All reviews of published articles are made public. This includes manuscript files, peer review comments, author rebuttals and revised materials. Note: This was optional for articles submitted before 13 February 2023.
Peer reviewers are encouraged (but not required) to provide their names to the authors when submitting their peer review. If they agree to provide their name, then their personal profile page will reflect a public acknowledgment that they performed a review (even if the article is rejected). If the article is accepted, then reviewers who provided their name will be associated with the article itself.
The authors successfully implemented the criticisms and comments raised by the reviewers during the revision period. Therefore, the manuscript can be accepted in its current form.
[# PeerJ Staff Note - this decision was reviewed and approved by Pedro Silva, a PeerJ Section Editor covering this Section #]
no comment
no comment
no comment
no comment
No comment
No Comment
No comment
The authors have responded to all my concerns and the manuscript is much clearer.
For further consideration, the authors must carefully and satisfactorily implement all referee comments.
[# PeerJ Staff Note: Please ensure that all review comments are addressed in a rebuttal letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. It is a common mistake to address reviewer questions in the rebuttal letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the rebuttal letter. Directions on how to prepare a rebuttal letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]
No comments
No comments
No comments
This study reports the differential expression of transcript genes in normal and keratoconic cornea. Over all, manuscript was well written with good scientific manner. Interesting to read by clinical scientist, those who working on corneal diseases and pathology. I have few minor corrections below.
1. Abstract: Results section Line no 39, Among differentially expressed genes were genes involved in collagen synthesis, the TGF-β, Hippo, Wnt signaling and other pathways linked to the extracellular matrix formation. Please rewrite the above sentence for better clarity for readers.
2. Key words: Please include Keratoconic cornea
3. Table-s1 – “The clinical characteristics and ophthalmic findings” could you please describe the clinical grades of KC like Amsler-Krumeich classification if not mentioned other method for classification.
- The article has multiple incidences of typographical and grammatical errors.
- It is unclear if the authors' knowledge of signaling pathways are weak or are just mis-identified. e.g. TGFB is called transcription growth factor instead of transforming growth factor.
- Literature review is incomplete in identifying the numerous studies that have identified candidate genes for keratoconus.
- An important candidate gene for keratoconus is LOX and the authors while studying this gene have failed to discuss the importance of the data or lack of thereof.
-
The experimental design is straight forward where it serves to secondary validate expression of genes identified by RNASeq previously. Techniques are apporpriate.
However, it would have been more prudent to identify SNPs for the genes associated which would have bolstered their study design rather than mere RNA expression.
The authors tend to over-interpret their data. The authors mention deregulation of multiple signaling pathways but actually do not demonstrate that. They indeed do demonstrate up/down regulation in a few genes that are pertinent to these pathways. However, there is perceived conflict in the data which is not given due discussion: e.g. Why is Smad7 (a negative regulator of TGFß pathway) down yet TGFBIP up? Are the expressions of genes down due to a inhibited activity of stromal cells or are they transcriptionally and translationally altered? It would have helped to correlate changes in gene expression with protein levels either via histochemistry or Western blots.
Authors further claim SLC4A11, Wnt 5a and other genes are not consistently altered within the disease group (see Fig. 1) i.e. large donor variability. Yet the authors talk about up/down regulation within the statistical treatment group which is not true.
The hirearchical clustering is organized haphazardly with not groupings between KTCN and non-KTCN samples. It would benefit with restructuring.
The paper is written in clear, unambiguous professional English. It uses a professional article structure with relevant figures and tables and the raw data is included as Supplementary data.
The introduction could be improved by expanding the rationale for the study. From the analyses conducted, it appears to be a replication study of previously reported findings (from these authors and others). However, the end of the introduction says the aim is to see if keratoconus patients can be distinguished from controls on the basis of gene expression in the cornea. It is not at all clear why this is the aim. Using corneal tissues to make a diagnosis of keratoconus does not sound useful when there are non-invasive diagnostic techniques that can be applied well before corneal graft is required. I think this study has confirmed some important pathways in keratoconus biology, using a second method to increase confidence in previous findings.
As noted above, the research question is defined, but I am not convinced that it is relevant or meaningful or what knowledge gap the study fills specifically. The manuscript should clarify this for the reader.
More detail is required on the methods for the choice of genes. For example, 23 genes were chosen from the previous RNAseq study, but it is not mentioned what criteria were used for inclusion. Is it a fold change? Top ranked genes? DE genes from specific candidate pathways? Similarly for the additional genes selected from the literature, how was it decided what to include and why?
This study reports a replication analysis of previous RNAseq data, using in independent method on the same patient specimens. This is valid and useful and provides increased confidence of the previous findings.
Further clarity could be achieved by indicating which of the genes in Table 1 were from the previous RNAseq study and which ones were added. The results should then be discussed in this context, indicating if any of the findings are novel (i.e. gene expression not previously studied).
The results should also be discussed in relation to the statistical significance. No adjusted p<0.05 was reported, yet it is claimed that all but 1 gene is confirmed as differentially expressed. The validity of this finding appears to be open to interpretation.
In Table 1, please indicate in the table description that these results are from the RTqPCR assay.
In Figure 1, please explain in the legend what the histogram is showing and if possible, clearly indicate which specimens are from keratoconus patients and which are from controls.
Please describe in the methods what range of indications for graft the non-KTCN patients have and discuss that in more detail specific to this study in the limitations section of the discussion.
All text and materials provided via this peer-review history page are made available under a Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.