My first revision

Thanks to the Chinese people my first critique was piece of cake.

It was a nice experience but mostly really funny. I was all the time laughing and smiling. 

I  was thinking that listening a presentation in English by a Chinese is fun but reading a draft manuscript which is supposed to get published in an even low impact factor journal was hilarious (όλα τα λεφτά που λέμε).

No offense to Chinese people. This post meant to be funny, and not racist. I apologize if I offended anyone.

Here is the critique modified by Dennis and Therese.

 

This manuscript (*********) by **** *., et al., investigates the role of protein A on Cell Carcinoma progression. The authors suggest that protein A downregulates protein B expression and thereby reduces invasiveness of cell carcinoma.

Unfortunately the study is an almost identical reproduction of a study recently published in **** by **** et al (*********** *********). **** et al demonstrated that S100A4 negatively regulates E-cadherin, and silencing of protein A reduces cell invasion, metastasis and proliferation of CC. Furthermore, **** presented a good correlation study with a large number of tissues (100 normal vs. 100 CC) and many clinico-pathological parameters (age, sex, histological grade, invasion depth, lymph node metastasis).

The study of **** et al., has several shortcomings in design, performance and presentation, just a few of which I mention.

1)                  There are inconsistencies between material and methods and results (e.g. 40 cryostat sections are mentioned in the material and methods that were used but in the table 48 samples are shown), between abbreviations used (EC109 is used in some cases and Eca-109 in other cases).

2)                  The given corresponding author email address does not match the person declared as corresponding author in the author list.

3)                  There is no figure legend to Figures 2. The legend to Figure 5 is effectively incorporated into that of Figure 4.

4)                  Materials and methods do not fully describe the experimental approach followed. The description is very general, incomplete and sometimes lacks scientific significance. Some examples are given. Sections are not normally “enrolled” for a study! Are each of the 40 (or 48) cryosections from different patients? Which were used for IHC (authors mention use of paraffin-embedded tissue for this), which for RT-PCR and which for WB? There is no patient description. The results are not scientifically rigid and lack information and the conclusions made are not based on the findings but on assumptions. The results of the immunohistochemistry analyses were processed differently for protein A (percent of stain) and protein B (intensity of staining). If correlations are to be made on the basis of stainings then the analytical approach for the different entities should be similar. What is the justification for setting ≥ 10% of stained cells as a criteria for positivity? For proper comparison/correlation analyses staining of protein A and protein B should be performed on serial sections. Better still would be to perform double staining using immunofluorescence techniques.

5)                  If the authors wish to attach importance of their in vitro observations to cell migration then a cell migration assay (e.g. wound assay or time lapse microscopy to monitor motility) should be added to the methodology. Migration and invasion do not necessarily occur concomitantly or by the same mechanisms.

6)                  The conclusions drawn are not supported by the data presented. Examples follow:

(a)                The first subtitle of the results “Increased Expression of protein A and protein B is Associated with Lymph Node Metastasis in CC” not appropriate. There is no information provided with respect to lymph node metastasis.

(b)                From the results in Figure 1 C no conclusive association between protein A and protein B expression can made; of the 5 non-metastatic materials 3 express protein B and 2 protein A. More patients need to be studied to obtain convincing data. This applies also to the data in Table 1.

(c)                The stainings in Figure 2 are not very convincing and the more important data on protein and transcript expression are not shown.

7)                  The data in Figure 5 is the only novel data in the study, but there are also shortcomings here. Protocols used to follow and document metastasis in vivo are not described. Have 8 (methods) or 6 (results) mice been used in the study per group? There seems to have animal experimentation on only a single occasion and should be reproduced on a second series of animalsGraphic presentation of node frequency is not sufficient; images of nodes should be presented. The authors should make a table of node occurrence in the different organs (per animal and/or total). Is the S100A4 up and downregulation still detectable after 21 days (e.g. by staining of the primary tumor or qPCR of sections)?

8)                  I advise obtaining linguistic assistance for future publications. This paper is at time exceedingly difficult and complicated to read and it is full of grammatical and typographical errors.