PONE-D-23-29960Identification and Characterization of Calcium Binding Protein, Spermatid Associated 1 (CABS1) in Selected Human Tissues and FluidsPLOS ONE

Dear Dr. Marcet-Palacios,  

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Abeer El Wakil, PhD

Academic Editor

PLOS ONE

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Additional Editor Comments:

Dear Authors,

The concept of the present study seems interesting. In this work, the authors address an interesting and original topic, which is the characterization CABS1 by using different approaches (immunohistochemistry, WB and bioinformatics) and it moves from the development of a technique just validated to obtain monoclonal and polyclonal antibodies. However, the manuscript in its actual form does not meet the standards set forth by Plos One. I highly recommend the authors to address precisely the reviewers’ concerns before delivering any decision.

Thank you,

Abeer El Wakil.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments about the manuscript:

“Identification and Characterization of Calcium Binding Protein, Spermatid Associated 1 (CABS1) in Selected Human Tissues and Fluids”

Calcium-binding protein associated with spermatid 1 (CABS1) is widely studied in spermatogenesis. Its mRNA is present in many other tissues, but with little information about it. The aim of the study presented here was to investigate the presence of this protein in different tissues (salivary glands, saliva, serum and testes) using polyclonal and monoclonal antibodies directed against different sections of the protein, prepared during from previous work, by Western blot, immunohistochemistry and bioinformatics.

This substantial work brings interesting and useful results. The manuscript nevertheless needs to be improved before considering its publication.

Page 5 “WB”: western blot? Write in full the first time the name appears in the text.

This remark is applicable throughout the manuscript. Please check this point. On the other hand, when the number of abbreviations is large (like here), it would be useful to provide a glossary of abbreviations.

Page 6. “Post-homogenization, samples were centrifuged”: specify the characteristics of the centrifugation: Speed (in number of g), duration.

Page 9. Immunohistochemical analyses. In the description of the technique, specify how the negative controls were carried out.

Page 9. “SMG“: write in full SMG (submandibular gland, I think?) with abbreviation between brackets, when it is encountered for the first time in the text. See note above.

Page 12, Figure 1 - Western blot analyses of human CABS1 in transient overexpression lysate (OEL) of HEK 293T cells and in control lysate (NCL) of HEK 293T cells transfected with empty plasmid: Is this really figure 1? it looks to me like figure 2. Please check and correct.

Page 12, Figure 2 – Estimate of the abundance of hrCABS1 in sections (range of kDa) of 1 D gel analyzed by mass spectroscopy: same: Is it not figure 3? Please, check and correct.

Page 13, Figure 3 – Western blot analyses of CABS1 in human female and male submandibular gland lysates. A: Is it not figure 4? Please check and correct.

Between Figures 3 and 5 (Figure 5 – Western blot analyzes of CABS1 in human (male) saliva), there is no Figure 4. Please check.

Page 15, at the bottom of the page, “Figure 5 – Western blot analyses of CABS1 in human (male) saliva.”: this figure has already been given in the text.

Page 17. Figure 7 – Immunohistochemical analyses of CABS1 in human testes. “(lower, 200X)”: use a scale bar instead of magnification because magnification varies depending on the size of the text sheet.

Page 18, line 1. “antibody or with isotype control antibodies were negative3: Specify the controls on material and methods (see comments).

Page 18, Figure 8 - Immunohistochemical analyses of CABS1 in human submandibular glands: use a scale bar on the pictures, and not a magnification (see remark above).

In the legend, “striated tubes“ are designed with “DD”, but with “SD” on the picture.

No abbreviation is associated to “excretory duct” in the legend. It is ED on the picture.

Reviewer #2: Dear Authors, the project idea of your manuscript is very ambitious because it aims to characterize CABS1 by using different approaches (immunohistochemistry, WB and bioinformatics) and it moves from the development of a laborious technique to obtain monoclonal and polyclonal antibodies. Albeit the study design seems well conceived considering the wide typology of human samples (tissues and fluid) to examine for the identification of CABS1, several flaws are present. The manuscript in fact is well described for the chapter of polyclonal and monoclonal antibodies production as well as for the mass spectrometry sequencing. Differently, the section of the manuscript referring to CABS1 characterization at level of tissues and fluids has scarce scientific basis considering the limited quantity of examined sample (human testis, blood and saliva), as well as an incomplete description of materials and method (western blotting). To demonstrate the expression of a protein at level of cellular elements at least 3-6 samples for each tissue are required. In the manuscript the authors refer to one single subject and there is not a statistical analysis. The authors should add more additional data on this, other that they have to add more detailed information on the cell lysate preparation from fluid (saliva) and on the specificity of the antibody. Moreover, in the chapter (HCABS1 in Serum) the authors should explain better the specific selection of the quantity of antibody and serum used for analysis. Moreover, I noticed some mistake in the number of the Figure 2 and its description in the text respect to the illustration. In addition, a part of western blot images of the Figure 2 is the same just reported in the thesis (title “The perplexity of calcium-binding protein, spermatid-associated 1 (CABS1): by Eduardo Alejandro Reyes Serratos, 2022) but the authors do not report this reference. The title of the two lanes reported in this figure is not clear and the authors have to modify it.

In addition, the authors should better specify the time of the execution of analysis on saliva samples considering that the samples were collected between 2017 and 2018 and indicate n. of samples tested. Regarding the immunohistochemical studies, the authors should indicate how many slides for samples were prepared for the analysis and how many observers (considering the intra- and inter-observer variability) were used. The authors have also to reorganize better the discussion that is sometimes confuse. The authors sometimes describe some aspects that are obvious (i.e. the role of Leydig cells in the production of androgens) and not necessitate to be described.

In addition, albeit the English form requires minor revision, a particular attention is due to the discussion where some sentences are not completely clear.

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Reviewer #1: No

Reviewer #2: No

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Identification and Characterization of Calcium Binding Protein, Spermatid Associated 1 (CABS1) in Selected Human Tissues and Fluids

PONE-D-23-29960R1

Dear Dr. Marcet-Palacios, 

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Abeer El Wakil, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: N/A

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments about the revised version of the mansucript:

“Expression pattern of germ cell markers in cryptorchid stallion testes”

The authors took into account the remarks I made following the first version. I thank them for that. For me, the article is ready to be published.

Reviewer #3: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

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