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: DIFFERENTIAL RESPONSES OF THE ACIDOBACTERIAL COMMUNITY IN THE TOPSOIL AND SUBSOIL TO FIRE DISTURBANCE IN PINUS TABULAEFORMIS STANDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is generally written in clear English and conforms to professional standards.  The article is well-organized, utilizing a consistent structure throughout the paper. The Materials and Methods, and Results sections in particular are very well organized and written with clear, direct language that facilitates understanding of the primary research findings. I also appreciate the inclusion of Table 1, which provides information about the edaphic variables in the research plots, and Table S2, which provides information about the vegetation structure. This information provides valuable ecological context, and I commend the authors for including it.

The primary weaknesses of the paper are that it contains insufficient background information to properly frame the research topic, and several of the figures require revision. Specifically, the Introduction and Discussion sections should be revised to more thoroughly review the current state of knowledge on the impacts of fire on the soil bacterial community. The authors should provide more detail about the role of Acidobacteria in ecosystems, and explain why the community structure of Acidobacteria sub-groups is important to ecosystems. For example, in lines 41-55 the authors briefly discuss the prevalence of Acidobacteria in soil environments, but the role of Acidobacteria in general, and specific Acidobacteria sub-groups on ecosystem processes is not discussed. It is thus unclear why the impact of fire on Acidobacteria is important to the ecosystem, and why the authors focus on the community structure of the various subgroups of Acidobacteria.

Additionally, the authors assess the strength of various edaphic and environmental variables as drivers of Acidobacteria community structure, including organic matter concentration, nitrate-nitrogen, soil moisture, pH, vegetation diversity, and tree structure. However, only background information regarding the role of pH in driving Acidobacteria community structure is provided in the introduction. Justification is not provided for the inclusion of the other variables in this study, and the manuscript should be revised to review the state of knowledge of these variables as drivers of Acidobacteria activity and community structure. Similarly, in the discussion, the authors describe environmental factors that appear to be driving changes in Acidobacteria communities, but the downstream effects of these changes is not given sufficient consideration. In both the Introduction and Discussion sections, the authors frequently make vague, generalized statements without providing precise, detailed examples in support (see specific comments below). The authors should revise to provide literature-based support of their claims.

Nearly all figures and tables require revision to formatting (mostly text size): see Specific Comments in a following section. Please provide an additional figure showing photos of the unburned and burned areas: this is very important for helping your reader see the visual impacts of the fire, associated with each severity level you classified.

Lastly, it is unclear why the authors focus solely on Acidobacteria after sequencing the entire bacterial community. Why did the authors focus only on Acidobacteria and not include other ecologically important and abundant phyla like Proteobacteria or Bacteroidetes? The approach should be explained and justified in the introduction.  Please also include a conclusions section or paragraph to clarify the take-away message from your study, and the implications for this research field.
Experimental design: The manuscript describes primary research within the aims and scope of PeerJ journals. The manuscript addresses three research questions that are clearly identified and prominent in the introduction, and these questions are clearly addressed by the results. The analytical methods utilized are described clearly and with sufficient detail, are technically sound and reproducible, and are within current methodological standards.

The paper would primarily benefit from providing some additional information about the experimental design, and site and soil characteristics. Specifically, the spatial distribution of the research plots needs clarification. For example, it is unclear whether the three plots within each severity level were located in a single area of contiguous burn severity, or whether the plots are located in separate severity “patches.” This should be clarified in the main text, and/or figure S1 should be revised to illustrate the pattern of burn severities across the research area and the distribution of the research plots. The mean and minimum distances between research plots should also be reported. Additionally, the authors should provide more information about the distribution of soil types across severity levels, and information about pre-fire vegetation structure. This would provide important context for the patterns of Acidobacteria community structure and organic matter content observed, and aid in interpreting whether these results are related to pre or post-fire conditions. If possible, representative photographs from each severity level should be included, which would provide context about pre and post-fire vegetation structure, and allow visualization of the fire’s impact on the soil.

The investigators clearly identify the knowledge gap they are investigating: to understand the impact of fire severity on the Acidobacteria community in soil. However, the relevance and importance of this topic is not given sufficient attention in the introduction, discussion, or conclusion sections of the manuscript, and additional information should be provided to justify the study (see comments in the basic reporting section above). The authors should also more directly state how their results contribute to filling this knowledge gap by including a conclusions section.
Validity of the findings: The investigators used straight-forward statistical techniques. Some minor clarification in the text is needed about how they implemented their t-tests when comparing different soil layers (see specific comments below). Although the authors used one-way anovas to evaluate differences among severity levels within soil depth increment, followed by t-tests to determine differences between depth increments within severity level, this could also be treated as a two-way anova followed by protected pairwise comparisons. The authors included appropriate controls, and the level of replication is acceptable, but additional information is needed about the location of the research plots relative to each other to ensure pseudo-replication is not a concern (see also comments in “Experimental Design” section above). The authors should revise the manuscript to include f- & p-values and t-statistics in the text, figures, and/or tables. Lastly, the authors should include a conclusions section that connects to the original question investigated.

Some important questions to consider relate to:
1) the scale of the severity assessments, and 2) the fact that the severity estimates were based on whole-stand characteristics and provide relatively low resolution of effects on the soil. For example, severity was assessed at a 20x20 m plot scale, but we know that there is a lot of spatial variability in fire effects. The authors should discuss whether 5 soil subsamples really represents a 20x20m area. Was severity totally consistent within each plot? Severity level may be more representative of effects on soil if it is assessed at the soil sample location(s). The data from this study also shows that fire may only affect soil bacteria in high-severity burns where all the organic matter has been consumed.

Additional references that may help strengthen the discussion include:
Hartford and Frandsen. 1992. When It's Hot, It's Hot... Or Maybe It's Not! (Surface Flaming May Not Portend Extensive Soil Heating). International Journal of Wildland Fire 2:139-144.
Kolka et al. 2014. Post-fire comparisons of forest floor and soil carbon, nitrogen and mercury pools with fire severity indices. Soil Sci. Soc. Am. J. 78:S58-S65
Kolka et al. 2017. Emissions of forest floor and mineral soil carbon, nitrogen and mercury pools and relationships with fire severity for the Pagami Creek Fire in the Boreal Forest of northern Minnesota. International Journal of Wildland Fire 26:296-305
Additional comments: Dear Authors,
This is a very interesting and novel study that provides important information about the effects of fire and burn severity in an understudied forest type. The specific comments below identify ways to help clarify the importance and value of your study.

Specific Comments:
Abstract:
Line 2: Many researchers consider the response of soil bacteria to fire to be a significant knowledge gap in the field. For example, see Pressler et al. 2018 (doi: 10.1111/oik.05738).
Line 10: How does the abundance of the Acidobacteria compare to other phyla? Was this the most abundant phylum?
Line 22: 15.6% is not “most of the variation.” Please revise for clarity.
Lines 23-23: What are the large gaps that still need to be explored? Specifically identifying these gaps will be a more effective concluding sentence for the abstract.

Introduction:
Line 33: What do you mean by “the balance of the ecosystem” and how do microorganisms regulate this balance? Can you also provide examples regarding the “crucial and unique role in energy flow and circulation of material?”
Line 37: The role of fire severity on microbial community structure and activity remains an open question. For example, Pressler et al. (2018) concluded that there are insufficient studies to draw strong conclusions about the role of fire severity on the bacterial community. Furthermore, one of the studies cited here (Weber et al. 2014) found inconsistent response of microbial communities to fire severity and the other (Hamman et al. 2007), found differences in microbial community structure related to fungi, not bacteria. This portion of the introduction could be rewritten to highlight the uncertainty that exists regarding bacteria response to fire severity and improve the justification for your study. Either way, this sentence should be revised to clarify that the impacts of fire severity on bacterial communities is far from a settled question.
Line 40: Please discuss why the effects of wildfire on specific bacteria phyla are important.
Line 44: Do the different Acidobacteria subgroups have different ecological roles? Here, it is important to explain why the structure of Acidobacteria community matters for the ecosystem, and thus, why your research is important.
Lines 49 – 51: Please explain how the oligotrophic characteristics of Acidobacteria affect ecosystem processes, define what eosinophilia is, and please be more specific about the phylogenetic diversity of the phylum.
Line 51: Delete “it should be pointed out that” from this sentence.
Line 51-55: How might the tolerance of Acidobacteria to different environmental conditions affect their response wildfire or ecosystem function after a fire? Do different sub-groups consistently show different responses to various environmental conditions?
Line 57: Please provide a reference of the statement that wildfire has especially variable results on Acidobacteria, or revise this sentence. I am skeptical that there is enough research available to draw conclusions about the impacts of wildfire on Acidobacteria relative to other phyla.
Lines 64-66: How does Acidobacteria vary in response to “sampling time, location, fire severity, and vegetation type” that leads you to this conclusion? How do the biological characteristics of Acidobacteria subgroups vary and how do these impact the response? Please provide additional detail and more references here.
Line 68-69: Why is knowledge of the response pattern at the subgroup level important? Again, this information is critical to justify this study.
Lines 70-72: These two sentences are out of place here and should be moved to the site description section of the Materials and Methods.

Methods:
Lines 86-87: Please provide more information about these soils. “Brown soil” and “cinnamon soil” will not be familiar to an international audience and more information about the characteristics should be provided here. At a minimum, please provide information about the typical textures, parent materials, horizonation, and pH of these soils.
Lines 91-92: The sentence “Soil samples were collected…” is out of place here. It could be moved to line 96, prior to “In each plot, five random soil cores…”
Line 94: Please provide a reference for the severity classification standard.
Line 94: Please include Table S1 in the primary text, not as a supplementary figure.
Line 94: Was severity classified for the entire 56.33 ha area that was burned? If so, what was the proportion that burned at high, moderate, and low severity? If not, how much of the burned area was assessed for severity?
Lines 94-95: Were the three plots in each burn severity area located in a single contiguous area of burn severity, or were they located in distinct burn severity patches that were separated by regions of variable burn severity?
Line 96: “Shady” is not an aspect, please provide the compass direction of the aspect.
Line 160-162: Is it correct that two separate ANOVAs were conducted, one for each soil layer? Please clarify.
Line 163-167: Was a single t-test performed (between depth increments, across all severity levels), or were separate t-tests performed to compare depths, within each severity level?

Results:
Line 185-186: The large difference in OM content at the high severity level, especially in the 10-20 cm layer is interesting, and requires further investigation and explanation. Although losses in soil OM following fire are not unusual, these losses are usually restricted to the upper few centimeters, because the heat pulse from fire generally does not penetrate very far into the mineral soil. This makes me suspect that the differences in OM content existed prior to the fire, and by extension, the observed differences in Acidobacteria community in the high severity plots may have existed prior to the fire. Is there any pre-fire soil data available from these plots? Is the soil type the same in the high severity plots as the other plots? Was the pre-fire plant community composition and structure similar to the other plots? At the very least, you should provide representative photographs from each severity level so the reader can assess whether the pre-fire plant structure was similar, and you should provide more background information about the soils. Please see the following for information about the impacts of fire on soil and soil OM:
Nave et al. 2011 (Ecological Applications 21(4), p 1189-1201),
Certini et al. 2005 (doi: 10.1007/s00442-004-1788-8),
Gonzalez-Perez et al. 2004 (doi: 10.1016/j.envint.2004.02.003)
Adkins et al. 2019 (doi: 10.1016/j.geoderma.2018.07.009).

Line 186-195: Please provide the specific f-values and p-values for each variable assessed with ANOVA. At a minimum, please also include p-values for each variable in Table 1.
Lines 190-195: This speculation about the reasons for differences in SM belongs in the discussion section, not the results.
Lines 192-194: Increases in SM may also be due to reduced uptake of soil water by plants.
Line 199: Please provide specific f- and p-values for each variable here and in Figure S2 (or put the f-statistics into a table).
Line 199-200: This sentence is redundant with the preceding sentence. Please delete.
Line 207: How does the proportion of reads belonging to Acidobacteria compare to other abundant groups? Was Acidobacteria the most abundant phylum?
Line 220: Please report specific f-values and p-values here and in Figure 2.
Lines 254-263: Please report r2 values and p-values for each correlation referenced in this paragraph.

Discussion:
Line 269: Please provide specific examples of the different environments.
Line 271: In the introduction you cite papers regarding the response of Acidobacteria to fire, but here you say the response in unknown. Please clarify what the knowledge gap is in regards to Acidobacteria response to fire here and in the introduction.
Line 273: Again, please clarify how the abundance of Acidobacteria compares to other phyla. Why is no information about other dominant phyla provided in this manuscript?
Line 276: Please summarize Zhang’s et al. (2014) findings and clarify how it compares to your results.
Line 278-280: You claim “a large number of studies have shown microbial diversity decreases significantly,” but only cite two studies here. Again, please see Pressler et al. 2018, who suggest there are not enough studies on bacterial communities post-fire to draw strong conclusions.
Line 285: Your findings are not consistent with these results. A recovery within 6 months is much faster than the recovery in 11 years observed by Xiang et al. (2014). Furthermore, your study assessing changes after a single wildfire is not comparable to Shen’s et al. study which assessed the response to repeated prescribed fires. Please update this section with more relevant references and clarify the differences in your results compared to other.
Lines 286-287: Because you do not have pre-fire and immediate post-fire Acidobacteria data, you cannot make the claim the Acidobacterial community recovered in 6 months. More likely is that the Acidobacteria community was not affected by the low and moderate severity fires. Most studies have indicated that it takes several years for the microbial community to recover, suggesting its unlikely that the Acidobacteria community recovered in 6 months.
Lines 289-290: Please revise this sentence to clarify that alpha diversity was higher in the topsoil than the subsoil in high severity plots, or whether it means that the alpha diversity was higher in topsoil in high severity plots than in the topsoil of the other plots.
Line 290 -293: Please provide additional detail about how edaphic and vegetable heterogeneity affects microbial competition for resources, and how this in turn impacts bacterial diversity. How might these processes impact Acidobacteria specifically?
Lines 294-295: Please summarize/provide cited examples about the ways in which microbial communities are impacted by aboveground vegetation and soil properties, and how fire impacts these properties.
Lines 296-297: Please explain how and why Acidobacteria promote nutrient cycling, plant growth, and soil restoration after disturbances. How does this differ from other bacteria phyla? Do different Acidobacteria subgroups play different roles?
Line 318: What specifically is the role that acidobacteria play in the nitrogen cycle?
Lines 321-330: In this section, please indicate that you are suggesting that a negative correlation with OM suggests oligotrophy.
Lines 321-330: Organic matter quality (e.g. carbon to nitrogen ratio) plays an equally or more important role as OM content in the colonization of soils of oligotrophs versus copiotrophs. For example, if OM is of low quality (e.g. high carbon to nitrogen ratio), we would expect to see relatively more oligotrophs regardless of OM content. You have not included data on OM C:N here, but you should at least mention the potential role of OM quality.
Lines 339-340: This sentence is redundant with the preceding portion of the paragraph. Please delete.
Lines 342-344: Consider assessing this hypothesis with your data
Lines 346-347: Please specify how these factors are beneficial for bacterial communities and how they might impact the differences you observed.
Lines 349-352: This is a weak conclusion to your paper, which presents an interesting & novel study. Please revise the text to conclude by highlighting your own findings, not the findings of others. Emphasize how your study adds valuable information to our understanding of fire in ecosystems around the world, and especially how it advances knowledge about fire's effects on forest soil. Make sure these are clear for your reader, so add this text in a separate conclusion section, or even in a conclusions paragraph.

Tables & Figures
Table 1: Please replace “Indices” with something more descriptive like “soil properties.”
Table 1: The soil depth column lists specific depths, but in the manuscript body you refer to these as “topsoil” and “subsoil.” Please use labeling in the tables that are consistent with the body text.
Table 1: Please spell out the full fire severity label rather than using abbreviations.
Table 1: Please add a column displaying the f-values and p-values for your ANOVA results.
Figure 1 Legend: Please also specify the forest type, time since fire, and define the abbreviations used in the x-axis.
Figure 3 Legend: Please define the abbreviations used in the figures and specify the different depths of topsoil and subsoil.
Figure 2: The figure text sizes should be greatly enlarged.  Please label the panels as a, b, c, and d. Move the panel titles to become the y-axis labels.
Figure 3: The figure text sizes should be greatly enlarged. The legend for panel b includes the incorrect labels.
Figure 4: I recommend moving the Figure 4 panels into Figure 3 as panels c and d (or into insets in panels a and b), and combining the captions. Consider following the format used by Robert et al. 2018  in Ecography 40:1-16, doi: 10.1111/ecog.03553 (https://onlinelibrary.wiley.com/doi/abs/10.1111/ecog.03553)

Supplementary Tables and Figure Captions: all captions should be revised to provide much more detail. They should all be written similarly to what is provided in the main manuscript figures and tables: remember that a reader should be able to understand & interpret each figure and table as a “stand-alone” unit, without the reader needing to rely heavily on the manuscript text.
Table S1: Please spell out the fire severity level rather than using single-letter abbreviations. Cite your source in the caption to clarify where this severity classification system came from (it looks similar to one by Ryan & Noste, but if you modified a published severity classification system, then please provide a clear rationale for how & why it was modified. If you used a published severity classification system, then just report the original classification descriptions for each category…the current descriptions in this table are very unclear. Please revise the criteria description for the moderate severity category to be more descriptive. Use the term “low-severity,” not “low-grade.” Revise “height” in the moderate severity description to read “high severity.” Capitalize “unburned” in the control description. Please include this table in the main manuscript rather than as supplementary information. What does “burned down” mean? Do you mean consumed? Charred but still present?
Table S2: Please spell out the fire severity level rather than using abbreviations. Please change the column heading “Burning degree” to follow the terminology used in other tables & the text.
Figure S1: Add a scale bar. Also please add another panel to this figure to show the location and distribution of your research plots within your study site.


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: DIFFERENTIAL RESPONSES OF THE ACIDOBACTERIAL COMMUNITY IN THE TOPSOIL AND SUBSOIL TO FIRE DISTURBANCE IN PINUS TABULAEFORMIS STANDS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Good.
Experimental design: Good.
Validity of the findings: Good.
Additional comments: The manuscript has revised well.

L185-189, 195-197: I don't agree with the response from the authors. If you don't want to make a special discussion or cannot find the place to put it, it means that this contents are not needed in this manuscript, thus it can be removed. If you have something to discuss, please do it in Discussion.

"So we don’t make a special discussion about the soil and vegetation characteristics in the discussion part. We've only made a brief discussion of the interesting phenomena in this part of the results. Besides, we haven’t found a suitable place in the discussion part to put this content."


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: DIFFERENTIAL RESPONSES OF THE ACIDOBACTERIAL COMMUNITY IN THE TOPSOIL AND SUBSOIL TO FIRE DISTURBANCE IN PINUS TABULAEFORMIS STANDS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revisions the authors have made to their manuscript have improved the quality and clarity of the paper. The revisions to the methods, results, and discussion sections are generally well-done and satisfy most of my comments. The figures have been adequately revised for the most part, and the inclusion of photographs of the severity levels are very helpful.  However, the paper still does not include sufficient background information to justify the study. This weakness is reflected by inadequate response of the authors to many of the changes suggested for the introduction. The authors still need to provide more detail about the role of Acidobacteria in ecosystems and explain why the community structure of Acidobacteria is important. It remains unclear why the impact of fire on Acidobacteria sub-groups is of ecological significance. Additionally, as mentioned in my previous review, the authors assess the strength of various edaphic and environmental variables as drivers of Acidobacteria community structure, including organic matter concentration, nitrate-nitrogen, soil moisture, pH, vegetation diversity, and tree structure. However, only background information regarding the role of pH in driving Acidobacteria community structure is provided in the introduction. Justification is not provided for the inclusion of the other variables in this study, and the manuscript should be revised to review the state of knowledge of these variables as drivers of Acidobacteria activity and community structure.
Experimental design: The authors have sufficiently addressed most of my comments regarding experimental design. However, more information is still needed regarding the spatial distribution of sample plots within the research area. For example, it is still unclear whether the three plots within each severity level were located in a single area of contiguous burn severity, or whether the plots are located in separate severity “patches.” The authors also did not provide information about the mean and minimum distances between research plots as I requested. Figure S1 still needs to be revised to include the locations of sample plots.
Validity of the findings: The authors have provided additional information about statistical methods and results, improving confidence in the validity of their findings. In a few instances, some additional information needs to be incorporated into the manuscript text (detailed in specific comments below) to aid in reader interpretation of results. Finally, as previously requested, and as detailed in the experimental design section above, more information is needed about the location of the research plots relative to each other to ensure pseudo-replication is not a concern. This information should be provided both in the materials and methods section of the manuscript and by revising Fig S1 to include more information.
Additional comments: Specific Comments:
The authors adequately addressed most of the issues I identified in the specific comments section of my previous review. Below, I provide follow-up responses to the many that have not been adequately addressed by the authors. Between lines beginning XXXX I have provided (1) my original reviewer comment, (2) the authors’ response, and (3) my follow-up response.

Original Reviewer Comment: Line 22: 15.6% is not “most of the variation.” Please revise for clarity.

Author Response: we rewrote this sentence. The revised sentence is “A variance partitioning analysis (VPA) showed that edaphic factors explained the most variation both in the topsoil (15.6%) and subsoil (56.3%). ” (Please see line 21 in the revised manuscript).

Reviewer follow-up: Please revise to “...edaphic factors explained the highest relative amount of variation in both the topsoil (15.6%) and subsoil (56.3%).

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Original Reviewer Comment: Line 33: What do you mean by “the balance of the ecosystem” and how do microorganisms regulate this balance? Can you also provide examples regarding the “crucial and unique role in energy flow and circulation of material?”

Author Response: “maintenance the balance of the ecosystem” means soil microbes perform certain functions to maintain the ecosystem stability, such as organic matter decomposition and driving soil nutrient cycling. At the same time, these physiological activities are also accompanied by material circulation and energy flow.

Reviewer follow-up: Please revise the manuscript text to clarify this to the reader. I suggest revising the sentence to read something similar to the information you have provided in your response to my original comment. For example, “As an important part of the forest ecosystem, soil micro-organisms maintain ecosystem stability through the role in driving organic matter decomposition and soil nutrient cycling.”

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Original Reviewer Comment: Line 37: The role of fire severity on microbial community structure and activity remains an open question. For example, Pressler et al. (2018) concluded that there are insufficient studies to draw strong conclusions about the role of fire severity on the bacterial community. Furthermore, one of the studies cited here (Weber et al. 2014) found inconsistent response of microbial communities to fire severity and the other (Hamman et al. 2007), found differences in microbial community structure related to fungi, not bacteria. This portion of the introduction could be rewritten to highlight the uncertainty that exists regarding bacteria response to fire severity and improve the justification for your study. Either way, this sentence should be revised to clarify that the impacts of fire severity on bacterial communities is far from a settled question.

Author Response: Thank you very much for your correction. Our original description is not rigorous and accurate. I wonder if I could quote the red part in your question. I think your summary is very concise and representative. If you allow me to quote your summary, please see lines 35-39 in the revised manuscript.

Reviewer follow-up: Yes, it is OK to use reviewer language to help clarify a manuscript. I recommend deleting the portion of the sentence that reads “… and Hamman et al. (2007) reported differences in microbial community structure related to fungi, not bacteria” since fungi is not a focus of your study. You could perhaps expand on this point to highlight the fact that most severity studies focus on fungi, rather than bacterial communities, but in its current form, this portion of the sentence is out of place.

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Original Reviewer Comment: Line 44: Do the different Acidobacteria subgroups have different ecological roles? Here, it is important to explain why the structure of Acidobacteria community matters for the ecosystem, and thus, why your research is important.

Author Response: Yes, different Acidobacteria subgroups have different ecological roles. This view is supported by the cited literature (Kielak et al., 2016) in my manuscript. Actually, I don’t think it is necessary to develop a description about the structure of Acidobacteria community and ecosystem. Firstly, in Kielak’s review article they have made a relatively comprehensive introduction about the Acidobacteria physiological and ecological characteristics. Secondly, our research focuses on the changing pattern of Acidobacteria subgroups to wildfire disturbance and tries to find the main driving factors. Moreover, we have made an emphasis that knowledge of the response pattern  (at the subgroup level) after a fire disturbance is still limited. However, the ecological functions of Acidobacteria subgroups in ecosystem are really important. So we make a discussion about the relationship between Acidobacteria subgroups and environmental factors.

Reviewer follow-up: This response is not satisfactory. Providing a description of the ecological roles of the Acidobacteria sub-groups is important for justifying this study. Simply providing a literature reference without summarizing the ecological roles and importance is not sufficient. In its current state, the introduction still does not provide adequate justification for this study. Please add a paragraph explain what is known about the ecological roles and characteristics of  Acidobacteria sub-groups.

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Original Reviewer Comment: Lines 49–51: Please explain how the oligotrophic characteristics of Acidobacteria affect ecosystem processes, define what eosinophilia is, and please be more specific about the phylogenetic diversity of the phylum.

Author Response: in lines 49-51, I didn’t say the oligotrophic characteristics of Acidobacteria will affect any ecosystem processes. However, the oligotrophic characteristics of Acidobacteria are indeed closely related to some ecosystem processes, such as the decomposition of organic matter and carbon cycling. But here I mainly want to bring out the following content that although Acidobacteria exhibits oligotrophy, not all subgroups are oligotrophs. So it is necessary to get a further insight into this phylum on the subgroup level.
Eosinophilia is an inaccurate description. We deleted it.
Liu et al. (2016) have made a more specific report about the phylogenetic diversity of the Acidobacteria. We just quote his viewpoint here.

Reviewer Follow-up: As you acknowledge in your response, oligotrophic characteristics are related to ecosystem processes. This is important context for your study, so please add the information provided in your response to the manuscript text. Furthermore, this portion of the manuscript does not mention that not all subgroups are oligotrophic. Please add this information to this portion of the manuscript, along with appropriate citations.

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Original Reviewer Comment: Line 51-55: How might the tolerance of Acidobacteria to different environmental conditions affect their response wildfire or ecosystem function after a fire? Do different sub-groups consistently show different responses to various environmental conditions?

Author Response: In lines 57-65 of the original manuscript, we had made an introduction about the different responses of Acidobacteria to fire disturbance. We speculated that in addition to objective factors, the different biological characteristics of the Acidobacteria subgroups were important factors leading to these inconsistent changes. Actually, previous studies found that different subgroups showed different responses to various environmental conditions. For example, Zhang et al. (2014) reported that the elevation gradient had different effects on Acidobacteria subgroups. The relative abundance of subgroup 1, 2, 3 increased with the altitude, and the relative abundance of subgroup 4, 5, 6, 16, 17 decreased with the altitude (Zhang et al., 2014). Furthermore, several researches also found that the effects of soil pH on Acidobacteria subgroups are not exactly the same (Jones et al., 2009; Turlapati et al., 2013; Kielak et al., 2016).
All these references can be found in the manuscript.

Reviewer Follow-Up: The specific information you provide in your response about how different sub-groups vary with environmental conditions is important context for your study that should be incorporated into the manuscript text. Although you provide the references in the manuscript, you could incorporate much more of the important information found within these references into the manuscript text.

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Original Reviewer Comment: Lines 64-66: How does Acidobacteria vary in response to “sampling time, location, fire severity, and vegetation type” that leads you to this conclusion? How do the biological characteristics of Acidobacteria subgroups vary and how do these impact the response? Please provide additional detail and more references here.

Author Response: this conclusion is based on the literature I have read. According to previous studies, we speculate that the different changes of Acidobacteria after burning are due to two reasons. One is objective reasons, such as sampling time, research location, different fire severity, and different vegetation types. The other is the biological characteristics of Acidobacteria subgroups themselves. As I mentioned in my previous answer different Acidobacteria subgroups showed different responses to various environmental conditions (Zhang et al., 2014; Jones et al., 2009; Turlapati et al., 2013; Kielak et al., 2016). However, these studies are all not related to fire. Shen et al. (2016) found minor change of Acidobacteria phylum, but a significantly lower abundance of subgroup 1 after burning. So there must be other subgroups increase after fire. Why are the changes in subgroups of the same phylum different? Combined with previous researches, I think it may be because different Acidobacteria subgroups have different biological characteristics. According to our result, we did find that the relative abundance of Acidobacteria subgroups vary inconsistently after fire (Fig.2). Correlation analysis also showed that the relationships between Acidobacteria subgroups and environment factors are inconsistent (Table S5; Table S6). I think these findings will help us to get a deeper understanding of Acidobacteria.

Reviewer Follow-up: You have not revised the manuscript to explain how Acidobacteria vary in response to “sampling time, location, fire severity, and vegetation type.” Some of information you provided here and in previous responses (e.g. variability in subgroup abundance with elevation) is relevant to addressing this issue, but this and additional information regarding how time, fire severity, and vegetation type influence Acidobacteria communities still needs to be added to the manuscript. If this information has not adequately been assessed by previous studies, you should discuss this in your introduction and explain the role your research plays in filling this knowledge gap.

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Original Reviewer Comment: Line 94: Please provide a reference for the severity classification standard.

Author Response: Thank you for your reminder. I forgot such an important reference. I have added a reference in the revised manuscript.
[1] Zheng Q., Cui, X. Y., and Di, X. Y. (2012). Effects of Different Forest Fire Intensities on Microbial Community Functional Diversity in Forest Soil in Daxing’anling. Scientia Silvae Sinicae. 48, 95-100.
Reviewer Follow-up: Please also provide this reference as an in-line citation in the methods section.

Original Reviewer Comment: Line 94: Was severity classified for the entire 56.33 ha area that was burned? If so, what was the proportion that burned at high, moderate, and low severity? If not, how much of the burned area was assessed for severity?
Author Response: Because of the spatial heterogeneity of fire, it is difficult to accurately calculate the burning area of different severities. According to the field investigation and our classification criteria for different fire severities, the area of high: moderate: low severity is roughly 5:3:1. we added this information in the revised manuscript (line 91).
Reviewer Follow-up: Please provide the relative proportions of different severity levels as percentages instead of as a ratio. Also, please explain how you estimated the relative areas.

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Original Reviewer Comment: Lines 94-95: Were the three plots in each burn severity area located in a single contiguous area of burn severity, or were they located in distinct burn severity patches that were separated by regions of variable burn severity?

Author Response: Every three plots were located in distinct burn severity patches.
The process of plots selection is as follows: field investigation of fired area→the fired area was divided into different parts (high, moderate and low severity areas) combined with the criterion→in every different part, we set three plots with similar slope, aspect and elevation.

Reviewer Follow-up: It is still unclear to me how the sample plots were distributed throughout the severity matrix. I.e., are the three high severity sample plots located in three distinct high severity patches? Or were the three high severity sample plots located in one single high severity patch? Please clarify this in the manuscript text. Adding more detail to figure S1 to show the entirety of the study area and the location of sample plots would also be of immense help.

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Original Reviewer Comment: Line 160-162: Is it correct that two separate ANOVAs were conducted, one for each soil layer? Please clarify.
Response: I think it is appropriate to apply two separate ANOVAs in different soil layers, respectively. Because the topsoil and subsoil were considered as two different parts, not a whole in this research.
Reviewer Follow-up: Revise the text to clarify that you performed separate ANOVAs for each soil layer in the manuscript text. It currently remains unclear in the text.

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Original Reviewer Comment: Lines 190-195: This speculation about the reasons for differences in SM belongs in the discussion section, not the results.

Author Response: Actually, we have made a discussion about the soil properties after fire in a paper we have published (Li et al., 2019). So we don’t make a special discussion about the soil and vegetation characteristics in the discussion part. We've only made a brief discussion of the interesting phenomena in this part of the results. Besides, we haven’t found a suitable place in the discussion part to put this content. We consider that this part of the content in the results does not affect the overall structure of the article. If not necessary, we want to leave this part of the content here.

Reviewer Follow-up: This belongs in the discussion. If necessary, revise the discussion to find a suitable place to include this information.

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Original Reviewer Comment: Lines 254-263: Please report r2 values and p-values for each correlation referenced in this paragraph.

Author Response: Thank you for your advice. However, the r values for each correlation had been shown in Table S5 & Table S6 and we also used clear identifications (“*” , “**” ) to point out the correlation on significant levels. So we don’t think it’s necessary to report r2 values and p-values for each correlation in manuscript again.

Reviewer Follow-up: Include r2 values and p-values in this paragraph as well so readers don’t have to navigate to a supplementary table to assess the strength of the relationships. This is an important component of your results and should be included in the main text.

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Original Reviewer Comment: Lines 296-297: Please explain how and why Acidobacteria promote nutrient cycling, plant growth, and soil restoration after disturbances. How does this differ from other bacteria phyla? Do different Acidobacteria subgroups play different roles?

Author Response: We rewrote this sentence to reduce ambiguity and the view of this sentence could be supported by the cited reference. Please see lines 299-301 in the revised manuscript.
Among them, Acidobacteria were found to play a role in promoting soil carbon cycling, as it can degrade complex plant derived polysaccharides such as cellulose and lignin (Huang et al., 2015).

Reviewer Follow-up: The sentence you provide in your response does not match the sentence in the manuscript. Revise manuscript to match the sentence provided in your response.

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Original Reviewer Comment: Lines 346-347: Please specify how these factors are beneficial for bacterial communities and how they might impact the differences you observed.

Author Response: Actually, we have made a brief explanation that why the change of canopy density will be beneficial for bacterial communities.
Further, changes in plant community structure, such as the decline in canopy density, increase the light availability and soil temperature in the forest, which are beneficial for the bacterial communities (Arunachalam & Arunachalam, 2000; Bolat, 2014).

Reviewer Follow-up: My original comment was not related to canopy density, rather it was regarding lines 346-347, where you mention climate change, soil metals, and soil enzyme activities. How are these factors relevant to your study? Revise manuscript accordingly.

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Original Reviewer Comment: Figure 1 caption: Please also specify the forest type, time since fire, and define the abbreviations used in the x-axis.

Author Response: The information of forest type and buning time have been declared in the M&M part (lines 82, 89).
We have made a supply in the figure 2 legend to define the abbreviations used in the x-axis.

Reviewer Follow-up: Please also specify forest type and time since fire in the figure caption, in addition to providing it in M&M lines 82 and 89.

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Original Reviewer Comment: Table S1: Please spell out the fire severity level rather than using single-letter abbreviations. Cite your source in the caption to clarify where this severity classification system came from (it looks similar to one by Ryan & Noste, but if you modified a published severity classification system, then please provide a clear rationale for how & why it was modified. If you used a published severity classification system, then just report the original classification descriptions for each category…the current descriptions in this table are very unclear. Please revise the criteria description for the moderate severity category to be more descriptive. Use the term “low-severity,” not “low-grade.” Revise “height” in the moderate severity description to read “high severity.” Capitalize “unburned” in the control description. Please include this table in the main manuscript rather than as supplementary information. What does “burned down” mean? Do you mean consumed? Charred but still present?

Author Response: The full names of fire severity levels have been shown in the table.
The relevant reference has been added now.
We made a revision of the moderate severity and replaced the inappropriate words.
“unburned” was capitalized in the control description.
We have adjusted this table to the main manuscript.
“burned down” means consumed.

Reviewer Follow-Up: Revise “burned down” to read “consumed.”

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Original Reviewer Comment: Figure S1: Add a scale bar. Also please add another panel to this figure to show the location and distribution of your research plots within your study site.

Author Response: The scale bar was added in fig. S1. We don't have more accurate remote sensing data. So more precise sample locations can not been shown in this figure.

Reviewer Follow-Up: The scale bar still does not appear in fig S1. Remote sensing data is not necessary to add more detail regarding the research area. Please provide an additional panel that shows the specific research area “zoomed in” to finer scale. Within this additional panel, add points to the map that show the location of the sample plots within the research area using either GPS or map coordinates. Use different symbology for the map points based on the severity level of each sample plot.


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

Input:
Title: THE ASSOCIATION BETWEEN MENTAL ROTATION CAPACITY AND MOTOR IMPAIRMENT IN CHILDREN WITH OBESITY—AN EXPLORATORY STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Review:
Mental rotation capacity in children with obesity is reduced, but only those with motor imairments

The authors present a mental rotation study with children with obesity and motor impairments    (OB-), with children without motor impairments (OB+) and with children with healthy weight (HW). The children were between 7-11 years old and they had to solve a mental rotation task with hands as stimuli and a motor task (M-ABC). The results show that the OB- group had significantly lower accuracy rates and inverse efficiency scores than the HW group. There was no difference between the HW group and the OB+ group. Due to this, the authors assume that the impaired motor impairment relates to the worse mental rotation performance in children with obesity.

In my opinion, the study is interesting and worth to be published in PeerJ. However, my major concern is the (limited) strength of the theoretical rationale and also the missing data of a cognitive processing speed measurement. For this, I suggest a major revision of the paper.

Introduction
•	The theoretical rationale could be ameliorated. The distinction of egocentric vs. object-based mental rotation task is missing and also the embodiment approach in mental rotation research.
•	Line 47/48:This focus contradicts the title of the study.
•	The following important paper is missing: Kaltner, S., Schulz, A., & Jansen, P. (2017). The association between obesity and mental rotation ability in an adolescent sample. Obesity Research & Clinical Practice, 11, 127-129.
•	Can the authors formulate direct hypotheses?

Methods &Materials
•	Are the children in the HW and obesity group matched by age and sex?
•	Why did the authors present the stimuli in a random time interval?
•	Line 164: “obesity” is missing
•	7 out of 25 in the HW group is a high number. What about the mental rotation performance of those children?
•	Line 191: Why did the authors choose these time windows for quick and late responses?
•	The calculation of the IES is rather unusual.
•	Did the authors correct for non-sphericity of the data?

Results
•	Line 240/241: p-values are missing.
•	Line 274: Is this significant after correction for multiple testing?

Discussion
•	The discussion is well written but I am missing the consolidation of the theoretical rationale.
•	There are some studies with children with Spina Bifida showing an impaired mental rotation performance. Those studies might be relevant.
•	The authors mention that cognitive processing speed was not measured. This is an important point especially because they emphasize the importance of motor planning processes. The authors should elaborate on this point.


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

Input:
Title: THE ASSOCIATION BETWEEN MENTAL ROTATION CAPACITY AND MOTOR IMPAIRMENT IN CHILDREN WITH OBESITY—AN EXPLORATORY STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Some minor English editing required.
Sufficient literature incorporated, however a few references need to be added to support some statements.
Professional article structure - YES.

Some results do not appear to be linked to the aims of the study.
Experimental design: Aims of study need to be better aligned to the results presented or the results should be amended to support the original stated aims. Specific notes provided in the attached document.

The building of the rationale in the introduction is not supported by the methodological approach used.  Please see comments regarding the need to add an additional group (that you have currently excluded for analysis).
Validity of the findings: Statement of main findings are flawed by methods of analysis and decision to exclude a required group for comparison (Healthy weight children with impaired motor skills).
Additional comments: Thank you for the opportunity to review ‘Mental rotation capacity in children with obesity is reduced, but only those with motor impairments’. This is an interesting paper using quantitative methods to explore the mental rotation capacity in children with obesity, and specifically their ability to use motor imagery as a proxy of motor planning and control.

I have provided a separate document with line by line comments for your consideration.  Should you be able to make these major amendments I would encourage you to resubmit a revised version 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: THE ASSOCIATION BETWEEN MENTAL ROTATION CAPACITY AND MOTOR IMPAIRMENT IN CHILDREN WITH OBESITY—AN EXPLORATORY STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Please see comments to the authors
Experimental design: Please see comments to the authors
Validity of the findings: Please see comments to the authors
Additional comments: Review revision:
Mental rotation capacity with obesity is reduced but only in those in motor impairments

The authors revised their manuscript in some points according to my suggestions. However, I have some concerns, so I can not recommend publication of this manuscript as it stands.

1.	First of all, the authors should not formulate post hoc hypotheses according to their data but in my opinion it is necessary to provide a theoretical rationale for the formulation of hypotheses BEFORE gathering the data. Because the authors have not done this, their research seemed not to be theoretical driven, which have to doubt the worthness of publishing this study in a journal like PEERJ.
2.	In my opinion, the differentiation in egocentric and object-based transformation due to the stimuli used is not correct, please consider the work of Voyer, Jansen, & Kaltner, 2017 in QJEP.
3.	The post-hoc analysis of the 7 HW children is not included in the manuscript. I also agree with reviewer 2 regarding this point. This point should be regarded seriously.


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

Input:
Title: THE ASSOCIATION BETWEEN MENTAL ROTATION CAPACITY AND MOTOR IMPAIRMENT IN CHILDREN WITH OBESITY—AN EXPLORATORY STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Please see comments in attached document from Reviewer 2.
Experimental design: Please see comments in attached document from Reviewer 2.
Validity of the findings: Please see comments in attached document from Reviewer 2.
Additional comments: Overall, the majority of issues have been addressed by the authors.  A few concerns remain regarding some responses and these have been added to the attached document. Please see comments in attached document from Reviewer 2. Should the authors be able to address these outstanding concerns I would support this manuscript being published. I would like to thank the authors for their efforts to address previous comments and thank them for their important contributions to this field.


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

Input:
Title: THE ASSOCIATION BETWEEN MENTAL ROTATION CAPACITY AND MOTOR IMPAIRMENT IN CHILDREN WITH OBESITY—AN EXPLORATORY STUDY
Review round: 3
Reviewer: 1

Output:
Basic reporting: I appreciate the explanation of the authors.
Please integrate: An explorative study in the title.
Experimental design: fine
Validity of the findings: fine
Additional comments: No additional comments


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

Input:
Title: PROSAPOSIN IN SEMINAL PLASMA ON THE DAY OF OOCYTE RETRIEVAL IS ASSOCIATED WITH NORMAL FERTILIZATION AND EMBRYO DEVELOPMENT IN IN VITRO FERTILIZATION CYCLES
Review round: 1
Reviewer: 1

Output:
Basic reporting: I found a typo in line 113, "grad" was "grade".
Experimental design: The authors said "The primary outcome of the study was normal fertilization rate" in line 123. Do we need the word "normal" here?
I think just "fertilization rate" is enough.
Validity of the findings: I could not understand the statement in Results section, line 179-180 "With adjustment of semen volume, the prosaposin concentration was significantly associated with pregnancy". Where I should refer the data in Table 6? And in Table 6, I could not find the mark "*" indicates significant at P<0.05. The authors should make clear the basis for making that statement.

The discussion is lengthy and not clear, so why don't the authors summarize it a little shorter? For example, the fourth paragraph is able to combine to the first paragraph about the nature of prosaposin.
In the line 251, the authors claimed "...between male fertility and ART outcomes...". However, in this study, the authors included only IVF cases. I recommend the authors to reconsider the use of the word "ART".
Additional comments: I read this manuscript with great interest. It is very difficult to find a marker in semen to predict pregnancy, because the cases of infertility are variable and complicate. Therefore, I am not surprised that the authors could not find significant correlation to fertilization. On the other hand, the authors found the correlation embryo development. I think this result mean that prosaposin supports the role of sperm function for early development of embryo.


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: PROSAPOSIN IN SEMINAL PLASMA ON THE DAY OF OOCYTE RETRIEVAL IS ASSOCIATED WITH NORMAL FERTILIZATION AND EMBRYO DEVELOPMENT IN IN VITRO FERTILIZATION CYCLES
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: mention below
Additional comments: Dear Editor-in-chief
As you send to me a manuscript entitled " Prosaposin in seminal plasma on the day of oocyte retrieval is associated with normal fertilization and embryo development in in vitro fertilization cycles. "
I reviewed this MN, it need minor revision.
The manuscript was written well and there were little grammatical mistakes.
Because the aim of this manuscript is evaluate the level of Prosaposin in the semen, and this protein produced by sertoli cells and epithelium of epidydyme. These cells have been controlled by FSH hormone, so it is necessary to check the level of FSH hormone in male partners to check the function of these cells that contribute to synthesis of Prosaposin, while the authors check the FSH hormone in female partners.


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: PROSAPOSIN IN SEMINAL PLASMA ON THE DAY OF OOCYTE RETRIEVAL IS ASSOCIATED WITH NORMAL FERTILIZATION AND EMBRYO DEVELOPMENT IN IN VITRO FERTILIZATION CYCLES
Review round: 2
Reviewer: 1

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: PROSAPOSIN IN SEMINAL PLASMA ON THE DAY OF OOCYTE RETRIEVAL IS ASSOCIATED WITH NORMAL FERTILIZATION AND EMBRYO DEVELOPMENT IN IN VITRO FERTILIZATION CYCLES
Review round: 2
Reviewer: 2

Output:
Basic reporting: the manuscript is written well and is an applied one.
Experimental design: well.
Validity of the findings: the results that obtained in this manuscript are novel and explain the hypothesis.
Additional comments: the manuscript entitled" Prosaposin in seminal plasma on the day of oocyte retrieval is
associated with normal fertilization and embryo development in in vitro fertilization cycles". was reviewed. the authors justify the items that informed before and improve it . so it seems the manuscript is suitable for publishing.


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: CELLULAR COMPONENTS IN TUMOR MICROENVIRONMENT OF NEUROBLASTOMA AND THE PROGNOSTIC VALUE
Review round: 1
Reviewer: 1

Output:
Basic reporting: In present study authors present an analysis of cellular makeup of the TME in neuroblastoma (NB) and proposed a cell risk score model to predict the prognosis of NB. Overall the work is very interesting and well-presented. Is it clear and concise paper with precise title, brief and concise abstract and sufficient introduction explaining the idea behind the project. Figures are sharp with legends that explain the figures.The paper is very interesting and the rationale for this work is solid. Presented results are largely novel and the manuscript is suitable for publication in the journal.
Several points:
In the present article, authors found that both Th1 and Th2 cells correlated with poor outcomes of NB. This should be further discussed, due to opposite roles of Th1 and Th2 cells in tumor biology and antitumor immunity.
The authors should consider analyzing ratios of immunocompetent cells of interest. For instance, correlation of Th1/Th2 ratio (xCell score) with outcomes of NB should be estimated and discussed.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: CELLULAR COMPONENTS IN TUMOR MICROENVIRONMENT OF NEUROBLASTOMA AND THE PROGNOSTIC VALUE
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The tumor microenvironment (TME) represents a complex network, and plays pivotal role in cancer progression and therapy. In this study, the authors detailed the cellular components of TME in neuroblastoma through gene expression data. Obtained data suggested that pCRS model provide a basis for treatment selection of high risk patients or targeting cellular components of TME in neuroblastoma.
Overall, the manuscript is very interesting and the rationale for this work is solid. The manuscript is recommended for publication.


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

Input:
Title: CELLULAR COMPONENTS IN TUMOR MICROENVIRONMENT OF NEUROBLASTOMA AND THE PROGNOSTIC VALUE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors suggested that cell composition in neuroblastoma microenvironment was markedly different among different prognostic groups and that the cell components of TME in NB affected patient survival.This model was constructed to provide a more powerful predicting of disease outcome of NB patients. Considering morbidity and mortality this investigation is up-to-date. Also, it is well structured, with sufficient field background, easy to read and understand.The literature references are correctly stated.
Experimental design: This is an in silico investigation. Gene expression profiles and clinical data were downloaded from available datasets. Hypothesis were well defined and meaningful, based on literature data. Investigation has been performed on sufficient number of patients, according to high ethical standards, without personal data presentation. Methodology was well described and detailed.
Validity of the findings: It is well known that the microenvironment of the different types of malignant tumors is very powerful predicting factor that can influence disease outcome. In silico analysis are powerful tool, but it is desirable to correlate results with pathohistological and clinical studies to give a more accurate picture of real-life events.
Additional comments: On the basis of all the above mentioned, I consider the paper is suitable for publication in a journal PeerJ.


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

Input:
Title: CELLULAR COMPONENTS IN TUMOR MICROENVIRONMENT OF NEUROBLASTOMA AND THE PROGNOSTIC VALUE
Review round: 1
Reviewer: 4

Output:
Basic reporting: The reporting is clear, unambiguous, and with professional English language used throughout. The Intro & background is adequate to show the context. The literature is well referenced & relevant. The figures are relevant, high quality, well labelled & described.
Experimental design: Rigorous investigation is performed using a high technical & ethical standard.
Methods are described with sufficient detail & information to be replicate.
Validity of the findings: Meaningful replication encouraged is rationale & benefit to literature is clearly stated.
All underlying data have been provided; they are robust, statistically sound, & controlled.
Additional comments: I commend the authors for their extensive data set, compiled over many years of detailed fieldwork.

Please, add some abbreviations missed, as aDc,cDc,iNK, OOB ...etc and place the significance of pCRS abbreviation found in discussion section at the place first time mentioned.


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: MOTORNEURON: AN OPEN-SOURCE R TOOLBOX FOR TIME-DOMAIN MOTOR UNIT ANALYSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is generally well written and easy to follow. Appropriate literature references are provided, and the context and scope of the article are discussed. All requirements regarding the provided subject data are fulfilled. My main concern regarding the overall structure is that the current interweaving of methodological description, toolbox description, and example R code makes it difficult to find methodological details.

1. While I appreciate the authors' considerable effort put into making the toolbox accessible to new R users, I would prefer a clear distinction between the presentation and discussion of the implemented methods, and example code. Currently, these two aspects are strongly overlapping in the text body, which can make it hard to find the technical details of a method. Would it be an option to separate these matters - mathematical description of the implemented methods on the one hand, and description of their implementation and example code on the other hand - into distinct sections of the article?

2. Generally, references are given where necessary. Still, I would have appreciated more references in several sections. In particular:
a) In the discussion, you write that frequency domain methods will be added in the future. If these are relevant, they should also be mentioned in the introduction.
b) For the different peak determination methods, there is one reference given for each of them. However, it is currently not obvious what exactly this reference refers to - e.g., in line 188, the reference (Keen et al. 2012) could refer either to the whole paragraph or only to the method of using a default peak. If you implemented the whole method following this one reference, please state that clearly in the beginning of the paragraph.
c) Also, as these peak determination methods appear to be known in the literature, it would be useful to provide some references to works (other than the original paper) where these methods have been used.
d) While I am not an expert in this field, it appears to me that there might be newer references that should be cited, e.g., Kline and De Luca (2016): "Synchronization of motor unit firings: an epiphenomenon of firing rate characteristics not common inputs."

3. I am not sure whether Figure 3 is of any utility to the reader and would advocate to remove it.

4. Is equation numbering with letters (line 196) journal standard? It seems very unusual and looks odd (also, because the equation number is to the left of and close to the equation).

5. There seems to be some mistake in the code listing in lines 277-292. Or are all the "$"s and "`"s intentionally there?

6. Several minor errors / suggestions:
Lines 70-71: Something is wrong or missing in this sentence.
Line 85: "The objective..." of this article/paper?
Line 154: "Recurrences" should be "recurrence" (singular).
Line 302: R is not a "scripted code" but a software.
Line 320: "flexibility" should be "flexible".
Experimental design: The authors describe a toolbox which implements several standard methods currently used in the field of motor unit synchronization analysis. The toolbox, including a sample data set, is freely available and open source, and the authors' laudable aim is to further reproducibility and standardization in this field. My main concern regards the presentation of the technical methods implemented in the toolbox, which is currently very brief and would benefit from more detailed description.

There are some details of the implemented methods that are not clear to me. While I understand that this is not a methodological paper, I believe that a precise explanation of the methods implemented in the toolbox is crucial, preferably using standard mathematical notation instead of textual descriptions (e.g. in line 196). In particular:
a) How exactly is the cumsum plot generated? How is it possible that the cumulative sum of a purely positive variable (bin counts divided by mean count) decreases at any point? (See Fig. 2A. and also the example in the toolbox.)
b) What exactly does "(considered as <=-60 and >=60ms)" mean? (Lines 176-177, 187)
c) Determining the peak boundaries as the 10%-90% range (line 185) seems counterintuitive - looking at the graph in Fig. 2A, I would expect this to yield a very unreasonable peak width. Could you elaborate and/or add an example application of this method?
d) What is the justification for assuming a default peak (line 188) if no significant peak can be detected? Doesn't this distort the subsequent analyses?
e) I do not understand the current description of the z-score method. What kind of uniform distribution do you mean? Uniform over which range? How is this related to a "shuffled" histogram? What does it all have to do with z scores? (Lines 191-193)
f) Is it really true that the histogram of independently firing motor units is expected to be completely "flat" (line 193)? I would assume the distribution to be falling off for large first-order distances, as it is e.g. very unlikely that the closest discharge time of the second MU is >1s away.
g) Related to f): Is there a justification for the choice of the +/-100ms range (e.g. line 175)? Is it reasonable across all muscles, subjects and conditions? Is it possible to adjust this value in the toolbox?
h) Is your usage of the term "cross-correlation" actually correct? At no point in the analysis, a correlation seems to be calculated, and the plots are also not what is typically understood as a "correlation diagram".
i) The definition of the "order" of an interval can currently only be found in the caption of figure 1. It should also be part of the main text.
Validity of the findings: I commend the authors on making their toolbox very easily available and accessible, including the provided sample data set. I encountered two issues while following the examples provided in the paper:

1) The command  "motor_unit_1 <- as.vector(subset(Sample_data, select = Time, motor_unit_1  == 1))" returned something of type list (not vector), and the subsequent call to "mu_synch" failed with an error message ("'motor_unit_1' and 'motor_unit_2' must be vectors."). I did "motor_unit_1 = motor_unit_1$Time" and the same for MU 2, and that fixed the issue.
2) The call "mu_synch(motor_unit_1, motor_unit_2, method = c("Zscore", "Cumsum"), order = 1, binwidth = 0.001, plot = TRUE)" did not result in any plot being shown.
Additional comments: I commend you for making these efforts to increase reproducibility and standardization of studies in this field. The toolbox looks useful and very well-documented, and you made it very easy to get started using it. If you can fix the current technical limitations of the paper, this will be a useful contribution.


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: MOTORNEURON: AN OPEN-SOURCE R TOOLBOX FOR TIME-DOMAIN MOTOR UNIT ANALYSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: Some experimental data is presented (Fig. 2 and 3), but the origin of this data is not explained. Although it is not the primary scope of the data, I would suggest a brief explanation of how the data presented was obtained. Also, a more systematic presentation of the values obtained for the different synchronization indexes in the presented data should be included.
A good example may be the McGill et al. "EMGLAB: an interactive EMG decomposition program" J Neurosci Methods 2005, which in a similar way presented software for iEMG decomposition, but (in my opinion) had a better integration of experimental data in the manuscript.
Validity of the findings: No comments
Additional comments: Overall, the manuscript is well written and provides a clear overview of the software. Apart from the suggestion to include more experimental data, the authors should consider a few additional comments:

- The cited review paper by Farina & Negro 2015 (and other work by that group) argues against the use of correlation between single pairs of motor units and suggests population-based analyses. I think this point should be emphasized, and possibly discussed as a future add-on to the software.

- It is a reasonable point that synchronization analysis usually requires expensive software, but the authors seem to overlook the fact that the same expensive software is typically required to obtain single motor unit spike trains (decomposition). In that sense, decomposition free-ware needs also to be developed in order to fulfill the ambition of enabling motor unit analysis for those without access to expensive software. This point should be discussed.

- In many articles using the CIS index, an additional parameter describing whether the peak in the histogram can be considered significant or not ("significant peak") is included. Here, significance is based on the peak in the Cusum. The authors may consider including this parameter as well.

- A prerequisite for valid estimation of synchronization is a critical number of decomposed spikes in the two spike trains. With too few spikes, a reasonable cross-histogram cannot be obtained. To the best of my knowledge no specific limit has been established, but I would suggest that the authors include typical number of spikes per spike train (used in previous studies) in the manuscript, in order to emphasize the importance to new users.


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: MOTORNEURON: AN OPEN-SOURCE R TOOLBOX FOR TIME-DOMAIN MOTOR UNIT ANALYSES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper is well written but it is not a research paper. There are no hypotheses or results.
Experimental design: This is a technical paper that describes a software package that is likely to make a worthwhile contribution to investigations of motor unit firing behavior. However, it is not a research paper and so does not really seem to fall into the Aims and Scope of the journal. I'm afraid I don't have any previous experience with this journal to know if this lack is disqualifying. If it is, then perhaps the authors could consider the Journal of Neuroscience Methods or the IEEE Transactions on Neural Systems and Rehabilitation.

There is no original research, no research question, no investigation, and no methods.
Validity of the findings: There are no findings.
Additional comments: The paper is good and worthwhile, but it is not a research paper and therefore does not seem to fall within the scope of 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: MOTORNEURON: AN OPEN-SOURCE R TOOLBOX FOR TIME-DOMAIN MOTOR UNIT ANALYSES
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: I would like to thank the authors for this strongly improved version. Most of my previous concerns have been addressed and I only have a few final comments.

126-127 Can you provide a reference for or very brief explanation of this "template matching technique"?

150-153 "if motor unit action potential trains are independent, then there is no relationship between the firing of one motor unit and the firing of the other and each latency or recurrence interval (e.g. -2 ms, +1 ms, or +6 ms) has an equal probability of occurring"
This statement in its current generality is not true, as I already indicated in my first review. For illustration just assume that both MUs fire independently at firing rates 20Hz and 30Hz, then it is very unlikely to observe a recurrence interval greater than, say, 100ms. In general, the distribution of the recurrence interval of two independently firing motor units will depend on the firing rates of the two motor units and not be uniform. I am unsure how this can be reconciled with the methods described here - maybe the distribution is approximately uniform for recurrence intervals below some threshold?

171-174 Thanks a lot for the improved description of this method. Just to make sure that I understand things correctly now: I assume "cumsum histogram" should be "cumsum graph" and refers to Fig. 2A? Then if I look for the 10%-90% range of the y axis in Fig. 2A I still end up with a really wide "peak", going from something around -0.08s up to maybe 0.02s. Is it just that the method does not work for this example (which would be helpful to mention) or do I still misunderstand something?

186-187 The same comment as above applies regarding the distribution of the recurrence interval of independently firing motor units.

185-198 What is the "shuffling" required for? Wouldn't it be equivalent to just compute mean and standard deviation of the original histogram data directly, without reshuffling? Also, the z-score is commonly defined as the difference of a sample from the population mean, divided by the standard deviation, and it is not identical to a confidence interval. In other words, a sample has z-score=2 if it is 2 standard deviations away from the mean. Under the assumption of a Gaussian distribution (is this justified here, i.e., is the amplitude distribution of bin counts Gaussian?), 95% of the data have z-score<=2 (asymptotically). The authors might want to revise this section to improve statistical rigor.
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: MOTORNEURON: AN OPEN-SOURCE R TOOLBOX FOR TIME-DOMAIN MOTOR UNIT ANALYSES
Review round: 2
Reviewer: 2

Output:
Basic reporting: -
Experimental design: My comments to the previous version of the manuscript have been addressed.
Validity of the findings: -
Additional comments: My comments to the previous version of the manuscript have been addressed.


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

Input:
Title: MOTORNEURON: AN OPEN-SOURCE R TOOLBOX FOR TIME-DOMAIN MOTOR UNIT ANALYSES
Review round: 2
Reviewer: 3

Output:
Basic reporting: The paper is well written and self contained. Raw data is shared in an associated web page.
Experimental design: The paper is mostly a technical paper to present a software package. But it does present data used to test the software, The methods are described in sufficient detail.
Validity of the findings: The data provided are sufficient for the purpose of demonstrating the software, and they also serve to make the point that synchronization indices can vary considerably when computed by different methods.
Additional comments: 26  "and investigate the diﬀerences in motor unit synchronization produced by diﬀerent methods in a case study example."  A single case study does not really constitute an investigation. Perhaps you could change this to something like "and present a case study showing how much the same synchronization index can differ when different methods are used to compute it."

348.  "According to results from Keen et al. (2012), this 1 impulse per second difference represents 471% of the standard deviation of CIS in the FDS."  For some reason, your results fall way, way outside the results reported by Keen et al. Therefore it probably doesn't make much sense to characterize the difference in terms of those results. Can you just use the 45% difference figure you used in the Abstract?

Read-me file. One possible point of confusion for readers of the motoRneuron.docx read-me file is that the cross-correlation histogram shown there (which is the one computed from the sample data in the code repository) is quite different from the one shown in Fig. 2 of the paper. First, the histogram in Fig. 2 has a relatively flat baseline count across its entire width, whereas the one in the read-me file falls off toward both ends. Presumably this was because the read-me histogram is based only on first-order intervals, whereas the one in Fig. 2 is based on first and higher order intervals. This should be explained in the read-me file.

Second, the central peak in Fig. 2 is compact and distinct as one often sees in short-term synchronization papers, whereas the central peak in the read-me histogram is diffuse and difficult to delineate visually. Perhaps this is the result of having too few recurrence intervals in the dataset, so that the statistical fluctuations in the bin counts are not sufficiently averaged out? In any event, some discussion about the difference between the central peaks in the two histograms is needed in the read-me file.