Discussion, revision and decision

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Decision

Verified with reservations: The content is scientifically sound, but has shortcomings that could be improved by further studies and/or minor revisions.

Dr. Bañuelos: Verified manuscript

Dr. Morris: Verified with reservations

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Revision

Response to Reviewer 1 (Dr. Bañuelos)

1. 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!

Our Response: Thank you so much for your thoughtful review. We have added a larger new introduction section of the paper (paragraphs 1-5 in the latest draft are new) that outlines the general importance of the topic, the Canadian context, details on Dalhousie University, and our overall thesis statement (i.e., most decisions were to improve the external communication value of grades). Moreover, we have added three new student quotes form the Dalhousie Gazette to build a stronger picture for student reactions, and to build a better case for our overall thesis statement (i.e., that changes in grading were often to increase the external communication value of grades). Moreover, throughout we have added some details on the overall funding trajectory for institutions in Canada that created some pressure to standardize grading. We think that these changes have improved the manuscript.

1. 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.

Our Response: This comment revealed that two additional pieces of context were needed for the introduction: (a) some national context for higher education policy in Canada and (b) some extended description of Dalhousie University when compared to other universities in Canada. To this end, two new paragraphs have been added to the paper (paragraphs 2 & 3 in the current draft).

Notably, Jones (2014) notes that “Canada may have the most decentralized approach to higher education than any other developed country on the planet” (pg 20). With this in mind, any historical review of education policy is by necessity specific to province and institution – that is, the information can be placed in its context, but resists wide generalization to the country as a whole. In the newest draft, we tried to describe the national, provincial, and institutional context in some more detail in paragraphs 2 & 3.

1. 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?

Our Response: We have added a new paragraph (paragraph 4) to the paper discussing the sources in some more detail. It is true that the verbatim discussions are frequently absent from the record, especially earlier in history – or if they exist, we have not found them! Instead, we frequently are reviewing meeting minutes or committee reports, which are summaries of discussions. As we now note in the paper, “Thus, the sources used showed what policy changes were implemented, when they were implemented, and a general sense of whether there was opposition to changes; however, there were notable gaps in faculty and student reactions to grade policy changes, as these reactions were frequently not written down and archived.”

This gap was most apparent in the Senate minutes around the 1940s, where I (the first author) could not find any direct discussions of why changes were implemented. Under the 1937-1947 heading, we more clearly indicate that the rationale for the changes was absent from the Senate minutes during this period. I add some further speculation on why these records might be absent, based on summaries from Waite (1998b); specifically, the university president of the time often made unilateral decisions, circumventing Senate, which might account for why the changes are absent from the records.

This will hopefully make the limitations of what can be learned from this approach more apparent.

1. 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.

Our Response: To our knowledge, there isn’t any comparable report to what we’ve written here documenting the transition from British “divisions” to American “letter grades” in Canadian Universities, making our report novel in this regard. It might well be that a similar historical arc exists in many of the 223 public and private universities in Canada, but we don’t believe such data exists in any readily accessible way – excepting perhaps undergoing a similar deep dive into historical documents at each respective institution! So, we do not have the answer to your question: “Are there any data on how many Canadian undergraduates enrolled in British versus American graduate programs over time?” However, we did add one reference which provided a snapshot point of comparison in 1960, noting in the paper “Baldwin (1960) notes that the criteria for “High First Class” grades in the humanities was around 75-80% at Universities of Toronto, Alberta, and British Columbia in 1960, suggesting that Dalhousie’s system was similar to other research-intensive universities around this time.” That said, there are a few major national events related to the funding of universities in Canada that we have elaborated on in the text to address the spirit of your recommendation for describing the national context:

a) In the “Late 1940s” section of the paper, we added: “Though Dalhousie had an unusually high proportion of veterans enrolled relative to other maritime universities during this period (Turner, 2011), the Veteran’s Rehabilitation Act was a turning point for large increases in enrollment and government funding Canada-wide, at least until the economic recession of the 1970s (Jones, 2014).”

b) In the 1990s, there were major government cuts to funding, creating challenging financial times for the university. We discuss the funding pressures that likely contributed to standardization of grading during this time by saying the following in the 1980s-2000s section: “Starting in in the 1980s-1990s there were major government cuts to university funding nation-wide, with the cuts becoming more severe in the 1990s (Jones, 2014; Higher Education Strategy Associates, 2021). Because of the nature of the funding formulas, cuts in Nova Scotia were especially deep. Beyond tuition increases, university administrators knew that obtaining external research grants, Canada Research Chairs, and scholarship funding was one of the few other ways for a university to balance budgets, so there was extra pressure to be competitive in these pools. […] The increased standardization was likely related to increased financial pressures at this time – standardization is an oft-employed tool to deal with ever-increasing class sizes with no additional resources.”

c) In the 2010s section of the paper, we added context to how universities in country-wide have become increasingly dependent on tuition fees for funding: “Following the 2008 recession, federal funding decreased again (Jones, 2014; Higher Education Strategy Associates, 2021); however, this time universities tended to balance budgets by increasing tuition and international student fees. This trend towards increased reliance on tuition for income is especially pronounced in Nova Scotia, which has the highest tuition rates in the country (Higher Education Strategy Associates, 2021). Thus, the university moved closer to a “consumer” model of education, so it makes sense that a driving force for standardization was student complaints.”

1. 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.

Our Response: In the introduction section, paragraph 4, we describe our sources in more detail including what is likely missing from the record and why we used them. Regarding the length of the reference list, we did add ~12 new references to the list in the course of making various revisions, which partially addresses your concern. Beyond this though, it’s worth noting that some of the sources more extensive than they seem, even though they don’t take up much space in the reference list (e.g., there is one entry for course calendars, but this covers ~100 documents reviewed!). Moreover, there were many dead-ends in the archives that are not cited (e.g., reviewing 10 years of Senate minutes in the 1940s produced little of relevance), so the reference list is curated to only those sources where relevant materials were found.

Reviewer response to revisions

The new introduction to the piece addresses many of my previous questions about the authors’ general arguments, the Dalhousie context, and the source material. Thank you for addressing these! Reading this version, it is much clearer that the key argument is that standardized, centralized grading practices were “to improve the external communication value of the grades, rather than for pedagogical reasons” (p. 6). I also really enjoyed the added quotes from students in the Dalhousie Gazette.

The authors’ response to Reviewer 2 really gave me a better sense of why they wrote this piece and also helped me to more clearly put my finger on what was troubling me in the first round. It still reads a little like a report for an internal audience – which is just fine and, in fact, can be extremely useful for historians of the future. But, as Reviewer 2 notes, this means it does not really seem like a piece of historical scholarship. I do worry that shaping it into this form would take an extensive revision and might not be in the spirit of what the authors intended to do.

A different version of this article might start with this idea that grades were standardized for external audiences and in response to financial pressures. It would then develop a richer story behind the sudden importance of these external audiences and the nature (i.e. source, type) of financial pressures Dalhousie was facing. It would highlight the impact such changes had on students and their future careers/graduate experiences. It could then connect these trends to other similar changes for external audiences and the increasing interconnectedness of American, Canadian, and British systems through graduate education. It might even turn to sociological theories of organizational change and adaptation and make an argument for when (historically) similar forms of decoupling were likely to occur in the Canadian higher education system. Finally, it might connect these grading changes to current trends – including accusations of grade inflation and accepted best practices for measuring learning outcomes.

But, it doesn’t seem that the authors necessarily want to do this, which I can understand and respect. I think there is enormous value in a piece of scholarship like this existing – both for internal audiences and for future historians. Indeed, imagine if every university had a detailed history of its grading policies like this available somewhere online! Comparing such practices across institutions would certainly tell us a lot about why grading currently looks the way it does.

Decision changed

Verified manuscript: The content is scientifically sound, only minor amendments (if any) are suggested.

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Response to Reviewer 2 (Dr. Morris)

The authors dove headfirst into Dalhousie’s archives, unpacking the subtle shifts in grading policy. Their work seems to be comparable to archaeologists, digging deep beneath mountains of primary sources to find nuggets of clues into Dalhousie’s grading evolution. I particularly liked when the authors were able to link these changes to student voices, as seen in moments when they referenced student publications.

Ultimately, I kept coming back to one main comment that I wrote in the margins: “So what?” I would humbly suggest that the authors reflect on why this history matters to them. Granted, they do this in the conclusion, where they touch on Schneider & Hutt’s argument that grades evolved to increasingly be a form of external communication with audiences beyond school communities. Sure. But I want more. I wanted to see a new insight that this microhistory of Dalhousie significant to the history of Canada or the history of education more generally.

If the authors are so inclined, there might be several approaches to transform this manuscript. I would suggest the following. First, instead of tracing the entire history of grading at the institution, choose one moment of change that you think is the most important. Perhaps in the 1920s and the lack of transparency in grading, or the post-war shift toward American grading. Second, show me – don’t tell me – what Dalhousie was like at this moment. Paint a picture of the institution with details about student demographics, curriculum, educational goals, the broader town, etc. Make the community come alive. Show me what makes Dalhousie unique from other institutions of higher ed. Once you establish that picture, perhaps you could link the change in grading practices to subtle changes at the university community, thereby establishing a before and after snapshot. This will require considerable amounts of work, and the skills of a historian. You will have to find primary and secondary sources that go far beyond what you’ve relied on thus far.

In the end, I found myself wanting the authors to humanize this manuscript, meaning I wanted them to show me that changes in grading practices have tangible effects on real-life human beings. A humanization of their research would mean going narrower and deeper; or, in other words, eliminating much of what they have documented.

However, if that is too tall of an order, I would ask that the authors clarify for themselves who this manuscript is for. Is this a chronicling of facts for an internal audience at Dalhousie’s faculty, alumni, and students? Fine. But my guess is that even members of the Dalhousie community want to read something relatable.

I am suggesting revisions, although not because of objective errors. History is more of an art, in my opinion. With that in mind, I would suggest that the authors paint a more vivid picture (metaphorically) of Dalhousie, showing me how changes one moment of change in grading practices impacted the lives of human beings.

Our Response: Thank you very much for taking the time to read our paper and provide your thoughts and recommendations. It may be helpful to begin by describing why I (the first author) decided to write this paper. Ultimately, I wrote this paper to satisfy my own personal curiosity and to connect with other people at my own place of employment by exploring our shared history. At present day, Dalhousie has a letter grading scheme with a standardized percentage conversion scheme that all instructors used. I wanted to know why this particular scheme was used, but I quickly realized that nobody at Dalhousie really knew how we ended up grading this way! There was an institutional memory gap, and a puzzle that was irresistible to me. So, I wrote this paper for the most basic of all academic reasons: Pure curiosity. I do very much recognize that the subject matter is very niche, perhaps too niche for a traditional journal outlet. Thus, my publishing plan is to self-publish a manuscript to the Education Resources Information Center (ERIC) database and a preprint server as a way of sharing my work with others who might be interested in what I found. Nonetheless, I believe in the importance and value of peer review, especially since I am writing in a field different than most of my scholarly work. That is why I chose PeerRef as a place to submit, so that I could undergo rigorous peer review to improve the work while still maintaining the niche subject matter and focus that drives my passion and curiosity for the project. Of course, if you feel the whole endeavor is so flawed that it precludes publication anywhere, then we can consider this a “rejection” and I will not make any further edits through PeerRef.<br /> The core of your critique suggested that I should write a fundamentally different paper on different subject matter. While I don’t necessarily disagree that the kind of paper you describe might have broader appeal, it would no longer answer the core research question I wanted an answer to: How has Dalhousie’s grading changed over time? So, I must decline to rewrite the paper to focus on a single timeframe as recommended. All this said, I did try my best to address the spirit of your various concerns to improve the quality of the manuscript. Below, I will outline the various major changes to the manuscript that we made to improve the manuscript along the lines you described, while maintaining our original vision for the structure and focus of the paper. The specific changes are outline below:

a) Two new paragraphs (now paragraphs 1-2 of the revised manuscript) were added to explain the “so what” part of the question. Specifically, we describe why we think the subject matter might be of interest to others and summarize the general dearth of historical information on grading practices in Canada as a whole.

b) Consistent with recommendations from the other reviewer, we now state a core argument (i.e., that most major grading changes were implemented to improve the external communication value of the grades) earlier in the introduction in paragraph 5 and describe how various pieces of evidence throughout the manuscript tie back to that core theme.

c) In an attempt to “humanize” the manuscript more, we added more student quotes from the Dalhousie Gazette throughout the paper so that readers can get a better sense of how students thought about grading practices at various times throughout history. Specifically, three new quotes were added in the following sections: 1901-1936, late 1940s, 1950s-1970s. We also added this short note about the physical location where grades used to be posted: “Naturally, this physical location was dreaded by students, and was colloquially referred to as “The Morgue” (Anonymous Dalhousie Gazette Author, 1937).”

d) Early in the paper, we describe why we chose Dalhousie and the potential audience of interest: “As employees of Dalhousie, we naturally chose this institution as a case study due to accessibility of records and because it has local, community-level interest. The audience was intended to be members of the Dalhousie community; however, it may also be a useful point of comparison for other institutions, should similar histories be written.”

e) We have described some of the limitations of our sources in paragraph 4, which may explain why the manuscript takes the form it does – it has conformed to the information that is available!

f) We have linked events at Dalhousie to the national context in some more detail, by detailing some national events related to the funding of universities in Canada. See our response to Reviewer 1, #4 above for more details on the specific changes.

g) Consistent with your stylistic recommendations, we have changed various spots throughout the paper from the present tense (e.g., “is”) to the past tense (e.g., “was”), and were careful in our new additions to maintain the past tense, when appropriate. If there are any spots that we missed, let us know the page number / section, and we will make further changes, as necessary.

h) We retained the first person in our writing – this may be discipline-specific, but in Psychology (the first author’s home discipline), first person is acceptable in academic writing. If you feel strongly about this, we can go through the manuscript and remove all instances of the first person, but we would prefer to keep it, if at all possible.

Hopefully this helps address the spirit of your concerns, and I look forward to hearing your thoughts in the second round of reviews.

Decision changed

Verified with reservations: The content is scientifically sound, but has shortcomings that could be improved by further studies and/or minor revisions.

Author response

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To: Reviewer: Heikki Vapaatalo, MD, PhD, Emeritus professor of Pharmacolog

Dear reviewer Thank you very much for the insightful suggestions, the manuscript improved a lot with the changes performed. Please find the point-by-point answer to the raised questions. In the main text, all changes are highlighted in yellow. I hope that with the changes made the new version is suitable for publication.

Best regards Valquiria Bueno

General assessment

The study is interesting and the title promises for me more than the MS finally contains.

Answer: The manuscript is part of a project aiming to study ACE1 and ACE2 expression in cells from the immune system of aging and young adults. These initial results suggest that ACE1 (and probably ACE2) plays somehow a role in the process of aging.

The background, question and the aim are relevant as explained in the introduction.

Answer: We included a piece of information in the “Introduction” trying to link ACE1 expression in tissue cells and age-related diseases, as it follows:

ACE1 has been suggested to influence age-related diseases (i.e. Alzheimer’s, sarcopenia, cancer) but the associated mechanisms are still under investigation. ACE1 polymorphisms were correlated with susceptibility to Alzheimer’s disease (AD). [15, 16] In addition, it was shown recently that in normal aging ACE1 expression is increased in brain homogenates and this expression is unchanged in early stages of AD. [17] Regarding sarcopenia, Yoshihara et al. [18] found a weak correlation between ACE polymorphism and physical function. In cancer (gastric or colorectal), patients presented higher expression of ECA1 in tumor when compared with healthy tissues. [19, 20]

The major criticism concerns the small size of the material (subjects, n=6), the small age difference (64-67 years) and the lack of younger controls.

Answer: We agree that the small number of studied subjects is a limitation of this study. In spite of the interesting results suggesting that ACE1 expression could be linked to the health status, it was not possible to perform correlation analysis due to the small sample size. Even though there is a small chronological difference between the subjects, the biological aging is very different among them and reflects the genetics, lifestyle, nutrition and comorbidities. Another limitation is the lack of younger controls to compare with the subjects studied. Our next steps are to include younger controls, to increase the number of studied subjects, and if possible to get samples from older subjects (i.e. 70-80, 80 and more years old)

Minor notes:

1)Title: Angiotensin converting enzyme (ACE) expression in leukocytes of older adults

Answer: We evaluated only ACE1 expression, and thus, title, abstract, and main text were changed to ACE1 instead of ACE. We decided to change to title for: Angiotensin converting enzyme (ACE) 1 expression in leukocytes of adults from 64 to 67 years old

2)Introduction: The last chapter, the Author should explain in more detail, how references 11-14 suggest that “ACE play an important role in the aging process”. Does this mean, that ACE is somehow regulating the aging process or in increasing age ACE -levels are changed?

Answer: References 11-14 shows that age-related diseases occurring in older adults are associated with changes in the immune system. To complete the text we added:

ACE1 has been suggested to influence age-related diseases (i.e. Alzheimer’s, sarcopenia, cancer) but the associated mechanisms are still under investigation. ACE1 polymorphisms were correlated with susceptibility to Alzheimer’s disease (AD). [15, 16] In addition, it was shown recently that in normal aging ACE1 expression is increased in brain homogenates and this expression is unchanged in early stages of AD. [17] Regarding sarcopenia, Yoshihara et al. [18] found a weak correlation between ACE polymorphism and physical function. In cancer (gastric or colorectal), patients presented higher expression of ECA1 in tumor when compared with healthy tissues. [19, 20]

Material and Methods:

The N-value of the subjects should be mentioned here, as well the relation of females/males.

Answer: Text was correct as suggested Blood was collected from adults (n=6, four females and two males) aged 64-67 years old in 2015.

Do the Authors really regard 64-67 “older age” nowadays?

Answer: Nowadays the most common term used for individuals older than 65 years is “older adults”.

Why first many years later the assays have been done in comparison to the collection of the blood? Are the samples still useable, not destroyed?

Answer: Samples are part of UNIFESP Biobank and have been kept in adequate conditions. We wanted to test cells from a period anterior to COVID-19 and those samples were the only ones that attended our purpose. We compared samples used in this study with fresh blood samples (cell viability and percentage of CD4+, CD8+ and CD19+) and the results showed good preservation of the cells.

Did the subjects have some diseases and/or drugs because the possibly were from hospital sample bank?

Answer: Samples are part of UNIFESP Biobank, but unfortunately we do not have information about diseases and medicaments.

Express the company details similarly than Amersham, cities and countries.

Answer: Changes were done as required ACE CD143 FITC (R&D Systems, Inc, Minneapolis, USA)

Results:

“Table 1 shows that older adults…..” The comparison between the present data and historical studies belongs to the Discussion.

Answer: Changes were done as required

Give also individual ages and gender of the subjects in the table 1.

Answer: The manuscript version sent to medrxiv@medrxiv.org had age and gender on tables, but due to their request, any possible variable that could identify the studied individual had to be removed. That is why in the present version these variables are not shown.

What means p-values here? Compared with which or interindividual differences in the particular variable? Should be explained

Answer: We used p-value for interindividual differences in each variable since individuals age differently (biological aging) and thus, physiological parameters could be affected by genetics, lifestyle, nutrition, and comorbidities. It is now explained in materials and methods

The numbering of tables and the text seems to me confusing. Only three tables, but in the text mentioned four. Number 4 does not exist.

Answer: For some reason table 2 is missing in the main text, please find the new version with Table 2 included

It would be good to have a list of abbreviations used in the description of the cell types for an unfamiliar reader.

Answer: In each figure and table we are now providing a description of cells evaluated.

Discussion:

A major part of the discussion deals with previous publications and not meaning or clinical significance of the present findings and comparison between the present and earlier studies.

Answer: The discussion was changed as suggested:

Our results show that for the studied population, chronological aging and biological aging don´t go at the same pace. Even individuals having a small chronological difference (64 to 67 years old), they are heterogeneous for physiological parameters such as glucose, urea, glycated hemoglobin (Hbglic), and C-reactive protein (CRP). Changes in the same functional parameters have been reported by Carlsson et al. [22] and Helmerson-Karlqvist [23] in healthy older adults. Carlsson’s study [22] found that CRP value was 2.6 with a coefficient variation of 1.4% whereas in our study, it was observed higher values of CRP in 5 out of 6 individuals. Increased CRP levels has been associated with inflammaging and our findings show that the studied population has changes in functional parameters which are likely associated with an inflammatory profile. [24] The link between RAS and inflammation has been suggested but its role is not completely clear under physiological and pathological conditions. [25, 26] In addition, the association between ACE1 altered expression in tissues (brain, muscle, heart and vessels) and the development and progression of age-related conditions such as Alzheimer’s, sarcopenia, and cardiovascular disease has been suggested but results are controversial. [17, 27, 28, 29, 30] There are few studies showing the association between ACE1 expression in cells from the immune system (monocytes, T cells) and the progression of kidney and cardiovascular disease. [9, 8, 31, 32]. Therefore, considering the lack of information on this issue, we questioned whether ACE1 (CD143) was highly expressed in cells from the immune system during the aging process. We found that ACE1 was expressed in almost 100% of T (CD4+, CD8+) and B lymphocytes and in all phenotypes of these cells. In non-lymphoid cells, ACE1 mean expression was 56,9%. In agreement with our findings, independent studies showed that T cells from healthy donors and monocytes from patients with congestive heart failure expressed ACE1, but there was no investigation on cell phenotype. [25, 26]. Our study is the first to show that either inexperienced (naive) or fully activated (memory) cells expresses ACE1. Our findings suggest a that the expression of ACE1 in lymphoid and non-lymphoid cells reflects the health status since our studied population presented changes in physiological parameters and high levels of ACE1 expression on immune cells. Previous independent studies showed that patients with unstable angina [32] or acute myocardial infarction [33] presented higher expression of ACE1 in T cells and dendritic cells than controls subjects. In addition, markers of the cell (lymphoid and non-lymphoid) functional status such as inflammatory or growth factors production could be modulated by ACE inhibitors (ACEi). Accordingly, mononuclear leukocytes from healthy subjects incubated with endotoxin exhibited high levels of tissue factor activity which was reduced in the presence of captopril in a dose-dependent pattern. This result could be related to the antithrombotic effect of ACEi. [34]. In patients with congestive heart failure, immune cells cultured with LPS secreted high levels of the pro-inflammatory TNF-alpha and these levels were significantly reduced in the presence of captopril. [35]

In those previous studies, also ACE2 has been reported, why not studied here?

Answer: Our next studies will be focused on ACE1 and ACE2 expression in cells from the immune system in both younger and older adults.

In the limitations, the Authors fairly mention the real problem: The small sample size, and I would like to say lack of younger subjects.

Answer: we agree with the limitations pointed and the text was changed as required:

This study have limitations such as the small sample size and the lack of young adults for comparison. As an example, the subject with the highest CRP and albumin also exhibited a high percentage of ACE1 expression on T (CD4+, CD8+), B and non-lymphoid cells in addition to the lowest percentage of CD4+ naive cells, and the highest percentage of CD8+ terminally differentiated (EMRA) and DN B cells. However, due to the small sample size it was not possible to associate the high expression of ACE1 on immune cells with inflammaging and immunosenescence. It would bring important information to correlate physiological parameters/health status with ACE1 expression and to find out whether age and associated chronic diseases could lead to increased ACE1 expression.

The COVID-19 point even tempting today, is too far from this study and unnecessary. Answer: Our point was to emphasize the negative impact of chronic diseases for the outcome of aging population during a viral infection and how ACE1/ACE2 expression could bring information to diagnosis and treatment. Therefore, we would like to maintain this piece of information.

Linguistic checking would improve the MS. Answer: We checked for possible linguistic mistakes

Reviewer, Heikki Vapaatalo:

I read with pleasure the very detailed answers to my comments.

I very warmly recommend acceptance of this MS for publications without any further notes.

Decision changed:

Verified manuscript: The content is scientifically sound, only minor amendments (if any) are suggested.

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To: Reviewer: Calogero Caruso

Dear Prof.Caruso Thank you very much for the revision of this manuscript. It is a privilege to have a manuscript reviewed by a research with high expertise on the field of ageing. Please find the answers to your questions and in the main text the changes in bold.

Sincerely yours,

Valquiria Bueno

The paper is essentially anecdotal because it studies the cells of 6 subjects without any comparison with other age groups. There is also a serious limitation because beyond the age and sex there is no information on the donors (how and why they were recruited, what drugs they took, etc.).

It is really a limitation to have only 6 individuals for the study, but they were the only ones fitting in the proposal of the manuscript. The samples were from a central bank of cells at UNIFESP and participants were considered “healthy” but there was not further information in addition to what we displayed on the tables of the manuscript. They were not living on homecares or hospitalized.

Our aim was to evaluate samples from individuals aged 60-69 years previously to COVID-19 and/or vaccination. In addition, there were no samples in the same conditions (PBMCs, -80oC) of young individuals and using fresh blood could bring a result that could not be compared mainly regarding to myeloid cells and B cells as is follows in the below reference. Braudeau C, Salabert-Le Guen N, Chevreuil J, Rimbert M, Martin JC, Josien R. An easy and reliable whole blood freezing method for flow cytometry immuno-phenotyping and functional analyses. Cytometry B Clin Cytom 2021;100(6):652-665. doi: 10.1002/cyto.b.21994.

Our goal from now on is to expand this study with young and old adults samples since it is important to understand whether ageing is associated with an increase in ACE expression on immune cells.

-To infer that chronological and biological ages do not match is inappropriate in the absence of the above information.

This piece of information regarding chronological and biological age was required by another reviewer. I agree that the concept does not match without more information on the donors. However, the information is now referenced and should be considered when older adults are studied. Vasto S, Scapagnini G, Bulati M, Candore G, Castiglia L, Colonna-Romano G, Lio D, Nuzzo D, Pellicano M, Rizzo C, Ferrara N, Caruso C. Biomarkes of aging. Front Biosci (Schol Ed) 2010;2(2):392-402. doi: 10.2741/s72. PMID: 20036955.

-However, the paper is of some interest because there are few studies on the topic.

Thanks for this positive comment. Few studies on the topic was the reason why we decided to send the manuscript for publication even though there were some important information on the donors missing and limited number of individuals.

Essential revisions that are required to verify the manuscript

1) Although we do not have data on donors, placing an age and gender column in all tables adds a minimum of useful information for the reader.

The first table submitted with age, but for requirement of MedRxiv, gender and age could no be linked to the metabolic results to preserve the anonymity of the donors.

2) Inflamm-ageing means low grade of inflammation. The value of CRP 23.1 suggests acute inflammation (also because albumin has high values, while in chronic inflammation its values decrease). Therefore the Ly averages do not have to take this subject into account.

Thank you for this comment. In a review of literature it was found an article (below) with CRP variation from 0.1 to 19.8 (Heumann Z, Youssim I, Kizony R, Friedlander Y, Shochat T, Weiss R, Hochner H, Agmon M. The Relationships of Fibrinogen and C-Reactive Protein With Gait Performance: A 20-Year Longitudinal Study. Front Aging Neurosci 2022;14:761948. doi: 10.3389/fnagi.2022.761948). There is also an article from your group showing CRPs <5g/dL and >5g/dL (Cancemi P, Aiello A, Accardi G, Caldarella R, Candore G, Caruso C, Ciaccio M, Cristaldi L, Di Gaudio F, Siino V, Vasto S. The Role of Matrix Metalloproteinases (MMP-2 and MMP-9) in Ageing and Longevity: Focus on Sicilian Long-Living Individuals (LLIs). Mediators Inflamm 2020;2020:8635158. doi: 10.1155/2020/8635158) that will be used to discuss how ageing impacts CRP levels. Considering the already small number of donors, data were maintained and statistics (mean + SD) with and without 23.1 mg/dL are now shown.

This will be the new version (discussion) about CRP Carlsson’s study [22] found that CRP value was 2.6 with a coefficient variation of 1.4% whereas in our study, it was observed higher values of CRP in 5 out of 6 individuals. In addition, it was shown by Cancemi et al. in an evaluation of individuals from 40 years to older than 95 years (long-living) that CRP increases in an age-dependent manner. Increased CRP levels has been associated with low grade of chronic inflammation (inflammaging) and our findings show that the studied population has changes in functional parameters which are likely associated with an inflammatory profile. [24] However, an individual presented CPR 23.1 mg/dL suggesting acute inflammation instead, but as all donors were not hospitalized or living on homecares, this sample was considered as part of the study. Another study evaluating gait speed found CRPs varying from 0.1 to 19.8mg/dL (Front Aging Neurosci 2022;14:761948.). Our study has an important limitation that is the lack of data on donors such as the use of continuous medicaments or sarcopenia, hypertension, cognition, among others, and thus it was not possible to correlate CRP with age-related conditions.

Table 1. Updated

Other suggestions to improve the manuscript The authors write that their findings suggest that ACE1 could play a role in several processes linked to aging including the generation and activation of autoimmune cells, due to the experimental evidence that inhibitors of ACE suppress the autoimmune process in a number of autoimmune diseases such as EAE, arthritis, autoimmune myocarditis. [49] They do not appear to have these findings in their paper. So, it needs to change the sentence.

Sentence changed to: According to experimental evidence, ACE inhibitors suppress the autoimmune process in a number of autoimmune diseases such as EAE, arthritis, autoimmune myocarditis. [49] Extrapolating these findings to our results, it is possible to suggest that ACE1 play a role in several processes linked to aging including the generation and activation of autoimmune cells.

Rviewer: Calogero Caruso

Decision changed:

Verified manuscript: The content is scientifically sound, only minor amendments (if any) are suggested.

Discussion, revision and decision

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Decision

Verified manuscript — The content is scientifically sound, only minor amendments (if any) are suggested.

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Revision

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Reviewer: Dacre Knight

(1) Included the World Health Organization (WHO) and the National Institute for Health and CARE Excellence (NICE) definitions of PASC. See: Lines 51; 332-339

(2) The PECO criteria is listed (and not just implied) in the body of the manuscript. See: Lines 207-237.

Decision changed — Verified manuscript: The content is scientifically sound.

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Reviewer: Yin Qianlan

(3) The purpose of the study was revised for clarity. See: Lines 114-171

Reviewer did not respond. Therefore, a third reviewer (Daniel Griffin) was asked to review the manuscript. They gave the decision, verified manuscript.

Discussion, revision and decision

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

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Responses to the reviewers

Reviewer Charlotte Martial

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

1-The work does not cite relevant and sufficient literature. For instance, the authors do not cite a recent publication which aimed to induce OBE using hypnosis (Martial et al., Scientific Reports, 2019).

Reply: We re-read the procedure of this paper we were aware of, but we didn’t find any reference related to an OBE induced by hypnotic suggestions. Here follows an excerpt of the procedure as reported in the cited paper: “The hypnotic instruction included a 5-min induction procedure with eye fixation (ultimately closing the eyes) and progressive muscle relaxation. Permissive and indirect suggestions were used to develop and deepen the hypnotic state. The participant was then invited to re-experience their NDE memory (20 min) and their pleasant autobiographical memory (20 min) in a counterbalanced order, ..”

Thanks for the reply. I do not agree, though. As described in the paper, Martial and colleagues (2019) focused on two prototypical features of the NDE during the hypnosis session, that is, feeling of peacefulness and out-of-body experience. Consequently, I think it is highly relevant for the present paper.

Reply: we accepted this suggestion and added a paragraph about this application of hypnosis.

2-Another example: The authors could have at least briefly discussed Parnia’s AWARE prospective study reporting OBE in NDE experiencers (Parnia et al., 2014).

Reply: Thank you for this suggestion, now added in the introduction

3-Besides, they sometimes cite some inappropriate references; I would like to invite the authors to cite rigorous and serious articles in the field. For example, they used Jourdan (2011) reference to draw a parallel with NDEs, however, Jourdan (2011) is not a reference article in the field…

Reply: Jourdan’s (2011) paper “Near Death Experiences and the 5th Dimensional Spatio-Temporal Perspective. Journal of Cosmology, 14, 4743-4762 deals with NDE. It is not clear why it cannot be considered a reference article in the field.

Dr Jourdan and most of his articles are scientifically controversial. They are many other references more rigorous that you could cite in order to be scientifically stronger.

Reply: we have a different opinion about Jourdan’s scientific findings. As written in the paper, we referred to Jourdan’s paper only as a source to draw the questions to be used with the participants when in the OBE status.

4- Importantly, it is worth mentioning that, so far, no rigorous empirical scientific study has shown evidence of veridical perceptions during OBE; according to me, it is not clear in the manuscript.

Reply: On pag. 2 we added “and verify by a third-person perspective what they declare to perceive in this particular state of consciousness”.

I thank the authors. However, this is still not clearer in the manuscript. I suggest that the authors clearly state that so far, no rigorous empirical scientific study has shown evidence of veridical perceptions during OBE. This is highly important for a manuscript aiming to study OBE in laboratory settings.

Reply: added this suggestion at the end of the Introduction

5-Some limitations of the study are not mentioned in the discussion section

Reply: On pag. 14 there is a whole paragraph “Study limitations”.

Well, let me clarify: some of the limitations of the study are not discussed in the manuscript.

Reply: the “Study limitations” is added on pag. 15

6- Some details of the study are missing. Notably, the description of the Table 6 is incomplete: what do the bold numbers mean

Reply: we revised the presentation of the data in Table 6 and clarified why some data were presented in bold.

7- Another example: how did they recruit the participants –who are co-authors of the paper? Are they researchers?

Reply: We have revised the section related to the participants’ selection

8- page13: what do they mean by “general consensus”? This is not clear to me

Reply: We revised the text in “The answers of all five participants to the eleven questions presented in Table 4 and 5, were quite similar in content”

9-The conclusions they draw in the discussion are not based on the present findings; they extrapolate

Reply: Given in the paper there is not a “Conclusions” paragraph, to what “conclusions” do you refer to?

I mean the interpretation of their results … As an example, based on what do you conclude that “…in this state of consciousness they can act without the limitations that the physical body imposes on motion and perception especially with regard to vision”. It is not clear that you here mean that their subjective experience is related to their imaginary. Please reformulate.

Reply: This is not a conclusion, it is a simple summary of participants’ phenomenological reports “….from all of the participants’ reports, it appears that in this state of consciousness they can act ….”

what do you mean by “a three-dimensional universe”?

Reply: we corrected this sentence that now is “Time seems to be absent or similar to a sequence of video-clips, suggesting that they are living in a four-dimensional universe, very similar to that described as the “Block Universe” which represents space-time as a fixed whole in which past and future events already exist in the space-time continuum, in accordance with the special theory of relativity”.

Based on which data can you conclude that what they experienced is “very similar to or the same as that described in NDEs”.

Reply: we deleted this sentence

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Reviewer Aminata Bicego

Decision — Verified with reservations: The content is scientifically sound, but has shortcomings that could be improved by further studies and/or minor revisions.

Thank you for your accurate review and suggestions.

Abstract :

L13 : what do the authors mean by “hypnotic induction”, is it hypnotic experience ? During a hypnosis session, the therapist starts by an induction and then makes some suggestions according to the goal that has been decided. It is not clear to what the “hypnosis induction” refers to.

Reply : We changed « hypnotic induction » as « hypnotic experience », as suggested.

L16 : “under hypnosis” should be changed as it suggests that the individual who is in hypnosis is passive. I would suggest “in hypnosis”.

Reply : changed as suggested

Introduction: L 54-56 : there has been some recent literature on hypnosis and OBEs or NDEs. It would be interesting to add newer literature on that topic.

Reply : we searched with google scholar if there were new studies about hypnosis and OBEs, but we didn’t find any. The were two studies related to hypnosis and NDEs, but these experiences are not related to our study (see also our response to reviewer Charlotte Martial).

L75 : if the study’s aim is to confirm Tressoldi and Del Prete (2007), then this study should be explicitly explained in detail. That way the reader understands better the present study.

Reply : We added some further details related to Tressoldi and Del Prete (2007) study, as suggested.

L76 : could the authors specify the suggestion used.

Reply : the suggestions used in the study are presented in the Supplementary Materials

L78 : the induction is not the only and principal part of the hypnotic sessions that impacts an individual perceptions but rather the suggestion that is used. This paragraph could be clearer in terms of methodology

Reply : the suggestion to induce an OBE in Tressoldi and Del Prete (2007) study, are too long to be added in the text, but are very similar to that used in the present study.

L79 to L95 : this should be in methods.

Reply : moved in the Methods under the paragraph « Study aims »

Materials and Methods : L110 : can the authors define and detail “clinical level of medical or psychiatric disease”.

Reply : we added « compatible with the clinical criteria defined in the main international clinical guidelines, such as DSM V or ICD-10,

L111 : took, should be written “take”. Throughout the manuscript there are language mistakes that have to be checked.

Reply : we checked the syntax as suggested.

L111 : can the authors be more specific on the “personal experience with hypnosis” ? What do you mean by experience ?

Reply : In Table 1, we changed the experience value adding the number of previous hypnotic sessions.

Procedure : in general the procedure is not very clear. Some results appear in the section when they should be in the result section

Reply : we moved the sentence « In total, twenty-eight sessions were necessary to complete the study. Eighteen sessions were carried out by phone with the participants at home” in the results section.

L125 : Can the authors explain why some participants had one or two sessions ?

Reply : In the Procedure we wrote "We placed the images in two different rooms as long as it was possible to require the identification of two different images in a single session. In only two cases, the participants had sufficient time and concentration to identify two images in a single session reducing their overall sessions to four instead of six"

L127 : Can the supplementary Material be numbered. L129 to 132 : Have all participants been seen in real life? Were all of them called? If not, was it always the same people in person or on the phone? This part should be more specific.

Reply : In the Results section we clarified that « In total, twenty-eight sessions were necessary to complete the study. Eighteen sessions were carried out by phone with the participants at home »

L130 : hypnotized should not be used. Same comment as comment L16.

Reply : corrected as suggested

L140 : was the order of the picture position randomized ? If so it should be mentioned here.

Reply : we wrote « …the six target images and randomized their order of presentation, the room where each one was placed and their orientation, either facing up or upside down »

L164: Who did the authors know that the participants where in an OBE state ? What were the criteria ? Especially on the phone ?

Reply: On pag. 5, we clarified that the hypnotist referred to the participants phenomenological account in response to the suggestions described in the Supplementary Material.

L174 : did the authors create the questions ? Do they come from a questionnaire ? This should be specified.

Reply : we clarified « devised by the authors of this study »

L188 : this should be in results Reply : moved in the Data analysis paragraph as suggested.

L191 : “... and give suggestion...” : It is confusing to use that word, comments, might be more appropriate.

Reply : corrected as suggested.

L198: what are the eleven questions ?

Reply : we corrected and added « all participants were emailed the same six questions related to their perceptual and cognitive experience when in OBE..

L208 : all the questionnaires used should also be described in material. The reader has no information on the minimal phenomenal selfhood, nor has he information about the “characteristics of spatial and temporal perception reported in NDEs”.

Reply: all questions were original and devised by the authors. None questionnaire of other authors was used.

L221 : how did the 3 decoys were selected ?

Reply : we added « low arousal pictures »

L225 : I only see 2 authors, not three.

Reply : we corrected as « co-author LP and another independent judge affiliate with the EcvanLab… »

L234 : a section with the statistical analyses should be written before the results.

Reply : we added a section « Statistical method »

L241 : 52.4 % is not metionned in the table, this is confusing.

Reply : Sorry, we missed the overall results in the last line, now added

L245 : this should be in the statistical analyses part, it is not a result per se.

L260 : the table 3 should be more specific: define ES + formula, CI, BF, H1, H0. A table should be able to be read by itself.

Reply : In the Table 3 presentation and title, we clarified all the statistics

L277 : This part merits some clarifications : Did the approval from the Ethical committee approved this part ? If so, did the participants signed an informed consent ?

Reply: The Ethical approval included the informed consent to be signed by participants

What should they refer to when they answered the questions ? Did the ones that had an OBE had to refere to that episode ? And those who did not life an OBE ? What was the experience of reference ?

Reply: Participants induced in OBE, should refer to their phenomenological experience in this state included the period when they were requested to identify the targets.

It there is no reference for the controls without an OBE experience then is seems logical that they do not answer like the others.

Reply : controls participants were selected among those who have only read about OBEs

L304 : the % is not correct, is should be 46.6%

Reply : Corrected

L338 : With what material was the comparison made ? With the material from Jourdan (2011) or the participants that were contacted after the study ? This part should be clearer If the comparison is made with Jourdan, then a table with the similarity and differences could be added.

Reply : We changed the title of Table 6 in order to clarify its data.

L382 : “but his aim was not to confirm his knowledge, but to compare it with the participants’ experience” : this was not mentionned before. It is hard to understand as we have no information in the hypnotist knowledge or the comparison that is mentionned. Could the authors clarify ?

Reply : The hypnotist knew very well the OBE phenomenology (now added in his description on page 4. However, the questions for participants were formulated in order not to confirm the hypnotist’s previous knowledge.

L387 : Another limitation it the response expectancy during hypnosis. There is a large literature on the subject this should be discused in the limites. More so because all the subject had good knowledge about OBEs

Reply : In the paragraph « Reliability of participants’ reports », we discuss precisely these issues.

L412 : Is it acceptable to disclose the participants identity ?

Reply : we deleted their names

Page 5 : The authors mention in a foot note that some other informatio will be avaiable in a future publication but the publication has since been published. Furthermore, that publication is cited in the bibliography this is confusing.

Reply : we corrected this discrepancy

Table S1 : it is hard for the reader to understand to what the table refers to. GAPED has to be explained.

Reply ; The GAPED acronym is described in the Materials paragraph on pag. 6

Discussion, revision and decision

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Author response

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To: Adam Marcus, co-founder Retraction Watch & Alison Abritis, PhD, researcher at Retraction Watch

Major Problems: I found serious deficits in both for this article, and thus I have serious concerns as to the usefulness of this article. Therefore, I have not proceeded in a line-by-line, as I consider the overall problems to be grave enough to require attention and revision before getting to lesser items of clarity.

I would like to point out that the authors show a marvelous attention to their work, and they have much to contribute to the field of retraction studies, and I do honestly look forward to their future work. However, in order for the field to move ahead with accuracy and validity, we must no longer just rely on superficial number crunching, and must start including the complexities of publishing in our analyses, as difficult and labor-intensive as it might be.

We do not consider that our article presents serious problems nor that it would be useless.

It is possible that a different view on the subject, some tendency to forbearance (understandable) for the difficult life of the publishing industry, along with some difficulties in understanding the ideas presented in the article, may have led to a series of points of view that we would like to comment on below.

We would first like to thank the reviewers for their comments, some of which will allow us to improve and nuance, using objective elements, the analysis of this bumpy field represented by the ecosystem of retracted publications. Because we have based our study on data from freely accessible sources of information, we will not insist too much on commenting on this issue.

The authors stated that they used the search protocol (and therefore presumably the same dataset) as described in Toma & Padureanu, 2021, and do not indicate any process to compensate for its weaknesses. In the referenced study, the authors (same as for this article) utilized a PubMed search using only “Retracted Publication” in Publication Type. This search method is immediately insufficient, as some retracted articles are not bannered or indexed as retracted in PubMed. This issue is well-understood among scholars who search databases for retractions, and by now one would expect that these searches would strive to be more comprehensive.

A better method, if one insists on restricting the search to PubMed, would have been to use Publication Type to search for “retracted publication,” and then to search for “retraction of publication,” and to compare the output to eliminate duplications. There are even more comprehensive ways to search PubMed, especially since some articles are retitled as “Withdrawn” – Elsevier, for example, uses the term instead of “Retracted” for papers removed within a year of their publication date – but do not come in searches for either publication type. Even better would have been to use databases with more comprehensive indexing of retractions.

In an ideal world, if any effort were to be made, it would be aimed at better indexing and managing existing databases, not at generating query strategies to make up for their shortcomings.

Thank you very much for the suggestions on the search strategy. We do not consider that the use of "Retracted Publication [PT]" should be compensated in any way but, if it should be compensated, we wouldn't want to add "Retraction of publication". We consider that using a search protocol more specific to systematic reviews is not very useful in our case: data are added/updated continuously (sometimes late), incorrect indexing can be corrected, the number of retracted articles increases from month to month; the same strategy can give different results at different times regardless of its complexity. Putting extra effort into detecting problematic articles without knowing the benefit but expecting it only highlights issues that can be improved at the publisher/editor(content delivery) and database level(indexing).

The dataset analyzed is a snapshot of a particular time interval and nothing more. Even during the analysis we found, in the case of one publisher, the addition of details to the initially incomplete retraction notes. Hence the need for follow-up studies. Therefore in the case of retractions, unlike the reviewer, we prefer an approach based on simple and easily reproducible strategies, widely accessible sources of information, and several steps. The first step in this strategy is the "number crunching" stage which includes this article.

1. The authors are using the time from publication to retraction based on the notice dates and using them to indicate efficacy of oversight by publishers. However, this approach is seriously problematic. It takes no notice of when the publisher was first informed that the article was potentially compromised. Publishers who respond rapidly to information that affects years/decades old publications will inevitably show worse scores than those who are advised upon an article’s faults immediately upon its publication, but who drag their heels a few months in dealing with the problem.

Indeed, the article uses the time between publication and retraction(exposure time – ET) as one of the SDTP score components for assessing editorial/publisher performance. Data on when a publisher or editor has been informed of problems with an article, apart from being relatively rare, is not a substitute for a retraction note. Moreover, the use of such information may induce a risk of bias.

We mention in the article the need to use reporting standards for retraction notes, and one element that might be useful is, indeed, the date on which the publisher or editor was informed of problems with an article. Unfortunately, as the author of this review knows very well, information precedes investigation; the retraction note contains (or should contain) much more data than the initial information about the quality problems of an article.

Our article aims to suggest a score for measuring publication performance in the context of retracted articles that would also allow an assessment of the dynamics of the activity of correcting the scientific record and, more importantly, how publishers engage in post-publication quality control. ET is only one component of this score.

It is quite clear from the data presented in the article that a publisher/journal that emphasizes systematic back-checking will have an increasingly longer average lifespan of retracted articles, logically higher than one that does not do this type of checking. We don't see precisely where the reviewer thinks there is a problem: once the checking is done, the ET will decrease, and a publisher that takes concrete steps to correct the literature will ultimately have a better reputation. This does not mean that a higher ET is laudable, it suggests that there is a post-publication quality control but also that the peer review process has let problematic articles through and that the control of these articles has been carried out late. This is an argument for more active involvement of publishers (as potential generators of editorial policies) in post-publication control.

Second, there is little consistency in dealing with retractions between publishers, within the same publishers or even within the same journal. Under the same publisher, one journal editor may be highly responsive during their term, while the next editor may not be. Most problems with articles quite often are first addressed by contacting the authors and/or journal editors, and publishers – especially those with hundreds of journals – may not have any idea of the ensuing problem for weeks or months, if at all. Therefore, the larger publishers would be far more likely to show worse scores than publishers with few journals to manage oversight.

It is exactly this inconsistency that we highlight in the article. Differing policies, attitudes, and responsiveness does not mean that a publisher cannot/should not ask questions about the effectiveness of internal processes and resources used for post-publication quality control or the implementation of uniform measures across journals in its portfolio.

Third, the dates on retraction notices are not always representative of when an article was watermarked or otherwise indicated as retracted. Elsevier journals often overwrite the html page of the original article with the retraction notice, leaving the original article’s date of publication alone. A separate retraction notice may not be published until days, weeks or even years after the article has been retracted. Springer and Sage have done this as well, as have other publishers – though not to the same extent (yet).

Historically, The Journal of Biological Chemistry would publish a retraction notice and link it immediately to the original article, but a check of the article’s PDF would show it having been retracted days to weeks earlier. They have recently been acquired by Elsevier, so it is unknown how this trend will play out. And keep in mind, in some ways this is in itself not a bad thing – as it gives the user quicker notice that an article is unsuitable for citation, even while the notice itself is still undergoing revisions. It just makes tracking the time of publication to retraction especially difficult.

We used the same date for all articles in our study (the one listed in PubMed), thus ensuring a uniform criterion for all publishers. If this date was not in PubMed we used the date from the retraction notes on the journal website but this was for a small number of articles. How different publishers handle retraction processes or the delay with which these are published is primarily related to internal editorial procedures, and these delays are reflected in the ET. In our experience, most articles retracted by Elsevier are available online, supplemented, and not replaced by retraction notes, which we think is an excellent policy.

1. As best as can be determined, the authors are taking the notices at face value, and that has been repeatedly shown to be flawed. Many notices are written as a cooperative effort between the authors and journal, regardless of who initiated the retraction and under the looming specter of potential litigation.

Shown to be flawed by who? Indeed, in our study, we refer to the retraction notes published by the journals. The fact that they are incomplete or formulated under the threat of litigation only supports our view that publishers and editors need to make a more significant effort to correct the biomedical literature, including avoiding litigation when the retraction note clearly describes the reasons for retraction. The way the retraction note is worded should be an editorial prerogative and should primarily aim at correcting scientific literature, not at appeasing egos, careers, or financial interests.

Trying to establish who initiated a retraction process strictly by analyzing the notice language is destined to produce faulty conclusions. Looking just at PubPeer comments, questions about the data quality may be raised days/month/years before a retraction, with indications of having contacted the journal or publisher. And yet, an ensuing notice may be that the authors requested the retraction because of concerns about the data/image – where the backstory clearly shows that impetus for the retraction was prompted by a journal’s investigation of outside complaints. As an example, the recent glut of retractions of papers coming from paper mills often suggest the authors are requesting the retraction. This interpretation would be false, however, as those familiar with the backstory are aware that the driving force for many of these retractions were independent investigators contacting the journals/publishers for retraction of these manuscripts.

Once again, the author of this review does not seem to fully understand our study, apparently favouring information published on third-party websites over that the journals officially assumed. The retraction notes represent the material available to a researcher doing documentation on a particular topic. The clarity and information contained in the note is the editor's or publisher’s responsibility, reflecting their performance and concern for the integrity of the science. Interpretation of a retraction note/analyzing an article occurs in this context. Not everyone has time for further investigation or to search third-party sites for information that is, with a notable exception, the result of a selection bias.

Assigning the reason for retraction from only the text of the notice will absolutely skew results. As already stated, in many cases, journal editors and authors work together to produce the language. Thus, the notice may convey an innocuous but unquestionable cause (e.g., results not reproducible) because the fundamental reason (e.g., data/image was fabricated or falsified) is too difficult to prove to a reasonable degree. Even the use of the word “plagiarism” is triggering for authors’ reputations – and notices have been crafted to avoid any suggestion of such, with euphemisms that steer well clear of the “p” word. Furthermore, it has been well-documented that some retractions required by institutional findings of misconduct have used language in the notice indicating simple error or other innocuous reasons as the definitive cause.

We understand your point of view and the situations presented may be accurate. However, from our point of view, the only valid reference remains the retraction note published on the journal's website. The existence of wording difficulties and various other problems that may arise are more likely to do with a tendency of the reviewer to make excuses for journals reluctant to indicate precisely what the reasons for retracting the article are. There are plenty of retraction notes in which the images with problems (including whether they were plagiarized, reused, manipulated, fabricated, etc.) are indicated with great precision, there are equally plenty of notes in which the word plagiarism is used without hesitation, indicating the sources, how they were informed, what was plagiarized. No matter how many hesitant publishers/editors there are, it should not be forgotten that there are many journals/publishers who take their role seriously, acknowledge and learn from their mistakes, thus providing a real service to the scientific community.

The authors also discuss changes in the quality of notices increasing or decreasing in publishers – but without knowing the backstory. Having more words in a notice or giving one or two specific causes cannot in itself be an indicator of the quality (i.e., accuracy) of said notice.

"Knowing the backstory" is not part of our objectives, and neither is assessing the quality of the retraction notes. This is also very difficult to do due to the lack of an accepted standard format. We are trying to propose a score composed of several parameters resulting from existing (or non-existing) data in the retraction notes so that we can have a picture of retractions at publisher level. Knowing the backstory is not relevant, reading and interpreting the official retraction note is relevant.

1. The authors tend to infer that the lack of a retraction in a journal implies a degree of superiority over journals with retractions. Although they qualify it a bit ( “Are over 90% of journals without a retracted article perfect? It is a question that is quite difficult to answer at this time, but we believe that the opinion that, in reality, there are many more articles that should be retracted (Oransky et al. 2021) is justified and covered by the actual figures.”), the inference is naive. First, they have not looked at the number of corrections within these journals. Even ignoring that these corrections may be disproportionate within different journals and require responsive editorial staff, some journals have gone through what can only be called great contortions to issue corrections rather than retractions.

We believe that this is a case of reviewer confusion generated either by the insufficiently precise wording of the text or a lack of understanding of our study objectives. We are trying to point out that more than 90% of the journals in the NLM catalogue-PubMed subset have not retracted a single article. We are not trying to say that journals without retracted articles are superior to the others. As explained in the article, we referred to retraction notes, not corrections.

Second, the lack of retractions in a journal speaks nothing to the quality of the articles therein. Predatory journals generally avoid issuing retractions, even when presented with outright proof of data fabrication or plagiarism. Meanwhile, high-quality journals are likely to have more, and possibly more astute, readers, who could be more adept at spotting errors that require retraction.

Of course, the quality level of articles in a journal is not determined by the number of articles removed.

Third, smaller publishers/journals may not have the fiscal resources to deal with the issues that come with a retraction. As an example, even though there was an institutional investigation finding data fabrication, at least one journal declined to issue a retraction for an article by Joachim Boldt (who has more than 160 retractions for misconduct) after his attorneys made threats of litigation.

Threats of lawsuits are instead a failure of a publisher/journal to adapt to the realities of the publishing business or to the risk of misconduct. This is something that needs to change.

Simply put, the presence or lack of a retraction in a journal is no longer a reasonable speculation about the quality of the manuscripts or the efficiency of the editorial process.

We have not attempted to suggest this, we have only analyzed the retracted articles and their associated retraction notes. On the other hand, the way a journal/publisher handles the retraction of problematic articles still reflects, to some extent, the quality/performance of the editorial processes.

1. I am concerned that the authors appear to have made significant errors in their analysis of publishers. For example, they claim that neither PLOS nor Elsevier retracted papers in 2020 for problematic images. That assertion is demonstrably false.

This is wrong. In our dataset, there are eleven PLOS articles related to human health with the publication year 2019 and 2020. None of these have images as retraction reasons.

Regarding the 21 Elsevier articles published in 2020, there is nothing in the retraction notes to indicate that the article was retracted because of the images. In 2 retraction notes there is mention of the comments made by Dr. Bik (The Tadpole Paper Mill - Science Integrity Digest) but the text of these (retraction notes) stops at the authors' inability to provide the raw data underlying the article.

Our study is based only on the content of the retraction notes published and assumed by the journal, not on opinions/comments appearing on other sites, which, for unknown/unmentioned reasons, are not officially assumed in the retraction note. Therefore, we consider the statement in the review to be questionable at best, as the use of material other than the retraction notes has severe implications for the internal and external validity of the study and the suggestion to use such methods is, in our opinion, wrong. We would also like to draw attention to the fact that many retraction notes are explicitely mentioning the request to provide raw images and the authors' inability to provide them.

Anyway, as far as images are concerned, our article suggested that there are publishers which seem to adopt image analysis technologies faster than others. The numbers are not really relevant in this case but the trend is: it describes the publishing activity complexity better than the numbers.

Reviewer response

We appreciate the authors’ zeal in standing by their work.

In regard to the deficits in the search process, the author states, “We do not consider that the use of ‘Retracted Publication [PT]’ should be compensated in any way but, if it should be compensated, we wouldn't want to add ‘Retraction of publication’”

There is a lack of appreciation for the complexities of indexing retracted materials in an indexing site such as PubMed. To have a comprehensive search, one should not be choosing to use either “Retracted Publication [PT]” OR “Retraction of Publication [PT].” One would use both, and then filter out the duplicates, because some retractions are indexed by retraction notices, some only have “Retracted” added to the indexed title and the publication type changed to “Retracted Publication.” Use of only one or the other guarantees that the search is far less comprehensive than it should be.

The authors state, “In an ideal world, if any effort were to be made, it would be aimed at better indexing and managing existing databases, not at generating query strategies to make up for their shortcomings.”

There is at least one database (http://retractiondatabase.org) that has a far more comprehensive indexing of retractions and is publicly available for use.

In Item 3, where it is pointed out that retraction notices themselves are inaccurate and cannot be taken at face value as to the reason behind the retraction, the authors responded, “Shown to be flawed by who?” — By an article cited in the manuscript:

Fang, Ferric C.; Steen, R. Grant; Casadevall, Arturo (2012): Misconduct accounts for the majority of retracted scientific publications. In Proceedings of the National Academy of Sciences of the United States of America 109 (42), pp. 17028–17033. DOI: 10.1073/pnas.1212247109.

“To understand the reasons for retraction, we consulted reports from the Office of Research Integrity and other published resources (7, 8), in addition to the retraction announcements in scientific journals. Use of these additional sources of information resulted in the reclassification of 118 of 742 (15.9%) retractions in an earlier study (4) from error to fraud.” Followed by “These factors have contributed to the systematic underestimation of the role of misconduct and the overestimation of the role of error in retractions (3, 4), and speak to the need for uniform standards regarding retraction notices (5).”

The authors then choose to state that it is the “editorial prerogative” – and that when notices “are incomplete or formulated under the threat of litigation [it] only supports our view that publishers and editors need to make a more significant effort to correct the biomedical literature, including avoiding litigation when the retraction note clearly describes the reasons for retraction.”

Following our attempt to explain why understanding the real reason behind a retraction is important to study the publication of notices, the authors respond: “Once again, the author of this review does not seem to fully understand our study, apparently favouring information published on third-party websites over that the journals officially assumed.”

First, yes, we do understand the study. We read a lot of these. Second, the “third-party websites” we prefer include the Office of Research Integrity and the Retraction Watch blog, where background investigations into the causes of retraction notices are described. If the authors are challenging the reference to PubPeer, keep in mind that journals initiate investigations based on comments on that website, and have taken to citing the website in their notices.

Had the authors not chosen to categorize the reasons for retraction, their reasoning may have had more support – but they did, and in doing so, by just using the notice with no further review, their findings address only the notice itself, with no context.

We recommend that the manuscript be substantially revised with strong attention to the comments we made in our original review.

Discussion, revision and decision

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Decision - Verified with reservations

Charles A. Schumpert: Verified manuscript

Max Shokhirev: Verified with reservations

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Author response and revisions

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Author response to reviewer Max Shokhirev

Dear Dr Shokhirev,

Thank you so very much for having reviewed my paper; your comments have been very helpful for me to improve it. Please find attached two identical copies of my revision of the paper (changes are highlighted in one of the copies) and a supplementary file that relates to one of your comments. Please see below my response to your comments when applicable.

Does the work cite relevant and sufficient literature? Some, but seems to be very limited in terms of biological literature.

I recognize and acknowledge this issue. It will be addressed in some of the specific responses below.

Are the conclusions adequately supported by the results? No

I understand this question and its answer as the theory presented in the paper (the conclusions) not providing new, proof-of-principle evidence (the results). That is correct, the theory is based only on existing evidence (most importantly, the already described age-dependent "transcriptional noise"), it is consistent with it and, importantly, it provides (in the new revision) three additional, experimentally testable predictions.

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 would argue the theory is not based on a superficial understanding of the biology of senescence as it is currently established—in fact it is not based on the current biology of senescence at all. The theory is actually based on my previous theoretical work where I show (supported by real data analysis) how the constraints on transcription start site-adjacent histone crosstalk that are explicitly uncorrelated with transcriptional levels associate strongly with cell differentiation states—even more strongly than those constraints correlated with transcriptional levels (an association which is expected and already described in previous work). This in turn suggests the existence of an additional, higher-order type of biologically meaningful information conveyed by histone crosstalk, with this information is by definition uncorrelated with the information for precise epigenetic control of transcriptional levels. I called this information "hologenic" because it associates to cell differentiation trajectories necessary for the development of the multicellular individual as as a whole while being explicitly uncorrelated with the transcriptional levels. My previous work showed that the capacity for hologenic information in histone crosstalk grows at the expense of that for epigenetic information (this is necessary for the development of the multicellular individual). I emphasize that the theory proposed here relies on only two assumptions: (i) the overall histone crosstalk remains statistically constant in magnitude throughout adulthood and (ii) the hologenic component of the overall histone crosstalk increases at the expense of the epigenetic component throughout adulthood (with the exceptions depicted in Fig. 1b). These assumptions are of course not to be taken for granted, so in the new revision of the paper they are presented as predictions that can falsify the theory. I will further elaborate on these assumptions in my specific responses below.

All the above being said, the version of the paper you reviewed apparently gives the wrong impression that the theory is a comprehensive description of the senescence process with all its complexity. It is not. It is not surprising then that the theory appears to oversimplify the explanatory power of mechanisms known to be part of the senescence process when in reality they are claimed—this is a big theoretical claim I am making—to be the consequence of the primary cause of senescence. It is only this primary cause what the proposed theory is all about. For these reasons and thanks to your observation, I stated this distinction explicitly in the new subsection 1.2 "Scope" and also modified the title of the paper accordingly.

I recognize and acknowledge that the paper does not present new data or experimental results in direct support of the theory—something that arguably makes it less compelling to be considered let alone to be tested experimentally. But I do wish to point out that a scientific theory, must "only" (i) effectively explain the phenomena it is aimed to explain (in this case, the primary cause of senescence), (ii) be consistent with existing observations/results (most importantly, with the well described age-dependent "transcriptional noise" in this case), and (iii) provide non-trivial, experimentally testable predictions that can falsify it. I have tried to make up for the lack of preliminary supporting evidence by adding three new straightforward, experimentally testable predictions that can falsify it. Importantly, two of said predictions (C and D in subsection 2.6) relate to precisely how senescence can be slowed down, even stopped, or accelerated—and with associated effects in terms of resistance/propensity to carcinogenesis—in any non-human species, because the direct testing requires genome editing. In this context, I have no problem granting that the prior probability of predictions C and D being verified experimentally is exceedingly small. On the other hand, the experimental verification of predictions C and D would arguably be a game-changing result in terms of the fundamental understanding of the senescence process, however unlikely this scenario is a priori. In this context, I submit to you that the extraordinary nature of predictions C and D in both theoretical and practical terms (I reiterate, this is not to say predictions C and D will be verified) more than makes up for the lack of preliminary evidence for the theory. The prospect of slowing down or even stopping the senescence process may catch the attention of at least some research groups with genome-editing capabilities, given that the gene edits described in predictions C and D are very specific. (You can find more about predictions C and D in my response to your comments related to the subsection 2.4 of the paper you reviewed).

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).

Thank you for pointing this out. I have included the suggested review articles as references and, most importantly, I tried now to clarify the scope of the theory in a new subsection (1.2 Scope). The scope of the theory is in a sense, very limited: it is indented to explain only the beginning of the causal chain of age-related changes we identify as aging at the multicellular-individual level. In other words, it is about what fundamentally triggers the process. On the other hand, such a scope (i.e., describing the first cause) is quite ambitious in the sense that it should allow, at least in principle, for the manipulation of the process (either slowing it down or accelerating it). I will come back to this point later in my response.

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).

I am not a native English speaker, and one the first things I did when addressing this problem was to study the associated terminology in the literature. Unfortunately, this terminology is not particularly monolithic. In some articles, the age-dependent, progressive dysfunction undergone by multicellular individuals once they reach their mature form is referred to as "senescence" (PMID 1677205, 6776406, 12940353, 22884974, among others), "biological aging" (PMID 31833194, 33982659, 34700008, among others), or even simply as "aging" (PMID 24862019, 34990845, 31173843, among others). In this context, I decided to stick to "senescence" mainly because (i) it is only one word and (ii) unlike "aging", "senescence" directly and unambiguously implies time-dependent dysfunction or decay. At any rate, to distinguish the term from cellular senescence/cellular aging I created a Glossary in the paper where these terms and others are clarified to avoid confusion.

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.

I am glad we agree that senescence is poorly understood (especially in comparison to cellular senescence). Unfortunately, some colleagues in the community interpret this as saying the research been done on the topic is worthless—it is not—when it is really pointing out the phenomenon largely lacks falsifiable theories, let alone an already tested falsifiable theory (with experiments failing to falsify it).

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.

What you point out here is very important, thank you. In a remarkable piece of research, Kumar and colleagues showed that core nucleosomal histone post-translational modification (hPTM) profiles are able to predict transcript abundance levels with very high accuracy (R~0.9, ref. 33). The constraints on hPTMs underpinning this predictive power (in turn underpinned by DNA-histone octamer interactions)—as well as those constraints on hPTMs that are explicitly uncorrelated to transcriptional levels—are central to the theory proposed. As stated in the new section 1.2, the complex cascade of changes/interactions characterizing senescence escapes the scope of the theory. In this context, most of the types of regulation you mention are under this theory not actually regulation in a "teleological" (the quotes are meant to avoid alienating the reader) sense but rather types of propagation/amplification of truly regulated/dysregulated changes. One of my goals when developing this theory was to try shift attention from "molecule A-collides with molecule-B, which collides to..." into higher-order constraints and trying to explain phenomena such as the well-known, age-dependent "transcriptional noise", which under the theory presented should be understood as senescence itself. Furthermore, I maintain the relative slow progress we have made in understanding phenomena such as cancer and senescence (in spite of the abundance of high-throughput data) comes from

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 my previous work, "hologenic" information content is defined as being uncorrelated with (i.e., orthogonal to) changes in transcriptional levels, in the same way "epigenetic" information has been defined (traditionally and for good reason) as being uncorrelated with changes in the DNA reason. Hologenic information content emerges when proliferation-generated extracellular gradients of secreted molecules start to being used to perform regulatory work (after being transduced) on the histone crosstalk of each cell's nucleus.

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.

This comment of yours (referring to complex yet specific signaling pathways and interactions) clearly shows I did a poor job (if not utterly failed) in conveying that the epigenetic and hologenic components must be understood in chromatin-wide terms. Yes, the random variables used to define both components in the sum decomposition of Eq.1 are defined with respect to a single, generic transcription start site, but these random variables take their respective values from data for all transcription start sites in the nucleus. This is why the terms Eq. 1 and the log-ratio in Eq. 2 must be understood as chromatin-wide terms. Again, this approach intends to shift attention from specific (however important) molecular mechanism to higher-order, information conveying hologenic/epigenetic constraints (whose imbalance are proposed to trigger senescence as proposed in this theory). The chromatin-wide nature of the hologenic and epigenetic components is not an obvious consideration but it is a very important one, so it is now explicit (twice) in the revised text and I thank you for bringing this to my attention.

For a statistically invariant level of overall histone crosstalk C(X1,...,Xn) in Eq. 1, a growth in magnitude of the hologenic (i.e., non-epigenetic) component must be accompanied by a decrease in magnitude of the epigenetic component and vice versa. This chromatin-wide trade-off might not hold, as you suggest, only if the overall histone crosstalk C(X1,...,Xn) varies significantly (in particular, if it varies significantly throughout adulthood). If, in fact, the overall histone crosstalk C(X1,...,Xn) varied significantly throughout adulthood the proposed theory would make no sense whatsoever. Mathematically, C(X1,...,Xn) is finite and upper bounded by ΣH(Xi) - max H(Xi) (where H(Xi) is the marginal Shannon uncertainty of Xi), and one can further expect that the overall histone crosstalk represented nu C(X1,...,Xn) remains statistically invariant for a number of reason, chief among them the massive chromatin instability that would ensue if it indeed C(X1,...,Xn) varied. For this reason, I included the C(X1,...,Xn) time-invariance as an additional prediction for the falsifiability of the theory.

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.

I went through the use of these terms in the paper and, unfortunately, I cannot reduce them to just one term or dispense with them altogether without losing rigor for the theory. Because of this I decided to include them in the Glossary, hoping that it will make any reader recognize that their respective uses in the text are actually not interchangeable.

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.

This response to this observation can be found in the new Fig. 2 and with greater detail in Supplementary File 1. In summary, the data analysis approach is basically the same used by Kumar et al. (ref. 33). That is, with tandem RNA-seq/ChIP-seq data obtained from the same cell sample a table can be constructed with the rows representing the transcription start sites (TSSs) in the genome and the columns displaying the normalized ChIP-seq signal for each hPTM in different positions relative to the respective TSS (variables X1,...,Xn in the paper) plus a column with the respective measured transcript abundance for each TSS (variable Y).

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.

This is a great observation, thank you. Yes, the correlation in Eq.3 relates strictly to chronological age (in other words, to time). The correlation must hold for somatic cells of the same type according to the theory; now this condition is explicit in the text. In this context, some cell types and tissue may senesce (see new Fig 1c, center) faster than others as you point out; in this case the associated slope is predicted to be steeper, whereas cell types that senesce relatively slower the slope should be gentler. Only in species displaying negligible senescence the hologenic/epigenetic log-ratio should remain constant (i.e., zero slope, as depicted in Fig. 1b, blue curve), or fluctuate significantly in species displaying "reversible" development (Fig. 1b, magenta curve).

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).

The trade-off between capacity for hologenic and epigenetic information within a constant overall histone crosstalk magnitude—in particular, the growth of the former at the expense of the latter throughout adulthood generating a dysfunctional imbalance—is arguably the cornerstone of the theory in fundamental terms. Whatever my response was to your comments about subsection 1.4, this crucial trade-off cannot be taken for granted, however compelling the arguments are. In this context, there is no better solution than putting the hologenic/epigenetic trade-off to the test (see prediction B for falsifiability of the theory, also further detailed in the new revision of the paper). Realistically, however, I expect prediction B to be tested (and the hologenic/epigenetic log-ratio quite thoroughly examined) only if predictions C and D are verified. In that scenario, it will be interesting to see whether the hologenic/epigenetic log-ratio increase may be steeper in some tissues (which should then explain why those tissues senesce faster than others).

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.

I can see now how it seems contradictory because I was saying the capacity for hologenic information (which is about transcriptional levels being accurate for the multicellular individual as a whole) increases up to the point of being dysfunctional at the multicellular-individual level. Here I failed to convey that said dysfunctional outcome derives from the concurrent decrease of capacity for epigenetic information, not from the increase of capacity for hologenic content per se). Thank you for bringing this to my attention. I decided to remove this subsection altogether because the hologenic/epigenetic trade-off is covered in greater detail in the next subsection.

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.

My intention here is to establish a fundamental contrast between the group of diseases we call cancer and senescence using the difference between the concepts of accuracy (average closses of the actual values to a target value) and precision (variance of the actual values, also added to the Glossary). In this context, since cancer is an almost canonical example of gene dysregulation (transcriptional accuracy is lost because capacity for hologenic information is lost), we can understand senescence as transcriptional overregulation in the sense of too much capacity for hologenic information gained over time at the expense of capacity for epigenetic information, thereby losing transcriptional precision). I added a third panel "c" to Fig. 1 to clarify the proposed contrast between cancer and senescence, in terms of impaired transcriptional regulation (i.e., inaccurate up to dysfunction in cancer and imprecise up to dysfunction in senescence).

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).

I am not including examples of mechanisms propagating dysfunctional changes in the senescence process because this theory is not about adding one more possible mechanism to the collection of well-described mechanisms associated to senescence. The theoretical claim I am making here is that the sequence of steps described in subsection 2.4 constitute the primary cause that triggers senescence throughout the multicellular individual's adulthood. This is of course an extraordinary theoretical claim and, as the great Carl Sagan pointed out, as such requires extraordinary evidence (or, in this theoretical paper, an extraordinary prediction aimed to obtain said evidence).

This is why I also added two predictions, C and D, to the now completely rewritten subsection 2.6 "Falsifiability". These predictions are extraordinary in that they provide extremely specific sufficient conditions for either slowing down or accelerating the senescence process in any non-human species. For this reason (i.e., adding predictions C and D) I have duly updated the "Competing interests" section of the paper. [Note: in a separate paper I explore in greater depth the theoretical underpinnings of predictions C and D.]

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.

Thank you for your suggestion, I organized a Discussion section accordingly, placing the predictions for falsifiability in it. Additionally, I changed “Senescence’s ultimate cause” for “Senescence’s proposed ultimate cause”, clarifying also in the Scope subsection 1.2 that ultimate/proximate is used only in terms of the concepts of causality as introduced and named by Ernst Mayr.

Following your advice, I removed the subsection “Can senescence be stopped?” Now, I believe this theory is bound to alienate at least some colleagues in the biological community (among those who read the paper, that is). As you point out, the theory addresses only one small aspect of the senescence process, but it is not any small aspect. It is about what triggers the process or, equivalently, what is the initial link of the causal chain leading to senescence with all its mechanistic complexity. This implies all other proposed primary causes would, simply by logical exclusion, be incorrect.

One of the reasons I developed this theory in the first place is that I failed to find even a single falsifiable description proposing a primary cause of senescence that integrates Mayr's proximate and ultimate concepts of causality. To be clear, this is not a shortage of explanatory accounts for the senescence process; it is a shortage of experimentally falsifiable ones. There are scientific fields where falsifiability is inherently difficult or probably impossible to meet, such as paleoclimatology. But that is not the case in biology—certainly not in developmental biology. This is why I agree with you in that the senescence process is still poorly understood in fundamental terms (which by no means implies all work in the field is worthless).

Forgive me for the following related digression/personal note: Since I started writing this paper I faced the dilemma of how many research works on this topic should I cite, given there is plenty (even considering this is not a review-type article). We scientists are only human and as such we very much like our work being cited in terms of current knowledge. That changes, however, when the explanatory account we like the most (or even worse, the explanatory account we ourselves proposed) is being cited to acknowledge its existence but the lack of predictions to falsify the "theory" is also pointed out (our reaction changes probably because unfalsifiable explanatory accounts can be always dismissed as just storytelling, however compelling the story might be). These rather petty emotional responses of ours have of course nothing to do with the advancement of science. Yet, many scientific journals—however prestigious, and particularly in the life sciences—routinely publish articles the word "theory" describing the work in the title and/or body when there is no prediction to be found for falsifiability. As a student, this is when I learned the interests of the scientific publishing industry (as we know it) and those of science are completely uncorrelated—not really surprising since their respective goals are so different. In this context, I found the PeerRef initiative/model very interesting since it aims to focus purely on the scientific content as opposed to essentially non-scientific considerations.] In the previous revision of the paper I offered only one prediction to falsify the theory, which is arguably cumbersome—and therefore arguably not too appealing—for experimentalist colleagues to test. For that reason I added three predictions (A, C, and D) which are straightforward to test in the laboratory. In this context, I am hoping the scientific community will be open to consider and to test falsifiable theories, however alienating they might be. Especially when we are dealing with a phenomenon for which paradigmatically accepted explanatory accounts is, at least to the best of my knowledge" all we currently have. Last but certainly not least, I wish to express again my gratitude for all the time you devoted to review my paper. Whether or not the theory it presents resists falsification attempts, I firmly believe the paper itself is now better than it was before thanks to your feedback.

Reviewer response

In general, it seems the paper is improved and includes quite a bit of additional clarification and qualifying statements.

The scope is also much more constrained and it is clear that the author is sticking only to proposing a possible theory, which is fine with me, albeit not as exciting as it might have been if the author had gone through and provided real-world examples or evidence. It still bothers me that the work is trying to implicate a very specific mechanism for senescence (chromatic cross-talk and regulation), since now it is clear that the work is mostly a theoretical exercise without much basis in established biology. In other words, chromatin cross talk might be an example of one way that this could happen among others (e.g. other epigenetic regulation or lack thereof). As a theoretical work it is fine as long as you agree that it is sufficient for the scope of the journal

Decision changed - Verified with reservations: The content is scientifically sound, but has shortcomings that could be improved by further studies and/or minor revisions.

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Author response to reviewer Charles A. Schumpert

Dear Dr Schumpert,

I wish to thank you for having reviewed my paper; your comments have been very encouraging for the effort of moving my theory forward. Please find attached two identical copies of my revision of the paper (changes are highlighted in one of the copies) and a supplementary file that relates to one of your comments. Please see below my response to your comments when applicable.

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.

Thank you for kind words, but I have to admit any and all brilliance that may be found in the write-up must be credited to my wonderful editor Angelika Hofmann. Regarding the assumptions, this theory relies on two critical ones, which of course cannot be taken at face value. This is why these assumptions inform prediction B (in the new revision). There are now four experimentally testable predictions to falsify the theory; hopefully some will be appealing to experimentalist colleagues.

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.

One of the problems I faced when writing this paper was how many different explanatory accounts (there is plenty) I was going to cite provided I would have to underscore their lack of testable predictions for falsifying them—which in time becomes dangerously close of being downright unfalsifiable. As Wolfgang Pauli famously said, unfalsifiable theories are not even wrong. In other words, the problem was how many readers I was going to alienate while having no intention to do so. Navigating academia's social ocean is not an easy task, at least not to me, so I decided to compromise. To be clear, I am by no means claiming my theory is correct but underscoring that (i) it can be tested experimentally and (ii) explains the known age-dependent, cell-to-cell transcriptional noise that I argue should be regarded as senescence itself in fundamental terms.

Discussion, revision and decision

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Discussion and Revision

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Author

Reviewer 1 (Takehiko Ogawa, Yokohama City University, ogawa@yokohama-cu.ac.jp ):

We had already suggested in the "Limitations" section on page 10 of the manuscript-PDF testis transplantation experiments to test biological functionality for future studies: https://www.biorxiv.org/content/10.1101/2021.10.12.464060v1.full.pdf

However, these experiments will require new cell lines, new funding, more resources, more logistics, and new ethics approval which we are considering for future studies. We believe that the observations described in our manuscript are valuable to the scientific community and are a basis to conduct further studies.

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Reviewer 2 (Dr. Pradeep G Kumar, Rajiv Gandhi Centre for Biotechnology, and kumarp@rgcb.res.in )

There are no line numbers in the manuscript-PDF but the pages are numbered: https://www.biorxiv.org/content/10.1101/2021.10.12.464060v1.full.pdf Since the pages of the manuscript-PDF are numbered, we think that citation of the manuscript is unproblematic. The text of the manuscript has been checked by multiple experienced authors and found not to require the suggested changes.

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Reviewer (Takehiko Ogawa)

I think that authors admit the limit of the study, which I think is serious. I think the paper has its own value with limitation as almost always in most cases. I would like to take “verified with reservation” as my final decision.

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Decision

Takehiko Ogawa: Verified with reservations

Pradeep G Kumar: Verified manuscript

Verified with reservations

Discussion, revision and decision

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Revision

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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.

The objective of the paper was not to address a research question but to report on a more recent set of PubMed retractions due to insufficient/old information available in the papers published on this subject(PubMed, not WoS retracted articles). One of the initial objectives was to analyze the dynamic of image related retractions(a relatively new subject), a subject for which the information is at least scarce if non existing. We have also studied the impact of retracted research via two citations databases (Google Scholar and Dimensions) and tried to represent the variability of this impact when the author country is being considered. At this time, the paper is the second biggest serie of PubMed retracted articles.

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.*

There was an error from our part, thank you very much for pointing this.

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.*

We have revised the article and added the informations related to previous research on this subject. Thank you so much for this suggestion.

Discussion, revision and decision

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Discussion and Revision

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Author response

We would like to thank the reviewers for their valuable comments. Below we provide pointwise response and the changes made in the revised manuscript.

To Dr. Jyotsnamayee Sabat

Pt-13: 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?

All the Indian isolates available in GISAID for the period 27th Jan – 27th May 2020 were download and considered for analysis. NO state-wise selection was done.

To Dr Parvin Abraham

Pt-12: The dataset is only from 27th Jan – 27th May 2020. Maybe they can include more Numbers.

The period of data collection was restricted to 27th Jan – 27th May 2020 to basically understand the variations observed across different states of the country during the early phase of pandemic. Also, we are interested in assessing the impact of lockdown in containing the spread of COVID19 and state-specific subclusters, if any.

To Hurng-Yi Wang:

Pt-13: 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.

The period of data collection is restricted to 27th Jan – 27th May 2020 to basically understand the variations observed across different states of the country during the early phase of pandemic. Also, we are interested in assessing the impact of lockdown in containing the spread of COVID19 and state-specific subclusters, if any.

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.

   In the revised manuscript we give the sign of DDG predicted using the tools I-Mutant2.0 and the MUpro along with the respective confidence scores. In I-Mutant2.0, the sign of protein stability change predicted and reliability index (which provides confidence to the prediction) are now incorporated in Table-1. Similarly, the sign change and confidence scores given by MUpro on using SVM and NN based models have been incorporated. We expect all the models to give same results, except in cases where the predictions may be hard to make. This has now been explicitly mentioned in the Materials and Methods section: “In I-Mutant2.0, the sign of DDG is based on SVM classifier, and the associated confidence score is given by the reliability index. On the other hand, MUpro provides sign change prediction using two models, one SVM-based and the other using Neural Networks. InTable-1, the predicted sign of DDG by I-Mutant2.0 and MuPRO along with the respective confidence scores is reported.”

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

   There indeed was some redundancy in the information available in the Materials and Methods section and in the section “Analysis of the Mutational Profile of Indian Isolates”. We have now edited the Materials and Methods section appropriately and deleted the para under the above- mentioned section.

3. 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.

   For 20A we define the haplotype comprising four co-occurring mutations D614G, C241T, C3037T, and C14408T. Similarly, six co-occurring mutations C6312A, C13730T, C23929T, C28311T, C6310A (S2015R) and C19524T are shown to be associated with subclade I/A3i. Together as a set, these are useful in identifying clusters or group of isolates with similar mutational profile. However, those that are non-synonymous mutations are likely to have some individual impact on the overall stability of the respective protein. And so, we have presented both these results. To address this point, we have added a sentence at the end of Materials and Methods section and is reproduced below: “While we report individual effects of mutations on protein stability, some of the mutations in a haplotype may not be under natural selection and are just hitchhiking mutations.”

4. 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.

   We have now redrawn the diversity plots in Figure 3 and Figure 8, (labelled Figure 2 and Figure 4, respectively, in the revised manuscript) and are shown below. We have introduced horizontal lines to show the height of the divergence line at variant positions discussed in the manuscript, and these are also marked with the same colour in corresponding subplots for comparison.

In the revised manuscript, Figures 9 and 10 are now Supplementary Figures 2c and 2d respectively. The new figure legends are: Supplementary Figure 2: The sequences carrying the mutations a) C5700A b) C23929T c) C18877T d) G29830T are depicted in yellow colour. Figure 10 (now Supplementary Figure 2(d)) is now re-plotted, and we have removed the blue dot corresponding to ‘C’ since no samples from India had this variation.

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

   It has now been added in the manuscript: Supplementary Figure 2(c) – On Pg-9, in the first line under the heading “Identification of novel subclade I/GJ-20A and unique mutations in Maharashtra”. Supplementary Figure 2(d) – On Pg-11, in the last paragraph under the heading “Identification of novel subclade I/GJ-20A and unique mutations in Maharashtra”.

2. 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.

   From Supplementary Figure 1 clade 20A (shown in ‘Green’) is predominantly observed in Gujarat (178/201) and the distribution of clade 19A (shown in ‘Blue’) is high in Telangana (75/97), followed by Delhi (55/76), Maharashtra (31/80), and Tamil Nadu (19/34). Four mutations, C6312A, C13730T, C23929T, and C28311T are reported to be associated with subclade I/A3i, which is India-specific subclade of 19A. These co-occurring mutations are found in ~32% of Indian samples sequenced (till 31st May 2020). Only 5 isolates of this subclade were observed after May in India with the last one dated 13th June 2020 (according to data available in Nextstrain). This indicates that the spread of subclade I/A3i had been largely contained during lockdown with efforts of contact tracing and quarantining the infected individuals. Also, Telangana and Delhi isolates cluster together due to shared I/A3i mutations, primarily due to the Tablighi Jamaat congregation that occurred just before lockdown was announced. Similarly, clade 20A defining mutations were observed to occur in ~ 90% of Gujarat samples. Due to the countrywide lockdown from 25th March 2020, this clade and its sub-clusters were localized in the state, defined by Gujarat-specific mutations, e.g., I/GJ-20A.

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Hurng-Yi Wang:

I agree to change to Verified manuscript.

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Decision

Verified manuscript

Dr. Abraham: Verified manuscript

Dr. Sabat: Verified manuscript

Dr. Wang: Verified manuscript