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: DEVELOPMENT AND VALIDATION OF A NOMOGRAM TO PREDICT POSTOPERATIVE PULMONARY COMPLICATIONS FOLLOWING THORACOSCOPIC SURGERY
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I read with interest the manuscript by Wang et al, and appreciate the opportunity to provide feedback. Here are my comments:

1) Do you have the data regarding blood loss volume or the use of transfusion during operation?
2) Although authors mentioned about the relation between thoracoscopic surgery and postoperative complications, they did not compare thoracoscopic surgery with thoracotomy.
3) This study cohort included various types of surgery such as mediastinal and esophageal surgery. I think that the extent of lung resection could greatly influence on the incident of pulmonary complications. Therefore, authors should examine the surgical types in univariate and multivariate analysis for PPC.


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: DEVELOPMENT AND VALIDATION OF A NOMOGRAM TO PREDICT POSTOPERATIVE PULMONARY COMPLICATIONS FOLLOWING THORACOSCOPIC SURGERY
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment. Good clear reporting
Experimental design: Well defined objectives
Validity of the findings: To elaborate how will this normogram impact clinical care
Additional comments: nil


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: DEVELOPMENT AND VALIDATION OF A NOMOGRAM TO PREDICT POSTOPERATIVE PULMONARY COMPLICATIONS FOLLOWING THORACOSCOPIC SURGERY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The changes made are appreciated and have improved the paper. This study should be of some interest to readers.
Experimental design: no concerns
Validity of the findings: no concern
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: A CHIMERIC VECTOR FOR DUAL USE IN CYANOBACTERIA AND ESCHERICHIA COLI, TESTED WITH CYSTATIN, A NONFLUORESCENT REPORTER PROTEIN
Review round: 1
Reviewer: 1

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


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

Input:
Title: A CHIMERIC VECTOR FOR DUAL USE IN CYANOBACTERIA AND ESCHERICHIA COLI, TESTED WITH CYSTATIN, A NONFLUORESCENT REPORTER PROTEIN
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The article describes the formation of a plasmid compatible with E. coli and in the cyanobacterium Synechocystis sp. PCC 6803, the plasmid pMJc01 can be introduced by electroporation into the bacterium Synechocystis sp. PCC 6803, and the plasmid carries the sequence coding for cystatin protein which is a papain inhibitor protein, which can serve as a reporter to evaluate the activity of regulatory elements in this type of bacteria.  It is also postulated that it can be used as a tool for the expression of proteins in Synechocystis and others related-bacteria. However, I believe that it should be demonstrated more convincingly that cystatin is present in Synechocystis and that it has inhibitory activity.

147: next morning; replace for next day.

148: Why is it necessary to gradually increase the concentration of Kanamycin in the case of Synechocystis sp.?

179:  L21_RBS*regulatory element. what does the asterisk mean?

207: What is LBK? please specify in the text.

257:   Why in the papain inhibition assay, to test for the presence of the inhibitor (cystatin) was measured at 550 nm, in the original protocol was measured at 520 nm.

284: Indicate in Figure 1 the unique restriction sites (MCS if exist) that may be useful to exchange for others the promoter regions or gene of interest.

370 :  In the Figure 4b. In the original activity assay the enzyme activity increases with increasing amount of enzyme used (ANALYTICAL BIOCHEMISTRY 47, 280-293 (1972)) and in the presence of inhibitor the activity decreases as the amount of inhibitor increases, as measured by Abs at 520 nm, as shown in reference 38. Why in the  figure 4b the Abs increases as the amount of inhibitor (cystatin) increases, shouldn't it decrease?.

373: What do you mean by "chassis"? is the plasmid backbone?

386: mark with an arrow the GFP protein band in the  figure 5b.

393: In the Figure 4C shows the relative copy number of the pMJc01 plasmid, which is higher than the pPMQAK1 plasmid, but this is not related to cystatin expression. The figure legends shows :"Evaluation of relative plasmid copy number in Synechocystis sp. PCC 6803 with cystatin reporter". It is essential to show convincingly the enzymatic activity of cystatin in Synechocystis, which is the main objective of the article. the authors point out that the cystatin gene carries a histidine-tag, so it would also be necessary to demonstrate its presence with an anti-histidine western blot.

408-409: The authors establish : "The amount of cystatin detected was approximately 5-fold higher in cells transformed with pMJc01 than in cells transformed with pPMQAK1 (Fig. 4c). but the figure shows that it is the relative amount of plasmid that is not related to cystatin expression.

448 : this is speculative "We attributed this to an unknown interaction with P lacUV , which controls the expression of replication proteins in the RSFmob-I replicon" please clarify.


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

Input:
Title: A CHIMERIC VECTOR FOR DUAL USE IN CYANOBACTERIA AND ESCHERICHIA COLI, TESTED WITH CYSTATIN, A NONFLUORESCENT REPORTER PROTEIN
Review round: 2
Reviewer: 1

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


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

Input:
Title: A COMPARISON OF DICECT AND HISTOLOGY FOR DETERMINATION OF NASAL EPITHELIAL TYPE
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper is clearly written, well referenced, professional, and altogether well reported.
Experimental design: This paper has excellent experimental design.
Validity of the findings: I have no doubt about the validity of these findings.
Additional comments: In this fantastic paper, Smith and colleagues study the olfactory epithelium of two bats comparing diceCT and histological methods.  This paper is excellent, and should be published largely as it is.  Frankly, I find it hard to find substantial fault in any aspect of this paper, but, in an effort to provide SOME constructive feedback, there are a couple of notes that might be worth mentioning.  By no means should any of these modifications be required, and, in my opinion, the paper should be accepted essentially in its current form with one notable caveat: as the authors know, I know absolutely nothing about epithelial tissues!  If a colleague with that expertise finds faults, those could/should be taken seriously.

With that said, here are some thoughts:

1)	There are some inconsistencies in tabs and spaces throughout the paper.  In our era of copyediting, this does not really bear mentioning, but I am grasping at straws to find flaws here!

2)	It might be worth highlighting that the investigators chose to increase the concentration of iodine rather than the duration of staining of their larger specimen for their diceCT methods.  To my knowledge, the jury is still out for which of these yields better results (I’m not aware of whether this sorely needed comparative study has been accomplished, but I would love to collaborate with any of the authors on such an investigation!), but in our anecdotal experience, we believe that increasing duration instead of concentration causes less shrinkage.  Since shrinkage was explicitly of interest in this study, it is probably worth mentioning this as a caveat, though, since there really is no scientifically definitive answer, this is certainly not a flaw of the current investigation.  If the authors are aware of a reason to choose concentration over duration, then it would be valuable for the field if they would mention that!

3)	On a related note, the investigators should probably mention the potential shrinking effects that the specimens have undergone during their decades of storage.  The two recent papers by Leonard and colleagues (though about muscles) speak to this.  Of course, we have no idea what the cumulative effect of decades of preservation, then diceCT, then slide mounting has on these tissue dimensions, and this is certainly not a disqualifying aspect of this paper, but it is probably worth mentioning as a caveat.

4)	As the investigators note, it is problematic that some of their tissues of interest approach their voxel sizes.  Indeed this is a problem all of us face.  It might be worth mentioning that surely technology will solve this problem: CT resolution is getting better and better as are staining regimes and hopefully too digital dissection software.  This problem is important but temporary.

5)	The authors note that histology has important uses that are beyond what we can do with CT, but they don’t note that, theoretically, serial histology slices could be used by those of us who do volumetric based “digital dissection” too.  This was important in the early 3D anatomy programs (e.g., I think that the first digital cadavers were made using serial slices that could then be resliced in any plane?), and may still be the way that digital cadavers are assembled.  I haven’t seen us anatomists do this (e.g., making stacks from slides and resampling their planes in 3D space), but we could, right?  And maybe we should?

6)	Arguably the only thing that, in my opinion, MUST be added to this paper is a better highlight of some of the truly important findings of this paper that the authors never mention: for instance, to my knowledge, this is the best (if not first?) study to demonstrate a) how well diceCT can be used on specimens that have been stored for DECADES in fluids (though I would appreciate more details about how these specimens were initially preserved and stored) and b) that diceCT specimens can subsequently also be histologically stained.  Both of these are seriously important pieces of information!  DiceCT is resource intensive both in terms of materials and time and I have been reluctant to try it on specimens that were not optimal.  Specifically, I was nervous that the long-term storage might have made the tissues less permeable to the iodine.  And I am astonished that the iodine and its subsequent de-stain did not undermine the ability to histologically stain those tissues.  Maybe other people have found both of these previously, though I am unaware of those findings (I do feel like I read about old museum specimens in diceCT, but I can’t remember where), and it is certainly worth highlighting that this paper shows that these methods do indeed work in these specimens and in combination.

Again, this paper is fantastic, and I look forward to seeing it published. – Adam Hartstone-Rose


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

Input:
Title: A COMPARISON OF DICECT AND HISTOLOGY FOR DETERMINATION OF NASAL EPITHELIAL TYPE
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	Clear and unambiguous, professional English used throughout
The manuscript is written in correct English. General suggestion I have is that the goals and methods need to be edited by the authors to focus the language to better orient the readers to the anatomy, describe the particulars of each test type and make the relevance of the paper clearer. More details on this are below.

2.	Literature references, sufficient field background/context provided
No comment

3.	Professional article structure, figures, tables. Raw data shared
The article follows an acceptable format.

The figures serve the text in general. More needs to be done to orient the reader to the anatomical context of the figures, and the figures need to be labeled and annotated better to help clarify the tests performed by the authors. For example, I think the annotations the authors made on the mucosa types need to be made more prominent and evident. Fig S1 shows the sights of sampling, and Fig.S2 shows some boundaries of their annotations, but none of this is present in the main figures. I believe it needs to be. All figures are insufficiently labeled. Here are a number of suggestions regarding the figures:
Figure 1:
	---Please clarify for the reader where in the skull we are in these sections.
	---What does v in vETII mean?
	---Do ETI and ETII correspond to anatomical terms? I think then ETII would be ventral to ETI? If they don’t correspond, make that clear that these are labels for this study alone.
	---Labels and lines are displaced in a number of places. Epiturbinal is outside the nasal cavity, for example.
	---What is ET vs ETI or ETII or vETII?
	---Define NpD in the legend.
	---Note the white lines with no labels.
	---Is that the cranial cavity? If so, please label it for orientation.
Figure 2:
	---On this or another introductory main figure, label parts of mucosa as background for readers and because the various features are central to the different tests the authors use: Epithelium, cilia, borders of lamina propria for both olfactory and respiratory mucosa, etc.
Figure 3:
	--- Radiopacity is mentioned in Fig. 3. In the text, radiopacity is also referred to in terms of glandular tissue. If possible, please feature and label this in this figure or another in order to illustrate for the reader what kind of assay the authors use for their blind annotations.
	---The legend says that the ‘turbinal is almost completely radiopaque’. This is confusing because “turbinal” is bone. Using turbinal interchangeably as just the bone and the entire structure with accompanying soft tissue can be confusing.
	---Some labels seem to be displaced, e.g. GL
	---Bowman’s gland is BG and GL on various figures.
Figure 4:
	---Perhaps this figure could include a comparison between blind test #1 and #2 to show the difference in the results when the added criterion of radiopacity is added.  This would help focus the conclusions of this article: the success of using diceCT in lieu of histology and the limitations of basing estimates from diceCT on epithelial thickness alone.
Figure S1:
	---Which specimen is this?
	---It’s not clear to me if c and d are meant to be olfactory mucosa or were chosen as respiratory mucosa locations. Please clarify in the legend and text.
Figure S2:
	---Perhaps enlarge S2b to include all four annotations and omit c.
	---Again, make it clearer, by coloring or something what is meant by perimeter.
	---Label lamina propria on diceCT image.
	---Where is S2i from in S2h? Please label S2i with features that distinguish it from olfactory tissue in S2g. They look very similar. For example, what distinguishes respiratory kinocilia, which is mentioned as an indicator in the legend, from OE cilia?
Figure S3:
        ---Please make it clear which trial this is.

4.	Self-contained with relevant results to hypotheses
I think this study stands on its own and is an appropriate unit of publication.
Experimental design: 1.	Original primary research within Aims and Scope of the journal.
I believe this article does meet the aims and scope of the journal.

2.	Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.
I think the authors accomplished their research goal, and it is relevant and meaningful. I also think the authors could edit the Introduction and Abstract to better focus the readers’ attention to the fact that previous studies using diceCT did not test the reliability of diceCT-generated annotations in relationship to histological ground truth by performing blind annotations, leaving an important gap that they begin to fill with this article.
The end of the Conclusion reminds us that using diceCT to identify and estimate mucosal perimeters would quicken the work of researchers like Yee, 2016 and Craven, 2010, etc in quantitative studies of the link between OE surface and ecological variables and airflow models and olfactory function. These examples and other publications on anatomical correlates of olfactory function give particular relevance to this current study, and perhaps these might be referenced in the Introduction as well to better illustrate the necessity of this study.

3.	Rigorous investigation performed to a high technical & ethical standard.
I think this investigation was performed with rigor and to a high technical and ethical standard.

4.	Methods described with sufficient detail & information to replicate.
The methods may need to be focused and pared down to define each test and clarify the protocol steps.  The use of radiopacity in estimating tissue perimeters needs to be clarified so as to be replicable. Authors might also clarify how indicators of glandular tissue are identified and used in the blind annotations.
Validity of the findings: 1.	Impact and novelty not assessed. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.
Although this article contributes new anatomical data on the nasal cavities of two bat species, it seems to be first and foremost a methods paper. Because its small sample is small and criteria are sometimes qualitative, it an initial study that establishes an good first step toward a working protocol for discerning and quantitatively estimating relative surface areas of mucosal types in mammal noses from diceCT images.

2.	All underlying data have been provided; they are robust, statistically sound, & controlled.
I suggest the authors speak to their sample of two individuals, why it was necessary and how a goal of future work might be to increase the number of specimens from a single species in order to statistically verify their results.

3.	Conclusions are well stated, linked to original research question & limited to supporting results.
The Results section needs clarification in a number of places. First, the subsections should more clearly track the series of tests/trials set up in Materials and Methods and reinforce each question at the beginning of each subsection. This will facilitate clarity and narrative style. Figures need to be referenced more often in the Results section to to reinforce the results visually. Referencing tables is not enough.  The Discussion section is more narrative and flows better.

---  Paragraph starting Line 212:  start paragraph with a sentence to reiterates question for the reader; Clarify annotation type here; define NT, vestibule, free margins; clarify what “five locations” correspond to; refer to figures; clarify when the text is referring to OE and when to non-OE.

----Paragraph starting Line 231: Again, reinforce question and trial type for reader in first  sentence; artefactual; clarify what’s been measured here; name “two structures”;  is this paragraph repeated by the next?

---Line 264: Subheading could be more informative
---Line 284: Clarify for the reader how to discern on Fig.3c that the lamina propria is highly glandular. I don’t see it clearly.
---Line 285: Label small patches of non-OE in Fig. 3. I don’t recognize them.
---Line 289: The subheading is awkward.  I’m confused by Trial 1.  Did the previous results have anything to do with Trial 1?  If not, it would be helpful to make it clear that all Results up to this point were histologically-informed and that this is the first departure from that.
---Line 292: The figures references didn’t seem to match the text.
---Line 295: Avoid vague descriptions like ‘both structures.’
---Line 302: The summary of the repeated measures ANOVA was not clear to me. Also, which types does “annotation type” include here?
---Line 307: I couldn’t find Table S3, S4 or S5.
---Line 320: Subheading doesn’t seem to summarize Trial 2.
---Line 321: ‘Entire mucosa’ could mean various things.
---Line 325: The second type of blind annotation is referred to here as ‘combined criteria of epithelial and overall mucosal thickness.’ This doesn’t describe the criteria well.
---Line 330: Name ‘both regions.’
---Line 368: Mucosa and epithelium are sometimes used interchangeably and sometimes as distinguishing identifiers. Lines 368-371 settle that puzzle, so this needs to appear sooner in the text.
---Line 395: Name the ‘two structures.’
---Line 417: The use of the word turbinals needs clarifying throughout. For example, here, “turbinals are supported by especially thin bone, and such structures may shrink to a greater extent than other surfaces with more substantial support.” the turbinal seems to be referred to as a whole unit supported by bone, whereas I understand a turbinal to be the skeleton itself. There are other examples of this.

4.	 Speculation is welcome, but should be identified as such.
I see no speculation in this article.
Additional comments: Just by way of summarizing what I took away from the article:

In “A comparison of diceCT and histology for determination of nasal epithelial type” Smith et al. test the potential for diffusible iodine-based contrast-enhanced computed tomography (diceCT) to replace traditional methods, such as microCT and histology with a non-destructive method for identifying olfactory mucosa in mammalian nasal cavities. The authors chose to study two specimens of bats, one an adult fruit bat and the other a full-term fetal vampire bat. They designed two methods of blind annotation of olfactory mucosa on the diceCT images, one based mucosal thickness alone (an indicator known in the literature) and one based on mucosal plus relative radiopacity (brightness here) and the putative presence of a dense presence of radiopaque glandular cells in the lamina propria underlying the epithelium. This was done at various constant anatomical landmarks on the ethmoturbinals and nasal septum throughout the image series. The authors successfully aligned three image types (histological sections, microCT and diceCT) and used a histologically-informed estimate of olfactory mucosa against which to judge their blind estimates. They concluded that in the posterior extent of the nasal cavity, that is, caudal to the most anterior extent of the ethmoturbinals, the blind estimates of olfactory mucosa perimeter from diceCT images based on thickness alone were not significantly different from estimates from histological samples. Rostral to this, the relationship fell apart due to highly vascularized, thick respiratory epithelium. This shows the limits of this one method of annotation. When radiopacity was taken into consideration, blind estimates of olfactory mucosa from diceCT closely matched those done from the histological sections throughout the nasal cavity. The authors also confirmed and quantified tissue shrinkage resulting from histological and iodine-stained specimens.

This study successfully defines the potential and some of the limits of using diceCT to discern and quantify olfactory and respiratory mucosa and will likely quicken the work of researchers interested in the mechanics and evolution of respiration and olfaction by guiding them in the use of diceCT, particularly with rare specimens that need to be investigated non-destructively.

My main suggestion is that the authors consider giving the reader a firmer footing in each question posed in the article and the particulars/criteria of each attending test. The language can be pared to focus on just that. I spent some time while reading the article having to double check where I was in the skull, the mucosa, and in the sequence of goals/tests. Please clarify this, label every term referenced, reinforce the goal and particular criteria of each step, It needs to be easier to follow.

Additionally, please acknowledge the small sample and the qualitative nature of a criterion such as radiopacity and reassure the reader that this is the first step toward working out a replicable protocol for this important work.


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

Input:
Title: A COMPARISON OF DICECT AND HISTOLOGY FOR DETERMINATION OF NASAL EPITHELIAL TYPE
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Smith, et al. investigate methodological differences of inferring olfactory versus non-olfactory epithelia in the nasal cavity of bats using histology, µCT, and diceCT. As diceCT methods launch the field of comparative anatomy into directions never before possible, it is critical to note the assumptions and effects of diceCT on the detailed anatomy that can is observed from histology. Despite diceCT going “mainstream” in 2016, very few studies have actually properly compared histology and diceCT within the same specimen, let alone for olfactory epithelia. Thus, I commend the authors for their efforts in doing so and advocate that this study be published if authors can address a few of my concerns below.

Major comments:
First, and perhaps this is a major-minor comment, there can be some broader implications discussed or introduced. For example, many studies estimating olfactory epithelia use turbinate reconstructions directly from bone (Van Valkenburgh, et al. 2011, Journal of Anatomy; Martinez, et al. 2020, PNAS). These studies often acknowledge that they are making a large assumption that there is direct correlation between the olfactory tissue and the turbinates, but this is rarely quantitatively verified. I feel like this study offers that opportunity to more clearly articulate this. Establishing these boundaries is essential for the use of OE studies in specimens where soft tissue specimens are not available. I suggest integrating this more into the narrative of the study.

My second concern regards length of staining time. Seven days for both specimens (albeit at different concentrations) concerns me that increased visibility of neurons and glands within the non-olfactory epithelia are minimal relative to Yohe, Hoffmann, and Curtis (2018), which stained in double the concentration for up to 15 weeks. Also, I do imagine that the thickness and density of the Cynopterus is significantly greater than that of the embryonic Desmodus, which again would affect the rate of diffusion.  While I do not think this affects the ability to detect the very bright olfactory epithelium, I do think there is much commentary on the presence of these mucosal glands as guiding the researcher for non-OE. Yet, from the figures I do not see any good examples of this and I wonder if this is due to undersaturation of iodine in the specimens. While I do not expect the researchers to repeat the study, I think a bit of discussion is warranted in the context of comparing to other diceCT studies using bat specimens (i.e., Hedrick, et al. 2018, Hall, et al. 2021 Evolution). I also recommend in the future for the researchers to explore longer stain times or to continuously refresh the iodine solution until the solution no longer discolors (suggesting more complete diffusion).

Third, unless I missed it, the authors do not comment on the effect of the diceCT stain on the histology. I know that it was unstained in sodium thiosulfate, but did this really remove all of the iodine? Could any be detected in the histological sections? Several of the authors have many publications using H&E on bats...how did this staining compare to previous (e.g., in coloration)? The reason I bring this up (outside of genuine curiosity), is that often times people discuss how they think the iodine will affect the histology with the assumption it would negatively do so, and then fail to ever do the histology because of this unwarranted assumption. I would like to see the authors comment on this in the discussion, as it would minimize poorly established rumors as to whether or not acceptable histological staining and sectioning is possible after diceCT.

Minor comments:
L61: I think a couple of sentences introducing the types of epithelia in the mammalian nasal cavity is warranted, along with what types of cells, general anatomical features, etc. are associated with these different types.

L159: “dice CT” should be one word

L302: “nut” should be “but”

L546: Typo in authors name...”Davie” should be “Davies”

Scale bars on the supplement Fig 1 would be appreciated


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

Input:
Title: A COMPARISON OF DICECT AND HISTOLOGY FOR DETERMINATION OF NASAL EPITHELIAL TYPE
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I appreciate how meticulously the authors considered the reviewers' comments and made multiple positive changes to the paper. I believe the authors took an already very good paper and made it even stronger. This will be a wonderful contribution.


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

Input:
Title: A COMPARISON OF DICECT AND HISTOLOGY FOR DETERMINATION OF NASAL EPITHELIAL TYPE
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors did a great job at responding to both my concerns and the concerns of other reviewers. I have no further comments and look forward to the manuscript being published.


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 PREDICTIVE ROLE OF PREOPERATIVE SERUM GLUTAMATE DEHYDROGENASE LEVELS IN MICROVASCULAR INVASION AND HEPATOCELLULAR CARCINOMA PROGNOSIS FOLLOWING LIVER TRANSPLANTATION—A SINGLE CENTER RETROSPECTIVE STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The manuscript would benefit from editing to correct English grammar.

2. In Table 1, please provide mean or median values for the lab test results, in addition to the ranges.

3. In Figure 1A, please provide a box plot or dot plot instead of a bar graph. Box and dot plots provide far more information about the distribution of the results, which will aid interpretation by the reader.
Experimental design: 1. In Figure 2, the authors should compare GLDH with MVI to predict survival.
Validity of the findings: No comment.
Additional comments: No additional comments.


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

Input:
Title: THE PREDICTIVE ROLE OF PREOPERATIVE SERUM GLUTAMATE DEHYDROGENASE LEVELS IN MICROVASCULAR INVASION AND HEPATOCELLULAR CARCINOMA PROGNOSIS FOLLOWING LIVER TRANSPLANTATION—A SINGLE CENTER RETROSPECTIVE STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: This is a well written manuscript that addresses a relevant subject matter.  The ability for GLDH to predict MVI and poor long-term survival for HCC after liver transplant could profoundly effect the selection of transplant recipients.

However, the normal reference range for GLDH is between 2 U/L and 10 U/L with an upper limit of normal of 11 U/L.  As the cut-point for GLDH to predict MVI and poor long-term survival for HCC after liver transplant is 7.45 U/L.  It is unclear to this reviewer how GLDH is able to discriminate outcomes in this normal range.  Furthermore, GLDH is released following hepatocyte injury.  How do subtle changes in GLDH caused by drug-induced liver injury or other hepatotoxic events in these patients affect the interpretation?  My primary concern is that the cut-point for GLDH is in normal range for the biomarker.

It may be worthwhile to evaluate the correlation between GLDH and ALT.  This may allow an understanding for the role of hepatocellular injury and an increase in GLDH in these patients.  We have previously evaluated both ALT and GLDH in patients post transplant and have observed a disconnect between these serum biomarkers.  It is not clear if this observation is linked to the authors observation.
Additional comments: no comment


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

Input:
Title: THE PREDICTIVE ROLE OF PREOPERATIVE SERUM GLUTAMATE DEHYDROGENASE LEVELS IN MICROVASCULAR INVASION AND HEPATOCELLULAR CARCINOMA PROGNOSIS FOLLOWING LIVER TRANSPLANTATION—A SINGLE CENTER RETROSPECTIVE STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Reporting is acceptable.
Experimental design: Design is acceptable.
Validity of the findings: Findings are acceptable as reported.
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: RELATIONSHIP BETWEEN HIGHER EDUCATION TEACHERS’ AFFECT AND THEIR PSYCHOLOGICAL ADJUSTMENT TO ONLINE TEACHING DURING THE COVID-19 PANDEMIC: AN APPLICATION OF LATENT PROFILE ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Lack of structure and clarity. Can be improved in the writing.
Experimental design: Good methodology.
Can be improved in procedure, can be improved in description of instruments
Validity of the findings: The discussion is not rich in content because it lacks structure and clarity to the reader.
The article is novel and of interest.
Additional comments: The manuscript is of interest and I consider it necessary for the authors to improve the manuscript in aspects such as the following:
-It is recommended to clarify and structure the abstract to provide clearer study conclusions.
- It is considered necessary to provide the introduction section with a greater structure and coherence. It would be appropriate to join the ideas by linking one with another.
- A further justification of the objectives of the study is needed.
-It is necessary to justify the selection of study participants, indicate reasons for inclusion, exclusion ...
- It is necessary to improve the methodology section because the procedure followed has not been clear.
- Please add sample questions from the questionnaires used.
- The discussion section add limitations.
- The discussion is not rich in content because it lacks structure and clarity to the reader.


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: RELATIONSHIP BETWEEN HIGHER EDUCATION TEACHERS’ AFFECT AND THEIR PSYCHOLOGICAL ADJUSTMENT TO ONLINE TEACHING DURING THE COVID-19 PANDEMIC: AN APPLICATION OF LATENT PROFILE ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: In methods, the authors, please explain more about the convenience sampling method, there remains the doubt if it is a snowball sampling or the authors chose the institutions.

The PANAS scale was validated for the Chinese population? please include the data.

Excellent statistical analysis was performed to validate the survey on psychological adjustments to online teaching.
Validity of the findings: In the discussion section, it is observed that there is little discussion about university professors. The discussion is focused on the characteristics found in the study and what could have been in any job. What makes working with university professors particular?
Additional comments: In the abstract, authors are encouraged to include the proportion of each latent class in the abstract. I think it is too ambiguous to say that one latent class is more prominent than another.

The second paragraph says, " Although China’s first large-scale online teaching program has achieved (perhaps even exceeded)," the phrase in parenthesis is not known if it is the authors' opinion or Dauguang's reference.

The introduction is engaging and complete about the issues of latent profile analysis and how affective characteristics can influence subjects. However, there remains an essential gap about university teachers, their characteristics, why to study them; university teachers were (before COVID-19) a job with risks in their health...and many more questions.

In the discussion section, it is observed that there is little discussion about university professors. The discussion is focused on the characteristics found in the study and what could have been in any job. What makes working with university professors particular?

The title of table 2 is very general. It is suggested to change the title to one related to the analysis shown.
In Table 4 and the statistical analysis section, the post-hoc test used is not indicated.

In Figure 1, I understand that these are averages. If so, the authors should include the standard deviations.

Authors are suggested to standardize the term "teacher." According to the study, a correct term should be "professor."


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: RELATIONSHIP BETWEEN HIGHER EDUCATION TEACHERS’ AFFECT AND THEIR PSYCHOLOGICAL ADJUSTMENT TO ONLINE TEACHING DURING THE COVID-19 PANDEMIC: AN APPLICATION OF LATENT PROFILE ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article has improved with the revisions made
Experimental design: The article has improved with the revisions made
Validity of the findings: The article has improved with the revisions made
Additional comments: The article has improved with the revisions made


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: INTEGRATIVE ANALYSIS OF ISO-SEQ AND RNA-SEQ DATA REVEALS TRANSCRIPTOME COMPLEXITY AND DIFFERENTIALLY EXPRESSED TRANSCRIPTS IN SHEEP TAIL FAT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors studied the tail fat content trait in cross-breed Hu sheep (DHH as fat-tail and DDH as thin-tail). Transcriptome analysis was performed using long reads (generated by PacBip platform) and short reads and authors tried to identify novel splicing variants, lncRNAs, and differentially expressed genes/transcripts. They also investigate alternative polyadenylation events.
Experimental design: All of my comments could be found in the attached pdf file. My edits are also highlighted in track changes.
Validity of the findings: There are a few issues regarding validity:
1- First, you said you identified 9001 novel gene loci (Line 188) but you didn't give any proof that they are really novel genes. You must either provide evidence for this claim or rewrite the sentence.
2- The authors performed differential expression analysis and reported DEGs/DETs but didn’t perform qRT-PCR for validation. Transcriptome analysis definitely should be accompanied by a validation qRT-PCR. Please consider it.
Additional comments: 1- Methods are not well-detailed. More details should be added.
2- Please check all abbreviations and give explanations in the first appearance. Also, give explanations for all abbreviations in the tables and figures. tables should be self-informative.


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: INTEGRATIVE ANALYSIS OF ISO-SEQ AND RNA-SEQ DATA REVEALS TRANSCRIPTOME COMPLEXITY AND DIFFERENTIALLY EXPRESSED TRANSCRIPTS IN SHEEP TAIL FAT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors used RNA-Seq to generate a transcriptomic data of tail fat tissue of sheep.
Novel transcripts in sheep tailed fat were identified. Differential expressed transcripts (DETs) between thin- and fat-tailed sheep were identified. GO and KEGG enrichment analysis were implemented to investigate the potential function of DETs. The big concern is about the used methods for aligning the reads against genome as well as lncRNA prediction method, which is not robust enough. Also, discussion is not appropriate and have to be enriched.
Experimental design: The manuscript needs to be revised by considering the following recommendations.

1-	Line 35: Authors claimed that for the first time they revealed the transcriptomic complexity of the tail fat of sheep!!! There are several studies in this filed with the same subject.

2-	Why only one sample was sequenced by Iso-Seq? As it is obvious isoforms and transcripts are different between thin and fat-tailed sheep. When all samples are pooled, how can you identify specific isoforms. In other words, how is it possible to differentiate isoforms/transcripts of each treat? Although Iso-Seq can sequence long transcripts, but in this study, authors have to sequence at least one sample per treat to be sure about the results.

3-	The filtering pipeline for lncRNA perdition is a little bit easy going and only CAPT software is used. For example, always several coding potential prediction tools have to be used and the results of all tools consider. Also, open reading frame prediction is the other step to remove some transcripts with coding potential. Why did not use these steps?

4-	Bowtie2 is used to align the reads against the genome? Why? As the data is RNA-Seq, a splice aware aligner, such as HISAT, have to be used.

5-	Differentially expressed mRNAs were not discussed and the results of their enrichment analysis are not provided. In discussion different aspects of the results have to be interpreted. I think that discussion need to be enriched.

6-	RNA-Seq samples have to be deposited in available databases like NCBI and the accession number of the data be mentioned in the manuscript.

7-	There are assorted typos and grammatical errors that need to be corrected. Totally, the manuscript has to be revised by a native speaker. For example:
a.	Line 117: “(Sage Science, MA, USA) was use …” have to be “(Sage Science, MA, USA) was used”
b.	Line 120: “(Life Technologies, CA, USA) was used” have to be “(Life Technologies, CA, USA) were used”
Validity of the findings: The results of RNA-Seq did not validate by Real-Time PCR, as it is needed.
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: INTEGRATIVE ANALYSIS OF ISO-SEQ AND RNA-SEQ DATA REVEALS TRANSCRIPTOME COMPLEXITY AND DIFFERENTIALLY EXPRESSED TRANSCRIPTS IN SHEEP TAIL FAT
Review round: 2
Reviewer: 1

Output:
Basic reporting: Dear Authors,

Thanks for revising the manuscript according to my comments. The majority of the raised points are addressed properly. I am still not totally convinced about the number of newly identified genes/transcripts. However, in the revised version, the limitations of the study were acknowledged, and the extra information provided in the tables and the supplementary file could also help the readers to decide about the soundness of the results.
The manuscript reads better and the English language is acceptable but it is not perfect. Given the limited number of published papers in sheep transcriptomics, I am happy to endorse the manuscript for publication in PeerJ.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: No additional comments


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

Input:
Title: INTEGRATIVE ANALYSIS OF ISO-SEQ AND RNA-SEQ DATA REVEALS TRANSCRIPTOME COMPLEXITY AND DIFFERENTIALLY EXPRESSED TRANSCRIPTS IN SHEEP TAIL FAT
Review round: 2
Reviewer: 2

Output:
Basic reporting: After checking the original text and its response carefully, I think that the current quality of the paper is suitable for publication in peer J. This manuscript is written in a standard manner, scientific issues are explained clearly.
Experimental design: There are indeed some problems with the experimental design. The fat tail trait is a complex quantitative trait. It mainly includes two main traits: fat deposition trait and long and short tail trait. Hu sheep is a short fat-tailed sheep breed, while Dorper sheep is a long thin-tailed sheep breed. Therefore, I think it would be more meaningful if choosing a short thin-tailed sheep breed such as Tibetan sheep as a control in this study.It is more scientific for studying fat trait.
Validity of the findings: The results of the research data are relatively reliable, and the analytical methods are appropriate, so the conclusions obtained are relatively reliable.
Additional comments: N.A.


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: PUNICALAGIN, A POMEGRANATE COMPOUND, INDUCES APOPTOSIS AND AUTOPHAGY IN ACUTE LEUKEMIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This paper reports data on antileukaemic effects of punicalagin, being a pomegranate compound, evaluated in in vitro studies with the use of NB4 and MOLT-4 leukemic cell lines. Although the manuscript presents some potentially interesting results, there are a number of points concerning their presentation and discussion that need to be addressed.

Specific points
1. “Introduction” and Discussion” sections require some reediting to avoid repetitive information found in both sections.
2.  It would be worth to include an additional figure presenting the chemical structure of punicalagin.
3. “Materials and methods” section – providers of punicalagin, daunorubicin and some chemicals used in the study should be given.
4. “Results” section, lines 157-162, 163-164 and “Discussion” section, lines 231-232 – to demonstrate that punicalagin acts synergistically with daunorubicin, an isobolographic analysis should be performed.
5. “Results” section, line 163 – the statement: “The normal PBMCs were less affected than leukemic cells” requires more detailed comment.
6. “Discussion” section, lines 213-214 – the p-values should be shown to demonstrate the correctness of the statement that the cytotoxic effect of punicalagin was higher on NB4 than MOLT-4 cells.
7. Some sentences/terms require careful revision, e.g. i. Abstract, lines 22-23: “The mRNA expression of apoptosis and autophagy was determined using reverse transcriptase-quantitative PCR”; ii. “Materials and methods” section, line 110 : “… apoptosis cells were measured by FACScantoII flow cytometer …”; iii. “Results” section, line 183: “… we examined the alteration of apoptotic genes”; iv. legend to Figure 6: “ … mRNA expression level …”.
8. Figure 4C – given the observed differences in apoptotic effects of punicalagin on NB4 and MOLT-4 cells,  it would be more interesting to present the percentages of early apoptotic, late apoptotic and necrotic cells, respectively rather than just showing the total percentage of apoptotic cells.
9. Some English and editorial work should be performed.

Minor points
1. Table 2 and Table 3 – it should be added that the “mean ± S.E.M” values are given in %.
2. Legend to Figure 7 – the given full name of APAF-1: “apoptotic peptidase activating factor 1” should be replaced by “apoptotic protease activating factor 1”.


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

Input:
Title: PUNICALAGIN, A POMEGRANATE COMPOUND, INDUCES APOPTOSIS AND AUTOPHAGY IN ACUTE LEUKEMIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: 1) The in vitro anti cancer properties of phenolic compounds contained in pomegranate juice and in particular of punicalagin have benn largely related in the past. In particular, the cytotoxic and proapoptotic effect of punicalagin on MOLT-4 cell line, one of the two leukemic models described here, have already been reported (Dahlawi, H. 2013 J SCI NUTR 1(2): 196-208).
2) The authors should provide some insights into the physiologic relevance of the punicalagin concentrations used. The complex metabolism of ellagitannins in the gastrointestinal tract strongly decreases their bioavailability, rendering their presence in the blood very unlikely. (Espin, JC 2007 J Agric Food Chem 55(25):10476-10485).
3) In the methods section, the authors do not mention in which buffer the chemical compounds were dissolved.
4) Related to the previous point, it is not mentioned whether the in the control groups the cells were treated with vector buffer at similar concentration as in the treatments groups, or whether cells were just cultured in normal culture medium.
5) The authors do not provide any information about the design of the primers used (home-designed or from literature, in this case cite sources), on their amplifications efficiencies (an essential data to assess whether the deltadelta Ct  method can be applied without efficiency corrections) and on stability of the housekeeping gene across treatments.
6) The STITCH analysis based on such a little set of data may lead to overinterpretations.
7) The term synergy to describe the effect of punicalagin in combination with daunorubicin is misused. Indeed, the cytotoxic effect of the two compounds used in combination is evel lower than the sum of the two used separately.
8) in the first figure the authors should discuss the absence of a significant difference between 24 and 48 hours of tretments at similar concentrations.
Validity of the findings: The data presented clearly demonstrate that treatment of cells with punicalagin, alone or in combînation with chemotherapeutic drug, induces NB4 and MOLT4 cell lines apoptosis and autophagy. Nevertheless, the insights provided in this study, as compared to the state of the art, is very limited. The authors omitted to discuss the relevance of the experimental conditions applied and the suggestion to use punicalagin as an anticancer drug, in this case for the treatment of leukemia, based on the presented data, exceeds the level of simple and reasonable speculation and should be omitted.
Additional comments: The topic covered by the study presented in this article is interesting, but researchers active in the field of polyphenols effect on different tissues/cells type in vitro should always carefully design their studies considering the bioavailability of the compounds in vivo, in order to remain in a range of physological relevance.


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: PUNICALAGIN, A POMEGRANATE COMPOUND, INDUCES APOPTOSIS AND AUTOPHAGY IN ACUTE LEUKEMIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In the revised version of the paper the Authors included modifications that followed all detailed remarks and suggestions of the reviewer. The revised version of the manuscript may be accepted for publication after suitable linguistic and editorial corrections.


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: PUNICALAGIN, A POMEGRANATE COMPOUND, INDUCES APOPTOSIS AND AUTOPHAGY IN ACUTE LEUKEMIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: Compared to the first version, the authors completed the article with some of the information requested. Nevertheless, some of the concerns were not answered. For example, nowhere is mentioned how the primers were designed and no information is provided on the efficiency of the different amplification systems. Moreover, the term synergy is still misused.
Validity of the findings: The work presented does not provide insights into the topic addressed. Changes implemented in the article are not sufficient to render it suitable for publication. Despite previous comments suggesting to eliminate speculations on the possible use of punicalagin in treatment of acute leukemia, the authors insist. In this version, they go even further in speculation suggesting the possible use of deliver vehicle molecules to provide punicalagin to target organs.
Additional comments: The changes introduced in this new version does not provide any qualitative improvement to the article. Both the experimental design and the arguments in the "discussions" section of the results remain questionable.


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: PUNICALAGIN, A POMEGRANATE COMPOUND, INDUCES APOPTOSIS AND AUTOPHAGY IN ACUTE LEUKEMIA
Review round: 2
Reviewer: 3

Output:
Basic reporting: Sufficient literature review was done, however the findings of different studies were just incorporated in the writing without analyzing and synthesizing them. There is lack of coherence in the flow of the article. Clearer problem statement, research gap and novelty of this study should have been included in the introduction.
Experimental design: No comment
Validity of the findings: The data are presented clearly and in organized manner by the authors. However, the claims of the findings could have been stronger if there was a proteomics study done on the proteins of interest. Given that the main claim of the study is that punicalagin induces apoptosis and autophagy, it is crucial to provide sufficient evidence through relevant experimental design. Some of the important points to consider are given below:

Important Points
1.	Combined cytotoxic effect of punicalagin and daunorubicin was studied in MTS but not in the rest of the experiments. Authors must justify the reason to utilize punicalagin (only) treatment for most part of the study instead of combination therapy as tested in MTS.
2.	IC50 was achieved for both 24 and 48-hour time points with small difference (eg. 57.1 and 53.5 μg/ml in NB4 cells for 24 and 48 h of treatment respectively). Provided there’s no significant difference in IC50 between the two time points, the authors did not mention why the following experiments only used IC50 of punicalagin achieved at 48 hours.
3.	Gene expression does not prove the existence of the proteins (we can’t be so sure if they are translated unless tested) or their concentrations. It is only an indirect conclusion. Hence, to confirm and quantify proteins of interest, western blot or proteome profiling must have been performed. Please refer studies with similar findings that had done proteomic works (Ganesan, Sinniah, Chik, & Alshawsh, 2020; Wang et al., 2013).
4.	Analysis through STITCH is a great aiding tool but it cannot be used as an evidence of protein interaction between punicalagin and NB4 or MOLT-4 in this study as STITCH provides a generalized analysis, not cell-specific.
5.	The claim that punicalagin enhances effect of daunorubicin (line 234-235) needs to take into consideration of the mechanism of actions of these 2 different compounds and possible combined side effects given that the combination treatment is only studied in MTS in this study and not throughout.
6.	Reference for line 251, “The induction of apoptosis by natural products has been reported in leukemic cell” is missing.
7.	The discussion part could be elaborated better because there is more literature review than discussion of the results of the study. For example, lines 294-303 are more suitable to be included in the introduction instead of discussion.
8.	There are a few grammatical errors and incorrect sentence structures that the authors need to rectify.
Additional comments: The study explores a very interesting subject and the authors have made genuine efforts to achieve the aim of this study. However, more attention should have been given to the study design to be more comprehensive in investigating the apoptotic and autophagic activity of punicalagin in acute leukemia.


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: PUNICALAGIN, A POMEGRANATE COMPOUND, INDUCES APOPTOSIS AND AUTOPHAGY IN ACUTE LEUKEMIA
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
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: DIFFERENTIAL P16 EXPRESSION LEVELS IN THE LIVER, HEPATOCYTES AND HEPATOCELLULAR CELL LINES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript deals with p16 expression levels in different cell models, aiming to discuss the implications of expression differences. The manuscript contains several items which need to be corrected. The Introduction Section contains relevant information.
Raw data is shared by the authors; however, it seems that Table 1 and Figure 2 present the same data.
Experimental design: The authors used appropriate standard qRT-PCR technique and described the cell culture methods in sufficient detail.
Validity of the findings: The raw data Table contains all necessary values for the calculation of ΔCt. To see the real differences in p16 expression the relative quantification of the gene expression should be presented in the Figure and further discussed.

Besides the descriptive Statistics, the authors should also perform the basic analysis of the obtained values to test for the group differences.
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: DIFFERENTIAL P16 EXPRESSION LEVELS IN THE LIVER, HEPATOCYTES AND HEPATOCELLULAR CELL LINES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled "Differential p16 expression levels in the liver,
hepatocytes and hepatocellular cell lines (#63307)" is a well written manuscript.The article explores the expression of the p16 gene in various liver cell lines as well as in liver tumor models using RT-qPCR technique. The literature references are sufficient with respect the field. In the results, they observed that there is different expression of p16 depending on the cell types or liver tissues studied. The authors conclude that the different expressions of p16 in different cell cultures or tumor tissues could perhaps be due to metabolic changes of each cell type studied.
In my opinion, it is necessary to study the expression of the p16 protein in order to be able to correlate it with the expression of the gene in this study.
Experimental design: The study seems relevant to me, as well as the question that the researchers ask themselves. However I believe that Western blot or immunohistochemical studies using antibodies against p16 are necessary to study the expression of the protein in different cell cultures and in liver tumor tissues.
Validity of the findings: I think the study is relevant since p16 is an important protein in the study of fibrosis, senescence and cancer in the liver. However, I believe that the study of the p16 gene has to be complemented with the expression of the protein.
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: DIFFERENTIAL P16 EXPRESSION LEVELS IN THE LIVER, HEPATOCYTES AND HEPATOCELLULAR CELL LINES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors additionally improved the text according to reviewers' comments.
Experimental design: Authors additionally improved the methods according to reviewers' comments.
Validity of the findings: Based on the additional experimental data the findings are discussed further.
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: ON THE ENIGMA OF PALAENIGMA WRANGELI (SCHMIDT), A CONULARIID WITH A PARTLY NON-MINERALIZED SKELETON
Review round: 1
Reviewer: 1

Output:
Basic reporting: This MS uses a correct profesional English. The MS is also well structured. Regarding the addional files, there is an image on the first page of peerj-60056-S4_Article_S4 that should be improved as it is blurred.
I recommend to give the information recorded on the Material chapter in a table.
Finally, there are several issues that should be addressed before its publication. Please see the PDF.
Experimental design: Reseach well exposed with rigorous study of the specimens under CT.
The method has a detailed description.
Validity of the findings: It is a meaningul study of a conulariid species neglected for almost 150 years. It is the first time that has been revisited with new technology, giving new information about its microstructure.
Additional comments: I am very glad the authors wrote this MS. It is a well-written, needed, and useful MS of an unusual conulariid species. The authors, however, need to improve the Palaeoecology chapter as the justification they use is not completely right.


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: ON THE ENIGMA OF PALAENIGMA WRANGELI (SCHMIDT), A CONULARIID WITH A PARTLY NON-MINERALIZED SKELETON
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting of the article is well done (see comments for further details).
Experimental design: The experimental design could use some work (see comments for further details).
Validity of the findings: The validity of some findings are uncertain, or at least needs to be further elaborated on (see comments for further details).
Additional comments: Review of Kröger et al, P. wrangeli
The manuscript “On the enigma of Palaenigma wrangeli (Schmidt), a conulariid with a partly non-mineralized skeleton” sheds new light on an enigmatic fossil that has been known about for sometime, but requires more advanced analysis than traditional paleontological studies could provide. The authors use their microCT method well, illustrating the fossil in fine detail. The microstructure and morphology are the paper’s strongest attributes and make the most compelling case for the author’s conclusions. The ESEM and EDS data have some of the largest gaps and do not contribute well to the stated conclusions (even though I agree with them!). Overall this is good work and the authors have shown new details of an obscure organisms. I would recommend publication after heavy modification.
See below for further comments, ranked by importance then line.
1. EDS Data Concerns: The EDS derived data is the thorniest of issues for this manuscript. It is the weakest instrument argument in the paper, but the sole reason for it even being in the MS (showing the presence of phosphate) is critical to the inferred taxonomy of the fossil. For examples of standard presentations of EDS data I would recommend looking at any article on the subject from James Schiffbauer at the University of Missouri, USA. EDS issues from the manuscript are as follows:
1a: Operating Conditions. What software and operating conditions were used during EDS collection? What kind of BSE detector was used? Was it a ‘copper wafer’ or ‘cone’ style? Why was that detector used? Why did you choose those and why did you use BSE rather than just SEM imagery? Likely it was for image clarity using ESEM conditions, but it wasn’t stated. I was impressed by the crispness of the ESEM images!
1b: Point Counts: Why are there only 2 points analyzed? Yes, there are thousands of counts for each point, but only two points is far to few to account for errors. These errors range from matrix (or other layers) inclusion or cover, time-of-flight issues, angle of materials, differing fossil preservation, or even differing vapor concentrations from the ESEM conditions. I completely understand that rare and limited fossils, with limited surface exposure, can be difficult to image, but it seems like there is more space that can be sampled. The more sampled points, the more robust your conclusions can be! If more sampling does occur, a cross-comparison with the known outgroup (the conulariids) should be considered.
1c: Context: Where are these EDS points taken? It is not clear what specimens these EDS points come from. The sample imaging spots are too zoomed in to give the reader any context to where they are on a specimen nor are they pointed out on the imaged specimens from Fig. 7. If possible, EDS maps are a great resource to show material differences in fossils.
2. CT Figures in Space: Presenting 3D data via 2D media is ALWAYS a hassle and it takes a few goes to get it right. The CT data (which is really microCT) presented here is outstanding and the details of the fossils in figures 2-6 are readily apparent. However, there are times when it is not clear how the fossils are rotated because the reader is not as familiar with the materials as the authors (again, been there). The best way to illustrate 3D specimen movement is with the tried-and-true XYZ cross (with the different coloured arrows). The author’s have already standardized the main viewing axis that most CT imagery is viewed from, so adding the XYZ parts in shouldn’t be too difficult. It can also be added to Figure 2 even though that is in a slice/cut form (which, again, has great resolution).

3. Preservation/taphonomy: This is not a critical issue for the manuscript, but it is related to many of the other topics of importance. Are there any other phosphatic organisms from the same samples (or rocks) that can be sampled with the EDS to show a similar preservation style, or at least a consistent signal in the analytical equipment? Can the matrix they have been found in support the preservation that the manuscript concludes then?
Line comments:
Line 22: This is actually microCT and should be mentioned as such throughout the MS.
31: Non-italicized P. wrangeli. Make sure to re-check MS for correct formatting.
37: This is a great opener, though the citation gets in the way a bit.
38: The description of the fossil is great, but a reconstruction (even old vs new) is missing as well as a regular light photograph. Those figures do take up space and are not critical, but they give the readers context on this enigmatic fossil.
109: This should start a new sub-section, as this is focused on collection and specimen management more than ‘methods’ proper. In my copy of this the link to the geo-collection is all there and not a hyperlink, which could be odd formatting, but should be made sure is fixed before publication.
119: We all know what a PDF is so that’s a bit odd. The author’s don’t state that every image in the PDF is a JPG do they? If that’s wording that ICZN requires, then so be it.
150: Given how much text is written to describe these specimens, images should probably be provided. I agree with the authors that the MS doesn’t need require them, but they could be put in the Supplemental Materials.
207: Is there a way to show the degrees of the pillars in the MS images?
210: This is a commentary more than suggestion: have the authors thought of doing an ontological analysis of this material? That would be interesting to see how each section was added on and what that could say about growth in these organisms.
250: Can the empty spaces be seen on any CT slices? If so, they should be pointed out or a cross-section shown.
275: Are there any other examples of microbial-fungal (maybe even boring sponge?) post-mortem damage on conulariids or other phosphatic organisms from the Ordovician? If so, they should be compared, otherwise this is just kind of hanging out there.
280: I see what you did there. Good opening, but I had to go back to the intro to get it.
300: The EDS data needs some work to make conclusion #1
310: The proposed new family requires way more description than is provided here. Maybe the authors are working on a more formal description in another MS, but without the rest of the usual description work this could cause taxonomic trouble. The section could end well with just the sentence from line 309 “Taking the general similarities…”.
344: I mostly agree with this conclusion, but this is the weakest of the conclusions reached. There is evidence of mud/soft substrate holdfasts for conulariids, but NOT at the sites with P. wrangeli, so a correlation is going to be hard to setup. Is there any evidence of any kind (from other fossils maybe) in the sampled rocks for a soft substrate? This conclusion needs refining.


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: ON THE ENIGMA OF PALAENIGMA WRANGELI (SCHMIDT), A CONULARIID WITH A PARTLY NON-MINERALIZED SKELETON
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is an interesting manuscript that describes some very worthwhile detailed instrumental analyses of an unusual fossil species. The MS is relatively short and clear, and the historic introduction is pleasant and nicely told. The science seems to be solid, but I do have some suggestions about how the results should be communicated.

First of all, the English could be improved somewhat by an edit. Although the writing is mostly good and clear, I did find that it was clumsy and non-idiomatic in quite a few places through the text. Here are a few examples:
Line 52 -
“it would have been easy to be found by the experienced and dedicated fossil hunters.”
Line 74 - “give an opportunity to take the mysterious species under a new scrutiny”
Line 91 - “subjected for microstructural observation”
Line 244 - “This is similar as in conulariids”

Perhaps more importantly, this paper could be far more impactful if the figures are made more accessible to the reader (more "user-friendly"). In the early part of the paper, it would be extremely helpful to have a figure showing photographs of some of the fossils under normal lighting conditions. The images produced using X-ray computed tomography etc. are very strong, and the 3-D videos are beautiful, but I found I wanted to know what these unusual fossils look like to the naked eye, and I had to do a Google search for photographs early in my reading of the paper. Such photos should be included.

In the Morphology section, it would be very useful to have a diagram showing salient structural features. Since this is not a “standard” conulariid, I found that I was a bit “at sea” reading the text and trying to relate it to the images derived from tomography and SEM. A labelled diagram would help a lot; it would also be helpful to have annotations or labels added in appropriate places on the photographs.

Late Ordovician apical structures are also documented in Robson and Young, 2013:
ROBSON, S.D. AND G.A. YOUNG. 2013. Late Ordovician conulariids from Manitoba, Canada. Journal of Paleontology, 87(5):775-785.
Experimental design: The research question is very well defined, and the authors have chosen instrumental approaches that allow them to look at the fossil's structures in considerable detail. Palaenigma wrangeli is a very unusual fossil that was not well understood, so this work does clearly address a gap in our knowledge of conulariid-like forms. I am not able to comment on the details of the instrumental work, or whether it is described in sufficient detail, as I have little experience of tomography and very limited SEM experience.
Validity of the findings: Overall, the findings seem to be sound and useful. Considering the systematic affinities of this remarkable fossil, it seems entirely reasonable that the authors have assigned it to a new family.

My one question on the findings relates to Lines 336-344. If the extreme apices are not known, how can the original life habit be determined? Either I am missing something, or I don’t understand the logic in this paragraph - the authors need to review this and try to make it clearer.
Additional comments: I have a couple of remarks, in addition to what is stated in the above sections:

Line 62 -  Tetradium is generally not considered to be a coral, nowadays, and is quite possibly a rhodophyte. Please look at Steele-Petrovich, 2009: https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1502-3931.2008.00146.x

Line 338 - The “1” is missing from “Brood, 1995”


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: ON THE ENIGMA OF PALAENIGMA WRANGELI (SCHMIDT), A CONULARIID WITH A PARTLY NON-MINERALIZED SKELETON
Review round: 2
Reviewer: 1

Output:
Basic reporting: The text is clear and well structured, with enough bibliographical references.
The images and videos have good quality.
Experimental design: It is a rigorous invstigation and well supported.
Validity of the findings: It is the review of an exisitng species and the creation of a new family (Palaenigmaidae fam. nov.). It is important for advancing in the conulariid research.

The conclusions are well stated and correct.
Additional comments: The text has been improved regarding the previous version, including the palaecology chapter.


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

Input:
Title: INTEGRATED BIOINFORMATICS ANALYSIS REVEALS DYNAMIC CANDIDATE GENES AND SIGNALING PATHWAYS INVOLVED IN THE PROGRESSION AND PROGNOSIS OF DIFFUSE LARGE B-CELL LYMPHOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Manuscript #62901 performed bioinformatic analysis on the gene expression profiles of diffuse large B-cell lymphoma (DLBCL) patients from the Gene Expression Omnibus database. By comparing to non-tumor controls, the authors claimed that they identified 15 differentially co-expressed core hub genes in DLBCL. Such analysis might be very helpful for understanding the mechanisms, guiding the diagnosis and therapy of related diseases.
The language needs to be improved. There are a lot of grammar errors, and some phrasings make comprehension difficult. For example, lines 213-215 and 227 are difficult to understand. The names of different types of T cells are normally written as CD4+ memory T cells, CD8+ T cells, and regulatory T cells, instead of the expressions in lines 235-246.
Labels in the figures are not clear, especially Figure 1, 2, 5, 6, 8, 11, 12.  It is almost impossible to read the labels in these figures and get any detailed information. The descriptions in the main text are also not clear enough. For example, the authors explained the method they used for figure 1C but did not explain what information the readers can get from this figure. There are similar problems in the other figures too.
Experimental design: The experimental design and descriptions are clear. The authors used many different methods to do the analysis and presented large amounts of data in the manuscript. It might be necessary to compare the data with other tumors to see if these 15 hub genes are specifically upregulated in DLBCL or are generally upregulated in different tumors.
Validity of the findings: The authors need to include some other types of tumors as controls to see if their findings are unique to DLBCL. Some of the figures seem not relevant to the main conclusions, for example, the immunohistochemistry data in figure 13 showed the localization of the proteins in the tissues, however, I don't think it is relevant to the claims made by the authors. In addition, the images are not clear and it is hard to tell the differences among different samples.
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: INTEGRATED BIOINFORMATICS ANALYSIS REVEALS DYNAMIC CANDIDATE GENES AND SIGNALING PATHWAYS INVOLVED IN THE PROGRESSION AND PROGNOSIS OF DIFFUSE LARGE B-CELL LYMPHOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the manuscript is well written, provides the necessary background information.
Experimental design: This paper performed bioinformatics analyzes, trying to reveal some potential diagnosis, prognosis markers and novel therapeutic targets of diffuse large B-cell lymphoma (DLBCL). The authors first retrieved the raw gene expression profile from the public database, identified the differential gene expression (DGE) between the DLBCL patients and non-cancer controls, and analyzed the function and pathway. They then analyzed the infiltrating immune cell types in the tumor microenvironments and found that the T cells CD4 memory activated, T cells CD8, and macrophages were the most represented cell fractions within the DLBCL microenvironment. They further constructed the protein-protein interaction (PPI) network and finally identified 15 core hub genes, which were associated with COVID-19.
Validity of the findings: However, there are some major concerns need to be addressed.

1.	A major concern, however, is about the novelty and biological significance of the results. Most of the findings were revealed previously, for example, the upregulation of p53 and KRAS genes, the immune cells infiltration profile in DLBCL patients. The paper should focus on a discussion about their novel findings and the potential biological significance of those genes.
2.	The relationship between the 15 core hub genes and COVID-19 seems far-fetched based on the limited discussion provided in the manuscript. The authors should provide more convincing evidence/discussions regarding this conclusion.

Minor comments:

1.	Figures are too blurry to see the details. High resolution figures should be provided.
2.	The core hub genes and other major findings should be highlighted in the illustrations, so that they can stand out from thousands of genes clearly and the readers can notice them easily.
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: INTEGRATED BIOINFORMATICS ANALYSIS REVEALS DYNAMIC CANDIDATE GENES AND SIGNALING PATHWAYS INVOLVED IN THE PROGRESSION AND PROGNOSIS OF DIFFUSE LARGE B-CELL LYMPHOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Manuscript #62901 performed bioinformatic analysis on the gene expression profiles of diffuse large B-cell lymphoma (DLBCL) patients from the Gene Expression Omnibus database to identify differentially expressed genes in DLBCL. The revised manuscript is overall well written, provided a good background introduction. The language has been greatly improved. However, there are still some minor grammar errors and typos. Please proofread the manuscript carefully.
Experimental design: The experimental design and descriptions are clear. The authors used different methods and analyzed the data from multiple aspects. They identified the differentially expressed genes, analyzed the protein-protein network, and also looked into the tumor-infiltrating immune cell profiles in DLBCL.
Validity of the findings: The authors used many different methods to do the analysis and presented large amounts of data in the manuscript. The results are overall well explained and conclusions are well stated.
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: INTEGRATED BIOINFORMATICS ANALYSIS REVEALS DYNAMIC CANDIDATE GENES AND SIGNALING PATHWAYS INVOLVED IN THE PROGRESSION AND PROGNOSIS OF DIFFUSE LARGE B-CELL LYMPHOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: While I still have some reservations about the phrasing and explanation of some of the results, the overall conclusions are clear and acceptable.
Experimental design: No comment
Validity of the findings: The revisions have addressed my major questions regarding the novelty of this MS, and the core genes have been highlighted clearly.
Additional comments: The manuscript is now suitable for publication.


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

Input:
Title: GOING ROGUE: WHAT SCIENTISTS CAN LEARN ABOUT TWITTER COMMUNICATION FROM “ALT” GOVERNMENT ACCOUNTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: >> clear english

Fairly ok.

>> literature references and context

Poor.

>> article structure, figures, tables. data

Structure is okay, but not the content.

>> results to hypothesis

The main analysis done by authors are summarised in lines 117-124.

The second point on the statistical work to determine factors that drive attention is a valid one, and the authors have done this job reasonably well, except that they have both intercept and all levels of a categorical variable in their model.

The first point is baseless. They have compared nothing -- they have only used specific methods (already existing) for specific tasks. Next, I do not understand what is "novel" in testing that a certain non-parametric test can be "applied".
Experimental design: >> original primary research; within scope of journal

Yes, the topic is within scope of journal.

>> research question well defined; explains filling of knowledge gap ??

The authors claim that there are very few studies in microblogging analysis that have combined both qualitative text analysis with more quantitative network analyses. They claim that mixed-method approaches have become common in health care research -- it seems that authors are inspired by the usage of mixed-method in some health related papers.

The authors say that quantitative microblogging analyses have followed two main forms: network analyses and unsupervised algorithms modeling, and continue to claim that the usage of such methods outside computer science fields has been limited. I do not understand how the authors are claiming such things -- may be they have read several papers in all fields.

In lines 73-84, the authors describe their knowledge of what methods have been applied to microblogging studies. In my opinion, the authors have very limited knowledge on what methods have been applied to understand various issues in microblogs like Twitter. A wide range of simple to sophisticated methods in areas of statistics, machine learning, networks, time series, etc. have been used already. I do not understand why the authors say that "the methods employed do not extend beyond those described above" in line 82-84.

In lines 99-100, the authors say that "there have been multiple studies to confirm automated sentiment coding as an effective and time-saving proxy for manual coding", but they "have been unable to find any mention of their use singly or in conjunction for social media text and interest analyses". This is a surprising statement -- there are tons of papers out there on Twitter data, which have used sentiment analysis -- on large data sets like Twitter nobody does manual coding of sentiments!!

So, upto this point, it clearly shows that the authors are trying to fill a gap of "applying some menthods" to microblogging analysis. They think that they are the first ones to apply such methods -- and its not true. So they are not filling any knowledge gap which they claim in the title of the paper as "quantitative methods". First, they have not developed any method. Second, methods which they are "applying" to their dataset can not be claimed as a "new method".

Next, in line 108, they write "To test methods against a real dataset...". They have not developed any method, I do not understand what they are testing. In line 118, they say they are "testing the applicability of non-parametric test". Any method, in general, can be applied to contexts that lie within the assumption of the method. I don't understand what the authors are testing.

In line 120-121, the authors say that they are interested to "determine what elements of a tweet make it more likely to be shared and accrue likes". This is the only statistical thing with some hypothesis that the authors are really doing in this paper. Everything else they have written about "quantitative methods" is baseless, naive, and shows that they have no idea of existing literature.

Again, in line 125, the authors mention "In testing these methods...". Frankly there is no method they are testing -- they are only applying existing methods to a microblogging context, which they think is very novel.

>> data

The data collected is of good quality.

>> rigorous investigation, ethical standard

The authors have used GAMM to investigate the effect of tweet sentiment and various features of tweet on attention (retweet, likes).

There are 7 types of sentiments in their analysis -- afraid, angry, etc. In Table 1, they have used all 7 types and they also have the intercept term! Usually when a variable has N categories, N-1 categories are included in the model if the model has an intercept. This is to avoid multicolinearity.

>> methods described with sufficient detail & info to replicate

In line 279, the authirs say "GAMM was an effective method for identifying underlying variables that could predict if a tweet would be liked or retweeted". The only method they have used is GAMM, and now they are saying it is an effective method -- to what other methods are they comparing GAMM to?
Validity of the findings: >> impact and novelty

The only results which are interesting are (1) tweets categorized as “angry” were both significantly more likely to be retweeted and to be liked, (2) words including “peer reviewed,” “knowledge,” and “verify” were associated with extremely high levels of retweets per tweet, and (3) tweets containing links to actual peer-reviewed papers were less likely to be retweeted or liked than those with other links.

The authors have expressed their intuition for such observation in the discussion section.

In my opinion, these are the "real results" from the authors in this paper. To me, these results are surprising and novel. The entire paper should have been written around these results. I do not know why the authors emphasized a lot on trying to write about their novelty on "quantitative methods" -- there is none in their work.

>> underlying data have been provided; replicable

With the list of Twitter accounts provided in supplementary, a researcher can ideally collect that data from Twitter and replicate the authors' study.

>> well stated conclusions; linked well to research question; limited to supporting results

The title of the paper is "Quantitative methods and perspectives..". Frankly, I do not see what new methods have been proposed in the paper. The authors have only used existing methods to analyse a particular hypothesis.

The abstract is poorly written. The last line of abstract -- "Overall, our results highlight analytical methods to quantify audience behaviour in microblogging platforms, informing possible pathways for science communications" -- is completely misleading. The authors have only used some methods to analyse their hypothesis. This does not mean that they are quantifying or measuring behaviour -- they are only investigating the nature of a particular behaviour.
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: GOING ROGUE: WHAT SCIENTISTS CAN LEARN ABOUT TWITTER COMMUNICATION FROM “ALT” GOVERNMENT ACCOUNTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is written with a professional use of English (but I am not a native speaker). References about previous studies justifying the goal of the study, methods used are included. Maybe, authors could try to cite more recent papers because the number of papers published analysing Twitter data has increased in last times.
The structure of the paper follows the standard indicated by the journal. It contains an introduction section, material & methods, results, discussion and conclusions. I recommend review the structure of material & methods because it contains 5 subsections and, for example, subsection 2.5 is summarized in 6 lines (maybe it is not necessary too many subsections). In addition, the results section should be reviewed because, for example, when describing Table 1 I think that some parts are missing.
Figures are relevant to check results, nevertheless, I would try to improve the resolution because they are quite difficult to analyze due to the size of the image (specifically, Figure 1 with CCA).
Experimental design: The paper is within the Aims and Scope of the journal.
The abstract of the paper states two main objectives. First, to compare communication strategies and test the applicability of different tests, methods. Second, to analyze what elements of a tweet gain attention. In my opinion, after reading the paper, the general idea that the reader gets is how to use different methods to analyze data from microblogging. I mean, the second objective of the paper is less present until the section of results. I think that during the introduction this part should be emphasized a little more because it seems that the important/novelty is the use of CCA, GAMM, etc and not where alt accounts matter, whether the content of the tweet matters, etc. As I said, It is not until the results or even discussion section, when reader notes the importance of sentiments, etc (and is one of the goals of the paper)
Regarding the material & methods section, in my opinion, authors describe in a good way (with full of details), the gathering of data, cleaning process, etc.
Validity of the findings: I think that this study presents novelty results and give ideas for future analyses using Twitter data, for example, new statistical analyses that can be employed from other fields.
I would recommend to author to specify the main limitations of their analysis. In addition, I would like to know if these results could be generalized to alt and official accounts but considering other issues and not the environmental issues? I would also like to know what is the future direction?
Additional comments: Thank you for providing the opportunity to review the manuscript. I think that this paper is interesting and novel analyzing microblogging data paying special attention to methods but also the phenomenon of alt vs. official accounts.