Peer review report

Reviewer: Nidia Bañuelos Institution: University of Wisconsin-Madison email: nbanuelos@wisc.edu

General comments

Questions of how and why grading practices change over time, how students, faculty, and administrators respond to grades, and the external pressures on grading practices (e.g. war, graduate school requirements) are inherently interesting! The authors have clearly done a careful job of tracking these – often minute, and likely, difficult to follow – changes at Dalhousie University. The manuscript is well-written and relatively easy to follow.

My biggest concern, reflected in the more detailed comments below, is that the authors could do a better job of explaining to the reader why these changes are interesting, important, and relevant to historians of higher education more broadly – even those who aren’t at Dalhousie. They do some of this at the very end of the paper and, indeed, this summing up of their findings and explanation of their relevance was my favorite part of the manuscript. I would suggest reorganizing the paper so that these bigger takeaways appear in the introduction and so that the reader is reminded of them at each major section break of the paper. For example, when the authors present a quote from a student who is concerned that grades have little to do with learning outcomes, they might remind us that one of their main arguments is that “decisions about university grading schemes had very little to do with actual pedagogy” (p. 15).

As it is written, the manuscript sometimes reads like a list of facts about grading changes. But, I think a reframing that focuses on the general importance of these changes could make the entire piece more engaging. More on this below…

Section 1 – Serious concerns

• Do you have any serious concerns about the manuscript such as fraud, plagiarism, unethical or unsafe practices? No

• Have authors’ provided the necessary ethics approval (from authors’ institution or an ethics committee)? not applicable

Section 2 – Language quality

• How would you rate the English language quality? High quality

Section 3 – validity and reproducibility

• Does the manuscript contain any objective errors, fundamental flaws, or is key information missing?

While I don’t notice any “objective errors”, I do think the paper has a major flaw (i.e. little explanation of the broader significance of this case study) and could benefit from additional information about the institutional context, the archival material, and external influences on grading trends. (Please see below.)

Section 4 – Suggestions

• Based on your answer in section 3 how could the author improve the study?

I. Most importantly, I would like to see an introduction that explains the authors’ general arguments about grading changes – including the trajectory of these changes at Dalhousie and why this arc contributes to our knowledge of the history of higher education more broadly. Then, the authors might continually remind us of the arc they present at the outset of their paper – especially when they are highlighting a piece of evidence that illustrates their central argument. To me, the quotes from students and faculty responding to grading changes are among the most interesting parts of the paper and placing these in additional context should make them shine even more brightly!

II. I’d like to read a little more about Dalhousie itself – why it is either a remarkable or unremarkable place to study changes in grading policies. Is it representative of most Canadian universities and thus, a good example of how grading changes work in this national context? Is it unlike any other institution of higher education and thus, tells us something important about grades that we could not learn from other case studies? I don’t think this kind of description needs to be particularly long, but it should be a little more involved than the brief sentences the authors currently include (p.3, paragraph 1) and should explain the choice of this case.

III. I’d also like to know more about the archival materials the authors used. The authors mention that they drew from “Senate minutes, university calendars, and student newspapers” (p. 3), but what kinds of conversations about grades did these materials include? At various points, the authors engage in “speculation” (e.g. p.4) about why a particular change occurred. This is just fine and, in fact, it’s good of the authors to remind us that they are not really sure why some of these shifts happened. But, they might go one step further and tell us why they have to speculate. Were explicit discussions of grading changes – including in inter- and intradepartmental letters and memo, reports, and other documents – not available in these archives? Why are these important discussions absent from the historical record?

IV. At various points, the authors make references to the outside world – for example, WWII (p. 5), the Veteran’s Rehabilitation Act (pp. 6-7), and British versus American grading schemas (p. 6). But, these references are brief and seem almost off-handed. I know space is limited, but putting these grading changes in their broader context might help make the case for why this study is interesting and important. Are the changes in the 1940s, for example, related to the ascendance of one national graduate education model over another (e.g. American versus British)? Are there any data on how many Canadian undergraduates enrolled in British versus American graduate programs over time? If so, I would share any information you might have on these broader trends.

Similarly, the authors make brief mention of the internal reaction to grade changes – quoting students or faculty minutes. But, it would be wonderful (space permitting) to have even more information the internal impact of these changes. Did they change faculty instructional practices? Did they seem to have any effect on students’ orientation to their learning? Did standardization reflect an increasing interdependence of departments, or did it contribute to their lessening autonomy? If the archival record doesn’t permit us to know these things, then this might be a limitation the authors note at the end of the manuscript. I noticed that the authors reference a secondary source on Dalhousie student experiences repeatedly (Waite, 1998). Even a little more from this text or another secondary source like it could help the reader better understand the impact of grade changes.

• Do you have any other suggestions, feedback, or comments for the Author?

This is a very nitpicky concern that doesn’t fit well elsewhere, so please take it with a grain of salt. I was surprised at the length of the reference list – it seemed quite short for a historical piece! I wonder, again, if more description of the archival material - including why you looked at these sources, in particular, and what was missing from the record – would help explain this and further convince the reader that you have all your bases covered.

Section 5 – Decision

Requires revisions: The manuscript contains objective errors or fundamental flaws that must be addressed and/or major revisions are suggested.

Peer review report

Reviewer: Champion Deivanayagam

Institution: University of Alabama at Birmingham

email: champy@uab.edu

General comments

Summary of the study: The authors begin the manuscript describing an effort to understand the various sizes of DMBT1 protein, namely variations in the copy numbers of SRCR repeats based on its DNA sequence variations among various groups. In this effort they have identified/chosen three major groups namely European (states in Figure 1of the manuscript they are European-Americans in Utah), African (Yoruba of Ibadan, Nigeria) and Asian (Chinese from Beijing). The observed high D value shown in Figure 1, they contend is the evidence for balancing selection (which this Reviewer has no expertise on). Based on this Tajima scores, they were able to identify two haplotypes, and this led to them arriving at SNP (rs11523871).

Now comes the interesting part of parsing the copy number variations of DMBT1’s SRCR domains within these two haplotype clades, where they conclude that the SRCR copy numbers are population specific. Then looking at tissue specific expression of DMBT1, where they observe higher expression levels in some tissues such as lung, small intestine, colon, and minor salivary gland. More interestingly they observe that there exists no linear relationship that exists among the alleles and concluding that there is no plausible explanation for protein expression, but the selection locus rs11523861 somehow is related at balancing selection.

In an attempt to determine the copy number variations, a small set of samples (8) were collected from saliva and analysed. In table 1 they summarize these findings, where they observe four different isoforms. They report that there is a strong linear relationship, but do not present a figure or other details, except statistical parameters to convince the reader. From here, they step into establishing alternative splicing as a possibility, where they use the H292 lung cell line model, and report in past tense that the H292 cell line was homozygous for the 11/10 SRCR domain repeats (Figure 4). While they could not conclude, it mentions that alternative splicing may play a minor role.

Finally, this study attempts to use the results from the classic Stromberg lab study published in 2007, which enumerated various properties of Gp340, and classified them into 4 groups, and enumerated their affinities for various carbohydrates, Lewis antigens and compared the oral and lung Gp340. The authors here use western blots to determine if the short allele is different from the normal one. They use two knock-outs of surface components on S. mutans, SpaP and Cnm to show that the shorter alleles display no binding. In the same set of experiments, they also show that S. mutans adheres to mono and dimeric amylase.

Finally, they conclude that some of the variations in adherence may be due to the carbohydrates present on the different DMBT1.

Section 1 – Serious concerns

• Do you have any serious concerns about the manuscript such as fraud, plagiarism, unethical or unsafe practices? No
• Have authors’ provided the necessary ethics approval (from authors’ institution or an ethics committee)? Yes

Section 2 – Language quality

• How would you rate the English language quality? Low to medium quality, but I understand the content

Section 3 – validity and reproducibility

• Does the work cite relevant and sufficient literature? Yes
• Is the study design appropriate and are the methods used valid? Yes
• Are the methods documented and analysis provided so that the study can be replicated? Yes
• Is the source data that underlies the result available so that the study can be replicated? Yes
• Is the statistical analysis and its interpretation appropriate? Yes to a large extent
• Is quality of the figures and tables satisfactory? No
• Are the conclusions adequately supported by the results? No
• Are there any objective errors or fundamental flaws that make the research invalid? Please describe these thoroughly. Yes

Section 4 – Suggestions

• In your opinion how could the author improve the study?

Additional experiments are needed to support the conclusions.

Major Concerns:

The research work presented here appears highly disjointed at times. While they begin this study to evaluate the need for copy numbers and how it could have been a part of selection process induced by a variety of factors, their analysis leads to Supplementary figure 1, where they identify two clades. From here they offer some evidence for the choice of rs11523861 to study the balancing selection. However, no concrete evidence is presented and/or discussed except peripherally. This Reviewer understands the effort, which is very interesting, however, without additional analysis the current form of results does not offer any new insights, and so the title is highly misleading.

At this point, they move into CNV’s, whereby their own admission have used a very small sampling of 8 individuals and extrapolate their results to be conclusive. Further sampling and additional studies are necessary to complete this section.

One solid set of results shown here are from the H292 lung cell line model, where they report different variations of the repeats. However, all analysis stops here, and conclusion is derived that alternative splicing is ruled out, but for a minimal role. Once again, the same pattern exists here to do an experiment, without further analysis and additional data, conclusions are drawn.

Finally, from sequence analysis the authors step into protein-based assays to expose the role of S. mutans adherence on the presence and absence of its surface proteins SpaP and Cnm. Their conclusion that Cnm is the major adherent protein is also out of one single experiment. More importantly, their analysis of I-IV alleles (without explaining them), an extension of a previous well cited article, results in a conclusion that smaller allelles (???) do not adhere to saliva. In Table 1, the molecular weights of these isoforms are in alignment with the 14 SRCR domains, but for their carbohydrate decorations, with one exceptions of sample 6. Also, this Reviewer is not sure if this table corresponds to the 8 samples, they have used for CNV analysis and/or for the smaller/larger alleles they allude to in the western blot study.

In summary this appears to be a manuscript that is not only disjointed, but also not detailed enough to warrant publication at this stage. If they could do additional analysis on each of the points raised, it would elevate the research and conclusions.

Minor concerns: The manuscript is extremely poorly written and needs major revamping in order to produce a more concrete publication.

Figure numbers and legends are often mixed up both in the text as well as in the legends. Figure 5 - Differential binding of S.mutans by DMBT1 isoforms in saliva: Overlay of individual saliva phenotypes with DMBT1 size isoforms I- IV with A) a biotinylated S. mutans SpaP A, Cnm strain and B) with DMBT1-specific antibodies. The positions of DMBT1, mono- and dimer amylase, and acidic PRP co-receptors are marked by arrows”. This bold line is not related to this figure but to the supplementary figure.

There is no marker in these figure 5A so indicate the specific molecular weights. The isoform IV on the last lane seems to lightup with the lower MW DMBT1 (allele?). However, the conclusions presented show that lower isoforms do not bind well is contrary to the results presented here.

Supplementary figure 2 should be in the main manuscript, even though this is not a new result, in combination with the other two, authors can summarize the results.

Abbreviations should be in the expanded form the first time.

Why do the authors use the SpaP A instead of SpaP?

What do they mean by a biotinylated S. mutans SpaP A, Cnm strain?

• Do you have any other feedback or comments for the Author?

These copy numbers have always been fascinating not only on DMBT1 but on other proteins, yet to date we don’t have a handle on the type of selection. If these authors could provide additional analysis on why and how balance selection could have played a role it would be very important and could be extended to numerous other proteins.

Section 5 – Decision

Requires revisions

Viki Male on Twitter: “@jtmayes3 @kevinault Let’s begin by taking the 172,000 number at face value. About 190,000 ppl have been vaccinated during pregnancy in the US. So if that were true it’s a miscarriage rate of 90%… 1/ https://t.co/cXYXDv1UgB” / Twitter. (n.d.). Retrieved February 2, 2022, from https://twitter.com/VikiLovesFACS/status/1487313040785686528

US government moves to end daily COVID-19 death reporting by hospitals. (n.d.). World Socialist Web Site. Retrieved February 2, 2022, from https://www.wsws.org/en/articles/2022/01/15/hhhs-j15.html

Dr Duncan Robertson. (2022, January 31). Cases will increase today. The way that cases are counted is changing. A short thread. 🧵 https://coronavirus.data.gov.uk/details/whats-new/record/af008739-ffa3-47b8-8efc-ef109f2cfbdd https://t.co/WY655My1RV [Tweet]. @Dr_D_Robertson. https://twitter.com/Dr_D_Robertson/status/1488115542263271427

Peer review report

Reviewer: Jinbao Liao Institution: Jiangxi Normal University. email: jinbaoliao@163.com

Section 1 – Serious concerns

• Do you have any serious concerns about the manuscript such as fraud, plagiarism, unethical or unsafe practices? No
• Have authors’ provided the necessary ethics approval (from authors’ institution or an ethics committee)? not applicable

Section 2 – Language quality

• How would you rate the English language quality? Low to medium quality. I understand the content

Section 3 – validity and reproducibility

• Does the work cite relevant and sufficient literature? Yes
• Is the study design appropriate and are the methods used valid? No
• Are the methods documented and analysis provided so that the study can be replicated? No
• Is the source data that underlies the result available so that the study can be replicated? Yes
• Is the statistical analysis and its interpretation appropriate? Yes
• Is quality of the figures and tables satisfactory? Yes
• Are the conclusions adequately supported by the results? Yes
• Are there any objective errors or fundamental flaws that make the research invalid? Please describe these thoroughly. No

Section 4 – Suggestions

• Do you have any feedback or comments for the Author?

Three basic models are used to study complex systems: dynamical system modelling, agent-based model, and complex networks. Dynamical system modelling uses top-down modelling ideas (modelling with macroscopic variables; based on mean-field ideas). The agent-based model uses bottom-up modelling ideas (modelling with micro variables; based on individual simulation ideas). Complex networks lie between dynamical system modelling and agent-based model (each individual interacts with each other, and the interactions are linked into edges to form a complex network). The paper 'Metacommunity research can benefit from including context-dependency' uses the agent-based model framework (NetLogo).

The agent-based model framework in this paper incorporates four dimensions: inter-habitat differences (heterogeneity), dispersal (dispersal rate on the probability of colonizing rather than general mobility), specialization (breadth of species response to collection of diverse habitats), species Interactions (competition, predation, mutualism, parasitism et al. mutualism, parasitism et al.), and these dimensions have been more or less extensively studied in the dynamical system framework and the complex network framework. Therefore, the authors should discuss in detail how agent-based model framework has advantages over the most common frameworks currently used for ecosystem modelling (dynamical systems, complex networks).

For example, for the dynamical system framework, recent studies suggest incorporating six modules (such as species interactions, dispersal, demography, evolution, environment and physiology) into the model to predict biodiversity (Norberg et al., 2012, Nature Climate Change; Urban et al., 2016, Science). For the complex network framework, early theoretical studies explored the effects of multiple relationship types or dispersal on complex networks (Holland & Hastings, 2008, Nature; Mougi & Kondoh, 2012, Science; Allesina & Tang, 2012, Nature).

Moreover, the idea of agent-based model is derived from the theory of complex adaptive systems (e.g., Kondoh, 2003, Science), and the core idea of this framework is that adaptability creates complexity. Many studies have focused on the evolution of dispersal strategies within single species (Cote et al., 2017, Ecography), while theoretical studies on the evolution of dispersal in meta-communities remain rare. For the dispersal dimension (a key dimension of this paper), do authors consider the evolution of dispersal strategies.

Finally, after reading this ms, it is really unclear what the model is and how to simulate the model. Maybe you should describe it in details following the ODD protocol for describing individual-based models (Grimm et al. 2006).

Section 5 – Decision

Requires revisions: The manuscript contains objective errors that must be addressed

Peer review report

Reviewer: Dr. Pradeep G Kumar Institution: Rajiv Gandhi Centre for Biotechnology email: kumarp@rgcb.res.in

Section 1 – Serious concerns

• Do you have any serious concerns about the manuscript such as fraud, plagiarism, unethical or unsafe practices? No
• Have authors’ provided the necessary ethics approval (from authors’ institution or an ethics committee)? not applicable

Section 2 – Language quality

• How would you rate the English language quality? High quality

Section 3 – validity and reproducibility

• Does the work cite relevant and sufficient literature? Yes
• Is the study design appropriate and are the methods used valid? Yes
• Are the methods documented and analysis provided so that the study can be replicated? Yes
• Is the source data that underlies the result available so that the study can be replicated? Yes
• Is the statistical analysis and its interpretation appropriate? Not applicable
• Is quality of the figures and tables satisfactory? Yes
• Are the conclusions adequately supported by the results? Yes
• Are there any objective errors or fundamental flaws that make the research invalid? No

Section 4 – Suggestions

• In your opinion how could the author improve the study?

• Do you have any other feedback or comments for the Author?

Text requires improvement at some places (citing them is difficult as pages and lines are not numbered). Some examples are given below:

.......was associated with molecular markers of the PGC...... (was associated with the expression of molecular???)

Primordial germ cells (PGCs), the developmentally first founder cells of the germline, are induced from epiblast cells on around embryonic day (E)6.25 by external signals,

Furthermore, it was shown for that male mouse ES cells cultured in serum that subjected to a chemical intervention, including a timed exposure to a combination of the a SIRT1 inhibitor Ex-527, the a DNA methyltransferase inhibitor RG-108 and the an electrophilic redox cycling compound tert-butylhydroquinone (tBHQ), was associated with induced the expression of molecular markers of the PGC

Section 5 – Decision

Verified manuscript

Peer review report

Reviewer: Takehiko Ogawa Institution: Yokohama City University email: ogawa@yokohama-cu.ac.jp

Section 1 – Serious concerns

• Do you have any serious concerns about the manuscript such as fraud, plagiarism, unethical or unsafe practices? No
• Have authors’ provided the necessary ethics approval (from authors’ institution or an ethics committee)? not applicable

Section 2 – Language quality

• How would you rate the English language quality? High quality

Section 3 – validity and reproducibility

• Does the work cite relevant and sufficient literature? Yes
• Is the study design appropriate and are the methods used valid? Almost Yes
• Are the methods documented and analysis provided so that the study can be replicated? Almost Yes
• Is the source data that underlies the result available so that the study can be replicated? I think, Yes
• Is the statistical analysis and its interpretation appropriate? Not applicable
• Is quality of the figures and tables satisfactory? Almost Yes
• Are the conclusions adequately supported by the results? No
• Are there any objective errors or fundamental flaws that make the research invalid? Please describe these thoroughly.

I would like to request authors to test the validity of cells with spermatogonia-like morphology (CSM) as spermatogenic stem cells. For this, transplantation of CSM into the seminiferous tubules in the testis of recipient mice is necessary. This may be an additional work, but authenticity of CSM as SSCs cannot be claimed without such experiment. They may claim that in case of ES cells most experiments do not perform blastocyst injection experiment to confirm the pluripotency of ES cells. However, culture system for ES cells has a long history and was established as robust method to maintain their pluripotency. It is actually the 2iL system as shown in this study as well. In case of SSCs or GS cells, the culture system is still fragile in my understanding, compared to the 2iL. The spermatogenic ability that SSCs and GS cells have could be lost during the cultivation in many cases. In particular, chemical intervention shown in this study could disturb cell’s intrinsic system, which could make almost anything happen. The enhanced expression of Lhx1 also could be an aberrant result of such turmoil in the cells. I do not insist that is the case, but intentionally taking a position to be extremely skeptical to the results. In order to dispel such doubts, it is necessary to do the transplantation experiments and prove that the CSM maintained the spermatogenic ability as SSCs.

Section 4 – Suggestions

• In your opinion how could the author improve the study?

Readers would wonder what the CSM is exactly. This naming means that CSM could be SSCs but possibly not, although they look like SSCs. It was honest naming but confusing in what the CSM really is. In order to start the experiment with CSM, authors should clear such ambiguous point. Thus, I recommend a transplantation experiment as stated above.

• Do you have any other feedback or comments for the Author?

In Page 6, it is described that medium change twice daily with a third of the volume at hour 8 and 16 was critical for the appearance of CSM. In case of the medium change with half of the volume every 12 hours, it was written that the CSM did not appeared at all. Authors argued that endogenously produced soluble factors might be the cause. I wonder what extent of difference could be there between the two method of culture medium change, regarding to the concentration of endogenously produced soluble factors. A simulation of the change in concentration of such hypothetical substances might help the speculation.

Section 5 – Decision

Requires revisions

Peer review report

Reviewer: Yuan Junpeng Institution: National Science Library, Chinese Academy of Sciences. email: yuanjp@mail.las.ac.cn

Section 1 – Serious concerns

• Do you have any serious concerns about the manuscript such as fraud, plagiarism, unethical or unsafe practices? No
• Have authors’ provided the necessary ethics approval (from authors’ institution or an ethics committee)? not applicable

Section 2 – Language quality

• How would you rate the English language quality? High quality

Section 3 – validity and reproducibility

• Does the work cite relevant and sufficient literature? Yes
• Is the study design appropriate and are the methods used valid? Yes
• Are the methods documented and analysis provided so that the study can be replicated? Yes
• Is the source data that underlies the result available so that the study can be replicated? Yes
• Is the statistical analysis and its interpretation appropriate? Yes
• Is quality of the figures and tables satisfactory? Yes
• Are the conclusions adequately supported by the results? Yes
• Are there any objective errors or fundamental flaws that make the research invalid?

The research question of this article is not clear enough, and this paper is more like a report than a research paper. Since a lot of research about retraction haven been published, many characteristics of retraction have been analysed. There seem not enough new messages comes from this article.

In addition, as ‘exploratory research’ defined by the title, the use of full data for analysis is more in line with the objectives of the title, instead of excluding other disciplines and restricting the analysis to human health. If the author’s goal is to analyse the characteristics of human health-related retractions, it is recommended to limit it in the title. The current topic is too general.

Section 4 – Suggestions

• In your opinion how could the author improve the study?

It is recommended that the author properly point out what have and haven’t been done in this topic, and their specific contribution to the existing knowledge, so as to show the innovation of the research.

It is recommended that the author clarify the research objectives and modify the title more in line with the content.

• Do you have any other feedback or comments for the Author? No

Section 5 – Decision

Requires revisions

Peer review report

Reviewer: Hurng-Yi Wang Institution: Institute of Ecology and Evolutionary Biology, National Taiwan University email: hurngyi@gmail.com

Section 1 – Serious concerns

• Do you have any serious concerns about the manuscript such as fraud, plagiarism, unethical or unsafe practices? No
• Have authors’ provided the necessary ethics approval (from authors’ institution or an ethics committee)? not applicable

Section 2 – Language quality

• How would you rate the English language quality? Medium quality

Section 3 – validity and reproducibility

• Does the work cite relevant and sufficient literature? No
• Is the study design appropriate and are the methods used valid? No
• Are the methods documented and analysis provided so that the study can be replicated? Yes
• Is the source data that underlies the result available so that the study can be replicated? Yes
• Is the statistical analysis and its interpretation appropriate? Yes
• Is quality of the figures and tables satisfactory? No
• Are the conclusions adequately supported by the results? No
• Are there any objective errors or fundamental flaws that make the research invalid?

Section 4 – Suggestions

• In your opinion how could the author improve the study?

• Do you have any other feedback or comments for the Author?

Agarwal and Parekh analyzed 685 SARS-CoV-2 isolates collected during 27th Jan - 27th May 2020 from India and described the distribution of virus strains and mutations across the country. While the information might be valuable to some local readers, the results are mainly descriptive and the data are a bit out of date. In addition, I have the following comments.

1. Some details of the methods are lacking. For example, the MUpro provides two methods, it is necessary to specify which method was used in the analysis. The confidence score of each prediction should also be provided. Besides, some results from I-Mutant and MUpro were conflicting, the authors may want to discuss the discrepancy.

1. The “Analysis of the Mutational Profile of Indian Isolates” should be moved to Materials and Methods.

1. The authors provided lengthy discussion about the effect of each mutation in some lineages, such as 20A and I/A3i. However, as these mutations are tightly linked, the effect of each individual mutation is difficult to access. It is possible that some of the mutations are just hitchhikers. They may want to address this alternative point.

1. Several figures are confusing and lack detail. The diversity plots of Figure 3 and Figure 8 are hard to be precisely compared to the mutations that occurred among different plots. Phylogenetic trees, as well as their figure legends, are confusing, especially Figure 9 and Figure 10. For Figure 9, it is impossible to tell which mutation site had changed from C to T. For Figure 10, spots depicted in yellow are both position 29827 A>T and position 29830 G>T, green spot only notes as G, but A29827 is not mentioned in the figure. Furthermore, the mutation position of blue spot C cannot be found.

1. Figure 9 and Figure 10 were not mentioned inside the text.

1. The Top 10 mutations in PCA analysis are the mutations in 20A and I/A3i. It is reasonable to observed a clear association of the clusters with the clades. It is not clear, however, how these distribution correlate with lockdown, contact tracing and quarantine measures.

Section 5 – Decision

Requires revisions

Peer review report

Reviewer: Dr. Jyotsnamayee Sabat Institution: Regional VRDL, RMRC(ICMR). email: jyotsnasabat@yahoo.com

Section 1 – Serious concerns

• Do you have any serious concerns about the manuscript such as fraud, plagiarism, unethical or unsafe practices? No
• Have authors’ provided the necessary ethics approval (from authors’ institution or an ethics committee)? not applicable

Section 2 – Language quality

• How would you rate the English language quality? Good quality

Section 3 – validity and reproducibility

• Does the work cite relevant and sufficient literature? Yes
• Is the study design appropriate and are the methods used valid? Yes
• Are the methods documented and analysis provided so that the study can be replicated? Yes
• Is the source data that underlies the result available so that the study can be replicated? Yes
• Is the statistical analysis and its interpretation appropriate? Yes
• Is quality of the figures and tables satisfactory? Yes
• Are the conclusions adequately supported by the results? Yes
• Are there any objective errors or fundamental flaws that make the research invalid?

No such application was observed.

Section 4 – Suggestions

• In your opinion how could the author improve the study?

They have analysed it in-depth and presented nicely.

• Do you have any other feedback or comments for the Author?

I want to know how the representative sequences were selected for different states. Is it based on no of sequences submitted or positivity rate of a particular region.

Section 5 – Decision

Verified manuscript

Peer review report

Reviewer: Parvin Abraham Institution: MIMS Research Foundation, Calicut, Kerala, India. email: parvinabraham@gmail.com

Section 1 – Serious concerns

• Do you have any serious concerns about the manuscript such as fraud, plagiarism, unethical or unsafe practices? No
• Have authors’ provided the necessary ethics approval (from authors’ institution or an ethics committee)? not applicable

Section 2 – Language quality

• How would you rate the English language quality? High quality

Section 3 – validity and reproducibility

• Does the work cite relevant and sufficient literature? Yes
• Is the study design appropriate and are the methods used valid? Yes
• Are the methods documented and analysis provided so that the study can be replicated? Yes
• Is the source data that underlies the result available so that the study can be replicated? Yes
• Is the statistical analysis and its interpretation appropriate? Yes
• Is quality of the figures and tables satisfactory? Yes
• Are the conclusions adequately supported by the results? Yes
• Are there any objective errors or fundamental flaws that make the research invalid? Please describe these thoroughly. No

Section 4 – Suggestions

• In your opinion how could the author improve the study?

The dataset is only from 27th Jan – 27th May 2020. Maybe they can include more Numbers.

• Do you have any other feedback or comments for the Author? No

Section 5 – Decision

Verified manuscript

Peer review report

Reviewer: Dr. : Peter Dahlberg Institution: SLAC national laboratory email: pdahlb@slac.stanford.edu

Section 1 – Serious concerns

• Do you have any serious concerns about the manuscript such as fraud, plagiarism, unethical or unsafe practices? No
• Have authors’ provided the necessary ethics approval (from authors’ institution or an ethics committee)? not applicable

Section 2 – Language quality

• How would you rate the English language quality? Low to medium quality. I have added several comments to section 4 as suggested edits.

Section 3 – validity and reproducibility

• Is the reason for developing a new method explained? Yes
• Is the description of the method technically sound? Yes
• Are sufficient details provided so that the method can be replicated? No
• Is the source data that underlies the result available so that the study can be replicated? not applicable
• Is quality of the figures and tables satisfactory? Yes
• Are the conclusions adequately supported by the results? No
• Are there any objective errors or fundamental flaws that make the research invalid? Please describe these thoroughly. No

Section 4 – Suggestions

• In your opinion how could the author improve the method?

The manuscript describes the progressive refinement method for sparse recovery. This approach uses minimal RAM while producing a finely discretized output for high density 3d fluorescence localizations. Furthermore, the approach does not require the PSF to be translationally independent. This is an assumption that is often made that simplifies the computation, but does not account for field dependent aberrations. There are several comments that I believe would improve what is overall strong manuscript. The most serious of which is addressed in 1a below.

1. The two main claims of the manuscript are that the PRIS method requires less RAM than a brute force approach and that the algorithm functions for spatially varying PSFs. Neither of these claims are supported directly by the text or figures. a. Perhaps I missed it, but there were no field dependent aberrations in the simulations. If this is the case, how exactly has it been demonstrated that the approach works well for a spatially varying PSF? Because this is a central claim of the PRIS method, I think it is worth implementing. b. The authors describe a scenario of brute force solving of the inverse problem requiring 152.6 GB, while I have no doubt that the PRIS approach would require less RAM, the authors do not make it clear how much less. While I know that the reduction will depend on the exact implementation and the data at hand, a rough comparison of the RAM requirements would be helpful for the reader.

2. Following algorithm 1, the authors introduce the “shrink” operator. A one line description would be helpful of what this operator does. As I understand it, it is a threshold of the output to keep the data output sparse.

3. Following algorithm 1 and 2, the authors describe the use of “kicking.” They do a nice job of giving a brief description of what the “kicking” does (improve the convergence speed), but I am concerned because neither algorithm 1 or 2 shows kicking. The kicking is wrapped up in another conditional statement that is not shown in either algorithm. This is confusing for the reader. Perhaps a parenthetical should be added stating that the kicking is not shown in the algorithm.

4. The code for the PRIS method should be made available publicly, both so the results can be replicated and also so that others can use the approach.

• Do you have any other feedback or comments for the Author?

Additionally, I have some minor text/figure edits

1. In the title, the acronym PRIS is defined as “progressive refinement method on sparse recovery” however in the text it is “progressive refinement method for sparse recover” I think the wording in the text is correct.

2. 5th paragraph of the introduction: “In principal” should be “In principle”

3. Paragraph preceding section 3: “in case if a species” I think should read “in case a species”

4. Throughout the figures, dark red, dark green, and dark blue are used over black and this is very difficult to see. For example, dashed red line in figure 2 on the right hand side, the cy5 and cy3 labels in figure 7, the dark blue box in figure 8.

5. Throughout the figures there are also a lot of small symbols used. For example, Figure (a)-4 there are small (red?) marks on top of a red heat map. These are extremely difficult to see clearly.

6. Figure 6c, it is very challenging to see differences in the distributions of points. I think this data would be better represented if additional histograms were shown.

Section 5 – Decision

Requires minor revisions

Peer review report

Reviewer: Dr. Christopher H. Bohrer Institution: NIH/NCI email: bohrerch@nih.gov

Section 1 – Serious concerns

• Do you have any serious concerns about the manuscript such as fraud, plagiarism, unethical or unsafe practices? No
• Have authors’ provided the necessary ethics approval (from authors’ institution or an ethics committee)? not applicable

Section 2 – Language quality

• How would you rate the English language quality? High quality

Section 3 – validity and reproducibility

• Is the reason for developing a new method explained? Yes
• Is the description of the method technically sound? Yes
• Are sufficient details provided so that the method can be replicated? No
• Is the source data that underlies the result available so that the study can be replicated? No
• Is quality of the figures and tables satisfactory? Yes
• Are the conclusions adequately supported by the results? No
• Are there any objective errors or fundamental flaws that make the research invalid? Please describe these thoroughly. No

Section 4 – Suggestions

• In your opinion how could the author improve the method?

1. The approach is nice, but I really think they should highlight the advantage of their approach --- that is, perform a simulation with imperfect optics then apply the traditional methodologies as well as their own to show their superiority.

2. The comparison to previous methodologies is nice, but the fact that they are different simulations with different parameters is a major problem --- for example, the photons used in their simulations are higher than used in the previous studies. Therefore, if they are going to compare, it should only be done if the methodologies were applied to the same data.

3. A user guide with an example, walking through the specifics would aid this work greatly. For instance, it is unclear how one obtains the different matrices given their data --- though this is likely within the references. Additionally, if they want others to use the methodology, this is a must!

4. Finally, though I don’t think they necessarily need to do this, but utilizing real experimental data to validate their approach would be nice. For instance, investigate the structure of the nuclear pore complex with the different methodologies --- a standard within the field.

• Do you have any other feedback or comments for the Author? No

Section 5 – Decision

Requires revisions

Peer review report

Reviewer: Deyou Qiu Institution: Chinese Academy of Forestry. email: qiudy@caf.ac.cn

Section 1 – Serious concerns

• Do you have any serious concerns about the manuscript such as fraud, plagiarism, unethical or unsafe practices? No
• Have authors’ provided the necessary ethics approval (from authors’ institution or an ethics committee)? not applicable

Section 2 – Language quality

• How would you rate the English language quality? High quality

Section 3 – validity and reproducibility

• Does the work cite relevant and sufficient literature? Yes
• Is the study design appropriate and are the methods used valid? Yes
• Are the methods documented and analysis provided so that the study can be replicated? Yes
• Is the source data that underlies the result available so that the study can be replicated? Yes
• Is the statistical analysis and its interpretation appropriate? Yes
• Is quality of the figures and tables satisfactory? Yes
• Are the conclusions adequately supported by the results? Yes
• Are there any objective errors or fundamental flaws that make the research invalid? Please describe these thoroughly. No

Section 4 – Suggestions

• In your opinion how could the author improve the study? No

• Do you have any other feedback or comments for the Author?

The resolution of Fig 3 is not good, could you pls improve it?

Section 5 – Decision

Requires revisions

Peer review report

Reviewer: Max Shokhirev Institution: Salk Institute for Biological Studies email: maxshok@gmail.com

Section 1 – Serious concerns

• Do you have any serious concerns about the manuscript such as fraud, plagiarism, unethical or unsafe practices? No
• Have authors’ provided the necessary ethics approval (from authors’ institution or an ethics committee)? not applicable

Section 2 – Language quality

• How would you rate the English language quality? High quality

Section 3 – validity and reproducibility

• Does the work cite relevant and sufficient literature? Some, but seems to be very limited in terms of biological literature.
• Is the study design appropriate and are the methods used valid? Yes
• Are the methods documented and analysis provided so that the study can be replicated? not applicable
• Is the source data that underlies the result available so that the study can be replicated? Yes
• Is the statistical analysis and its interpretation appropriate? Yes
• Is quality of the figures and tables satisfactory? Yes
• Are the conclusions adequately supported by the results? Yes
• Are there any objective errors or fundamental flaws that make the research invalid? How could the author improve the study?

The author has laid out a theoretical argument for senescence as a tradeoff between information capacity between epigenetic and non-epigenetic content.“A constraints-based theory of senescence: imbalance of epigenetic and non-epigenetic information in histone crosstalk.” This work is interesting, but is based on a superficial understanding of the biology underlying senescence/aging, makes several dangerous oversimplifications and assumptions, and does not provide any data or analysis to support the theory. I’ve laid out my comments for each section below:

Sections 1.1-1.3

The author only mentions the Hayflick limit as a biological reference for senescence. There is a very rich body of literature on senescence and aging that is completely overlooked here. The author should include additional references to reviews for senescence and aging to orient the reader to the complexity of these biological processes (e.g. PMC8658264, PMC7846274). Please clarify what you mean by senescence vs aging for both cells and individuals. Senescence is a natural biological process that cells/organisms use to turn off cell replication due to damage (e.g. telomere shortening, double-stranded breaks, etc.). Other cells can also facilitate this process through signaling (e.g. immune cells or contact inhibition). Aging is typically thought of as an organismal phenomenon, which is still poorly understood but is theorized to include tradeoffs (as you describe in section 1.2). It is also accepted that aging is cell, tissue, and organism specific. Since you talk about senescence and aging across both biological scales, it is important to define exactly what your theory pertains to.

Section 1.4

The author posits that senescence is an imbalance in information contents of histone post-translational modifications around transcription start sites. This is just one level of regulation, albeit an important one. The author seems to completely overlook many other types of regulation (e.g. microRNA, lincRNAs, metabolic/energetic constraints, non-proximal regulation at enhancers, higher ordered structure of the chromatin, post-translational regulation of proteins, and etc.). How can all of these other important levels of regulation fit into this theory? All have been implicated in senescence/aging in some form or another. The author further suggests that histone crosstalk information content can be decomposed into two unrelated components: epigenetic and non-epigenetic. The non-epigenetic component is described as “hologenic information content,” which stems from a previously published work by the author. Non-epigenetic is confusing in this context since really this is information content that stems from the synergies of individual cells to form a whole, e.g. the emergent information content that comes from many cells working together (or at least this is how I understand the underlying theory). This information content is important for the general maintenance and survival of the organism. The author should clarify this point further, since this seems to be one of the fundamental assertions being made in the paper. For example, bringing in the descriptions used in section 2, can further clarify these central points. In addition, the author states: “ Moreover, the sum decomposition in Eq. 1 implies that the growth in magnitude (bits) of the hologenic (i.e., non-epigenetic) component must be accompanied by a decrease in magnitude of the epigenetic component.” This is not necessarily true, since signaling is a separate biological process from the regulation of gene expression. In other words, both can increase or decrease simultaneously. For example, a healthy non-senescent immune cell can upregulate very specific transcriptional programs that lead to very complex signaling and extra-cellular interactions. You can argue that both represent an increase in information content for both the epigenetic and non-epigenetic “hologenic” components. In addition, as cells naturally senesce they are programmed to turn off cell-cycling while upregulating autophagy and repair processes. They may not upregulate extracellular signaling at this time, which would seem to contradict the author’s theory/statement. In this case, the simplification that all cells are the same is dangerous because it overlooks the tradeoff of information contents between cells. It also ignores important repair pathways (senescence being one of them), to deal with cells that have dysregulated their natural processes over time. It also overlooks the important action of immune cells that work to get rid of cancer and poorly-functioning cells. Also, it seems crosstalk, correlation, capacity, and content, are used interchangeably. Please clarify that these are all the same, or use one of these terms to avoid confusion.

Section 1.5

The author provides a general approach for measuring the log of the ratio of epigenetic and non-epigenetic capacities for a particular histone modification at three positions (i,j,k), and for some measured abundance of mRNA Y. Since we typically measure abundance of a particular modification genome-wide, and the mRNA level for tens of thousands of genes, how would a realistic equation look like (i.e. one that has 10k mRNA levels, and 10k histone positions)? In addition, the author does not explain how to combine correlations across multiple histone modifications. Please expand this section to make it relevant for real-world genome-wide measurements since this will be important for falsifying the theory. Since public datasets are available (e.g. the aging atlas https://doi.org/10.1093/nar/gkaa894), the author should show an example of how a dataset might be used to falsify or demonstrate the theory in more detail.

Section 2.1

The author uses correlation of the log ratio of the epigenetic and non-epigenetic content with age as a readout of “reassignment” of crosstalk/contents, arguing that for cancer cells this correlation should be essentially zero. This seems like an oversimplification of the “reassignment” process since senescence may occur in phases across the age of a cell/organism, and since there might be both increases and decreases in the log ratio of contents due to natural biological processes and variability. Would it not be better to measure the sum of changes in the log ratio or the difference between the log ratios at different ages?

In addition, the biological age of a cell/tissue/organism can vary. For example, stem cells may have negligent aging, while other cells might age relatively quickly. Again, the author should clarify the context of age: are we measuring strictly chronological age correlation? Should we consider different correlations for each cell/tissue in the organism? What about tradeoffs in information content between cell types and tissues? In other words, it is unclear how the theory should be applied to biological systems.

Section 2.2

The author argues that senescence is an emergent property of the loss of information content for epigenetic histone crosstalk and an increase in information content of “hologenic” information content (e.g. cell signaling and anti-tumor signaling). I believe this premise does not stem from the reality of biological systems (see my comments for section 1.4). Also, this section seems to be contradicting the author’s conclusions and is very confusing. The author seems to argue that there is both more AND less constraint at the multi-cellular level (organismal)? Please clarify or remove this section.

Section 2.3

Senescence as transcriptional overregulation is vague. Here the author is arguing that as epigenetic constraint decreases, you have a decrease in precision (e.g. loss of regulation), but then you have a competing global or hologenic increase in constraints, which constrains the expression of genes for the overall benefit of the organism. A shift toward global constraint.

Section 2.4

This seems to be describing an illustrative real-world example? This section is incredibly specific and again only focuses on one possible mechanism and does not include any measured data or analysis. Please preface this section to explain that this is just one of many possible examples. Again, it will be good to provide other examples looking at other aspects of aging biology (not just histone modifications).

Section 2.5-2.9,3

This seems to be a general discussion. It would be easier to organize these sections into one discussion section for added clarity. Again, I would recommend not talking about sweeping statements like “Senesensce’s ultimate cause” and “Can senescence be stopped?” since this theory only addresses one small aspect of the biology underlying aging and senescence and does not address the heterogeneity of aging. These topics are controversial and should be addressed very carefully to avoid alienating the biological community.

Section 4 – Decision

Revisions required

Peer review report

Reviewer: Charles A. Schumpert Institution: University of South Carolina email: schumpca@email.sc.edu

Section 1 – Serious concerns

• Do you have any serious concerns about the manuscript such as fraud, plagiarism, unethical or unsafe practices? No
• Have authors’ provided the necessary ethics approval (from authors’ institution or an ethics committee)? not applicable

Section 2 – Language quality

• How would you rate the English language quality? High quality

Section 3 – validity and reproducibility

• Does the work cite relevant and sufficient literature? Yes
• Is the study design appropriate and are the methods used valid? Yes
• Are the methods documented and analysis provided so that the study can be replicated? not applicable
• Is the source data that underlies the result available so that the study can be replicated? Yes
• Is the statistical analysis and its interpretation appropriate? Yes
• Is quality of the figures and tables satisfactory? Yes
• Are the conclusions adequately supported by the results? Yes
• Are there any objective errors or fundamental flaws that make the research invalid? Please describe these thoroughly.

Overall the manuscript is written brilliantly and provides excellent context to the audience about a complex theoretical biological concept. No flaws can be found, although one could argue against a few of the points in the assumptions used to construct the theory, there’s nothing illogical or irrational.

Section 4 – Suggestions

• In your opinion how could the author improve the study?

The writing of the paper makes it easy to read, which can sometimes be a challenge with theoretical biology manuscripts. Potentially adding a bit more context on the various theories of aging may help demonstrate the marriage of the ideas into the theory he constructed.

• Do you have any other feedback or comments for the Author? No

Section 5 – Decision

Verified manuscript

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report with data collection

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That's an interesting hypothesis. I wouldn't have thought histamine was involved. Though, histamine being a stimulant, it also makes some sense. I'd have thought the primary mechanism is serotonin blockade, which would work in part by dopamine dis-inhibition as mentioned here.

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