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

Input:
Title: THE FEASIBILITY OF PREDICTING GROUND REACTION FORCES DURING RUNNING FROM A TRUNK ACCELEROMETRY DRIVEN MASS-SPRING-DAMPER MODEL
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: General comments
This study investigated the accuracy of MSD model to calculate trunk acceleration and to estimate GRF with the obtained trunk acceleration. This is an interesting study, and generally well written. I enjoyed reading this manuscript. One major concern is that the acceleration measured using trunk mounted accelerometer may not represent the actual trunk acceleration. If the acceleration measured using trunk mounted accelerometer is not equal to the actual trunk acceleration measured using a motion capture system or other device, inaccurate estimation of GRF possibly came from the inaccurate trunk acceleration.

Specific comments
L98: Please add the velocity of running in this previous study.

L126: “2 m from the centre of the force platform” is not clear. Please specify the locations of the photocell gates.

L282: Please eliminate this last sentence.


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

Input:
Title: THE FEASIBILITY OF PREDICTING GROUND REACTION FORCES DURING RUNNING FROM A TRUNK ACCELEROMETRY DRIVEN MASS-SPRING-DAMPER MODEL
Review round: 1
Reviewer: 2

Output:
Basic reporting: Model naming.  The authors estimate model parameters from measured trunk acceleration as well as from measured GRFs.  It becomes somewhat confusing in the results and discussion to keep track of which source data were used to estimate the parameters (example line 214/215.  These parameters were calculated using GRFs rather than truckacc). Suggest using an abbreviation or some sort of means to distinguish which model parameters are being discussed (GRFparameters or ACCparameters).  Along these lines, including a table that lists the specific model parameters determined by each model explored would be very helpful.

Literature references well represented.  One omission is in the paragraph starting on line 65.  The authors state that monitoring GRFs are limited to the lab.  Line 71 alludes to estimating GRFs but does not include a reference.  I suggest including references here (Full disclosure that I’m suggesting referencing work I’m directly involved with (https://doi.org/10.1371/journal.pone.0099023).  While this may be biased, I do believe that this reference is helpful in this article since GRFs are estimated from acceleration using statistical methods.  This method is currently limited to peak vertical GRFs only.  The work the authors presented here is targeting complete resultant GRF waveforms from acceleration and therefore is of interest to folks working in the area of portable methods to quantify GRFs.)

Line 78 and 85:  Sentences on these 2 lines seem to contradict each other.  Suggest clarifying wording.  Line 78 states that a single accelerometer is inadequate to derive GRF waveforms while line 85 proposes a single sensor would be a useful tool.  Possibly add to line 78 that ‘to date’ a single sensor has been inadequate but would be brilliant if feasible…

Figure 3:  Suggest including how ‘extreme outliers’ were determined?  How many of these outliers were excluded?

Figures 3 and 4:  Include information about the source of the representative data. Single subject?  Multiple subjects?  Single stride?  Average of multiple strides?
Experimental design: Figure 1 illustrates the overall MSD model.  If the acceleration from the trunk (a1) was measured, why wasn’t the acceleration of the leg (a2) measured as well to further enhance the parameter estimations?  The purpose of the approach proposed in the paper is to have a method to estimate GRF that does not need a FP.  Adding a second accelerometer/GPS unit seems to maintain that purposes while also providing an ‘easy’ means to quantify additional variables from the model.

Line 121:  Add a space after ‘approval’ and before ‘for’.

Line 121:  No details were included regarding the warm up period.   Please provide information about the warm up completed by subjects.

Line 127:  The use of 4 trials at each speed seems small.  Suggest adding information about how 4 trials was the chosen number of trials to complete.  Additionally, how were unsuccessful contacts with the force plate handled?  Were these data included?  Trial repeated?

Line 128:  Please include how dominant leg was determined.

Line 135:  Suggest including more information about the Minimax device used.  How does it report raw accelerations?  Are they processed using proprietary algorithms?

Line 137:  For this study, the GPS device was placed on the dorsal part of the upper trunk.  Suggest adding information about how this location was chosen.  Seems like this position may be subjected to more rotational accelerations that other locations on the trunk?  What steps were done to ensure tight coupling between the elastic vest and the subject

Line 142: Assuming the last sentence is referring to only stance phase data being collected for GRFs? If so, please specify in the sentence.

Line 163 and on:  Please specify that resultant GRF was estimated. (for example, line 170 states ‘..GRF acting on the MSD-model…;.  This should specify resultant GRF.)

Line 176:  You specify that ‘for each trial’ model parameters were optimized.  In subsequent sentences and figures, it’s unclear if the calculations being discussed are also for each trial or were potentially averaged/grouped somehow (example line 183 discussing GRF predictions).  Please clarify possibly with a general sentence early in the methods that all calculations discussed were for each trial.  Similarly, the figures include ‘representative’ examples but it is unclear if these examples are for a particular subject, several subjects, all subjects?  Please include more details.
Follow up question: If I understood correctly, each trial was analyzed independently.  Was there any analysis done to look at variations in the parameters calculated for a subject across his 4 trials?  Between subjects differences?
Validity of the findings: Overall a well thought out and thorough discussion of the results.  One point that keeps arising for me is it seems that based on your results, ‘like predicts like’.  Model parameters predicted from GRFs, predict GRFs well.  Model parameters predicted from accelerations, predict accelerations well.  The predicted GRFs in Figure 3 look like very similar to the accelerations with a phase lag, yes?  After reviewing Figure 3 and reading through the explanations as to why the model predictions failed when using parameters identified from the trunkacc to predict GRFs, I’m left to wonder if it’s just a simple ‘like predicts like’ versus the trunk accelerations measured do not represent the a1?  Along these lines, it seems it would be useful to explore the overall feasibility and precedent (if there is any?) of predicting GRFs with accelerations or predicting accelerations with GRFs?

Line 332: change ‘is’ to ‘are’
Additional comments: Generally, the article is very well written and fairly clear, especially considering the number of variables and complexity of the model(s) explored.


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

Input:
Title: THE FEASIBILITY OF PREDICTING GROUND REACTION FORCES DURING RUNNING FROM A TRUNK ACCELEROMETRY DRIVEN MASS-SPRING-DAMPER MODEL
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed previous comments and questions very well.  I appreciate the time and effort put into editing the manuscript to improve the clarity.  One question/comment remains for me.

The 8 model parameters.  Thank you for including abbreviations to distinguish the source of the model parameters.  This helped clarify immensely for me.  One issue remains however.  As currently written, there is no way for the reader to have any idea of the differences in the model parameters, even a ballpark difference.  Since the specific parameters are critical to the estimations, it seems as though these should be reported.  While I understand the authors concerns about potentially confusing readers by reporting two sets of these parameters, for transparency to the reader that might be interested in these values, the parameters should be reported.  If another researcher is attempting to apply these findings to their own outcomes, the parameter specifics are critical.
Experimental design: No comments.
Validity of the findings: Please include some details of how RMSE was calculated.  Was every data point used to calculate overall RMSE?  Peak values?
Additional comments: No 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: CHEMICAL COMPOSITION, ANTIOXIDANT AND ANTITUMOR ACTIVITIES OF SUB-FRACTIONS OF WILD AND CULTIVATED PLEUROTUS FERULAE ETHANOL EXTRACTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript should be deeply revised both for English and for the description of the results appearing quite confuse; moreover many typing mistakes are present all in the text, especially in the list of references.
It is necessary to compare results with previous research, in the manuscript is not clear presented the previous reports of antioxidant capacity, such as reports by autors Alam et al : Consequence of the antioxidant activities and tyrosinase inhibitory effects of various extracts from the fruiting bodies of Pleurotus ferulae.
Experimental design: Figures are excellent designed, but according my opinion it will be better if the Table 3 and 4 will be merged. It will be easier to compare results of GC-MS analyses. It will be necessary to upload one of chromatograms.
Since that some methods are well known (Determination of polysaccharide content, Determination of polyphenol content, Determination of total flavonoid content, Reducing Power), maybe it can be more effective if you add only reference without detailed  explanation and add modification.
The PCA analyses of the content of polysaccharides, flavoinoids and polyphenols, as well as results of antioxidant capacity and anticancer effect an be useful tool to explain and  for connecting these results, which are really insufficient explained.
Validity of the findings: The subject of manuscript is very current and has not been sufficiently explored according to literature data.Furthermore, authors did not compared results with previous reported. These results are very notable, but the effect of measured chemical compounds on bioactive effect is not explained. Also,it is necessary deeper explanation of GC-MS results, in order to detect the difference, which can be cause for different bioactive activity of analyzed samples.
Additional comments: Introduction must to be more informative and included previous reports about chemical composition and bioactive effects of Pleurotus ferula.
Page 1, line 39-44; please delete the sentences since they are little informative in this context.
Page 2, line 57; please check abbreviation EA-C (also in further text)
Page 2, line 59-63; please replace these sentences to conclusion or delete.

Material and methods

Page 2, line 66; please add the source of cultivated and wild fungus
Page 2, line 74; it is not clear the production process of sub-fractions. What is dissolved in DMSO?
Page 5, line 152: Can you explain the sample preparation for GC-MS or add reference.
Page 5, line 164: Which program did you use for statistical analyses?

Results

The explanation of results is not in agreement with the stat analyses. The authors have serious mistakes in the part The polysaccharide, polyphenol and flavonoid contents of PFEE-W and PFEE-C and sub-fractions.
Page 7, line 170-172; Polysaccharide content in Pe-W is lower than that in Pe-C and Ea-W and Ba-W are similar with Ea-C and Ba-C, respectively.
This sentence is not correct, since that the polysaccharide content of these samples are Pe-W (9.764), Ea-W (20.36), Ba-W (40.64), PFEE-C (54.87), Pe-C (20.84), Ea-C (16.14), and Ba-C (38.51). Please check also the letter that showed significant difference. Why is significant difference marked with b for sample PFEE-W (38.46±1.005b)and not with a ?
Page 7, line  173-174. The explanation of results did not in agreement with results of the stat analyses, since that according to presented results Ea-C and BA-W are marked with some letter, thus these are not significant different.

Page 8,line 204; Why did you choose these samples (Pe-W  and Pe-C) for GC-MS analyses?


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: CHEMICAL COMPOSITION, ANTIOXIDANT AND ANTITUMOR ACTIVITIES OF SUB-FRACTIONS OF WILD AND CULTIVATED PLEUROTUS FERULAE ETHANOL EXTRACTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: The authors compared extracts from WT and cultured PF and showed that there were no significant difference in inhibition of  tumor cell growth, induction of apoptosis and ROS.
It is  important and useful for development of nature anticancer drugs and healthy food usage. However, the Ms lacks control  samples and some results and explanation is not very convincing.
Non cancer cell line should be included in all experiments.
line 149,  WAS  DCFH-DA directly added to the culture, or cells were harvested before they were stained? give details of the experiment
line 176-177  delete ” The results indicated that PFEE-W/C and sub-fractions might have
177 different antioxidant and antitumor activities due to their different contents of some components”  due to at the moment data cant indicate anything.
The authors should do statistical analysis for data presented in Table 2 and table3

Fig 3 B, pls show summarized data in addition to representative imagesThere are many grammar errors.
The explanation for ROs change  does not make sense.
Line 261-262, the authors claimed that the effect of Ea-W and Ea-C on ROS is due to their antioxidant activity. It is contradict.  ROS  induces apoptosis.  Not because of apoptosis results in ROS increase. The authors should clarify and demonstrate  it.
There are many grammar errors.


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: CHEMICAL COMPOSITION, ANTIOXIDANT AND ANTITUMOR ACTIVITIES OF SUB-FRACTIONS OF WILD AND CULTIVATED PLEUROTUS FERULAE ETHANOL EXTRACTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Minor suggestions:

1.	In the Materials and Methods section under Preparation of wild and cultivated P. ferulae extractions, it states on line 67, “100 g powders of wild and cultivated P. ferulae were extracted three times using 1 L of 95% (v/v) ethanol”- done to obtain PFEE-W and PFEE-C extracts and then on line 72, “Then PFEE-W and PFEE-C were successively extracted with equal volume of petroleum ether”- where they obtain the sub-fractions. The meaning of the word “extracted” appears to be different in the 2 contexts and is confusing. The authors need to fix the wording in these lines.

2.	Describe what B16, Eca-109, BCG823, and HeLa cells are in the Cell lines and cell culture part of the Materials and Methods section. For e.g. B16 mouse melanoma cells.

3.	In Figure 3B, the nuclear morphology is hard to discern. However, it is better in the raw file. The authors are encouraged to make an inset within Figure 3B containing a magnified portion of the figure to facilitate easy visualization of the nuclear morphology.
Experimental design: In this manuscript titled “Chemical composition, antioxidant and antitumor activities of sub-fractions of wild and cultivated Pleurotus ferulae ethanol extracts”, Yang. Y et al. compare the bioactivities of organic extracts of wild and cultivated P. ferulae edible mushroom. This manuscript builds on earlier evidence that P. ferulae extracts possess antioxidant and antitumor properties (Alam N et al 2012, Wang W et al 2014, Choi DB et al 2004). In particular, they analyze the polysaccharide, polyphenol, and flavonoid composition in P. ferulae ethanol extracts and in the petroleum ether, ethyl acetate, and n-butanol sub-fractions of the ethanol extracts. Furthermore, the authors measure the antioxidant potential of the extracts using DPPH scavenging and reducing power assays. Ethanol extracts and sub-fractions of wild and cultivated P. ferulae display a dose-dependent increase in antioxidant activity. The extracts are then tested for antitumor activity on mouse and human tumor cell lines. Higher concentrations of wild P. ferulae extracts show more dramatic effect on cell viability than cultivated P. ferulae extracts, particularly in Eca-109 and HeLa cells. The authors provide evidence that P. ferulae extracts potentiate the release of reactive oxygen species (ROS) in the tumor cells that triggers cell death.

Some noteworthy aspects of the study:
1.	Use of Cisplatin and DMSO as controls in cell viability experiments.
2.	Detailed descriptions of experimental methods.
3.	Analysis of apoptotic and necrotic cell populations in treated cells.
4.	GC-MS analysis to determine chemical composition of petroleum ether sub-fractions of wild and cultivated P. ferulae ethanol extracts.
Validity of the findings: However, the authors need to address the following concerns to publish the manuscript:

1.	According to the Preparation of wild and cultivated P. ferulae extractions in the Materials and Methods section, ethanol extracts were evaporated to remove ethanol and then further treated with petroleum ether, ethyl acetate, and n-butanol. It is not clear whether these solvents were also evaporated prior to reconstitution in DMSO. In addition, line 74 should mention that the extracts were dissolved in 100 percent DMSO.
        a.	If the solvents petroleum ether, ethyl acetate, and n-butanol were not evaporated, then it must be shown either by experimental evidence or literature citation whether these solvents have any effect on DPPH scavenging, reducing power, and cell viability.

2.	In the Results section, Polysaccharide, polyphenol and flavonoid contents of PFEE-W and -C and subfractions, the authors state on line 171 “Polysaccharide content in Pe-W is lower than that in Pe-C and Ea-W and Ba-W are similar with Ea-C and Ba-C, respectively.”

The polysaccharide content in Pe-W is lowest compared to all other sub-fractions. The statement is not clear and does not correlate with the numbers on Table 1.

3.	In Figure 1, DPPH scavenging and reducing power assays, there is no reference positive control (a known compound with antioxidant properties). It is hard to assess the importance of these results in the absence of a reference. The authors must either provide experimental data using a reference positive control or draw comparison to other compounds that have antioxidant properties in the Discussion section.

4.	The authors analyze the differences in fatty acid content of petroleum ether sub-fractions of wild and cultivated P. ferulae using GC-MS and claim that the differences in the constituents may cause different antitumor activities. The authors need to explain this further in the Discussion citing studies that show antitumor activity of compounds containing various fatty acids.

5.	In Figure 4A, the authors show data that Ea-W and Ea-C sub-fractions can induce ROS production in HeLa cells. The control used in these experiments is 0.4% DMSO. DMSO itself appears to induce ROS production at 8h, 12h, and 24 timepoints. The authors must add untreated cells as an additional control to show the baseline level of ROS at these timepoints. The authors are encouraged to find other literature where dynamic changes in ROS has been reported and include in the Discussion section.
Additional comments: No additional comments


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

Input:
Title: CHEMICAL COMPOSITION, ANTIOXIDANT AND ANTITUMOR ACTIVITIES OF SUB-FRACTIONS OF WILD AND CULTIVATED PLEUROTUS FERULAE ETHANOL EXTRACTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Reporting has improved with the addition of correct descriptions of the experimental methods and reagents as well as detailed discussion of data.
Experimental design: Minor issues:

The authors have carried out additional experiments to include ascorbic acid as a positive control for data presented in Figure 1. However, the authors have not discussed how the Pleurotus ferulae extracts compare to the positive control. In addition, Figure 1 legend needs to include a key for "Vc".
Validity of the findings: The authors' inclusion of positive and negative controls have strengthened the validity of the results.
Additional comments: The authors have adequately addressed reviewers' concerns.

The manuscript is suitable for publication upon correction of minor issues.


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: AN EXPLORATION OF THE EFFECT OF PROPRIOCEPTIVE KNEE BRACING ON BIOMECHANICS DURING A BADMINTON LUNGE TO THE NET, AND THE IMPLICATIONS TO INJURY MECHANISMS
Review round: 1
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: LINE 48: When you first write abbreviations, you must describe it.
LINE 72-74: It would have been better to attach the photo the article” Reflective markers were placed on the shank and thigh using the Calibrated Anatomical System Technique” and” To enable the fitting of the brace, the thigh and shank marker clusters were placed above and below the brace respectively”
LINE 78: Please upload the photo ”knee braced”
LINE 81: How is “standardised warm-up”?
LINE 83: It would have been better to have clear pictures of how to do the test.
LINE159: Please list numerous studies for ”This has been previously seen in several studies during step descent (Selfe et al., 2011)”
 Table1: What does HS mean? Please write the full term.
 It is better to refer to the results of Table 2 and 3 for more positive brace effect
The English language 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: AN EXPLORATION OF THE EFFECT OF PROPRIOCEPTIVE KNEE BRACING ON BIOMECHANICS DURING A BADMINTON LUNGE TO THE NET, AND THE IMPLICATIONS TO INJURY MECHANISMS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Meet journal's standards.
Experimental design: Experimental design is acceptable, with sufficient detail to replicate.
Validity of the findings: Data is robust, statistically sound, & controlled.
Additional comments: 1. In the introduction part, second paragraph, line 42-43, “This may provide important information for players and coaches to assist in the decision making in training when considering shot performance and return to sports post injury.” What does “this” refer to? I suggest to combine this sentence with the previous one, for instance, “…which may give a greater understanding of injury risk factors and provide important information for players and coaches to assist in the decision making in training when considering shot performance and return to sports post injury.”

2. In the introduction part, third paragraph, line 48, what does “PFP” refer to? If this is the first abbreviation, please mark the full name before giving the abbreviation.

3. In the introduction part, third paragraph, line 49, “knee abduction moments have also been shown to be an important contributor to symptoms.” Grammar mistakes exist in this sentence, which should be replaced by “knee abduction moment has also been shown to be an important contributor to symptoms.” Please modify.

4. In the discussion part, third paragraph, line 154-155, “This would indicate a reduction the medial compartment contact force.” grammar mistakes exist in this sentence, which should be replaced by “This would indicate a reduction in the medial compartment contact force.” Please modify.

5. In the discussion part, fourth paragraph, line 164-165, “…whereas movement control and strategy during the lunge tasks was affected.” Grammar mistakes exist in this sentence, which should be replaced by “…whereas movement control and strategy during the lunge tasks were affected.” Please modify.

6. In the discussion part, last paragraph, line 171-172, “it is not possible the extrapolate these findings to elite players.” Grammar mistakes exist in this sentence, which should be replaced by “it is not possible to extrapolate these

findings to elite players.” Please modify.

7. Grammar mistakes were not mentioned completely, please pay more attention and check it carefully throughout this manuscript.

8. Please pay more attention on the format of this manuscript and make revision based on the journal guidelines.


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: AN EXPLORATION OF THE EFFECT OF PROPRIOCEPTIVE KNEE BRACING ON BIOMECHANICS DURING A BADMINTON LUNGE TO THE NET, AND THE IMPLICATIONS TO INJURY MECHANISMS
Review round: 1
Reviewer: 3

Output:
Basic reporting: manuscript is reasonably well written - language. but some parts are missing
Experimental design: is ok, power analysis - sample size estimation needed and reliability of experimental protocol and analysis is required.
Validity of the findings: I have some concerns - see specific comments
Additional comments: General comments
In this manuscript, a study investigating the effects of a knee brace and fatigue on the biomechanics of a forward lunge to the net in badminton is presented. The study design is rather complex as three factors (with and without brace, pre and post fatigue and backhand and forehand lunge) were to be investigated by testing 16 subjects repeatedly while fatiguing them during each of two sessions. The paper as such is well written and the study itself is relevant and might add to the knowledge in regard to fatigue effects and knee injury potential in badminton. There are, however, major concerns in regard to the methods section which appears incomplete. In particular, there is no mention, reference or discussion on the reliability of the marker placement protocol which are crucial factors. I also feel that the results section is quite heavy and not easy to digest. Too many mean values are presented in the tables. While not being wrong as such it becomes very tedious to read. I would also prefer to see some moment curves instead, to highlight the time curves where significant differences occur. Rather stick to a presentation with main and significant findings (as indicated above – in relation to specific hypotheses), like in Tables 3 and 4.  The discussion appears to contain some misconceptions and remains on a very general level. This needs to be substantially improved.

Specific comments
Abstract (first time listed in the review document):
Line 5	specify the type of post hoc test used.
Line 10	… showed significantly lower …
Third last 	From the wording it is not immediately clear if the clinical assessments were done at pre and post or only once each session, reword!
Second last	what it meant by an ‘improved loading pattern’ needs to be defined.

Introduction:
Line 48	Acronym PFP needs to be introduced.
Line 56	Aim is not quite clear: you now list fatigue, lunge strategy and knee bracing as factors. It should read ‘lunge direction, forehand and backhand’. Further, you list a number of parameters to be looked at while some clear hypotheses would make it easier for the reader but also yourselves how to present results and so on. Provide specific hypotheses which can be tested statistically.
Methods:
Line 72	Spelling of several authors is erroneous, correct here and in the reference list.
Line 74 	More information is needed how the data were further processed in visual 3D, e.g., which model was used or did you use a custom model? How did you extract peak values etc.? How reliable is your experiment – the calculations? This is needed to assess if the differences are, e.g., clinically relevant.
Line 75	Why these and why different cut-off frequencies. Provide a reason or explain how you estimated these?
Line 78	… the laboratory on two occasions …
Line 89	Following the fatigue protocol, participants …
Line 90/91	Neither here nor in the abstract it is (grammatically) 100% clear if the additional tests were only carried out once per session or pre and post fatigue. If the latter (which your results indicate), it needs to be indicated how long it took from end of fatiguing protocol to the tests, also how long until the 5 lunges were performed. A definition of fatigue is missing. Did you test for fatigue, e.g., force measured, perception tests? Also many more details are needed. Did the markers stay on the subjects for the whole session? Did you control if the moved or fell off  (not sure if they did but it may happen due to sweating - please comment)?
Line 98	Referring to a comment above: I presume that SPSS was only used to statistically compare results (hence the title of this paragraph should rather read 'statistical analysis', while the data analysis in visual 3D should be more detailed.
Results:
Line 108	you only report main effects but shouldn’t you first report interactions? Or were none of them significant?
More general	I don’t think it is pleasant to be simply referred to the tables. I gather it would be clearer to concentrate the tables to the hypothesis-derived numbers or merely list significant findings. You can then indicate in the text %-differences or F statistics as recommended in many other journals. – The results appear comprehensive but as if you compared almost everything possible and ran statistics to check where you might find differences. Apart from the fact that you inflate the number of comparisons and with that the chance for type 1 errors you should also present a sample size estimation of sorts.
Discussion:
Line 127 	… net moments … ??
Line 143, end	‘… plane’ or better ‘… in the sagittal and transverse planes’.
Line 146	This thought appears unrelated. I don't think that a lunge would lead to hyperextension of the knee, also you mention that there is an increase knee flexion, that would be a contradiction in itself. So how is this possible?
Try to rework the discussion much more specifically. What differences did you identify and what do they mean in regard to joint loading etc.?
Line 149	… five times higher risk for ACL injury.
Line 151	There seems to be a misconception. An extension moment does not imply a hyperextension of the knee.
Line 154	… reduction of the medial compartment …
Line 157	… mechanical realignment …
Line 171, end 	… not possible to extrapolate …
Line 177	Finally, proprioceptive …
Line 178	This is a somewhat long shot: You investigated the effect of braces, how does this play into how to develop training strategies?


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: AN EXPLORATION OF THE EFFECT OF PROPRIOCEPTIVE KNEE BRACING ON BIOMECHANICS DURING A BADMINTON LUNGE TO THE NET, AND THE IMPLICATIONS TO INJURY MECHANISMS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.
Experimental design: Original primary research within Aims and Scope of the journal.
Validity of the findings: Conclusion are well stated, linked to original research question & limited to supporting results.
Additional comments: Revised version is suitable to publish.


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: FEEDING RESPONSE OF THE TROPICAL COPEPOD ACARTIA ERYTHRAEA TO SHORT-TERM THERMAL STRESS: MORE ANIMAL-DERIVED FOOD WAS CONSUMED
Review round: 1
Reviewer: 1

Output:
Basic reporting: This submission is well written and adequately referenced. I do, however, recommend restructuring the article to make it more clear. For instance, it took me a while to understand where each calculation was coming from: I recommend more clearly separating the analysis of feeding on the basis of cell counts from those based on molecular work. This will not require a large re-write, just the insertion of some sub-headings.
Experimental design: The preparation procedures described subsequent to Line 149 is not very clear to me. Do I understand that the authors took four volumes of water and condensed that down on to a 0.47um filter to concentrate natural seston and plankton, then resuspended that material into a volume of filtered water equivalent to one of the original four volumes? If so, while the concentration procedure may not affect diatoms and detritus etc, it would gravely affect more delicate protozoans such as ciliates, ameboids and euglenoids, which would surely decompose once hitting the filter.  By what factor is the particulate material in natural water concentrated?
Moreover, the authors introduce 50 Acartia erythraea into an experimental container of only 200mls, which corresponds to a concentration of 250,000 per cubic metre. It is not stated whether only adults were used in the experiment, but since the mesh size used in collection was 505um it is likely that only late copepodites and adults were available. In any case, this represents a massively inflated concentration of copepods from natural field densities (which were?) and could have implications for the interpretation of the results.
Otherwise, the protocols adopted for the experiments were perfectly standard.
At line 159 it is stated that a 50ml water sample was collected and preserved with Lugols, but there is no information on how the plankton were enumerated.
I was surprised to read that copepods preserved in Lugols were used for molecular analysis – I would have thought that the iodine would have compromised the extraction of nucleic acids. I’d like to see this clarified in the text – though I accept that this paper seems to be part of a body of work by the senior author in which these issues may have been addressed. That said, I’d say this contribution ought to stand on its own.

At line 228 the authors introduce a parameter called the Chao-1 index. I hadn’t heard of this before, and all the diversity indices used ought to be described in the Methods section.
Validity of the findings: I was attracted to review this manuscript because I found the principal finding – the trend toward carnivory in warmer temperatures – to be very interesting in the broader context of a warming world. Just how much the effluent from power stations affects local plankton communities depends on the oceanography of the discharge area. If tides and currents are appreciable, I would think that such effects would be extremely localized.

That said, the findings are of general interest even though they are placed in the context of local effects, and the molecular analyses of prey taxa are broadly applicable and very welcome.
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: FEEDING RESPONSE OF THE TROPICAL COPEPOD ACARTIA ERYTHRAEA TO SHORT-TERM THERMAL STRESS: MORE ANIMAL-DERIVED FOOD WAS CONSUMED
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, this manuscript does provide a clear hypothesis and description of how it was tested, but lacks a distinct amount of detail in key sections of the manuscript.

 For example, the introduction lacks a sense of depth and clarity as to the importance of short-term increases in water temperatures on the physiology of copepods in particular. Changes in water temperature ay affect copepod physiological processes directly, by altering metabolic rates and energetic demands, or indirectly through alterations in the availability of prey. On Lines 85-87 the authors state “Elevation of temperature within a moderate range could promote the growth and metabolic rates of copepod, but if the temperatures exceed this range, the physiological state of copepods might be changed.” However, this statement needs to be expanded upon, and there needs to be a more explicit link drawn between increases in water temperature and the specific physiological changes that may occur, how they may occur, and why. For example, why are the authors looking specifically at feeding responses? Is this due to the presumed direct effects of temperature on the physiology of copepods, or indirectly through dietary switching as suggested in Lines 93-103. While both of these are clearly important processes, each should be explained in more detail, and a clearer separation of the two is necessary.

Furthermore, more context about thermal effluent and its effects on surrounding environments is needed. How prevalent are coastal power plants in tropical areas? How often does thermal discharge occur, and how drastically is the water temperature increased? How fast does this change occur, and how long does the water stay elevated? These are important details that will help inform the reader not only the magnitude of the problem at hand, but also about the validity of the experimental design. This could be emphasized in the discussion as well, since it was highlighted at the forefront of the manuscript. What are the implications of potential dietary shifts, given the fact that most organisms are facing not only increased temperatures due to global climate change, but also due to anthropogenic impacts?

Lastly, while Figure 2 is clear and demonstrates the authors’ data well, Figure 1 is slightly difficult to interpret, and somewhat cumbersome.
Experimental design: Generally speaking, details are missing from the description of the experimental design that may be important to the reader. For example, the authors state on Lines 132-133 that mature females were separated out from the samples, but then do not state if they tested females, males, or a combination of both.

It is also unclear if the authors ensured equal concentration of prey cells in each treatment, as the concentration of prey could influence feeding rate as well. While this did not bias results, since the authors calculated grazing rate using the initial cell count of each vial individually, the density of prey could influence copepod behavior in the absence of a temperature effect. If this was indeed the case, and I have misinterpreted the authors, I apologize, but perhaps this is an indication that it should be made clearer.

Similarly, the authors report Simpson, Shannon, and Chao indices, yet make no mention of these in the methods section.
Validity of the findings: Generally, the interpretation of the data by the authors is scientifically sound and well supported. Indeed, the authors provide great detail for their interpretations of the results, and are clear in the purported mechanisms that may have contributed to what was quantified. Overall, I found this section of the paper most pleasing to read.
Additional comments: While the writing does not inhibit the understanding of the reader, this manuscript should be reviewed and edited substantially by a native English speaker to correct many of the grammatical and structural errors of the text. This would improve the flow of the text, and might help mediate any confusion on the readers’ part.


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: FIRST STEPS TOWARDS MITOCHONDRIAL PAN-GENOMICS: DETAILED ANALYSIS OF FUSARIUM GRAMINEARUM MITOGENOMES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The approach and methods are well described and the work is thoroughly conducted.  However, the narrative of the manuscript is often difficult to follow and disjointed in certain places.
Experimental design: The manuscript reports the use of a pan-genomic approach to analyze the mitochondrial genomes of 24 strains of Fusarium graminearum. The use of pan-genomes is novel approach for the analysis of mitochondrial genomes, and the manuscripts describes its effectiveness in assessing genome variability and the use of pooled sequencing.
Validity of the findings: The pan-genomic approach and use pools data sets has utility in the characterization of mitochondrial genomes, especially with the increase in NGS data. The sections of the paper that describe these approaches are well done, although some details of the mapping, coverage analysis, and coverage gaps are difficult to follow and would benefit by having additional descriptive statements.  In contrast, the sections of the manuscript that focus on the use mitochondrial regions as barcodes for fungal populations fails to provide appropriate citations and lacks convincing evidence to support the claim that introns could be used to track the spread of a specific fungal population. The descriptions of group I intron mobility, in general, lacked important details and glossed over well-established concepts. The variation of group I intron content in fungal mitochondrial genomes is well documented, and there are numerous reports in the literature that high rates of transmission among fungal and plant mitogenomes. Thus the idea that there are ‘introns unique to a single population’ has not, to my knowledge, been substantiated. With the demonstrated ability of mobile group I introns to spread horizontally among strains of different vegetative compatibilities groups and even different genera (and on rare occasions, different kingdoms), it cannot be presumed that a natural population would have a specific set mitochondrial introns, let alone that an individual intron would be an accurate marker to track their spread. The manuscript would be greatly enhanced if it were primarily focused on the pros and cons of the pan-genome approach to analyze mitogenomes and potential use of pooled DNAs and remove or minimize the sections that discuss the possible use of introns as markers for a population.
Although the authors point out the limitations of using pooled data sets to identify SNPs, short indels, and linkage between markers, they underestimate the importance of retaining this information in understanding the origin and spread of specific mobile (and immobile) introns. The influence of mobile introns on genetic diversity of fungal mitogenomes goes well beyond their presence or absence. The co-conversion of SNPs flanking the insertion site associated with intron homing is a major contributor to the mtDNA diversity. The polymorphisms flanking intron insertion sites are generated via homologous recombination between different haplotypes or by error-prone repair mechanisms. The use of pooled data sets would not only have limited utility in characterizing these genetic differences, it would also potentially complicate the identification of the extant origin of a mobile intron.
Additional comments: Minor Comments
Abstract:
The term ‘recombination’ needs be defined. The authors seem to apply the term to describe recombination between mitochondrial genomes, and make no mention that homologous recombination is inherent to intron mobility. The term needs to specifically defined throughout.
The sentence stating that the ‘mitogenome of F. graminearum is recombining… indicating that the mitogenome recombination is a common phenomenon in Fusarium” is unnecessary. I cannot think of a fungal genus (nor any other eukaryote outside of metazoans) in which mitogenomes are not recombining, and this statement makes it seem as if this is a something in question, which it is not.
Line 46: Other citations to studies in which mitochondrial genomes are used to monitor species distribution are need. The Harrion, 1989 reference refers to animal genomes that have notoriously low levels of recombination.  In addition, it would be worthwhile to point out the major challenges in applying one approach to all mitogonomes due the exceptional number of differences in size, diversity, and mechanisms of variation across kingdoms.
Line 50: The sentence is misleading; successful genotypes reflect the compatibility of nuclear and mitochondrial genomes, not just nuclear genotypes.
Line 58: The sentence is misleading; the use of mito genes as barcodes have mostly been abandoned in phylogenetic studies of fungal species.
Lines 194-196: Linear Model. It doesn’t seem necessary to include a model to quantify something when ‘percent intron’ would suffice (although, admittedly, this is first instance this reviewer has seen this metric applied).
Line 254: “However, all the strains had a unique mitochondrial genome sequence”. Vague statement. Is this referring to a single region?
Lines 256, 260 & 263: “Supplementary Table ” # missing in parentheses.
Lines 263-267: The explanation is too simplistic, and the statements should be removed or expanded to include a more complete description of the evolutionary events that could have produced the variability. Again, the meaning of the term ‘recombination’ is inadequately described. Intron homing could affect some intergenic regions, as the extent of flanking site co-conversion can exceed the length of exons.
Lines 367-377: I found this section to be difficult to follow. Why would pooling data reduce coverage of mito or nuclear copies? Why invoke PCR methods? How would NUMTs affect the results? The next sentence is very convoluted and difficult to understand. The final sentence of the paragraph also needs further explanation.
Lines 386- 393: See previous comment concerning the ubiquitous nature of recombination in fungal mitogenomes.
Lines 393-395: See previous comments and use of introns to track strains or populations
Lines 395-396: What do you mean by ‘tagged’?
Lines 396-397: This seems to imply that you would have to track a population over time to see the appearance of a new intron. Not sure how this would be accomplished.
Lines 400-405: What is being described is a ‘twintron’. These have been reported in numerous lineages in papers dating back to the early 1990s. Modify description and add citations.
Lines 406-407: This sentence should be removed, unless it can be further substantiated.
Lines 409-419: The Conclusion section should be re-written to focus on the novel approaches used to analyze the mitogenome of F. graminearum. The statements about the use of introns to track populations should be removed.


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: FIRST STEPS TOWARDS MITOCHONDRIAL PAN-GENOMICS: DETAILED ANALYSIS OF FUSARIUM GRAMINEARUM MITOGENOMES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors present a well written document in clear and easily understandable English. A sufficient amount of background information is provided in the Introduction, which also clarifies the context and scope of the study. Some small corrections are suggested in the attachment.
Some minor work is needed in the reference list. Some journals are abbreviated, others not, and some journals are both abbreviated and unabbreviated.
The document conforms to the traditional and professional structure of a scientific article, and the tables and figures are of publication quality. All raw data have been made available on public databases.
Experimental design: The sampling (i.e., isolates) was sufficient to support the assertions in the manuscript, and so were the in silico analyses.
Validity of the findings: The findings are valid for a selected assemblage of isolates from Fusarium graminearum. However, the results are exciting and should in be replicated for other Fusarium species. The potential use of mt-introns to track the movement of fungal pathogens around the world is a particularly exciting suggestion, and other groups should start investigating the effectivity and cost-effectiveness of such an approach.
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: FIRST STEPS TOWARDS MITOCHONDRIAL PAN-GENOMICS: DETAILED ANALYSIS OF FUSARIUM GRAMINEARUM MITOGENOMES
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: To the editor of PeerJ
The manuscript “First steps towards mitochondrial pan-genomics: Detailed analysis of Fusarium graminearum mitogenomes” is a research paper that presents an improved version of the F. graminearum mitochondrial genomes and a paper that describes the use of pooled sequencing in assessing sequence diversity. The manuscript included sufficient background information and discussion however a minor revision is suggested before the manuscript can be published
L58: please clarify how NGS and new analysis methods have resolved the issue of cox1 and barcoding? Yes, NGS allow for more genomes to be sequenced and for populations to be analysed, however not sure if this will replace DNA barcoding?
L264-267: recombination should be a separate section and should be discussed in more detail.
Discussion general comments: In order to improve the overall flow of the discussion, the authors should follow the layout of the conclusion. Therefore, start with a discussion about the genome, the introns, recombination and all, and then have a section that describes the sequencing methods.


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: MORPHOLOGICAL CHARACTERIZATION AND STAGING OF BUMBLE BEE PUPAE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors are native English speakers, so, for me, the English is clear and professional, making easy to understand and read the text. They cited the best references in the area and the Introduction and Discussion proved enough background.

**The figures and schemes present high quality, but some figures need scales.

**The research is a master peace of Bombus pupal development, my unique concern is when the authors compare the development of queens of Apis meliffera with Bombus species. I recommend to eliminate the statement:  " The rationale for why a honey bee queen would take less time is/ 557 interesting to consider, and perhaps could be due to their caste being determined more by diet/ 558 quality, differential selection for developmental rate by caste, or alternative physiological /559 mechanisms for generating castes." This is not the objective of the research and the authors can't speculate that.
Experimental design: The research is a master peace of the morphological aspects of the pupal development in Bombus and presents high potential to be used for the scientific community not only for genetic studies, but for toxicological ones as well.
Creative methodology, sufficient detailed, providing enough information for replication.
Validity of the findings: The research is novelty and presents direct impact on many areas, as in molecular regulation of development and developmental toxicology. These bees are serious under threat, not only in USA (B. affinis) as in worldwide. The detailed knowledge of the larval and pupal development of this genus is extremely important for monitoring abnormal conditions and to understand the molecular mechanisms of Bombus development.
Additional comments: Nothing do add.


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: MORPHOLOGICAL CHARACTERIZATION AND STAGING OF BUMBLE BEE PUPAE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Robust keys are available in a range of bumble bee species that are often used for studying the morphology of the adult queens, workers, and males. While less is known about the larvae, there is still some literature available on the morphology and development of larvae (with most studies being limited to Bombus terrestris). These have already been used to some degree to study development and changes in gene expression in the larvae of a handful of bumble bee species. However, very little is known about the changes in morphology, and the timing and progression of development in bumble bee pupae, and this study is an attempt to bridge that gap in the literature. In my view the authors have done this very successfully, and as such I would like to recommend that this article be accepted for publication, with the caveat that they consider a few relatively minor revisions.

In general, the written English is of a high standard, and was very easy to follow throughout. However, there were one or two minor grammatical mistakes that the authors should correct before their final submission. I noticed a few sections where the authors confuse tenses, for example on line 86 the sentence ‘…pre-pupa shed the larval cuticle…’ should read ‘…pre-pupa sheds the larval cuticle….’ While this may seem like a very minor issue, I noticed several instances where the authors make similar mistakes (other examples include lines 110, where ‘is’ should be replaced with ‘are’, line 126 which should read ‘…develop for four days…’, line 246, which should read ‘…on the coxae and trochanters…’, line 434, which should read ‘…there are also significant differences between…’, line 537 should read ‘… larger workers taking longer to develop than smaller ones...’, line 564 should read ‘that override the effects of size’) I would therefore advise a very careful rereading of the text to remove any similar errors.

In addition, they may need to clarify one or two sections, for example on line 151 I was unclear what they meant by ‘the same source of fresh pollen’. If they meant that the pollen arrived with the colony, or that they bought the pollen from Koppert, then they should state this more clearly.

The authors provide an extensive list of citations, and mostly cite the relevant literature in the field, however they very occasionally make points without providing relevant examples. Therefore I would like to see a few further citations to back up their points on lines 73 (sentence starting ‘For example,’), 207 (Sentence starting ‘Previous studies…’, those studies need to be cited) and 516 (sentence starting ‘Many studies…’ these studies should also be cited). They should recheck their introduction and discussion to ensure there are no other statements made that would usually require citations.
The structure of the article is fine, and follows a logical order. While they have a large number of figures, and a large proportion of them are photos, these represent the primary data, and are quite usual for a study of this kind. I would encourage the authors to ensure the figures are presented in the correct order because currently figure 7 is first described on line 266 whereas figure 6 is not described until line 367. I suggest that the authors check for any other discrepancies like this and correct them.

On lines 422-429, the authors describe the ranges of pupal durations for each of the described castes. While they state that the means and standard errors are provided on table S1, I would prefer them to also include the means and standard errors for each of the values described on line 424. Furthermore, they need to ensure that they are consistent in the units they use. For example, in the text in this section they refer to the timings of pupal duration in days, whereas in table S1 they use a different metric (I presume they use hours in the table, but it does not state this, so that also needs to be corrected). Some of their statistical reporting could be improved in this section too, for example on line 426 they say that pupal duration is significantly longer at the lower temperature of 29, but they don’t describe the magnitude of the effect (or range of effects). I didn’t find the results presented on lines 427-429 very clear so I think they should re-write that section, expand on any of the results presented if necessary, and should reference the appropriate statistical tests in support of each of their conclusions. Likewise for lines 430-439, some of the results presented don’t reference the appropriate statistical justification, or the figures or the tables, and the authors need to ensure that the language used should present these results as clearly as possible.

Finally, the sentence starting on line 563 did not make sense to me and I think it needs to be re-written to make their point more clear. I should also say that despite the inference that body size is ‘driving’ development time, I think a plausible argument could easily be the converse of this, i.e. that development time is actually driving changes in body size, or that a third factor is driving the changes in those two variables.
Experimental design: The methods section was generally highly detailed, and I felt like they provided sufficient information for the study to be easily replicated. My understanding of the methods is that they cut open the cocoons, then follow the developmental progress of all the individual that were found to be either silk-spinning larvae or pre-pupae (Line 155-163). However, it is not clear what (if anything) they do when they first open cocoons that already contain pupae (including late-stage pupae). My guess is that these were ignored or discarded, but this needs to be stated clearly, together with the sample sizes which include the numbers of cocoons opened, the numbers that were monitored from larvae/pre-pupae and the numbers that already contained pupae. On the other hand, if they used the cocoons that already contained pupae for the experiment, then they need to provide more detail about the sample sizes of pupae that were monitored from different starting points, and how they were able to distinguish pupae that were different ages.

In the sentence starting ‘Based on measurement…’ on line 221, the authors categorize the adult workers and males as small, medium, or large depending on the ITS. It’s not clear whether those size ranges mean anything biologically, or were based on the mean and ranges of the adults that they sampled, or were just arbitrarily chosen. It would be helpful if they could state this, and/or provide literature citations to back up why they classified adults into those particular size ranges. This is important because many of their results and conclusions were dependent on the sizes of the adult individuals that they were measuring. In addition, my guess is that they determined those size categories retroactively, once the pupae they were monitoring had eclosed as adults. Once they had eclosed, the authors then analysed data separately for pupae that were known to develop into different sized males and workers. If they did categorize them this way then I would like them to directly state this (or otherwise make it clearer if I have interpreted this wrongly).
Validity of the findings: Overall, I felt like the data were robust, and will inevitably be of wider use to the field in general. They were able to state their main conclusions, and these were well supported by their results. During some sections they speculate where their results might be more broadly applicable to researchers studying other bumble bee taxa, but are clear where their data might have limitations, for example on line 473 where they state that these data might only be applicable to Pyrobombus species.

I found the section from lines 515-520 to be unnecessary. They state (without citation) that many studies use the production of, and eclosion from, cocoons to define pupation and that this is a problem because pupation doesn’t occur until sometime after the cocoons are made, and ends some time before the new adults eclose. However, the authors do not provide any new data to show when the precise timing of pupation should be defined, and do not explicitly state any firm conclusions as to what future studies should do to avoid the issues they claim are problematic. For these reasons I would recommend they simply remove that paragraph.
Additional comments: Overall I thought the study was good, and assuming the authors consider the points and suggestions that I have raised, it should easily meet the criteria for publication in PeerJ.

I had a brief comment about one of findings of the study. On line 556 the authors speculate on the reasons why larger bodied honeybee queens develop more quickly than workers, whereas in bumble bees they found that it was the other way around. One likely reason for this, which they elude to but don’t explicitly state, is that honey bee queens are under immense selection pressure to develop quickly, because the first queen to emerge will usually take-over the colony, and then kill any rival queen-destined larvae and pupae while they are still developing in their cells. There is no analogous process to this in bumble bees, as queens do not take-over the colony, and simply disperse once they have eclosed. Therefore, there is no reason why bumble bee queens should develop more quickly than workers which is why bumble bees have a more simple linear relationship between body size and development time compared to honey bees.


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: MORPHOLOGICAL CHARACTERIZATION AND STAGING OF BUMBLE BEE PUPAE
Review round: 1
Reviewer: 3

Output:
Basic reporting: Tian and Hines investigated morphological changes during pupal development of bumble bees and provided criteria for staging. The English language is clear and professional. The manuscript is very well structured, including high quality supporting files (figures, tables and so on). Cited references are OK.
Experimental design: The study is original because it improves the current staging criteria applied for Bombus pupae using more morphological details, thus filling some scientific gaps. The work is technically and ethically appropriate. The methodology and experiments are well described and designed, respectively.
Validity of the findings: Results, statistics, discussions and conclusions are relevant and robust.
Additional comments: Minor suggestions:

- Change title to: Staging of bumble bee pupae based on morphological criteria

- Replace Bumble bees (Hymenoptera: Bombus) by Bumble bees (Hymenoptera: Bombinae)

- Replace "de F Michelette E, and Soares A" by "Michelette EF, and Soares AEE"


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 DIETARY SUPPLEMENTATION WITH LACTOBACILLUS ACIDOPHILUS ON THE PERFORMANCE, INTESTINAL PHYSICAL BARRIER FUNCTION, AND THE EXPRESSION OF NOD-LIKE RECEPTORS IN WEANED PIGLETS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study mainly investigated the effect of dietary supplementation with Lactobacillus acidophilus  on the performance, intestinal physical barrier function, and the expression of NOD-like receptors in weaned piglets. The results are positive.
Experimental design: the author should provide the reference for the bacterial concentration in this study.
Validity of the findings: Conclusion are well stated, linked to original research question & limited to supporting results.
Additional comments: 1. the author should provide the reference for the bacterial concentration in this study.
2. in the table3, average daily weight gain was markedly enhanced, while no difference was noticed in final body weight. Please explain!
3. in fig1, the authors indicated that TGs were tested, while only zo1 and occludin were studed in this study, claudin family also should be incuded. In addition, mRNA in insufficient for investigating TGs as the protein abundances and the distribution in the intestine play a key role in the gut barrier function.
4. for the signalling study, western blot should conducted rather than PCR.


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 DIETARY SUPPLEMENTATION WITH LACTOBACILLUS ACIDOPHILUS ON THE PERFORMANCE, INTESTINAL PHYSICAL BARRIER FUNCTION, AND THE EXPRESSION OF NOD-LIKE RECEPTORS IN WEANED PIGLETS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Some English language corrections are required. For example: (Line 198) “Weaning piglets will appear gastrointestinal physiological disorders….” The meaning is unclear and needs clarification
Intro and background show clear context, and is referenced with recent, relevant publications.
The structure conforms to PeerJ
Figures are relevant, high quality, and described. Labelling in the legends is confusing in respect to the replication. It was thought 8 animals were used in each group therefore n=8 and not n=3.
Raw data is supplied, but is incomplete. The raw data files relating to the qPCR results are either incomplete, or inaccurate. For example: in this study 16 animals were used with samples being taken from two regions (jejunum and ileum) of the intestine. For each tissue region, the expression of 10 genes was examined in triplicate by qPCR. Therefore, it would have been expected to find in the raw data files information appertaining to this. 10 genes, each measured in triplicate = 30 values. Multiply this by the 16 animals tested = 480 values per tissue resulting in a total of 960 values. However, all that is supplied in the raw data file is information relating to three technical replicates of the same sample and therefore n=1.
Experimental design: Original primary research with clearly defined research question. The knowledge gap has been identified and addressed. The study has sufficient technical replication (n=8 animals per group), and was conducted ethically.
Methods used require additional information to be replicated and also clarity is required. For example: (Line 125) “All piglets were weighed on day 14”. Unsure if this means day 14 of age, or post weaning, or something else. Please clarify.
For cDNA production and qPCR methodology the thermal profile used has not been described. It is also unclear whether SYBR or probes were used. Additionally, the reference gene used for the qPCR analysis has not been identified. It could be assumed that β-actin was used, but this needs to be clarified.
Validity of the findings: The results are confusing in the context of the number of replicates used. For example lines 116-119 states “sixteen crossbred healthy piglets were divided into 2 groups into eight replicate pens (n=8 each group)”. In line 160 for the qPCR it mentions “three replicates were performed for each reaction and results expressed as 2-ΔΔCT values”. This indicates technical replication of the same sample (n=1) and therefore intra-variation only. This only provides evidence on the ability to pipette accurately rather than any variation that may exist between individual animals within the groups. This also suggests that tissue from only a single animal from each group was used for downstream qPCR analysis and comparison. If this was indeed the case, how was it decided which animals were used for the analysis?
The conclusion is linked to the original research question, and is supported by the results provided. However, considering that the results are unclear it is not possible to determine how accurate this conclusion is.
Additional comments: Lactobacillus acidophilus is clearly beneficial in enhancing the growth performance of weaned piglets and reducing serum diamine oxidase activity. This work by Wang and colleagues provides evidence that an L. acidophilus strain isolated from pigs has a positive impact on the intestinal epithelial barrier by inducing the expression of specific TJ genes, and inhibiting the expression of genes of major signalling pathways. Overall the study is of a good design, with some interesting results obtained. I have some comments for further improvement of the manuscript.
Major points
1.	In this study a total of 16 piglets were used which were divided into two groups, therefore 8 animals in each group. For the calculations of values for dietary treatments on growth performance (Table 3) and DAO and D-lactate (Table 4), you have clearly indicated that the  values obtained were from all replicate animals in each group (n=8). Unfortunately, how the qPCR data was obtained and used in figures 1 and 2 is not clearly described. In line160 the authors state “three replicates were performed for each reaction and the results expressed as 2-ΔΔCT values”. This indicates technical replication of the same sample (n=1). This also suggests that tissue from only a single animal from each group was used for downstream qPCR analysis and comparison. If this was indeed the case, how was it decided which animals were used for the analysis? Could you please clarify what samples were used and what analysis was undertaken as it is currently unclear?
Minor points
1.	Line 64: Authors described “The occludin protein blocks the cell gap”. It could be suggested to change this to “The occludin protein blocks the gap between cells”
2.	Line 67: Remove “several” from “involved in several immune…”
3.	Line 68: Remove “an” from “making it an important….”
4.	Possibly include information relating to the importance of D-lactate and DAO in the introduction. As it is, there is no explanation as to why DAO was measured and the importance of D-lactate was not mentioned until the discussion
5.	Line 108: The authors describe the “L. acidophilus strain used in this study as being isolated from the excrement of a healthy piglet”.  However, how this bacterium was identified/characterised as L. acidophilus has not been described. If this information has previously been published include a reference, if not briefly explain how this strain was identified as such.
6.	The thermal profile used for the qPCR needs to be described. Also, identify the reference gene used for the analysis.
7.	Line 198: “Weaning piglets will appear gastrointestinal physiological disorders….” The meaning is unclear and needs clarification.
8.	Line 213: Remove “for example” at the end of the sentence


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

Input:
Title: EFFECTS OF DIETARY SUPPLEMENTATION WITH LACTOBACILLUS ACIDOPHILUS ON THE PERFORMANCE, INTESTINAL PHYSICAL BARRIER FUNCTION, AND THE EXPRESSION OF NOD-LIKE RECEPTORS IN WEANED PIGLETS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Some English language corrections are required. For example: (Line 221) “Weaned piglets will appear slow growth, a low feed utilization rate, and gastrointestinal physiological disorders such as diarrhea”. I suggest changing this to “Some weaned piglets have slow growth, a low feed utilisation rate …..”

Intro and background show clear context, and is referenced with recent, relevant publications.
The structure conforms to PeerJ
Figures are relevant, high quality, and described. Labelling in the legends has been clarified and relates back to the main text. Raw data is supplied.
Experimental design: Original primary research with clearly defined research question. The knowledge gap has been identified and addressed. The study has sufficient technical replication (n=8 animals per group), and was conducted ethically.
Methods used have been clarified and could be repeated.
Validity of the findings: The conclusion is linked to the original research question, and is supported by the results provided.
Additional comments: Lactobacillus acidophilus is clearly beneficial in enhancing the growth performance of weaned piglets and reducing serum diamine oxidase activity. This work by Wang and colleagues provides evidence that an L. acidophilus strain isolated from pigs has a positive impact on the intestinal epithelial barrier by inducing the expression of specific TJ genes, and inhibiting the expression of genes of major signalling pathways. Overall the study is of a good design, with some interesting results obtained.


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: SYNTHESIS, CHARACTERIZATION AND ANTIBACTERIAL ACTIVITY OF SILVER NANOPARTICLES USING RHAZYA STRICTA
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comments
Experimental design: no comments
Validity of the findings: no comment
Additional comments: 1. Overall, the manuscript is concise, clear and well written. Introduction provides a good, comprehensive background of the topic that makes the reader a clear understanding of the work. However, the author needs to give some background about previous work regarding synthesis and application of any other metallic nanoparticles using the same plant, Rhazya stricta(if studied), with appropriate references, which may further potentiate the authors work.
2. The methods and experimental techniques used in the study for synthesis and characterizations are generally appropriate for the study.
3. The results obtained are coherent with the objectives defined. However, the author should demonstrate more clearly that the results obtained in current study are consistent with the results found in previous studies (mainly for synthesis section). Discussion regarding other metallic nanoparticles (if any) using same plant in previous studies will be appreciated.
4. References need to be formatted. Within text citation should be according to journal guidelines. Either the abbreviation or full name of the journal should be mentioned in complete references throughout. Moreover, some references include bold text that must be corrected.
The manuscript is recommended for publication if author responds to above comments properly as these issues cannot be overlooked.


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: SYNTHESIS, CHARACTERIZATION AND ANTIBACTERIAL ACTIVITY OF SILVER NANOPARTICLES USING RHAZYA STRICTA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Peer J: Manuscript 28748v1
Manuscript title Synthesis, characterization and antibacterial activity of silver nanoparticles using Rhazya stricta by green synthesis.
The manuscript reports on "Synthesis, characterization and antibacterial activity of silver nanoparticles using Rhazya stricta by green synthesis" looks very interesting and fascinating and will help for further studies related to activities Rhazya stricta by green synthesis. The article looks suitable and I suggest for the acceptance. The idea looks novel and supported very well by experimental results, however, I suggest following minor comments before publication.
1.	Generally, the abstract and the explanatory part of the paper are well presented, however;
The background looks too long please shorten it by modifying other sentence or remove the last sentence. “They are widely used in diagnostics, therapeutic and pharmaceutical industries. Studies have shown potential inhibitory antimicrobial, anti-inflammatory and anti-angiogenesis activities of AgNPs”.
2.	The last sentence of the result should be like a conclusion and should be more concise. Please remove the future prospect from the result section. “AgNPs can be used for enhancing the efficacy and retention time of phytochemicals, however, in-vivo, preclinical and clinical studies are required to completely address its therapeutic potential in various diseases”.
3.	Please remove the repeated words from the following consecutive sentences.
Currently, green synthesis of NPs is gaining significant attention over the physical and chemical methods, because it exhibits greater catalytic activity and reduces the use of costly and lethal chemicals (Ponarulselvam et al., 2012; Verma&Mehata, 2016). NPs synthesized by the green method are highly stable, environment-friendly, biocompatible, cost-effective, less toxic and safe for the diagnostic and therapeutic purpose (Ponarulselvam et al., 2012; Rajasekharreddy& Rani, 2014; Verma&Mehata, 2016).
4.	Please put a space between numbers and units. Sucha 147g etc
5.	Please look for the references and make a uniform style.
Experimental design: Experiments are designed well and materials and methods are described with sufficient detail & information are properly written.
Validity of the findings: The data are meaningful, and backgrounds research literature are clearly described. The conclusion is well drawn for future perspective.
Additional comments: The data are meaningful, and backgrounds research literature are clearly described. The conclusion is well drawn for future perspective.


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: SYNTHESIS, CHARACTERIZATION AND ANTIBACTERIAL ACTIVITY OF SILVER NANOPARTICLES USING RHAZYA STRICTA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Check the general comments
Experimental design: Check the general comments
Validity of the findings: Check the general comments
Additional comments: The manuscript entitled “Synthesis, characterization and antibacterial activity of silver nanoparticles using Rhazya stricta” seems to be interesting. The manuscript is of acceptable quality after minor revisions.
The authors should address the following points before the manuscript is accepted
Generally, the abstract and the explanatory part of the paper are well presented, however;
1.	Currently, the area of nanotechnology is a fresh front line of science and technology that manipulates matter at the nanometer scale (1-100nm change to 1-100 nm) so what is the actual size of the synthesized nanoparticles and how it has been confirmed in this study.
2.	To date, a lot of literature has reported on biological synthesis of Silver nanoparticles using plants as well as microorganisms including bacteria and fungi, so what was the condition of the plant that has been used in the synthesis of NPs.
3.	The author needs to explain the Fourier Transform Infrared Spectroscopy (FTIR) in Materials and Methods.
4.	The author needs to write the trade name and company name as well as dose for cefipime.
5.	How the author choose the concentration for the time dependent effect in this study.
6.	The last sentence should be corrected ‘Future studies are needed to uncover the underlying mechanism of bacteriostatic or bactericidal effects to green synthesized AgNPs’.
7.	In references there is dots before the last name, it is the format of the references or mistakes, author should take care for the uniformity of references.


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: SYNTHESIS, CHARACTERIZATION AND ANTIBACTERIAL ACTIVITY OF SILVER NANOPARTICLES USING RHAZYA STRICTA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Manuscript ha been well revised.
Experimental design: Experimental part is ok now
Validity of the findings: Ok
Additional comments: Acceptable after revision


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: SYNTHESIS, CHARACTERIZATION AND ANTIBACTERIAL ACTIVITY OF SILVER NANOPARTICLES USING RHAZYA STRICTA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article is clear with proper literature reviews of the background knowledge, figures and tables are properly arranged, and results are well discussed.
Experimental design: Experimental section is properly arranged. changes are made according to suggestion.
Validity of the findings: Finding are discussed properly.
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: SYNTHESIS, CHARACTERIZATION AND ANTIBACTERIAL ACTIVITY OF SILVER NANOPARTICLES USING RHAZYA STRICTA
Review round: 2
Reviewer: 3

Output:
Basic reporting: accept
Experimental design: ok
Validity of the findings: ok
Additional comments: accept


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: SYNTHESIS, CHARACTERIZATION AND ANTIBACTERIAL ACTIVITY OF SILVER NANOPARTICLES USING RHAZYA STRICTA
Review round: 2
Reviewer: 4

Output:
Basic reporting: The basic reporting looks good however leaving ample of scope for improvements.
Experimental design: The Experiments are quite pertinent to the topic.
Validity of the findings: The findings seems to be good however the element of novelty is missing. Mere choosing new plant species for the synthesis of silver nanoparticles and reporting antibacterial activity is not worthy of publication.
Additional comments: In the present manuscript the authors have reported synthesis of silver nanoparticles using plant extract of Rhazya stricta and later employed them for the antibacterial studies. Though the authors have satisfactorily addressed the comments of the previous reviewers, still some concerns need to be addressed.
1.	Several papers have been published on the green synthesis of silver nanoparticles, their characterization and their biological effectiveness studied in in vitro models or their dye degradation potential. They generally differ in the choice of plant extract used in the nanoparticle preparation, the bacterial species for antibacterial studies or the dye that is being shown to degrade using nanoparticles. Though the work reported in the manuscript appears technically sound and well written, in my opinion, it does not appear to offer anything more useful than that in the other similar papers except the use of new plant extract. There is an urgent need to move ahead of just reporting another plant/bacteria/ yeast for green synthesis of silver nanoparticles. There should be studies that could report the in vivo effects of these nanoparticles. A simple study using disc diffusion is not sufficient at this time point. Here, it would be worth publishing if the authors could include some studies for deciphering the mechanism by which the silver nanoparticles reported here are exerting the antibacterial effect. Also a look inside the bacterial cells for the morphological or anatomical changes due to exposure to silver nanoparticles would be worth publishing.
2.	Figure 3- the size values shown in the figure are not clear, hence it’s difficult to conclude about the size of nanoparticles.
3.	Figure 5- the peaks corresponding to fcc of silver should be labeled.
4.	Figure 6-Significant peaks should be labeled in the FT IR spectra and if possible they should be overlayed to facilitate understanding.
5.	Figure 8,B,D- all the error bars look same, it’s not clear how many times the experiment was done. The label of y-axis is also missing.
6.	Figure 9-B again the y-axis label is missing and the no. of experiments done is not clear or mentioned.
7.	Please refer these articles in order to improve the presentation of the figures.
a.	Synthesis and in vitro antineoplastic evaluation of silver nanoparticles mediated by Agrimoniae herba extract. Qu D, Sun W, Chen Y, Zhou J, Liu C. Int J Nanomedicine. 2014 Apr 15;9:1871-82. doi: 10.2147/IJN.S58732. eCollection 2014.
b.	Green synthesis of silver nanoparticles using Prosopis juliflora bark extract: reaction optimization, antimicrobial and catalytic activities. Arya G, Kumari RM, Gupta N, Kumar A, Chandra R, Nimesh S. Artif Cells Nanomed Biotechnol. 2018 Aug;46(5):985-993. doi: 10.1080/21691401.2017.1354302. Epub 2017 Jul 18.


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: SYNTHESIS, CHARACTERIZATION AND ANTIBACTERIAL ACTIVITY OF SILVER NANOPARTICLES USING RHAZYA STRICTA
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Though the manuscript has been improved as suggested but the first point remains unaddressed. Even the authors have agreed that this study is incomplete and they are pursuing the experiments in the lab and would be interested in publishing at some later point, with those results.
As per my understanding the present manuscript does not seem to have sufficient novelty for publication and hence I am not in the favor of acceptance for publication. I would encourage the authors to submit the manuscript only after incorporating the results of experiments that deal with deciphering the mechanism of antibacterial effect of the AgNPs.


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: IN SILICO ENGINEERING OF PSEUDOMONAS METABOLISM REVEALS NEW BIOMARKERS FOR INCREASED BIOSURFACTANT PRODUCTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Figure 2A should be edited in order to improve readability, (probably name of reactions can be hidden since they are listed separately).
Experimental design: The work here reported is well described and the method used are innovative, however I would suggest the following point to address in order to improve the quality of the manuscript:

- the new production rate of rhamnolipids should be compared not only to that predicted by the previous version of the model but also to the one obtained through P. aerugionosa model (from which reactions have been taken).

- a detailed list of genes and reactions identifiers added to the model is missing, such data should be provided alongside a functional description of genes added.

- line 75, no reason for such a transfer of genes is reported, could you provide any example  of production of rhamnolipids in other strains by mean of the named genes?
Validity of the findings: The manuscript would benefit of a  comparison between P. putida and P. aeruginosa performance in rhamnolipids production and would eventually support the suggested P. putida  engineering.
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: IN SILICO ENGINEERING OF PSEUDOMONAS METABOLISM REVEALS NEW BIOMARKERS FOR INCREASED BIOSURFACTANT PRODUCTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English is professional, but be careful to include all necessary articles. There are a few missing 'the's scattered through the text.
Experimental design: The claims of novelty in the introduction are not justified, as the authors cite several other studies that offer more rigorous assessments (both in silico and in vitro) of optimized rhamnolipid production than what they here show (particularly Wittgens et al. 2011., but also Tiso et al. 2016. and many more papers optimizing rhamnolipid production in P. aeruginosa and stutzeri). I do not feel that the study fills an identified knowledge gap.

There is also far too little detail in the methods to assess how the modeling was performed. The provided supplemental code is incredibly bare and inadequate, and the 'flux_balance' function as presented is not, to my knowledge, a part of the Cobra Toolbox which they claim to use in this analysis. I therefore can't run the minimal code they present to even figure out what is going on, though I can optimize their provided model using other Cobra Toolbox functions. There are also many complex implementations of multi-objective optimizations that they here seem to bypass in favor of the multi-level approach used by Angione et al. 2015, and the rigor of the former approaches in addition to the lack of methods detail is concerning. Furthermore, the lack of description regarding the 'flux distribution' they are analysing with PCA makes me think that they have not performed any flux sampling, flux variability analysis, or other approaches that would ensure that they have obtained a fixed solution versus one of the many possible flux distributions which may fulfill their constraints. This issue must be addressed for any findings to be valid.
Validity of the findings: As the addition of the rhamnolipid pathway to the P. putida model is a straightforward and relatively easy process, the main scientific advances claimed by this study seem to be 1) the multi-level optimization balancing growth and rhamnolipid production (which I cannot properly assess given the above methods issues, and there are easier ways to perform this by tracing the pareto front of the two objectives) and 2) the PCA assessment of the 'flux datasets.' I have trouble following the PCA analysis, particularly what 2B is showing and how it relates to the PCA. They furthermore do not even address Figures 2D-E in the text. They also provide little discussion or contextualization of the PCA results, likely because the reactions identified are not particularly interesting or relevant to rhamnolipid production.
Additional comments: Calling PCA machine learning is technically accurate, but quite a stretch in common practice given the much more advanced machine learning being implemented in biology today.


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: IN SILICO ENGINEERING OF PSEUDOMONAS METABOLISM REVEALS NEW BIOMARKERS FOR INCREASED BIOSURFACTANT PRODUCTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors extensively improved the quality of the manuscript by including litterature references as a background  and a clear work rational in the introduction.
 They performed the suggested comparison and edited the figures in order to improve readability.


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: COMPARING ENZYME ACTIVITY MODIFIER EQUATIONS THROUGH THE DEVELOPMENT OF GLOBAL DATA FITTING TEMPLATES IN EXCEL
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article compares arranged equations with classical equations of enzyme kinetics. It is not clear what is compared: sometimes the author mentions an arrangement of equations and in other cases he mentions a partial inhibition mechanism. If the problem is the mechanism, he should mention that a equation deduced from a partial inhibition mechanism will sure be better describing the partial inhibition and better when fitting to experimental data where the system is affected by partial inhibition. If this is not the case, it is hard to understand how a arranged equation will fit better than the original. Maybe the author is proposing a new fitting method, however, it was not explicitly mentioned.
Experimental design: There is no description of the experimental design, methods and analysis of the data.
Validity of the findings: The main conclusion was that the modifier equation was able to model in good agreement the experimental data. How obvious is that an arranged equation will fit as well as the original equation? I think that we have two options:
i) The arranged equations are as good as the original equations when used as mathematical models. Unless the author is proposing a new fitting method (which is not explicitly mentioned), the article lacks scientific merit.
ii) The author did not properly nor clearly described the objective of this work to ensure the correct understanding when reading the manuscript.
 In addition, as the methods were not described it is difficult to evaluate the quality of the results.
Additional comments: The main objective of the article was not clearly exposed. What the author intend to do? What intend to demonstrate? What is the improvement when using the arranged equations?
The methodology was not described. The methods used to test the ability of the arranged equations to fit experimental data were not described. In the same way, the methods to compare the fitting quality were not described. Indeed, there are a lot of information, calculations and calculation methods implicit in the results that were not described. Surely, a significant improvement of the manuscript is possible adding this information. As an example, the Bayesian information criterion (BIC) was calculated and used to evaluate the data. It is necessary to describe the calculation and usefulness of the BIC in the data evaluation process.
It is necessary to add all this vital information to improve tha manuscript.

Some comments:
1. The author continuously define enzymatic activity as Vmax. This definition is wrong because the enzymatic activity is obtained from an experimental assay in standard conditions which result is not necessarily the Vmax. Indeed, the Vmax can be related to the enzymatic activity, however, the enzymatic activity cannot be related to Vmax. Just in the case that the substrate concentration is saturated (S0 >> Km), the obtained reaction rate will be approximately the Vmax.

2. It is usually found in books and articles the classification of enzymatic inhibition as competitive, non-competitive, uncompetitive and mixed. Exposing the types of inhibition in this way can drive to a misunderstanding because the non-competitive inhibition is a subclass of mixed inhibition. Considering this the types of inhibition has to be exposed as competitive, uncompetitive and mixed inhibition. When the mixed inhibition presents the same value for both inhibition constants, the competitive (Ki) and the uncompetitive (Ki’) components, it means Ki = Ki’, the inhibition is denominated non-competitive. It is important to expose a correct classification of the types of inhibition to finish with the eternal misunderstanding.

3. In line 76 the author indicated that the equations presented are unable to describe partial inhibition. This is correct. However, the inability to describe the partial inhibition is due to the origin of those equations which were obtained from reaction mechanisms describing total inhibition and not partial inhibition. Thereby, the inability in question was wrongly related to those equations when it should be related to the mechanisms proposed to derive those equations.

4. In line 86 the author exposed that the catalytic activity of an enzyme is defined as the coordinates Km and Vmax. As I commented above, the enzymatic activity is obtained from an experimental assay in standard conditions. In this case the author has to talk about “reaction rate”, which is the dependent variable in all the exposed rate equations (v). It is true that the activity is obtained through empirical determination of a reaction rate at certain operating conditions, however, a reaction rate is not necessarily the enzymatic activity.