Sample Review

Modulated Expression Of Subcortical Proteins By Serotonin And Noradrenaline Reuptake Inhibitors RtR23 and RtR57 In A Rat Model Of Chronic Stress

NewReview Manuscript Summary

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Manuscript Overview
This interesting report examines the effect of SNRI compounds RtR23 and RtR57 on levels of subcortically expressed high abundance proteins in an anhedonic rat model of chronic stress. This content review from AJE focuses on suggestions we believe the authors may want to address prior to journal submission. * While the reviewers believe this paper is a valuable contribution to its field, that importance is not made apparent enough in the Abstract. A concluding statement incorporating a comment on the newness and potential impact of the findings would help to engage a journal reader. * The Introduction can be shortened and made more concise. * The Methods are carefully described, however, the reviewers pointed out some areas (pcr reaction, statistics, etc.) that might benefit from rewriting as suggested in the review. * The Results section also requires rewriting for clarity. Equally important is the need to correct discrepancies regarding several data points listed in figures, which are indicated. * The Discussion section could easily be made more informative by placing the authors' results in the context of previously published studies more so than in the current paper. A more complete comparison of the authors' data to literature reports would help the reader to understand the newness of the findings and how they extend the work of others. * Clearly stated Conclusions at the end of the main text would leave the reader with the most important messages to take away from this research report. NewReview at American Journal Experts thanks the authors for the opportunity to offer this pre-submission peer review. We wish you the best of luck with rewriting and submitting this impressive work to a highly regarded journal.

Detailed Reviewer Comments by Section

Title page
The title of this paper appropriately reflects the purpose of this study, i.e., to investigate the impact of SSNRIs RtR23 and RtR57 on expressed products in a rat stress model. The title page information is currently incomplete, which may have been intentional for this pre-submission review. As a reminder, prior to submission, please include: full authorship with

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affiliations, corresponding author with contact information, key words (usually up to 5), and a running title (usually no more than 40 characters and spaces). The word count and funding sources also may be required by your intended journal. Please consult the journal's Instructions for Authors on for details.

Abstract
The abstract is much too brief (3 sentences). Consider including opening sentences that emphasize the motivation for this study based on relevant background literature. In one or two sentences indicate the important issue(s) not addressed in previous reports of others that relate to this class of compounds and the described behavioral model. What then is the objective of the present study that follows logically from that preceding background information? Did the authors test a hypothesis? If so, please state the hypothesis. Briefly indicate the experimental approach and summarize the overall results. At the end of the abstract please provide a concise conclusion that can be drawn from the results. Any subsequent corrections or changes to the methods, results, discussion and/or conclusions based on the comments below should also be changed in the abstract to maintain consistency.

Introduction
The current Introduction is quite long and detailed. The purpose of this section is not to provide a general introduction a concept or field of interest, but to introduce the rationale for the specific experiments that were performed, and the hypothesis that drives the studies. This is to be done within the context of relevant background material. Consider removing summaries of previous reports that do not directly relate to the current research (e.g., paragraphs 4 and 5; first two sentences of paragraph 6). Such changes would help make the Introduction more focused. The authors should consider emphasizing the relevant literature on the association between SNRI-induced subcortical proteins during behavioral challenges with reference to models similar to those of the present report. Indicate the advantages of using RtR23 and RtR57 for the present purposes. How is this relevant to the authors' interest in mechanisms related to stress and drug action? Based on the background information, what new data are you seeking by means of the present study, and why is it important? Towards the end of the Introduction, a clearly stated objective is necessary. Overall, the introductory information should help to clarify the authors' motivation for carrying out these studies, the newness of the data and then highlight the potential importance of the findings to the field.

Experimental methodology
The experimental section is very well written. However, several of the techniques raise some issues that should be addressed.

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Prior to describing animal studies, please include a statement of approval from your institutional committee on the ethical treatment of animals in research. Name the committee and the institution. The quantification method used for real-time PCR appears to be the deltadelta Ct method, which is a standard method for relative quantification of transcript levels using real-time PCR. The text statement is, "The cycle number for each candidate gene ...." If this is in fact the deltadelta Ct method, it should be described as such to aid the reader's understanding and to help ensure reproducibility. Please provide references. If that method was used, should the "reference gene" be changed to the "reference sample"? Typically, there are two levels of controls (the first is the internal control gene, the second is the comparison against the external control sample). In the current report the statistical methods are incorporated into other methods. The stats are more clearly presented as a separate section towards the end of the Methods in which all specific tests are listed. The P value for significance must be specified. Also please include the statistical software package, the version and the manufacturer. Paired t tests may not be the optimal statistical test of choice to properly assess significance of these data. Since data presented (e.g., see Fig. 2) incorporate two variables (time [days] and drug) the authors might consider a two-way ANOVA instead of paired t tests. The exclusive use of t-tests in the present study may have led to overestimation of statistical significance. In this regard, the authors may want to analyze their data once again. Additional points to consider: The number of samples and number of repetitions per experiment and means are not indicated, but should be (expressed as mean value S.D. (n = X)). Both "mM" and "mmol" as used units. We suggest you convert everything to "mM" for consistency. Please reference methods when details are not provided to ensure reproducibility.

Results
The results section is very comprehensive. Significant changes can be highlighted in the text of the results (with P values indicated), but avoid repeating the detailed data from the tables (as in current paragraphs 2, 4 and 8). There are several statements (page 9) describing the side effects of both SNRI compounds at doses that were higher than used in the first set of experiments (Figs. 1-4) although the expression levels of markers remained relatively unchanged (Table 5, Fig 5). These additional data are interesting and should be included in the Discussion. The time points chosen for analysis appear insufficient to support the trend line shown for

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marker expression (Fig. 3). The authors extrapolated a straight line between 12 and 24 hours but no data is shown. Overall, this time point expression data would be more convincing by including measurements at baseline and 3 hour intervals up to 24 hours. Finally, the results section should focus on DHC8 and DHC9, which are the main products of interest. However, paragraph 7, which mentions DHC79 results "(data not shown)" seems out of place and can be omitted.

Tables and figures

General comments: The reader may find it difficult to understand the intended meaning of some figures because legends are very brief or not included. Please make certain figure titles and legends clearly describe the meaning of the data. The same is true of tables. Readers should be able to have a general understanding of a figure or table without having to refer to the text. Please check your journal instructions to see if the following are required for tables and figures legends: all abbreviations defined once again; if significance is reported, the specific statistical test for each analysis is indicated; P value for significance is included. Specific comments: * Figure 2: the axis labeled is missing (presumably "day"). In the legend clarify whether this is, presumably, the day after first administration of drug. * Figure 3: discussed above (Results). * Figure 4: In Figure 4C, 300 pmol of RtR23 produces an effect of about 70% whereas in 4D this same dose of RtR57 produces a little more than 80% effect (outside the range of experimental error). Given the similarities between the compounds, an explanation seems necessary. The different results were not mentioned in the Results or discussed in the next section. * Figure 5: the bands are not identified. Please mark them on the figure and describe them in the legend. Also, controls should be included in this figure. * Figure 6: the legend does not state which assay was used to obtain the results.

Discussion
We suggest rewriting the first paragraph as a brief summary of results and how they address the objective of the research. Subsequently, the authors should include a discussion of their experiments as compared to similar published studies. Comparisons between the authors' data and published works should highlight similarities and differences when they occur, with possible explanations for the latter. Include citations for all statements of fact. The authors may want to consider including a direct comparison of data on the effects of RtR23 and RtR57 (behavioral, biological) since, although related structurally, these compounds did not

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consistently produce similar effects (a point for further study), which was mentioned above (Figure 4). Also high RtR doses (either one) that impacted behavior did not further affect expression levels of HAPs. Is this a novel or unexpected finding? Is this observation contrary to the authors' suggestion that the high abundance proteins they describe are relevant to stress or drug action? What suggested mechanisms might explain these observations? Bear in mind, however, that causality cannot be inferred from descriptive studies.

Conclusions
Currently, the paper lacks strong conclusions. The "Conclusions" listed at the end of the Discussion are actually a summary of the results (which can be deleted). The Conclusion section should try to convey the potential impact of your results in two to three sentences (i.e., summarize what the results mean rather than what the results are). What potential impact will your data have on your area of study? Is it possible your results, potentially, will have relevance to a clinical situation? Overall, the Conclusions section is the authors' opportunity to tell the reader why the results are important.

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