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: GIANT CELL TUMOR STROMAL CELLS: OSTEOBLAST LINEAGE-DERIVED CELLS SECRETE IL-6 AND IL-10 FOR M2 MACROPHAGES POLARIZATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a well writing manuscript with scientific sound. The article structure and figures presentation are organized in a clear and unbiased manner. The only concern from the reviewer is the gramma errors and typos in the manuscript. The authors should carefully go through the manuscript to revise the typos.
Experimental design: The goal of this manuscript is to identify the effects of giant cell tumor stromal cells in giant cell tumor progression. This is an interesting study and important to the field. To address this scientific question, the authors isolated GCTSCs and compared them with normal BMMSCs. In addition, they identified IL-6 and IL-10 secreted by GCTSCs in regulation of tumor macrophage polarization. The research aims and experimental designs  were well defined, and results were relevant to their hypotheses.
Validity of the findings: The authors identified the role of GCTSCs in regulation of tumor macrophage polarization. In addition, IL-6 and L-10 were discovered as the potential underlying mechanisms in this regulations. However, how IL-6/IL-10 can promote M2 macrophage polarization was not fully discussed. Please discuss more detail potential mechanisms in the paper.
Additional comments: This is an important and novel study. However, it is not clear how to apply these findings to clinical applications. The authors should carefully discuss and link their discoveries to future clinical trials and studies.


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

Input:
Title: GIANT CELL TUMOR STROMAL CELLS: OSTEOBLAST LINEAGE-DERIVED CELLS SECRETE IL-6 AND IL-10 FOR M2 MACROPHAGES POLARIZATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comments
Additional comments: In the present study, the authors investigated the effects of GCT stromal cells (GCTSCs) on proliferation, apoptosis, and polarization of human macrophages. The results indicated that GCTSCs expressed a similar panel of MSC-related markers but had higher proliferation and relative lower diﬀerentiation abilities compared with BMMSCs. Meanwhile, GCT CD14+ monocytes contain an increased proportion of CD206+ M2 macrophages as compared to normal human blood CD14+ monocytes. In addition, co-culture of GCTSCs with macrophages promoted M2 macrophage polarization and increased the release of IL-6 and IL-10 from GCTSCs. These findings suggest that GCTSCs contribute to GCT pathogenesis possibly by promoting M2 macrophage polarization via increased secretion of secret IL-6 and IL-10.  The reviewer has the following major points that need to be carefully addressed:
1)	Lines 90-91, it is stated that “Normal MSCs were obtained from the bone marrow of C57 mice as described previously.”  It is questionable to use murine BMMSCs as compared with human GCTSCs in term of their immune profiles, proliferation, and multipotent differentiation capabilities.
2)	Lines 92-96, as is known, exogenous M-CSF is usually required to culture and maintain CD14+ monocytes isolated from peripheral blood. It is unclear whether this growth factor was included in the present study.
3)	In Figure 2b, the expression of a panel of MSC-related makers, STRO-1, SSEA-4, CD146, CD73, and CD90 was detected in GCTSCs by immunofluorescence (IF) staining, but NOT in BMMSCs. To support the conclusion, paralleled experiment should be performed in in BMSCCs. Meanwhile, the quality of IF is concerned with a relatively high background signal in nuclei while a relatively weak signal for CD146 and CD73. Flow cytometry analysis should be more convincing as was shown in Figure 2a.
4)	Figure 4a showed that CD14+ monocytes from GCT and peripheral blood contain 85.05% and 61.15% of CD206+ M2 macrophages, respectively. However, in Figure 2b, why was the percentage of CD206+ M2 macrophages among GCT CD14+ monocytes less than 10%? After co-culture with BMMSCs and three strains of GCTSCs, the proportion of CD206+ M2 macrophages in blood-derived CD14+ monocytes were 8.6%, 11.0%, 11.6%, 19.1%, respectively, which were much lower than the basal percentage (61.15%) of CD206+ M2 macrophages.  How to explain such discrepancies?
5)	As is known, IL-10 is one of the major anti-inflammatory cytokines secreted by M2 macrophages. It is unknown whether induced M2 macrophages also contribute to the elevated IL-10 levels in the co-cultured GCTSCs and macrophages.
6)	It cannot be definitely concluded that GCTSCs contribute to M2 macrophage polarization through elevated secretion of IL-6 and IL-10 unless neutralizing antibodies for IL-6 and IL-10 were utilized in 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: GIANT CELL TUMOR STROMAL CELLS: OSTEOBLAST LINEAGE-DERIVED CELLS SECRETE IL-6 AND IL-10 FOR M2 MACROPHAGES POLARIZATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The present manuscript entitled “Giant cell tumor stromal cells: Osteoblast lineage-derived cells secrete IL-6 and IL-10 for M2 macrophages polarization” describes the role of giant cell tumor stromal cells (GCTSCs) in the macrophage polarization by releasing of IL-6 and IL-10. Authors also investigated the mesenchymal stem cells (MSCs) characters of GCTSCs and the eﬀect of GCTSCs on macrophage proliferation and apoptosis.

The manuscript includes interesting and informative outcomes, however, this manuscript seems to be somewhat superficial and need further investigations to confirm the relation of macrophages polarization and IL-6, IL-10 released by GCTSCs. Therefore, the following revisions should be addressed to be able to publish.

1. Most of the experiments of this manuscript was based on the comparison of BMMSC and GCTSCs. However, the GCTSCs were isolated from human samples while the BMMSC were obtained from C57 mice. The results would be more reliable if the authors used human BMMSC as control.
2. The evidence that GCTSCs affect macrophages polarization by secreting IL-6, IL-10 was weak. Please do more experiments to testify this statement, for example, down-regulate IL-6, IL-10 of GCTSCs and investigate how it affects the polarization of macrophages.
Experimental design: What’s the purpose to test the proportion of M2 macrophage in human blood in figure 4?
Please add description or discussion.
Validity of the findings: no comment
Additional comments: Comments to the Author:
The present manuscript entitled “Giant cell tumor stromal cells: Osteoblast lineage-derived cells secrete IL-6 and IL-10 for M2 macrophages polarization” describes the role of giant cell tumor
stromal cells (GCTSCs) in the macrophage polarization by releasing of IL-6 and IL-10. Authors also investigated the mesenchymal stem cells (MSCs) characters of GCTSCs and the eﬀect of GCTSCs on macrophage proliferation and apoptosis.
The manuscript includes interesting and informative outcomes, however, this manuscript seems to be somewhat superficial and need further investigations to confirm the relation of macrophages polarization and IL-6, IL-10 released by GCTSCs. Therefore, the following revisions should be addressed to be able to publish this work on this journal.

There are some issues that the author should clarify as follows:
1. Most of the experiments of this manuscript was based on the comparison of BMMSC and GCTSCs. However, the GCTSCs were isolated from human samples while the BMMSC were obtained from C57 mice. The results would be more reliable if the authors used human BMMSC as control.
2. The evidence that GCTSCs affect macrophages polarization by secreting IL-6, IL-10 was weak. Please do more experiments to testify this statement, for example, down-regulate IL-6, IL-10 of GCTSCs and investigate how it affects the polarization of macrophages.
3. Please modify the title of the manuscript as the research didn’t provide convincing evidence of the key role of IL-6, IL-10.
4. What’s the purpose to test the proportion of M2 macrophage in human blood in figure 4?
5. The language should be revised, as there were some unclear points that was distracting and impedes the scientific presentation. Moreover, proofreading is needed since there are several grammatical errors and misspelling words in the manuscript.


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

Input:
Title: GIANT CELL TUMOR STROMAL CELLS: OSTEOBLAST LINEAGE-DERIVED CELLS SECRETE IL-6 AND IL-10 FOR M2 MACROPHAGES POLARIZATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors addressed all my concerns.


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: GIANT CELL TUMOR STROMAL CELLS: OSTEOBLAST LINEAGE-DERIVED CELLS SECRETE IL-6 AND IL-10 FOR M2 MACROPHAGES POLARIZATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have carefully addressed all the reviewers' comments and revised the manuscript accordingly. Therefore, the manuscript is acceptable for publication in this journal.


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

Input:
Title: GIANT CELL TUMOR STROMAL CELLS: OSTEOBLAST LINEAGE-DERIVED CELLS SECRETE IL-6 AND IL-10 FOR M2 MACROPHAGES POLARIZATION
Review round: 2
Reviewer: 3

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


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

Input:
Title: ADDITIONAL RECORDS AND STRATIGRAPHIC DISTRIBUTION OF THE MIDDLE EOCENE CARETTOCHELYID TURTLE ANOSTEIRA PULCHRA FROM THE UINTA FORMATION OF UTAH, NORTH AMERICA
Review round: 1
Reviewer: 1

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: Dear Editors, Dear Authors,

this manuscript pertains to the description of new shell material of the somewhat enigmatic fossil turtle Anosteira pulchra from the Uintan of North America. The authors correctly highlight that this turtle has only been known by few previous occurrences. I am therefore very pleased to see that the authors were able to pull together so many specimens from different institutions to paint a more meaningful picture of this turtle. It is also wonderful to see that so many museums take their mandate so seriously of providing the scientific community access to unpublished public specimens in their care. I only wished this practice was more widespread.

All in all, this is a well-written manuscript that provides important new information. The geological section and stratigraphic work, of course, are outstanding. I nevertheless note some minor weaknesses that should perhaps be addressed:

1) I notice a few problems with the figures:

a) The labels and objects in Figure 3 are too small, I fear. In addition, keep in mind that PeerJ maintains that unnecessary white margin on the left side of each page, which means that the figure will become even more difficult to read once published, unless the authors request specifically that the figure is published at full page length, which is often forgotten during the proofing stage. I, therefore, suggest rearranging the figure into portrait format and using larger labels. Figure 7 could profit from this as well.

b) I know that the figures are a little fuzzy due to compression associated with the submission process, but it seems to me that some pictures have poor focal depth (e.g., Fig. 3D, F) or are simply out of focus (Fig. 3M, N, R, S, T). I know that it is difficult to photograph small material, but amazing results can be achieved with a relatively basic SLR camera in combination with a photo stand, basic lighting, and focus stacking (an option included in Photoshop, so no special software is needed). If the authors are willing to give it another try, but need advice, I will gladly send instructions. Incidentally, I cannot judge the quality of the images in Figure 4, as even the high-quality version is terribly pixelated. I understand that it may not be practical to photograph material held in distant collections, so this is not the highest priority, as the figures certainly reveal what they are supposed to show.

2) I am a little disappointed by the taxonomic aspects of this manuscript for several reasons:

a) The authors state that the two named species from North America have traditionally been distinguished by differences to their neural formula, vertebrals, and the dorsal spines, but I am unaware of any literature pertaining to the dorsal spines, as these have not yet been described for A. ornata (and were barely highlighted for A. pulchra by Clark, 1932). Please clarify or modify.

b) The authors failed to highlight that the new material negates purported differences in the neural formula between A. ornata and A. pulchra, as both species now seem to have hexagonal neurals with short anterior sides. The authors nevertheless note that differences exist in regards to "additional vertebral scales that surround the costo-neural region," but I have no idea what they are talking about. Indeed, as far as I can tell, there are no reasons left to conclude that two species are present. I already alluded to this in Joyce (2014), Havlik et al. (2014), and Joyce et al. (2018). I, therefore, would like to encourage the authors to either highlight more clearly what they believe to be the remaining differences, or synonymize the two, or at least highlight that synonymy may be reasonable.

3) The authors compiled a wonderful set of stratigraphic data, but do not really use it to draw any conclusions. Are any correlations available that might reveal something about the paleoecology of Anosteira, for instance a correlation with certain depositional systems, faunas, floras, or climate signals?

4) I find the discussion regarding plastral kinesis to be suboptimal as is:

a) The authors claim that kinesis is present, but do not state where. Indeed, the only sentence were this is discussed seems to grammatically imply that kinesis in the peripherals, which is doubtful. I therefore suggest spelling out to the reader where they believe kinesis to be found.

b) The presence of kinesis in carettochelyids is not really new, so announcing its presence for this taxon should be muted. Also, the authors extensively cite the work of Bramble in regards to plastral kinesis, but I cannot find any discussion of kinesis in carettochelyids in the cited papers. A quick literature search suggests instead that plastral kinesis had previously been noted for carettochelyids by Meylan (1988, Peltochelys), Meylan and Gaffney (1989, Adocus), or Hutchison et al. (2004, Burmemys), to name a few.

c) The authors state with doubt that posterior plastral kinesis is present in the females of Allaeochelys. Why is there any doubt?

d) Indeed, given the published record of sexual dimorphism in regards to posterior plastral kinesis, why don't the authors flag similar variation found in their sample?

A small number of additional points are highlighted in the attached PDF.

All in all, the requested changes are rather minor. I therefore highly recommend publication with modifications.

With best regards,

Walter Joyce


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: ADDITIONAL RECORDS AND STRATIGRAPHIC DISTRIBUTION OF THE MIDDLE EOCENE CARETTOCHELYID TURTLE ANOSTEIRA PULCHRA FROM THE UINTA FORMATION OF UTAH, NORTH AMERICA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This report reveals the relative abundance of Anosteira pulchra (Carettochelyidae) based on numerous unpublished materials during the middle Eocene in North America. Thus, this will offer a significant data on the taxonomic diversity of non-marine turtles during the Paleogene.
Experimental design: A comparative figure of A. pulchra and A, ornata would be useful for readers, if any.
Validity of the findings: This report seems important, as carettochelyids are relatively rare turtles in the fossil record in North America. This reveals the relative abundance of this family based on numerous unpublished materials during the middle Eocene in this area.
Additional comments: This report seems very important for understanding the morphological and stratigraphic diversities of hitherto little known A. pulchra. I hope this will be published very soon.


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: ADDITIONAL RECORDS AND STRATIGRAPHIC DISTRIBUTION OF THE MIDDLE EOCENE CARETTOCHELYID TURTLE ANOSTEIRA PULCHRA FROM THE UINTA FORMATION OF UTAH, NORTH AMERICA
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is an interesting and well-written manuscript. Several minor suggestions are indicated here (see General comments for the authors)
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This is an interesting and well-written manuscript. Several minor suggestions are indicated here:
- Avoid using the abbreviation “NALMA” in the abstract, moving “North American Land Mammal Age (NALMA)” to the introduction.
- Some abbreviated generic names are used to start sentences both in the abstract and elsewhere in the manuscript (e.g. “…North America. A. pulchra is…”, “…taxa (Gilmore, 1916). B. inflata has…”). No generic name should be abbreviated at the beginning of a sentence.
- At the end of the introduction the authors indicate: “However, targeted collecting in recent years of Uintan herpetofauna in a measured stratigraphic framework has yielded 95 carettochelyid specimens”. I suggest completing this sentence indicating how many of these specimens were unpublished.
- Abbreviation section: Please divide the information into two subsections, each with a title (i.e., Institutional abbreviations; Anatomical abbreviations)
- Newly Referred Specimens subsection of the Systematic Paleontology section: add some more information in the text, at least referring to the number of individuals.
- Figures 3 and 4: I suggest not including specimen numbers in the figures: that information is indicated in the captions, and that should be enough.
- Figure 3: I think that the font size used for the abbreviations of the plates is very small.
- Caption of Fig. 6: Abbreviations should be removed from here, as they are indicated in the section Abbreviations.
- Figure 7: I suggest adding a scale to know the size of all the specimens, and not just of one of them. In addition, I believe that the photographs of some specimens of which a drawing is supplied have not been added in this work, but have not been published in previous papers either: Please add photograph of all specimens of which a drawing is provided.

I'm available for any additional clarification that authors and editors may need.

Cordially,

Adán

--
Dr. Adán Pérez-García

http://dfmf.uned.es/biologia/personal/aperez/

Grupo de Biología Evolutiva, Facultad de Ciencias, UNED
Madrid, Spain


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: ADDITIONAL RECORDS AND STRATIGRAPHIC DISTRIBUTION OF THE MIDDLE EOCENE CARETTOCHELYID TURTLE ANOSTEIRA PULCHRA FROM THE UINTA FORMATION OF UTAH, NORTH AMERICA
Review round: 2
Reviewer: 1

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: I read the revised manuscript and the rebuttal latter and feel overall content with the implemented changes. I, therefore, suggest publication as is.
One final point: the layout team of PeerJ will likely squeeze the figures to only fill about two-thirds of the page to maintain the white margin of the left. If this is done, be sure to request that the figures are expanded to fill the full page. Thanks.
Walter Joyce


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: VALANGINIAN OCCURRENCE OF PELOMEDUSOIDES TURTLES IN NORTHERN SOUTH AMERICA: REVISION OF THIS HYPOTHESIS BASED ON A NEW FOSSIL REMAIN
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript entitled “Valanginian occurrence of Pelomedusoids turtles in northern South America: Revision of this hypothesis based on a new fossils remains” is concerned with the description of a new shell fragment of turtle from Rosablanca Formation (Early Cretaceous, Colombia). This discovery allows the author assigns to the described specimen to Pelomedusoides. Overall, this brief article is a good contribution with nice figures and interesting data. The text is clear and well written and the references are updated.
Experimental design: Previously, on the basis of fragmentary carapace bones (one left costal 5 and a left peripheral 9?) recovered from Rosablanca Formation (Late Valanginian) at northeast of Zapatoca town, Colombia,  Cadena (2011) suggested the assignation of these carapace remains to Podocnemidoidea taking into account the inguinal buttress that medially extends onto the visceral surface of the costal bone 5. Lately, Romano et al (2014) suggested that this character is highly variable between Pelomedusoids and in Testudines. Considering these carapace fragments from Early Cretaceous Colombia as undiagnostic

The new discovery of a shell fragment (a right portion of the shell bridge including a mesoplastron and peripherals 5-7) from Rosablanca Formation allows to Edwin Cadena to attribute this specimen to Pelomedusoides Incertae Sedis on the basis of proportions and shape of mesoplastron bone. I concur with this determination because previously Lapparrent de Broin (2000) in her phylogenetic analysis of Pelomedusoids recognized a rounded and lateral mesoplastron as a derived character of this pleurodiran clade.

The fossil material (UR-CP-0025) studied by Cadena is properly housed in Paleontological Collection of the Facultad de Ciencias Naturales from Universidad del Rosario, in Bogotá. The author for carapace estimation length of the fragmentary exemplar of Rosablanca Formation (UR-CP-0025) used two large Podocnemis expansa specimens and he set the scale to the one provided in the photos of the specimens
and measured the maximum length of both mesoplastra, peripherals 6, 7, and the total length of the carapace, and he established a simple linear regression. For comparative
purposes, the author illustrated the right portion of the shell of UR-CP-0025 and several extinct and extant panpleurodiran species.
Validity of the findings: Although the described turtle material is so fragmentary, preventing a detailed taxonomic determination, it is relevant due to the Valanginian mesoplastron outline and proportions suggest Pelomedusoids affinities, this record represents the earliest Pelomedusoids worldwide.
Additional comments: The manuscript entitled “Valanginian occurrence of Pelomedusoids turtles in northern South America: Revision of this hypothesis based on a new fossils remains” is concerned with the description of a new shell fragment of turtle from Rosablanca Formation (Early Cretaceous, Colombia). This discovery allows the author assigns to the described specimen to Pelomedusoides. Overall, this brief article is a good contribution with nice figures and interesting data. The text is clear and well written and the references are updated.
Previously, on the basis of fragmentary carapace bones (one left costal 5 and a left peripheral 9?) recovered from Rosablanca Formation (Late Valanginian) at the northeast of Zapatoca town, Colombia,  Cadena (2011) suggested the assignation of these carapace remains to Podocnemidoidea taking into account the inguinal buttress that medially extends onto the visceral surface of the costal bone 5. Lately, Romano et al (2014) suggested that this character is highly variable between Pelomedusoids and in Testudines. Considering these carapace fragments from Early Cretaceous Colombia as undiagnostic

The new discovery of a shell fragment (a right portion of the shell bridge including a mesoplastron and peripherals 5-7) from Rosablanca Formation allows to Edwin Cadena to attribute this specimen to Pelomedusoides Incertae Sedis on the basis of proportions and shape of mesoplastron bone. I concur with this determination because previously Lapparrent de Broin (2000) in her phylogenetic analysis of Pelomedusoids recognized a rounded and lateral mesoplastron as a derived character of this pleurodiran clade.

The fossil material (UR-CP-0025) studied by Cadena is properly housed in Paleontological Collection of the Facultad de Ciencias Naturales from Universidad del Rosario, in Bogotá. The author for carapace estimation length of the fragmentary exemplar of Rosablanca Formation (UR-CP-0025) used two large Podocnemis expansa specimens and he set the scale to the one provided in the photos of the specimens and measured the maximum length of both mesoplastra, peripherals 6, 7, and the total length of the carapace, and he established a simple linear regression. For comparative
purposes, the author illustrated the right portion of the shell of UR-CP-0025 and several extinct and extant panpleurodiran species.

Although the described turtle material is so fragmentary, preventing a detailed taxonomic determination, it is relevant due to the Valanginian mesoplastron outline and proportions suggest Pelomedusoids affinities, this record represents the earliest Pelomedusoids worldwide.

Minor comments
In lines 149-150 Edwin Cadenan says “… the primitive condition exhibited by Platychelyidae and Cretaceous members of Pan-Chelidae (Fig. 3A–C, G–I)” should said “ … the primitive condition exhibited by Platychelyidae, Cretaceous and Paleocene members of Pan-Chelidae (Fig. 3A–C, G–I)”    (see Paleocene panchelids with mesoplastra in Bona, 2006, Bona and de la Fuente, 2005)

In line 217,  in reference list,  Edwin Cadena says “ de la Fuente, MS, Umazana, AM , Sterli J, and Carballido, J.L. 2011…”, should said “ de la Fuente, MS, Umazano, AM , Sterli J, and Carballido, J.L. 2011…”


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: VALANGINIAN OCCURRENCE OF PELOMEDUSOIDES TURTLES IN NORTHERN SOUTH AMERICA: REVISION OF THIS HYPOTHESIS BASED ON A NEW FOSSIL REMAIN
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an interesting and well-written manuscript. Several minor suggestions are indicated here (see general comments)
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This is an interesting and well-written manuscript. Several minor suggestions are indicated here:

- I suggest considering the geographical figure as Figure 1 instead of 2, and citing it for the first time in the last paragraph of the Introduction.
- I do not understand why the carapace length estimation is limited to the comparison with a single extant form. The shell morphology of the Valanginian taxon is unknown. Therefore, I suggest also add comparisons with other pleurodires with different proportions and morphologies of the carapace. In this sense, I would also include extinct forms (as is the case, among others, of Francemys, recognized in this work as having a similar evolutionary degree).
- I suggest moving the statement indicated as “Remarks” in the “Systematic Paleontology” section to the “Dicussion”, and justifying it with references.
- The author indicates “Another transformation of mesoplastra inside Pan-Pleurodira is their complete lost in Dortokidae, Araripemydidae”. However, I suggest changing this for “… complete lost in some Dortokidae, some Araripemydidae”: mesoplastra are present in the dortokid Eodortoka and in the araripemydid Taquetochelys.
- Figure 1: I suggest using a dashed line to represent the broken edges: in the current version of the drawings it is not possible to know if, for example, the anterior margin of the mesoplastron, or the lateral margin of the peripherals, are or are not broken.
- Caption of Fig. 1: Please, complete the following sentence, indicating the age and locality: “UR-CP-0025 Pelomedusoides shell bridge fragment FROM…”
- Caption of Fig. 2: Please also add more information in the sentence “Geographic location of the fossil finding STUDIES HERE, CORRESPONDING TO… FROM…”
- Figure 2: I suggest a separation between the map of South America and that of Colombia like that between those of Colombia and the Santander Department.

I’m available for any additional clarification that author and editors may need.

Sincerely,

Adán

--
Dr. Adán Pérez-García

http://dfmf.uned.es/biologia/personal/aperez/

Grupo de Biología Evolutiva, Facultad de Ciencias, UNED
Madrid, Spain


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: VALANGINIAN OCCURRENCE OF PELOMEDUSOIDES TURTLES IN NORTHERN SOUTH AMERICA: REVISION OF THIS HYPOTHESIS BASED ON A NEW FOSSIL REMAIN
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: The data that have been provided are not robust: a simple linear regression using only two specimens do not provide any statistical power. There is a huge confidence interval, resulting in a unreliable estimate. The linear model for the estimate is imprudent. To improve this, I suggest remove shell size stimatives from manuscript or redo the analysis by measuring as many especimens as possible.
Additional comments: Comments and suggestions throughout the manuscript are attached in the following PDF document: peerj-reviewing-49823v1.


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: VALANGINIAN OCCURRENCE OF PELOMEDUSOIDES TURTLES IN NORTHERN SOUTH AMERICA: REVISION OF THIS HYPOTHESIS BASED ON A NEW FOSSIL REMAIN
Review round: 2
Reviewer: 1

Output:
Basic reporting: This manuscript contains relevant information about the early history of Pelomedusoides. In addition, it is well structured, with a well-written text and good images that provide clear comparative osteology.
Experimental design: No comment.
Validity of the findings: The description of the material is consistent with that illustrated and compared in the manuscript. Furthermore, the author improved the data by measuring new specimens, as suggested. The conclusions are clear and have now been pointed out after the discussion.
Additional comments: This manuscript contains relevant information about the early history of Pelomedusoides. In addition, it is well structured, with a well-written text and good images that provide clear comparative osteology.

The description of the material is consistent with that illustrated and compared in the manuscript. Furthermore, the author improved the data by measuring new specimens, as suggested. The conclusions are clear and have now been pointed out after the discussion.

I congratulate the author for the excellent work  and I am available for any clarification.

Sincerely,

Natália.


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: HIGH FAT-FED GPR55 NULL MICE DISPLAY IMPAIRED GLUCOSE TOLERANCE WITHOUT CONCOMITANT CHANGES IN ENERGY BALANCE OR INSULIN SENSITIVITY BUT ARE LESS RESPONSIVE TO THE EFFECTS OF THE CANNABINOIDS RIMONABANT OR Δ(9)-TETRAHYDROCANNABIVARIN ON WEIGHT GAIN
Review round: 1
Reviewer: 1

Output:
Basic reporting: The written expression in this manuscript makes the content and arguments difficult to access and even appear to be logically flawed at times. Some examples where the language could be improved are:
1.	The title of the manuscript, High fat-fed GPR55 null mice display impaired glucose tolerance without concomitant changes in energy balance or insulin sensitivity but are less responsive to the effects of the cannabinoids rimonabant or Δ(9)-tetrahydrocannabivarin on weight gain’, implies that GPR55 is NOT responsible for THCV mediated ‘weight gain’ whereas the authors concluded in the abstract that THCV and rimonabant ‘reduced weight gain’, and this effect was in part mediated by GPR55.
2.	The statement ‘By contrast with the wildtype mice, the fall in blood glucose after giving insulin to the knockout mice reached statistical significance in the THCV-treated mice (Figures 9B, 9D) but there was no effect of genotype on the fall in blood glucose concentration with any treatment (see Figure 8D for absolute values)’ needs to be rephrased to make the argument easier to follow. Such confusing and ambiguous statements have appeared throughout the manuscript and they make the content of the manuscript difficult to comprehend.
3.	In ‘The increase in energy expenditure elicited by rimonabant in knockout mice was not significant when compared to either control knockout or wild-type dosed mice’, the terms ‘control knockout’ and ‘wildtype dosed mice’ should be clarified.
4.	The authors have also used the following sentence multiple times in the figure legend and it is not clear what does ‘all values are given per mouse’ actually mean.
5.	It was stated in line 187-190 that ‘Food intake (results not shown), energy expenditure (Figure 5), body composition measurements (Figure 6) and fat pad weights (Table 3) also confirmed the negative results of the preliminary study’, here ‘the negative results of the preliminary study’ should be specified.

The introduction has addressed relevant background and briefly reviewed some of the key publications in the topic area. However, it was reported in a 2019 study (Ruz‐Maldonado et al), which examined the effect of Rimonabant and its analogue AM251 on insulin secretion using wildtype and GPR55 knockout mice, that Rimonabant stimulates insulin secretion via a GPR55-independent pathway. This is in direct contrast to the findings in the current manuscript that rimonabant reduced plasma insulin levels in wildtype but not GPR55 knockout mice, which suggest that the effect on plasma insulin was mediated via GPR55. The Ruz-Maldonado study also demonstrated that Rimonabant stimulates insulin secretion whereas the current study concluded that Rimonabant reduced plasma insulin levels. Although differences in in vivo and in vitro experimental settings may impact on the results, the Ruz-Maldonado study is highly relevant to the current study, should be referenced, and the discrepancies between the two studies should be addressed in this manuscript.

Figures in their current state are of very low resolution and axis labels are not visible at all in many places.
Experimental design: Despite a relatively well established role for GPR55 in regulating islet function and insulin secretion in vitro, GPR55 knockout mice do not have an apparent metabolic phenotype. It is therefore, interesting to examine whether there are any metabolic abnormities in GPR55 knockout mice under conditions of increased metabolic demand such as during diet induced obesity. The research topic of this manuscript is within the scope of the journal and the research question is of importance. However, the current manuscript has significant flaws in the design of the study, lacks accurate and sufficient details in descriptions of experimental design and methodology and failed to present some of the key results (e.g. insulin secretion/concentration and insulin content, detailed in the beginning of this review). As a result, it is very difficult to interpret the findings of this study and the study, as it stands, lacks sufficient evidence for a coherent conclusion for the research question to be drawn.

With the exception of detailed description of experimental animals, the authors offered absolutely no detail on how all of the metabolic parameters under investigation were conducted. All methodologies have been referred to previously published papers but some methods, such as the insulin tolerance tests, were in fact not described in the corresponding reference (i.e. Wargent et al 2013 was provided as a reference for insulin tolerance tests in line 137-138. However, there are two Wargent et al 2013 papers on the reference list and none of these two papers have any description on insulin tolerance test).

In terms of the experimental design, it wasn’t clear at all why various metabolic parameters were measured at particular stages of HFD feeding and comparison of these parameters, measured at different times during the HFD feeding, have been used to draw conclusions from the study. For instance, the authors speculated that ‘energy expenditure’, being ‘measured during days 25 to 29 only, may not have been a reflection of the whole period of the study’. ‘Technical reasons’, as explained by the authors, can not account for the lack of careful consideration when designing the study. Another example is that in table 2, it was stated that ‘Food intake was measured daily when the mice were between 6 and 15 weeks of age, body composition by DEXA and NMR when they were 14 and 15 weeks old respectively, energy expenditure when they were 10 to 11 weeks old, and locomotor activity when they were 13 weeks old’. The rationale for taking these measurements at these specific times should be explained. It should also be noted that under conditions of diet induced obesity, these parameters are undergoing continued change and adaptative responses towards prolonged diet modification are of great physiological relevance. As such, body composition measured when mice are 14 and 15 weeks old may not directly reflect what had happened in energy expenditure and locomotor activity (which were again measured at different time points) when mice are 10 weeks old. Furthermore, in figure 3, GTTs and ITTs were conducted ‘at age 13 weeks’ following HFD feeding since presumably age 6, whereas it was stated in Figure 9 legend that ‘there was no significant difference in the insulin tolerance curves on day 38 between the vehicle-treated knockout mice and the vehicle-treated wild-type mice (Figures 9A and 9B)’. In fact, inconsistent descriptions such as ‘Fat pad weights were measured AT TERMINATION ON DAY 56’ (table 3 legend) vs ‘our mice WERE KILLED AT 15 WEEKS OF AGE’ (line 276-277) are present throughout the manuscript; such discrepancies and/or inconsistencies in the description of key technical details make it very difficult to decipher the results obtained in this study.

Also, ‘basal’ measurements of all metabolic parameters between the wildtype and the GPR55 knockout mice should be reported to exclude any potential phenotypic differences at the start of HFD feeding.
Validity of the findings: In this manuscript entitled ‘High fat-fed GPR55 null mice display impaired glucose tolerance without concomitant changes in energy balance or insulin sensitivity but are less responsive to the effects of the cannabinoids rimonabant or Δ(9)-tetrahydrocannabivarin on weight gain’, Wargent and colleagues described their study investigating the impact of high fat diet (HFD) feeding on the metabolic parameters of the GPR55 knockout mice. Although GPR55 knockout mice fed HFD displayed impaired glucose tolerance, no significant differences in body weight, adiposity, energy balance, locomotor activity, insulin sensitivity between wildtype and GPR55 knockout mice have been seen. The authors speculated, in the discussion, that impaired insulin secretion upon GPR55 deletion could account for the impaired glucose tolerance. However, it was described in the result that ‘pancreatic insulin concentration and pancreatic total insulin content were not affected by genotype irrespective of diet’ (data from these experiments weren’t presented and it wasn’t clear how these were measured and hence the difference i.e. is the ‘pancreatic insulin concentration circulating insulin levels and if so, basal or stimulated?). In addition, Figure 3B demonstrated that plasma insulin levels in GPR55 knockout mice fed HFD was significantly higher 30 minutes after glucose injection. This is not coherent to the lack of changes in ‘insulin concentration and insulin content’ described earlier, and is not a plausible explanation to support the hypothesis that impaired insulin secretion in GPR55 knockout mice accounts for the glucose intolerance in these mice. The authors also examined whether the beneficial effects of THCV and Rimonabant on energy balance and glucose homeostasis in HFD fed wildtype mice (reported by other publications) are modulated by GPR55 deletion. Although the effect of rimonabant on reducing body weight was impaired in HFD-fed GPR55 knockout mice, the authors did not obtain conclusive evidence to explain the mechanisms involved in this.

The authors have also used flawed arguments such as:

The most likely explanation for the effect of genotype on body weight gain in response to rimonabant and THCV is that the compounds elicited a lower increase in energy expenditure in the knockout than the wild-type mice. These differences WERE NOT STATISTICALLY DIFFERENT, but the differences in the means may be sufficient to account for the differences in body fat content (line 353-356);

and

Other workers (Lipina et al., 2019) have reported that GPR55 knockout mice have impaired insulin sensitivity but this may be because their mice displayed increased adiposity. In fact, although they found that blood glucose fell significantly in wild-type but not knockout mice following administration of insulin, they did not find a significant difference between genotypes (line 303-306).

The authors attempted to conclude that GPR55 interacts with insulin signalling in a more direct way than via increased fat mass. To draw such a conclusion, it is absolutely essential to include data from experiments directly measure insulin secretion. This should both be conducted by measuring insulin secretion and insulin content from islets isolated from HFD-fed wildtype and GPR55 knockout mice, and by measuring plasma insulin before and after a glucose challenge. Indeed, the findings in figure 3B where GPR55 knockout mice had a greater insulin levels in response to glucose injection seem to be directly contradict to this conclusion.
Additional comments: Although it is a potentially interesting research question, the manuscript at its current state requires a significant revision in terms of both the study design and the way the results are interpreted and the way this study is reported.


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: HIGH FAT-FED GPR55 NULL MICE DISPLAY IMPAIRED GLUCOSE TOLERANCE WITHOUT CONCOMITANT CHANGES IN ENERGY BALANCE OR INSULIN SENSITIVITY BUT ARE LESS RESPONSIVE TO THE EFFECTS OF THE CANNABINOIDS RIMONABANT OR Δ(9)-TETRAHYDROCANNABIVARIN ON WEIGHT GAIN
Review round: 1
Reviewer: 2

Output:
Basic reporting: The figures are mostly very well presented, although a few need to be clearer – these are annotated in the text but specifically relating to figure 4 which is ineligible.
Experimental design: No comment.
Validity of the findings: The major findings of this paper are (i) that GPR55 KO mice fed a high fat diet show impaired glucose tolerance, and (ii) that the cannabinoids, THCV and rimonabant, have lesser effects in GPR55 KO animals with respect to weight gain, therefore, suggesting that the actions of these drugs are partly mediated via the GPR55. Whilst for most of the figures, statistical analyses are appropriate, there are some instances (eg. table 3), where a different statistical test should be performed.

Furthermore, the authors conclude that glucose tolerance is impaired in KO mice compared to the WT and preliminary data do support this. However, in later figures, where WT vehicle and KO vehicle are compared, these should show the same genotype differences. For example, in figure 9, if you directly compare the WT vehicle and KO vehicle curves, do they show the same differences as in figure 2 - this is important, particularly given that this is one of the major conclusions and the title of the manuscript. This also relates to a genotype comparison in figure 8 panel C.
Additional comments: The manuscript is generally well written and with clear aims/objectives and conclusions. The main conclusions of this study are that (i) GPR55 KO mice fed a high fat diet show impaired glucose tolerance, and (ii) that the cannabinoids, THCV and rimonabant, have lesser effects in GPR55 KO animals with respect to weight gain, therefore suggesting that the actions of these drugs are partly mediated via the GPR55.

Overall, the conclusions of the study are supported by the data presented. However, I have a few general points which should be addressed:
1.	What is the rationale behind using only male mice? It would be useful to state this in the methods. Have you conducted any similar analyses in female mice, and do they show similar genotype differences?
2.	Direct comparisons between the effects of the two treatments, THCV and rimonabant, cannot really be drawn (e.g. when discussing figure 4) without PK/PD data showing relative levels of exposure to each compound. Furthermore, data to show the efficacy of each drug at the GPR55 in vitro would be useful. This data may already be published, but it would enhance the current manuscript to compare the action of these two ligands at the GPR55 in vitro. This leads me to the question - is rimonabant having a greater effect in this dataset because it is more efficacious at the GPR55 or is the exposure higher to this ligand in vivo? The introduction and/or discussion should consider this point, and perhaps the statements around a greater effect of rimonabant on weight gain could be softened.
3.	Generally, it would be useful to ensure that statistical analyses and results of such analyses are outlined in every figure legend. There are some inconsistencies in the way that statistical comparisons are described.
4.	When comparing treatment effects and genotype effects, such as in table 3, a one-way ANOVA is not appropriate. These data should be analysed using a two-way ANOVA.
5.	It would markedly improve the manuscript if bar graphs were replaced with scatter charts so that the variability of data within each experiment is displayed.
6.	In all figures, are data displayed as means -/+ SEM or SD? This needs to be stated in the figure legends, as this is important when considering the statistical analyses.
7.	In figures 7 and 8, are there any effects of the two drugs on the KO? The trend seems to be similar for the WT, but this is not displayed as a significant effect? In figure 8 panel C, the comparison between WT vehicle and KO vehicle should be significant as this would be the same as in panel A – is this the case?
8.	For figure 9, if the authors were to directly compare the WT and KO control curves – would these show similar differences to earlier figures? I.e. do they support earlier conclusions and in fact the title of the manuscript?


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

Input:
Title: HIGH FAT-FED GPR55 NULL MICE DISPLAY IMPAIRED GLUCOSE TOLERANCE WITHOUT CONCOMITANT CHANGES IN ENERGY BALANCE OR INSULIN SENSITIVITY BUT ARE LESS RESPONSIVE TO THE EFFECTS OF THE CANNABINOIDS RIMONABANT OR Δ(9)-TETRAHYDROCANNABIVARIN ON WEIGHT GAIN
Review round: 2
Reviewer: 1

Output:
Basic reporting: In my previous review, I suggested that scatter charts could replace bar charts to display the variation between individual mice and I still think that this would markedly improve the presentation of the data.

In all figures, are data displayed as means -/+ SEM or SD? This needs to be stated in the figure legends, as this is important when considering the statistical analyses.
Experimental design: Previously I questioned the rationale for the sole use of male mice. I wonder if it is possible to state the reasons why comparisons have not been drawn in female mice as well.
Validity of the findings: In my previous review, I raised the following points:

1. It is difficult to discuss a comparison between the efficacy of two treatments, THCV and rimonabant, without consideration of the relative exposure levels. Is rimonabant having a greater effect in this dataset because it is more efficacious at the GPR55 or is the exposure higher to this ligand in vivo. This should be addressed in the discussion.

2. What is the explanation for why the WT vehicle control and KO vehicle control plasma insulin levels (Figure 8C) are not statistically different. Is this the same data as displayed in Figure 8A? Where the KO vehicle control is statistically higher than the WT vehicle control 30 minutes after the glucose load?

3. In figure 9 have the WT and KO control curves been directly compared and do any genotype differences here support the earlier conclusions in this manuscript?
Additional comments: The majority of my previous comments have been addressed, but there remain a few points outlined above that should also be considered.


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: EFFECTS OF 12-WEEK CADENCE RETRAINING ON IMPACT PEAK, LOAD RATES AND LOWER EXTREMITY BIOMECHANICS IN RUNNING
Review round: 1
Reviewer: 1

Output:
Basic reporting: See general comments
Experimental design: See general comments
Validity of the findings: See general comments
Additional comments: The study assessed the effect of cadence retraining on impact related variables and lower limb kinematics and kinetics. Overall the study is well written and examines an important research question. I have suggested some changes to improve the reporting of the manuscript. My main concern relates tot the presentation of results, where I am unclear as to what statistical tests have been used and reported. Please see specific comments in the points below.

1. Abstract – it would be nice see some data reported in the results section of the abstract. If you are tight on word limit perhaps present the most important results. Please see my comments in relation to statistical analysis and results as to what is appropriate to include in the results.

Introduction
2. Line 44 – please change patellofemoral joint pain synthesis to patellofemoral pain – as that is the current description of this condition. Also injuries to the lower leg have bene reported to be just as common as the knee in more recent publications. Please document this (e.g. Buist et al. BJSM 2010: 44:598-604; Franke et al. JOSPT 2019: 518-529).

3. Lines 46-52: In this section please differentiate those studies that have shown an association with injury (i.e. retrospective studies) to prospective studies which provide a greater level of evidence. In other words, is there prospective evidence for excessive impact forces and loading rates as a risk factor for running injuries?

Methods
4. Line 99 – you have indicated sampling rate twice in this sentence – please remove one duplicate.

5. Lines 111-118: Please provide more details in regard to the sequence of your experimental protocol. For example, were markers placed on participants and static calibration performed before or after the 5 min run on the treadmill?

6. Line 132: The Alfredson reference seems to be the incorrect reference here

7. Lines 135-137: How did the researchers ensure compliance to the retraining protocol? You mention that cadence was uploaded, but what was the action if participants were not achieving the desired cadence?

8. Line 138: please provide details on the weekly group training.What did you do in these sessions?

Results
9. Overall for your results you should be presenting the interaction effect as your main finding, not the time effects. If there was truly an effect of gait retraining on your variables there should be group differences at follow-up. Reporting changes over time as treatment effects are misleading. I am little confused as to how you present these group differences, for example is the p-value reported for impact peak the interaction effect, or the result of the independent t-test? You have reported in your statistics that independent or paired t-tests were used when a significant interaction was found, yet you haven’t reported where interactions were found, so what is the justification for following up with these tests? As per your methods if you did not find interaction effects you should not be reporting these post-hoc tests.

10. It would be useful if you could also present the 95% confidence interval of the differences for group effects (i.e. impact peak, step length, CoG excursion). Also please provide some magnitude of effect for the difference (i.e. ES)

11. Did you not find an interaction effect for cadence? The figure indicates that it may have been close to significance? Given this is your intervention it is surprising there was no group differences at follow-up.

Discussion
12. As per my comment in the results, overall in the discussion you need to indicate that only peak impact forces, step length and C0G excursion exhibited a group by time interaction. Thus these are the only variables that demonstrated differences related to time and group allocation. Much of your discussion is focused on variables that showed a time effect and this should be reported with some caution.

13. Line 242: you still have not indicated how you controlled compliance to the cadence retraining so find it hard how you can suggest this is a reason for the reported differences. Please provide details in the methods I regard to how you ensured compliance with the increased cadence.

14. Line 245: In the results you indicate a reduction of 12% in peak impact forces, which is different to that reported here.

Line 307: You need to temper your conclusions based upon my comments related to your statistical 15. analysis and presentation of results.


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: EFFECTS OF 12-WEEK CADENCE RETRAINING ON IMPACT PEAK, LOAD RATES AND LOWER EXTREMITY BIOMECHANICS IN RUNNING
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors are highlighting an important topic in running injury research especially gait retraining with emphasis on cadence retraining. There is evidence in the literature demonstrating that ( Willy et al.2016, Hobara et al. 2012 ) increasing cadence above one’s natural step rate appears to result in an immediate reduction in load rates. Willy et al. 2016 used a wireless accelerometer as a cue for 8 training sessions, to increase preferred step rate by 7.5% and this was effective at reducing impact forces, peak hip adduction and eccentric knee joint work in healthy runners.Hafer et al. also showed that that increased running cadence using cadence retraining reduces kinematics and kinetics that have been tied to overuse running injuries.
The introduction lacks clear justification for performing the study. The authors do not provide any deficiencies in the running literature to justify a need to do this study.
There is no clear distinction between increase in cadence or decrease in step length that is the focus of the study.  Hypothesis is not specific and focused enough.
 There is no justification for the amount of variables that were chosen for analysis in this study (kinematics and kinetics)

The raw data and the figures were helpful to understand the data.
The description of the data in the results in the data was muddled because of the lack of clarity if it was talking about the data within the CAD group or CON group (pre and post) or comparing the CAD and CON group.
Experimental design: There is no discussion about sample size calculations.
There is no information about how the randomization was performed for the two groups. If any of the investigators were blinded and who performed the randomization.
The marker set used for the gait analysis was not described.
There is no justification for using the 12 week retraining period or the 7.5% increase in cadence. They have cited a study but why they felt this protocol was appropriate for their study was not stated.
It is not clear why the runners ran on the treadmill prior to data collection .
Were three running trials enough to collect enough data points ? It wasn't mentioned for how many seconds every trial was performed. The assessment was performed at fixed speed but retraining occured at preferred speeds.The authors do not provide details of the weekly group training sessions .
There were many(atleast 17) biomechanical variables analyzed in this study and except the impact forces or loading rates there was no justification  for the rest of the variables. Which plane were the peak moments of hip, knee and ankle analyzed or was it an overall support moment?
 Some variables like vertical momentum at initial contact,maximum extension/dorsiflexion angle during the stance phase;maximum flexion/plantar flexion angle during the stance phase;centre of gravity,sagittal plane distance between ankle and knee/ ankle and hip  at initial contact are variables which have not been previously reported in the running literature. The validity of the overall findings is questionable without knowing the impact of these variables on injury status in runners. I am not sure if centre of gravity is what the authors use for centre of mass which is used in the running literature.

T-tests to look at differences between the two groups were not performed. Height of the participant has been shown to impact step length.
Validity of the findings: The authors do a good job using tables and figures to describe the data. The discussion section introduces the role of foot strike pattern which tends to shift the focus from cadence retraining . Previous literature has highlighted the role of forefoot strike pattern and decrease in average and peak loading rates( healthy runners and runners with tibial stress fracture)(Boyer et al., 2014; Shih et al., 2013,Yong et al.2018; Futrell et al.2018)
The novelty of this study is not clearly demonstrated by the authors. It rather seems like a validation of other studies or a replication of the protocol.
Additional comments: Abstract:
Line 17: Please use Injuries not injury status
Line 19: during running
Results section: This section needs numbers and p-values. It is very wordy.
Conclusion: Needs to be more impactful and not be similar to the introduction.
Introduction:
Line 36: Please provide the name of the report
Line 40: Please ensure that the citation is accurate. Typically, all running studies reference the RunningUSA.org for the statistics on participation
Line 44: Patellofemoral pain syndrome
Line 47: Consider using the word Impact rather than affect
References were nor provided for: Line 40, Line 49
Line 48-49: Please make sure you change it to running injuries everywhere rather than running injury status
Please rewrite the sentences for grammar corrections:
Lines 52: compared “ to “ healthy runners
Lines 58: decreased
Lines 59: indicate
Lines 59-65: There is no clear distinction between increase in cadence or decrease in step length that is the focus of the study.
Lines 73-77: There is no justification for the amount of variables that were chosen for analysis in this study (kinematics and kinetics)
Lines 81-83: The hypothesis needs to be clear and specific
Lines 85-95: Please perform sample size calculation and describe the randomization protocol. Was there any blinding of the investigators who performed data analysis and collected the data? Please specify if it was lower limb musculoskeletal injuries.
Line 100: Please describe which marker set was used.
Line 104: Please remove the word force before ground reaction force data
Line 112: Please describe why you had them run on treadmill
Retraining Protocol: Please describe why you did not collect data at preferred speed if they ran for 12 weeks at their preferred speed. What was the difference in speeds ? Decrease in running speed can lead to decrease in kinetic forces and viceversa. Please describe the group training sessions
Data processing: Please justify your  reasons for selecting so many variables for analysis. There are many variables which are not commonly used in the running literature(vertical momentum at initial contact,maximum extension/dorsiflexion angle during the stance phase;maximum flexion/plantar flexion angle during the stance phase;center of gravity,sagittal plane distance between ankle and knee/ ankle and hip  at initial contact ).  Does Center of gravity refer to center of mass? Which plane were the peak moments of hip, knee and ankle analyzed or was it an overall support moment?
Please perform t-tests for demographic variables between two groups. Height of the participant can impact step length.
Line 192-193: Revise for grammar.
Line 220,223,225: which group?
Line 240: Please explain why you think your study did not see the increase in cadence that you expected( 5.7% vs 7.5%) specifically if any biomechanical adaptations can explain this
Please link your results to cadence retraining and kinetic parameters linked to running injury rather than focusing on foot strike pattern. Can you describe how the results of the study can be used for future studies especially in participants with running injuries.


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: EFFECTS OF 12-WEEK CADENCE RETRAINING ON IMPACT PEAK, LOAD RATES AND LOWER EXTREMITY BIOMECHANICS IN RUNNING
Review round: 1
Reviewer: 3

Output:
Basic reporting: I commend the authors on undertaking and submitting an article on a very clinically relevant topic which should be of interest to readers.

The article examines the effect of a 12-week cadence retraining program on impact forces and lower extremity biomechanics. Whilst the authors' study findings do support the original research goals, their conclusion that reducing impact forces may decrease the risk of impact-related running injuries needs further exploration in the introduction. The link between running injuries and impact forces remains unclear which needs to be indicated.

The article is well written although some sections, including tables and figures, need further detail or clarification and I have included specific comments in the General Comments section below which I hope the authors will find helpful.
Experimental design: The research is within the Aims and Scope of the journal.

However, further elaboration of the introduction to the topic is required to justify the study and research question. Additionally, the link between impact forces and running related injuries needs to be explored further to justify any of the conclusions made in the abstract. The study primarily attempts to answer whether increasing running cadence can decrease loading rates in runners and should likely focus on this. There are additional studies, such as Yong et al. (2008) and Chan et. al (2018) which have reviewed the impact of gait retraining, including increasing cadence, on loading rates in runners and should be referenced within the article. This will strengthen the authors' findings and can help to justify the association with injury reduction.

Additionally, the methodology requires additional detail. There should be further justification regarding the chosen sample size and elaboration of the participants running experience and training history, where possible. This will assist with the application of the research findings.
Validity of the findings: There are some concerns regarding the methodological quality in terms of the reliability of assessing running gait over a force platform where participants have to place the foot. Further information on the experimental set-up would help.

Additionally, the validity and reliability of CONDOON app and use phones to measure running metrics will need to be included in the study to justify the findings.
Additional comments: INTRODUCTION
Line #42-43: “In addition, 19.4% to 79.3% of long-distance runners experienced 43 injuries” – Can you further elaborate why the large variance; for example, novice runners or those who run increased distance show higher injury rates.
Line #42-43: “whilst 63% of them had a history of lower extremity injury” – This is unclear who it is referring to. There is no reference to this in van Gant et. al. 2007
Line#44: “such as patellofemoral joint pain synthesis” – Remove word “synthesis”
Line#49: Can please you reference running retraining studies
Line#55: “Increasing the running cadence or decreasing the step length” - Remove “the”
Line#56: “at a certain speed” – What speeds
Line#58-59: “decreased” – Grammatical error. Changed to past tense
Line#60: “the cadence or reducing the step length” - Remove “the”. Also, increasing cadence and reducing step length are the same.
Line#66: “In regard” – Change to in regards
Line#67: Is 6 weeks defined as a “long term change”
Line#68-72: “Hafer et al. (2015) did not find significant changes in impact forces in another cadence retraining intervention, but the loading rates decreased after 6-week cadence retraining with a 10% increase in cadence. In addition, reductions in peak loading rates and average loading rates were observed after an eight-session cadence retraining intervention with a 7.5% increase in cadence”
-	Consider re-wording this clearer and separate the two studies.
Line#72: “cadence retraining modifies” -  If it decreased say it decreased loading rates
Line#75-77: “Assessing these additional variables may result in better understanding of cadence retraining on running biomechanics and its associated risk of injuries”
-	Are you assessing the risk of injury? You need to elaborate on the link between impact forces and running injuries.

METHODS
Good description
Line#89: How did you determine that participants were rearfoot strikers?
Line#102-103: Rewrite as 90cm x 60cm x 10cm
Line#111: Remove “that is”. Write at baseline or commencement and at the end of the program.
Line#115: I would be interested to see the experimental design in the appendix with a description of the run-up and force plate location. This is a limitation of the study as you are assessing running biomechanics with a limited run-up with a relatively small area for foot placement.
Line#121: How did you determine “preferred speed”
Line#123: Need to provide some data regarding the reliability and validity of CODOON running app in tracking running volumes, speed and cadence. How accurate are phones in measuring cadence? Have you controlled for the positioning of the phone and where will it be held during trials? ie. Held in hand or strapped to the arm.
Line#124” “The participants were asked to run outdoors for three times” – remove word “for”
Line#126 – Grammatical. Consider rewriting
Good summary of data processing and statistics. Recommend further statistical assessment prior to publication to validate the analysis.

RESULTS
Line#191-192: Consider summarising more clearly.
Line#197: “CAD: 23.3+/-22.9 km/week” – There appears to be an error in standard deviation calculations. I could not validate these numbers as only patient training characteristics at baseline were reported in the supplementary material. If not, can you explain the large variance in the training of the CAD group?

DISCUSSION
Consider elaborating some of the arguments in the introduction rather than introducing new arguments in the conclusion.

Lines#247-251: These arguments should be included in the introduction and can be referred back to in the discussion.
Lines#258-259: Consider re-wording
Lines#269-271: Do we have evidence that reducing loading rates can decrease injuries? Yong et al. (2008) “Acute changes in foot strike pattern and cadence affect running parameters associated with tibial stress fractures” has also explored this and should be referenced in the text.
Lines#275-277: Needs reference. Additional grammatical errors. Consider revising sentence.
Lines#296-297: “Although cadence increased by 5.7% in the CAD group after 12-week retraining, the 297 magnitude was only 170.5 step/min, leaving additional space for increasing cadence”
-	You have not reported on normative cadence values for runners. Why is there additional space? Also is there a ceiling effect or where increasing cadence can have a negative effect or reduce running economy. One participant increased to a cadence of 193 steps/minute.

CONCLUSION
Lines#309-311: “Increasing the cadence as a retraining method may reduce the risk of impact-related injuries” - Again, needs further link in the introduction to justify this.
Lines#310-312: “Meanwhile, the foot became “flatter” when its position was closer to the CoG at the initial contact after training, providing the mechanical explanation for the decreased impact forces” Elaborate on the mechanical explanation. Also, consider the change in vertical oscillation (excursion).
Lines#312-315: “Hence, cadence retraining can lead to the biomechanical adaptation of the entire lower extremity to the running pattern” – I am unsure what you are trying to get across here

REFERENCES
The references have some consistent errors. Consider removing DOI unless required.
Lines#331 and 408 have minor author errors.

TABLES AND FIGURES:
Table 1:
Elaborate or use key/legends groups (CAD and COG)
Label standard definitions
Table 2:
Consider using a line chart to illustrate
Table 3:
Variables are not labelled on the chart and it is difficult to refer to the note below. Consider full label on the table and in landscape mode.
Needs consistent use of decimal places
Need to label standard deviations
Figure 2-5:
Need to elaborate abbreviations (eg. POI Lah)
Consider expanding CAD and CON


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: EFFECTS OF 12-WEEK CADENCE RETRAINING ON IMPACT PEAK, LOAD RATES AND LOWER EXTREMITY BIOMECHANICS IN RUNNING
Review round: 2
Reviewer: 1

Output:
Basic reporting: My overall comments about the Introduction: You haven’t highlighted the need for the study, You just summarized the available literature and It seems like because lower extremity biomechanics has plethora of variables available for assessment, you are picking the one’s that have not been highlighted. I mentioned it in first review , please justify the need for the study and I feel this still lacks that.
Experimental design: The calculation of variables and statistical tests performed are valid.
My suggestion would be that If you still want to include all your variables( kinematics and kinetics) could you use linear regression techniques to evaluate which variables predict impact forces and loading rates?
Validity of the findings: The Discussion revolves around Impact forces, Loading rates , foot angle and some kinematic variables. In my opinion, It should focus more on mechanisms of changes and differences between the two groups . There is no mention about changes in control group and the differences seen with CAD group.

I would recommend rewriting the limitations.

The conclusion was strong and focused on the big picture points from the study and that is why I would recommend rewriting the paper to focus on the changes in impact forces, loading rates rather than including all the 30 variables.
Additional comments: Thank you for your resubmission and for addressing the comments. It is more clear now with what the study intends to do but I am still not sure about the need to include 30 variables ( kinematics and kinetics) to evaluate the changes of a 12 week cadence retraining program. In your Introduction, you suggest " insufficient evaluation of variables and small sample size of previous studies as the need to perform this study but your study also has a small sample size and you did not address why all the variables you included in your study are important to evaluate.
I have made specific comments below but I would recommend the focus of the paper be strictly on the kinetic variables(Impact forces and loading rates and how CoG and foot angle play a role to support your findings) You should also highlight your 12 week retraining program and how the compliance tracking and meeting participants was the novel part in this study.

Specific Comments:
Abstract:
Lines 16-17 : You only focus on Impact forces and vertical loading rates and then lines 18-19 you talk about Impact forces and lower extremity biomechanics. I would recommend being consistent both the times
Lines 22: Please describe if the control group ran without any changes in cadence
Lines 24: Please mention in the Cadence training group so we know you are talking about this group. If in the results you are including the data for some variables then you have to include it for all variables especially if all of them are significant.
Line 31: Grammar, it should be knee range of motion.
Line 33: I would include the % increase in cadence in the sentence.
Line 34: Grammar, I would recommend using induce not induced. I would also recommend using the word “leads to” instead

Introduction:
Lines 41-43: 5.83 million runners vs 50 million recreational runners Is a huge difference in participation numbers. Can you cite the either the numbers for participation in running events or recreational runners every year for both the countries.
Lines 43-45: I would consider removing the sentence on running has more injuries than other activites, it sounds redundant. You have already provided injury numbers.
Lines 43-46: I would move this sentence to after the Messier reference. In addition, 19.4% to 79.3% of long-distance runners experienced lower extremity injuries (van Gent et al. 2007).
Line 48: I would recommend not using the word in recent publications. Instead use literature or there is data to suggest
Line 51: Grammar, I would recommend using influence not impact
Line 52-53: I would recommend using gait retraining studies instead of in running retraining strategies
Lines 57-60: Please rewrite this sentence to indicate specifically what is the point that you are trying to make
Lines 60-66: You talk about Impact forces, then you jump in to energy absorption then you talk about Impact forces again till line 70. Line 70-75 you talk about in general all effects of increasing cadence . I would recommend bringing everything together and justifying why al these things are important and how are they related to your study. in my opinion,listing all changes in gait variables doesn’t support your paper, it seems like you are doing a review of literature rather than telling the reader why these variables are important.
Line 69: Be consistent using terminology: stick to decrease step length or reduced step length. They mean the same thing but it’s important to be consistent.
Line 76: I would take out the word long term
There is confusion if you are focused on impact forces or the impact phase of running or both
Lines 83-86: I would specificially list how many participants were there in those studies and why you think that is a small number especially considering your study had 12 in the experimental group. Why do you think 6 weeks is a short training intervention time? Do you think it is practically feasible to retrain someone’s gait for more than 6 weeks and is there literature to support that you need more than 6 weeks for gait retraining to occur?  Please explain what do you mean by Insufficient evaluation of variables.
Please rewrite these three lines. It is a very long sentence and it’s not clear what is that point that you are trying to make.
Lines 86-88: Please explain why you need more variables to understand cadence retraining and injury risk. Do you think the variables that you have listed from lines 60-75 are not enough ? Are there any variables linked to Injury that these papers don’t talk about ?
Lines 89: You haven’t talked about why 12 weeks is important.

Methods:
Lines 98-99: Why did you choose effect size 0.6?
Lines 102: Which Simple randomization procedure
Lines 103-106: Which limb did you calculate the foot angle ? Can you include the mean of the foot angle ?
Line 127 : What is test vest?
Line 138: Add “ protocol “ after cadence retraining
Lines 145-146: how did the runners know they were running with a 7.5% increase in cadence?
Lines 147-149: Rewrite this sentence
Lines 157-163: It is not clear what is the purpose of the weekly retraining group? Do you think it is feasible to expect runners to come in every week and why did you choose dynamic stretching and what muscles were targeted?
Lines 169: Why did you choose 7Hz for filtering the markers?
Results
Lines 226-Lines 233: I would recommend using the values and changes in cadences and step length in this paragraph
Line 240:  Please change to CAD group “were” significantly

Discussion:
Lines 278: Change from in the present study was to “ were”
Neal et al. observed higher cadence post training ( 7.5% compared to your study 5.7%) Please talk about why you think there was a difference
Line 305-308: I would consider removing this because your study did not look at Injured runners and this doesn’t support your study. Instead focus on cadence retraining and impact on loading rates and mechanistically explaining how cadence retraining changes these variables.
Lines 320-321: How much was the change in cadence, please be specific.
Lines 320-334: I would recommend talking how your study contributed to the literature rather than restating the literature. I would focus more on mechanisms because its discussion section rather than highlighting the literature.
Lines 336: This is unclear. Do you mean step frequency ? That is not magnitude. This is the first time you mention step frequency. Also, there are no norms in the literature to suggest numbers for preferred step frequency.
Lines 338: Please explain step by step approach especially after you suggest the 12 week training improved compliance and had a longer retraining phase compared to other studies.


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

Input:
Title: EFFECTS OF 12-WEEK CADENCE RETRAINING ON IMPACT PEAK, LOAD RATES AND LOWER EXTREMITY BIOMECHANICS IN RUNNING
Review round: 2
Reviewer: 2

Output:
Basic reporting: Numerous grammatical errors remain within the manuscript.
Some examples are: Line #46: "bene", Line #63: "to related", Line #64 “CoG” Abbreviation but on 1st reference. I would recommend a full 3rd party grammatical review prior to publishing.

There are some areas that still require reference updates.
Line #41: "2017" - Needs updated reference.
Line #55-56: These are case series so please indicate accordingly.

The figures and tables are significantly improved.
Experimental design: I thank the authors for expanding on their methodological design.

A few smaller concerns are as follows;

Line #64. Derrick used CoM as a description, which is common with biomechanical studies on running. It would be helpful to see how you determined or estimated CoG. What this from a hip marker? If so, please include.

Line #124: The anatomical locations of the marker set could be described further in the appendix ao that the protocol could be reproduced.

Line #140: It remains unclear why 7.5% was chosen. This needs further justification for why it was chosen over 10%. Referencing other studies provided support but you still need clear reasoning.

Line #172. The impact variables are well described. However in Line #229 "impact forces" is used but it is unclear which variable is being referred to. This theme continues in the results section (Lines #231-234) and discussion (Lines #278)
Validity of the findings: There is one conclusion that needs further refinement.

Lines #281-283. "This finding indicated that the change in the vertical momentum of CoG was reduced during impact attenuation" - This conclusion appears incorrect. Velocity would not change as it was measured at initial contact. Any decreases would more likely be due to less centre of mass displacement.
Additional comments: The authors have done a good job answering the reviewers' concerns and amending the manuscript.

This shows a significant improvement in many areas. Readability may be improved with an external grammatical review as indicated above.


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

Input:
Title: EFFECTS OF 12-WEEK CADENCE RETRAINING ON IMPACT PEAK, LOAD RATES AND LOWER EXTREMITY BIOMECHANICS IN RUNNING
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors have done a good job answering the reviewers’ concerns and making changes to the manuscript. There is an improvement in many areas especially with the Introduction to focus on kinetics( impact forces and loading rates) and highlighting why they chose the 7.5% increase in cadence and the 12 week cadence retraining period.
I am still concerned about including other lower extremity variables(kinematics) in the paper.Please change average and maximum loading rates to vertical instantaneous load rates (VILR) and vertical average load rates (VALR) everywhere in the manuscript.
I am also concerned about the discussion section as some of the points have still haven't been changed or explained from previous submissions.
Experimental design: Methods section was adequate . Please check my general comments for minor changes.
Validity of the findings: Please review my general comments for the discussion and conclusion section.
I am still concerned that your discussion and conclusion doesn't highlight what your study found and how it links to the results.
Please focus on your study rather than only citing previous research and those results.
Additional comments: Abstract:
Please include results from the control group also in the results.
Please rewrite the conclusion to reword lines 37-39

Introduction
Line 42 feels incomplete. I recommend adding form of physical activity or exercise in China and across the world.
Lines 43-44: Change the grammar. 5.83 million participants ran in 1,581 marathon events  in China in 2018 . I would just check to make sure 1581 marathon events or races that include all distances( 5k,10k,half and full marathon)
Line 69: I would remove the word "immediate"
Line 71: Change "In regard" to "With regards to"
Line 82-83: Please elaborate on what do you mean by " reduce risks of rapid transition"
Line 83-85: I would recommend deleting lines 325-326. It sounds repetitive
Lines 87-88 are also a repetition from lines 75-78. I would recommend moving those lines here.
Methods
Line 103: Please change "was included " to "were included"
Line 106: Please change "running" patterns to "foot strike" patterns
Line 109: Please take out the word "visibly"
Line 129: Please add "gait retraining" program
Line 137: Please change " metatarsus" to "metatarsals"
Line 180-185: Futrell et al.2018 calculated vertical instantaneous load rates (VILR) and vertical average load rates (VALR). Please ensure that you mention these specific terms.
Line 185: Please add " We also calculated " instead of kinetic variables because impact peak and loading rates are also kinetics
Lines 187-188: Please only mention flexion and extension. All lower extremity joints are not dorsiflexion and plantar flexion, only ankle joint is.
Line 197: Please change "were" to "was" or change the sentence to " The mean and standard deviations were calculated for all variables"
Line 222-223: Please report changes if any in cadence in control group
Line 225-226 : Please indicate the values of step length in control group
Lines 230-237: Please refer to my earlier comments about terminologies
Lines 256-257: Please align this with study aims
Lines 258: Please remove "cadence retraining could decrease running impact force variables"or explain how you came to this conclusion.
Lines 264-266: Please reword this to explain why Neal et al.2018 had greater improvements in cadence compared to Hafer at al. 2015 and present study.
Lines 267-270: Please reword this to clearly explain your study variables VS Hobara et al. 2012 study values. It's confusing when you say impact peak decreased by 10.2% but then you mention it decreased by 12% in the CAD group compared to the control group.
Line 278: Please remove "recently"
Line 278-282: This section is muddled. What is subtle heel strike. What do you mean by change from rear foot to non-rearfoot strike. How is that subtle. Please provide values of the change in rear foot angles
Lines 285-293: Please refrain from using terms like " Positive effect on reducing risk of running injuries" and highlighting other studies that don't focus on gait retraining and its effects on loading rates and impact peaks.
Lines 294: Please change "touchdown" to "initial contact"
Line 295-301: This is repetition because you already included changes in foot angle in the earlier section. The study is not focused on foot strike pattern and or foot angle. Please reword this.
Line 302: please provide a reference
Lines 302-303: Please mention specific change in cadence
Lines 304-306: Please reword this to " The increase in cadence in the CAD group decreased the peak knee flexion angle and vertical excursion of the CoG.
Lines 307-308: Please remove this sentence "Thus, reduction in the vertical excursion of the CoG may be mainly attributed to the knee joint" or provide a justification based on your study results why you think this is the case, just because peak knee flexion angle was different doesn't justify that it is the reason why change in vertical COG was observed.
Lines 309-310: Your study did not " acutely increase the cadence" Your study was a gait retraining study over 12 weeks.
Lines 327-329: Please remove this sentence because your discussion did not look at vertical velocity of Cog and impact forces. Your discussion focused only on change in foot angle. Also, please refrain from including new terms, please be consistent with terminologies. Through out the manuscript you mention rear foot, non-rearfoot , subtle but now you include " flattening"

Table 2: Please include pre and post values of  impact forces and load rates in Table 2
Lines 371: Futtrell reference has been deleted from the manuscript reference section. Please add it again.
Lines 390: Han T. 2019. Marathon statistics in China. THIS REFERENCE IS INCORRECT. PLEASE CITE IT ACCURATELY


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: EFFECTS OF 12-WEEK CADENCE RETRAINING ON IMPACT PEAK, LOAD RATES AND LOWER EXTREMITY BIOMECHANICS IN RUNNING
Review round: 3
Reviewer: 2

Output:
Basic reporting: See General comments
Experimental design: See General comments
Validity of the findings: See General comments
Additional comments: I commend the authors on accepting the feedback and for making significant improvements to their article. As mentioned previously the article is on a very clinically relevant topic which should be of interest to readers. Well done.

I have a few minor suggestions to improve the paper prior to accepting.

Line #45. 2019 is not a reference. Update this to the author (Running USA).
Line #129. Remove i.e.
Line#289. "runners with high loading rates are more likely to develop injuries compared with those with low loading rates" We don't know this. One study is retrospective (Davis). The other reports symmetry angle of impact peak to be different between injured and non-injured runners. Consider revising this line but I think the argument is logical.
Line#298-9.  "+10% of their preferred cadence".  "+30% of their preferred cadence" Is this and increase?


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: APPLICATION OF CARBON DIOXIDE TO THE SKIN AND MUSCLE OXYGENATION OF HUMAN LOWER-LIMB MUSCLE SITES DURING COLD WATER IMMERSION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors prepared a very interesting study, focused on a useful recovery method. In my opinion, this research is useful in the training recovery programs and the main findings should be used in athletes' recovery.

In general, the English language is good, while some parts of the Introduction and discussion should be re-written, in terms of some language errors.

The literature references were prepared correctly, while I suggested adding a few other references to the Introduction and Discussion. In general, the authors presented the main findings from previous studies.

The article structure, including figures and table, is correct. The figures are very clear.

The hypothesis should be clarified, but the main hypothesis is consistent with the results.
Experimental design: This study is original research, with an interesting research design. In my opinion, an additional section should be added - including study design. This would increase the value of this paper. For sure, it would be more clear for the reader.

I have marked in the comments for the authors - the aim of the study should be more specifically included in the introduction.

Ethical standards are included. The authors described the approval and the Helsinki Declaration.

The methods and measurements are very specifically described.
Validity of the findings: The research protocol is described as correct, but the research design should be more precise. The study is very interesting and fills the lack of knowledge in recovery and water immersion.

The results are very well presented and described. The figures are clear, without any errors.

Conclusions are understandable but should be re-written, including the main findings and main aim of this study.
Additional comments: Major comments:

Introduction:
First of all, the introduction should be written more logical. In this current version it is not very clear. Please re-arrange this section according to my comments and suggestions.
1.	CWI – review of previous paper, including information about different protocols – temperatures and duration, and physiological mechanism – a clear statement.
2.	Carbon dioxide (CO2) – What are the main outcomes of apply  CO2 in cold water therapy?
3.	Connect CCWI with near-infrared spectroscopy – especially in the first part of this paragraph, the reader may found lack of information.
4.	Explain, why CCWI is important in recovery.
5.	Hypothesis and clear goal of the study.

Line 56-64: In my opinion, the first paragraph should include a smooth description of cold water therapy and recovery. Please provide more information according to physiological mechanisms following CWI. Please provide publication review according to different possibilities to apply CWI in recovery. Please check papers written by Šrámek et al. (2000), Bleakley et al. (2010) and Ihsan (2016).

Šrámek, P., Šimečková, M., Janský, L., Šavlíková, J., & Vybíral, S. (2000). Human physiological responses to immersion into water of different temperatures. European journal of applied physiology, 81(5), 436-442.
Bleakley, C. M., & Davison, G. W. (2010). What is the biochemical and physiological rationale for using cold-water immersion in sports recovery? A systematic review. British journal of sports medicine, 44(3), 179-187.
Ihsan, M., Watson, G., & Abbiss, C. R. (2016). What are the physiological mechanisms for post-exercise cold water immersion in the recovery from prolonged endurance and intermittent exercise?. Sports Medicine, 46(8), 1095-1109.

Line 75: Please provide a statement connecting CWI and near-infrared spectroscopy. This two paragraphs should be connected and logical.

Line 73-92: It is not known, what is the reason to use CCWI? Please explain in the last part of the introduction. Please add also about different temperatures for CWI. It should be shown a analysis of prior studies.

Materials and methods:
1.	Have you calculated sample size and  power for this study? It would be worth to add, what is the minimal study group needed.
2.	Were the participants physically active?
3.	Have you considered included and excluded criteria?
4.	What is the reason to prepare this research - CWI and CCWI with temperature 20oC?

Line 117-118: “The subjects were not informed whether the water was CCWI or CWI.” Please provide in sub-section - Participants' characteristics or a special “study design”. It would be worth to explain the study design and the main metrological aspects.

Discussion
Line 316-317: “Using NIRS, this study examined two common assumptions in the application of CO2 rich cold water immersion to change in skin- and muscles-blood perfusion related to hematocrit,” Could you please explain this statement. I have not found any information about relation with hematocrit before, in the introduction. Please explain and clearly this.

Please discuss the effect of CWI using protocols with different temperatures. In previous papers you may find few reviews, e.g. Bleakley and Davison, 2010.

Study limitations:
Please indicate point by point limitations, with brief explanation. In my opinion this section should be re-arranged and clarified.
None post-exercise state should be marked as the major limitation with explanation.

Athletic application of CO2-rich water immersion
Please indicate more specifically practical implications.

Conclusions
In my opinion, the conclusions should be re-write to a more consistent form.

Minor comments:

Abstract:
Line 28-29: “Cold therapy has a disadvantage in that a cold stimulus can induce vascular constriction in arterial and venous capillaries”. Please re-arrange this sentence.

Introduction:
Line 56-57: “Cold therapy has a disadvantage in that a cold stimulus can induce vascular constriction in arterial and venous capillaries”. Please re-arrange this sentence.

Line 57-59: “As a result, the decreased blood flow in both peripheral and deep vasculature would lead to a delayed accumulation of substances that are related to muscle damage and muscle fatigue after competitive sports events.” Please consider to move this sentence at the end of the paragraph.

Line 65-67: “Over the last three decades, researchers observed that the effects of carbon dioxide (CO2) hot water depend mainly on not only the cutaneous vasodilation but also the muscle vasodilation elicited by the CO2 that diffuses into the subcutaneous tissues through the skin layers”. Please re-arrange this sentence. Grammatically should be improved.

Line 72-74: “We hypothesized that the arterial and venous vasodilation in both superficial and inside-muscle layers of different muscles would be significantly augmented by enriched CO2, even though during CWI the cold stimulus induces vascular constriction.” This sentence should be moved to the end on the introduction. Try to connect with the aim of the study.

Materials and methods:

Line 114, 116: Please do not begin sentence with – “He …” “his”

Line 127-137: Move to the Introduction

Statistical analysis:

I would recommend adding eta square to ANOVA analysis.

Conclusions:
Line 515: Please change term „leg” to “lower extremity”.