Compiled on March 24th, 2017

Rubriq scorecard

Manuscript: Treatment of XX-induced hepatotoxicity through upregulation of XX

Client: John Smith

Reviewer 1

This paper has a very low chance of publications until the Western blots are corrected; they do not look clear. Dose-response data may not be a deal breaker but it would help. The authors need to discuss cause and effects in greater detail. Did XX cause autophagy or did the toxicity caused by XX cause autophagy, as is widely believed? I do not think this paper is sufficiently strong to change current opinions.

Reviewer 2

Overall, this is an interesting study on the effects of XX on mitochondrial autophagy. Because the field that will be interested in this study is somewhat small, the potential impact is limited. That said, most of the techniques used were state-of-the-art and were done well. However, the use of single methods to address a question leads to over-interpretation and some inappropriate conclusions. These issues could be improved with the addition of several new experiments.

Reviewer 3

My overall impression of this manuscript is that it will be suitable for publication with extensive editing. The overall message of the manuscript is missed due to the errors in the figures and legends. These errors need to be resolved and the manuscript checked for correspondence between the figures, legends and the with the manuscript text in general. Overall the science is sound, although some points of the results will need to be refined according to the edited figures and legends.

Quality of Research

Hypothesis, Objective, Rationale– 7.1

Key points

Meets all criteria
Rationale is unclear
Objective/hypothesis is not supported by background
Objective/hypothesis is not stated
Other - see comments

Reviewer 1

Many of the references were in obscure or lower tier journals and not in front line journals needed to justify a novel hypothesis. The references could be upgraded for a bigger impact.

Reviewer 2

The hypothesis is not directly stated, although it is inferred.
The authors do a good job of stating background information to lay the foundation for their studies.

Reviewer 3

The hypothesis/objective meet most of the criteria. However, the rationale and hypothesis could be more clearly stated in the last paragraph of the introduction. The introduction lacks background material on XX. XX is only mentioned in the last paragraph of the introduction. Thus, it is not clear from the introduction why the author is evaluating the role of XX. An overview of the role of XX in mitophagy would help ready understanding.

Methods and Data– 7.3

Key points

Meets all criteria
Missing essential references
Design/techniques not up-to-date
Missing important details for reproducibility
Missing some experimental controls
Inappropriate statistical analyses
Missing an important experiment
Approach/data not consistent with objectives/hypothesis
Other - see comments

Reviewer 1

The authors need to justify ip dosing with XX; this is not the human route of exposure.
The XX data need a dose-response study to be stronger.

Reviewer 2

The conclusions drawn are too strong considering the single method used to address the question. For example, only the use of XX is used to inhibit mitophagy after XX treatment. While this is usually an effective method, genetic blockade (e.g., siRNA against an ATG protein) or at least another inhibitor (e.g., chloroquine) should have been used to strengthen the conclusion. This note applies to several points in the paper.
The description of some of the methods is excessive and could be reduced.

Reviewer 3

The methods are missing a few key elements as follows:
1) it would be helpful to define XX as a XX inhibitor, etc.,
2) there is no mention of the antibody for XX in the western blot section, and
3) a reference should be given for the dosages of XX in the in vitro and in vivo studies. Are the dosages of XX relevant to other literature in this area? Are these dosages acute or close to physiological levels?
The animal treatment groups are not clearly defined in the methods. The order of the methods and the subtitles for the methods should be changed, as it is hard to follow the flow of the study based on the current order. The authors should give references for the in vivo utilization of Midivi-1. Lastly, the authors should consider the addition of another autophagy marker in addition to XX.

Interpretation– 6.5

Key points

Meets all criteria
Does not adhere closely to the data
Biased or overstated interpretation
Leads to inaccurate conclusions
Not supported by the data
Other - see comments

Reviewer 1

The authors put much importance on modest differences in gel density.
Some crucial data need to be rephotographed for clarity as noted.

Reviewer 2

Some interpretations are not well supported by the data presented. This includes the degree of mitochondrial fragmentation and the involvement of mitochondrial biogenesis.
The authors should also present other potential explanations for the effects of XX on liver toxicity other than effects on mitochondria.

Reviewer 3

The interpretation of the data is consistent with the results that are presented. However, the results section is confusing, and the figure legends do not match the figures. Therefore, it is difficult to follow the results section.

Quality of Presentation

Title, abstract, and introduction– 8.0

Key points

Meets all criteria
Title is inappropriate
Background/rationale is missing from abstract or introduction
Methods are missing from abstract
Conclusions are missing from abstract
Hypothesis/objective is missing from abstract or introduction
Results are missing from abstract
Other - see comments

Reviewer 1

Dose-response data may not be a deal breaker but it would help.
The authors need to further discuss cause and effects. Did XX cause autophagy or did the toxicity caused by XX cause autophagy (as is widely believed)?
I do not think this these data are strong enough to change opinions.

Reviewer 2

The very end of the introduction could do a better job of framing the hypothesis without literally stating the question. As it stands, it is a somewhat awkward way of leading into the rest of the manuscript.
The rest of the introduction is quite good.

Reviewer 3

The abstract is missing a description of the methods. This may not be (but often is) required by the journal. The rationale and hypothesis in the introduction are not clear and need to be edited. The introduction is missing background on key factors that are evaluated in the manuscript, such as XX.

Results (text)– 7.0

Key points

Meets all criteria
Excessive repetition of data (e.g., from tables or figures)
Poorly organized or not succinctly presented
Missing significance indicators (e.g., p-values)
Not focused on objectives
Other - see comments

Reviewer 1

Dose-response studies would improve the impact of this paper.

Reviewer 2

The results section is not clear, particularly towards the end. It becomes very confusing and does not flow well. I would recommend trying to write the results as more of a story, rather than just stating the results matter-of-factly.
The results should include a very brief rational for why the experiments were conducted and how the results lead to the next set of experiments.
There should be descriptions of all of the data that are presented. For example, the Western blots for the mitochondrial fission/fusion machinery are not described (other than XX).

Reviewer 3

The results section is not clear due to the figure legends not matching the figures. It would be helpful if the authors would include additional subtitles in this section that are descriptive of the data. The authors mention protein XX but do not say that this is XX.
The following does not belong in the results section: “In summary, XX-induced mitochondrial fragmentation requires the core mitochondrial fission machinery, and mitochondrial fission is required for mitophagy during XX-induced hepatotoxicity.”
Once figures/legends are corrected, the authors should revise the text of the results section to match the data presented.

Results (figures, graphs, and tables)– 2.4

Key points

Meets all criteria
Problems with ordering, numbering, titles, or labels
Data presentation is inappropriate for the experiments performed
Images are of poor quality
Figures are too complex, confusing, or unclear
Captions, footnotes, or legends are incomplete or missing
Missing important data
Other - see comments

Reviewer 1

Figures 1, 2, 5 and 6 were missing. Figure 7A needs to be replaced with a better gel photo. It looks like it was not the best gel run for all 3 bands. This figure will hurt chances for publication.
Also, XX needs to be rerun because the XX band is weak, and that will alter the ratio data. This is important and not simply cosmetic.

Reviewer 2

Overall, the figures need a lot of work, as they are confusing and do not adequately show many of the conclusions that are drawn from them.
Data for XX should be shown rather than just referred to.
Figures showing the quantification of mitochondrial morphology need to be included. These could be actual measurements of individual mitochondrial length averaged between the experimental conditions, or they could be blinded grouping of longer and shorter mitochondria. Just showing a few sample images is not sufficient.
In figure XX, the other subcellular fractions should be shown to determine the degree to which XX translocates to the mitochondria after XX treatment.
More than one siRNA should be used for XX to control for off-target effects.
The Western blots for XX are not clear and certainly are not of sufficient quality for quantification.
The numbering of many of the figures is off, and it is very difficult to determine which figure the text is referring to. For example, there are two figure 7’s in the manuscript, and the EM from the in vivo studies is not included. I will not state all of the problems here , but great care should be taken to ensure that the figures match the descriptions in the text.

Reviewer 3

The figures and legends do not correspond. It is difficult to tell if the authors have 8 or 9 figures. These need to be thoroughly reviewed and edited.
General comments:
1) XX—The authors should provide better quality images for XX and XX. The current images are not convincing.
2) It is not clear why the authors term XX as a mitochondrial contaminant and then use this a control for XX-II protein expression levels. The authors should consider utilizing the ratio of XX-II/XX-I, and 3) XX—the altered morphology induced by XX is hard to discern from the figure. Authors should consider adding arrows.
The authors should clearly explain why some factors are not described well that were evaluated in the context of the study (i.e., XX, XX, etc).

Discussion– 6.4

Key points

Meets all criteria
Missing concise and accurate summary of results
Missing discussion of potential limitations
Biased commentary
Insufficient comparison to relevant literature/previous results
Not consistent with objective/hypothesis
Other - see comments

Reviewer 1

Alternate explanations were not addressed, and the limitations of the study design were not addressed.

Reviewer 2

The discussion section does not flow well and could be improved. For example, the second paragraph seems very out of place in the context of the rest of the discussion.
Do not make direct references to the figures in the discussion. A more broad and general referral to the results is a better approach.

Reviewer 3

Overall, this section is good and discusses important concepts with the exception of the limitations of the study.
Some important references are missing in paragraph number 2 and in other places. When you state ‘previous studies have indicated’ a reference(s) is required.
It is difficult to assess the content of this section due to the mistakes in the legends and figures.

Conclusions– 7.0

Key points

Meets all criteria
Missing take-away statements
Vague, overstated, or understated applicability
Not consistent with discussion or objectives
Not supported by the data
Other - see comments

Reviewer 1

An alternate explanation was not addressed - i.e., cell death results in reduced mitochondria and ATP.

Reviewer 2

Overall, the conclusions drawn from the presented data are too strong and not fully supported. With one exception (the comment on mitochondrial biogenesis – see below), none of the conclusions are inappropriate; rather, experiments using a single inhibitor or a single siRNA are deemed conclusive, where further verification should be conducted before moving on.
The conclusion that mitochondrial biogenesis is not involved is not appropriate. No data were shown for this, and mitochondrial biogenesis was not directly addressed. That this wasn’t included is not a problem, but stating that it is not involved is not appropriate.

Reviewer 3

The conclusion lacks a clear take-away message about the relevance of the findings.
The conclusion is missing text addressing clearly 'why this study was important and implications to human health'.

References– 6.1

Key points

Meets all criteria
Problems with ordering or numbering
Not focused on current/latest literature
Do not provide informative context for Introduction/Discussion
Do not support objectives
Missing key references
Other - see comments

Reviewer 1

In need of major formatting. Some references are all caps and some are in lower case, some journals are abbreviated and some are written out, some authors are all included and some are one author with et al. These styles must be made consistent with the target journal style sheet. As noted above, many journals are obscure and not top tier.

Reviewer 2

The references are appropriate and up-to-date.

Reviewer 3

The manuscript is missing key references.

Writing– 7.2

Key points

Meets all criteria
Incorrect level of depth in some or all sections (too brief or too lengthy)
Poor/unscientific word choice or grammar
Writing lacks clarity, focus, or organization
Other - see comments

Reviewer 1

The writing was clear andthere was some degree of repetition in the Introduction and Discussion, which clarified the authors' opinions.

Reviewer 2

The methods sections contains excessive depth and could be shortened.
The results could be improved by telling more of a story rather than just stating the results.
The content of the discussion section is good, but the writing lacks flow and seems to jump from one topic to another too rapidly.
The abstract and introduction (except for the final paragraph) are well written.

Reviewer 3

Overall, the writing lacks some clarity. Clear subtitles in the results section would assist the reader in understanding the flow of data and findings.
The lack of correspondence between the legends and figures needs careful evaluation.
Clarity should be improved with correct figures and legends.

Novelty & Interest

Novelty– 6.0

Key points

New technique, method, or approach (proof of principle)
High
Med
Low
N/A
New question, theory or hypothesis (totally new idea)
High
Med
Low
N/A
New result, discovery, or perspective/synthesis (proves an established idea)
High
Med
Low
N/A

Reviewer 1

The molecular mechanism of XX toxicity is unknown, and autophagy of mitochondria is unproven.

Reviewer 2

While this is, to my knowledge, the first description of XX-induced mitophagy, many of the mechanisms by which this occurs were already known.
Although the use of the mouse model is very good, the rest of the studies focus on only one cell line.

Reviewer 3

This study is novel form the standpoint that few studies really address XX-induced mitophagy and the potential mechanism involved in the process. The manuscript adds new information to the current literature concerning XX toxicity and its effects on mitochondria. The mechanism for mitophagy has been previously demonstrated. Manuscript will be best suited to a journal in the field of toxicology rather than a more general field.

Interest– 6.1

Key points

of broad interest to researchers in this field and other fields
of broad interest within the field
of moderate interest within the field
of interest to a small group within the field
of limited interest

Reviewer 1

If this idea holds up, it will be of interest to researchers in metal toxicity.

Reviewer 2

How XX induces mitochondrial defects is a fairly specific question that will only be of interest to a relatively small group of scientists.

Reviewer 3

This manuscript will be of interest to investigators in the field of XX-induced cellular toxicity. The findings would be of lower interest to researchers in other fields as the mechanisms have been previously established in other systems.

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