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: TRICHOPLAX ADHAERENS REVEALS A NETWORK OF NUCLEAR RECEPTORS SENSITIVE TO 9-CIS-RETINOIC ACID AT THE BASE OF METAZOAN EVOLUTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Review on the manuscript by Novotný et al. “Trichoplax adhaerens reveals an endocrine-like network sensitive to 9-cis-retinoic acid at the base of metazoan evolution”

In Srivastava et al. 2008, four different nuclear receptor (NR) family transcription factors were reported to be encoded in the genome of Trichoplax adhaerens, a basal animal that is morphologically very simple. The four Trichoplax family members, RXR (NR2B), HNF4 (NR2A), COUP (NR2F) and ERR (NR3B), show a high degree of similarity to their human homologs. NRs are normally defined as ligand-activated transcription factors that allow the regulation of target genes by small lipophilic molecules such as hormones (thyroid hormones, steroids), morphogens (retinoic acid) or dietary components (fatty acids).
In the present article, Novotný et al. explore the role of NRs in Trichoplax. First, they show that one of the four NRs of Trichoplax, the retinoid X receptor homolog (TaRXR), is highly conserved (about 66% identity to vertebrate homologs) and is able, like the human homolog, to bind to 9-cis-retinoic acid (9-cis-RA) in the low nanomolar range. To show this, they have used an assay with radioactive 9-cis-RA established earlier (e.g. used to describe the affinity of the RXR of the cnidarian Tripedalia cystophora by Kostrouch et al. 1998). Novotný et al. also show that 9-cis-RA increases the expression of a malic enzyme they had identified in Trichoplax. They suggest that this could reflect a natural regulation of this metabolic enzyme by activated TaRXR. Moreover, the authors observed a change of the expression pattern of three of the four NRs induced by 9-cis-RA. Apparently, the authors also observed a difference in the expression patterns of NRs when they compared animals of different sizes. In the last part of the manuscript, the authors report that the food source has an impact on the size and growth rate of Trichoplax. I have to admit that I did not quite understand why these observations have been added to the manuscript about NRs and the effect of 9-cis-RA on TaRXR. The observations on the effect of food on Trichoplax are not really connected with the rest of the report. Somehow, when I read the abstract, I thought that addition of 9-cis-RA to the culture would have changed the shape, size or behaviour of the animals – it almost goes without saying that this would be very interesting. Have such experiments been performed and no changes were observed? Do the authors think that components of the algae might be ligands for the NRs? Another possibility to link the two sections would have been to test for changes of the expression level of different NRs upon feeding the animal with different algae.

Overall, I find the observations presented in the manuscript interesting, but several aspects were not entirely clear to me. I therefore suggest that the authors re-analyze their data and carry out additional experiments before considering publication of their findings. Moreover, I do not agree with the authors that the results indicate the presence of an “endocrine-like network” in Trichoplax. Please note that the current title of the manuscript does not really make sense. The term “endocrine-like network probably refers to the possible network of NRs found in the study, but the term endocrine appears a bit far-fetched as it implies the presence of an endocrine system in the animal, for which the present study does not provide clear evidence.

Major points that need to be revised:

1) In the Introduction, the authors do not make sufficiently clear why they have studied the NRs of Trichoplax adhaerens in particular. For example, they write “that Life on Earth began 4.1 to 3.5 billion years ago (Bell et al. 2015) with the appearance of the first unicellular prokaryotic organisms that subsequently evolved, in part, to multicellular lifeforms forming the kingdom Metazoa that have specialized tissues for digestion, regulation of homeostasis, locomotion, perception, analysis of the environment and reproduction.” This is obviously not correct: animals are eukaryotic organisms. Several eukaryotic lineages are multicellular: animals, fungi, plants and others. Here it would be advisable to shortly explain what we know about the evolution of NRs and their ligands in animals (and other eukaryotes), for example as has been done in Kostrouchova & Kostrouch 2015, for example. Trichoplax with its relatively simple set of NRs represents an animal at the base of the animal kingdom and could therefore shed light on an early set of NRs in animals, if that was the aim of the study. I think that this point is currently more clearly outlined in the Discussion, a section of the manuscript written in a clearer fashion than the rest of the manuscript in my opinion.

2) Remarkably, Trichoplax has a set of NRs that are highly similar to human NRs, in particular TaRXR is highly conserved. The conservation is shown by a sequence alignment, but I did not understand why only few organisms are represented in the alignment (and in the tree). Why, for example, are model cnidarians like Nematostella or Hydra not included? Don’t they have the particular receptor? What about sponges and ctenophores? What about bilaterians? This should be explained and the data set, if necessary, expanded. I suggest to improve the representation of the sequence alignment by clearly indicating the domains (DBD & LBD) and residues involved in 9-cis-RA. If these residues were indicated, the few non-conserved residues do not need to be outlined in the text (line 294). Check for example Gutierrez-Mazariegos et al.. 2016. Evolutionary diversification of retinoic acid receptor ligand-binding pocket structure by molecular tinkering. R. Soc. open sci. 3:150484 (PMID: 27069642). It also seems that the authors are unaware of a study, in which the set of Trichoplax NRs was investigated already (Baker ME. 2008. Trichoplax, the simplest known animal, contains an estrogen-related receptor but no estrogen receptor: Implications for estrogen receptor evolution. Biochem Biophys Res Commun 375:623–627. PMID: 18722350). By the way, I did not understand in the manuscript (line 280) whether the known TaRXR sequence (Srivastava et al. 2008) was used or whether the authors generated a revised/improved sequence of the TaRXR. If so, what exactly was improved?
Furthermore, I suggest to omit the phylogenetic analysis. As far as I understood, the important point was to show that the 9-cis-RA binding site is conserved between man and Trichoplax. Probably there is a protein structure that can be used to discuss the conservation and binding site. A tree that is based only on a few sequences does not provide much information and might be biased, because only selected sequences have been used. I also suggest to omit Table 1, because a sequence comparison based on a few Blast search results does not provide much information. For both data sets, a more rigorous analysis would be necessary. Again, the authors should compare their results to the above mentioned study by Baker ME. 2008.

3) In the section about 9-cis-RA binding, the authors only state that 9-cis-RA binds with high affinity and specificity, but do not explain in the text nor in the figure legend how the experiments have been performed. Currently, the reader has to go to the Method section to find out that this was done using radioactive 9-cis-RA (how was radioactivity measured?) which was added to recombinant proteins expressed in E. coli. Why isn’t the purity of the proteins shown by SDS-PAGE, for example? Was TaRXR used as a GST fusion protein or was the cleaved protein used in the experiments shown? How does the affinity measured compare to the affinity of 9-cis-RA to the human (or others) protein reported in earlier studies? That “this was clearly observed in repeated experiments” (line 319) should not be stated in the Results, rather the figure legend should make clear that the experiments were carried out several (how often?) times, also by adding error bars.

4) The upregulation of the malic enzyme in Trichoplax has been demonstrated by quantitative PCR (Fig. 4). However, no control of a housekeeping gene like actin or another enzyme has been performed to make sure that really similar amounts have been used. This also concerns the experiments shown in Fig. 5 and experiments mentioned only in the text, where the authors report about different sizes of animals. Moreover, as no error bars are shown, it seems that the experiments have not been repeated sufficiently. What I did not understand was why the data in Fig. 5 are not given, as in Fig. 4, as copies/µl? What are the values on the y-axis in Fig. 5? Furthermore, the data shown in Fig. 5 suggest that the effect of 9-cis-RA on Trichoplax was strongest at 1 nM concentration. But why then were the experiments shown in Fig. 4 only carried out at 3 nM and at higher concentrations, where, according to the experiments shown in Fig. 5, rather opposing effects of 9-cis-RA have been observed?

5) The date shown in Fig. 5 are interpreted by the authors as an expression network induced by 9-cis-RA. It remained unclear to me, however, whether the authors imply that 9-cis-RA elicits its effect solely via binding to TaRXR, which in its activated form then activates the expression of its own gene and the genes of the other NRs, or whether they think that 9-cis-RA can also interact with the other NRs at nanomolar concentration. Binding experiments with 9-cis-RA on other purified Trichoplax NRs could be carried out to prove that TaRXR is the only nM-affinity receptor for 9-cis-RA. Another possibility might be that the Trichoplax NRs could form heterodimers.

6) In the section about expression pattern of NRs (reported in the text and in Fig. 5) in Trichoplax, the authors write (line 346) "From the experience we gained by culturing T. adhaerens, as well as from the previous experiments we knew that the culture conditions can dramatically influence phenotype. Having the possible developmental functions of the ancestral NRs in mind, we raised the question whether the expression patterns of the NRs reflect changes in phenotype." To me it is not entirely clear what this statement is supposed to mean. Why wasn't the expression level of different NRs established first on a normal, large enough population of animals with different sizes (i.e. before selecting animals with different sizes)? Is the difference in the expression profile in small and big (large) animals really significant (33% of RXR compared to other NRs in big and 24% in small animals)? And most importantly: what was counted as big and what as small animal? How was the size of the animals determined? Which animal population, the big or small or both sizes, showed an increase to 51% after treatment (line 354)? In the Methods it is written that the animals were treated only during sunny days. Is this an important information as all 9-cis-RA binding experiments have been performed in the dark? I explicitly ask this, because in the following part of the manuscript, it is reported that the feeding conditions have a visible impact on the animals. This statement is not supported by a rigorous analysis, however. Some pictures of animals in different shapes and colours are shown in Fig. 6, which, in my opinion, solely document that the animals can vary, but it remains unclear whether the variation was induced by a certain feeding condition. It also remains unclear whether the poor growth of Trichoplax on Chlorella (Fig. 7), for example, is due to the low nutritive value or composition of that particular algae or simply because this algae is not the preferred food source of Trichoplax or because the algae is better protected against being captured by the animal. In that case, Trichoplax would just eat less. In Fig. 8, I did not understand what exactly was plotted. In some panels it might be the number of animals and in others the area, as there is no legend given. Have the growth experiments been carried out only once for each condition, because there is again no error bar given (Fig. 7 and Fig. 8)?
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: TRICHOPLAX ADHAERENS REVEALS A NETWORK OF NUCLEAR RECEPTORS SENSITIVE TO 9-CIS-RETINOIC ACID AT THE BASE OF METAZOAN EVOLUTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Novotney et al studied an ancestral Trichoplex RXR ortholog and find that it is activated by 9-cis-RA leading to the expression of other nuclear receptors.  This is a neat report.  To my mind it is the first example of a nuclear receptor ortholog in a basal metazoan being activated by RA.

Comments
1. The effect of algal extract on gene expression is very interesting.  Phytochemicals are known to activate the estrogen receptor.  Thus, I have a bias for phytochemicals also activating RXR.  However, phytochemicals could activate pathways that increase the synthesis of retinoids or other novel endogenous molecules that are ligands for RXR.  This is a strong alternative possibility.  Considering the strong sequence similarity between Trichoplax RXR and human RXR, the extract should affect human RXR action, an experiment that would provide support for Novetney et al.

2. RA binding to RXR regulating other nuclear receptors in Trichoplax is very interesting.  Activation occurs at 1 nM with a clear decrease at higher concentrations.  Two thoughts: first, assays at 0.5 nM and 0.1 nM would be of much interest.  Regulation at these concentrations would increase the impact of this research.  Second, a comment on the decreased activity at relatively low concentrations of RA [3nM] is appropriate.  It suggests to me that RA is not the endogenous ligand and thus, Trichoplax does not have metabolic pathways to regulate RA homeostasis as is found for steroid homeostasis in vertebrates.

3. I think the Introduction could be better focused on early metazoans, such as Trichoplax, Jellyfish and sponges.  The discussion of invertebrates such as C. elegans and Drosophila could be condensed and moved to the final Discussion or eliminated.  The strength of this paper is its data on the simplest animals.  Trichoplax contains 4 nuclear receptors, in contrast to C. elegans and Drosophila which are more complex and not as ancient.

4. In 2008, I published a paper in BBRC [Trichoplax, the simplest known animal, contains an estrogen-related receptor but no estrogen receptor: Implications for estrogen receptor evolution 375, 623-627 (2008)] analyzing ERR in Trichoplax.  As part of my analysis I did BLAST searches that showed that Trichoplax contained orthologs of RXR, HNF4 and COUP.  Like Novotney et al, my BLAST searches found that Trichoplax RXR had very strong [unexpected] sequence similarity to human RXR.

5. The strong conservation of Trichoplax RXR ortholog and mammalian RXR deserves some comment.  When I was studying ERR in Trichoplax, I was surprised at the strong similarity between Trichoplax RXR and human RXR.  The BLAST scores were off the charts.  In vertebrates, RXR functions as a heterodimer with several nuclear receptors.  The requirement to bind several diverse nuclear receptors would be expected to constrain the RXR sequence.  RXR forms heterodimers with Ecdysone receptor.  You also cite evidence that jellyfish RXR forms heterodimers with TR.  This indicates that heterodimers of RXR evolved early in metazoan evolution, which could explain the constraints on sequence and the strong similarity of Trichoplax RXR and human RXR.  At least that is one simple explanation.
Moreover, I think there should be a comment about RXR acting as a homodimer instead of a heterodimer, with a transition to heterodimer in the evolution of more complex metazoans.  This also implies that functioning of RXR in heterodimers of nuclear receptors was an important event in constraining sequence changes in RXR.  Sometime in the future it would be worth determining if Trichoplax RXR forms heterodimers with human TR.

6. Line 95 mentions that an ancestral NR possessing gene regulatory capacity, may have been an unliganded molecular regulator.  This is, to me, a reasonable possibility.  Do the authors have a reference to this hypothesis?

7. Line 99.  Is this a jellyfish?  I would like a little more description of the gene and what was found.

8. Line 101 replace homology with identity.

9. Line 38, 125 and numerous other places.  I think that orthologs are more accurate than homologs.  To me, two genes are orthologs if they are descended from the same gene by speciation.  Usually they have the same function, although I think it likely that that some orthologs may acquire a new function over time.  As I understand it, the main point of this manuscript is that Trichoplax has a gene that has the same ligand binding activity at human RXR, as well as strong sequence identity.  Thus, Novotny et al are proposing that the Trichoplax gene is an ortholog of vertebrate RXR.  Also consider replacing homolog with ortholog in other places in the manuscript.


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: TRICHOPLAX ADHAERENS REVEALS A NETWORK OF NUCLEAR RECEPTORS SENSITIVE TO 9-CIS-RETINOIC ACID AT THE BASE OF METAZOAN EVOLUTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: Good.

The title should be changed as the data does not show an "endocrine-like network". It shows potential components of a potential network but there is no proof of connectivity and no proof of endocrine-likle function.
Experimental design: Good
Validity of the findings: Good
Additional comments: Review of “Trichoplax adhaerens reveals an endocrine-like network sensitive to 9-cis-retinoic acid at the base of metazoan evolution (#6219)”

Thanks to the authors for submitting this paper. The English is excellent and concise, the figures and headings are clear, and the subject interesting. I suggest the paper in accepted for publication with some minor changes. I have attached a copy of the PDF with changes in notes on the document but these are also detailed below. My main criticism of the manuscript is that parts of the discussion (and some of the results section) try to push potential ideas and suggestions of function a little too far. As such some of the conclusions should be softened and some of the rhetoric about potential function should be removed.

In addition the title must be changed to reflect the comments made in the conclusions (the data does not show an "endocrine-like network". It shows potential components of a potential network but there is no proof of connectivity and no proof of endocrine-likle function.)

See pdf for accurate corrections…
L 104 -  NR3 should be NR3B (There are probably better refs but this is clear in Vogeler 2014; DOI: 10.1186/1471-2164-15-369)
L137 -  correction (using becomes implemented in)
L139 – please clarify number of animals in each extraction
L168 – I think g. Not xg.
L209 – GPS coordinates have too many decimal places.
L283 – Delete “a high”
L294 - These two lists are the same.
L308 - With Nuclear receptors I do not find these whole gene identities particularly informative. I would much prefer to see Identity figures for each of the LBD and DBD alongside the whole gene identity, as these give much better idea of functional variability between species.
L383 – replace “more affecting” with “influencing”
L384 – insert more “… more than”
L400 - insert known “arrangement known with”
L402 – Dispose? Do you mean display? Or perhaps "...are disposed with..."
Comments
L423 - I agree but I suppose it depends on the assumed phylogenetic position of the phylum Placozoa (as mentioned above).
L438-L458 The evidence you have presented is convincing. However (and you have eluded to this), it is still not proof enough and there is a substantial link missing here in cause and effect relationship.

I think you need to soften your stance, clarify exactly what is proven and perhaps suggest what needs to be done experimentally to prove the signalling cascade you are hypothesizing.

To clarify...

Proven...
RXR binds 9-cis-RA.
9-cis-RA exposure increases expression of metabolic gene.

Hypothesis (not proven)...
9-cis-RA binds RXR and induces expression of metabolic gene.

L460 - This title should be changed in line with comment below (L479) (The data only shows a connection in expression levels upon treatment. It does not demonstrate a regulatory connection or formation of a network)

L479-481 - I find this a stretch. The data only shows a connection in expression levels upon treatment. It does not demonstrate a regulatory connection.

L507-L510 - The hormone story is perhaps a bit of a stretch (a hormone is a signalling molecule produced by the animal itself; food can mimic a hormone but it isn't a hormone).  A more likely hypothesis would be that certain food contains essential nutrients at levels that allow T.a. to grow more efficiently. This is the case for almost all algal feeding inverts and is well characterised in commercial bivalve species.

L511-512 This is a really nice (and probably the most important) finding.

L512-L516 This is the logical next step but the work in this paper does not go this far. I think you should soften the rhetoric in the second half of this paragraph. Or simply even delete it.

L529 - What endocrine-like network? There is no endocrine like network proven. Can you write this instead "In conclusion, the presence of functional NRs in T. adhaerens supports ..."


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: TRICHOPLAX ADHAERENS REVEALS A NETWORK OF NUCLEAR RECEPTORS SENSITIVE TO 9-CIS-RETINOIC ACID AT THE BASE OF METAZOAN EVOLUTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: -
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: TRICHOPLAX ADHAERENS REVEALS A NETWORK OF NUCLEAR RECEPTORS SENSITIVE TO 9-CIS-RETINOIC ACID AT THE BASE OF METAZOAN EVOLUTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This is a much improved manuscript, starting with the new title.  They have made numerous edits in response to the other reviewers, and this improves the manuscript.   Although the authors decided not to do additional experiments that I suggested, the data and conclusions are sufficient to merit publication; that is, I think this is a useful contribution to the literature.  It is a long manuscript covering both molecular studies and feeding studies.  This may influence the readership.

I hope there will be follow-up studies of experiments suggested in my original review.


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

Input:
Title: MULTI-SCALE IMMUNOEPIDEMIOLOGICAL MODELING OF WITHIN-HOST AND BETWEEN-HOST HIV DYNAMICS: SYSTEMATIC REVIEW OF MATHEMATICAL MODELS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clearly written. Manuscript's structure is appropriate.
Experimental design: Goal and justification of this study, as a systematic review, are clearly stated. Methodology of systematic review is well presented.
Validity of the findings: Conclusions are well stated.
Additional comments: 1.	Assuming viral shedding as part of the basic viral dynamics model (line128, page 7; Table 2) is uncommon. A more representative example is found in Perelson 2002, Modelling viral and immune system dynamics, Nature Reviews.
2.	Do not use "et al." when there are only two authors; e.g. "Martcheva and Li" instead of "Martcheva et al" (line 134, page 8)—also in lines 140,153, 181, 220, 222, 238.
3.	Adding a formulation of model's summary for within host scale models for "HIV evolution" (lines144-149) and "HIV and therapeutic interfering particles" (lines 158-164) would improve the introduction of models (similar to Tables 2, 3, etc.).
4.	Note that Saenz & Bonhoeffer (2013) also consider acute, latent and late stages of HIV infection, so this should be mentioned in the corresponding section (lines 177-185).
5.	In section "HIV transmission rate as a function of viral load" (lines 212-233), mention the different types of functions that are used (e.g., linear, Hill's).
6.	A better reference for the sentence "The viral load (and thus the transmission rate) is high during the acute and late stages of HIV infection while being low during the latent stage" (lines 214-216) is: Hollingsworth et al. 2008, HIV-1 transmission by stage of infection, Journal of infectious diseases.
7.	A few typos in References list (e.g., "hiv" in line 376).
8.	Models diagrams (Tables 2, 3, 4, 5, 6, 7) should include state variables when stating rates, otherwise they are inconsistent (e.g. "k" is not a rate in the same sense as "d").
9.	The reproduction rate of T related to Tm is missing in the model diagram of Table 5.
10.	Note that not all studies considered in the manuscript used the function r*V(t) as implied in Table 8.


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: MULTI-SCALE IMMUNOEPIDEMIOLOGICAL MODELING OF WITHIN-HOST AND BETWEEN-HOST HIV DYNAMICS: SYSTEMATIC REVIEW OF MATHEMATICAL MODELS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: The manuscript is well organized. Thank authors for writing the manuscript carefully.

1. The manuscript is a review article. The review was done in a systematic way as described in lines 102 and in Figure 2.
2. The authors reviewed 9 articles selected from 89 found in the PubMed database. The inclusion and exclusion criteria were clearly defined in the METHODS section (line #98).
3. The authors summarized the results found in those selected papers and stated in the RESULTS section starting at line # 109. One of the key findings is the within host viral load that enhances between host infection. They also highlighted the impact of super infection, antiretroviral therapy, drug resistance, treatment at early or late stages on HIV infection and prevalence.
4. The authors though saw the increased complexity of the multi-scale modeling, but found significant public health impact of these models.
5. This review should be helpful for further studies to eliminate the long time persisted disease HIV-AIDS.

With regards to your concern “regarding the assessment of the experimental design or the validity of the findings were provided”- The authors of the manuscript did not conduct any experiments. So, there was no issue of validation of findings. However, they have followed a systematic review process that I mentioned in my opinion (1,2) above.

Based on my findings I have no objection to accept this manuscript for publication.


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

Input:
Title: MULTI-SCALE IMMUNOEPIDEMIOLOGICAL MODELING OF WITHIN-HOST AND BETWEEN-HOST HIV DYNAMICS: SYSTEMATIC REVIEW OF MATHEMATICAL MODELS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors are making a systematic review of the use of immunoepidemiological mathematical models of HIV. Overall, they found 9 articles where such models were used. Although the paper contains interesting pieces of information, the basic reporting would require modifications. A clear definition of the objectives is likely the most important issue, followed by a need for synthesis and better cohesion between the elements of this paper.
Without a clear understanding of the review’s objectives, it was difficult to assess both the relevance and adequacy of the results. I assumed that the authors meant to review how, in the mathematical models, the within-host evolution of HIV was considered to affect the transmission of the disease at the population level. To that regard, it was difficult to find this information in the paper. In terms of cohesion, working on the link between the pieces of information that are reported would help, as the sentences sometimes gave the impression to be “floating”, like bullet points. A clearer definition of the objectives would greatly help in that regard, as the information could be linked back to these objectives throughout the paper and hence give a better sense of direction to the reader. The authors could also consider discarding some of the information that is given, depending on these objectives.

In terms of the context, the authors should consider elaborating on the clinical relevance of immunoepidemiological models. Maybe a good way would be to describre models that consider, separately, the within-host or the between-host HIV dynamics, especially the questions they were able to answer but mostly those that cannot be answered because they are not considered altogether. Finding more precise and clinical research questions relative to what is written in lines 77-80 could help improve motivation. Also, is it the first time immunoepidemiological models are reviewed in the context of HIV?

As for the Results section, is it possible that some sentences from the Results were statement of facts or data-driven conclusions (e.g. lines 133-134, 159-160, 178-180, 187, 191-192), rather than model elements of the reviewed paper? If this is the case, these sentences would be a better fit in the Introduction. I would avoid dividing the results in very short sections and work on synthesizing the information. Some statements were ambiguous, e.g. sentences like “… can be modeled similar to co-infection (Metzger et al. 2011)… (line 161)” left me to wonder if the referenced authors are the ones stating that it could be modeled, or that they actually included this element in their model and, if so, if they were the only one considering TIPs. I believe important that the authors identify these sentences and make more precise statements. I suggest systematically reporting the frequency of papers having considered which specific model element and why they have done so. Maybe a table with one line per model and check marks for elements considered (columns) could help?

Table 1 contained the most interesting information. I suggest elaborating more on the important aspects of it. Consider discarding Figure 1, Tables 2 to 9, which did not bring much to the reading, unless they can be related to an objective. Table 9 could help with motivation (see above comment), but I would also add why it is important to assess these questions.
Experimental design: The general methodology seems adequate for a systematic review. However, I was wondering if the authors considered using MeSH terms, from PubMED; this is a great tool to find all terms related to concepts. Also, I suggest looking at other databases, as mathematical models can be underrepresented in PubMED.
Validity of the findings: I was unable to relate the discussion to the objectives, for the issues stated above.
Additional comments: No further 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: MULTI-SCALE IMMUNOEPIDEMIOLOGICAL MODELING OF WITHIN-HOST AND BETWEEN-HOST HIV DYNAMICS: SYSTEMATIC REVIEW OF MATHEMATICAL MODELS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: All my previous concerns/comments have been satisfactorily answered. I have no further comments.


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

Input:
Title: MULTI-SCALE IMMUNOEPIDEMIOLOGICAL MODELING OF WITHIN-HOST AND BETWEEN-HOST HIV DYNAMICS: SYSTEMATIC REVIEW OF MATHEMATICAL MODELS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors made modifications which improved my understanding of the nature of the reported results. Some of my concerns remain. My main concern pertained to the clarity of the stated objectives (see below for details). To this end, I understand that no modification was made by the authors. Consequently, my troubles relating the reported results to the objectives remain. Of relatively lesser importance is the text’s lack of direction. A lot of information is given and it would help a lot if the authors explained, before the Results section, how they chose which information to report and the purpose of reporting this information. I also had a bit of difficulties finding the pieces of information which I thought most relevant to the paper.

I leave it to the editor to decide if the latter issues are important enough to be tackled.

Objective
To be more precise, I struggled understanding the last part of this sentence: “The objective of this study is to conduct a systematic review of multi-scale HIV immunoepidemiological models to infer the synergistic dynamics of HIV prognoses at the individual level and the transmission dynamics at the population level.” Is this what the authors meant: “The objective of this study is to conduct a systematic review of multi-scale HIV immunoepidemiological models to better understand how the evolution of the virus within the hosts could impact the transmission of the disease between individuals, and vice-versa.” ?

Please clarify in the text.

Results
Most of the Results section focuses on technical aspects of the modeling approach. What is the general purpose of presenting this information? Please provide a rationale in the text. In my mind and if I understand the objectives, the authors wished to inform the synergy between within- and between-host dynamics. If this is the case, then model inferences can inform this synergy (Table 1), not the modeling approach. The modeling approach, on the other hand, informs the reader of the validity of model inferences (see suggestion below for details). Although it is quite possible that this was the authors’ intent in presenting the modeling approach, how the results are presented does not help the reader make the appropriate assessment of inferences.

Accordingly, here are some modification suggestions:

-	Most models have a great number of underlying assumptions (e.g. homogeneous populations, deterministic/stochastic nature of a relationship, etc.). Please mention why the specific model assumptions were chosen to be reported in the objectives. As an example, you could write: “Inferences from mathematical models are sensitive to model assumptions. To enable a better assessment of the validity of inferences, we will report the model assumptions that we believe had a larger impact on model predictions.”

-	In the Results section, it would be advisable not to separate data-driven facts, model assumptions and model inferences, and make it clear which is which. As an example based on lines 275-279 (this is not meant to be accurate!): “There is evidence suggesting that deploying therapeutic interfering particles in even a small proportion of infected individuals reduces the prevalence of HIV to low levels. (ref=longitudinal study) This is thought to be due to TIPs’ ability to transmit between hosts and and target high-risk groups. (ref=source of this opinion) In one immunoepidemiological model, this was modelled by assuming that the susceptible population infected by TIP carriers become TIP carriers themselves, and those have one-half the transmission rate of a non-TIP carrier.(ref=model paper) This assumption was supported by clinical data. Results from analyzing this model suggest that TIP may reduce the challenges of ART therapy and vaccines, and can be complementary to both.(ref=model paper) This is due to [particular aspect of the model dynamics] and adds to the current knowledge ...”

-	If I understood your objectives correctly, I suggest describing the most important information relative to your objectives in the text rather than in a table (e.g. Table 1). I would put less relevant technical aspects of the modeling approach in Appendix.
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: AN EXPLORATION OF STRATEGIES USED BY DRESSAGE HORSES TO CONTROL MOMENTS AROUND THE CENTER OF MASS WHEN PERFORMING PASSAGE
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment in general, please add abbreviations for table in legend
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This paper studies the biomechanical variables which influence pitching moments around the COM in passage in elite dressage horses. It uses comprehensive recording and analysis to understand the background for the high biomechanical skills of three elite dressage horses.
There are two points regarding the study object and presentation of the results which should be considered:
1.	“balance control”: In the abstract, introduction and discussion the term balance or balance control, balance management is used. You also defined this term in the abstract as the ability to control nose up/nose down pitching moments. This might be a quite limited part of the general locomotor balance of the horse. To my knowledge the FEI does not exactly define this term “balance”. While this study focusses on the sagittal-plane motion, the transversal plane movements might be of even more importance especially during slow gaits. As balance might mean generally “a state of equilibrium“ and the capacity to maintain postural stability, it might be nice to discuss these aspects in your discussion in more detail.
2.	Results: Table 1: Please provide the explanations for the abbreviations in the table legend.


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: AN EXPLORATION OF STRATEGIES USED BY DRESSAGE HORSES TO CONTROL MOMENTS AROUND THE CENTER OF MASS WHEN PERFORMING PASSAGE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please see the annotated PDF for minor comments on phrasing.

I find there are limited references within the article, particularly in the introduction. It is not mentioned that this topic is understudied, so I am surprised that there are not more citations. I don’t believe in including irrelevant citations for the sake of it, but some mention of limited references, or inclusion of more support for discussions of trot and pitching moments in other movements may be beneficial. This would also help to demonstrate how this research fits with the broader field.

It would be more logical to define pitching moments in the introduction, as it is an important aspect of the study, as opposed to the discussion (Lines 219-221).

The figures are clear and appropriate to the article; however, I’m not sure why they are subplots of the same figure. It would be clearer to have two distinct figures, and these should also be referred to specifically in the text. Figure 1 is referred to, but not 1A or 1B.
Experimental design: There are minor phrasing comments within the annotated PDF to clarify the Materials and Methods section, but overall it contains sufficient detail.

The research fits within the Aims and Scope of the journal as stated on the website, and the research question is fairly well defined. The statement “some dressage horses fail to reach the highest levels…” (Lines 65-67) is interesting and a nice rationale for the study, but is buried within the introduction.

The authors use the term “elite” as well as “highly-trained” dressage horses. I think there needs to be a clear definition of these terms. The use of only three horses seems very small for “highly-trained” dressage horses, but it more acceptable if “elite” is referring only to Olympic competitors. There should also be acknowledgement of the small sample size before the end of the discussion, which could be accomplished by defining the criteria for inclusion.
Validity of the findings: I believe the methods and analysis are sound, and the results presented are reliable. I particularly like the supplementary video.

The discussion and conclusions are appropriate to the results of this study, but there are aspects of the discussion that I think could be improved.

The first sentence of the discussion states that dressage “…involves maintaining the trunk in an uphill (nose-up) orientation” (Line 201). This is a key factor of the pitching moments discussed in the paper, but is not mentioned before the discussion. This should be brought to the attention of the reader in the introduction, and would also assist with identifying the purpose of the study.

The manuscript overall is focused on healthy horses performing a difficult movement; the mention of disease (Line 211) seems off topic and unnecessary. I think it should be removed.

I don’t think it’s necessary to mention that there are no significant differences between breeds when all the individuals were the same breed (lines 262-265).

The final sentence of the conclusions refers back to lines 65-67, which I have already stated is where I believe the key rationale for this study to be. But, as it’s not mentioned within the discussion, this last sentence seems oddly placed. I recommend highlighting this more within the introduction and discussion.

The discussion overall could be brought together more effectively. It reads a little disjointed, each result is discussed separately, rather than passage overall. There could be greater comparison between passage and trot, as this is briefly mentioned but not clearly compared. For example, lines 227-229, the comparison here is not clear (I’m not sure what “controlled by manipulating both […] GRF moments” means in relation to the trot).

There is also little mention of the three balance strategies that are highlighted earlier in the manuscript. They are included in Table 1, but then not discussed. This would be a good way to bring together all the information from the discussion to an overall mechanism for balance in the passage.
Additional comments: Overall, I think this is a well written, PeerJ appropriate manuscript. The methods are ethical and appropriate, the data is sound, and raw data is provided as supplementary information. There are areas that could be improved to demonstrate the research question that this manuscript is aiming to address.


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: SLOWER EEG ALPHA GENERATION, SYNCHRONIZATION AND “FLOW”—POSSIBLE BIOMARKERS OF COGNITIVE IMPAIRMENT AND NEUROPATHOLOGY OF MINOR STROKE
Review round: 1
Reviewer: 1

Output:
Basic reporting: It is not immediately obvious in the abstract that stroke patients where tested at both sub-acute and chronic stages. Please add additional detail to make this clear.

The authors have used "ɑAVG" in equations for calculation of both weighted average alpha frequency (line 173) and probability density estimate (line 182). Is this an error?

Groups are represented by different shades of blue in all figures, and to my eyes, the light blue used for the control group is almost too faint to comfortably see. Consider using a different color scheme.
Experimental design: No comment
Validity of the findings: Age comparisons between groups were made for stroke patients in the sub-acute, but not chronic stage. Was the age of stroke patients at follow-up in the chronic stage still matched to controls? Potentially important given well described effects of advanced age on EEG alpha (as mentioned in the Discussion, lines 452–458).

Most significantly for the the coherence measures, it seems likely that volume conduction is a major biasing factor. Also a potential issue is the use of a common reference, in this case, the average of C3 and C4 (for example, see Nunez et al. 1997 Electroencephalogr Clin Neurophysiol 103:499–515; Nolte et al. 2004 Clin Neurophysiol 115:2292–2307). It is recommended the authors consider using analytical methods that limit these confounds (e.g. imaginary coherence).
Additional comments: No further comments.


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

Input:
Title: SLOWER EEG ALPHA GENERATION, SYNCHRONIZATION AND “FLOW”—POSSIBLE BIOMARKERS OF COGNITIVE IMPAIRMENT AND NEUROPATHOLOGY OF MINOR STROKE
Review round: 1
Reviewer: 2

Output:
Basic reporting: LANGUAGE: Mostly good usage of English language throughout. Some errors e.g. “neurophysiological” wrongly used in place of “neuropsychological” (lines 88, 119); “above the overall” (twice, in line 368) should be changed to “overlying the”, or similar; “for the” is missing from “compensate for the lesion” (line 57 & elsewhere). The first sentence of Results in the Abstract (lines 43-46) is too long and complex, and needs to be separated into two sentences (at least).

INTRO, BACKGROUND, CONTEXT & LITERATURE: The Introduction provides a reasonable summary of literature relevant to this study, although with some exceptions and some shortcomings. In particular, the chief focus of this study is on EEG alpha activity. However the authors state in the Intro that “alpha and theta frequency could be of interest for the screening of post-stroke cognitive impairment” (line 116). Why then is theta activity not analyzed in more detail in this study? In addition the Schleiger et al (2014) study is cited, but the authors overlook the finding from that study that delta/alpha ratio (DAR) was found to correlate with post-stroke cognitive outcomes. Why was DAR not analyzed in this study? The authors also overlook that the Schleiger et al (2014) found alpha power from all electrodes (not frontal or posterior specifically) to correlate with post-stroke cognitive outcomes – but it seems that this study only analyzed frontal or posterior electrodes. The Intro and manuscript also is lacking a citation of Schleiger et al (2017) which reports findings that are more recent and relevant to this manuscript: including re peak alpha frequency and slowing of alpha after stroke. So more justification is needed in the Intro for the authors’ choice of EEG parameters. Or – more parameters (e.g. DAR, or lower-frequency / slower alpha range) could be analysed.
Line 83: post-stroke cognitive impairments are not always, and not only, consequences of frontal “impairments”/damage specifically. For example, damage to other brain regions – e.g. medial temporal, parietal cortex – can also results in cognitive impairments.

STRUCTURE:  appears appropriate.
FIGURES: appear appropriate; possibly too many (7) figures?
RAW DATA: I’m not aware as to whether or not the raw EEG data are available?
Experimental design: SCOPE: appears to be within journal scope.

RESEARCH QUESTION: I think the question and primary aim is not very well defined or identified. We have stroke and control samples. From the stroke sample we have two assessment times – mean 9.7 days and mean 13.7 months after stroke. Was EEG recorded at both times? This is ambiguous (line 151). Stroke symptoms (by NIHSScale) and cognitive function (by MoCAssessment) were assessed at both times. It remains unclear as to whether the focus is on: controls v stroke? Longitudinal changes in stroke EEG? Correlations between (early) stroke EEG and outcomes (MoCA / NIHSS?) from stroke? Instead there seems an over-emphasis on EEG analyses and parameters. I believe the authors need to more clearly identify and state what is their primary research question and aim; also what is the secondary (and perhaps tertiary) question of aim.

STANDARDS OF INVESTIGATION: No issues (apart from lack of focus of primary question and aim).

METHODS DETAIL: Generally adequate, although a few missing details. Was EEG recorded at both assessment times? - this is ambiguous (line 151). Was (C3+C4)/2 the online, or offline, reference technique (or both)? In any case – this is unusual. Common average reference is mentioned in a later section (line 202), but was this applied for ALL EEG analyses and parameters? Was it average of all 19 electrodes used, or average of only the 8 electrodes analyzed? Were ONLY the 4 frontal and 4 posterior electrodes analysed (line 164)? – the authors say “particularly” but I think they mean “only”? Was relative power calculated relative to 1.1 – 50 Hz power, or other range (line 168-9)? For each frequency band was power summed or averaged? What was the frequency resolution of the FFT output?
Validity of the findings: Results seem inconclusive, mainly because they are from a small, specific sample of stroke patients: 9 patients with Right middle cerebral artery (RMCA) stroke, who had minor strokes (NIHSS < 5). This is acceptable for this journal, but this disclaimer needs to be stated. The “benefit”, or implications, for the literature could be proposed. For example – these findings from a (small) sample of RMCA patients, could not be extrapolated to LEFT MCA cases (even if the RMCA sample size was large).

DATA: the main issue is the small and specific nature of the sample (see above).

CONCLUSIONS: Need to be tempered (i.e. disclaimer added) as appropriate given that small, specific sample of stroke patients  - 9 patients with Right middle cerebral artery (RMCA) stroke, who had minor strokes (NIHSS < 5) – were studied. Some conclusions or interpretations seems to general or perhaps speculative – e.g. re “alpha flow” (lines 375-7; 471; 59) and lines 449-451. Some speculation is ok (see below) but this should be presented as speculative proposal(s) rather than “firm, conclusive or final”.
Discussion paragraph (lines 452-8) seems quite irrelevant and could be deleted (or shortened).

SPECULATION: The conclusions and claims of the authors, as they stand, need disclaimers or caveats (as recommended above) and thus should be considered relatively speculative at this early stage.
Additional comments: I encourage the authors to revise the manuscript as recommended - this will result in an improved version of what is a quite interesting study and manuscript.


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: SLOWER EEG ALPHA GENERATION, SYNCHRONIZATION AND “FLOW”—POSSIBLE BIOMARKERS OF COGNITIVE IMPAIRMENT AND NEUROPATHOLOGY OF MINOR STROKE
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: With regard to volume conduction and use of a common reference biasing coherence measures: I am willing to accept that the findings of this study are still valid without altering the analyses. Nevertheless, the potential influence of these confounds is still a limitation that needs to at least be acknowledged in the Discussion.
Additional comments: Nil


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: SLOWER EEG ALPHA GENERATION, SYNCHRONIZATION AND “FLOW”—POSSIBLE BIOMARKERS OF COGNITIVE IMPAIRMENT AND NEUROPATHOLOGY OF MINOR STROKE
Review round: 2
Reviewer: 2

Output:
Basic reporting: Reporting is generally satisfactory. I recommend just two improvements:
[1] The second-last sentence of the Discussion (lines 499-504, tracked version) is simply too long, and thus it also becomes potentially confusing. Please break this down into at least two sentences.
[2] The revised Aim at end of Intro (lines 141-145) is improved, however it remains too long and thus, the aims seem to diffuse or lacking adequate focused. Please deconstruct this into at least two sentences - the first should contain the principal/ primary aim, and the second sentence can state secondary/ supplementary aims.
Experimental design: No further comments.
Validity of the findings: No further comments.
Additional comments: I feel the authors have done a reasonable job in revising their manuscript in response to comments from myself and the other reviewer. If the two very long sentences (at end of Intro and end of Discussion) can be revised and improved (following my comments above) i believe this manuscript will then be suitable for publication. I wish the authors well in their future research in this field.


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: PREDICTING STIMULATION-DEPENDENT ENHANCER-PROMOTER INTERACTIONS FROM CHIP-SEQ TIME COURSE DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: I think that in general the article is well written, in clear professional English. Literature is in general cited sufficiently (with one exception described below). However, some details are missing and especially the figures need improvement.

Major points:

My main point of criticism here is about the quality of the figures and the lack of explanation about what they are showing. The reader basically has to make too many assumptions to be able to interpret them with confidence.

- Fig. 1: what are the ranges shown? Center of the cluster +/- SD? What are the numbers above each plot? The number of regions in the cluster? What is the Y axis? Log fold change? Why are 12 clusters shown in each subplot? Another problem is that many clusters seem practically indistinguishable from each other (see for example the four top plots in Fig. 1a). Should these really be considered as different clusters? They might be merged, I feel.
- For Fig. 2e, people will wonder why the density is 0 for the smaller distances. I guess this is because of the way intergenic enhancers were defined here. It would be better to indicate this in the figure; for example: mark the region (+/-300bps?) where enhancers would be filtered out.
- For Fig. 3: the colors in Fig. 3a are not clear. Adding gene symbols to the genes might help the understanding. Fig. 3b-g: Adding some distance between the training and test panels would make things more intuitive. An explanation is needed about what “PolII”, “ER”, etc on top of each plot means, as well as what “NB” stands for, and what is the meaning of “distance” and “data”. Please also explain what the error bar means. At present, all of this has to be guessed by the reader. There appears to be a “3” in Fig. 3f, third panel, which should be removed. Finally, at present 5 panels are shown for training data, and only 1 for test data. It would make more sense to show performance on the test data in more detail, rather than on the training data, as this performance gives a better idea of the general performance on new data.

Minor points:

- In November 2016, several papers (see Cell Volume 167 Issue 5, including Spurrell et al., Cell, 2016; Javierre et al., Cell, 2016; etc) were published by the BLUEPRINT Consortium/ International Human Epigenome Consortium (IHEC) describing analysis of promoter-enhancer interactions using promoter capture Hi-C in multiple cell types and conditions. I think a reference to these studies and their expected impact in the future should be added to the Introduction.
- I think that no reference was given for MACS. This should be added.
- p4 line 157. “The method is similar to k-means but can achieve much better optimisation of the k-means objective function than the standard EM algorithm.” As far as I understand k-means and the EM algorithm are two different things, and k-means does not use EM. I would recommend removing this sentence, or providing a citation that supports this statement.
- P8 line 320: what are “0.66, 0.47, and 0.35”? Is this correlation? A more clear explanation is necessary.
Experimental design: I think that the paper described original research, with a well defined and clear problem and aim. The computational methodology is described in sufficient detail and is quite interesting.

However, there are some issues about the newly produced ChIP-seq data (see below).

Major point:
- New ChIP-seq data (ER-alpha, H3K4me3, and H2AZ) was produced, but details about the experimental procedures are completely missing from the Materials and Methods section. How were antibodies obtained? How about sequencing libraries, buffer solutions, concentration, cell counts, etc?

Raw data appears to have been submitted to NCBI GEO, however, the data is still set to private, and no reviewer URL was provided, so I can not comment on the data itself.

Minor points:

- Were control samples produced for the ChIP-seq data? If no, some discussion about how this could influence the downstream analysis would be useful. Differences in chromatin accessibility can result in non-specific peaks which are hard to remove without using control samples. Some ChIP-seq peaks might therefore simply reflect open chromatin structure, and promoter-enhancer correlations could reflect similar changes in chromatin accessibility, rather than purely binding by ER-alpha or Pol2-II.
- In the Materials and Methods section (p5 line 201~) the assumption is made that each enhancers regulates a single gene. I think that this is in practice a reasonable assumption, as it becomes computationally impossible to consider more combinations of enhancer-to-promoter interactions. However, it would seem quite reasonable and doable to add the case where an enhancer does not regulate any of the genes to the model. It would be quite interesting to see what happens if this case (“I(0)_j”?) is included. Does it change the assignments? Will you end up with many enhancers having no apparent target promoter?
Validity of the findings: In my opinion the findings are valid, though they are not so easy to interpret with confidence from the figures (see comments above). Improving the quality of the figures are their explanation will improve this point.
Additional comments: Some additional (brief) discussion of the approach and results might be of interest for people working on related topics.
- Currently you are considering only the correlation between consistent features (for example: ER-alpha at the promoter vs ER-alpha at the enhancer). However, it might be possible that a feature at the promoter is better correlated with a DIFFERENT feature at the enhancer (example: Pol-II at the promoter vs ER-alpha at the enhancer).
- Time-lagged correlation has been used for biological network inference in the past. Here, you are not considering this. Some discussion on this would be nice (e.g. would this be computationally difficult? Would this be biologically relevant here?)
- (Somewhat related to the above) H3K4me3 levels are typically low at enhancers. Leaving out this data seems to not affect accuracy very much. Other modifications (H3K27ac?) might have been more promising. Some discussion could increase the interest of the publication.


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

Input:
Title: PREDICTING STIMULATION-DEPENDENT ENHANCER-PROMOTER INTERACTIONS FROM CHIP-SEQ TIME COURSE DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors have developed a Bayesian classifier model which integrates genomic distance with a correlation of time-course ChIP-seq data to predict physical enhancers-promoter interactions.
The article is well written, and includes sufficient references in introduction to demonstrate the research goal. The methods are clearly described.
However, the legend of each figure should be written more carefully. For example, the definition of the shaded regions indicate in Fig 1, the error bars in Fig 3, and the number of data points in each panel in Fig 3, should be explained in legend.
Please refer MACS in the references (line 127).
Experimental design: The proposed model is evaluated using various data including ChIA-PET, Hi-C and GRO-seq data, while the scope of investigation performed is limited in enhancers-promoter interactions of stimulation-dependent genes for estrogen in MCF7 cells.
(Minor comment) Because MACS version 1.4.2 is outdated, the authors should use MACS2 for peak-calling, while the conclusion of the article may hold.
Validity of the findings: The conclusion of the article is robust in the data used for evaluation. However, the article focuses the interactions between promoters and intergenic enhancers only, while there are quite a few “genic” enhancers in the genome. Enhancer-enhancer interactions also exist. The discussion is necessary about how the proposed model works for these kinds of interaction. Furthermore, because this method can capture stimulation-dependent genes only, it should be estimated what percentage of interactions were captured by this model against all interactions captured by ChIA-PET assay.
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: PREDICTING STIMULATION-DEPENDENT ENHANCER-PROMOTER INTERACTIONS FROM CHIP-SEQ TIME COURSE DATA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Complete lists of predictions should be provided as Suppl Tables.
Some texts in the figures are very small to read.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Dzida et al. developed a method to predict enhancer-promoter interactions from ChIP-seq time course data. They used ChIP-seq data of ERalpha, Pol II, H2AZ, and H3K4me3 at eight different time points after E2 stimulation in MCF7 cells, and showed, by comparing with GRO-seq for validation, that the method is able to successfully predict distally regulated target genes. In short, I find the use of time course data of several ChIP-seq experiments significantly improve the prediction of enhancer-promoter interactions in genes regulated by ERalpha. However, there are some issues the authors should address in this manuscript.

1. Pg 8, Fig 1. There is no description about blue and red lines in the figure legend. And also, why are there 12 graphs in each panel? What are the numbers at the top of each graph (I guess these are number of instances, but they are not explained in the figure legend.)? Please elaborate more on these points.
2. Pg 8, Line 313. The authors says “..., there is a substantial overlap in the positive and background distance densities due to a large separation…” (in Fig 2e). I don’t see the substantial overlap in this graph. Please clarify or explain in more detail about this. And also, what is the unit for distance (x-axis)? The numbers, especially for the exponential part, are too small to read.
3. Suppl Figs should not be numbered as 1, 2, 3, ..., but as s1, s2, s3, ....
4. Suppl Figs s1, s2, s9, s10, and S11 are not mentioned in the main text.
5. Pg 5, Line 252. I guess ‘TPR’ means ‘True Positive Rate’. Write it out in full the first time.
6. Pg 10, Fig 3a. It is hard to discriminate green and gray lines. What is the difference in thickness of these lines? X-axis must be genomic coordinate, but the unit is not clear in the figure.
7. Pg 10, Fig 3f. What is ‘3’ at the lower right corner of the ‘Pol II, ER’ panel?
8. Suppl Figs s3-6. Why are the chromosomes divided into odd and even ones?
9. Please provide complete lists of predictions as Supplementary Tables (for example, those mentioned in ‘Validation of ER-regulated target gene predictions’ in Pg 11).


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: PREDICTING STIMULATION-DEPENDENT ENHANCER-PROMOTER INTERACTIONS FROM CHIP-SEQ TIME COURSE DATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The issues raised by me (and also by the other reviewers) been addressed by the authors. Especially, the clarity of the figures has been strongly improved.
Experimental design: The authors have added a detailed description of the experimental procedures of he ChIP-seq data. I think that the manuscript is acceptable for publication now.
Validity of the findings: Thanks to the improved quality of the figures and explanation of the methods, the validity of the findings can be more objectively judged. The findings of the paper are valid and suitable for publication.
Additional comments: I appreciate the efforts made in improving the quality of the manuscripts. The paper us suitable for publication now.


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: PREDICTING STIMULATION-DEPENDENT ENHANCER-PROMOTER INTERACTIONS FROM CHIP-SEQ TIME COURSE DATA
Review round: 2
Reviewer: 2

Output:
Basic reporting: In the revised manuscript, the authors have added new experiments and explanations, and some of my concerns were satisfied, but the authors should add the discussion about the points below for publication of this manuscript. I agree with the authors that "complementary approaches to infer enhancer-promoter interactions by exploiting readily available sources of genomic data, such as ChIP-Seq and RNA-Seq data, are of interest." However, the advantages and the limitations of the proposed method against chromosome conformation capture techniques are not clearly discussed.
Experimental design: No comment
Validity of the findings: ・The line 445-447 “Our model can therefore serve as a complementary approach to chromosome conformation capture techniques and offers insight into context-specific, and cell-type specific transcriptional regulation.” This method can only capture the interactions between the promoter and the intergenic enhancer for stimulation-dependent genes, while ChIA-PET and Capture Hi-C can obtain all types of interactions. Moreover, this method used ten time points for each TF, resulting in the requirement of many ChIP-seq samples, which is not cost-effective and is also technically challenging. Such limitation should be discussed in Discussion section.
・The discussion about the minimum time points required the proposed model is also necessary.
・The authors’ comment “We also limited attention to enhancers outside of gene regions. This was mainly due to the nature of our data, since we are using Pol-II as one of our features and this mark is found across the whole body of transcribing genes.”  Is this a limitation of the proposed method, or the data used (Pol2)? If there is a dataset of other TF, the genic enhancers can be investigated?
・The definition of “context specific enhancer-promoter interactions” in the title and the abstract is ambiguous, and no explanation in the manuscript. If this means “stimulation-dependent enhancer-promoter interactions”, the authors should replace it.
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: PREDICTING STIMULATION-DEPENDENT ENHANCER-PROMOTER INTERACTIONS FROM CHIP-SEQ TIME COURSE DATA
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the revised manuscript, the authors properly responded to all the points raised in the initial round of the review. I don't have any further concerns, and would like to recommend its publication in PeerJ.


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

Input:
Title: ADVANCES IN THE STUDY OF NODAVIRUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In overall, the manuscript was written with clear and unambiguous English. There are some points that need to be addressed. The references were sufficient.
Experimental design: This manuscript is a review article.
Validity of the findings: This manuscript is a review article.
Additional comments: Advances in the study of nodaviruses
	This review manuscript described nodaviruses in various aspects including transmission, classification, immune response and diagnosis using molecular and immunological techniques. Overall, the manuscript thoroughly explain in detail; however, there are some points that should be added for completeness. The whole M.S. should be edited by native English speaker.
1.	Line 86: The RGNNV has been shown to infect tilapia (Keawcharoen,J, et al., J. Fish Dis. 38, 49-54 (2015). This information should be added.
2.	Line 293: The monoclonal antibody specific to MrNV was reported (Wangman et al., Dis. Aquat. Org. 2012, 98(2):121-31). The information based on this report should be mentioned.
3.	Line 143: What is a well-established cell line?  The detail is needed.

Minor points
-	The “Mr” in “MrNV” should be italicized throughout the manuscript.
-	Lines 27, 207: use “Histopathology..”
-	Line 75-76: use “…….infected fishes. Neither alpha-nor beta-nodavirus………(NaveenKumar et al., 2013). Another type of nodavirus includes…….
-	Line 78: use “…PvNNV should be categorized…”
-	Line 79: use “…compare with that of both….”
-	Line 165: use comprise of
-	Line 167: use “Both of which ….”
-	Line 213: use “lymphocytes”
-	Line 225: use “…with that of members…”
-	Line 246: italicize “in situ” and use “..nested RT-PCR…”
-	Line 274: use “..higher than that of …”
-	Line 279: use “…lower than that of …”
-	Line 422: change “vertebrate” to “fishes”


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: ADVANCES IN THE STUDY OF NODAVIRUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This review is well-written.
Literature should be implemented.
Experimental design: Not applicable.
Validity of the findings: Not applicable.
Additional comments: The manuscript is a review about Nodavirus. Paper is well written and clear.
Respect to the first statements about recent reviews in the field there are some good reviews not included covering distinct aspects:
Doan QK, Vandeputte M, Chatain B, Morin T, Allal F. Viral encephalopathy and retinopathy in aquaculture: a review. J Fish Dis. 2017 May;40(5):717-742.
Costa JZ, Thompson KD. Understanding the interaction between Betanodavirus and its host for the development of prophylactic measures for viral encephalopathy and retinopathy. Fish Shellfish Immunol. 2016 Jun;53:35-49.
Reshi ML, Su YC, Hong JR. RNA Viruses: ROS-Mediated Cell Death. Int J Cell Biol. 2014;2014:467452.
Hong JR. Betanodavirus: Mitochondrial disruption and necrotic cell death. World J Virol. 2013 Feb 12;2(1):1-5.
Chen YM, Wang TY, Chen TY. Immunity to betanodavirus infections of marine fish. Dev Comp Immunol. 2014 Apr;43(2):174-83.
Chaivisuthangkura P, Longyant S, Sithigorngul P. Immunological-based assays for specific detection of shrimp viruses. World J Virol. 2014 Feb 12;3(1):1-10.
But also other reviews related to viral diseases, immunity, etc. in aquaculture species during the last decade. Thus, the starting point of this review is not adequate.
The topic is certainly interesting and merits further research since nodaviruses are actually producing probably the largest economic loss in the productive sector.
Regarding insect nodaviruses, there is only scarce explanation and this could be either deleted from the paper or reinforced with further information (detection, immunity, vaccines…).
Comments:
-	Line 100. The number of infected fish species is much larger. Please, revise it.
-	Line 104. Starting from this point include the latin name of all animal species in the text.
-	Line 143. The cell line name is lacking.
-	Line 320. This reference in not appropriate for this.
-	Line 340. Classification of Mx as an AMP is not adequate and consistent. This belongs to the type I IFN response rather to the AMP repertoire.
-	Section related to the immunity, both fish and prawns, needs to be improved and better organized. It is not clear whether the cited studies area about gene expression or functional and this is an important issue to be denoted. References in this section need to be further revised and better selected. For example,  important references should be included:
doi: 10.1016/j.fsi.2009.11.008.
doi: 10.1016/j.dci.2012.02.007
doi: 10.1016/j.fsi.2011.03.018


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: ADVANCES IN THE STUDY OF NODAVIRUS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The English is clear and unambiguous.
Experimental design: The review included many updated data.
Validity of the findings: The conclusion was drawn as shown in Fig. 1.
Additional comments: All of the questions were properly responded. I have no further comments.


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

Input:
Title: ADVANCES IN THE STUDY OF NODAVIRUS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The paper is OK.
Experimental design: NA
Validity of the findings: Ok
Additional comments: The manuscript has been improved accordiingly.


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: A PROBE-BASED QRT-PCR METHOD TO PROFILE IMMUNOLOGICAL GENE EXPRESSION IN BLOOD OF CAPTIVE BELUGA WHALES (DELPHINAPTERUS LEUCAS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: 1. Although the present system shows some potentials to evaluate health conditions of captive beluga whales, it is better to combine other clinical pathological analysis results (e.g., complete blood count (CBC), serum chemistry panel) with the qPCR results to validate the system.
2. The limitations of the present study should be discussed. Changes on RNA level don't guaranty protein changes, especially when the gene changes a little.
3. The rationale selecting the 6 specific genes remain unclear. Both interleukin and respective receptor are crucial for downstream signaling pathway activation. Why some interleukin genes were selected, while a specific interleukin receptor was examined?
Additional comments: In the present study, the author investigated several inflammatory genes in captive beluga whales by quantitative RT-PCR. Generally, this is an interesting study and important to whale health monitoring in aquariums. Several other concerns need to be addressed before publication.
1. Animal backgrounds including place, age, sex and health conditions need to be included in a separated table, which is more straightforward to the readers.
2. The term of "A novel universal probe library quantitative reverse transcription polymerase chain reaction" in the title was confusing which needs to be revised. It should be careful to define the library as a universial library given its application area remain to be determined.


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: A PROBE-BASED QRT-PCR METHOD TO PROFILE IMMUNOLOGICAL GENE EXPRESSION IN BLOOD OF CAPTIVE BELUGA WHALES (DELPHINAPTERUS LEUCAS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Line 65, ‘the short half-lives of leukocyte gene transcripts encoding surface and secreted messengers’ can be replaced with ‘the short half-lives of leukocyte biomarkers’.

2. Line 70, ‘mRNA level’ should be plural.

3. Include the full name of cDNA at Line 93 instead of Line 118.

4. Line 124, Cq is the abbreviation of crossing point.

5. Line 134, add ‘the’ before ‘target gene’.

6. Line 152, “NV of IL-12 in sample A-I was 3.03, demonstrating that the transcript level of IL-12 was elevated.” The authors need to say, “NV of IL-12 in sample A-I was 3.03 and that of the median was XXX, demonstrating that the transcript level of IL-12 was elevated.” Make similar changes for other genes.

7. In Figure 1, does the error bars represent standard deviation?
Experimental design: 1. Immunological gene expression changes mainly in leukocytes after exposure, why the authors used whole blood cells instead of leukocytes? The authors need to validate the use of the whole blood cells or to use isolate the leukocytes.

2. There are commercially available ELISA kits to detect these cytokines, and ELISA doesn’t require blood cell separation, cell lysis or qRT-PCR. It is possible that those kits for human or mice cannot detect the cytokines from beluga. There could be one reason why people want use qRT-PCR instead of ELISA. Have the authors tested if the commercial kits work for beluga?

3. While it is clear that the qRT-PCR method is able to measure the expression levels of the genes, it fails to demonstrate its capability to evaluate the health status of the whales.

4. Line 138, ‘Elevated expression levels of IFNγ and decreased levels of TNFα in C-II were suggested as normal responses to social stressors in the environment, as previously observed in a killer whale study.’ How about other whales? How do the authors know they don’t suffer from social stress? Also, the authors took 4 samples from the same whale, why it is seen in only one sample.
Validity of the findings: 1. Did the authors observe normalized value outliners in healthy samples, too? Please explain if there is.

2. The authors observed seven outliners but can only explain four of them, please explain why they thought it was enough. Also, if the elevated IL-12 expression in A-I whale suggested activated immune response, why they failed to observe its elevated expression in other 3 unhealthy samples. Same questions for IFN-γ, IL-2Rα, and TNF-α.

3. The authors used normalized value (NV) in this work. However, this term is not commonly used in qRT-PCR analysis. I suggest using ‘relative expression’. Why the authors define NV as Log2(ETCqT/Geomean (ER1CqR1 & ER2CqR2))?

4. What does ‘threshold value’ in Table 1 mean? How did the authors calculate it? People use the threshold cycle (Ct) in qRT-PCR analysis, but the ‘threshold’ here is clearly not Ct.

5. Line 70, it is real-time PCR but it doesn’t enable real-time gene expression measurement. The assay is off-line.

6. Line 144, why the authors use the median of NVs instead of the mean?
Additional comments: The paper described a method to quantify immunological gene expression in whole blood cells of beluga whales. The authors extracted mRNAs from the blood cells of healthy and unhealthy whales and compared the expression of six relevant genes. The authors attempted to find the correlations between the change of certain gene expression and health conditions. The paper is well organized, and easy to read. Probe-based qRT-PCR, such as Taqman, is a widely used method to evaluate gene expression, so the authors cannot claim it is a novel method. The authors need to avoid the use of 'novel' unless they can prove its novelty. Additionally, the authors need to improve the qRT-PCR analysis part and the discussion is weak.

1. Line 102 and Line 112, add the address information of the manufacturers.

2. Line 112, the authors need to put the data in supporting information.

3. Line 144, ‘Gene transcript levels were used to establish three arbitrary categories: IL-2Rα (highly transcribed; median NV < 5), IL-10 and TNFα (moderately transcribed), and IFNγ, IL-4 and IL-12 (lowly transcribed; (median NV > 8).’ The sentence can be removed.

4. Line 152, “NV of IL-12 in sample A-I was 3.03, demonstrating that the transcript level of IL-12 was elevated.” The authors need to say, “NV of IL-12 in sample A-I was 3.03 and that of the median was XXX, demonstrating that the transcript level of IL-12 was elevated.” Make similar changes for other genes.

5. SYBR Green was once widely used in qRT-PCR, but nowadays more and more people use probe-based Taqman. The Roche’s universal probelibrary is a new method, but it was not first introduced in this work.

6. Line 215, the most commonly used qRT-PCR analysis method is ΔΔCq, not ΔCq.


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: A PROBE-BASED QRT-PCR METHOD TO PROFILE IMMUNOLOGICAL GENE EXPRESSION IN BLOOD OF CAPTIVE BELUGA WHALES (DELPHINAPTERUS LEUCAS)
Review round: 1
Reviewer: 3

Output:
Basic reporting: This was an interesting study on the immunology of captive belugas, providing preliminary data which may improve the health and welfare of captive cetaceans in the future.

Although the authors have done a very good job as (presumably) non-native English speakers with the language of this paper, I would still suggest that they request a native English speaker to proof-read the text throughout and to name this person in the Acknowledgements section, according to PeerJ/ICMJE criteria. Overall, the English is good, however, occasional sentences, such as on lines 33-34, lines 65-68, line 54, etc. could be rewritten to ensure their meaning is clearer.

In my opinion, the title is too long. I do not believe that anyone reading this paper needs the whole PCR procedure “spelling out”, surely this could be shortened by using qRT-PCR or RT-qPCR.

The Introduction provides a good background to the subject. Literature is current and relevant.

Perhaps it would be better to separate the Results and Discussion. This is the section order requested in the Instructions to Authors and it would appear quite straightforward to split this section.

Although Figure 1 is clear and relevant, I am not sure that “note” is the correct name for the symbol used for sample C-I, perhaps this could be changed to a more obvious and more commonly-used symbol to avoid confusion.
Experimental design: The research is within the scope of PeerJ.

Research question: clear. It is stated that immunological data on cetaceans is limited and research is needed.

Ethics: I have concerns about the “voluntary” aspect of the blood collection. While the authors state that international guidelines were followed (and I am sure that this is the case), these are not cited and I have been unable to find any such guidelines on the internet (I am sure they exist, but would like the authors should cite them directly). It is repeatedly stated in the results sections that beluga C showed reluctance to the “voluntary” blood draw and I feel that, when dealing with such sentient animals, this needs to be addressed and further explained by the authors.

M&M: While it is clear from the raw data that 16 blood samples were taken in total (i.e. 4 from each animal), and not 16 from each beluga, this is not so obvious in the M&M section. Suggest rewriting slightly. I would also suggest stating how much blood was taken from each animal, presumably at least 1ml was taken to allow for the exact measurement of the 500 µl EDTA-blood to be preserved.

Primer & probe design: Please state in the text that all gene accession numbers listed in Table 1 are from bottlenose dolphins.

Results: Were any of the results statistically significant? Particularly the differences between the sick and healthy animals. I realise, however, that the low number of samples probably makes a robust statistical analysis impossible.
Validity of the findings: Although 2 reference genes were used, they are not included in the results. Surely, if these genes were the control, they should at least be mentioned and not just included in the raw data.

Conclusions: While I would agree that studies of this kind are important in an attempt to improve the health of captive cetaceans, I would suggest to the authors that they tone down their final conclusions and at least add a statement that their results are based on only two clinically symptomatic beluga and so may not be representative of the general cetacean population.
Additional comments: Throughout:
Shouldn’t RNAlater be written with a trademark symbol?

Materials & Methods:
Line 83: The beluga are in captivity, not in “human care”, which sounds rather strange.
Line 100: Was gDNA contamination confirmed or excluded? Perhaps the absence of contamination was confirmed?
Line 101: This is the first mention of cDNA so I would add the definition (complementary DNA) here rather than on line 118.

Results & Discussion:
Line 146: I would suggest that the opposite of highly transcribed is poorly (not lowly) transcribed.
Lines 150-157: This text appears to repeat what is already shown in Figure 1. If there are any results in particular that the authors would like to highlight then I would do this here, but it doesn’t seem necessary to repeat all the information that is shown in the figure. Perhaps add the reference gene results here?
Line 186: Were any tests for possible viruses carried out on Beluga C? It would be interesting to know what was the cause of the symptoms, plus different pathogens could have different effects on the immune system.
Line 191: Perhaps add here that IL-4 is known to play an important role……….in humans, rather than just stating this as if the reference refers to cetacean research.


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: A PROBE-BASED QRT-PCR METHOD TO PROFILE IMMUNOLOGICAL GENE EXPRESSION IN BLOOD OF CAPTIVE BELUGA WHALES (DELPHINAPTERUS LEUCAS)
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: All concerns have been addressed.


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: A PROBE-BASED QRT-PCR METHOD TO PROFILE IMMUNOLOGICAL GENE EXPRESSION IN BLOOD OF CAPTIVE BELUGA WHALES (DELPHINAPTERUS LEUCAS)
Review round: 2
Reviewer: 2

Output:
Basic reporting: No further comments.
Experimental design: Thank you for adding the reference to animal handling guidelines. Could you please explain your comment in the response letter “When the animal showed reluctance to the voluntary blood draw, the trainers suppose some thing happens”? Perhaps you mean the trainers stopped the blood draw procedure and did some other training activity? It would be good to add this to the Materials and Methods section of the text if this is the case.
The addition of Table 1 is a great help to the readers in understanding when blood samples were taken.
Validity of the findings: No further comments.
Additional comments: Lines 93-94: As the authors explained in their response letter, blood samples are taken from the fluke of the animal. As Beluga C was reported to have vesicles on the fluke perhaps it is important to mention that the area of blood draw may have influenced this animal’s “reluctance”. As such, I would suggest adding the following (or similar) to the blood sample method description “Sixteen blood samples……..were obtained via venipuncture of the fluke on a monthly basis…..”
Line 94: Please change to “Within five minutes of 1-2 mL blood being collected, 500µl…..”
Throughout: While RNAlater has been changed on line 96, ideally it should be corrected throughout the document.
Line 207: As the authors stated in their responses that another research group is currently working on virus investigation, it would be informative for readers to mention here that this research is ongoing
Table 1: please add “(years)” to the Age column
Figure legends: For clarity ,I would add that the outliers refer to clinically symptomatic animals, e.g. “…..blood samples from clinically healthy (n=12) and symptomatic (n=4, outliers) belugas.”


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: THE SPANISH VERSION OF THE FATIGUE ASSESSMENT SCALE: RELIABILITY AND VALIDITY ASSESSMENT IN POSTPARTUM WOMEN
Review round: 1
Reviewer: 1

Output:
Basic reporting: An article whose findings are important to those with closely
related research interests. This manuscript present and discusses the validation of a scale to assess fatigue during the puerperium period. As author explain in the discussion section, this may be an apropiatte scale to be used in different time points besides the early postpartum period and helpful to assess fatigue evolution along all the postpartum

As english is not my (reviewer) mother language , I would suggest an expert english language review.
Experimental design: Reserch question is well designed and methodology used is adequate. Methods are well described and easy to understand.

Hipotesis stated by authors is fulfilled.
Validity of the findings: Analisys is well performed and is confirmatory. However as a suggestion to authors I would reccomend to obviate the exploratory analisis on item #4 and #10. It is already demonstrated and confirmed the saturation of these two items, and this exploratory analys may be considered as redundant.
Additional comments: Manuscript is well written and easy to understand.
Exploratory analysis  for items #4 and #10 may be considered as redundant. Please consider reviewing
This scale may be a good instrument to assess fatigue during postpartum period in Spain, and useful for  international comparisons
Minor comments:
Line 122: authors use the acronym  BSES-SF for the first time, please explain
Line 135: it seems a wording mistake " closet o 0,95" , as i understand it is intended to be " close to 0,95". please confirm or correct


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: THE SPANISH VERSION OF THE FATIGUE ASSESSMENT SCALE: RELIABILITY AND VALIDITY ASSESSMENT IN POSTPARTUM WOMEN
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors have developed a clear and rigorous scientific paper, with clear development and adequate language. The introduction is complete and allows the reader to be correctly placed in the context of the research. The references cited are useful and up-to-date.

Complete graphic resources are provided and raw data is supplied.

I suggest you take note of the following points about your figures and tables:

Figure 1 - Write "Spanish" (as proper noun).
Table 1 - Write "Table 1" (in English) instead of "Tabla 1" (Spanish).
Table 2 - Add "#" symbol at ítem 4 and ítem 10. They can also do the same in the lines 142, 201, 210 and 217.
Table 3 - Review spelling: "standard deviation".
Experimental design: It is an original investigation with a clear purpose, which respects ethical codes, and whose method is simple and easily applicable.
Validity of the findings: The conclusions are clearly related to the research approach. The validity of the results will allow an application of the scale in the daily consultations.
Additional comments: I suggest you take note of the following format and spelling points:

Abstract - Review spaces between numbers, words and fulls stops.
Line 33 - Close quotation marks at the end of the sentence,
Line 40 & 41 - I suggest you to link the expresions "fear and duration of labour" in one sentence.
Line 74, 101 & 104 - Review spaces between words and brackets.
Line 105 - Review spelling "maintain".
Line 110 - Delete accent "version". Delete dead space at the end of the sentence.
Line 111 & 112 - Review agreement between singular/plural in these sentences.
Line 132 - Review spelling "and" ; "comparative".
Line 145 - Review full stop of the sentence.
Line 158 - Tabulate the beginning of the sentences.
Line 159 - Delete innecesary full stop in the middle of the sentence.
Line 162 & 164 - Review dead spaces.
Line 179 - Write "molesta" and "preocupada" in italics (Spanish words)
Line 203 - Full stop at the end of the sentence required.
Line 216 - To maintain uniformity, I suggest you to to write "table 3" according to the other styles you use; "Table 3".
Line 244 - Write "molesta" and "preocupada" instead of "molesto" and "preocupado" (maintain uniformity).
Line 253 - Review space between full stops and words.
Line 270 & 271 - Write "Model 1" and "Model 2" instead of "model 1" and "model 2" (maintain uniformity).