEDICAL STUDENTS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The latest version of the submitted manuscript has improved in language, however, minor points of medical (education) terms should be noted as follows.

1. Line 94, since it is the first time that M.D. appeared, I would prefer using "with/holding a Doctor of Medicine Degree" or similar expressions.
2. Line 136, I think by using "Diagnostic Syllabus", the authors are actually trying to say "Physical Examination Course Syllabus".
3. Line 224, maybe a past participle of "have hypothesized" is better than the current past tense used.
Experimental design: The authors mentioned in the Method part that the Angoff method with 5 judges was usedto determine the competence score, the latter of which is a kind of cut-off score.  This helps clarify the approach they used, although 5 judges is the minimum acceptable number of judges involved in a standard setting. Anyway, it's acceptable.
Validity of the findings: The authors have extended their Discussion part in the current version of manuscript. Lines 195-206 has provided improved comparsion of their results with other DP related studies.
Additional comments: Please see to the minor language issue, I think the manuscript is otherwise okay for acceptance.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FUNCTIONS OF 1,25-DIHYDROXY VITAMIN D3, VITAMIN D3 RECEPTOR AND INTERLEUKIN-22 INVOLVED IN PATHOGENESIS OF GOUT ARTHRITIS THROUGH ALTERING METABOLIC PATTERN AND INFLAMMATORY RESPONSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study is very valuable to the medical field as well as to the general public.  The contribution of Vit D and its metabolites is becoming increasingly important in disease control.

Major issues:
•	Although the reasoning behind the study is clear after reading the complete paper, the problem statement is not clearly formulated in the introduction section. For example, it is stated that gout is a problem in human health, but not how much (epidemiology) and how (symptoms).  It is mentioned that MSU deposits, but not where, and that gout is triggered by fluctuations in uric acid, but the reason why this happens is not mentioned.  Apart from the ending sentence, to provide more insight into the disease, how could this study contribute to medicine? Could it perhaps improve treatment? Enhance diagnosis?

•	I do feel that the writing style can be improved. For example, may sentences begin with the word ‘And’. Section 2.1 line 66 ‘In’ should be ‘in’.  Abbreviations are not defined upon first mention (section 2.2 line 79 for example), and some, such as KEGG and TB are not defined at all.

•	Writing is not always clear, for example, Introduction line 45 – cytokines trigger inflammation, but is inflammation a known symptom of gout? Line 45 – Vit D inhibit inflammation, which form/metabolite of Vit D?

•	Minor issues:
•	Change the ‘vitamin receptor’ keyword to Vitamin D receptor’
•	I would suggest that full descriptions, and not abbreviations, are used in the title of the paper.  These abbreviations might not be standard and known to the whole scientific community.
•	Section 2.2 line 78 delete ‘what’s more’
•	Line 273 – Pseudomonas aeruginosa should be in italics.
Experimental design: Major issues:

•	Patient information can be described in more detail (section 2.1) – were all patients and controls hospitalised? Where were controls recruited? In which country is this Hospital? It is mentioned in line 63 that the criteria had to be met – it will add content to the section if these criteria could be mentioned here. Patient received ‘regular’ treatment – does this refer to ‘normal’ or ‘chronic’ treatment? It will be good if it is also mentioned here that controls and patients were aged and sex matched. Did controls also provide written consent? How were samples collected – by a doctor in hospital, were patients fasting, what time of day, etc.

•	No detail is provided on the quantification, zero-replacement, normalisation and cantering of the untargeted GC-MS data.  Also no information on the similarity hit for annotation of compounds or signal to-noise-ratio used.

•	The PCA and methods for identification of important metabolites from the untargeted data are not described in the methods section.

Minor issues:

•	Is TRIzol a kit?

•	It is mentioned that PCR was done in duplicate, was the average used in statistical analyses?

•	Add G-force and time to centrifugation steps.

•	It is stated that 1 mg sample was used, should this perhaps be 1 mL?

•	It is not clear if the proteins were removed after the cold extraction, before the evaporation.

•	Line 119 – ‘a two sided p<0.05’ please add the test, ANOVA or t-test.

•	Baseline characteristics are presented as mean or median – I would suggest using the same value.
Validity of the findings: Major

•	It is concerning that only 3 samples from 2 groups were used for untargeted analyses.  The rationale behind this is not clear. Metabolomics power calculations would indicate that these groups are not statistically viable.  Conclusions made can therefore only be speculations.  In addition, all 3 groups of samples should have been included to make concrete conclusions.  This is of major concern, and I suggest that GC-MS analyses are done on all samples in the cohort, or as an alternative, the importance of the metabolomics data and the conclusions made should be made less prominent, and describe only as a pilot study.

•	It is not clear how the important metabolites were identified, based on which criteria.  The reason for doing pathway analyses twice, with different sets of metabolites, is also unclear.

•	It should be mentioned whether or not the correlations between CRP and IL-22 and VD3 are similar for all 3 groups of participants.

Minor:

•	In line 132 it is mentioned that any other co-morbidities were documented, although only diabetes and hypertension are noted – were these the only co-morbidities?
•	Please define STRING
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FUNCTIONS OF 1,25-DIHYDROXY VITAMIN D3, VITAMIN D3 RECEPTOR AND INTERLEUKIN-22 INVOLVED IN PATHOGENESIS OF GOUT ARTHRITIS THROUGH ALTERING METABOLIC PATTERN AND INFLAMMATORY RESPONSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript has been well planned and is in the main clearly written and understandable. The authors are commended for their ability to report the vast amount of data generated effectively in a relatively succinct manner. The language employed is generally of good quality with only a few colloquial errors in the manuscript.

Referencing in the main is of a high standard. The authors employ references well throughout the manuscript. The one area where referencing may appear more weak is in the introduction. The references employed are acceptable. I however, feel that more references here alongside the explanation for why the study was conducted would be valuable.

The figures and tables used within the manuscript are of high quality. The normal conventions has been adhered to and thus the comprehensibility of the figures are high. In general the structure of the manuscript and figures contribute highly to the good readability thereof.
Experimental design: The experimental design of this study is based on a well identified knowledge gap. The methodology employed is acceptable in the main, including high technical and ethical standards being employed. There is some discomfort however, in the fact that the authors did not appear to have performed any sort of sample size calculation, nor any calculation of the statistical power of their findings. This is highly regrettable, as the findings appear very promising. The repeatability thereof, along with the generalizability thereof is however, in doubt due to the small sample size. The discussion of methods and results is good, but the conclusion should reflect on the fact that this may only serve as a preliminary study, based on the small sample size.
Validity of the findings: The authors have discussed findings well and in line with the data provided. All bench and calculated data has been provided in a clear and usable manner. The only criticism remains the small sample size that is not justified or discussed in the manuscript.
Additional comments: This is to my mind an excellent piece of work. The only detracting factor, being the sample size. In all other aspects I was impressed with the high standards achieved by the authors.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FUNCTIONS OF 1,25-DIHYDROXY VITAMIN D3, VITAMIN D3 RECEPTOR AND INTERLEUKIN-22 INVOLVED IN PATHOGENESIS OF GOUT ARTHRITIS THROUGH ALTERING METABOLIC PATTERN AND INFLAMMATORY RESPONSES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The reviewer is happy with the changes made.
Experimental design: I still feel that this is just a pilot/proof of concept study in terms of the metabolomics analyses, and although it was added and the end of the manuscript that the limitation is the small sample size, it should also be mentioned in the methodology section that the metabolomics section is only a pilot study and that the conclusions made are just hypothetically at this stage.  With high throughput systems such as the Leco GC-TOFMS, 58 samples can easily be analyzed and aligned using the statistical compare function of ChromaTOF.  It should also be added in the methodology that pooled samples were used, since, at present, it is indicated that only 3 patient samples were include per group, and the pooling is not mentioned at all.  Also, the reasoning behind analyzing aliquots of a pooled sample as opposed to using individual patient samples should explicitly be mentioned.  The use of a pooled sample is not advisable for disease metabolomics, since, in practice, this lowers your sample cohort to 1 sample per group and it does not include individual variation at all.

PCA is still not described in the methods section, but the PCA result is discussed in the discussion section.
Validity of the findings: The reviewer is happy with the changes
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FUNCTIONS OF 1,25-DIHYDROXY VITAMIN D3, VITAMIN D3 RECEPTOR AND INTERLEUKIN-22 INVOLVED IN PATHOGENESIS OF GOUT ARTHRITIS THROUGH ALTERING METABOLIC PATTERN AND INFLAMMATORY RESPONSES
Review round: 2
Reviewer: 2

Output:
Basic reporting: Upon reviewing the amended manuscript, it is clear that the authors accepted the review comments from both reviewers and have done their best to comply with the suggestions of the reviewers. This is highly appreciated. It appears that shortcomings in the original manuscript, pertaining mostly to a limited introductions, some lack of description in the methods and some acknowledgement of the small sample size, has all been appropriately addressed by the authors. I am pleased to report, that with this being considered, the manuscript now appears suitable for publication in my opinion. The use of language and  literature is of a acceptable quality. The methodology used and the data is available and clear. The conclusion is supported by the data and contributes to the knowledge in the field.
Experimental design: The experimental design is in my opinion rigorous and as I have indicated is now more clear and understandable. The data analysis is now also more clearly explained.
Validity of the findings: Findings are meaningful and well stated. The only concern is the relatively small sample, size, which authors acknowledge. The findings are still meaningful in my opinion and may lead to further research.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FUNCTIONS OF 1,25-DIHYDROXY VITAMIN D3, VITAMIN D3 RECEPTOR AND INTERLEUKIN-22 INVOLVED IN PATHOGENESIS OF GOUT ARTHRITIS THROUGH ALTERING METABOLIC PATTERN AND INFLAMMATORY RESPONSES
Review round: 3
Reviewer: 1

Output:
Basic reporting: The reviewer is happy with the changes made
Experimental design: The reviewer is happy with the changes made
Validity of the findings: The reviewer is happy with the changes made
Additional comments: The reviewer is happy with the changes made


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: STRESS-REGULATED ELEMENTS IN LOTUS SPP., AS A POSSIBLE STARTING POINT TO UNDERSTAND SIGNALLING NETWORKS AND STRESS ADAPTATION IN LEGUMES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors have submitted a review on stress-regulated elements involved in stress adaptation signaling in Lotus spp. This is a very interesting and missing piece of information in legumes. A list of interesting examples of diverging stress-related behaviors among the Lotus species are reviewed.
Experimental design: The survey methodology should be improved (see below)
Validity of the findings: The reported information are important to postulate interplay between different biotic and abiotic stresses and responsive elements, paving the way for the obtainment of legume stress resilient genotypes. The review is well written and the reading flows nicely.
See also general comments below
Additional comments: I have two major criticisms.
The L. japonicus MG20 and Gifu are the two accessions mostly used as model systems for fundamental studies. The authors report the Sato et al. 2006 reference (web site: http://www.kazusa.or.jp/lotus/index.html) where information and tools are provided. I found quite strange that all over the review, the authors never cite the Mun et al. 2016 paper (web site: https://lotus.au.dk/) , where some innovative tools are also provided. In particular, the co-expression toolkit is something that the authors should have exploited to support (if possible) some of the interesting correlations they report, aimed to identify putative protein-protein interactions playing roles in the stress-related signaling pathways.
Another missing point is the lack of information on the pathways affecting the flowering time in Lotus spp, the TF involved and the correlation with stress conditions. This is a crucial phenotypic treat for biomass production in forage legumes and the MG20 accession, which is known as an early flowering accession, could provide important clues.

Minor points
I would suggest to divide the two main sections, Transcription Factors and Chaperons, redox….. in subtitled paragraphs. The subtitles of the TF section could be the names of the different TF families analyzed. In the case of the second section, I would suggest to subtitle with the different kind of stresses analyzed (e.g. Salt stress, drought…).
On my view, the paragraph on the CBS motifs (lines 294-302) could be eliminated. There are too poor information reported in literature to state their roles in stress response pathways.
Line 108, In vitro in vivo. Italic


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: STRESS-REGULATED ELEMENTS IN LOTUS SPP., AS A POSSIBLE STARTING POINT TO UNDERSTAND SIGNALLING NETWORKS AND STRESS ADAPTATION IN LEGUMES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This contribution is a review of bibliography of the proteins and genes involved in stress signaling in several Lotus spp (as expected, mainly from the model L. japonicus). The focus is on stress signaling networks and the paper is organized in two major blocks: "transcription factors" and a miscellaneous section including  "chaperones, redox signals and proteins of unknown function". There are abundant studies on transcription factors and more scattered information on the other types of proteins. I would make "hormones" a separate section, as they cannot be included in the second block. The authors briefly describe polyamines, strigolactones, ethylene and ABA. The description of results on these hormones needs to be expanded and should encompass other hormones and defense-related compounds such as jasmonic acid and salicylic acid. For clarity and easier reading, the authors should consider also making a specific block for antioxidants, which can include information on thioredoxins and phytochelatins from the second block, as well as reports on ascorbate metabolism, thiol metabolism, superoxide dismutases and catalases. mention to reactive oxygen and nitrogen species (RONS) is also lacking. These reactive species are clearly involved in stress signaling. There are several papers linking these enzymes and metabolites in the response to drought and salt stress. English is quite good and the text can be read without problems, but the paper needs to be revised by a native speaker because there are numerous errors of syntax and grammar. A double-check for correct citations in the text and literature secion needs also to be conducted (for instance, citation of Xie et al. 2019 is not included in the bibliography section). Also, the paper requires a thorough revision of English language.
Experimental design: The article is within the scope of the journal. The survey of literature is unbiased but incomplete. Studies on antioxidant defenses in response to stress and on additional  hormones and growth regulators are lacking. Also, in most cases it is unclear if the data come from non-nodulated plants or from nodulated plants. That is important because nodulation may make a major difference in sensitivity/tolerance to abiotic stress. Blocks of information (sections) need to be reorganized. Also, there seems to be little connection between the sections. It would be desirable to try to put the review of genes/ proteins into the context of a metabolic and regulatory framework. In this respect, one or two figures addressing the pathways of stress defense and signaling would facilitate reading and would make the review more appealing.
Validity of the findings: The novelty of the literature survey is justified as it includes both the model Lotus japonicus and other Lotus spp. (L. tenuis and L. corniculatus) that are relevant as cultivated plants or in natural ecosystems. The conclusions that L. japonicus can be used as a model to study stress signaling is straightforward. However, a similar study could have been performed using another model legume such as Medicago truncatula and its related crop species M. sativa. In other words, it is fine to focus on Lotus but similar conclusions could have been obtained by examining literature of molecular stress responses in Medicago. The review is of interest to a broad readership, ranging from plant physiologists and agronomists to geneticists and molecular biologists.
Additional comments: The review is a literature survey of Lotus spp. in response to stress. The idea and timing for this review are fine, but the paper needs reorganization, expansion and some "glue" between sections. One or preferably two figures are required to provide a global picture of the genes/protein being reported. This would also enhance the interest and understanding of the review. Correct correspondence of citations between text and bibliography section needs to be revised. English needs also to be heeded.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: STRESS-REGULATED ELEMENTS IN LOTUS SPP., AS A POSSIBLE STARTING POINT TO UNDERSTAND SIGNALLING NETWORKS AND STRESS ADAPTATION IN LEGUMES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have properly responded to all the comments and submitted an improved version of the review. I'm fully satisfied with the revised manuscript.
Experimental design: The study design has been improved
Validity of the findings: The review summarizes an important issue in legumes
Additional comments: The authors have properly responded to all the comments and submitted an improved version of the review.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INTERNET RISKY BEHAVIOURS AMONG YOUTH WITH VISUAL IMPAIRMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is well written.
Experimental design: The paper investigates the risks faced by visually impaired adolescents when accessing the Internet. The study protocol is robust.
Validity of the findings: The findings are factual and useful.
Additional comments: The study is useful and the paper is well written. You may consider more thorough statistical analysis in your future papers.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INTERNET RISKY BEHAVIOURS AMONG YOUTH WITH VISUAL IMPAIRMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear English: The article is mostly written in understandable English; however, there are a number of minor glitches that I've identified under Section 4. General comments and provided suggestions for edits.

Literature: The article does give some background to the issues examined in it; however, there are some areas where more background information would be useful. In particular, you could describe how the Internet use of blind and partially-sighted youth differs in practical terms from youth without visual impairments (e.g. screen reading software, large print, zooming functions?) This would be useful for those less familiar with these technological solutions and their limitations. In addition, there are some definitional issues etc. that I've commented on specifically in Section 4 below.

Data, structure, figures: The data have been shared and the tables and general structure of the article seem fine.

Self-contained: Yes, the article is self-contained. Objectives stated instead of hypotheses - from my point of view, this is fine.

Reference list: There are quite a few sources in Polish. While I'm not sure about this journal's style requirements for these situations, it would be helpful for readers unfamiliar with Polish to have their names translated into English in brackets after the original Polish name.
Experimental design: Original research: The article does report original primary research.

Research questions: Objectives are meaningful but could be a bit more specific than they currently are.

Technical and ethical standard: The technical standard of the article seems fine, and informed consent was obtained. My only reservation is whether the consent process sufficiently took into account the special needs of the participants (see Section 4 below).

Methods described with sufficient detail: This seems to be the case.
Validity of the findings: The data are provided, and conclusions are generally supported by the data. However, the lack of non-VI control group reduces the usefulness of the findings, which the authors have acknowledged in the Limitations. If there is any comparable research on non-VI youth in Poland, comparisons should be made with it. The authors might also recommend conducting a replication study featuring participants with and without VI for the sake of data comparability (this would also be a great study for them to carry out themselves).
Additional comments: Title and terminology in the text: since adolescence is defined by WHO ( https://www.britannica.com/science/adolescence ) as the age range of 10-19 years, whereas "young people" are defined as those who are 10-24 years old, considering that your sample has people up to the age of 24 years, it would be better to use the term "youth" or "young people" instead of "adolescents" in your title and throughout your manuscript where references are made to your own participant group. "Boys" and "girls" could be replaced with "male  youth" and "female youth" (as uncountable nouns) to correspond with this terminology (alternatively, "male students" and "female students" are fine and you have already used these at times).

l. 68-69: It would be good to provide brief (a line or two) contextual information about the EU Kids Online project (aims, number of countries and participants, etc.) to enable readers unfamiliar with it to see why it's so relevant to your study

l. 84 It would be good to provide a definition for electronic aggression the first time you introduce it; it would also be helpful if you can explain how you see it as same or different than cyberbullying, for example (with appropriate citations to the literature)

l. 87 "and also about of people aged": some percentage missing here?

l. 90 please clarify what you mean by the term "techno-discursive design"

l. 93 "...in a way that cannot be controlled" - this is quite a sweeping statement, how can you be sure?

l. 96 please define the terms flaming and trolling in brackets

l. 101 after reference endnote [12] might be a good place to split the paragraph, which is a bit long

l. 110-115 it would be good to contextualize the studies by stating where they were conducted

l. 131-132 They may be scarce, but if you are aware of some, it would be good to cite a few here to show what's already been done in this field

l. 145 On lines 152-153 you stated the questionnaires were in Braille or large print - what about the participant information sheet and consent form? Were they in an accessible format? Do you have any reflections on best practice in obtaining consent from young people with visual impairment?

l. 154 "The mean time of Internet sessions..." - did you count activities both on computers and mobile devices? Please state in the text.

l. 156 This line talks about uploading MP3s, whereas line 193 talks about downloading them. Did you actually ask about both and do you think there is some relevant risk related to either activity?

l. 209 "p= Chi2=12.596;" - is something missing or out of place here?

l. 244 "In the ordinal regression model..." - In order not to confuse readers, it might be clearer if you state "In the second ordinal regression model..."

l. 248-251 The sentence "The odds for participants..." is confusing, please check - especially at "were 2.60 (p<.05) compared"

l. 255 "In the next ordinal regression model" -> "In the final ordinal regression model"

l. 257-259 "The odds for participants..." again a confusing sentence, please check

l. 273 "competences" -> "competencies"

l. 278-280 "Previous study revealed that using online chat..." - curious finding, do you have any speculation as to why seeking disability-related information or participating in support groups lowered well-being, when they are in principle supposed to do the opposite?

l. 330 "teaching digital competences" -> "when teaching digital competencies"

Limitations: It's good that you have noted the limitation about the lack of comparable data here - nevertheless, I wonder if there are no studies conducted with Polish youth without VI using any of the same variables as the present study. If there were any, comparison with the present study's findings would be most useful for giving some indication as to whether adolescents with VI are at an increased risk, decreased risk, or equal risk of suffering negative consequences from their Internet use when compared with adolescents without VI.

l. 396 "bahaviours" -> "behaviours"

l. 424 "Waszyngton" -> "Washington"

p. 21, 23, 25 "Each data point activities faced by students" - confusing, consider rewording


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INTERNET RISKY BEHAVIOURS AMONG YOUTH WITH VISUAL IMPAIRMENT
Review round: 1
Reviewer: 3

Output:
Basic reporting: - Clear and unambiguous, professional English is used, with some exceptions which can easily be corrected.
- The description of the general state of research could be more detailed. In this regard, important terms should be better defined.
- The manuscript would benefit from a more thorough structuring.
- No hypotheses are mentioned.
Experimental design: - The investigation seems to have been conducted rigorously and to a high technical and ethical standard, but some important details regarding the procedure of data collection are missing.
- No experiment is conducted. It could be described as quasi-experimental design, but data is not analysed as such.
- Research question should be stated more clearly and be integrated better into the general state of research part.
- Methods are not described in sufficient detail.
Validity of the findings: - Discussion and conclusion would benefit from a more thorough link with the current state of research.
- For several arguments in the discussion, it is not clear how they relate to the reported data.
Additional comments: The authors address the question how young participants with visual impairments engage in different types of internet risk behaviors. The topic is of great importance and interest for the research community as well as for practitioners. The data set is quite impressive, with a total sample of 490 students answering a self-administered questionnaire. In addition, the manuscript is in most parts well written and understandable.

However, the manuscript also contains various elements that can be criticized and have potential for improvement. The most important improvement would imply a stronger focus on a specific research question which is based on a clear research gap. This would also include the formulation of directed hypotheses at the end of the theory section of the manuscript. I would have expected a clear statement of the research gap and the research question at the beginning of the manuscript, followed by a more detailed and better organized review of the state of the art in the domain. In this regard, the line of argument could be strengthened with a clearer focus on the research question and the specific user group. Additionally, terms and concepts are often mentioned very briefly and are not defined thoroughly (e.g. flaming, trolling, incivility), which is an issue for the understanding of the study details and the integration of the findings into the general state of knowledge. The review of previous research should be more detailed in order to be able to understand the knowledge this study adds to the general state of research.

The method section could be structured in sub-headings (e.g. participants, measures and instruments, procedure, data analysis). It was not clear to me why the authors refer to the term ‘study group’. This somehow implies that there is also some sort of control group – which seems not to be the case (unfortunately) in this study (as compared to the ‘pre-study’ in which two user groups were compared [30]). The measures used in this study should be described in more detail. All the questions asked should be mentioned, including the answering options - other researchers should be able to replicate this study based on the information presented in the method section.

As no hypotheses have been formulated, it is quite difficult to comment on the data analysis section.
The discussion section reads in large parts as a repetition of the results section and the integration of the findings into the larger context of the general state of research could be improved. Additionally, I would suggest not to end the manuscript with the limitations (which should be reported in the discussion section).


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INTERNET RISKY BEHAVIOURS AMONG YOUTH WITH VISUAL IMPAIRMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have successfully resolved several issues identified by each reviewer. However, the manuscript appears not to have been thoroughly proofread, copyedited, or edited for correct grammar. I've identified all of these issues in Section 4 below. If the authors resolve those issues I think it should be publishable then.
Experimental design: There are minor details that need to be added - see Section 4.
Validity of the findings: The article is in much better shape after the previous edits with regard to this area.
Additional comments: Here are my detailed line-by-line suggestions and questions:

l. 50	I see "risky Internet use" was changed to "risk Internet use". The original wording is more grammatically correct, please revert back to that.

l. 71 	Missing a space between "youth" and "with"

l. 77-78	"provide a high quality, independent and comprehensive research findings" -> "provide high quality, independent and comprehensive research findings"

l. 80	"an  integrated" -> "an integrated" (delete 1 excess space)

l. 85-87	"Social networking services (SNS), video/audio conferencing tools, games, movie watching and listening to music are used" -> "Using social networking services (SNS) and video/audio conferencing tools, playing games, watching movies and listening to music are done"

l. 118-120 For clarity, please cite the source immediately after the sentence giving the definition of electronic aggression. Just wondering - the definition looks exactly like the textbook definition of cyberbullying. If you consider electronic aggression and cyberbullying to be the same thing, it might be good to state this to link your work to the broader cyberbullying research.

l. 121 	"study  showed" -> "study showed"; "off-line  about" -> "off-line, about" (remove 1 excess space in both cases, add comma in the latter)

l. 122 	"person  aged" -> "person aged" (remove excess space)

l. 123 	"of an electronic aggression" -> "of electronic aggression" (electronic aggression is an uncountable noun, hence no article)

l. 123 	"about 14% of older students have received sexual messages and 7% of participants have been perpetrators of cyberbullying or hate speech." -> Clarify which study's participants were described

l. 129 	"media content" -> "media content." (add missing full stop)

l. 129-131 The added sentence added isn't quite clear yet. It still needs a brief definition of "discursive power" (maybe in brackets after the term) and "automatically equate visibility or popularity" seems to be missing the thing that they're being equated with?

l. 133	The word "sometimes" should be before the word "encourage" ... or should it be ", sometimes in an uncontrollable way" ?

l. 140 	"harassment)[15]" -> "harassment) [15]"  (Add missing space)

l. 175-176	Add missing line break between paragraphs

l. 177	"Youth with physical disability is excluded" -> "Youth with a physical disability are excluded"

l. 179 	"for  Internet" -> "for Internet" (delete excess space)

l. 189 	"to see better the content" -> "to see the content better"

l. 223 	"youth  with" -> "youth with"; "Another  preliminary"  -> "Another preliminary" (delete excess spaces)

l. 231 	"risk  behaviours" -> "risk behaviours" (delete excess space)

l. 232	"poor-sighed.  [32, 34].  Rresearch" -> "poor-sighted [32, 34]. Research" (delete excess spaces and full stop, correct spellings)

l. 234 	"such as: gender," -> "such as gender," (no comma after "such as")

l. 241 	"The following hypothesis " -> "The following hypotheses " (use plural form because more than one hypothesis is stated)

l. 248 	"perpetrators  of" -> "perpetrators of"

l. 249-250	"all types of  electronic aggression (victim, perpetrator, witness)" -> "all forms of engagement in electronic aggression (victim, perpetrator, witness) "

l. 309	"to International" -> "to the International"

l. 319-320	Add missing line break between paragraphs or combine into the same paragraph, as appropriate.

l. 324	"survey.  The" -> "survey. The" (delete excess space)

l. 326	"why  490" -> "why 490" (delete excess space)

l. 327 	"recruited" -> "recruited." (add missing full stop)

l. 329-330	"known as the sensitive data" -> "considered sensitive data"

l. 330-331	Add missing line break between paragraphs or combine into the same paragraph, as appropriate.

l. 351-443	Add missing line breaks between paragraphs or combine into the same paragraph, as appropriate.

l. 354-355	"the sociodemographic variables such as: gender, age (birth year) and type of school were collected." -> "questions on sociodemographic variables included gender, age (birth year) and type of school."

l. 357-358	"The time of the online activities on computer and mobile devices was estimated based on participants’ declarations." -> "Time spent on online activities on computers and mobile devices was estimated on the basis of participants’ declarations."

l. 366	"(downloading the software" -> "(downloading software"

l. 373	"performing three roles: perpetrators, victims, and witnesses." -> "with three roles: perpetrators, victims, and witnesses." (being a victim or witness aren't really "performed")

l. 377	"comprised five-point Likert scale" -> "used a five-point Likert scale"

l. 438	"behaviors" -> "behaviours" (so far the article used UK spelling - check consistency throughout the article).

l. 449 	"and  almost" -> "and almost" (delete excess space)

l. 451 	"students  both" -> "students both" (delete excess space)

l. 457-459 Should the Chi-Square figures be reported for differences between male and female students?

l. 460	"to  almost" -> "to almost" (delete excess space)

l. 491 	"downloaded the software" -> "downloaded software"

l. 495-496	"and younger students more often resorted to hacking than older students [(N=8, 5.3% vs N=10, 3.1%; Chi2=5.652; p=0.059)]."  - since the Chi-Square was only marginally significant and the difference was only two students per group, this might be better to leave out.

l. 499	"N=1.3%," - something's missing here

l. 500-502	Again, very small differences in absolute figures. Consider including Chi-Square figures if significant and deleting if non-significant.

l. 507	"threating" -> "threatening others"; also, what behaviors does "offending" include? Or should it be "threatening or offending others"?

l. 516 	"downloading the software" -> "downloading software"

l. 518 	"on  schooldays" -> "on schooldays"

l. 560 	"often  compared to" -> "often than"

l. 593 	"in the adulthood among" -> "in adulthood among"

l. 594	"group.  The" -> "group. The";  "for  social" -> "for social" (delete excess spaces)

l. 595	"which confirms our assumption" - with regard to which hypothesis?

l. 610 	"high comparing to Polish non-VI peers " -> "as among Polish non-VI peers"; "The hypothesis regarding gender disparities" - you numbered the hypotheses  in the introduction, so it'll be good to state the number of the hypothesis in question here.

l. 612 	"downloading the software" -> "downloading software"

l. 613-614 "the males examined viewed websites with sexual content more often than females." -> "the male participants of this study viewed websites with sexual content more often than their female counterparts."

l. 676 	"making intimate relationships" -> "forming intimate relationships"

l. 679 	"especially that" -> "especially given that"

l. 682	"threating or offending" -> "threatening or offending others"

l. 686 	"statistically relevant," -> "statistically significant"

l. 687	"are also" -> "were also"; when you say that there were no statistical differences between genders, and yet you describe such a difference, does this mean that there was a difference in the sample but it was not generalizable to the population due to non-significant chi-square tests? Please clarify.

l. 691	"eg. unpleasant" -> "such as unpleasant"

l. 694	"i.e." -> ", namely"

l. 695	"by invisible audience" -> "by an invisible audience"

l. 726-727	"self-administrated form of survey was used in order to enable anonymity." -> "a self-administrated survey was used to enable anonymous participation in the study."

l. 733-734	"the universal study protocols" -> "universal study protocols"

l. 752	"digital competences" -> "digital competencies"

References:	Please check once more to ensure correct copyediting in keeping with the journal's style

Overall: 		Please copyedit and proofread the manuscript carefully before submitting the revised version. It may be helpful to view the file without tracking 	turned on before submitting it, or even print out the manuscript for final inspection, to see minor copyediting issues more clearly.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: AEROMONAS HYDROPHILA INFECTION INDUCES TOLL-LIKE RECEPTOR 2 (TLR2) AND ASSOCIATED DOWNSTREAM SIGNALING IN INDIAN CATFISH, CLARIAS MAGUR (HAMILTON, 1822)
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. Fish gene names should be italicized, with all letters in lower-case. Protein symbols are not italicized, but only the first letter should be capitalized. All the gene and protein symbols in this manuscript need to be corrected.

2. In section 2.8, the method for primer specificity analysis is not provided, which is inconsistent with the title of this section.

3. If the authors want to keep section 3.3 as a part of Result section, more results should be provided. Currently, only the amplification efficiency of primers is shown, with no data showing the specificity of the primers (melt curve or gel image). Alternatively, section 3.3 can be removed and only Table 3 is kept and attached to section 3.4.

4. Line 85: there is a missing "a" between "play" and "vital"

5. Line 354: "higher" should be "highest"
Experimental design: 1. Line 212: The authors used ef1a (italic) as the internal control for qPCR. Why is this gene selected? Although ef1a (italic) is a housekeeping gene, one cannot directly use it as internal control unless its expression level has been already shown to be unchanged by certain stress (in this case, A. hydrophila). Therefore, please provide references or experimental evidences to support it.
Validity of the findings: No comment
Additional comments: Since this is the first study of indian catfish tlr2 gene function and this gene was shown to be upregulated by A. hydrophila, it would be nice to also test whether tlr2 expression is affected by PGN, LPS, etc. As the authors mentioned (Line 369-372), the response of tlr2 to A.hydrophila might be due to these components, but it is not clear in Indian Catfish.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: AEROMONAS HYDROPHILA INFECTION INDUCES TOLL-LIKE RECEPTOR 2 (TLR2) AND ASSOCIATED DOWNSTREAM SIGNALING IN INDIAN CATFISH, CLARIAS MAGUR (HAMILTON, 1822)
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. English should be properly scrutinized.
2. Some references are lacking (see attached PDF file with notes).
3. Article structure, figures, tables and raw data are acceptable, but should provide more data for a full-length article.
Experimental design: Experimental design is fine, but should explain why it was Gram-negative bacteria (Aeromonas hydrophila) instead of Gram-positive bacteria was used in challenge trail when most of the references indicated that TLR2 is responsible for sensing ligands from Gram-positive bacteria.
Validity of the findings: Many of the questions and suggestions are included in the PDF file uploaded.
Additional comments: More functional studies on Clarias magur TLR2 (mTLR2) is more than welcome, such as subcellular localization of mTLR2 in fish cells, protein-protein interaction of mTLR2 and potential adaptor proteins (MyD88, TIRAP, TIRF and Sarm1), and ligand specificity of mTLR2 to known TLR2 ligands (can be done using luciferase assay)


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: AEROMONAS HYDROPHILA INFECTION INDUCES TOLL-LIKE RECEPTOR 2 (TLR2) AND ASSOCIATED DOWNSTREAM SIGNALING IN INDIAN CATFISH, CLARIAS MAGUR (HAMILTON, 1822)
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have addressed my concerns, and I recommend publication in the journal.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: AEROMONAS HYDROPHILA INFECTION INDUCES TOLL-LIKE RECEPTOR 2 (TLR2) AND ASSOCIATED DOWNSTREAM SIGNALING IN INDIAN CATFISH, CLARIAS MAGUR (HAMILTON, 1822)
Review round: 2
Reviewer: 2

Output:
Basic reporting: Figs. 1 and 2 should be corrected.
Experimental design: Good.
Validity of the findings: This is a interesting manuscript, but efforts are required to make this manuscript with a better quality.
Additional comments: The authors have made much progress on making this manuscript with a better quality for publication. However, some points are needed to be corrected.

1.	PRR should not in italics and in lower case (e.g. lines 16, 17 ,19, 38 and many places in the manuscript).
2.	MyD88 should not in italics and lower case in line 20 (it is the MyD88 protein to pass the signal to the downstream, not the gene itself, so do for other places not just for MyD88).
3.	LRR should not in lower case (e.g. lines 26, 27). It is protein we are talking about here.
4.	TIR should not in lower case (e.g. line 28).
5.	TLR2, TLR1 and TLR6 should not in italics and in lower case in lines 71 and 72.
6.	Fig. 1 is incorrect. Please make sure the number of LRRs are only 6 of it in mTLR2, which I found quite hard to believe (Fig. 1). Also, TLR2 should have LRR-NT. It is worth to check Quiniou et al 2013 (DOI: 10.1007/s00251-013-0694-9). The supplementary data of this paper provides each LRR for every TLRs found in channel catfish. Simply make alignment with catfish TLR2, you can easily find other LRRs that you can’t identify using SMART software.
7.	Fig. 2 is incorrect. Please identify each LRR manually according to the definition of LRR, not just use online software since online software may give biased results due to software settings.
8.	The authors stated that “Unlike mammal, the tlr2 in fish has extended functional attributes to recognise Gram-negative bacteria and LPS in addition to its known ligands, and Gram-positive bacteria.”. But there is no direct evidence to prove this point. Upregulation of tlr2 genes does not mean it was activated. For your information, a paper was published in 2018 (https://www.frontiersin.org/articles/10.3389/fimmu.2018.01413/full) and they show NOD1 is one of the receptor for LPS recognition. However, we can’t exclude the possibility that TLR2 might play a role in this recognition, but more evidence is needed to demonstrate this point.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: AEROMONAS HYDROPHILA INFECTION INDUCES TOLL-LIKE RECEPTOR 2 (TLR2) AND ASSOCIATED DOWNSTREAM SIGNALING IN INDIAN CATFISH, CLARIAS MAGUR (HAMILTON, 1822)
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors have responded well and have answered all the questions raised. Additionally, the corrections have been properly panted in the new version of manuscript. I think it is now ready to be process for publication.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FIRST CHARACTERIZATION OF THE PROBIOTIC POTENTIAL OF LACTIC ACID BACTERIA ISOLATED FROM COSTA RICAN PINEAPPLE SILAGES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The work is novel and relevant.
Experimental design: The study was well desgined.
Validity of the findings: The data are convincing and the conclusion justified.
Additional comments: The present study described the isolation of several lactic acid bacteria (LAB) from Costa Rican pineapple silage and characterization of their probiotic potential. As a result, an LAB strain, Lacticaseibacillus paracasei_6714, was identified as a promising probiotic candidate. The work was well-designed and presented.

Major concerns:
1.	The antimicrobial activity needs to be evaluated using more LAB strains of different species.
2.	The safety of the LAB isolates needs to be tested.
3.	In vivo experiments are suggested to evaluate the probiotic ability of the LAB.
4.	Well-identified reference LAB strains need to be included as controls in all the assays.
5.	The novelty and innovation of the study should be highlighted.
6.	The manuscript should be revised by native English speakers.

Minor points:
1.	The layout of the tables and figures should be optimized.
2.	The references should be a consistent style.
3.	Line 1, lactic acid bacteria
4.	Line 33, species
5.	Lines 144-145, A 1.5- kb fragment…..the primer pair 27F/1492R
6.	Line 273, a 1-kb
7.	Lines 278 and 295, HeLa
8.	Lines 327-328, L. paracasei
9.	Line 331, GenBank
10.	Line 397, 0.3%


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FIRST CHARACTERIZATION OF THE PROBIOTIC POTENTIAL OF LACTIC ACID BACTERIA ISOLATED FROM COSTA RICAN PINEAPPLE SILAGES
Review round: 1
Reviewer: 2

Output:
Basic reporting: See notes to authors
Experimental design: ok
Validity of the findings: ok
Additional comments: Line 60: use the WHO/FAO 2022 or ISAPP 2014 definition of probiotics, and cite either source.
Line 63: to colonize the gut is not a prerequisite for probiotics, was not even demonstrated for any strains. Colonization is only temporary, please comment.
Line 101: this is not true, many more studies were conducted in southamerica, especially in Brazil by Prof. R. Schwan https://pubmed.ncbi.nlm.nih.gov/?term=Schwan+RF&cauthor_id=32869919
Line 108: this is not true either, many other were conducted in brazil with LAB from coca: https://pubmed.ncbi.nlm.nih.gov/?term=brazil+cocoa+lactic+acid+bacteria please update.
The introduction is too long, almost 3 pages, it should be reduced by half and transfer the remaining text to discussion.
How many samples were used for isolation? How many colonies were obtained from each sample for further identification? How many isolates failed to be identified as LAB? What other species non LAB were found?
Line 328: was RAPD applied to say that the strains were really different strains? Most isolates from the same sample are indeed same strains, so it is important to indicate in a table if isolates were obtained from the same sample, or if RAPD was done to make sure that the seven strains are really 7 different strains or copies of the same isolate.
Line 453: this sentence: “and one of few studies obtaining LAB with biotechnological potential from agro-industrial waste in Latin America” shows a limited knowledge of what was published, please deleted or update it.
Tables: do not express results of survival as %, show the colony counts before and after exposure ot acidity, lysozyme, bile salts, etc.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FIRST CHARACTERIZATION OF THE PROBIOTIC POTENTIAL OF LACTIC ACID BACTERIA ISOLATED FROM COSTA RICAN PINEAPPLE SILAGES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Most issues were addressed
Experimental design: ok
Validity of the findings: ok
Additional comments: Still authors must consider to refer to isolates, not to strains, unless they show by RAPD that isolates are indeed different strains.

In particular in this paragraph:

       A total of twelve different LAB morphotypes were isolated from twenty pineapple silages with
334 increasing levels of urea. Considering the 16S rRNA sequence and pheS gen the isolates
335 correspond to L. paracasei (seven strains), Lentilactobacillus parafarraginis (two strains),
336 Limosilactobacillus fermentum (two strains), and W. ghanensis (one strain)


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: MAPPING THE HIDDEN DIVERSITY OF THE GEOPHAGUS SENSU STRICTO SPECIES GROUP (CICHLIDAE: GEOPHAGINI) FROM THE AMAZON BASIN
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study was well-designed and successfully executed, using modern community standards of molecular species delineation in combination with morphological data in Amazonian fishes. The main conclusion is well supported, for the existence of six morphologically distinct but as yet undescribed species of i/Geophagus/-i.

My only suggestion, which I’m sure the authors will agree, is to modify the last sentence of the Abstract to: “Our results highlight the importance of combining DNA and morphological data in biodiversity asessment studies especially in taxonomically diverse tropical biotas."
Experimental design: The research question, methods and interpretations are excellent, sufficient described, and meet community standard for the field.
Validity of the findings: The results are interpreted reasonably in light of the methods employed.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: MAPPING THE HIDDEN DIVERSITY OF THE GEOPHAGUS SENSU STRICTO SPECIES GROUP (CICHLIDAE: GEOPHAGINI) FROM THE AMAZON BASIN
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this study by Ximenes et al. present an interesting and a well-written study on the species diversity of the cichlid group Geophagus in the Amazonian River basin in South America. The authors use an impressive dataset of mitochondrial cox1 marker and carryout several single locus species discovery methods to understand and report the diversity of this group. I have only minor comments which are directly commented on the pdf of the manuscript which is attached herewith.
Experimental design: no comments
Validity of the findings: no comments
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: MAPPING THE HIDDEN DIVERSITY OF THE GEOPHAGUS SENSU STRICTO SPECIES GROUP (CICHLIDAE: GEOPHAGINI) FROM THE AMAZON BASIN
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment. The paper was well done and had no such deficiencies.
Experimental design: No comment. The experimental design was good.
Validity of the findings: Findings were valid and clearly explained.
Additional comments: This paper treats species delimitation in the Cichlid genus Geophagus in the Amazon basin. The authors use a single locus species delimitation method approach based on the DNA barcoding gene COI to test species delimitation based on broad sampling and a relatively large number of samples of Geophagus. They also examined morphospecies from photos and museum specimens.

The overview of the problem and the approach they took is good, the references cited are appropriate and the methods are well done with the authors using several different sequence based methods to delimit the species. They find some described species cannot be justified as separate species, many misidentified specimens in collections, and several putative new species. The distribution of each species is presented in the Discussion. They end the paper discussing the conservation implications of recognizing putative undescribed species given the environmental problems plaguing the Amazon basin like dam construction.

I found the paper quite well done. It treats an important biological problem and makes an important contribution to moving knowledge of the genus forward. The paper will also likely serve as a valuable reference for other researchers wishing to conduct similar studies on other taxa.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EPICURATOR: AN IMMUNOINFORMATIC WORKFLOW TO PREDICT AND PRIORITIZE SARS-COV-2 EPITOPES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors have reported a curation workflow for selecting epitopes with desired immunity. The manuscript sticks to the theme and is easily understandable.

The authors have cited most of the tools that they have used, except a few times. Like in the case of IEDB, authors have mentioned that they have used IEDB version 2.25. Though, the most recent version was published in 2018, and in 2019.
This is important in this case, because the older will surely have no epitopes from SARS-CoV2, which was discovered only in 2020.

In the table 2, it is very surprising that out of so many epitopes reported in the literature, the proposed approach failed to any of the epitopes in envelop protein and captured only 15 in the spike proteins. While we know that these proteins are the most immunodominant and therefore chosen for many vaccines running successfully.
Experimental design: The design follows the standard practices for epitope selections. It is impressive to see that the authors have been very transparent in describing each method alongwith the threshold/model used for prediction.

While performing conservation analysis, the authors relied on Blast, which might not be perfect tool, because of its design. The Blast is designed to search local alignment and the authors seem to apply it for global coverage.

Though at the end of the manuscript, I was expecting to see a table with final filters and sequences, which is missing.
Validity of the findings: In terms of validity, the authors have used a set of tools, and no experimental validation is done.
Another validation is that the breadth and length of epitopes were recently published to describe a bigger picture of the immune response (doi: 10.1016/j.chom.2021.05.010) was not reflected in their analysis.
There are many such reports published and authors have not shed any light on the comparison of those studies against theirs.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EPICURATOR: AN IMMUNOINFORMATIC WORKFLOW TO PREDICT AND PRIORITIZE SARS-COV-2 EPITOPES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Authors have aimed to develop the workflow to predict the epitopes of SARS-CoV-2.  According to the obtained results, a lot of epitopes were predicted and some of them were concluded to detect for the first time. In my opinion, there are some shortcomings of this study.

 In this study, NetMHCpan and NetMHCIIpan were used to predict MHC-1 and MHC-2 epitopes (Figure 1). Both of them already work under the IEDB database and this database has been frequently used in a lot of studies predicting epitopes of SARS-CoV-2 until now. Depending on this, it is not clear how these new epitopes were predicted although limited alleles have been used. In my opinion, if authors examine more articles conducted in this field, I think that authors can recognize that these new epitopes have been detected before.  Several articles that are not cited in this study are listed below with a limited search, and this shows that more investigations should have been done.  As a result, this study is not suitable for publication yet.

- https://www.nature.com/articles/s41467-020-16505-0

- https://www.nature.com/articles/s41598-020-79645-9

- https://www.nature.com/articles/s41598-020-73371-y
Experimental design: Experimental design lack of originality.
Validity of the findings: Findings are not novel completely
Additional comments: The manuscript is not valuably acceptable for publication


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EPICURATOR: AN IMMUNOINFORMATIC WORKFLOW TO PREDICT AND PRIORITIZE SARS-COV-2 EPITOPES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript titled “EpiCurator: an immunoinformatics workflow to predict and prioritize SARS-CoV-2 epitopes” introduces a pipeline that combines different specialized prediction tools into a single workflow. This pipeline is used for predicting SARS-CoV-2 epitopes, with a focus on the lineages observed in Brazil for the purpose of enabling vaccine design and development targeted at the Brazilian population. The manuscript makes predictions which can be experimentally verified by other groups once they are made publicly available.

While the core idea behind the method is decent, and the results of the analysis and corresponding data are sufficient and adequate, the reporting style and grammar are not clear and need major improvements. The manuscript needs to be re-written, using the help of a person who is fluent in English.

There are some studies done in the labs of Shane Crotty and Alessandro Sette, that have extensively studied the role of T-cell immune responses to SARS-CoV-2 infections and vaccination. Since, the manuscript describes about including T-cell epitopes, in addition to B-cell epitopes, including a short paragraph providing a background of T-cell immune responses to SARS-CoV-2 infections would be good. There are also several studies on neutralizing antibody responses to SARS-CoV-2 infection/vaccination that show the effect of the mutations observed in the variants of concern, on the immune response, and these also need to be mentioned in the introduction. A brief mention about a new nanoparticle-based vaccine developed at the Institute for Protein Design currently in Phase 3 clinical trials at South Korea, would also be nice to include.
Experimental design: More emphasis needs to be placed on how the vaccine designed through these approaches will offer an advantage over current SARS-CoV-2 vaccines. Additional explanation/description of current SARS-CoV-2 vaccines for a bit of background and context could also be included.

The methods are detailed and easy to understand (but again- the sentences need to be rephrased and grammar corrected). It is important to specify that the findings made here, once validated, can be used for designing a vaccine that is specific to the Brazilian population (and not a universal vaccine), against the variants currently circulating in Brazil, since the genome sequences used are from samples isolated from Brazil.

Additional details are needed in the Methods and Results section to describe how the epitopes that were found to be highly conserved in Brazilian variants were also searched in  other lineages.

Describe how the PepDock server models the peptide structures and the HLA-peptide complexes, and generates binding free energy values
Validity of the findings: The findings made in this study are predictions and will have to be experimentally validated. The fact that the study is able to predict epitopes that are already experimentally validated or present in the Immune Epitope Database reflects the reliability of the predictions.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EPICURATOR: AN IMMUNOINFORMATIC WORKFLOW TO PREDICT AND PRIORITIZE SARS-COV-2 EPITOPES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have improved on language and data presentation. I believe that in terms of references, authors have missed out on some of the most important references in their manuscript, like IEDB (database paper or analysis resource paper), GISAID are used a number of times, but not cited properly.
Experimental design: The authors have stated clearly that the manuscript is of computational profile and so is the design or validation.
Validity of the findings: The authors have provided data and also compared their analysis with the ones done previously.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: GENOME-WIDE ANALYSIS OF THE CALS GENE FAMILY IN COTTON REVEALS THEIR POTENTIAL ROLES IN FIBER DEVELOPMENT AND RESPONSES TO STRESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: PeerJ MS#65037
The manuscript by Feng et al. describes the identification and analysis of callose synthase (CalS) genes in the 4 cotton genomes. They characterized 27, 28, 16 and 15 CalS from G. hirsutum, G. barbadense, G.raimondii and G. arboretum, respectively, by BLAST search the cotton genome. Phylogenetic analysis of these CalS genes in combination of those from Arabidopsis revealed 5 clades with distinct gene structures. Selection analysis indicate that these CalS proteins have undergone purify selection during cotton evolution. Further expression analysis and cis element annotation enable the authors to connect the expression CalS genes with hormonal and abiotic stress responses.
The manuscript is generally well-written and contains certain amount of work. The results were clearly described and presented.
I have a few minor points to improve the manuscript:
1. Line 14, “Callose synthase” describe a gene and should be italicised.
2. Line 23 and elsewhere in the MS, change GHCalSs into GhCalSs.
3. Line 48-50, please be aware the space between the parentheses and the text.
4. Line 59, change “significant” to “important”.
5. Line 59-66, I found myself had a problem in seeing the key question or aim of this study from this paragraph. I would suggest the authors to re-phrase this paragraph.
6. Line 67, “donors” to “genome donors”
7. Line 90, please explain why “incomplete sequences were deleted”
8. Line 110, please define the promote more accurately, “upstream the CalS gene is obscure”.
9. Line 120, in the section of “RNA isolation and qRT-PCR analysis”, please include all the details of how the experiments were conducted, for example, how much RNAs were used as templet for reverse transcription, were the specificity of qRT-PCR primers verified by PCR-Sequencing and Standard Culve, as such, the authors should be aware the transparency of the experiments.
10. Line 177, “chromosomes” to “chromosome”
11. Line 185, “upper and lower ends..” to “upper and lower arms..”
12. Line 207, I appreciated the authors’ efforts in mining the cis-elements in the regulatory regions of all the CalS genes. Nonetheless, a control gene/genes with related function should be set up for comparison.
13. Line 220-221, all these CalS gens at best can be categorized as  “abiotic/hormonal responsive” genes based on the presence of corresponding cis-elements in the promoters but rather “abiotic/hormonal signaling”.
14. Line 224, “researches” into “studies”.
15.  Line 221-222, the last sentence of this paragraph makes no sense from the data presented, I would suggest to delete it.
16. Line 231, “the above hypothesis”, which hypothesis?
17. Line 242-247, the information of this paragraph is somehow misleading, please re-phrase.
18. Line 255, “materials” into “samples”
19. Line 260, “part” into “role”
20. Line 262-263 and elsewhere in the text and figure legend, latin names should be italicised.
21. Line 280, “distributed” into “identified”
22. Line 286, “short-day induced ABA increased” into “short-day induced ABA biosynthesis/signaling”


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: GENOME-WIDE ANALYSIS OF THE CALS GENE FAMILY IN COTTON REVEALS THEIR POTENTIAL ROLES IN FIBER DEVELOPMENT AND RESPONSES TO STRESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: non comment
Experimental design: no comment
Validity of the findings: Conclusions were made, however,  the conclusion of "CalS gene family in cotton reveals their potential roles in fiber development and responses to stress validation“ lacks evidences from function aspects.
Additional comments: The paper presents the data analysis for the genome sequence of cotton, and the conclusion of "CalS gene family in cotton reveals their potential roles in fiber development and responses to stress validation“ were based on sequence analysis. The  evidences from function aspects, such as Gene Ontology, KEGG pathway can help.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: GENOME-WIDE ANALYSIS OF THE CALS GENE FAMILY IN COTTON REVEALS THEIR POTENTIAL ROLES IN FIBER DEVELOPMENT AND RESPONSES TO STRESS
Review round: 2
Reviewer: 1

Output:
Basic reporting: As far as I can see, the authors have made a great efforts in revising the manuscript. All my concerns have been adequately addressed. I dont have further comments.
Experimental design: N/A
Validity of the findings: N/A
Additional comments: As far as I can see, the authors have made a great efforts in revising the manuscript. All my concerns have been adequately addressed. I dont have further comments.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: GENOME-WIDE ANALYSIS OF THE CALS GENE FAMILY IN COTTON REVEALS THEIR POTENTIAL ROLES IN FIBER DEVELOPMENT AND RESPONSES TO STRESS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: Figure 8 is interesting. However, this figure should be revised. The gene numbers should be presented in this figure rather than only the significant degree. The significant level of each functional terms can be presented by the node size of each point. The GO bubble figure from clusterprofile package can help(OMICS. 2012 May; 16(5): 284–287).
Validity of the findings: no comment
Additional comments: no comment


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FEM: MINING BIOLOGICAL MEANING FROM CELL LEVEL IN SINGLE-CELL RNA SEQUENCING DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript, on the whole, is straightforward and easy to read; there are a few instances of phrasing which the authors could modify to reduce ambiguity, which have been noted in the PDF as comments. The structure of the paper is suitable, and the figures are legible. However, the figure captions require additional information to clarify the results. These instances have been noted in the PDF.
For bulk sequencing analysis, there have been multiple approaches devised to transform a gene expression matrix to a functional expression matrix at the study-level or single sample-level (e.g. GSVA, ssGSEA, singscore) which the authors clearly appreciate given they have referenced these methods. However, these methods have been applied to scRNA-seq, and further there are additional methods designed specifically for scRNA-seq (e.g. Vision, padog2) to address some of the issues authors detail as motivation for developing their FEM method. It’s clear that the FEM method is distinct from these other methods, however it would be helpful for readers to understand what is unique about the FEM method in the context of other scRNA-seq tools (see Zhang et al. 2020 https://doi.org/10.1016/j.csbj.2020.10.007 for a benchmarking study of these tools applied to scRNA-seq).
Finally, one issue that needs to be addressed is whether the authors intend FEM to replace or complement GEM for downstream analysis. From their methods section and the Github repository, the FEM method is a pathway activity score (PAS) calculation method which does not perform cell clustering, classification, or differential analysis (the authors use Seurat for this). This is not abundantly clear in the introduction and methods, so clarification would be useful.
Experimental design: The manuscript is within the research scope for the journal, and the question they seek to address is relevant to the field. The authors are eager to demonstrate the utility of the FEM approach contrasted with a traditional GEM approach to scRNA-seq analysis, and use public datasets from 3 different tissue sources to highlight how FEM can be used in lieu of GEM and complementing FEM approaches. Benchmarking of PAS schemes for scRNA-seq has been done before, and it’s not in the scope of this type of manuscript to benchmark against other approaches. However, the authors could provide more detail about the results of their comparison between FEM and GEM. Specifically, figures 3D and 4C show overlaps of FEM and GEM clustering/classification, but the captions and figures need more detailed descriptions to help the reader understand the results.
The authors use the terms “FC-DEF” and “GC-DEF” but do not define the acronyms, which makes it difficult to understand what the authors are referring to. The authors state, “ 257 If the two groups of cells are far apart on GEM, but are close or partially overlapped on FEM, it indicates that they are different subtypes of the same cell type, or the two groups of cells may perform similar functions.” This is an interesting proposal, however it isn’t clear from Figure 4 or the text how FEM does this; clarification and expansion of this concept would be very helpful.
With respect to how they use Seurat, the authors state, “The feature number selection, scaling ratio, PCA, and clustering parameter selection were appropriately adjusted according to circumstances” which makes it difficult for readers to recapitulate their analysis. A detailed workflow of 1) how to use the FEM python package on GitHub and 2) how to use Seurat downstream of FEM is needed.
In Figure 6, the authors present reanalysis of bulk RNA-seq datasets from GEO, however the methods for how they arrived at these specific gene sets from the GEO datasets is not in their methods section.
Validity of the findings: The scRNA-seq datasets presented are previously published and publicly available. They have defined their method for FEM calculation and scoring scheme in the methods, use appropriate pathway and ontology genesets (Reactome and GO), and downstream of FEM use appropriate methods for scRNA-seq clustering and differential analysis (Seurat). As stated in the previous section, a description of their Seurat method is needed to fully evaluate their results. The authors figures and results are appropriate for their stated question at the outset. However, the authors’ introduction states that FEM can be a replacement for GEM, while in their discussion, the authors state that FEM is complementary to GEM. I think from their results, the latter is a better description, which would require a modification of the introduction. Further, the discussion section is somewhat short; it would be to the authors’ benefit for them to expand on the utility, simplicity, and broad functionality of the FEM approach. The authors have implemented a classic enrichment test (Fischer’s test) to calculate a pathway activity score (PAS). It’s simple analysis in an elegant fashion and the authors should expand on this further.
Additional comments: In their manuscript “FEM: mining biological meaning from cell level in single-cell
RNA sequencing data”, Liu et al present a method to transform gene-level information to pathway-level information for scRNA-seq clustering and analysis using the classic Fisher’s Exact test. This method can help circumvent one of the main pitfalls of scRNA-seq, namely the sparsity of data with respect to genes annotated. By transforming scRNA-seq data to a Functional Expression Matrix (FEM), the effect missing data (i.e. genes not captured or annotated in a scRNA-seq study) can be minimized by investigating single cell transcriptomics at the pathway or ontology/function level. The authors clearly highlight the rationale for this type of approach to complement a traditional scRNA-seq pipeline demonstrate its utility. While this manuscript and approach is promising, additional information with respect to the 1) methods of analysis following FEM (i.e. clustering and differential expression), 2) comparison between FEM and GEM approaches (e.g. how to interpret overlapping cell cluster assignments in Figure 3D, 4C), and 3) how an FEM approach can aid in the characterization of potential cell-type specific heterogeneity. With further details on the specific methods used downstream of FEM, expanded interpretation and clarification of the results (primarily the complementarity of FEM with GEM), and a longer discussion detailing the method in the context of the wider scRNA-seq field and the utility of the FEM approach, this manuscript would be in a good position for publication at PeerJ.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FEM: MINING BIOLOGICAL MEANING FROM CELL LEVEL IN SINGLE-CELL RNA SEQUENCING DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1)	At number of places in Materials and Methods and Results sections, there is spacing errors for references. Authors can correct spacing on Line 100, 101, 103, 123, 156, 270, 278
  Line 75, delete symbol  “( “ before quality control,
2)	In Figure 1: Please correct the spelling error of algorithm in the image 1A
3)	In Figure 1C Fene is mentioned. Do the authors mean gene or there is a full name for Fene that is required to be included in manuscript?
The legend of Figure 1C describes FEM, however, authors have not included FEM in the image of Figure 1C
4)	On line 194, author states  ‘Optimization of the algorithm can be illustrated using the symbols in 2.4.2.’
It is not clear what is 2.4.2 referred to?
5)	At number of places, structure and idea stated are hard to understand. Specifically, the sections of Results and Discussion are written with poor editing of English language. There are grammatical errors as well as meaning of the sentence are hard to gauge.
Results section lacks explanation on subsection of figures. Authors needs to explain results in a systematic manner for the better understanding of the data.
In Discussion section, line 293: the sentence meaning is hard to undertand…"Sometimes, evaluate the impact of some biological effects on the cell clusters is necessary, such as the impact of cell cycles on cell-type clustering results"
I suggest if authors can take help from colleague and improve the overall English used in the manuscript.
6)  The figure legends are very brief and lack necessary details to understand data. There is scope of describing the legends in a precise and detailed manner. Furthermore, the name of plots ‘t-SNE’ or any other plots as used should explicitly reported in figure legend. Lastly, information on statistical analysis parameters should be included in figure legends of Figure 3, 4, 5, and 6

7) Colors used in Figures 3 and 4 are difficult to read for comparing the data in t-SNE plot and the tabular representation of GO-based FEM cluster. I recommend if the color co-ordination of particular cluster (cell type) can be kept similar in both t-SNE plot and Cell type heading that describes various cell types.
For example acinar cell in Figure 3C image is red, but the color of the same cell in cluster 3 or header of the table is blue.

8) Line 265, NC cells are mentioned. It is not clear what category of cells are being referred NK or DC cells from Figure 5? Authors can please correct this error.
Experimental design: 1)	The description of idea of using FEM and GEM clusters on line 142, point 4 does not align well with interpretation of same message shown in the pictorial representation of Figure 1 D. Authors needs to clarify how differentially expressed genes are identified using FEM.

2)	 Although there is a comparative analysis of GO-based FEM cluster with ground truth cell data (Figure 3, 4 and 5), it is not clear what is the detail evaluation metrics used for collecting ground truth cell data versus GO-based FEM cluster. Authors should describe these details in the methods section.

3)	As mentioned by the author in line 285, “Figure 6 shows….. method proposed in this study.”
Can authors please clarify the source of the bulk RNA-seq data  that is presented in Figure 6 ?
Also, authors can be more specific about the extent to which this validation study overlap with the traditional method they are referring to.
I would also recommend revising the legends of Figure 6 as they lack details on comparison of t-SNE plots in the Figure 6A and 6B.
Validity of the findings: 1) Authors have reported t-SNE plots that compare clustering based on GEM and FEM for PBMC dataset in Figure 4, however, they have skipped similar comparison for the data represented in Figure 3.
GEM and FEM based t-SNE plots for liver and pancreas data should be included as a part of comparison of additional cell type in addition of PBMC dataset.

2) The first paragraph and last paragraph of discussion contradicts each other i.e authors claim of using FEM analysis instead of GEM for sc-RNA-seq. There is a scope of improvement of Discussion section.. if authors can structure the ideas in a more systematic manner for giving reader explicitly potential uses and limitations of FEM based algorithm.

3) Line 254: Authors claim that the overlap seen for different cells in PBMC can be interpreted as a functional similarity of some group of cells versus others. Can authors support this claim by describing in detail which cell types showed overlap and how they are functionally related with supporting literature.
Additional comments: Authors have considerably explained the idea of FEM algorithm and tested data-sets from three different sources to test the algorithm. This method eliminate the need of an additional filtering step, which can likely introduce a bias. Furthermore, this methods relies on GEM and FEM based matrix that can be utilized to interpret diverse set of scRNA -seq data in a more meaningful manner. The rationale of this study is promising, however, authors fails to describe many important details that are required for  interpretation of the data from this study.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FEM: MINING BIOLOGICAL MEANING FROM CELL LEVEL IN SINGLE-CELL RNA SEQUENCING DATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The reporting of this manuscript is decidedly improved from their first submission. The authors have responded to the comments and critiques from reviewers, and I believe this manuscript meets the reporting guidelines and standards for PeerJ.
Experimental design: The authors have expanded on the description of their FEM algorithm method and included the necessary code to use the algorithm in Github. I believe their FEM method can offer a unique insight to the analysis of sc-RNA-seq data and they have described how their approach fits within the context of the field, and the specific questions it can uniquely address.
Validity of the findings: The authors have used multiple publicly-available datasets to test their method and compared it with a standard method in the field, along with the parameters used for the comparison. This is illustrative of the utility of their method.
Additional comments: The authors have sufficiently and painstakingly addressed all the comments from the reviewers. They have clearly defined the scope, need for, and utility of their FEM method for the analysis of single-cell RNA-seq data. I am very pleased with how they have described their approach within the context of available gene set enrichment methods, and I find the insights gleaned from their approach unique and interesting.

A few minor comments:
- In figures 3, 4, and 5, panel A shows the GEM-based clustering results while panel B shows the FEM-based clustering result. I would recommend clarifying this in the caption for 3B, 4B, and 5B as well as possibly putting "GEM" and "FEM" in some fashion on panels A and B, respectively, for figures 3-5.
- It would be useful to include the GitHub link in the introduction and/or the method description.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FEM: MINING BIOLOGICAL MEANING FROM CELL LEVEL IN SINGLE-CELL RNA SEQUENCING DATA
Review round: 2
Reviewer: 2

Output:
Basic reporting: Authors have worked substantially towards improving readability of the manuscript with clearly stating applicability of their algorithm for analysis of scRNA-seq data. They have made much clear distinction of how this FEM-based is different from traditionally used GEM-based method.

Minor improvement is recommended in the English editing: There are sentences with excessive use of "however" even in the place where its use is not required.
Experimental design: Lastly, author needs to clarify or clearly state in manuscript how other researcher can use this algorithm and what are the exact parameters used in each software they used. Specifically,  the section "GEM and FEM fusion analysis" (starting line 263) is more general and lacks required parameters of the method.
Validity of the findings: -Not Applicable- revised
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: LYOPHILIZED CELL-FREE SUPERNATANTS OF LACTOBACILLUS ISOLATES EXHIBITED ANTIBIOFILM, ANTIOXIDANT, AND REDUCES NITRIC OXIDE ACTIVITY IN LIPOPOLYSACCHARIDE-STIMULATED RAW 264.7 CELLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear and professional English.
Experimental design: Sound.
Validity of the findings: Valid.
Additional comments: The authors studied the antibiofilm, antioxidant, and antiinflammatory activities of lyophilized cell-free supernatants of lactobacillus isolates. The manuscript is scientifically sound. Methods and conclusions are appropriate.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: LYOPHILIZED CELL-FREE SUPERNATANTS OF LACTOBACILLUS ISOLATES EXHIBITED ANTIBIOFILM, ANTIOXIDANT, AND REDUCES NITRIC OXIDE ACTIVITY IN LIPOPOLYSACCHARIDE-STIMULATED RAW 264.7 CELLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study is a interesting approach to probiotic properties of lactobacilli against biofilms.

The english is clear with sufficiente background and references in Itroduction section.

It is missing statistical analysis in the Materials and Methods section and no limitations are recognized by the authors. They started with four pathogenic bacteria but only evaluate antibiofilm properties with the two Gram-negative bacteria.

Bold statements are done in Discussion section and repetition with Introduction section was made.
Experimental design: The manuscript is within aims and scope of the journal.

The research question is well defined and relevant.

The Materials and Methods and Discussion sections need major revisions by the authors.
Validity of the findings: The findings are worth to be publish in the journal. However, major revisions are needed before acceptance.

The conclusions showed bold statements and no limitations were recognized by the authors.
Additional comments: I did all my comments and suggestions in the annotated PDF file of the manuscript in attachments.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: LYOPHILIZED CELL-FREE SUPERNATANTS OF LACTOBACILLUS ISOLATES EXHIBITED ANTIBIOFILM, ANTIOXIDANT, AND REDUCES NITRIC OXIDE ACTIVITY IN LIPOPOLYSACCHARIDE-STIMULATED RAW 264.7 CELLS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Authors should improve the literature background.
Experimental design: Nitric oxide reduction is not enough to prove anti-inflammatory effects. Authors should provide more evidence regarding anti-inflammatory activities by ELISA, for instance.
Validity of the findings: Statistics should be presented. How was the data processed? Which statistical test was used? It should be provided.
Additional comments: “Lyophilized Cell-Free Supernatants of Lactobacillus Isolates Exhibited Antibiofilm, Antioxidant, and Anti- inflammatory Activities in Lipopolysaccharide stimulated RAW 264.7 cells” is an interesting paper that brings new evidence of the biotechnological potential use of postbiotics in vitro. This topic is current and attractive. However, it needs some improvements before being considered for publication.

Title
-	Why did the authors affirm that the postbiotics had anti-inflammatory activity? Nitric Oxide alone cannot prove that. This way, the title should be changed.
Abstract
-	Some parts of the results contain methodology steps. It should be removed. Also, the conclusion should be more specific and focused.
Introduction
-	Authors should present this reference in the introduction:
-	Xu X, Qiao Y, Peng Q, Shi B, Dia VP. Antioxidant and Immunomodulatory Properties of Partially purified Exopolysaccharide from Lactobacillus Casei Isolated from Chinese Northeast Sauerkraut. Immunol Invest. 2021 Jan 8:1-18. doi: 10.1080/08820139.2020.1869777. Epub ahead of print. PMID: 33416001.
Materials and Methods
-	Authors should evaluate the production of key cytokines (such as TNF-α and IL-10) to prove that they had any anti-inflammatory activity. It is not possible to conclude that they have anti-inflammatory activities just performing Nitric Oxide by the Griess reaction.
-	It is not clear why the authors chose 16 h to perform the Griess reaction.
-	Authors should include how they evaluated the data. The statistical analysis should be added in the Materials and Methods section.
Results
-	Table 1 – The name of the four bacteria used in this assay should be added in the table legend.
-	Figure 1 – What are the controls for this experiment? What the authors used as a positive control of antibiofilm inhibition activity? Also, the quality of the image should be increased.
-	Figure 5- There is no statistical difference between the tested groups? It is not clear in the graphic.
Discussion
-	Authors should explore further why postbiocts were more effective regarding eradication than inhibition.
-	Again, it is not possible to affirm that the postbiotics have anti-inflammatory effects just by evaluating the NO production. How about the key pro-inflammatory cytokines?

Conclusion
-	The conclusion should be more focused and be clear about what this paper brings of novelty to the literature.