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: WASH REGULATES THE OXIDATIVE STRESS NRF2/ARE PATHWAY TO INHIBIT PROLIFERATION AND PROMOTE APOPTOSIS OF HELA CELLS UNDER THE ACTION OF JOLKINOLIDE B
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors have investigated the anti-tumor effects of JB at different concentrations on HeLa cell line as a model for cervical cancer. Although tumoricidal activity of JB has been demonstrated previously in other cell lines, the authors have attempted to test the impact of WASH protein expression on the effectiveness of JB. This aspect of the paper and the rationale behind testing the WASH mediated mechanism of JB action is poorly explained and needs further support. The authors have included certain references in discussion (lines 284-288), which would benefit from being moved to the introduction for clarification. The connection between WASH and Nrf2/ARE pathway also need further explanation and clarification (lines 297- 304 could be stated as a hypothesis in introduction). Language is professional and satisfactory but can be polished a bit more to enhance clarity of text.
The figures and tables in general need to be improved with respect to their labeling. Figures in the current version are extremely blurry and difficult to follow (labeling especially). Figure 1 does not add to the text or data in any way and as such can be deleted.
The authors have not explained their observations and interpretations in adequate detail. The results section needs to be expanded with additional comments on the impact of presence or absence of WASH protein in the two stable cell lines used. The authors also have frequently misinterpreted the results with no raw data/ values to support their conclusions. E.g. Fig3: authors have provided no explanation on how necrosis was quantified or make no comment on potential impact of WASH protein. Fig4: authors claim significant reduction in proliferation when the % decrease is less than 0.2% for either scramble or shWASHa groups and the error bars at time point 24hr are practically overlapping. Fig5: The flow plots are extremely poor resolution and make it impossible to judge the actual labels and data. Additionally, the authors show that BrDU positivity is greater in shWASH cells with increasing JB concentration but conclude that JB effectively suppresses proliferation in the absence of WASH protein which is the opposite of what the data suggest. Fig 6: Flow plots for panel c and g are practically identical and gating is inconsistent across the panels. This raises questions regarding the data validity. In many ways, the article fails to meet the standards and would need to be reassessed with clearer figures including raw data.
Experimental design: The authors need to improve explanation of the rationale behind (1) using only one cell line (2) rationale for testing impact of WASH protein and connection of WASH with Nrf2/ARE pathway (3) hypotheses that are being tested by each experiment. Certain methods used for assessing cell proliferation and apoptosis are redundant and do not add to the overall evidence presented. The authors have not described the experimental methods adequately and do not follow a universal method of describing cell densities across various set ups. E.g. line 139 states number of cells per well as 0.5x 10^5 which should be corrected to 5x10^4. Or on line 145, simply state the total number of cells per well as the concentration of cells is irrelevant to the set up. Line 141 only states that manufacturers protocols were followed with no details provided. Authors should add some basic information for each method performed in addition to stating manufacturer guidelines for reference. In the methods section, the authors have described using WST1 assay platform to measure cell viability but in discussion line 282, authors state the use of MTT which is a different assay- please clarify which assay was used. For statistical testing, the authors merely state the software used but not the actual statistical tests such as Student T test or ANNOVA etc. These details along with specific information regarding groups being tested is critical in evaluating the significance of the results.
Validity of the findings: The underlying raw data need to be provided by the authors to assess the validity of these findings. There is no clear indication or conclusion regarding the mechanistic impact of WASH protein on effect of JB and as such the manuscript fails to deliver data and conclusions that the experimental system was designed for.
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: WASH REGULATES THE OXIDATIVE STRESS NRF2/ARE PATHWAY TO INHIBIT PROLIFERATION AND PROMOTE APOPTOSIS OF HELA CELLS UNDER THE ACTION OF JOLKINOLIDE B
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is clear and easy to read.
The introduction section needs to be revised as mentioned in comments.
WASH needs to be describe more in detail i.e. it function, relevance for cancer, protein structure/subunits.
The representation of data can be improved as stated in comments.
Rational for implicating WASH in JB sensitivity is not described.
Experimental design: Methods section needs improved description as described in comments.
Validity of the findings: The data are encouraging but are preliminary.
Data interpretation need to be revisited. Conclusions of the data need to be explained further.
Additional comments: In the manuscript submitted by Hong et. al., the authors report the effect of Jolkonilide B on HeLa cells. They report that WASH silenced cells have heightened cell death in response to JB. The data are encouraging, but there are several concerns as listed in Major and Minor concerns.
Major
1.	The data are currently only performed on one cell line, HeLa. In order to extrapolate the results to cervical cancer, additional cell lines are needed. Also the current data indicates that WASH status determines JB sensitivity and therefore cervical cancers need to be screened for WASH levels before JB is considering it for treatment options.
2.	Jolkinolide structure had been previously published in the literature and can inserted as a reference instead of representing as Fig1.
3.	The authors use the name WASH in some instance and WASHa in other. Please provide the office NCBI Gene name and use that designation consistently throughout the manuscript. Also, it is suggested that the authors explain the rationale behind why they suspected that manipulation of WASH would sensitize cells to JB.
4.	Please provide antibody details (company and catalog number) for WASH protein.
5.	Check the figure label for Fig 3 a and d. They are control (non-JB) but the labels indicate JB.
6.	The authors mention WASH protein sinking in the figures and legends. It is suggested to describe this in the methods section.
7.	For better representation of the data in Figure 4, normalize the data from time 0 of all mentioned concentration to 100% and indicate % cell viability of the different treatment conditions.
8.	In Figure 5 the authors use BrdU incorporation to assess proliferation. BrdU is incorporated into dividing cells. The authors previously show more cell death shWASH in 0.5mM of JB than 0.25mM. BrdU appears to be higher in shWASH in 0.5mM of JB which is not consistent with other reported data/images of cell death. Also shControl cells have lesser BrdU incorporation than treated cells. Please explain.
9.	Cell cycle data also indicates increased cells in S phase of cell cycle in JB treated cells. This is consistent with the BrdU data. The authors are suggested to explain these observations.
10.	The cytokine profiling data indicated increased expression of IL1α, IL-6 and IL-8 at basal level in shWASH cells and is not statistically higher upon JB treatment of these cells. Further IL-6 increases only in 0.25mM but not 0.5mM. This data indicates that WASH silencing regulates cytokine gene expression and the results must be revised to indicate this rather than stating that JB influences cytokine and inflammation/immune cell function.
Minor
1.	For technical correctness, please use the format “shWASH” instead of ShWash/ShWasha throughout the manuscript.
2.	revise introduction for accuracy. The authors state Cancer is one of the deadliest diseases and cause for highest mortality rates for humans. This statement is not correct.
“The top global causes of death, in order of total number of lives lost, are associated with three broad topics: cardiovascular (ischaemic heart disease, stroke), respiratory (chronic obstructive pulmonary disease, lower respiratory infections)” Ref:
https://www.who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death
3.	Replace purinomycin to puromycin in Ln 110


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: WASH REGULATES THE OXIDATIVE STRESS NRF2/ARE PATHWAY TO INHIBIT PROLIFERATION AND PROMOTE APOPTOSIS OF HELA CELLS UNDER THE ACTION OF JOLKINOLIDE B
Review round: 2
Reviewer: 1

Output:
Basic reporting: Please see additional comment
Experimental design: Please see additional comment
Validity of the findings: Please see additional comment
Additional comments: Regarding the response to #1 Major concern, it is suggested that the authors include a line in the discussion to mention that analysis of WASH in other cervical cancer cell lines would be necessary to before its consideration for therapeutic purposes.

With respect to #3, since the official gene ID lists WASHC1 as the official symbol with alternate names of WASH1 and WASH, it is necessary to list all these three versions next to the gene ID for clarity.

Please remove the word “artificial” to describe HeLa cell line in Methods section 2.4

All other concerns have been addressed.


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

Input:
Title: FIVE DECADES OF CHANGE IN SOMATIC GROWTH OF PACIFIC HAKE FROM PUGET SOUND AND STRAIT OF GEORGIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is adequately presented and clearly structured. The different sections are easy to follow and coherent. The figures and tables are also clear and nicely presented. Raw data is available for checking and seems suitable.
Experimental design: The research question and framing of the introduction is well-crafted and sums up well the background, theory and expected contribution behind this analysis, particularly the changes in growth rate and their timing in the different populations. I have however a few concerns and/or questions (some of which are detailed in the next section).

First, being more familiar with its Atlantic counterpart, how accurate is Pacific hake aging using otolith increments, and has there been any validation experiments done prior? I’m asking since European hake aging using otoliths has recently become more and more problematic and even removed from current stock assessment in favor of Age-Length Keys. The main issue was a consistent inter-reader overestimation / underestimation when looking at increments over 3 years old, and an overall zonation pattern proven to be less straightforward than initially thought. In the present manuscript the various source used to reference otolith aging of Pacific hake are old and correspond to the onset of similar work on European hake before it was refuted.

I’m also curious about some of the methodological choices made for the growth back-calculation using otolith increments. Why wasn’t the biological intercept method modified from Fraser Lee used instead of the base one? In terms of results it consistently performs equally or better than the FL method, but has the advantage of basing it on real measurements so that the emergence of proportionality between fish and otolith growth is correctly estimated. The issue with FL is that it uses a statistically determined intercept based on the relationship between otolith and fish length (showed in Appendix 1), which is directly dependent on the samples available and in particular should be treated carefully for growth estimates beyond the age classes represented in the data.

Here there is a reasonable number of samples with a good R2 but the populations are not equal. For example, the Strait of Georgia population stops at age 2 when it comes to available samples, yet a large portion of this manuscript discusses growth during first summer. Linear proportionality between fish and otolith growth is known to be particularly unreliable in the younger ages, especially when those are only derived from the back-calculation model. I’d be careful with including 1st summer back calculations for SoG, or at least I’d like to see more mentions of this potential limitation either in methods or in the discussion.
Validity of the findings: The discussion did a good job to provide a summary of the results and recontextualize them with the research questions. A clear and concise conclusion is also provided at the end to summarize the key findings, encompassing both the modelling results on the factors driving growth dynamics. Overall, it is a good quality work but there remains some issues.

I don’t have much to say about the first part of the analysis. The multivariate analysis is suitable and well presented, and shows interesting temporal and inter-populations differences. I am somewhat conflicted on other aspects and in particular the following modelling approach.

First, regarding the discussion of trace elements in the growth modelling. Ba signals have been identified in nutrient-rich waters, and given the particular dynamic of SoG and PS there is certainly a high likelihood for upwelling induced chemical signals that positively correlate with growth. The authors discuss the possible positive effect of freshwater through the negative correlation between growth and Sr, but it would have been good to discuss it in light of the Ba signal too, given that in both SoG and PS there is a strong dependence on freshwater influx from the nearby rivers for bringing in nutrients.

Second, the analysis could have benefited from more consideration of the fishing factors. The idea of fishery-induced physiological changes, notably an earlier maturation and smaller / growth, is adequately brought in the discussion, but lacking from direct analysis. In both populations the authors show a difficulty separating the different models due to low delta AICs, and a generally large proportion of unexplained variance. It would have been interesting to test those models after including some form of fishery-associated factors, for example F. I suspect they would have been easier to separate and would have added stronger signals. In turn, it could have also become interesting to contrast the influence of harbour seal predation on density-dependent growth with fishing-induced density release.
Additional comments: There is very little mention of limitations throughout the manuscript, either experimental or methodological. The GLM results reported lack statements on uncertainty and confidence, if would be good to report at least CIs in the different plots from figure 6 and 7.

A model equation should be included in the method section for clarity (I suggest the most complex model as reference). Out of curiosity, did the authors consider a GLMM (with random effects) instead? Given the natural variability in individual growth trajectories, it is usually quite important to include at least a fish-specific random intercept.

Regarding elemental values, there is mention of averaging them but no further details. Were those averaged per age? Per summer zone? Over the whole otolith? It should be specified in the text as well as in the legend of figures 6 and 7.

L.126 : add Lc = length at capture to the equation description

Table 1: add a divider between the two population as it can be confusing to read from SoG age 6 to PS age 0. Eventually a sub-total for each pop.

Figures 6 and 7: add confidence intervals


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: FIVE DECADES OF CHANGE IN SOMATIC GROWTH OF PACIFIC HAKE FROM PUGET SOUND AND STRAIT OF GEORGIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Review of the manuscript peerj-69048

Manuscript tittle: “Five decades of change in somatic growth of Pacific hake from Puget Sound and Strait of Georgia”

Chittaro et al. wrote an interesting manuscript comparing the somatic growth of two populations of the Pacific hake in North America. They use different descriptors (predator abundance, otolith microchemistry and temperature proxies) to understand the drivers of differential somatic growth between the populations and through time within the populations. The dataset is robust and the statistical analysis are sound. Therefore, the authors presented interesting results and discussed them properly. I believe that the manuscript can be accepted for publications in PeerJ after minor revisions. Specific comments by section presented below:

Lines 43 to 44 – It is not usual to start the introduction with a question. I recommend the authors to rephrase this sentence.

Line 126 – Why did the authors used Fraser-Lee method instead of more recent and potentially more precise methods for back-calculations from otoliths? See Vigliola & Meekan 2009 (The Back-calculation of fish growth from otoliths).
Lines 173 to 174 – It would be better to have the chemical symbol of the elements instead of their names.
Line 235 – The authors could cite the R packages they used, once Rstudio it is just an UI from R software.

Figures 4 and 5 should use colors that are colorblind friendly. Also, I wonder if wouldn’t be clearer to have the information of winter and summer presented differently, because the somatic growth during winter is much smaller than during summer, so it is harder to identify changes in growth for winter when presented in the same axis as the summer values.
Experimental design: The experimental design is good and the research questions are well defined and the methods are also clearly described.
Validity of the findings: The results are interesting and have broad impact (it can be used as a comparison to other fish populations elsewhere). The statistical analysis were robust and the interpretation of the results well discussed.
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: FIVE DECADES OF CHANGE IN SOMATIC GROWTH OF PACIFIC HAKE FROM PUGET SOUND AND STRAIT OF GEORGIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No additional comment from the initial review. Overall, I commend the authors for the clear and thorough review work on their manuscript. I appreciate the detailed answers and considerations given to each point, and the incorporation of most of the more important comments within this reviewed manuscript. There isn't much to be added, though I'd still like to voice my concerns about aging of Merluccius species using otoliths (see next section).
Experimental design: The inclusion of the biological intercept has solved most of my issues with the otolith growth model. I am thankful for the addition of a growth equation, as well as the integration of Ba in the chemistry results.

While I appreciate the technical reports sent as support for an otolith-based individual aging used in assessment, I am still somewhat skeptical of its validity in an hake species given that there haven't been modern validation experiments to back it up beyond the 1985 paper provided here. Indirect validation through tracking of cohorts is a fine approach, but it is sensitive to biases when human interpretation is straightforward but have no biological meaning. Here the analysis relies on estimates of yearly growth based on the interpretation of annual rings: my point is that without any direct experiment of otolith deposition, the ages and subsequent growth estimates could be wrong

The European hake was for a long time assessed similarly, and issues with aging only arose in the last two decades when it was realized the deposition pattern was less straightforward than anticipated, and age estimates were consistently wrong especially in young fish. I therefore recommend the authors to be careful of any aging done on the Merluccius genus, and alternatively would encourage new validation experiments to see if aging of Pacific hake using otolith is indeed biologically plausible.
Validity of the findings: No additional comment
Additional comments: No additional 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: FIVE DECADES OF CHANGE IN SOMATIC GROWTH OF PACIFIC HAKE FROM PUGET SOUND AND STRAIT OF GEORGIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: Chittaro et al. amended the all the issues pointed by both reviewers and the editor, therefore I believe that the manuscript can be accepted for publication in Peerj.
Experimental design: The manuscript was well designed and analyzed. The results are sound and they were interpreted correctly.
Validity of the findings: The authors reported an interesting case of analysis in fish somatic growth, reporting data on growth during winter season, which is not usually done. They findings can be useful for the scientific community.
Additional comments: No additional comments


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

Input:
Title: ASSESSING AVAILABILITY OF EUROPEAN PLANT PROTECTION PRODUCT DATA: AN EXAMPLE EVALUATING BASIC AREA TREATED
Review round: 1
Reviewer: 1

Output:
Basic reporting: Ok
Experimental design: There's no experiment
Validity of the findings: See below my comments
Additional comments: The authors estimated the area applied with PPPs in Ireland and identified the most used AIs. The paper reads more like a review rather than a research paper.
Although a national-based estimate is a good first step, assessing contamination level and exposure of non-target organisms require a much finer scale data than national-level data. The dynamic, fate, transport, and residues of pesticides, like any other chemicals, depend largely on soil properties and climatic conditions. Also spatially distributed data is important for understanding the potential exposure of non-targeted organisms to the AIs. If the pesticide were applied in places where biodiversity is low, then the risk for non-target organisms getting in-contact with the pesticide can also be low. Without spatial data, it is difficult to do a meaningful risk assessment. The authors should consider compiling subnational pesticide usage data.
 Line 378-409: The risk assessment was not correctly conducted. First of all, the LC50 is expressed as concentrations. How did you convert the application rate into concentrations in water, soil, and atmosphere? How did you decide the High, low and medium risk? Some AIs are not soluble or have high adsorption to soil, thus, these chemical are very unlikely to get to water even though they might be very toxic to fish. Similarly, some AIs have very low volatilization, thus may not even get to the atmosphere. Following my comment above, risk assessment is only meaning when it is done with spatially distributed data and by considering the physiochemical properties of the AIs, the soil properties, and the environmental conditions (temperature, rainfall, groundwater, proximity to surface water, etc). I recommend removing the risk assessment part from this manuscript and also removing it from the title. Otherwise, the risk assessment has to be done properly.
Other comments:
Line 47: FAOSTAT records about 4 million tonnes of pesticides usage globally in 2015. I think it should be 3 billion kgs (not tonnes) in 2009. Please check and update the figure. FAOSTAT provides data up to 2019.
Line 91 – 114: Besides FAOSTAT and EU commission, USGS reports usage of individual AI in different cropping systems at county level. PESTCHEMGRID also estimates the individual AI usage globally (including Ireland) in different cropping systems at georeferenced grid level. I think these databases should be mentioned.
Line 197: “…. grasslands alone account for 99.4% of this crop type” – Which crop type?
Line 199-200: Is the fraction of land applied with PPPs coincide with the fraction of organic farming?
Line 206: “These represent 44%, 32% and 24% of the area where herbicides are applied.” I do not understand this sentence. Do you mean PPPs rather than herbicides?
Line 209 – 212: How do these results compared to FAOSTAT in terms of unit total mass and how do they compare against PESTCHEMGRIDS in terms of both mass and surface area?


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: ASSESSING AVAILABILITY OF EUROPEAN PLANT PROTECTION PRODUCT DATA: AN EXAMPLE EVALUATING BASIC AREA TREATED
Review round: 1
Reviewer: 2

Output:
Basic reporting: The english is clear and the paper reads well. Literature referenced is appropriate, although balance in relation to a summary of the regulatory system would be helpful. The title possibly overstates the scope of the paper, the main output of which is the assessment of data availability and recommendations for improvement to support better interpretation of comparability and impact.
Experimental design: The comparison of usage data between European MS, and recommendations for improvement of recording, to allow comparison of impacts is relevant and meaningful. The recommended proxy of basic treated area  I don't think is adequately demonstrated to be an appropriate comparator nor are the associated maximum and minimum treated areas. Additional comments have been added to the annotated PDF. The comparison of commonly used PPPs  against regulatory ecotoxicological endpoints isn't novel and basing this comparison on basic areas of top 5 PPPs doesn't adequately demonstrate an advantage to other methods of comparison such as use of pesticide impact indices using PPP weight. The methods are not quite described with sufficient detail to fully understand or replicate.
Validity of the findings: The analysis of the available data fully support the recommendations for the need for consistent data collection and reporting and this is an important and valuable message. The recommendation on the suitability of basic area to be a suitable proxy for use is not adequately supported in my opinion, either for use as a comparison of usage between MS or to indicate comparative implications for impact on non-target organisms.
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: ASSESSING AVAILABILITY OF EUROPEAN PLANT PROTECTION PRODUCT DATA: AN EXAMPLE EVALUATING BASIC AREA TREATED
Review round: 2
Reviewer: 1

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


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

Input:
Title: SEEDS HARVESTED DURING MOWING FROM SEMI-NATURAL GRASSLANDS AS AN AD HOC BUT EFFECTIVE SOLUTION FOR GRASSLAND RESTORATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Review of the manuscript by Perera et al. “Seeds collected accidentally from semi-natural grasslands as an ad hoc but effective solution for grassland restoration”. (#72060)

Restoring species-rich grasslands is a key topic in conservation in agricultural and urban landscapes. A variety of techniques have been proposed and tested under field conditions. One approach is to collect seeds that remain in mowing machines when cutting species-rich semi-natural grasslands. This seed material can be quite rich in valuable grassland species depending on the community type used for harvesting. In contrast to the transfer of hay from species-rich grasslands, the content of germinable seeds apparently is higher when using this seed generation technique, and the amount of biomass to be transported is lower. This little compact study investigates the germination success of “accidently” collected grassland seeds during a single mowing event in July in pot and field experiments. The results show that, even though the proportion of germinated seeds was comparably low (42 %), a considerable number of characteristic species of species-rich grasslands was restored, and the method thus seems to be applicable in cases, where species-rich grasslands are mown and the hay shall not be used for restoration. The advantage of this approach is primarily the higher species numbers included and the likely higher intraspecific diversity represented by the seeds compared to commercially available seed mixtures. Moreover, it is a cheap approach. Thus, the study seems to add some valuable observations on seed collecting in grassland conservation management.
Experimental design: The experimental design seems to be sound.
Validity of the findings: Even though the seeds were collected only from a single meadow and during a single moving event, the results are of some interest to conservation managers. The statistical analysis is sound and the data are presented quite well. I agree with most of the authors’ conclusions and recommend publishing after a final linguistic check.
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: SEEDS HARVESTED DURING MOWING FROM SEMI-NATURAL GRASSLANDS AS AN AD HOC BUT EFFECTIVE SOLUTION FOR GRASSLAND RESTORATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well-written and concise. References are appropriate and well supporting the message.
The results are of high scientific quality.
Experimental design: In general the experiments are well conducted, I found however some issues, which should be clarified:

l141 some additional info would be fine here. For example which type of substrat was used for the sowing (?potting soil? Soil collected from the restoration sites?), and what was the watering regime? How was the sowing executed? Was there a soil covering applied after sowing?
l151-161 – Not clear what are these treatments and why are these important for the sowing experiment?
l192 It is a pity that rototilled but non-sown plots were not established as control for local dispersal and seed banks. It would be nice to see which species were those which were present in the vegetation of the surroundings and also were established in the sown plots (of course they were also sown).
Validity of the findings: The manuscript covers an important and practice-driven approach and adds information to a so far neglected aspect of grassland restoration.
Additional comments: l46 – maybe “high quality plant material” would be better here
l86 – instead of “employed” use “applied”
l206 These figures for pure seeds are really high – where were 5kg seed material attached to a mover? Some photos would be interesting to see how the experiments were implemented or how this material looks like.


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: SEEDS HARVESTED DURING MOWING FROM SEMI-NATURAL GRASSLANDS AS AN AD HOC BUT EFFECTIVE SOLUTION FOR GRASSLAND RESTORATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: I am not a native English speaker, but the manuscript seems to be written in good English. The problem of the importance of meadows in the environment and the problems associated with their creation have been well described. The list of references includes 33 positions, which seem to cover well the basic knowledge about the research topic. The paper is supplemented by 3 figures and a table. All of them important for the manuscript. The work structure is clear and compliant with PeerJ standards.
The title of the work does not fully correspond to the content. It would be advisable to include in the title that the seeds were harvested during mowing. The word "accidentally" is ambiguous.
The work also requires some editing corrections. Describing the units in which the frequency is represented would be advisable.
Fig. 1B. Lack of b for J20 and M21 for the first week
Fig. 3 in the description (), Fig. 1 [] standardize to be uniform
References format in List of references are different than in the authors' guidelines. Dates in brackets, "&" instead of "and".
Experimental design: The research questions and hypotheses are well formulated. Material and methods are well described, but more information can be added with respect to the seed extraction method. The type of mower should be described in more detail, as not all types of mower will allow you to collect seeds. The quality of the seeds was examined in detail and with the use of appropriate statistical methods.
Validity of the findings: The supplementary material contains two tables with raw data. The raw data has been compiled clearly and is complete. In table S1, enter the frequency and cover units. There should be a point as the decimal separator in table S2.
The conclusions are properly formulated. The benefits of using seeds collected from the machines are presented, but also the limitations are underlined. It has rightly been suggested that the method may be a valuable addition to the standard methods of creating multi-species meadows.
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: SEEDS HARVESTED DURING MOWING FROM SEMI-NATURAL GRASSLANDS AS AN AD HOC BUT EFFECTIVE SOLUTION FOR GRASSLAND RESTORATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: After the corrections, no comment
Experimental design: after improvement no comment
Validity of the findings: no comment
Additional comments: After minor revision article should be accepted


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: CHANGES IN THE CORE SPECIES OF THE ANT-PLANT NETWORK OF OAK FOREST CONVERTED TO GRASSLAND: REPLACEMENT OF ITS ANT FUNCTIONAL GROUPS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Even though I am not a native English speaker, I noticed some language errors and sentences that can be improved to facilitate understanding. Therefore, I suggest an English revision of the article.
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: This study focused an interesting topic on ant-plant mutualistic interactions, which was not well-studied so far (in temperate ecosystems). The analyses are overall well-described with suitable methodology and interesting conclusions. In the introduction and materials and methods sections, sentences are well-organized and suitable references are provided. But the authors should overall revise the texts in the results and discussion section, including figure captions, because of awkward sentences and lack of explanation. Finally, even though I am not a native English speaker, I noticed some language errors and sentences that can be improved to facilitate understanding. Therefore, I suggest an English revision of the article.

Therefore I would suggest "Major revision" to be acceptable for the PeerJ.

I have a few minor comments which I list below:

Introdution; L118-123: Okay, I see this being explored here and at the beginning of your discussion you address the issue of functional groups, which I think is fantastic and which is something that greatly enriches the study. However, you do not address throughout the methodology that the study ants classified into functional groups, likewise this is not explored as expected in the results. Finally, I suggest you present as supplementary material this ant functional group classification.

Methodology; L131-133: I think you have to add the map of study area to this paper. Many readers of this journal does not know Mexico geography. I hope that you will be able to add the map of study area so that people all over the world can understand it.

L156-157: Haven't samples been established independently along the transects? Does every transect correspond to a large sample? I understand that for the intended analyses, this may not have significant implications, however, I believe you have to detail this information here.

L176-178: So the ants were collected, I imagine via manual collection right? Using tweezers? Entomological vacuums? This has to be made clear in your methodology. Please rephrase and add this information a few paragraphs above.

Results: I miss the biological part here. I suggest starting by exploring the ant and plant community. I would start with that part. How many interactions were most frequent throughout the study? Which were unique to each environment? You present some of this information throughout the paragraphs, but I find it difficult to follow the presentation of the results as it is, please reformulate.
Also, the part of classifying ants into functional groups that is presented throughout the discussion should be presented here. Please rephrase this.

Discussion: Discussion too extensive and partly difficult to follow. I believe that following the same objective logic as presented in the methodology and results would be fundamental here. Part of what is presented in some paragraphs can be properly presented in the results and the text reformulated here.

L334-338: As I mentioned before, I miss the presentation of the results referring to this classification of the sampled species into functional groups. Some of what you explore here throughout the discussion (in the third and fourth paragraphs) I expected to appear earlier in the results section. This will allow you to summarize some of the unnecessary information presented here.

L458-459: How many? I expected this information to be properly explored in the results section.

L459-475: I understand that part of what is presented here has already been presented earlier in the results, and that it can be discussed in another way. I suggest rephrasing and summarizing this information.

Figure 1: Use the letters (a) and (b) to distinguish the environments, and reformulate the legend.

L668-670; Figure 2 legend: Please inform here in the legend that the species name codes correspond to those presented in table 1, this is essential.

Table 1: I suggest some reformulations in this table. Present a first column with the name of the family, a second with the name of the species - which must contain the appropriate scientific name of the species with author and year -, a column with the code (use "code") and finally a column distinguishing the type of environment. And use an “X” to indicate the occurrence of the species. This will make the table more adequate and even with less unnecessary information.

Specific comments:
L29: replace “the nested structure and specialization level” with “network properties”

L87: replace “in” with “on”

L88: remove "a" from the phrase "a not well-connected groups of species"

L95: replace: “in temperate ecosystems than in tropical ones” with “in temperate than in tropical ecosystems”

L105: remove space before "Therefore"

L108: replace “work” with “study”

L329: replace “As expected, the” with “The” [I particularly don't like to start a paragraph like this, it gives little or almost no weight to the study's findings. It could be something like hypothesized, hence the whole meaning changes. However, as they preferred not to work with hypotheses, or not to leave this formulated in the study (which would easily be possible) I make this small suggestion for a change.]

L406: remove space before " Disturbance"

L406-409: Checking the English of this part, I found it confusing. I suggest rephrasing to allow proper understanding of the information.

L426: replace “ants” with “ant”

L477: replace “As expected, the” with “The”

L488: replace “The above” with “Our 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: CHANGES IN THE CORE SPECIES OF THE ANT-PLANT NETWORK OF OAK FOREST CONVERTED TO GRASSLAND: REPLACEMENT OF ITS ANT FUNCTIONAL GROUPS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a good manuscript have explained and fundamentally sound research. It is well written and have a coherent structure that is able to address your research in descriptive way.
Introduction:
This section is well described the importance of research as well the objectives. Background information is very clear but there is lack of hypothesis in the paper. Generally, there is need to remove a paragraph from the introduction as it looks more appropriate in the discussion to support the results, it have also mentioned in general comments section. Although citations are not latest as I have suggested some latest articles to must cite it, which will make it better. References are mentioned in the annotated attached file which will be open in adobe reader.
Experimental design: This part is well written, and each point of experiment layout has been clearly described by the author. Few comments for improvements have been given in the general comments section. Please add the map of study and it is good to add the table of climatic conditions of the studied region.
Validity of the findings: Well explained and appropriate results according to the research objectives. Discussion have needed some grammatically improvement, and also requires more citations to support the results in discussion.
Please remove figure and table numbers from the discussion.
Figures:
Figures require more improvement in description of each figure.
Additional comments: 1-	In line “60” change the font size of “essentially”.
2-	In line “72” change “Contribute to community” to “Contribute in community”.
3-	In line “87” the word “changes can have” should replace by “changes may have ”
4-	In line “87” “and can even cause” should replace by “and also cause”
5-	Lines# 105 to 111 should be the last lines of INTRODUCTION. Though ,it would seem more better in the end.
6-	Lines 118 to 123 may use in the discussion chapter rather than in introduction. It would support the results in that chapter.


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: CHANGES IN THE CORE SPECIES OF THE ANT-PLANT NETWORK OF OAK FOREST CONVERTED TO GRASSLAND: REPLACEMENT OF ITS ANT FUNCTIONAL GROUPS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript evaluated the difference in the structure of facultative ant-plant networks (metrics for network and species levels) between an Oak Forest and grassland in the same region. The authors went several times to the field to record the interactions and, though they don’t have spatial replicas, they have a good picture of ants foraging on plants at these two types of vegetation. I suggest here some points that I guess have the potential to improve the quality of the manuscript.
Authors are not consistent with the use of “plant-ant” or “ant-plant” (e.g., line 152), although they used most of the times “plant-ant”. In the literature “ant-plant” and also “animal-plant” are the most used terms. If the authors have a special reason to use plant-ant it could be justified, but if not, I strongly recommend the use of ant-plant (please check throughout the manuscript).
Here I summarize my main concerns, but they are detailed in the following pages: (i) One of my most important concerns is related to what the authors consider as a land-use change because, in my opinion, they are actually comparing two vegetation types. They argued that the grassland is recovering in the last 80 years ago, so it is not an ordinary land-use change, it is established vegetation through this time. To do the comparisons between land-use changes, the authors would need to define replicas obtaining a measure of the variation within the Oak Forest and the grassland. Even better when it is possible to proceed with the BACI (Before After Control Impact), one can tell that this is the real effect of the land-use change. In the case of the study presented here, they can only infer how the networks are structured in different vegetation types or different habitat conditions. (ii) Authors use several examples of other types of biotic interactions, not properly compared with a facultative ant-plant interaction network, like pollination, etc. I suggest focusing on ant-plant interactions and/or co-occurrences, which are a good number of publications available, and they can mention other networks, as pollination, briefly, in a couple of sentences. (iii) Also, and related with the latter, authors mentioned in the M&M that they observed and recorded each type of interaction between ants and plants but they never mentioned it in their results. I strongly recommend exploring this data, because this will improve the strength of this manuscript. (iv) Authors need to introduce and present hypotheses for each of the metrics they measured, i.e., bring the biological meaning for each of the metrics they used. In the last paragraph of the introduction, they presented basically a hypothesis related to the nested structure of networks and in the M&M and Results they present several other interesting metrics, but they have to tie them with the purposes of the work. (v) The authors mentioned a couple of times about functional groups based on a previous work of Andersen (2000) but I was not able to see which ants belong to these groups and how they evaluated it in the present manuscript. (vi) There is a lack of description of some statistical analysis they used to compare metrics, and also more details regarding the statistics of each test. (vii) The discussion needs deep improvement. I used the first two paragraphs to give some examples of the concerns that I noticed. Also, the authors need to stress each of the results they discuss, avoiding repeating the discussion of the same topic more than once. (viii) Authors argued that in grassland there is a resilient network and it is difficult to return to the interactions of the oak forest. However, the species that occupy these sites are related to the vegetation structure. So, if in the grasslands it is possible to recover the same habitat structure of the oak forest, maybe the ants that occupy these sites will also change.

1 – Title: I strongly recommend including some results/conclusions of the study in the title. My opinion is that a “comparison” is less attractive to the reader.

2 – Abstract: This section needs deep improvement.
2.1 – (Lines 24-26) The definition of nestedness in the second sentence of the abstract and with a lower connection with both prior and next sentences seems inappropriate. Besides, this definition is a bit confusing.
2.2 – (Lines 28-30) This is one of my main concerns. Change in land-use sounds like an experiment with before and after. They have three transects of samplings (pseudoreplicates) merged in the same matrix and the networks derived from it. So, they have no replicas of grasslands and oak forests, and they cannot infer about change in this sense. They have a comparison of two different environments, in terms of vegetation structure and other characteristics. Also, the history of this past intervention is not well explained (How was this intervention? See my comments for the method section). Finally, I have some concerns related to the aims/hypotheses, but please see my comments in the introduction section.
2.3 – (Lines 34-36) Based on some network metrics authors inferred about resilience and based on that they conclude that “it will be more difficult for it to return to its original state”. Is the original state related to the network structure and ant-plant interactions or the vegetation structure? The authors also used this idea in the last paragraph of the paper (Line 487).
2.4 – (Lines 36, 37) More vulnerable than what? Compared with tropical? Based on what? It is not the conclusion of this study.

3 – Keywords: This paper explores network analysis, ant-plant interactions, different vegetation structures, habitat conditions, and other subjects. I think that keywords more related to the main points of the paper should be better than the species names of some organisms studied.

4 – Introduction: Some aspects of the work were not well introduced, and some topics are disconnected from the main proposal of this study. Please see below some examples of aspects that deserve attention.
4.1 – (Line 46): Sentence without citation. I found some others sentences that the authors missed citing properly. Please check it carefully.
4.2 – (Line 49, 50): This sentence is not related to the subject of the study and also there is no citation. I recommend removing it.
4.3 – (Line 53): “land-use change” instead of “land use”?
4.4 – (Lines 54, 55): The most important for what? Maybe there is some information missing here.
4.5 – (Lines 59, 60): Sentence without citation.
4.6 – (Line 63): “has made it possible” is weird, please rephrase it.
4.7 – (Lines 64, 65): Is there a specific reason to use "plant-ant" instead of "ant-plant"? I can tell that it is widely used "ant-plant" in scientific publications.
4.8 – (Line 73): Why there is resistance to species loss? How does it work? It needs to be better explained and tied with your work.
4.9 – (Lines 80-82): How it is stated seems like it was not explored before, but several works evaluated it.
4.10 – (Lines 88, 89): And what it means or what are the consequences of it in biological terms?
4.11 – (Line 94): Cite examples or a review that explored it.
4.12 – (Lines 95-97): Sentence without citation.
4.13 – (94-101): Along with the introduction, and especially in this paragraph, authors based a lot on other mutualistic networks non-ant-plant. However, there are fundamental differences between other mutualistic networks authors mentioned and ant-plant networks. Also, as the authors have mentioned, within ant-plant interactions there are lots of variations in the predictable network when they are mutualistic or not. My suggestion is to focus on ant-plant networks, only mentioning other networks to put the results in the context of the big picture, for example, at the end of the discussion or the very beginning of the introduction.
4.14 – (107, 108): I do not agree. There are lots of papers that evaluated how ant-plant networks change with disturbances. Authors can search, for example, on Google Scholar for [ant plant disturbance] or [ant-plant anthropogenic] and they will find some publications, as a whole book dedicated to this subject (organized by Oliveira and Koptur), and some articles that investigate the fire, dam, anthropogenic, clearcutting, forest fragmentation, etc. After I made this comment, I saw that the authors cite one of these papers, my point here is as the authors stated seems like no one ever evaluated how disturbance affects ant-plant networks.
4.15 – (Line 110): Here is one of my main concerns, the aims/hypotheses. The authors chose to consider a priori that the structure of both networks would be nested. On the other side, the authors have no hypotheses/expectations for the all-other metrics they measured. If the authors analyze data, they have to introduce some information about this analysis and they have to state a biological expectation for that analysis.
4.16 – (Line 110): Seems out of context an introduction referring a lot to mutualistic networks and then the authors are studying facultative ant-plant networks. First of all, what is a “facultative ant-plant network”? In this system, plants are protected by ants? I suggest being clear about how the information regarding the other networks (mutualistic) can be used to a facultative ant-plant network and the limitations of these comparisons.
4.17 – (Line 112): Maybe “disturbance loving species” sounds weird.
4.18 – (Lines 113 & 117): Emer et al., 2017 is not in your reference list. It is very serious, I did not check all references, but I suggest the authors do it carefully.
4.19 – (Line 126): As I mentioned in my comment 2.2, the original network sounds like a before-after comparison. Here you have two different vegetation types, with no replicas.

5 – M&M: Lacks important information, needs deep improvement.
5.1 – (Lines 136-146): I miss a better description of the study area. The tall of the oak trees, the understory of the oak forest, the height of the grass, the shadow/sunny at these areas, etc. If the different vegetation types are interspersed among them or if they are in continuous patches, etc.
5.2 – (Line 169): “foraging” instead of “frequenting”?
5.3 – (Lines 161-170): Authors spent a long-time doing fieldwork and recording the ants and the resource they were foraging on. However, they do not explicitly explain which types of interactions they consider, and how they explored them (I didn’t find any information regarding types of interactions in this paper). They have to describe each of them in detail and present them in the Results. The types of interactions between ants and plants can open an interesting and novelty perspective to this system, nicely strengthening this manuscript; e.g., Costa FV, Mello MAR, Bronstein JL, Guerra TJ, Muylaert RL, Leite AC, et al. (2016) Few Ant Species Play a Central Role Linking Different Plant Resources in a Network in Rupestrian Grasslands. PLoS ONE 11(12): e0167161. https://doi.org/10.1371/journal.pone.0167161.
5.4 – (Lines 172-176): Split these ideas into two sentences, it is large and confusing.
5.5 – (Line 179): If it is in English there is no accent in Mexico. But I suggest keeping the original name of the institution in this case, keeping the name of Mexico country in its Spanish form.
5.6 – (Line 180): Here you can mention that the ants and plants were identified by specialists and include them in the acknowledgments. Also, in the acknowledgments, the authors cited again these names and in addition W. Mackay, not cited here.
5.7 – (Line 181): Please provide the data related to the curation of the specimens.
5.8 – (Line 190): “i” and “j” in italic.
5.9 – (Line 191): If authors have the weighted data, why not use the WNODF; e.g., Britton, N., Neto, M. A., & Corso, G. (2016). Which matrices show perfect nestedness or the absence of nestedness? An analytical study on the performance of NODF and WNODF. Biomath, 4(2), 1512171.?
5.10 – (Lines 198, 199): The higher the D, they are more generalist? Is it compared among them or there is a scale?
5.11 – (Lines 198-218): As I said before, there are no expectations for these analyses. In the last paragraph of the discussion, authors need to bring the reasons they are analyzing those metrics, and the biological meaning of each of them must be introduced in the introduction section.
5.12 – (Lines 231-233): The definition of generalist and specialist core species is proposed by Dáttilo, according to the appropriate equation. These other authors didn't mention the core species under the same terms.

6 – Results: First of all, authors need to present the basic results of their study, as how many species of ants and plants, belong to what subfamily and family, etc. Also, they have to present the exploratory data, as the frequency of ants and plants on each type of vegetation, etc. Also, I recommend being consistent with the use of two or three decimal degrees, for example, NODF = 48.47 (Line 242) and SA = 0.422 (Line 253).
6.1 – (Lines 244, 245): How did the authors get this P? Which statistical test is that? It is a serious concern. All statistical analyses must be presented in the data analysis section. In addition, the simple P does not tell too much about the statistical test.
6.2 – (Lines 245-247): This seems like a discussion and interpretation of the results.
6.3 – (Lines 271-318): Especially for the subsection “Species-level metrics”: Impossible to follow these results in the text, I suggest including a table or tables presenting them.
6.4 – (Line 313): Is P. imparis more central than others?
6.5 – (Lines 320-325) The same as the comment 6.1: How those comparisons were made?

7 Discussion: It needs improvement. For example, sometimes they mentioned the results in “network” terms but did not contextualize them in biological terms. Also, I found some possible misinterpretations of some results.
7.1 – (Line 331): What is the possible cause of this result? What it does mean in biological terms?
7.2 – (Lines 333, 334): Checking the results, I found that both were low Oak: 0.15 and Grassland: 0.36 (niche overlap of ants), so there is no a greater tendency of ant specialization in grassland because even if one is higher than another one, both are low (less than 0.5). Or is it another result?
7.3 – (Lines 344-348): Is this a specialist/generalist matter or is it related to the abundance of nests? How it can be controlled/explored?
7.4 – (Lines 349, 350): I did not understand why invasive species are mentioned.
7.5 – (Lines 352-355): Where are the results of your work related to different resources ants foraging on? (Comment #5.3)
7.6 – (Lines 341-364): In this paragraph, authors cited several studies not connecting them with their results; and at the end (Lines 361, 362) they re-state a result in one sentence, but lacking connection with the literature cited and the biological meaning of this result.
7.7 – (Line 366-412):
Table: The current table could bring basic information (e.g., frequency) and the metrics at the species level (e.g., d’, who is the core species, etc.), and also the codes. A table with only codes is not useful for the reader.

Figures:
Figure 1: A photo of one road is not representative of the habitat the authors did the samples – or the road was the transect?
Figure 2: Include on the caption of this figure where the reader can find the names of the species based on the codes presented.
Experimental design: I included all information in the section "Basic reporting".
Validity of the findings: I included all information in the section "Basic reporting".
Additional comments: I included all information in the section "Basic reporting".


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: CHANGES IN THE CORE SPECIES OF THE ANT-PLANT NETWORK OF OAK FOREST CONVERTED TO GRASSLAND: REPLACEMENT OF ITS ANT FUNCTIONAL GROUPS
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: CHANGES IN THE CORE SPECIES OF THE ANT-PLANT NETWORK OF OAK FOREST CONVERTED TO GRASSLAND: REPLACEMENT OF ITS ANT FUNCTIONAL GROUPS
Review round: 2
Reviewer: 2

Output:
Basic reporting: I have a suggestion in the title
"Changes in the core species of ant-plant network from an oak forest to induced grassland: replacement of ant functional groups"
I suggest to replace your title with the above mentioned because in the present title authors have used more than enough "an" which seems to be ambiguous and grammatically imperfect.
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: CHANGES IN THE CORE SPECIES OF THE ANT-PLANT NETWORK OF OAK FOREST CONVERTED TO GRASSLAND: REPLACEMENT OF ITS ANT FUNCTIONAL GROUPS
Review round: 2
Reviewer: 3

Output:
Basic reporting: REVIEWER 3
I am revising the updated version of this manuscript. Now, I found an improved manuscript in general. The authors did a good job in order to address my concerns, however, I still have comments that need improvement.
One important general comment that must be clear is that the authors are not evaluating “mutualistic networks” as they are stating at the beginning of their Abstract; authors have to check the entire MS on both terminology and concept/theory/background/state of the art and change it.

I have some additional comments that I list as follow:
Comment/Response 3: I have made a search on Google Scholar for “ant plant network disturbance” and “ant plant network disturbance cooccurrence” and I have found some papers that match the purpose of the authors’ study and they can read and potentially use to cite and some papers that the authors already have cited, but can also be useful to contextualize the study:
https://doi.org/10.1111/1365-2656.12820
https://doi.org/10.1007/s10980-018-0747-4
https://doi.org/10.1007/s00114-019-1614-0
https://doi.org/10.1007/s10841-020-00216-4
https://doi.org/10.1111/cobi.12045
https://doi.org/10.1111/ens.12407
https://doi.org/10.1111/ecog.04531
My point is, avoid to using studies that did not evaluate comparable network interactions the authors are evaluating.

Comment/Response 4: As I suggested before, I strongly recommend exploring this data, in terms of data analysis and/or network analysis. Table 2 is not easy to follow, but different colors for different interactions presented on the networks or other visual/analytical approaches would be very welcome.

Comment/Response 5: I have one comment regarding the hypotheses. It is not clear why authors started mentioning “ants associated with nectar and/or hemipteran resources tend to be mutualistic, and mutualistic networks have shown to be nested”. The authors did not evaluate plants with EFNs, although very few plants had EFNs. Authors are evaluating ants foraging on the vegetation, it is definitively not a mutualistic relationship.

Comment/Response 32: I do not see the point of stressing the theory of mutualistic/facultative and not contextualizing to authors’ work. My point is to let clear to the reader what is the connection of these ideas and concepts to their target.

Comment/Response 38: Authors included the citation Costa et al. 2016 and do not cite that paper. Again, this is a very serious concern. It is not acceptable for any publication this kind of mistake. In comment 34 from my first review I highlighted that they had the same issue and they said that they “We apologize, we made another revision of the literature”; as the authors can see, it seems that the bibliography revision needs to be better done, it definitively deserves more attention.

Comment/Response 56: I completely disagree. One thing is that the network has a value different from the null model, the other thing is the value of the metric. They have completely different meanings. The value of the metric is low for both treatments as I said before, authors cannot argue that there is a greater specialization of one in relation to another if both values are lower than 0.5. Please check the meaning of the null model and what do mean being different from the null models.

Comment/Response 61: And where is the new photo?
Experimental design: Please see section 1.
Validity of the findings: Please see section 1.
Additional comments: Please see section 1.


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: CHANGES IN THE CORE SPECIES OF THE ANT-PLANT NETWORK OF OAK FOREST CONVERTED TO GRASSLAND: REPLACEMENT OF ITS ANT FUNCTIONAL GROUPS
Review round: 3
Reviewer: 1

Output:
Basic reporting: Now I found an improved manuscript in which the authors solved the concerns I raised. I would like to congratulate the authors that did a really great job. I hope the authors agree that they have now a strong and clear manuscript, ready to be published.
Experimental design: NA
Validity of the findings: NA
Additional comments: NA


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: INSIGHTS INTO MOBILE GENETIC ELEMENTS AND THE ROLE OF CONJUGATIVE PLASMID IN TRANSFERRING AMINOGLYCOSIDE RESISTANCE IN EXTENSIVELY DRUG-RESISTANT ACINETOBACTER BAUMANNII AB329
Review round: 1
Reviewer: 1

Output:
Basic reporting: Authors conducted a well designed study, well controlled and this study gives molecular information about the behavior of Acinetobacter baumannii which is one of the most important microorganisms due its ability to adquire or to develop resistance to several antibiotics
However some annotations must be done such as to include MICs of antibiotics and to clarify some points in the document.
Experimental design: Experimental desing is well conducted and disegned, just some momments.
In Supplementary material more than to include resistance interpretation, MICs must be included and to remove some antibiotics. Besides, to mention wich document was used to define breakpoint.
A. baumannii has intrisic resistance Ceftriaxone, cefotaxime, tetracycline, therefore those dont represent clinical information o microbiological decisions (to review EUCAST intrisic resistance and CLSI M100)
Line 146: to change Bertini by Bertani.
Transconjugated selection is no clear, quote- Transconjugants were selected on LB plates containing the following components: 250 μg/ml sodium azide; 50 μg/ml ticarcillin or 250 μg/ml sodium azide; 20 μg/ml kanamycin. Plates were prepared with (1) 250 sodium azide, (2) ticarcillin and (3) or sodium azide (again). To clarify this point.
In table 2, to include MICs of Ak resistance after transconjugation. Besides to remove those antibiotics that are intrinsically resistant
Validity of the findings: No comment
Additional comments: Line 32: In silico must be written in italics.
Line 55: after bla OXA-23 in subscript
Line 58: after bla, GES-11 in subscript
OXA-23 was the most common gene present in several mobile genetic elements such as ISAb4, ISAb10, Tn2006, Tn2007 and Tn2008 inorder to have a better imagen of how common was this element to change redaction, Suggestion: blaOXA-23 was found in..... in this way is easier to follow the findings
Line 56: ISAba4 and ISAb10, after nomenclature of insetion sequence (IS) Aba in italics.
Line 297: In silico in italics
Some elements that confers intrisic resistance such as OXA-51 and ADC (a AmpC) must be included in discussion.


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: INSIGHTS INTO MOBILE GENETIC ELEMENTS AND THE ROLE OF CONJUGATIVE PLASMID IN TRANSFERRING AMINOGLYCOSIDE RESISTANCE IN EXTENSIVELY DRUG-RESISTANT ACINETOBACTER BAUMANNII AB329
Review round: 1
Reviewer: 2

Output:
Basic reporting: The english should be revised.
The quality of the figures should be improved.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript “Insights into mobile genetic elements and the role of conjugative plasmid in transferring aminoglycoside resistance in extensively drug-resistant Acinetobacter baumannii AB329” describe the genomic analysis of an A. baumannii strain causing an outbreak in Thailand. The analysis reports the identification of two plasmids, one of them a conjugative megaplasmid, and several prophages, genomic islands and insertion sequences. They conclude that all these MGEs are responsible for the acquisition and transference of antimicrobial resistance in this strain.
The work is well elaborated and offer a general vision of the role of all these MGEs in the antimicrobial resistance acquisition.
Minor comments:
1-	Line 36: the authors should include the definition of conjugative plasmid, as this is an important part of the work.
2-	Line 40: which is the relevance of the plasmid GR6? Is this included in one of the 21 lineages? Please, give more information about this plasmid.
3-	Line 43: define prophage.
4-	Line 46: as this sentence is writen it seems that the 177 strains were analysed in this work. Please write it in a way that it is well understood.
5-	Line 48: include the definition of genomic island.
6-	Line 53: include the definition of insertion sequence.
7-	Line 62: the authors should include more information about this outbreak.
8-	Line 133: the authors should indicate the tool employed to identify the genomic island.
9-	Line 152: the first section of the results would be better structured if it were divided into two parts, one including the antibiotic susceptibility and other with the genome sequence.
10-	Line 160: the authors should include the genome size of the plasmids.
11-	Line 161: The number of ORFs and tRNA are the total of the chromosome or include the plasmids? The authors should explain it.
12-	Line 178: which is the difference between shell genes and cloud genes?
13-	Line 251-260: the authors should base this hypothesis in previous published works. Include reference that reinforce your hypothesis.
14-	Figure 1b: this figure is elongated. Please, improve it.
15-	Figure 2: in this figure is very difficult to read the gene names.
16-	Figure 4. Improve the figure quality.
Major comments
In the section of Conjugative transfer of aminoglycoside resistance gene of plasmid pAB329b, the authors determine the transference of the aminoglycoside resistance by conjugation, but the don´t explain if this transference is only of the gene or the complete plasmid. Besides, if the transference only implies the gene, is this due to the effect of a transposase?


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: INSIGHTS INTO MOBILE GENETIC ELEMENTS AND THE ROLE OF CONJUGATIVE PLASMID IN TRANSFERRING AMINOGLYCOSIDE RESISTANCE IN EXTENSIVELY DRUG-RESISTANT ACINETOBACTER BAUMANNII AB329
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript was clear described. and it also contain enough information and backgroud. and its structure was accptable. also the raw data was shared.
Experimental design: The research question was well designed. and the methods here is ok to replicate.
Validity of the findings: whole genome sequencing and othere experiment was performed. and the result support their hypothesis.
Additional comments: Line 57: Tn2009(OXA-23) was missing
Line 81: the strain used in this study is quite old. it would be difficult to represent the current situation.
Line 82: did AST followed CLSI? which version?
Line 130: which paster version server was used in this study? 2?
Line 163: how about the allele variant of ST86 to ST2? it should be stated in the manuscript.
Line 215: the detail of the loss of aph3-VIa should be analysis.
The easyfig figure should be improved. e. g. the blastn length should be set.


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: INSIGHTS INTO MOBILE GENETIC ELEMENTS AND THE ROLE OF CONJUGATIVE PLASMID IN TRANSFERRING AMINOGLYCOSIDE RESISTANCE IN EXTENSIVELY DRUG-RESISTANT ACINETOBACTER BAUMANNII AB329
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors have made all changes according suggestions. Work is clearer and fluid.
Experimental design: Authors have made all changes according suggestions in methodology. Work now is clearer and fluid.
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: INSIGHTS INTO MOBILE GENETIC ELEMENTS AND THE ROLE OF CONJUGATIVE PLASMID IN TRANSFERRING AMINOGLYCOSIDE RESISTANCE IN EXTENSIVELY DRUG-RESISTANT ACINETOBACTER BAUMANNII AB329
Review round: 2
Reviewer: 2

Output:
Basic reporting: The questions were all addressed.
Experimental design: The questions were all addressed.
Validity of the findings: The questions were all addressed.
Additional comments: The questions were all 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: SHORT PHYSICAL PERFORMANCE BATTERY AS A PREDICTOR OF MORTALITY IN COMMUNITY-DWELLING OLDER ADULTS: A LONGITUDINAL STUDY IN THE BRAZILIAN AMAZON REGION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article was written using clear and technically correct language. Literature background is sufficient and supports the hypothesis and the research problem. All complementary information and files were well provided, making the study reproducible and reliable. Methods were well described and the results support both objectives and conclusion.
Experimental design: The knowledge gap was presented with enough literature support, which pointed to the relevance of the results for clinical practice in Gerontology and Geriatrics. Methods was appropriatedly described and seem feasible and reliable, in accordance with the best practice in epidemiology research.
Validity of the findings: Findings is novel and relevant for clinical practice in Gerontology and Geriatrics. The SPPB is widely used in research and practice, then the information about its cutoff point and the relationships with mortality increase the interest with regard older adult's physical functioning despite chronic diseases.
Additional comments: Please, check the reference citation in the line 65. There is a typo.


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: SHORT PHYSICAL PERFORMANCE BATTERY AS A PREDICTOR OF MORTALITY IN COMMUNITY-DWELLING OLDER ADULTS: A LONGITUDINAL STUDY IN THE BRAZILIAN AMAZON REGION
Review round: 1
Reviewer: 2

Output:
Basic reporting: - This is an observational, analytical cohort study with a significant sample of elderly people. The topic is not new, but it contributes with some data to the area of gerontology.
- English is adequate and understandable.
-Tables and figures were presented properly.
- References used are updated.

ABSTRACT
- Line 18- They must not state that studies were not carried out, due to the speed of publications currently. I suggest “We did not find Brazilian longitudinal studies that aimed to analyze the predictive capacity and accuracy of the SPPB in the mortality of the elderly in the community”.
- Line 21- The wording of the objective is a little confusing. I suggest: “This study aimed to analyze the predictive capacity and accuracy of the SPPB in the mortality of the elderly in the community, as well as to determine cut-off points for men and women”.
- Line 22 -Use only the abbreviation SPPB, since its abbreviation has already been defined previously.
- Include in the methodology which level was considered statistically significant or indicative of significance.
- Line 26 - Include in the methodology which level was considered statistically significant or indicative of significance.

INTRODUCTION
- Many studies on the importance of the SPPB test were discussed. I suggest decreasing and increasing the number of solid references that reinforce the contextualization of the theme. Why is the SPPB test an important screening tool for the elderly? As a suggestion, it is possible to add more about the various changes associated with aging, bringing more physiological information about what happens. Even so, the reason for choosing this test should be reinforced, in contrast to other tests, in which environment it has been used (home, hospital, research).
- Line 50 - I suggest citing “Various studies” rather than “Various authors”. And include at least three studies in the reference.
-Line 65 - Correct this reference in the text and in the final references. It is misquoted. You should also include it within the same parentheses as the previous authors.
- Line 76 - Correct the reference as mentioned above.
Experimental design: METHODOLOGY
- Original research, with a defined research question. Well-described statistical analysis.
- The procedures performed were described, but there is no information regarding the training of the researchers responsible for data collection (to ensure the quality of the evaluation protocol), nor if the evaluations were applied on the same day and if the participants practiced a recognition of the test before the main assessment.
- Design and sample: Correct to “Analytical observational cohort study”.
- Inclusion and exclusion criteria: I suggest reversing the wording. "Elderly individuals aged 60 years or older, able to walk, with or without gait aids, were included in this study." It is not necessary to quote the informed consent form again. The wording of Figure 1 is confusing. I suggest improving. Only 370 elderly people were followed from 2017 to 2020?
- Physical performance (independent variable): What was the cut-off point used in the sit-to-stand test and in the gait test?
Validity of the findings: RESULTS
- All underlying data has been provided; are robust, statistically sound and controlled.
- In Table 1 and 2, I suggest placing a margin in the classifications of each variable, it is better to observe.
- On lines 176 and 177: Point out that it was statistically significant even though it has the p-value.
- In the paragraph of Figure 3: The p value was significant for both curves, however the classification of the area under the curve was not presented. I suggest reading “Metz CE. Basic principles of ROC analysis. Semin Nucl Med.1978;8(4):283-98.” and present the result of the area according to the classification proposed by the author.

DISCUSSION
- First paragraph of the discussion: The purpose of the study has already been mentioned. The first paragraph of the discussion should point out the most relevant findings of the study and contribution to the area. Include some references that reinforce these findings.
- Line 201: As already mentioned we shouldn't point this out, due to the speed of publications nowadays. It's a risky statement. If it is the will of the authors, it is possible to indicate that so far there are still few studies. It may be interesting to comment on the strengths of the study as well, such as originality and methodological rigor.
- Line 207: Change "It is worth mentioning" to "We emphasize that", so as not to be repetitive.
- Line 208: As already mentioned we should not point this out, due to the speed of publications nowadays.
- Line 233: We cannot say this, due to the speed of publications currently.
- Line 238: This paragraph became vacant in the middle of the discussion. I suggest linking to the results found in the search.

- Line 247: In this paragraph the same happened as in the introduction. Studies that used the SPPB are brought up. But within the clinical and practical context, what is the importance of the results found? I suggest pointing out that these variables cannot be used indiscriminately, as there are biopsychosocial factors that involve balance, gait and strength, such as individual health characteristics, intrinsic and environmental factors. Bring references and briefly discuss other variables involved.
- Line 255: In this paragraph the same thing happened as in the introduction. Studies that used the SPPB are brought up. Little discussion about the results.
- Excellent closing of the discussion and conclusion of the study.
Additional comments: Overall the article is well written. I highlight the organization of the methodology and presentation of the results. However, the introduction should be improved, as well as the discussion, which should address the importance of the data found and not just the similarity with 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: SHORT PHYSICAL PERFORMANCE BATTERY AS A PREDICTOR OF MORTALITY IN COMMUNITY-DWELLING OLDER ADULTS: A LONGITUDINAL STUDY IN THE BRAZILIAN AMAZON REGION
Review round: 1
Reviewer: 3

Output:
Basic reporting: Literature references, sufficient field background/context provided. Only in the Introduction: Lines 75-76 it is necessary to put which studies.
Professional article structure, figures, tables. Raw data shared.
Self-contained with relevant results to hypotheses.
Experimental design: Well-defined, relevant, and meaningful research question. It is stated how the research fills an identified knowledge gap.
Important suggestions in methodology:
It is necessary that the methods need to be written with more details: How were the tests applied? face-to-face or over the phone? It needs to be clearer how the evaluation occurred in 2017.
It is important to make it clear in the methodology that the contact in 2020 was only to know the deaths, at the beginning of the methodology (lines 104-108 are a little confusing). In this same paragraph, there is no need to put the results of deaths in the methodology, because they already are in the results. It is clearer to put lines 104-108 together with the item project and sample, before the inclusion and exclusion criteria.
Line 117: It is Important to describe how many repetitions are necessary to do in the sit-to-stand test.
Validity of the findings: Results: appropriated.
All underlying data has been provided; they are robust, statistically sound, and controlled.
Discussion: Meaningful replication encouraged where rationale and benefit to literature is clearly stated.
It is necessary to put references in lines: 201-203; and 238.
Conclusion: The conclusion must be properly formulated. It must be related to the original question investigated, and must be limited to those supported by the results. Is it important to further argue a little more the conclusion. About how it can be used, or how important the study was. Needs for further studies? What does this study bring positive? Make an overview of the main results.
Additional comments: I congratulate the authors on tackling this extremely important topic. It is extremely important that we increasingly have tests for the clinical evaluation of the elderly, especially because it can lead to the early identification of individuals with increased risk for various health situations.


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: SHORT PHYSICAL PERFORMANCE BATTERY AS A PREDICTOR OF MORTALITY IN COMMUNITY-DWELLING OLDER ADULTS: A LONGITUDINAL STUDY IN THE BRAZILIAN AMAZON REGION
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments.
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: Authors responded to requests.


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: SHORT PHYSICAL PERFORMANCE BATTERY AS A PREDICTOR OF MORTALITY IN COMMUNITY-DWELLING OLDER ADULTS: A LONGITUDINAL STUDY IN THE BRAZILIAN AMAZON REGION
Review round: 2
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.
Literature references, sufficient field background/context provided
Professional article structure, figures, tables. Raw data shared
The requested topics have been reviewed by the authors
Experimental design: Original primary research within Aims and Scope of the journal.
Methods described with sufficient detail & information to replicate.
The requested topics have been reviewed by the authors
Validity of the findings: The requested topics have been reviewed by the authors
Additional comments: I congratulate the authors on tackling this extremely important topic. I consider the article accepted.
It is extremely important that we increasingly have tests for the clinical evaluation of the elderly, especially because it can lead to the early identification of individuals with increased risk for various health situations.


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: CD14 AND CD26 FROM SERUM EXOSOMES ARE ASSOCIATED WITH TYPE 2 DIABETES, EXOSOMAL CYSTATIN C AND CD14 ARE ASSOCIATED WITH METABOLIC SYNDROME AND ATHEROGENIC INDEX OF PLASMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: This manuscript by Pérez-Macedonio et. al. explores serum exosomal proteins viz. cystatin C, CD26 and CD14 from patients with T2DM. The authors have used ELISA to determine protein concentrations and electron microscopy for structural analysis of exosomes. The findings reported in this clinical study are new, however, there are major concerns related to the study design and data that need to be addressed prior to consideration for publication.
Major comments:
1.	What was the rationale for selecting and analyzing only three exosomal proteins? The authors need to clearly define the rationale behind their hypothesis. There are several other exosomal proteins that are altered in individuals with T2DM in addition to Cystatin C, CD26 and CD14.
2.	What do the authors mean by “MetS Components”? Please include a clear definition of these components and cite literature to substantiate definition.
Validity of the findings: 3.	A major shortcoming of this study is unequal gender distribution. Since 86.4% of the population is females, the conclusions/findings of this study can only be validated in females and do not apply equally in males. Hence, the authors are encouraged to either increase the number of males to an equal proportion to females or change the title and conclusions specific to female population only.
4.	Individuals with T2DM are significantly older and this can be a confounding factor on the data. What are the levels of these proteins (Cystatin C, CD26 and CD14) in serum exosomes of non-diabetic age-matched controls?
5.	Figure 1B: It appears that the levels of proteins on western blot are different between individuals with and without T2DM. Therefore, a quantification with an appropriate loading control (or total protein) is required to better understand the data presented.
6.	Figure 1A: The authors have concluded that exosomes from individuals with T2DM are smaller. Was there a difference in total number of exosomes in the two groups?
7.	Table1: The blood glucose of individuals with T2DM ranges from 89-205 indicating both patients with well controlled and poorly controlled blood glucose. What treatment were these patients on? It would add value to the publication of this group is further classified in 1. Normoglycemic and 2. Hyperglycemic individuals.
8.	Table 3: Cystatin-C is not significantly associated with T2DM or AO. Moreover, Cystatin C levels in exosomes are not changed in diabetic individuals. This makes the title misleading and therefore the authors are encouraged to modify title and conclusions accordingly.
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: CD14 AND CD26 FROM SERUM EXOSOMES ARE ASSOCIATED WITH TYPE 2 DIABETES, EXOSOMAL CYSTATIN C AND CD14 ARE ASSOCIATED WITH METABOLIC SYNDROME AND ATHEROGENIC INDEX OF PLASMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The Authors has presented a well written and nicely organized manuscript on levels of CD14, Cystatin C and CD26 in serum exosomes and their association with type 2 diabetes, metabolic syndrome and atherogenic index of plasma. This study is important first step in understanding their use as biomarkers before their extensive characterization. I commend the authors for their extensive data collected for this manuscript. Overall, I enjoyed reading this manuscript; though before publication.

I have the following recommendations that need to be addressed to improve the quality of the manuscript.
In my opinion the authors need to provide more details in the introduction particularly explaining the terms such as atherosclerotic risk factors, atherogenic index of plasma etc., as well as roles of CD14, Cystatin C and CD26 in the pathogenesis of diabetes and CVD.
Experimental design: The authors need to explain the purpose of testing these marker in exosomes rather than testing them in serum or plasma directly. What are the advantages and disadvantages? Is the purpose to show that theses proteins are getting transported throughout the body through exosome if so please explain that in more detail in the manuscript.

The authors mentioned that number of circulating EV are increased in patients with T2D and vascular complications, so I would suggest the authors to include a table or figure showing if there are any differences observed in no. of exosomes isolated in patients with T2D vs without T2D.

Please clarify if the correlation performed in this study was for both subject groups combined or with disease group alone. My suggestion would be to perform two separate correlation for both groups.
Validity of the findings: Even though p-values for some of Correlation between exosomal proteins and different atherosclerotic risk factors is < 0.05 the Spearman's regression coefficient is < 0.25 which is considered to be very weak or no correlation which means minimal relationship between vaiables. Authors need to provide clarification regarding this.
Additional comments: Minor comments:
•	In Table 1 the authors did not report cholesterol as medians (p25th-p75th).
•	In discussion: fix the spacing issue between line 215 and 216


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: CD14 AND CD26 FROM SERUM EXOSOMES ARE ASSOCIATED WITH TYPE 2 DIABETES, EXOSOMAL CYSTATIN C AND CD14 ARE ASSOCIATED WITH METABOLIC SYNDROME AND ATHEROGENIC INDEX OF PLASMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper is well-structured and clearly written. There are a few minor issues that need to be fixed further.
(1)The conclusion section of the abstract contains some of the content of the results section. The conclusion section may need to be more precise and clear.
(2)The relationship of Cystatin C with diabetes/MS/AIP is well defined by previous studies. A short paragraph might be needed added to the introduction to explain why the determination of exosomal cystatin C, CD14 and CD26 was chosen as the detection indicators, and whether there was a correlation between these indicators or some consistent pathophysiological mechanisms.
Experimental design: Overall, this section is well designed. There are two issues that may lead to bias in the analysis. The first is that the two groups of populations are over-represented by women, and the second is that the diabetic group is older. It might be better to discuss it in the discussion.
Validity of the findings: Two small issues may be needed to be further clarified.
(1)In the correlation analysis, CD26 of exosomes was not correlated with blood glucose, lipid profile and metabolic syndrome. However, CD26 is significantly elevated in diabetic patients, and the reasons for this may need to be analyzed in the discussion.
(2)AIP score was calculated from TG and HDL-C. There are two points in Table 3 that may require further clarification. The first is why neither TG nor HDL-C was correlated with CD14, but AIP score was a correlated factor for CD14. The second is that AIP score may not be suitable for inclusion in regression analysis at the same time as TG and HDL-C.
Additional comments: No further comments.


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

Input:
Title: CD14 AND CD26 FROM SERUM EXOSOMES ARE ASSOCIATED WITH TYPE 2 DIABETES, EXOSOMAL CYSTATIN C AND CD14 ARE ASSOCIATED WITH METABOLIC SYNDROME AND ATHEROGENIC INDEX OF PLASMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Please modify Table 1 as per comment below:
7. Table1: The blood glucose of individuals with T2DM ranges from 89-205 indicating both patients with well controlled and poorly controlled blood glucose. What treatment were these patients on? It would add value to the publication of this group is further classified in 1. Normoglycemic and 2. Hyperglycemic individuals.
When comparing controlled and uncontrolled T2D individuals, no significant differences were observed among the exosomal proteins studied, only an increase in CD14 was identified in uncontrolled diabetics, however, it was not significant. Therefore, we conclude that the effect on dysregulation of exosomal proteins is mainly due to diabetes, regardless of diabetic control. On the other hand, when we evaluated a generalized regression model, the effect of uncontrolled diabetes on CD14 increase was not significant. Thus, we considered that it is not necessary to change Table 1, since there was no statistical difference between controlled and uncontrolled diabetic individuals and exosomal proteins, however, we leave it to the reviewer to consider whether Table 1 in the manuscript should be modified.
Experimental design: No further comments
Validity of the findings: No further comments
Additional comments: No additional comments


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

Input:
Title: CD14 AND CD26 FROM SERUM EXOSOMES ARE ASSOCIATED WITH TYPE 2 DIABETES, EXOSOMAL CYSTATIN C AND CD14 ARE ASSOCIATED WITH METABOLIC SYNDROME AND ATHEROGENIC INDEX OF PLASMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: No Comment
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: No Comment