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: IDENTIFICATION AND EXPRESSION ANALYSIS OF THE SUCROSE SYNTHASE GENE FAMILY IN POMEGRANATE (PUNICA GRANATUM L.)
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are some other papers that already dealt with different aspects of Sucrose metabolism in Pomegranate species (Meletis et al., 2018; Poudel et al., 2020). They should have been included in the discussion in order to provide a sufficient field context.
Experimental design: The study mostly relies on published data. The qPCR analyses presented in this study cannot be considered a “verification” of the data of Luo et al., 2020. Even if is the same var. and, possibly, the same plants, they were carried out in different experiments times.
Validity of the findings: The bioinformatic methodology used in this study is not up-to-date. Firstly, the TopHat read mapper has entered low maintenance since 2016 as it is now largely superseded by HISAT2 (https://ccb.jhu.edu/software/tophat/index.shtml). So the RNA-seq analysis should be entirely reconsidered. Moreover, I would suggest the authors not use FPKM (fragments per kilobase of exon model per million mapped reads) for transcript quantification since it should be used for comparing within-sample expression transcript expression levels. While TPM (Transcripts per Million) effectively normalizes for the differences in the composition of the transcripts in the denominator rather than simply dividing by the number of reads in the library. So that TPM is considered a more reliable parameter when comparison across samples is carried out.
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: IDENTIFICATION AND EXPRESSION ANALYSIS OF THE SUCROSE SYNTHASE GENE FAMILY IN POMEGRANATE (PUNICA GRANATUM L.)
Review round: 1
Reviewer: 3

Output:
Basic reporting: In the study titled with “Identification and expression analysis of sucrose synthase gene family in pomegranate (Punica granatum L.)” by Longbo Liu et al. reported the identification of five sucrose synthase (SUS) genes from the pomegranate genome. With the phylogenic and motif analysis, they found that the PgSUS family were highly conserved during evolution. The author also predicted the protein structure and cis-regulatory elements of PgSUS1-5 genes and further investigated the spatial expression profiles of PgSUS family genes with public RNA-seq data and verified with Q-PCR.

The manuscript contains some interesting analysis; however, it is clear the author does not have a strong grasp of the English language and this makes it hard to follow. The author needs to improve the overall writing of manuscript with either professional English language editing service or benefit from someone of native English-speaking areas.

The overall structure of the manuscript conforms to journal standards and discipline norm except the discussion section. The literatures in introduction and other sections were well referenced & relevant with the correct format. Most figures were presented and showed in high quality and well labelled with raw data.
Experimental design: The experimental design including material preparation and bioinformatic software application in the manuscript is suitable to define questions which the author wants to demonstrate. The author described the methods with sufficient detail & information to replicate. At the same time, the author should present the methods in a more precise or accurate way which will make it more friendly to broader readers.
Validity of the findings: The most important finding in this manuscript is the identification of five PgSUS genes from the available genome pomegranate sequence. All the underlying data have been provided and statistically sound and controlled. Most conclusions are well stated, linked to the original research questions and supported by the results provided in the manuscript.
Additional comments: 1)In the identification of PgSUS genes section, the author used six Arabidopsis SUS protein as the query sequences and obtained 19 candidates. After that, five candidates were excluded as they are carrying the sucrose-phosphatase domain. Could the author explain in detail in either results or method sections about how they filtered out the other 9 candidates? What were their criteria and definition of “redundant sequences”?

2) In my opinion, the figure 3 delivered very limited amount of information and should be placed in supplementary information or deleted from the main manuscript. Had the author also predicted the structure of SUS proteins from other species? If yes, are there any structure similarities between them? The author should provide this information if possible.

3) I would suggest the author to combine the figure 6 and 7 as both figures were used to demonstrate the spatial expression profile of five PgSUS gene.  The heatmap data in figure 6 clearly indicates that certain PgSUS gene showed a tissue specific expression pattern. As the x-axis value is quite different in current figure 7 plot which make it impossible to compare the expression level of different PgSUS genes in the same tissue. Could the author re-organize or replot the data figure 7 so that it is clear to evaluate the five different PgSUS genes in the same organ?

4)I would suggest the author to rewrite the discussion section as it is very long and impossible to read. There are lots of detailed results information in this section and make is more like a supplement of results instead of discussion.

Minor points:
1)The last sentence in method sections of abstract is misleading, the author actually obtained the expression profiles of five PgSUS from previously published data. The author should clarify it.

2)Line 73-79, the description about SUS gene numbers in different species need further modification to make it more comprehensible, not confusing.

3)Line 178-179, The sentence “Each cDNA solution diluted 10 times was used as qPCR templates” is very confusing. The author should provide the exact amount of starting total RNA sample for cDNA synthesis.

4) The author should unify the description of quantitative real-time PCR as either qPCR or qRT-PCR.

5)Line 198-199, the sentence “Chromosomes 2, 4, and 6 contained only one gene, and chromosomes 8 contained two genes, respectively.” is improper and misleading. It could be change into “Among them, one single SUS gene was originated from Chr2, 4 and 6 respectively, while the rest two was located in Chr8”.


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: IDENTIFICATION AND EXPRESSION ANALYSIS OF THE SUCROSE SYNTHASE GENE FAMILY IN POMEGRANATE (PUNICA GRANATUM L.)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The new version of the manuscript is in line with the provided suggestions.
Experimental design: The new experimental design description is clear and scientifically solid.
Validity of the findings: The findings presented in this study have a good relevance for sucrose metabolism investigation in pomegranate research being a spillover for the improvement of this crop through breeding programs.
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: IDENTIFICATION AND EXPRESSION ANALYSIS OF THE SUCROSE SYNTHASE GENE FAMILY IN POMEGRANATE (PUNICA GRANATUM L.)
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors figured out most of the issues I concerned and made detailed responses to my comments. In view of the improved manuscript with much better quality, I suggest the publication of this work in this journal.
Experimental design: No comment.
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: KIR GENE CONTENT IMPUTATION FROM SINGLE-NUCLEOTIDE POLYMORPHISMS IN THE FINNISH POPULATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The structure and writing follow standard conventions and are easy to understand. The figures are appropriate and support the text. The literature references are also appropriate and sufficient. The supporting data is sufficient and open.

The goals and claims are clear and modest. The appropriate amount of evidence and discussion are provided.
Experimental design: The manuscript describes an investigation to improve Finnish blood bank services by developing an efficient and accurate SNP panel and genotyping workflow. It is well defined, relevant, and meaningful. The claims do not exceed the design. The methods meet or exceed high technical and ethical standards and, with a few small improvements, contain enough information for reproduction.
Validity of the findings: The goal is clear. The methods and data reflect that goal, have been provided, and are appropriately detailed. The conclusions are also in line with the other sections and are appropriate.
Additional comments: I recommend these items be addressed.

a)	The github test doesn’t run for me
Rscript ./src/run_KIR_imputation.R     "./results/models"     "./test/simulated_ref.raw"     "./test/simulated_ref.bim"     "./test/output"
Error: No models found! Stopping.
I'm running R 4.0.3 on OS X 11.6.
b)	The link on line 94-95 needs altering. It looks like there is an erroneous space in the URL.
c)	I’m not quite sure about the meaning of the sentence in 117-118. Why have this cutoff? How many genotypes were affected (not counted) because of it? Does this have to do with why the overall cohort size is 818, but the evaluation cohort is less than half of that? It seems to me that all testing individuals/genes should be considered in the accuracy reporting, or the reasons should be explained more.
d)	The figures and tables should have “n=” information in them or their captions.
e)	The text doesn’t state the 12 genes that were genotyped nor why they were selected. It appears this decision was based on IPD-KIR gene definitions, excluding the frameworks. Is his correct?
f)	Did the validation genotyping confirm the assumption that every individual contains every framework gene?

These items are suggestions.

g)	The SNPs were from the FinnGen project, but that is the only information provided about how/why these markers were selected. It would be valuable if more information could be written or referenced about how the SNPs for the KIR region were included in the panel. Of special interest is how markers for B haplotype genes were selected.
h)	The text does not mention how many SNPs are used by the algorithm. It is 743, I believe. It would aid the text to add this information and break down the SNP counts by gene. Do the gene names in the SNP names reflect the genes they originate from, mark, or both?
i)	It would be nice if the github page would describe the expected input, in addition to referencing the publication. Also, the required packages.
j)	The submission metadata says there are 3 figures, but I believe there are only 2 (and 1 table).
k)	The web page referred to in lines 134-135 displays two studies: EGAS00001005457 and EGAD00010002206?. They look the same. Why are there two, and what are the differences?


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: KIR GENE CONTENT IMPUTATION FROM SINGLE-NUCLEOTIDE POLYMORPHISMS IN THE FINNISH POPULATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall the manuscript is well-written. I did have a problem with the paragraph in the introduction encompassing lines 48-60. First, in line 50 the authors state the A haplotype has 'functional' 3DL3, etc genes, but then later in the paragraph goes on to state that many of the 2DS4 genes encode non-funtional receptors. 'Functional' should be removed. Second, the genes listed are neither activating or inhibitory, instead they encode activating or inhibitory receptors. The text should be modified to reflect this. For example, '...harbours several activating KIR genes...' should be changed to '..harbours several genes encoding activating KIR...'.

With regard to references, in line 70 they state that KIR*IMP is the only other known imputation method available, but then in the Results section (line 155) they mention a second method, kpi. Reference to it should be included in the introduction.
Experimental design: The method is well-described and appears highly accurate for the populations in which it was tested. Data, code and models are available. It will be interesting to see how well this method will perform when presented with data from more diverse populations.
Validity of the findings: The authors have properly stated that their training and testing focused on European populations and that adding geographically/ethnically diverse populations may impact the accuracy of their predictive model.

In the discussion of results for 2DL5, 2DS3, and 2DS5, the authors comment on positional variability. Along with that comes the possibility of 0-4 copies in any individual. It would be helpful if the authors could comment on how that may affect the accuracy of their prediction. In the same vein, there are known haplotypes that contain duplication of segments of the KIR region. Are the authors aware of any such haplotypes in their dataset and how that copy number variation (3 copies were two maximum are expected) would affect their results?
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: KIR GENE CONTENT IMPUTATION FROM SINGLE-NUCLEOTIDE POLYMORPHISMS IN THE FINNISH POPULATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is well written, and structured in a way that it's straightforward to follow the rationale. The introduction provides sufficient context, and the amount of cited literature is appropriate. Figure are very clear and nicely presented.
Experimental design: The authors describe a novel method to impute KIR gene presence from SNP genotyping data. The study was well designed and executed, and all methods are described with sufficient detail. The authors rightly state that there is only one SNP-based approach for KIR imputation thus far, which requires an upload of individual-level genetic data to an online server. This is often not possible due to genetic privacy regulations, and thus the authors' efforts present an important and welcome contribution to the field of immunogenetics. One possible issue is the exclusion of patients based on probability scores before calculating imputation accuracy (see general comments).
Validity of the findings: The conclusion is well supported by method description and results. The authors clearly state that their method was developed for the Finnish population. However, it seems that the workflow itself can be replicated in other populations relatively easily, and I'm sure others, including yours truly, would appreciate the authors' point of view on such efforts. It would be great to add a paragraph to the discussion explaining what would be required to translate this to populations of different genetic ancestry.
Additional comments: 1) Numbers in Figure 2E do not add up to 409, as I would have expected based on Figure 1. How did the author lose 8% of their cohort? The description of how posterior probability was used is not clear to me (Lines 161-166). Did the authors remove individuals according to a certain threshold, before reporting accuracy estimates? If yes, this warrants a clear description and more discussion. One might argue that low PP for a significant fraction of the cohort is indeed a measure of accuracy, and that it's not appropriate to simply disregard that. My preference would be a more conservative approach, first reporting accuracy independent of this filtering, and then explaining that PP can be used to remove likely false results from a cohort one intends to analyze, which in turn leads to very high accuracy for the 92% of individuals that can be considered.

2) Figure 2b might require some clarification, since it appears as if the accuracy estimates for the novel software vs. kpi ("WGS (external)") were from the same evaluated data set, which is not the case. So it is not a direct comparison, but the figure shows independently generated accuracy estimates from different populations.

3) A short description of how the reference data was generated (apart from the name of the vendor) would be appreciated.


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: KIR GENE CONTENT IMPUTATION FROM SINGLE-NUCLEOTIDE POLYMORPHISMS IN THE FINNISH POPULATION
Review round: 1
Reviewer: 4

Output:
Basic reporting: no comment
Experimental design: The study is designed in a standard way of other SNP imputation-based papers.
The method is straight forward and comparisons with existing software are clearly proven.
The limitation of the panel is clearly stated
Validity of the findings: Internal validation is performed and high accuracy is achieved, however; the review is concerned about the problem of overfitting and validation with an independent dataset is necessary to validate the accuracy of the reference.
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: KIR GENE CONTENT IMPUTATION FROM SINGLE-NUCLEOTIDE POLYMORPHISMS IN THE FINNISH POPULATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: I find the requested changes to be acceptable.
Experimental design: I find the requested changes to be acceptable.
Validity of the findings: I find the requested changes to be acceptable.
Additional comments: I find the requested changes to be acceptable.


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: KIR GENE CONTENT IMPUTATION FROM SINGLE-NUCLEOTIDE POLYMORPHISMS IN THE FINNISH POPULATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript text did not match the rebuttal text for the requested correction of line 50 (and the remainder of the paragraphJ. I would suggest amending as follows for clarity.

The KIR gene cluster on the human chromosome 19q13.4 encodes fifteen relatively homologous KIR genes and two pseudogenes, constituting two main haplotypes: A and B (https://www.ebi.ac.uk/ipd/kir/sequenced_haplotypes.html). The group A haplotype consists of functional KIR3DL3, KIR2DL3, KIR2DL1,  KIR2DL4, KIR3DL1, KIR2DS4 and KIR3DL2 genes of which all except KIR2DS4 encode inhibitory KIR. The group B haplotype, on the other hand, is more diverse being characterised by the presence of at least one of KIR2DS2, KIR2DL2, KIR2DL5, KIR2DS5, KIR3DS1, KIR2DS3 or KIR2DS1 genes (Bashirova et al., 2006). Thus, the group B haplotype harbours several  genes encoding activating KIR, whereas the only activating receptor, KIR2DS4, encoded by  group A is a non-functional truncated variant in a significant proportion of Caucasians (Maxwell et al., 2002; Bontadini et al., 2006), rendering about 40% of group A homozygotes solely inhibitory. Approximately 55% of haplotypes are mixtures between group A and B (Middleton & Gonzelez, 2010), making the haplotype strucure highly variable in the population. Allelic diversity within KIRs is equally high with at least a few hundred  known polymorphisms (https://www.ebi.ac.uk/ipd/kir/stats.html), which can affect class I ligand affinity (Carr, Pando & Parham, 2005; Frazier et al., 2013).
Experimental design: The authors have addressed my concerns.
Validity of the findings: No additional 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: KIR GENE CONTENT IMPUTATION FROM SINGLE-NUCLEOTIDE POLYMORPHISMS IN THE FINNISH POPULATION
Review round: 2
Reviewer: 3

Output:
Basic reporting: The revised sections of the text are clear and well written.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I appreciate the addition of a training script to the repository that will allow training building custom imputation models.

Figure 1 now states that 807 samples were included, and 50% randomly assigned to training and test set. This still doesn't explain the sum of 405 in the confusion tables (Figure 2E). It's a minor discrepancy (I'd expect 403 or 404 based on the 50/50 assignment), but it should be explained.


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: ANTIGENIC PROPERTIES OF THE SARS-COV-2 NUCLEOPROTEIN ARE ALTERED BY THE RNA ADMIXTURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript by Kolesov DE et al investigates the effect of RNA admixture on antigenic properties of SARS-CoV-2 nucleoprotein (NP) in ELISA-based antibody capture assay. The authors have purified  NP (and its N-terminal domain, NTD) using E. coli-based expression system and used the proteins as such or after RNA removal as antigen in the ELISA assay to detect the reactivity against SARS-CoV-2 positive serums. Authors claim that RNA-free NP is significantly better antigen in ELISA test compared to RNA-admixtured-NP.
	The overall study is interesting as it studies the effect of NP’s purity on its antigenic properties. However, the manuscript in the current form suffers from several issues related to the write-up, experimental design, figures and data presentation, and interpretation of results.

1. The Introduction section of the manuscript provides an overview of current state of the art of SARS-CoV-2 tests, importance of NP as antigen for testing, and difficulties with getting pure preparations of NP. However, despite providing valuable insights, these discussions are overly long and impede with the smooth readability. The portions of the Introduction section discussing serological tests (lines 42–60), nucleoprotein (NP) (lines 61–76), NP admixtures (lines 77–89) should be re-phrased to make it concise while retaining the information provided in order to make the flow of the manuscript smooth and focused. Lines 90–94 are unnecessary since authors have not compared S antigen with N antigen in the current study. These lines could either be removed or more tightly integrated with lines 83–89 in order to make author’s motivation for the current study clearer/more sound.

2. The language used throughout the manuscript could be improved for better readability and to eliminate ambiguous/unclear sentences. For example, there are minor English problems on lines 20, 21, 26, 28, 30, 37, 41, 50, 51, 97, 99, 125, 127, 138, 140, 141, 152, 154, 155, 160, 183, 192 which can be easily fixed by using appropriate article/propositions/term.

3. Introduction section has some lines stating facts/possibilities without citing a literature reference. For example, statements in lines 34, 35 and 38 need suitable recent literature references. Similarly, statements ending or stated in lines 48, 81, 90 also need appropriate references.

4. Some of the figures are not high resolution or clear as discussed below:

4 (i). Fig 1A could be made higher resolution to avoid pixelation.

4 (ii). The resolution of Fig 1D and 1E is so poor that it is not possible to distinguish red curve from blue. Additionally, the text details on x-axis and y-axis as well as other details on top of each of the figures are in too small font to read.

4(iii). In Fig 2C, the x- and y-axes details are too small to read.

4 (iv). Fig 2E does not mention the molecular weight of the peaks.

4 (v). Fig 2D and Fig S2 are identical. It is not clear what is the purpose of authors to show the same figure twice.

4 (vi). In Fig S3 and S4, peaks have not been marked with the molecular weight.

4 (vii). Raw gel pictures in the supplementary folder have not been annotated. Therefore it is not clear which gel belongs to which experiment and what to conclude from the gels pictures.
Experimental design: 1. The authors have not included any negative or positive control for their NP antigen in ELISA assay against the sera tested. The lack of a negative control (such as any globular non-NP protein) limits the specificity claim of their NP antigen. On the other hand, lack of a positive control (such as an established NP-based test antigen) makes it difficult to assess the effectiveness of their NP antigen in real-world testing scenario compared to the established NP-based tests.

2. Authors have used deca-His tag (line 100) for their NP purification unlike hexa-His tag routinely used  in the protein purification community. Authors have not mentioned why they went high on His numbers. This is important because it will not only affect the net charge on NP but might also promote association with negatively charged nucleic acids. Ideally, a His-free purification system should have been employed given the high propensity of NP to associate with nucleic acids as mentioned by authors on line 83–85.

3. There are inconsistencies/missing details in the Materials and Methods section:

3 (i). The restriction/cloning sites on line 97 is PciI/HindIII, on line 99 it is NcoI/HindIII, while on Fig 1A, it is Xba1/HindIII. Authors should clarify this discrepancy in the restriction sites.

3 (ii). The amino acid sequences of NP, NTD, or CTD has not been provided. Authors should provide the complete amino acid sequences in Supplementary Information and also mention residue numbers for NTD and CTD fragments in the manuscript lines 106 and 108 respectively.

3 (iii). How were NTD and CTD purified has not been mentioned in Protein purification sub-section (lines 124–136).

3 (iv). Line 132 mentions high imidazole (250mM) for elution. Was imidazole buffer-exchanged before use as antigen?

3 (v). Gel destaining should be briefly mentioned in SDS-PAGE analysis sub-section (lines 137–142).

3 (vi). It is not clear what was the purpose of keeping high imidazole (100 mM) in size exclusion chromatography (line 145).

3 (vii). How many total positive and negative sera sample were obtained should be clearly mentioned in the ELISA subsection (lines 149–151).

3 (viii). It is not mentioned if the sera only contained IgG or had IgM along with IgG.

3 (ix). Details of UV measurements (lines 186–187) intercalating dye experiment (lines 198–199)  should be provided in the Materials and Methods section.

3 (x). No mass spectrometry data has been shown for NP-NTD.
Validity of the findings: The Results section should provide additional information/elaboration of results:

1 (i). There are several lower molecular weight bands on gel in Fig 1C in 250mM imidazole lane in both gel. Clearly, author’s NP preparation has contaminants even after RNA removal. Authors should clarify how these impurities do not affect their results. The peak-width in mass spectrum in Fig 2C is pretty broad suggesting several peaks hidden in the main peak. It appears that NP preparation in the manuscript suffers from some kind of proteolysis resulting in several smaller molecular weight fragments which show up in gel and mass spectrum as pointed out above.

1 (ii).  Lines 200-204, the claim that NP preparation was free from DNA and RNA has not been supported by data/fig in the manuscript.

1 (iii). Authors claim that three N-terminal amino acids are cleaved by proteases during purification (lines 214–217). Could this be avoided by use of protease inhibitors during purification?

1 (iv). Lines 234–237, authors need to discuss salt effects on NP polymerization.

1 (v). Lines 248–252 have seemingly contradictory statements.

1 (vi). In Fig S5, the ODs are not substantially different for NP and NP-RNA+NaCl curves. Lines 254–256, however mentions significant difference between the two. This suggest obvious wrong statistics done on this set of data. This should be corrected.

1 (vii). The r-values are much better for S1-NP#1, S1-NP#2, S1-RBD, RBD-NP#1 and RBD-NP#2 pairs compared to any combination of author’s NP (RNA free or otherwise) with either NP#1, NP#2 or RBD (Fig 4). This suggests that authors NP preparation discussed in this manuscript is worse than above literature reported antigens. Authors should comment on this in lines 266–279.

2. The Discussion section needs literature references for the statements in line 294 (“Although the general method….NP is well known”) and 295 (“was described previously”).

3. The claim made in line 313–314 “developed a purification procedure that is fully scalable” is not substantiated as there is no data in manuscript proving the scalability of the procedure.

In light of above concerns, the validity of findings is not beyond questions.
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: ANTIGENIC PROPERTIES OF THE SARS-COV-2 NUCLEOPROTEIN ARE ALTERED BY THE RNA ADMIXTURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, Kolesov et al reported that the two steps of RNA removal (RNAse A treatment of the crude E.coli lysate and subsequent selective RNA elution by the 2 M NaCl solution) results in the purification of RNA-free NP that represents better signal-to-noise ratio when used in ELISA. The author further delineates the usability of the full-length NP and N-terminal domain antigen devoid of RNA in antibody capture ELISA. They also tested the control panel of SARS-CoV-2 positive serum samples and compared the positivity of ELISA using NP or NP-RNA with other conventional antibody tests. Consequently, they found that the complete removal of RNA form NP during the purification may significantly improve its antigenic properties, and the absence of the RNA in NP preparations should be monitored during the production of antigen.
Although the article is well written and interesting, and reports what could be potentially important in COVID-19 antibody testing, the overall studies are still preliminary and proposed hypothesis is not well verified with current data. Additional experiments with proper controls are needed before the manuscript can be accepted for publication. My specific comments are listed below.
Experimental design: Figure 1C, D
Since 2M NaCl is used for RNase+, please make the same comparison for RNase-. Also, only 2M NaCl is used, please consider other concentrations. It would be possible to compare NaCl with other chaotropic salts such as NaBr, NaClO4 or Cl3COONa (https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793(83)80675-9). Please provide the data showing the size of NP-RNA.

Figure 2A, B
This reviewer think that NP-CTD should be included as a negative control in the figure to verify the hypothesis that the authors claimed.

Figure 3A, B, C
Again, please add the data of NP-CTD as a negative control. Alternatively, authors may use other non-RNA binding proteins

Figure 4
There is no data regarding NP#1 and NP#2 tests employing NP or NP-RNA.
It would be helpful if the authors cite the reference showing the correlation between antibodies targeting NP and S1/RBD in COVID-19 patient sera.
Validity of the findings: Figure 1C, D
I'm not sure what the non-binding lane indicates. Please add an explanation.

Figure 2C
Since it is not known whether RNA exists or not in the full length NP, please add an explanation.

Figure 2D, E
In Fig1, 260nm is Red and 280nm is Blue, but the colors are reversed in this figure. Please unify the colors to make readers understand easily.

Figure 3D, E
It would be easier to see if the data of NEG and POS are described separately.
Additional comments: This article is very interesting, but I think data of NP-CTD is essential as a control.


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: ANTIGENIC PROPERTIES OF THE SARS-COV-2 NUCLEOPROTEIN ARE ALTERED BY THE RNA ADMIXTURE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript needs to be improved in terms of writing. there are several grammatical errors.

current statistics about the infection and mortality can be included in the manuscript.
Experimental design: overall the study is well planned and has good results but some more experiments need to be performed to support the hypothesis.

1. Western Blot analysis to confirm the expression of nucleoproteins

2. DLS and native page analysis to further support the oligomeric state of the protein.

3. mass spectrometric analysis methods need to be included in a detailed manner along with a citation.

4. To support the ELISA result, the author can perform the dot-blot far-western analysis to find out the interactions.
Validity of the findings: this will helps in exploring the possibility of using nucleoprotein as an antigenic protein or some part of the protein for eliciting the response against the virus.

the result and discussion sections of the manuscript is properly written.
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: ANTIGENIC PROPERTIES OF THE SARS-COV-2 NUCLEOPROTEIN ARE ALTERED BY THE RNA ADMIXTURE
Review round: 2
Reviewer: 1

Output:
Basic reporting: All the concerns raised have been addressed by the authors in the revised version of the manuscript.
Experimental design: The revised manuscript has addressed all the concerns raised in this section of the review.
Validity of the findings: The revised manuscript has addressed all the concerns raised in this section of the review.
Additional comments: The authors have adequately addressed all the concerns raised in the review. I appreciate the authors for their time and effort.


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: ANTIGENIC PROPERTIES OF THE SARS-COV-2 NUCLEOPROTEIN ARE ALTERED BY THE RNA ADMIXTURE
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment.
Experimental design: no comment.
Validity of the findings: no comment.
Additional comments: The authors have addressed my original concerns and substantially revised their manuscript. I think it is much improved.
The only minor issue is that the authors used only 2M NaCl for the dissociation of RNA. This could be further addressed in their future studies.


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: ANTIGENIC PROPERTIES OF THE SARS-COV-2 NUCLEOPROTEIN ARE ALTERED BY THE RNA ADMIXTURE
Review round: 2
Reviewer: 3

Output:
Basic reporting: The Authors have answered all the queries.
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: UVARIOPSIS DICAPRIO (ANNONACEAE) A NEW TREE SPECIES WITH NOTES ON ITS POLLINATION BIOLOGY, AND THE CRITICALLY ENDANGERED NARROWLY ENDEMIC PLANT SPECIES OF THE EBO FOREST, CAMEROON
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written and presents a new species of Annonaceae. The new Uvariopsis dicaprio has already a nomenclatural issue, the name Uvariopsis ebo was invalidly published as a preprint by the same authors of this manuscript. I suggest that this information is included in the section “notes”, since it is an important information about the taxonomy of this new species.
Experimental design: The authors inferred the extent of occupancy and the area of occurrence but forgot to indicate the method it was inferred. I suggest including it in the section “Material and methods”.
Validity of the findings: The description of the new species Uvariopsis dicaprio is relevant for the conservation of the Ebo forest and for the scientific community.
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: UVARIOPSIS DICAPRIO (ANNONACEAE) A NEW TREE SPECIES WITH NOTES ON ITS POLLINATION BIOLOGY, AND THE CRITICALLY ENDANGERED NARROWLY ENDEMIC PLANT SPECIES OF THE EBO FOREST, CAMEROON
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Adjustments to be made and suggestions please check the pdf file. Please check the end of the document.
Experimental design: .
Validity of the findings: .
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: UVARIOPSIS DICAPRIO (ANNONACEAE) A NEW TREE SPECIES WITH NOTES ON ITS POLLINATION BIOLOGY, AND THE CRITICALLY ENDANGERED NARROWLY ENDEMIC PLANT SPECIES OF THE EBO FOREST, CAMEROON
Review round: 2
Reviewer: 1

Output:
Basic reporting: After revision I have no additional comments to make. The manuscript is well written and should be accepted.
Experimental design: The metodology is the usual for this kind of study.
Validity of the findings: The manuscritp is relevant for the area of plant taxonomy.
Additional comments: I have 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: UVARIOPSIS DICAPRIO (ANNONACEAE) A NEW TREE SPECIES WITH NOTES ON ITS POLLINATION BIOLOGY, AND THE CRITICALLY ENDANGERED NARROWLY ENDEMIC PLANT SPECIES OF THE EBO FOREST, CAMEROON
Review round: 2
Reviewer: 2

Output:
Basic reporting: See note to editor
Experimental design: See note to editor
Validity of the findings: See note to editor
Additional comments: See note to editor


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: PSYCHOMETRIC PROPERTIES OF THE VALUING QUESTIONNAIRE IN A SPANIARD SAMPLE AND FACTORIAL EQUIVALENCE WITH A COLOMBIAN SAMPLE
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: Material & Methods
3. I think the description of the procedures (lines 191 to 199) could be presented before the participants (line 139).
4. What was the method used to estimate the sample size of this study?
5. What are the inclusion and exclusion criteria of this study? How many participants accepted participated in this study?

Instruments
6. Can the authors add the VQ’s alpha of the original study, as they do with the other scales?
Validity of the findings: Discussion
The authors could discuss the practical implications of the current study.
Additional comments: Psychometric properties of the Valuing Questionnaire in a Spaniard sample and factorial equivalence with a Colombian sample

I am very grateful for the opportunity to review this manuscript. I would like to congratulate on addressing an important instrument in ACT model, The Valuing Questionnaire (VQ). In my opinion, this study is relevant and is well structured, with research question well defined, relevant, and meaningful. The statistical analyses are appropriate to the aims of the study, and the results are interesting. There are some issues that I have outlined below.

Abstract
1. The abstract is well structured and presents relevant information. I just have a minor suggestion. In methods please add the following information: “The VQ was administered to a Spaniard sample of 846 adult participants from general online population”.

Introduction
2.The introduction is very complete. It was presented a good background, and objectives and hypothesis of this study was clear. I just have a question: In first paragraph of introduction, the authors use the term “middle-level term”. What this means?

Material & Methods
3. I think the description of the procedures (lines 191 to 199) could be presented before the participants (line 139).
4. What was the method used to estimate the sample size of this study?
5. What are the inclusion and exclusion criteria of this study? How many participants accepted participated in this study?

Instruments
6. Can the authors add the VQ’s alpha of the original study, as they do with the other scales?

Statistical and Psychometric Analyses
The authors presented a logical and adequate description of the statistical analyses.

Results
7. What is the correlation between two factors of the scale?

Discussion
8.   I think it would be convenient to start the discussion with a sentence that would inform us about what this scale measures.
9. The authors could discuss the practical implications of the current 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: PSYCHOMETRIC PROPERTIES OF THE VALUING QUESTIONNAIRE IN A SPANIARD SAMPLE AND FACTORIAL EQUIVALENCE WITH A COLOMBIAN SAMPLE
Review round: 1
Reviewer: 2

Output:
Basic reporting: In my view, the manuscritp is well-written in English. The introduction section is well structured, although additional information is mainly required to strengthen the choice of this questionnaire beyond its broad use (see additional comments, section: introduction).
Experimental design: The study meets all ethical standards required for human research.
The contribution of the study, justification and research question are well defined. The objective is clear.
The type of design adopted in the study is missed. Anyway, the needed steps for validation were adequately followed.
The authors described the materials and method in an accurate and valid manner. However, some psychometric analyses are not run (see additional comments: section: method). In the same vein, the multi-group analysis of invariance is uncompleted by excluding strict invariance.
The data anlyses are in line with the objective raised.
Validity of the findings: Conclusions are consistent with the objective raised, data analyses run and results obtained. However, practical implications are needed to highlight the utility of the study (see additional comments: section: conclusions). A point underlined was to gather evidence supporting cross-cultural inviariance of the questionnaire.
Additional comments: Below, a series of comments are proposed to improve the quality of the content and methodological aspects of the manuscript.

Introduction:
Comment 1. Lines 59-60: Please, consider the inclusion of a brief definition for each one of the six core processes described in psychological flexibility.

Comment 2. Lines 87-88. The authors indicated that three systematic review studies were carried out about self-reported measures of values. However, it is unknow what they added the study. Please, more information is required on these studies.

Comment 3. Lines 90-93. The authors listed a series of measures of values to finish with the valuing questionnaire. There is a need to strengthen the choice of this questionnaire beyond its broad use. In this sense, specify the advantages or benefits that this questionnaire presents in measuring values regarding the other instruments mentioned.

Comment 4. Lines 95-105. Great work in describing the instrument to be validated.

Method
Comment 5. Design. Please, specify the type of design adopted for this study. According to Ato et al. (2013), an instrument design was followed by analysing the psychometric properties of a measurement instrument.

Comment 6. Participants. Please, add the type of method used to recruit and select the participants.

Comment 7. Line 149. This sentence “They responded to an anonymous Internet survey distributed through social media” may be removed given that similar information was found in the procedure subsection.

Comment 8. Instruments. The stem that preceded to each instrument is missed.

Comment 9. Procedure. More information on the online questionnaire’s administration process is required. To illustrate, type instructions and guidelines given to fill the online survey, strategies followed to control social desirability in completing the survey, or average time for completion.

Comment 10. Statistical and Psychometric Analysis. Please, indicate the statistical treatment followed for multivariate and univariate outliers.

Comment 11. Statistical and Psychometric Analysis: Line 216-218: Assessment of RMSEA. Please, add p-value to have additional criteria to judge the RMSEA performance (Kline, 2016, page 273-276).

Comment 12. Statistical and Psychometric Analysis: Multi-group analysis of invariance. The most commonly used methodological proposals to test invariance consistently indicate four types of invariance to be examined (e.g., Gregorich, 2006; Milfont & Fisher, 2010; Putnick & Bornstein, 2016). It is, therefore, needed to include strict invariance into the two multi-group analyses run.

Comment 13. Statistical and Psychometric Analysis: Internal consistency. It is required to include cut-off points for interpretation of Cronbach’s alpha and McDonald’s omega. Indeed, it is recommended to clarify if the interpretation of these coefficients is made on the basis of mean scores or using 95%confidence interval of this mean score.

Comment 14. Statistical and Psychometric Analysis: Discriminant validity. It is required to estimate some criterion to analyse the instrument’s discriminant validity. It is, therefore, recommended to compute heterotrait-monotrait ratio of correlations (Henseler et al., 2015). This criterion displays a good level of discrimination among factor when values are as high as .85 (Henseler et al., 2015).

Comment 15. Statistical and Psychometric Analysis: Convergent validity. It is advisable to estimate average variance extracted as a convergent validity evidence (Martínez-García & Martínez-Caro, 2009). This measure is shown to be acceptable with values equal to .50 or higher (Hair et al., 2018)

Comment 16. Statistical and Psychometric Analysis: Criterion validity: Correlations used to estimate convergent validity are more suitable to provide criterion validity evidence. In this same vein, it is suggested to consider partial correlations controlling for country, gender or age in order to gain statistical power in the type of evidence given. Moreover, the instrument’s criterion validity is missed.
Results
Comment 17. Dimensionality: Line 258. This sentence “we chose the two-factor model due to its greater parsimony” is confusing for me. Please, specify the criterion followed to affirm that the two-factor model was more parsimonious. Parsimony indices (e.g., CAIC) were not reported.

Comment 18. Table 1. Goodness-of-fit measures: If the 2/ df coefficient is estimated for both models, its value is higher than 5, cut-off point considered as minimally acceptable (Bentler, 1989). Particularly, this coefficient is 5.46 for the two-factor model, and 5.54 for the two-factor model with correlated error terms.

Comment 19. Measurement invariance: Lines 266-269 and Table 2: In my view, there is an incoherence between the sentence “changes in CFI and NNFI indexes were equal to or greater than -.01” and differences below .010 in CFI and NNFI. Please, revise this point.

Comment 20. Validity evidence based on relationships with other variables. This section has been described in the data analysis subsection as convergent validity. Please, revise this point to gain coherence and consistence.

Discussion

Comment 21. It is needed to compare the results obtained in this study with those reported in the previous validation studies on the VQ. To illustrate: lines 309-310. “Regarding internal consistency, the VQ showed appropriate Cronbach's alpha and McDonald's omega values for both Progress (.85) and Obstruction (.84)”.

Comment 22. It is required to include a discussion about the validity evidence based on relationships with other variables. In addition, it is needed to discuss the results regarding differences by gender and country.

Conclusions:
Comment 23. lines 350-352. Practical implications require to be improved.

References
Ato, M., López-García, J. J., & Benavente, A. (2013). A classification system for research designs in psychology. Annals of Psychology, 29(3), 1038–1059. https://doi.org/10.6018/analesps.29.3.178511
Bentler, P. M. (1989). EQS structural equations program manual. BMDP Statistical Software.
Gregorich, S. E. (2006). Do self-report instruments allow meaningful comparisons across diverse population groups? Testing measurement invariance using the confirmatory factor analysis framework. Medical Care, 44(11 Suppl 3), S78–S94. https://doi.org/10.1097/01.mlr.0000245454.12228.8f
Hair, J. F. J., Black, W. C., Babin, B. J., & Anderson, R. E. (2018). Multivariate data analysis (8th ed.). Cengage Learning EMEA.
Henseler, J., Ringle, C. M., & Sarstedt, M. (2015). A new criterion for assessing discriminant validity in variance-based structural equation modeling. Journal of the Academy of Marketing Science, 43(1), 115–135. https://doi.org/10.1007/s11747-014-0403-8
Kline, R. B. (2016). Principles and practice of structural equation modeling (4th ed.). The Guilford Press.
Martínez-García, J. A., & Martínez-Caro, L. (2009). Discriminant validity as a scale evaluation criterion: Theory or statistics? Universitas Psychologica, 8(1), 27–36.
Milfont, T. L., & Fisher, R. (2010). Testing measurement invariance across groups: Applications in cross-cultural research. International Journal of Psychological Research, 3(1), 111–121. https://doi.org/10.21500/20112084.857
Putnick, D. L., & Bornstein, M. H. (2016). Measurement invariance conventions and reporting: The state of the art and future directions for psychological research. Developmental Review, 41, 71–90. https://doi.org/10.1016/j.dr.2016.06.004


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: PSYCHOMETRIC PROPERTIES OF THE VALUING QUESTIONNAIRE IN A SPANIARD SAMPLE AND FACTORIAL EQUIVALENCE WITH A COLOMBIAN SAMPLE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript was improved. Congratulations! I have a minor suggestion in the introduction:
on the last page of the introduction, I suggest you replace the word "Unfortunately" to "However". We need to pay attention to the words used in scientific manuscripts.
Experimental design: How did the researches ensure that participants who answered the questionnaire belonging to Sample 1 did not also complete the questionnaire belonging to Sample 2?
Validity of the findings: no comment
Additional comments: The manuscript was improved. Congratulations!


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: PSYCHOMETRIC PROPERTIES OF THE VALUING QUESTIONNAIRE IN A SPANIARD SAMPLE AND FACTORIAL EQUIVALENCE WITH A COLOMBIAN SAMPLE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors made a great labour in responding to every comment raised by me. The manuscript is well structured. The introduction have a goold guiding thread and the study contribution is clear. The objectives are clear.
Experimental design: The method section is well structured and contained all information necessary to replicate this study. Indeed, the data analyses is aligned with the proposed objective and the aurhos have complemented construct validity evidence by considering the suggested coefficents for discriminant and convergent validity. Similarly, the study desing is also clear owing to the authors followed the desing proposed.
Validity of the findings: The results are clear and provided an answer to the objective. The discussion is well structured and suitably argued the results obatined wiht the ones in previous studies. The conclusions relied on the found results and provided a response to the objective of the study.
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: ASSESSMENT OF MICROBIOTA DIVERSITY IN DENTAL UNIT WATERLINE CONTAMINATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is of a great interest for the present journal, the topic addressed is something original and therefore it is considered highly important for us. However, there are some sections that should be reviewed to make the article more accessible to an international audience.

First of all, it is the reviewer idea that the whole manuscript should be revised by a native English speaker to make the text more fluent and avoid any type of grammar or syntactical mistakes.

The abstract is a key section of your work, it should give a proper overview to the reader, it should be captivating and extremely precise about the information displayed in the text. It is the reviewer idea that the whole abstract should be rewritten to properly fit its core features.
Experimental design: INTRODUCTION:

 This section depicts properly the main topic’s background although there might be some parts that should be revised
1.	First of all, it is the revisor idea that this section could be implemented by better explaining each method previously described or each machine used to detect the type of bacteria.
2.	From line 75 to 79, please explain better what the authors mean by referring to high-throughput sequencing methods based on bacterial 16S rDNA.

MATERIALS AND METHODS:

This is the most crucial part of any kind of original research. A bad methodology is something that is clearly not acceptable for any type of journal. The methodology presented in the present article is very well presented although it could be better structured. The division in paragraphs is a useful tool to describe a step-by-step methodology, and the authors decided to do so in their manuscript. However, it is the reviewer idea that a more precise division, even in sub sections, could help the understanding of the current methodology.

	DISCUSSION:

The discussion section is a crucial part for the manuscript. It is the point where the author’s hypothesis has to be confirmed by the produced data and results.
However, in the present sections it seems that this kind of structure is not fully represented, therefore it is the revisor idea that this part of the article must be improved and implemented.
Validity of the findings: .
Additional comments: Overall, the manuscript represents a good specimen over the evaluation of such common situation regarding the microbiota diversity in dental unit waterlines. It is therefore of a great interest for the present journal, however some extensive English revisions, some paragraph implementations and enrichments should be addressed before the submission procedure goes on.


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: ASSESSMENT OF MICROBIOTA DIVERSITY IN DENTAL UNIT WATERLINE CONTAMINATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Dear Authors,
Thank you for submitting your article to this prestigious journal.
The manuscript is well presented but it presents some issues:

Abstract: Abstract should be revised and make it more appealing.

Keywords: To ensure a properly research in medical databases, use MeSH terms to find  appealing keywords that could be helpful in finding your article.

 “Additionally, in 2015, nine children were infected by Mycobacterium after having a pulpotomy at a pediatric dentistry practice in Georgia, United States (Peralta et al., 2015). Microorganisms in DUWLs, especially opportunistic pathogens, are potential risk factors for medically compromised or immunocompromised patients receiving dental treatment. The bacterial concentrations in some studies have far…” Please be more specific citing more articles
Introduction should be extended.
AIM should be revised
Materials and Methods and Results sections are well written and presented
In the statistical analysis do the authors provide a descriptive analysis of the sample? Why do the authors have used a non parametric test such as the Kruskal–Wallis H test?

This indicates that microbial contamination in DUWLs requires more attention, since microorganisms in dental treatment water can be swallowed or inhaled by patients via water flow or aerosols (Spagnolo et al., 2020). We also found no significant difference between water samples from the morning (before patient treatments) and afternoon (after all patient treatments). Authors should better describe this findings.
Discussion section should be entirely revised and make it more interesting to the reader.
Conclusion must be entirely revised making it more appealing and expanded.
English spell revision is necessary.

Check if it necessary the references section. (For e.g. PMID is not necessary).

Best Regards.
Experimental design: .
Validity of the findings: .
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: ASSESSMENT OF MICROBIOTA DIVERSITY IN DENTAL UNIT WATERLINE CONTAMINATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The study evaluated the bacterial concentrations, diversity, and community structure of microorganisms present in dental unit waterlines (DUWLs) in a dental hospital’s periodontics, endodontics, and prosthodontics departments through cultivation and high-throughput sequencing method. Results demonstrated that the bacteria concentrations and microbiome composition is influenced by the characteristics of dental specialty. And some potentially human-pathogenic bacteria were detected in the DUWLs. The authors thus concluded that validated disinfection protocol should be treated to control microbial contamination in DUWLs among different departments.

In the Discussion part, the different distributions of bacteria in water samples among the departments and the potentially human-pathogenic bacteria detected in different specialties are discussed. The citation is not enough, and there is a lack of comparison with some important and recent papers.
Such as:
1. Distinct Microbial Community of Accumulated Biofilm in Dental Unit Waterlines of Different Specialties. Fan C, Gu H, Liu L, Zhu H, Yan J, Huo Y. Front Cell Infect Microbiol. 2021 Jun 17;11:670211. PMID: 34222041
2. Pyrosequencing analysis of bacterial diversity in dental unit waterlines. Costa D, Mercier A, Gravouil K, Lesobre J, Delafont V, Bousseau A, Verdon J, Imbert C.Water Res. 2015 Sep 15;81:223-31. PMID: 26072020
3. High throughput sequencing-based analysis of microbial diversity in dental unit waterlines supports the importance of providing safe water for clinical use. Zhang Y, Ping Y, Zhou R, Wang J, Zhang G.J Infect Public Health. 2018 May-Jun;11(3):357-363. PMID: 28988812

There are some grammatical errors or missing words throughout the text, it would be better to improve the English language.  For example, Line 235 “Bacteria may had different” should be “Bacteria may have different”, Line 280 “analyse” should be “analyze”. In addition to some grammatical errors, there are a number of inadequate usage of spaces. Some examples include Line 93 “prosthodontics department(Table S1)”, Line 173 “Proteobacteria(Fig.1A) ”, Line 274 “used  cultivation”, Line 280 “used  cultivation”, Line 290 “validated  disinfection”.
Experimental design: About the sample collection method in the study, there are 2 kinds of methods to collect the sample. The 5 ml water samples were used to further culture and count the bacteria concentration, while the 250 ml water samples were used for the sequencing. Why don’t use the same type of water sample for the test of bacteria concentration and sequence? Besides, for the count of CFU, literature usually used the R2A plate, please explain the reason for choosing the BIH plate.
The general information of sampling Dental Chair Units could influence the bacteria composition. It will be better to provide the general information of Dental Chair Units, such as Operating years, Age Average patients per month, Number of patients on the sampling day.

The author described that the raw data have been submitted to NCBI, but I couldn’t find the deposit under the provided accession number PRJNA690183. Please explain the detail of the database, such as NCBI Sequence Read Archive (SRA) database.
 There is error in the department column of the supplement_Data_S1:  there is “4”, which may be “3”, as there is only “1=PE,2=EN,3=PR”.
Validity of the findings: The experiments are obtained using good technology and bioinformatics analysis.
Figure 1 shows the results of Bacteria composition in the periodontics, endodontics, and prosthodontics departments. It is better to give a comparison of the bacterial composition of different classification levels in different departments.

Table 3 shows the observed species, but the method part describes Ace, which is inconsistent.

In Figure 4, the texts are blurred, and the resolution needs to be improved.
Additional comments: The study is interesting and useful for the clinic control of risk infection and provides detailed information about the bacterial diversity among different specialties. And I think the manuscript needs minor changes as mentioned above.


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: ASSESSMENT OF MICROBIOTA DIVERSITY IN DENTAL UNIT WATERLINE CONTAMINATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: It is the reviewer idea that the changes added to the present manuscript are correcting and adjusting the aim of the present work, making it more understandable for the scientific community.
It is for this reason that I accept the manuscript in present form, without needing further changes.
kind regards and Merry Christmas
Andrea Del Giudice
Experimental design: .
Validity of the findings: .
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: ASSESSMENT OF MICROBIOTA DIVERSITY IN DENTAL UNIT WATERLINE CONTAMINATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have made sufficient revisions and addressed the comments. All the changes performed in order to answer all the reviewers have helped to improve the paper.  And I suggest that this paper be accepted without further modification.
Experimental design: Please see details in Basic reporting.
Validity of the findings: Please see details in Basic reporting.
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: CORTICOSPINAL EXCITABILITY REMAINS UNCHANGED IN THE PRESENCE OF RESIDUAL FORCE ENHANCEMENT AND DOES NOT CONTRIBUTE TO INCREASED TORQUE PRODUCTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: I took the traditional approach and included everything below
Experimental design: I took the traditional approach and included everything below
Validity of the findings: I took the traditional approach and included everything below
Additional comments: Frischholz et al. conducted a nice, comprehensive study looking into corticospinal excitability as related to residual force enhancement.  Very nice background and historical context! The work is well presented, and would be a great addition to PeerJ.  This group does a fantastic job methodologically to control for muscle fascicle excursions via ultrasound recordings, and the methods/results are presented very clearly. .  I present here a few points for the authors to consider:

1) Flexors and extensor muscle groups have differing neural inputs (e.g., incidence of PICs), it’s quite possible that results in the DFers will not match results in the PFers – therefore, differing results to that of Sypkes et al. is not surprising, nor controversial. This point could be raised if warranted.  Regarding, “rFE is not linked to changes in CS excitability”, when presenting these statements, consider framing in the context of the muscle group tested.  There is considerable data on the history-dependence of force that would indicate CS excitability is altered.  Taken together, I would suggest softening any definitive statements throughout the paper not directly related to what the authors measured, or the PFers.

2) “making rFE a muscle mech property rather than a combined neuromuscular property”. Given the body of literature on the HD of force, this statement seems obvious.  I would suggest that CNS excitability can be altered by rFE (and FD), and during voluntary contractions, depending on activation level etc, rFE has been shown to also differ in magnitude.  There is no doubt rFE is a property of muscle. The quoted statement would make an uninformed reader think this was a new finding.  This comment also applies to Line 152-154, and the conclusion.  Something like this may be helpful, I’ll leave it up to the authors:  “rFE is indeed an intrinsic property of muscle. While our results indicate no alterations to CS excitability, others show that rFE (and rFD) can modulate CS excitability, this may be dependent on muscle group tested, and level of neuromuscular activation”

3) 2nd Aim (Line 139): It’s not clear to me that this aim is trying to achieve.  If someone was performing a voluntary contraction during the SP of an evoked MEP, wouldn’t that temporary reduction in force output affect your measures? This aim may need to be clarified.

Line 89: Consider Jakobi et al. (PMID: 32347154 ) to support the single MU statement
Line 337 – did the outliers present with similar CS values?
Line 346 – not matching EMG will affect the evoked CS values, was this accounted for?
The authors considered the limitations of their methods, and presented it very transparently.

Great work! Reviewed by: Geoff Power


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: CORTICOSPINAL EXCITABILITY REMAINS UNCHANGED IN THE PRESENCE OF RESIDUAL FORCE ENHANCEMENT AND DOES NOT CONTRIBUTE TO INCREASED TORQUE PRODUCTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: With the exception of some minor errors and a small number of specific sentences (noted in the General Comments), the text is well-written and the English will be easily understood by an international audience. Most of the relevant literature is cited and the authors’ rationale for the study is clear.  I have no concerns with the quality and labelling of the figures.
Experimental design: While I have serious concerns about the validity of the reanalysis that led to the first aim of the research study (see next section), the intent is clear and the authors explain how both aims address a knowledge gap. The research was conducted carefully and includes the relevant methodological details needed to replicate the experiment. My only concern about experimental design relates to the setting of subthreshold and suprathreshold TMS intensities; i.e., the authors report intensities relative to maximal stimulator output so it would appear there were no criteria based on the actual physiological responses (e.g., percentages of AMT or, in the case of suprathreshold stimulation, a particular MEP amplitude).
Validity of the findings: The raw data have been provided and the interpretations are appropriate, for the most part. I am troubled by the process of including and excluding participants based on the presence of rFE. Looking at the raw data, there were two participants (#2 and 14) who were included in the subthreshold analyses because they displayed rFE across 10 sets of contractions but excluded from the suprathreshold analyses because rFE was not evident with 2 sets of contractions. Conversely, three participants (#8, 10, and 11) were included in the suprathreshold analyses even though they failed to show rFE across the more robust subthreshold protocol. I find both of these scenarios concerning, particularly the second one. For example, in the subthreshold protocol, a participant could show a mean rFE across 10 contractions but fail to display rFE with several combinations of just 2 contractions (meaning, they would be excluded from the suprathreshold protocol if those were the only 2 contractions). Please justify this approach to inclusion and exclusion.

A second point regarding the raw data, there are instances when participants have a value for the STR or REF condition but not both. How did the authors handle these cases when performing statistical analyses? Assuming these participants were excluded, the authors should make it clear in the manuscript how many participants were included for each variable. For example, it would appear that only 6 participants were included in the analysis of MG EMG suppression.

As alluded to in my comment for the previous question, I believe the reanalysed data to be invalid. In the case of reanalysing the data from the 2018 study by Sypkes and colleagues to normalise the MEP to Mmax, the analysis itself is correct but it is unnecessary and the interpretation is incorrect. Normalising the MEP to the CMEP accounts for both spinal and peripheral contributions to the MEP, which means the procedure isolates cortical excitability in each participant. This method to evaluate cortical excitability is far superior to a comparison of mean values of MEP and CMEP normalised to Mmax. As such, the interpretation put forth by Sypkes and colleagues (reduced spinal excitability and unchanged cortical excitability) is accurate and the present authors incorrectly state that those data support increased cortical excitability. The second reanalysis conducted by the authors involved pooling the data from two studies (Hahn et al., 2012 and Sypkes et al., 2018) and is even more problematic than the first reanalysis. Measures of neural excitability (i.e., MEP, CMEP, and, to a lesser extent, Mmax) are highly dependent on the conditions of the task, which makes it inappropriate to pool data collected during maximal contractions (Hahn et al., 2012) with data collected at a moderate intensity (Sypkes et al., 2018). Furthermore, the responses can differ greatly between muscles (e.g., Giesebrecht et al., 2010 vs. Gandevia et al., 1999 or Martin et al., 2006 vs. Oya et al., 2008), which makes it inappropriate to pool data from the soleus (Hahn et al., 2012) and TA (Sypkes et al., 2018). Given these critical issues with the reanalysed data, I do not believe the reanalyses have a place in the manuscript. This will have significant implications on the rationale of the study and would require the elimination of Figure 1.
Additional comments: General note
Although well written, the Introduction is far too long. I consider two double-spaced pages to be typical but the present Introduction is nearly three times that length. Given the issues with the reanalysed data, eliminating all of that text would be a good place to start efforts to shorten the Introduction.

Introduction
- lines 53 & 58: This section of the text refers to isolated muscle fibres so I suggest the authors choose a word other than “recruitment” because of its association with motor units during voluntary contractions.
- lines 86-93: In addition to the Altenburg study, the authors should also report the single motor unit data from the 2020 study by Jakobi et al.
- line 111: Replace “was” with “were”
- lines 124-127: I agree with the authors that it is beneficial to have a large rather than small number of MEPs; however, the cited recommendations relate to responses collected from a relaxed muscle. The variability is considerably greater during relaxation compared to a submaximal contraction (although variability increases again during very strong or maximal contractions), which undermines the relevance of the reported numbers.
- lines 127-135: See earlier comments about the validity of the analysis on the pooled data. Also, the present study has only 9 (subthreshold data) or 10 (suprathreshold data) participants so I would encourage the authors to reconsider an emphasis on the sample size of the previous studies.
- line 129: Has anyone tried to replicate the results? I think there is an important distinction between an inability to replicate findings and a lack of an attempt.
- lines 131-134: What are the units for the CMEPs and MEPs?

Methods
- lines 204-205: It becomes clear later but I suggest the authors make it clear that MVA refers to EMG rather than torque output
- lines 212-216: See my comment about TMS intensities in the Experimental Design section
- lines 231-233: Given that plantar flexor torque reflects the contribution of SOL, MG, and LG, why did the authors use only SOL EMG as feedback to the participants?
- line 257: Replace “peak-to-peak” with “peak”
- line 268: I believe this should refer to Figure 3 rather than 2
- lines 270-272: What was the rationale for only 2 sets of contractions for the suprathreshold protocol?
- lines 273-275: How many trials were excluded? Based on the raw data, it would appear a few of the participants (#1, 3, 7) had significant fatigue development by the time they completed the suprathreshold protocol
- lines 306-307: It is explained a couple of sentences later but it should be made clear here the correlations refer to EMG suppression and torque for each contraction type rather than a correlation between REF and STR torque, which is how it reads at present
- line 307: Replace “was” with “were”
- lines 308-309: Again, make it clear what two variables are being compared with the correlation
- lines 311-313: Why did the authors take this approach to account for the disparity in absolute torque between REF and STR rather than compare the relative decrease in torque for the two contraction types?
- lines 322-323: The end of this sentence is difficult to follow so I suggest rewriting the explanation of determining the end of the SP

Results
- line 341: What do the authors mean by “largely”? Was there some formal (statistical) assessment of their ability to match the target?
- line 372: Delete “your results here”

Discussion
- line 377: If I understand the meaning of the sentence correctly, this should be “decreased” not “increased torque production”
- line 383: See the comment for line 341
- lines 385-386: What is the basis for considering 2% to be a negligible difference? I am not suggesting that is necessarily a meaningful difference but some participants had a mean rFE of ~2% so feel there should be some support for this decision
- line 407: Replace “supports” with “support”
- line 411: As noted in the Limitations section, the MEP is not normalised to the Mmax so “corticospinal” is not technically correct because the MEP reflects excitability of the entire motor pathway
- lines 411-413: See earlier comments about reanalysed data
- lines 417-420: Merton’s data were plotted as absolute forces but the relationship of superimposed twitch size to contraction strength is less about absolute force/torque and more about the activation of the motoneurone pool
- lines 420-423: In light of the previous point and the fact that EMG output was matched for the two contraction types, I question if the superimposed twitch data can be used as they are in this sentence. At a minimum, the speculative nature of this statement needs to be clearer.
- line 429: See the comment for line 411
- lines 431-434: The authors correctly note that spinal modulations cannot be excluded but fail to note that cortical modulations also cannot be excluded
- lines 435-436: Given the earlier comments about the superimposed twitch data, I do not think they can be used to exclude a modulation of spinal excitability
- lines 436-440: Mean Mmax amplitude was not different between REF and STR in the study by Sypkes and colleagues; however, a closer examination of Figure 3 suggests this reflects offsetting increases and decreases in different participants. As such, I think it is still important to normalise MEPs to Mmax in individual participants
- lines 440-442: See earlier comments about the interpretation of the superimposed twitch data
- lines 442-446: While I don’t believe it was necessary to obtain complete TMS input-output curves, the authors are understating the limitation to their approach of “ensuring MEPs could increase with increasing stimulation intensity”. Whether TMS intensity is set relative to a threshold (AMT or RMT) or to obtain a particular MEP size (relative to Mmax to ensure a comparable activation of the motoneurone pool), it is important to have some criterion that is applied in all participants

Tables
- Table 2 legend: To match the legend of Figure 5, make MEP and SP plural
- Table 2: Should MEP amplitudes be in mV rather than V?

Figures
- Figure 5: Should y-axis units be mV rather than V?


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: CORTICOSPINAL EXCITABILITY REMAINS UNCHANGED IN THE PRESENCE OF RESIDUAL FORCE ENHANCEMENT AND DOES NOT CONTRIBUTE TO INCREASED TORQUE PRODUCTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: General Comments
This manuscript provides evidence that residual force enhancement during isometric plantar flexion contractions, is not modulated through corticospinal pathways, and is therefore likely a muscle mechanical property rather than a combined neuro-muscular phenomenon. The manuscript is well written and concise, and provides the reader pertinent information to reach the conclusions stated. However, minor changes are required regarding structure of sentencing that lead to use of the methodology, which are described below:

Abstract:
Line 21: “Following active muscle stretch…..”. The use of the term active muscle stretch in this context, I believe refers to a voluntarily active muscle being passively stretched with a dynamometer. However, when first read it seems a little confusing, and could be perceived as an activation of the muscle to reposition the joint angle voluntarily (i.e. joint angle was changed through voluntary activation of the muscle). Can you please restructure to make this more apparent that an active muscle during the STR condition is passively stretched into position, similar to the opening sentence of introduction.

Methods:
Line 171: Was the computer monitor positioned directly in front of the participants? Please clarify.
Line 183: Were the electrodes placed parallel to the orientation of the muscle fibers for each muscle? Please clarify?
Line 212-216: The reduction and increase of stimulator output following AMT appears to be arbitrary, as currently written. Do the stimulator percentages reported correspond to average stimulator outputs used throughout the experiment for the participant pool?. Please report the percentage that the stimulator was reduced or increased for each participant (i.e. stimulator output was reduced 15% from AMT for subthreshold stimulation, and increased……).
Line 241: “muscle were actively stretched…..”. Would this not be a passive stretching of the muscle by the dynamometer, while maintaining muscle activation?. Please clarify.
Results:
Line 341: “largely managed to maintain….”. Rephrase this statement please. According to your inclusion criteria, EMG was maintained between 25-35%. This sentence makes it appear otherwise.
Discussion:
Line 383: Again please rephrase this sentence. Either the participants maintained contraction levels within the inclusion criteria or they did not.
Limitations
Line 435: This sentence should be removed. The previous sentence outlines that changes may have occurred within a differing structure than outlined in the current experiment. Therefore a conclusion should not be made, or restructured.


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: CORTICOSPINAL EXCITABILITY REMAINS UNCHANGED IN THE PRESENCE OF RESIDUAL FORCE ENHANCEMENT AND DOES NOT CONTRIBUTE TO INCREASED TORQUE PRODUCTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: please see below
Experimental design: please see below
Validity of the findings: please see below
Additional comments: The authors addressed my initial concerns, and IMHO provided a balanced response to the rest of the reviewers. There are some points we do not see 100% eye to eye on, but given the thoughtful reply I am happy to agree to disagree.
All the best
Geoff Power


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: CORTICOSPINAL EXCITABILITY REMAINS UNCHANGED IN THE PRESENCE OF RESIDUAL FORCE ENHANCEMENT AND DOES NOT CONTRIBUTE TO INCREASED TORQUE PRODUCTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Thank you to the authors for their detailed responses to my comments and the careful edits to the manuscript, which I view as much improved. Before providing a few comments about the revised manuscript, I will revisit some comments from the original review and the authors’ responses .

Original comment:
As alluded to in my comment for the previous question, I believe the reanalysed data to be invalid. In the case of reanalysing the data from the 2018 study by Sypkes and colleagues to normalise the MEP to Mmax, the analysis itself is correct but it is unnecessary and the interpretation is incorrect. Normalising the MEP to the CMEP accounts for both spinal and peripheral contributions to the MEP, which means the procedure isolates cortical excitability in each participant. This method to evaluate cortical excitability is far superior to a comparison of mean values of MEP and CMEP normalised to Mmax. As such, the interpretation put forth by Sypkes and colleagues (reduced spinal excitability and unchanged cortical excitability) is accurate and the present authors incorrectly state that those data support increased cortical excitability. The second reanalysis conducted by the authors involved pooling the data from two studies (Hahn et al., 2012 and Sypkes et al., 2018) and is even more problematic than the first reanalysis. Measures of neural excitability (i.e., MEP, CMEP, and, to a lesser extent, Mmax) are highly dependent on the conditions of the task, which makes it inappropriate to pool data collected during maximal contractions (Hahn et al., 2012) with data collected at a moderate intensity (Sypkes et al., 2018). Furthermore, the responses can differ greatly between muscles (e.g., Giesebrecht et al., 2010 vs. Gandevia et al., 1999 or Martin et al., 2006 vs. Oya et al., 2008), which makes it inappropriate to pool data from the soleus (Hahn et al., 2012) and TA (Sypkes et al., 2018). Given these critical issues with the reanalysed data, I do not believe the reanalyses have a place in the manuscript. This will have significant implications on the rationale of the study and would require the elimination of Figure 1.

Authors’ response part 1:
We have now removed the reanalysis and Figure 1 from the manuscript. However, this is because we think the reanalysis is problematic because the supplementary material of Sypkes et al. does not provide the M-waves corresponding to the MEPs.

New comment:
Obviously, I approve of the authors’ decision to remove the reanalysis element of the manuscript; however, I am a bit concerned that their sole reason was the absence of M-wave data from the Sypkes study. Even if those data were present, as I pointed out in my original comment, there are fundamental flaws associated with combining data collected at different contraction intensities and from different muscles. I am all in favour of trying to combine similar data to improve statistical power, but the two studies in question are too dissimilar to justify an attempt at merging the datasets.

Authors’ response part 2:
We do not see why the normalization of MEPs to CMEPs should be “far superior” to normalizing both CMEPs and MEPs to their corresponding Mmax. Rather, we actually think that analysing MEPs using CMEPs as the ratio of MEP/CMEP is problematic, unless MEPs and CMEPs are perfectly matched in size so that they can be assumed to represent the exact same motoneuron pool, and, more importantly, when M-waves do not vary between conditions. This is demonstrated in the table below (Table 1, see the reference condition). In the first virtual test condition, MEPs and CMEPs are reduced by the same amount between reference and test conditions (from 10 to 5), which results in a MEP/CMEP ratio of 1 indicating reduced spinal excitability, but unchanged cortical excitability (similar to Sypkes et al. 2018). However, as the Mmax corresponding to the MEPs changed as well (the M-wave MEP became smaller by 20% between reference and test conditions, whereas the M-wave CMEP was unchanged), the normalised MEP/CMEP ratio goes up to 1.25. This virtual finding would subsequently indicate reduced spinal excitability, but increased cortical excitability. In the second virtual test condition, CMEPs are reduced between reference and test conditions (again from 10 to 5), but MEPs are unchanged (similar to Sypkes et al. 2018). While the reduced CMEPs would again indicate reduced spinal excitability, the increased MEP/CMEP ratio of 2 (as expected based on reduced CMEPS, but unchanged MEPs) would indicate increased cortical excitability. However, this calculated ratio might be biased by M-waves varying between conditions.

New comment:
In terms of my comment about normalizing the MEP to the CMEP being far superior to separate comparisons to Mmax, when each of the MEP, CMEP, and Mmax are collected under the same conditions (and the MEP and CMEP are a similar proportion of Mmax size in the control condition, which should always be the aim), there is no benefit to normalizing MEP to Mmax. In this scenario, any change to Mmax, CMEP/Mmax, and MEP/CMEP values indicate changes in peripheral, spinal, and cortical excitability, respectively and nothing of interest comes from MEP/Mmax because corticospinal excitability is less insightful than separate measures of spinal and cortical excitability. In the Sypkes study, the Mmax and MEP were collected in one contraction and the CMEP in another, with the CMEP and MEP at the same time point in each contraction. In retrospect, it would have been best to collect Mmax data from both contractions, which would align with the design portrayed in Table 1 (alternatively one could collect MEP and CMEP in one contraction and CMEP and Mmax in another or all three in a single contraction). However, given the fluctuations in EMG or torque within a contraction (not including the effects caused by different stimuli), there is a case to be made that collection of Mmax and MEP (or Mmax and CMEP) a second or more apart in the same contraction is not necessarily a better comparison than one made across contractions. In the scenario illustrated in Table 1, the Mmax differs by 20% across two contractions of the same type, yet the comparison of MEP/Mmax to CMEP/Mmax assumes (relies on the idea) that spinal excitability is affected similarly in both contractions. I think the authors would agree* that spinal compared to peripheral excitability is far more likely to differ between two like contractions, which means their portrayal of MEP/Mmax vs. CMEP/Mmax as a superior method to MEP/CMEP is misleading. *In their answer to my comment about line 411 of the original manuscript, the authors suggest that they did not collect Mmax because it has been shown not to differ between control and RFE conditions, which means there is even less reason to believe it would differ substantially (e.g., 20%) between two control or two RFE contractions. Ultimately, no scenario allows us to test the different portions of the motor pathway under identical conditions and we have to live with the inherent limitations of any option. Given my concern with the soundness of the argument put forth in the revised Introduction and the associated supplemental Table (see new comment on the revised manuscript), I believe it will only create confusion and mislead readers to include these new elements of the manuscript.

Authors’ response part 3:
Accordingly, when CMEPs are reduced (as in Sypkes et al. 2018), but the MEP/CMEP ratio remains unchanged, the MEPs would need to decrease as well. While this is of course a theoretical consideration, this was not the case in Sypkes et al. 2018, where MEPs were unchanged between conditions. Accordingly, we think that the increased MEP/CMEP ratio from Fig. 3 F in Sypkes et al. (2018) did not achieve significance because the study was underpowered.

New comment:
In terms of absolute size, in most cases, the MEPs did decrease as well, which is why the MEP/CMEP ratio remained unchanged. The authors state “MEPs were unchanged between conditions” but, as absolute MEPs were not provided in the Sypkes article, I believe the authors mean the MEP/CMEP ratio was unchanged (see new comment on lines 108-113 of the revised manuscript). I fully concede that a larger sample size may have led to a statistically significant increase in the MEP/CMEP ratio. That said, if we are working with hypotheticals, it is pretty clear from Figure 3F in the Sypkes study that two participants are responsible for the authors’ perceived trend in the data; i.e., a larger sample size of participants more similar to the rest of group could make the MEP/CMEP line between ISO and RFE even flatter. Who knows?

Authors’ response part 4:
In the revised manuscript, we now show that the previous studies were underpowered, which is why we doubt their findings. On an important note, this is not just related to the Sypkes et al. (2018) study, but also to our own previous study (Hahn et al. 2012). Further, the aim of our study was not to disprove the findings from Sypkes and/or Hahn, but to better understand the neural nature of rFE. Accordingly, we do not care whether corticospinal excitability is changed or unchanged, but we are just interested in valid results and we think reviewer#2 should also be open minded to results not necessarily supporting their own view.

New comment:
I absolutely agree that the studies, like nearly all in human neuromuscular physiology, would benefit from a larger sample size. My original and new comments about the merging of data and advancing the benefits of the MEP/Max vs. CMEP/Mmax comparison have nothing to do with my perceptions of what the data should show (in this case, I have no preconceptions), they were/are based on the validity of the results and arguments put forth by the authors. Like you, I do not care whether an RFE state causes increases or decreases in cortical or spinal excitability. If a study is conducted carefully, the data are what they are.

General comments on the revised manuscript:

Introduction
- lines 105-108: As stated earlier, I think the authors are overstating the challenges of interpreting the MEP/CMEP ratio and overselling the benefits of the MEP/Mmax ratio.
- lines 108-113: I maintain that this interpretation is incorrect because it only makes sense if the data provided in the Sypkes study were absolute CMEPs and MEPs. However, the data provided were normalized not absolute potentials. That is CMEP/Mmax (not CMEP) was reduced and MEP/CMEP (not MEP) was unchanged. As the comparison of MEP/CMEP ratio between conditions represents an isolated measure of cortical excitability, I fail to see how that the authors can reinterpret an unchanged MEP/CMEP ratio to suggest an increase in cortical excitability.

Methods
- lines 323-324: I suggest the authors reword “was surprising assuming no difference”.


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: CORTICOSPINAL EXCITABILITY REMAINS UNCHANGED IN THE PRESENCE OF RESIDUAL FORCE ENHANCEMENT AND DOES NOT CONTRIBUTE TO INCREASED TORQUE PRODUCTION
Review round: 3
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: Thank you for allowing me to view the revisions.
My final review was summarized in the previous round
nice work.
Geoff


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: CORTICOSPINAL EXCITABILITY REMAINS UNCHANGED IN THE PRESENCE OF RESIDUAL FORCE ENHANCEMENT AND DOES NOT CONTRIBUTE TO INCREASED TORQUE PRODUCTION
Review round: 3
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Thank you to the authors for their carefully-considered responses to the last review. Although none of the authors’ arguments won me over to their point of view, I respect their opinions and am happy to conclude the debate. Congratulations.