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: NEUROMEDIN U AND NEUROTENSIN MAY PROMOTE THE DEVELOPMENT OF THE TUMOUR MICROENVIRONMENT IN NEUROBLASTOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: This paper used open data of gene expression to explore why stage 4 vs. stage 4S neuroblastoma are different. The authors chose two out of the 25 hub genes that were differentially expressed at higher levels in stage 4 NB and confirmed their association with invasiveness of the BE2 cells, a neuroblastoma cell line with MYCN amplficiation.

A major issue is that MYCN amplification is a very strong driving factor of pathogenesis and worse prognosis in neuroblastoma but this factor was not considered nor analyzed.

Minor points:

There are some typo's in the text

Abstract: "Neuromedin U (NMU) and neurotensin (NTS) were verified to be elevated in stage 4," -- Please specify how they were verified

Fig. 4. Panels B and D: The authors may mean inva"d"ing cell number in the Y axis.

Line 250: "Determining the reasons underlying these changes in TME from INSS stage 4S to stage 4 is difficult" -- Most clinical cases of stage 4 neuroblastoma are not changed from Stage 4S neuroblastoma. They are probably different diseases with different biology. This sentence should be revised.
Experimental design: Lines 101-105: Please specify why these two datasets were chosen. Please specify which kind of gene expression platform was used and which kind of data (e.g. microarray or whole transcriptome) was analyzed. Please cite publications related to these datasets. Please confirm how many tumor samples were obtained before chemotherapy. Please describe more about the clinical background of these datasets, e.g. age, stage, treatment protocol, country/region, etc.

Line 110: Please explain what is |logFC| and why this criterion was chosen.

Line 126: Please explain how the authors perform the Oncomine analysis. Are the results of Oncomine using the two datasets or different datasets?
Validity of the findings: Lines 195-197: Please explain more about why NMU and NTS were identified or chosen as the major genes to be reported. Were they the only DE genes that were
A Figure of the workflow to identify these genes can be very helpful.

Figure 2A, right panel: Why are NMU and NTS labeled in pink? What are the differences between these two genes and the other genes? What's the difference between the left and right panels?
Figures 2B-2C: What does the scale mean? What do the error bars mean? Are these two genes the top two genes with the highest relative expression level in stage 4 NB in the Oncomine database? How big was the difference?
Please also revise Figure Legend to be more clear.

Figures 3A-3B. Which stage are these tumors?
Figures 3C-3D. Why were all tumors shown are stage 4? Can the authors compare stage 4 vs. stage 4S?
Additional comments: Stage 4 neuroblastoma is a heterogeneous group. Because MYCN-amplified vs. MYCN-non-amplified neuroblastoma are the two prominent subgroups in stage 4 neuroblastoma, the authors are advised to further stratify or compare among MYCN amplified, MYNC non-amplified, and stage 4S tumors.

The workflow about how and why the two genes were chosen to be reported should be explained more clearly.


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: NEUROMEDIN U AND NEUROTENSIN MAY PROMOTE THE DEVELOPMENT OF THE TUMOUR MICROENVIRONMENT IN NEUROBLASTOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors of the present study revised the manuscript according to the comments included in the peer review.
Experimental design: Regarding the comments for the Methods section, the authors have added in revised manuscript the missing information and have responded to all the suggestions and corrections included in the peer review.
Validity of the findings: Regarding the comments for the Results section I still have two points.

Minor point:
In Figure 4 (A-B) the western blot image shows an increase of proteins after silencing with siNMU and siNTS compared to the control siRNA and the housekeeping protein GAPDH contrary to what is stated in the figure legend and elsewhere in the manuscript. This should be revised.

Major point:
The authors have postulated that NMU and NTS influence the interaction of cancer cells and immune cells combining both literature data and immune cells infiltration analysis. However, the work is only descriptive and in several instances no mechanism was proposed to explain this observation. It would therefore be extremely helpful and advance the study considerably, if the authors could provide a much more detailed analysis, preferably accompanied by dedicated experimental data, focused in supporting the proposed hypothesis before publication can be considered.
In addition, the authors performed a functional study in which they evaluate the effects of NMU and NTS in the invasion and migration of neuroblastoma SK-N-BE(2) cells. The observation is interesting but not explained in the study. Authors should investigate and discuss this evidence in more detail.
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: NEUROMEDIN U AND NEUROTENSIN MAY PROMOTE THE DEVELOPMENT OF THE TUMOUR MICROENVIRONMENT IN NEUROBLASTOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised version is much more clear and informative about the methods and results. The issue of MYCN was mentioned in introduction (lines 265-266) but was not analyzed.

Figure 1 and 1A Legend: Please clarify that the 216 DEGs were compared between stage 4 and 4S.

Figure 2A and related text (e.g. Lines 335-336): Please further describe how did the Oncomine verification work -- Was it derived from microarray datasets? How many samples/patients? How many were stage 4/4S?
Experimental design: I suggest to add a summary table of Supplemental Table 1 to summarize the numbers of key clinical and genomic characteristics of the samples enrolled (e.g. between stage 4 vs. 4S, how many had MYCN amplification, were diagnosed at older age, and had the other genomic alterations [when available]?)
Validity of the findings: The effect of MYCN amplification was not assessed in this analysis. The DEG may be driven by MYCN amplification as this alteration was usually enriched in stage 4.
I suggest the authors to compare the the expression of the two genes between MYCN amplified vs. non-amplified samples among the stage 4/4S samples that were enrolled for analysis and present the result in a figure panel or a supplemental figure.

Lines 924-934: A concise review of the function of NMU and NTS in normal cells and/or cancers and their potential roles in NB should also be discussed.
Additional comments: Thank you for making the thorough 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: NEUROMEDIN U AND NEUROTENSIN MAY PROMOTE THE DEVELOPMENT OF THE TUMOUR MICROENVIRONMENT IN NEUROBLASTOMA
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
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: NEUROMEDIN U AND NEUROTENSIN MAY PROMOTE THE DEVELOPMENT OF THE TUMOUR MICROENVIRONMENT IN NEUROBLASTOMA
Review round: 3
Reviewer: 2

Output:
Basic reporting: The authors have clarified that the 2 hub genes was not associated with MYCN amplification.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thanks to the authors' efforts to make the revision. I would recommend that the paper is ready for publication.


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: PROMOTING EFFECT OF RAPAMYCIN ON OSTEOGENIC DIFFERENTIATION OF MAXILLARY SINUS MEMBRANE STEM CELLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear and unambiguous, professional English used throughout
Introduction needs more details.
-	In the literature there is a paper by Isomoto et al: "Rapamycin as an inhibitor of osteogenic differentiation in bone marrow-derived mesenchymal stem cells" published in 2007 in the Journal of Orthopedic Science, I suggest the authors should better describe the different studies that demonstrate how rapamycin induces osteogenic differentiation in stem cells.
-	Rows 69-70; this paragraph should be reworded as several papers report the role between autophagy and osteogenic differentiation of BMSCs even if not MSMSCs (see Pantovic A, Krstic A, Janjetovic K, et al. Coordinated time-dependent modulation of AMPK / Akt / mTOR signaling and autophagy controls osteogenic differentiation of human mesenchymal stem cells. Bone. 2013; 52 (1): 524-531. doi: 10.1016 / j.bone.2012.10.024, Vidoni C, Ferraresi A, Secomandi E, et al Autophagy drives osteogenic differentiation of human gingival mesenchymal stem cells. Cell Commun Signal. 2019; 17 (1): 98. Published 2019 Aug 19. doi: 10.1186 / s12964-019-0414-7; Wan Y, Zhuo N, Li Y , Zhao W, Jiang D. Autophagy promotes osteogenic differentiation of human bone marrow mesenchymal stem cell derived from osteoporotic vertebrae. Biochem Biophys Res Commun. 2017; 488 (1): 46-52. Doi: 10.1016 / j.bbrc.2017.05.004 )
-	Since the authors carry out their ex vivo experiments with biosynthetic scaffolds, I would also suggest describing how the combined presence of synthetic scaffolds (Bio-Oss) and rapamycin promotes osteogenic differentiation (see Lee KW et al. Rapamycin promotes the osteoblastic differentiation of human embryonic stem cells by blocking the mTOR pathway and stimulating the BMP / Smad pathway. Stem Cells Dev 2010; 19: 557–568. Carpentieri A. et al. "Rapid Rapamycin-Only Induced Osteogenic Differentiation of Blood-Derived Stem Cells and Their Adhesion to Natural and Artificial Scaffolds. "Stem cells international vol. 2017 (2017): 2976541. doi: 10.1155 / 2017/2976541).
-	In Discussion, rows 271 and 320 the role of Runx2, as well as epigenetic changes, during osteogenic differentiation induced by rapamycin and scaffold have recently been described. Literature should be added (see Gambacurta A. et al. Human osteogenic differentiation in Space: proteomic and epigenetic clues to better understand osteoporosis. Sci Rep. 2019; 9 (1): 8343. Published 2019 Jun 6. doi: 10.1038 / s41598-019 -44593-6).

-	Structure conforms to PeerJ standards
-	Figures are good quality, well labelled and described.
-	Raw data are supplied
Experimental design: -	Original primary research within Scope of the journal.
-	Research question well defined, relevant & meaningful.
-	Rigorous investigation performed to a good technical & ethical standard.
-	Methods described with sufficient detail & information to replicate.
Validity of the findings: -	The impact and novelty are evaluated positively
-	All underlying data have been provided; they are robust, statistically sound, & controlled.
Additional comments: The paper entitled "Promoting effect of rapamycin on osteogenic differentiation of maxillary sinus membrane stem cells" by Yan-Jun Lin and co-authors describe how rapamycin is able to induce stem cells osteogenic differentiation (in this study maxillary sinus membrane stem cells) and suggest a relationship between osteogenic differentiation and autophagy. The design of this study shows sufficient data in order to confirm the potential role of rapamycin in osteogenic differentiation and how there is a strong link with the autophagy process.
In conclusion, I feel that this paper, after revision, is of a high enough scientific bases to be considered for publication in 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: PROMOTING EFFECT OF RAPAMYCIN ON OSTEOGENIC DIFFERENTIATION OF MAXILLARY SINUS MEMBRANE STEM CELLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Manuscript is well written and clearly articulated.

Background is mostly appropriate, however, there is a lack of detail as to the effects of rapamycin on other MSC populations and genetic models where the role of mTORC1 in lineage determination and osteogenesis has been examined in detail.

Structure conforms to PeerJ standards

Figures are ok and legends mostly ok.
Experimental design: Research question:
The effects of rapamycin and mTORC1’s role in osteogenesis had been examined in detail in human and rodent cells and rodent models. In this manuscript, the authors use rat MSMSCs as a model system to examine the effects of rapamycin on osteogenesis. More detailed studies as to the differentiation potential of human MSMSCs has been performed previously but not using rat cells.
The link to autophagy is tenuous at best and at no point do the authors test the hypothesis that rapamycin promotes osteogenesis in rat MSMSCs by suppressing mTORC1-dependent autophagy. For the osteogenic assays, the experiments haven’t been performed where the cytostatic effects of rapamycin have been accounted for and there is no justification for the rapamycin concentrations used in the study.
Validity of the findings: As pointed out herein, there are a number of limitations to the experiments conducted that should be addressed by the authors:

Can the authors justify the use of a single dose of rapamycin? Rapamycin/FBK12 has high affinity for mTORC1 and may be considered irreversible which could be used to justify a single dose. However, the authors provide no supportive data to this effect (test activation status of p70S6k and/or 4E-BP1 +/- Rapamycin time course). A single dose may be insufficient to inhibit mTORC1 for the duration of the assay which could affect the interpretations of the study findings.

In terms of the dose of rapamycin, these are not justified. Rapamycin has an IC50 for inhibition of mTORC1 in the low nanomolar range (<1nM). Therefore, in terms of the experimental design, the authors should show the concentrations of Rapamycin that inhibit mTORC1 is rat MSMSCs by performing a dose response and western blotting for the activation status of mTORC1 substrates.

The authors have quantified alizarin red staining as a readout of osteogenesis/mineralisation. While this method does provide some indication, it is not considered to be an accurate method and the authors would be better placed to measure acid solubilised mineral formation and normalise to total cell number.

With respect to the Alkaline phosphatase and alizarin red quantitation, the assays do not consider the effects of Rapamycin on cell proliferation. Rapamycin is cytostatic and thus the signal for the ALK P and ARS must be normalised to cell number as the differences in the staining could be attributed to changes in cell number.

With respect to the ectopic bone assay, the authors provide no quantitation (histomorphometry using Image J or Osteomeasure) of the explants. Thus, comments such as “rapamycin markedly induced bone-like tissue” are very subjective and the authors should perform a more detailed analysis of the explants which may provide some insight into how rapamycin may function to promote osteogenesis in rat MSMSCs (i.e. is there more osteoblasts? If not, then an increase in OB function?).

In terms of induction of autophapy, the authors provide TEM analysis showing putative autophagosomes and qRT-PCR data showing induction of Benc1 and LC3 expression. In terms of the autophagy field, these two measures are deemed insufficient to conclude induction of autophagy and the authors would need to provide, at the very least, LC3 I/II protein levels in response to rapamycin.

The authors have suggested that Rapamycin promotes autophagy which is a mechanism by which rapamycin promotes oteogenesis. However, as stated by the authors in the final discussion, no experiments were performed to test this hypothesis. Moreover, there is no correlation between the level of induction of autophagy (Fig. 2C; highest induction at 1uM) and osteogenic induction (Fig. 3C; 1uM has poor effect).

In terms of experimental details, these appear to be ok. The authors should make sure that the full name of the chemical (for example MDC (Monodansylcadaverine) is defined.

In terms of basic characterisation of rat MSMSCs, it would be of interest to enumerate the number of CFU-F’s in this tissue which would be a good addition to Figure 1.
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: PROMOTING EFFECT OF RAPAMYCIN ON OSTEOGENIC DIFFERENTIATION OF MAXILLARY SINUS MEMBRANE STEM CELLS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: My 3 major issues with the initial version of the manuscript was a lack of justification for the concentrations of Rapamycin used, the potential cytostatic effects of rapamycin and the lack of evidence linking the rapamycin-mediated induction of autophagy with the pro-osteogenic response.

The authors have decided not to provide any dose-responses and instead quote other MSC literature, which is in a different cell context. Given that this is the first study using rat MSMSCs, a rapamycin dose-response seems appropriate.

With respect to the cytostatic effect of rapamycin, the cytostatic effects may be limited given that the cells are only treated for 4 hours and the differentiation assays go for several weeks. Nonetheless, as a basic measurement of the cellular response to rapamycin, a short proliferation assay (3-4 days) would provide useful information about how this cell population responses to drug treatment (given the potential clinical applications).

With respect to the pro-osteogenic response of rapamycin being related to autophagy, the authors have revised the discussion to make it clearer that no conclusions can be drawn from their data to suggest this concept. As I noted previously, the pro-osteogenic response and stimulation of autophagy do not appear to correlate (i.e. at the dose of rapamycin that stimulates maximal autophagy (1mM), the osteogenic response is diminished).

With respect to quantitation of the in vivo ectopic bone formation, if the authors fail to provide any quantification of this data, then they must refrain from using any language that suggests the data has been quantified ie “more significant” (line 262) and ‘markedly induced” (line 258). Moreover, ImageJ (Fiji plugin) and Osteomeasure are widely used for histomorphometry and the authors shouldn’t dismiss these approaches. The impact of their in vivo model would be more compelling if new bone area or osteocycte numbers or osteocyte numbers/bone area were measured.

Overall, the authors could mitigate some of these issues by expanding the section in the discussion where they describe the current limitations of 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: PROMOTING EFFECT OF RAPAMYCIN ON OSTEOGENIC DIFFERENTIATION OF MAXILLARY SINUS MEMBRANE STEM CELLS
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript describes, for the first time, the effects of rapamycin on the osteogenic potential of a unique stem cell population, maxillary sinus membrane stem cells. In the revised manuscript, the authors have detailed the limitations of their study such that a reader will be sufficiently informed as to the limitations of the data.


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: T-COMPLEX PROTEIN 1 SUBUNIT ZETA-2 (CCT6B) DEFICIENCY INDUCES MURINE TERATOSPERMIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript submitted by Yang et al. show that CCT6B, a testis-specific member of the Chaperonin- Containing Complex, is required for proper development of spermatozoa. More specifically, the authors show that sperm of CCT6B knockout mice have decreased motility and abnormal head morphology. I commend the authors for their extensive data set, compiled over multiple experimental approaches and of detailed analyses. For the most part, the manuscript is clearly written in professional, unambiguous language. However, there are a few items that should be attended to improve the manuscript before Acceptance.

Main edits:

The resulting cct6b gene mutation generated by crispr is not clearly defined.  The authors mention that "an in-frame deletion in exon 4 of the Cctb6 gene" was generated, but an in-frame deletion would not result in the absence of protein and transcript described later in the text.  Please revise this statement if you meant "out of frame" and provide a figure (main or supplementary) that describes the resultant mutant sequence and predicted mutant protein product generated by this mutation.  Most likely, since transcript levels decrease, this mutation generates a premature stop codon that make the transcript suitable for non-sense mediated decay.  Please clarify.

Lines 92-95: Please include dilutions and incubation times used for each antibody.

Lines 121-127: Please specify whether the reverse transcription reaction was performed using oligo(dT), random primers, or both. This is relevant because 18S rRNA (which is not polyadenylated) is being used as normalization control.

Minor edits:
Line 54: I believe TCP1 should be included in the following sentence: “The CCT2-8 subunits share a moderate degree of sequence identity (~30%)...”

Line 121: Regarding samples being “maintained on ice at all times”, please clarify whether this is during or after the RNA extraction, or both.

Line 122: “RN” should be “RNA”

Line 161: There is a typo on “antigen retrieval. a microwave oven for 10 min”

Line 194: What is meant by “SA”?

Line 210: Typo in "we", do you mean "was"?

Line 246: "Downregulation" of other cct genes should be edited to "decreased transcript abundance" because it is not clear whether this is due to a physiological side-effect or a direct participation in transcriptional regulatory networks.  In addition, it should be specified that this is only about a 10% decrease.

Line 253:  Define what is meant by CCR?

Line 273:  When mentioning "material", do you mean "motility"?

Cct6b is written as Cctb6 in multiple occasions.  Please revise.
Experimental design: no further comment
Validity of the findings: no further comment
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: T-COMPLEX PROTEIN 1 SUBUNIT ZETA-2 (CCT6B) DEFICIENCY INDUCES MURINE TERATOSPERMIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: See general comments
Validity of the findings: See general comments
Additional comments: The authors have evaluated the knockdown of CCT6B, which is involved in microtubule assembly, showing an impairment in mice spermatogenesis, namely in progressive motility. It is a very interesting paper, but I have some concerns about the specificity of knockdown using Cas-9. There is a high homology between CCT6B and the others CCTs, and probably targeting the exon 4 of CCT6B may also affect the other CCTs. If exon 4 sequence is CCT6B-specific I suggest to carry out an alignment of sequences demonstrating the specificity. This alignment can be inserted as supplementary figure. In fig. 8, it is speculative to state that downregulation of CCT6B induces downregulation of CCT4 and CCT7 because the possible unspecificity of knockdown and several times the protein expression may not accompaign the mRNA expression.
Also, it is not clear the knockdown of CCT6B. For example, in fig. 2D appears a band with high intensity. Which protein correspond this band? The antibody is unspecific?
The expression of CCT6B should be determined by western blot and not only by qPCR as shown in Fig. 1B.
The article is well writing but there are some mistakes throughout the text that should be reviewed. For example:
- Line 122: it misses “A” in “total RN”
- Line 194: “SA” such


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: T-COMPLEX PROTEIN 1 SUBUNIT ZETA-2 (CCT6B) DEFICIENCY INDUCES MURINE TERATOSPERMIA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well structured and organized, the figures are relevant, and all the most relevant information presented with sufficient detail in figures and tables (also supplemental). The results presented are relevant to the hypothesis raised.

Authors must improve figure legends as further information is necessary to interpret per see with no need to read the full respective text in the results sections. Namely:
Legend figure 1A - age of animals specifically in this part of the figure
Legend figure 2D - everybody knows what GAPDH is, but a clear reference should still be made. Also, a comment on what is the band above CCT6B.
Legend figure 3 - clarify what stages VIII and XI are.
Legend figure 4 - provide n for this experiments.
Legend figure 6 - insufficient; insert identification of what is in each image and the conclusions from analysing them.
Legend figure 7 - what is the n? What was used as a control gene?

The manuscript is written clearly and presents no major issues in what concerns the usage of English. Some minor typos must be corrected as follows:
- line 20 (abstract) - "...gene expression win these animals..."
- line 194 (results) - "... synchronous, SA such,..."
- line 237 (results) - "... differences we observed..."

Literature references are sufficient. Raw data is provided.
Experimental design: The research described in the manuscript is relevant and novel. It fits the scope of the journal.

The research question per see is well identified, namely identifying the role of cct6b in microtubule assembly in spermatogenesis, given that it is solely expressed in the testes. The experimental work was designed accordingly to confirm the research hypothesis.

The authors performed the experiments using mostly characterization techniques; all were appropriate to the research question. Nevertheless, some details of the methods are insufficiently described. For instance, no details are provided on the dilutions of the antibodies used, which makes difficult the easy and rapid replication of the western blot and immunohistochemistry experiments.
Validity of the findings: The authors replicated experiments to confirm data described in the literature and referred to in the introduction section—namely data presented in figure 1A. While the authors confirming previously published data is relevant in this case - mostly because the reference was more than 20 years old - it would be sufficient to indicate it in the text or reference the original publication close to the confirmation data.

The results are generally well described and presented, and all necessary raw data provided. Still, there are two major issues with the results that the authors should clarify:
1 - The data presented in figure 1D is not clear to us (even considering the raw data provided). The control protein GAPDH band intensity in Cct6b-/- animals is lower than in the Cct6b+/+; does this mean that the loading is inferior in this lane and explains the absence of the CCT6B band? What is the band above Cct6b? Why is the antibody recognizing this band? What are the remaining bands presented in the raw data blot for CCT6B? How sure are the authors that no CCT6B protein is expressed and hence that the KO experiment was completely successful?
2 - On the data provided in Figure 7 about the decrease in the expression of Cct4 and Cct7 in the absence of Cct6b. The decrease is residual when compared to controls. What is the biological significance of this decrease? Does it translate at the protein level? The authors should discuss/explain this. Furthermore, what is the similarity in the sequence between these Cct4 and 7 genes compared to Cct6b? In the introduction, the authors indicate an overall 30% identity. Is it possible that the 5bp deletion designed for Ccct6b affects the Cct4 and 7 genes?

This and other aspects should be further explained and explored in the discussion section, which is short and misses relevant aspects.
Additional comments: The research described in the manuscript is relevant and novel. The experiments are appropriate for the research hypothesis. Nevertheless, some aspects need clarification from the authors.


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: T-COMPLEX PROTEIN 1 SUBUNIT ZETA-2 (CCT6B) DEFICIENCY INDUCES MURINE TERATOSPERMIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed my comments and I find the current version of the manuscript acceptable for publication.  Thank you and congratulations on your work.
Experimental design: no comment
Validity of the findings: no comment
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: T-COMPLEX PROTEIN 1 SUBUNIT ZETA-2 (CCT6B) DEFICIENCY INDUCES MURINE TERATOSPERMIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors addressed the issues in the section "1. Basic reporting" raised by this reviewer to the v0 of the manuscript in the revised (v1) version of the manuscript.
Experimental design: The authors addressed the issues in the section "2. Experimental design" raised by this reviewer to the v0 of the manuscript in the revised (v1) version of the manuscript.
Validity of the findings: The authors addressed the issues in the section "3. Validity of the findings" raised by this reviewer to the v0 of the manuscript in the revised (v1) version of the manuscript.
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: TERATOLOGICAL CHANGES IN POSTEMBRYOS OF ERATIGENA ATRICA OBTAINED BY THE APPLICATION OF ALTERNATING TEMPERATURES ON SPIDER EMBRYOS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: Overall, the manuscript is written very well. The only major issue I found was the complete absence of imaging techniques in the methods section. The experimental design seems very sound, the rest of the methods were detailed, but I would recommend that full details of the preparation and imaging techniques, including types/brands of microscope be included in the methods.
Validity of the findings: This is a very interesting study and would be of interest to the wider scientific community.
I believe this study on E. atrica will provide important reference material for this particular species is widespread throughout Europe and one of the most abundant synanthropic species, ideal to compare against for studies such as climate change, or new alien, and potentially invasive species arriving in Europe, particularly those from warmer climates.
Additional comments: Line 74: I would avoid the term "It is a known fact that" and begin the sentence with "In the natural environment, spider....."


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: TERATOLOGICAL CHANGES IN POSTEMBRYOS OF ERATIGENA ATRICA OBTAINED BY THE APPLICATION OF ALTERNATING TEMPERATURES ON SPIDER EMBRYOS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Most of my specific comments are on an attached annotated copy of the manuscript.

One suggestion not included on that copy concerns the designations of walking legs and neuropils used in Figure 2. The symmetry evident in the subesophageal ganglia (ventral nerve cord) in Fig. 2C indicates that the left n4/L4 is part of the same metamere as the right n5/L5. Likewise, the left n3/L3 appears to be from the same metamere as the right n4/L4. If the authors agree about these correspondences, then I suggest that the structures currently designated n2/L2 and n3/L3 in Fig. 2 (and discussed in the text with these designations) be re-designated as n2A/L2A and n2B/L2B, respectively. In so doing, the right n4/L4 and n5/L5 would become n3/L3 and n4/L4, respectively, thus providing them with the same designation as their metamere counterparts on the left side of the prosoma. This will emphasize the common metameric origin of these structures.
Experimental design: no comment
Validity of the findings: The authors are well positioned to interpret the histological results, having done this kind of analysis for many years. I would just ask them to consider, and discuss in the paper, how confident they are that their interpretation of polymely in the Fig. 2 individual is correct. The small neuropil evident in Fig. 2D does demonstrate that, at the very least, there is a distortion to n2 not present in any other neuropils, but whether this constitutes a separate neuropil, reflecting a supernumerary leg fused with the first two walking legs, is something I am not yet entirely convinced of based on what is presented. But the authors may be in possession of more data (e.g., in the form of additional sections between those shown in Figs. 2C and 2D) that gives them confidence that the neuropils they designate n2 and n3 are indeed distinct and not fused. For example, if intervening sections reveal little or no continuity between n2 and n3, then I would agree they are separate and unfused neuropils, making a solid case for a supernumerary leg being part of the fused assemblage. If the authors do have additional observations that lend greater certainty to their claim that polymely exists in this individual and that fusion of legs is not reflected by fusion in the CNS (including between what they designate n2 and n3), then it would be helpful if they could describe these observations. If they do not have such supporting observations, then they might consider raising the possibility in their Discussion that the fused limb contains only two legs (despite the difference in thickness of the two free ends on the fused assemblage) and the “n3” neuropil is part of a distorted n2.
Additional comments: I enjoyed reading your paper and seeing the beautiful histology and was especially interested to read about the "boundary model" as it potentially relates to the Fig. 2 specimen.

As mentioned above, most of my specific suggestions are on an annotated copy of the manuscript, entered using the Track Changes feature in Microsoft Word. Please do take the time to fully consider them since that is where I invested most of my effort. Since PeerJ only allows a PDF to be uploaded, feel free to contact me (mark.townley@unh.edu) if you wish me to send my annotated version of the manuscript as a Word document, where it is easier to accept any suggestions you agree with.


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: TERATOLOGICAL CHANGES IN POSTEMBRYOS OF ERATIGENA ATRICA OBTAINED BY THE APPLICATION OF ALTERNATING TEMPERATURES ON SPIDER EMBRYOS
Review round: 1
Reviewer: 3

Output:
Basic reporting: For the most part clear and unambiguous, professional English was used throughout the text.  A few places of clarification for the reader would be helpful for someone not in the field.  Perhaps define teratology in the first sentence for readers that aren’t familiar with the term.  Terminology defining the nature of each defect is well described in the introduction, but this may also be useful information to repeat in table 1 for readers that are not familiar with the terms. Line 106 wording “ a new interesting cases” should be reworded to remove the a or the s from cases.

Authors mention in line 74 that “…spiders are exposed to a range of factors which can affect their development causing various morphological deformities.” Could the author list some examples perhaps in parentheses of these factors and provide a reference to these factors (specifically ones other than temperature)? Additionally, has this temperature effect of teratogenicity been seen in other spiders, arthropods, or other classes of invertebrates?

In line 95 it is mentioned that histological analyses of the nervous system were performed in the authors’ previous studies.  It is mentioned then in the next paragraph that various abnormalities were observed depending on the defect.  It would benefit the reader to know the nature of these defects in the introduction to support what the authors will be looking for in the current study.

The article is professionally and conventionally structured.

Raw data is shared, and no statistical analyses were performed or needed.

Figures are clear.  However, it would be of interest to readers to add a few things to the figures and table for ease of interpretation.  For the table, definitions for the abnormalities could be included in a column.  Perhaps additional columns, showing percentages of these abnormalities in the authors previous studies could also be useful for comparison.   Arrows pointing to the various abnormalities in the figures would be useful to the reader.  While some are obvious, absent or rudimentary limbs take longer to pick out.  In the bicephalous larva images a dotted line demarcating where the two heads meet would be useful. For the final figure, it would be useful to have a diagram of the normal layout of the neuropils and a wildtype to compare to (more on this later).
Experimental design: This study represents original primary research within the Aims and Scope of PeerJ.
This is an exploratory study with a clearly defined research question - What are the physical teratogenic effects of extreme temperature treatments on embryogenesis if applied at early stages?

While the question is clear and methods appropriate, relevance and the gap of knowledge this research fills are not clearly stated. What relevance does this have to the natural underpinnings of developmental biology/teratology, particularly if the results are not consistent and these abnormalities seem to occur at random (new abnormalities in this study not seen in many other studies with the same treatment)?  This could be easily rectified with a clear statement of relevance in the introduction.  There is some mention of this in the discussion.  In line 285, the authors state that developmental defects, can “contribute to a better understanding of developmental mechanisms in invertebrates.” The authors argument would be strengthened if they could provide the next steps in using the information provided by their study to understand developmental processes.

One question I had reading the introduction was whether the temperatures used in the lab studies were ones that might be experienced in the natural environment.
Majority of the methods are described with sufficient detail & information to replicate. Authors should clarify whether one cocoon from each of the 24 females was used.  One might expect that the genetic background of some cocoons might predispose them to being susceptible to temperature shifts.  This is controlled for in splitting the embryos equally into treatment groups.  Perhaps in the discussion, authors could address the possibility that embryos having abnormalities came from the same breeding pair.  A cool future study could be to keep the embryos separated by cocoon to trace genetic susceptibility. Are those individuals with complex anomalies (more than one of the anomalies listed) double counted?  For those with complex anomalies. Are there any combinations that occur together with more frequency?

Figures represent individuals with complex abnormalities.  Are the defects as severe in individuals with single abnormalities?  Perhaps the authors could include an additional figure with a representative image for each of the single abnormalities.

Of top priority, normal wildtype spider should be shown for comparison in all figures including sectioned images.
Validity of the findings: All underlying data have been provided.  Statistical analysis is not needed but as described earlier some additional numbers would be useful for comparison (types of abnormality combinations in complex abnormalities, percentages from previous studies, etc.)

The study is well controlled by splitting embryos from each egg sac equally into treatment groups.  However as previously mentioned, a control wildtype spider should be shown for comparison in all figures.

One of the most well studied areas in spider developmental biology is body segmentation and leg development. While it is mentioned in line 234, that temperature shifts could induce changes in the genes responsible for these processes, it is unclear how.  Authors are encouraged to explore this literature in the discussion section to propose some gene families that may be altered (such as hox genes) and the effects that temperature may have on protein function.  The space limitation argument in line 238 doesn’t make sense as an alternative to the segmentation gene idea, but seems more of an explanation of how these defects in segmentation and patterning manifest in the various defects seen.  Damage to a particular cell type might be a better alternative to specific protein malfunctions and would be further supported by the boundary model that is described in detail.

Places whether the authors provide speculation is clear and conclusions are not overstated.
Additional comments: The manuscript entitled “Teratological Changes in the Eratigena atricla larvae obtained as a result of the application of alternating temperatures on spider embryos.” is an interesting continuation of the researchers work looking at the effect of extreme temperatures on embryogenesis.  This study is scientifically sound, however addressing some minor points could strengthen the impact of the paper.  I am suggesting that it be accepted upon minor revisions.  Of highest priority is updating the figures with wildtype control images.


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: TERATOLOGICAL CHANGES IN POSTEMBRYOS OF ERATIGENA ATRICA OBTAINED BY THE APPLICATION OF ALTERNATING TEMPERATURES ON SPIDER EMBRYOS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Congratulations on this work, it is an excellent research paper.


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: TERATOLOGICAL CHANGES IN POSTEMBRYOS OF ERATIGENA ATRICA OBTAINED BY THE APPLICATION OF ALTERNATING TEMPERATURES ON SPIDER EMBRYOS
Review round: 2
Reviewer: 2

Output:
Basic reporting: This is a review of a revision prepared by the authors. The authors have done a conscientious job taking the comments and suggestions of the reviewers into account in preparing their revised manuscript.

As with the original manuscript, most of my specific recommended changes are included on an annotated version of the revision that I submit with this review. These are all minor with one exception: in three instances there is a discrepancy between the view (ventral or dorsal) stated for a figure in the text versus what is stated in the figure legend (one says dorsal, the other says ventral). I would just ask the authors to review these carefully and be certain that text and figure legend are in agreement, and that both of them agree with what is seen in the actual photomicrographs.
Experimental design: No comment
Validity of the findings: An earlier concern regarding one aspect of the interpretation of the histologically-examined postembryo has been addressed very satisfactorily in the revised manuscript.
Additional comments: I have no further reservations regarding the publication of this paper. I would only ask the authors to consider the suggestions I give in the accompanying annotated manuscript and accept those they agree with.


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: TERATOLOGICAL CHANGES IN POSTEMBRYOS OF ERATIGENA ATRICA OBTAINED BY THE APPLICATION OF ALTERNATING TEMPERATURES ON SPIDER EMBRYOS
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: THank you for taking the comments into account for your publication.  I think that adding additional discussion on molecular basis of the abnormalities and including the wildtype spider for comparison really strengthened the paper.


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: NEW INTERPRETATION OF THE CRANIAL OSTEOLOGY OF THE EARLY CRETACEOUS TURTLE ARUNDELEMYS DARDENI (PARACRYPTODIRA) BASED ON A CT-BASED RE-EVALUATION OF THE HOLOTYPE
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a descriptive work in which new anatomical information about a relatively poorly known taxon is provided, based on the revision of its holotype. The descriptions are adequate.
I recommend the publication of this work with new data on Arundelemys. I have made some minor suggestions in the attached PDF.
Experimental design: The methodology used to carry out this study is adequate, and it has been explained in detail. I have made a comment regarding a suggestion about this in the PDF.
Validity of the findings: The results are adequate, being based on the new data obtained through the redescription of the Arundelemys holotype.
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: NEW INTERPRETATION OF THE CRANIAL OSTEOLOGY OF THE EARLY CRETACEOUS TURTLE ARUNDELEMYS DARDENI (PARACRYPTODIRA) BASED ON A CT-BASED RE-EVALUATION OF THE HOLOTYPE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript presents a redescription of Arundelemys dardeni based on µCT reconstructions, as part of an effort by this group of coauthors to improve our basic anatomical knowledge of paracryptodiran turtles. The structure and writing of the manuscript are flawless (only a few typos are identified in the annotated PDF), all figures are high quality and relevant to the discussed subjects and the raw data is supplied in an online repository.
Experimental design: Classic thorough anatomical description, with relevant comparisons to other taxa.
Validity of the findings: Some structures are reinterpreted, new traits are described (e.g., head scute system) and everything is well based on the digital reconstruction presented in the manuscript, and the reader can confirm all observations in the original tomographic images, which are provided in an online repository.
Additional comments: I congratulate the authors for this straightforward, yet comprehensive redescription of this important taxon (and for the general effort to gather more information on paracryptodires as well). I provided only a few corrections of typos in the annotated PDF and have no issue with the interpretations presented in the manuscript, except for the homology hypothesis of some of the head scutes.

The midline series seem to be non-problematic in your hypothesis, as it is in all of the previous publications developing on this system. The more lateral scutes are a bit messier (aside from F, I and J-series, which, again, are undoubtedly identified). You suggested Mongolochelys scute identification in Sterli & de la Fuente 2013 is mistaken regarding D and H, but I believe the identification misrepresented in that study is that of Meiolania, instead: scutes D and H are switched there. In Proganochelys and Mongolochelys D is anterior to H, overlaying the parietal and postorbital, and contacting medially G and H, anteriorly F, laterally E and posterolaterally C. The H, on the other hand, on both taxa is posterior to D and overlays the squamosal, not reaching G nor F anteriorly. If we change H for D in Meiolania's identification this is the same pattern exactly, but if we switch D and H in Mongolochelys, it should also be changed in Proganochelys too. Further, in all subsequent publications (Rabi et al. 2013, 2014; Li et al. 2019) D is always in contact with F and G and never reaches scute A. In all previous publications scute H contacts X and A medially (except in Li et al. 2019 in which it is not identified, but there seems to be a posterior bifurcation of the X-D sulcus that could indicate a tentative H scute, again, between D, X and A as well). For these reasons, I think your scute D is better identified as H.
In all other studies, the only scutes to reach the orbit are F and J, but in your hypothesis, you have two differences: ?H and E also reach the orbit. In previous studies, F scutes are the only to be (i) restricted to the postorbital and (ii) margin the orbit posterodorsally. Both criteria are met by your E scute, which I propose is more parsimoniously interpreted as another of the F series. That would leave a ?H scute between two F-scutes. The three criteria you used to identify it as H also apply to an F-scute (in Annemys levensis one F also contacts G medially; Rabi et al. 2014). That leaves C as another doubt. C is only identified in Sterli & de la Fuente (2013), and it reaches E anteroventrally, D anteriorly, H medially and B posteriorly. It never reaches the scutes X or F (which would happen if you agree with my alternative interpretation). If we identify it instead as D, that would fit perfectly the original system: it contacts F anteriorly, X medially and H posteriorly.
To sum up, the alternative identification I propose is: scutes H, E, C and D in your hypothesis become F', F'', D and H.
Needless to say, homology hypotheses are just hypotheses, and you can disagree with my alternative interpretation. I consider it more parsimonious, though, and think you should at least consider it.

The authors are free to contact me if something is not clear in my assessment.
All the best regards,
Gabriel Ferreira


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: LIPID ACCUMULATION OF CHLORELLA SP. TLD6B FROM THE TAKLIMAKAN DESERT UNDER SALT STRESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is written clearly, however  proof-editing  is required.

Introduction refer to some relevant studies , but insufficiently covers recent discoveries on Chlorella species isolated from arid areas.

Article is well structured . Raw data are shared.
Experimental design: Research question is defined: how high salt concentration affect gene expression of the desert isolated of Chlorella. Physiological characterization included measurement on a few parameters.
Methods are well described.
Validity of the findings: The manuscript describes the effect of increasing NaCL concentration in the nutrient medium on the transcriptomic response of Chlorella sp. TLD6B.
Two concentrations were included in the transcriptomic study, designated as low (0.1) and high (0.8M).
Based on OD measurements , the growth was not substantially affected by 0.1M NaCl but was inhibited at 0.8 M NaCl.
Cultivation experiment rises several questions and concerns over selection of conditions.
The experiment duration is very long for the basic physiological experiment.
Considering long lag and long duration it is not clear when growth rates were determined.
The culture supplemented with high salt did not grow .
Total lipids increased insignificantly from the day 0 while carbohydrates severely decreased at 0.8 M.
What are other major components of biomass in this case?
Additional comments: Have you performed phylogenetic analysis of Chlorella sp. TLD6B?
Have you examined if cells are viable after a prolong exposure to salt stress?
The results indeed show that under high salinity level, the desert Chlorella sp. modulates gene expression of several fatty acid biosynthesis and TAG biosynthesis genes as well some genes of central carbon metabolism. And, the manuscript can be reorganized and rewritten to emphasize the effect on transcriptome. Based on the physiological data presented,  the relevance to biodiesel production potential is questionable.


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: LIPID ACCUMULATION OF CHLORELLA SP. TLD6B FROM THE TAKLIMAKAN DESERT UNDER SALT STRESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The novelty of this study lies in the fact that the authors have investigated the salt stress-related responses of Chlorella sp. in terms of gene expression and metabolic pathways using transcriptomic analysis coupled with physiological changes like that of biomass, sugar, and lipid content. This provides an insight into the salt-adaptation mechanisms of this microalgae thereby contributing to the basic knowledge of algal physiology. Despite the above-mentioned highlighting aspects, a lack of an overall clarity, structure, and coherence in several parts of the manuscript including introduction, methods, results and discussion significantly lowers the standard/quality of the manuscript.

1. The language of the manuscript needs to be improved substantially. Grammar- and vocabulary-related errors are quite frequently noted throughout the manuscript. For example, the phrases like “distributes”, “researches”, and “at home and abroad” in Lines 51-55 reflects poor standard of professional English.

2. The context of the study is quite unclear from the introduction. Thorough justification is required as to why we need to study the relationship between salinity stress and lipid accumulation in Chlorella sp.  TLD6B. The authors have not provided sufficient references to present a clear background to their study especially in terms of highlighting the gaps in knowledge pertaining to the effect of high salinity on lipid accumulation in microalgae (maybe Chlorella in particular) and changes at the physiological and molecular/ gene level.

3. Figures:
a. Relevance: Table 1 and Figure 1 represent the same dataset with the table highlighting the significant differences between the relevant values. Significant differences are denoted by uppercase and lowercase letters which is quite confusing while trying to interpret the table. It is better to have the most prominent differences highlighted in the figure rather than the table.
b. High quality: The resolution of Figures 3-5 and 7 needs to be enhanced.
c. Labelling and description: Incorrect for y-axis in Figure 1C, legend labels inconsistent with color scheme in Figure 3, the different parts of the legend need to be consistent throughout all three bubble plots in Figure 4, statistical representations in the scatterplot of Figure 5 needs to be rectified.

4. Raw data in all supplied files have been presented in a clear and readable format. However, the files for Table 1 and Figure 1 provide the same information. This needs to be verified. Raw data supplied for Table 2 does not show how the coefficient of correlation was determined between biomass and other parameters keeping in mind different NaCl concentrations at each time point. Raw data file for Figure 5 does not include the negative values as represented in part A of figure for RT-qPCR results. The authors need to resolve these above-mentioned inconsistencies in the raw data provided.
Experimental design: 1. The manuscript does demonstrate original research pertaining to the lipid accumulation in microalgae under salt stress conditions. The study fits well within the scope of the journal but, it is highly disappointing to note that the authors have not presented a clear research question that can resolve any potential gap in current knowledge. Examples are as follows:
Lines 47-50: The authors have presented one long and complicated sentence here which does not clearly indicate findings related to either lipid accumulation or lipid metabolism. The terms “lipid accumulation” and “lipid metabolism” have not been appropriately placed in the text and increases confusion to the reader throughout the introduction.
Lines 57-60: The sentence “After millions of years…” seems too hyped up. Please revise.
Lines 63: An introduction to the aspect of performing transcriptomic analyses of microalgae that can accumulate lipids would be more benefiting to highlight research gaps and enhance the overall standard of the manuscript.
Lines 66-68: This example of Chlamydomonas sp. seems irrelevant here especially when there are other references relating to Chlorella sp. itself.
Line 72: The authors state that their selected strain Chlorella sp. TLD6B from the desert has higher salt tolerance than freshwater Chlorella sp. based on their unpublished results. This is a strong statement considering that no evidence has been provided in this regard and that it is known that certain freshwater algae can tolerate slightly higher levels of salinity.

2. The description of the methods needs to be significantly improved in terms of the structure i.e. sequence of workflow, parameters assessed, data collection and analysis especially in the sections prior to RNA sequencing, transcriptome and gene expression analyses. Certain parts of the methods include irrelevant information while other parts of the methods require more technical details.

For example, in the first subsection of the methods i.e. “Algae strain and culture conditions”, in Lines 80-81, the authors strongly state that Chlorella sp. TLD6B was collected from the desert. This contradicts the latter half of the same sentence where an algal strain was isolated from a mixed environmental sample which was identified later as Chlorella sp. TLD6B. The environmental conditions such as temperature, salinity or light intensity during sample collection in the desert is missing here.
Lines 86-88: The authors need to provide detailed information on the type of light used in the laboratory. This can affect the physiology and lipid accumulation in algae. Other missing aspects here are the final salinity of the BBM medium Table S1 provided as a supplementary file, showed that 25 mg of NaCl was included during preparation. However, the original BBM medium composition contains 0.4M NaCl. Please clarify. Also the information pertaining to final volume in each flask per treatment is missing here.
Line 94: Usually analysis of algal physiology includes not only the biomass but also the morphological changes like changes in cell size/volume, changes in photosynthetic parameters, concentration of pigments, and changes in the mode of growth owing to salinity-stress in this context. Did the authors observe such differences in physiology in their culture?
Line 97: Please rectify the volumes mentioned for the tubes versus culture added.
Line 100: Can the authors explain why freezing in liquid nitrogen is necessary for dry weight measurements?
Lines 104-107: Brief descriptions of the method used to calculate values of Dcw% for carbohydrate and lipids is required for clarity.
Line 116: What do the authors mean by “ based on results of preceding studies…”? There is no reference cited here.
Lines 121-123: The authors have not provided any prior justification to why the drought-stressed Chlorella sp. TLD6B needs to be prepared here. This seems irrelevant here.
Line 156-157: Statistical analysis needs to include more details especially as to how correlation coefficients were determined between the physiological and biochemical indices.
Validity of the findings: The findings of this study have been very poorly presented i.e. lack of organization, and an overall coherent flow. The novelty of the study has also not been well discussed by the authors. It is incredibly difficult to understand clear trends from the physiological/biochemical datasets and the RNA-related datasets. For example,

Lines 162-192: The terms “cell growth rate”, “specific growth rates”, “cell growth” have been used interchangeably in several instances. Likewise, for the terms “carbohydrate consumption” versus “carbohydrate concentration” and “lipid concentration” versus “lipid productivity”.
The authors need to be aware of using the right terminology at each instance.

Lines 193-203: Significant correlations are highlighted well in Table 2 but not presented well in the text here. This paragraph needs to be more concise.

Line 232-234 and 242-244: Don’t these lines indicate the same message? It is better to avoid repetition. Likewise, for Lines 238-242 and Lines 247-252.

Line 254: The terms “NaCl1 VS CK18” and “NaCl2 VS CK18” have been introduced here for the first time. Please clarify.

Lines 310-311: The readers would benefit more from the description and/or comparison of different salt-tolerant mechanisms found in microalgae or different strains of Chlorella sp. itself.

Lines 317-318: This conclusion cannot be inferred from the presented findings since the investigated Chlorella sp. TLD6B was not compared to other microalgal strains in the same study and experimental conditions. Please revise.

Lines 323-324: What do the authors mean by “appropriate salt treatment…” ? If the highest lipid accumulation was observed at 0.2M NaCl, then this is the typical freshwater conditions ! Please verify. Also, how is salt treatment related to lowering the production cost of biodiesel?

Lines 379-380: It is probably the lack of clarity in the results and discussion but the authors need to be careful while mentioning that “salt stress could regulate carbon flow between starch biosynthesis and lipid biosynthesis in Chlorella sp. TLD6B”.
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: LIPID ACCUMULATION OF CHLORELLA SP. TLD6B FROM THE TAKLIMAKAN DESERT UNDER SALT STRESS
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1. The manuscript contains some grammatical errors and needs to be proof read by a native/near native English speaking person. Some examples where the language could be improved include lines 47-50, 53-57, 313-314, 329-333 – the current phrasing makes
comprehension difficult.
2. There were many format problems throughout the manuscript, such as missing spaces in the word. For example, line 15: Chlorellahas; line 16: droughttolerance; line 17: beencultivated; line 23: andfatty; line 71: Itis; line 72: hasa; line 68: productivityin. Please check carefully.
3. Too many long and complicated sentences were included in the manuscript, such as line 47-50, line 325-329, line 329-333 and so on.
4. Some abbreviates were not explained, such as FPKM,DEGs, TAG.
5. The raw data of transcriptome should be deposited in the public database, such as NCBI.
6. The method needs more detail, such as the parameter for denovo assembly and annotation.
7. Figure 3 and 4 should be replaced by GO and KEGG enrichment of 2802 DEGs common shared by sample NaCl1 and NaCl2.
8. Figure 6: Genes and their transcriptional expression involved in fatty acid and TAG
biosynthesis pathways under different salt treatments. However, the expression level was not showed in the figure.
Experimental design: Authors mentioned that low salinity can promote the accumulation of lipid in Chlorella sp. However, why low salinity can promote the accumulation of lipid should be discussed. In other means, whether the accumulation of lipid would affect the salt tolerance of Chlorella sp. and how?
Validity of the findings: This research elaborated the relationship of salt tolerance
and lipid accumulation in Chlorella sp., which promotes the utilization of Chlorella sp. TLD6B.
Additional comments: no comment


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: LIPID ACCUMULATION OF CHLORELLA SP. TLD6B FROM THE TAKLIMAKAN DESERT UNDER SALT STRESS
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is a revised version of the manuscript. English has been substantially improved.
Experimental design: Please see comments to the authors.
Chlorella is a fast-growing species and generally, experiments can be perg\formes within several days, not weeks.
Validity of the findings: The validity of the transcriptomics results seems reliable.
Additional comments: The revised manuscript has been significantly amended, in particular in writing.
However, the authors have to better justify the study design (e.g. long lag and slow growth in control culture) and correct a few issues.
You note several times an increase in oleic acid. Where the data are shown?
389-390 (Jung et al., 2000a). Schwartzbeck et al. (2001) used transgenic technology to inhibit FAD
Fatty acid desaturases (FAD) are a group of enzymes with different substrate specificity. You shall not generalize by naming just FAD. Please be more specific.
You indicate certain molecular species of chloroplast glycerolipids in Fig. 7. Any data behind this presentation.


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: LIPID ACCUMULATION OF CHLORELLA SP. TLD6B FROM THE TAKLIMAKAN DESERT UNDER SALT STRESS
Review round: 2
Reviewer: 2

Output:
Basic reporting: N/A
Experimental design: N/A
Validity of the findings: 1. The accession number of transcriptomic raw data was not showed in th revised manuscript.
2. The conclusion number is different from abstract. Authors need to rephrase for the conclusion section.
Additional comments: This version of the manuscript has been greatly improved. However, some mistakes are still present in the manuscript.
1. Use italics for the names of Chlorella in the whole manuscript, such as line 56. Checked throughout the manuscript.
2. Line 60: delete surplus 'the'
3. The accession number of transcriptomic raw data was not showed in th revised manuscript.
4. The conclusion number is different from abstract. Authors need to rephrase for the conclusion section.
5. Where are legends for the supplemental materials?


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: PAEONIFLORIN IMPROVES FUNCTIONAL RECOVERY THROUGH REPRESSING NEUROINFLAMMATION AND FACILITATING NEUROGENESIS IN RAT STROKE MODEL
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this paper the authors reported that Paeoniflorin represses neuroinflammation and facilitates neurogenesis in rat stroke model and might be a potential drug for the therapy of ischemic stroke.
In particular they reported that Paeoniflorin improved the functional recovery, facilitated neurogenesis and vasculogenesis, repressed microglia activation, repressed JNK and NK-κB signaling activation in rat stroke model and repressed microglial viability and inflammatory cytokines production in vitro.

However, these data are not new since there are numerous papers in the literature that, using exactly the same animal model, demonstrated that Paeoniflorin is neuroprotective against ischemia-induced brain damage by inhibiting the inflammatory response mediated by NF-KB. (See Guo et al., 2012; Zhi Liu et al.,  2005; Xiao et al 2005; Tang et al., 2010; Zhang et al., 2015; Zhang et al., 2017; Wu et al., 2020 etc etc).
The only new data is that concerning neurogenesis and vasculogenesis.

Moreover, the analysis of the data is also incorrect since the expression of the various markers observed in the ipsi-lateral hemisphere should be normalized for the same analysis conducted in the contra-lateral hemisphere as internal control.
Experimental design: The Methods are not described with sufficient detail & information to replicate;
for examples it is not specified how long after the ischemic damage the analyzes were conducted. The authors refer to timing only with respect to neurological tests.

The analysis of most of the data is incorrect:
1) the expression of the various markers observed in the ipsi-lateral hemisphere should be normalized for the same analysis conducted in the contra-lateral hemisphere as internal control.
2) the reduction of IL1b, TNFa, IL6 induced by Paeoniflorin in BV2 cells (Fig 4B-D) should be normalized for the number of cells since they observed a reduction of BV2 viability after Paeoniflorin treatment (Fig4A). Moreover they cannot exclude that Paeoniflorin might be toxic for BV2 cells so it is not correct to say that Paeoniflorin inhibits BV2 activity.
Validity of the findings: The data reported are not new since there are numerous papers in the literature that, using exactly the same animal model, demonstrated that Paeoniflorin is neuroprotective against ischemia-induced brain damage by inhibiting the inflammatory response mediated by NF-KB. (See Guo et al., 2012; Zhi Liu et al.,  2005; Xiao et al 2005; Tang et al., 2010; Zhang et al., 2015; Zhang et al., 2017; Wu et al., 2020 etc etc).
The only new data is that concerning neurogenesis and vasculogenesis.
Additional comments: No comment


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: PAEONIFLORIN IMPROVES FUNCTIONAL RECOVERY THROUGH REPRESSING NEUROINFLAMMATION AND FACILITATING NEUROGENESIS IN RAT STROKE MODEL
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is written in a clear English.
The introduction needs more details, since there is not any information regarding the interplay between stroke  and neurogenesis, neither between inflammation, in particular at central nervous system level, and neurogenesis. The authors should mentioned the potential effects of stroke and polaridez microglia on neurogenesis, and the background according to whom they decided to explore the level of adult neurogenesis in their experimental model.

The paper has an acceptable format, however the figures should be improved. In particular, the details of the scale bar used should be insert in Figure 2 and 5.
In the text it has been reported that behavioral tests have been performed with 5 animals per groups (which is a very small sample for behavioral assessment),while in the raw data file on the first and second page have been reported the results of 3 animals per groups. Which is the correct number?

Did the author run a power analysis to calculate the minimum sample size required for such  experimental design?
Experimental design: The behavioral data have been obtained with animals treated with two doses (5mg/kg and 10 mg/kg) and both were effective following 3 days of treatment, however the other analyses were performed comparing only the vehicle with the higher dose, could the authors explain such decision?  In addition, for the behavioral results should be rub an ANOVA analysis since the data consist in repetitive measures between subjects.

Part of the analyses were not performed on the sham group, could the authors provide such data or explains such decision?

The authors should specify the number of subjects per group used for the immunofluorescence and molecular analyses.

The authors should provide more information regarding the timeline of the experiments; in particular, it is not clear in which timepoint the molecular analyses (e.g. ELISA and immunofluorescence staining) were performed.
Validity of the findings: Although the aim and the impact of this work have been appropriately stated, discussion and conclusions should be improved adding the potential molecular and cellular mechanisms underlying the treatment effects. Again, should be implemented the interplay between stroke consequences, inflammation and neurogenesis levels, and the therapeutic action of the treatment.
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: PAEONIFLORIN IMPROVES FUNCTIONAL RECOVERY THROUGH REPRESSING NEUROINFLAMMATION AND FACILITATING NEUROGENESIS IN RAT STROKE MODEL
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: no comment
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: DEVELOPMENT OF AN INEXPENSIVE MATRIX-ASSISTED LASER DESORPTION—TIME OF FLIGHT MASS SPECTROMETRY METHOD FOR THE IDENTIFICATION OF ENDOPHYTES AND RHIZOBACTERIA CULTURED FROM THE MICROBIOME ASSOCIATED WITH MAIZE
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a well-written, well-organized and timely manuscript.  The manuscript is remarkably clear overall and conforms to all relevant professional standards.  The introduction is well-written, but  focusing more clearly and directly on the most interesting question would enhance the manuscript.  Certainly, exploration of the maize microbiome among different varieties and plant locations is interesting, but the manuscript contains a somewhat “buried headline” – the fact that the majority of isolates they studied were not able to be accurately identified by the rather ubiquitous biotyping workflow is very important and likely of great interest to the diverse readers of PeerJ.  The need for methods to enhance rapid MALDI-based approaches to explore non-clinical realms of the microbiome are very much needed, and this paper directly addresses that need; however, this research question/need could be more fully and clearly foregrounded and underscore the coherent nature of this part of the body of work.  Figures are clear, and raw data are available.  A table summarizing the number of isolates analyzed, the number of isolates per library, etc. would be a helpful addition.
Experimental design: The work is certainly within the scope of PeerJ, and the research questions are intriguing and relevant; however, they should be better focused as detailed above (more clearly focusing on and underscoring the need for rapid MALDI-based approaches to environmental microbiome analysis).  The analysis appears appropriately rigorous and quantitative largely, but some details are not included (or difficult to locate), including numbers of replicates per sample/library, number of laser shots, raster spots, etc.
Validity of the findings: The findings seem valid and well-substantiated by the data presented, but additional background/rationale is needed in some areas (see comments in marked up manuscript attached).  Combining the results and discussion sections may facilitate the authors’ ability to better contextualize the findings.
Additional comments: Overall, this is a timely manuscript and notably well-written and clear.  The concept of MTUs is rather intriguing (and somewhat novel), but the concept warrants greater focus (again, foregrounding) and clear explanation (early in the manuscript) its significance and all methods used to define an MTU.  The discussion section has a nicely concise sentence or two summarizing how this is calculated/used, but this ought to be included earlier in the manuscript (perhaps even discussing the need for something akin to an MTU in the Introduction) and more clearly explained in less jargon-laden prose for the benefit of the diverse readers of 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: DEVELOPMENT OF AN INEXPENSIVE MATRIX-ASSISTED LASER DESORPTION—TIME OF FLIGHT MASS SPECTROMETRY METHOD FOR THE IDENTIFICATION OF ENDOPHYTES AND RHIZOBACTERIA CULTURED FROM THE MICROBIOME ASSOCIATED WITH MAIZE
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the introduction, there is little or no time spent on the benefits of increased resolution in phenotyping bacteria, which is a big limitation of 16S identification (which lumps lots of functionally distinct bacteria into the same taxonomic bin).

In the discussion, I don’t think you need to spend several paragraphs listing all the different types of bacteria that you observed (unless you can speculate at the level of bacterial phenotype on why a genus like Pseudomonas was grouped into different MTUs), rather try to explain how this method could yield phenotypic data about the microbes (eg. a difference in spectra may reflect production of growth promoting hormones by beneficial vs neutral strains) or talk about the advantages and potential of expanding it to help study other plant associated microbes. Please discuss whether Maldi-TOF technology is commercially available at reasonable prices to most researchers.

I have made various other comments or suggestions on the attached PDF, which I hope you'll find helpful.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Your study is an important one, and you are beginning to build resources that the plant-microbiome community needs, thank you. The scientific importance of this paper, is its development of a method (and a database?) for better identification and characterization of plant-associated bacteria, however the authors present it more as a study of maize microbial ecology. Beginning with the title, I think your paper could benefit from a restructuring of the orientation, for example remaking it as something like this: “Development of an inexpensive matrix-assisted laser desorption – time of flight mass spectrometry method and database for the identification of rhizobial and endophytic bacteria”. Maize microbes were really just a model system, and shouldn't be built up into something too much more. I think that you should also try to better introduce and discuss more about what the differences in the metabolic spectra might mean and how it might be developed in the future to help better characterize the phenotypes of plant-associated bacteria. For example, this paper (Clark, Chase M., et al. "Coupling MALDI-TOF mass spectrometry protein and specialized metabolite analyses to rapidly discriminate bacterial function." Proceedings of the National Academy of Sciences 115.19 (2018): 4981-4986.) reported examples of how changes in a bacteria's functional proteins alter the MALDI-TOF profile. I think you should likewise add and restructure the introduction and discussion to cover some of the points I mention in the "Basic Reporting" area. I have made various other comments or suggestions on the attached PDF, which I hope you'll find helpful.


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: USING A MOBILE HEALTH APP TO IMPROVE PATIENTS’ ADHERENCE TO HYPERTENSION TREATMENT: A NON-RANDOMIZED CLINICAL TRIAL
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: This study used quasi experimental design. It is well conducted, but there are some issues that I think should be addressed.

1- It would be good if the authors explain some details about validity and reliability of the used questionnaires.

2- The manuscript does not clearly describe the sample method used (e.g., random) in the study to recruit participants.
Validity of the findings: 1- The major issue with the study is the low number of participants and high withdraw rate which make it difficult to gain applicable clinical conclusion about the use of the app, where effectiveness needs to be evaluated for longer time.

 2- Table 1 shows that most participants in the intervention group were above 50 years old. I think there is a significant difference between people who less than age of 50 and those who are over 50 in term of their experience, views and engagement of using such those apps. I would like a word about this in the Discussion section and whether this influence the study result or not.

3- There is no recommendation for future studies in this area. I would be interested to read your thoughts on future research in this area.
Additional comments: This study is quasi experimental study aimed to evaluate the effectiveness of an app on medication adherence to hypertension treatments and how users experience influence the outcomes. It is well conducted, but there are some issues that the authors need to be addressed.


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: USING A MOBILE HEALTH APP TO IMPROVE PATIENTS’ ADHERENCE TO HYPERTENSION TREATMENT: A NON-RANDOMIZED CLINICAL TRIAL
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: 2 issues in the Conclusion:
1. Lines 246, 247 - "Regular monitoring by professionals encouraged users to pay more attention to their self-monitoring. " Even though the mHealth app contains an interaction function that enables patients directly communicating with healthcare providers, the authors did not provide any related results supporting this assumption (causation or correlation). I don't think the authors can draw this conclusion based on this study design.
2. Lines 249,250 - "This study also revealed that considering interaction aspects and design implications is imperative to achieve better health outcomes. " As I stated above.
Additional comments: This is a non-randomized open-label trial investigates the effect of the mHealth app on patients with hypertension. Previous studies demonstrated positive effects of mHealth over hypertension management (Medline: 32311957, Medline: 29710289, Medline: 30431384). Here I address several issues that I concern about:
1. The app was designed to record blood pressure, weight, waist circumference, height, sleep, mood, and engagement in physical activities. Is it possible to include these data, especially BP, in this manuscript to strengthen the conclusion?
2. Method, Line 153 -" Loss to follow-up was due to participants’ loss of interest and inability to provide data for analysis." What does "inability to provide data for analysis" mean? It appears to be the most significant reason that participants dropped out.
3. Discussion, Lines 191,192 - "Akoko et al. (2017) found that factors related to the patient and the health service providers showed significant associations with adherence. " what factors?


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: USING A MOBILE HEALTH APP TO IMPROVE PATIENTS’ ADHERENCE TO HYPERTENSION TREATMENT: A NON-RANDOMIZED CLINICAL TRIAL
Review round: 1
Reviewer: 3

Output:
Basic reporting: Introduction section
1. The statement in lines 34-36 needs to be more specific. I would suggest the authors specify which hypertension diagnostic threshold that they referred to when they argued that “one-third of patients with hypertension have not been diagnosed”. For example, the American Heart Association updated its hypertension diagnostic threshold from 140/90 mmHg to 130/80 mmHg. However, the new guideline has not been adopted in many developing countries. The following article documented the situation in China.
Ni, Z., He, J., Wang, J.G., Cao, J.P., Yang, Q., Wu, B., & Shaw, R.J. (2019). “Chinese
Physicians’ Perspectives on the 2017 American College of Cardiology/American Heart
Association Hypertension Guidelines”. High Blood Pressure & Cardiovascular Prevention,
26(3), 247-57. https://doi.org/10.1007/s40292-019-00321-9

2. The word “feasibility” in line 39 is not clear to me. The authors may need to rephrase the sentence – “Adherence to treatment relies on several factors, such as feasibility” – to make it easier to understand.

3. In the manuscript, “adherence to hypertension treatment” is a key concept. I would suggest the authors give a clear definition of it.

4. In the Introduction section, the authors listed several factors related to poor adherence to hypertension treatment, but in fact poor adherence to hypertension treatment is a complex issue relevant to a lot of factors, including patients’ age, educational level, number of prescribed medications, social-economic status, number of comorbidities, lack of awareness and etc. To strengthen the manuscript, more evidence should be studied, and more references should be cited to give a deeper and broader explanation of the reasons of poor adherence to hypertension treatment. The following is an article that reported several factors related to poor adherence to cardioprotective medications (including antihypertensive medications), which may help the authors to better understand why adherence to hypertension treatment is a public health issue.
Cardioprotective medication adherence among patients with coronary heart disease in China: a systematic review https://heartasia.bmj.com/content/11/2/e011173

5. The English language of this manuscript needs to be improved, particularly the third and fourth paragraphs of the Introduction section. For example, in lines 57-59, the sentence – “Technology such as mHealth apps can help patients remember to check blood pressure regularly, take medications and adopt healthy habits, all leading to better treatment adherence” –  could be changed into “MHealth apps can remind patients of healthy habits, such as checking blood pressures regularly and taking medications as prescribed.” The sentence in lines 63-64 – “User experience studies can act as the force to investigate and explicate the needs and purposes of users of an mHealth app in providing the patient an exceptional health-care experience” – is difficult for me to understand.

6. In line 75, the sentence – “We conducted a quasi-experimental study (non-randomized, controlled, open-label) involving data collection before and after 12 weeks of follow-up” – is not clear to me. I would suggest the authors change it into “We conducted a quasi-experimental study (non-randomized, controlled, open-label) and collected participants’ data at enrollment and 12 weeks after our mHealth intervention.”

7. The first and fourth paragraphs in the Introduction section are not coherent. Also, in the fourth paragraph, the authors mentioned two concepts, “user experience” and “longitudinal approach”, but they were not described in a logical way and were a little difficult to understand.

Methods section
1. In lines 126-127, the sentence “During recruitment, we helped participants who had difficulties with the app, giving feedback and more detailed instructions.” is difficult for me to understand.

2. The authors mentioned that “the time frame was based on the study of Neumann et al (2015).” I would suggest the authors briefly mention the rationale that Neumann chose 12 weeks as the time frame.

3. The eligibility criteria of participants are ambiguous. In line 92, the criterion #1 is “current and ongoing medical monitoring and follow-up regarding hypertension treatment.” Who is going to monitor and follow-up? Healthcare providers or participants themselves? In line 94, it says that the criterion #3 is “ability to measure blood pressure periodically.” There are several ways of measuring blood pressure, such as using an electronic blood pressure cuff and a mercury sphygmomanometer. The authors should specify which measurement they refer to.

4. In the Statistical analysis section, the authors mentioned that “We analyzed the UEQ’s responses using its own data analysis tool”. What does “its own data analysis tool” mean?

Results section
1. In line 178, the phrase “to improve the preventive usefulness of the collected data” might need to be rephrased to make it easier for readers to understand.

2. In the Table 1, “20 to 29 years” may need to be changed into “20 to 39 years”.

Discussion section
1. The authors argued that “The age group of this study may have positively influenced the study’s adherence rate, according to the findings of Daniel and Veiga (2013), who observed that increasing age was associated with a higher probability of adherence to the recommended treatment.” This statement needs more evidence to support. Using one study’s finding to support that the age has positively influenced the study’s adherence rate is not convincing. For example, there are studies documented that increased age could decrease patients’ medication adherence.

2. The second paragraph of the Discussion section may need to be rephrased to improve its clarity. For example, “….to improve the preventive usefulness of the collected data….” is difficult for readers to understand.

Conclusion section
1. The Conclusion section should be more concise. Some of the content, such as “As better adherence implies several underlying improvements related to the diverse treatment factors, this can be considered a cornerstone of the success of digital interventions” could be moved to Discussion section.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript reported a non-randomized clinical trial in which an app was used to help patients with hypertension to improve adherence to hypertension treatment. This manuscript is a good start. To improve the quality of the manuscript, the authors could consider using university-based editorial services to improve the English language of 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: USING A MOBILE HEALTH APP TO IMPROVE PATIENTS’ ADHERENCE TO HYPERTENSION TREATMENT: A NON-RANDOMIZED CLINICAL TRIAL
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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: USING A MOBILE HEALTH APP TO IMPROVE PATIENTS’ ADHERENCE TO HYPERTENSION TREATMENT: A NON-RANDOMIZED CLINICAL TRIAL
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors reported the study clearly with sufficient information.
Experimental design: The authors addressed all the provided comments
Validity of the findings: The authors did their best to address all provided comments
Additional comments: The authors addressed all the provided comments. Thank you.


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: FOOD INSECURITY INCREASES ENERGETIC EFFICIENCY, NOT FOOD CONSUMPTION: AN EXPLORATORY STUDY IN EUROPEAN STARLINGS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Excellent, bar a few small quibbles.
Experimental design: Excellent. It is acknowledged and discussed that each individual study is relatively small but meta-analyses are run across each experiment.
Validity of the findings: Robust, within the recognised limits of the sample sizes and the heterogeneity of the various experiments.
Additional comments: This report is a masterclass in undertaking, analysing and reporting on experimental studies. The experiments are clearly explained and complimentary, the limits to the study are clearly explained in an upfront and honest way and demonstrate strong stats literacy, and throughout the manuscript the authors explicitly recognise the limitations of the work based on sample size and various methodological constraints. One thing that possibly could be explored a little more, though I appreciate that this would somewhat go against the approach taken of looking across the studies together given that each individual study has low predictive power, is why there is such marked variation in outcome variables particularly between replicate cages in the same experiments, when this is the case. Finally, the links between humans and (other) animals adds an important additional dimension to the study.

Some minor comments and observations:
L49 – Surely there are studies quantifying food intake in people experiencing different levels of food insecurity.
L50 – Why past tense?
L118-122 – Here, I’m unclear what exactly the three experiments are (having re-read these sentences several times).
L134 – reinforcements of what, exactly?
L317, and indeed the rest of the paragraph – unclear. What is the variable of behaviour (with units) being used in this study? (It seems to be one variable? And about roosting?)… L332 – perhaps made clear here, but a bit late on.
L433 – The use of p = 0.05 for ‘significant or not significant’ is inherently problematic (as I appreciate you will know) and highlighted here. Would you have concluded that body mass and competition were important moderators here if e.g. p = 0.049?  Play around with your data and you will find that just marginal tweaks to the measurement will create this amount of difference in p. Better to interpret this p value as ‘some evidence for an effect’?
Figure 4 shows some considerable variation in effect size (and direction) across the experiments, including across aviaries within experiments. For example, (A) experiment 1 aviary 3; (B) experiment 1 aviary 1. How different the findings would have been, and the conclusions drawn, had e.g. a single experimental type or single aviary been used.  This is not a criticism, but shows the value of multiple replicates and experimental types, and then good, clear figures to show the results.