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It was corrected in manuscript
| 2 | 1 |
Figure 6 Bacterial EPSs are normally composed of repeating units. How can the authors ascertain that mannose does not come from mannans in yeast extract? A 2D COSY spectrum would be necessary to confirm the assignments made on the 1D: e.g., H2- Man is not normally found in the range 3.2-3.5 ppm (the statement ll. 495-496 is wrong). The abbreviation for glucose is Glc.
| 1 | 2 |
biom12060834_makarova
| 1 |
A new discussion of 1D and 2D NMR spectra was added and corrected.
| 2 | 1 |
l. 500 The chemical shift range is more than from 1 to 5. ll.
| 1 | 2 |
biom12060834_makarova
| 1 |
According to the suggestion, section numbering has been checked thoroughly in the revised version of the manuscript.
| 2 | 1 |
l. 537 No section 3.6.2
| 1 | 2 |
biom12060834_makarova
| 1 |
As suggested by the reviewer, the necessary revision in done in the manuscript.
| 2 | 1 |
l. 636 Glc and Gal
| 1 | 2 |
biom12060834_makarova
| 1 |
As suggested by the reviewer, the information is corrected and revised now.
| 2 | 1 |
l. 683-684 Food-grade oils are not aliphatic and aromatic hydrocarbons.
| 1 | 2 |
biom12060834_makarova
| 1 |
Suggestion has been included in the revised version of the manuscript
| 2 | 1 |
l. 725 possibility of future
| 1 | 2 |
biom12060834_makarova
| 1 |
As suggested by the reviewer, minor errors throughout the manuscript has been revised now.
| 4 | 1 |
The English language was improved, but there are still errors.
| 3 | 2 |
biom12060834_makarova
| 1 |
int. We agree with some of the comments that you made. However, we would like to remind you that the main purpose of this manuscript is not the complete elucidation of the EPS chemical structure but also the optimization of the EPS production, and characterization of the EPS functional properties. At the same time, we provided different chemical properties like: sugar identification and its molar ratio, molecular weight, functional group analysis and thermal stability of the EPS, and this journal has recently published other papers with similar or less chemical information on the EPS, for instance: Kuo, H.-C.; Liu, Y.-W.; Lum, C.-C.; Hsu, K.-D.; Lin, S.-P.; Hsieh, C.-W.; Lin, H.-W.; Lu, T.-Y. ; Cheng, K.- C. Ganoderma formosanum Exopolysaccharides Inhibit Tumor Growth via Immunomodulation. Int. J. Mol. Sci. 2021, 22, 11251. https://doi.org/10.3390/ijms222011251 Fetsiukh, A.; Conrad, J.; Bergquist, J.; Timmusk, S. Silica Particles Trigger the Exopolysaccharide Production of Harsh Environment Isolates of Growth-Promoting Rhizobacteria and Increase Their Ability to Enhance Wheat Biomass in Drought-Stressed Soils. Int. J. Mol. Sci. 2021, 22, 6201. https://doi.org/10.3390/ijms22126201.
| 4 | 1 |
Why was linkage analysis not performed? Bacterial EPSs are normally composed of repeating units. Based on the 1H NMR spectrum, the purity and/or heterogeneity of the EPS is questioned. (I was referring to peak intensities that are not uniform. The new spectra emphasize this even more: for example, I count at least 10 H1/H2 cross peaks of different intensities on the COSY.) The authors added a COSY spectrum but did not analyze it. Chemical shift similarity is not sufficient to identify monosaccharide and linkage in a polysaccharide repeating unit. The evidences for the partial structure given are not convincing. If the authors do not add methylation analysis and perform full NMR spectral analysis, the claims about structure determination should be removed from the manuscript. (I was referring to the chemical method by methylation.). They deleted erroneous information about mannose residues, but came up with new conclusions about mannose identity and linkages based solely on chemical shift of anomeric proton/carbon.The information contained in the other 2D spectra was not exploited to provide structural information.
| 3 | 2 |
biom12060834_makarova
| 1 |
On the other side, we performed the 1D and 2D - NMR analysis as complementary information to the other techniques in the present work. Such analysis was performed to try to replace the traditional acid hydrolysis, methylation/acetylation and GC-MS used in glycoside analysis. On the opposite to NMR, we do not have access to this analysis, and we could not find it as a service.
| 4 | 1 |
Why was linkage analysis not performed? Bacterial EPSs are normally composed of repeating units. Based on the 1H NMR spectrum, the purity and/or heterogeneity of the EPS is questioned. (I was referring to peak intensities that are not uniform. The new spectra emphasize this even more: for example, I count at least 10 H1/H2 cross peaks of different intensities on the COSY.) The authors added a COSY spectrum but did not analyze it. Chemical shift similarity is not sufficient to identify monosaccharide and linkage in a polysaccharide repeating unit. The evidences for the partial structure given are not convincing. If the authors do not add methylation analysis and perform full NMR spectral analysis, the claims about structure determination should be removed from the manuscript. (I was referring to the chemical method by methylation.). They deleted erroneous information about mannose residues, but came up with new conclusions about mannose identity and linkages based solely on chemical shift of anomeric proton/carbon.The information contained in the other 2D spectra was not exploited to provide structural information.
| 3 | 2 |
biom12060834_makarova
| 1 |
The HPLC and NMR results from the present work confirmed the heterogeneity of the EPS (3 different sugar moieties with different molar ratios), which agrees with the other reported mannan polysaccharides. The 1H and 13C NMR chemical shifts are comparable with literature reports of other similar EPS or oligosaccharides, where complex structures are identified by NMR too (Casillo et al., 2021; Chatterjee et al; 2018). Besides, our 2D-NMR analysis supported EPS heterogeneity, identifying different types of monomer linkages.
| 4 | 1 |
Why was linkage analysis not performed? Bacterial EPSs are normally composed of repeating units. Based on the 1H NMR spectrum, the purity and/or heterogeneity of the EPS is questioned. (I was referring to peak intensities that are not uniform. The new spectra emphasize this even more: for example, I count at least 10 H1/H2 cross peaks of different intensities on the COSY.) The authors added a COSY spectrum but did not analyze it. Chemical shift similarity is not sufficient to identify monosaccharide and linkage in a polysaccharide repeating unit. The evidences for the partial structure given are not convincing. If the authors do not add methylation analysis and perform full NMR spectral analysis, the claims about structure determination should be removed from the manuscript. (I was referring to the chemical method by methylation.). They deleted erroneous information about mannose residues, but came up with new conclusions about mannose identity and linkages based solely on chemical shift of anomeric proton/carbon.The information contained in the other 2D spectra was not exploited to provide structural information.
| 3 | 2 |
biom12060834_makarova
| 1 |
Your mention of the non-uniform peak intensity confirming the three different sugar units found. This is because the signal intensity of NMR peaks is related to an -H abundance. Different monomer ratios are responsible for this variation and support our findings. Also, the 10 H1/H2 cross peaks observed in the COSY spectra supported our findings of EPS heterogeneity, given the following facts: 1) We found 3 different types of sugars by HPLC. 2) We identified by NMR at least 2 types of linkages α 1-2 Man-Man and alfa 1-4 Man-Man monosaccharides. 3) There are other 2 sugars for which the β- type of linkage was identified but not the exact sugar position. 4) Anomeric protons shifts from units at the reducing end of polysaccharide differs from the one in another position. In conclusion, there are different linkage possibilities (plus already identified) to different monosaccharides in different ring positions. This gives rise to a multiplicity of signals (not fully identified in this work), which could produce different H1/H2 correlations.
| 4 | 1 |
Why was linkage analysis not performed? Bacterial EPSs are normally composed of repeating units. Based on the 1H NMR spectrum, the purity and/or heterogeneity of the EPS is questioned. (I was referring to peak intensities that are not uniform. The new spectra emphasize this even more: for example, I count at least 10 H1/H2 cross peaks of different intensities on the COSY.) The authors added a COSY spectrum but did not analyze it. Chemical shift similarity is not sufficient to identify monosaccharide and linkage in a polysaccharide repeating unit. The evidences for the partial structure given are not convincing. If the authors do not add methylation analysis and perform full NMR spectral analysis, the claims about structure determination should be removed from the manuscript. (I was referring to the chemical method by methylation.). They deleted erroneous information about mannose residues, but came up with new conclusions about mannose identity and linkages based solely on chemical shift of anomeric proton/carbon.The information contained in the other 2D spectra was not exploited to provide structural information.
| 3 | 2 |
biom12060834_makarova
| 1 |
About the COSY spectrum discussion, only a minor and partial discussion is presented that can be found below the Scheme in manuscript: “In the COSY spectrum, it was found its α-configuration by low-field H-1 signal at δ 5.34 and correlated with H-2 (δ 4.0), H-6 (δ 3.98 and 3.85), and H-5 (δ 4.12) (Speciale et al., 2022).”
| 4 | 1 |
Why was linkage analysis not performed? Bacterial EPSs are normally composed of repeating units. Based on the 1H NMR spectrum, the purity and/or heterogeneity of the EPS is questioned. (I was referring to peak intensities that are not uniform. The new spectra emphasize this even more: for example, I count at least 10 H1/H2 cross peaks of different intensities on the COSY.) The authors added a COSY spectrum but did not analyze it. Chemical shift similarity is not sufficient to identify monosaccharide and linkage in a polysaccharide repeating unit. The evidences for the partial structure given are not convincing. If the authors do not add methylation analysis and perform full NMR spectral analysis, the claims about structure determination should be removed from the manuscript. (I was referring to the chemical method by methylation.). They deleted erroneous information about mannose residues, but came up with new conclusions about mannose identity and linkages based solely on chemical shift of anomeric proton/carbon.The information contained in the other 2D spectra was not exploited to provide structural information.
| 3 | 2 |
biom12060834_makarova
| 1 |
Regarding the TOCSY and HMBC mentioned in the methods, they are now deleted from the manuscript, as suggested.
| 4 | 1 |
Why was linkage analysis not performed? Bacterial EPSs are normally composed of repeating units. Based on the 1H NMR spectrum, the purity and/or heterogeneity of the EPS is questioned. (I was referring to peak intensities that are not uniform. The new spectra emphasize this even more: for example, I count at least 10 H1/H2 cross peaks of different intensities on the COSY.) The authors added a COSY spectrum but did not analyze it. Chemical shift similarity is not sufficient to identify monosaccharide and linkage in a polysaccharide repeating unit. The evidences for the partial structure given are not convincing. If the authors do not add methylation analysis and perform full NMR spectral analysis, the claims about structure determination should be removed from the manuscript. (I was referring to the chemical method by methylation.). They deleted erroneous information about mannose residues, but came up with new conclusions about mannose identity and linkages based solely on chemical shift of anomeric proton/carbon.The information contained in the other 2D spectra was not exploited to provide structural information.
| 3 | 2 |
biom12060834_makarova
| 1 |
Finally, we kindly request the reviewer to consider that this is the first approach to studying a Chilean hot spring EPS isolation and its functional characterization. We also mention that further work to fully reveal this EPS structure will be necessary using higher resolution NMR spectra combined with chemical techniques. This work has given partial structural elucidation, which can be considered a previous background for other research on this topic.
| 4 | 1 |
Why was linkage analysis not performed? Bacterial EPSs are normally composed of repeating units. Based on the 1H NMR spectrum, the purity and/or heterogeneity of the EPS is questioned. (I was referring to peak intensities that are not uniform. The new spectra emphasize this even more: for example, I count at least 10 H1/H2 cross peaks of different intensities on the COSY.) The authors added a COSY spectrum but did not analyze it. Chemical shift similarity is not sufficient to identify monosaccharide and linkage in a polysaccharide repeating unit. The evidences for the partial structure given are not convincing. If the authors do not add methylation analysis and perform full NMR spectral analysis, the claims about structure determination should be removed from the manuscript. (I was referring to the chemical method by methylation.). They deleted erroneous information about mannose residues, but came up with new conclusions about mannose identity and linkages based solely on chemical shift of anomeric proton/carbon.The information contained in the other 2D spectra was not exploited to provide structural information.
| 3 | 2 |
biom12060834_makarova
| 1 |
To better understand EPS chemical structure, further 2D NMR experiments were performed and results are presented inside the manuscript. In short, they allow to clearly identify the D-mannose α-(1→2) and α-(1→4) linkages in EPS. These results suggest they are the prevailing units of the EPS backbone making it a branched one. Also, β-glucopyranose and β-galactopyranose structures were identified and the acetylation was confirmed.
| 2 | 1 |
To my deep regret the structural characteristic of EPS is the weak point of this publication.
| 1 | 2 |
biom12060834_makarova
| 1 |
According to the composition of EPS no metal was found to be attached with the structure of purified EPS. Likewise, in this study the monomeric composition was analysed to demonstrate the structure of purified EPS, where no metal was found to be attached with the chemical structure of it. This is also mentioned and referred in the revised version of the manuscript.
| 2 | 1 |
It is advisable, in my opinion, to provide information on the content of heavy metals in the EPS, since the authors plan to offer this product for the food industry in the future.
| 1 | 2 |
biom12060834_makarova
| 1 |
As suggested by the reviewer, this keyword is removed from the revised version of the manuscript.
| 2 | 1 |
Line 42: It is premature to include the term "structure" in keywords at this stage.
| 1 | 2 |
biom12060834_makarova
| 1 |
Thank you so much dear reviewer for mentioning this po The correction has been made now in the revised version of the manuscript.
| 2 | 1 |
Line 526-527: The proposal should be reformulated. According to Figure 7. the activity of the ascorbic acid is higher than the activity of EPS produced by CamB6.
| 1 | 2 |
biom12060834_makarova
| 1 |
As suggested by the reviewer, the non-pertinent references has been checked throughout the manuscript and unnecessary references are removed too.
| 2 | 1 |
The manuscript is very long: it contains a lot of information and many references not always pertinent to EPS characterization.
| 1 | 2 |
biom12060834_perova
| 1 |
As suggested by the reviewer, now the title include the production optimization too and the new title is “Optimization and characterization of a novel exopolysaccharide from Bacillus haynesii CamB6 for food applications”.
| 2 | 1 |
Optimization of production constitutes an important part not mentioned in the title.
| 1 | 2 |
biom12060834_perova
| 1 |
As suggested by the reviewer, the acceptability for food application has now been mentioned with appropriate refences (Nicolaus et al., 2010; Kambourova et al., 2018; Gongi et al., 2022) in the revised version of the manuscript.
| 2 | 1 |
Acceptability for food applications was not discussed.
| 1 | 2 |
biom12060834_perova
| 1 |
According to reviewer´s suggestion, the language of the manuscript is thoroughly revised.
| 2 | 1 |
English language should be revised thoroughly. There are inconsistencies in singular-plural concordance between subject and verb as well as noun and pronoun. Verb tenses should be checked. Articles (mostly definite, but also indefinite) are often missing and sometimes superfluous.
| 1 | 2 |
biom12060834_perova
| 1 |
As suggested by the reviewer, the consistent of the word had been checked throughout.
| 2 | 1 |
EPS vs. EPSs in plural form should be consistent throughout.
| 1 | 2 |
biom12060834_perova
| 1 |
Constant abbreviation had been checked throughout in the revised version of the manuscript.
| 2 | 1 |
Use a consistent abbreviation (l or L) for liter (including milliliter and microliter) throughout.
| 1 | 2 |
biom12060834_perova
| 1 |
Accoding to the suggestion of the reviewer, Table 1 is revised and both coded – noncoded values are now included.
| 2 | 1 |
l. 160 There is no coded value in Table 1.
| 1 | 2 |
biom12060834_perova
| 1 |
Both the tables (table 1 and table 2) have been placed in the proper place where it expected in the revised manuscript.
| 2 | 1 |
l. 165 Table 2 where Table 1 expected
| 1 | 2 |
biom12060834_perova
| 1 |
The suggested correction has been done in the revised version of the manuscript
| 2 | 1 |
l. 169-170 thirty instead of thirteen ?
| 1 | 2 |
biom12060834_perova
| 1 |
The corrected has been made now.
| 2 | 1 |
l. 177 i instead of 0 in second term?
| 1 | 2 |
biom12060834_perova
| 1 |
The revision has been made as suggested.
| 2 | 1 |
l. 228 GPC defined on l. 231
| 1 | 2 |
biom12060834_perova
| 1 |
as suggested by the reviewer, PEG has been defined in the revised version of the manuscript
| 2 | 1 |
l. 230 PEG undefined
| 1 | 2 |
biom12060834_perova
| 1 |
The volume is now added in the revised version of the manuscript.
| 2 | 1 |
l. 247 Volume of 0.2 mM ethanolic DPPH solution?
| 1 | 2 |
biom12060834_perova
| 1 |
Suggestion has been included in the revised version of the manuscript throughout.
| 2 | 1 |
255, 286, and 441 et al.
| 1 | 2 |
biom12060834_perova
| 1 |
This to kindly mention to the reviewer, the map is the collection site of the EPS producing Bacillus haynesii CamB6. This is a first such report from this collection site, the map is necessary to keep in the manuscript.
| 2 | 1 |
Figure 1 What is the significance of the tick mark labels on the maps? Are these maps really necessary?
| 1 | 2 |
biom12060834_perova
| 1 |
Figure 2 has been modified as suggested by the reviewer.
| 2 | 1 |
Figure 2 No x-axis label Units not specified
| 1 | 2 |
biom12060834_perova
| 1 |
As suggested by the reviewer, Figure 3 is now modified with specified units of the axes and also in the figure legend.
| 2 | 1 |
Figure 3
| 1 | 2 |
biom12060834_perova
| 1 |
All the Suggestion has been included in the figure legend of revised version of the manuscript
| 2 | 1 |
417 and 432 30.0 instead of 3.0 ?
| 1 | 2 |
biom12060834_perova
| 1 |
To avoid the redundancy of the figures as suggested by the reviewer, figure 4 is removed from the revised version of the manuscript.
| 2 | 1 |
Units are not specified on the axes labels or in the legend. Figure 4 This figure is redundant as it gives the same information as Figure 3. Tick label values for glucose and yeast extract are different.
| 1 | 2 |
biom12060834_perova
| 1 |
As per the suggestion given by the reviewer, section numbering has been checked thoroughly in the revised version of the manuscript
| 2 | 1 |
433, 434, 454, 458, and 507 First section 3.6 (and its subsections) should be section 3.5.
| 1 | 2 |
biom12060834_perova
| 1 |
This has been checked now and revised.
| 2 | 1 |
l. 471, 600, 678, and 718 Reference format is different; corresponding references could be missing in the list.
| 1 | 2 |
biom12060834_perova
| 1 |
We performed 2D-NMR analysis , which allows us to determine exactly the α-Manp linkage, which is the major sugar component, and the β- structure of Galp and Glucp.
| 2 | 1 |
l. 480 Why was linkage analysis not performed?
| 1 | 2 |
biom12060834_perova
| 1 |
This is a heteropolysaccharide composed of 3 different sugars as determined for HPLC, which is highly common in this kind of polysaccharides isolated from bacterial strains (10.3390/foods11020156)
| 2 | 1 |
Figure 6 Bacterial EPSs are normally composed of repeating units. How can the authors ascertain that mannose does not come from mannans in yeast extract? A 2D COSY spectrum would be necessary to confirm the assignments made on the 1D: e.g., H2- Man is not normally found in the range 3.2-3.5 ppm (the statement ll. 495-496 is wrong). The abbreviation for glucose is Glc.
| 1 | 2 |
biom12060834_perova
| 1 |
There are several earlier report where different concentration of yeast extract is added to the culture media for optimized EPS production, and the resultant EPS also concisted of mannan. However, there is no report that the mannan came from yeast extract (DOI: 10.1016/j.ijbiomac.2019.09.139, DOI: 10.1023/B:WIBI.0000033068.45655.2a). The same applies for our study too. In addition, the yeast mannan have β-(1→4) linkage and the one found in this work have α-linkage type. This allows us to conclude they are different polysaccharides.
| 2 | 1 |
Figure 6 Bacterial EPSs are normally composed of repeating units. How can the authors ascertain that mannose does not come from mannans in yeast extract? A 2D COSY spectrum would be necessary to confirm the assignments made on the 1D: e.g., H2- Man is not normally found in the range 3.2-3.5 ppm (the statement ll. 495-496 is wrong). The abbreviation for glucose is Glc.
| 1 | 2 |
biom12060834_perova
| 1 |
Several 2D-NMR spectra were performed to confirm 1H and 13C chemical shift of EPS structure, and their analysis was added to the manuscript.
| 2 | 1 |
Figure 6 Bacterial EPSs are normally composed of repeating units. How can the authors ascertain that mannose does not come from mannans in yeast extract? A 2D COSY spectrum would be necessary to confirm the assignments made on the 1D: e.g., H2- Man is not normally found in the range 3.2-3.5 ppm (the statement ll. 495-496 is wrong). The abbreviation for glucose is Glc.
| 1 | 2 |
biom12060834_perova
| 1 |
It was corrected in manuscript
| 2 | 1 |
Figure 6 Bacterial EPSs are normally composed of repeating units. How can the authors ascertain that mannose does not come from mannans in yeast extract? A 2D COSY spectrum would be necessary to confirm the assignments made on the 1D: e.g., H2- Man is not normally found in the range 3.2-3.5 ppm (the statement ll. 495-496 is wrong). The abbreviation for glucose is Glc.
| 1 | 2 |
biom12060834_perova
| 1 |
A new discussion of 1D and 2D NMR spectra was added and corrected.
| 2 | 1 |
l. 500 The chemical shift range is more than from 1 to 5. ll.
| 1 | 2 |
biom12060834_perova
| 1 |
According to the suggestion, section numbering has been checked thoroughly in the revised version of the manuscript.
| 2 | 1 |
l. 537 No section 3.6.2
| 1 | 2 |
biom12060834_perova
| 1 |
As suggested by the reviewer, the necessary revision in done in the manuscript
| 2 | 1 |
l. 636 Glc and Gal
| 1 | 2 |
biom12060834_perova
| 1 |
As suggested by the reviewer, the information is corrected and revised now
| 2 | 1 |
l. 683-684 Food-grade oils are not aliphatic and aromatic hydrocarbons.
| 1 | 2 |
biom12060834_perova
| 1 |
Suggestion has been included in the revised version of the manuscript
| 2 | 1 |
l. 725 possibility of future
| 1 | 2 |
biom12060834_perova
| 1 |
As suggested by the reviewer, minor errors throughout the manuscript has been revised now.
| 4 | 1 |
The English language was improved, but there are still errors.
| 3 | 2 |
biom12060834_perova
| 1 |
Dear reviewer, thanks for your valuable comments for improving the quality of this paper. We will answer in the following paragraphs because all comments were related to the same topic.
| 4 | 1 |
Chemical shift similarity is not sufficient to identify monosaccharide and linkage in a polysaccharide repeating unit. The evidences for the partial structure given are not convincing. If the authors do not add methylation analysis and perform full NMR spectral analysis, the claims about structure determination should be removed from the manuscript. They deleted erroneous information about mannose residues, but came up with new conclusions about mannose identity and linkages based solely on chemical shift of anomeric proton/carbon.The information contained in the other 2D spectra was not exploited to provide structural information.
| 3 | 2 |
biom12060834_perova
| 1 |
We agree with some of the comments that you made. However, we would like to remind you that the main purpose of this manuscript is not the complete elucidation of the EPS chemical structure but also the optimization of the EPS production, and characterization of the EPS functional properties. At the same time, we provided different chemical properties like: sugar identification and its molar ratio, molecular weight, functional group analysis and thermal stability of the EPS, and this journal has recently published other papers with similar or less chemical information on the EPS, for instance: Kuo, H.-C.; Liu, Y.-W.; Lum, C.-C.; Hsu, K.-D.; Lin, S.-P.; Hsieh, C.-W.; Lin, H.-W.; Lu, T.-Y.; Cheng, K.- C. Ganoderma formosanum Exopolysaccharides Inhibit Tumor Growth via Immunomodulation. Int. J. Mol. Sci. 2021, 22, 11251. https://doi.org/10.3390/ijms222011251 Fetsiukh, A.; Conrad, J.; Bergquist, J.; Timmusk, S. Silica Particles Trigger the Exopolysaccharide Production of Harsh Environment Isolates of Growth-Promoting Rhizobacteria and Increase Their Ability to Enhance Wheat Biomass in Drought-Stressed Soils. Int. J. Mol. Sci. 2021, 22, 6201. https://doi.org/10.3390/ijms22126201.
| 4 | 1 |
Chemical shift similarity is not sufficient to identify monosaccharide and linkage in a polysaccharide repeating unit. The evidences for the partial structure given are not convincing. If the authors do not add methylation analysis and perform full NMR spectral analysis, the claims about structure determination should be removed from the manuscript. They deleted erroneous information about mannose residues, but came up with new conclusions about mannose identity and linkages based solely on chemical shift of anomeric proton/carbon.The information contained in the other 2D spectra was not exploited to provide structural information.
| 3 | 2 |
biom12060834_perova
| 1 |
On the other side, we performed the 1D and 2D - NMR analysis as complementary information to the other techniques in the present work. Such analysis was performed to try to replace the traditional acid hydrolysis, methylation/acetylation and GC-MS used in glycoside analysis. On the opposite to NMR, we do not have access to this analysis, and we could not find it as a service.
| 4 | 1 |
Chemical shift similarity is not sufficient to identify monosaccharide and linkage in a polysaccharide repeating unit. The evidences for the partial structure given are not convincing. If the authors do not add methylation analysis and perform full NMR spectral analysis, the claims about structure determination should be removed from the manuscript. They deleted erroneous information about mannose residues, but came up with new conclusions about mannose identity and linkages based solely on chemical shift of anomeric proton/carbon.The information contained in the other 2D spectra was not exploited to provide structural information.
| 3 | 2 |
biom12060834_perova
| 1 |
The HPLC and NMR results from the present work confirmed the heterogeneity of the EPS (3 different sugar moieties with different molar ratios), which agrees with the other reported mannan polysaccharides. The 1H and 13C NMR chemical shifts are comparable with literature reports of other similar EPS or oligosaccharides, where complex structures are identified by NMR too (Casillo et al., 2021; Chatterjee et al; 2018). Besides, our 2D-NMR analysis supported EPS heterogeneity, identifying different types of monomer linkages. Casillo, A., Fabozzi, A., Russo Krauss, I., Parrilli, E., Biggs, C. I., Gibson, M. I., … Corsaro, M. M. (2021). Physicochemical Approach to Understanding the Structure, Conformation, and Activity of Mannan Polysaccharides. Biomacromolecules, 22(4), 1445–1457. doi:10.1021/acs.biomac.0c01659. Chatterjee, S., Mukhopadhyay, S. K., Gauri, S. S., & Dey, S. (2018). Sphingobactan, a new α-mannan exopolysaccharide from Arctic Sphingobacterium sp. IITKGP-BTPF3 capable of biological response modification. International Immunopharmacology, 60, 84–95. doi:10.1016/j.intimp.2018.04.039 •
| 4 | 1 |
Chemical shift similarity is not sufficient to identify monosaccharide and linkage in a polysaccharide repeating unit. The evidences for the partial structure given are not convincing. If the authors do not add methylation analysis and perform full NMR spectral analysis, the claims about structure determination should be removed from the manuscript. They deleted erroneous information about mannose residues, but came up with new conclusions about mannose identity and linkages based solely on chemical shift of anomeric proton/carbon.The information contained in the other 2D spectra was not exploited to provide structural information.
| 3 | 2 |
biom12060834_perova
| 1 |
Your mention of the non-uniform peak intensity confirming the three different sugar units found. This is because the signal intensity of NMR peaks is related to an -H abundance. Different monomer ratios are responsible for this variation and support our findings. Also, the 10 H1/H2 cross peaks observed in the COSY spectra supported our findings of EPS heterogeneity, given the following facts: 1) We found 3 different types of sugars by HPLC. 2) We identified by NMR at least 2 types of linkages α 1-2 Man-Man and alfa 1-4 Man-Man monosaccharides. 3) There are other 2 sugars for which the β- type of linkage was identified but not the exact sugar position. 4) Anomeric protons shifts from units at the reducing end of polysaccharide differs from the one in another position. In conclusion, there are different linkage possibilities (plus already identified) to different monosaccharides in different ring positions. This gives rise to a multiplicity of signals (not fully identified in this work), which could produce different H1/H2 correlations.
| 4 | 1 |
Chemical shift similarity is not sufficient to identify monosaccharide and linkage in a polysaccharide repeating unit. The evidences for the partial structure given are not convincing. If the authors do not add methylation analysis and perform full NMR spectral analysis, the claims about structure determination should be removed from the manuscript. They deleted erroneous information about mannose residues, but came up with new conclusions about mannose identity and linkages based solely on chemical shift of anomeric proton/carbon.The information contained in the other 2D spectra was not exploited to provide structural information.
| 3 | 2 |
biom12060834_perova
| 1 |
About the COSY spectrum discussion, only a minor and partial discussion is presented that can be found below the Scheme in manuscript: “In the COSY spectrum, it was found its α-configuration by low-field H-1 signal at δ 5.34 and correlated with H-2 (δ 4.0), H-6 (δ 3.98 and 3.85), and H-5 (δ 4.12) (Speciale et al., 2022).”
| 4 | 1 |
Chemical shift similarity is not sufficient to identify monosaccharide and linkage in a polysaccharide repeating unit. The evidences for the partial structure given are not convincing. If the authors do not add methylation analysis and perform full NMR spectral analysis, the claims about structure determination should be removed from the manuscript. They deleted erroneous information about mannose residues, but came up with new conclusions about mannose identity and linkages based solely on chemical shift of anomeric proton/carbon.The information contained in the other 2D spectra was not exploited to provide structural information.
| 3 | 2 |
biom12060834_perova
| 1 |
Regarding the TOCSY and HMBC mentioned in the methods, they are now deleted from the manuscript, as suggested.
| 4 | 1 |
Chemical shift similarity is not sufficient to identify monosaccharide and linkage in a polysaccharide repeating unit. The evidences for the partial structure given are not convincing. If the authors do not add methylation analysis and perform full NMR spectral analysis, the claims about structure determination should be removed from the manuscript. They deleted erroneous information about mannose residues, but came up with new conclusions about mannose identity and linkages based solely on chemical shift of anomeric proton/carbon.The information contained in the other 2D spectra was not exploited to provide structural information.
| 3 | 2 |
biom12060834_perova
| 1 |
Finally, we kindly request the reviewer to consider that this is the first approach to studying a Chilean hot spring EPS isolation and its functional characterization. We also mention that further work to fully reveal this EPS structure will be necessary using higher resolution NMR spectra combined with chemical techniques. This work has given partial structural elucidation, which can be considered a previous background for other research on this topic.
| 4 | 1 |
Chemical shift similarity is not sufficient to identify monosaccharide and linkage in a polysaccharide repeating unit. The evidences for the partial structure given are not convincing. If the authors do not add methylation analysis and perform full NMR spectral analysis, the claims about structure determination should be removed from the manuscript. They deleted erroneous information about mannose residues, but came up with new conclusions about mannose identity and linkages based solely on chemical shift of anomeric proton/carbon.The information contained in the other 2D spectra was not exploited to provide structural information.
| 3 | 2 |
biom12060834_perova
| 1 |
To better understand EPS chemical structure, further 2D NMR experiments were performed and results are presented inside the manuscript. In short, they allow to clearly identify the D-mannose α-(1→2) and α-(1→4) linkages in EPS. These results suggest they are the prevailing units of the EPS backbone making it a branched one. Also, β-glucopyranose and β-galactopyranose structures were identified and the acetylation was confirmed.
| 2 | 1 |
To my deep regret the structural characteristic of EPS is the weak point of this publication. Fourier transform infrared spectroscopy (FTIR) and NMR spectroscopy data can only be regarded as preliminary. According to the 1H NMR spectrum, it can be assumed that EPS has a complex branched structure. Probably, to establish its structure, it is necessary to use chemical methods (hydrolysis, methylation, etc. ), as well as two-dimensional NMR spectroscopy.
| 1 | 2 |
biom12060834_perova
| 1 |
According to the composition of EPS no metal was found to be attached with the structure of purified EPS. Likewise, in this study the monomeric composition was analysed to demonstrate the structure of purified EPS, where no metal was found to be attached with the chemical structure of it. This is also mentioned and referred in the revised version of the manuscript.
| 2 | 1 |
EPC is isolated from a thermal spring of volcanic origin, which contains a set of various elements, including heavy metals. It is advisable, in my opinion, to provide information on the content of heavy metals in the EPS, since the authors plan to offer this product for the food industry in the future.
| 1 | 2 |
biom12060834_perova
| 1 |
As suggested by the reviewer, this keyword is removed from the revised version of the manuscript.
| 2 | 1 |
Line 42: It is premature to include the term "structure" in keywords at this stage.
| 1 | 2 |
biom12060834_perova
| 1 |
Thank you so much dear reviewer for mentioning this point. The correction has been made now in the revised version of the manuscript.
| 2 | 1 |
Line 526-527: The proposal should be reformulated. According to Figure 7. the activity of the ascorbic acid is higher than the activity of EPS produced by CamB6.
| 1 | 2 |
biom12060834_perova
| 1 |
This is not the case; HSA and the other fluids tested as inductors were added to the growth medium (LB) medium, and the cells were allowed to grow in these conditions for 5 h at 37ºC. After that, the cells were collected, and RNA was extracted. Since HSA does not reach the cytosol, at least not in an intact manner, hydrolysis seems not possible during cell growth.
| 2 | 1 |
It is known that HSA can bind RNA and DNA and has RNA hydrolyzing activity. In this way your experiment in par 2.2. may lead to not all RNA after incubation with such amount of albumin. That is why some of your effects may have other explanations.
| 1 | 2 |
biomedicines10030600_makarova
| 1 |
It is also worth mentioning that the presence of HSA in the growth medium produced a reduction only in some mRNA species. These results speak against an unspecific hydrolyzing effect.
| 2 | 1 |
It is known that HSA can bind RNA and DNA and has RNA hydrolyzing activity. In this way your experiment in par 2.2. may lead to not all RNA after incubation with such amount of albumin. That is why some of your effects may have other explanations.
| 1 | 2 |
biomedicines10030600_makarova
| 1 |
We also want to underscore that all RNA samples were checked after extraction. Agarose gels electrophoresis confirmed the integrity of RNA and lack of DNA contamination.
| 2 | 1 |
It is known that HSA can bind RNA and DNA and has RNA hydrolyzing activity. In this way your experiment in par 2.2. may lead to not all RNA after incubation with such amount of albumin. That is why some of your effects may have other explanations.
| 1 | 2 |
biomedicines10030600_makarova
| 1 |
Finally, the article provided by the reviewer describes HSA hydrolysis of extracellular, not intracellular, RNA.
| 2 | 1 |
It is known that HSA can bind RNA and DNA and has RNA hydrolyzing activity. In this way your experiment in par 2.2. may lead to not all RNA after incubation with such amount of albumin. That is why some of your effects may have other explanations.
| 1 | 2 |
biomedicines10030600_makarova
| 1 |
A follow-up project that will be submitted for publication in the near future extends the studies of the effects produced by human fluids, and their components will include the experiments demonstrating a bactericidal effect of cefiderocol.
| 2 | 1 |
Cefiderocol is a cidal antibiotic. It would be of great interest if for the key results the authors would also provide MBC and / or time-resolved kill-curves. I believe this kind of data would be of great relevance for assessing the clinical relevance of the observed effects.
| 1 | 2 |
biomedicines10030600_makarova
| 1 |
We thank the reviewer for their suggestion and have now computed AMPA-NMDA ratios and included a phrase in lines 424-425 that summarises the statistics for the comparison of AMPA-NMDA ratios between the GluN2B variants (WT and mutants).
| 2 | 1 |
Since mutations can affect NMDA receptor localization, data on whether AMPA/NMDA ratios changed would be informative. It follows from the methodology that AMPA currents were recorded on a regular basis.
| 1 | 2 |
brainsci12060789_makarova
| 1 |
We thank the reviewer for this excellent suggestion. We have now measured the decay time constants for the NMDA-EPSCs before and after TCN-201. The graph of the data is shown in a new figure in the supplement, Fig. S4. The finding is insightful, and so we have added a paragraph relating to it in the results section, lines 520-536.
| 2 | 1 |
Please add a chart about tau changes in Figure 3.
| 1 | 2 |
brainsci12060789_makarova
| 1 |
In answer to the last comment here from the reviewer, the time scale in Fig. 3A is correct – the examples chosen are representative of the group data in terms of the amount of block by TCN-201, but these examples all happen to have faster decays than the examples shown in Figures 1 and 2. In any case, to be more consistent with the formatting in some of the other figures, we have lengthened the scale bar and increased the time proportionally.
| 2 | 1 |
Please verify if the time scale is correct in Fig. 3А.
| 1 | 2 |
brainsci12060789_makarova
| 1 |
This is an excellent idea. We have added an additional (final) main text figure, Fig. 7, illustrating how the molecular defects could converge on similar NMDA-EPSCs. We have explained the model in the accompanying figure legend with reference to supporting evidence in other figures.
| 2 | 1 |
A scheme summarizing how different molecular defects can converge on similar NMDAR-mediated EPSCs would be helpful in the discussion
| 1 | 2 |
brainsci12060789_makarova
| 1 |
We have added further discussion relating to the pathogenesis of neurodevelopmental disorders arising from GRIN2B mutations in lines 765-771 of the manuscript.
| 2 | 1 |
A small commentary on the possible functional significance of the identified mutation properties for the pathogenesis of certain diseases would be interesting.
| 1 | 2 |
brainsci12060789_makarova
| 1 |
It is hard to know exactly why the measurements of the internal control, untransfected neuron appear to vary between the experimental groups. While an effort was made to try and evoke synaptic responses with a consistent stimulus voltage setting, the effectiveness of the stimulus for evoking an NMDA-EPSC depends on, among other things, the condition of the stimulating electrode (e.g. resistance, stray capacitance, bubbles etc). It was rarely practical to record all mutant conditions in the same experiment, so variation in the stimulating electrode condition over the course of this series of experiments could potentially lead to some apparent differences between untransfected neurons with respect to their mean NMDA-EPSC peak amplitude and charge transfer. What this does serve to illustrate though is how important it was for us to use untransfected neurons as an internal control.
| 2 | 1 |
Even though fig.2aiii shows a comparable relative peak amplitude, which indicates both GOF and LOF rescue NMDA-EPSC in Grin2b-/- neurons, it looks to me that the absolute amplitude in fig.2aii shows a quite big difference in un-transferred groups which are supposed to be comparable, not as consistent as shown in fig.1ci. How to explain this discrepancy?
| 1 | 2 |
brainsci12060789_makarova
| 1 |
The supplementary figures were within the same PDF so we are not sure why the author had trouble gaining accessing to the figures. Assuming the reviewer had trouble understanding the contents of the supplement, we have tried to simplify somewhat the text in the supplementary figure legends.
| 2 | 1 |
The supplementary figures are inaccessible.
| 1 | 2 |
brainsci12060789_makarova
| 1 |
We thank the reviewer for this comment. Indeed, there were many long sentences and the abstract did not capture all of our findings. We have rewritten the abstract to accommodate the reviewer’s suggestions. The abstract is now 311 words long (almost 40 words shorter than the original abstract), has shorter sentences, and includes some summary of the findings reported in all the main figures.
| 2 | 1 |
The abstract is too long, which does not actually abstract the whole content very well. There are also a number of sentences that are so complicated for easy read. This part should be substantially polished into a shortened and precise style.
| 1 | 2 |
brainsci12060789_makarova
| 1 |
We thank the reviewer for their suggestion and have now computed AMPA-NMDA ratios and included a phrase in lines 424-425 that summarises the statistics for the comparison of AMPA-NMDA ratios between the GluN2B variants (WT and mutants).
| 2 | 1 |
Since mutations can affect NMDA receptor localization, data on whether AMPA/NMDA ratios changed would be informative. It follows from the methodology that AMPA currents were recorded on a regular basis.
| 1 | 2 |
brainsci12060789_perova
| 1 |
We thank the reviewer for this excellent suggestion. We have now measured the decay time constants for the NMDA-EPSCs before and after TCN-201. The graph of the data is shown in a new figure in the supplement, Fig. S4. The finding is insightful, and so we have added a paragraph relating to it in the results section, lines 520-536. In answer to the last comment here from the reviewer, the time scale in Fig. 3A is correct – the examples chosen are representative of the group data in terms of the amount of block by TCN-201, but these examples all happen to have faster decays than the examples shown in Figures 1 and 2. In any case, to be more consistent with the formatting in some of the other figures, we have lengthened the scale bar and increased the time proportionally.
| 2 | 1 |
Please add a chart about tau changes in Figure 3. Please verify if the time scale is correct in Fig. 3А.
| 1 | 2 |
brainsci12060789_perova
| 1 |
This is an excellent idea. We have added an additional (final) main text figure, Fig. 7, illustrating how the molecular defects could converge on similar NMDA-EPSCs. We have explained the model in the accompanying figure legend with reference to supporting evidence in other figures.
| 2 | 1 |
A scheme summarizing how different molecular defects can converge on similar NMDAR-mediated EPSCs would be helpful in the discussion
| 1 | 2 |
brainsci12060789_perova
| 1 |
We have added further discussion relating to the pathogenesis of neurodevelopmental disorders arising from GRIN2B mutations in lines 765-771 of the manuscript.
| 2 | 1 |
A small commentary on the possible functional significance of the identified mutation properties for the pathogenesis of certain diseases would be interesting.
| 1 | 2 |
brainsci12060789_perova
| 1 |
It is hard to know exactly why the measurements of the internal control, untransfected neuron appear to vary between the experimental groups. While an effort was made to try and evoke synaptic responses with a consistent stimulus voltage setting, the effectiveness of the stimulus for evoking an NMDA-EPSC depends on, among other things, the condition of the stimulating electrode (e.g. resistance, stray capacitance, bubbles etc). It was rarely practical to record all mutant conditions in the same experiment, so variation in the stimulating electrode condition over the course of this series of experiments could potentially lead to some apparent differences between untransfected neurons with respect to their mean NMDA-EPSC peak amplitude and charge transfer. What this does serve to illustrate though is how important it was for us to use untransfected neurons as an internal control.
| 2 | 1 |
Even though fig.2aiii shows a comparable relative peak amplitude, which indicates both GOF and LOF rescue NMDA-EPSC in Grin2b-/- neurons, it looks to me that the absolute amplitude in fig.2aii shows a quite big difference in un-transferred groups which are supposed to be comparable, not as consistent as shown in fig.1ci. How to explain this discrepancy?
| 1 | 2 |
brainsci12060789_perova
| 1 |
The supplementary figures were within the same PDF so we are not sure why the author had trouble gaining accessing to the figures. Assuming the reviewer had trouble understanding the contents of the supplement, we have tried to simplify somewhat the text in the supplementary figure legends.
| 2 | 1 |
The supplementary figures are inaccessible.
| 1 | 2 |
brainsci12060789_perova
| 1 |
We thank the reviewer for this comment. Indeed, there were many long sentences and the abstract did not capture all of our findings. We have rewritten the abstract to accommodate the reviewer’s suggestions. The abstract is now 311 words long (almost 40 words shorter than the original abstract), has shorter sentences, and includes some summary of the findings reported in all the main figures. The abstract now reads as follows: GRIN2B mutations are rare but often associated with patients having severe neurodevelopmental disorders, including a varying range of symptoms such as intellectual disability, developmental delay and epilepsy. Patient symptoms likely arise from mutations disturbing the role that the encoded NMDA receptor subunit, GluN2B, plays at neuronal connections in the developing nervous system. In this study, we have investigated the cell-autonomous effects of putative gain- (GoF) and loss-of-function (LoF) missense GRIN2B mutations on excitatory synapses onto CA1 pyramidal neurons in organotypic hippocampal slices. In the absence of both native GluN2A and GluN2B subunits, functional incorporation into synaptic NMDA receptors was attenuated for GoF mutants, or almost eliminated for LoF GluN2B mutants. NMDA receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) from synaptic GoF GluN1/2B receptors had prolonged decays consistent with their functional classification. Nonetheless, in the presence of native GluN2A, molecular replacement of native GluN2B with GoF and LoF GluN2B mutants all led to similar functional incorporation into synaptic receptors, more rapidly decaying NMDA-EPSCs and greater inhibition by TCN-201, a selective antagonist of GluN2A-containing NMDA receptors. Mechanistic insight was gained from experiments in HEK293T cells, which revealed that GluN2B GoF mutants slowed deactivation in diheteromeric GluN1/2B, but not triheteromeric GluN1/2A/2B receptors. We also show that a disease-associated missense mutation, which severely affects surface expression, causes opposing effects on NMDA-EPSC decay and charge transfer when introduced into GluN2A or GluN2B. Finally, we show that having a single null Grin2b allele has only a modest effect on NMDA-EPSC decay kinetics. Our results demonstrate that functional incorporation of GoF and LoF GluN2B mutants into synaptic receptors and the effects on EPSC decay times are highly dependent on the presence of triheteromeric GluN1/2A/2B NMDA receptors, thereby influencing the functional classification of NMDA receptor variants as GoF or LoF mutations. These findings highlight the complexity of interpreting effects of disease-causing NMDA receptor missense mutations in the context of neuronal function.
| 2 | 1 |
The abstract is too long, which does not actually abstract the whole content very well. There are also a number of sentences that are so complicated for easy read. This part should be substantially polished into a shortened and precise style.
| 1 | 2 |
brainsci12060789_perova
| 1 |
We concur that a brief description of the literature regarding EIH in FM is warranted and have added information to the discussion as follows: “Previous research regarding the effects of acute bouts of exercise on pain sensitivity in FM patients is largely equivocal. Some studies have demonstrated a hypoalgesic effect [5,24] while other studies show either no changes in pain perception or an exacerbation of pain [25–27].”
| 2 | 1 |
However, the authors need to compare their findings to other research evidence showing impaired EIH and/or descending inhibition in patients with FM - especially considering that their results did not support EIH in the controls.
| 1 | 2 |
brainsci6010008_makarova
| 1 |
With respect to the reviewer’s comment regarding the lack of EIH in controls, we have included some additional information to demonstrate the consistency of our findings with previous research. The passage now reads: “However, it appears that the exercise bout as prescribed was not a sufficient stimulus to induce a decrease in pain sensitivity in our pain-free controls. This is consistent with previous literature regarding the effects of acute exercise on pain in healthy controls, which typically finds that higher intensity exercise is necessary to elicit a hypoalgesic response [33].” Additionally, we want to again mention that our study was not primarily designed to induce EIH in controls; it was designed to examine brain responses to pain following exercise and thus we wanted to ensure that FM patients could complete the exercise bout. Exploring brain responses associated with EIH in healthy individuals would be an interesting future direction.
| 2 | 1 |
The authors merely mention that the exercise stimulus may have been insufficient for the controls to show EIH without critically discussing what such a conclusion would mean (eg, FM patients have better EIH mechanisms than the controls).
| 1 | 2 |
brainsci6010008_makarova
| 1 |
With respect to the reviewer’s comment on generalizability, we agree that our exclusionary criteria were stringent and have added the following sentence to our limitations section. “Further, we excluded individuals with diagnosed mental health conditions and those who were taking medications that could impact pain or the interpretation of brain responses. As such our results may not apply to FM patients with comorbid conditions.”
| 2 | 1 |
Also, the discussion section should be revised to acknowledge that many patients with FM would have been excluded from participation in this study due to major depression and medication consumption so there may be concerns about the generalizability of the sample, BUT the authors appropriately compared the Fibromyalgia Impact Questionnaire scores of their sample to normative data and found no significant difference.
| 1 | 2 |
brainsci6010008_makarova
| 1 |
Lastly, with respect to the comment about controlling for physiological noise, the following statements have been added. “Though the evidence is equivocal, it has been suggested that cardiovascular mechanisms may be involved in the hypoalgesic response to exercise [34] and thus statistically controlling for these effects may have influenced the interpretation of our results. However, without controlling physiological differences between EX and QR the neuroimaging data would have been very difficult to interpret.” I don’t see that the reference actually indicates studies of activities of daily living so the authors should revise the sentence or add an appropriate reference.
| 2 | 1 |
In addition, the authors might want to consider and mention that their method of controlling “physiological noise” might have influenced the results because cardiovascular reactions to exercise were, at one time, proposed to be a mechanism of EIH.
| 1 | 2 |
brainsci6010008_makarova
| 1 |
This sentence has been revised to better reflect the reference used.
| 2 | 1 |
P. 1, L. 40 – I don’t see that the reference actually includes studies of activities of daily living so the authors should revise the sentence or add an appropriate reference.
| 1 | 2 |
brainsci6010008_makarova
| 1 |
We agree. We consulted with each physician with respect to type and dosage of medication, including whether this was a low, moderate or high dose. We also double-checked each medication and dose with the Physician’s Desk Reference (60th-65th editions). We have added the following information to the methods section “Medication information and dosage were supplied by the patient and their physician and dosage levels (low, moderate, high) were determined through both physician consultation and use of the Physician’s Desk Reference (65th edition).
| 2 | 1 |
P. 2, L. 18 – A reference for the determination that a dosage of antidepressants was “high” should be provided.
| 1 | 2 |
brainsci6010008_makarova
| 1 |
This addition has been made.
| 2 | 1 |
P. 3, L. 15 – The time post-exercise before scanning was provided, but not the time post-quiet rest.
| 1 | 2 |
brainsci6010008_makarova
| 1 |
The suggested change has been made.
| 2 | 1 |
P. 6, L. 5 – “Elevations” should be revised to “higher” so that readers do not erroneously believe that a pre-scan application of heat stimuli was administered.
| 1 | 2 |
brainsci6010008_makarova
| 1 |
Thank you for catching this omission. This was an oversight on our part and these effect sizes are now included on page 11.
| 2 | 1 |
P. 6, L. 12 - I am uncertain why the authors only report the effect sizes of group differences for the first run.s
| 1 | 2 |
brainsci6010008_makarova
| 1 |
These analyses were intended primarily for descriptive purposes. However, we recognize the need to control for multiple comparisons in order to reduce the risk of making Type I errors. Therefore, we created 3 families including 2 correlations each (one for each of the significant regions) and performed a Bonferroni correction, making the critical alpha level for significance 0.025. This has been clarified in the statistical analysis section.
| 2 | 1 |
However, in the latter case the authors need to address the issue of multiple correlations. Changes in pain sensitivity after exercise versus rest were significantly correlated with changes in activity in DLPFC (exercise vs. rest).
| 1 | 2 |
brainsci6010008_makarova
| 1 |
This change has been made.
| 2 | 1 |
Nine individuals in each group were included in neuroimaging analyses, this should be indicated in the abstract.
| 1 | 2 |
brainsci6010008_makarova
| 1 |
This correction has been made.
| 2 | 1 |
In table 3 the subheading "Peak X, Y, X" needs correction.
| 1 | 2 |
brainsci6010008_makarova
| 1 |
We agree that this would add valuable information to the discussion section and have added the paragraph shown below discussing previous work using neuroimaging to understand the effects of exercise on the brain.
| 2 | 1 |
Consider discussing the results in relation to previous studies on exercise and neuroimaging
| 1 | 2 |
brainsci6010008_makarova
| 1 |
A growing number of studies have begun to employ neuroimaging methods to better understand the impact of exercise on the brain both longitudinally and acutely. For example, Smith and colleagues [37] conducted fMRI scans pre and post an exercise training program in older adults with mild cognitive impairment and found that exercise improved neural efficiency during cognitive tasks post-intervention. Structural MRI has also been used to show the neuroprotective effects of regular exercise in older adults with respect to preservation of brain volume[38]. In contrast to using neuroimaging to track changes in the brain over time, neuroimaging during and immediately following exercise presents some unique challenges due to artifacts associated with movement and the physiological underpinnings of many neuroimaging methods (e.g. BOLD response). EEG has been used most extensively to explore the effects of exercise on cortical activity [39]. PET and fMRI have also been used, though to a much lesser extent. For example, Boecker and colleagues used PET to demonstrate the effects of a long-distance run on opioid release in the brain and Janse Van Rensberg and colleagues used fMRI to examine brain responses to nicotine craving following 10 minutes of moderate intensity cycling. Our study adds to this important body of literature by using fMRI to show that an acute bout of moderate intensity exercise improved brain mechanisms underlying pain modulation in patients with chronic pain and further highlights the potential benefits of utilizing neuroimaging technology to better understand the more immediate effects of exercise on the human brain.
| 2 | 1 |
The findings are novel and may be compared to previous studies of exercise and neuroimaging in fibromyalgia.
| 1 | 2 |
brainsci6010008_makarova
| 1 |
We concur that a brief description of the literature regarding EIH in FM is warranted and have added information to the discussion as follows: “Previous research regarding the effects of acute bouts of exercise on pain sensitivity in FM patients is largely equivocal. Some studies have demonstrated a hypoalgesic effect [5,24] while other studies show either no changes in pain perception or an exacerbation of pain [25–27].”
| 2 | 1 |
However, the authors need to compare their findings to other research evidence showing impaired EIH and/or descending inhibition in patients with FM - especially considering that their results did not support EIH in the controls. The authors merely mention that the exercise stimulus may have been insufficient for the controls to show EIH without critically discussing what such a conclusion would mean (eg, FM patients have better EIH mechanisms than the controls).
| 1 | 2 |
brainsci6010008_perova
| 1 |
With respect to the reviewer’s comment regarding the lack of EIH in controls, we have included some additional information to demonstrate the consistency of our findings with previous research. The passage now reads: “However, it appears that the exercise bout as prescribed was not a sufficient stimulus to induce a decrease in pain sensitivity in our pain-free controls. This is consistent with previous literature regarding the effects of acute exercise on pain in healthy controls, which typically finds that higher intensity exercise is necessary to elicit a hypoalgesic response [33].” Additionally, we want to again mention that our study was not primarily designed to induce EIH in controls; it was designed to examine brain responses to pain following exercise and thus we wanted to ensure that FM patients could complete the exercise bout. Exploring brain responses associated with EIH in healthy individuals would be an interesting future direction.
| 2 | 1 |
However, the authors need to compare their findings to other research evidence showing impaired EIH and/or descending inhibition in patients with FM - especially considering that their results did not support EIH in the controls. The authors merely mention that the exercise stimulus may have been insufficient for the controls to show EIH without critically discussing what such a conclusion would mean (eg, FM patients have better EIH mechanisms than the controls).
| 1 | 2 |
brainsci6010008_perova
| 1 |
With respect to the reviewer’s comment on generalizability, we agree that our exclusionary criteria were stringent and have added the following sentence to our limitations section. “Further, we excluded individuals with diagnosed mental health conditions and those who were taking medications that could impact pain or the interpretation of brain responses. As such our results may not apply to FM patients with comorbid conditions.”
| 2 | 1 |
Also, the discussion section should be revised to acknowledge that many patients with FM would have been excluded from participation in this study due to major depression and medication consumption so there may be concerns about the generalizability of the sample, BUT the authors appropriately compared the Fibromyalgia Impact Questionnaire scores of their sample to normative data and found no significant difference.
| 1 | 2 |
brainsci6010008_perova
| 1 |
Lastly, with respect to the comment about controlling for physiological noise, the following statements have been added. “Though the evidence is equivocal, it has been suggested that cardiovascular mechanisms may be involved in the hypoalgesic response to exercise [34] and thus statistically controlling for these effects may have influenced the interpretation of our results. However, without controlling physiological differences between EX and QR the neuroimaging data would have been very difficult to interpret.” I don’t see that the reference actually indicates studies of activities of daily living so the authors should revise the sentence or add an appropriate reference.
| 2 | 1 |
In addition, the authors might want to consider and mention that their method of controlling “physiological noise” might have influenced the results because cardiovascular reactions to exercise were, at one time, proposed to be a mechanism of EIH.
| 1 | 2 |
brainsci6010008_perova
| 1 |
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