Doubleness / Division: Freddie seems to code-swotch frequently, he can switch up between two personalities very quickly (aka North vs South).

This is indeed something that needs to be done. It is really sad to see that these companies are not allowing for such diverse perspectives. I also think it should be a genuine change rather just for PR. Showing diverse faces but failing to incorporate their different backgrounds is just as bad.

The biggest question here is "Why do white people always go against black people even when they have done nothing wrong?" to me the answer to that question is because white people feel there self lesss superior under black people so they try to take advantage of there life by bringing them down with false rumours. White people wont code google to help back people so they wont anticipate such results to appear.

From my understanding of this it goes to show that people coded google to be racist. Its like all the things that google was programed to do and the way it was to respond to searches it would aim at black people being bad.

"One kind of person" is the part of this statement that speaks the loudest to me. This does not only apply to this specific field, but to all jobs that produce a service that is available to a wide and diverse audience. How can I, as a member of a minority, expect my perspective to be reflected in any way in this service/product when none of the producers are anything like me? They are unable to relate to me and thus fail to account for my views. There needs to be someone who can represent me amongst the producers, at least to an extent. A voice that will speak against the discrimination or offensive elements of the service or product that the 'white male' would never notice.

We need to have people who are open minded about cultures and how content posted online can offend people especially those in minority groups. There NEEDS to be representation in the workforce to make a contribution to putting an end to the racial and mysogynistic perceptions presented on the Internet.

Everything looks good, I didn't see where you used the HTML comment to explain your coding/highlight interesting code

I would have used an element selector in the external style sheet to add the styles. This way it is only one line of code and keeps your CSS out of your HTML which is always ideal.

Is he saying that everyone who has morals does not have reason? If that is what he is saying, I disagree because people who have morals use reason to create them.

Split the response into two separate code blocks

Use Request/Response at all places in the code samples

Request in Sample code

Replace Example Request with Request in the Sample code

same comment as Emandate

Create a payment checkout form for customers to make Authorization Transaction and register their mandate. You can use the Handler Function or Callback URL.

EDA tool that quickly generates reports.

I think you have added extra link tag for css file. gone through all code and i felt it you have added extra tags .

This line may be long. You can write each sentence new line. If they are in the same p element, I think it does not effect your code structure.

in this code you may want to format your code so it looks something like this

student bio

</header>

I have read all code and you have written all required elements that assigned in instructions.

This looks great to me but I still don't really understand everything I'm looking at.

Your use of sections seems very sensible. I just used line breaks to space mine but semantically I think this is more sensible.

I see you did that extra fancy bit to make the email link go to the subject line. Very nice

And your comment seems appropriate - to help the collaborators understand your reasoning

Can I ask how you decide how long to make each line of text with the

? Is it an aesthetic choice or just for ease of being able to read each line more easily while you're working?

I would love to be able to suggest something but I'm not knowledgeable enough at this point to see a way to improve upon this.

Three type of social capital.

Author Response

Reviewer #1 (Public Review):

This is a well-done analysis using the very robust Swedish national population registry.

The study strengths include large size, prolonged follow-up, and use of two comparison populations.

Thank you for the encouraging comments on our study.

The main limitations which need to be addressed by the authors are accounting for reverse causality, namely if a psychiatric illness (PI) developed before or about the same time as the CVD. The much steeper risk relationships early after a CVD event are so suggestive. Some further analyses to tease out those with clearly NO PI before CVD would be in order.

Thank you for the comment. Previous studies have consistently reported an association between psychiatric disorders and CVD [1,2], thus, we agree that reverse causality may, in principle, explain some of the observed results indicating a rise in incident psychiatric disorders after incident CVD, particularly during the immediate period. Yet, it is reasonable to assume that a diagnosis of a lifethreatening disease, such as CVD, is in many cases a traumatic experience resulting in an immediate rise in risks of psychiatric disorders. Others have reported such associations e.g. after natural disasters and we have indeed observed such a pattern in our previous work, e.g., after cancer diagnosis [3]. However, we agree that reverse causality cannot be excluded and may partly contribute to the highly increased risk of psychiatric disorder immediately after CVD diagnosis. Indeed, some of these patients may have been attended for their psychiatric disorders in primary care before the incident CVD. As the Patient Register only captures in- and outpatient hospital care, we have conducted an additional analysis, also excluding individuals with previous prescriptions of psychotropic drugs (ATC codes: N05, N06) before their incident CVD – thereby adding a detection of patients with prevalent mental health problems attended by primary care. The results show similar point estimates (Supplementary Appendix Table S5, listed also as below) thus not supporting the notion that reverse causality is a major concern. Furthermore, the association is noted up to 28 years after CVD diagnosis, which is unlikely due to reverse causality.

We have now added our motivation for this additional analysis on the Method (Page 9), as below. “Because the Swedish Patient Register includes only information related to specialist care, we might have misclassified patients with a history of milder psychiatric disorders diagnosed before index date attended only in primary care. To account for the reverse causality of having undetected psychiatric disorders or symptoms before the incident CVD, we performed a sensitivity analysis additionally excluding study participants with prescribed use of psychotropic drugs before the index date (ascertained from the Swedish Prescribed Drug Register including information on all prescribed medication use in Sweden since July 2005), and followed the remaining participants from 2006 to 2016.”

Second, for the robust matched cohort design, the authors age and sex matched each patient with 10 individuals from the general population and then also stratified their model by the matching variables. Could adjusting for matched factors in such cohort studies re-introduce bias into these estimates?

Thank you for the comment. Adjusting for matching factors should provide estimates with the same validity as using a stratified model. In our study, we matched individuals diagnosed with a CVD with their unaffected full siblings as well as 10 randomly selected, unexposed individuals, on the same age and sex, without such diagnosis. As controlling for matching variables is recommended when there are additional confounders [1,2], we used a stratified Cox model commonly applied in family-based studies [3,4].

References:

1.Sjölander A, Greenland S. Ignoring the matching variables in cohort studies - when is it valid and why? Stat Med. 2013 Nov 30;32(27):4696-708.<br /> 2.Mansournia MA, Hernán MA, Greenland S. Matched designs and causal diagrams. Int J Epidemiol. 2013 Jun;42(3):860-9.<br /> 3.D'Onofrio BM, Lahey BB, Turkheimer E, Lichtenstein P. Critical need for family-based, quasiexperimental designs in integrating genetic and social science research. Am J Public Health. 2013 Oct;103 Suppl 1(Suppl 1):S46-55.<br /> 4.Song, H., Fang, F., Arnberg, F. K., Mataix-Cols, D., de la Cruz, L. F., Almqvist, C., ... & Valdimarsdóttir, U. A. (2019). Stress related disorders and risk of cardiovascular disease: population based, sibling controlled cohort study. bmj, 365.

Third, the range of PIs associated with CVD is a lot broader than would be expected or unexpected (eg eating disorders!).

Thank you for the comment. We agree with the reviewer that the strong association between CVD and incident eating disorders is somewhat surprising although the link between cardiovascular risk factors (e.g. obesity) and binge eating have indeed been reported [1,2]. We have now performed the analysis on the association between first-onset CVD and following incident eating disorder, additionally excluding individuals with a history of psychotropic medication use. We found that the associations became even stronger after this exclusion (Supplementary table 5). It is possible that individuals suffering their first CVD indeed drastically alter their lifestyle, in some cases resulting in dysfunctional eating and may therefore be vulnerable to eating disorders. Given that the evidence assessing the risk of eating disorder among CVD patients is still limited, our study adds a valuable piece of knowledge on this regard and calls for further investigations to better understand this association.

References:

1.Mitchell JE. Medical comorbidity and medical complications associated with binge-eating disorder. Int J Eat Disord. 2016 Mar;49(3):319-23.<br /> 2.Bulik CM, Sullivan PF, Kendler KS. Medical and psychiatric morbidity in obese women with and without binge eating. Int J Eat Disord 2002;32:72–78.

Lastly, the authors should try to account for secular changes in smoking and alcohol consumption or BMI over the study period. In particular, while Sweden never had very high smoking rates (due to Snus) alcohol use within specific cohorts might have both affected CVD risk (particularly stroke) and PI risk. Examining trends in for example liver cirrhosis over the study time period might help or use sales/consumption data. The authors do recognize a limitation in being unable to adjust for smoking, alcohol, and adiposity.

Some additional analyses to address these points and some more caution in the discussion are required.

Thank you for the comment. As the reviewer points out, we do recognize the potential unmeasured influence of lifestyle factors (e.g. smoking and alcohol consumption) on the studied associations as these data are not collected in the Swedish registries. However, the associations between CVD and psychiatric disorders were quite stable across calendar time, although somewhat stronger by the end of the observation period. The evidence does not suggest a drastic change in lifestyle factors in Sweden during the latter part of the observation period except for a slight increase in alcohol consumption [1,2] and liver cirrhosis [3]. Although we find it implausible that such underlying secular trends in lifestyle are a major contributor in the reported associations, we have now conducted additional analyses, excluding individuals with alcoholic cirrhosis of liver (ICD-10 code: K70.3) or COPD (chronic obstructive pulmonary disease, ICD-10 code: J44) as a proxy for heavy drinking or smoking. The results remained virtually unchanged.

We have now added reasons for stratified analysis by calendar years in Method (Pages 8-9), and as below:

“We performed subgroup analyses by sex, age at index date (<50, 50-60, or >60 years), age at follow-up (<60 or ≥60 years), history of somatic diseases (no or yes), and family history of psychiatric disorder (no or yes). We also performed subgroup analysis by calendar year at index date (1987-1996, 1997-2006, or 2007-2016) to check for potentially different associations over time (i.e., due to lifestyle factors that changed over time, including smoking and alcohol use).”

We found somewhat higher risk of psychiatric disorder observed in recent calendar years than earlier years (as in shown Supplementary Table S3).

We found similar associations between first-onset CVD and incident psychiatric disorder with and without excluding individuals with a history of alcoholic cirrhosis of liver or COPD, used as a proxy for heavy drinking or smoking. The table has now added as Supplementary Table S8, and also shown as below).

We have now added justifications in Method (Page 10) and in Discussion (Page 21), and as below: In method, Page 10:

“To account for potential impact of unmeasured confounding due to lifestyle factors, we performed a sensitivity analysis excluding individuals with a history of alcoholic cirrhosis of liver (ICD-10 code K703) or chronic obstructive pulmonary disease (COPD, ICD-10 code J44), as proxies for heavy drinking or smoking.”

In Discussion (Page 21):<br /> “although we found similar results with and without excluding individuals with a history of liver cirrhosis or COPD, as proxies for heavy drinking or smoking (Supplementary Table S8). We did not have direct access to hazardous behaviors that could potentially modify this association, and therefore cannot exclude the possibility of residual confounding not fully controlled for in the sibling comparison.”

References:

1.Statista. https://www.statista.com/statistics/693505/per-capita-consumption-of-alcohol-in-thenordic-countries/. Retrieved on 19 Aug.<br /> 2.Alcohol and Drug Report. Nordic Baltic Region. https://www.nordicalcohol.org/swedenconsumption-trends. Retrieved on 19 Aug. 3.Gunnarsdottir SA, Olsson R, Olafsson S, Cariglia N, Westin J, Thjódleifsson B, Björnsson E. Liver ;cirrhosis in Iceland and Sweden: incidence, aetiology and outcomes. Scandinavian journal of gastroenterology. 2009 Jan 1;44(8):984-93.

Reviewer #2 (Public Review):

Shen et. al investigated the associations between CVD and subsequent risk of psychiatric disorders using a prospective study design. The authors also performed subgroup analysis by sex, age at cohort entry and at follow-up, calendar year, history of somatic diseases, family history of psychiatric disease, and finally assessed the potential role of psychiatric comorbidity in cardiovascular mortality in CVD patients. The main takeaway of the analyses are the increased risk of psychiatric disorders in CVD patients compared to the different comparison groups.

Though the study uses nationwide registers in a prospective study design setting, there are some methodological flaws with respect to study design.

For assessing the primary aim the authors chose a rather unusual starting point by preselecting the exposure (CVD) group, rather than depicting the nationwide cohort of the general population followed up for a disease outcome with each category having exposed and unexposed individuals. Assuming that the population comparison group comes from the same study population as CVD patients, it is not clear why a similar strategy of study design as those cited in the manuscript (Zhang et. al 2015, Kivimäki et. al 2012, Godin et. al, 2012) was not followed. Similarly, one would expect sibling comparison group w.r.t outcome (psychiatric disorders) and not for exposure (CVD).

Thank you for the comment. As correctly pointed out by the reviewer, we used a matched cohort design, both in the population- and sibling comparison. We firstly identified a nationwide cohort of general population who were born after 1932 and were residing in Sweden 1987-2016. We then identified all exposed individuals with first-ever diagnosis of CVD and matched population controls from this same nationwide population.

A matched cohort design is applied here due to the strong confounding effects of some variables, e.g., age and sex, on the studied association between CVD and risk of psychiatric disorder. Exact matching on age and sex in our study makes the exposed and unexposed groups comparable and relief the confounding effects from matching factors in the design phase. Another practical viewpoint for why we use a matched cohort is a straightforward understanding of the comparison between exposed and unexposed groups being always at the same time, providing measures (such as risks and rates) during the follow-up period that are easily interpreted. Further, we have used this matched cohort design in many of our previous works [1,2] to maintain an identical design in both sibling and population comparison, so that the point estimates can be directly compared. The matched cohort design generates results of equal validity of the more conventional cohort design suggested by the reviewer [3] but has the additional quality of making the results from the various cohorts (here: population- and sibling comparison) more comparable. Our study therefore takes advantage of using a siblingcontrolled matched cohort, which is indeed a cohort design recommended for family-based studies [4] and provides results with similar validity as a full cohort.

We have now added a sentence and a reference in Method to motivate the use of matched cohort design (Page 7).

“We constructed a sibling-controlled matched cohort to control for the familial confounding according to guidelines for designing family-based studies.24”

We have now updated the flowchart to add a box in the top reflecting the source population where both groups were identified from, shown in Supplementary Figure S1.

References:

1.Song H, Fang F, Arnberg FK, Mataix-Cols D, Fernández de la Cruz L, Almqvist C, Fall K, Lichtenstein P, Thorgeirsson G, Valdimarsdóttir UA. Stress related disorders and risk of cardiovascular disease: population based, sibling controlled cohort study. BMJ. 2019 Apr 10;365:l1255.<br /> 2.Song H, Fang F, Tomasson G, Arnberg FK, Mataix-Cols D, Fernández de la Cruz L, Almqvist C, Fall K, Valdimarsdóttir UA. Association of Stress-Related Disorders With Subsequent Autoimmune Disease. JAMA. 2018 Jun 19;319(23):2388-2400.<br /> 3.Sjölander A, Greenland S. Ignoring the matching variables in cohort studies–when is it valid and why?. Statistics in medicine. 2013 Nov 30;32(27):4696-708. 4.D'Onofrio BM, Lahey BB, Turkheimer E, Lichtenstein P. Critical need for family-based, quasiexperimental designs in integrating genetic and social science research. Am J Public Health. 2013 Oct;103 Suppl 1(Suppl 1):S46-55.

Reviewer #3 (Public Review):

Shen et al. investigated the relationship between the diagnosis of cardiovascular disease (CVD) and subsequent diagnosis of psychiatric disorders using national databases and health records over a 30year period in Sweden. They also investigated the association between the diagnosis of psychiatric disorder and subsequent CVD-related mortality. Comparisons were made between participants diagnosed with CVD and siblings without CVD, and between the CVD participants and random age- and sex-matched controls from the general population.

They show that diagnosis of all types of CVD investigated was associated with increased risk of all types of psychiatric disorders considered, both in comparison to non-CVD siblings and general population controls. They also showed that diagnosis of psychiatric diagnosis subsequent to CVD diagnosis was associated with greater CVD-related mortality.

A key strength of this study is the use of national databases and populations, as it has allowed for sufficiently large numbers for important subgroup analyses investigating specific types of CVD and psychiatric disorders. In addition to disease and disorder subtypes, the authors have investigated many other factors that may be important for understanding these relationships, including time of diagnosis during follow-up, year of diagnosis, age of participant, and various comorbidities. The duration of follow-up is another important strength of this study, as is the use of sibling controls to mitigate the potential confounding effect of genetic and early-life environment.

However, while it is acknowledged as a limitation by authors, the lack of lifestyle data is a notable weakness of the study. The authors allude to causal inference in the abstract and discuss controlling for important confounding factors, but this is somewhat undermined by not being able to account for lifestyle factors, particularly since there are shared biological pathways such as inflammation linked to both CVD and many psychiatric disorders. As such, the associations reported in this study are potentially influenced substantially by unmeasured confounding related to lifestyle factors.

Overall, this is important data, and the conclusions around these findings supporting surveillance of psychiatric disorders in individuals diagnosed with CVD due to its association with increased risk of mortality may be of interest to clinical settings.

Thank you for the very positive comments.

Now, we (I, at least) barely ever think of "the web" as bundles of code, but CERN's vision for the web makes sense in context as a research organization. It's almost like a hyper-detailed Javadoc for experiments, code, machines, and those that work with them.

They have the same structure.

Often we have discussions about the language we use at home, on the playground and in the classroom. Using this terminology and teaching students the differences may impact how they communicate for learning.

When to simplify a line of code?

A class is a code template for creating objects. Objects have member variables and have behaviour associated with them.

I see this as setups/

This is a massive failure of infrastructure (and education/"professionalism"—by and large, most people whose careers are in operating or maintaining Web infrastructure don't haven't been inculcated into or adopted the sort of "code of ethics" that sees this as a failure).

The thing might just be for something like the Internet Archive to get into training or selling professional services for handling companies' "Web presence, done the right way". (This is def. take some organizational restructuring, however.) I'd like to see, for example, IA-certified partner organizations that uphold the principles described here and the original vision for the Web, and professional associations that work hard at making sure the status quo improves a lot over what's common today (and doesn't slide back).

To make the text more readable, I recommend adding lines between different content to better organize the code.

I'm having a hard time seeing anything wrong with your code! I've read it over a few times and at the moment nothing stands out that I can see needs to be fixed!

The descendent elements of the body element should be displayed visually. Preceding whitespace between the parent and child block elements makes allows for programmers to visually organize the code.

Different list items should be on different lines to make reading the code easier.

Great coding, i did not found anything wrong with this code.

your code looks good! Don't forget, when using the word I to refer to yourself it is always a captial I :)

I think your code looks good :)

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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Reply to the reviewers

Reviewer #1 Virant and colleagues have devised well-thought-out experimentation and analysis pipelines to obtain unbiased measurements of kinetochore protein counts and distances from the centromeric histone known as Cnp1 in fission yeast. My concerns with this study are mainly regarding the clarity of some of the data analysis strategies and data presentation. The authors should be able to address these concerns without new experimentation.

We would like to thank reviewer 1 for their valuable comments. We have addressed all comments and highlighted the changed sections in our revised manuscript.

1. Segmentation of individual centromeres: In general, the authors are to be commended for including a detailed description of their procedures and analysis method. However, it wasn't readily clear to me exactly how they segmented individual centromeres. The lack of a consistent offset between the fluorescence spots corresponding to the protein of interest and Cnp1 in image in Figure 1 makes this issue even more confusing. It will help to display representative segmented individual kinetochores either in the main figure or a supplementary figure.

We thank reviewer #1 for this comment and highly appreciate that they value our effort for detailed method descriptions. We strongly agree: Correct segmentation and best-possible visualization are crucial for our analyses. We hope that the following clarifications help to understand our work better:

All images of the manuscript are reconstructed from localization files using Rapidstorm, which linearly interpolates the localizations on a subpixel grid and fills the pixels based on the distance between the localization and the center of the main subpixel bin to avoid discretization errors (Wolter et al., 2012). These images are then overlaid with a Gaussian blur corresponding to their localization uncertainty. In our opinion, this procedure gives the most realistic image impression of the data and results in reconstructed images that as best as possible mimic real fluorescence images. This is in our opinion a very important aspect when presenting SMLM data in reconstructed images as it is crucial that one - when looking at those images - does not over- or under-interpret the SMLM data. We extended the explanation in the manuscript: “For visualization, we aimed to reconstruct SMLM images that neither over- nor under-interpret the resolution of the SMLM data and resemble fluorescence images as closely as possible. Localizations were tracked together using the Kalman tracking filter in Rapidstorm 3.2 with two sigma, and the NeNA value used as sigma. Images were then reconstructed in Rapidstorm 3.2 with a pixel size of 10 nm. Rapidstorm linearly interpolates the localizations and fills neighboring pixels based on the distance between the localization and the center of the main subpixel bin to avoid discretization errors (Wolter et al., 2012). These images were then processed with a Gaussian blur filter based on their NeNA localization uncertainty in the open-source software ImageJ 1.52p (Schindelin et al., 2012). Importantly, images were only used for image representation purposes, all data analysis steps were conducted on the localization data directly (see data analysis).”

Importantly, individual centromeres were segmented not on the images but on the localization data directly which we visualized in a self-written 3D visualization software that has the functionality to e.g. zoom in and out and to switch or overlay channels. This flexible visualization tool allowed us to make the best-possible informed decisions for the cluster selections and pairing of POI/cnp1CENP-A pairs. We extended our explanation by: “For several manual steps, localizations were visualized in a custom software, which allows to zoom in/out flexibly and to switch/overlay between the sad1/POI/ cnp1CENP-A channels. Using this tool, individual localizations could be selected and classified. For channel alignment, localizations belonging to the same fiducial marker in all three channels were grouped together. Cells with visible kinetochore protein clusters in the focal plane were selected and classified as individual region of interests (ROIs) and all clusters were annotated. cnp1CENP-A clusters were paired together with corresponding POI clusters. Whenever there was any doubt whether two clusters belonged to the same kinetochore or whether a cluster represented a single centromere region or several, the clusters were discarded. Two exemplary data sets can be found in the zip-file Supplementary Data 1. The annotation work was quality-checked by cross-checking the annotation of two different persons.” Maybe interesting to add is that we initially and extensively tried several ways to fully automate the annotation. As all tested routines could not reach the quality of manually annotated data and had to a large extend be manually rechecked and corrected, we in the end directly annotated the data manually. We were rigorous in case of doubt: “Whenever there was any doubt whether two clusters belonged to the same kinetochore or whether a cluster represented a single centromere region or several, the clusters were discarded (manuscript page 10, section Visualization and Manual Analytics).”

Furthermore, the reviewer is right, we didn’t include too much visual data/results into our manuscript. We now showcase some examples for our annotation. In the zip-folder “Supplementary Data 1”, the reviewer will find two csv SMLM data examples of annotated localization data of cells with a mitotic spindle that passed the quality checks of drift control and channel overlay. For the first example, a cell with dam1 as POI, all 6 kinetochores are visible and all were grouped and separated from noise. One can also nicely see (due to the large distance of dam1 to cnp1CENP-A) which of the six belong to which spindle by the spatial orientation of the cnp1CENP-A and dam1 cluster to each other, and both groups of three have a pair that is spatially closer to the wrong pole, important for question 2 below. The second example, with spc7 as the example POI, has only 5 visible clusters. A closer look shows that one of them has unusual dimensions and a high number of localizations, representing most likely two overlapping centromeric regions. Thus, for this cell, only 4 kinetochore pairs were annotated for further analysis. Also, the cluster pairs are overlapping much more, so a direct decision to which spindle they belong to gets difficult.

Finally, we realized that the example cell used for figure 1 which we chose a long time ago for representative purposes actually is a dataset that did not pass the final quality controls of drift correction and channel overlay. Thus, it is not part of the data that was used for the results of this work. While it is pretty embarrassing that we did not realize earlier, we are really grateful for the reviewers’ question about it. We now replaced it by another example which nicely represents the data that went into the analysis and also represents the biological heterogeneity, not only in offset but also e.g. in shape: In this work, we simplify by ignoring any shape in our current analysis and only use cluster centroid distances. Kinetochore POI cluster shapes are currently investigated in a more detailed follow-up study.

1. Use the mixture coefficient: The authors use the coefficient λ to create a mixture model for the Bayesian inference of distances. The description provided in the methods section is not sufficient for an average reader to understand how this coefficient is ultimately used (I had to look up the code and then the Stan manual for a superficial understanding of this procedure). It will be very helpful to flesh out this part of the model. Similarly, notation for the model that they use should be included either in the Methods or in supplementary data so that casual readers can get some understanding of the model.

We added the following sentence to explain the significance of the mixture coefficient:

“The coefficient then corresponds to the prior probability that the centromere is attached to the first spindle pole.”

We are not sure whether we understand the last sentence of this comment correctly. We added more explanations and definitions to the Stan code (see kinetochore.stan) to make it easier to understand. However, the section “Distance calculation” is intended to give the reader a full understanding of all relevant parts of the model, a look at the code should therefore not be necessary. The Stan implementation of the model uses non-centered parameterization, which might make it appear more complex than what is described in the text. However, this is an implementation detail that is intended to make the posterior more well-suited for Monte Carlo estimation and does not change the underlying statistical model. It should therefore be of no concern to casual readers. Finally, we prepared a new Supplementary Figure S6 to visualize the model for all readers.

1. The regional centromeres in fission yeast can incorporate varying levels of Cnp1 depending on its expression level (e.g. see Aravamudhan et al. 2013 Current Biology, Joglekar et al. 2008 JCB). Much of this "extra" Cnp1 is likely to be incorporated at sites distal to the Cnp1 molecules that directly nucleate the kinetochore. Therefore, the centroid of Cnp1 molecules is likely to be "shifted" to some extent from the foundation of the kinetochore. Any shift in the Cnp1 centroid will be important especially when comparing the fission yeast measurements with the budding yeast data. The authors should ascertain whether such a shift can be detected by comparing the budding yeast and fission yeast measurements.

The reviewer is absolutely right, there is an active discussion in the field to which extend there are cnp1CENP-A molecules at distal sites and thus not part of the platform for the kinetochore structure. While different quantification methods in the literature partly disagree in cnp1CENP-A numbers, we have no indications that our own assessment by PALM imaging is wrong. In this study, we get a very stable (Suppl. Figure S9 b) cnp1CENP-A read-out by PamCherry1, which agrees with our Lando et al. 2012 study (a single color study with a different, much simpler protocol and using a different microscope with different settings and analysis routines, mainly using mEos2 but also some SI data on PamCherry1 for counting cnp1CENP-A molecules). Furthermore, the recombinant fusions in the native locus of cnp1CENP-A are stable and the strains show no signs of growth or phenotypic defects.

While we therefore can argue that we see a native level and undisturbed distribution of cnp1CENP-A, we nevertheless do not know how much of this cnp1CENP-A is involved in building up the kinetochore. What we believe to know is the following:

1. With our ChipSeq data in Lando et al. 2012 we explored the distribution and read-out hits of cnp1CENP-A within the outer repeats as well as the inner centromeric region of all three chromosomes (Figure 3 and SI in Lando et al. 2012). While cnp1CENP-A is highly populated within the ~ 10 to 15 kb large inner centromeric regions, there are less detections in the outer regions. Thus, while ChipSeq is not a quantitative method, we believe it’s showing the correct trend with some cnp1CENP-A in the outside regions but most cnp1CENP-A localizing in the inner region. We believe that in overexpression studies (like e.g. done both in Joglekar et al. 2008 & Aravamudhan et al. 2013), this most likely will differ (but we did not experimentally explore this).
2. We generally would argue that our distances are rather accurate using a symmetry and compaction argument. The about 10kb inner regions are a roughly 3.4 µm long DNA strands at a linear 1-dimensional scale but in vivo are highly compacted. Total kinetochore sizes as seen in EM data for mammalian cells are “approximately 250 nm wide and 80 nm deep, with an electron-opaque inner plate juxtaposed to the centromeric chromatin, a translucent gap layer, and an electron-opaque, chromatin-distal outer plate apparently embedding the plus ends of spindle microtubules” (Musacchio and Desai, Biology 2017, 6(1), 5; 10.3390/biology6010005) and for * pombe also in the 200 nm range (Ding et al., J Cell Biol (1993) 120 (1); 10.1083/jcb.120.1.141). We evaluated the cnp1CENP-A cluster shapes as seen in our SMLM data. The clusters show a major axis length of 218 ± 88 nm and a minor axis length of 110 ± 29 nm on average. Taking into account our NeNA localization precisions, this is in nice agreement with the EM data measuring the lateral extend of the kinetochore structure. All together, we would argue a) that there is no reason or any indication in the literature that cnp1CENP-A not directly involved in kinetochore nucleation preferably gets incorporated on only one of the distal sites and thus would cause an asymmetry. We rather would argue that they are randomly inserted on both sides at low level and thus keep the symmetry needed to determine the center of the cnp1CENP-A cluster involved as the kinetochore platform. We also would argue b) that the structure is highly compacted and thus errors caused by additional cnp1CENP-A molecules will be small in respect to our resolution. We cannot completely exclude that there is such an effect that would increase the measured distances, however, and given all other sources of error (drift correction, channel alignment, cluster selection etc.) this most likely is not the main factor in defining the widths of the posterior distributions as we obtain them (Suppl. Figure S7). This argument is supported by the fact that we do not see any indication of such an effect for cnp1CENP-A-close proteins. We carefully checked fta2, fta7 and cnp20 data and also included some examples for the reviewer (see innerPOIexamples.zip). We hope that the reviewer agrees with us that there is no indication for a systematic asymmetric offset between the POI and cnp1CENP-A clusters. Finally, our distance numbers nicely agree with the distances the Ries group has measured for S. cerevisiae in the co-submitted manuscript. They a) have a point centromere with presumably only one kMT and b) did not use cnp1CENP-A *as their reference, they used spc7KNL1 (spc105).

Virant and colleagues present a rigorous single-molecule localization-based analysis of the kinetochore protein copy number and organization within the fission yeast kinetochore. Although the fission yeast kinetochore has been extensively studied, the spatial organization of its kinetochore components has remained uncharacterized. This manuscript addresses this deficiency, and in concert with the budding yeast study, highlights the conserved and diverged features of the kinetochore in the two yeast species. Therefore, this manuscript will be of great interest to the kinetochore and cell division field.

We would like to thank the reviewer again for their very helpful and highly constructive review.

Reviewer #2

In this study, Virant and colleagues have applied single molecule localization microscopy to map the positions of proteins in the pombe kinetochore. This has not been reported previously and this study is both well-conducted and the data appear solid. They also use a modification of this technique to assess the stoichiometry of kinetochore proteins. The results that they obtain are broadly in line with several previous studies that use other methodology but not in fission yeast nor to this level of detail. There are some important novel conclusions from this work. I would like the authors to address the following concerns prior to publication:

We would like to thank Reviewer #2 for their appreciation of our work and their helpful remarks regarding our manuscript which we will answer below.

1. It is not clear to me why the sad1-Scarlet-I signal in Figure 1C, displays a grid-like pattern? This must be an artefact of image collection or processing. Could the authors explain this pattern since this may affect the ability to find a centroid position of this signal?

Thanks a lot for this comment. Yes, the grid-like pattern the reviewer observed is an artefact from image processing when compiling the exemplary composite 3-color images. We revisited the raw movie data and changed the procedure to produce the exemplary images to avoid this ugly artifact (which did not influence our data analysis as it is not present in the raw movies used for centroid determinations). Please note: we also changed the example cell for figure 1 due to reasons explained in the answer 1 to reviewer 1.

1. It is my understanding that the distances reported are based on the positions of the proteins in one dimension, along the spindle axis, consistent with other studies and as illustrated in Figure 1b. This should be clearly stated in the results section.

The model underlying our Bayesian inference is that cnp1CENP-A-POI and one of the two sad1 spindles are all on one “mitotic” axis, along the kinetochore microtubule. BUT the orientation of this mitotic axis is NOT necessarily parallel to the spindle axis. See Figure 1a, the in red drawn spindle axis is not necessarily parallel to the green drawn microtubules connecting the kinetochores to the spindle poles. (Please note, thanks to this comment, we found that our original sketch of Figure 1a was misleading and corrected for that.). Figure 1b in this respect is a bit misleading as only one spindle pole is shown. The slight difference between the kinetochore and spindle axis cannot be visualized with only half a spindle. For answering the reviewers comment no. 5, we also now plotted the measured offset from and relative position of cnp1CENP-A cluster centroids to the spindle axis, see below.

1. The distances between proteins in this study are measured during anaphase, whereas the distances measured in cerevisiae previously were in both metaphase and anaphase (Joglekar et al 2009) and in the accompanying manuscript (Cielinski et al) in metaphase. In the comparison of distances, it would be worth describing how the mitotic stage may have affected distances, since Joglekar et al, found significant positional changes in cerevisiae kinetochore proteins from metaphase to anaphase.

We would like to thank the reviewer for this comment. It sparked a longer discussion to precisely disentangle the cell cycle states. We afterwards went carefully through the literature again and added a column to Table S4 stating for which cell cycle phase(s) the individual works were conducted which we believe is highly useful when comparing the different data.

Discussing with the Ries group we made sure that we indeed measured the cells in the same state and that we in our manuscript made mistakes in defining it correctly. Important for S. pombe is, that while it is not possible to decide for 100% between metaphase and anaphase A, we can safely exclude anaphase B so that we can state that we did not image anaphase B: Supplementary Figure S10 shows that all spindle distances measured are smaller than one nuclear diameter which is given by 2-3 µm (MacLean 1964, 10.1128/jb.88.5.1459-1466.1964; Tda et al 1981, 10.1242/jcs.52.1.271) plus we ourselves measured a nup132 strain in early G2 phase and obtained 2.4 µm ± 0.19 µm (data not shown)). This is nicely in line with Joglekar et al. 2009 (this paper actually has a very good SI figure on exactly this topic, see Fig. S1) and corrected the manuscript accordingly. Thanks for pointing this out to remove this lapse in definitions.

__ 4. __It is hard to interpret the POI copy numbers in terms of each kMT. I am assuming that each cluster measured represents a single pombe kinetochore, containing 2-4 kMTs? If we assume that each pombe kinetochore can contain 2, 3, or 4 kMTs, then we might expect to see a trimodal dataset, I am guessing this was not seen in the data? Would it be possible to estimate protein numbers per kMT in Table 2, as done for the Cielinski et al study? I realize this would require an estimate of the number of kMTs per kinetochore. Alternatively, the authors are resolving individual kMTs, in which case this should be made clear.

Yes, the reviewer is absolutely correct, the clusters are associated with 2-4 kMT (as nicely resolved in Ding et al, J Cell Biol (1993) 120 (1); 10.1083/jcb.120.1.141). Thus, we can assume that 2-4 kinetochore structures are also involved per centromeric region. In our current analysis, we have to work with the average of 2-4 kMTs. The shapes of POI and cnp1CENP-A clusters we have in the SMLM data are definitely diverse, and we plan to extract more data on spatial distribution in the future, perhaps even at the level of individual centromeric regions, but we did not systematically explore shapes in this work. Thus currently, we cannot give a precise answer how individual regional kinetochores look like at the level of a single kinetochore, but we strongly agree with the reviewer that this will be highly interesting to explore further. In this manuscript, therefore, to compare the stoichiometries between the point centromere of S. cerevisiae and the regional centromere of S. pombe, we used ratios as given in Suppl. Table S4. These ratios provided us with comparable results across a wide range of literature since the ratios are only calculated internally for each study and not across studies (which would lead to compatibility issues).

For this study, we also labeled MT via atb2. Unfortunately, the SMLM experiments were very difficult as atb2 is also present everywhere else in the nucleus, in particular at the dense central MT bundle (see image below, white sad1-mScarlet-I, blue PamCherry1- cnp1CENP-A, and red mEos3.2-A69T-atb2). Thus, we could not resolve such fine details as single fibers for the kMTs: Most kMTs overlapped with the central fiber and due to the dense central MT bundle of atb2, the data of the atb2 channel could not be read-out neither in a quantitative nor complete way and we could not extract which percentage of atb2 molecules we actually successfully recorded in the SMLM data. Thus, especially visualizing fine fibers was difficult and the images obtained do not meet our quality standards – but the exemplary one below is maybe nevertheless informative in a qualitative way. Our current idea would be to use ExM plus SMLM, but this work would be a stand-alone study requiring the set-up and optimization of such a protocol.

1. The same kMT issue may affect the measurements of distances. Each pombe kinetochore contains multiple kMTs and it is not clear whether these would align perfectly on the spindle axis. Did the authors see anything in their data that would support the notion that individual kMTs are aligned on the axis (as illustrated in Figure 2) or whether they are slightly separated? This is itself a potentially important result.

It is important to note that we did not measure the distance in projection of the spindle axis (defined as sad1-sad1 centroid axis), see also question 2. We can show in our data that the main microtubule bundle between the spindles is angled to the kinetochore microtubules that connect the centroids of our three-color channels for each centromeric region in a sad1-POI-cnp1 axis. For sad1, we cannot simply determine which one of the two spindles is the correct one, thus we implemented a mixture model for our Bayesian model, see also answers to reviewer 1).

While in the budding yeast literature it has been measured by EM that there are only small angles of up to 6° between the two axes present (Joglekar et al. 2009, 10.1016/j.cub.2009.02.056, Figure S1), Ding et al. 1993 showed larger deviations for S. pombe. Our data agrees with these findings. In the new Suppl. Figure S12, we plotted the height of all measured cnp1CENP-A centroids, normalized to the spindle lengths to represent the angular distribution between the spindle and kMT axes and in absolute nanometer distances to show that most kinetochores are in direct vicinity to the central bundle and only few show heights larger than 150 nm (also technically important as our focal z-range is ~600 nm).

1. In all measurements of kinetochore protein intensity (both in this study and previous studies) there seems to be significant variation in the data for individual kinetochores, even for S. cerevisiae, which supposedly has a fixed number of the kMTs. The coefficient of variation is ~ 0.5 in the data shown in Table 2. Could the authors discuss the variability in POI copy numbers since it either reflects an inability to measure protein levels accurately or that there is some flexibility in kinetochore protein stoichiometry (or in this case differing numbers of kMTs per kinetochore - see point above)?

Regarding the variability in our counting data we indeed expect a mixture of biological and technical nature in line with what the reviewer argues above but intuitively would lean towards the latter, being technically limited by the read-out precision of quantitative PALM imaging using fluorescent proteins, which have finite maturation- and read-out efficiencies and possess limited signal-to-noise contrast. When discussing this comment and how we possibly could support our intuition by evidence, we realized that the main argument to be made is that the FtnA oligomer is a biologically highly defined structure and that the variance we obtain for our FtnA calibration standard indeed also directly can serve as a proxy for our technical variance. Using the results of 21.63 counts ± 10.2 STD for the 24mers and of 7.27 counts ± 2.72 STD for the 8mers, we thus can estimate that the technical variance causes a coefficient of variance of 0.35 to 0.5, thus almost completely explaining the experimentally seen coefficient of ~ 0.5. we added this argument to our manuscript by “The POI protein copy numbers as given in Table 2 show a large coefficient of variation. To assess to which extend this variability reflects a technical inability to measure protein levels accurately or some flexibility in kinetochore protein stoichiometry (e.g. due to differing numbers of kMTs per kinetochore), we can use the data of the FtnA oligomer counting standard: The FtnA oligomer is a biologically highly defined structure. Thus, our FtnA measurements can directly serve as a proxy for the contribution of the technical inaccuracy of our PALM imaging and analysis strategy to the variance. Using the results of 21.63 counts ± 10.2 STD for the 24mers and of 7.27 counts ± 2.72 STD for the 8mers, we can estimate that the technical inaccuracy causes a coefficient of variance of 0.35 to 0.5, thus almost completely explaining the experimentally seen coefficient of ~ 0.5 for our POI data (Table 2). Due to this high technical inaccuracy, we cannot resolve sub-populations of possibly different kinetochore structures (and thus POI copy numbers) on 2-4 kMTs in our current counting data (Supplementary Figure S9).”. Secondly, as the reviewer also pointed out earlier, we have a biological variance of 2-4 kMTs and thus must assume smaller and larger kinetochores on individual centromeres. Due to the high technical variance, we nevertheless cannot resolve sub-populations in our current counting data (please also compare to the data in Supplementary Figure S9).

Minor points:

Delete "an" from "...structure at an about 100 nm resolution" (page 3).

Thanks!

In Figure 2 the proteins in the schematic are color coded, but it is not clear what the coloured proteins are in all cases. Would it be possible to color code the adjacent text, e.g. Spc7 in orange.

Thanks for this suggestion, adjusted figure accordingly.

Also in this figure, the POI copy numbers are indicated by color coding of the data points. However, the points will likely be too small in the final figure for these colors to be clearly visible. Perhaps copy numbers could be indicated in another way or the "mean value" boxes could be larger?

We tested several options and decided to adjust the widths of the boxes.

Please define "N" in Table 2. e.g. N = number of kinetochores measured.

We added this information to the caption: “N number of centromeric regions analyzed.”

Reviewer #2 (Significance (Required)):

This manuscript, together with an accompanying one from Cielinski et al., are nice complementary studies that provide the first single molecule localization studies of the yeast kinetochore. Although other labs have used super-resolution methods to study individual kinetochore proteins; both of these new studies map distances between many proteins at the kinetochore and thus are able to produce maps of the overall kinetochore structure. Like the previous study using standard resolution methods (Joglekar et al, 2009. Current Biology 19, 694-699); these studies will likely provide a benchmark for future studies on eukaryotic kinetochore architecture, including those in mammalian systems. Additionally, this work will appeal to super-resolution microscopists.

My expertise is as a yeast kinetochore cell biologist.

We would like to thank Reviewer #2 again for their appreciation of our work and their valuable remarks and discussion points which improved our manuscript substantially during the revision phase.

Additional comments for both reviewers:

As the co-submitted manuscript from Cieslinski et al. 2021 re-analyzed all their distances and numbers during the revision phase, we updated the comparisons in Suppl. Table S4, S5 and our summary text: 1) their cnn1 distance measurement got corrected and no shows no deviation anymore to our data. Also, their protein copy numbers changed slightly. So we changed the summary from “Additionally, two organism-specific differences surfaced: cnp20CENP-T (cnn1) is located between spindle pole and cnp1CENP-A in our case (at similar distance as fta2CENP-P and fta7CENP-Q), whereas Cieslinski et al. position cnn1 (and mif2) behind cnp1CENP-A. Furthermore, the ratio of cnp20CENP-T to COMA is 1:0.9 in our case and 1:2.1 for S. cerevisiae“ to “Importantly, one substantial organism-specific difference for the inner kinetochore strategy surfaced: The ratio of cnp20CENP-T to COMA is 1:0.9 in our case and 1:2.0 for S. cerevisiae.” 2) Additionally, we were able to add a discussion about their new measurement of ask1 of the dam1 complex. Ask1 is a protein of the DASH ring. Their new distance measurement of S. cerevisiae ask1 fits to the distance we measure for S. pombe dam1 and thus supports our discussion that, for S.pombe, the C-terminus of dam1 localizes at the DASH ring and not at the ndc80 heads like for S. cerevisiae. We added this sentence to the summary: “Furthermore, the S. cerevisiae work measured the position of ask1, a protein of the DASH ring. Their positioning of S. cerevisiae ask1 is consistent with the distance we measured for S. pombe dam1 and thus directly supports our reasoning that the C-terminus of S. pombe dam1 is localized to the DASH ring and not to the ndc80 heads (like for S. cerevisiae).”

We found that we – very unfortunately - did a calculation mistake ourselves and used the inverse of the correction factor (multiplied with 0.9 instead of dividing with 0.9) when correcting our SMLM localizations to absolute protein counts. Thus, the numbers we gave in Table 2 and the color bar in Figure 2 were wrongly converted. We now corrected for this lapse.

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Referee #2

Evidence, reproducibility and clarity

In this study, Virant and colleagues have applied single molecule localization microscopy to map the positions of proteins in the pombe kinetochore. This has not been reported previously and this study is both well-conducted and the data appear solid. They also use a modification of this technique to assess the stoichiometry of kinetochore proteins. The results that they obtain are broadly in line with several previous studies that use other methodology but not in fission yeast nor to this level of detail. There are some important novel conclusions from this work. I would like the authors to address the following concerns prior to publication:

1. It is not clear to me why the sad1-Scarlett-I signal in Figure 1C, displays a grid-like pattern? This must be an artefact of image collection or processing. Could the authors explain this pattern since this may affect the ability to find a centroid position of this signal?
2. It is my understanding that the distances reported are based on the positions of the proteins in one dimension, along the spindle axis, consistent with other studies and as illustrated in Figure 1b. This should be clearly stated in the results section.
3. The distances between proteins in this study are measured during anaphase, whereas the distances measured in cerevisiae previously were in both metaphase and anaphase (Joglekar et al 2009) and in the accompanying manuscript (Cielinski et al) in metaphase. In the comparison of distances, it would be worth describing how the mitotic stage may have affected distances, since Joglekar et al, found significant positional changes in cerevisiae kinetochore proteins from metaphase to anaphase.
4. It is hard to interpret the POI copy numbers in terms of each kMT. I am assuming that each cluster measured represents a single pombe kinetochore, containing 2-4 kMTs? If we assume that each pombe kinetochore can contain 2, 3, or 4 kMTs, then we might expect to see a trimodal dataset, I am guessing this was not seen in the data? Would it be possible to estimate protein numbers per kMT in Table 2, as done for the Cielinski et al study? I realise this would require an estimate of the number of kMTs per kinetochore. Alternatively, the authors are resolving individual kMTs, in which case this should be made clear.
5. The same kMT issue may affect the measurements of distances. Each pombe kinetochore contains multiple kMTs and it is not clear whether these would align perfectly on the spindle axis. Did the authors see anything in their data that would support the notion that individual kMTs are aligned on the axis (as illustrated in Figure 2) or whether they are slightly separated? This is itself a potentially important result.
6. In all measurements of kinetochore protein intensity (both in this study and previous studies) there seems to be significant variation in the data for individual kinetochores, even for S. cerevisiae, which supposedly has a fixed number of the kMTs. The coefficient of variation is ~ 0.5 in the data shown in Table 2. Could the authors discuss the variability in POI copy numbers since it either reflects an inability to measure protein levels accurately or that there is some flexibility in kinetochore protein stoichiometry (or in this case differing numbers of kMTs per kinetochore - see point above)?

Minor points:

Delete "an" from "...structure at an about 100 nm resolution" (page 3).

In Figure 2 the proteins in the schematic are color coded, but it is not clear what the coloured proteins are in all cases. Would it be possible to color code the adjacent text, e.g. Spc7 in orange. Also in this figure, the POI copy numbers are indicated by color coding of the data points. However, the points will likely be too small in the final figure for these colors to be clearly visible. Perhaps copy numbers could be indicated in another way or the "mean value" boxes could be larger?

Please define "N" in Table 2. e.g. N = number of kinetochores measured.

Significance

This manuscript, together with an accompanying one from Cielinski et al., are nice complementary studies that provide the first single molecule localization studies of the yeast kinetochore. Although other labs have used super-resolution methods to study individual kinetochore proteins; both of these new studies map distances between many proteins at the kinetochore and thus are able to produce maps of the overall kinetochore structure. Like the previous study using standard resolution methods (Joglekar et al, 2009. Current Biology 19, 694-699); these studies will likely provide a benchmark for future studies on eukaryotic kinetochore architecture, including those in mammalian systems. Additionally, this work will appeal to super-resolution microscopists.

My expertise is as a yeast kinetochore cell biologist.

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Referee #1

Evidence, reproducibility and clarity

Virant and colleagues have devised well-thought-out experimentation and analysis pipelines to obtain unbiased measurements of kinetochore protein counts and distances from the centromeric histone known as Cnp1 in fission yeast. My concerns with this study are mainly regarding the clarity of some of the data analysis strategies and data presentation. The authors should be able to address these concerns without new experimentation.

1. Segmentation of individual centromeres: In general, the authors are to be commended for including a detailed description of their procedures and analysis method. However, it wasn't readily clear to me exactly how they segmented individual centromeres. The lack of a consistent offset between the fluorescence spots corresponding to the protein of interest and Cnp1 in image in Figure 1 makes this issue even more confusing. It will help to display representative segmented individual kinetochores either in the main figure or a supplementary figure.
2. Use the mixture coefficient: The authors use the coefficient λ to create a mixture model for the Bayesian inference of distances. The description provided in the methods section is not sufficient for an average reader to understand how this coefficient is ultimately used (I had to look up the code and then the Stan manual for a superficial understanding of this procedure). It will be very helpful to flesh out this part of the model. Similarly, notation for the model that they use should be included either in the Methods or in supplementary data so that casual readers can get some understanding of the model.
3. The regional centromeres in fission yeast can incorporate varying levels of Cnp1 depending on its expression level (e.g. see Aravamudhan et al. 2013 Current Biology, Joglekar et al. 2008 JCB). Much of this "extra" Cnp1 is likely to be incorporated at sites distal to the Cnp1 molecules that directly nucleate the kinetochore. Therefore, the centroid of Cnp1 molecules is likely to be "shifted" to some extent from the foundation of the kinetochore. Any shift in the Cnp1 centroid will be important especially when comparing the fission yeast measurements with the budding yeast data. The authors should ascertain whether such a shift can be detected by comparing the budding yeast and fission yeast measurements.

Significance

Virant and colleagues present a rigorous single-molecule localization-based analysis of the kinetochore protein copy number and organization within the fission yeast kinetochore. Although the fission yeast kinetochore has been extensively studied, the spatial organization of its kinetochore components has remained uncharacterized. This manuscript addresses this deficiency, and in concert with the budding yeast study, highlights the conserved and diverged features of the kinetochore in the two yeast species. Therefore, this manuscript will be of great interest to the kinetochore and cell division field.

Heading missing for the code below. Always have a 'Sample Code' heading for all API codes

Also, looks like there is a formatting issue in the code here...

annoying limitation

have to come up with unique (and unchanging?) URIs up front

otherwise (if type is omitted), this restrictive "about:blank" URI is assumed by default

Loved the fact that you've added comments in the code.

i saw the code and its seems perfect however i didn't know about the hyperlink with the mail one because i don't know that we could use our mail also in hyperlink. so i got new thing to learn and the code is perfect and everything is easy to read.

Perfect example of why they need someone to manage the backlog and let the dev team focus!

Add comment to explain your code or important things

Reviewer #3 (Public Review):

Noel et al provide a neural representational account of three brain areas in a virtual, visual navigation task paradigm especially designed to achieve a closed action-perception loop closely resembling natural behaviour. The authors recorded hundreds of neurons from three monkeys while the animals were engaged in the task where latent cognitive variables like distance travelled and distance to target continuously changed. The authors build on their previous work where they robustly characterized animal behaviour on this task paradigm. Here, they aim to find neural codes of dynamic, latent variables and report a mixed and heterogeneous profile of task variable coding distributed across the two brain areas in the parietal cortex (MSTd and area 7a) and one in the prefrontal cortex (dlPFC).

Major strength: Multi-area recording and the close-loop behavioural paradigm are major strengths of this study. The robust model-based analysis of neural data strengthens the paper even more. The correlation of coupling between MSTd and dlPFC and behaviour, albeit in a coarse time scale (of sessions), is particularly interesting and makes the paper strong by quantitatively relating behaviour to neural activity.

Major weakness: The paper mainly gives a long list of what task variables the three brain areas code for along with measures of connectivity between areas. Although this is a valuable contribution to the field, the study is not designed to test predictions of specific computational hypotheses. Towards the end of the paper, the authors bring up the two alternate mechanisms: vector-coding vs distance-coding, but only as a speculation. These two hypotheses could have been developed further at the outset to make specific predictions for neural dynamics and subsequently be tested in their data. This will likely lead to richer findings going beyond representations of task variables. Nevertheless, the findings presented in the paper are surely novel and exciting.

Impact: The main impact of the paper is neurophysiology under a novel, naturalistic behavioral paradigm. The data, both behavioral and neurophysiological, is rich and has potential to test predictions of more fine-grained computational hypotheses. However, the observation that MSTd codes for latent variables is not as surprising as the authors claim. Given the recent observations of heterogeneous variables represented in brain areas traditionally thought to be highly specific (e.g. locomotion variables in V1, mixed coding in EC etc.), it is not surprising to find latent variables in a 'traditionally' sensory area, especially in a continual behavioral paradigm where many variables are changing and are correlated.

Based on their previous work and this work, the authors mention multiple times the task strategy and its embodied nature. While the authors conclusively show the involvement of eye movement in solving the task, it is difficult to imagine a concrete definition of an embodied task strategy without clear alternate hypotheses. How would the animals behave if their eye movements were prevented? Worse performance (like humans did in their previous paper) or unable to perform (akin to a bird unable to fly without wings) or a different strategy? What should we predict based on the neural observation reported here? The impact of this paper would be greater if the authors bring up these questions and provide some speculations rooted in neurophysiological observations.

Upon clicking the link, the customer should enter their country code and phone number.

Use must sparingly.

"Refactoring is a systematic process of improving code without creating new functionality that can transform a mess into clean code and simple design."

https://refactoring.guru/es/refactoring

This entire selection here is a great example of how content can be made more ‘understandable’. The use of categories on the top of the page is a great way of allowing individuals to track where they are, find content, and navigate. When individuals select a specific category, such as ‘Paralympic Games’, the background of the selection changes to a yellow colour in comparison to the unselected categories, where the background remains white, allowing one keep track of which category they are viewing. This is very important for individuals living with ADHD, autism or dyslexia.

This icon over here in the right top corner, is an incredible example of content that is robust. This means, this webpage is capable of operating and being accessed on a wide range of devices, including assistive technologies. When one clicks on this icon, they are able to view the many accessible features this webpage offers, such as screen readers, bigger text, and text spacing. Additionally, this webpage also has an option to make the content dyslexia friendly.

I don't see anything wrong with section of the code of ethics. Although I can see how this can be seen as a struggle of equality. A Social Worker might feel conflicted as to when the right time is to intervene in a situation. That balance of power is completely one sided. If the social worker in question has a biased for, or against their client, that could shape the future of someones life permanently, for better or worse. I think this section could be fleshed out a little as it's a little vague on what determines a threat to the greater view of society. Harming one person's future for something they haven't, and never were planning on doing is causing harm. At the same time not informing the authorities based on your own intuition, and then having the client do something negative and harmful, is a violation of the Code.

I feel as though this also raises question of power and structural inequality as different access to services because of barriers is being discussed. Why are certain groups because of cultural and socioeconomic differences not granted the same access to services? Why must these groups face such barriers when trying to access a helpful tool for them to receive necessary services? How did this begin? I think once again this excerpt can go more in depth of how structurally these inequalities exist. This can be done by possibly elaborating on what these barriers are and why they represent structural inequality. Social workers aim is to uncover these barriers. Therefore, the Code of Ethics should identify more of these as well.The power this has on the lives of clients I feel needs to be more explained more in depth. For example, the Code of Ethics should go more in depth of the power conflict between these oppressed groups and the others. Furthermore, the impact of these differences because of the structural inequality embedded in our society can be explained more as a whole.

In this excerpt, under the category of cultural competence, I feel as though this excerpt represents an example of structural inequality and power but it could go more in depth as well. This excerpt is identifying that biases to unfortunately take place within the field. As we know, these biases are unfortunately deeply embedded in society, but our job as social workers is to break these biases down and uncover them. I just feel as though the Code of Ethics should go more in depth in discussing this bias and why it is prevalent. This excerpt just simply states that bias exists and social workers should reflect on themselves to examine their biases in effect. If this excerpt mentioned more of how these biases arise, where they come from, why they exist, and exactly what ways social workers can understand it, I feel as though this piece would be more informative. I do not think it sufficiently discusses the severity of structural inequality and how it affects these clients. It does not touch on the problem behind having bias enough. The power dynamic can be touched on more as well by showing how bias is a result of a group of people discriminating against another. Thus making the discriminated group feel inferior. I think in touching on structural inequality and power the importance of understanding cultural differences should be fore-fronted more as well.

The issue here seems to be the transitivity issue. If method A calls B which in turn calls C, then if C adds a new checked exception B needs to add it even if it is just proxying it and A is already handling it via "finally". This seems like an issue of inference to me. If method B could dynamically infer its checked exceptions this wouldn't be as big of an issue.

You also probably want effect polymorphism for the exceptions so you can handle it for higher order functions.

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Reply to the reviewers

Manuscript number: RC-2022-01594

Corresponding authors: Hidehiko Kawai and Hiroyuki Kamiya

1. General Statements [optional]

We would like to extend our gratitude to the Editor and both Reviewers for their constructive and insightful comments to our manuscript. We deeply appreciate the Reviewers’ careful consideration of our work, in result of which we think the paper has greatly improved. Below, we have responded to all points raised by the Reviewers.

2. Point-by-point description of the revisions

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

The analysis of mutations in mammalian, including human, genomes has been of interest for many decades. Early DNA sequencing technologies enabled direct identification of mutations in target genes provided that the mutant genes could be readily isolated. This requirement stimulated the development of shuttle vector plasmids that carried a mutation marker gene and could replicate in both mammalian and bacterial cells. These were used in experiments in which the plasmids, treated with a mutagen, would be passaged through mammalian host cells after which the progeny plasmids were introduced into an indicator bacterial strain. Colonies with mutant marker genes could be distinguished by color or survival, the plasmids recovered, and the sequence of the mutant gene determined. The shuttle vector plasmid that became the most widely used contained as the marker the supF amber suppressor tyrosyl tRNA gene positioned in the plasmid such that deletion mutations associated with mammalian cell transfection were selected against. Although various improvements have been introduced since its introduction in the mid-1980s, including bar codes to distinguish independent from sibling mutations (in the early 1990s), the basics of the system have been maintained, and it and variations are still in use. The Kamiya group has made several adjustments to the supF shuttle vectors, including the construction of indicator bacterial strains based on survival of bacteria containing mutant supF genes (the initial system relied on colony color). They have published many studies of mutagenesis by various agents, error prone polymerases, etc. In the current submission they describe a comprehensive approach to identifying mutations in the supF gene that exploits Next Generation Sequencing technology that can identify the full spectrum of mutations including those that escape detection in phenotypic screens. The study is exhaustive and presents a methodical validation of each component of their approach. They report UV induced mutations, the mechanism of which has been well characterized in previous literature. They also describe a category of multiple mutations, which had been observed in the early work with the supF plasmids, and whose relationship to UV photoproducts is most likely indirect.

*We thank the Reviewer for their very insightful feedback to our manuscript and their positive assessment. We have added some discussion points based on the essential references mentioned in the Reviewer’s comments, which we believe made the explanation of our study more complete. *

Major comments: This manuscript presents a technical advance on the use of the supF mutation reporter system. The extent of the validation of each component of the system, including the bar code is rigorous. Their data on the nature and location of UV induced mutations are in very good agreement with previous studies with supF and other reporter genes, a further validation of their approach. Their discussion of the mechanism of the UV induced mutations is in accord with prior work from other laboratories. However, their interpretation of the multiple mutations, although reasonable in invoking a role for APOBEC deamination of cytosines (see eLife. 2014; 3: e02001 for another discussion of this issue), overlooks a much earlier study on the same topic that showed that nicks in the vicinity of the marker gene are mutagenic and can induce multiple mutations (Proc Natl Acad Sci 1987 84:4944-8). It would be useful for the authors to consider their data on the multiple mutations in the light of the earlier analysis. Furthermore, a check to verify the covalently closed circular integrity of the plasmid preparations would be an important quality control and could reduce the mutagenesis observed in 0 UV controls.

We thank the Reviewer for the valuable comments that made our manuscript clearer and more emphatic. We are hereby addressing all of the Reviewer’s concerns. The available data accumulated from previous studies have proved the high sensitivity of the supF assay as a mutagenesis assay, which now has been clearly supported by the results in the current study. We believe that this NGS assay will be able to fulfil the data requirements to tackle many questions related to mutagenesis, thanks to the simplicity and cost-effectiveness of the procedure. However, to meet the experimental objectives, the preparation and analysis of the library are crucially important procedures in the stages of initial setting up of the assay. The covalently closed circular integrity of the vector library is definitely one of the important points we should pay attention to when performing this assay. After the construction of the BC12-library, we have to check the quality of the library by agarose gel electrophoresis. The background mutation frequency and the sequence of the library itself (uploaded as described in the DATA AVAILABILITY section of this manuscript) also needs to be analyzed by NGS before the experiment. We are also routinely constructing the double-stranded shuttle vector from a single-stranded circular DNA with a variety of site-specific damaged oligonucleotides. The treatment with T5 exonuclease followed by purification is absolutely essential to decrease the background mutation frequency. Without the treatment with the exonuclease, cluster mutations may be increased under specific experimental conditions. For this study, we carried out the conventional supF assay using the BC12-library purified after T5 exonuclease treatment. However, in this case the process of purification slightly increased the mutant frequency of the BC12-library to about 2 x10-4 (corresponding to 1x10-6/bp).Therefore, when setting up the essay, we have to consider the background control that we will need for the data analysis. In response to the Reviewer’s comments, we have now added the following paragraph in the DISCUSSION section:

Page 16, line 25:

”5) For the supF assay, spontaneous cluster mutations at TC:GA sites were often observed, and it was well illustrated in an earlier study that a nick in the shuttle vector was a trigger for these asymmetric cluster mutations (54). Therefore, we need to be aware of the quality of each library and how it affects the outcome of each analysis, especially for detection of very low levels of mutations. Depending on the purpose of the experiments, in the preparation of covalently closed circular vector libraries it is essential to eliminate the background level of mutations. In fact, the in vitro construction of the library of double-stranded shuttle vectors from single-stranded circular DNA requires the process of treatment with T5 Exonuclease, which drastically decreases background mutations.”

Minor points The authors state that only 30% of the base sequence of the supF gene can be "used for dual-antibiotic selection on the indicator E. coli". An earlier review (Mutation Res 220: 61,1989) indicated that within the mature tRNA region single or tandem mutations had been reported at 87% of sites, using the colony color assay. The direct NGS analyses would be indifferent to phenotype, and one would expect the maximum number of mutable sites would be recovered from this approach. It would be helpful for an explicit statement regarding the number of mutant sites to be in the Discussion, as this should strengthen the case for the NGS strategy.

We thank the Reviewer for the helpful comment. These are important points we should indeed mention. This method will complement previous data, and especially the data from titer plates will provide us with non-biased mutation spectra for the whole analyzed region. We have now explained in detail about the coverage of mutation spectra in the DISSCUSSION section.

Page 14, line 14:

“The mutation spectra of single or tandem base-substitutions for inactive supF genes identified by using the blue-white colony color assays were comprehensively summarized in an earlier review article, and it was noted that the mutations were detected at 86 sites within a 158-bp region covering the supF gene (54%) and at 74 sites within the 85-bp mature tRNA region (87%), thus demonstrating the great sensitivity of the supF assay system for analysis of mutation spectra (19). However, obtaining reliable datasets by the conventional supF assay requires skill and experience, especially for studies where the mutations of interest are induced with low frequency. The method has been advanced by the construction of indicator bacterial strains with different supF reporter genes which allow selection based on survival of bacteria containing mutant supF genes. However, the fact that the supF phenotypic selection process relies on the structure and function of transfer RNAs that may be differently affected by different mutations means that the improvement of the efficiency of the selection process may cause loss of coverage of the mutation spectra, as it is under our experimental conditions, where the coverage is about 30% (19,20).”

Page 15, line 4:

“From this point of view, we believe that we can secure a sufficient number of experiments to improve the accuracy of the analysis and to confirm the reproducibility of the experiments. Furthermore, the data from colonies grown on titer plates provides us, at least in principle, and with the exception of large deletions and insertions, with non-biased mutation spectra for the whole analyzed region.

Supplementary Figure 1 shows the organization of 8 supF reporter plasmids. Were these discussed in the text and employed in the experiments? It was not clear in the text.

We thank the Reviewer for the helpful comment. It was indeed not clear which vectors we used and why we constructed a series of vectors. Now, we have added the vectors we used for the constructions of the library and each experiment in the RESULTS and MATERIALS AND METHODS sections. Since this is quite important for us and, we believe, the readers, we also added the explanations in the DISCUSSION section, detailing why we have constructed a series of shuttle vectors, as follows:

Page 19, line 36:

“Mutational signatures identified in cancer cells are emerging as valuable markers for cancer diagnosis and therapeutics. Innumerable physical, chemical and biological mutagens, including anticancer drugs, induce characteristic mutations in genomic DNA via specific mutagenic processes. The mutation spectra obtained here by using the presented advanced method were in good agreement with accumulated data from previous papers where the conventional method had been used, with the advantage that our method provided less-biased mutation spectra data. As described above, the datasets presented here highlighted novel mutational signatures and also cluster mutations with a strand-bias, which could be associated with the processes of replication, transcription, or repair of DNA-damage, including a single strand break (a nick). In this study, eight series of supF shuttle vector plasmids were constructed, as presented in Supplementary Figure S1; however, the analysis was carried out using N12-BC libraries prepared from either pNGS2-K1 (Figures 1-4) or pNGS2-K3 (Figures 5-10). The pNGS2-K1/-A1/-K4/-A4 and pNGS2-K2/-A2/-K3/-A3 vector series contain an M13 intergenic region with opposite orientations relative to the supF gene, which allows us to incorporate specific types of DNA-damage at specific sites in the opposite strand of the vector library. Also, the pNGS2-K1/-A1/-K3/-A3 and pNGS2-K2/-A2/-K4/-A4 vector series contain the SV40 replication origin, which enables bidirectional replication and transcription, at opposite sides of the supF gene. Although this is still preliminary data, it is notable that the spontaneously induced mutations for the different vectors in U2OS cells were not significantly different. Therefore, the here presented mutagenesis assay with NGS, by using these series of libraries, can be applied in many different types of experiments to address both quantitative and qualitative features of mutagenesis. It is possible to design series of libraries containing DNA lesions or sequences suitable for the investigation of specific molecular mechanisms, such as TLS, template switching, and asymmetric cluster mutations.”

CROSS-CONSULTATION COMMENTS Comment on the issue raised by Reviewer #2 regarding plasmids with unrepaired DNA damage introduced into E. coli after passage through U2OS cells: treatment of the plasmid harvest with Dpn1 eliminates un-replicated plasmid DNA. Also, SV40 T antigen drives run away replication of the plasmids, which contain the SV40 origin of replication. This greatly dilutes plasmids with remaining UV photoproducts.

Reviewer #1 (Significance (Required)):

Significance This is a comprehensive description of a technical advance for the analysis of mutations based on the most widely used system for reporting mutations in mammalian, including human, cells. As costs for NGS decline it is likely to become the approach of choice.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

In this manuscript, the authors developed a novel mutagenesis assay by combining the conventional supF forward mutagenesis assay with NGS technology. The manuscript is well written, providing design, methods, and results of the experimental system in very much details, which this reviewer highly evaluates. However, the manuscript may be too long and could be more concise. In addition, this reviewer is afraid that main figures seem difficult to fit printed pages (especially multi-paneled figures of large size, such as Fig. 5 through 8). The authors should re-organize the figures by reducing size and/or moving partly to supplementary information.

We thank the Reviewer for the helpful comments to our manuscript. It is true that the multi-paneled figures were too large, and we have now re-analyzed and optimized most of the figures by reducing size, transferring to Supplementary Figures, and separating one figure into two. Although the number of Figures and Supplementary Figures have now increased, we believe that it has become easy to follow for readers and to fit printed pages. *We considered carefully the Reviewer’s remark about the length of the manuscript, but we feel that the text was already as concise as we could make it, and we have already left out some more detailed explanations. *

1. Some UV-induced DNA damage (typically CPD) is repaired only slowly in human cells, so that the replicated plasmid DNAs recovered from U2OS cells may still contain damage and possibly induce mutations in E. coli after transfection. As the result of high sensitivity of NGS analysis, it is worried that such mutations could be also included in the results. To obtain even more accurate mutational characteristics in mammalian cells, the authors could consider to treat the DNA samples with photolyases before transformation of E. coli. The authors could consider to discuss on this point.

*We thank the Reviewer for the helpful comment, indeed Dpn I treatment is one of the very important procedures for avoiding analysis bias. We have now expanded the explanation why the libraries have to be treated with Dpn I, as follows: *

Page 11, line 4:

“the libraries were extracted from the cells, and treated with dam-GmATC-methylated DNA specific restriction enzyme Dpn I to digest un-replicated DNAs that contain UV-photoproducts.”

1. It is quite intriguing that multiple mutations in a single BC clone tend to occur in the same DNA strand. Is there any trend in a distance between the mutated sites? Considering participation of TLS polymerases in the first round of replication, it may be interesting if multiple DNA lesions occur in relatively close positions so that TLS polymerases elongate the DNA strand without switching back to replicative polymerases.

We thank the Reviewer for the valuable and insightful suggestions for this assay. We have analyzed the positions of SNSs in multiple-mutations shown in Supplementary Figures S11 and S12. As the reviewer mentioned, we may be able to address the mechanisms of TLS switching in mammalian cells by using this assay. In this study, the obtained non-biased mutation spectra of multiple mutations may not be enough for the static analysis, but our results indicate that multiple mutations were induced at relatively close positions. It would be interesting if we could address the mechanisms of TLS polymerase switching. We believe that the accumulation of large numbers of non-biased mutation spectra will provide us with growing opportunities to address more questions in mutagenesis. We have now added the Supplementary Figures S11 and S12, as well as the following discussion points:

Page 14, line 6:

“5) The distance between two SNSs in multiple mutations induced by UV irradiation was relatively shorter than the theoretically expected based on the sequence (Supplementary Figures S11 and S12).”

Page 18, line 27:

“In addition, the positions of SNSs in the multiple mutations were closer to each other compared to the theoretically expected positions (Supplementary Figures S11 and S12), which may reflect switching events involving TLS polymerases. It should be noted that the presented data for the distance between two SNSs in the multiple mutations was analyzed from the data from selection plates in order to secure a sufficient number of mutations, and therefore, there may be a bias due to hot spots associated with the selection process. However, the results from the limited number of mutations from the titer plates are similar to these from the selection plates. It can be proposed that this assay may also be applied for analysis of TLS polymerases in mammalian cells.”

1. This reviewer is wondering whether the results of mammalian cells are influenced by transcription-coupled repair in this experimental system. Because the SV40 replication origin functions as bidirectional promoters, the supF region may be transcribed in U2OS cells so that DNA damage on transcribed strands may be removed more efficiently than non-transcribed strands. Please comment on this, if relevant.

*We thank the Reviewer for the insightful comments. This issue is also very important and interesting, and should be addressed in the mutagenesis research. That is exactly the reason why we presented series of vectors for the assay in this paper. The SV40 replication origin has an effect on the background mutations, which this is also dependent on the experimental conditions. However, this needs to be confirmed by further studies. We hope the idea for these constructions will be helpful for many laboratories. We have now added the following parts in the DISCUSSION section. *

Page 18, line 36:

“Mutational signatures identified in cancer cells are emerging as valuable markers for cancer diagnosis and therapeutics. Innumerable physical, chemical and biological mutagens, including anticancer drugs, induce characteristic mutations in genomic DNA via specific mutagenic processes. The mutation spectra obtained here by using the presented advanced method were in good agreement with accumulated data from previous papers where the conventional method had been used, with the advantage that our method provided less-biased mutation spectra data. As described above, the datasets presented here highlighted novel mutational signatures and also cluster mutations with a strand-bias, which could be associated with the processes of replication, transcription, or repair of DNA-damage, including a single strand break (a nick). In this study, eight series of supF shuttle vector plasmids were constructed, as presented in Supplementary Figure S1; however, the analysis was carried out using N12-BC libraries prepared from either pNGS2-K1 (Figures 1-4) or pNGS2-K3 (Figures 5-10). The pNGS2-K1/-A1/-K4/-A4 and pNGS2-K2/-A2/-K3/-A3 vector series contain an M13 intergenic region with opposite orientations relative to the supF gene, which allows us to incorporate specific types of DNA-damage at specific sites in the opposite strand of the vector library. Also, the pNGS2-K1/-A1/-K3/-A3 and pNGS2-K2/-A2/-K4/-A4 vector series contain the SV40 replication origin, which enables bidirectional replication and transcription, at opposite sides of the supF gene. Although this is still preliminary data, it is notable that the spontaneously induced mutations for the different vectors in U2OS cells were not significantly different. Therefore, the here presented mutagenesis assay with NGS, by using these series of libraries, can be applied in many different types of experiments to address both quantitative and qualitative features of mutagenesis. It is possible to design series of libraries containing DNA lesions or sequences suitable for the investigation of specific molecular mechanisms, such as TLS, template switching, and asymmetric cluster mutations.”

1. page 13: Please check whether the description of Fig. 9C is correct (6th line, graph on top; 9th line, bottom graph).

We thank the Reviewer for carefully checking our manuscript, it was mislabeled in the text. Now, following the Reviewer’s comments, most figures have been changed from the figures in the previous submission. We appreciate the careful review.

CROSS-CONSULTATION COMMENTS Reviewer #1 gives quite relevant comments as an expert of the mutagenesis field. It would improve this manuscript greatly for the authors to make appropriate modifications according to his/her suggestions.

Reviewer #2 (Significance (Required)):

It is quite convincing that this method has a great potential to give much more extensive information on mutational characteristics, most importantly, by eliminating the bias caused by phenotypic selection. Therefore, this work certainly must be worth being published in an appropriate journal.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

The analysis of mutations in mammalian, including human, genomes has been of interest for many decades. Early DNA sequencing technologies enabled direct identification of mutations in target genes provided that the mutant genes could be readily isolated. This requirement stimulated the development of shuttle vector plasmids that carried a mutation marker gene and could replicate in both mammalian and bacterial cells. These were used in experiments in which the plasmids, treated with a mutagen, would be passaged through mammalian host cells after which the progeny plasmids were introduced into an indicator bacterial strain. Colonies with mutant marker genes could be distinguished by color or survival, the plasmids recovered, and the sequence of the mutant gene determined. The shuttle vector plasmid that became the most widely used contained as the marker the supF amber suppressor tyrosyl tRNA gene positioned in the plasmid such that deletion mutations associated with mammalian cell transfection were selected against. Although various improvements have been introduced since its introduction in the mid-1980s, including bar codes to distinguish independent from sibling mutations (in the early 1990s), the basics of the system have been maintained, and it and variations are still in use.

The Kamiya group has made several adjustments to the supF shuttle vectors, including the construction of indicator bacterial strains based on survival of bacteria containing mutant supF genes (the initial system relied on colony color). They have published many studies of mutagenesis by various agents, error prone polymerases, etc. In the current submission they describe a comprehensive approach to identifying mutations in the supF gene that exploits Next Generation Sequencing technology that can identify the full spectrum of mutations including those that escape detection in phenotypic screens. The study is exhaustive and presents a methodical validation of each component of their approach. They report UV induced mutations, the mechanism of which has been well characterized in previous literature. They also describe a category of multiple mutations, which had been observed in the early work with the supF plasmids, and whose relationship to UV photoproducts is most likely indirect.

Major comments:

This manuscript presents a technical advance on the use of the supF mutation reporter system. The extent of the validation of each component of the system, including the bar code is rigorous. Their data on the nature and location of UV induced mutations are in very good agreement with previous studies with supF and other reporter genes, a further validation of their approach. Their discussion of the mechanism of the UV induced mutations is in accord with prior work from other laboratories. However, their interpretation of the multiple mutations, although reasonable in invoking a role for APOBEC deamination of cytosines (see eLife. 2014; 3: e02001 for another discussion of this issue), overlooks a much earlier study on the same topic that showed that nicks in the vicinity of the marker gene are mutagenic and can induce multiple mutations (Proc Natl Acad Sci 1987 84:4944-8). It would be useful for the authors to consider their data on the multiple mutations in the light of the earlier analysis. Furthermore, a check to verify the covalently closed circular integrity of the plasmid preparations would be an important quality control and could reduce the mutagenesis observed in 0 UV controls.

Minor points:

The authors state that only 30% of the base sequence of the supF gene can be "used for dual-antibiotic selection on the indicator E. coli". An earlier review (Mutation Res 220: 61,1989) indicated that within the mature tRNA region single or tandem mutations had been reported at 87% of sites, using the colony color assay. The direct NGS analyses would be indifferent to phenotype, and one would expect the maximum number of mutable sites would be recovered from this approach. It would be helpful for an explicit statement regarding the number of mutant sites to be in the Discussion, as this should strengthen the case for the NGS strategy. <br /> Supplementary Figure 1 shows the organization of 8 supF reporter plasmids. Were these discussed in the text and employed in the experiments? It was not clear in the text.

CROSS-CONSULTATION COMMENTS

Comment on the issue raised by Reviewer #2 regarding plasmids with unrepaired DNA damage introduced into E. coli after passage through U2OS cells: treatment of the plasmid harvest with Dpn1 eliminates un-replicated plasmid DNA. Also, SV40 T antigen drives run away replication of the plasmids, which contain the SV40 origin of replication. This greatly dilutes plasmids with remaining UV photoproducts.

Significance

Significance:

This is a comprehensive description of a technical advance for the analysis of mutations based on the most widely used system for reporting mutations in mammalian, including human, cells. As costs for NGS decline it is likely to become the approach of choice.

I guess this is the basis of modern code version control and colab, or what now commonly known as github. Although, the programming fields seem to adopt it more than "law and public relations" as Nelson imagined.

This is a smart way to save storage space. By allowing common text, code, etc to reside in common memory while letting the differences reside in their own memory space.

The use case for this in the tech industry is paramount. Modern code review systems utilize this extensively to keep track of changes.

drinkagec According to the code chunk below.

visible-web-page looks to have been published and/or written on 2022 June 26.

I emailed Omar a few weeks earlier (on 2022 June 7) with with a link to plain.txt.htm, i.e., an assembler (for Wirth's RISC machine/.rsc object format) written as a text file that happens to also allow you to run it if you're viewing the text file in your browser.

(The context of the email was that I'd read an @rsnous tweet(?) that "stuff for humans should be the default context, and the highly constrained stuff parsed by the computer should be an exceptional mod within that", and I recognized this as the same principle that Raskin had espoused across two pieces in ACM Queue: The Woes of IDEs and Comments Are More Important Than Code. Spurred by Omar's comments on Twitter, I sent him a link to the latter article and plain.txt.htm, and then (the next day) the former article, since I'd forgotten to include it in the original email.)

Social permission floating through the inheritance of access codes. This is really interesting!

A fine example of Morse Code communication.

Just a thought...I think a major issue parents are having with these books being in the library is that there is no rating system in place to help children know what they will find inside a book. "You can't judge a book by its cover" has never been a truer saying. For example, my eleven-year-old is an advanced reader and is constantly looking for new books to read. He has long since moved from the MG reading level to YA, and this has presented some problems. A few months ago, he found a sci-fi book in the YA section that looked like a decent read. Knowing about the issue of pornography, he brought the book down to me to double check before he took it home. It was by an author I'd never heard of, but by the cover and summary on the back, it looked like a decent read (space exploration, scientific experiments, etc.--totally a book my science-brained math whizz kid would enjoy). I told him it looked fine, but then on a whim cracked it open to the middle. I kid you not, I opened directly to a page with major nudity, sex, and drug use. The content was definitely inappropriate for my poor 11-year-old, who is still nowhere close to hitting puberty! And this is the major problem I believe parents are having with these books in the library: kids don't know what they're going to find inside a book. I would love to see public schools (could be a state-by-state thing) adopt some kind of system to inform readers what they will find inside their YA and MG books. This could be some kind of code placed on the back (N for nudity, D for drug use, S for sex, etc.) so that children aren't unintentionally exposed to content they don't want. There are a few websites online that claim to rate books, but they are not widespread, and many of them require membership fees or their rating systems are wonky. If there were a shared database with accurate information on book content that librarians and school board members could pull from when they select books for their shelves, I think there would be fewer issues with parents trying to ban books.

• OK
• INTERESTING

• INTERESTING

• V8

Down with efficiency! I have to go through all these links

I wonder how much Artificial intelligence and virtual reality enhancements will affect the role of the learner designer. I know we will have to learn the platform, its technology but at what level. Will we have to learn how to code to be able to do an effective learning environment.

Aside from the line break suggestion, I think the overall code itself is looking great :)

Something that I find really helpful for being able to review my own code is making sure new elements, like h1 and p are on separate lines in visual studio

A better tittle is "Introduction to Econometrics with Base R". You should justify and explain why you use base R in this book rather than tidyverse, despite the fact that there could be several reasons you could use tidyverse rather than base R: 1) Many of the things you do throughout the book could be done more easily with tidyverse. 2) tidyverse is more used than base R. 3) it is much easier to understand tidyverse code.

Very useful resource but it risks being outdated at publication because it is based on base R.

This focus is exactly why I started blogging some 21+ years ago and why I'm feeling the ache of less frequent postings now.

Creating compounds is not as simple as knowing their components

The producers interview Sonia Ben Ouagrham-Gormley in the bio-defense program at George Mason University. ("I study weapons of mass destruction, particularly biological weapons.") She notes that there is more to creating a compound than knowing its ingredients. There is a science and an art to it as well. So although the source code and the data sets are open access, it still takes chemistry know-how to create the compounds.

Great job, Cat. Additions to the skeleton code look good.

This area of the Code of Ethics raises questions for me about how you might handle a situation because it says that there may be no simple answers which I agree with. But like it says in the passage if you go back to the ethical values, standards and principles and apply it to the tough situation it can make it easier. I believe if I went through those steps there can be a simple answer for most situations throughout social work even though the tough times.

This area in the Code of Ethics seems relevant a situation that I have experienced doing volunteering or field work because every type of work that I have volunteered in or done relating to social work field experience was on the behalf of vulnerable and oppressed individuals and groups of people. That is why I like the field of social work because I get to help people in issues of oppression. poverty, unemployment etc. When I volunteered with habitat for Humanity, we worked with oppressed families that were homeless and in poverty and provided the adults and their children free clothes, shoes, food etc. to help them in their tough situations that they were facing. I start my internship with PRAB next month but at the orientation she explained that we will be helping predominately Hispanic 3rd and 6th graders afterschool to get their homework done and set them up for success. Black and Hispanic students do not always get the attention that they need especially if they live in low-income neighborhoods and not the best areas but that is what we are there for to encourage them and help them with their school needs. When it said, "Social workers strive to ensure access to needed information, services, and resources; equality of opportunity; and meaningful participation in decision making for all people". It is the truth because as social workers we make sure every good opportunity is presented and we strive to ensure that all individuals and family's needs are met regardless of what situation they are in.

I feel as though this excerpt which discusses ethical obligations coming in conflict with agency policies raises question for me in handling situations with clients. The Code of Ethics states that if a reasonable resolution does not seem attainable, the social worker should seek out proper consultation. I feel as though this allows for some gray area to be present. This is because as always the social worker should use this Code of Ethics as a guide to their practice, but if a supervisor is stating to handle a specific instance in a certain manner it can become difficult to navigate the path to success. For example, say I am in a session with a client and am faced with a situation that puts my ethical obligation in conflict with a policy my agency holds, some decisions need to be made right on the spot when dealing with a client like as the dialogue is taking place. Based on the Code of Ethics, should I handle a situation by stopping and contacting my supervisor for consultation, although what they say can be breaking an ethical obligation? I feel as though this may break rapport with a client and breaking an ethical obligation is a truly severe issue. Therefore, this area of the Code of Ethics does raise question for me on how to best handle a situation like this when it arises. If proper consultation is not accessible at that moment and a decision needs to be made right away I question how to handle a situation like this as my obligations ethically and with the agency are put to the test.

This specific area of the Code of Ethics is relevant to a situation that I have experienced in my field work for various reasons. First off, I encountered a situation when working with a client, with my supervisor present, that required me to keep this value and ethical principle in mind. In this instance, the client was expressing to me the way her family dynamic is run based on her cultural views. Although, my family dynamic may not be run the same way as my client's, in correspondence with the ethical principle of respecting her inherent dignity and worth, I used this information to be mindful of her individual differences and understand how this might play a role in her situation. This is an example of treating my client not only with respect but seeing how culturally her family operates their household. Furthermore, I could identify the emotional impact expressing this situation had on my client so I handled her feelings with empathy and compassion, as requested of us as social workers. As the session progressed, the client expressed difficulty in fully finding her drive when completing tasks in her daily routine. Due to this, I prepared self motivating activities and strategies for the client to visualize ways to push herself to complete these tasks. Specifically, we worked together on a time management chart where the client mapped out her schedule. In this, I was attempting to promote her socially responsible self-determination. By helping the client find her inner drive, I was attempting to instill in her that she was not only contributing to her personal self-determination but also to the social community around her as well. I hoped that helping her visualize this, it would make her want to act in positive ways and see how her inner self is a wonderful piece to the puzzle of her community around her. Moreover, this falls into enhancing her capacity and opportunity to change. My client expressed to my supervisor and I that it was difficult for her to pinpoint exactly what areas she wanted to work on and she felt like she needed assistance with. Through our discussion of her strengths, her interests, her hobbies, and especially her goals, I took note that the client began to open up more as the discussion went on and find her way of speaking on her struggles. This is a relevant example of respecting the dignity and worth of my client because in acknowledging her struggles, I am showing that I am not only a listener to her but a guide to her as well that seeks to help her find her inner desires. I constantly used phrases like, "I can absolutely see how you feel that way.." as a means of reassuring the client she is heard and respected. In my discussion with my client, the piece of the Code of Ethics that states, "seek to enhance clients capacity and opportunity to change" I think is especially relevant. This is because in my session with the client I aimed to have her strive to seek out just how much potential she truly possesses through conservation and self-reflection activities I provided. By giving my client strategies towards positive change, I felt as though this was representative of my client being able to pinpoint her needs while also understanding just the positive strategies she can use to meet those needs. In terms of the opportunity to change, in our session we talked a lot on this change towards a positive mindset and the ways she can do that on her own time as well, which I think also falls into this ethical principle.

Who wouldn't worry. To test out

I have looked over this code and found nothing wrong with it

I don't any issues with your code, but might recommend the use of capitals in your Title, headers and paragraphs :)

your code looks good :)

I have looked over this code and found nothing wrong with it.

I don't see anything wrong with your code!

I have looked over this code and found nothing wrong with it.

I've looked over your code, and I don't see any issues!

The heading and the paragraph should be written on different lines for better readability. While this is a simple web page with only two elements, on a more complicated website, having multiple elements on the same line makes code editing harder.

Moving "<p>This is Ezra speaking.</p>" to it's own separate line below "<h1>Hello World</h1>" would provide for easier readability when looking at the code.

I went through the code and everything looks great.

code format maybe?

Nothing wrong here that I can see, but I am personally wondering is it best to enter the code in between this many lines instead of one after another?

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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Reply to the reviewers

In this study we reveal that, in both mice and humans, the metabolic benefits of caloric restriction (CR) are sex- and age-dependent. Through a systematic review of the literature, we show that sex differences have been largely overlooked by previous CR research, a finding that Reviewer 1 highlights as “an important point”. Our results have critical implications for understanding the fundamental biology linking diet and health outcomes, as well as translational strategies to leverage the therapeutic benefits of CR in humans.

We thank the reviewers for their helpful appraisal of our manuscript, which Reviewer 2 highlights as “a very well written paper”. Reviewer 1 emphasised the translational relevance of our findings and commented on the “systematic” nature of our study. They noted that it “was well performed”, ”is a valuable and important contribution to the field”, and “will elicit great interest in the scientific and public readership.” Indeed, the importance of sex as a biological variable is the focus of a September 2022 news feature in Nature (https://www.nature.com/articles/d41586-022-02919-x), underscoring the timeliness and relevance of our findings. Our response to the reviewers comments is outlined below, including the changes we have incorporated in a revised version of our manuscript.

Reviewer 1 – Major Comments:

__A) The clinical part is definitely the weak spot in the study. I don't think that the data should be omitted, but the authors should be very careful in interpreting the data. Obvious limitations apply to this part, which need to be more directly addressed in the abstract and discussion. It feels like the data from the small-scale clinical trial is exaggerated. __The clinical study was conducted by Prof Alex Johnstone’s group at the Rowett Institute of Nutrition and Health, University of Aberdeen. Her group are experts in the study of dietary interventions for weight loss. The study was conducted to a high standard and therefore we have the utmost confidence in the conclusions drawn from our analysis of this data.

As we discuss in response to the reviewer’s other points below, the clinical study was primarily designed to address other outcomes and we analysed the data retrospectively to investigate if sex and age affect CR-induced weight and fat loss. This explains some of the limitations that the reviewer mentions, e.g. the relatively low numbers of younger males, and the focus on overweight and obese subjects. As requested, we have now addressed these limitations as follows:

1. Updated the abstract to clarify that the data are from overweight and obese subjects.
2. Updated the results to emphasise that we did a retrospective analysis of CR in overweight and obese subjects (lines 396-398).
3. Performed an additional ANCOVA analysis to test if baseline adiposity or BMI contribute to the sex differences in body mass, fat mass or fat-free mass (new Supplementary Figure 11); see Reviewer 1 Major Point D below.
4. Updated the ‘Limitations’ section of the Discussion to highlight the retrospective nature of the human study (lines 746-748).
5. Updated the Methods to again clarify the retrospective nature of the analysis (lines 884-885). __B) It is important to mention in the abstract and the discussion that the human data came from obese participants. This might well influence the findings from human data. __The human subjects were overweight or obese; this was previously stated in the methods section (line 885) and in the discussion (lines 509-511). To further clarify this, we now also mention it in the Abstract (lines 52-53) and have reiterated it in the Discussion (line 744). Importantly, the fact that humans still show age-dependent sex differences in fat loss, even when overweight and obese, supports our conclusion that this age effect in mice is not simply a consequence of aged mice being fatter than younger mice. We refer to this as the ‘baseline adiposity’ hypothesis (lines 500-518 of the Discussion). In response to point D below, we have also analysed if the loss of fat mass or fat-free mass is influenced by adiposity or BMI at baseline (pre-CR). Our analyses show that neither of these parameters explain the sex differences in loss of fat mass or fat-free mass (see new Supplementary Figure 11).

__C) It is very important to calculate the % calorie restriction of the human participants achieved throughout the CR study. This is crucial information to compare it to other studies. __We have updated the Methods (lines 906-909) to explain the basis for the weight loss diet, as follows: “Participants had their basal energy requirements determined and each participant was then fed an individualised diet with a caloric content equivalent to 100% of their resting metabolic rate (Table 3). This approach was taken to standardise the diet to account for individual energy requirements and energy restriction.” We have also updated Table 3 to show the caloric intake for males and females. Note that RMR accounts for ~60-70% of total daily energy expenditure (TDEE) in adults (Martin et al., 2022), so the diet in our study would give a daily caloric deficit of around 30-40% from baseline TDEE.

__D) Since there is quite a wide range in the BMIs of the participants, can the authors also stratify against BMI? __We have done this against both baseline BMI and against baseline fat mass (the latter to further test the ‘baseline adiposity’ hypothesis). We present this data in an updated Supplementary Figure 11. We find that, in males but not in females, baseline BMI or fat mass are significantly associated with the changes in fat mass or fat-free mass: surprisingly, individuals with higher baseline fat mass or BMI show less fat loss and a greater loss of fat-free mass during CR. Importantly, males and females do not significantly differ in the relationships between baseline fat mass (or BMI) and loss of fat mass or fat-free mass. This further supports our conclusion that the sex differences in fat loss are unrelated to differences in baseline adiposity. We report this in lines 409-411 of the Results and lines 513-515 of the Discussion.

__E) There is no mention of any pre-study registration online of the clinical part (e.g. _gov_). Was this done? __This study was done before pre-registration was a requirement for clinical trials. We retrospectively analysed the study data to investigate if sex and/or age influence the outcomes. In the updated manuscript we now state this on lines 884-885 of the Methods, as well as in the Results (line 396) and Discussion (lines 746-748).

__F) In the methods section the authors write "Participants were informed that the study was funded by an external commercial sponsor...". This is important information, and this is not mentioned anywhere else in the paper. Can the authors clarify this point? A commercial sponsor would, in my view, qualify for a conflict of interest that needs to be mentioned. __We have updated the Declaration of Interests section to clarify this as follows: “The human weight loss study was funded by a food retailer; however, the company had no role in the data analysis, interpretation or conclusions presented in this paper.”

__G) How did the authors determine the group sizes for the clinical part? I have some doubts about the sub-group sizes. It would be valuable information if the authors had a statistical analysis plan prior conducting the study. It appears a bit, like the sub-groups were chosen at random, to match findings of the mouse data. Otherwise, there should have been a better allocation within the sub-groups (especially age). __We agree that larger group sizes would have been preferable. This limitation reflects that the study was not originally designed to test age and sex effects on CR outcomes, but instead was analysed retrospectively to investigate the impact of these variables. As mentioned above, we have updated the text of the manuscript to highlight the retrospective nature of the analyses. In the Discussion, under ‘Limitations’, we also highlight the fact that relatively few younger subjects are included in the human study (lines 744-745).

__H) *There's a big problem with the age stratification of the male participants in the clinical data. If I'm correct there are only 5 males 45 groupings.

__I) The applied protocol for CR in mice is known to provoke long fasting phases and probably elicits some effects through fasting alone, rather than the caloric deficit. There are some papers out addressing this (e.g. by deCabo, Lamming). The authors should not dismiss this fact and at least address it in their discussion. Also, given this fact, it would be thoughtful to include a database-search - not only regarding CR - but also regarding various types of intermittent fasting protocols in humans and animal studies (similar to what the authors did in the supplemental figure). __We agree on the importance of highlighting recent studies demonstrating that prolonged daily fasting contributes to the outcomes of typical ‘single-ration’ CR protocols. We have added a new paragraph to the Discussion to address this (Lines 710-719).

Regarding the second point, we feel that including a new literature search that addresses not only CR, but also intermittent fasting, is beyond the scope of the current manuscript. However, this is a very good idea and would be worth addressing in a future standalone review article. We have also updated our source data to include all data from our literature reviews, to help if other researchers wish to analyse according to fasting duration or other variables.

__J) Did the authors monitor the eating time of the mice? __We have since done this in new cohorts of mice fed using the same CR protocol. We find that the mice consume their food within 2-3 hours, consistent with other CR studies. We have now mentioned this in the Methods section (lines 867-868).

__K) While CR certainly has a lot of health benefits in rodents and humans, it should be advised to raise the cautious note that it may not be beneficial for everyone in the general population. For some groups of people and in some cases (e.g. infectious diseases, pregnancy) even CR with adequate nutritional intake of micro/macronutrients might be disadvantageous. This should be mentioned clearly, as the topic gets more and more "hyped" in public media and online. __We now highlight this important point in the opening paragraph of the introduction (lines 65-67).

__L) There is no indication of how the authors dealt with missing data. Statistically this can be very important, especially in cases with a low number of data points. __In the Methods section we previously explained (lines 846-848) that “Mice were excluded from the final analysis only if there were confounding technical issues or pathologies discovered at necropsy.” No data had to be excluded from our human study and we have now stated this in the Methods (lines 897-898). For analyses involving paired or repeated-measures data (e.g. time courses of body mass or blood glucose), if data points were missing or had to be excluded for some mice then we used mixed models for the statistical analysis. We have now updated this information in the ‘Statistical analysis’ section of the Methods (lines 1047-1048). Because of the large numbers of mice used in our studies, analyses remain sufficiently well powered even if some data points were missing or had to be excluded.

__M) Key data from qPCR should be followed up by western blots or other means. If this was done and there was no effect, the authors should report this. Also, is there any evidence or the possibility to support these findings regarding pck1 and ppara in human samples? __As requested, we will next use Western Blotting to assess the expression of proteins encoded by the transcripts that show sex and/or diet differences within the liver (Fig. 6A). These data will be reported in our fully revised manuscript.

Regarding effects of CR on PCK1 and PPARA expression in human liver samples, no human CR studies have taken liver biopsies for downstream molecular analysis. Recent studies of the GTEx database confirm that hepatic gene expression in humans is highly sexually dimorphic (Oliva et al., 2020). We checked PCK1 and PPARA in the GTEx database and found that, in the liver, each of these transcripts is expressed more highly in females than in males (https://www.gtexportal.org/home/gene/PCK1 & https://www.gtexportal.org/home/gene/PPARA). While this is the opposite to what we observe in our ad libitum mice (Fig. 6A), it demonstrates that sex differences in these genes’ hepatic expression do occur in humans. The effect of CR on their hepatic expression, and whether this differs between males and females, remains to be addressed.

N): I think it would be very valuable to analyse the sex-differences in lipolysis directly in fat tissues. The authors concentrated on differences in hepatic mRNA profiles, but there's an obvious possibility and gap in their story. ____We agree that this would be informative. In the Discussion we cite previous research identifying sex differences in adipose lipolysis and lipogenesis and explain how this fits with our findings (lines 567-574). Since submitting our manuscript, we have begun experiments to investigate sex differences in the effects of CR on lipid metabolism and molecular pathways in adipose tissue. However, these analyses are extensive and ongoing, so we feel strongly that attempting to include them in our present paper would not only substantially delay publication, but also overload what is already a very extensive paper. Therefore, we plan to report our findings in future publications.

__O) Given the relatively low n and sometimes small effect sizes I fear that some of their findings won't be reproduced by other labs. Were the (mouse) data collected all at once in one cohort or did the authors pool data from different cohorts/repeats? __We presume the reviewer means ‘relatively high n’, as most of our mouse analyses used large group sizes. The mouse data were pooled from across multiple cohorts, with ANOVA confirming that the same sex-dependent CR effects were observed within each cohort. This reproducibility across multiple cohorts is a clear strength of our study because it demonstrates the robustness of our findings. Importantly, the sex differences in fat loss, weight loss and glucose homeostasis were still observed in our much-smaller cohort of evening-fed mice (Fig. S5-S6) (n = 5-6), demonstrating that large sample sizes are not needed for other researchers to detect these effects.

Reviewer 1 – Minor comments:

__a) The discussion is very extensive, and I suggest compressing the information presented there to make it more easily readable. __We have removed some text that was more speculative, such as the paragraph discussing a possible role for ERalpha. We have also revised wording elsewhere to state things more succinctly. However, given the scope of our study we feel we cannot substantially cut down the Discussion without compromising the interpretation of our findings. We note the Reviewer two’s comment that “This is a very well written paper” and feel that attempting to compress the extensive information in the Discussion would compromise, rather than help, the readability.

__b) There is some confusion present in the literature regarding the nomenclature of CR/fasting interventions. Recently some reviews have summarized the different forms (e.g. Longo Nature Aging, Hofer Embo Mol Med, ...) and the authors should address this briefly. Especially the applied CR intervention in ____mice overlaps with intermittent fasting. __We have updated the Discussion (lines 710-719) to explain how our single-ration CR protocol also incurs a prolonged intermittent fast, and how this fast per se may contribute to metabolic effects.

c): The order of the subpanels in Figure 9 (and other figures where B is below A and so on) is confusing. Please rearrange or indicate in a visual way which panels belong to each other.

We disagree that the order of subpanels is confusing: the panels are clearly labelled, and we find it most logical to have the absolute values shown in the top row (panels A, C and E), with the corresponding graphs of fold changes shown beneath each of these (panels B, D and F). This allows the reader to quickly compare the absolute vs fold-change data for each readout. If we had panels A-C on the top row and D-F on the second row, then the connection between graphs 9C and 9D would be less clear and comparable.

d): Did the authors also measure cardiovascular (e.g. blood pressure) parameters? There is some evidence out there that there is an age/sex dependency during fasting/CR. This would be a nice add-on to the rather small clinical data here.

We did measure various cardiovascular parameters for our mice but find, unlike for the metabolic outcomes, these generally don’t show sex or age differences. In our human study we measured blood pressure and heart rate before starting CR and at weeks 3 and 4 post-CR. For this response to reviewers we have summarized these human data in Figure R1. The data show that CR decreases blood pressure and heart rate in males and females (Figs. R1A-E). In the younger age group (We have decided to not include these data in the current study because we feel it is already extensive and is focused on metabolic outcomes. We instead plan to report the cardiovascular outcomes (from both humans and mice) in a separate paper.

__e) What was the decision basis for stratifying the human data into 45 years? __We used 45 years as the cutoff point because this is the age when, in women, oestrogen levels begin to decline (this point was stated in lines 491-492 of the Discussion, and we now reiterate it in lines 414-415 of the Results).

__f) The part on aging starting in Figure 7 comes quite surprising and it is not clearly linked to the data before. A suggestion here would be to smooth the transition in the text and the authors could again perform a literature search regarding age-of-onset for CR/fasting interventions in mice and humans. __We have added a sentence to smooth the transition to these studies (lines 363-364). We had previously done a literature search to identify the age of onset of CR interventions in mice and humans. We summarise the findings of this search in lines 452-470 and 484-495 of the Discussion. We have also updated the source data so that it includes the our review of the CR literature, allowing other researchers to interrogate this data.

g) At the first mention of HOMA and Matsuda indices, the effect direction should be put into physiological context.

We now mention this in lines 231-232 of the Results.

h) There is no mention of how the PCA analyses were conducted.

We have updated the Methods to explain that the PCA analyses were done using R. We have updated the source data to include the outputs from these analyses, as well as the underlying code. These data and code are now available here https://doi.org/10.7488/ds/3758.

i) Were the mice aged in-house in the authors' facility or bought pre-aged from a vendor? Is it known how they were raised? If bought pre-aged, were female and male animals comparable?

We bred and aged all mice in house. Males and females were littermates from across several cohorts. Therefore, there are no concerns about lack of comparability resulting from environmental differences.

j) Very minor note: I think that "focussed" has become very rarely used, even in British English. I don't know about the journal's language standards, but I would switch to the much more common "focused".

We have updated to ‘focused’ as requested.

k) Figure 6B/F (PCAs) should indicate the % difference of each dimension.

We have updated the figures to show the % variance accounted for by each principal component. We have also updated the figure legend to specify this.

l) Limitations section: Maybe tone down on "world-leading mass spec facility". This sounds like an excuse and this statement is unsupported and doesn't add anything valuable to the section. Other limitations would include the low n, as mentioned above and the mono-centric fashion of the mouse and human experiments.

We have addressed these points as follows:

• Toned down the description of our mass spec facility (they are renowned for expertise in steroid hormone analysis, so we our original text was intended to highlight that our facility are not novices for this).
• Regarding the low n for some of the human groups, we now highlight this on lines 744-745 of the Discussion.
• We have added a new paragraph to the Discussion (lines 710-719) explaining the limitations of our CR protocol, i.e. that includes elements of both CR and intermittent fasting. Reviewer 2:

__Point 1: This is a very well written paper. __We thank the reviewer for this kind comment.

__Point 2: Since the authors fed the animals in the morning, this is likely the reason for energy expenditure to be different in the CR vs ad lib groups. Although the authors do study the effects of night v day feeding and saw no change in the outcomes regarding weight, this fact I think should be mentioned somewhere. Also, figure 4A is expressed a W while all the other graphs are in kJ. I think it would be nice to see it all consistent. __Regarding the first point, we agree that time of feeding can influence when energy expenditure is altered, but most studies show that CR decreases overall energy expenditure regardless of time of feeding. For example, Dionne et al studied the effects of CR on energy expenditure, administering the CR diet during the night phase (Dionne et al., 2016). They found that CR mice have lower energy expenditure in the day but not in the night (Figure 3C in their paper), which is the opposite to our findings (Figure 4C). However, total energy expenditure in their study remains decreased with CR. This goes against the reviewer’s suggestion that feeding the animals in the morning “is likely the reason for energy expenditure to be different in the CR vs ad lib groups”. We have updated our manuscript (Lines 576-581) to clarify this.

Regarding the second point, we have updated Figure 4A to express the data in kJ (showing the average kJ, per hour, at each time point). The figure legend has been updated to reflect this.

__Point 3: For all the graphs, can you make the CR groups bold and not filled as it is hard to see the lighter colours. __We have updated the graphs so that the CR groups are represented by solid lines, rather than dashed lines.

__Point 4: I know many investigators use them, but I am not sure how relevant HOMA-IR and the Matsuda index are in mice since they were specifically designed for humans. __The issue of whether it is ‘correct’ to use HOMA-IR and/or Matsuda index in mice is often debated in the metabolism field. Importantly, we are not using the absolute values for HOMA-IR or Matsuda in the same way that they are used in humans; instead, we are comparing the relative values between groups because these are still physiologically meaningful. We discussed this with Dr Sam Virtue, an expert in mouse metabolic phenotyping (Virtue and Vidal-Puig, 2021), who agrees on their usefulness in this way.

__Point 5: Something also to note is the fact that all the glucose uptake data is under basal conditions. Just because there are no differences in the basal state does not mean that there are no differences after a meal/during an insulin stimulation. I think that this needs to be discussed and the muscle and fat not completely discounted as a player in the differences seen. __We agree that CR can enhance insulin-stimulated glucose uptake but our OGTT data suggest that it is effects on fasting glucose, rather than insulin-stimulated glucose uptake, that contribute to the sex differences we observe. We have now updated the Discussion (lines 608-613) as follows, “CR enhances insulin-stimulated glucose uptake (82) and it is possible that this effect differs between the sexes. However, our second relevant finding is that, during an OGTT, CR decreases the tAUC but not the iAUC, highlighting decreases in fasting glucose, rather than insulin-stimulated glucose disposal, as the main driver of the improvements in glucose tolerance.”

References cited in Response to Reviewers:

Dionne, D.A., Skovso, S., Templeman, N.M., Clee, S.M., and Johnson, J.D. (2016). Caloric Restriction Paradoxically Increases Adiposity in Mice With Genetically Reduced Insulin. Endocrinology 157, 2724-2734. 10.1210/en.2016-1102.

Martin, A., Fox, D., Murphy, C.A., Hofmann, H., and Koehler, K. (2022). Tissue losses and metabolic adaptations both contribute to the reduction in resting metabolic rate following weight loss. Int. J. Obes. 46, 1168-1175. 10.1038/s41366-022-01090-7.

Oliva, M., Muñoz-Aguirre, M., Kim-Hellmuth, S., Wucher, V., Gewirtz, A.D.H., Cotter, D.J., Parsana, P., Kasela, S., Balliu, B., Viñuela, A., et al. (2020). The impact of sex on gene expression across human tissues. Science 369, eaba3066. 10.1126/science.aba3066.

Virtue, S., and Vidal-Puig, A. (2021). GTTs and ITTs in mice: simple tests, complex answers. Nat Metab 3, 883-886. 10.1038/s42255-021-00414-7.

Minor point, as this code works perfectly. But putting this comment on its own line will make it more readable

Code looks good. Love the colour green

very nice one line css code there nothing wrong in it and also love the green color

This could be in next line which would make the code attractive.

indentation will help for the code be readable when the code is larger

for a cleaner code, let new tags begin on a new line.

as far as i know this is the clear code and i haven't find any errors in it and it is easily readible and easy to understand. you have done the great work.

This is just a brilliant code and I am not able to find any mistake.

Describes how to build electron app AppImage from source.

Section 22 in The Indian Penal Code “Movable property”.—The words “movable property” are intended to include corporeal property of every description, except land and things attached to the earth or permanently fastened to anything which is attached to the earth.

Section 3, Transfer of Property Act “attached to the earth” means— (a) rooted in the earth, as in the case of trees and shrubs; (b) imbedded in the earth, as in the case of walls or buildings; or (c) attached to what is so imbedded for the permanent beneficial enjoyment of that to which it is attached;

Section 2(6) of Registration Act "immovable property" includes land, buildings, hereditary allowances, rights to ways, lights, ferries, fisheries or any other benefit to arise out of land, and things attached to the earth or permanently fastened to anything which is attached to the earth, but not standing timber, growing crops nor grass.

Section 24 in The Indian Penal Code “Dishonestly”.—Whoever does anything with the intention of causing wrongful gain to one person or wrongful loss to another person, is said to do that thing “dishonestly”.

Section 23 in The Indian Penal Code “Wrongful gain”.—“Wrongful gain” is gain by unlawful means of property to which the person gaining is not legally entitled. “Wrongful loss”.—“Wrongful loss” is the loss by unlawful means of property to which the person losing it is legally entitled.

reassurance of lack of possibility for run-time shenanigans

RE-NUMBERED - PREVIOUSLY 1.14

Someone is going to need to eventually explain their convoluted reasons for labeling this a downside.

Sure, strictly speaking, a bigger source tree is bad, but delegating to package.json and npm install doesn't actually change anything here. It's the same amount of code whether you eagerly fetch all of it at the same time or whether you resort to late fetching.

Almost none of the hypothetical benefits apply to the way development is handled in practice. There was one arguably nice benefit, but it was a benefit for the application author's repo host (not the application author), and the argument in favor of it evaporated when GitHub acquired NPM, Inc.

No, those are orthogonal. Whether you obtain the code for your dependency* at the same time you clone your repo or whether you get it by binding by name and then late-fetching it after the clone finishes, neither approach has any irreversible, distinct consequences re bugs/fixes.

* and it still is a dependency, even when it's "vendored"...