Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HEARING ASSESSMENT DURING DEEP BRAIN STIMULATION OF THE CENTRAL NUCLEUS OF THE INFERIOR COLLICULUS AND DENTATE CEREBELLAR NUCLEUS IN RAT
Review round: 1
Reviewer: 1

Output:
Basic reporting: Entirely adequate.
Experimental design: Methods organization could be improved.
The section describing DBS was buried in the ABR method section. It should have been a separate and more complete section, as it was in the Smit et al., (2016) research report.
The low n (5 / group) of the present study sets it up for failure to find significant effects. This appears particularly true since non-parametric statistical tests, which do not take advantage of the repeated-measures design, were used. The authors should explain their choice of statistical tests and consider the issue of adequate statistical power to determine significance.
Although it wasn’t well explained (but should have been), apparently DBS was delivered throughout the epoch devoted to ABR acquisition. Hence there was a need to remove stimulus artifacts, as shown, but not thoroughly explained, in Figure 2. Apparently the DBS, both high-frequency and low, were delivered without controlling phase relationship to the ABR acquisition. That experimental choice should be justified. DBS periods were either 10 msec or 100 msec. ABR inter-stimulus intervals were 45 msec, while the ABR recording periods were 50 msec. Rather than let these events wander in and out of phase, a more tightly controlled protocol could have been used. For example, if the ABR-eliciting sound pulses were out of phase with DBS, then the DBS stimulus artifact would not have to be filtered. This would eliminate the loss of data produced by EP filtering.
Validity of the findings: Perhaps the most important results appear in Figure 4. In my opinion it would have been more informative to show thresholds, or ABR signal levels (such as RMS), separately for waves I through V. This, because it is surprising that DBS in the IC would not affect ABR wave V amplitudes, since the IC is a major source of wave V potentials. It wasn’t clear to me how the wave V/I amplitude ratios, reported in Table 1, addressed the issue of amplitude effects. Were all of the Table 1 data collected with sound pulses at 90 dB (SPL)? This was implied in Methods, but not clear. If yes, what about the dynamic range of wave V (and other wave components)? Furthermore, if both wave I and wave V diminished, the V/I ratio would not change.
On the topic of threshold: Threshold is just one point of the dynamic range of evoked potentials. In this context Schaette (Schaette, 2014) and others have introduce the concept of “hidden hearing loss.” A more sensitive measure of loss would be to quantify the evoked response strength over the entire range of stimulus levels, for each test frequency and/or each ABR wave (I – V). Root mean square (RMS) is often used for that purpose. Another advantage of the dynamic-range approach is that thresholds do not have to be determined by experimenter judgment.
One level of DBS (100 µA) was tested. Since this level was shown in the preceding tinnitus study (Smit, et al., 2016) to be effective in tinnitus attenuation, perhaps this is adequate to show that the tinnitus attenuation did not derive from a hearing threshold shift.. However, the value of the present study would have been improved if a range of DBS levels had been tested… analogous to testing a drug at more than one dose.
Additional comments: The primary value of the present research might be the addition of the DCBN component. Note the published experiment (Smit et al., 2016) showed that DBS in the IC did not affect PPI. On this basis one would have to conclude that IC DBS did not interfere with central auditory processing. This would be true because PPI depends not only upon a functioning auditory brainstem (and ear), but also to some extent, a functioning auditory forebrain (Du et al., 2011). The point is that the prior published finding diminishes the knowledge added by the present work. Nevertheless, demonstrating an effect, or lack of effect, on the ABR has value.
References
Du, Y., Wu, X., Li, L. 2011. Differentially organized top-down modulation of prepulse inhibition of startle. J Neurosci 31, 13644-53.
Schaette, R. 2014. Tinnitus in men, mice (as well as other rodents), and machines. Hear Res 311, 63-71.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HEARING ASSESSMENT DURING DEEP BRAIN STIMULATION OF THE CENTRAL NUCLEUS OF THE INFERIOR COLLICULUS AND DENTATE CEREBELLAR NUCLEUS IN RAT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is written in clear English and is well structured. The hypotheses are clearly explained, reasonable and supported by sufficient amount of existing work in the literature. The structure of the result reflects what is expected from the hypotheses tested by the authors.
Experimental design: In this article, the authors test the potential of deep brain stimulation to treat tinnitus in two brain targets. Both targets are theoretically relevant and untested, moreover, they chose to implant the electrodes in the area, which is involved directly in auditory input processing and another area, which is not involved, but remains relevant based on lesion studies.
The scientific is well formulated and directly tested.
The investigation was performed rigorously and the authors have considerable experience with deep brain stimulation experimental design. This particularly visible in the method description section, in which is all relevant experimental details are well explained to allow for data reproduction.
Validity of the findings: Although the data are mostly not significant, the authors performed strict scientific observation of their data collection. The data set is robust and is discussed adequately in the discussion. Statistical tools have been used appropriately and the data set has been rigorously and thoroughly explored.
Additional comments: The article is well written and the authors discussed objectively their findings. The use of the ABR is welcome and provide an objective assessment of the efficiency of their method. Although the data set does not suggest the chosen targets to be relevant for treating tinnitus, this work is scientifically executed and deserve its place in te litterature, as it is very informative theoretically and technically.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HEARING ASSESSMENT DURING DEEP BRAIN STIMULATION OF THE CENTRAL NUCLEUS OF THE INFERIOR COLLICULUS AND DENTATE CEREBELLAR NUCLEUS IN RAT
Review round: 2
Reviewer: 1

Output:
Basic reporting: Acceptable.
Experimental design: Acceptable.
Validity of the findings: Acceptable.
Additional comments: This revised manuscript has been substantially improved. My questions have been satisfactorily answered.
The descriptive data, e.g., Fig 4, certainly appear equivalent between treatment conditions (i.e., brain stimulation) and groups (recording site). This, of course, supports the conclusions.
Suggested minor improvements.
The authors discuss the importance of recording electrode placement for ABR acquisition. Equally, or perhaps more important is consistency of sound presentation. Sound presentation features should be more precisely explained, how was the speaker presenting sound to the right ear (lines 146 – 147) arranged and standardized between recording sessions? Our experience shows that more ABR between subject variance can be attributed to sound stimulus variation than to electrode placement.
Line 176. Use “data” as a plural noun.
Line 276. Substitute “why” for “that.”
Although not that important for the present findings, the discussion paragraph considering why HF-DBS might disrupt tinnitus but not exteroceptive hearing (lines 291 -298) could be improved. In this paragraph HF-DBS is initially considered as having broad volume impact. Later in the paragraph it is hypothesized that HF-DBS might disrupt tinnitus because of a focal effect, leaving parallel pathways carrying external sound information unaffected. Overall this appears contradictory. A related consideration (maybe too distant to be included) is that most researchers now consider the pathophysiological underpinning of tinnitus to be broadly distributed in the CNS.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: VOOMDDA: DISCOVERY OF DIAGNOSTIC BIOMARKERS AND CLASSIFICATION OF RNA-SEQ DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: In summary, this study was well planned, efficiently organized, and competently reported. The manuscipt presentation and literacy standard are generally high.
Experimental design: Some of the real RNA-sequencing datasets were download from public database. They may produce from different laboratories. Measurements are affected by laboratory conditions, reagent lots and personnel differences. Did the author takes the batch effect into account? Will this affect your predicted results?
Validity of the findings: In evaluation process paragraph (line 428-429), the data were randomly split into two parts as training (70%) and test (30%) set. We know that test errors may differ for different train/test splits.(line 436).How dose the user determine the appropriate split ratio?
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: VOOMDDA: DISCOVERY OF DIAGNOSTIC BIOMARKERS AND CLASSIFICATION OF RNA-SEQ DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Zararsiz et al. introduced a method which combined voom and nearest shrunken centroids/diagonal discriminant classifiers together to perform classification of RNA-seq data. The methods are interesting, particularly for its possible application in the discovery of diagnostic biomarkers based on RNA-Seq data. Nevertheless, there are several weaknesses that should be improved. My major concerns and specific comments are listed below.

1) The manuscript was not very well written, containing few typos and formatting/grammar errors. The equations lists in the manuscript seem to be correct but not easy to follow, detailed mathematical annotations are encouraged to be included.
Experimental design: 2) The purposed method VoomDLDA/VoomDQDA or VoomNSC didn't show superior performance compared to conventional approaches, in both simulation (Fig3) and real data. It will thus limit the application in practice.

3) There is large paragraph descriping data normalization in the Materials & Methods section. However, the final results showed that expression data from raw read counts, median ratio normalization or TMM normalization are basically identical (Fig3 & Fig4), any explanation for that?
Validity of the findings: 4) For the purpose of discovering diagnostic biomarkers, it would be more relevent to train the model to prioritize the known biomarkers. Unfortunately, I'm not ware the author performed analysis of the kind in the manuscript.

5) The author split the data into training and test sets with 70% and 30%, is it part of cross validation, please clarify.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: VOOMDDA: DISCOVERY OF DIAGNOSTIC BIOMARKERS AND CLASSIFICATION OF RNA-SEQ DATA
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The paper introduces a novel data normalization, processing, classification, and feature selection method for RNA-Seq count data, by combining Voom normal linear modeling method with NSC (nearest shrunken centroids) and other classification methods. The presentation and experimentation is comprehensive. The authors utilize both simulated and real datasets and compare their results with some of the commonly used classification methods.

I commend the authors for making their source code available. The method is also made available via a web service.

I do not have any major concerns. I have listed the following points which would improve the publication:

* In the abstract, keep background info to a minimum (RNAseq advantages not necessary to list in abstract) and highligh the contributions more.

* The authors call their method voomNSC in the abstract, but voomDDA in the web link and the title. Use one name for the method, consistently. What does voomDDA stand for? If voomDDA is the umbrella term, the abstract should explain that rather than just voomNSC.

* Expand the Voom acronym in the abstract ("variance modeling at the observational level")

* In intro, authors introduce Voom as a good recently developed method and list its advantages. But a motivation for extending Voom is missing. What does Voom not handle that this paper is addressing?

* SVM seems to perform well, but the main criticism of the authors is its inability to produce sparse models. SVM is frequently applied in combination with a feature selection strategy (e.g., forward selection or backward elimination). Performing SVM without feature selection is an unfair comparison.

* In Equation 1: what is X.i ?

* In Tables, mark the winners in bold.

* Include other classifiers (e.g., SVM) also in the webtool.

* Network analysis and GO analysis may not be appropriate for the selected classifier genes. Sparse set of genes that are good for classification would mask other coregulated and functionally relevant genes that are necessary for meaningful downstream analysis.

* The web address in pdf is hyperlinked to "http://www.biosoft.hacettepe.edu.tr/voomDDA/.%20". The extra .%20 causes problems.

* Heatmap, Network, and Go features seem to require the user having to run VoomDDA analysis first. Disable Heatmap/Network/Go until VoomDDA is executed, or automatically execute it.

* Proofread the paper for syntax and writing errors. Some errors are:
* counts which [are] obtained from
less advancements: awkward phrasing
we benefit from the delta rule: replace "benefit"
lead to obtain[ing]
thenumber, usingdeseq, parameterthat, thenormal, miRNAswith, andlung, isapplied


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: VOOMDDA: DISCOVERY OF DIAGNOSTIC BIOMARKERS AND CLASSIFICATION OF RNA-SEQ DATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: All my concerns were well answered.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: VOOMDDA: DISCOVERY OF DIAGNOSTIC BIOMARKERS AND CLASSIFICATION OF RNA-SEQ DATA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have largely addressed my comments. The revised manuscript is better and I am happy with the content of this manuscript.
Experimental design: As above.
Validity of the findings: As above.
Additional comments: As above.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ANTI-APOPTOTIC PROPERTIES OF CARBON MONOXIDE IN PORCINE OOCYTE DURING IN VITRO AGING
Review round: 1
Reviewer: 1

Output:
Basic reporting: The work is well written and presented and the data are clear.

Insufficient citations of the literature. The authors should be more comprehensive of the literature as it relates to reviews cited (e.g. Motterlini) as well as some of the seminal papers showing the effects of HO-1/CO to mediate apoptosis (Brouard, Zuckerbraun, et al.).

The results are relevant to the hypotheses.
Experimental design: The experimental design is reasonable, but not terribly innovative given the available tools by which to study the effects of HO-1/CO on apoptosis. The authors use an outdated CORM (CO Releasing Molecule) and are missing important controls that include the metal core and the inactive compound.

The use of a phamacologics to block HO-1 should be complemented with siRNA validation given the off-target effects of Zn-PP.

The use of immunohistochemistry should be validated with PCR or Western blot data to show that there is true changes versus artifact of the staining and specificity of the antibodies.

From a scientific standpoint, none of the data is terribly novel given the enormous literature base. Adding a cell target for CO would strengthen the work and increase the enthusiasm that this will be an advance for the field.
Validity of the findings: Data is not terribly robust with the majority of findings dependent on immunostaining. Validation with qPCR or western blot is necessary. Given that both HO-1 and HO-2 are changing almost identically draws question as to whether the antibodies are specific. No controls are provided. HO-2 is typically constitutively expressed and if there is induction, this could be an important contribution.

Conclusions are reasonable, but again based on limited data sets that confirm a large literature base. Adding more detail on cell targets would be a stronger advance for the field.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ANTI-APOPTOTIC PROPERTIES OF CARBON MONOXIDE IN PORCINE OOCYTE DURING IN VITRO AGING
Review round: 1
Reviewer: 2

Output:
Basic reporting: This work studied the role of CO as an antiapoptotic molecule in porcine oocytes. The paper is easy to read and the basic ideas are clear. The english and references are appropiated. As metioned the structure is fine, but the some figures are dificult to understand and some results needs to be more robust.

Specific comments:

Page 7, line 38, please change "animal species" for "mammals"
Page 9, line 97, please change "carbon monoxide" for "CO"
Page 9, line 126, why above do you use ethanol and acetic acid and then changed to paraformaldehyde to fix the cells ?

In figure 2, change HO-1 for HO-2

Figures 4 and 5 are difficult to understand, can you show your results in a clearer way ?

In all the figures, please change the A, B, C nomenclature for more traditional *,**,*** for statitical significance.

Why in the text you add the mean values without error or standar deviation ?

Is the entire HO-1 localized in the nucleus or is a fragment of it, which could be recognized by the antibody ?

I have some concerns in relation with the HO inhibitor used in this work. In my undestanding (and as you commented in the discusion secction) the Zn-PP IX is a HO inhibitor but also inhibits other enzymes and receptors such as for example: interleukin-1 receptor, GCS, and all the NOS isoformos. Therefore, the results obtained could be subtantially improved if iRNA agaist HO is used.

Additionally, is suggested to use another CO donors with different structures in order to compare your results (e.g. CORM-A1). This because CORM-2 as well known produces CO, but also produce several other molecules, are you confident that any of these additional molecules are not doing anything in your model ?
Experimental design: The experimental design is fine, just minor comments, see above. The only concern is that the pharmacology can be improved (see comment above)
Validity of the findings: The results are novel, but they are not so robust, they need more work in terms of pharmacology and use of iRNA.

Conclusions are well stated and not to speculatives.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ANTI-APOPTOTIC PROPERTIES OF CARBON MONOXIDE IN PORCINE OOCYTE DURING IN VITRO AGING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The responses of the authors were reasonable. unfortunately, the data in figures 3 and 10 are not complete. The authors need to show side by side the changes in HO-1 and caspase 3 cleavage.   In figure 11, why is the dose response curve showing increased apoptosis with higher amounts of CORM at 24h?
Experimental design: reasonable except for the comments above about including comparators.
Validity of the findings: In light of the newly added data, it is difficult to see how the conclusions are being made and now need additional data.
Additional comments: Overall, the responses are adequate, but the data are somewhat incomplete (see above).


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ANTI-APOPTOTIC PROPERTIES OF CARBON MONOXIDE IN PORCINE OOCYTE DURING IN VITRO AGING
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript have been improved. The authors asnwered to all my main concerns.  However, still remains some point to be improved.

In the introduction between lines 72 and 88 is given the same (more or less) information compared to the paragraph between lines 89-97. Please fuse them in only one paragraph.

Line 238, change "they were" by we

In Results. Figure 2 must be the new figure 1.Therefore the sentece between lines 199 and 205 should be

"The objective of the experiment was to prove the presence of HO-1 and HO-2 proteins in meiotically mature porcine oocytes (MII) and oocytes exposed to in vitro aging for the period of 24, 48 and 72 hours. HO isoforms are present in porcine oocyte (FIG 1, ex figure 2), and their expression gradually increases during in vitro aging. In case of HO-1, the signal was predominant in the nucleus/perichromosomal area (Fig 2). On the contrary, in HO-2 the signal was primarily observed mainly in the oocyte cytoplasm (Fig. 3)."

In Discussion: if Zn-PP IX can inhibits the NOS...why you do not try in inhibit the NOS with L-name or other and see if the effect is similar or complementary to the CO-induced effect.
Experimental design: NA
Validity of the findings: NA
Additional comments: NA


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF ASTRAGALUS POLYSACCHARIDE ON THE ADHESION-RELATED IMMUNE RESPONSE OF ENDOTHELIAL CELLS STIMULATED WITH CSFV IN VITRO
Review round: 1
Reviewer: 1

Output:
Basic reporting: It is a solid, well-written article on the effects of Astragalus polysaccharide on porcine endothelial cells. Figures are good, literature adequate.
Experimental design: The article and data are original enough to be published in the journal. The experimental design is well prepared, experiments are good and results are sound. The only problem is the lack of definition of the Astralagus polysaccharide. As it is crucial for the investigation, the respective information has to be added.
Validity of the findings: Data are find, sound and interesting. Conclusion is fully based on results. List of references is adequate and relevant.
Additional comments: An overall solid paper with potential clinical and/or industrial interests has one problem -  the lack of definition of the Astralagus polysaccharide. As it is crucial for the investigation, the respective information has to be added. It is not possible to publish biological study without attempting the fully describe the material used in the study.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF ASTRAGALUS POLYSACCHARIDE ON THE ADHESION-RELATED IMMUNE RESPONSE OF ENDOTHELIAL CELLS STIMULATED WITH CSFV IN VITRO
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. The introductions need more details about the relationships between ECs cells, hemorrhagic diseases, and the factors tested in this studies.
2. In the Discussion section, please refer to related figures in text. The authors did that in the Results section, but they are also needed in the Discussion section.
Experimental design: 1.	The authors need to indicate the catalog numbers or concentrations of the antibodies used in this study.
2.	In Line 84, did the authors add serum and antibiotics to the medium?
3.	In Lines 93-95, please write the amounts of HPR-labeled ABC and DAB were added into each well.
4.	In Line 240-243, it looked like P-selection expression occurred later than E-selection expression in Figures 2A and 2B. Since the WPBs were released quickly, the authors cannot observe that kind of difference through qRT-PCR, which is used to study the changes at transcriptional level. The authors need to explain more clearly about their proposed mechanisms.
5.	In Lines 366-367, what does “puce staining” mean?
6.	In Lines 369-370, the authors need to refer the different bars to the culture conditions. Also do this in figure legends 3 and 4. Also, add “either” before “medium alone”, and replace “and CSFV plus APS” by “or CSFV plus APS”.
Validity of the findings: 1.	My only major concern is the claims about APS’s functions. In this work, the authors studied the gene expression profiles related to cell adhesion and immune response, but did not do functional assays to confirm APS’s protective role during CSFV infection nor use APS to treat hemorrhagic infection.
2.	In the abstract, the authors concluded that APS is a potential adjuvant for CSFV vaccine. However, in the last paragraph of Introduction, the authors claimed that the adjuvant part was studied in their previous work, while the aim of this study was to identify a method for hemorrhagic treatment. Please make the conclusion clear and consistent throughout this manuscript.
Additional comments: The paper studied the immunomodulatory effects of Astragalus polysaccharide, the key active component extracted from a traditional Chinese medicinal herb, on endothelial cells’s immune response to classical swine fever virus. The authors stimulated the ECs with CSFV in the presence of APS and investigated the gene expression profiles of key factors related to cell adhesion, cell growth and immune response. They observed that the addition of APS could downregulate the expression of selectins, tissue factor, TLR-4, interferon and TGF beta, thus indicating APS’s potential as an adjuvant for CSFV vaccine.

The authors used qPT-PCR and ELISA to quantify gene expression, which is commonly used method in this area; the statistical analysis part is good.

1.	In Lines 369-370, the authors need to refer the different bars to the culture conditions. Also do this in figure legends 3 and 4. Also, add “either” before “medium alone”, and replace “and CSFV plus APS” by “or CSFV plus APS”.
2.	In Line 371, RT-PCR is real-time PCR here, the “real-time” can be deleted.
3.	In Lines 371 and 377, it is “SYBR Green” not “SYBgreen”.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF ASTRAGALUS POLYSACCHARIDE ON THE ADHESION-RELATED IMMUNE RESPONSE OF ENDOTHELIAL CELLS STIMULATED WITH CSFV IN VITRO
Review round: 1
Reviewer: 3

Output:
Basic reporting: In the study entitled ‘Effects of astragalus polysaccharide on the adhesion-related
Immune response of endothelial cells stimulated with CSFV in vitro’, the authors have studied the effects of Astragalus polysaccharide on porcine endothelial cells. They have quantified the mRNA expression levels of E and P-selectins ,TNF, VGF and cytokines after CSFV infection and the effect of APS either by pretreatment or addition along with CSFV. The have also studied the levels of cytokines in these cultures by ELISA.  Overall, the experiments are well formulated but the manuscript lacks a detailed discussion of the results and interpretation of differences observed at different time points after stimulation. The authors should discuss their observations in the light of what has already been published in the literature in case of CSFV and other viral infection. APS has been observed to regulate the levels of immunomodulating compounds in many different cell types before (Zhou L, Sci Rep, 2017; Xue H, Sci Rep, 2017; Wu S, Sci Rep, 207 etc), whether these effects on Endothelial cells are specific for CSFV or true for most viral infection should be stated. Also, APS has been shown in previous studies to have an effect on many different cell types including myocardial functions, neuronal cells, macrophages etc, therefore, in general, its effects on various different cell types have been studied and its use as an adjuvant in vaccines against viral infections may be generic and not specific for CSFV and endothelial cells.
Experimental design: Line 42-46) -The basis of the study that author’s state in introduction is that vascular endothelial cells play a role in recruitment of immune cells and influence the outcome of immune response, but not sufficient literature is cited to support this.
Many different cell types are involved in innate immune defense in viral infections, and the authors have cited their own study on the effect of Astralagus polyscachharide on CSFV infection in peripheral blood mononuclear cells, published in Plos One in 2012. But they fail to convince in the introduction and discussion that ECs are infact important cells involved in primary line of immune defense against CSFV infection. Other such as Borca M,  2008 (Virus Res), Dong X, 2013 (Virology J), Glaude , 2008 (J virol) have studied CSFV infection in different cell types and should be cited.
(Line 51-70)- The background on Astragalus polysaccharide is hugely similar to the previous study the authors have published on the role of APS in PBMCs (Zhuge ZY, 2012, Plos One) and should be re written.
Validity of the findings: No comments
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF ASTRAGALUS POLYSACCHARIDE ON THE ADHESION-RELATED IMMUNE RESPONSE OF ENDOTHELIAL CELLS STIMULATED WITH CSFV IN VITRO
Review round: 2
Reviewer: 1

Output:
Basic reporting: Generally, my review of the new version remains the same: An overall solid paper with potential clinical and/or industrial interests has one problem - the lack of definition of the Astralagus polysaccharide. As it is crucial for the investigation, the respective information has to be added. It is not possible to publish biological study without attempting the fully describe the material used in the study.

The authors revised or explained all requests from the second reviewer., however, in case of my request, nothing really changed. Line 73 does not explain anything. Additional references support the claims, we there is still a need to explain the material, i.e., composition, purity  etc.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: From the reviewer;s pioint of view, nothing really changed. Line 73 does not explain anything. Additional references support the claims, we there is still a need to explain the material, i.e., composition, purity  etc.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF ASTRAGALUS POLYSACCHARIDE ON THE ADHESION-RELATED IMMUNE RESPONSE OF ENDOTHELIAL CELLS STIMULATED WITH CSFV IN VITRO
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The revised version addresses all my concerns and it is well-written. I recommend to accept the manuscript for the publication on PeerJ.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF INVASION HISTORY ON PHYSIOLOGICAL RESPONSES TO IMMUNE SYSTEM ACTIVATION IN INVASIVE AUSTRALIAN CANE TOADS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, I thought the article was well written and set up very well with previous research. I only have a couple of comments:

Ln 70: "These responses are predicted (and have been
70 shown) to be down-regulated in invasive populations (Lee & Klasing 2004; Lee et al. 2005)." Some examples of types of immune responses and what taxa would be helpful here.

Ln 71: What does standing immune responses mean? Does this mean constitutive?
Experimental design: Ln 194: I assume the authors mean 30 ul of Luminol.
Validity of the findings: Ln 115: Add the direction of the relationship.

In the table, the effect of RBCs is significant. Is this due to the autoflorensce of the RBCs? Just a brief sentence explaining this may be helpful for those not familiar with the technique.

Low sample size, but it is acknowledged by authors and conclusions are framed appropriately.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF INVASION HISTORY ON PHYSIOLOGICAL RESPONSES TO IMMUNE SYSTEM ACTIVATION IN INVASIVE AUSTRALIAN CANE TOADS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is clear and very well written. Title and abstract are both informative. Introduction supports the study and adequately refers to pertinent literature. However, the logic flow of the introduction should be improved to explain more effectively the main hypothesis of the study (see specific comments below).  The structure of the manuscript conforms to PeerJ standards. Figures and tables are all nice and properly labelled. Raw data are provided.
Experimental design: The research is within the scope of the journal and the research questions are quite clearly stated (but see specific comments). The experimental protocol has been carefully selected not only to address the specific questions of the study but also to propose a novel effective procedure to investigate immune pathways. Since a previous work (Brown et al. 2015) which investigated the same aspect (immune response) in the same two populations of cane toad did not account for inter-individual variation, the study also represents a further step in our comprehension of invaders’ eco-immunology. The research protocol is rigorous and numerous details are provided to replicate the study.
Validity of the findings: Although the main prediction of the study (that is higher less costly immune response in toads from the invasion front) is not confirmed by the results, the findings of the manuscript are still valid with several suggested explanations to support the null outcome. Also, limitations of the study are clearly recognized (e.g. it did not “account for differences in environmental effects prior to collection”, L393-394) and future more advanced studies are proposed. Data is controlled and statistics used is adequate. The sample size is small and this could partially obscure the results but the limitation is well recognized in the manuscript. Conclusions are limited to supporting results and speculations are recognized as such. However, I feel that the second part of the discussion is a little bit too long (one or two paragraphs should be shortened or removed) and speculative. Only most valid speculations should be incorporated (see specific comments).
Additional comments: General comments

In this study, immune response and glucocorticoid response are experimentally induced in two Australian invasive populations of cane toads (one from the core and one from the periphery of the invasion) by injecting an antigen lipopolysaccharide. The study is very interesting and well supported by the exceptional literature regarding this invasive species. Although the results are not fully conclusive (no difference detected between the populations), the robustness of the protocol and the novelty of an immune response measurement in vivo make its publication recommended. Although I do not have any major revision to recommend, I have added some comments below that I hope could improve the readability of the manuscript, especially in the introduction section.

Specific comments

L38 - add “wild-caught” before “toads”?

L55-75 - The first part of the introduction, although logic and well structured, is a little bit misleading. The authors state correctly that a down-regulation of the costly systemic inflammatory response is expected in invasive populations (or in the populations at the invasion front); however eco-immunology theory predicts also that less costly immune pathways should be up-regulated by invasive populations or species (see for example “humoural” response in Lee and Klasing 2004). The authors, later in the text, hypothesize exactly this up-regulation (also in accordance to a similar study conducted by Brown et al. 2015 that compared the same two invasive populations). Therefore, they should clarify here (and not later, at L89-91) that two divergent eco-evolutionary scenarios (downregulation of costly immune pathways and upregulation of less costly immune pathways) are expected in invasive (or invasion front) populations.  This should be helpful, especially for a general reader, to comprehend the aim of the study, its assumptions and also the main prediction reported at L129-132.

L73 – “in human neutrophils” Could you find more references, not necessarily in humans, that support this?

L76-77 “To Australia from Hawaii” - The cane toad did not arrive in Australia directly from the native range (South America) but rather it was moved from one area to another (e.g. Hawaii) until it established in Australia. It means that during its invasion history, it has been exposed to many different groups and sources of pathogens. Could have this promoted a strong less costly response of the immune system that become canalized (less plastic) in the invasive Australian populations? Since you are not comparing a native pop with an invasive population but two invasive populations, this should be somewhere stated in the manuscript, maybe in the discussion.

L90-91 I would suggest “less costly” instead of “not costly”. Any immune response should have a cost, although minimal.

L 104 “because this antigen is no longer part of a live organism, mutation and replication are not possible”. Any reference for this?

L107-108 “We applied this experimental method to clarify immune function in different populations of invasive cane toads.” This sentence is too generic, please rephrase.

L139-140 Could you please add the geographic coordinates for the two sites of capture (Cairns and Oombulgurri)? Also, a map of Australia with the cane toad invasion, although not essential, could help the reader to visualize introduction point and current invasion front.

L290-291 “Contrary to our predictions, responses to LPS exposure did not differ significantly between toads from the invasion front population versus the range core population”. Your prediction was more specific than that because you were expecting an up-regulation; please rephrase.

L291-293 “Our predictions were based upon the energetic costs associated with mounting immune responses while undergoing enemy release.” This sentence is too generic, please rephrase. Also, as many different types of immune responses can occur and some are not costly, I would suggest being more specific across the whole manuscript for example distinguishing between systemic inflammatory responses and humoral responses (as stated in Lee and Klasing 2004).

L310-319 This could be summarized in only two sentences.

L 330 “More mobile toads”. Please clarify this for a general reader. Do they move more frequently? Do they move longer?

 L 370-376 Since Brown et al. 2015 did not detect any sex effect, this paragraph could be removed because it does not add anything essential to the interpretation of your results.

L574 Please fix reference (space issue)

In the tables. Significant results in bold? Also, p-value is not defined anywhere in the manuscript.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: QUALITATIVE STUDY OF HEALTHCARE PROVIDERS’ CURRENT PRACTICE PATTERNS AND BARRIERS TO SUCCESSFUL REHYDRATION FOR PEDIATRIC DIARRHEAL ILLNESSES IN KENYA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear English:
Overall, the language used was clear, unambiguous, and reflects current professional standards. Some minor grammatical errors. Specifically, place a comma after using "despite" when followed by a contrasting clause (ex. lines 52 & 95). Additionally, some of language included may benefit from minor revision (examples below).

Line 110: Use of the phrase “in our experience” seems very anecdotal. Though there may be very little, if any, research regarding the use of NGs, consider including literature that looks at which rehydration practices are most used in Kenyan healthcare settings. While your experiences are more than valid, offering a more concrete reference, if available, would strengthen your point.

Line 113: “There is no literature” is a very strong statement. While I am sure your review of the available literature was exhaustive, you may want to avoid such an absolute. Consider using “based on an exhaustive (or extensive) review of the literature…” or “No literature pertaining to x was identified”.

References:
Most of your references are 6 or more years old. One is almost 30 years old. While many seem relevant to the current study, consider including literature published in the last 5 years.

Line 92: You use a CDC reference from 2011. I would encourage you to use a more current reference. There are mortality estimates--those cited in CDC reports--that have been published in the last 3 years.

Line 94: To my knowledge, cholera does not drive diarrheal rates among infants and young children in Kenya. Consider referencing the pathogen(s) that drive diarrheal rates among this population in this setting. The Global Enteric Multicenter Study has a number of published papers that could provide some additional information.

Figures and tables:
Table 1 could use some additional development. Consider including more demographic/background information in table 1, if available.

Self-contained:
Overall, the paper was strong and well-written. However, there are several areas of the paper that could benefit from additional consideration. Specifically, the methods and results sections (discussed below).
Experimental design: Original research:
The research is original and fits well within the scope of the journal.

Research question:
The research question is clear and fills a important knowledge gap in the literature.

Rigorous investigation:
Based on the information included, the research meets high technical and ethical standards. However, I do have one question regarding consent.

Line 126: Since your study population was limited to pediatric care providers, all of whom are likely literate, why did you only collect verbal consent? Verbal consent is generally reserved for studies exploring socially sensitive/taboo topics or for populations where literacy is limited. Were there barriers to collecting written consent?

Methods:
When writing up a methods section, I generally work to answer the who, when, what, where, and how of the study. While simplistic, it serves to ensure that at least the basic components of the methods section are articulated. While you clearly address the where, who, and what - where (western Kenya), who (44 healthcare providers at various facilities around MTRH and AMPATH), what (a case-scenerio and semi-structured interviews), you do not provide any information as to “when” the study was conducted, “how” participants and health facilitates were selected for inclusion or “how” much time each participant contributed to the study. Further, I do not see any reference to your sampling technique. Though based on the description offered, it seems as convenience sampling was used. This should be made explicit. Noting the sampling techniques used, even when describing a study with a small sample, provides the reader with a better frame to interpret the results.  Also, please describe the number of healthcare facilities in the region and the number you planned to include in the study.

Line 148-156: The description of the study population needs to be improved. As noted, be sure to include a more developed description of sampling techniques used. As it stands, it is unclear if you selected either the research sites or the individual participants based on convenience, purposive, snowball, and/or random sampling. Though you have a small sample, this information in necessary for framing the results presented. Also, would community health workers receive training regarding NGs and/or would they typically be expected to administer them or be familiar with the process. If this is not included in their training, their responses may skew the results. Lastly, how many healthcare facilitates sit within a certain radius of MTRH and AMPATH and how many of these healthcare facilities were represented in data collection?
Validity of the findings: Once other comments are considered, you may want to update the discussion and conclusion accordingly.
Additional comments: Results:
You seemed to lump all providers together thereby failing to acknowledge the varying levels of training received. Community health workers (CHWs) receive far less training than clinicians. Moreover, given the level of training received by CHWs, should they be expected to be familiar with the administration and/or use of NGs for rehydration or even the related WHO guidelines? There are arguably 3 or 4 levels of providers with regard to training. However, the results section, as currently written, does not seem to consider the extent to which provider training may influence NG use. You essentially treat all providers as equal leaving me with several answered questions: Were specific provider types more likely to describe NGs as less effective? Were specific provider types more likely to be familiar with the WHO guidelines? Did this change according to practice type (i.e. providers at a referral hospital are more likely to use the procedure)? Other similar considerations are offered below.

Line 266: How many providers out of 44 were unfamiliar with the guidelines? Again, did you look at this according to provider type? Or practice type?  Further, is it possible that the results were impacted by other factors? How long did each provider practice? How certain are you that these approaches/techniques were taught? At some point prior to the study, did all providers receiving training on the administration of NGs? Was this influenced by provider type?  Additionally, nurses represented almost half of the participants included. How might this impact the study results?

Additional general comments:

Title: Rehydration can occur both within and outside of healthcare settings. Your title should reflect your focus on healthcare providers/healthcare settings.

Line 104: You suggest that NGs can be applied before arrival to the hospital. Given the settings and the more typical modes of transportation used when trying to access healthcare, who would be applying the NGs before arrival. As written, it implies that non-healthcare providers, those involved in transport, could perform the procedure.

Line 164: Consider writing out emergency department rather than using the acronym “ED”.

Line 175: Every interview is not likely to be a “key-informant” interview in the strictest interpretation of the term. I would encourage you to remove that language as it does not add anything to the sentence and may distract the reader from the main point you are trying to make.

Line 183: See comment regarding line 126.

Line 204: Be specific about the type of triangulation referenced (investigator triangulation).

Line 209: In general, you should avoid beginning a sentence with a number. Spell out the number 44.

Line 313: Consider this statement in concert with the statement regarding line 110. None the studies you allude to are clearly references above.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: QUALITATIVE STUDY OF HEALTHCARE PROVIDERS’ CURRENT PRACTICE PATTERNS AND BARRIERS TO SUCCESSFUL REHYDRATION FOR PEDIATRIC DIARRHEAL ILLNESSES IN KENYA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language is good, fluent, correct and clearly understood.
Experimental design: There are 3 main problems with the study:
1.	According to Good Clinical Practice (GCP) one needs an approval for a study, and informed consents, for studies dealing with patients. This study does not deal with patients: it deals with caregivers. This study is a questionnaire for caregivers. Well, it is the same as examination, so it needs neither approval, nor informed consents.
2.	The sample size is 44 caregivers. The reader has no idea about the population size of caregivers out of which the sample was taken, and its distribution. It could be that the sample was too small, and hence the study may not make any conclusion regarding the population of caregivers in Kenia. Moreover, the authors did not explain how they chose the participants in the sample, so even the authors have no idea if the sample that they chose is representative.
3.	According to WHO guidelines, one does NOT use NG tube after failing an attempt placing IV line; rather, one uses NG tube BEFORE even trying the first attempt placing IV line! So, the questionnare should be changed, all the study should be redone.
Validity of the findings: The findings can not be discussed, as there are the study is mal-designed.
Additional comments: I am sorry, but the manuscript is not acceptable for publishing anywhere, due to serious design mistakes.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: MECHANISMS OF ACTION AND  IN VIVO ANTIBACTERIAL EFFICACY ASSESSMENT OF FIVE NOVEL HYBRID PEPTIDES DERIVED FROM INDOLICIDIN AND RANALEXIN AGAINST  STREPTOCOCCUS PNEUMONIAE
Review round: 1
Reviewer: 1

Output:
Basic reporting: ... stated in 'General comments for the author'
Experimental design: ... stated in 'General comments for the author'
Validity of the findings: ... stated in 'General comments for the author'
Additional comments: In the manuscript #18224v1 by Jindal and colleagues “Mechanisms of action and in vivo antibacterial efficacy assessment of five novel hybrid peptides derived from Indolicidin and Ranalexin against Streptococcus pneumoniae”, authors aim to investigate the mechanisms of action and the therapeutic efficacy of the hybrid peptides. Among other things authors assessed the effects of those peptides on the integrity of the pneumococcal cell wall/membrane. They also measured by DNA retardation assay, the impact of the peptides on the integrity of, and binding to genomic DNA of those microbes. The in vivo-toxicity and antibacterial activity was evaluated on ICR male mice. Authors have found that the hybrid peptides caused significant morphological alterations in Streptococcus pneumoniae and disrupted the integrity of the cell wall/membrane. The rapid release of ATP from pneumococcal cells after one hour of incubation suggests that the antibacterial action for the hybrid peptides is based on membrane permeabilization and damage. The DNA retardation assay revealed that at 62.5μg/ml all the hybrid peptides were capable of binding and preventing the pneumococcal genomic DNA from migrating through the agarose gel. In vitro synergy was observed when pneumococcal cells were treated with combinations of hybrid peptides with each other and with conventional drugs erythromycin and ceftriaxone. The in vivo therapeutic efficacy tests revealed that the hybrid peptide RN7-IN8 could improve the survival of pneumococcal-infected mice, as 50% of the infected mice survived up to 7 days post-infection. In vivo antibacterial efficacy of the hybrid peptide RN7-IN8 was significantly improved when combined with the standard antibiotic ceftriaxone. The presented results suggest that breaching the cell wall/membrane is likely the principal mechanism of action of the hybrid peptides. Furthermore, the hybrid peptides may affect DNA synthesis and gene expression. This is potentially-interesting manuscript, however it requires a lot of improvements.

Improvements suggestions:

1.)
Manuscript’s English language is so bad that often it is difficult to understand the meaning of the sentences (even in the summary). The manuscript MUST BE edited by a native English scientist prior to resubmission.

2.)
Please state in the figure legends 3 and 4, from how many experiment-repetitions the data has been obtained.

3.)
The data displayed in figure 3 is poorly presented. It should be rearranged and preferably split into 2 subfigures (with controls included in both), so that the ATP-release is seen as time kinetics.

4.)
The discussion needs to be expanded, especially regarding possible molecular mechanism(s) of the interactions of the dipeptides with bacterial membranes. Authors may get inspired by recent papers by Ghavami et al (doi: 10.1111/j.1582-4934.2008.00129.x), and Savelyeva et al., (doi: 10.1007/978-1-4471-6458-6_10).


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: MECHANISMS OF ACTION AND  IN VIVO ANTIBACTERIAL EFFICACY ASSESSMENT OF FIVE NOVEL HYBRID PEPTIDES DERIVED FROM INDOLICIDIN AND RANALEXIN AGAINST  STREPTOCOCCUS PNEUMONIAE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the English is acceptable. However, the first sentence shows an unsual structure.

Reference style needs to be optimized (capitals in journal titles)

The authors use hybrid AMPs but do not explain what sort of hybrids they used; a reference to a previous paper is not enough information for a reader.

Introduction and Discussion need to be optimized; do not repeat information in both parts.
Experimental design: Quality of EM pictures is low and not very informative.

The effect of AMPs is time and dose dependent; provide such data.

ATP release: provide time and dose dependent data

AMPs can work within minutes; proide data for 10, 20, 40 min

DNA binding: why do you assume that AMPs bind to DNA; explain your rationale; Fig 4 is not convincing; do you have a positive control for a DNA binding agent?

Kaplan Meyer curves:
Better than control would be: healthy animals without infection
Not untreated: better infected animals without antimicorobial treatment
Validity of the findings: Without dose and time dependent data, this paper is rather premature. It needs more laboratory work to be conclusive.
Additional comments: Without dose and time dependent data, this paper is rather premature. It needs more laboratory work to be conclusive.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: MECHANISMS OF ACTION AND  IN VIVO ANTIBACTERIAL EFFICACY ASSESSMENT OF FIVE NOVEL HYBRID PEPTIDES DERIVED FROM INDOLICIDIN AND RANALEXIN AGAINST  STREPTOCOCCUS PNEUMONIAE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors of this report present us with an interesting study.  The contribution of this paper proves significant in determining the action mechanism of new antimicrobial peptides. The investigation about new molecules with antimicrobial activities is currently under study.
Introduction to this paper is comprehensive, and it provides a clear support for both the theoretical aspects and the research objectives. Literature reference is sufficient to support the study. The article structure is clear and figures and tables are appropriately used.

Some suggestions and points that need clarify are in the revised version.
Experimental design: The article is an original study, the experiments assays are appropriately designed, however in some cases the materials and methods section requires more detail and more clarity in order to be useful to future readers.
The authors should be providing information about the reproducibility of findings. How many experiments were carry out?
Validity of the findings: No comment
Additional comments: The article is original and was appropriately discussed. The comments and suggestions to make it better are in revised version (pdf).


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: MECHANISMS OF ACTION AND  IN VIVO ANTIBACTERIAL EFFICACY ASSESSMENT OF FIVE NOVEL HYBRID PEPTIDES DERIVED FROM INDOLICIDIN AND RANALEXIN AGAINST  STREPTOCOCCUS PNEUMONIAE
Review round: 2
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: I am satisfied with the revision.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: MECHANISMS OF ACTION AND  IN VIVO ANTIBACTERIAL EFFICACY ASSESSMENT OF FIVE NOVEL HYBRID PEPTIDES DERIVED FROM INDOLICIDIN AND RANALEXIN AGAINST  STREPTOCOCCUS PNEUMONIAE
Review round: 2
Reviewer: 2

Output:
Basic reporting: My main concern and criticism has not been adressed.

Introduction and discussion are not consice enough; omit information which is not necessary to understand this paper
Experimental design: My concern addressed the quality of the experiments.
It demands that some experiments need to be  repeated and proper controls need to be used.
Validity of the findings: without controls the results remain difficult to interpret
Additional comments: the quality of the figures is still very bad; colours cannot be seen.
Figures are very crowded and not well designed


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: MECHANISMS OF ACTION AND  IN VIVO ANTIBACTERIAL EFFICACY ASSESSMENT OF FIVE NOVEL HYBRID PEPTIDES DERIVED FROM INDOLICIDIN AND RANALEXIN AGAINST  STREPTOCOCCUS PNEUMONIAE
Review round: 2
Reviewer: 3

Output:
Basic reporting: The basic report is include in general comments for the author
Experimental design: No comments
Validity of the findings: No comments
Additional comments: The second version of the paper “Mechanisms of action and in vivo antibacterial efficacy assessment of five novel hybrid peptides derived from Indolicidin and Ranalexin against Streptococcus pneumoniae” was appropriately revised.  However, some improvements are required.
1.	Figures: The figure 2 should be described appropriately on the figure legends.  In the actual figure 3 the authors should be include standard deviation and explain carefully the data of significant differences.
2.	I suggest that the authors review the use of abreviations of Units following the international system of Units. Por example: abbreviation for “hora” should be “h”. Use the space between number and unit.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: MECHANISMS OF ACTION AND  IN VIVO ANTIBACTERIAL EFFICACY ASSESSMENT OF FIVE NOVEL HYBRID PEPTIDES DERIVED FROM INDOLICIDIN AND RANALEXIN AGAINST  STREPTOCOCCUS PNEUMONIAE
Review round: 3
Reviewer: 1

Output:
Basic reporting: I am satisfied with the revision
Experimental design: I am satisfied with the revision
Validity of the findings: I am satisfied with the revision
Additional comments: I am satisfied with the revision


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INTEGRATION OF LNCRNA–MIRNA–MRNA REVEALS NOVEL INSIGHTS INTO OVIPOSITION REGULATION IN HONEY BEES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is flawed because it did not make clear the major question and the hypothesis of the study, neither in the abstract nor in the introduction and/or discussion sections.

The authors did not cite literature already published in Apis mellifera, which make important relationships for the robustness and support of this manuscript:

(1) ovarian activity and microRNAs (Macedo et al., 2016 - Insect Mol Biol. 25(3):216-26.);

(2) vitellogenin and microRNAs (Nunes et al., 2013 - J Exp Biol. 216(Pt 19):3724-32.);

(3) EcR-mRNA-microRNAs (Mello et al., 2014 - Front Genet. 5:445.);

(4) caste and microRNAs (Guo et al., 2013 - PLoS One. 8(12):e81661.; Shi et al., 2014 - Apidologie. 46:35);

(5) ovary, caste, mRNA, long noncoding RNAs (Humann and Hartfelder, 2011, Insect Biochem Mol Biol. 41(8):602-12.; Humann et al., 2013. PLoS One. 8(10):e78915.; Jayakodi et al., 2015 - BMC Genomics. 16:680.; Lago et al., 2016 - Insect Biochem Mol Biol. 79:1-12.).

Figures, tables and legends are not clear and should be improved. For example: What does A, B and C mean in Figure 1?
Experimental design: In my opinion, the research would fill an identified knowledge gap if question was well defined (but it is not well defined) and if data were appropriately analyzed. Methods are not described with sufficient information to be reproducible by another investigator. For example:

(1) how lncRNA's target genes were predicted?

(2) how many genes were validated by qPCR (5 lncRNA as in line 165 and 298? or 9 as in "result sheet" of Table S1? or 10 (used actin as reference gene?) as in "primer sheet" of Table S1?

(3) What does microarray mean in line 170?

(4) Line 152: What does "trans role is to identify each other by the expression level" mean?
Validity of the findings: Sequencing data appear to be of sufficient quality and preliminary analyzes were well performed. However, the experimental design of comparisons to know whether a gene is differentially expressed is unclear. What experimental design was used: reference design? loop design? saturated design? others? They used DESeq to compare any two groups (lines 132-135) / discriminate between two comparison groups (lines 179-180), however I'm not convinced that this is the best form of analysis. I would have agreed with authors if they had been performed all possible pairwise comparisons. If they have done all them, this is not clear from the text of the manuscript.

Finally, the authors performed an unsupervised hierarchical clustering analysis, which is fine (great job!). But they need to clarify in more detail about the use of each approach and the parameters used in each of them. How lncRNA's targets were predicted? About the construction of networks, the authors assumed two expression patterns: (1) up-regulated lncRNA / mRNA versus down-regulated miRNA or (2) down-regulated lncRNA / mRNA versus up-regulated miRNA. In what literature have the authors based themselves on assuming such premises or supporting such expression patterns?

In my view, conclusion are not well stated and not limited to supporting results. The queen's reproduction regulation was not elucidated (lines 375-378). The conclusion does not present the answer of a scientific question (knowledge gap). There are many speculations, but they have not been identified as such.
Additional comments: Dr. Xiao Chen and colleagues investigated the expression of genes (mRNA, miRNA and lncRNA) in queen ovaries undergoing four different contexts () in order to understand the molecular events associated with prolificacy. Despite the quality data, the written information in the manuscript is vague, generic, confusing or inconclusive. In general, it seems more a making-list + long descriptions than the expected interpretive deepening of the data.

Below, some points and suggestions:

1 - Prolificacy is an argument used in the abstract, in the beginning of the introduction and in line 340 of the discussion. I suggest that the authors use the data generated to explain this term "molecularly".

Throughout the text, it is very confusing to understand in which situation a gene is up- or down-regulated. The authors should make clear which is the reference sample, and always declare something like that: "up-regulated in situation A compared to situation B (or situations B, C and D)", for example.

It is not clear from the text what the new insights are. The found GO terms are too vague and / or generic (for example, system and organism development / metabolic process / etc.) so that the knowledge can not be advanced/deepen.

Line 129: Did miRNA target prediction use only 3'UTRs of target genes? Please, clarify it.

Lines 160-170: About Quantitative qPCR, how many genes were really validated? I observed primers designed for Gene ID 726542 (histone H3) but no results. Also, if authors used oligodT primer, it means that all validated genes are polyadenylated, are not they?

Line 290: network of ovaries activation or network of oviposition recovery? Please, clarify it.

Lines 331-352: microRNAs such as let-7, miR-316, miR-252, miR-1, miR-133, miR-375, miR-31a, miR-92a, miR-3049, miR-3718 should be better (Nielsen et al., 1994), and the results obtained in the present study were similar to those reported in the present study (Macedo, et al., 1991, Insec Mol Mol Biol 25 (3): 216-26). Exp Biol. 216 (Pt. 19): 3724-32.).

In addition, please also observe all the criticisms / comments / suggestions presented in the sections above: 1. Basic reporting, 2. Experimental design, 3. Validity of the findings.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INTEGRATION OF LNCRNA–MIRNA–MRNA REVEALS NOVEL INSIGHTS INTO OVIPOSITION REGULATION IN HONEY BEES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English in the manuscript needs to be improved. In many instances the language does not make the meaning of the sentences unclear, but it would still be improved considerably by having a native English speaker review the manuscript before resubmission.

An example is lines 346-347: “Ame-miR-315 down regulated in ovaries activation and target 253 mRNAs in the network.” This should say: “Ame-miR-315 is down regulated in the ovary activation samples and targets 253 mRNAs in the network” It could also say “… regulated in activated ovaries and targets…”
Along these lines, while “ovaries” is the plural of ovary, when referring to a sample type (e.g. ovary activation) it should be singular because it is a category. This is done in the introduction and the figure legends, but not in the rest of the manuscript.

There are some cases in which it is unclear what the meaning of a sentence is.
Line 152: “Trans role is lncRNA to identify each other by the expression level.” This sentence doesn’t make sense. A google search reveals that this is used multiple times, virtually verbatim in several publications over the last 3 years. These other papers seem to be making the distinction between cis and trans effects that lncRNAs can have (acting on neighboring genes, cis, or acting on distant genes, trans). However, “identify each other by the expression level” is completely unclear. How does a lncRNA “identify” something based on expression level? The remainder of the paragraph says that the authors looked for correlations between the expression levels of lncRNAs and coding genes. If the first sentence is meant to state this, it would be better stated as something like: “The potential trans role of lncRNAs (acting on non-neighboring genes) can be assessed by correlating expression levels between lncRNAs and mRNAs.”
Importantly, this sentence and the sentence following it are nearly identical to those used by other authors, particularly Lu et al., (2016). I understand that some technical phrases are difficult to rewrite and I am not accusing the authors or plagiarism, but it is important to rework these ideas into your own words. This case is particularly poignant because the sentence is completely ambiguous and has been passed down over several papers.

I have also found the interpretation to be difficult due to potentially ambiguous use of the term “target.” Targeting has a directionality to it and finding that X targets Y doesn’t mean that Y targets X.
Lines 74-75: “…lncRNAs can also be directly targeted by miRNAs for cleavage.”
	This line is clear and is directly supported by the citations, they show that miRNAs target the lncRNAs (miRNA acts upon the lncRNA), not the other way around.
Lines 155-156: “…lncRNAs and mRNAs which linked by the same target miRNAs, with…” In this case, the sentence states that the lncRNAs and mRNAs both target (act upon) one particular mRNA. As far as I am aware this is not the direction in which this works, mRNAs and lncRNAs are targeted by miRNAs (miRNAs act upon the others) and not the other way around. The Fan et al., (2015) paper supports this, the lncRNAs can influence mRNA expression level by interfering with the miRNAs that normally act upon that mRNA, but they interfere by being targeted by the miRNAs, not by targeting the miRNAs.
	I think that the Fan et al., 2015 reference does a good job in making this clear in their language (“lncRNAs acting as miRNA targets”).

The article contains many other grammatical, spelling, and formatting problems. Some of these should be caught by spell-checking software (e.g. line 156 “up-regualted”). There are a number of cases in which spaces are omitted (lines 126, 128, Figure 1 and Table 2 legends). These may be due to pdf formatting, but should be checked. Similarly, in the note of the Table 2 legend, the “Q” group seems to have a copy/paste error “Q, group of virgin queens normal oviposition queens;”.

Results section, Lines 234-296. These 6 paragraphs are tedious to read, if only because they are largely the same format and just report the two main patterns (up/down/up or down/up/down) in the three ovary states (activated, inhibited, recovery). Could these be incorporated into a table rather than being written out? If this can be done, it would make the results, and therefore the paper, much more concise and would make it easier to focus.
	Line 259-260: What is meant by “lncRNAs target miRNAs from 2 to 3” or “miRNAs target mRNAs from 9 to 18”? Does it mean that the lncRNAs have from 2 to 3 target miRNAs? (however, see note above about use of “target”) If this is the case, then it should be spelled out. E.g. lncRNAs act as targets for 2 to 3 miRNAs.

Figure 1: Why does the mRNA heat map show all 3 samples for each treatment, but the others only show a single column for each treatment?

Supplemental files: I appreciate the supplemental files as it makes it much easier to assess the results. However, it would be helpful to have a single file that includes all the details about the supplemental tables, including explanations of headers in those files. The header explanations could be included as a separate sheet in the table workbooks.

I didn’t look at every entry in every supplemental file, but I found two errors. The first sheets in Supplemental Tables 3 and 5 have rows that only contain FBgn names and they look like a pasting error (S3 row 2687, S5 rows 1644 and 1646). The rest of the files should be checked for these types of errors.
Experimental design: This work is original primary research that fits within the aims and scope of PeerJ. Overall, the experimental design and data collection appear rigorous. However, the methods need to be more clearly spelled out. I lay out some questions that should be addressed in a new version.

Lines 151-159. What are some of the specifics of constructing a network?
1)	Line 155-156: This states that lncRNAs and mRNAs were linked by the same target miRNA (presumably they are targeted by the same miRNA). However, lines 224-229 states that the network was constructed based on the correlation analysis, which looked just at expression levels. Which is the case?
2)	What does it mean to have correlated expression? Do they have to have the same pattern (e.g. both upregulated), or do they have to have the same magnitude within a pattern (e.g. each shows a 3.5 fold upregulation)?
3)	How were targets determined? Line 154 says that miRNA targets were determined using miRanda, but what parameters were used?
4)	Why wasn’t GSTAr.pl run like Fan et al., 2015? That seems like an important analysis to determine whether a lncRNA is a decoy vs a target of a miRNA. While the manuscript isn’t explicitly about decoy vs target, the authors ultimately want to conclude that lncRNAs are acting to regulate mRNAs through this network, and they seem to rely on the lncRNAs acting as decoys.
5)	Lines 93-96: Were these ovaries from a single queen? It seems like this is the case, but explicitly stating it would be helpful.
6)	Lines 131-138: It is never explicitly stated what comparison is used for the DE analysis. Based on the categories of DE used in Figs 2-4, it seems that over- and under-expression are based on comparisons with the virgin queens (i.e. overexpression means: Q>V, C>V, R>V and underexpression means: Q<V, C<V, R<V). This should be made clear in the methods.
Validity of the findings: Apart from the language issues and making the results more concise, my chief concern is the network analysis interpretation. Here is my understanding based on the manuscript:

RNAs were considered to be in the same network if they showed one of the two patterns lncRNA-miRNA-mRNA (up-down-up or down-up-down). lncRNA and mRNA seem to also be linked by being targeted by the same miRNA (line155-156, see comment in experimental design section).

I think that this network analysis makes sense when considering the expression levels, but I think it goes awry when trying to look at functional classifications because it begs the question (i.e. it is circular). GO terms and KEGG pathways were determined for lncRNAs and miRNAs by looking at the functions of their targets, which are mRNAs. These mRNAs should be already in the network, since the network is defined in part by the shared targets (one miRNA targeting a lncRNA and an mRNA). If this is correct, then that means that any functional analysis of lncRNAs or miRNAs should find considerable overlap between these RNAs and the mRNAs. This leads to spurious conclusions, like lines 204-205: “The result revealed that the lncRNAs and mRNAs interacted with each other and promoted the ovaries activation and the subsequent oviposition process.” This is exactly what you would expect based on the analysis regardless of any actual interaction. I’m not saying they didn’t interact, but the only evidence of that is the shared miRNAs and the expression pattern, the functional analysis adds no information.
	I think that the functional analysis can stay, if done solely at the mRNA target level and not attempting to compare it with the lncRNA or miRNA. This will still be helpful for understanding what the genes in these networks do, but it can’t be used to support the existence of the network itself.

More broadly speaking, I think the manuscript can be improved with a bit more discussion of what these processes mean for ovary activation and development. There are a lot of GO terms and KEGG pathways stated in the results and it would be helpful to have a more in depth biological discussion. The current manuscript relies heavily on the network analysis, but I think this should play a smaller supporting role. It seems that the main story should be about the biological processes involved in ovary regulation and the network analysis is a method of finding what functions these ncRNAs might have.
Additional comments: In the manuscript, “Integration of lncRNA–miRNA–mRNA reveals novel insights into reproductive regulation in honey bees,” Chen et al. report on expression levels of long-noncoding RNAs, miRNAs, and mRNAs in the ovaries of queen honey bees in four conditions of egg laying. These are: virgin queens not laying eggs, normal queens laying eggs, caged queens that are restricted from laying eggs, and recovering queens that were restricted but have been allowed to begin laying eggs again. They analyzed expression regulatory networks by comparing expression levels and potential targets between these categories of RNAs and by comparing the predicted functions of these genes (using GO and KEGG analyses). I think that this project is very exciting, that the experiments and data are solid, and that the results will be very useful for our understanding of honey bee reproductive biology and how it is regulated. However, I have concerns about the analyses and presentation of the work. I recommend that the manuscript undergo major revisions to address these concerns and to make the manuscript more concise.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INTEGRATION OF LNCRNA–MIRNA–MRNA REVEALS NOVEL INSIGHTS INTO OVIPOSITION REGULATION IN HONEY BEES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript was substantially improved. However, language still needs many tweaks and improvements. Several sentences remain vague, confusing or meaningless. This makes it difficult to read and consequently to understand the manuscript.
Experimental design: Language needs to be improved to allow full understanding of the text.
Validity of the findings: Language needs to be improved to allow full understanding of the text.
Additional comments: Language needs to be improved to allow full understanding of the text.

Also, please provide nucleotide sequences (or GenBank accession number) of all data named as TCONS_?

How can authors know that many TCONS_ sequences are genuine lncRNA?

To my knowledge, 20E is a symbol used for 20-hydroxyecdysone.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INTEGRATION OF LNCRNA–MIRNA–MRNA REVEALS NOVEL INSIGHTS INTO OVIPOSITION REGULATION IN HONEY BEES
Review round: 2
Reviewer: 2

Output:
Basic reporting: 1	The English has been improved in the manuscript, removing many ambiguous sentences. However, there are still many grammatical errors throughout.

2	Lines 42-44: The first sentence makes a very broad statement with no citation included. What are the biological constrain(t)s that are going to be ameliorated? If it’s the complex genetic architecture, then this is circular reasoning. These sentences together state that the way to ameliorate the problem of complex genetic architecture of reproduction is to better understand the genetics of reproduction.

3	Lines 65-66: This is another very broad statement with no citation.

4	Figures and tables: The network figures (Figures 2 and 3) are large, crowded and not readable. If these kinds of network figures are included, they should be scaled down or altered to guide the reader through the network. As it stands, they are simply too complex to serve as a main figure. If the full network needs to be included, then it should go in the supplement. Figure 4 is still very large, but this one at least shows that many of the genes in the pathways are DE in this study.
Table 3 isn’t a table, it’s a list. Again, if the authors feel this is necessary then it should be in the supplement. In that case, it can be converted into a useful format (spreadsheet) so that each gene has it’s own row, so that readers can easily sort or copy IDs.

5	In my previous review, I brought up concerns about performing functional analyses (GO and KEGG) on the lncRNA and miRNA, because those analyses rely on using networked partners of those RNAs (they use the mRNAs). This was point #19 in the previous review. While the authors did clarify their methods for making the network and don’t use the functional analyses to support the existence of a network, they still performed the functional analyses on both lncRNAs and miRNAs and proceed to discuss how similar the results of those analyses are to the results of the mRNA analysis. I don’t see any way that the results could not overlap because the three RNA types have already been shown to be in a network (based on expression correlation). The function of the lncRNAs and miRNAs are defined by the mRNAs with which they interact, so of course they overlap. If there is something I’m missing on why this analysis was performed and why it’s discussed, then that needs to be made very clear in the manuscript. It currently is not.

6	Lines 329, 365, and 431: None of these sentences should use the term “proved”, these are scientific hypotheses that have been supported.
Experimental design: The authors have improved the description of the methods significantly.
Knowledge Gap. I think that the work attempts to fill a gap in our knowledge of the genetic regulation of oviposition in honey bees. I’m not so sure that they spell out that knowledge gap very well (see comments above regarding lines 42-44).
The manuscript fits within the aims and scope of the journal.
Validity of the findings: I appreciate the addition of the QTL for ovariole number, I think it improves the strength of the current conclusions. However, the language makes it hard to determine what are new conclusions and what are conclusions from previous work. Lines 179-181 make it sound like this was a QTL analysis performed for this manuscript, but it was the work of Linksvayer and Graham. I don’t think this is intentional, but simply stating that it was “previously localized to…” would clarify.
This same issue comes up a lot in the discussion. I appreciate that the authors are discussing their findings in relation to what is known, but it is not always clear when new conclusions are being made and when they are simply stating the previous conclusions. The use of the past tense also makes this more difficult (e.g. lines 587-588, “Nej was considered…”)
Additional comments: I appreciate the fuller discussion of the the genes in relation to previous work, however I still feel that the manuscript could be improved by having a common thread for the reader to focus on throughout the discussion. In some ways the discussion feels like a list of facts about these pathways without being tied back together into a proper conclusion. I understand that a lot of this work results in new candidate genes to be further investigated, but more comprehensive conclusions would help to guide the reader.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INTEGRATION OF LNCRNA–MIRNA–MRNA REVEALS NOVEL INSIGHTS INTO OVIPOSITION REGULATION IN HONEY BEES
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors adequately addressed all of my questions/suggestions (as well as those of the other reviewer), clarifying them satisfactorily and significantly improving the quality (and also the language) of the manuscript. The study increases our understanding of the molecular relationships between the non-protein-coding layers of bee genome and the reproductive biology of Hymenoptera. I recommend that the revised version of the manuscript be accepted for publication at PeerJ.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A RANDOMIZED CONTROL TRIAL FEASIBILITY EVALUATION OF AN MHEALTH INTERVENTION FOR WHEELCHAIR SKILL TRAINING AMONG MIDDLE-AGED AND OLDER ADULTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Authors have done a wonderful work describing their feasibility study which would help them carry out a larger randomized controlled trial in future. The manuscript is very well written in professional English.

I have few minor specific comments which are detailed below:
L40: Please mention if the values in L41 are Mean+/-SD or Mean+/-SEM or something else?
L47: "questionnaire."
L148: Consider replacing "reticence" with something simple.
L219: "was randomly assigned"
L224: "the group"
Figure1: Consider reducing the size of Figure 1
L258: "were 44"
L306: "wheelchair usage might"
Table 2: What is DC1, DC2, HUI. Consider describing them in a footnote of this Table.
L366: "training) were tabulated"
L371: Use the Greek symbol alpha
L390: Why there is n=7 in Table 3's title and not n=9? Also in Table 3, to be consistent consider using 8.0 and 26.0 for the range.
L455: "expositions"
L464: "identifies that"
L482: "Moving forward,"
L516: "frustration with his/her"
L517: "attend the post-treatment"


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A RANDOMIZED CONTROL TRIAL FEASIBILITY EVALUATION OF AN MHEALTH INTERVENTION FOR WHEELCHAIR SKILL TRAINING AMONG MIDDLE-AGED AND OLDER ADULTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. The introduction of this paper reviewed sufficient past related research and have a clear field background for this study.

2. Article structure, figures, tables, and data are provided appropriately.
Experimental design: 1. This study which used 2 x 2 factorial design RCT, recruited 18 participants with a retention rate of 94% and practiced their mHelath MWC skills training program for older adults met the good technical standard in experimental design.

2. The method of this study was also described clearly with detail information to replicate.
Validity of the findings: 1. The results of EPIC Wheels group post-treatment questionnaire showed that participants had high satisfaction of the program, however, there is a need to state how those questionnaires were distributed and administrated to clarify if participants felt free and comfortable to give their feedback without being afraid of ruining their patient-trainer relationship.
Additional comments: 1. According to the definition of WHO and ageing and aged research, most developed world countries have accepted the chronological age of 65 years as a definition of 'elderly' or older person.  However, in this study, participants were aged 55 and older. Please reconsider if the title still remind the use of "older adults" or only use the participants who are aged 65 and older.

2. Gender always plays an important role on the level of involving interventions or programs in ageing and aged related studies. Please add gender information and possible demographic background (age distribution, race,...etc.) to understand the overall look of the participants.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RELATIONSHIP BETWEEN HEMOGLOBIN GLYCATION INDEX AND EXTENT OF CORONARY HEART DISEASE IN INDIVIDUALS WITH TYPE 2 DIABETES MELLITUS: A CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Results:
-	The authors might consider to create a flowchart to visualize the process of the number of patients screened, number of patients excluded, excluded
-	Can the authors show which antidiabetic drug were the patients on?
-	The last sentence (line 173) should be deleted from the result section and inserted in the discussion.
-	The authors presented a univariate regression analysis for the association between MVD and LAD disease with HGI. The authors need to perform a multivariate regression analysis including well known confounders for CAD severity (e.g. hypertension, hyperlipidemia etc.). Otherwise this is a strong limitation for the study.
-	The authors might consider to OR and 95% CI for MVD and LAD disease using fasting glucose or HbA1c alone in comparison to HGI.
Experimental design: Methods:
-	Patients had measurement of HGI 3 months before coronary angiography. What were the qualifying reasons for enrollment? How many patients had angina, angina equivalent, SOB (eg. According to CCS and NYHA classification)? What measures were used to schedule an angiography (e.g. positive stress test, high likelihood of CAD estimated by pre-test probability).
-	Please clarify, whether patients with existing CAD were excluded or not. If not excluded, add a raw in table 1 showing the percentage of these patients in each HGI category.
-	Describe in detail who you calculated the predicted HbA1c.
-	Describe the statistical methodology in more detail. Specifically, the regression analysis
Validity of the findings: The findings described by the authors are of particular finding given the fact that HGI easy to calculate and widely available.
However, the authors have to address some issues raised by this reviewer (see below).
Additional comments: Drs. Cheng et al. performed a nice cross sectional study of patients with DM scheduled for angiography. The authors calculated the HGI before angiography and found an association to CAD severity (MVD and LAQD disease).
The paper is well written and carefully performed. Nevertheless this reviewer has the following comments:
Abstract
-	Describe the method for calculation of HGI in the abstract.
Methods:
-	Patients had measurement of HGI 3 months before coronary angiography. What were the qualifying reasons for enrollment? How many patients had angina, angina equivalent, SOB (eg. According to CCS and NYHA classification)? What measures were used to schedule an angiography (e.g. positive stress test, high likelihood of CAD estimated by pre-test probability).
-	Please clarify, whether patients with existing CAD were excluded or not. If not excluded, add a raw in table 1 showing the percentage of these patients in each HGI category.
-	Describe in detail who you calculated the predicted HbA1c.
-	Describe the statistical methodology in more detail. Specifically, the regression analysis
Results:
-	The authors might consider to create a flowchart to visualize the process of the number of patients screened, number of patients excluded, excluded
-	Can the authors show which antidiabetic drug were the patients on?
-	The last sentence (line 173) should be deleted from the result section and inserted in the discussion.
-	The authors presented a univariate regression analysis for the association between MVD and LAD disease with HGI. The authors need to perform a multivariate regression analysis including well known confounders for CAD severity (e.g. hypertension, hyperlipidemia etc.). Otherwise this is a strong limitation for the study.
-	The authors might consider to OR and 95% CI for MVD and LAD disease using fasting glucose or HbA1c alone in comparison to HGI.
Discussion:
-	The authors mentioned that HGI reflects an excess of advanced glycosylation end products. Since there is a difference to HbA1c levels, the authors should devote some lines to address these differences. Essentially he relation between HbA1c on one side and advanced glycosylation end products on the other side with CVD severity.
-	Since the marker easy to calculate, the authors should describe clinical implications of the study findings.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RELATIONSHIP BETWEEN HEMOGLOBIN GLYCATION INDEX AND EXTENT OF CORONARY HEART DISEASE IN INDIVIDUALS WITH TYPE 2 DIABETES MELLITUS: A CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous English is used throughout the text. References are relevant, but most are older than 10 years. More recent references should be used, if available in the literature. Overall, the paper is clear and make simple and significant correlations. Other commentaries may be found in the .pdf document provided.
Experimental design: Experimental design is overall clear and reproducible. Other commentaries may be found in the .pdf document provided.
Validity of the findings: Other commentaries may be found in the .pdf document provided.
Additional comments: The work is simple and very clear. If reproduced broadly, this correlation between HGI and CHD in T2D patients may provide an efficient tool for diagnosis purposes worldwide. Please refeer to my other commentaries in the .pdf document for further suggestions.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RELATIONSHIP BETWEEN HEMOGLOBIN GLYCATION INDEX AND EXTENT OF CORONARY HEART DISEASE IN INDIVIDUALS WITH TYPE 2 DIABETES MELLITUS: A CROSS-SECTIONAL STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments.
Experimental design: The study design is accurate. However the description of the estimation of predicted HbA1c is still not clear. The authors need to provide regression  coefficients etc. in the graph (figure 1) and explain the where d the numbers in the equation (predicted HbA1c = 0.008 x FPG + 6.28) come from.

Furthermore the reviewers recommendation of insertion of a multivariate OR 95%CI was only commented by the authors and not inserted in the revised version of the paper.
Validity of the findings: The study addresses an important patient population. In such patients developing tools for  better patient care are crucial.
Additional comments: The reviewers comments were only partly addressed in the revised version of the manuscript.
Essentially the authors should avoid discussing their finding in the result section (165-171 of the PDF version of the revised manuscript).
Despite attempts to address the reviewers comment regarding the predicted HbA1c equation, the authors did not provide an understandable explanation in the manuscript. Please provide information where the numbers in the equation come from!