Identification of Biochemical Differences in White and Brown Adipocytes Using FTIR Spectroscopy
Round 1
Reviewer 1 Report
Line 46 page 2 " Despite the severity of obesity, numerous fundamental research have been conducted but none of them used FTIR spectroscopy." This is not clear as there are multiple references related to FTIR imaging of adipose tissues.
- Aboualizadeh E, Carmichael OT, He P, Albarado DC, Morrison CD and Hirschmugl CJ (2017) Quantifying Biochemical Alterations in Brown and Subcutaneous White Adipose Tissues of Mice Using Fourier Transform Infrared Widefield Imaging. Front. Endocrinol. 8:121. doi: 10.3389/fendo.2017.00121
- Baloğlu, F. K. , & Severcan, F. (2018). Characterization and Differentiation of Adipose Tissue by Spectroscopic and Spectral Imaging Techniques. In (Ed.), Adipose Tissue. IntechOpen. https://doi.org/10.5772/intechopen.75156
- Küçük Baloğlu, “Biophysical characterization and diagnosis of obesity from adipose tissue by Fourier transform infrared spectroscopy and imaging,” Ph.D. - Doctoral Program, Middle East Technical University, 2017.
- Diletta Ami, Paolo Mereghetti, Andrea Foli, Masayoshi Tasaki, Paolo Milani, Mario Nuvolone, Giovanni Palladini, Giampaolo Merlini, Francesca Lavatelli, and Antonino Natalello ATR-FTIR Spectroscopy Supported by Multivariate Analysis for the Characterization of Adipose Tissue Aspirates from Patients Affected by Systemic Amyloidosis Analytical Chemistry 2019 91 (4), 2894-2900. DOI: 10.1021/acs.analchem.8b05008.
- Zhang H, Wang Q, Zhang K, Liu R, Fan S, Wang Z. Estimation of postmortem interval using attenuated total reflectance: Fourier transform infrared spectroscopy in adipose tissues. J Forensic Sci Med 2019;5:7-12
Author Response
I attached a file and please refer to it.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript is further evidence of the wide range of applications of infrared spectroscopy.
My main objections are formal.
In the Materials and Methods section, % values are given everywhere without exception, but nowhere is it specified what kind of %. This should be added (lines 54, 55, 57, 61, 63, 66, 67, 74, 75).
Some abbreviations are not explained: T3 (line 61) DMEM (line 63), PBS (line 76).
In line 86 the formula is incorrect, Ca2F is correct.
Line 90 - the pixel size is not um, it is um.
Line 90 - OPUS software version number is missing.
The scaling of the figures is very wrong. Even at 150x magnification the captions are not readable.
I suggest to change the black and white figures to colour, the shades of black and grey are difficult to see.
Table 1 is redundant as it can be read from Figure 2. It should be summarised in the text. Since the table evaluates on the basis of the derivative curve, it would be useful to include the deiverted spectra in addition to the raw spectra.
The same is the case with Table 2 - both the editing and the title are incorrect. Figures 3 and 4 should be explained in the text instead of Table 2. Here again, I miss the derivative spectra next to the raw spectra.
Figure 6 is missing the name of the x-y axis of the two-dimensional graph and both graphs are missing the numerical values of the PC-s.
In the Discussion section, there is unnecessary repetition between lines 208-216
Author Response
I attached a file and please refer to it.
Author Response File: Author Response.pdf
Reviewer 3 Report
This paper addresses a recognition process of adipocytes by FTIR spectroscopy. Major cells classes (immatures, white, brown) are concerned even if the context of application (diagnostic, biopsy, metabolic follow up of cultured celles, ... ???) is not clearly established.
Now concerning the spectral analysis, it presents a numbers of flaws. Among these:
No data about the PCA are presented, especially these components itselves
The AI/AII ratio is widely commented but water still contributes to the signal at 1640 cm-1 : Full spectra must by displayed to check any variations in bound water of dried cells.
AI and AII are associated to protein peptide bonds : How they may inform about degradation (L238-239) ? There are thousands of proteins in any cell : How can one study protein spectific secondary structures (L251-252)?
Spectra graphs are much to small to be analysed, Tables as these in the paper are very unusual...
Did baseline subtractions have been performed ? Normalisation ? How : MSC ?
PCA is a first step. Discriminant analyses with variables reduction must be performed to provide a signature (Lasso, RandomForest)
Did the FTIR experiments have been reproduced ? If so 3 times ?
The interest of the paper will be assessed if a much deeper description of the spectral data is presented.
Author Response
I attached a file and please refer to it.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
all comments were answered to satisfaction by the authors
Author Response
We replied to all questions at major revision.
Reviewer 2 Report
For percentage terms, the type of percentage is still missing. Percentage by weight, percentage by volume? This needs to be clarified.
Author Response
Please see the attachment.
Author Response File: 
Author Response.docx
Reviewer 3 Report
The question of hydration was relative to cells water content. Opus correct only for atmosphere water VAPOUR.Supplementary data are not enough informative: Must be displayed with the proper figure labels.
ALL raw spectra (why only 2 are displayed
The principal components
The question of hydration was relative to cells water content. Opus correct only for atmosphere water VAPOUR.
Author Response
Please see the attachment.
Author Response File: Author Response.docx
