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: EFFECT OF OXIDE SCALE STRUCTURE ON SHOT-BLASTING OF HOT-ROLLED STRIP STEEL
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, this is an interesting inquiry into steel oxidation, how storage time affects the structural evolution of the oxide scale, and how the structure impacts the optimal descaling approach. It needs significantly more detail in the methods to support its conclusions, and perhaps some additional thought on whether observations are attributable to composition or morphology.

Language use is decent. Needs some work in a few areas, see general comments section for specifics. Title should be reorganized, if possible, for readability and clarity – I recommend “Effect of oxide scale structure on shot-blasting of hot-rolled strip steel.”

The introduction needs a little bit of readability work; but the literature cited seems relevant and the establishment of the background – oxide scaling approaches and their uses and limitations – is strong and provides good detail. In several places the authors say that “research is not sufficient” on particular topics. It would strengthen the paper to explain how this insufficiency impacts productivity, and/or how this insufficiency will be addressed by the present research.

Most figures are of acceptable quality. Interpretation and results could be improved through the inclusion of some additional data, specific recommendations below. Scale bars are included on all images. I believe there is a repeated typographical error in Figure 1, I recommend the authors carefully review figures and tables to catch any other such errors. I would like to see significantly more detail on SEM and XRD settings either in the captions or in the main body of the text, as well as details on sample preparation. The omission of these elements considerably weakens the conclusions – see section 3 of this review.
Experimental design: The introduction could do a better job of framing the research question. It was clear by the end of the paper what the question was, but I would have liked a clearer picture at the outset of why the “storage time” or “air-cooling time” is relevant in the broader scope of steel oxide layer removal. Would it be better for the authors’ stated goals of efficiency and lowering emissions to have a short storage time or a long one, perhaps considering the manufacturing and supply chain? More discussion on this topic would help frame the purpose of the paper for the reader.

My concerns about rigor and detail are constrained to the analytical methods and sample preparation, which I will cover in detail in the following sections.

Replication of these findings will be difficult without additional information on the samples themselves as well as the analysis methods. A thermal history of the steel samples during hot-rolling would help. This is partially addressed in the introduction, but never fully explained. A simple figure showing representative dwell temperature vs. time for Q235 during the hot-rolling process would make the article more accessible to the general materials science audience, not just those versed in steel manufacturing.
Validity of the findings: My main concerns with this paper have to do with the robustness and controlled nature of the data presented. I believe the data is legitimate and supports the authors’ conclusions (with some exceptions, see Section 4), but because these conclusions depend purely on interpretation of SEM images and XRD phase IDs, much more background is needed. General improvements for presented figures:

• XRD data should include:
         o Sample preparation & mounting
             Were samples powdered/pulverized? How? How much was analyzed?
                • If not, how were the samples mounted? How were orientation effects filtered from the data?
         o Instrument model & make
         o Tube/anode material and/or x-ray wavelength
             Tube excitation parameters (KV/mA) would also be preferable
         o Calculated/literature phase data (“stick patterns”) and source thereof (ICDD, COD, card number, etc.)

• SEM data should include:
         o Sample preparation (sectioning, mounting)
         o Accelerating voltage
         o Working distance
         o Imaging mode (SEI, BSEI)
             If a backscattered electron image (BSEI):
                • Imaging mode (i.e. compositional vs. topographical)
                • Tilt angle

This information can be supplied either in the Experimental Methods section or in the figure captions. Detailed review on individual figures can be found in the general comments.

I believe the compositional assertions about the eutectoid phase must be identified as speculation, without the inclusion of additional data or literature support.
Additional comments: Figures

Figure 1. Desaling -> Descaling

Figure 3. Labeling the figures with their approximate air exposure time (1 year vs. 1 week) will help readability. Combining this figure with some of the later cross-sectional analysis may also help readability.

Figure 4. In addition to the general notes from section 3: the y-axis should be in English, I am assuming this is “Intensity (counts)” or similar. The data could use a background correction, as there is some scattering contributing to the background in the 10-30 2θ region. Additionally, there are some phase-identification discrepancies that would be resolved by the inclusion of the literature patterns – with ICDD or COD card catalog numbers – on the figure itself. This typically requires reproduction in color, so alternative presentations may be necessary if color is not available.
	Peak ID inconsistencies that need correction:
	30° 2θ – M, O, or both?
	38° 2θ – M or O?
	58° 2θ – M, O, or both?
	90° 2θ – M or I?
The primary issue with this figure is the claimed ID of a F3O4/Fe phase. The data as presented show that the authors have observed an Fe phase (having a tight correlation with Q235 literature patterns) and a magnetite (Fe3O4) phase in both samples. However, in order to identify a eutectoid (i.e. a solid solution of two phases) more careful analysis of peak shifts and peak intensities would have to be performed, as the interatomic distances should change as a result of varying Fe inclusion, compared to the parent phases. For the record, I do not believe that the presence or non-presence of the eutectoid phase is necessary to explain the differences in behavior of the oxide scale, so references to this phase could be removed, the inconsistencies above could be corrected, and the data could be presented as-is.
The important difference I do see is that Figure 4B shows a higher percentage of the Fe phase, but without further explanation of the samples’ preparation and mounting, there is no way to tell that the variance in the Fe phase is not simply due to variation arising from the presumed pulverization/grinding process, or possibly due to the thicker deposit of oxides (i.e. higher mass fraction) in the No. 2 group. Inclusion and discussion of the sample preparation would strengthen the central claims about the different oxidation times requiring different descaling methods.

Figure 5. This figure could be improved by adding a reference surface, i.e. before blasting.

Figure 6. This figure could be combined with Figure 5, so that the surface and cross-sectional views are next to each other. The claims about No.2/6B having “better combination with the basal body” in line 199 could use additional support, this does not seem obvious to me from the image. An alternate theory could simply be that the No.1 film is more cohesive and densified, therefore when shot-blasted, it comes off in larger flakes rather than being crushed into smaller pieces. A higher magnification image or, better, a compositional BSE image showing a density gradient in this zone might provide evidence of this “better combination” claim. HRTEM would provide conclusive proof, but these experiments are difficult and lengthy.

Figure 7. Additional notes about the imaging modes are needed – 7A is clearly a compositional BSE image but 7B may be topographical BSE, or even SEI. SEM firing parameters and working distance are needed to allow for replication.

Figure 8. The claim about “color uniformity” on lines 213-214 is only true if this is a compositional BSE image. Please specify, the contrast variations at edges indicate that these are SE images, which carry no information about composition. Additionally, if these are compositional BSE images, these contrast variations will need to be addressed in the text.

Figures 9-12. I would recommend combining these for direct comparison – figures 9 and 11 can be combined so that they can be compared side-by-side. Same with 10 and 12. The imaging parameters included in figures 10/12 should be included for other images in the manuscript, though usually these are in the caption or the Experimental Method section. The data would also be stronger if EDS mapping of Fe for these regions were included. Also – figure 9/11 does a better job (but still not entirely conclusive) demonstrating the “better combination” claim from Figure 6. This is the type of magnification needed, preferably with before-and-after images.

Table 1. How was the chemical composition determined?

Table 2. How was position in the sample determined? Was this paired with a depth-profiling technique? Details are needed!

Text

Line 19 – explain what “the related researches are not sufficient” means
Line 33 – specify that we are talking about steel.
Line 45 – “impact of water is not sufficient” – specify, for what?
Line 72-73 – English usage
Line 90 – “research… on the blasting descaling effect is not sufficient” – specify what information is missing – what do we not know that this paper will address?
Line 106-107 – include thermal history of the samples here
Lines 116-147 – include sample preparation, analysis method settings, additional detail from figure commentary here
Line 131 – unless an equation is going to be shown using these parameters, symbols are not necessary
Lines 154-155 – Why does the sample with the longer oxidation time have a thinner scale? I would like to see some discussion of density here, not just composition.
Line 157 – this “well combined” claim shows up several times, it is not obvious from the data presented. Either provide more analytical support or remove.
Line 216-218 – the behavior described here could also result from poorly-densified, morphologically heterogeneous oxide crystals, rather than a layered structure. Layering should be clear from the cross-sectional SEM in Figure 3, but it simply looks disorganized and poorly densified. I think this is the real conclusion from the data – somewhat counterintuitively, well-formed, highly dense oxide scales are easier to shatter and remove under shot-blasting.
Line 225 – don’t state that results are accidental. Just describe what is learned from the high-magnification image, and what is learned from lower-mag/EDS.
Lines 246-252 – I think this will need to be rewritten, unless the data more conclusively demonstrates the presence of this eutectoid phase.
Lines 253-259 – it seems odd to spend much of the paper showing how the No.1 oxide scales are more completely removed and then to turn around here and say that the descaling will be less efficient and more expensive. I am not sure this is what the authors intend.


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: EFFECT OF OXIDE SCALE STRUCTURE ON SHOT-BLASTING OF HOT-ROLLED STRIP STEEL
Review round: 1
Reviewer: 3

Output:
Basic reporting: I felt that the written structure of the article generally followed acceptable practice
Experimental design: The abstract suggested that the properties of the scale would explain the effectiveness of the descaling process.

I was disappointed by the very limited experimental design of the investigation which only looked at two aging conditions of the steel and its surface scale which was 1) no aging and 2) aging at ambient after one year.  I cannot believe that manufacturers or consumers would want to wait for a year to be able to effectively descale these materials so that they could avoid various other methods including pickling.

This work did not directly compare shot blasting and pickling to show the difference.  In the methods section, the experimental equipment was described where the process parameters were detailed to include 1) angle of impingement, 2) velocity, 3) shot diameter and 4) amount of shot used.  Only the last variable was apparently used, and no other attempt was done to determine it other factors might have contributed to the success of the investigation.  There was no optimization of the process claimed in the conclusions because there were no other factors that were tested.  There was only one type of shot blasting material used.
Validity of the findings: I was looking forward to see the properties of the scale explained by the morphology and chemical composition, however it did not go far enough.  Only the average chemical composition of the scale was used as a characterization.  The Fe / O phase diagram was referred to in the quoted references, but was not discussed in enough detail to establish what was expected in the present investigation.  Obviously the scale present in fresh material was not in equilibrium, and it was implied that material stored for a year was nearly in equilibrium.  If it was important to the success of the investigation to produce the equilibrium phase and distribution, why then was no attempt made to accelerate the formation of the equilibrium product?

While the morphology was shown in layer thickness, there was no quantitative expression of grain size or other distribution of the phases present.  No attempt was made to assess the mechanism of adhesion or bonding or the strength of the adhesion of the scale layers to help explain the observed behavior.  I would imagine that a micro shear test or bend tests as described for tension descaling would be appropriate methods to derive the properties of the scale needed for this characterization.

The introduction reviewed an extensive list of competing descaling processes used in the industry compared to shot blasting, but no attempt was made to make an assessment of the relative cost, material aging or logistical tradeoffs between these methods.  There was also no pass / fail criteria expressed to help judge the ultimate success of failure of the results.
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: EFFECT OF OXIDE SCALE STRUCTURE ON SHOT-BLASTING OF HOT-ROLLED STRIP STEEL
Review round: 2
Reviewer: 1

Output:
Basic reporting: Rewritten sections are improved and clearer. I am satisfied with the changes, with the exception of the XRD data, see section 2.
Experimental design: I do not feel that my commentary on the XRD data has been appropriately addressed.

The authors indicate the samples are simply attached to the XRD sample holder and scanned, without any processing. This results in the potential for changing intensities due to orientation of the crystallites within the sample. Generally samples are powderized before XRD analysis. Without this step, there is no way to be assured that the entire pattern of any particular phase is appearing, because the method for phase identification depends on having the analyzed crystallites randomly oriented within the sample volume. In the specific case here - an oxide film growing off of a flat substrate - it is likely that grains will grow in a particular direction, resulting in preferred (and unknown) orientations within the sample. It introduces many questions about the data. This should be addressed in the experimental method - how these data were refined to address the orientation questions. If they were not refined, they should be.

In the rebuttal letter, the authors mention PDF cards. I'm assuming this means ICDD data - please include the card numbers for the specific patterns used somewhere in the article.

The authors say the X-ray tube is "ceramic" - this may be the housing, but the actual anode material is, in virtually all cases, a metal target. What I am really asking for here is which wavelength(s) the samples were illuminated with. If it is standard Cu k-alpha (1.541 angstroms) this should be stated somewhere. The name of the instrument is insufficient, most suppliers offer many tube options, though 95% of the instruments have Cu anodes. This is critical, along with the PDF numbers, for repeatability and comprehension of the data.

Finally, the peak identifications themselves are inconsistent from Figure 4A to Figure 4B. This may be a result of the orientation issues mentioned above, which can cause peaks in literature powder patterns to not appear in scans. However, if that is the case, additional data must be presented to justify assigning a peak to one pattern in one sample and a separate pattern for the second. The most egregious cases are, again, as noted in my previous review:

30° 2θ - assigned to both M/O in 4A, assigned to O in 4B.
38° 2θ - assigned M in 4A, assigned O in 4B.
58° 2θ - assigned to both M/O in 4A, assigned to M in 4B.
90° 2θ - assigned I in 4A, assigned M in 4B.

The authors claimed to address these in the rebuttal, but the uploaded images have not been changed. Additionally, they simply assign 3 of the peaks to both patterns (highly improbable, based on what I've seen of Fe3O4 and Fe2O3 patterns in the literature), and I believe they misidentify the peak at 90 as Fe - I suspect the actual Fe peak is at 82, again, based on my own very limited searching of the literature. Lastly, the literature patterns themselves are not shown in the image nor referenced in the document. There is additionally a prominent peak at 33° 2θ assigned to phase O in 4A that disappears in 4B, despite still identifying the O phase for that sample. If the authors simply include the literature patterns (as "sticks" underneath the scan itself, or in a separate chart) on the image, these questions are resolved, and the phase ID will be more-or-less obvious.

The reason the discussion above (on powders vs. bulk) is relevant here, is that it is not easy to determine that the disappearance of the peak at 33 is because the sample lacks Fe2O3, or because the Fe2O3 crystals are oriented in such a way that this peak is not revealed in that sample. Similarly the changes in intensity for the I phase may be related to preferred orientation, as the ratio between the two peaks does appear to change from sample to sample - it is simply not possible to know, without further data treatment and explanation in the paper.

Finally, the eutectoid phase mentioned still has no literature pattern nor phase ID. I am not familiar with this subfield, so "eutectoid Fe/Fe3O4" may be a misnomer, but generally, this refers to a solid solution, not the mere simultaneous presence of two distinct phases (this would be something like "heterogeneous polycrystal" not eutectic or eutectoid). In my opinion, it may be better to rely more (if not entirely) on the EDS data from Table 2 to make the point about the oxygen composition of the scale and how it affects shot peening.

On the other hand, I feel the electron microscopy work has been clarified well, and have no further issues.
Validity of the findings: See above critique of the XRD data. Otherwise, I feel the paper is valid.
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: LIMITED PROTEOLYSIS OF PYRANOSE 2-OXIDASE RESULTS IN A STABLE AND ACTIVE COMPLEX
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript presented in a clear form, well referenced and good context. The work flow is straightforward and results are well described.

Minor comments:
Line 123: 53 check citation format

Figure 3 has an embedded legend as "Figure 1", a possible editing error.

Figure 3 a) and b) are mainly the same at different time points. Both can be a single figure, maybe merging lines 2 and 7 from 3 b).

Legend of Figure 4 is confusing and should be rewritten for clarity.

Figure 5 will be clearer if full length and proteolyzed POx lines are shown with solid lines, whereas markers in dashed or thinner lines.
Experimental design: . Did secondary structure content analysis of full and proteolyzed POx match with other results? Authors discussed secondary structure content, but values are not presented nor the deconvolution method used is described. How do the HT values behave? How many spectra were recorded or averaged? CD measurements are valuable for testing the main objective of this research, but authors use a visual inspection of the CD spectra as evidence.

Did the authors made blank reactions on enzyme activity without POx? I think an enzyme kinetics approach will give an insight of the effect of removing loops to the enzyme's turnover or substrate affinity.
Validity of the findings: The major goal of this research is to reduce the enzyme's size, without losing its enzymatic activity for further applications. But the authors do not expect that proteolysis by trypsin alters the active site or its surroundings significantly. Actually, they assume that two small loops of six residues are removed. I think it is not clear whether this is a proof of concept if the protein is not significantly size reduced or its active site is more exposed. In fact, the main finding is that two flexible/exposed loops can be removed without singnificantly loss of POx function.
Additional comments: From the biochemical approach of the experimental work, this manuscript is more suited for the biological and environmental section of this journal, than the materials science one.


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: LIMITED PROTEOLYSIS OF PYRANOSE 2-OXIDASE RESULTS IN A STABLE AND ACTIVE COMPLEX
Review round: 1
Reviewer: 2

Output:
Basic reporting: Islam et al present the effect of reducing the size of pyranose 2-oxidase on stability and enzymatic activity. Using trypsin to remove flexible structural regions; however, most of the claims about this being a superior method and tool for electrochemical applications do not seem to be adequately supported. Lastly, the presentation of the experimental data needs to be substantially improved.
Experimental design: - The manuscript needs information on replicates and analysis. For all experiments, please provide number of replicates and what the errors are. This is critical for evaluation of the data. This needs to be fully evaluated and the data shown.

- The authors present some potentially interesting data in this work, however, there is no detailed biochemical and biophysical analysis of the proteolyzed enzymes. Some data are not clear with awkward phrases here and there.

- Only 1 proteolyzed enzyme was showed similar kinetic rate with WT? The authors state that this method is effective. I would challenge this concept by the authors expressed and purified all proteolyzed enzymes, which is by no means a low-effort endeavor.

- Figure 7: I would like to see the kcat and KM values and the error in these values.

- Which step in the catalytic cycle of proteolyzed enzyme is rate-limiting? For completeness it would be good to present the full kinetics parameters of proteolyzed enzyme.
Validity of the findings: - This manuscript could benefit greatly by a more focused effort on biochemistry and biophysical properties of particular proteolyzed enzymes, or a broadening of the experimental effort by identifying proteolyzed enzymes with a clearer effect on performance.

- Author should explain why proteolyzed enzymes can convert similar efficiently with WT?

- Results are a bit overselling and there is relatively little discussion about the usefulness/success rate of the applied method.

- The paper attempting to rationalise the observations is rather speculative and it is not clear what we have learned from the current study about making small proteins stable.
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: LIMITED PROTEOLYSIS OF PYRANOSE 2-OXIDASE RESULTS IN A STABLE AND ACTIVE COMPLEX
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors take into account the reviewers comment. Their revised manuscript improved its clarity to state the major findings and future perspectives.


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: LIMITED PROTEOLYSIS OF PYRANOSE 2-OXIDASE RESULTS IN A STABLE AND ACTIVE COMPLEX
Review round: 2
Reviewer: 2

Output:
Basic reporting: This revised version is more clearer finding than the one before.
Experimental design: -
Validity of the findings: -
Additional comments: No additional comments


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

Input:
Title: CROSS-COUPLING POLYMERIZATION AT IODOPHENYL THIN FILMS PREPARED BY SPONTANEOUS GRAFTING OF A DIAZONIUM SALT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study by Marshall et al looks at the surface-initiated Kumada catalyst transfer polymerization of polythiophene after grafting of 4-iodobenzenediazonium tetrafluoroborate on gold substrates. Overall the study is interesting, well done and worthy of publication. A few minor comments should be addressed prior to publication:

Line 54. Sentence ends with “it” consider revising.

Figure 5 – more details on the CV (electrodes, solution salt concentration, etc..) should be include in the caption.

Line 163 define “PEPPSI”
Line 175 and 178: Figure 6. Figure 6 “a” should be included for clarity.
Line 178 this is the film after polymerization correct? Not before? This is not clear and should be identified.
Line 187 define “EDS”
Experimental design: good experimental design
Validity of the findings: L:114. XPS analysis: how does XPS provide thickness measurements? It provides an elemental compositional analysis. Where does the 2.5nm come from? The author should describe the assumptions used to make this analysis an provide more details (either in main text or in ESI).

For the CV (L152) can the authors comment on the stability of the films through repetitive CV measurements? Are the responses consistent after 5, 10 ,50 cycles? Is Figure 5 the first run?
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: CROSS-COUPLING POLYMERIZATION AT IODOPHENYL THIN FILMS PREPARED BY SPONTANEOUS GRAFTING OF A DIAZONIUM SALT
Review round: 1
Reviewer: 2

Output:
Basic reporting: * The Results & Discussion section would be easier to follow if a brief description of the experimental procedures used to prepare the samples were added to the beginning of each section (preparation of aryl iodide surfaces before line 112, polymerization conditions between the sentences in Line 156)

* Throughout the manuscript, there are points where added detail would make the science easier to follow, for example:
	* Abstract (lines 7-8): "thick, well-defined polythiophene thin films" -- what range of thicknesses? what is meant by well-defined?
	* Line 55: "the surface produced is highly variable" -- what aspect of the surface is variable?
	* Line 56: "Thiol SAMs are quite reproducible" -- what aspect of the SAMs is reproducible?
	* Line 131: "...coverage...consistent with a closely packed ferrocene monolayer." -- what coverage level or range is reported in the cited reference?
	* Line 136: "the surface coverage...was significantly lower" -- state measure value to show how much lower.
	* Line 137: "the peak-to-peak separation...indicates a slower rate" -- state the two different peak-to-peak separation values so they can be directly compared.
	* Lines 146-150: "An important ancillary finding of this work..." -- This discussion is confusing. Sources for the standard procedure should be cited. What role does reaction time play? What specific method for preparing thienylmagnesium chlorides was used in this work? How much lower is the observed surface coverage if an excess of isopropylmagnesium chloride is used? Because the substrates are immersed into a solution of the Grignard reagent, presumably the concentration of thienylmagnesium chloride is much larger than the concentration of surface-bound aryl halides regardless of the preparation method used. Is the implication that the excess isopropylmagnesium chloride can also react with the surface-bound aryl halides? Were any ratios of iPrMgCl:FcCH2ThBr between 2 and 3 examined to find the lowest ratio where complete conversion could be observed (presumably this would lead to the highest yields)?
	* Line 165: "highly uniform" -- How was the uniformity of the film assessed?
	* Line 180: "low-polydispersity" -- How was the dispersity of the particles assessed?
	* Line 187: "the brush thickness on the 2D surface is close to the particle radius." -- How were thickness and particle radius assessed? How close were the values to each other?
	* Fig 6b is described as showing "a regular distribution of narrow-size distribution PT particles" -- how were the regularity of the distribution and the dispersity of the particles assessed?
	* Line 205: "The film produced by the reduced PEPPSI Pd catalyst was not as thick nor the reaction as controlled..." -- What is meant by "controlled" here? Does it refer to molecular weight? dispersity? fraction of chains bound to the surface rather than in solution? A clearer description would be helpful.

* Figure 3: PEPPSI is a catalyst (Pd + ligand), not a ligand, so the definition of "L" is not accurate
* Figure 5: The caption should specify that the sample used for the CV in part (b) is on an ITO surface whereas the one in part (a) is on a gold surface.
* Lines 154-156: "Reaction...with surface-bound...catalysts has been established as a state-of-the-art technique" -- References should be cited (or re-cited) here.
* Line 174: Extraneous "(C)"?
* Figure 6: The caption for Figure S1 describes the film in Figure 6b as being cracked, but that is not mentioned in the caption to Figure 6.
* Figure 7: The caption for Part B does not appear to be correct.
* Lines 129-130/216: The ferrocene-functionalized thienyl Grignard reagent is described as being "well-known" earlier in the text, but in the experimental section the reader is referred to a prior publication (20% yield in 4 steps from dibromothiophene) from the author.

* Figure S1: The caption refers to Figure 7b (IR spectra) instead of 6b. The main conclusion from these images seems to be that there is no Au/S/C/Pd in the cracked area of the film.
* Figure S2: It is difficult to tell what conclusion is being supported by the element map--is there supposed to be a correlation between areas of S & C density? Dots corresponding to Au seem most abundant--would this be expected for a thick film of polymer? In the spectrum at the bottom, it would be useful if the region where N atoms were expected to appear were labeled so that the loss of nitrogen during grafting can be easily confirmed.
* Figure S3: The text cites this figure as evidence that a highly uniform film was prepared. It is difficult to tell from the SEM image, but it could also be interpreted as being made up of smaller fibrillar units that do not look particularly regular. Some more explanation in the text would help.
* Figure S4: This figure is confusing. The starting material (SM) should be identified as FcCH2ThBr. What was the TLC solvent system? The intensity of the baseline spot seems to increase with reaction time--is this important? The ratio of reagents should be clearly defined (equivalents of iPrMgCl to FcCH2ThBr).
Experimental design: The experiments are mostly well-designed, but the absence of any characterization data for the key molecule, 4-iodobenzenediazonium tetrafluoroborate, is very concerning. Basic characterization data should be included.

Additionally, there are several points where control experiments would be useful for supporting the conclusions:
	* Line 121: "XPS of the spontaneously grafted film is consistent with a thin layer of C sp2 atoms with some adventitious carbon, including residual carbonate from oxidative cleaning at 288.5 eV." - XPS of the substrate after cleaning but before grafting would be useful to confirm the source of the sp2 C signal. (Also, Figure 4c should be be referred to here.)
	* Lines 134-136: It is not clear that using trimethoxysilane grafting to an ITO surface should be directly comparable to benzenediazonium salt coupling to a gold surface. Is there any literature to support the idea that they should have comparable levels of surface coverage? There are also likely to be differences in efficacy of coupling to in iodophenyl surface (diazonium/Au system) vs a bromophenyl surface (silane/ITO system). Controls with a bromophenyldiazonium salt/Au surface and/or with an appropriate iodophenyl thiol/Au surface would be more likely to reveal more about these systems.
	* Line 195: XPS (or other analysis of the film) after sonication and chloroform extraction would confirm that the film remained intact after the particle removal process.

Line 263: The "rigorous cleaning" procedure is described here to be extraction with boiling chloroform, but in the main text (Lines 194-195), the procedure is described as including both sonication in acetone and chloroform extraction.
Validity of the findings: The presentation of the findings is generally reasonable, but it leaves open a number of questions:
* Line 114: "thickness of the thin film of 2.5 nm, consistent with other reported spontaneously grafted films." A monolayer of iodophenyl groups should be much thinner than 2.5 nm (< 1 nm)--can the author comment on the accuracy of this value and what it implies about surface coverage if it is accurate? References should be cited here for other reported films.
* Lines 115-116: The measured 1:8 I/C ratio is significantly lower than the expected 1:5 ratio (implying that only 62.5% of phenyl rings still have an iodine that can participate in grafting reactions). Can the authors comment on what this would imply about potential surface coverage?
* The studies with the ferrocene probe are used to suggest the formation of a closely packed monolayer--how does this finding relate to the later conclusions about polymer surface coverage?
* Line 159: How were sample colors determined and compared? Can the color change be influenced by the gold surface? Citing absorption spectra here would be helpful and more quantitative.
* Lines 179-182: "A reasonable explanation for these...features is that they were formed from Pd(O) catalyst centers that were physisorbed onto the substrate surface" -- If this is true, then it should also happen in the ferrocene studies--what effect would excess Pd catalyst have on the surface coverage studies? Could physisorbed catalyst react with thienylmagnesium chloride and/or thienyl bromide to either give detectable solution species or excess surface-bound ferrocene?
* Lines 183-185: "[upon precipitation] the active catalyst site was inaccessible and polymerization effectively ceased." -- Is this necessarily true? This would depend upon the porosity of the particles formed upon polymer precipitation, the location of catalyst sites (particle interior vs particle surface), and solubility of monomer in polymer particle. The intended initiation sites are also bound to an insoluble material (the gold substrate), so it is difficult to argue that polymerization will occur from the gold surface and not from a polymer particle.
* Lines 187-188: "the brush thickness on the 2D surface is close to the particle radius, implying that the features may have grown as part of the same process." -- It is not clear why this would be true. By the authors' arguments, the thickness of the brush should depend upon polymer growth rate, while the particle size should depend upon whatever stage of agglomeration the precipitating solution polymer chains (which can grow from both ends and should have a different degree of polymerization than the surface bound chains) achieve by the time they adsorb to the substrate--these seem to be two very different factors.
* Lines 196-198: "...microscopy of the substrate...reveals a roughly 1 micron thick, regular film of polythiophene..." -- If the assumption is that each surface-bound aryl iodide results in one polymer chain and that there is a high density of aryl iodide sites, does a thickness of 1 micron fit with accessible molecular weights for polythiophene prepared by Pd catalysts? My instinct is that preparing polythiophene chains with a chain length of around 1 micron would mean reaching much higher molecular weights than can typically be prepared by these methods.
* Line 232: The yield for the key molecule 4-iodobenzenediazonium tetrafluoroborate is described as "0.090 g, 28%", but 0.090 g of the product (317.82 g/mol) corresponds to 0.283 mmol of product, which, starting from 2.5 mmol of 4-iodoaniline, seems to correspond to an 11.3% yield. The absence of any additional characterization data is concerning.
Additional comments: This manuscript describes some potentially interesting results, but there is significant room for improvement in the interpretation and presentation of the results. All of the areas of concern seem addressable if the authors are allowed sufficient time for revisions.


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: CROSS-COUPLING POLYMERIZATION AT IODOPHENYL THIN FILMS PREPARED BY SPONTANEOUS GRAFTING OF A DIAZONIUM SALT
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper is well written and well referenced. Figures and table are good.
Experimental design: The experimental design is presented in detailed procedures. Methods are described in detail and information will be easy to replicate.
Validity of the findings: The findings are supported by experimental evidence.
Additional comments: This manuscript reports the synthesis of polythiophene films by surface-initiated Kumada cross-coupling polymerization. The experimental methods are well described and the analysis of the polymer film by SEM is proper because it allows the determination of the film thickness. I appreciate the originality of the reported work but I do not see much potential in terms of potential applications. By using non-substituted thiophene as the monomer, there is no regioregularity problem.

The authors should determine the optical bang gap from the UV-vis spectrum of the film shown in Figure 7. From the manuscript, it is not clear if the polythiophene film thickness can be controlled. The authors should discuss if the film thickness can be controlled because this is very useful.

The authors could try to adjust the method to grow the polythiophene film on transparent ITO which will allow the characterization of the film to determine optical and electronic properties. The molecular weight of the polythiophene cannot be determined because non-substituted polythiophene is insoluble in organic solvents. I would see a lot more value to this work if 3-hexylthiophene would be used as the starting monomer to generate P3HT. This would also allow a discussion of regioregularity.

The authors recognize the formation of polymer in solution which is physisorbed on the surface of the film. The authors should try to analyze the films using TMAFM analysis to determine the rms. The morphology of the film is important, and TMAFM analysis would give some answers regarding the surface morphology of the polythiophene films.

The authors could try  to measure the conductivity of the film upon doping with iodine using a four point probe.


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

Input:
Title: EFFECTS OF ALKALI TREATMENT ON THE MECHANICAL AND THERMAL PROPERTIES OF SISAL/CATTAIL POLYESTER COMMINGLED COMPOSITES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is clear and written using professional English language.
The article is quite interesting and I thank you the authors for providing extensive references of similar work done by others. If there is a comment I would like to make is that the general reference listed on line 79 (another study) and line 93 (In an investigation) could be improved by adding either the tittle of the study or investigation on question or just the author involved, despite of the fact that it is well reference (4) and (1) respectively.
I would like to add that I have not found Tables S1 listed on line 221 and Table S2 listed on line 344. The figures are very illustrative and reveal well the findings.
Experimental design: The methodology used is consistent with a comprehensive coverage of the subject.However, I would like to state that the tittle mentions the mechanical and thermal properties, and, only the thermal conductivity was tested. I expected to see more than just one thermal property. Nevertheless, the research is quite meaningful and methods are well described with sufficient information to replicate.
Validity of the findings: I commend the authors for their extensive data set and again comparison with other similar work performed by others. This comparison makes it easier for the reader to understand what to expect from the study. I would like to mention that perhaps there is an error on line 218 in which mentions 26.17 ± 8.33 and 35.17 ± 54.96 tex. I think it might be 26.17 ± 8.33 tex and 35.17 ± 5.496 tex.
Conclusions are well stated and limited to supporting results. Overall the study is well performed, well understood, data have been provided and they are statistically sound.
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: EFFECTS OF ALKALI TREATMENT ON THE MECHANICAL AND THERMAL PROPERTIES OF SISAL/CATTAIL POLYESTER COMMINGLED COMPOSITES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript “Effects of alkali treatment on the mechanical and thermal properties of sisal/cattail polyester commingled composites” is a well-written and comprehensive report. The authors provide a good review of relevant literature and compare their own results to those presented in relevant studies.

- The authors should add scale bars to all microscopic images
- In the paragraph “fractography studies” the authors refer to Figure 2 but describe Figure 3. Please change reference to Figure 3.
- The authors are inconsistent with respect to the number of decimals reported for modulus and strength values and should revise accordingly.
- For the caption of Figure 3, I suggest adding a comment that clarifies that fracture surfaces are shown.
- The unit “MPa” is reported as “Mpa” several times, e.g. in lines 283, 287, and 290.
Experimental design: The experiments were designed to thoroughly investigate the posed research question, and the conclusions are supported by data. All methods and experimental procedures are well described.
Validity of the findings: Conclusions are generally supported by data and the authors compare their own findings to results reported in relevant studies. A few things to address:
- The authors discuss that the thermal conductivity does not change significantly between composites with treated and untreated fibers. This contradicts the statement in the conclusion that thermal properties were enhanced for composites contain treated fibers. The authors should change the conclusion so that it matches their findings.
- The impact strength data was not provided in the form of a graph or raw data, numbers are mentioned in the text.
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: KINETIC AND THERMODYNAMIC ANALYSES OF THE CORROSION INHIBITION OF SYNTHETIC EXTRACELLULAR POLYMERIC SUBSTANCES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Language is satisfactory, when all other basic issues are completely unsatisfactory.

(1) The authors misunderstand the origin of Equations which they apply to treat the data.

(2) The authors' background in kinetics of corrosion processes is very poor.

(3) Presentation of electrochemical data and corresponding quantities is misleading.

(4) There is no any scientific result or hypothesis. Instead, we see a number of observations for very complex and less characterized system accompanied by incorrect data treatment, see below.
Experimental design: The arrangement of experiment is more or less traditional for corrosion research, based mostly on the measurements of polarization curves. Very important information about the type of carbon steel is absent (the authors report only elemental composition, but do not consider phase composition which is crucial for corrosion behavior) and escape to describe the samples surface pretreatment (which is crucial for kinetics of interfacial processes).

The most dramatic problem is the choice of the inhibitor under study. For practical applications, this mixture can probably work, but it is impossible to consider the mixture of 5 substances in terms of adsorption isotherms. Any of the components is characterized by certain isotherm when adsorbs in the absence of other species. However the adsorption from the mixture is typically competitive, and cannot be considered in any additive manner. The parameter of Langmuir adsorption isotherm has a meaning of surface-adsorbate interaction energy and cannot be extracted from the data for mixtures.

For FTIR experiments, I failed to understand under what conditions these spectra were registered. To consider FTIR information in the context of adsorption from solution, one needs to measure the spectra in situ. The usual problem in this case is the overlap of bands for solute and adsorbate, and there are many specific techniques to separate these contributions, but nothing is written about this principle technical issue. Most probably Fig.8 demonstrate the spectra of the mixture as is, so it can tell us nothing about participation of certain functional groups in adsorption.

There is no indication of solution pH, which is important in respect to protonation constants for acids. The adsorption properties of organic acids and their anions are completely different.
Validity of the findings: All the findings resulting from the treatment of polarization curves are senseless because of the following reasons.

(0) There are no linear parts in Tafel plots, so I wonder how the authors extrapolated the anodic and anodic curves in their Tafel plots to obtain I_corr. I can assume that one of the reasons of non-linear behavior is the Ohmic drop because the authors use a huge (5cm^2) electrode, and the currents in rather wide potential intervals are above 1 mA. For any electrochemical experiments, much smaller electrodes must be applied, and Ohmic contribution should be examined from the very beginning.

(1) Arrhenius Eq (2) is valid for the rates of particular processes, and cannot be applied to I_corr, which presents combination of at least two (anodic and cathodic) coupled processes. It is very easy to see (Figs 1-3) that the inhibitor effects on cathodic and anodic branches are different, so the contribution in I_corr are also different for solutions of various concentrations.

(2) It is impossible to determine the surface coverage from I_corr using Eq. (4) or even from the currents at cathodic and anodic branches because the rates of reactions are not obligatory proportional to adsorbate-free surface. In particular, anodic dissolution includes chloride adsorption step, which can preferentially occur at some active centers. Adsorption of large molecules like carbohydrates does not obligatory suppress the adsorption of low-molecular species participating in corrosion. For hydrogen evolution, the rate can be proportional either to teta, or to teta^2, depending on the nature of the limiting step.

(3) It is impossible to apply Langmuir isotherm to high surface coverages (0.69 an even much higher) because this isotherm assumes the absence of lateral interactions in adlayer. These interactions are unavoidable for molecules with any functional groups. It is also impossible to apply Langmuir isotherm to large molecules which tend to poly-layer adsorption. Moreover, before starting with any isotherm one needs to clarify whether the adsorption is reversible or irreversible. Correspondingly, 'adsorption part' is completely mistaken.

(4) I can assume that 'local electrolytic cells' mentioned by the authors is the notion related to local galvanic pairs. However this mechanism of corrosion is not universal, it can take place only for strongly inhomogeneous surface and depends on distribution of minor components in the steel sample. In any case, in NaCl solution the basic mechanism is related to chloride, not to any galvanic pairs.

(5) The data tabulated by the authors do not allow to determine any quantities with the accuracy presented now (up to five significant digits). What is tabulated for K (three points in Table 4) does not allow to calculate anything even with low accuracy.
Additional comments: If the authors consider their mixture as useful for the practical corrosion protection, they should arrange standard corrosion tests instead of measuring polarization curves.

If the authors plan to develop corrosion science and to publish articles, they should start from the very beginning.


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: KINETIC AND THERMODYNAMIC ANALYSES OF THE CORROSION INHIBITION OF SYNTHETIC EXTRACELLULAR POLYMERIC SUBSTANCES
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this work English is suitable, clear and technically correct.
However the introduction is not enough described and does not clearly introduce the innovative aspects of the study and the literature can be more descriptive on the actual background. (The authors shoud be careful when citing the literature with a specific program since sometimes the references can be missing)
The structure of the article is not good in the results part where no description is given and should be more emphasized, maybe with a joint results and discussion section.
The number of figures is adequate for the present study and are relevant for the clarity of the paper, however they can be more thoroughly described throughout the text helping the reader to follow the line of the work. In particular figure9 is not described in the text
The results could be relevant to the work if more elaborated
Experimental design: The present study is within the scope of the journal, could be of a particular scientific interest and is quite innovative for the use of synthetic EPS as corrosion inhibitors.
The aim of the work should be more emphasized in terms of applicability of the inhibitor in the introduction section.
The experimental part could be better defined. The authors should better clarify the methods used for statistical reproducibility and repeatability. If the authors used the same sample and performed replicated electrochemical analyses, it is possible that the results obtained where not comparable to each other since the analyses induced a change from the original electrochemical conditions and an anomalous variability. If the authors used different samples, they should explicit the number of replicates more clearly in order to have a good data dispersion, a statistical validation and a significance of the measurements. Why did the authors choose a range of temperatures from 25 °C to 70°C? Can the authors report it in the experimental section to justify the choice of temperature range?
Do the authors think that 6 hours is enough to define the behavior of the systems in presence of inhibitors? It would be important to justify it.  Did the authors evaluate the Fe release inside the solution to verify the inhibition effect of the polymer?
Validity of the findings: The impact and the novelty of the work is undeniable but the work lacks some rationale in the methodology used. For instance in the section where the authors discussed the properties of synthetic EPS is a good introduction part but is not supported by the results previously described, thus should be moved or both in the introduction and in the methodology section.
The data provided in the text can be questionable if the Tafel experiments were performed always on the same sample. The analyses changed the interface and the behavior described does not reproduce the actual conditions, inducing an anomalous variability. It would be better perform experiments using for each condition a 'new' sample (3 samples for each condition).
Some parts of the results are missing and are described in the discussion section. I would suggest to join the results and discussion section in order to have a text that results more clear to the reader.
Additional comments: In this work the author investigated the role of specific synthetic EPS to be evaluated as corrosion inhibitors for mild steel in an anaerobic chloride-rich environment. The present study is within the scope of the journal, could be of a particular scientific interest and is quite innovative for the use of synthetic complex polymers that can be found in nature. However the work needs more thorough investigations and is not enough elaborated to be published as a scientific article.
I suggest the authors to rewrite the article focusing on the results and discussion section, clearly indicating the results ad describing the trends found and after that trying to justify the data obtained according to the parameters chosen.
In terms of scientific rigor, the units should always be the same throughout the paper (use K or °C and not both)


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: PREPARATION, THERMAL STABILITY AND ELECTRICAL TRANSPORT PROPERTIES OF VAESITE, NIS2
Review round: 1
Reviewer: 1

Output:
Basic reporting: Helena et al. reported the transport properties of NiS2 densified by the Hot-press method. They also compared the results with the band calculation. It is important work to clarify the intrinsic bulk properties in NiS2 with large sample dependences. I will be agreement with the publication in PeerJ, if you revised below points.

1) If the values of Seebeck coefficients, resistivity or power factor were already reported in thin film or single crystal study, the authors should note them and compare with those in your hot-pressed NiS2.

2) NiS2 is known to show magnetic ordering at 30-40 K. It is not clear that your hot-pressed samples exhibit a magnetic transition in the temperature dependence of resistivity in figure 7. Since the sharpness of the anomalies by transition may be closely associated with the quality of your samples, authors should show the resistivity data around transition temperature, more clearly, for example by using the enlarged view.
Experimental design: Totally, experimental procedure is well explained in detail in this manuscript. I just think that the authors need to mind below one point.

Before hot-press, the authors synthesized the NiS2 samples (as grown samples). What is the properties of your as-grown sample? I recommend to show the properties for comparison, for example, lattice constant, compositions and electrical resistivity (or the activation energy) of your as-grown sample.
Validity of the findings: 1) The authors showed the band calculation of NiS2 with metallic band structures. I am not sure that this work by authors is the first report. If it was already reported, the authors should explain the originality of your band calculation.

2) The authors described that the NiS2 is good starting points for a new family of thermoelectric materials. However, it is not evidently, for readers, that the values of Seebeck coefficients and power factor in your hot-pressed NiS2 are large or not. I suggests these values of typical examples of thermoelectric materials are described in the introduction.
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: PREPARATION, THERMAL STABILITY AND ELECTRICAL TRANSPORT PROPERTIES OF VAESITE, NIS2
Review round: 1
Reviewer: 2

Output:
Basic reporting: meet the standards
Experimental design: Original research with reasonable technical level.
Validity of the findings: Fig. 6 and Fig. 7 showed that the samples sintered by different temperatures exhibited different transport properties. It should be further confirmed and given explanation.
Additional comments: This is a new phase and the transport properies are worthy to be studied more deeply.


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

Input:
Title: A MICROFLUIDIC APPROACH FOR SEQUENTIAL ASSEMBLY OF SIRNA POLYPLEXES WITH A DEFINED STRUCTURE-ACTIVITY RELATIONSHIP
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this submission, Loy et al. describe the use of a microfluidic assembly approach to produce functionalized siRNA polyplexes. Improving control during the bottom-up manufacturing of polymer-nucleic acid particles is a worthwhile aim that could help solve the issues of batch-to-batch variability and convoluted structure-function relationships that persist. By incorporating sequence-defined oligocations and sequential chip-based assembly steps, the authors have made a nice step toward achieving fully defined polyplex micromanufacturing. The language is clear throughout, appropriate context is provided through referenced literature, and the experimental methods are both appropriate and well-described. Unfortunately, the modularity and adaptability of the system and its components is a bit of a double-edged sword - the many combinations of assembly conditions and components can cause confusion. This could be alleviated by editing some of the figures to improve clarity.
Experimental design: The methods chosen by the authors are appropriate, well-described, and well-controlled. The statistical analysis is transparent and thorough. However, there are a few issues with the results and their presentation that hinder interpretation.

1) Portions of the results are derived from nanoparticles assembled by loading ‘core polyplexes’ onto a microfluidic chip for subsequent functionalization (most of the characterization figures). Other results involve nanoparticles generated entirely on a chip from their molecular constituents (e.g. the MTT and RNAi figures). It is not clear without delving deep into the text which data is derived from which assembly paradigm. It is conceivable that different degrees of condensation could affect the ratio of available:embedded cholanic acid binding sites, thereby changing the effective molar ratio of lipid anchors and PEG ligands provided. Ideally, the characterization assays and functional assessments would be done with identical batches of particles. At the very least, the figures and captions should clearly indicate which assembly method was used in each specific case. Without such disambiguation, the results can neither be accurately interpreted nor reproduced.

2) Figure 1 is extremely confusing. It should be redrawn with descriptive labels in the figure itself instead of the caption only. Many of the acronyms used are only explained much later in the text. The rationale for including LA and LAE in the study is not discussed until Line 474, nearly halfway through the manuscript. Abbreviations like DP48F are not immediately obvious. Figure 1B should be changed to clarify that it illustrates two different assembly schemes. It also lists “Shell-ligand” where everywhere else it is called “Lipid anchor-ligand”. The abstract refers to optimized PEG lengths of 36, but the methods and Figure 1 refer only to 12, 24, 48, etc. It is not clarified until much later that this sum includes both the lipid anchor and PEG ligand. An unambiguous system should be adopted early and used consistently throughout. Fig 1C & D could be moved elsewhere in the text, perhaps nearer to the sections describing the results using the CON assembly system.
Validity of the findings: The adequate sample sizes and acceptable variability of the data enable robust statistical comparison. The trends identified by the authors are generally supported by the results. A few additional controls would strengthen some of the structure-function relationships identified.

3) It should be confirmed that the physicochemical characterizations are performed on stabilized and equilibrated particles. If size and charge continue to change over time, comparisons between samples are less reliable.

4) It is not stated whether the 500 ng dose/well was optimized. With zero toxicity observed in most conditions, it would be interesting to see a dose escalation test. Not only would this perhaps enable more robust gene silencing, but it would elucidate any differences in cytotoxicity between formulations. It would also be nice to see a validation of FA activity following microfluidic assembly.

5) The authors should speculate on the reasons a U-shape effect is observed in gene silencing relative to PEG ligand length.
Additional comments: Minor Issues:
- A MALDI analysis of the CO starting material could help validate its synthesis and monodispersity.
- Consider representing hydrodynamic diameter as Dz instead of Dh, as it is derived from the intensity-weighted Z-average.
- Figures scales should be adjusted to fit the data (e.g. Figure 2, Figure 3A).
- The asterisks/lines in Figure 5 are ambiguous. Consider using alternate symbols or tick marks to indicate significance between groups.
- The methods provided for the gel shift experiments indicate 4uL of loading dye was used, but the sample volume is not stated.
- Channel heights should be included in Figures S10 and S11
- Cell source is not provided. Are the KB cells derived in-house or obtained commercially?
- Consider using the more-standard PDI instead of pdi
- The role of acetone in the synthesis should be clarified - is it to retard siRNA compaction (line 524) or facilitate lipid anchor deposition (line 476)?
- Consider adding densitometry analysis to the gel results, as the reader is asked to compare relatively minor differences in band intensity

Typos:
Line 77: _ _
Line 97: oppositional
Line 229: hepes should be capitalized
Line 367: to-scale
Line 464: lipdid
Line 494: and
Line 296/301/305: well(s) is more standard than pocket(s)


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

Input:
Title: A MICROFLUIDIC APPROACH FOR SEQUENTIAL ASSEMBLY OF SIRNA POLYPLEXES WITH A DEFINED STRUCTURE-ACTIVITY RELATIONSHIP
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript is well written. There is a nice general introduction of the topic and the results section begins with a first section in which the function of the different part of each compound is well explained and made accessible to non-specialist. All important information are given making possible to reproduce the work and possibly to use the described technology. Note that some figures and nomenclature are misleading, and I strongly advise to revise the manuscript to take this into account (see my General comments to the authors).
Experimental design: Overall, the experiments are well conducted. My only main concern was about the decorrelation between the siRNA used in the ex vivo study and the reporter monitored. Indeed, it is not clear for me why the authors measure luciferase activity whereas they used an siRNA targeting GFP. Indeed, if measuring luciferase activity, it would have made more sense to treat cells with siRNA targeting Luciferase gene; or if targeting GFP gene, why not measuring GFP fluorescence? This is a minor comment, but it would be great if the authors justify their choice?
Methods section is in general well written and complete. Especially schematic of the microfluidic devices are given as supporting material which is essential for being able to reproduce the results and use the technology. Yet, couple of minor (yet important) information are missing and should be added (see General comments below).
Validity of the findings: The conclusions and explanations proposed are in line with the results obtained from the experiments. Overall, I agree with all the conclusions exposed by the authors. Moreover, when necessary results were confirmed by an independent approach (e.g. Microscopy and FRET; electrophoresis and Ethidium Bromide assay…).
Additional comments: In their manuscript, Loy et al present a new class a transfection agent made of three blocks together with the possibility of using microfluidics to generate homogeneous nanoparticles made two to three components and characterized by controlled structure and composition. The originality of these reagents is the use of a lipid layer to self-assemble the particle through non-covalent interaction. Moreover, the authors introduce a microfluidic device allowing for preparing multilayer nanoparticles of controlled size and dispersity made of three different components in a single step. This is an elegant application of microfluidics and I’m pretty supportive to this work. However, prior to accepting this manuscript for being published in PeerJ-Materials Science, I would recommend the following points to be addressed.

Major points
1. Perhaps the biggest issue with this paper is the lack of consistency between the figures and the text regarding compounds naming. For instance, on Line 562-565 the authors talk about LA-DP48F but the reader needs to understand that this corresponds to P60F (12 Eos from LA + 48 Eos from DPF) on the figure. I acknowledge that an explanation is given in Lines 491-493, but still I have been unsure several times about this point. Another example is on line 692 where the authors state that “…reached their base at DP24F…”. Again, the reader has to understand that, if I’m correct, the authors are not talking about the bars labelled P24F, but rather P36F (ie 24 EOs from the PEG-ligand + 12 EOs from the anchor). If my understanding of the discrepancy between the text and the figure is correct, I would definitely ask the authors to change one of them and use a single nomenclature throughout their manuscript and figure.

2. I have been confused with the figures (Figure 4A and S15) showing FRET results. Indeed, in the captions, the authors say that “FRET: excites Atto488 (485 nm), measures CY5 (680 nm). This is misleading for me because we do not know (unless digging the information from the Method section) if this set of wavelengths was apply only to FRET or to all the measurement. Therefore, I would propose that the authors up-date the figures and add next to the name of the experiment (e.g. Atto488) the set of wavelengths they used for this particular measurement (in this case “Atto488 (ex./em.; 485-20 nm / 535-25 nm)”).

3. The authors mention several times “conventionally prepared core”. Even though, methods section informs that this corresponds to manually prepared nanoparticles, I think it would be wise that clarify this point the first time the sentence is used in the result section (probably line 540).

Minor points
1. In general, I wondered how efficient the transfection agents used in this work are with respect to other commercial ones frequently used for cell transfection (e.g. lipofectamine and Fugene). Do the authors have any clue on that? Such a benchmarking might be of great interest for biologist readers and would allow to better justify these new developments.

2. Line 94: The authors state that PRINT method has only advantages. If so, why searching for alternatives? It might be useful to cite disadvantages/limitations of PRINT as well.

3. Line 360: could the author give the supplier of the fluoro-silane they used?

4. Lines 363-364: the authors use a plasma treatment to bind their PDMS device onto glass slides. It is known that the gas conditions as well as the type of plasma cleaner are important as well. Can the authors indicate if the plasma was generated from air or from oxygen? Also, model and supplier of the plasma cleaner should be given.

5. Line 517: the authors mention that “additional efforts” are required in case 25% acetone is used. Could they specify what these efforts correspond to?

6. Lines 523-524: acetone was used to retard siRNA compaction. Could the authors shortly explain why this is necessary and how it works?

7. Lines 571-572: “CO in HBG with and without 50% acetone… LA or LAE with and without 50% acetone… with and without their respective PEG-ligands…”. From this sentence I was expecting 16 conditions to be tested. However, it turns out that only 8 conditions are found on Fig S14. Therefore, either data are missing or the reader should understand “CO combined with LA or LAE with and without 50% acetone… with and without their respective PEG-ligands…”, in which case the sentence should be corrected.

8. On figure S13.A, third panel, the point at 10 mL/hr indicates a size reliably twice higher than the other points. Do the authors have an explanation to propose for this striking behavior?

9. Figure S17. It might be useful for a broad readership if the authors could explain how the Ethidium Bromide displacement assay they used works.

10. Transfection assessment. I found odd that the authors used an siRNA targeting GFP mRNA and monitor Luciferase activity as a readout. Why not directly quantifying GFP fluorescence or using siRNA targeting Luciferase mRNA? I think an explanation should be given.

11. It is argued that Glutamic acids (E) were included in LAE to increase attachment. Yet, this point is not addressed anymore in the final discussion of the article which left the reader with an open question on “how useful was finally the addition of Es”. I would suggest that the authors had a statement on this point in their discussion.

12. Line 815: The authors say that T-junction is best for large scale preparation. How about their microfluidics? Could this technology also be used for large scale preparation?

13. In the text, or at least in the caption of figure 1, it would be great to explain what HBG stands for and why using this particular buffer.

Typos:
Line 123: “for or for” should read “for”
Line 675: repeated “in”
Caption Fig. 1: “lipdid” should read “lipid”


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

Input:
Title: A MICROFLUIDIC APPROACH FOR SEQUENTIAL ASSEMBLY OF SIRNA POLYPLEXES WITH A DEFINED STRUCTURE-ACTIVITY RELATIONSHIP
Review round: 2
Reviewer: 1

Output:
Basic reporting: On the whole, the authors do a nice job of using a chemically-defined delivery system and controlled assembly to demonstrate structure-function relationships. The characterization is comprehensive, using a host of relevant methods (DLS, TEM, FRET, heparin challenge, etc.). Combined with the thorough and transparent analysis of cytotoxicity and luminescence silencing efficiency, the work will represent a nice addition to the field. I am comfortable with its publication in the current form.
Experimental design: With this resubmission, the authors have clarified murky portions of the text and included a number of new experimental results that give further validation and context to their findings. The revisions resolve the bulk of the ambiguities that existed in the previous version. In particular, the confusion regarding the covalently-attached PEG ligands (CON conditions) has been resolved by separating its discussion and creating Figure 6.
Validity of the findings: No comment
Additional comments: Minor suggestions: The author’s may wish to explain their selection of folic acid as a targeting ligand for readers outside the delivery field (e.g. potentially upregulated in cancer cells, implicated in endocytosis).


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: FACILE IMINE SYNTHESIS UNDER GREEN CONDITIONS USING AMBERLYST® 15
Review round: 1
Reviewer: 1

Output:
Basic reporting: The work is written confidentially
Experimental design: Could be interesting to use also aliphatic aldehydes or other aliphatic ketones in addition to the cyclic one 1i.
Validity of the findings: The procedure has several advantages including simplicity, irreversibility and the green and environmentally friendly conditions (room temperature, recyclable catalyst). Moreover immines are important intermediates in the synthesis of biologically active N-heterocyclic products and in industrial synthetic processes.
Additional comments: The work reports a simple and efficient procedure to obtain imines. A variety of aromatic aldehydes containing electron-withdrawing and electron-donating groups, with aliphatic and aromatic ammines can be employed; could be interesting to use also aliphatic aldehydes or other aliphatic ketones in addition to the cyclic one 1i.
I think that the manuscript can be published in PeerJ because it has several advantages including irreversibility and the green and environmentally friendly conditions (room temperature, recyclable catalyst).
Some typing errors:
In the line 15 of abstract is reported “aliphatic and aromatic aldehydes” change with “aromatic aldehydes” and also in line 120 change “aldehydes” with “aromatic aldehydes”.
In line 43 the reference could be added: “Green Chemistry Theory and Practice Paul T. Anastas and John C. Warner Oxford University Press 1998”
Line 108: change “Scheme 1” with “Scheme 2”
Line 185: change “Subash, B.” with “Subash, B.;”
Line 195: change “Kodumuri, S.” with “Kodumuri, S.;” and “Gajula, K.S.” with “Gajula, K.S.;”
Schemes and tables:
 note a of Table 2: change “benzaldehyde:amine” with “1a:2a”
 Scheme 2:  change “5hrs” in “5h”
Supporting information:
line 25: change “748 ” with “748. ”
lines 52 and 72: change “H NMR” with “1H NMR”
lines 80:  superscript  1 in “1H NMR”
put lines 82 and 83 (white solid and IR) before line 81


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: FACILE IMINE SYNTHESIS UNDER GREEN CONDITIONS USING AMBERLYST® 15
Review round: 1
Reviewer: 2

Output:
Basic reporting: Dr. Giovanna Bosica and coauthors described the synthesis of imine by condensation of primary amines with aliphatic and aromatic aldehydes and cyclohexanone under environmentally-friendly solventless heterogeneous catalysis. They reported that Amberlyst® 15 is a performant heterogeneous catalyst in this transformation. Furthermore, the commercial availability and recyclability of this catalyst, the ease of separation from reaction mixture and versatility, makes this synthetic process an innovative and interesting approach for the synthesis of imine derivatives.
The paper is generally well referenced but, in my opinion, when critical points are reported to describe the disadvantages of other procedures (rows 38-41), related references should be added.
Anyway, some changes to improve the quality of this paper are required.
Preliminary experiments (table 1 and 2) should be more detailed, and the related results reported in just one table (Table 1 + Table 2).
The header of all tables (all named as "table 1") and the caption of the schemas (all named as "Scheme 1") need to be corrected.
Cyclic aliphatic amines have a nitrogen comprised in the ring (i.e., piperidine, pyridine, etc....), cyclohexylamine is just a primary aliphatic amine with a cyclic substituent (row 103).
To evaluate the effectiveness of using this amine, the reaction with cyclohexanone must also be compared with other amines. Further data could be obtained using cyclohexylamine also with other carbonyl substrates.
Instead of “catalyst’s physical state” (row 121), the term “catalyst morphology” is preferable.
In reference 2, an author is missed (Wei Zhang). Please check.
In reference 11, the article title is “Hβ Catalyzed condensation reaction between aromatic ketones and anilines: To access ketimines (imines)”. Please check.
Some changes in the Supporting Information file are also required.
Some signals that refer to a singlet are reported as multiplet and/or with ppm expressed as a range (i.e., methoxy and tBu groups, etc….), why? (For example, see compounds 3c, 3d, 3e, 3f, 3k ….).
The 1H-NMR data of compound 3i are reported in reverse order of all the others.
The 1H-NMR data reported for compound 3j, in my opinion, don’t fit structural characteristics; furthermore, the indicated reference (18), refer to the 4-nitro derivative NMR data; please check.
Compound 3k was described with one more aromatic hydrogen (5 ArH instead of 4 ArH); a comma is missed before 8.20 (row 49).
Compound 3m: a comma instead of “and” is required (row 59).
Compound 3p: the J for peaks at 8.21 and 8.70 ppm are missed (row 73).
Compound 3r: (row 81), please erase the bracket after Hz; IR data should be moved before NMR data; the signal of iminic hydrogen is probably missed.
Experimental design: No comment
Validity of the findings: The authors have to check carefully NMR data and the way they have to be reported. Scanned spectra could be added to the Supporting information file in order to help the reader.
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: FACILE IMINE SYNTHESIS UNDER GREEN CONDITIONS USING AMBERLYST® 15
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript titled “Facile imine synthesis under green conditions using Amberlyst® 15” describes the synthesis of sixteen aldimines obtained from de reaction of arylaldehydes with alkyl/aryl amines catalyzed by Amberlyst® 15 at room temperature under solventless conditions. Still, one example of a ketimine is presented. In my opinion, the reaction concept is well known as previously published by several authors (J. Org. Chem., 1971, 86, 1570, Synthesis 1985; 679, Tetrahedron Letters 1997, 38, 2039, Synlett 2004, 2135, J. Chem. Res. 2005, 299, Synthesis 2006, 1652, etc). I have several observations that justify why the manuscript cannot be accepted for publication on PeerJ Journal at this moment. Please, see the following points.
1)	I did not fully understand from the Table 1 and 2 why Amberlyst-15 was chosen as the optimal catalyst. Overall yields of 3a in the case of acid alumina, M K-10, molecular sieves, and anhydrous MgSO4 were higher.
2)	For me, the section “Results and Discussion” is poor, and it is limited to aryl aldehydes and few amines.
3)	General Procedure: The purification method needs to be better elucidated. Which products were purified by column chromatography? Which were crystallized? Details on crystallization method need to be provided when appropriate.
4)	One of the steps in typical procedures for the synthesis of compounds 3 is washing the reaction mixture with diethyl ether. Authors should add the information concerning the amount of diethyl ether used in this step.
5)	Supporting information: The supporting information has twenty-one references with no need.
6)	Authors should thoroughly check the values of spin-spin coupling constants through all the Supporting Information taking into account the fact that the spin-spin coupling constants of the protons, which are coupled to each other, should be the same.
7)	The melting point analysis is missing for compounds 3o, 3p and 3r.
Experimental design: No comment.
Validity of the findings: Totally, I don’t think this work has big impact for organic synthesis research. The synthesized compounds are known and the manuscript lacks novelty. So, I do not recommend this manuscript to be published in PeerJ.
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: FACILE IMINE SYNTHESIS UNDER GREEN CONDITIONS USING AMBERLYST® 15
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript considered most of the reviewers’ comments. The Tables have been corrected and more literature data has been added. Despite the unfortunate failure of the instrument, which did not allow the insertion of the preliminary experiments with other aldehydes/aliphatic ketones, many substrates are reported and characterized.
Experimental design: I think that the manuscript is suitable for publication in Peer J in the present form because represents a very simple, eco-friendly, efficient, and alternative approach to the synthesis of imines.
Validity of the findings: The ease of use and recoverability of Amberlyst 15® makes the catalyst a viable cheap alternative
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: FACILE IMINE SYNTHESIS UNDER GREEN CONDITIONS USING AMBERLYST® 15
Review round: 2
Reviewer: 2

Output:
Basic reporting: Dr. Giovanna Bosica and coauthors submitted the revised manuscript “Facile imine synthesis under green conditions using Amberlyst® 15” accordingly to reviewer’s suggestions.
Unfortunately, there are still problems with the 1H-NMR data reported for compound 3j.
The chemical shifts of aliphatic protons do not correspond to the isobutyl group of 3j and they are in absolute disagreement with those reported in reference 20.
In my opinion, the derivative 3j is neither the N-(3-nitrobenzylidene)-2-methylpropan-1-amine nor N-(4-nitrobenzylidene)-2-methylpropan-1-amine. The authors have to check the starting reagents and repeat the experiment.
Furthermore, I did not find copies of selected scanned spectra in the Supporting information file.
Row 73: round bottom flask.
Row 155: (ref 2) please add a semicolon between P. and Zhang.
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: FACILE IMINE SYNTHESIS UNDER GREEN CONDITIONS USING AMBERLYST® 15
Review round: 2
Reviewer: 3

Output:
Basic reporting: In my previous review, several were the comments made to the authors. The authors have properly revised the manuscript and SI which are better now.
Therefore, I would recommend accepting this manuscript in its current form.
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: GC-MS ANALYSIS OF PHYTOCHEMICAL COMPOUNDS OF OPUNTIA MEGARRHIZA (CACTACEAE), AN ENDANGERED PLANT OF MEXICO
Review round: 1
Reviewer: 1

Output:
Basic reporting: In the manuscript entitled as GC-MS analysis of phytochemical compounds of Opuntia megarrhiza (Cactaceae), an endangered plant of Mexico the authors describe the identification and characterization of the phytochemical compounds present in wild individuals of O. megarrhiza using Gas Chromatography Coupled to Mass Spectrophotometry. In this study, the authors reported 53 phytochemical compounds, 18 have previously identified with some biological activity. The variety of metabolites presents in O. megarrhiza justifies the medicinal use of this plant in traditional medicine and highlight it as a source of phytochemical compounds with potential in medicine and biotechnology.
The journal PeerJ Organic Chemistry publishes articles exploring subjects including biochemistry, polymers, green chemistry, photochemistry, and synthetic organic chemistry. I believe that the present study is in accordance with the profile of the journal, that the manuscript is relatively free of error and demonstrates a quality of the paper in terms of scientific value. Therefore, I recommend this manuscript for publication in PeerJ Organic Chemistry after consideration of the issues and corrections.

1) The whole manuscript is clearly written in professional, unambiguous language. The introduction section is full of information about the phytochemical compounds, however, I think you should do an update in some references as at lines 37-43, to show the topic is more current. Some examples: Sci Rep 11, (2021) 10041 (https://doi.org/10.1038/s41598-021-89437-4); Food Chemistry 362 (2021) 130196 (https://doi.org/10.1016/j.foodchem.2021.130196); Biotechnology Reports 30 (2021) e00633 (https://doi.org/10.1016/j.btre.2021.e00633).

2) I strongly recommend that the authors draw the chemical structures to the demonstrate the phytochemical compounds with biological activities (in the Tables 6 and 7). In this Tables (6 and 7) you should exclude the Similarity (%) and RT (min) columns, they are a duplicate information.
Experimental design: 1) Some studies in current literature report the use of LC-MS–MS to identification and characterization of components in plant extracts. With this kind of Cactaceae plant, could the authors use this technique? Or is the GC-MS the most appropriate for this situation?

2) The method was described with sufficient detail and information to replicate. However, the authors need to correct some abbreviations of the solvents. Methanol is correctly represented as MeOH, not MeOTH. Still, acetone isn’t CH₃CH₃, this is ethane molecule. The correct acetone representation is (CH3)2CO.

3) In some point of the manuscript (line 159, 172 and go on) the authors starting described the phytochemical constituents from the CH3CN extract. Is this information correct? The use of acetonitrile wasn’t described in the Materials and Methods section.
Validity of the findings: 1) Only the compounds and their respective activities were reported in the Discussion section, and the obtained results support the medicinal use of this plant in traditional medicine. However, the authors should explore more the importance of why find out what kind of phytochemical compounds are present in O. megarrhiza. For example, you should discuss about a future developing of new drugs, new chemical studies, or about the encouragement of planting this species once this one is listed as endangered in the Red List of the IUCN.

2) In the line 198 the authors described the “the presence of 18 phytochemical compounds with biological activities, with similarity values of identification above 93%.” Considering the tables 1-4, there are only 15 phytochemical compounds with similarity values of identification above 93%. Is this count correct? Still, a short sentence about the similarity values of identification and if there are a percentage acceptable to characterize a molecule, should be add in the discussion.

3) For better molecular characterization, the MS spectrum of compounds at the Table 6 and 7, should be add in the Supplemental files.
Additional comments: Please correct following typos and errors:
- change from 5ºC to 5 ºC (line 129)
- change from 300ºC to 300 ºC (line 129)
- the number 3 in the compound is subscript (e.g. CHCl3) (lines 144, 159, 172, 188 and go on)
- the letter H in 2H-Pyran-2-one (line 162) is in italic form.
- change from MetOH3 to MeOH (lines 185, 186, 192 and go on, Figure 3, Table 5)
- change from CHCl to CHCl3 (line 193)
- the quality of Figure 2 should be improved.


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: GC-MS ANALYSIS OF PHYTOCHEMICAL COMPOUNDS OF OPUNTIA MEGARRHIZA (CACTACEAE), AN ENDANGERED PLANT OF MEXICO
Review round: 1
Reviewer: 2

Output:
Basic reporting: The MS “GC-MS analysis of phytochemical compounds of Opuntia megarrhiza (Cactaceae), an endangered plant of Mexico”, authored by Arturo De-Nova and Pablo Delgado-Sánchez et al., describes the identification and characterize the phytochemical compounds present in individuals of O. megarrhiza using the gas chromatography coupled to mass spectrometry. The authors used organic solvents to prepare the extracts and 53 phytochemical compounds were identified. The protocol used in the chemical characterization agrees with literature procedures. However, the text is not clear and need to an extensive revision for types. The quality of figures and chemical structures also need to be improved. After reading of the manuscript, it is possible to identify a confusion in the nomenclature/abbreviation of the solvents used to prepare the extracts, which makes it difficult to understand the text. This work could be a relevant contribution for Chemistry of Natural Products, but I don’t recommend the acceptance of this manuscript in Peer J. this way. I suggest a careful review of the text and addition of a biological application study for the extracts before a new submission.
Experimental design: The research agrees with the Scope of the journal. However, in some cases the methods are not described with sufficient details or information to replicate. I suggest a detail review.
Validity of the findings: The results obtained are promising. However, they need to be better discussed. I suggest adding another reference parameter to complete the characterization of the compounds present in the extracts since the similarity of the spectra in many cases is less than 75%. This comment applies to the results described in tables.
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: GC-MS ANALYSIS OF PHYTOCHEMICAL COMPOUNDS OF OPUNTIA MEGARRHIZA (CACTACEAE), AN ENDANGERED PLANT OF MEXICO
Review round: 1
Reviewer: 3

Output:
Basic reporting: While the work is interesting and relevant regarding the phytochemical analysis of Opuntia megarrhiza, I have some concerns about the manuscript that make me not to recommend publication in its current form. Thus, it needs major revisions before publication, including English language improvement.
Experimental design: In my opinion, the main concern is the tentative identification of the extracted phytochemicals based only on the similarity approach from the Mass Spectral Libraries. Although the libraries are an important tool to help in the identification, additionally analysis should be used. At least, I recommend a Linear retention index analysis and comparison with standard samples.
Validity of the findings: The authors mention that the extracts were filtered using two different membrane filter (PTFE and PVDF) and as consequence, the chemical composition of the extracts are different. Thus, the authors should clarify the difference between both membranes and discuss the results regarding the composition.

Regarding the chromatograms showed in the Figure 2, some of the labeled peaks are nearly in the noise, which difficult the appropriated identification of the phytochemicals.
I also suggest improving the discussion comparing the identified compounds and the identified compounds reported in other studies for the Opuntia Species

The authors reposted that a” exhaustive search” was made for the biological activity based in the literature. But, if possible, a small biological activity screening against bacteria or fungi will considerably improve the study. As suggestion too, due to the large number of cited references in the manuscript (more the 100 references) a revision manuscript about the phytochemical composition and biological activity from Opuntia Species could be organized.
Additional comments: Some minor revision to improve the manuscript are suggested as follow:
Mass analysis is a spectrometric technique and not a ”Spectrophotometry” technique. Also, use the word “technique” instead of the word “technic”.
In the Abstract, the sentence “and they were analyzed in a mass spectrometry detector with eletronic impact” could be replaced with “and they were analyzed by gas chromatography-electron impact-mass spectrometry”.
The introduction should be revised. It is very repetitive.
In line 30, the sentence seems incomplete. “127 general…”
In line 71, the authors should replace de “UV resonance“, with “UV-Vis spectrophotometry”. UV-Vis spectrophotometry is not a resonance spectroscopy.
Line 113: correct “past” to “paste”.
Line 114: The for methanol is MeOH and not MeOTH or MetOH3 as shown in Table 5. Check all the acronyms along the manuscript. Also, check all the molecular formulas in the mains text and Tables, such as for acetone which represented as CH3CH3
Line 132: Correct the misspelt word “specters”
All the compounds nomenclature should be revised.
The symbol for mass/charge (m/z) is always italicized. Check it in the main manuscript and Tables


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: GC-MS ANALYSIS OF PHYTOCHEMICAL COMPOUNDS OF OPUNTIA MEGARRHIZA (CACTACEAE), AN ENDANGERED PLANT OF MEXICO
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the manuscript entitled as “GC-MS analysis of phytochemical compounds of Opuntia megarrhiza (Cactaceae), an endangered plant of Mexico” the authors described the identification and characterization of the phytochemical compounds present in wild individuals of O. megarrhiza using Gas Chromatography Coupled to Mass Spectrometry. In this study, the authors reported 53 phytochemical compounds, 19 have previously identified with some biological activity. The variety of metabolites presents in O. megarrhiza justifies the medicinal use of this plant in traditional medicine and highlight it as a source of phytochemical compounds with potential in medicine and biotechnology.
I believe that the present study is in accordance with the profile of the journal, that the manuscript is well written and after all changes performed by the authors, by adding current literature, improve de discussion, and included the chemical structures and MS spectrum of compounds in Supplementary, I have seen an improvement in the quality of the manuscript. Thus, I consider this article accepted for publication in PeerJ Organic Chemistry.