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: SLEEP APNEA DETECTION FROM A SINGLE-LEAD ECG SIGNAL WITH AUTOMATIC FEATURE-EXTRACTION THROUGH A MODIFIED LENET-5 CONVOLUTIONAL NEURAL NETWORK
Review round: 1
Reviewer: 1

Output:
Basic reporting: In their manuscript “Obstructive sleep apnea detection from single-lead 1 ECG signal with automatic feature-extraction through improved LeNet-5 convolutional neural network“, the authors used a standardized dataset (PhysioNet Apnea-ECG dataset) and applied convolutional neural network, a component of artificial intelligence which can automatically learn the underlying rules and features of objects.
- Wording and grammar need to be improved. I propose to get this manuscript proof read by a native speaker.
- If I understand the manuscript correctly, the authors were the first to use CNN in the field of sleep apnea detections. This could be further clarified in the manuscript.
Experimental design: - This is not a specialized artificial intelligence journal. Please explain further how exactly CNN works.
- The authors mention, that OSA episodes were detected. Were just “obstructive” sleep apnea episodes or also “central” and “mixed” apnea episodes detected. I would propose to avoid the use of “OSA” and to use “sleep apnea episodes” instead. Please provide further PSG data: Obstructive vs central apneas. Sleep apnea episode durations…
Validity of the findings: Methodological comments:
- The method is not tested prospectively in an additional dataset, which limits the novelty and the clinical implications.

Statistical comments:
- One limitation is the small dataset to validate/ develop the algorithm.
- Are there statistical/ methodological rules to assess the minimal size of the validation cohort?
Additional comments: The manuscript is interesting and of potential clinical importance. However, there are several points (including revision of wording and grammar) which should be considered in a revised version of the manuscript. The introduction is too long and should be shortened to better focus on the hypothesis and goal of the study.


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

Input:
Title: SLEEP APNEA DETECTION FROM A SINGLE-LEAD ECG SIGNAL WITH AUTOMATIC FEATURE-EXTRACTION THROUGH A MODIFIED LENET-5 CONVOLUTIONAL NEURAL NETWORK
Review round: 1
Reviewer: 2

Output:
Basic reporting: -The article is written in professional English. However, it has some gramatical mistakes that should be amended and/or revised.

-The article includes sufficient background with references. However, the advantages of deep learning (CNN) over traditional approaches could be better explained.

-The article has an appropriate structure, including figures and tables. Raw data is also indicated and code is available. The only suggestion is that the different lines in the code files could contain more text comments in such a way that the readers can understand it better.
Experimental design: -Authors indicate how the research fills an identified knowledge gap. In this sense, they indicate at the end of the introduction that they propose an improved Le-Net with adjacent ECG segments for OSA detection. However, I would indicate explicitly the hypothesis and the goal of the study, since it could enhance the manuscript's readability.

-Methods are in general well described. Nonetheless, I would add a figure/scheme that illustrates the preprocessing stage (lines 110-122).
Validity of the findings: -Data employed in this study come from a well-known database (Apnea-ECG). However, this database has some limitations:
i). There is no differentiation between apneas and hypopneas.
ii).Annotations are made for each 1-min segment. However,in most of the databases annotation files for apneas/hypopneas contain the start and end of the events. Thus, a 1-min segment could contain more than one apnea/hypopnea event, or an apnea/hypopnea event could occur in two different 1-min segments. Thus, the approach of labeling each 1-min segment as apnea or non apnea may not be applicable to other databases. Nonetheless, authors indicate as a future goal the necessity of using another datasets.

-The conclusion stated in the abstract is that their method achieves similar or better resuts in comparison with existing works in the literature. The conclusion is derived from the results. However, I think that this conclusion could be better stated. The conclusion of the study should be that, based on the results obtained, their proposal (a deep learning approach based on a modified LeNet-5 with adjacent ECG segments) is useful for OSA detection.
Additional comments: -Lines 55-57. With respect to the following text“Among them, the ECG signal is the most concerned signal because it can show the physiological characteristics of OSA occurrence and facilitates recording using a wearable device.”  Other signals can also be recorded using a wearable device (pulse rate and blood oxygen saturation). Authors should justify better the use of ECG instead of other signals from polysomnography.

-Authors should indicate the advantages of deep learning methods (such as CNN) over traditional approaches around line 76.

-Authors state that use an improved Le-Net in the title and introduction. I think that it would be more correct to say that a modified version of Le-Net is used.

-Line 91. Authors indicate at the end of the introduction that they propose an improved Le-Net with adjacent ECG segments for OSA detection. However, I would indicate in a more explicit way which is the hypothesis and the goal of the study.

-Lines 98-109. Authors should indicate that an OSA minute means that there is an apnea or hypopnea in this minute. This would enhance readability.

-Lines 110-122. Authors should include a figure/scheme in order to better explain this process. This would help to understand it. Do authors mean distance between R peaks when they say RR intervals?  Do authors mean values of the R peaks when they say RR amplitudes? In the line 112, authors could put an explanation such as “ (five 1-min segments in total) ” after “the labeled segment and its surrounding ±2 segments of the ECG signal”.

-Lines 124-132. Authors should indicate which properties of a signal CNN can measure in such a way that CNN are useful for signal analysis.

-Lines 157-161. Authors should use references to support statements 1) to 4).

-Lines 183-190. The use of positive and negative predictive values and the use of positive and negative likelihood ratios could also be useful to assess the performance of your proposal.

-It should be more clear that 5 minutes of the ECG signal are used to predict the presence of apneas and hypopneas in each 1-min segment.

-Authors should analyze the reasons why their CNN approach may have obtained better results than the machine learning classifiers the as well as the results reported by previous studies. That is to say, which properties of the CNN and the RR features have made their method obtain better results.

-Line 268. I think that it should be ”Table 5” instead of ”Table 3”.

-There are some problems of the Apnea-ECG database that could affect when using the CNN trained in another databases: i) an apnea/hypopnea event could occur in the middle of two 1-min segments; ii) a 1-min segment could contain more than an apnea/hypopnea event.  These are limitations of the study.

-Authors say that their method should be applied to other datasets as a future goal. In this sense, there is another public database the St. Vincent's University Hospital / University College Dublin Sleep Apnea Database (https://physionet.org/pn3/ucddb/) that contains ECG recordings.  In the ucddb database, apnea/hypopnea events are indicated in the annotation text files by the start and duration of the event. In this respect, it would be really interesting to see the performance of the CNN model to detect apneas, hypopneas and estimate the AHI of the patients of the ucddb database.

- The conclusion of the study should be that, based on the results obtained, their proposal (a deep learning approach based on a modified LeNet-5 with adjacent ECG segments) is useful for OSA detection.


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: SLEEP APNEA DETECTION FROM A SINGLE-LEAD ECG SIGNAL WITH AUTOMATIC FEATURE-EXTRACTION THROUGH A MODIFIED LENET-5 CONVOLUTIONAL NEURAL NETWORK
Review round: 2
Reviewer: 1

Output:
Basic reporting: -English language has been improved. There are still some grammatical mistakes but not as many as in the original version of the manuscript.
Experimental design: -Authors indicate their proposal in the introduction of the study. However, they should indicate explicitly the hypothesis and the goal of the study (we hypothesize that, the hypothesis of this research is, our goal is, the main objective of the study is ….), since it could enhance the manuscript's readability.

-Methods are in general well described. Nonetheless, in methods and results sections it seems that authors are using only Apnea-ECG database, whereas in the discussion they also comment the validation on the UCDDB database.  Authors only comment the use of the UCDDB database in the discussion section. I think that this study uses two public databases: Apnea-ECG an UCDDB. Thus, the use of both datasets should be indicated in the materials and methods section of the manuscript. In addition, results of the UCDDB recordings should also be included in the results section of the manuscript. This would really give to the readers the idea that this research has employed both datasets.
Validity of the findings: -Authors have proven the validity of their methodology using two different databases: Apnea-ECG database and UCDDB database.
Additional comments: -Despite authors effort to address my comment, the hypothesis and the goal of the study care not explicitly indicated in the manuscript. In this sense, they should indicate explicitly the hypothesis and the goal of the study (we hypothesize that, the hypothesis of this research is, our goal is, the main objective of the study is ….), since it could enhance the manuscript's readability.

-Lines 113-114: “Therefore, the labeled segment and its surrounding ±2 segments of the ECG signal (five 1-minute segments in total) are extracted as a whole for further processing” I would include this explanation in the figure 1 that authors have included in the manuscript following my previous review.

-Authors only comment the use of the UCDDB database in the discussion section. I think that this study uses two public databases: Apnea-ECG an UCDDB. Thus, the use of both datasets should be indicated in the materials and methods section of the manuscript. In addition, results of the UCDDB recordings should also be included in the results section of the manuscript. This would really give to the readers the idea that this research has employed both datasets.


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: CURCUMIN PLAYS A SYNERGISTIC ROLE IN COMBINATION WITH HSV-TK/GCV IN INHIBITING GROWTH OF MURINE B16 MELANOMA CELLS AND MELANOMA XENOGRAFTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: See the general comments
Experimental design: See the general comments
Validity of the findings: See the general comments
Additional comments: In this manuscript, Li and co-workers applied curcumin to HSV-TK/GCV gene therapy and found that curcumin can enhance the bystander effect mediated by gap junction. Furthermore, the in vivo study showed promising inhibition of the xenograft growth. Based on the results from in-vitro and in-vivo studies, the authors claimed that the findings provided new insight into the combination therapy and might provide a novel therapeutic strategy to increase gene therapy efficacy. The paper is well written, and the experimental results are solid and convincible. However, the manuscript is lacking discussion on the drawbacks of curcumin which has been demonstrated as both a PAINS (pan-assay interference compounds) and an IMPS (invalid metabolic panaceas).

Curcumin is a PAINS because it shows positive results in most drug discovery assays but fails to advance as drug leads. So far, curcumin has not been successful in any clinical trial. Moreover, the current research has found that curcumin exhibits some confounding behaviors including covalent labeling of proteins, metal chelation, redox reactivity, aggregation, membrane disruption, spectral interference, and structural decomposition (J. Med. Chem. 2017, 60, 1620−1637). There are a few questions the authors need to address in the manuscript.

1.	The solubility of curcumin in the water-based buffers is very poor and is pH- and temperature-dependent. Typically, the highest concentration of curcumin in water is ~10 μM. In practice, curcumin can only dissolve in water at acidic pH and high temperature. Although there are some studies reported the dissolving of curcumin in water at ambient temperature with higher concentration, it turned out that the curcumin either degraded or aggregated during the dissolving process. The authors need to show the details of the preparation of curcumin solutions and provide the evidence that the curcumin is the actual major components in the solutions.

2.	Curcumin is very unstable and the T1/2 is less than 5 min both in vitro and in vivo (J. Pharm. Biomed. Anal., 1997, 15, 1867−1876 and J. Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2007, 853, 183−189). Therefore, the positive assay results may due to other products from degradation rather than curcumin.

3.	Curcumin forms chemical aggregates (colloidal) under common biochemical assay conditions. The previous study showed that a critical aggregation concentration is ~17 μM by DLS (ACS Chem. Biol. 2015, 10, 978−988). The enzymes involved in the biochemical assays may interact with the chemical colloids, thereby changing the actual protein concentrations which leads to false positive assay results. The authors should check the aggregation level of the curcumin sample before the assays (i.e. by DLS). Otherwise, the authors should discuss the reasons if they think the chemical aggregation won’t affect the assay results.

4.	The authors claimed that the bystander effect was enhanced because of the improvement of GJIC by curcumin. However, several studies have demonstrated that the curcumin has strong interaction with phospholipids and alter bilayer properties (ACS Chem. Biol., 2014, 9, 1788−1798). Also, curcumin can interact with membrane proteins and change their functions. The authors need to discuss whether the double-fluorescence dye transfer assay in this study can rule out the possibility of membrane malfunction caused by curcumin.

5.	In another study (Pharm. Res., 2015, 528–537), the researchers have already tested the effect of curcumin on HSV-TK/GCV gene therapy as well. They designed a deliver peptide to transport both the HSV-TK/GCV gene and curcumin simultaneously to the target spots to overcome the poor bioavailability, solubility, and stability of curcumin. They also found that curcumin induced cell death efficiently both in vitro and in vivo which is the same as the conclusion drawn in this manuscript. Please cite this paper and discuss what is novel in your study compared with that one.

6.	The reference didn’t organize very well. There is a total of 38 papers cited in the manuscript, but there are only 37 papers in the reference section. Please correct this.


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: CURCUMIN PLAYS A SYNERGISTIC ROLE IN COMBINATION WITH HSV-TK/GCV IN INHIBITING GROWTH OF MURINE B16 MELANOMA CELLS AND MELANOMA XENOGRAFTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Basic reporting is clear and unambiguous.
Sufficient literature is cited.
The manuscript has a professional structure, figures, and tables.
The manuscript has relevant results.
Experimental design: The original primary research is within the aims and scope of the journal.
The research question is well defined and relevant.
The methods are described in sufficient detail.
Validity of the findings: The results are novel.
The underlying data have been convincingly provided with robust, statistical controls.
The conclusions are well stated.
Additional comments: Line31: Please change (CX)32 to CX32
Line 73: Please use the standard scientific language for Curcuma longa (italicize it).
Line 200: Please show the data for antiproliferation effect by mixing the B16TK and B16WT cells.


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: CURCUMIN PLAYS A SYNERGISTIC ROLE IN COMBINATION WITH HSV-TK/GCV IN INHIBITING GROWTH OF MURINE B16 MELANOMA CELLS AND MELANOMA XENOGRAFTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: See the general comments
Experimental design: See the general comments
Validity of the findings: See the general comments
Additional comments: In the resubmission, the authors discussed the drawbacks of curcumin and addressed the questions from previous reviewers properly. This study demonstrated that the curcumin was able to enhance the bystander effect of HSV-TK/GCV system by improving the gap junction intercellular communication function. Although the degradation of curcumin was inevitable and quantitative control of curcumin concentration in the in-vivo study was infeasible,  the authors overcame these issues by careful design of the control experiments. Overall, the resubmission is a well-written and solid paper, I recommend that the manuscript should be accepted for publication in its present form.


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

Input:
Title: IDENTIFICATION OF PIVOTAL LNCRNAS IN PAPILLARY THYROID CANCER USING LNCRNA–MRNA–MIRNA CERNA NETWORK ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: I suggest that you improve the first sentence of your Introduction (line 45). Using “most of the human genome transcribes RNAs that do not code for proteins” sounds more appropriate.
On line 47, the authors say that “NcRNAs can be divided into two classes: small ncRNAs and long ncRNAs (lncRNAs)”. This affirmation is over simplified and I suggest a better definition, looking in the innumerous reviews about ncRNAs
On line 58 the citation is not in agreement with the idea. Double check the references
The English language should be improved to ensure that a better understand your text. Review your writing, including lines 62, 65, 81, 141– the current phrasing makes comprehension difficult.
Line 63 – the bibliographic review is out of date. A quick search in PubMed shows 19 papers in 2019 with regard to PTC and lncRNA.
Experimental design: “The RNA sequencing expression data of 3 PeerJ reviewing PDF | (2019:04:36581:0:1:NEW 15 Apr 2019) Manuscript to be reviewed 74 datasets (GSE33630, GSE3467, GSE3678) were obtained from GEO”
                     All the datasets were generated through array.

“DESeq2 package[10] was used to normalize 75 read counts of gene expression in two GEO datasets”
              DESeq2 was used to normalize array data?

“Construction of lncRNA-miRNA-mRNA network of PTC”
                   For the construction of the ceRNA network no statistical method was used to evaluate the expression ratio of lncRNA-miRNA-mRNA. Only direct prediction of target is not enough to establish a ceRNA network.
Validity of the findings: Figure 3: This network representation is very poorly represented. Cannot identify nodes or edges of the network.

Line 151 – The authors write “Therefore, our results suggested that NR2F1-AS1 may play an important role in the progression and development of PTC and the cancer genes related pathways”. There is no data in this study to support this assumption.
Additional comments: The idea of the paper is good, but not well elaborated. There are language mistakes, references could be more updated and the analysis is poor. Nice figures, but without proper interpretation, mean nothing.


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

Input:
Title: IDENTIFICATION OF PIVOTAL LNCRNAS IN PAPILLARY THYROID CANCER USING LNCRNA–MRNA–MIRNA CERNA NETWORK ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: - The authors have used a clear, ambiguous language, some editing is required.
- Sufficient background of microRNAs and lnRNAs is provided.
- Additional background about PTC can be included. Like statistics (deaths... or incidence) to justify why this disease was chosen for this study.
- The authors have shared necessary figures, tables and raw data.
- The results are relevant to the hypothesis and are clearly stated.
Experimental design: - The research findings are original and in line with the scope of the journal.
- Hypothesis is unique and stated. The authors investigate lcRNAs in papillary thyroid cancer using comprehensive network analysis techniques.
- They also use good techniques (Both Gene Ontology annotation and KEGG pathway enrichment analysis) to prove their point.
- Methods are described well and are comprehensive as well.
- The investigation seems to of high technical standard.
Validity of the findings: - The authors state that 4 lncRNAs were identified as pivotal lncRNAs in in PTC. These are novel findings and also very relevant to the field.
- Additionally, co-relation of lncRNAs with Survival has been shown.
- They also showed that lncRNA regulated PI3K-Akt signaling pathway, focal adhesion, which are very crucial and interesting findings.
- Please include in discussion the potential functional effect of akt/FAK pathway activation. Do you expect potential role metastasis/migration ?
- Since these findings are derived through pathways analysis, the authors may have included some in vitro functional studies for one of the lnRNAs eg. SLC26A4-AS1 to prove that these results are impactful.
- Results and conclusions are well stated. Appropriate controls are used.
- The authors do discuss the significance of their findings, including lncRNA NR2F1-AS1 regulating hepatocellular carcinoma.
- Please add a paragraph how these findings would affect globally in terms of translational/clinical research. Would it help to identify lnRNAs as biomarkers for patient selection? can these be targeted? Have they been studied as biomarkers of response?
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: ANKLE AND MIDTARSAL JOINT QUASI-STIFFNESS DURING WALKING WITH ADDED MASS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper is clearly written with just a few minor grammatical errors. The paper provides a relatively thorough overview of research on human leg stiffness and joint quasi-stiffness during walking and running, although there is too much emphasis on leg stiffness during running that is not clearly relevant to the present study (see below). It also discusses results in the context of relevant research, although a more nuanced discussion of foot biomechanics models is warranted (see below). For the most part the article is structured appropriately, although the authors do not mention a significant component of their analysis until the discussion (see below), and thus do not present appropriate hypotheses or methods for this analysis. Outside of this, the results presented are appropriate. However, the authors should share more of their raw data, in particular data from individual subjects for all steps analyzed, including basic kinematic and kinetic waveform data used to calculate study variables.
Experimental design: This paper presents original research within the scope of PeerJ. The stated research question is to test the effects of added mass on quasi-stiffness of the ankle and midfoot joints, although the broader purpose of this goal is too vague. The reasoning behind the hypothesis of this study, that quasi-stiffness will increase with added mass, is also not explained. The study tests the research question thoroughly, although a few key details are missing from the methods (see below).
Validity of the findings: The results of the study appear robust, although the authors never state whether their data are actually normally distributed, which would determine the appropriateness of their use of repeated measures anovas. In case the data are not normally distributed for some tests, the authors could always used Wilcoxon Signed Rank tests. Conclusions are appropriate given data, although I have some issues with how the authors interpret their results with regard to the windlass mechanism and the midtarsal locking mechanism (see below).
Additional comments: In framing their research question, the authors discuss the literature on how leg stiffness is modulated during running in response to features of the terrain such as surface compliance and irregularity. However, these studies aren’t really relevant to the present research, as running is a mass spring gait in which using a specific leg stiffness is important to things like maintaining a constant CoM displacement and foot contact time. It is not clear how these things relate to walking, where leg stiffness is likely not constant throughout a step, and the importance of modulating leg stiffness is not as straightforward. On a related note, the authors point out that the ankle has been shown to be the most important contributor to overall leg stiffness, although to my knowledge this has only been demonstrated for mass-spring ‘gaits,’ like hopping and running. I recommend that the authors avoid discussing their research question in the context of overall leg stiffness during walking, and instead focus on how understanding the factors that dictate ankle and midfoot quasi-stiffness will improve our understanding of walking gait mechanics.

The authors do not provide a thorough enough description of what quasi-stiffness is, or why it’s important. They should provide a simple definition of quasi-stiffness, including how it’s calculated, and how it differs from other measurements of stiffness. They should also explain why quasi-stiffness is interesting and important for leg function, as well as what the broader implications of understanding quasi-stiffness is for things like prosthetics design and musculoskeletal health. More specifically, what do they see as the importance of quantifying quasi-stiffness with added mass?

The authors also need to provide some rationale for their hypotheses. Why should quasi-stiffness go up with added mass? The kinematic and kinetic data presented in Huang and Kuo (2014 JEB) could be used as a basis for predictions for the ankle. For the foot, the authors should draw on our current understanding of foot biomechanics. For this, they could use previous studies of foot kinetics or EMG (e.g. Sanchis-Sales et al. 2016 JAPMA; Kelly et al 2015 J R Interface), but should makes sure that their predictions are grounded in a biomechanical model of how the foot should function given added mass.

I believe that the authors should go a little further in interpreting their results as to the broader significance of this study. For instance, in finding that ankle and midtarsal stiffness goes up with added mass, what does this tell us about gait mechanics that we didn’t already know? Alternatively, what are the implications for prosthetic design, and/or musculoskeletal injury?

A major organizational issue in this study is that the authors do not mention testing the effects of the windlass mechanism and arch height on midtarsal joint quasi-stiffness until the discussion. This is an important part of this study, and the authors have included it in the abstract and conclusions. The authors should present predictions as to how the windlass mechanism and arch height will affect midtarsal quas-stiffness in the introduction, and describe all analyses and results for this part of the study in the appropriate sections. This is potentially a very interesting element of this study, and so needs to be set up and described properly. The authors are missing some key methodological detail concerning these analyses, including how they measured MTP joint angle, and statistics (are these data normally distributed?). They should also present a graph showing average MTP joint motion with relevant midtarsal dorsiflexion and plantarflexion quasi-stiffness phases highlighted.

In discussing the relevance of their findings to windlass mechanism, the authors should acknowledge that the contribution of the windlass mechanism to midtarsal dorsiflexion quasi-stiffness are likely to be small. Their analyses indicate that during this period, the MTP joints only undergo 0-10 degrees excursion, which may not have a big effect on tension in the plantar aponeurosis, which drives the windlass mechanism (Caravaggi et al 2010 JEB). The MTP dorsiflexion that occurs during plantarflexion quasi-stiffness may be more important. If so, these results suggest that the windlass mechanism does not have a major effect on midtarsal quasi-stiffness. However, the authors largely neglect intrinsic foot muscle activity as a possible major contributor to foot quasi-stiffness, as has been argued recently (e.g. Kelly et al., 2014, 2015, J R Interface). Although they can’t test this, they should discuss it as a possibility.

The authors suggest the midtarsal-locking mechanism may contribute to differences in midtarsal quasi-stiffness between participants, but do not test this idea. Considering they do not test it, and this model of human foot function has little empirical support (see Tweed et al., 2008 The Foot), I suggest that they do not discuss it.

Minor Issues:

lines 55-56 – Explain exactly what is meant by ‘structures distal to the foot.’

lines 57-59 – Explain more clearly how foot quasi-stiffness relates to moment arm length of the plantarflexors and ground reaction force, and what the implications are for gait (e.g. Takahashi et al 2016 Sci Rep).

Throughout, Sanchis-Sales et al (2016) is incorrectly cited as Sales et al. Please correct.

The authors should provide illustrations of the marker sets they used, how they defined kinetic foot segments, and where the joints between the foot segments are located. The description in the text is not clear enough. The authors also seem to be missing some detail about how pelvis and thigh segments are defined (only two markers/segment?), but this isn’t relevant to the present study.

lines 133-135 – The definition of the ankle joint doesn’t make sense. Why define it as motion between leg and forefoot segments, rather than leg and rearfoot segments? Or is this a typo?

lines 141-143 – What are the ankle and forefoot ‘rockers’?

The authors need to provide a much more thorough description of how they defined the ‘linear portions’ of the angle moment curves. Is there any way that they can ensure that these definitions were objective? This is another reason to include all of the raw data. Additionally, it would be useful to see the approximate timing of these phases on the moment and angle results figures, as in Sanchis-Sales et al (2016). At the very least, provide some indicate of the percentage of stance when these phases occur.

How many steps were analyzed per subject per condition?

line 173 – Change ‘ankle’ to ‘midtarsal’.

line 219 – Should this be quasi-stiffness in ‘dorsiflexion’ at midtarsal joint?

Standing midtarsal angle may not be a great proxy for arch height, as it is prone to error from marker placement, and it doesn’t take into account how much the arch deforms under weight-bearing relative to its height when unloaded. The authors should discuss this limitation of their study.


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

Input:
Title: ANKLE AND MIDTARSAL JOINT QUASI-STIFFNESS DURING WALKING WITH ADDED MASS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The report is very well written, is concise, clear and unambiguous. There is adequate reference to the literature and the authors have included most of the relevant papers in this area. The authors have written a thorough introduction that justifies the need for the study.

The figures are well presented and well described.

The discussion could be restructured slightly. At present it is “joint quasi-stiffness” then “Kinematics and kinetics” then back to “midtarsal joint quasi-stiffness”. Perhaps aligning all the discussion around joint-quasi stiffness together might help with the flow of the discussion.
Experimental design: The research question is well defined and well justified. There is a clear gap in knowledge that this research addresses.

Methodology is generally appropriate and well designed. The study is adequately powered and the investigators should be commended on this.

Calculating quasi stiffness for the mid-foot and ankle during the loading and unloading phase seems unusual. Quasi stiffness would typically be measured during the loading (dorsiflexion) phase when negative work is being done at each respective joint. Given that stiffness is defined as the magnitude of deformation of a given applied force, calculating stiffness during unloading (recoil of the arch) is hard to define, as the mid-foot is not deforming, but rather returning to its original shape, as force declines. Has this approach been applied previously for similar measures?

In regard to the quasi stiffness measure of the mid-foot. I understand the difficulties associated with these calculations. Particularly with reference to assigning forces to various foot segments when multiple segments are in contact with the ground. This is particularly difficult in walking when the heel is on the ground for a considerable period of time. When I look at the angle-moment curves for the mid-foot I wonder if the large stiffness values for the mid-foot are an artifact of assuming zero moment until the COP passed the joint axis. There is obviously a moment generated about this joint prior to this point, as there is considerable midfoot dorsiflexion that occurs prior to the registration of any joint moment. I wonder if a simpler approach may be to fit a straight line between the midfoot angle at foot contact and the angle at peak midfoot moment? While this may seem less informative, it likely helps to avoid the pitfall of assuming the midfoot to be excessively stiff.
Validity of the findings: Refer to above comment pertaining to measures of quasi stiffness.

The authors report considerable variability in midfoot dorsiflexion quasi stiffness. Could they please clarify if this is variability in stiffness between individuals, or variability in the response to increased load carriage within individuals? If the former is the case, is it possible that this variability may be a consequence of the method used to quantify midfoot DF quasi-stiffness? See comment above about how assuming zero mid-foot joint moment until the COP progresses beyond the joint axis might lead to over-estimations in quasi-stiffness. Eg. those with faster COP progression under the foot will have reduced stiffness, as the joint moment is registered earlier in the stride cycle. Those with delayed COP progression under the foot have increased stiffness, as a considerable amount of midfoot dorsiflexion has already occurred before a joint moment is even calculated. This would be counter-intuitive, but might be an artifact of the calculation.

The additional investigation about the contribution of the windlass mechanism to midfoot stiffness is problematic. The Windlass mechanism is proposed to stiffen the arch in late stance, prior to propulsion. However, the data presented suggests no relationship between MTP joint range of motion and midfoot stiffness in the plantar flexion phase, when presumably the Windlass mechanism would be acting. In fact, the findings seem to contradict the effectiveness of the Windlass mechanism, as the midfoot is actually more complaint in the PF phase than the DF phase. This finding is also similar to the findings of Welte et al. (J. Royal Society interface, 2018). Although, this could also be related to the method of calculating midfoot quasi stiffness (see comments above). If the authors would like to determine the role of the Windlass in stiffening the mid-foot, an alternative solution might be to plot the MTP and midfoot joint angles and angular velocities. This may help to show that MTP joint rotation commences prior to midfoot plantarflexion, hence providing some evidence for the Windlass changing midfoot kinematics.

The authors describe large subject to subject variability in data. It would be fantastic to be able to present this data as supplementary or supporting data. For example, the authors could present angle-moment plots for the midfoot and ankle quasi stiffness for each individual, as supplementary data.
Additional comments: Specific comments
Line 142 – what are “the ankle and forefoot rockers”
Line 173 – should “peak ankle moment” be “peak midtarsal moment” ?
Line 186-187 – It is unclear what this sentence means. Please re-word to improve clarity
Line 193 – The moment is “produced” about a joint, rather than “experienced”
Line 237 – Why was the secondary analysis only performed on the 0% bodyweight condition?
Line 257 – Based on bone pin studies (see Nester et al. 2009) there really is no evidence to support the notion of mid-tarsal joint locking.


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: ANKLE AND MIDTARSAL JOINT QUASI-STIFFNESS DURING WALKING WITH ADDED MASS
Review round: 2
Reviewer: 1

Output:
Basic reporting: This article is generally well-written and organized, with a few minor grammatical errors throughout the text. I suggest the authors carefully re-read and edit the manuscript to remove these errors. The introduction and background set-up the goals and hypotheses of the study nicely, and are generally well-referenced. I have a few suggestions for additional literature that the authors may choose to reference below. The article is structured appropriately, and the figures are all excellent and easy to interpret. The authors now provide all of the source data that would be necessary to re-run analyses.
Experimental design: This study represents a novel contribution to our understanding of the factors that influence joint stiffness in the ankle and foot. The investigation is rigorous, and the methods are clearly described.
Validity of the findings: Results are valid and interesting, and interpretations are generally appropriate. I have one minor issue with the interpretation of the windlass mechanism results that I wish for the authors to consider (see below), but overall I think the conclusions of the study are warranted.
Additional comments: I believe the authors have done a fantastic job of revising this article, and feel that they have adequately addressed most of my previous issues. My one remaining issue is that I’m still skeptical that <10 degrees of MTP dorsiflexion could stiffen the foot appreciably via the windlass mechanism. I appreciate the argument extended by the authors regarding the findings of Caravaggi et al. 2010, but suspect that the 4% PA strain observed in that study was probably driven more by bending of the arch than MTP motion. Indeed McDonald et al. (2016 PlosOne 11: e0152602) found that during running, MTP motion accounted for less than 15% of the observed strain in the PA, with the remainder due to compression and the concomitant lengthening of the foot. While that study looked at running and not walking, I think it remains to be demonstrated that the relative contribution of the MTP joints to PA strain is substantially different in walking. I also have a hard time accepting that differences on the order of 2 degrees of MTP motion could play a substantial role in the observed differences in midtarsal stiffness between unweighted and weighted conditions. Nevertheless, the results of this study are difficult to explain otherwise, and so I think the authors have provided one possible interpretation. I think the authors should use these results to call for more research on the relative effects of small changes in MTP joint position on PA tension and midtarsal stiffness.

Concerning their findings on the absence of a relationship between arch height and midtarsal stiffness, it would be useful if the authors could provide some qualitative assessment of the range of arch heights they sampled. The arch height measurement method that they used provides values that are difficult to interpret, and the sources of error in this method (that they acknowledge) indicate that their findings can only be interpreted tentatively. Were there subjects in this study who could be classified as possessing ‘flexible flat foot’, or other extremes of the arch height spectrum? Their finding of a lack of relationship is surprising, since very high arches are anecdotally associated with rigid feet, and flat feet are anecdotally associated with highly compliant feet. Do these data allow the authors to contradict these common assumptions? This provides another opportunity to call for further research, especially research using arch height as a continuous variable.
I have a few more minor issues that I hope the authors will consider:
lines 52-60 - I think the explanation of quasi-stiffness provided here could use a little bit more clarification. In particular, it seems to me that part of what joint quasi-stiffness is telling us is how muscles are being used to resist motion at a joint. During gait, a joint does not have a single inherent quasi-stiffness value, but instead its stiffness will change based on how muscles are being used at a given time in stance. Thus, quasi-stiffness provides a measure of how muscle can be used to tune the stiffness of a joint for a given task (or for a given phase in gait.)
line 61 – This statement is too general. Farley and Morgenroth 1999 only found that the ankle was a major contributor to leg stiffness during hopping, but not any other tasks. Furthermore, they didn’t quantify quasi-stiffness, they quantified limb and ankle stiffness more generally. The authors should adjust this sentence accordingly.

lines 80-82 – Reference 22 (Zifchock et al) only shows a relationship between arch height and static arch stiffness, and this relationship is very weak. Furthermore, Holowka et al (2018 Sci Rep 8: 3679) found no correlation between static foot stiffness measurements and foot stiffness measurements during walking, so the connection between arch height and foot stiffness in the literature is tenuous at best. I think the authors should adjust these sentences accordingly.

There is an error in the authors names in reference 21.


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: ANKLE AND MIDTARSAL JOINT QUASI-STIFFNESS DURING WALKING WITH ADDED MASS
Review round: 3
Reviewer: 1

Output:
Basic reporting: As stated in my previous review, I believe this article is well-written and organized. The grammatical errors have been corrected, and the article include appropriate references. The figures are excellent and appropriate. All source data is provided.
Experimental design: As previously stated, I think this study represents a novel contribution to our understanding of the factors that influence joint stiffness in the ankle and foot. The investigation is rigorous, and the methods are clearly described. I am pleased to see the authors have identified and amended an error in their prior analysis. I have one lingering issue about how they calculated midtarsal dorsiflexion stiffness, which I detail below.
Validity of the findings: I believe that the results are valid and interesting. Correcting the error in the prior analysis leads to results that make a lot more sense to me, and bolster some of the original conclusions of the study. While some conclusions have to be changed, these changes strengthen the findings of the paper in my view.
Additional comments: Fixing the analysis error in the prior submissions leads to results that make more intuitive sense to me, and so I commend the authors for identifying this issue and adjusting the paper accordingly. This makes the results and interpretation much more straightforward in my opinion. I just have one substantive issue remaining, which concerns the calculation of midtarsal joint dorsiflexion stiffness. In lines 148 to 149 the ‘dorsiflexion phase’ is defined as spanning the period from peak plantarflexion to peak dorsiflexion at each joint. However, for the midtarsal joint, Figure 2B shows this phase beginning at a somewhat arbitrary point, not at the peak joint plantarflexion. So, how exactly was ‘dorsiflexion phase’ determined for the midtarsal joint? Was the phase actually defined as beginning when the CoP crossed anterior to the midtarsal joint (when the moment could be calculated)?

lines 61-63 – I still disagree with the authors’ use of ‘quasi-stiffness’ to describe the ankle measurements used by Farley and Morgenroth. Their estimate was based on a single moment in stance phase of a hop, and was not calculated by fitting a slope to the joint angle-moment relationship. Therefore, their measurement does not fit the definition of quasi-stiffness provided at the beginning of this paragraph, and also does not match the calculation used by the authors in this study. Thus, I think the authors should remove ‘quasi’ here to avoid giving readers the false impression that Farley and Morgenroth used a similar method of measuring stiffness as that used in this study.

line 277 – Change ‘Body’ to ‘Added’.


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: NEGATIVE EMOTIONAL STATE SLOWS DOWN MOVEMENT SPEED: BEHAVIORAL AND NEURAL EVIDENCE
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are some minor typos and language issues throughout the document. For example:

Abstract-Conclusion – “emotional processing consuming more resources” should specify cognitive resources.
Line 58 – “Neural condition” was used instead of “neutral condition”.
Line 152 – should say “researchers”
Line 324 – “For the sixth action task, the interference effect may attribute to the induced negative emotion.”
Line 416 – “Study have showed that attention-deficit individuals evoked lower CNV amplitude than normal.”
Line 435 – “Thus, the total number of action tasks was a little much. there might be a fatigue effect.”
Line 439 – “Maybe we can increase the number of action tasks (like 12 action task) after each image viewing and reduce total emotional images.”

I’m unsure of the relevance of EPN and LPP (Line 367), as they weren’t discussed in the Introduction, or mentioned in the Methods as EEG components of interest. Did the authors also examine EPN and LPP? If so, this should be reported, and there should be an appropriate statistical correction for multiple comparisons.

I encourage the authors to consider plotting the data in Figure 2-3 such that the raw scores can be observed for all time points e.g. by using scatter plots with box plots. Efforts made to improve data transparency will certainly aid future interpretations of the findings. An exploration of the benefits of transitioning away from bar graphs can be read here:
https://doi.org/10.1371/journal.pbio.1002128
and
https://garstats.wordpress.com/2016/03/09/one-simple-step-to-improve-statistical-inferences/
Experimental design: The article is written to address the question of movement speed, particularly with regards to sport. However, the experiment design is better suited to examine response time, which is also a very important factor for athletes. In the present study, participants are required to perform a relatively simple button press task in which the limiting factor is not likely to be finger movement speed, but rather reaction time to visual stimuli. Perhaps this distinction needs to be made clearer in the Introduction.

To aid researchers who wish to replicate this research, the exact image file names used from the CAPS and IAPS databases should be provided in Supplementary Materials.
Validity of the findings: Line 170 – It appears as though the images extracted from CAPS were mixed with those from the IAPS. Did the authors conduct statistical testing to ensure that the valence and arousal ratings of the images from both databases were approximately equivalent? Additionally, it would be useful to know the standard deviations for valence and arousal ratings of the negative and neutral images.

Regarding EEG analyses, were EEG epochs checked for noise artefacts? If so, how many trials were removed on average per participant? Did the authors perform an independent component analysis (ICA) to remove muscle and blink artefacts?

The statistical analysis makes use of a 2 x 2 rmANOVA. However, it seems to me that a general linear model with a categorical factor of emotional valence (negative vs neutral) and a continuous factor of task sequence (1st through to 6th) would be more appropriate. This has added benefit of incorporating data from the 2nd, 3rd, 4th and 5th cued-action tasks, thereby improving estimates of the effect.

Line 316 – The difference between 1st and 6th action tasks is interpreted in the manuscript as competition for cognitive resources during the 1st task, which dissipates by the 6th task. However, it is possible that at least part of the increase in speed from 1st to 6th task is a practice effect. This could be checked by examining the discrepancy in response time between 1st and 6th trials for the first 75 images displayed as compared to the last half of the images. A practice effect would show greater discrepancy 1st - 6th actions within the first half of images compared to the last half due to ceiling effects. A distractor effect would reveal relatively consistent discrepancies throughout the trials. A general linear model using continuous data for the action sequence tasks could be used to quantify the strength of distractor and practice effects using the following fixed factors:
•	Emotional Valence (negative vs neutral)
•	Action sequence (1st, 2nd, 3rd, 4th, 5th, and 6th)
•	Order (continuous labelling of images in the order displayed. Can also be turned into a categorical variable by labelling images as halves/tertiles/quartiles)
An analysis of this type would provide additional support for the discussion in Line 324 e.g. “for the sixth action task, the interference effect may attribute to the induced negative emotion”.

Line 235 – It’s not clear to me why a one-way rmANOVA was used to analyse averaged P1 amplitudes. If the data is grouped using one factor (emotion valence) with two levels (negative and neutral), a paired t-test would be appropriate. The same applies to averaged theta oscillations (line 247).
Additional comments: Line 338 – is it not also possible that negative stimuli reduce the availability of cognitive resources for the 1st action task as well as the 6th, thus consistently slowing response time? This appears to be the most parsimonious explanation that doesn’t require invoking pre-activation of motor systems through negative imagery. More exposition is needed to make the reader understand why this simple explanation doesn’t fit the observed data.

Line 347 – “Behavioral findings that movement speed was significantly slower in the first action task than in the sixth action task was also remarkable.” I would tone down the language here (i.e. the word ‘remarkable’). Especially as this result is entirely in line with expectations – participants show improved response times with repetition on the action tasks due to practice effects. This is also supported by the CNV results, which showed improved attention and preparation in later action tasks relative to earlier tasks.

Line 370 – “This might be due to the interference of the action task.” I’m not sure what this sentence refers to, it does not seem to relate directly to the preceding sentence.


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: NEGATIVE EMOTIONAL STATE SLOWS DOWN MOVEMENT SPEED: BEHAVIORAL AND NEURAL EVIDENCE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Title: Negative emotional state slows down movement speed: behavioral and neural evidence

The manuscript by Li and Colleagues analyzed the effect of emotions on movement speed. The authors presented emotional pictures to the subjects during EEG recording and evaluated the speed of these subjects after each picture. The authors reported that EEG data showed increased theta oscillation and larger P1 amplitude in response to negative than to neural images. Furthermore, participants exhibited significantly slower movements in the negative emotional condition than in the neutral condition.

In general, the manuscript has intriguing results and merits publication. However, there are also some points that should be clarified before publication.

The introduction is well written I do not have any critics for the introduction part.
Experimental design: Materials line 176: The results of SAM should be given in the results section with more information, including the means, Standard deviations, and comparisons between negative and neutral emotions with p values.

Line 224: FCz was re-referenced to the average of TP9 and TP10. It is not clear why the authors re reference the data to these specific electrodes?

Line 233: “P1 at PO3, PO4, PO7 and PO8 within the time window of 190-230-ms was selected as target ERP”  and line 245 “The POz,PO7,and PO8 electrode were selected for the analysis of the evoked theta oscillation (5–9Hz) within the time window of 100- 200-ms”. This point is the weakest point of all paper. The authors just select few electrodes to analyse and these electrodes are not same for the analysis of P1 and theta. Although authors give reference to Aftanas work as a rationale it is still not understood. There are many other papers which analyze the event related oscillations in the whole topology. There could be hemispheric differences or dominance of occipital location in comparison to frontal region. Although the theta responses are the dominant rhythm of frontal locations during emotional pictures theta responses also increase in parietal and occipital locations. The authors only analyze the parietal and parietal-occipital and they even do not analyze the occipital electrodes (O1, Oz, O2). This point is an important limitation of the study.

Line 360 Although the authors only analyze parietal electrodes they discuss the importance of occipital electrodes. The analysis should also include occipital electrodes the authors then may discuss this point.
Validity of the findings: It will be nice to see the results of correlation analysis between the  SAM scores and theta responses and as well as the speed of movement.

The additional SPSS files presented as a data file does not help to the reviewers like me who cannot read and talk Chinese.
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: NEGATIVE EMOTIONAL STATE SLOWS DOWN MOVEMENT SPEED: BEHAVIORAL AND NEURAL EVIDENCE
Review round: 2
Reviewer: 1

Output:
Basic reporting: There remain a few minor typos in the document:

Line 109: speed might become faster than violate this rule
Line 130: Brain circuits involved in emotional processing are activated during the movement of athletics by trained athletes.
Line 178: …negative images were significantly differed from neutral images.
Line 168: After data preprocessing, a subject with many additional movements were removed.
Line 277: We used [the] scatter plots with box plots to improve data transparency in Fig.2B.
Line 344: Our findings are in line with previous study claiming.
Line 346: The significantly decrease of negative emotion.
Line 363: …the interference effect for the first action task can be explained by biological evolutionary perspective.
Line 368: Once people were instructed to approach a negative stimulus, the movement speed will be slowed down.
Line 387: …the practice effect on the sixth action task will make participants to perform the sixth action task faster.
Line 390: …behavior data results of 2×6 rmANOVAs add us understand more comprehensively.
Line 425: …greater dedication of attentional resources and well movement preparation
Line 449: …with neutral condition elicited larger CNV than negative condition.
Line 453: Study have showed that attention-deficit individuals.

The use of the word “besides” to begin a sentence is a touch too colloquial/informal. It can also be misinterpreted to mean either “additionally…” or “but more to the point…”, where the former is the continuation of a thought, and the latter is a partial negation.

The legends in Figure 3, 4, and 5 says “neural” instead of “neutral”
Experimental design: I very much appreciate that the authors re-analysed the data using a 2 x 6 rmANOVA in addition to the original 2 x 2 rmANOVA.
Validity of the findings: The Discussion is well thought through, and the findings appear to be well-supported by the data.
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: NEGATIVE EMOTIONAL STATE SLOWS DOWN MOVEMENT SPEED: BEHAVIORAL AND NEURAL EVIDENCE
Review round: 2
Reviewer: 2

Output:
Basic reporting: Authors have addressed my remarks adequately, and I have no other comments. The manuscript merits publication in its revised form.
Experimental design: Authors have addressed my remarks adequately, and I have no other comments.
Validity of the findings: Authors have addressed my remarks adequately, and I have no other comments.
Additional comments: Authors have addressed my remarks adequately, and I have no other comments.


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

Input:
Title: IDENTIFICATION AND CHARACTERIZATION OF CIRCRNAS AS COMPETING ENDOGENOUS RNAS FOR MIRNA-MRNA IN COLORECTAL CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: Yuan W et al have identified some circRNAs as putative candidate diagnostic biomarkers or potential therapeutic targets for colorectal cancer (CRC). To achieve this, they compared the expression profiles of circRNAs in CRC and the adjacent non-tumorous tissues using high-throughput sequencing. They found 50,410 circRNAs in the CRC tissue and the adjacent non-tumor tissues, of which 33.7% (16,975) are new. Interestingly, they also uncovered that six circRNAs associated with CRC could play important roles in carcinogenesis as competing endogenous RNA (ceRNA) for regulation of miRNA-mRNA network. However, to make the story more compelling, the authors need to provide additional information and validations that could directly test their model.

Specific comments:

1) Figure 1C: you indicate in this figure that the BIRC6 host gene produces the highest numbers of circRNAs isoforms. Interestingly, it was described in other studies that BIRC6 overexpression is a predictor of poor prognosis in colorectal cancer. What about your data (mRNA and miRNA differentially expressed, circRNAs…), could you discuss about this point?

2) About the previous point, could you also please include in your study an excel file that contains the lists of differentially expressed miRNAs and mRNAs?

3) The current phrasing in your paragraph “Interaction between differentially expressed circRNAs, miRNAs and mRNAs” (lane 177 to 186) makes comprehension difficult. Could you make it easier to understand? Especially when we compare the different numbers shown in Figures 3C, 3D and 3E with the numbers mentioned in this paragraph. In addition, could you also add an Excel file containing the list of the mature miRNAs and the list of mRNAs?

4) I suggest that you improve the description and the discussion relative to the figures 4B and 4C to provide more justification for your study (negative/positive survival factors…). For example, is a low expression of hsa_circ_0067163 associated with a poor survival due to a low expression of TPM2 and high expression of has-miR-424-5p? For the other examples, it seems that the other circRNAs are up-regulated which in return should increase the expression level of the target genes and thus the survival curves.

5) Finally, could you also validate in cells your model about the Networks Regulated by circRNAs by using an overexpression strategy and/or a RNA interference strategy with siRNA or shRNA directed to the back-splicing site of the different circRNAs and then quantify the expression levels of the miRNAs and target mRNAs?

Minor comments:

L49: replace “More” by more.
L72-73: the text is highlighted in yellow.
L79: are you sure about the bioanalyzer chips RNA 7500 or did you used RNA pico or nano Chips?
L98: the base of the logarithm is missing in your formula.
L113: replace “miRNAsprediction” by miRNAs prediction.
L132: only chromosome 1 and chromosome 2 produced more than 4,000 circRNAs.
L146-148: you need to rephrase the sentence.
L152: replace “we foundthat” by we found that.
L160: in the description of the figure 2B you indicate p-values while in the volcano plots you mentioned q-value (the same for figure 3A and 3B).
L169: instead of “(Fig. 3)”, you have to precise Fig 3A and 3B.
L173: miRNAs or pre-miRNAs?
L222: instead of brain (65,731) it should be testis (15,996).
L223: Since in normal brain tissue it was identified 65,731 circRNAs and you identified 50,410 circRNAs in colorectal tissues, you should change your statement about “most abundant” in your sentence.
L240: Competing endogenous RNA (ceRNA). You have to indicate the definition of ceRNA at the lane 35 when you used it for the first time in your text.
L249: I did not find hsa-mir-96-5p in your ceRNA network of the selected "hot-spot" circRNAs (Figure 4B).
Figure 1C: replace “Relatuve Frequency” by Relative Frequency.
Figure 2C and lane 159: replace “CPC tissues” by CRC tissues.
Figure 2C: replace “exontic 94” by exonic 94.
Figure 3D: replace “targe mRNA” by target mRNA.
Experimental design: As mentioned in the previous section, supplemental data/figures are required to strengthen the manuscript.
Validity of the findings: As mentioned in the previous section, supplemental data/figures are required to strengthen the manuscript.
Additional comments: No additional comments


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

Input:
Title: IDENTIFICATION AND CHARACTERIZATION OF CIRCRNAS AS COMPETING ENDOGENOUS RNAS FOR MIRNA-MRNA IN COLORECTAL CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Comments to the authors

In general, aberrant expressions of genes are observed in cancers. Circular RNAs (circRNAs) are known to be generated by splicing of precursor messenger RNAs (pre-mRNAs). Therefore, it is assumed that featured circRNAs exists in each cancer. Indeed, the authors have found 98 featured circRNAs in Colorectal cancer (CRC) in this manuscript (Supplemental file 2). If the featured circRNAs function as sponges or inhibitors of their interacting microRNA (miRNA), the miRNA-targeted mRNAs would be controlled through the expression level of the featured circRNAs in CRC, probably resulting in malignant cell transformation in this cancer. Based on this idea, the authors have attempted to find the circRNAs-miRNAs-mRNA network in CRC. However, the data are not enough to demonstrate the network in this manuscript. In particular, explanations for the data are totally insufficient to inform the author’s thought based on the data. Several concerns are enumerated below.

1. Figure 2A – There is no explanation about CRC-D-C, B, A and CRC-C-A, B, C. The author should clear which lanes are the normal samples or the CRC samples.

2. Figure 2E – The lines between the circles are illegible. The authors should describe the relationship between this network analysis and the CRC.

3. Figure 3C and D – What is “DE_” ? What is pre-miRNA? What is target mRNA? Nothing explains about them in detail in Figure legends. It is difficult to understand the description of this data on lines 178-186. The authors would show the flow chart concerning this analysis.

4. Figure 4A – What are host genes of these circRNAs? Are the host genes included in the differential expression of mRNAs between the normal and the CRC samples?

5. Figure 4C – What is different between blue and red lines? The authors should describe this point in Figure legends.

6. Finally, the authors should perform some wet experiments to confirm their dry analysis. For instance, they could perform the luciferase reporter experiments using the reporter gene bearing the binding sites of miRNA which could interact with a specific circRNAs in CRC. These experiments would be useful for confirming the circRNAs-mRNAs-mRNA network in CRC.

Minor point
7. line 151 – Is Fig. 1C correct? The author should check this point.


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

Input:
Title: IDENTIFICATION AND CHARACTERIZATION OF CIRCRNAS AS COMPETING ENDOGENOUS RNAS FOR MIRNA-MRNA IN COLORECTAL CANCER
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: Yuan et al. have found various misregulated circRNAs in colorectal tissue samples impacting the expression level of hundreds of miRNAs and mRNAs. Among the 6 top-misregulated circRNAs, they have shown that a regulation network (as ceRNA) is involved in pathogenesis in CRC. These data suggest that those 6 circRNAs could be used as potential diagnostic biomarkers.
However, authors's conclusion are not fully supported by direct evidence and need more decisive experiments.

Major Points:

1) It will be interesting to test if one of the top 6 deregulated circRNAs act as a miRNA sponge. Hsiao et al. (Cancer Research, 2017) have shown that circCCDC66 act as miRNA sponge for specific miRNAs with a nice experiment.

2) Can you analyse the expression level of miRNAs interacting with circRNA by RT-qPCR and also mRNAs targeted by those miRNAs still by RT-qPCR with and without siRNA against your top misregulated circRNAs?

Minor Points:

1) Line 49: change More by more
2) Line 113: miRNA prediction in the title
3) Line 152: found that
4) Line 153: circ instead of cicr
5) Fig 1C Relative Frequency
6) Fig 2C Exonic
7) Can the authors test the effect of downregulation of upregulated circRNA (with siRNA) on CRC proliferation with MTT assay (or another one)
Additional comments: No additional comments


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

Input:
Title: IDENTIFICATION AND CHARACTERIZATION OF CIRCRNAS AS COMPETING ENDOGENOUS RNAS FOR MIRNA-MRNA IN COLORECTAL CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: the authors took notice of the recommendations and also provided us supplementary files requested.

However, it could have been great if they performed some wet lab validations as requested by all the reviewers. For example, to validate the new circRNAs but most importantly to validate the circRNAs- miRNA-mRNA network (these validations could have been done at least in cell lines).

Moreover, I totally understand the point raised by the authors concerning the grants/funding issues… It is complicated for all of the scientific community. However, even if this manuscript is “only” a preliminary study and the fact that other journals allow that, it is not an argument. Indeed, we are talking about science.

I appreciate that the authors described the shortcomings of this manuscript in the discussion section. However, in order to avoid misleading the reader and without wet lab validations of the model, they should have done that since the beginning by talking about correlative study, putative diagnostic biomarker or which could play important roles…

Overall, this manuscript has still a good potential:
-	circRNAs profiling in CRC and the adjacent non-tumorous tissues using high-throughput sequencing.
-	miRNA and mRNA expression profiles retrieved from the TCGA database.
-	the correlative study (circRNAs- miRNA-mRNA network).

Specific comments:

•	in PDF, the resolution of the figures is bad in particular when we try to zoom in.
•	L79: size of the font is different.
•	L111: replace “miRNA sprediction” by miRNAs prediction.
•	L180 -188: I suggest that you improve the description of this paragraph (Interaction between differentially expressed circRNAs, miRNAs and mRNAs) to make it easier to understand.
•	L197: you should verify but it seems that only 3 (instead of 6) ceRNAs related mRNAs were significantly correlated with the survival time of CRC patients. It is true that several parameters can regulate the expression level and/or the stability of miRNAs, that's why some wet lab experiments should have been done to validate your network. For example, when the expression of hsa_circ_ 0023990 is high, the survival time is improved in patients due to high expression of SOX1. Unfortunately, the expression level of the corresponding miRNA (hsa-miR-3065) is also high. The same for ITGBL1 and hsa-miR-20a.
•	 L201: you should replace “associated with” by “correlates with”
Experimental design: the recommendations are mentioned in the previous section
Validity of the findings: the recommendations are mentioned in the previous section
Additional comments: No additional comments


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

Input:
Title: IDENTIFICATION AND CHARACTERIZATION OF CIRCRNAS AS COMPETING ENDOGENOUS RNAS FOR MIRNA-MRNA IN COLORECTAL CANCER
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Comments to the authors

The authors have answered in a satisfactory manner to raised issues in most of the comments. While the quality of manuscript has largely improved, a few issues still remain unclear. The concerns are enumerated below.

1. Figure 4A – The top 3 down-regulated circRNAs (circPIEZO1-3, hsa_circ_0067163 and hsa_circ_0140188) in CRC are derived from PIEZO1, TPRA1 and DMD genes, respectively, whereas the 3 up-regulated circRNAs (hsa_circ_0002632, hsa_circ_0001998 and hsa_circ_0023990) come from STIL, FUT8 and NOX4 genes, respectively. Expectedly, the analysis of the differentially expressed mRNAs in the CRC shows that the expression of DMD gene is decreased, while that of NOX4 gene is elevated in the CRC (additional_file_4), suggesting that the production of hsa_circ_0140188 and hsa_circ_0023990 is due to the expression amount of the host genes. This is a reasonable event. On the other hand, the other host genes (PIEZO1, TPRA1, STIL and FUT8) do not include in the list of the differentially expressed mRNAs in the CRC (additional_file_4). Therefore, the authors should explain how to alter the expression of circPIEZO1-3, hsa_circ_0067163, hsa_circ_0002632 and hsa_circ_0001998 in the CRC.

2. Figure 4B – The authors mentioned that low expression of hsa_circ_0067163 associated with a poor survival due to a low expression of TPM2 via the function of hsa-miR-424-5p in the new version of the manuscript on lines 242-243. However, the hsa-miR-424-5p is also targeted by hsa-circ_0001998 which is one of the up-regulated circRNAs in the CRC (Figure 4B). Therefore, it is unclear why the expression of TPM2 is decreased in the CRC. The authors should describe this discrepancy concerned with the sponge effect of hsa_circ_0067163 and hsa-circ_0001998 to hsa-miR-424-5p in the CRC.

Minor point
3. Figure 4C – It is still unclear what is blue and red lines. There is no description regarding the lines in the figure legends of the new version of the manuscript. The annotation of the lines is too small in the graph.


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

Input:
Title: IDENTIFICATION AND CHARACTERIZATION OF CIRCRNAS AS COMPETING ENDOGENOUS RNAS FOR MIRNA-MRNA IN COLORECTAL CANCER
Review round: 2
Reviewer: 3

Output:
Basic reporting: Authors should bring some minor modifications to their article:

line 47: Change "diseasee" by disease
line 111: correct the title of paragraph
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Authors should bring some minor modifications to their article:

line 47: Change "diseasee" by disease
line 111: correct the title of paragraph


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 NEW RECORD OF KELP LESSONIA SPICATA (SUHR) SANTELICES IN THE SUB-ANTARCTIC CHANNELS: IMPLICATIONS FOR THE CONSERVATION OF THE “HUIRO NEGRO” IN THE CHILEAN COAST
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The macroalgal community has been strangling for years with the taxonomy of Lessonia species for this region. The macroalgal taxonomy and biodiversity research will highly benefit from this study’s discoveries.

However, the manuscript needs minor revisions and thorough proofreading. Below are highlighted the points to be addressed.

•	The word “macroalga” is mentioned through the text but the majority of the studies use the word “macroalgal”, e.g. Line 24 and Line 34: “macroalga” conservation should be “macroalgal” conservation, and Line 41: “macroalga” genera should be “macroalgal” genera.

•	References format throughout the manuscript should be checked again and make sure they all are consistent, e.g. Line 46: et al. is not italicized even though it is in the rest of the manuscript, and Line 54: a comma is missing before the date.

•	Line 55: It would be better if “17° to 41°” are mentioned with locations as in the lines 57 and 58, e.g. “Peru (17°S) to Peru (17°S)”, for better understanding.

•	Line 68: Since when is the Katalalixar National Reserve a marine protected area (MPA)? Some info about how long is this MPA running (since when) would be useful.

•	Line 75: It would be beneficial for the reader if you briefly mention what is the Humboldt Current and why is it important for the taxonomic composition and biogeography.

•	Line 76-79: Fig. 1a could be mentioned along those lines.

•	Line 162: It is more accurate to say “we increase the knowledge of the species richness” instead of “we increase the species richness”.

•	Line 164: It is wiser to say, “we hypothesize that” or “we predict that” … “will move southward in the near future”.

•	Line 224: “spawning ground” is a more accurate term.


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 NEW RECORD OF KELP LESSONIA SPICATA (SUHR) SANTELICES IN THE SUB-ANTARCTIC CHANNELS: IMPLICATIONS FOR THE CONSERVATION OF THE “HUIRO NEGRO” IN THE CHILEAN COAST
Review round: 1
Reviewer: 2

Output:
Basic reporting: It is an exploratory study, without hypothesis, that reports the presence of a kelp species (Lessonia spicata) in the sub-Antarctic Magellanic channels using morphological and phylogenetic analysis. The manuscript is written with appropriate technical English.
The theoretical justification of the study is adequate. The bibliographical references are appropriate. I suggest to review all the literature cited in the manuscript. For example: "Vega et al. 2016 "(line 207), and" Suhr 1839 "(line 148) has no bibliographical reference. The reference "Fraser et al. 2009 "(line 260) is not cited in the manuscript. The reference "McCarthy C. 1998" on line 294 is incomplete. "Sernapesca 2019" (line 203 and 205) in capital letters. The year of some references is in parentheses (line 357 and 360). "Yoon et al. 2001 "in the legend of Table 1 has no reference.
The structure of the article is adjusted to the format of the PeerJ. The figures and tables are clear and have appropriate legends. Raw data is available and accessible. In the legend of figure 4, it is necessary to differentiate the text of the phylogenetic tree (a) from the cladograms of ITS sequences (b).
Experimental design: no comment
Validity of the findings: Results.
In the text of the manuscript are the same data presented in table 2 (lines 132-135). In addition, there is an error in the holdfast diameter of a individuals collected in the Castillo Channel (9 cm in the text and 5 cm in the table). I suggest first describing the results of the comparative analysis between Lessonia species (figure 3, see line 150-154 discussion); Then, I suggest complementing the analysis with the results of table 2 (see line 135 and 136, not including average measurements).
In molecular analysis, I suggest first describing the results of the phylogenetic tree (figure 4a) and then the results of the cladogram (figure 4b). Why in the tree of figure 4b does only LesA appear?

Discussion.
I suggest moving part of the first paragraph of the discussion (line 150 to line 157) has results - morphological analysis. Another sentence of this paragraph could be part of the results of the phylogenetic analysis: "Intraspecific divergence of L. spicata ..." (Lines 155-157).
I also suggest moving (or copying) “This is first confirmed report of L. spicata…” (line 159-160) to the beginning of results (line 130). Leave "Fig2a" only in results.
I suggest briefly discussing "the bank of algal microscopic forms" (Hoffman & Santelices 1991 MEPS 79: 185-194) as a potential mechanism of population persistence of L. spicata in high latitudes (see lines 189-194).
Additional comments: The recording of Lessonia spicata in the sub-Antarctic channels is an important finding for the conservation and management of the kelp species of the South American Pacific. In this context, the justification of the problem in the manuscript is well established and the methods are adequate.
However, the results should describe the figures and tables of the morphological and phylogenetic analyzes with concluding sentences; following the numerical order of figures and tables. In this context, in the discussion there are phrases that should be in the results section. In addition, citations and references should be reviewed in depth.
Finally, it would be very interesting to briefly discuss the hypothesis of the "bank of algal microscopic forms" as a potential mechanism of persistence of populations of L. spicata in high latitudes. What would be the eventual effect on the phylogeny?


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 NEW RECORD OF KELP LESSONIA SPICATA (SUHR) SANTELICES IN THE SUB-ANTARCTIC CHANNELS: IMPLICATIONS FOR THE CONSERVATION OF THE “HUIRO NEGRO” IN THE CHILEAN COAST
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
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: YAK RUMEN MICROBIAL DIVERSITY AT DIFFERENT FORAGE GROWTH STAGES OF AN ALPINE MEADOW ON THE QINGHAI-TIBET PLATEAU
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors do an excellent job of introducing their model system and ruminant and frame the relevance of their study well. Readability is reasonable, but the text contains a small, but significant, number of typos and formatting issues including spacing near commas. I would recommend these be addressed prior to acceptance. Figure quality is sufficient for publication. Availability of data fits acceptable standards, but fastq files would be preferable.
Experimental design: The manuscript fits within the journal scope and addresses an area of limited knowledge; yak ruminal ecology across seasons on pasture with distinction between sexes. The experimental design is limited to the harsh environmental constraints of the system studied and contains replicable detail. From the text it appears 10 experimental animals (even sex-split) were grazed for nine months in a herd of 30 animals. These parameters are sufficient to support the authors’ conclusions regarding rumen microbial ecology and function. Sample collection and storage protocols followed well-established norms.
Validity of the findings: Presented data and statistical treatment of data are robust. The authors address both the amplicon-derived ecology and taxonomy-predicted function of the yak rumen microbiome as well as forage nutritional composition in great detail. As a largely discovery-oriented, incremental manuscript, the authors provide a baseline reference for future inquiries into yak microbial ecology or the effects of range seasonal phenology on the yak rumen microbiome.
Additional comments: I believe future work would benefit from the inclusion of fungal amplicon sequencing (ITS).

Your treatment of the microbial sequence data is robust, but I would recommend exploring Love et al., 2014 for the purposes of differential abundance between groups and Koh et al,. 2017 for relating microbial data to continuous covariates, like forage quality indicators, if you have not already done so.


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: YAK RUMEN MICROBIAL DIVERSITY AT DIFFERENT FORAGE GROWTH STAGES OF AN ALPINE MEADOW ON THE QINGHAI-TIBET PLATEAU
Review round: 1
Reviewer: 2

Output:
Basic reporting: We know that It is difficult to do experiment use yaks in Qinghai Tibetan plateau. However the importance of the present experiment is not so high, because we know that when the diet changes the microbiota in the rumen change accordingly. We do research we can come to a conclusion that will guide the herdsman to farm yaks and increase economic income or protect the grassland, however neither the aims come true.
Experimental design: It is said in line 87 that ten healthy yaks were selected, then in line 88 thirty cattle without supplementary feeding were allowed to graze, I do not know how the experiment was designed.
Validity of the findings: The importance of the findings of the study is not so high I think.
Additional comments: Line 13 and line 31 P<0.05   P<0.05  P < 0.05 , please keep the format in line with the requirement of the journal. And other places in the whole paper. Line 285 reference et al or et al ? check the whole paper please.
Line 50   line 54 reference  &   or  and   ?  check the paper carefully other places
Line 100   24 hours   or  h   ?
Line 105  you did not show the  pH value of the rumen liquid, it is not necessary to declaim how to determine the pH value.
Line 111-112  how to determine the CP EE ADF NDF,  add the references ! AOAC ? id 984.02 ? van soest 1991 to determine NDF ADF
Line 133-134  I suggest you use SAS to analyze data and do not use GLM model.
Table 1 you should present SEM and P value of data
Tabel 2-7 why you present reg (%)  g (%) in the tables ?
Table 3-5  the phylum  family and genus,  the  importance of the three tables are not so high, why you do not present order class ? or just delete family.
Fig 4  why you only present the difference between female and male ? what about the difference among three seasons you investigated, that is the important point ?


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

Input:
Title: YAK RUMEN MICROBIAL DIVERSITY AT DIFFERENT FORAGE GROWTH STAGES OF AN ALPINE MEADOW ON THE QINGHAI-TIBET PLATEAU
Review round: 1
Reviewer: 3

Output:
Basic reporting: The study is both interesting and focused on the real changes of the rumen in the animals studied. However, the structure and presentation of the manuscript could be better if the authors improve several items.
Tittle should emphasize characteristics of the study more than the techniques because it is not a paper describing a new methodology, it is a paper that describes microorganisms. It would be better to consider the species of the animal in the title, but in the paragraphs use the word yak. The phrase ‘during different phenological periods in the montane grasslands of the Qinghai-Tibet Plateau’ of the line 10-11, it is adequate to be included in the title.
Authors did not select the best keywords for the study. For example, the word forage had to be considered. I suggest making a new selection of keywords.
Regarding introduction, the authors did not describe in detail previous studies about the abundance of microorganisms in the rumen of yaks. Perhaps the information of the rumen of the animal living in the same zone could support the reason of the study.
Regarding grammar, authors used capital letters after a comma, did not respect the space after a full-stop, the expression et al was not written in cursive. Sometimes they used verbs in present when they should be written in past. Authors should revise and correct the entire manuscript.
Line 18 The most abundant phyla were, not are.
Line 18- 19 The phyla Bacteroidetes and Firmicutes are mentioned twice.
Line 20 The word respectively is not adequate in this context. Authors should mention all the percentages related to the phyla or re-write the sentence.
Line 26-27 The ruminal microbial flora was, not is.
Line 30 The word abundance would be better in this case, you use the word changes twice in the same statement.
Line 42 It is not clear if this climate change is referred about differences in the weather, or in the process that is still in discussion if it is real o not.
Line 46 Components of plants affect the nutritional state, and this situation affects animal growth.
Line 49 Archaea do not produce volatile fatty acids, they use them to produce methane. Authors should consider the real role of the microorganisms in the rumen.
Line 53-64 Authors should mention the microorganisms that have been detected in previous studies.
Line 62 If there are not several studies of the microorganisms in rumen, authors should emphasize the use of high- throughput sequencing of their research. DNA pyrosequencing is related to the platform 454 of Roche. Currently, there are several platforms to sequence DNA of microbial communities. The word high- throughput sequencing is better to use in this case.
Line 69 Define what is a short period of time would help to support the importance of to do a study for a long time.
Line 70 Provide a short definition of phenological periods.
Line 71 Delete the word therefore.
	Why did the authors select only forage as food in the study? It is not clear if the animals can eat other different kinds of food.
Experimental design: Authors mentioned the three stages of the forage: regreening, grass and withered grass. It would be better the use of abbreviations for these words in all the manuscript. In addition, the description of the information they would be to use the same order to describe the three stages. For example, first they could mention to regreening, then grass and the last withered. These suggestions to make easier to read the manuscript since in some paragraphs is a little difficult. If the authors possess photographs of the forage in the stages mentioned that could support the idea mentioned above.
Line 88 Which was the role of thirty cattle?
Line 94 To know the proportion (in percentage) of every species could be interesting for the discussion of the results. It will be adequate to consider if it has been described the components of the walls of the plants that constitute the forage and to find a relation with the results obtained in the prediction and the abundance of the microorganisms.
Line 96- 101 Authors mentioned the sessions in the abstract, they should relate samples with them in this part of the methodology.
Line 105 The stomach of ruminants possesses different zones. It would be interesting to know the zones that have been described in yaks and where samples were taken. It is not clear if the methodology to take samples was described previously or it was a procedure implemented in this study.
Line 106 Begin the statement with the word twenty, not 20.
Line 109 How many measurements did the authors do?
Line 115 Mention the number of samples that were analysed.
Line 117 If you analyse bacteria, you analyse prokaryotic no eukaryotic.
Line 119 It is necessary to mention the reference of the primers.
Line 124 It is necessary to mention the database used to do the taxonomic classification.
Line 132 Add the title; statistical analyses.
Validity of the findings: Authors presented Venn diagram, but they do not describe in the results the figure.
The tables present the term pooled-SEM or observed species that were not mentioned in the manuscript.
Authors did not present bar-charts of the relative abundance that would help to understand better the results.
Some statistical analysis can compare physicochemical parameters and abundance of microorganisms, such as Canonical Correspondence Analysis, that were not considered and could give information of the most important components of the forage that had influence in the microorganisms.
The prediction of the metabolic function is adequate; however, I consider the focus in the abundance of microorganisms is most important because they are a real result, not a prediction.
Line 138- 148 Authors mentioned the significant differences of the results; however, they did not compare the differences among the samples. For example; a presented 10% more protein than B. Authors should rewrite the paragraph and consider the differences detected among the samples.
Line 146- 148 If authors know the percentage of each plant, they should incorporate the information into tables.
Line 152 To describe in methodology what animals were selected. In methodology is only specified 10 yaks and another group that was not described in detail.
Line 160 It is better to use the word gender in this case, not sex.
Line 160-162 If the gender of animal presented differences, this would be part of the abstract.
Line 171 Why did the authors consider only the 15 most abundant species? Microbial communities always possess microorganisms in a low abundance as well as uncultivated microorganisms. It is adequate to mention them because they can possess a high activity and an important role in the degradation of the organic matter, even though they are not present in a high abundance or their function is not known.
Line 185 It is better to write; of different genera, no sexes.
	   To relate genera with phyla could help to understand the taxonomic groups that present more changes. Families would also be associated to a phylum.
Line 253- 263 This paragraph would be in the introduction because it is the reason of the selection of specific times to take samples.
Line 274 There are previous studies that describe pathways to degrade cellulose in Firmicutes and Bacteroidetes.
Line 294 Synergistetes is related to syntrophic associations with archaea to produce methane.in anaerobic environmental.
Line 312 What is the meaning is sex x stage?
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: YAK RUMEN MICROBIAL DIVERSITY AT DIFFERENT FORAGE GROWTH STAGES OF AN ALPINE MEADOW ON THE QINGHAI-TIBET PLATEAU
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors use clear, professional English to convey a thorough background literature introduction, methods used, results obtained and a discussion of findings. Article formatting, figures, tables, and raw data are sufficient for publication. In the future, please always provide sequence files as ".fastq" (fasta files with base quality information) rather than ".fasta" (no base quality information).

The conclusions of the study are well-aligned with the scope of the work. Namely, that this is an initial exploration (small N) of seasonal and sex-based changes in yak rumen microbiomes on QTP range and that findings are of primary utility to the development of more robust future inquiry.
Experimental design: The presented work is original and within scope.

The research question presented is "how do bacterial composition and bacterial composition-inferred function of the yak rumen differ across season and sex on the QTP?" This question is presented as meaningful given the importance of yak as livestock to the agricultural region of the QTP and the importance of the rumen microbiome to efficient production as detailed in non-yak ruminants.

The methods used meet technical and ethical standards.

Technical methods are described in sufficient details as to be replicable.
Validity of the findings: Given the largely preliminary nature of the study and the complexities of long-term rangeland-based animal trials, I believe that the design is sufficiently robust to support the authors' conclusions.

Figures and data included in the submission appear to match and are treated in a statistically sound manner with the notable exception of the NCBI accession quoted under the data availability header. SRP 169600 is not associated with your study as far as I can tell. This must be amended.

Conclusions made are not overstated, address the research question, and identify future directions based on challenges discovered in the course of study.
Additional comments: I acknowledge the challenges the authors faced in conducting a long-term on-range trial. I hope to see the lessons learned from this initial investigation applied to focused future work with QTP yak livestock.