Metabolomics of Pseudomonas aeruginosa Treated by Atmospheric-Pressure Cold Plasma

Round 1

Reviewer 1 Report

See the attached file.

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

General Comments: The study written by Xu et al, presents a potential good process. However, the manuscript is written in an extremely poor manner. There are significant grammatical errors and structural errors in the manuscript. Also, the methodology is very poorly explained (No QC metabolites mentioned, no Internal standard mentioned, statistics poorly represented etc.).

The results are written in a poor manner. What were the outputs of these experiments? This is not clearly articulated. The discussion is completely missing. I suggest that the authors consult to professional english writers or native english writers within their team to improve the overall writeup.

I will not recommend the article to be published in its current condition. It needs massive improvements and corrections, and needs to almost re-written completely (except Introduction part to some extent).

 

Specific comments:

Line 84: The grammar of writing temperature is that there should be no space between the value and degree sign (E.g. 121 °C should be corrected to 121°C)

Line 86-102: Putting dot points is not required. Simple paragraphs should be sufficient.

 Line 92: Please shorten '20 minutes' to '20 min' here and throughout the manuscript

Line 111: The font of °C is not uniform. Please make sure that the font remains uniform throughout.

Line 112: Please indicate which metabolites were used as QC samples. This is important. The authors can present this a table in Supplementary section (QC metabolite, %RSD).

Line 113: methoxamine is incorrect. The correct term is Methoxyamine HCl. Also, it is very important to name suppliers for the chemicals (Product ID, Vendor, City, Country)

Line 116: Please expand BSTFA. Also V/V (all capital letters) is incorrect. It should be v/v 

Lines 122-124: Please put full information regarding the column condition. E.g. Mass spectrometer model, column length (is it 15m or 30m?)

Line 136: Here the figure is referred to as FIG 3. However, elsewhere, the figures are indicated as Figure 1, Figure 2 etc (see line 66). Please make this uniform throughout the article.

Line 141: N2+(B-X) is incorrectly written. Please correct the subscripts and superscripts.

Line 169-170: What is the internal standard used. This is not mentioned. Also, what is the statistical variation of IS concentration (% relative standard deviation).

Lines 159-164: This section must be transferred to Materials and methods section

Line 172-186: This section must be transferred to Materials and methods (Please put a separate Statistical analysis part in Materials and Methods section to incorporate all sections related to statistics e.g %RSD, p-value, FDR, etc.). Please refer to the work of Karpe et al for better clarity of this section (https://doi.org/10.3390/metabo11060380)

Line 172: Were any statistical tools (or software) were used for OPLS-DA and other multivariate analysis? If yes, this must be mentioned. If done in MS Excel, it must be clarified.

Line 194: Figure 6B is incorrectly used. The R2 and Q2 values are already indicated in Table 1, so there is no need for Figure 6B. In this type of figure, a score scatter plot (Figure 6A) is coupled with a loading scatter plot (indicating metabolite spread across the plot). 

Line 204-218: This is too descriptive and is not needed. It can be paraphrased in 1-2 sentences at the most (if needed to be explained at all). In fact, Table 1 tells a better story than what is mentioned in these lines.

Line 222-226: This part must go to Materials and methods section

Line 226-229: This is poorly written. Please restructure the sentence.

Line 227: Please address the 'attached attached Excel table' phrase. What is referred to here? 

Line 230: What is the purpose of VIP legends for Figure 7? This does not make any sense. The plot needs to be redrawn. Please elaborate on Figure 7 caption.

Line 235-236: Please use full form of VIP. Also, the VIP values are not represented here. The whole section related to VIP is missing! Please address this.

Line 246: The usage of the term 'differential' is in error. It should be either 'different' or 'various'. The term 'differential' is used in terms of expression.

Line 253 (Figure 8 and elsewhere): Captions are very poorly used. The items in this figure are not explained at all. For example, what does the dendrogram refer to? What do the color coding refer to? What is the magnitude of each color change (normalized change or Fold change)? What do the terms 1T and C refer to? Formatting of metabolites is not uniform (Why does Cellobiose written as cellobiose 2? Some metabolites are in CAPS, some are not). The plot needs to be correctly made.

Line 257-261: This is poorly written. The sentence must be restructured. 

Line 262: Table 2 caption must indicate a full story as a standalone item. In the table, just a dash (-) is sufficient for metabolites with no KEGG ID. Fold change can be presented in upto 4 places as well. p-value (raw or FDR adjusted) are not presented. Metabolite names are not uniform. One concerning question is that if Table 2 present significant metabolites, what is the use of Figure 8? From a reader's point, they present the same data.

Line 263-271: The inclusion of Pearson's correlation here (Figure 9 as well) does not make any sense. What does it add to the story?

Line 274-282: What does the phrase 'attached Excel' refer to? This is not clear.

 

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

General Comments:

The article has improved  to some degree, but still needs a significant improvement before it actually drives the impactful story. I will repeat this, "The plasma treatment part and its metabolic output" is a great story to tell, especially as there are very few biochemical indicators as to how he plasma treatment changes the biochemical mechanism of pathogens in the water.  

However, the story telling is still the weak point of this manuscript. The results section is still filled with lot of obsolete content and, there is some less material in Discussion section. This is still unaddressed and needs to be reflected in the manuscript to make this study more impactful. I would suggest the authors to refer to some of the published manuscripts in MDPI's journal 'Metabolites' so that they get a gist of how these studies, and were the current study lacks. 

Specific Comments:

Line 50: Replace 'dielectric barrier discharges(DBD)' with 'dielectric barrier discharges (DBD)'. (Put a space before the bracket).

Line 115 and elsewhere: Pseudomonas aeruginosa and other biological name s must be italics

Lines 127-128:  Methoxyamination hydrochloride is incorrect. The name is methoxyamine hydrochloride

Line 126: If the QC samples referred by the authors are simply the combined aliquot of individual samples, then it is to be referred as Pooled biological quality control (PBQC) samples. If only QC samples are mentioned, it generally refers to individual metabolite standards which are used at a pre-determined concentration levels, individually or in a mix refer to 

Line 130: more information is needed for the chemicals and reagents. This has not been addressed (Product number, Manufacturer, City, Country). For example, FAMEs have been mentioned, but which FAMEs, odd numbered, even numbered or both? This needs to be specified ad for that, product number is important.

Line 138: Please correct the term slitless to splitless

Lin 154-160: Which platform was used for univariate analysis? Was it SIMCA? I ask this because SIMCA is a good platform to do multivariate statistics, but it has some limitations regarding univariate analysis (not saying that it cannot do it). The volcano plot (Figure 7) which is a univariate output, does not appear to be a SIMCA analyzed work. This part is still not completely addressed! 

Line 193: Even if the authors refer to the article of Burns and Walker (2019) for IS and normalization processes, it is very important that these items are always indicated in the manuscript! This has not been done. Also, what was the variability in IS measurement? This is still unaddressed.

Line 211-216 and similar: This is a repeat and still not addressed! The statements made here are matter of fact statements which SIMCA dashboard tells when an analysis is done. The authors have to put it in their own words what do these graphs mean in reference to their test. A simple copy-paste type work is not enough and not necessary (E.g. why would I as a reader will need to know what an ordinate or an abscissa mean? I can see that plainly in the graph. I do not want to read them as texts. Same with the shape and color. Why would these appear in text formats when I can see them clearly in the figure?). 

Figure 7 and Table 2: This is still unresolved. Both the table and figure tell same story, the table being more informative. The Volcano plot therefore, becomes obsolete. Due to this, I had mentioned in my previous review to keep only one of them (preferably the table). Same thing goes for Figure 8. The authors are giving the information which they have already provided in Figure 7 and Table 8. Therefore, Figure 8 becomes an obsolete item. At the best, it can go in the Supplementary materials section, but it does not add anything to the story of the main manuscript. Also, please make the fonts in Table 2 consistent. Some metabolites start with a Capital letter, while some start with small letter. Also, terms like 'Melatonin 2' is to be expressed in 'Melatonin' (same for others).

Line 315: Replace 'Many scholars' by 'Studies'.

Line 317-318: This line seems ambitious. lease restruture it. A good way is to begin with something like, "To our knowledge there is an absence of studies which have tested the effects of plasma treatment on bacterial metabolism".

Line 329: Replace 'Organoheterocyclic Compounds' by 'organoheterocyclic compounds'.

Line 330: Please add the relevant reference here.

Line 335: When the authors mention, ' most obvious changes', what do they refer to ? Were these pathways found to be upregulated or were they downregulated? Things like must be clarifies, with a reasoning. 

 

 

 

 

Author Response

Please see the attachment

Author Response File: Author Response.docx